THE AYURVEDIC PHARMACOPOEIA OF INDIA

THE AYURVEDIC PHARMACOPOEIA OF INDIA PART - I VOLUME - IX Government of India Ministry of AYUSH 2016 Published by ... सत्यमेव जयते First Edition. THE ...

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THE AYURVEDIC PHARMACOPOEIA OF INDIA PART - I VOLUME - IX First Edition

सत्यमेव जयते

Government of India Ministry of AYUSH 2016

Published by

PHARMACOPOEIA COMMISSION FOR INDIAN MEDICINE & HOMOEOPATHY GHAZIABAD

THE AYURVEDIC PHARMACOPOEIA OF INDIA PART - I VOLUME - IX First Edition

Government of India Ministry of AYUSH 2016

Published by

PHARMACOPOEIA COMMISSION FOR INDIAN MEDICINE & HOMOEOPATHY GHAZIABAD

PDH.81.Pt.I.Vol.IX 2000-2015-(DSK-II) © 2016, Pharmacopoeia Commission for Indian Medicine & Homoeopathy Ministry of AYUSH, Government of India

On behalf of

:

Government of India Ministry of AYUSH, AYUSH Bhawan, B Block, GPO Complex, INA, New Delhi - 110 023

Published by

:

Pharmacopoeia Commission for Indian Medicine & Homoeopathy PLIM Campus, Kamla Nehru Nagar, Ghaziabad-201002 (U.P.) India

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LEGAL NOTICES In India, there are several laws dealing with drugs for which monographs with quality standards and certain other requirements are prescribed. These monographs should be interpreted subject to the restrictions imposed by these laws wherever they are applicable. In general, the Drugs and Cosmetic Act, 1940; the Dangerous Drugs Act, 1930; the Poisons Act, 1919; Drugs and Magic Remedies (Objectionable Advertisement) Act, 1954; the Narcotic Drugs and Psychotropic Substances Act 1985 and the Biodiversity Act, 2002; all as amended from time to time, along with the Rules framed thereunder, should be consulted to ensure that the provisions of such laws are being complied with. Under the Drugs and Cosmetics Act, the Ayurvedic Pharmacopoeia of India, represented by its Parts and Volumes is the book of standards for substances included therein and such standards are official. If considered necessary these standards can be amended and the Pharmacopoeia Commission for Indian Medicine & Homoeopathy is authorized to issue such amendments. Whenever such amendments are issued, the specific Ayurvedic Pharmacopoeia of India intended thereby would be deemed to have been amended accordingly.

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GENERAL NOTICES Title: The title of the book is “Ayurvedic Pharmacopoeia of India, Part-I, Volume-IX.” Wherever the abbreviation “API, Pt.-I, Vol.-IX” is used, it stands for the same and for the Supplements or Amendments thereto. Name of the Monograph: The name given on top of each monograph is in SaÆsk¤ta as mentioned in the Ayurvedic classics and/or Ayurvedic Formulary of India (AFI) and will be considered Official. These names have been arranged in alphabetical order in English. If a preparation is intended to be stored over a period of time, deterioration due to microbial contamination may be inhibited by the addition of a permitted preservative. In such circumstances the label should state the name and the concentration of the preservative and the appropriate storage conditions. Mere presence of a monograph in the Pharmacopoeia shall not qualify the ingredient as a drug. The primary purpose of the monographs is to specify the quality parameters that can be employed to assess the “fitness for use” for the desired purpose which could be as a drug, dietary supplement, cosmetic or a functional food. The ingredients mentioned in this Pharmacopoeia may be prepared and their quality assessed as per the methods mentioned in the respective monographs. These ingredients may be regarded as Pharmacopoeial grade, even if they are to be used for nontherapeutic purposes. Introductory para: Each monograph begins with a Definition in an introductory paragraph. For drugs of plant origin, the part used has also been specified. The requirements given in the monographs are not framed to provide against all impurities, contaminants or adulterants; they provide appropriate limits only for possible impurities that may be permitted to a certain extent. Material found to contain an impurity, contaminant or adulterant which is not detected by means of the prescribed tests in the Appendix 2 are also to be considered as impurity, should rational consideration require its absence. Hydro-alcohol: 50 per cent v/v of ethanol in purified water Yield of Extract: The yield of extract mentioned in the monographs is meant to be indicative only. This is so, as newer techniques for extraction are being developed having higher efficiencies. Further, extractive values and thus yields are known to exhibit a high degree of inherent variability due to seasonal, geographical, edaphic and ontogenic factors. Standards: For statutory purposes, the following shall be considered Official Standards: Definition, Identification, Quantitative parameters, Assay and Other requirements. Added Substances: An article for which a monograph has been recommended contains no added substances/excipients, except when specifically permitted in the individual monograph. Unless otherwise specified in the individual monograph, or elsewhere in the General Notices, such added substances/excipients shall be from the approved list of Drugs and Cosmetics Rules, under Rule 169 to enhance its stability, usefulness, elegance, or to facilitate its preparation. Such added substances shall comply with the quality indicated for it, shall be harmless in the amounts used, shall not exceed the minimum quantity required to provide their intended effect, shall not impair the therapeutic efficacy or the bioavailability and safety of the preparation and shall not interfere with the tests and assays prescribed for determining compliance with the official standards. Particular care should be taken to ensure that such substances are free from harmful viii

organisms. Though the manufacturer of an extract is given the freedom to use an added substance, the manufacturer must guarantee the innocuousness of the added substance. The manufacturer shall also be responsible to explain to the appropriate authority, if needed, regarding the purpose of the added substance(s). Meanings of Terms Alcohol: The term ‘alcohol’ without qualification means ethanol (95 per cent). Other dilutions of ethanol are indicated by the term ‘alcohol’ followed by a statement of the percentage by volume of ethanol (C2H6O/C2H5OH) required. Desiccator: A tightly closed container of suitable size and design that maintains an atmosphere of low moisture content by means of silica gel or phosphorus pentoxide or other suitable desiccant Drying and Ignition to Constant Weight: Two consecutive weighings after the drying or igniting operations do not differ by more than 0.5 mg, per g of the drug taken the second weighing, following an additional period of drying or of ignition, respectively appropriate to the nature and quantity of the residue. Ethanol: The term ‘ethanol’ without qualification means anhydrous ethanol or absolute alcohol. Filtration: Unless otherwise stated, filtration is the passing of a liquid through a suitable filter paper or equivalent device until the filtrate is clear. Freshly prepared: Made not more than 24 hours before use Label: Any printed packing material, including package inserts that provide information on the article Negligible: A quantity not exceeding 0.50 mg Solution: Where the name of the solvent is not stated ‘solution’ implies a solution in water. The water used complies with the requirements of the monographs on Purified Water. Temperature: The symbol ‘0’ used without qualification indicates the use of the Celsius thermometric scale. Water: If the term is used without qualification means Purified Water of the Pharmacopoeia. The term ‘distilled water’ indicates Purified Water prepared by distillation. Water-bath: A bath containing boiling water unless water at another temperature is indicated. Other methods of heating may be used provided the required temperature is approximately maintained but not exceeded. Capital Letters in the Text: The names of the Pharmacopoeial substances, preparations and other materials in the text are printed in capital initial letters, and these infer that materials of Pharmacopoeial quality have been used. Italics: Italic types are used for Scientific names of the plant drugs and microorganisms, and for some subheadings and certain notations of the chemical names. Italic types have also been used for words which refer to solvent system in TLC procedure, reagents and substances, processes covered under Appendices. Chemicals and Reagents, and Substances of Processes in Appendices have also been printed in italics. Odour and Taste: Wherever a specific odour has been observed, it has been mentioned as characteristic for that substance, but the description as ‘odourless’ or ‘no odour’ has generally been avoided in the Description where a substance has no odour. Where an ‘odour’ is said to be present, it is examined by smelling the drug directly after opening the container. If an odour is discernible, the contents are rapidly transferred to an open ix

vessel and re-examined after 15 minutes. If odour persists to be discernible, the sample complies with the description for ‘odour’, as a characteristic for that substance. The taste of a drug is examined by taking a small quantity of drug by the tip of a moist glass rod and allowing it to remain on the tongue. This does not apply in the case of poisonous substances. Powder: Drug substances are subjected to comminution during preparation. It is desirable that such powders maintain certain average particle size for effective processing. To provide for such situations, the fineness of a powder is given in terms of sieve sizes, using the BIS sieves as standard. The sieve sizes follow the latest revision of the BIS. For the convenience of users, the equivalents or nearest equivalent numbers according to the earlier BIS have also been given. Weights and Measures: The metric system of weights and measures is employed. Weights are given in multiples or fractions of a gram (g) or of a milligram (mg). Fluid measures are given in multiples of fraction of milliliter (ml). The amount stated is approximate but the quantity actually used must be accurately weighed and must not deviate by more than 10 per cent from the one stated. When the term “drop” is used, measurement is to be made by means of a tube which delivers 20 drops per gram of distilled water at 150. Identity, Purity and Strength: Under the heading “Identification”, tests are provided as an aid to identification and are described in the respective monographs and included. Herbal/Plant drugs should be duly identified and authenticated and should be free from insects, pests, fungi, microorganisms, pesticides, and other animal matter including animal excreta, should be within the permitted and specified limits for lead, arsenic and heavy metals, and show no abnormal odour, colour, sliminess, mould or any sign of deterioration. Herbal/plant drugs should be duly identified and authenticated and should be free from insects, pests and other animal matter including animal excreta, should have, within the permitted and specified limits, for fungi, microorganisms, pesticides, and heavy metals, and show no abnormal odour, colour, sliminess, mould or any sign of deterioration. Quantitative tests like total ash, acid-insoluble ash, water-soluble ash, alcohol-soluble extractive, water-soluble extractive, moisture content, volatile oil content and assays are the parameters upon which the standards of Pharmacopoeia depend. Except for Assays, which are covered under each monograph, the methods of determination for the others are given in Appendices, with a suitable reference in the monograph to the specific Appendix. An analyst is not precluded from employing an alternate method in any instance if one is satisfied that the method, which one uses, will give the same result as the Pharmacopoeial method described under assay. However, in the event of doubt or dispute the methods of analysis of the Pharmacopoeia are alone authoritative. Unless otherwise prescribed, the assays and tests are carried out at a temperature between 20 and 300. In the performance of an assay or any test procedure, not less than the specified number of dosage units or quantities should be taken for analysis. Proportionately larger or smaller quantities than the specified weights and volumes may be taken for substances under assay or test substances, Reference Standards or Standard Preparations, provided the measurement is made with at least equivalent accuracy and provided that any x

subsequent steps, such as dilutions, are adjusted accordingly to yield concentrations equivalent to those specified and are made in such a manner as to provide at least equivalent accuracy. Expression of Results: Total ash, acid-insoluble ash, water-soluble extractive, alcohol-soluble extractive, water content, content of essential oil and content of active principle are calculated with reference to the drug that has not been specially dried, unless otherwise prescribed in the monograph. In other words, all limits are thus proposed on “as such basis” unless specified otherwise. Limits for Heavy metals, Microbial load, Pesticide residues and Aflatoxins: Articles included in this volume are required to comply with the limits for heavy metals, microbial contamination, pesticide residues and aflatoxins prescribed in the individual monographs and wherever limit is not given in the monograph, they must comply with the limits given in the respective Appendices. The methods for determination of these parameters are given in the Appendices. Thin-Layer Chromatography (TLC): Under this title, the Rf values given in the monographs are not absolute but only indicative. The analyst may use any other solvent system and detecting reagent to establish the identity of any particular chemical constituent reported to be present in the test substance. However, in case of dispute the pharmacopoeial method would prevail. Unless specified in the individual monograph all TLC have been carried out on pre-coated Silica gel 60F254 aluminium plates. Reference Standards: Reference substance and standard preparation are authentic substances that have been verified for their suitability, for use as standards for comparison in some assays, tests and TLC. The reference standards, abbreviated as RS are issued by Pharmacopoeial Laboratory of Indian Medicine (PLIM). Quantities to be weighed for Assays and Tests: In all descriptions quantity of the substance to be taken for testing is indicated. The amount stated is approximate but the quantity actually used must be accurately weighed and must not deviate by more than 10 per cent from the one stated. Percentage of Solutions: In defining standards, the expression per cent (%) is used, according to circumstances, with one of the four meanings given below. Per cent w/w (percentage weight in weight) expresses the number of grams of active substance in 100 grams of product. Per cent w/v (percentage weight in volume) expresses the number of grams of active substance in 100 milliliters of product. Per cent v/v (percentage volume in volume) expresses the number of milliliters of active substance in 100 milliliters of product. Per cent v/w (percentage volume in weight) expresses the number of milliliters of active substance in 100 grams of product. Percentage of Alcohol: All statements of percentage of alcohol (C2H5OH) refer to percentage by volumes at 15.560. Solubility: When stating the solubilities of chemical substances the term “Soluble” is necessarily sometimes used in a general sense irrespective of concomitant chemical changes. Statements of solubilities, which are expressed as a precise relation of weights of dissolved substance of volume of solvent, at a stated temperature, are intended to apply at that temperature. Statements of approximate solubilities for which no figures are given, are intended to apply at ordinary room temperature. xi

Pharmacopoeial chemicals when dissolved may show slight physical impurities, such as fragment of filter papers, fibres, and dust particles, unless excluded by definite tests in the individual monographs. When the expression “parts” is used in defining the solubility of a substance, it is to be understood to mean that 1 gram of a solid or 1 millilitre of a liquid is soluble in that number of millilitres of the solvent represented by the stated number of parts. When the exact solubility of pharmacopoeial substance is not known, a descriptive term is used to indicate its solubility. The following table indicates the meaning of such terms:Descriptive terms

Relative quantities of solvent

Very soluble Freely soluble Soluble Sparingly soluble Slightly soluble Very slightly soluble Practically insoluble

Less than 1 part. From 1 to 10 parts. From 10 to 30 parts. From 30 to 100 parts. From 100 to 1000 parts. From 1000 to 10,000 parts. More than 10,000 parts.

Reagents and Solutions: Reagents required for the assay and tests of the Pharmacopoeia are defined in the Appendix showing the nature, degree of the purity and strength of solutions to be made from them. Therapeutic uses: Therapeutic uses wherever given are as mentioned in the API. Doses: The doses mentioned in the monograph are in the metric system, which are approximate conversions from classical weights mentioned in Ayurvedic texts. A conversion table is appended giving classical weights with their metric equivalents (Appendix 5). Doses mentioned in the API are intended merely for general guidance and represent, unless otherwise stated, the average range of quantities per dose which is generally regarded suitable by clinicians for adults only when administered orally. They are not to be regarded as binding upon the prescribers. Storage: Statement under the heading ‘Storage’ constitutes non-mandatory advice. The substances and preparations of the Pharmacopoeia are to be stored under conditions that prevent contamination and, as far as possible, deterioration. Precautions, that should be taken in relation to the effects of the atmosphere, moisture, heat and light, are indicated, where appropriate, in the individual monographs. Specific directions are given in some monographs with respect to the temperatures at which Pharmacopoeial articles should be stored, where it is considered that storage at a lower or higher temperature may produce undesirable results. The conditions are defined by the following terms. Cold- Any temperature not exceeding 80 and usually between 20 and 80. A refrigerator provides a cold place in which the temperature is maintained thermostatically between 20 and 80. Cool- Any temperature between 80 and 250. An article for which storage in a cool place is directed may, alternately, be stored in a refrigerator, unless otherwise specified in the individual monograph. Room temperature - The temperature prevailing in a working area. Warm - Any temperature between 300 and 400. Excessive heat- Any temperature above 400.

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Protection from freezing- Where, in addition to the risk of breaking of the container, freezing results in loss of strength or potency or in destructive alteration of the characteristics of an article, the label on the container bears an appropriate instruction to protect from freezing. Storage under non-specific conditions- Where no specific storage directions or limitations are given in the individual monograph, it is to be understood that the storage conditions include protection from moisture, freezing and excessive heat. Packaging and Containers: In general the ASU extracts should be packed in well closed container i.e. one that protects the contents from extraneous matter, moisture or loss of material under normal condition of handling. The preferred packaging for extracts are a primary cover made up of 12  polyester, 100  polyethylene and a secondary cover made up of 9 aluminium sandwiched between 2 layers of 12  polyester and 100  polyethylene. The tertiary package can be HDPE drums. The container is the device that holds the article. The immediate container is that which is in direct contact with the article at all times. The closure is a part of the container. The container is designed so that the contents may be taken out for the intended purpose in a convenient manner. It provides the required degree of protection to the contents from environmental hazards. The container should not interact physically or chemically with the article placed in it so as to alter the strength, quality or purity of the article beyond the official requirements. Prior to its being filled, the container should be clean. Special precautions and cleaning procedures may be necessary to ensure that each container is clean and that extraneous matter is not introduced into or onto the container. Light-resistant Container- A light resistant container protects the contents from the effects of actinic light by virtue of the specific properties of the material of which it is made. Alternatively, a clear and colourless or a translucent container may be made light-resistant by means of an opaque (light-resistant) covering and/or stored in a dark place: in such cases, the label on the container should bear a statement that an opaque covering or storage in dark place is needed until the contents have been used up. Well-closed Container- A well-closed container protects the contents from extraneous contamination and from loss of contents under normal conditions of handling, shipment, storage and distribution. Tightly-closed Container- A tightly-closed container protects the contents from contamination by extraneous liquids solids or vapours, and from loss or deterioration of contents from effervescence, deliquescence or evaporation under normal conditions of handling, shipment, storage and distribution. Single Unit Container- A single unit container is one that is designed to hold a quantity of the drug product intended for administration as a single finished device intended for use promptly after the container is opened. The immediate container and/or outer container or protective packaging is so designed as to reveal evidence of tampering, if any. Multiple Unit Container- A multiple unit container is a container that permits withdrawals of successive portions of the contents without changing the strength, quality or purity of the remaining portion. Tamper-evident Container- A tamper-evident container is fitted with a device or mechanism that reveals irreversibly whether the container has been opened. Labeling: In general, the labeling of drugs and pharmaceuticals is governed by the Drugs and Cosmetics Act, 1940 and Rules there under. xiii

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PHARMACOPOEIA COMMISSION FOR INDIAN MEDICINE & HOMOEOPATHY Pharmacopoeia Commission for Indian Medicine & Homoeopathy (PCIM&H) is an autonomous organization under Ministry of AYUSH, Govt. of India with a primary mandate to develop pharmacopoeial standards for drugs/formulations used under Ayurveda, Siddha, Unani and Homoeopathic systems of medicine. It serves as an umbrella organization for Ayurvedic Pharmacopoeia Committee (APC), Siddha Pharmacopoeia Committee (SPC), Unani Pharmacopoeia Committee (UPC) and Homoeopathic Pharmacopoeia Committee (HPC). Pharmacopoeial Laboratory for Indian Medicine (PLIM) and Homoeopathic Pharmacopoeia Laboratory (HPL) are its permanent supporting structures. The Commission was initially established as Pharmacopoeia Commission for Indian Medicine (PCIM) in the year 2010. In pursuance to the decision of Central Government, Homoeopathy was incorporated and the Commission was renamed as Pharmacopoeia Commission for Indian Medicine & Homoeopathy (PCIM&H) on 25th June 2014. Commission has a three-tier structure of Governance comprising of the General Body, Standing Finance Committee and Scientific Body. The Secretary, Ministry of AYUSH, Govt. of India is ex-officio Chairman of the Commission. Objectives 1.

Publication and revision of the Ayurvedic, Siddha, Unani and Homoeopathic Pharmacopoeia of India at suitable intervals and of such addenda or supplementary compendia during the intervening periods as may be deemed necessary; releasing the publications for public use from a date when they are to become official.

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Publication and revision of the Ayurvedic, Siddha and Unani Formularies of India, Homoeopathic pharmacopoeia as well as Homoeopathic Pharmaceutical Codex at regular intervals with a view to make it an authentic source of information on rational combination and use of medicines including their methods of preparation, therapeutic indications, adverse reactions, contra-indications, drug-drug interactions and similar issues concerning Indian medicines for safe use in humans and animals. Identification of Ayurvedic, Siddha and Unani formulations and Homoeopathic pharmacopoeia as well as Homoeopathic Pharmaceutical Codex with a view to develop their quality standards and to ensure quality and safety of ASU & H medicine.

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To nurture and promote awareness of quality in Ayurvedic, Siddha and Unani drugs/formulations, Homoeopathic pharmacopoeia as well as Homoeopathic Pharmaceutical Codex and drug research on ASU products and publish regularly or at suitable intervals other related scientific information as authorized under the rules and procedures of the Commission.

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Exchange information and interact with expert committees of the World Health Organization and other international bodies with a view to harmonize and develop the Ayurvedic, Siddha, Unani and Homoeopathic Pharmacopoeial standards to make those internationally acceptable.

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Arranging studies either under its own auspices or through collaboration with other institutions to develop standards and quality specifications for identity, purity and strength of raw materials and compound formulations and to develop Standard Operating Procedures for the process of manufacture included or to be included in the Ayurvedic, Siddha, Unani and Homeopathic Pharmacopoeia/formulary and its addenda or supplementary compendia or other authorized publications.

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Maintain National repository of authentic reference raw materials used in the manufacture of Ayurveda, Siddha, Unani and Homeopathic medicines for the purpose of reference and supply of reference standards to the stake holders at a price.

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To assign responsibilities described for Pharmacopoeial Laboratory for Indian Medicine and Homoeopathic Pharmacopoeia Laboratory under the Drugs & Cosmetics Act.

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Generate and maintain repository of chemical reference marker compounds of the plants or other ingredients used in standardizing Ayurveda, Siddha, Unani and Homeopathy medicines and supply them as reference standards to the stake holders on price.

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Furtherance of the provision of Chapter IVA of Drugs and Cosmetic Act, 1940 in case ASU drugs & 4A of Schedule II of Drugs & Cosmetics Act in case of Homoeopathy medicine and rules there under related to Ayurvedic, Siddha and Unani drugs and Homoeopathy medicine respectively.

10. Acting as a coordinating centre for analytical laboratories, industry and academia by encouraging exchange of scientific and technical information and staff and by undertaking sponsored funded research as well as consultancy projects. 11. Organizing national/international symposia, seminars, meetings and conferences in selected areas from time to time and to provide updated regular training to the regulatory authorities and stake holders. The General Body The General Body is the apex body and is responsible for overall governance of the Commission. Composition: i) Secretary, Ministry of AYUSH Chairman st Sh. Nilanjan Sanyal until 31 August, 2015; Sh. Ajit M. Sharan from 1st Sept., 2015 ii) Joint Secretary, Ministry of AYUSH Vice-Chairman - 1 st Sh. Raj Pratap Singh until 1 Dec., 2014 Sh. Anurag Srivastav until 1st Nov., 2015 Sh. Jitendra Sharma from 2nd Nov., 2015 iii) Chairman, Scientific Body, PCIM&H Vice-Chairman - 2 Prof. S. S. Handa iv) Secretary and Director General, ICMR Member Dr. Soumya Swaminathan v) Chairman, CII or his nominee Member Sh. Sumit Mazumder vi) Chairman, FICCI or his nominee Member Mr. Harshavardhan Neotia vii) Drugs Controller General (India) Member Dr. G. N. Singh viii) Central Drug Controller (AYUSH) Member ix) Adviser (Ayurveda), Ministry of AYUSH Member Dr. Manoj Nesari x) Adviser (Unani), Ministry of AYUSH Member Prof. Rais-Ur-Rahman xi) Adviser (Homoeopathy), Ministry of AYUSH Member Dr. N. Radha xii) Eminent ASU&H experts (one from each system) Members 1. Dr. Vaidya Balendu Prakash (Ayurveda Expert) Turner Road, Dehradun, Uttarakhand xvi

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2. Dr. V. Arunachalam (Siddha Expert) Dean, Santhigiri Health Care & Research Organization, Santhigiri Ashramam, Santhigiri P.O, Thiruvanathapuram-695589, Kerala 3. Dr. Mohd. Khalid Siddique (Unani Expert) Former DG, CCRUM, Jamia Hamdard Enclave, New Delhi 4. Dr. S. P. Singh (Homoeopathy Expert) Former Adviser (Homoeopathy), S R B, 68-C Shipra Riviera. Indirapuram, Ghaziabad-201014 One representative each of ASU&H Drug Manufacturers 1. Mr. Pramod Sharma (Ayurveda Industry) Managing Director, Shree Baidyanath Ayurvedic Bhawan (P) Ltd. Patna 800001. Bihar 2. Dr. M. K. Thyagarajan (Siddha Industry) IMPCOPS, Adayar, Chennai-600020 3. Dr. Ajmal K. P. (Unani Industry) Hermas Herbal Unani Pharmaceuticals, Chennamangallur, (PO) Mukkam, Calicutt-673602 4. Dr. P. N. Verma (Homoeopathy Industry) Scientific Advisor, Dr. Willmar Schwabe India Pvt. Ltd, Noida-201307 Director, PCIM&H Dr. Rajeev Kr. Sharma

Members

Member Secretary

The Standing Finance Committee All matters with respect to financial approvals are dealt by Standing Finance Committee. Standing Finance Committee is responsible for screening/appraising/evaluating the projects/works etc. of the Commission and recommend for the approval of these projects /works by the General Body. Composition: i) Joint Secretary (AYUSH) Sh. Raj Pratap Singh until 1st Dec., 2014 Sh. Anurag Srivastava until 1st Nov., 2015 Sh. Jitendra Sharma from 2nd Nov., 2015 ii) Chairman, Scientific Body Prof. S.S. Handa iii) Financial Adviser, M/o Health &Family Welfare Smt. Vijaya Srivastava iv) Central Drug Controller (AYUSH) v) Director, PCIM&H Dr. Rajeev Kr. Sharma

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Chairman

Vice-Chairman Member Member Member Secretary

The Scientific Body The Scientific Body is responsible for designing/preparing and according technical approval for all the scientific & technical works/projects and execution of these works/projects through different Pharmacopoeia Committees / other agencies, publication of validated Pharmacopoeia after obtaining the approval of the General Body. Composition: i) Eminent Scientist or an ASU&H Expert with significant experience in Pharmacopoeial work Prof. S. S. Handa ii) Chairman, Indian Pharmacopoeia Commission or his nominee Dr. G. N. Singh iii) Chairman, Ayurvedic Pharmacopoeia Committee Prof. V. K. Joshi iv) Chairman, Unani Pharmacopoeia Committee Dr. G. N. Qazi v) Chairman, Siddha Pharmacopoeia Committee Dr. G. Veluchamy vi) Chairman, Homoeopathic Pharmacopoeia Committee Dr. C. Nayak vii) Director General, ICMR or his nominee (ASU Drugs Expert) Dr. Soumya Swaminathan viii) Director General, CSIR or his nominee (ASU Drugs Expert) Dr. Girish Sahni ix) Director General, Central Council for Research in Ayurvedic Sciences Prof. K. S. Dhiman x) Director General, Central Council for Research in Unani Medicine Prof. Rais-Ur-Rahman xi) Director General, Central Council for Research in Siddha Prof. R. S. Ramaswamy xii) Director General, Central Council for Research in Homoeopathy Dr. R. K. Manchanda xiii) Central Drug Controller (AYUSH) xiv) Adviser (Ay.), Ministry of AYUSH Dr. Manoj Nesari xv) Adviser (Unani), Ministry of AYUSH Prof. Rais-Ur-Rahman xvi) Adviser (Homoeopathy), Ministry of AYUSH Dr. N. Radha xvii) Dy. Adviser (Siddha), Ministry of AYUSH Dr. K. Ravi (Joint Adviser) xviii) Professional expert drawn one each from ASU&H industry 1. Dr. K. Anil Kumar, Managing Director, Kerala Ayurveda Pharmacy Ltd. (KPL), Athani-683585, Aluva, Kerala 2. Dr. S. Jahir Hussain, Sidha Physician, QC Section, TAMPCOL, Alathur, Dist. Kanchipuram 3. Sh. Kafeel Ahmed, Manager, Tibbiya College Dawakhana, Aligarh Muslim University, Aligarh 4. Dr. Nishant Tripathi, General Manager, xviii

Chairman

Member Member Member Member Member Member Member Member Member Member Member Member Member Member Member Member Members

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M. Bhattacharya & Co. Pvt. Ltd. 73, N. S. Road, Kolkata-700 001 One expert each from ASU&H Academia and Regulatory Bodies 1. Dr. G. S. Badesha, Director (Ayurveda), Govt. of Chattisgarh, Raipur, Chattisgarh. 2. Dr. N. Kabilan, Associate Professor, The Tamil Nadu Dr. M. G. R Medical University, Anna salai, Guindy, Chennai-600 032 3. Prof. Yashmin Shamsi Faculty of Unani Medicine, Jamia Hamdard University, Delhi 4. Dr. S. K. Nanda, Director, National Institute of Homoeopathy, Kolkata-700106 Director, PCIM&H Dr. Rajeev Kr. Sharma

Members

Member Secretary

Execution of Pharmacopoeial Work Pharmacopoeia Committees The Scientific and technical work of the Commission is being executed through Ayurvedic, Siddha, Unani & Homoeopathic Pharmacopoeia committees under the supervision of the Scientific Body. The function of Pharmacopoeia committees is to prepare official formularies, Pharmacopoeias of single drugs and compound formulations, Pharmacopoeial codex and other technical documents related to standards for drugs. The present composition of ASU&H Pharmacopoeia committees is as below: I. 1.

2.

3.

4.

5.

6.

7.

8.

Ayurvedic Pharmacopoeia Committee Prof. V. K. Joshi Chairman B-6, New Medical Enclave, Nariya, Banaras Hindu University, Varanasi-221005 Adviser (Ay.): Dr. Manoj Nesari Member Ministry of AYUSH, AYUSH Bhawan, B Block, GPO Complex, INA, New Delhi- 110023 Director Member Pharmacopoeial Laboratory for Indian Medicine Kamla Nehru Nagar, Ghaziabad- 201002 Director General: Prof. K. S. Dhiman Member Secretary Central Council for Research in Ayurvedic Sciences (CCRAS) 61-65, Institutional Area, Opp. ‘D’ Block, Janakpuri, New Delhi- 110058 Prof. V. K. Kapoor Member 1473, Pushpak Complex, Sector-49 B, Chandigarh- 160047 Dr. Roopak Kumar Member Vice-President, Multani Pharmaceuticals Ltd., Analytical Division, Village:Makkanupur, Roorkee, Dist.- Haridwar- 237891 Prof. Karan Vashisth Member Former Head, University Institute of Pharmaceutical Science (UIPS), Chandigarh-160014 Ms. S. Satakopan Member th 7/4 Padmam Flats, 7 street Nanganallur, Chennai-600061

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9.

10.

11.

12.

13.

14.

15.

16.

17.

Dr. Malati Chauhan IPGT&RA, Gujarat Ayurved University, Jamnagar, Gujarat Dr. J. L. N. Sastry Head, Dabur Research & Development Center (DRDC), Dabur India Limited 22, Site IV, Sahibabad (UP) Dr. K. N. Dviwedi Head, Deptt. of Dravyaguna, I.M.S., Banaras Hindu University, Varanasi- 221005 Dr. S. K. Dixit B-3/402, Shivala, Varanasi-221005 Dr. S. S. Savrikar Prof. & HOD Rasashastra & Bhaishajya Kalpana Govt. Ayurvedic College & Hospital Solapur Road, Madhuban, Osmanabad, Maharashtra- 413501 Dr. C. K. Katiyar CEO Technical, Emami HCD, Emami Towers, 5th Floor, 687, Anandapur, E.M. Bypass, Kolkata-700107 Dr. P. M. Variar Arya Vaidya Sala, Kottakkal, Malappuram, Kerala-676503 Dr. Aditya Kaushik Head-Medical Regulatory Affairs Glaxo SmithKline Consumer Healthcare Ltd., Sector 32, Gurgaon-122001, Haryana Dr. Shailesh Nadkarni Shree Dhootapapeshwar Ltd. 135, Nanubai, Desai Road, Khetwadi, Mumbai- 400004

II.

Siddha Pharmacopoeia Committee

1.

Dr. G. Veluchamy 24, Chokkanathar Street, Karthikeyan Nagar, Maduravoyal, Chennai-600095 Deputy Adviser (Siddha): Dr. K. Ravi (Joint Adviser) Ministry of AYUSH, AYUSH Bhawan, B Block, GPO Complex, INA, New Delhi- 110023 Director Pharmacopoeial Laboratory for Indian Medicine Kamla Nehru Nagar, Ghaziabad- 201002 Director General: Prof. R. S. Ramaswamy Central Council for Research in Siddha (CCRS) SCRI, Anna Hospital Campus, Arumbakkam, Chennai-600106 Dr. Sharada Vasanth Former Research Officer (Chem.), SCRI, Chennai-600106 Dr. K. Balakrishna Former Research Officer (Chem.), SCRI, Chennai-600106

2.

3.

4.

5.

6.

xx

Member

Member

Member

Member

Member

Member

Member

Member

Member

Chairman

Member

Member

Member Secretary

Member

Member

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

III. 1.

2.

3.

4.

Prof. V. Gopal Principal, Govt. College of Pharmacy, Mother Teresa PG Research Institute of Health Sciences, Puducherry-605006 Dr. Sasikala Ethiraju Research Officer (Pharmacognosy), SCRI, Chennai-600106 Dr. P. Jayaraman Former Prof. of Botany, Presidency College, Chennai Dr. (Prof.) I. Sornamariammal Former Joint Director of Indian Medicine, Chennai Dr. P. Kumar Drug License Issuing Authority for ISM, Anna Hospital Campus, Arumbakkam, Chennai Prof. Jayprakash Narayanan Former Vice-Principal, Old NO. 55, New No. 70, Panchaliamman Koil Street, Arumbakkam, Chennai-600106 Dr. Kumaravel No. 25, II Street Ram Nagar, North Extension Vijayanagar, Velacherry, Chennai Dr. V. Kalidass Proprietor, Raja Siddha Marunthagam, 1/3, Dhermathupatty, Madurai- 625008 Dr. K. Vasanthira Prof. of Pharmacology, Stanley Medical College, Chennai Dr. G. Thiyagarajan Former Joint Director of ISM, 19/5, Arunachalapuram Street, Sandopalayam, Aminjikarai, Chennai Dr. T. Anandan 75, O Block, Ganapathy Colony, Anna Nagar (E), Chennai

Member

Member

Member

Member

Member

Member

Member

Member

Member

Member

Member

Unani Pharmacopoeia Committee Dr. G. N. Qazi, Vice-Chancellor, Jamia Hamdard University, Mehrauli Road, New Delhi- 110062 Adviser (Unani): Prof. Rais-Ur- Rahman Ministry of AYUSH, AYUSH Bhawan, B Block, GPO Complex, INA, New Delhi- 110023 Director Pharmacopoeial Laboratory for Indian Medicine, Kamla Nehru Nagar, Ghaziabad- 201002 Director General : Prof. Rais-Ur-Rahman Central Council for Research in Unani Medicine (CCRUM) 61-65, Institutional Area, Opp. ‘D’ Block, Janakpuri, New Delhi- 110058 xxi

Chairman

Member

Member

Member Secretary

5.

6.

7. 8.

9. 10.

11.

12.

13.

14.

15.

16.

IV. 1.

2.

3.

Dr. Rahul Singh Head, CQA Healthcare Products, Emami Group of Companies, Emami Towers, 5th Floor, 687, Anandapur, E.M. Bypass, Kolkata-700107 Dr. Arshad Khuroo, Plot GP-5, Sector-18, Udyog Vihar Industrial Area, Gurgoan-122001 Prof. Maninder Kaur UIPS, Punjab University, Chandigarh Dr. Deepak M. Sr. Manager, Natural Remedies, Plot No. 5B, Veersanda Indl. Area, 19th K.M. Stone, Hosur Road, Bangalore- 560100 Prof. Kunwar Mohammad Yusuf Amin Deptt. of Ilmul Advia, AMU, Aligarh Dr. Santosh Joshi Head R&D, Hamdard Laboratories, Meerut Road, Ghaziabad- 201010 Prof. Tajuddin Former Dean, Deptt. of Saidla, Faculty of Unani Medicine, AKTC, AMU, Aligarh Prof. M. Idris Head, PG Deptt. of Ilmul Saidla, Ayurveda & Unani Tibbia College, Ajmal Khan Road, Karol Bagh, New Delhi-110005 Dr. Asad Mueed Director, Hamdard (Wakf) Laboratories, Hamdard Building, 2A/3 Asaf Ali Road, New Delhi-110002 Dr. M. U. R. Naidu Plot No. 176/B 2, House No. 1-241, Santh Nagar Co. H. Society, Motinagar, Hyderabad- 500018 Prof. Zillur Rehman Former Dean, Faculty of Unani Medicine, AMU, Aligarh Dr. Absar Ahmad Principal Scientist, Biochemical Division, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune-411008

Member

Member

Member Member

Member Member

Member

Member

Member

Member

Member

Member

Homoeopathic Pharmacopoeia Committee Dr. C. Nayak, Ex-Director General, CCRH, New Delhi House No. 55-B, Block No. C- 4-D Janakpuri, New Delhi-110058 Adviser (Homoeopathy): Dr. N. Radha Ministry of AYUSH, AYUSH Bhawan, B Block, GPO Complex, INA, New Delhi- 110023 Director Homoeopathic Pharmacopoeia Laboratory, Kamla Nehru Nagar, Ghaziabad- 201002 xxii

Chairman

Member

Member

4.

5. 6.

7.

8.

9.

10.

Director General: Dr. R. K. Manchanda Member Secretary Central Council for Research in Homoeopathy (CCRH) 61-65, Institutional Area, Opp. ‘D’ Block, Janakpuri, New Delhi- 110058 Dr. B. N. Suhagia, Dean, Faculty of Pharmacy, Member Dharamsinh Desai University, Nadiad-387001, Gujarat Dr. Dilip Pannakadda, Member Head, Department of Pharmacy, National Institute of Homoeopathy Kolkata-700106, West Bengal Dr. (Mrs.) Shanta Mehrotra, Member Ex-Emeritus Scientist, NBRI, HIG-91, Aliganj Extension, Sector-E, Lucknow-226024 Dr. P. V. Santhosh Member Managing Director, Kerala State Co-operative Homoeopathic Pharmacy, XII/37 to 39, Mararikulam South, Pathirappally P.O., Alappuzha - 688 521 Sh. Mohd. Faiyaz Alam, Member Technical Manager, SBL. Pvt. Ltd. A/3, Site -4, Indl. Area Sahibabad, U.P. Dr. Sohan Singh, Member Former Vice-Principal, JSPS Homoeopathic Medical College, Hyderabad. House No 8-3-248/B/23/1, Opposite Times of India, Road No 3, Journalist Colony, Banjara Hills, Hyderabad-500034

PCIM&H Secretariat: Scientific and administrative support to Pharmacopoeia Committees extended by the followingDirector:

Dr. Rajeev Kr. Sharma

Joint Director:

Dr. Madhira Bhawanishankar

ASU&H:

Scientific Officers- Dr. V.Vijayakumar (Siddha), Dr. Nikhil Jirankalgikar & Dr. Vijay Gupta (Ayurveda)

Chemistry:

Principal Scientific Officer- Dr. S. C. Verma; Scientific Officer- Dr. Anupam Maurya

Pharmacognosy:

Principal Scientific Officer- Dr. Jayanthy A.; Scientific Officer- Dr. (Ms.) Nitin Rai; Research Assistant- Sh. Jeetendra Kumar Vaishya, Sh. Suman Halder

Pharmacology:

Scientific Officer- Dr. Ramachandran S.

Secretarial Assistance: Sh. Pankaj Nagdeve, Sh. Afzal Hashmi, Sh. Sanjay Singh Rawat, Sh. Ashish Kumar, Sh. Sunil Dahiya

xxiii

ACKNOWLEDGMENTS Pharmacopoeia Commission for Indian Medicine & Homoeopathy expresses the gratitude towards Prof. S. S. Handa, Chairman, APC (2009-2012) presently Chairman, Scientific Body of the Commission and other erstwhile members and co-opted Members of the Ayurveda Pharmacopoeia Committee for their continuous support in finalizing this volume. Thanks are reserved for Dr. S. K. Sharma, Vice Chairman APC, Ms. Savita Satakopan, Member APC, and Dr. M. M. Padhi, Deputy Director General, CCRAS, Former Director Incharge, PCIM for their enormous guidance, help, expert advice and critical views on the contents of this volume. The contributions made by the staff of the participating institutions viz., Natural Remedies Pvt. Ltd. Bangalore, Chemolids Pvt. Ltd. Vijayawada, Green Chem Pvt. Ltd. Bangalore, Sanat Product Pvt. Ltd. Bulendashar and Arjuna product Pvt. Ltd, Kochi associated with the APC project work for developing quality standards of plant drugs and their hydroalcoholic and water extracts are duly acknowledged. The Commission also sincerely thanks Dr. Pramila Pant, Assistant Director [Chem.]; Dr. Ravindra Singh, Assistant Director [Chem.]; Dr. Bishnupriya Dhar, Former Assistant Director [P’cognosy]; Dr. Renu Dixit, Ex-Consultant [Bot.] ; Dr. S. C. Verma, then Research Officer [Chem.], Dr. Shruti Khanduri, Research Officer [Ay.]; Dr. B. S. Sharma, Research Officer [Ay.]; Dr. Chhote Lal, Dr. A. K. S. Bhadoria; Ms. Talat Anjum, Research Officer [Bot.] and Mr. Chinmay Rath, then S.R.F [Bot.], Mr. Ashish Kumar, Ms. Meenakshi, Ms. Deepti Anand D.E.O.s and other associated officers, for their constant efforts in bringing out this volume. Thanks are also due to technical staff of Pharmacopoeial Laboratory Indian Medicine, Ghaziabad. Commission also acknowledges inputs and suggestions offered by Dr. Manoj Nesari, Adviser (Ay.), Prof. K. S. Dhiman, Director General, CCRAS and Dr. Anupam Srivastava, Research Officer, S-2 (Ay.) from Ministry of AYUSH. Efforts of the scientific staff of the Commission Dr. Madhira Bhawanishankar, Dr. S. C. Verma, Dr. Jayanthy A., Dr. Anupam Maurya, Dr. (Ms.) Nitin Rai, Dr. V. Vijayakumar, Dr. Nikhil Jirankalgikar, Dr. Vijay Gupta, Dr. Ramachandran S., Sh. Jeetendra Kumar Vaishya and Sh. Suman Halder are appreciated in bringing up this publication. In last, thanks are due to all those who have directly or indirectly contributed in the preparation of this volume.

xxiv

INTRODUCTION The Ayurvedic system of medicine is prevalent in India since the vedic period and as early as the dawn of human civilization. Though Ayurveda has undergone many changes in the course of its long history, it still remains the mainstay of medical relief to a large section of population of the nation. Due to urbanization and dwindling of forests, vaidya by and large is no longer self-contained unit collecting and preparing his own medicines as before. He has now to depend on the newly developed agencies like one collecting and supplying the crude drugs and the other undertaking mass production of medicines in the Ayurvedic pharmaceutical units run on commercial scale. In view of the new trend in Ayurvedic pharmaceutical field, Government of India considered it expedient to utilize the existing Drugs and Cosmetics Act 1940, to also control to a limited measure the Ayurvedic, Siddha and Unani drugs by amending the Act. The act was accordingly amended in 1964, to ensure only a limited control over the production and sale of these medicines namely:i) The manufacture should be carried under prescribed hygienic conditions, under supervision of a person having a prescribed qualification; ii)

The raw materials used in the preparation of drugs should be genuine and properly identified and

iii)

The formula or the true list of all the ingredients, contained in the drugs, should be displayed on the label of every container.

The Ayurvedic Pharmacopoeia Committee, (APC) constituted under the erstwhile Department of AYUSH (vide letter No. 5-5/CCRAS-2006/Tech/APC/Hqrs. dated 12th March, 2009) Ministry of Health and Family Welfare, Govt. of India initiated the exercise on present volume. This Pharmacopoeia Committee included Prof. S. S. Handa (Chairman), Dr. S. K. Sharma (Vice Chairman), Dr. G. S. Lavekar (Member Secretary until February 2010) and Dr. Ramesh Babu Devalla (Member Secretary) and other eminent experts in respective fields. The work was further carried out under auspices of PCIM&H and duly approved by its Governing Body.

xxv

SPECIAL INTRODUCTION TO VOLUME IX (EXTRACTS) Contrary to popular preparation, Ayurvedic therapeutic ‘modes’ or presentations in current usage have had a long history of development. Ayurvedic techniques of formulating compound mixtures (yogas) developed gradually from the pre-Vedic period through the Vedic, the SaÆhit¡, and the Sa´graha periods and continue to develop. In the SaÆhit¡ period, ancient indigenous science was at the peak of its glory and we find that almost all the pharmaceutical modes, now identified as ‘classical’ were known during this period. From the scanty evidences and interpretations available about the pre-Vedic period, one naturally concluded that the pre-Vedic Indian employed only a few pharmaceutical modes and that methods in pharmacy were rather simple. Raw materials were used in their crude form, whole, or at best comminuted, to assist application. Extraction was limited to expression of fresh juice or extracting by decoction. During the Vedic period preparation of medicines might have gained importance, as fire and pyre sacrifices were popular practices followed during the period, but complex formulations and combination of drugs did not appear during this period. Single drugs, their juices and pastes were the main forms in use. It is doubtful whether the formulation, in its contemporary sense, was practiced in the Vedic period. In the Vedas, we find praises of single drugs. No complex mixtures of medicines are traceable in the Vedic literature. We find that systematized information on Ayurvedic pharmacy appears in the compendia of the SaÆhit¡ period. It is in these texts that complicated compounding procedures as well as multi-ingredient formulations are recorded. Tips on formulations, concepts of compatibility and incompatibility among ingredients, systematic classification of preparations etc. are also available in these books. Though the oldest available Samhita contains references to almost all the classical pharmaceutical modes, it is not logical to conclude that all of them developed simultaneously. Hence, we may consider that primary preparations or basic modes were the most ancient. These work technically termed “KaÀ¡ya Kalpan¡”. Five primary preparations, such as expressed juice (Svarasa), paste (Kalka), decoction (Kv¡tha), cold infusion (á¢ta KaÀ¡ya) and hot infusion (Ph¡¸¶a KaÀ¡ya) are mentioned in the ancient classics. All these modes have very short shelf life and hence were prepared as and when needed. More stable forms generated as secondary and tertiary preparations or derived modes such as medicated fatty preparations (oils, gh¤ta, etc.), Jelly or semi solid preparations (Avaleha), fermented products (AriÀ¶a, Ësava etc.) & Pills (Gu¶ik¡) were yet another means of presentation of drugs. Apart from many other factors, fats, sugar and alcohol were known to be natural preservatives. During the Sa´graha period, we find from recorded literature that formulations based on metals and minerals gained usage. This is on par with the development of Ayurvedic iatro-chemistry known as Rasa¿¡stra. Some pharmaceutical modes such as syrups also appeared in Ayurvedic pharmacy. This was mainly due to the influence of Unani medicine that emanated from Middle East. In fact, it started even as early as the invasion of Alexander the Great, but attained great growth due to the active contribution by Moghuls in Medieval India. A gradual shift in practice from extemporaneous and need based production, to a more organized and planned long term production ensured ready medicines round the year. This trend was evident as early as the 10th century AD, but got established at the dawn of the 20th century, resulting in a change in the pattern of drug production, and improvement in its technology. Ayurvedic pharmacy by this time faced challenges from Western medicines and modern methods of pharmacy during the British rule. There were efforts to replace aqueous decoctions of indigenous medicines with tinctures but such changes faced stiff opposition from the conservative section of Ayurvedic physicians. xxvi

Present trend: Since the last half a century, Ayurveda has had to compete with modern medicines, which are proven to be quick-acting, strong and effective. Convenience and acceptability of these medicines by patients was another factor that necessitated the Ayurvedic fraternity to modernize. Many manufacturers shifted their traditional preparation like Va¶¢ and Gu¶ik¡ to compressed tablets and capsules. There were attempts to achieve greater shelf life for traditional medicines, by adopting newer techniques of extraction, chemical preservation and application of modern principles in Ayurvedic pharmacy. A recent trend in Ayurvedic manufacturing pharmacy aims at (1) enhancement of potency and reduction in bulk of dosage form (2) convenience in administering doses and (3) acceptability by improving palatability. This trend is an outcome of significant gains in knowledge of phyto-chemical contents of the source plant, and improved methods of assessing the pharmacological and therapeutic actions of such phytochemical contents. A direct development of this awareness is the introduction of extracts of plants as a more effective means of obtaining desirable results. EXTRACTS Extraction, as the term is used in Pharmacy, involves the separation of medicinally active portion from plant or animal tissues using selective solvents through standard extraction procedures. The general techniques of extraction of medicinal plants include maceration, infusion, percolation, digestion, decoction, hot continuous extraction (soxhlet), aqueous alcoholic extraction by fermentation (such as Ësava) counter current extraction (CCE), microwave assisted extraction, ultrasound extraction (sonication), supercritical fluid extraction (SFE), etc. Such extraction techniques separate out soluble plant metabolites leaving behind insoluble cellular marc. The product so obtained from plants are relatively complex mixtures of a number of groups of plant metabolites. Extracts are prepared by using an appropriate menstruum, with a view to extraction of active principles or at least elimination of the inert bulk. Hence, modernization of Ayurvedic drug industry is experimenting with various extraction techniques. More and more capsules and tablets appearing in the market are based on products using extraction techniques. Even liquids like syrups, medicated oils and other oral suspensions depend on the extracts. Extraction is essential to reduce the bulk of the drug material and enhance its potency, acceptability and convenience of administration of the drug. The purpose of standardized extraction procedures for crude drugs is to acquire the therapeutically desired portion and eliminate inert material, by treatment with a selective solvent known as menstruum. The extract thus obtained is used as a medicinal agent directly, or further processed to be incorporated in any dosage form such as tablet, capsule or syrup etc. Standardized extract for use in a pharmacopoeia indicates an extract having an acceptable limit of the given content, specified by a biomarker or chemical/analytical marker. The extract should specify the defined range for the constituents (biomarker or chemical/analytical marker). Dry extracts usually have a loss on drying or water content not greater than 5 per cent w/w, unless specified otherwise in any monograph. In the cases of standardized extracts, the presence and content of the inert permissible excipients including preservatives, if any, should be declared on the label. Extracts shall be free from solvents used for the extraction and shall comply with the respective limits (Appendix 3.8). Harmful and carcinogenic solvents shall not be used for extraction purposes. Extracts may be exposed to ethylene oxide for fumigation or low dose gamma radiation for the purpose of avoiding microbial contamination. In cases where the extracts are fumigated, the final extracts exposed shall meet residual levels of ethylene dioxide limits as applicable. Herbs treated with low dose of gamma radiations shall meet national regulations related to such a treatment and shall be labeled as per law. xxvii

Phyto-chemical Reference Standards as Markers: Extracts are usually complex mixtures of several chemical constituents. For a large majority of botanical extracts it is not known with certainty which of the various components is responsible for the reported pharmacological effect. It is generally believed that several constituents act synergistically to provide the reported effect. For articles for which compendial monographs are provided, certain chemical constituents of the article are chosen and quantitative test procedures for determining their content are provided. The choice of such constituents, known generally as marker compounds, is based on certain considerations. Currently, the following types of marker compounds are specified in compendial monographs and may be identified in raw materials: Active Principles: These are constituents that have proven clinical activity. A minimum content or range for the active principles is usually specified in the individual monograph. A quantitative determination of active principles carried out periodically during stability studies of dosage forms provides necessary information for arriving at suitable expiry dates. Active Markers: These are constituents that have known pharmacological activity contributing in some extent to its efficacy. However, the clinical efficacy for these constituents may not be proven. A minimum content or range for active markers is usually specified in individual monographs. A quantitative determination of active markers during stability studies of dosage forms provides necessary information for arriving at suitable expiry dates. Analytical Markers: Where neither defined active principles nor active markers are known, other constituents of the botanical extract amenable to quantitative determination are chosen. These markers aid in the positive identification of the article under text. In addition, maintaining a minimum content or a specified range of the analytical markers helps to achieve standardization of the plant extract and to arrive at a suitable expiry date during stability studies. Negative Markers: These are constituents that may have toxic or allergenic properties, rendering their presence in the botanical extract undesirable as for example β-asarone from Vac¡ (Acorus calamus). A stringent limit for such negative markers may be specified in individual monographs. Monograph on Plant Extracts: Monograph of an extract in the pharmacopoeia is to provide qualitative and quantitative standards of quality for the extract for its use as a food item or a food supplement, dietary supplement/ nutraceutical, as a drug and/or as an ingredient in cosmetics. Each of such use would need to comply with applicable regulations. As per quality criteria, the plant extract in its entirety, is defined as the active substance. Consequently, all relevant aspects of quality of an extract must be considered and these include plant material, solvent used for extraction, extraction technology employed and manufacturing equipment used. Methodology: Fifteen single plant drugs were selected for preparing aqueous and hydro-alcoholic dried extracts. Authenticity, purity and quality of each of the fifteen plant drugs and their powders was confirmed before preparing hydro-alcoholic extract and water extract. In order to make this volume self-containing and xxviii

comprehensive, monograph of the selected whole plant drug, hydro-alcoholic extract and water extract have been prepared. The work of preparing extracts using identical standard operating procedures was allotted to different extract manufacturers in two groups viz., Natural Remedies Pvt. Ltd. Bangalore, Chemolids Pvt. Ltd. Vijayawada and Green Chem Pvt. Ltd. Bangalore in one group and Sanat Product Pvt. Ltd. Bulendashar, Arjuna Products Pvt. Ltd, Kochi and Amsar Pvt. Ltd. Indore in other group. Both the groups are co-ordinated by Natural Remedies Pvt. Ltd. Bangalore. Raw material was procured in one lot and distributed among the three collaborators. Data generated was shared on three batches of each. Comprehensive reproducible data has been incorporated in the monographs. The monographs on fifteen plant drugs have been already included in earlier volumes of API, Part I. They have now been upgraded by the addition of Thin Layer Chromatography, finger print profiling using Phyto-Chemical Reference Standard (PRS). New addition of assay for the PRS has been also added to make the monograph comparable to international standards. Keeping in view the use of extracts in Ayurvedic formulations, the Drugs and Cosmetics 5th Amendment Rule, 2010 under 158 (B) clause IV issues guidelines with respect to AuÀadha Ghana (Medicinal Plant extracts- dry/wet) obtained from plants mentioned in books of First Schedule of the Act including aqueous, hydro-alcoholic and other than aqueous and hydro-alcoholic extracts. In the light of this latest amendment in the Drugs and Cosmetics Rule 158 (B) clause IV, the Ayurvedic Pharmacopoeia Committee considers it appropriate to prepare monographs on plant extracts and in this Volume IX of the API, Part I, standards for aqueous and hydro-alcoholic extracts are presented. To start with, 15 traditionally well-known medicinal plants have been selected and this volume contains upgraded monographs on 15 source plants, their aqueous and hydro-alcoholic extracts, thus comprising a total of 45 monographs, for ensuring their quality for use as drug ingredients. We offer this Volume for public use and welcome comments and criticisms to enhance its value in future revisions and additions.

xxix

ABBREVIATIONS FOR TECHNICAL TERMS 0

C cells containing pigments centimeter(s) cluster crystals of calcium oxalate concentrated diameter dilute gram per liter gram(s) hour(s) kilogram(s) litre(s) meta meter(s) micron (0.001mm) microgram per milliliter microliter micrometer milligram(s) milliliter per minute milliliter(s) millimeter minute(s) Molarity nanogram nanoliter nanometer Normality ortho para parts per billion parts per million phloem fibres rate per minute





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ccp cm clr con. dia. dil. g/l g h kg l m m  g/ml l m mg ml/min ml mm min M ng nl nm N o p ppb ppm phf rpm

xxx

Reference Standard Refractive index detector rosette crystals of calcium oxalate specific gravity starch grains Ultraviolet volume volume in volume volume in weight weight weight in volume weight in weight

_

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xxxi

RS RI detector ro sp.gr. sg UV vol v/v v/w wt w/v w/w

INDO-ROMANIC EQUIVALENTS FOR DEVANËGARÌ ALPHABETS + +É < <Ç = >ð @ñ B Bä +Éä +Éè Æ : Eò JÉ MÉ PÉ Ró SÉ Uô VÉ ZÉ \É ]õ `ö

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au

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ka

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kha

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xxxii

ÚA

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ta

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tha

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da

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ba

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CONTENTS FOREWORD PREFACE LEGAL NOTICES GENERAL NOTICES PHARMACOPOEIA COMMISSION FOR INDIAN MEDICINE & HOMOEOPATHY ACKNOWLEDGMENTS INTRODUCTION SPECIAL INTRODUCTION TO VOLUME IX (EXTRACTS) ABBREVIATIONS FOR TECHNICAL TERMS INDO-ROMANIC EQUIVALENTS OF DEVANËGARÌ ALPHABETS MONOGRAPHS Ap¡m¡rga 1. Ap¡m¡rga Hydro-alcoholic extract 2. Ap¡m¡rga Water extract 3. Asana 4. Asana Hydro-alcoholic extract 5. Asana Water extract 6. D¡ruharidr¡ 7. D¡ruharidr¡ Hydro-alcoholic extract 8. D¡ruharidr¡ Water extract 9. 10. Dh¡r¡ V¤kÀ¡mla 11. Dh¡r¡ V¤kÀ¡mla Hydro-alcoholic extract 12. Dh¡r¡ V¤kÀ¡mla Water extract 13. Ka¶uk¡ 14. Ka¶uk¡ Hydro-alcoholic extract 15. Ka¶uk¡ Water extract 16. MaµjiÀ¶Å¡ 17. MaµjiÀ¶Å¡ Hydro-alcoholic extract 18. MaµjiÀ¶Å¡ Water extract 19. MeÀa¿¤´g¢ 20. MeÀa¿¤´g¢ Hydro-alcoholic extract 21. MeÀa¿¤´g¢ Water extract 22. Meth¢ 23. Meth¢ Hydro-alcoholic extract 24. Meth¢ Water extract 25. Ni¤gu¸·¢ 26. Ni¤gu¸·¢ Hydro-alcoholic extract 27. Ni¤gu¸·¢ Water extract 28. Punarnav¡ 29. Punarnav¡ Hydro-alcoholic extract 30. Punarnav¡ Water extract 31. áallak¢ 32. áallak¢ Hydro-alcoholic extract xxxiii

iii v vii viii xv xxiv xxv xxvi xxx xxii 1 5 7 9 12 14 16 19 21 23 25 27 29 32 34 36 39 41 43 47 49 51 54 56 58 61 63 65 68 70 72 74

33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

áallak¢ Water extract áir¢Àa áir¢Àa Hydro-alcoholic extract áir¢Àa Water extract áu¸¶h¢ áu¸¶h¢ Hydro-alcoholic extract áu¸¶h¢ Water extract Svar¸apatr¢ Svar¸apatr¢ Hydro-alcoholic extract Svar¸apatr¢ Water extract Tulas¢ Tulas¢ Hydro-alcoholic extract Tulas¢ Water extract

Appendix - 1 1.1 Apparatus for Tests and Assays 1.1.1 Nessler Cylinders 1.1.2 Sieves 1.1.3 Thermometers 1.1.4 Ultraviolet Lamp (For general purposes and for chromatography work) 1.1.5 Volumetric Glassware 1.1.6 Weights and Balances Appendix - 2 2.1 Tests and Determinations 2.1.1. Microscopical identification of Botanical Substances 2.1.2. Net Content 2.1.3. Determination of Foreign Matter 2.1.4. Determination of Moisture content (Loss on Drying) 2.1.5. Determination of Total Ash 2.1.6. Determination of Water-soluble Ash 2.1.7. Determination of Acid-insoluble Ash 2.1.8. Determination of Alcohol-soluble Extractive 2.1.9. Determination of Water-soluble Extractive 2.1.10. Determination of pH Value 2.1.11. Determination of Total-soluble Solids 2.1.12. Determination of Volatile oil in drugs Appendix - 3 3.1 Test for Heavy Metals 3.1.1 Limits for Heavy Metals 3.1.2 Determination of Lead, Cadmium, Arsenic and Mercury 3.2 Microbial Limit Tests 3.2.1 Total Aerobic Microbial Count 3.2.2 Tests for Specified Microorganisms 3.3 Pesticide Residue 3.3.1 Test for Pesticides 3.3.2 Quantitative Analysis 3.4 Test for Aflatoxins xxxiv

76 78 81 83 85 88 90 92 95 97 99 102 104 109 109 109 109 109 109 110 110 111 111 111 113 113 113 113 114 114 114 114 114 115 115 117 117 117 117 117 122 124 127 128 129 129

3.5 Thin-Layer Chromatography (TLC) 3.5.1 Quantitative Measurement 3.6 Liquid Chromatography 3.7 Spectrophotometry 3.8 Test for Residual Solvent 3.9 Stability Testing and Shelf Life Determination 3.9.1 Scope and Objective 3.9.2 General Information on Stability 3.9.3 Selection Batches 3.9.4 Container and Closure system 3.9.5 Specification 3.9.6 Testing frequency 3.9.7 Storage condition 3.9.8 Evaluation 4 Reagents and Chemicals 5 Weights and Measures 5.1 Metric Equivalents of Classical Weights and Measures 5.2 Metric System

xxxv

130 132 133 133 134 136 136 136 136 136 137 137 137 138 139 145 145 146

 

API, Part-I, Vol.-IX (Extracts); Monographs 

APËMËRGA  Ap¡m¡rga consists of dried whole plant of Achyranthes aspera L. (Fam. Amaranthaceae); a stiff, erect, 0.3-0.9 m high herb, found commonly as a weed throughout India up to 900 m. Ap¡m¡rga contains not less than 0.002 per cent of oleanolic acid when assayed.

Fruit - Indehiscent, dry utricle enclosed within persistent perianth and bracteoles

Synonyms:

Root - Mature root shows 3-8 layered, rectangular, tangentially elongated, thin-walled cork cells; secondary cortex consisting of 6-9 layers, oval to rectangular, thin-walled, parenchymatous cells having a few scattered single or groups of stone cells; followed by 4-6 discontinuous rings of anomalous secondary thickening composed of vascular tissues; small patches of sieve tubes distinct in phloem parenchyma, demarcating the xylem rings; xylem composed of usual elements; vessels simple pitted; medullary rays 1-3 cells wide; small prismatic crystals of calcium oxalate present in cortical region and numerous in medullary rays

May£raka, Kharamaμjar¢, áikhar¢

Other/Regional

Seed - Sub-cylindric, truncate at the apex, round at the base, endospermic, brown b) Microscopic:

PratyakpuÀpa,

Language

Names:

Assamese: Chirchita; Bengali: Apamg; English: Prickly Chaff Flower; Gujarati: Aghedo; Hindi: Chirchita, Latjira; Kannada: Uttarani; Malayalam: Katalati; Marathi: Aghada; Punjabi: Puthakanda; Tamil: Nayuruvi; Telugu: Uttarenu; Urdu: Chirchita

Description: a) Macroscopic: Root - Cylindrical tap root, slightly ribbed, 0.1-1.0 cm in thickness, gradually tapering, rough due to presence of some root scars, secondary and tertiary roots present, yellowish-brown; odour, not distinct

Stem - Young stem shows 6-10 prominent ridges, which diminish downwards upto the base where it becomes almost cylindrical; epidermis single layered, covered by thick cuticle having uniseriate, 2-5 celled, covering trichomes and glandular with globular head, 3-4 celled stalk; cortex 6-10 layered, composed of parenchymatous cells, most of them containing rosette crystals of calcium oxalate; in the ridges cortex collenchymatous; vascular bundles lie facing each ridge capped by pericyclic fibres; transverse section of mature stem shows lignified, thin-walled cork cells; pericycle a discontinuous ring of lignified fibres; vascular tissues show anomalous secondary growth having 4-6 incomplete rings of xylem and phloem; secondary phloem consisting of usual elements form incomplete rings; cambial strips present between secondary xylem and phloem; secondary xylem consists of vessels annular, spiral, scalariform and elongately pitted, fibres elongated, lignified; pith wide consisting of oval to polygonal, parenchymatous cells

Stem - 0.3-0.5 cm in cut pieces, yellowish-brown, erect, branched, cylindrical, hairy, solid, hollow when dry Leaf - Simple, subsessile, exstipulate, opposite, decussate, wavy margin, obovate, slightly acuminate and pubescent due to the presence of thick coat of long simple hairs Flower - Arranged in inflorescence of long spikes, greenish-white, numerous, sessile, bracteate with two bracteoles, one spine lipped, bisexual, actinomorphic, hypogynous; perianth segments 5, free, membranous, contorted or quincuncial, stamens 5, opposite the perianth lobes, connate forming a membranous tube-like structure, alternating with truncate and fimbriate staminodes, filament short; anther, two celled, dorsifixed; gynoecium bicarpellary, syncarpous; ovary superior, unilocular with single ovule; style single; stigma capitate

1  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Leaf - Petiole - Shows crescent-shaped outline, having single-layered epidermis with thick cuticle; ground tissues consisting of thin-walled, parenchymatous cells containing rosette crystals of calcium oxalate; 4-5 vascular bundles situated in mid region Midrib - Shows a single layered epidermis, on both surfaces; epidermis followed by 4-5 layered collenchyma on upper side and 2-3 layered on lower side; ground tissue consisting of thin-walled, parenchymatous cells having a number of vascular bundles; each vascular bundle shows below the xylem vessels, thin layers of cambium, followed by phloem and a pericycle represented by 2-3 layers of thick-walled, non-lignified cells; rosette crystals of calcium oxalate found scattered in ground tissues Lamina - Shows single layered, tangentially elongated epidermal cells covered with thick cuticle having covering trichomes, similar to those of stem, on both surfaces; mesophyll differentiated into palisade and spongy parenchyma, palisade 2-4 layered of thick parenchyma; larger, slightly elongated in upper, while smaller and rectangular in lower surface; spongy parenchyma 3-5 layers thick, consisting of more or less isodiametric parenchymatous cells; idioblast containing large rosette crystals of calcium oxalate distributed in palisade and spongy parenchyma cells; stomata anisocytic and anomocytic, on both surfaces; stomatal index 4.59.0 on upper surface, 9.0-20.0 on lower surface; palisade ratio 7.0-11; vein islet number 7-13

Fig. 1: Powdered drug of APËMËRGA (Achyranthes aspera L.)

Identity, Purity and Strength: Identification: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using oleanolic acid as a reference standard. Test solution: Extract 2 g of substance by refluxing with 50 per cent methanol (50 ml x 3) for a period of 30 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 100-ml round bottomed flask. Add 20 ml of 2 M methanolic hydrochloric acid and reflux at 60700 on a water bath for 3 hours, cool and transfer the solution to a separating flask, extract with chloroform (25 ml x 3). Combine all the organic extracts and wash gently with water. Pass the combined chloroform extract through anhydrous sodium sulphate and evaporate the chloroform under vacuum. Dissolve the residue obtained in 10 ml of methanol. Standard solution: Dissolve 10 mg of oleanolic acid RS in about 10 ml of methanol.

c) Powder: Powder shows simple, multicellular, sharp or blunt ended, warty or smooth trichomes; lower epidermal cells of leaf showing sinuous walls, and upper with fairly straight walls, glandular trichomes with globular head of 3 or 4 cells, anisocytic and anomocytic stomata, thick-walled and thin-walled fibres with sharp or forked ends; cork tissue, fragments of pitted vessels; prismatic and rosette and sandy crystals of calcium oxalate scattered as such throughout or in idioblasts (Fig. 1) 2  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Visible after derivatisation Rf

residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

1.0

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with 50 per cent methanol (50 ml x 3) on a water bath for 30 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 100 ml round bottomed flask. Add 20 ml of 2 M methanolic hydrochloric acid and reflux at 60-700 on a water bath for 3 hours, cool and transfer the solution to a separating flask, extract with chloroform (25 ml x 3). Combine all the organic extracts and wash gently with water, pass the combined chloroform extract through anhydrous sodium sulphate and evaporate the chloroform under vacuum. Dissolve the residue obtained in 5 ml of methanol, transfer to a 10-ml volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, oleanolic acid RS in a 100 ml volumetric flask and dissolve in 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (100 mm x 3.0 mm, 2.5 m). Mobile phase: Filtered and degassed mixture of 33 volumes of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 900 ml of water, adding 1 ml of orthophosphoric acid and making up the volume to 1000 ml) and 67 volumes of acetonitrile. Injection volume: 20 l. Flow rate: 0.3 ml per min. Detection: UV 205 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of oleanolic acid in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

0.5

0.0 RS T Fig. 2: Thin-Layer Chromatogram of Ap¡m¡rga RS: Oleanolic acid, T: Test solution Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : toluene : formic acid (45.0 : 0.5 : 0.1). Dry the plate in air. Spray the plate with 10 per cent methanolic sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 2). Quantitative parameters: Foreign matter: not more than 2.0 per cent (Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 17.0 per cent (Appendix 2.1.5); Acidinsoluble ash: not more than 5.0 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 2.0 per cent (Appendix 2.1.8); Watersoluble extractive: not less than 12.0 per cent (Appendix 2.1.9)

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide 3  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Constituents: Oleanolic acid glycosides; ecdysterone, ecdysone, betaine, tritriacontanone, pentatriacontan-17-ol, 27-cyclohexylheptacosan7-ol, 16-hydroxy-26-methylheptacosan-2-one, 4methylheptatriacont-1-en-10-ol, etracontanol, βsitosterol, pentatriacontane, pentatriacontan-6one, hexatriacontane, triacontane, hentriacontane, octacosan-10-one, triacosan-4-one; lauric, myristic, palmitic, stearic, oleic, linoleic, arachidic and behenic acids

Properties and Action: Rasa: Ka¶u, Tikta;

Fig. 3: HPLC chromatogram of Ap¡m¡rga with Oleanolic acid as RS

Additional requirements:

Gu¸a: Sara, T¢kÀ¸a; V¢rya: UÀ¸a; Vip¡ka: Ka¶u; Karma: Chedana, Kaphahara, Medohara, P¡cana, V¡tahara

Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents.

Ap¡m¡rgakÀ¡ra taila, Gu·apippal¢, JyotiÀmat¢ taila

Important formulations:  Abhy¡ lava¸a, Therapeutic

Apac¢ (chronic lymphadenopathy/scrofula), Ar¿a (piles), Ka¸·£ (pruritis), Medoroga (obesity), á£la (pain), Udararoga (disease of abdomen)

Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard:

Dose: C£r¸a (powder): 3-6 g

API Oleanolic acid RS

4  

uses:

Ap¡m¡rgakÀ¡ra,

 

API, Part-I, Vol.-IX (Extracts); Monographs 

APËMËRGA HYDRO-ALCOHOLIC EXTRACT methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : toluene : formic acid (45.0 : 0.5 : 0.1). Dry the plate in air. Spray the plate with 10 per cent methanolic sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Ap¡m¡rga Hydro-alcoholic Extract is a dried and powdered extract prepared from Ap¡m¡rga (appropriately powdered). The extract contains not less than 0.08 per cent of oleanolic acid when assayed.

Method of Preparation: Take Ap¡m¡rga suitably sized (powder or pieces) in an extractor. Add 50.0 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under reflux at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 7 per cent.

Visible after derivatisation Rf 1.0

0.5

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using oleanolic acid as a reference standard. Test solution: Extract 2 g of substance by refluxing with 50 per cent methanol (50 ml x 3) for a period of 30 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 100-ml round bottomed flask. Add 20 ml of 2 M methanolic hydrochloric acid and reflux at 60-700 on a water bath for 3 hours, cool and transfer the solution to a separating flask, extract with chloroform (25 ml x 3). Combine all the organic extracts and wash gently with water. Pass the combined chloroform extract through anhydrous sodium sulphate and evaporate the chloroform under vacuum. Dissolve the residue obtained in 10 ml of methanol. Standard solution: Dissolve 10 mg of oleanolic acid RS in about 10 ml of

0.0 RS T Fig. 1: Thin-Layer Chromatogram of Ap¡m¡rga hydro-alcoholic extract RS: Oleanolic acid, T: Test solution

Quantitative parameters: Loss on drying: not more than 5.0 per cent (Appendix 2.1.4); Total ash: not more than 10.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.0-6.5 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-I)

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API, Part-I, Vol.-IX (Extracts); Monographs 

Other requirements:

Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of oleanolic acid in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with 50 per cent methanol (50 ml x 3) on a water bath for 30 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 100-ml round bottomed flask. Add 20 ml of 2 M methanolic hydrochloric acid and reflux at 60-700 on a water bath for 3 hours, cool and transfer the solution to a separating flask, extract with chloroform (25 ml x 3). Combine all the organic extracts and wash gently with water, pass the combined chloroform extract through anhydrous sodium sulphate and evaporate the chloroform under vacuum. Dissolve the residue obtained in 5 ml of methanol, transfer to a 10-ml volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, oleanolic acid RS in a 100 ml flask and dissolve in 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (100 mm x 3.0 mm, 2.5 m). Mobile phase: Filtered and degassed mixture of 33 volumes of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 900 ml of water, adding 1 ml of orthophosphoric acid and making up the volume to 1000 ml) and 67 volumes of acetonitrile. Injection volume: 20 l. Flow rate: 0.3 ml per min. Detection: UV 205 nm.

Fig. 2: HPLC chromatogram of Ap¡m¡rga hydro-alcoholic extract with Oleanolic acid as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Oleanolic acid RS

6  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

APËMËRGA WATER EXTRACT Ap¡m¡rga Water Extract is a dried and powdered extract prepared from Ap¡m¡rga. The extract contains not less than 0.01 per cent of oleanolic acid when assayed.

and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase : ethyl acetate : toluene : formic acid (45.0 : 0.5 : 0.1). Dry the plate in air. Spray the plate with 10 per cent methanolic sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Method of Preparation: Take Ap¡m¡rga suitably sized (powder or pieces) in an extractor. Add water, about 3 times the quantity of raw material and heat at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 9 per cent.

Visible after derivatisation Rf 1.0

0.5

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using oleanolic acid as a reference standard. Test solution: Extract 2 g of substance by refluxing with 50 per cent methanol (50 ml x 3) for a period of 30 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 100-ml round bottomed flask. Add 20 ml of 2 M methanolic hydrochloric acid and reflux at 60-700 on a water bath for 3 hours, cool and transfer the solution to a separating flask, extract with chloroform (25 ml x 3). Combine all the organic extracts and wash gently with water. Pass the combined chloroform extract through anhydrous sodium sulphate and evaporate the chloroform under vacuum. Dissolve the residue obtained in 10 ml of methanol. Standard solution: Dissolve 10 mg of oleanolic acid RS in about 10 ml of methanol. Procedure: Apply 10 l each of the test

0.0 RS

Fig. 1: Thin-Layer Chromatogram of Ap¡m¡rga water extract RS: Oleanolic acid, T: Test solution

Quantitative parameters: Loss on drying: not more than 5.0 per cent (Appendix 2.1.4); Total Ash: not more than 25.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.5- 6.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-II)

7  

T

 

API, Part-I, Vol.-IX (Extracts); Monographs 

test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of oleanolic acid in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with 50 per cent methanol (50 ml x 3) on a water bath for 30 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 100 ml round bottomed flask. Add 20 ml of 2 M methanolic hydrochloric acid and reflux at 60-700 on a water bath for 3 hours, cool and transfer the solution to a separating flask, extract with chloroform (25 ml x 3). Combine all the organic extracts and wash gently with water, pass the combined chloroform extract through anhydrous sodium sulphate and evaporate the chloroform under vacuum. Dissolve the residue obtained in 5 ml of methanol, transfer to a 10 ml volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, oleanolic acid RS in a 100-ml volumetric flask and dissolve in 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (100 mm x 3.0 mm, 2.5 m). Mobile phase: Filtered and degassed mixture of 33 volumes of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 900 ml of water, adding 1 ml of orthophosphoric acid and making up the volume to 1000 ml) and 67 volumes of acetonitrile. Injection volume: 20 l. Flow rate: 0.3 ml per min. Detection: UV 205 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the

Fig. 2: HPLC chromatogram of Ap¡m¡rga water extract with Oleanolic acid as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Oleanolic acid RS  

8  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

ASANA Asana consists of heart-wood of Pterocarpus marsupium Roxb. (Fam. Fabaceae); a moderate to large sized, deciduous tree, upto 30 m high and 2.5 m in girth, with straight clear bole, found mostly throughout Gujarat, Madhya Pradesh, Bihar and Orissa. Asana contains not less than 0.25 per cent of pterostilbene when assayed.

c) Powder: Powder shows vessels with bordered pits, fibre tracheids, tracheids, fragments of xylem rays and few crystal fibres, starch absent (Fig. 1).

Synonyms: B¢jaka, P¢tas¡ra, Asanaka, B¢jas¡ra Other/Regional Language Names: Assamese: Aajar; Bengali: Piyasala, Pitasala; English: Indian Kino Tree; Gujarati: Biyo; Hindi: Vijayasara, Bija; Kannada: Bijasara, Asana; Kashmiri: Lal chandeur; Malayalam: Venga; Marathi: Bibala; Oriya: Piashala; Punjabi: Chandan Lal, Channanlala; Tamil: Vengai; Telugu: Yegi, Vegisa; Urdu: Bijasar

Description: a) Macroscopic: Drug occurs as irregular pieces of variable size and thickness, golden yellowish brown with darker streaks, on soaking in water gives yellow colour solution with blue fluorescence; fracture strong, tough, very hard, moderately heavy difficult to break but brittle; taste astringent. Fig. 1: Powdered drug of ASANA{xe  "ASANA"} (Pterocarpus marsupium Roxb.)

b) Microscopic: Transverse section shows alternating bands of larger and smaller polygonal cells consisting of tracheids, fibre tracheids, xylem parenchyma and traversed by xylem rays, numerous xylem vessels distributed throughout, in singles or in groups of 23, showing tyloses in isolated preparations, vessels drum or barrel shaped with well-marked perforation rims and bordered pits, tracheids numerous, long, thick-walled with tapering ends and simple pits, fibre tracheids elongated, thick-walled with narrow lumen and simple pits, xylem parenchyma rectangular with simple pits, paratracheal surrounding vessels, xylem rays uni to biseriate, 35-7 cells high, prismatic crystals of calcium oxalate present in crystal fibres, starch absent

Identity, Purity and Strength: Identification: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using pterostilbene as a reference standard. Test solution: Extract 1 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg of pterostilbene RS in about 10 ml of methanol. Procedure: Apply 10 l 9

 

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase : ethyl acetate : nhexane (5.5 : 4.5). 254 nm

Visible after derivatisation

Assay:

Rf 1.0

0.5

0.0 RS

T

RS

T

Fig. 2: Thin-Layer Chromatogram of Asana RS: Pterostilbene, T: Test solution Dry the plate in air and examine under UV 254 nm. Spray the plate with anisaldehyde - sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 2).

Time (min) 0.01 5 10 20 25 30 35

Quantitative parameters: Foreign matter: not more than 2.0 per cent (Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 2.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 0.5 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 7.0 per cent (Appendix 2.1.8); Water-soluble extractive: not less than 5.0 per cent (Appendix 2.1.9)

Phosphate buffer (per cent) 85 45 35 35 45 85 85

Acetonitrile (per cent) 15 55 65 65 55 15 15

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 320 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the

Other requirements:

10  

Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (25 ml x 3) on water bath for 15 min each, cool and filter. Combine all the filtrates and transfer to a 100-ml volumetric flask and make up the volume with methanol. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, pterostilbene RS in a 100 ml volumetric flask and dissolve in about 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 500 ml of water, adding 0.5 ml of orthophosphoric acid and making up the volume to 1000 ml) and acetonitrile in the following proportions:-

 

API, Part-I, Vol.-IX (Extracts); Monographs 

API reference standard:

response for the analyte peak. Calculate the content of pterostilbene in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

API Pterostilbene RS

Constituents:

Pterostilbene, marsupsin, liquiritigenin, isoliquiritigenin, pterosupin, p-hydroxybenzaldehyde, 7,4'-dihydroxyflavone, (2R)-3-(p-hydroxyphenyl) lactic acid, propterol, marsupol, carpusin

Properties and Action: Rasa: Ka¶u, Tikta, KaÀ¡ya; Gu¸a: Laghu, R£kÀa; V¢rya: á¢ta; Vip¡ka: Ka¶u; Karma: Kaphamedovi¿oÀa¸a, Ke¿ya, KuÀ¶haghna, Rakta¿odhana, Ras¡yana, Stambhana, Tvacya  

Important formulations: Asanabilv¡di taila, Nyagrodh¡di c£r¸a

Therapeutic uses: P¡¸·u (anaemia), Prameha (increased frequency and turbidity of urine)

Dose: C£r¸a (powder): 3-6 g

Fig. 3: HPLC chromatogram of Asana with Pterostilbene as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

11  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

ASANA HYDRO-ALCOHOLIC EXTRACT shows a band corresponding to that of the standard solution (Fig. 1).

Asana Hydro-alcoholic Extract is a dried and powdered extract prepared from Asana (appropriately powdered). The extract contains not less than 0.5 per cent of pterostilbene when assayed.

254 nm

Visible after   derivatisation

Method of preparation: Take Asana suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under a reflux at a temperature between 80850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 6 per cent.

1.0

0.5

0.0

RS

T

RS 



RS: Pterostilbene, T: Test solution Fig. 1: Thin-Layer Chromatogram of Asana hydro-alcoholic extract

Identity, Purity and Strength: Thin-layer chromatography:

Quantitative parameters:

Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using pterostilbene as a reference standard. Test solution: Extract 1 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg of pterostilbene RS in about 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase : ethyl acetate : n-hexane (5.5 : 4.5). Dry the plate in air and examine under UV 254 nm. Spray the plate with anisaldehyde sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution

Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 9.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.07.0 (Appendix 2.1.10); Total soluble solids: not less than 85.0 per cent (Appendix 2.1.11) (Method-I)

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4) 12

 

Rf

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (25 ml x 3) on water bath for 15 min each, cool and filter. Combine all the filtrates and transfer to a 100-ml volumetric flask and make up the volume with methanol. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, pterostilbene RS in a 100-ml volumetric flask and dissolve in about 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 500 ml of water, adding 0.5 ml of orthophosphoric acid and making up the volume to 1000 ml) and acetonitrile in the following proportions:Time (min) 0.01 5 10 20 25 30 35

Phosphate buffer (per cent) 85 45 35 35 45 85 85

Fig. 2: HPLC chromatogram of Asana hydroalcoholic extract with Pterostilbene as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Pterostilbene RS

Acetonitrile (per cent)

 

15 55 65 65 55 15 15

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 320 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of pterostilbene in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. 13  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

ASANA WATER EXTRACT Asana Water Extract is a dried and powdered extract prepared from Asana. The extract contains not less than 0.1 per cent of pterostilbene when assayed.

Rf 1.0

Method of preparation: Take Asana suitably sized (powder or pieces) in an extractor. Add water, about 3 times the quantity of raw material and heat at a temperature between 80850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh and pack. The yield obtained is about 8 per cent.

0.5

0.0

Fig. 1: Thin-Layer Chromatogram of Asana water extract RS: Pterostilbene, T: Test solution

Quantitative parameters:

Identity, Purity and Strength:

Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 8.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.07.0 (Appendix 2.1.10); Total soluble solids: not less than 65.0 per cent (Appendix 2.1.11) (Method-II)

Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using pterostilbene as a reference standard. Test solution: Extract 1 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg of pterostilbene RS in about 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase : ethyl acetate : n-hexane (5.5 : 4.5). Dry the plate in air and examine under UV 254 nm. Spray the plate with anisaldehyde sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (25 ml x 3) on water bath for 15 min each, cool and filter. 14

 

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Combine all the filtrates and transfer to a 100 ml volumetric flask and make up the volume with methanol. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, pterostilbene RS in a 100 ml volumetric flask and dissolve in about 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 500 ml of water, adding 0.5 ml of orthophosphoric acid and making up the volume to 1000 ml) and acetonitrile in the following proportions:Time (min) 0.01 5 10 20 25 30 35

Phosphate buffer (per cent) 85 45 35 35 45 85 85

Fig. 2: HPLC chromatogram of Asana water extract with Pterostilbene as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents.

Acetonitrile (per cent) 15 55 65 65 55 15 15

Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Pterostilbene RS

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 320 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of pterostilbene in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

15  

 

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DËRUHARIDRË prismatic crystals of calcium oxalate; stone cells also found scattered in phloem ray cells in groups, rarely single, mostly elongated, a few rounded; secondary phloem, a broad zone, consisting of sieve elements and phloem fibres, traversed by multiseriate phloem rays; sieve elements arranged in tangential bands and tangentially compressed cells alternating with single to five rows of phloem fibres, secondary xylem broad consisting of xylem vessels, tracheids, xylem fibres and traversed by multi seriate xylem rays; xylem vessels numerous, small to medium sized, distributed throughout xylem region in groups or in singles, groups of vessels usually arranged radially; isolated vessels cylindrical with rounded or projected at one or both ends with spiral thickenings; fibres numerous, lignified, large, thick-walled with wide lumen, and pointed tips; xylem rays quite distinct, straight, multiseriate, consisting of radially arranged rectangular cells, each ray 30-53 cells high, 8-12 cells wide, a few ray cells containing brown contents

D¡ruharidr¡ consists of dried stem of Berberis aristata DC. (Fam. Berberidaceae); an erect, spinous, deciduous shrub, usually 1.8-3.6 m in height found in the Himalayan ranges at an elevation of 1000-3000 m, and in the Nilgiri hills in south India. D¡ruharidr¡ contains not less than 0.4 per cent of berberine when assayed.

Synonyms: Ka¶a´ka¶er¢, D¡rv¢ Other/Regional Language Names: Bengali: Daruharidra; English: Indian Berberry; Gujarati: Daruharidra, Daruhuladur; Hindi: Daruhaldi, Darhald; Kannada: Maradarishana, Maradrishina, Daruhaldi; Kashmiri: Ras ashud, Rasvat; Malayalam: Maramannal, Maramanjal; Marathi: Daruhalad; Oriya: Daruharidra, Daruhalidi; Punjabi: Sumalu; Tamil: Gangeti, Varatiu manjal; Telugu: Manupasupu; Urdu: Darhald

Description: a) Macroscopic: Drug available in pieces of variable length and thickness, bark about 0.4-0.8 cm thick, pale yellowish-brown, soft, closely and rather deeply furrowed, rough, brittle, xylem portion yellow, more or less hard, radiate with xylem rays, pith mostly absent, when present small, yellowishbrown when dried, fracture short in bark region, splintery in wood; taste bitter b) Microscopic: Stem - Shows rhytidoma with cork consisting of 3-45 rectangular and squarish, yellow coloured, thin-walled cells, arranged radially; sieve elements irregular in shape, thin walled, a few cells containing yellowishbrown contents; phloem fibres arranged in tangential rows, consisting of 1-4 cells, each fibre short thickwalled, spindle-shaped, lignified having wide lumen; half inner portion of rhytidoma traversed by secondary phloem rays; phloem rays run obliquely consisting of radially elongated parenchymatous cells, almost all phloem ray cells having single prismatic crystals of calcium oxalate, a few cells of rhytidoma also contain

Fig. 1: Powdered drug of DËRUHARIDRË (Berberis aristata DC.) 16

 

 

API, Part-I, Vol.-IX (Extracts); Monographs 

acid : Water (6.5 : 1.5 : 2.0). Dry the plate in air and examine under UV 366 nm. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 2).

c) Powder: Fine powder shows mostly fragments of cork cells, yellow coloured phloem fibres entire or in pieces, stone cells in singles or in groups, numerous prismatic crystals of calcium oxalate, xylem vessels having spiral thickenings, thick-walled, lignified xylem fibres and ray cells (Fig. 1)

Quantitative parameters: Foreign matter: not more than 2.0 per cent (Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 14.0 per cent (Appendix 2.1.5); Acidinsoluble ash: not more than 5.0 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 6.0 per cent (Appendix 2.1.8); Watersoluble extractive: not less than 8.0 per cent (Appendix 2.1.9)

Identity, Purity and Strength: Identification: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using berberine chloride as a reference standard. 366 nm

Other requirements:

Rf 1.0

Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

0.5

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (25 ml x 3) on water bath for 15 min each, cool and filter. Combine all the filtrates and transfer to a 100-ml volumetric flask and make up the volume with methanol. Dilute the solution to match the standard concentration. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, berberine chloride RS in a 100 ml volumetric flask and dissolve in about 25 ml of methanol and make up the volume with methanol. Dilute 5 ml of this solution to 25 ml. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: Silica CN (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of

0.0

RS T Fig. 2: Thin-Layer Chromatogram of D¡ruharidr¡ RS: Berberine chloride, T: Test solution Test solution: Extract 0.2 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg of berberine chloride RS in about 100 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase : n-butanol : glacial acetic 17  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

API reference standard:

35 volumes of acetonitrile and 65 volumes of water containing 0.3 per cent w/v of orthophosphoric acid. Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 346 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of berberine in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

API Berberine chloride RS

Constituents:

Berberine, palmatine, oxyberberine, oxyacanthine, karachine.

Properties and Action: Rasa: Tikta; Gu¸a: R£kÀa, Laghu; V¢rya: UÀ¸a; Vip¡ka: Ka¶u; Karma: DoÀap¡cana, Stanya¿odhana, StanyadoÀahara

Important formulations: A¿vagandh¡dyariÀ¶a, Bh¤´gar¡ja taila, J¡ty¡di taila, Khadir¡di gu¶ika, Khadir¡riÀ¶a, Triphal¡ gh¤ta

Therapeutic uses: Ëm¡tis¡ra (diarrhoea due to indigestion), Ka¸·£ (pruritis), Kapharoga (disease due to kapha doÀa), Kar¸aroga (disease of ear), Medoroga (obesity), Mukharoga (disease of mouth), Netraroga (disease of eye), Prameha (increased frequency and turbidity of urine), Írustambha (stiffness in thigh muscles), Vra¸a (wound)

Dose: C£r¸a (powder): 3-6 g

Fig. 3: HPLC chromatogram of D¡ruharidr¡ with Berberine chloride as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

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DËRUHARIDRË HYDRO-ALCOHOLIC EXTRACT 366 nm

D¡ruharidr¡ Hydro-alcoholic Extract is a dried and powdered extract prepared from D¡ruharidr¡ (appropriately powdered). The extract contains not less than 6 per cent of berberine when assayed.

Rf 1.0

Method of preparation: Take D¡ruharidr¡ suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under a reflux at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5.0 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 13 per cent.

0.5

0.0 RS T Fig. 1: Thin-Layer Chromatogram of D¡ruharidr¡ hydro-alcoholic extract RS: Berberine chloride T: Test solution Quantitative parameters: Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 12.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 6.0-8.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-I)

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using berberine chloride as a reference standard. Test solution: Extract 0.2 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg of berberine chloride RS in about 100 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: n-butanol : glacial acetic acid : Water (6.5 : 1.5 : 2.0). Dry the plate in air and examine under UV 366 nm. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the Prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4) Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (25 ml x 3) on water bath for 15 min each, cool and filter. 19

 

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Combine all the filtrates and transfer to a 100-ml volumetric flask and make up the volume with methanol. Dilute the solution to match the standard concentration. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, berberine chloride RS in a 100-ml volumetric flask and dissolve in about 25 ml of methanol and make up the volume with methanol. Dilute 5 ml of this solution to 25 ml. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: Silica CN (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 35 volumes of acetonitrile and 65 volumes of water containing 0.3 per cent w/v of orthophosphoric acid. Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 346 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak.

Calculate the content of berberine in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article. API reference standard: API Berberine chloride RS

Fig. 2: HPLC chromatogram of D¡ruharidr¡ hydro-alcoholic extract with Berberine chloride as RS

20  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

DËRUHARIDRË WATER EXTRACT 366 nm

D¡ruharidr¡ Water Extract is a dried and powdered extract prepared from D¡ruharidr¡. The extract contains not less than 4 per cent of berberine when assayed.

Rf 1.0

Method of preparation: Take D¡ruharidr¡ suitably sized (powder or pieces) in an extractor. Add water, about 3 times the quantity of raw material and heat at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5.0 per cent. Mill the mass and sieve the powder through 500 m mesh and pack. The yield obtained is about 16 per cent.

0.5

0.0 RS T Fig. 1: Thin-Layer Chromatogram of D¡ruharidr¡ water extract RS: Berberine chloride, T: Test solution

Quantitative parameters: Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 12.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 6.0-8.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-II)

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using berberine chloride as a reference standard. Test solution: Extract 0.2 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg of berberine chloride RS in about 100 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase : n-butanol : glacial acetic acid : Water (6.5 : 1.5 : 2.0). Dry the plate in air and examine under UV 366 nm. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (25 ml x 3) on water bath for 15 min each, cool and filter. Combine all the filtrates and transfer to a 100 ml 21

 

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Calculate the content of berberine in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

volumetric flask and make up the volume with methanol. Dilute the solution to match the standard concentration. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, berberine chloride RS in a 100 ml volumetric flask and dissolve in about 25 ml of methanol and make up the volume with methanol. Dilute 5 ml of this solution to 25 ml. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: Silica CN (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 35 volumes of acetonitrile and 65 volumes of water containing 0.3 per cent w/v of orthophosphoric acid. Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 346 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak.

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Berberine chloride RS  

Fig. 2: HPLC chromatogram of D¡ruharidr¡ water extract with Berberine chloride as RS

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DHËRË VÎKâËMLA c) Powder:

Dh¡r¡ V¤kÀ¡mla consists of dried fruit of Garcinia gummi-gutta (L.) Rob. syn. Garcinia cambogia (Gaertn.) Desr. (Fam. Clusiaceae); a small tree, common in evergreen forests of Western ghats, from Konkan southwards to Travancore, and in the Shola forests of the Nilgiris up to an altitude of 1800 m. Dh¡r¡ V¤kÀ¡mla contains not less than 5 per cent of hydroxycitric acid and not less than 5 per cent of lactone when assayed.

Shows isolated cells of mesocarp, containing dark reddish brown gummy exudates, prismatic crystals of calcium oxalate and starch grains; fragments of longitudinally cut spiral and annular vessels (Fig. 1)

Synonym: KÀ¢r¢ V¤kÀ¡mla Regional Language Names: English: Malabar Tamarind, Kokum, Butter Tree; Gujarati: Kokam, Kokan; Hindi: Kokam; Kannada: Murgin huli, Murgala; Malayalam: Panampuli; Marathi: Kokam, Ratamba, Amsol, Amsul, Ratambi; Oriya: Raktasrava; Tamil: Kodukkappuli; Telugu: Vrksamta, Simachinta

Description: a) Macroscopic:

Fig. 1: Powdered drug of Dh¡r¡ V¤kÀ¡mla (Garcinia gummi-gutta (L.) Rob.)

Fruits are ovoid, yellow or red when ripe and become black when dried. 6-8 grooves are seen up to the middle. Dried pieces of drug consists of longitudinal fragments of pericarp of various size and shapes strongly inwardly curved, boat or half moon shaped, dark brownish black, wrinkled irregularly and internally smooth. Odour characteristic, taste sour, astringent and slightly bitter

Identity, Purity and Strength: Identification: High performance liquid chromatography: Carry out liquid chromatography (Appendix 3.6) using (-)-hydroxycitric acid lactone and calcium (-)-hydroxycitrate as a reference standards. Test solution, Standard solution, Chromatographic system, Mobile phase, Injection volume, Detection and Procedure follow as mentioned under Assay. The chromatogram obtained with test solution shows peaks corresponding to the retention time of (-)-hydroxycitric acid lactone and (-)-hydroxycitric acid (Fig. 2).

b) Microscopic: TS of pericarp shows a layer of epicarp, composed of rectangular to tangentially elongated cells covered externally with thin cuticle; mesocarp very wide composed of 100 to 150 rows of parenchymatous cells of various size and shape which possess simple and compound starch grains and prismatic crystals of calcium oxalate; vascular bundles consists of phloem and xylem with spiral vessels, rectangular to irregular shaped parenchyma cells, traversing throughout the mesocarp but more prominently in inner zone of pericarp

Quantitative parameters: Foreign matter: not more than 2.0 per cent (Appendix 2.1.3); Loss on drying: not more than 20.0 per cent (Appendix 2.1.4); Total ash: not more 23

 

 

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than 8.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.5 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 20.0 per cent (Appendix 2.1.8); Water-soluble extractive: not less than 35 per cent (Appendix 2.1.9)

acid in the substance being examined from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Dh¡r¡ V¤kÀ¡mla 

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.5 g, accurately weighed, of the substance being examined and reflux with 25 ml of water (adjusted to pH 2.1 with sulphuric acid solution) on a water bath for 10 min and transfer to a 50-ml volumetric flask. Cool, filter and make up the volume with water (adjusted to pH 2.1 with sulphuric acid solution). Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, (-)-hydroxycitric acid lactone RS and 50 mg of calcium (-)-hydroxycitrate RS in a 25ml volumetric flask and dissolve in about 10 ml of water (adjusted to pH 2.1 with sulphuric acid solution) and make up the volume with water (adjusted to pH 2.1 with sulphuric acid solution). Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed 0.05 M sodium sulphate in water (adjusted to pH 2.3 with sulphuric acid solution). Injection volume: 20 l. Flow rate: 1.0 ml per min. Detection: UV 210 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peaks, identify the analyte peak using relative retention time. The relative retention time for (-)hydroxycitric acid lactone is 1 and for (-)hydroxycitric acid is about 1.1. Calculate the contents of (-)-hydroxycitric acid lactone and (-)-hydroxycitric

Fig. 2: HPLC chromatograms of Dh¡r¡ V¤kÀ¡mla with (-)-Hydroxycitric acid lactone and Calcium (-)-hydroxycitrate as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standards: API (-)-Hydroxycitric acid lactone RS and Calcium (-)-hydroxycitrate RS

Constituents:

(-)-Hydroxycitric acid, (-)- hydroxycitric acid lactone, citric acid, tartaric acid

Properties and Action: Rasa: Amla; Gu¸a: Laghu, R£kÀa; V¢rya: UÀ¸a; Vip¡ka: Amla; Karma: Ar¿oghna, D¢pana, Kapha-v¡tahara, Rucya, Sandh¡n¢ya, á£laghna, T¤À¸¡nigraha¸a

Therapeutic uses: Agnim¡ndya (digestive impairment), Ar¿a (piles), Gulma (abdominal lump), á£la (pain), Vibandha (constipation)

Dose: C£r¸a (powder): 3-6 g 24

 

 

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DHËRË VÎKâËMLA HYDRO-ALCOHOLIC EXTRACT Dh¡r¡ V¤kÀ¡mla Hydro-alcoholic Extract is a dried and powdered extract prepared from Dh¡r¡ V¤kÀ¡mla (appropriately powdered). The extract contains not less than 3 per cent of (-)hydroxycitric acid and not less than 14 per cent of (-)-hydroxycitric acid lactone when assayed.

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix3.3); Residual solvent: Complies with the prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Method of Preparation: Take Dh¡r¡ V¤kÀ¡mla  (Powder) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under reflux at a temperature between 80-85 for 3-4 hours. Filter the extract through an appropriate filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at 80 till the moisture is less than 7 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 38 per cent.

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.5 g, accurately weighed, of the substance being examined and reflux with 25 ml of water (adjusted to pH 2.1 with sulphuric acid solution) on a water bath for 10 min and transfer to a 50-ml volumetric flask. Cool, filter and make up the volume with water (adjusted to pH 2.1 with sulphuric acid solution). Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, (-)-hydroxycitric acid lactone RS and 50 mg of calcium (-)hydroxycitrate RS in a 25-ml volumetric flask and dissolve in about 10 ml of water (adjusted to pH 2.1 with sulphuric acid solution) and make up the volume with water (adjusted to pH 2.1 with sulphuric acid solution). Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed 0.05 M sodium sulphate in water (adjusted to pH 2.3 with sulphuric acid solution). Injection volume: 20 l. Flow rate: 1.0 ml per min. Detection: UV 210 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peaks, identify the analyte peak using relative retention time. The relative retention time for (-)hydroxycitric acid lactone is 1 and for (-)-

Identity, Purity and Strength: High performance liquid chromatography: Carry out liquid chromatography (Appendix 3.6) using (-)-hydroxycitric acid lactone and calcium (-)-hydroxycitrate as a reference standards. Test solution, Standard solution, Chromatographic system, Mobile phase, Injection volume, Detection and Procedure follow as mentioned under Assay. The chromatogram obtained with test solution shows peaks corresponding to the retention time of (-)-hydroxycitric acid lactone and (-)-hydroxycitric acid (Fig. 1).

Quantitative parameters: Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total Ash: not more than 7.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 2.0-3.5 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-I) 25  

 

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Additional requirements:

hydroxycitric acid is about 1.1. Calculate the contents of (-)-hydroxycitric acid lactone and (-)-hydroxycitric acid in the substance being examined from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standards: API (-)-Hydroxycitric acid lactone RS and Calcium (-)-hydroxycitrate RS  

Dh¡r¡ V¤kÀ¡mla Hydro-alcoholic extract

Fig. 1: HPLC chromatograms of Dh¡r¡ V¤kÀ¡mla hydro-alcoholic extract with (-)-Hydroxycitric acid lactone and Calcium (-)-hydroxycitrate as RS

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DHËRË VÎKâËMLA WATER EXTRACT Dh¡r¡ V¤kÀ¡mla Water Extract is a dried and powdered extract prepared from Dh¡r¡ V¤kÀ¡mla. The extract contains not less than 6 per cent of (-)hydroxycitric acid and not less than 20 per cent of (-)-hydroxycitric acid lactone when assayed.

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Method of Preparation: Take Dh¡r¡ V¤kÀ¡mla  (Powder) in an extractor. Add water, about 3 times the quantity of raw material and heat under reflux at a temperature between 80-85 for 3-4 hours. Filter the extract through an appropriate filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at 80 till the moisture is less than 7 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 38 per cent.

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.5 g, accurately weighed, of the substance being examined and reflux with 25 ml of water (adjusted to pH 2.1 with sulphuric acid solution) on a water bath for 10 min and transfer to a 50-ml volumetric flask. Cool, filter and make up the volume with water (adjusted to pH 2.1 with sulphuric acid solution). Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, (-)-hydroxycitric acid lactone RS and 50 mg of calcium (-)hydroxycitrate RS in a 25-ml volumetric flask and dissolve in about 10 ml of water (adjusted to pH 2.1 with sulphuric acid solution) and make up the volume with water (adjusted to pH 2.1 with sulphuric acid solution). Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed 0.05 M sodium sulphate in water (adjusted to pH 2.3 with sulphuric acid solution). Injection volume: 20 l. Flow rate: 1.0 ml per min. Detection: UV 210 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peaks, identify the analyte peak using relative retention time. The relative retention time for (-)hydroxycitric acid lactone is 1 and for (-)hydroxycitric acid is about 1.1. Calculate the contents of (-)-hydroxycitric acid lactone and (-)-

Identity, Purity and Strength: High performance liquid chromatography: Carry out liquid chromatography (Appendix 3.6) using (-)-hydroxycitric acid lactone and calcium (-)-hydroxycitrate as a reference standards. Test solution, Standard solution, Chromatographic system, Mobile phase, Injection volume, Detection and Procedure follow as mentioned under Assay. The chromatogram obtained with test solution shows peaks corresponding to the retention time of (-)-hydroxycitric acid lactone and (-)-hydroxycitric acid (Fig. 1).

Quantitative parameters: Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 7.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 2.0-3.5 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-II) 27  

 

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Additional requirements:

hydroxycitric acid in the substance being examined from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standards: API (-)-Hydroxycitric acid lactone RS and Calcium (-)-hydroxycitrate RS

Dh¡r¡ V¤kÀ¡mla Water extract 

 

Fig. 1: HPLC chromatograms of Dh¡r¡ V¤kÀ¡mla water extract with (-)-Hydroxycitric acid lactone and Calcium (-)-hydroxycitrate as RS

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KAÙUKË vascular bundles surrounded by single layered endodermis of thick walled cells; secondary phloem composed of phloem parenchyma and a few scattered fibres; cambium 2-4 layered; secondary xylem consists of vessels, tracheids, xylem fibres and xylem parenchyma, vessels vary in shape and size having transverse oblique articulation; tracheids long, parenchyma thin walled and polygonal in shape; centre occupied by a small pith consisting of thin-walled cells; simple round to oval, starch grains abundantly found in all cells

Ka¶uk¡ consists of the dried rhizome with root of Picrorrhiza kurroa Royle ex Benth. (Fam. Scrophulariaceae); a perennial, more or less hairy herb common on the north-western Himalayas from Kashmir to Sikkim. Rhizome is cut into small pieces. It contains not less than 6.0 per cent of bitters and not less than 4.0 per cent of sum of picroside I and picroside II when assayed.

Synonyms: Tikt¡, Tiktarohi¸¢, Ka¶urohi¸¢, Ka¶v¢, Matsya¿akal¡

Other/Regional

Language

Names:

Root - Young root shows single layered epidermis, some epidermal cells elongate forming unicellular hairs; hypodermis single layered; cortex 8-14 layered; consisting of oval to polygonal, thickwalled, parenchymatous cells; primary stele tetrach to heptarch, enclosed by single layered pericycle and single layered, thick-walled cells of endodermis; mature root shows 4-15 layers of cork, 1-2 layers of cork cambium; secondary phloem poorly developed; secondary xylem consisting of vessels, tracheids, parenchyma and fibres; vessels have varying shape and size, some cylindrical with tail-like, tapering ends, some drum shaped with perforation on end walls or lateral walls; tracheids cylindrical with tapering pointed ends; fibres aseptate, thick walled, lignified with tapering, blunt, chiesel-like pointed ends.

Assamese: Katki, Kutki; English: Hellebore; Gujarati: Kadu, Katu; Hindi: Kutki; Kannada: Katukarohini; Malayalam: Katukurohini, Katurohini; Marathi: Kutki, Kali kutki; Oriya: Katuki; Punjabi: Karru, Kaur; Tamil: Kadugurohini; Telugu: Katukarohini, Katki

Description: a) Macroscopic: Rhizome - 2.5 cm long and 4-8 mm thick, subcylindrical, straight or slightly curved, externally greyish-brown, surface rough due to longitudinal wrinkles, circular scars of roots and bud scales and sometimes roots attached; tip ends in a growing bud surrounded by tufted crown of leaves; at places cork exfoliates exposing dark cortex; fracture short; odour pleasant; taste bitter Root - Thin, cylindrical, 5-10 cm long, 0.05-0.1 cm in diameter, straight or slightly curved with a few longitudinal wrinkles and dotted scars, mostly attached with rhizomes, dusty grey, fracture short, inner surface black with whitish xylem; odour pleasant; taste bitter b) Microscopic: Rhizome- Shows 20-25 layers of cork consisting of tangentially elongated, suberised cells; cork cambium 1-2 layered; cortex single layered or absent, primary cortex persists in some cases, one or two small vascular bundles present in cortex;

Fig. 1: Powdered drug of KAÙUKË (Picrorrhiza kurroa Royle ex Benth.) 29

 

 

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plate with 10 per cent methanolic sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows bands corresponding to that of the standard solution (Fig. 2).

c) Powder: Powder dusty grey, shows group of fragments of cork cells, thick walled parenchyma, pitted vessels and single round to oval starch grains (Fig. 1)

Identity, Purity and Strength:

Quantitative parameters:

Identification:

Foreign matter: not more than 2.0 per cent (Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 7.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.0 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 10.0 per cent (Appendix 2.1.8); Water-soluble extractive: not less than 20.0 per cent (Appendix 2.1.9)

Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate Appendix 3.5) using picrosideI and picroside-II as reference standards. 254 nm

Visible after derivatisation

Rf 1.0

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4) 

0.5 (1)   (2)

(1) (2)

Assay of bitters: Carry out the assay by Gravimetry. Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (50 ml x 3) on water bath for one hour each, cool and filter. Combine all the filtrates, concentrate and evaporate to dryness under reduced pressure. Dissolve the residue in hot water and filter. Extract the filtrate repeatedly with 50, 50, 50, 25, 25 ml of ethyl acetate. Combine all the ethyl acetate extracts and filter and evaporate to dryness under reduced pressure. Dry the residue at 1000 for one hour and weigh the residue. Calculate the content of bitters from the weight of the residue and from the weight of substance taken for the test.

0.0 RS T RS T Fig. 2: Thin-Layer Chromatogram of Ka¶uk¡ RS: (1) Picroside-I and (2) Picroside-II, T: Test solution Test solution: Extract 1 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 5 mg each of picroside-I RS and picroside-II RS in 25 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : methanol : water (82.0 : 10.0 : 8.0). Dry the plate in air and examine under UV 254 nm. Spray the

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.2 g, 30

 

 

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accurately weighed, of the substance being examined and reflux with water (25 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 100-ml volumetric flask and make up the volume with water. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, each of picroside-I RS and picroside-II RS in 25 ml volumetric flask and dissolve in about 20 ml of water and make up the volume with water. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 17 volumes of acetonitrile and 83 volumes of water containing 0.1 per cent phosphoric acid. Injection volume: 20 l. Flow rate: 1 ml per min. Detector: UV 262 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the responses for the analyte peaks. Analyte Picroside-II Picroside-I

Calculate the content of picroside-I and picrosideII in the substance being examined from peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standards: API Picroside-I RS and Picroside-II RS

Constituents: Picrosides I, II and III, pikuroside, kutkoside, cucurbitacins, cucurbitacin glycosides, apocynin, androsin, picein, vanillic acid, veronicoside, minecoside, 6-feruloylcatalpol

Properties and Action: Rasa: Tikta; Gu¸a: Laghu; V¢rya: á¢ta; Vip¡ka: Ka¶u; Karma: Bhedin¢, D¢pana, Ëmapacan¢, Gulmaghn¢, Jvarahara, Ka¸·£ghna, Lekhan¢ya, Pittahara, Stanya¿odhana, á£lahara, áleÀmahara, ViÀaghna  

Relative retention time 1.0 2.5

Important formulations: Ërogyavardhin¢ gu¶ik¡, Mah¡tikataka gh¤ta, Sarvajvarahara lauha, Tiktaka gh¤ta

Therapeutic uses: D¡ha (burning sensation), Ëmajavara (fever due to indigestion), Ëmav¡ta (rheumatism), Jvara (fever), K¡mal¡ (Jaundice), KuÀ¶ha (diseases of skin), Pl¢hodara (splenomegaly), Sthaulya (obesity), áv¡sa (dyspnoea), ViÀamajvara (intermittent fever), Vra¸a (wound), Yak¤d¿otha (hepatitis)

Dose: C£r¸a (powder): 1-3 g Fig. 3: HPLC chromatogram of Ka¶uk¡ with Picroside-I and Picroside-II as RS

 

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KAÙUKË HYDRO-ALCOHOLIC EXTRACT chromatographic profile of the test solution shows bands corresponding to that of the standard solution (Fig. 1). 254 nm Visible after derivatisation Rf 1.0

Ka¶uk¡ Hydro-alcoholic Extract is a dried and powdered extract prepared from Ka¶uk¡ (appropriately powdered). The extract contains not less than 6 per cent of bitters and not less than 1 per cent of sum of picroside-I and picroside-II when assayed.

Method of preparation: Take Ka¶uk¡ suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under a reflux at a temperature between 80850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 20 per cent.

0.5 (1) (2)

(1) (2) (2) 0.0 RS T RS T Fig. 1: Thin-Layer Chromatogram of Ka¶uk¡ hydro-alcoholic extract RS: (1) Picroside-I and (2) Picroside-II, T: Test solution

Identity, Purity and Strength:

Quantitative parameters:

Thin-layer chromatography:

Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 7.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.0 per cent (Appendix 2.1.7); pH: 4.06.0 (Appendix 2.1.10); Total soluble olids: not less than 90.0 per cent (Appendix 2.1.11) (Method-I) 

Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using picrosideI and picroside-II as reference standards. Test solution: Extract 1 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 5 mg each of picroside-I RS and picroside-II RS in 25 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : methanol : water (82.0 : 10.0 : 8.0). Dry the plate in air and examine under UV 254 nm. Spray the plate with 10 per cent methanolic sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4) 32

 

 

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Analyte Picroside-II Picroside-I

Assay of bitters: Carry out the assay by Gravimetry. Take about 2 g, accurately weighed, of the substance being examined, and reflux with methanol (50 ml x 3) on water bath for one hour each, cool and filter. Combine all the filtrates, concentrate and evaporate to dryness under reduced pressure. Dissolve the residue in hot water and filter. Extract the filtrate repeatedly with 50, 50, 50, 25, 25 ml of ethyl acetate. Combine all the ethyl acetate extracts and filter and evaporate to dryness under reduced pressure. Dry the residue at 1000 for one hour and weigh the residue. Calculate the content of bitters from the weight of the residue and from the weight of substance taken for the test.

Relative retention time 1.0 2.5

Calculate the content of picroside-I and picrosideII in the substance being examined from peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.2 g, accurately weighed, of the substance being examined and reflux with water (25 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 100-ml volumetric flask and make up the volume with water. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, each of picroside-I RS and picroside-II RS in 25-ml volumetric flask and dissolve in about 20 ml of water and make up the volume with water. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 17 volumes of acetonitrile and 83 volumes of water containing 0.1 per cent phosphoric acid. Injection volume: 20 l. Flow rate: 1.0 ml per min. Detector: UV 262 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the responses for the analyte peaks.

Fig. 2: HPLC chromatograms of Ka¶uk¡ hydro-alcoholic extract with Picroside-I and Picroside-II as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standards: API Picroside-I RS and Picroside-II RS

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KAÙUKË WATER EXTRACT Ka¶uk¡ Water Extract is dried and powdered extract prepared from Ka¶uk¡. The extract contains not less than 8 per cent of bitters and not less than 5 per cent of sum of picroside-I and picroside-II when assayed.

charring. The chromatographic profile of the test solution shows bands corresponding to that of the standard solution (Fig. 1). 254 nm Visible after derivatisation

Method of preparation: Take Ka¶uk¡ suitably sized (powder or pieces) in an extractor. Add water, about 3 times the quantity of raw material and heat at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh and pack. The yield obtained is about 20 per cent.

0.5 (1) (2)

(1)  (2) 

0.0 RS T RS T Fig. 2: Thin-Layer Chromatogram of Ka¶uk¡ water extract RS: (1) Picroside-I and (2) Picroside-II, T: Test solution

Identity, Purity and Strength:

Quantitative parameters:

Thin-layer chromatography:

Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 5.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.0 per cent (Appendix 2.1.7); pH: 4.0-7.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-II)

Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using picroside-I and picroside-II as reference standards. Test solution: Extract 1 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 5 mg each of picroside-I RS and picroside-II RS in 25 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate: methanol : water (82.0 : 10.0 : 8.0). Dry the plate in air and examine under UV 254 nm. Spray the plate with 10 per cent methanolic sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay of bitters: Carry out the assay by Gravimetry. Take about 2 g, accurately weighed, of the substance being 34

 

Rf 1.0

 

API, Part-I, Vol.-IX (Extracts); Monographs 

examined, and reflux with methanol (50 ml x 3) on water bath for one hour each, cool and filter. Combine all the filtrates, concentrate and evaporate to dryness under reduced pressure. Dissolve the residue in hot water and filter. Extract the filtrate repeatedly with 50, 50, 50, 25, 25 ml of ethyl acetate. Combine all the ethyl acetate extracts and filter and evaporate to dryness under reduced pressure. Dry the residue at 1000 for one hour and weigh the residue. Calculate the content of bitters from the weight of the residue and from the weight of substance taken for the test.

Fig. 2: HPLC chromatograms of Ka¶uk¡ water extract with Picroside- I and Picroside-II as RS

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.2 g, accurately weighed, of the substance being examined and reflux with water (25 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 100-ml volumetric flask and make up the volume with water. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, each of picroside-I RS and picroside-II RS in 25-ml volumetric flask and dissolve in about 20 ml of water and make up the volume with water. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 17 volumes of acetonitrile and 83 volumes of water containing 0.1 per cent phosphoric acid. Injection volume: 20 l. Flow rate: 1 ml per min. Detector: UV 262 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the responses for the analyte peaks. Analyte Picroside-II Picroside-I

Calculate the content of picroside-I and picrosideII in the substance being examined from peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standards: API Picroside-I RS and Picroside-II RS  

Relative retention time 1.0 2.5

35  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

MAØJIâÙHË MaμjiÀ¶Å¡ consists of the dried root of Rubia cordifolia L. (Fam. Rubiaceae), a perennial herbaceous creeper or climber, with hooked prickles and whorls of four leaves, but without interpetiolar stipules, found throughout the country ascending to 3750 m. It contains not less than 0.04 per cent of rubiadin when assayed. Synonyms: Yojanavall¢, Vastraraμjin¢, Rakt¡

Medullary rays are uni- to multiseriate and oval to circular starch grains present in cortical and phloem parenchyma cells. c) Powder: Shows numerous fragments of cork, lignified xylem vessels, tracheids and fibres, raphides, clusters and sandy oxalate crystals, parenchyma with red content and starch grains (Fig. 1)

T¡mravall¢,

 

Other/Regional Language Names: Assamese: Phuvva; Bengali: Manjishtha, Manjith; English: Indian Madder; Gujarati: Manjitha; Hindi: Manjitha, Manjit; Kannada: Manjustha; Malayalam: Manjatti, Manchatti; Marathi: Manjishtha; Punjabi: Manjistha, Manjit; Tamil: Manatte, Manjitti; Telugu: Manjishtha

Description: a) Macroscopic: Root - Cylindrical, often surmounted by a knotty crown of root stock; about 2 to 9 cm in length and 0.2 to 0.6 cm in width; surface smooth finely striated longitudinally and occasionally grooved, often exhibiting lateral root scars; dark reddish brown both externally and internally. Fracture short, taste sweetish, acrid and disagreeable, odour pleasant

 

Fig. 1: Powdered drug of MAØJIâÙéË (Rubia cordifolia L.)

b) Microscopic: TS of root shows a well developed cork, consisting of 3 to 8 layered suberized radially arranged cells, occasionally filled with reddish brown content, followed by a cortex of 3 to 10 cell layers; some cortical cells filled with acicular and sandy crystals of calcium oxalate more towards periphery. Phloem 8 to 12 layers wide, consists of sieve tubes, companion cells and phloem parenchyma. Xylem consists of vessels, fibres, tracheids and xylem parenchyma. Vessels are broader towards the peripheral region of the xylem. The size of vessels vary from 30 to 270 m in length and 18 to 90 m in breadth.

Identity, Purity and Strength: Identification: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using rubiadin as a reference standard. Test solution: Extract 2 g of substance by refluxing with chloroform (25 ml x 3) for a period of 10-15 min each. Filter and concentrate the combined extract to dryness. Dissolve the residue in 2 ml of 36

 

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Other requirements:

methanol. Standard solution: Dissolve 1 mg of rubiadin RS in about 10 ml of methanol. 254 nm

Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

366 nm Rf 1.0

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (50 ml x 3) on water bath for 5-10 min each, cool and Filter. Combine all the filtrates, concentrate to 50 ml and transfer in a 100-ml volumetric flask and make up the volume with methanol. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, rubiadin RS in a 100-ml volumetric flask and dissolve in about 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 500 ml of water, adding 0.5 ml of orthophosphoric acid and making up the volume to 1000 ml) and acetonitrile in the following proportions:

0.5

0.0 RS T RS T Fig. 2: Thin-Layer Chromatogram of MaμjiÀ¶h¡ RS: Rubiadin, T: Test solution Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop upto 8 cm from the base of the plate using the mobile phase: toluene : ethyl acetate : chloroform : glacial acetic acid (10.0 : 5.0 : 1.0 : 2.5). Dry the plate in air and examine under UV 254 nm and under UV 366 nm. The chromatographic profile of the test solution shows a band corresponding to that of standard solution (Fig.2).

Time (min) 0.01 5 15 20 25 30

Quantitative parameters: Foreign matter: not more than 2.0 per cent (Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 12.0 per cent (Appendix 2.1.5); Acidinsoluble ash: not more than 0.5 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 3.0 per cent (Appendix 2.1.8); Watersoluble extractive: not less than 10.0 per cent (Appendix 2.1.9)

Acetonitrile (per cent) 35 50 80 85 35 35

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 278 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the 37

 

Phosphate buffer (per cent) 65 50 20 15 65 65

 

API, Part-I, Vol.-IX (Extracts); Monographs 

response for the analyte peak. Calculate the content of rubiadin in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

dihydroxy-2methylanthraquinone, 1,5- dihydroxy-2-methylanthraquinone, 3-prenyl5-methoxy-1,4- napthoquinone, 1-hydroxy-2methoxyanthraquinone, 1,4-dihydroxy-2-methyl5-(or 8)-methoxyanthraquinone, 1,3- dimethoxy2-carboxyanthraquinone

Properties and Action: Rasa: KaÀ¡ya, Tikta, Madhura; Gu¸a: Laghu, R£kÀa; V¢rya: á¢ta; Vip¡ka: Ka¶u; Karma: PittasaÆ¿amana, Sandh¡n¢ya, Var¸ya, Vra¸aropa¸¢

Important formulations: Methik¡di c£r¸a, Pal¡¿apuÀp¡sava, Yogar¡j¡sava

Therapeutic

Garbhap¡ta (abortion), Pakv¡tis¡ra (chronic diarrhoea), Tvakroga (skin disease), Vra¸a (wound), Vya´ga (dark shade on face due to stress and excessive exercise)

Fig. 3: HPLC chromatogram of MaμjiÀ¶h¡ with Rubiadin as RS

Dose: 3-6 g

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API references standard: API Rubiadin RS

Constituents: Rubiadin, anthraquinones, alizarin, purpurin, purpuroxanthin, ruberythric acid, 1,3-dihydroxy-2-ethoxymethyl-9,10nthraquinone, lucidin primeveroside, 2-methyl1,3,6-trihydroxy-9,10-anthraquinone 3-O- (6´-O-rhamnosyl(12)--glucoside, acetyl)furomollugin, rubilactone, 2-carboxymethyl-3prenyl-2,3-epoxy-1, 4-naphthoquinone, 1hydroxy-2-hydroxymethyl-9,10-anthraquinone, 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone, rubioncolin B, 1-hydroxy-2-methyl anthraquinone, nordamnacanthal, physcion, 1,4dihydroxy-6-methylanthraquinone, 1,438  

uses: Bhagna (fracture),

 

API, Part-I, Vol.-IX (Extracts); Monographs 

MAØJIâÙHË HYDRO-ALCOHOLIC EXTRACT shows a band corresponding to that of standard solution (Fig. 1).

MaμjiÀ¶h¡ Hydro-alcoholic Extract is a dried and powdered extract prepared from MaμjiÀ¶h¡ (appropriately powdered). The extract contains not less than 0.05 per cent of rubiadin when assayed.

254 nm

366 nm Rf 1.0

Method of Preparation: Take MaμjiÀ¶h¡ suitably sized (powder or pieces) in an extractor. Add 50.0 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under reflux at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 4 per cent.

0.5

0.0 RS T RS T Fig. 1: Thin-Layer Chromatogram of MaμjiÀ¶h¡ hydro-alcoholic extract RS: Rubiadin, T: Test solution

Quantitative parameters:

Identity, Purity and Strength:

Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 22.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.5-7.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-I)

Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using rubiadin as a reference standard. Test solution: Extract 2 g of substance by refluxing with chloroform (25 ml x 3) for a period of 10-15 min each. Filter and concentrate the combined extract to dryness. Dissolve the residue in 2 ml of methanol. Standard solution: Dissolve 1 mg of rubiadin RS in about 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop upto 8 cm from the base of the plate using the mobile phase: toluene : ethyl acetate : chloroform : glacial acetic acid (10.0 : 5.0 : 1.0 : 2.5). Dry the plate in air and examine under UV 254 nm and under UV 366 nm. The chromatographic profile of the test solution

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4) 

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, 39

 

 

API, Part-I, Vol.-IX (Extracts); Monographs 

accurately weighed, of the substance being examined and reflux with methanol (50 ml x 3) on water bath for 5-10 min each, cool and filter. Combine all the filtrates, concentrate to 50 ml and transfer in a 100 ml volumetric flask and make up the volume with methanol. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, rubiadin RS in a 100-ml volumetric flask and dissolve in about 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 500 ml of water, adding 0.5 ml of orthophosphoric acid and making up the volume to 1000 ml) and acetonitrile in the following proportions: Time (min) 0.01 5 15 20 25 30

Phosphate buffer (per cent) 65 50 20 15 65 65

Fig. 2: HPLC chromatogram of MaμjiÀ¶h¡ hydro-alcoholic extract with Rubiadin as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

Acetonitrile (per cent) 35 50 80 85 35 35

API reference standard: API Rubiadin RS

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 278 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of rubiadin in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

40  

 

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MAØJIâÙHË WATER EXTRACT 254 nm

MaμjiÀ¶h¡ Water Extract is a dried and powdered extract prepared from MaμjiÀ¶h¡. The extract contains not less than 0.02 per cent of rubiadin when assayed.

366 nm Rf 1.0

Method of Preparation: Take MaμjiÀ¶h¡ suitably sized (powder or pieces) in an extractor. Add water, about 3 times the quantity of raw material and heat at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 8 per cent.

0.5

0.0 RS T RS T Fig. 1: Thin-Layer Chromatogram of MaμjiÀ¶h¡ water extract RS: Rubiadin, T: Test solution

Quantitative parameters: Loss on drying: not more than 7.0 per cent, (Appendix 2.1.4); Total ash: not more than 18.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.0-6.5 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-II)

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using rubiadin as a reference standard. Test solution: Extract 2 g of substance by refluxing with chloroform (25 ml x 3) for a period of 10-15 min each. Filter and concentrate the combined extract to dryness.Dissolve the residue in 2 ml of methanol. Standard solution: Dissolve 1 mg of rubiadin RS in about 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop upto 8 cm from the base of the plate using the mobile phase:toluene : ethyl acetate : chloroform : glacial acetic acid (10.0 : 5.0 : 1.0 : 2.5). Dry the plate in air and examine under UV 254 nm and under UV 366 nm. The chromatographic profile of the test solution shows a band corresponding to that of standard solution (Fig. 1).

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4) 

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (50 ml x 3) on water bath for 5-10 min each, cool and filter. Combine all the filtrates, concentrate to 50 ml and 41

 

 

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transfer in a 100-ml volumetric flask and make up the volume with methanol. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, rubiadin RS in a 100-ml volumetric flask and dissolve in about 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 500 ml of water, adding 0.5 ml of orthophosphoric acid and making up the volume to 1000 ml) and acetonitrile in the following proportions: Time (min) 0.01 5 15 20 25 30

Phosphate buffer (per cent) 65 50 20 15 65 65

Fig. 2: HPLC chromatogram of MaμjiÀ¶h¡ water extract with Rubiadin as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents.

Acetonitrile (per cent) 35 50 80 85 35 35

Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Rubiadin RS

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 278 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of rubiadin in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

42  

 

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MEâAáÎ×GÌ MeÀa¿¤´g¢ consists of dried leaf of Gymnema sylvestre R.Br. (Fam. Asclepiadaceae), a large woody, much branched, climber, with pubescent young parts, found throughout India in dry forests upto 600 m. It contains not less than 8.0 per cent of gymnemic acids and not less than 2.0 per cent of gymnemagenin when assayed.

conjoint and situated in centre; rest of the tissue between collenchyma and vascular bundles consisting of polygonal thin-walled parenchymatous cells with intercellular spaces, a few having rosette crystals of calcium oxalate Lamina - Cuticle striated shows dorsiventral structure; epidermis with cells having beaded walls and trichome as in petiole and midrib; trichome consists of 3 to 6 cells nearly similar in width and variable in length, terminal cells blunt, most of them curved inwards from the leaf surface; palisade 1 or 2 layers; spongy parenchyma irregular, arranged with distinct intercellular spaces, rosette crystals of calcium oxalate present in this region; stomata paracytic, present only on lower surface; palisade ratio 7 or 8; stomatal index 20 to 25, and vein islet number 7 to 10

Synonyms: Madhun¡¿in¢ Other/Regional Language Names: Bengali: Medhasingi; English: Periploca of the Woods; Gujarati: Kaavalee, Medhasinge; Hindi: Gudmaar, Medhasingi; Kannada: Kadhasige; Malayalam: Cakkarakkolli, Madhunaashini; Marathi: Kaavalee, Medhashingi; Tamil: Sirukurunjan, Shakkaraikkolli; Telugu: Padapatri

Description: a) Macroscopic: Leaf simple, opposite, elliptical or ovate, petiolate, petiole 6 to 12 mm long and pubescent; lamina 3 to 6 cm long and 1 to 3 cm broad; acute or shortly acuminate; more or less pubescent on both sides, base rounded or cordate, venation reticulate; odour unpleasant; taste bitter and acrid, and leaves a benumbing sensation b) Microscopic: Leaf - Petiole  ‐ Nearly semi-circular in outline having a deep furrow, shows a single layered epidermis covered with thick cuticle; multicellular uniseriate trichomes present; cortex composed of 3 or 4 layers of collenchyma and 3 or 4 layers of thin walled parenchymatous cells with intercellular spaces; vascular bundle bicollateral, conjoint and 3 in number, one central larger and crescent shaped and 2 lateral and smaller in size; a few rosette crystals of calcium oxalate present in cortical region Midrib ‐  Epidermis and trichome as in petiole; epidermis followed by 2 or 3 layers of collenchyma adjacent to the lower surface; vascular bundle crescent shaped, bicollateral,

Fig. 1: Powdered drug of MEâAáÎ×GÌ (Gymnema sylvestre R.Br.) 43

 

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : methanol : formic acid (82.0 : 10.0 : 8.0). Dry the plate in air. Spray the plate with 10 per cent methanolic sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 2).

c) Powder: Light yellow; shows polygonal, thin walled parenchymatous cells, simple pitted fibres and vessels; laticiferous vessels embedded with granular contents, large and a few small rosette crystals of calcium oxalate, simple and compound starch grains, measuring 5 to 11 μ in dia (Fig. 1)

Identity, Purity and Strength: Identification: Thin-layer chromatography:

Quantitative parameters:

Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using gymnemagenin as a reference standard. Test solution: Extract 0.5 g of substance by refluxing with a mixture of 50 per cent methanol and 11 per cent potassium hydroxide solution (10 : 2), for a period of 1 hour. Add 1.8 ml of concentrated hydrochloric acid and reflux again for one hour on water bath. Cool and adjust the pH between 7.5 to 8.5 with 11 per cent potassium hydroxide solution. Dilute to 50 ml with 50 per cent of methanol and filter. Standard solution: Dissolve 10 mg of gymnemagenin RS in 10 ml of methanol.

Foreign matter:  not more than 2.0 per cent (Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 12.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 7.0 per cent (Appendix 2.1.8); Water-soluble extractive: not less than 28.0 per cent (Appendix 2.1.9)

Other requirements: Heavy metals:  Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Visible after derivatization Rf 1.0

Assay of gymnemic acids: Carry out the assay by Gravimetry. Take about 5 g, accurately weighed, of the substance being examined, in a100-ml round bottomed flask; reflux with 0.1 N sodium hydroxide solution (25 ml x 3) on a water bath for 10 min each. Cool and centrifuge, collect all the supernatants, concentrate to 25 ml and add dilute sulphuric acid drop wise till precipitate is completed. Centrifuge and wash the residue with cold water till the residue is acid free. Dissolve the residue in 50 ml of methanol and evaporate to dryness under reduced pressure. Dry the residue at 1050 for one hour and weigh the residue. Calculate the content of gymnemic acids from the weight of the residue. 

0.5

0.0

RS T Fig. 2: Thin-Layer Chromatogram of MeÀa¿¤´g¢ RS: Gymnemagenin, T: Test solution 44  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.5 g, accurately weighed, of the substance being examined, add 10 ml of 50 per cent methanol and 2 ml of 11 per cent potassium hydroxide solution, reflux for one hour on a water bath, cool and add 1.8 ml of concentrated hydrochloric acid and again reflux for one hour on a water bath. Cool and adjust pH between 7.5 to 8.5 with 11 per cent potassium hydroxide solution and make up the volume to 100 ml with 50 per cent methanol. Take 20 ml of this solution add 400 mg of polyamide (C-200), stir for one hour and filter the supernatant. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, gymnemagenin RS in a 50 ml volumetric flask and dissolve in about 25 ml of 50 per cent methanol and make up the volume with 50 per cent methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of acetonitrile and water containing 0.1 per cent of orthophosphoric acid in the following proportions:-

MeÀa¿¤´g¢

Fig. 3: HPLC chromatogram of MeÀa¿¤ng¢ with Gymnemagenin as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Gymnemagenin RS

Constituents:  Triterpenoid

saponins of gymnemic acid A, B, C and D with sugar residues such as glucuronic acid, galacturonic acid, ferulic and angelic acids attached as carboxylic acids. Several isopropylene derivatives of gymnemagenin, a hexahydroterpene, gymnemagenin, gymnemic acid. The leaves also contain betaine, choline, gymnamine alkaloids, inositol, d-quercitol. Hydrocarbons such as nonacosane, hentriacontane, tritriacontane, pentatriacontane, phytin, resin, tartaric acid, formic acid, butyric acid, γ-butyric acid, amino acids such as leucine, isoleucine, valine, alanine

Time Buffer Acetonitrile (min) (per cent) (per cent) 0.01 85 15 20 58 42 22 0 100 26 0 100 28 85 15 35 85 15 Injection volume: 20 l. Flow rate: 1 ml per min. Detection: UV 210 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of gymnemagenin in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Properties and Action: Rasa: Tikta, KaÀ¡ya; Gu¸a: Laghu, R£kÀa; V¢rya: UÀ¸a; Vip¡ka: Ka¶u; Karma: CakÀuÀya, D¢pana, Gulmaghna, Kaphahara, V¡tahara, á£laghna, ViÀaghna

45  

 

Important

API, Part-I, Vol.-IX (Extracts); Monographs 

formulations:

Ayask¤ti, M¤tasaμj¢van¢ sur¡, Mah¡viÀagarbha taila, Nyagrodh¡di c£r¸a

Therapeutic uses: Gulma (abdominal lump), KuÀ¶ha (disease of skin), Prameha (increased frequency and turbidity of urine), Sthaulya (obesity), áiraÅ¿£la (headache), Vidradhi (abscess)

Dose: C£r¸a (powder): 3-6 g

46  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

MEâAáÎ×GÌ HYDRO-ALCOHOLIC EXTRACT 10.0 : 8.0). Dry the plate in air. Spray the plate with 10 per cent methanolic sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

MeÀa¿¤´g¢ Hydro-alcoholic Extract is a dried and powdered extract prepared from MeÀa¿¤´g¢ (appropriately powered). The extract contains not less than 20 per cent of gymnemic acids and not less than 4 per cent of gymnemagenin when assayed.

Method of preparation:

Visible after derivatisation Rf 1.0

Take MeÀa¿¤´g¢ suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under a reflux at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 15 per cent.

0.5

0.0 RS T Fig. 1: Thin-Layer Chromatogram of MeÀa¿¤´g¢ hydro-alcoholic extract RS: Gymnemagenin, T: Test solution

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using gymnemagenin as a reference standard. Test solution: Extract 0.5 g of substance by refluxing with a mixture of 50 per cent methanol and 11 per cent potassium hydroxide solution (10 : 2), for a period of 1 hour. Add 1.8 ml of concentrated hydrochloric acid and reflux again for one hour on water bath. Cool and adjust the pH between 7.5 to 8.5 with 11 per cent potassium hydroxide solution. Dilute to 50 ml with 50 per cent of methanol and filter. Standard solution: Dissolve 10 mg of gymnemagenin RS in 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : methanol : formic acid (82.0 :

Quantitative parameters: Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total Ash: not more than 15.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.0 per cent (Appendix 2.1.7); pH: 4.5-7.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-I)

Other requirements: Heavy metals:  Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4) 47

 

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Assay of gymnemic acids:

Time (min) 0.01 20 22 26 28 35

Carry out the assay by Gravimetry. Take about 5 g, accurately weighed, of the substance being examined, in a 100 ml round bottomed flask; reflux with 0.1 N sodium hydroxide solution (25 ml x 3) on a water bath for 10 min each. Cool and centrifuge, collect all the supernatants, concentrate to 25 ml and add dilute sulphuric acid drop wise till precipitate is completed. Centrifuge and wash the residue with cold water till the residue is acid free. Dissolve the residue in 50 ml of methanol and evaporate to dryness under reduced pressure. Dry the residue at 1050 for one hour and weigh the residue. Calculate the content of gymnemic acids from the weight of the residue. 

Acetonitrile (per cent) 15 42 100 100 15 15

Injection volume: 20 l. Flow rate: 1 ml per min. Detection: UV 210 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of gymnemagenin in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Assay: Carry out the assay by liquid chromatography (Appendix 3.6).Test solution: Take about 0.5 g, accurately weighed, of the substance being examined, add 10 ml of 50 per cent methanol and 2 ml of 11 per cent potassium hydroxide solution, reflux for one hour on a water bath, cool and add 1.8 ml of concentrated hydrochloric acid and again reflux for one hour on a water bath. Cool and adjust pH between 7.5 to 8.5 with 11 per cent potassium hydroxide solution and make up the volume to 100 ml with 50 per cent methanol. Take 20 ml of this solution add 400 mg of polyamide (C-200), stir for one hour and filter the supernatant through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, gymnemagenin RS in a 50-ml volumetric flask and dissolve in about 25 ml of 50 per cent methanol and make up the volume with 50 per cent methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of acetonitrile and water containing 0.1 per cent of orthophosphoric acid in the following proportions:-

MeÀa¿¤´g¢ hydro-alcoholic extract

Fig. 2: HPLC chromatogram of MeÀa¿¤´g¢ hydroalcoholic extract with Gymnemagenin as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Gymnemagenin RS

48  

Buffer (per cent) 85 58 0 0 85 85

 

API, Part-I, Vol.-IX (Extracts); Monographs 

MEâAáÎ×GÌ WATER EXTRACT MeÀa¿¤´g¢ Water Extract is a dried and powdered extract prepared from MeÀa¿¤´g¢. The extract contains not less than 15 per cent of gymnemic acids and not less than 2.5 per cent of gymnemagenin when assayed.

10 per cent methanolic sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Method of preparation:

                     

Take MeÀa¿¤´g¢ suitably sized (powder or pieces) in an extractor. Add water, about 3 times the quantity of raw material and heat at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh and pack. The yield obtained is about 15 per cent.

Rf

1.0

0.5

0.0

Identity, Purity and Strength:

RS T Fig. 1: Thin-Layer Chromatogram of MeÀa¿¤´g¢ water extract RS: Gymnemagenin, T: Test solution

Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using gymnemagenin as a reference standard. Test solution: Extract 0.5 g of substance by refluxing with a mixture of 50 per cent methanol and 11 per cent potassium hydroxide solution (10 : 2), for a period of 1 hour. Add 1.8 ml of concentrated hydrochloric acid and reflux again for one hour on water bath. Cool and adjust the pH between 7.5 to 8.5 with 11 per cent potassium hydroxide solution. Dilute to 50 ml with 50 per cent of methanol and filter. Standard solution: Dissolve 10 mg of gymnemagenin RS in 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : methanol : formic acid (82.0 : 10.0 : 8.0). Dry the plate in air. Spray the plate with

Quantitative parameters: Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total Ash: not more than 20.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.0 per cent (Appendix 2.1.7); pH: 4.5-8.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-II)

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4) 49

 

Visible after derivatisation

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Assay of gymnemic acids:

Time (min) 0.01 20 22 26 28 35

Carry out the assay by Gravimetry. Take about 5 g, accurately weighed, of the substance being examined, in a 100 ml round bottomed flask; reflux with 0.1 N sodium hydroxide solution (25 ml x 3) on a water bath for 10 min each. Cool and centrifuge, collect all the supernatants, concentrate to 25 ml and add dilute sulphuric acid drop wise till precipitate is completed. Centrifuge and wash the residue with cold water till the residue is acid free. Dissolve the residue in 50 ml of methanol and evaporate to dryness under reduced pressure. Dry the residue at 1050 for one hour and weigh the residue. Calculate the content of gymnemic acids from the weight of the residue.

Acetonitrile (per cent) 15 42 100 100 15 15

Injection volume: 20 l. Flow rate: 1 ml per min. Detection: UV 210 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of gymnemagenin in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.5 g, accurately weighed, of the substance being examined, add 10 ml of 50 per cent methanol and 2 ml of 11 per cent potassium hydroxide solution, reflux for one hour on a water bath, cool and add 1.8 ml of concentrated hydrochloric acid and again reflux for one hour on a water bath. Cool and adjust pH between 7.5 to 8.5 with 11 per cent potassium hydroxide solution and make up the volume to 100 ml with 50 per cent methanol. Take 20 ml of this solution add 400 mg of polyamide (C-200), stir for one hour and filter the supernatant. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, gymnemagenin RS in a 50-ml volumetric flask and dissolve in about 25 ml of 50 per cent methanol and make up the volume with 50 per cent methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of acetonitrile and water containing 0.1 per cent of orthophosphoric acid in the following proportions:-

MeÀa¿¤´g¢ water extract

Fig. 2: HPLC chromatogram of MeÀa¿¤´g¢ water extract with Gymnemagenin as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Gymnemagenin RS

50  

Buffer (per cent) 85 58 0 0 85 85

 

API, Part-I, Vol.-IX (Extracts); Monographs 

METHÌ Meth¢ consists of seeds of Trigonella foenumgraecum L. (Fam. Fabaceae); an aromatic, 30 to 60 cm tall, annual herb, cultivated throughout the country. Meth¢ contains not less than 5 per cent of saponins and not less than 0.2 per cent of 4hydroxyisoleucine when assayed.

c) Powder: Powder shows groups of palisade parenchymatous and bearer cells in top and side views, aleurone grains, oil globules, endosperm and epidermal cells of testa (Fig. 1).

Synonym: Other/Regional Language Names: Assamese: Methi; Bengali: Methi; English: Fenugreek; Gujarati: Methi; Hindi: Methi; Kannada: Menthe, Mente; Kashmiri: Methi; Malayalam: Uluva; Marathi: Methi; Punjabi: Methi; Tamil: Ventayam; Telugu: Mentulu; Urdu: Methi

Description: a) Macroscopic: Seed oblong, rhomboidal with deep furrow running obliquely from one side, dividing seed into a larger and smaller part, 0.2 to 0.5 cm long, 0.15 to 0.35 cm broad, smooth, very hard; dull yellow; seed becomes mucilaginous when soaked in water; odour pleasant; taste bitter. b) Microscopic: Seed - Seed shows a layer of thick-walled, columnar palisade, covered externally with thick cuticle; cells flat at base, mostly pointed but a few flattened at apex, supported internally by a tangentially wide bearer cells having radial rib-like thickenings; followed by 4 to 5 layers of tangentially elongated, thin-walled, parenchymatous cells; endosperm consists of a layer of thick-walled cells containing aleurone grains, several layers of thin walled, mucilaginous cells, varying in size, long axis radially elongated in outer region and tangentially elongated in inner region; cotyledons consists of 3 to 4 layers of palisade cells varying in size with long axis and a few layers of rudimentary spongy tissue; rudimentary vascular tissue situated in spongy mesophyll; cells of cotyledon contain aleurone grains and oil globules.

Fig. 1: Powdered drug of Meth¢ (Trigonella foenum-graecum L.)

Identity, Purity and Strength: Identification: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using 4-hydroxyisoleucine as a reference standard. Test solution: Extract 2 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 2.5 mg of 4-hydroxyisoleucine RS in 10 ml of methanol. 51

 

   Visible after derivatisation

API, Part-I, Vol.-IX (Extracts); Monographs 

Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Rf 1.0

Assay of Saponins: Carry out the assay by gravimetry. Take about 5 g, accurately weighed, of the substance being examined and reflux with 50 per cent of methanol (50 ml x 3) on water bath for one hour each, cool and filter. Combine all the filtrates, concentrate and evaporate to dryness under reduced pressure. Add 25 ml of petroleum ether (400-600) to the residue and reflux for 10 min cool and decant the petroleum ether layer. Add 10 ml of methanol to the residue and dissolve, add 100 ml of acetone; filter the precipitate in a tared filter paper. Dry the residue at 800 for one hour and weigh the residue. Calculate the content of saponins from the weight of the residue.

0.5

0.0 RS T Fig. 2: Thin-Layer Chromatogram of Meth¢ RS: 4-Hydroxyisoleucine, T: Test solution Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: n-butanol : acetic acid : water (40 : 10 : 10). Dry the plate in air. Spray the plate with 1 per cent ninhydrin in methanol reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 2).

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.5 g, accurately weighed, of the substance being examined in a 25-ml of volumetric flask and add 2.5 ml of coupling solution (40 ml of acetonitrile : 8 ml of triethylamine : 12 ml of Water), sonicate until all the sample is dissolved. Add 125 l of phenyl isothiocyanate to the solution and sonicate for 5 min and make up the volume with methanol. Dilute 5 ml of this solution to 50 ml with mixture of 35 volumes of methanol and 65 volumes of water. Filter through 0.42 m membrane. Standard solution: Take about 2.5 mg, accurately weighed, 4-hydroxyisoleucine RS in a 10 ml volumetric flask and add 1 ml of coupling solution (40 ml of acetonitrile : 8 ml of triethylamine : 12 ml of Water), sonicate until the entire sample is dissolved. Add 50 l of phenyl isothiocyanate to the solution and sonicate for 5 min and make up the volume with methanol. Dilute 5 ml of this solution to 50 ml with mixture of 35 volumes of methanol and 65 volumes of water. Filter through 0.42 m membrane.  Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (100 mm x 3.0 mm, 5 m). Mobile phase: Filtered and

Quantitative parameters: Foreign matter: not more than 2.0 per cent (Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 4.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 0.5 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 5.0 per cent (Appendix 2.1.8); Water-soluble extractive: not less than 10.0 per cent (Appendix 2.1.9)

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: 52  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Additional requirements:

degassed gradient mixture of acetonitrile and water containing 0.1 per cent of orthophosphoric acid in the following proportions: Time (min)

Water containing 0.1 per cent orthophosphoric acid (per cent)

Acetonitrile (per cent)

0.01 30 32 36 38 45 0.01

80 30 0 0 80 80 80

20 70 100 100 20 20 20

Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API 4-Hydroxyisoleucine RS

Constituents: Graecunins H, I, J, K, L, M, N; trigofoenosides A, D, F, G; trigoneosides IIa, Ib, diosgenin. Trigoneosides Ia, IIb, IIIa, IIIb, Xa, Xb, XIb, XIIa, XIIb and XIIIa, yamogenin tetroside B and C, smilagenin, sarsa-sapogenin, yamogenin, tigogenin and neotigogenin, yuccagenin, gitogenin and neogitogenin, vitexin, saponaretin, homoorientin, vicenin-1 and vicenin-2. Seed oil contains octadecatrienoic acid

Injection volume: 5 l. Flow rate: 0.4 ml per min. Detection: UV 254 nm. Procedure: Inject 5 l of the standard solution and record the chromatogram. Inject 5 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of 4-hydroxyisoleucine in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Properties and Action: Rasa: Tikta; Gu¸a: Laghu, Snigdha; V¢rya: UÀ¸a; Vip¡ka: Ka¶u; Karma: D¢pana, Kaphahara, Rucya, V¡tahara

Important formulations: Mustak¡riÀ¶a, M¤tasaμj¢van¢ sur¡

Therapeutic uses: Aruci (tastelessness), Graha¸¢ (malabsorption syndrome), Jvara (fever), Prameha (increased frequency turbidity of urine)

Meth¢

Dose: C£r¸a (powder): 3-6 g

Fig. 3: HPLC chromatogram of Meth¢ with 4-Hydroxyisoleucine as RS 

53  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

METHÌ HYDRO-ALCOHOLIC EXTRACT Meth¢ Hydro-alcoholic Extract is a dried and powdered extract prepared from Meth¢ (appropriately powdered). The extract contains not less than 50 per cent of saponins and not less than 0.5 per cent of 4-hydroxyisoleucine when assayed.

chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1). Visible after derivatization Rf 1.0

Method of preparation: Take Meth¢ suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under a reflux at a temperature between 80850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 6 per cent.

0.5

0.0 RS T Fig. 1: Thin-Layer Chromatogram of Meth¢ hydro-alcoholic extract RS: 4-Hydroxyisoleucine, T: Test solution

Identity, Purity and Strength:

Quantitative parameters:

Thin-layer chromatography:

Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 6.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.0-7.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-I)

Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using 4-hydroxyisoleucine as a reference standard. Test solution: Extract 2 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 2.5 mg of 4hydroxyisoleucine RS in 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: n-butanol : acetic acid : water (40 : 10 : 10). Dry the plate in air. Spray the plate with 1 per cent ninhydrin in methanol reagent and heat at 1050 till the colour of the spots/bands appear without charring. The

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

54  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Assay of Saponins:

Time (min)

Carry out the assay by gravimetry. Take about 5 g, accurately weighed, of the substance being examined and reflux with 50 per cent of methanol (50 ml x 3) on water bath for one hour each, cool and filter. Combine all the filtrates, concentrate and evaporate to dryness under reduced pressure. Add 25 ml of petroleum ether (400-600) to the residue and reflux for 10 min cool and decant the petroleum ether layer. Add 10 ml of methanol to the residue and dissolve, add 100 ml of acetone; filter the precipitate in a tared filter paper. Dry the residue at 800 for one hour and weigh the residue. Calculate the content of saponins from the weight of the residue.

0.01 30 32 36 38 45 0.01

Injection volume: 5 l. Flow rate: 0.4 ml per min. Detection: UV 254 nm. Procedure: Inject 5 l of the standard solution and record the chromatogram. Inject 5 l of the test solution, record the chromatogram and measure the response for the analyte peak.Calculate the content of 4-hydroxyisoleucine in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.5 g, accurately weighed, of the substance being examined in a 25-ml of volumetric flask and add 2.5 ml of coupling solution (40 ml of acetonitrile : 8 ml of triethylamine : 12 ml of Water), sonicate until all the sample is dissolved. Add 125 l of phenyl isothiocyanate to the solution and sonicate for 5 min and make up the volume with methanol. Dilute 5 ml of thissolution to 50 ml with mixture of 35 volumes of methanol and 65 volumes of water and filter through 0.42 m membrane. Standard solution: Take about 2.5 mg, accurately weighed, 4hydroxyisoleucine RS in a 10-ml volumetric flask and add 1 ml of coupling solution (40 ml of acetonitrile : 8 ml of triethylamine : 12 ml of Water), sonicate until the entire sample is dissolved. Add 50 l of phenyl isothiocyanate to the solution and sonicate for 5 min and make up the volume with methanol. Dilute 5 ml of this solution to 50 ml with mixture of 35 volumes of methanol and 65 volumes of water and filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (100 mm x 3.0 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of acetonitrile and water containing 0.1 per cent of orthophosphoric acid in the following proportions:-

Meth¢ hydro-alcoholic extrtact

Fig. 2: HPLC chromatogram of Meth¢ hydro-alcoholic extract with 4- Hydroxyisoleucine as RS Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article. API reference standard: API 4-Hydroxyisoleucine RS 55

 

Water containing 0.1 Acetonitrile percent orthophosphoric (per cent) acid (per cent) 80 20 30 70 0 100 0 100 80 20 80 20 80 20

 

API, Part-I, Vol.-IX (Extracts); Monographs 

METHÌ WATER EXTRACT Visible after derivatisation

Meth¢ Water Extract is dried and powdered extract prepared from Meth¢. The extract contains not less than 20 per cent of saponins and not less than 0.2 per cent of 4-hydroxyisoleucine when assayed.

Rf 1.0

Method of preparation: Take Meth¢ suitably sized (powder or pieces) in an extractor. Add water, about 3 times the quantity of raw material and heat at a temperature between 80850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh and pack. The yield obtained is about 6 per cent.

RS T Fig. 1: Thin-Layer Chromatogram of Meth¢ water extract RS: 4-Hydroxyisoleucine, T: Test solution

Identity, Purity and Strength:

Quantitative parameters:

Thin-layer chromatography:

Loss on drying: not more than 8.0 per cent (Appendix 2.1.4); Total ash: not more than 12.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.0‐7.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-II)

0.5

0.0

Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using 4-hydroxyisoleucine as a reference standard. Test solution: Extract 2 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 2.5 mg of 4hydroxyisoleucine RS in 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: n-butanol : acetic acid : Water (40 : 10 : 10). Dry the plate in air. Spray the plate with 1 per cent ninhydrin in methanol reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay of Saponins: Carry out the assay by gravimetry. Take about 5 g, accurately weighed, of the substance being examined and reflux with 50 per cent of methanol (50 ml x 3) on water bath for one hour each, cool and filter. Combine all the filtrates, concentrate 56

 

 

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and evaporate to dryness under reduced pressure. Add 25 ml of petroleum ether (400-600) to the residue and reflux for 10 min cool and decant the petroleum ether layer. Add 10 ml of methanol to the residue and dissolve, add 100 ml of acetone; filter the precipitate in a tared filter paper. Dry the residue at 800 for one hour and weigh the residue. Calculate the content of saponins from the weight of the residue.

Assay:

Water containing 0.1 per cent orthophosphoric acid (per cent)

Acetonitrile (per cent)

0.01 30 32 36 38 45 0.01

80 30 0 0 80 80 80

20 70 100 100 20 20 20

Injection volume: 5 l. Flow rate: 0.4 ml per min. Detection: UV 254 nm. Procedure: Inject 5 l of the standard solution and record the chromatogram. Inject 5 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of 4hydroxyisoleucine in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.5 g, accurately weighed, of the substance being examined in a 25-ml of volumetric flask and add 2.5 ml of coupling solution (40 ml of acetonitrile : 8 ml of triethylamine : 12 ml of Water), sonicate until all the sample is dissolved. Add 125 l of phenyl isothiocyanate to the solution and sonicate for 5 min and make up the volume with methanol. Dilute 5 ml of this solution to 50 ml with mixture of 35 volumes of methanol and 65 volumes of water and filter through 0.42 m membrane. Standard solution: Take about 2.5 mg, accurately weighed, 4-hydroxyisoleucine RS in a 10-ml volumetric flask and add 1 ml of coupling solution (40 ml of acetonitrile : 8 ml of triethylamine : 12 ml of water), sonicate until the entire sample is dissolved. Add 50 l of phenyl isothiocyanate to the solution and sonicate for 5 min and make up the volume with methanol. Dilute 5 ml of this solution to 50 ml with mixture of 35 volumes of methanol and 65 volumes of water and filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (100 mm x 3.0 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of acetonitrile and water containing 0.1 per cent of orthophosphoric acid in the following proportions:-

Meth¢ water extract

Fig. 2: HPLC chromatogram of Meth¢ water extract with 4-Hydroxyisoleucine as RS Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article. API reference standard: API 4-Hydroxyisoleucine RS 57

 

Time (min)

 

API, Part-I, Vol.-IX (Extracts); Monographs 

NIRGUÛÚÌ a discontinuous ring in the distal region surrounding a central crescent shaped stele; a few smaller vascular bundles present below the crescent and two, or rarely three, bundles situated above the crescent.

Nirgu¸·¢ consists of dried leaves of Vitex negundo (L.) Dunal. (Fam. Verbenaceae), a large aromatic shrub or a small tree, upto 4.5 m in height, common throughout the country ascending to an altitude of 1500 m in the outer Himalayas. It is common in waste places around villages, river banks, moist localities and in the deciduous forests. Nirgu¸·¢ contains not less than 0.25 per cent of negundoside and 1.0 per cent agnuside when assayed. Other/Regional Language Names: Assamese: Aslak; Bengali: Nirgundi, Nishinda; Gujarati: Nagod; Hindi: Nirgundi, Sindur, Sambhalu; Kannada: Lakkigida, Nekkigida; Malayalam: Indranee, Nirgundi; Marathi: Nirgudi; Punjabi: Sambhalu, Banna; Tamil: Karunochchi, Nocchi; Telugu: Nallavavilli, Vavili; Urdu: Sambhalu, Panjangusht

Lamina - shows single layered epidermis having mostly unicellular hairs, bi and multicellular and glandular trichomes being rare; hypodermis 1-3 layered, interrupted at places by 4-8 palisade layers containing chlorophyll; mesophyll almost entirely of palisade cell layers, with isodiametric parenchymatous cells sparsely distributed in the spongy region, and present more at the edges of the lamina; a large number of veins enclosed by bundle sheath traverse mesophyll; stomata present only on the ventral surface, covered densely with trichomes; vein-islet and vein termination number of leaf are 23-25 and 5-7 respectively.

Description:

c) Powder:

Synonyms: Sinduv¡ra, áeph¡lik¡, N¢la

a) Macroscopic: Leaves born on a rachis 2.5-3.8 cm long; palmately compound, mostly trifoliate, occasionally pentafoliate; in trifoliate leaf, leaflet lanceolate or narrowly lanceolate, middle leaflet 5-10 cm long and 1.6-3.2 cm broad, with 1-1.3 cm long petiole, remaining two sub-sessile; in pentafoliate leaf inner three leaflets have petiole and remaining two sub-sessile; surface glabrous above and tomentose beneath; texture leathery. b) Microscopic: Rachis - T.S. shows single layered epidermis having a number of unicellular, bicellular and uniseriate multicellular covering trichomes and also glandular trichomes with uni to tricellular stalk and uni to bicellular head; cortex composed of outer collenchymatous tissue and inner 6-8 layers of parenchymatous tissue; collenchyma well developed in basal region and gradually decreases towards middle and distal regions; pericyclic fibres absent in the basal region of petiole but present in the form of

Fig. 1: Powdered drug of NIRGUÛÚÌ (Vitex negundo (L.) Dunal.) 58

 

 

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The powder shows bicellular trichomes and groups of vessels with scalariform thickenings besides tissue fragments comprising both thin and thick walled cells (Fig. 1).

profile of the test solution shows bands corresponding to that of the standard solution (Fig. 2).

Identity, Purity and Strength:

Foreign matter: not more than 2.0 per cent (Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 8.0 per cent (Appendix 2.1.5); Acidinsoluble ash: not more than 1.0 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 10.0 per cent (Appendix 2.1.8); Watersoluble extractive: not less than 20.0 per cent (Appendix 2.1.9)

Quantitative parameters:

Identification: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using negundoside and agnuside as reference standards. Visible after derivatisation

254 nm

Rf

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

1.0

Assay:

0.5 (1) (2)

Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 1 g, accurately weighed, of the substance being examined to a 100-ml round bottomed flask, reflux with methanol (50 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and make up the volume in a 100-ml volumetric flask. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, each of negundoside RS and agnuside RS in a 100-ml volumetric flask and dissolve in about 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 900 ml of water, adding 0.5 ml of orthophosphoric acid and making up the volume to 1000 ml) and acetonitrile in the following proportions:

(1) (2) 0.0

RS T RS T Fig. 2: Thin-Layer Chromatogram of Nirgu¸·¢ RS: (1) Negundoside and (2) Agnuside, T: Test solution Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : glacial acetic acid : water (8.0 : 1.0 : 1.0). Dry the plate in air and examine under UV 254 nm. Spray the plate with of anisaldehyde- sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic 59  

 

Time (min) 0.01 10 20 23 25 35

Phosphate buffer (per cent) 95 85 70 85 95 95

API, Part-I, Vol.-IX (Extracts); Monographs 

Acetonitrile (per cent) 5 15 30 15 5 5

Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standards: API Negundoside RS and Agnuside RS

Constituents:

Negundoside, nishindaside. agnuside, mussaenoside tetraacetate, mussaenoside pentaacetate, 6'-p-hydroxybenzoyl mussaenosidic acid, 2'-p-hydroxybenzoyl mussaenosidic acid, -phellandrene, sabinene, p-cymene, -terpinene, terpinen-4-ol, -caryophyllene, viridiflorol, -eudesmol, -pinene, -carene, limonene, camphene, citral, caryophyllene, methyl heptanone, linalool, camphor, 1,8-cineole, terpineol, geraniol, caryophyllene oxide, terpenyl acetate, geranyl acetate, benzaldehyde, cinnamaldehyde, 5-hydroxy-3,6,7,3',4'pentamethoxy flavone, 5,3'-dihydroxy-7,8,4'trimethoxy flavanone, 5,3'-dihydroxy-6,7,4'trimethoxy flavanone, 4,4'- dimethoxy-transstilbene, 5,6,7,8,3'4'5'-heptamethoxy flavone, 5-Odesmethylnobiletin, gardenin A, B, corymbosin

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 254 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and identify the analyte peaks using the relative retention times, as below. Analyte Relative retention time Negundoside 1.00 Agnuside 1.15 Calculate the content of negundoside and agnuside in the substance being examined from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Properties and Action: Rasa: Tikta, Ka¶u, KaÀ¡ya; Gu¸a: Laghu; V¢rya: UÀ¸a; Vip¡ka: Ka¶u; Karma: K¡saghna, K¤mighna, Kaphahara, V¡tahara, Vra¸a¿odhana

Important formulations: Da¿m£la taila, Mah¡v¡tavidhvaÆ¿ana rasa, Nirgu¸·¢ taila, Tribhuvanak¢rti rasa, Trivikrama rasa, V¡tagaj¡´ku¿a rasa, ViÀatinduka taila, Yak¤tpl¢h¡ri lauha

Nirgu¸·¢

Therapeutic uses: Aruci (anorexia), áv¡sa (dyspnoea), K¡sa (cough), K¤mi (helminthiasis), Prati¿y¡ya (coryza), Sandhi¿otha (arthritis), áotha (inflammation), V¡tavy¡dhi (disease due to v¡ta doÀa/neurological disease), Vra¸a (wound)

Fig. 3: HPLC chromatograms of Nirgu¸·¢ with Negundoside and Agnuside as RS

Dose: C£r¸a (powder): 3-6 g

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. 60  

 

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NIRGUÛÚÌ HYDRO-ALCOHOLIC EXTRACT Nirgu¸·¢ Hydro-alcoholic Extract is a dried and powdered extract prepared from Nirgu¸·¢ (appropriately powdered). The extract contains not less than 1 per cent of negundoside and 0.05 per cent agnuside when assayed.

without charring. The chromatographic profile of the test solution shows bands corresponding to that of the standard solution (Fig. 1). 254 nm

Visible after derivatisation Rf 1.0

Method of preparation: Take Nirgu¸·¢ suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under reflux at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a        (1) syrupy consistency and dry under vacuum (2) (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per    cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 14 per cent.

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using negundoside and agnuside as reference standards. Test solution: Extract 2 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg each of negundoside RS and agnuside RS in 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : glacial acetic acid : water (8.0 : 1.0 : 1.0). Dry the plate in air and examine under UV 254 nm. Spray the plate with of anisaldehyde-sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear

(1) (1) (2) (2) 0.0 RS T RS T Fig. 1: Thin-Layer Chromatogram of Nirgu¸·¢ hydro-alcoholic extract RS: (1) Negundoside and (2) Agnuside, T: Test solution

Quantitative parameters: Loss on drying: not more than 5.0 per cent (Appendix 2.1.4); Total ash: not more than 15.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.0-6.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-I)

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the prescribed limits, 61

 

0.5

 

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(Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Analyte Negundoside Agnuside

Assay:

Calculate the content of negundoside and agnuside in the substance being examined from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 1 g, accurately weighed, of the substance being examined to a 100-ml round bottomed flask, reflux with methanol (50 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and make up the volume in a 100-ml volumetric flask. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, each of negundoside RS and agnuside RS in a 100-ml volumetric flask and dissolve in about 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 900 ml of water, adding 0.5 ml of orthophosphoric acid and making up the volume to 1000 ml) and acetonitrile in the following proportions: Time (min) 0.01 10 20 23 25 35

Phosphate buffer (per cent) 95 85 70 85 95 95

Nirgu¸·¢ hydro-alcoholic extract

Fig. 2: HPLC chromatograms of Nirgu¸·¢ hydro-alcoholic extract with Negundoside and Agnuside as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents.

Acetonitrile (per cent) 5 15 30 15 5 5

Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standards: API Negundoside RS and Agnuside RS

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 254 nm. Procedure: Inject 20 l of the standard solution and record  the chromatogram. Inject 20 l of the test solution, record the chromatogram and identify the analyte peaks using the relative retention times.

62  

Relative retention time 1.00 1.15

 

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NIRGUÛÚÌ WATER EXTRACT 254 nm

Nirgu¸·¢ Water Extract is a dried and powdered extract prepared from Nirgu¸·¢. The extract should contain not less than 1 per cent of negundoside and 0.05 per cent agnuside when assayed.

Visible after derivatisation Rf 1.0

Method of preparation: Take Nirgu¸·¢ suitably sized (powder or pieces) in an extractor. Add water, about 3 times the quantity of raw material and heat at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 23 per cent.

0.5    

(1) (2) 0.0

   

Identity, Purity and Strength: Thin-layer chromatography:

RS T RS T Fig. 1: Thin-Layer Chromatogram of Nirgu¸·¢ water extract RS: (1) Negundoside and (2) Agnuside, T: Test solution

Quantitative parameters:

Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using negundoside and agnuside as reference standards. Test solution: Extract 2 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg each of negundoside RS and agnuside RS in 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate: glacial acetic acid : water (8.0 : 1.0 : 1.0). Dry the plate in air and examine under UV 254 nm. Spray the plate with of anisaldehyde sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows bands corresponding to that of the standard solution (Fig. 1).

Loss on drying: not more than 6.0 per cent (Appendix 2.1.4); Total ash: not more than 15.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 3.0 per cent (Appendix 2.1.7); pH: 4.0-6.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-II)

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

63  

(1) (2)

 

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Assay:

Analyte Negundoside Agnuside

Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 1 g, accurately weighed, of the substance being examined to a 100 ml round bottomed flask, reflux with methanol (50 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and make up the volume in a 100-ml volumetric flask. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, each of negundoside RS and agnuside RS in a 100-ml volumetric flask and dissolve in about 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of phosphate buffer (prepared by dissolving 0.14 g of Potassium dihydrogen orthophosphate in 900 ml of water, adding 0.5 ml of Orthophosphoric acid and making up the volume to 1000 ml) and Acetonitrile in the following proportions: Time (min) 0.01 10 20 23 25 35

Phosphate buffer (per cent) 95 85 70 85 95 95

Calculate the content of negundoside and agnuside in the substance being examined from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Nirgu¸·¢ water extract

Fig. 2: HPLC chromatograms of Nirgu¸·¢ water extract with Negundoside and Agnuside as RS

Acetonitrile (per cent) 5 15 30 15 5 5

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 254 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and identify the analyte peaks using the relative retention times, as below.

API reference standards: API Negundoside RS and Agnuside RS

64  

Relative retention time 1.00 1.15

 

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PUNARNAVË  thickenings; trachieds, thick-walled with simple pits; fibres aseptate, elongate, thick-walled with pointed ends; phloem occurs as hemispherical or crescent patches outside each group of xylem vessels and composed of sieve elements and parenchyma; a broad zone of parenchymatous tissue, in between two successive rings of xylem elements, composed of thin-walled, more or less rectangular cells arranged in radial rows; central region of root occupied by primary vascular bundles; numerous raphides in single or in groups present in cortical region and in parenchymatous and xylem tissue; starch grains simple measuring up to 11  in diameter and compound, having 2 to 4 components, found in abundance in most of the cells of cortex and xylem elements.

Punarnav¡ consists of dried root of Boerhaavia diffusa L. (Fam. Nyctaginaceae); a trailing herb with stout root stock and many diffused, slender, prostrate or ascending branches. The extract contains not less than 0.005 per cent of boeravinone B when assayed. Synonyms: Ka¶hill¡, áophaghn¢, áothaghn¢ Other/Regional Language Names: Assamese: Ranga Punanabha; Bengali: Rakta punarnava;  English: Horse Purslene, Hog Weed;  Gujarati: Dholisaturdi, Motostodo;  Hindi:  Gadapurna, Lalpunarnava; Kannada: Snadika, Kommeberu, Komma; Kashmiri: Vanjula punarnava; Malayalam: Chuvanna Tazhutama, Tavilama, Tlutama; Marathi: Ghetuli, Vasuchi muli, Satodimula, Punarnava, Khaparkhuti; Oriya: Lalapuiruni, Malipuruni; Punjabi: ltcit (Ial), Khattan; Tamil: Mukurattai (Shihappu); Telugu: Atikammamidi, Erragalijeru; Urdu: Surkh punarnava

c) Powder: Light yellow; shows vessels with reticulate thickenings or simple pits, fibres, fragments of cork cells, cells containing raphides of calcium oxalate and simple, rounded, starch grains, measuring 2.75 to 11  in diameter and compound starch grains having 2 to 4 components (Fig. 1.)

Description: a) Macroscopic: Root well developed, fairly long, somewhat tortuous, cylindrical, 0.2 to 1.5 cm in diameter; yellowish to brown; surface rough due to minute longitudinal striations and root scars; fracture short; odour not distinct; taste slightly bitter. b) Microscopic: Mature root shows anomalous growth; cork composed of thin-walled, tangentially elongated cells in the outer few layers; cork cambium 1 to 2 layers of thin-walled cells; secondary cortex consists of 2 to 3 layers of parenchymatous cells, followed by cortex composed of 5 to 12 layers of thin-walled, oval to polygonal cells; several concentric bands of xylem tissues, alternating with zone of parenchymatous tissue present below cortical region; number of bands vary according to thickness of root and consists of vessels, trachieds and fibres; vessels mostly found in groups of 2 to 8 in radial rows, having simple pits and reticulate

Fig. 1: Powdered drug of PUNARNAVË (Boerhaavia diffusa L.) 65

 

 

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Identity, Purity and Strength: Identification:

ash: not more than 3.0 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 5.0 per cent (Appendix 2.1.8); Water-soluble extractive: not less than 8.0 per cent (Appendix 2.1.9)

Thin- layer chromatography:

Other requirements:

Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using boeravinone B as a reference standard. Test solution: Extract 2 g of substance by refluxing with 50 ml of methanol for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 2 mg of boeravinone B RS in 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: toluene : ethyl acetate : formic acid (4.0 : 5.0 : 1.0). 254 nm

Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 5 g, accurately weighed, of the substance being examined and reflux with methanol (25 ml x 3) on water bath for 15 min each, cool and filter. Combine all the filtrates and transfer to a 100-ml volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 2.5 mg, accurately weighed, boeravinone B RS in a 100-ml volumetric flask and dissolve in about 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of water and acetonitrile in the following proportions:-

Rf 1.0

0.5

0.0 RS

T

 

Time (min) 0.01 5 15 20 23 25 30

Fig. 2: Thin-Layer Chromatogram of Punarnav¡ RS: Boeravinone B, T: Test solution

Dry the plate in air and examine under UV 254 nm. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 2). Quantitative parameters:

Acetonitrile (per cent) 20 45 80 80 45 20 20

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 280 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution,

Foreign matter: not more than 2.0 per cent (Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 15.0 per cent (Appendix 2.1.5); Acid-insoluble 66  

Water (per cent) 80 55 20 20 55 80 80

 

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API reference standard:

record the chromatogram and measure the response for the analyte peak. Calculate the content of boeravinone B in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

API Boeravinone B RS

Constituents: Punarnavoside, boeravinone C, liriodendrin; hypoxanthine-9-L-arabinofuranoside, eupalitin-3-O--D-galactopyranoside. Eupalitin, eupalitin-3-O--D-galactopyranosyl-(1´´´2´´)O--D-galactopyranoside, 3,3´5-trihydroxy-7methoxyflavone, 4´,7-dihydroxy-3´methylflavone, 3,4-dimethoxy phenyl-1-O--Dapiofuranosyl-(1´´3´)-O--D-glucopyranoside.

Properties and Action: Rasa: Madhura, Tikta; Gu¸a: R£kÀa; V¢rya: UÀ¸a; Vip¡ka: Madhura; Karma: Anulomana, M£travirecan¢ya, Ras¡yana, áothahara, V¡ta¿leÀmahara

Important formulations: Punarnav¡Àtaka

Fig. 3: HPLC chromatogram of Punarnav¡ with Boeravinone B as RS

kv¡tha c£r¸a, Punarnav¡di Punarnav¡sava, Sukum¡ra gh¤ta

Additional requirements:

Therapeutic uses: Dh¡tu kÀaya (tissue

Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents.

wasting), P¡¸·u (anaemeia), áotha (inflammation)

Dose: C£r¸a (powder): 3-6 g

Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

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PUNARNAVË HYDRO-ALCOHOLIC EXTRACT  254 nm

Punarnav¡ Hydro-alcoholic Extract is a dried and powdered extract prepared from Punarnav¡ (appropriately powdered). The extract contains not less than 0.025 per cent of boeravinone B when assayed.

Rf 1.0

Method of preparation: 0.5

Take Punarnav¡ suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under a reflux at a temperature between 80850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 10 per cent.

0.0  

RS T Fig. 1: Thin-Layer Chromatogram of Punarnav¡ water extract RS: Boeravinone B, T: Test solution

Quantitative parameters: Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 12.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 6.0- 8.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-I)

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using boeravinone B as a reference standard. Test solution: Extract 2 g of substance by refluxing with 50 ml of methanol for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 2 mg of boeravinone B RS in 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: toluene : ethyl acetate : formic acid (4.0 : 5.0 : 1.0). Dry the plate in air and examine under UV 254 nm. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Other requirements: Heavy metals:  Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the  prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 5 g, accurately weighed, of the substance being examined and reflux with methanol (25 ml x 3) 68

 

 

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on water bath for 15 min each, cool and filter. Combine all the filtrates and transfer to a 100-ml volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 2.5 mg, accurately weighed, boeravinone B RS in a 100-ml volumetric flask and dissolve in about 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of water and acetonitrile in the following proportions:Time (min) 0.01 5 15 20 23 25 30

Water (per cent) 80 55 20 20 55 80 80

Fig. 2: HPLC chromatogram of Punarnav¡ hydro-alcoholic extract with Boeravinone B as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents.

Acetonitrile (per cent) 20 45 80 80 45 20 20

Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Boeravinone B RS

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 280 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of boeravinone B in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

 

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PUNARNAVË WATER EXTRACT  254 nm

Punarnav¡ Water Extract is a dried and powdered extract prepared from Punarnav¡. The extract contains not less than 0.0025 per cent of boeravinone B when assayed.

Rf 1.0

Method of preparation: Take Punarnav¡ suitably sized (powder or pieces) in an extractor. Add water, about 3 times the quantity of raw material and heat at a temperature between 80–850 for 3-4 hours. Filter the extract through a filter (preferably10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh and pack. The yield obtained is about 8 per cent.

0.5

0.0  

RS T Fig. 1: Thin-Layer Chromatogram of Punarnav¡ water extract RS: Boeravinone B, T: Test solution

Quantitative parameters: Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 12.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 6.0-8.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-II)

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using boeravinone B as a reference standard. Test solution: Extract 2 g of substance by refluxing with 50 ml of methanol for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 2 mg of boeravinone B RS in 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase : toluene : ethyl acetate : formic acid (4.0 : 5.0 : 1.0). Dry the plate in air and examine under UV 254 nm. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 5 g, accurately weighed, of the substance being examined and reflux with methanol (25 ml x 3) on water bath for 15 min each, cool and filter. Combine all the filtrates and transfer to a 100-ml 70

 

 

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volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 2.5 mg, accurately weighed, boeravinone B RS in a 100 ml volumetric flask and dissolve in about 50 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of water and acetonitrile in the following proportions: Time (min) 0.01 5 15 20 23 25 30

Water (per cent) 80 55 20 20 55 80 80

Fig. 2: HPLC chromatogram of Punarnav¡ water extract with Boeravinone B as RS 

Additional requirements:

Acetonitrile (per cent) 20 45 80 80 45 20 20

Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Boeravinone B RS

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV, 280 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of boeravinone B in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

   

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áALLAKÌ (Exudate)  áallak¢ consists of exudate of Boswellia serrata Roxb. (Fam. Burseraceae), a moderate sized, deciduous tree, upto 18 m in height and upto 2.4 m in girth, commonly found in the dry forests from Punjab to West Bengal and in peninsular India. It contains not less than 4.0 per cent of βboswellic acid when assayed.

Identity, Purity and Strength: Identification: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using β-boswellic acid as a reference standard. Test solution: Extract 0.2 g of substance by refluxing with 50 ml of methanol for a period of 15 min. Filter and concentrate the extract to 25 ml.

Synonym: Kunduru Other/Regional

Language

Names:

Assamese: Sallaki; Bengali: Salai, Salgai; English: Indian Olibanum Tree; Gujarati: Shaledum, Saleda, Saladi, Gugal, Saledhi; Hindi: Salai, Labana; Kannada: Madimar, Chilakdupa, Tallaki, Maddi; Kashmiri: Kunturukkam, Samprani; Marathi: Salai cha dink; Punjabi: Salai Gonda; Tamil: Kundurukam; Telugu: Anduga, Kondagugi tamu; Urdu: Kundur

Visible after derivatisation Rf 1.0

Description:

0.5

a) Macroscopic: Drug occurs in stalactitic, transparent, tears forming agglomerates of various shapes and sizes, brownish-yellow, upto 5 cm long, 2 cm thick, fragrant, fracture brittle; fractured surface waxy and translucent; burns readily and emanates an agreeable characteristic, balsamic resinous odour; taste, aromatic and agreeable

0.0 RS T Fig. 2: Thin-Layer Chromatogram of áallak¢ RS: β-boswellic acid, T: Test solution

Identification - Trituration with water forms an emulsion; when immersed in alcohol (90 per cent) a tear of' áallak¢ is not altered much in form but becomes almost opaque and white; when a drop of con. H2SO4 is added to a freshly fractured surface, it becomes cherry red which, when washed with water changes to a white emulsion, turning later to a buff colour

Standard solution: Dissolve 10 mg of β-boswellic acid RS in 25 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: toluene : etthyl acetate : methanol (8.0 :1.5 : 0.5). Spray the plate with anisaldehyde - sulphuric acid reagent and heat at 1050 till the colour of the spots/ bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 2).

Fluorescence Test - Brownish-yellow colour in day light; aqueous extract under UV light (366 nm) light green and in (254 nm) shows dark blue colour; alcoholic extract under UV light (366 nm) is colourless and in (254 nm) shows light green colour 72  

 

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Quantitative parameters: Foreign matter: not more than 5.0 per cent (Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 10.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 8.0 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 45.0 per cent (Appendix 2.1.8); Water-soluble extractive: not less than 28.0 per cent (Appendix 2.1.9)

áallak¢ 

Fig. 3: HPLC chromatogram of áallak¢ with β-boswellic acid as RS Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article. API reference standard: API β-boswellic acid RS Constituents: Volatile oil (9 per cent), gum and resin; the volatile oil contains -thujene, -phellandrene, β phellandrene, -terpineol, limonene, camphene, myrcene, -terpene, p-cymene; active principles boswellic acids from resin viz., -boswellic acid, β boswellic acid, 3-O-acetyl- β -boswellic acid, 11keto- β -boswellic acid, 3- O- acetyl -11-keto-β boswellic acid. A diterpene alcohol serratol and four tetracyclic triterpene acids 3--acetoxytirucall-8,24dien-21-oic acid, 3-ketotirucall-8, 24-dien-21-oic acid, 3--hydroxytirucall-8, 24-dien-oic acid, 3 - β hydroxytirucall-8, 24-dien-21-oic acid Properties and Action: Rasa: Tikta, Madhura; Gu¸a: Guru, Snigdha, T¢kÀ¸a; V¢rya: UÀ¸a; Vip¡ka: Ka¶u; Karma: Balya, Kaphahara, Kaphapittahara, Raktastambhana, áothahara, V¡tahara Important formulations: Bal¡gu·£cy¡di taila, Bal¡ taila, J¢rk¡di modaka, Karp£r¡dyarka Therapeutic uses: Jvara (fever), Mukharoga (disease of mouth), Pitt¡bhiÀyanda (conjunctivitis due to pitta doÀa), Pradara (excessive vaginal discharge), áarkar¡meha (glycosuria), Sandhi¿£la (joint pain), á£la (pain), áv¡sa (dyspnoea) Dose: C£r¸a (powder): 1-3 g

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.4 g, accurately weighed, of the substance being examined and reflux with methanol (10 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 25-ml volumetric flask and make up the volume. Standard solution: Take about 10 mg, accurately weighed, β-boswellic acid RS in a 25ml volumetric flask and dissolve in about 10 ml of methanol and make up the volume with methanol. Chromatographic system: High performance liquid Chromatography. Column and stationary phase: Reverse phase C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 1 volume of water and 9 volumes of acetonitrile. Injection volume: 20 l. Flow rate: 1.0 ml per min. Detection: UV 205 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of, β-boswellic acid in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. 73  

 

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áALLAKÌ HYDRO-ALCOHOLIC EXTRACT  Visible after derivatisation

áallak¢ Hydro-alcoholic Extract is a dried and powdered extract prepared from áallak¢ (appropriately powered). The extract contains not less than 0.4 per cent of β-boswellic acid when assayed.

Rf 1.0

Method of preparation: Take áallak¢ suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under a reflux at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 45 per cent.

0.5

0.0 RS T Fig. 1: Thin-Layer Chromatogram of áallak¢ hydro-alcoholic extract RS: β-boswellic acid, T: Test solution

Quantitative parameters:

Identity, Purity and Strength:

Loss on drying: not more than 5.0 per cent (Appendix 2.1.4); Total Ash: not more than 4.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.0 per cent (Appendix 2.1.7); pH: 3.5-5.5 (Appendix 2.1.10); Total soluble solids: not less than 70.0 per cent (Appendix 2.1.11) (Method-I).

Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using βboswellic acid as a reference standard. Test solution: Extract 0.2 g of substance by refluxing with 50 ml of methanol for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg of β-boswellic acid RS in 25 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: Toluene : Ethyl acetate : Methanol (8.0 :1.5 : 0.5). Spray the plate with anisaldehyde - sulphuric acid reagent and heat at 1050 till the colour of the spots / bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4).

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.4 g, 74

 

 

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accurately weighed, of the substance being examined and reflux with methanol (10 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 25-ml volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, β-boswellic acid RS in a 25-ml volumetric flask and dissolve in about 10 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid Chromatography. Column and stationary phase: Reverse phase C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 1 volume of water and 9 volumes of acetonitrile. Injection volume: 20 l. Flow rate: 1.0 ml per min. Detection: UV 205 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak.Calculate the content of β-boswellic acid in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

áallak¢ hydro-alcoholic extract

Fig. 2: HPLC chromatogram of áallak¢ hydro-alcoholic extract with β-boswellic acid as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API β-boswellic acid RS

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áALLAKÌ WATER EXTRACT  Visible after derivatisation

áallak¢ Water Extract is a dried and powdered extract prepared from áallak¢. The extract contains not less than 0.1 per cent of β-boswellic acid when assayed.

Rf 1.0

Method of preparation: Take áallak¢ suitably sized (powder or pieces) in an extractor. Add water, about 3 times the quantity of raw material and heat at a temperature between 80850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh and pack. The yield obtained is about 12 per cent.

0.5

0.0 RS T Fig. 1: Thin-Layer Chromatogram of áallak¢ water extract RS: β-boswellic acid, T: Test solution

Identity, Purity and Strength:

Quantitative parameters:

Thin-layer chromatography:

Loss on drying: not more than 5.0 per cent (Appendix 2.1.4); Total ash: not more than 5.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.0 per cent (Appendix 2.1.7); pH: 3.5-5.5 (Appendix 2.1.10); Total soluble solids: not less than 65.0 per cent (Appendix 2.1.11) (Method-II)

Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using β-boswellic acid as a reference standard. Test solution: Extract 0.2 g of substance by refluxing with 50 ml of methanol for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg of βboswellic acid RS in 25 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: toluene : ethyl acetate : methanol (8.0 :1.5 : 0.5). Spray the plate with anisaldehyde -sulphuric acid reagent and heat at 1050 till the colour of the spots/ bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4) 

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.4 g, accurately weighed, of the substance being examined and reflux with methanol (10 ml x 3) on a water bath for 15 min each, cool and filter. 76

 

 

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Combine all the filtrates, concentrate and transfer to a 25-ml volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, β-boswellic acid RS in a 25-ml volumetric flask and dissolve in about 10 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid Chromatography. Column and stationary phase: Reverse phase C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 1 volume of water and 9 volumes of acetonitrile. Injection volume: 20 l. Flow rate: 1.0 ml per min. Detection: UV 205 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of β-boswellic acid in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

áallak¢ water extract

Fig. 2: HPLC chromatogram of áallak¢ water extract with β-boswellic acid as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API β-boswellic acid RS

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áIRÌâA present in middle and outer phloem region; phloem fibres, elongated, thick-walled, lignified, present in many concentric strips, mostly enclosed by crystal sheath throughout the middle and inner regions of phloem; crystal fibres having a number of septa, each chamber containing a single prismatic crystal of calcium oxalate; phloem rays numerous, radially elongated, somewhat wavy in outer phloem region and bi to multiseriate in the inner phloem region, being 2-5 cells wide and 7-25 cells high

áir¢Àa consists of stem bark of Albizia lebbeck (L.) Benth. (Fam. Mimosaceae), a large tree, common throughout the country, ascending to 1200 m on the Himalayas. It contains not less than 5.0 per cent of total polyphenols calculated as pyrogallol and not less than 0.1 per cent of catechin when assayed.

Synonyms: áukapriya, M¤dupuÀpa Other/Regional Language Names: Bengali: Sirish, Siris; English: Siris Tree, Lebbeck Tree; Gujarati: Shrish; Hindi: Siris, Shris; Kannada: Bagey, Bage Mara, Hombage; Malayalam: Vaka, Nenmenivaka; Marathi: Shirish; Oriya: Sersuan, Sirisha; Punjabi: Sirish, Sareehn; Tamil: Vakai; Telugu: Dirisena; Urdu: Siris 

c) Powder: Greyish-brown; shows large number of stone cells of different sizes and shapes from rhytidoma or cortex, prismatic crystals of calcium oxalate, crystal fibres, phloem fibres, cork cells and parenchymatous cells with starch grains (Fig.1).

Description: a) Macroscopic: Bark 1.5-2.5 cm thick, external surface dark brown, rough due to longitudinal fissures and transverse cracks, rhytidoma forming major part of bark and peeling off in flakes exposing buff coloured surface, middle bark brown, inner bark much fibrous, light yellow to grey; fracture, laminated in outer region and fibrous in inner region; taste astringent b) Microscopic: Mature bark about 2 cm thick, shows dead tissue of rhytidoma; cork consists of a few layers of thinwalled, transversely elongated and radially arranged cells; secondary cortex wide, composed of radially elongated to squarish, moderately thick-walled cells containing orange to reddish-brown contents; a few of the cells contain prismatic crystals of calcium oxalate; stone cells variable in shape and size, present in singles or in groups throughout the region; secondary phloem consists of sieve elements, phloem parenchyma, phloemfibres and crystal fibres, traversed by phloem rays; prismatic crystals of calcium oxalate present in most of the phloem parenchyma cells; tangential bands of ceratenchyma

Fig. 1: Powdered drug of áIRÌâA (Albizia lebbeck (L.) Benth.) 78

 

 

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Identity, Purity and Strength:

12.0 per cent (Appendix 2.1.4); Total ash: not more than 8.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.0 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 12.0 per cent (Appendix 2.1.8); Water-soluble extractive: not less than 6.0 per cent (Appendix 2.1.9)

Identification: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using catechin as a reference standard. Test solution: Extract 2 g of substance by refluxing with 50 ml of water for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg of catechin RS in 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: toluene : ethyl acetate : formic acid (1.2 : 1.8 : 1.0). Dry the plate in air and examine under UV 254 nm. Spray the plate with fast blue salt B reagent, and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 2). 254 nm

Other requirements: Heavy metals:  Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4).

Assay for total polyphenols: Carry out the assay by Spectrophotometry (Appendix 3.7). Test solution: Always prepare fresh solution. Take about 500 mg, accurately weighed, of the substance being examined in a 100-ml round bottomed flask, reflux with 50 per cent aqueous methanol (25 ml x 3) on water bath for 15 min each, cool, filter and make up the volume to 100 ml in a volumetric flask. Further dilute 5 ml aliquot to 50 ml with water in a volumetric flask. Standard solution: Take about 100 mg, accurately weighed, pyrogallol RS in a 100 ml volumetric flask and dissolve in about 50 ml of 50 per cent aqueous methanol and make up to 100 ml with 50 per cent aqueous methanol. Further dilute 5 ml to 50 ml with water in a volumetric flask. Procedure: Pipette out 2, 5, 7 and 10 ml of standard solution and 5, 7 ml of test solution to different 100-ml volumetric flasks. Add 40 ml of water, 5 ml of Folins reagent and 10 ml of 30.0 per cent sodium carbonate solution to each of the volumetric flasks. Allow to stand for 30 min. Make up the volume with water, shake and allow to stand for 20 min more. Read in a suitable spectrophotometer at 750 nm using water as blank. Prepare a calibration curve from the values obtained. Calculate the content of polyphenols in the sample from the absorbance using the calibration curve.

Visible after derivatisation Rf 1.0

0.5

0.0  

RS T RS T Fig. 2: Thin-Layer Chromatogram of áir¢Àa RS: Catechin, T: Test solution Quantitative parameters: Foreign matter:  not more than 1.0 per cent (Appendix 2.1.3); Loss on drying: not more than 79  

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Assay:

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 280 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of catechin in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 4 g, accurately weighed, of the substance being examined and reflux with water (50 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and make up the volume in a 100-ml volumetric flask. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, catechin RS in a 100-ml volumetric flask and dissolve in about 50 ml of water and make up the volume with water. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 900 ml of water, adding 0.5 ml of orthophosphoric acid and making up the volume to 1000 ml) and acetonitrile in the following proportions:Time (min) 0.01 18 22 25 30

Phosphate buffer (per cent) 95 75 75 95 95

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Catechin RS

Constituents: (+)-Catechin, saponins, (+)leucocyanidin, Albizia saponin A, Albizia saponin B, Albizia saponin C, acacic acid, albegenin

Acetonitrile (per cent) 5 25 25 5 5

Properties and Action: Rasa: Tikta, KaÀ¡ya, Madhura; Gu¸a: Laghu, R£kÀa; V¢rya: AnuÀ¸a; Vip¡ka: Ka¶u; Karma: TridoÀahara, TvagdoÀa, Var¸ya, ViÀaghna

Important

formulations:

B¤hanmaric¡dya taila, Devad¡rv¡riÀ¶a, Vajraka taila

áir¢Àa

Ayask¤ti, Da¿¡´ga lepa,

Therapeutic uses: K¡sa (cough), K¤mi roga (worm infestation), Ka¸·£ (pruritis), KuÀ¶ha (disease of skin), M£Àaka viÀa (rat poisoning), Netr¡b¡hiÀyanda (conjunctivitis), P¡m¡ (eczema), P¢nasa (chronic rhinitis), Prati¿y¡ya (coryza), SarpadaÆ¿a (snake bite), S£ry¡varta (chronic sinusitis), á¢tap¢tta (urticaria), áotha (inflammation), Ardh¡vabhedaka (migraine), áv¡sa (dyspnoea), ViÀamajvara (intermittent fever), Visarpa (erysipales), Vra¸a (wound)

Fig. 3: HPLC chromatogram of áir¢Àa with Catechin as RS

Dose: C£r¸a (powder): 3-6 g 80

 

 

API, Part-I, Vol.-IX (Extracts); Monographs 

áIRÌâA HYDRO-ALCOHOLIC EXTRACT 254 nm

áir¢Àa Hydro-alcoholic Extract is a dried and powdered extract prepared from áir¢Àa (appropriately powdered). The extract contains not less than 10 per cent of total polyphenols calculated as pyrogallol and not less than 0.1 per cent of catechin when assayed.

Visible after derivatisation Rf 1.0

Method of Preparation: Take áir¢Àa suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under reflux at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 21 per cent.

0.5

 

RS T RS T Fig. 1: Thin-Layer Chromatogram of áir¢Àa hydro-alcoholic extract RS: Catechin, T: Test solution

Quantitative parameters: Loss on drying: not more than 5.0 per cent, (Appendix 2.1.4); Total ash: not more than 10.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.0-6.5 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-I)

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using catechin as a reference standard. Test solution: Extract 2 g of substance by refluxing with 50 ml of water for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg of catechin RS in 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: toluene : ethyl acetate : formic acid (1.2 : 1.8 : 1.0). Dry the plate in air and examine under UV 254 nm. Spray the plate with fast blue salt B reagent, and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Other requirements: Heavy metals:  Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay for total polyphenols: Carry out the assay by Spectrophotometry (Appendix 3.7). Test solution: Always prepare fresh solution. Take about 500 mg, accurately weighed, of the substance being examined in a 100-ml round 81

 

0.0

 

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bottomed flask, reflux with 50 per cent aqueous methanol (25 ml x 3) on water bath for 15 min each, cool, filter and make up the volume to 100 ml in a volumetric flask. Further dilute 5 ml aliquot to 50 ml with water in a volumetric flask. Standard solution: Take about 100 mg, accurately weighed, pyrogallol RS in a 100-ml volumetric flask and dissolve in about 50 ml of 50 per cent aqueous methanol and make up to 100-ml with 50 per cent aqueous methanol. Further dilute 5 ml to 50 ml with water in a volumetric flask. Procedure: Pipette out 2, 5, 7 and 10 ml of standard solution and 5, 7 ml of test solution to different 100-ml volumetric flasks. Add 40 ml of water, 5 ml of Folins reagent and 10 ml of 30.0 per cent sodium carbonate solution to each of the volumetric flasks. Allow to stand for 30 min. Make up the volume with water, shake and allow to stand for 20 min more. Read in a suitable spectrophotometer at 750 nm using water as blank. Prepare a calibration curve from the values obtained. Calculate the content of polyphenols in the sample from the absorbance using the calibration curve.

Time (min) 0.01 18 22 25 30

Phosphate buffer (per cent) 95 75 75 95 95

Acetonitrile (per cent) 5 25 25 5 5

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 280 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of catechin in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 4 g, accurately weighed, of the substance being examined and reflux with water (50 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and make up the volume in a 100 ml volumetric flask. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, catechin RS in a 100 ml volumetric flask and dissolve in about 50 ml of water and make up the volume with water. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 900 ml of water, adding 0.5 ml of orthophosphoric acid and making up the volume to 1000 ml) and acetonitrile in the following proportions:-

áir¢Àa hydro-alcoholic extract

Fig. 2: HPLC chromatogram of áir¢Àa hydroalcoholic extract with Catechin as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard: API Catechin RS 82

 

 

API, Part-I, Vol.-IX (Extracts); Monographs 

áIRÌâA WATER EXTRACT 254 nm

áir¢Àa Water Extract is a dried and powdered extract prepared from áir¢Àa. The extract contains not less than 10 per cent of total polyphenols calculated as pyrogallol and not less than 0.1 per cent of catechin when assayed.

Visible after derivatisation Rf 1.0

Method of Preparation: Take áir¢Àa suitably sized (powder or pieces) in an extractor. Add water, about 3 times the quantity of raw material and heat at a temperature between 80850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 17 per cent.

0.5

0.0   

RS T RS T Fig. 1: Thin-Layer Chromatogram of áir¢Àa water extract RS: Catechin, T: Test solution

Quantitative parameters: Loss on drying: not more than 5.0 per cent (Appendix 2.1.4); Total ash: not more than 12.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.5-6.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-II).

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using catechin as a reference standard. Test solution: Extract 2 g of substance by refluxing with 50 ml of water for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 10 mg of catechin RS in 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: toluene : ethyl acetate : formic acid (1.2 : 1.8 : 1.0). Dry the plate in air and examine under UV 254 nm. Spray the plate with fast blue salt B reagent, and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Other requirements: Heavy metals:  Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4).

Assay for total polyphenols: Carry out the assay by Spectrophotometry (Appendix 3.7). Test solution: Always prepare fresh solution. Take about 500 mg, accurately weighed, of the substance being examined in a 100-ml round bottomed flask, reflux with 50 per cent aqueous methanol (25 ml x 3) on water bath 83

 

 

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acid and making up the volume to 1000 ml) and acetonitrile in the following proportions:-

for 15 min each, cool, filter and make up the volume to 100 ml in a volumetric flask. Further dilute 5 ml aliquot to 50 ml with water in a volumetric flask. Filter through 0.42 m membrane. Standard solution: Take about 100 mg, accurately weighed, pyrogallol RS in a 100ml volumetric flask and dissolve in about 50 ml of 50 per cent aqueous methanol and make up to 100-ml with 50 per cent aqueous methanol. Further dilute 5 ml to 50 ml with water in a volumetric flask. Filter through 0.42 m membrane. Procedure: Pipette out 2, 5, 7 and 10 ml of standard solution and 5, 7 ml of test solution to different 100 ml volumetric flasks. Add 40 ml of water, 5 ml of Folins reagent and 10 ml of 30.0 per cent sodium carbonate solution to each of the volumetric flasks. Allow to stand for 30 min. Make up the volume with water, shake and allow to stand for 20 min. more. Read in a suitable spectrophotometer at 750 nm using water as blank. Prepare a calibration curve from the values obtained. Calculate the content of polyphenols in the sample from the absorbance using the calibration curve.

Time (min) 0.01 18 22 25 30

Phosphate buffer (per cent) 95 75 75 95 95

Injection volume: 20 l. Flow rate: 1.5 ml per min. Detection: UV 280 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peak. Calculate the content of catechin in the substance being examined from the peak response of analyte. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Assay:

áir¢Àa water extract

Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 4 g, accurately weighed, of the substance being examined and reflux with water (50 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and make up the volume in a 100 ml volumetric flask. Filter through 0.42 m membrane. Standard solution: Take about 10 mg, accurately weighed, catechin RS and in a 100-ml volumetric flask and dissolve in about 50 ml of water and make up the volume with water. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed gradient mixture of phosphate buffer (prepared by dissolving 0.14 g of potassium dihydrogen orthophosphate in 900 ml of water, adding 0.5 ml of orthophosphoric

Fig. 2: HPLC chromatogram of áir¢Àa water extract with Catechin as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standard:   API Catechin RS 84

 

Acetonitrile (per cent) 5 25 25 5 5

 

API, Part-I, Vol.-IX (Extracts); Monographs 

áUÛÙHÌ arranged radially around numerous scattered, collateral vascular bundles, each consisting of a few unlignified, reticulate or spiral vessels upto about 70  in diameter, a group of phloem cells, unlignified, thinwalled, septate fibres upto about 30  wide and 600  long with small oblique slit like pits, numerous scattered idioblasts, similar to those of cortex, and associated with vascular bundles, also present, idioblasts about 8-20  wide and up to 130  long with dark reddish-brown contents: in single or in axial rows, adjacent to vessels, parenchyma of cortex and stele packed with flattened, rectangular, ovate, starch grains, mostly 5-15  - 30-60  long about 25  wide and 7  thick, marked by five transverse striations

áu¸¶h¢ consists of dried rhizome of Zingiber officinale Roxb. (Fam. Zingiberaceae), widely cultivated in India, rhizomes dug in JanuaryFebruary, buds and roots removed, soaked overnight in water, decorticated, and some times treated with lime and dried. áu¸¶h¢ contains not less than 0.75 per cent of total gingerols (sum of 6gingerol, 8-gingerol, 10-gingerol and 6-shogaol) when assayed.  Synonyms: MahauÀadha, Vi¿vabheÀaja, ᤴagavera

N¡gara,

Vi¿v¡,

Other/Regional Language Names: Assamese: Adasuth, Aadar Shuth; Bengali: Suntha, Sunthi; English: Ginger Root, Ginger; Gujarati: Sunth, Sundh, Suntha; Hindi: Sonth; Kannada: Shunthi; Kashmiri: Shonth; Malayalam: Chukku; Marathi: Sunth; Oriya: Sunthi; Punjabi: Sund; Tamil: Sukku, Chukku; Telugu: Sonthi, Sunthi; Urdu: Sonth, Zanjabeel

c) Powder:

Description: a) Macroscopic: Rhizome, laterally compressed bearing short, flattish, ovate, oblique, branches on upper side each having at its apex a depressed scar, pieces about 515 cm long, 1.5-6.5 cm wide (usually 3-4 cm) and 1-1.5 cm thick, externally buff coloured showing longitudinal striations and occasional loose fibres, fracture short, smooth, transverse surface exhibiting narrow cortex (about one-third of radius), a wellmarked endodermis and a wide stele showing numerousscattered fibro-vascular bundles and yellow secreting cells, odour agreeable and aromatic; taste agreeable and pungent b) Microscopic: Transverse section of rhizome shows cortex of isodiametric thin-walled parenchyma with scattered vascular strands and numerous isodiametric idioblasts, about 40-80  in diameter containing a yellowish to reddish-brown oleo-resin, endodermis slightly thick walled, free from starch, immediately inside endodermis a row of nearly cells, isodiametric

Fig. 1: Powdered drug of áUÛÙHÌ (Zingiber officinale Roxb.) Powder shows parenchyma cell groups containing yellow brown oleo-resinous bodies; occur also as fragments or in droplets. Starch grains abundant, simple, ovoid or sac shaped upto 30-50  in length and have a distinct hilum at the narrower end with 85

 

 

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concentric striations; septate sclerenchymatous lignified fibres in groups associated with vessels. Vessels mostly non-lignified (Fig. 1)

chromatographic profile of the test solution shows bands corresponding to that of the standard solution (Fig. 2).

Identity, Purity and Strength:

Quantitative parameters:

Identification:

Foreign matter:  not more than 1.0 per cent (Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 6.0 per cent (Appendix 2.1.5); Acidinsoluble ash: not more than 1.5 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 3.0 per cent (Appendix 2.1.8); Watersoluble extractive: not less than 10.0 per cent (Appendix 2.1.9). 

Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using 6-gingerol and 6-shogaol as reference standards. Test solution: Extract 1 g of substance by refluxing with 50 ml of methanol for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 5 mg each of 6gingerol RS and 6-shogaol RS in about 25 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : toluene : diethyl ether (3.0 : 6.0 :1.0). 254 nm

Visible after derivatisation

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (25 ml x 3) on water bath for 15 min each, cool and filter. Combine all the filtrates and transfer to a 100-ml volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, capsaicin RS in 25-ml volumetric flask and dissolve in about 10 ml of methanol and make up the volume. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 55 volumes of acetonitrile, 44 volumes of 0.1 per cent phosphoric acid and 1 volume of methanol. Injection volume: 20 l. Flow rate: 1 ml per min. Detection: UV 282 nm.

Rf 1.0

(2)

(2)

(1)

(1)

0.5

0.0 RS

T

RS

T

Fig. 2: Thin-Layer Chromatogram of áu¸¶h¢ RS: (1) 6-Gingerol and (2) 6-Shogaol, T: Test solution Dry the plate in air and examine under UV 254 nm. Spray the plate with anisaldehyde- sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The 86  

 

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API reference standards: API 6-Gingerol RS, 6-Shogaol RS and Capsaicin RS

Constituents:

6-Shogaol, 6-gingerol, zingiberol, - phellandrene, -zingiberene, arcurcumene, -bisabolene. Gingerenones A, B & C, isogingerenone B, hexahydrocurcumin, diaryl heptanoids, gingerdiols, 6-gingesulfonic acid, gingerglycolipids A, B & C, gingerenones, (+)-angelicoidenol-2-O--D-glucopyranoside, geraniol glycosides, -santalol, -eudesmol, nerolidol, farnesol, elemol, neral, geranial, - and -pinene, camphene, sabinene, myrcene, limonene, 1,8-cineole, aliphatic alkanes, alcohols, aldehydes, ketones, sulphides

áu¸¶h¢

Fig. 3: HPLC chromatogram of áu¸¶h¢ with Capsaicin as RS

Properties and Action: Rasa: Ka¶u, Tikta;

Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and identify the peaks using the relative retention times, as below. Analyte 6-Gingerol Capsaicin 8- Gingerol 10-Gingerol 6-Shogaol

Gu¸a: Laghu, Snigdha; V¢rya: UÀ¸a; Vip¡ka: Madhura; Karma: Anulomana, D¢pana, ËmadoÀaghna, H¤dya, Kaphaghna, P¡cana, Rucya, V¡takaphahara, V¤Àya

Relative retention time 0.8 1.0 1.6 4.0 2.04

Important formulations: Saubh¡gya va¶¢, Saubh¡gya¿u¸¶h¢, Trika¶u, Vai¿v¡nara C£r¸a

Therapeutic uses: Agnim¡ndya (digestive impairment), Ëdhm¡na (flatulence with gurguling sound), Ëmav¡ta (rheumatism), Gulma (abdominal lump), P¡¸·u (anaemia), P¢nasa (Chronic rhinitis), álipada (filariasis), áv¡sa (dyspnoea), Udararoga (disease of abdomen)

Sum the peak areas of 6-gingerol, 8-gingerol, 10gingerol and 6-shogaol in the substance being examined. Calculate the content of total gingerols from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Dose: C£r¸a (powder): 1-3 g

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

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áUÛÙHÌ HYDRO-ALCOHOLIC EXTRACT heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows bands corresponding to that of the standard solution (Fig. 1).

áu¸¶h¢ Hydro-alcoholic Extract is a dried and powdered extract prepared from áu¸¶h¢ (appropriately powdered). The extract contains not less than 1.5 per cent of total gingerols (Sum of 6-gingerol, 8-gingerol, 10-gingerol and 6shogaol) when assayed.

254 nm

Method of preparation: Take áu¸¶h¢ suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under a reflux at a temperature between 80850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 7 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 11 per cent.

Rf 1.0

     (2)

(2)

     (1)

(1)

0.5

0.0 RS T               RS T Fig. 1: Thin-Layer Chromatogram of áu¸¶h¢ hydro-alcoholic extract RS: (1) 6-Gingerol and (2) 6-Shogaol, T: Test solution

Identity, Purity and Strength:

Quantitative parameters:

Thin-layer chromatography:

Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 12.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.0-7.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-I)

Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using 6-gingerol and 6-shogaol as reference standards. Test solution: Extract 1 g of substance by refluxing with 50 ml of methanol for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 5 mg each of 6gingerol RS and 6-shogaol RS in about 25 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobilephase: ethyl acetate : toluene : diethyl ether (3.0 : 6.0 :1.0). Dry the plate in air and examine under UV 254 nm. Spray the plate with anisaldehyde- sulphuric acid reagent and

Other requirements: Heavy metals:  Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4) 88

 

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API, Part-I, Vol.-IX (Extracts); Monographs 

Analyte 6-Gingerol Capsaicin 8- Gingerol 10-Gingerol 6-Shogaol

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (25 ml x 3) on water bath for 15 min each, cool and filter. Combine all the filtrates and transfer to a 100-ml volumetric flask and make up the volume. Filter through 0.42 m membrane.

Relative retention time 0.8 1.0 1.6 4.0 2.04

Sum the peak areas of 6-gingerol, 8-gingerol, 10gingerol and 6-shogaol in the substance being examined. Calculate the content of total gingerols from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. áu¸¶h¢ Hydro-alcoholic extract

Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article. API reference standards:

Fig. 2: HPLC chromatogram of áu¸¶h¢ Hydro-alcoholic extract with Capsaicin as RS

API 6-Gingerol RS, 6-Shogaol RS and Capsaicin RS

Standard solution: Take about 5 mg, accurately weighed, capsaicin RS in 25-ml volumetric flask and dissolve in about 10 ml of methanol and make up the volume. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 55 volumes of acetonitrile, 44 volumes of 0.1 per cent orthophosphoric acid and 1 volume of methanol. Injection volume: 20 l. Flow rate: 1 ml per min. Detection: UV 282 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and identify the peaks using the relative retention times, as below.

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áUÛÙHÌ WATER EXTRACT áu¸¶h¢ Water Extract is a dried and powdered extract prepared from áu¸¶h¢.  The extract contains not less than 0.2 per cent of total gingerols (Sum of 6-gingerol, 8-gingerol, 10gingerol and 6-shogaol) when assayed. 

Method of preparation: Take áu¸¶h¢ suitably sized (powder or pieces) in an extractor. Add water, about 3 times the quantity of raw material and heat at a temperature between 80850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 7 per cent. Mill the mass and sieve the powder through 500 m mesh and pack. The yield obtained is about 16 per cent.

254 nm

Rf 1.0

  (1)

(1)

  (2)

(2)

0.5

0.0 RS

T

RS

T

Fig. 1: Thin-Layer Chromatogram of áu¸¶h¢   water extract RS: (1) 6-Gingerol and (2) 6-Shogaol, T: Test solution 

Quantitative parameters: Loss on drying: not more than 7.0 per cent (Appendix 2.1.4); Total ash: not more than 12.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 2.0 per cent (Appendix 2.1.7); pH: 4.0-7.0 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-II)

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using 6gingerol and 6-shogaol as reference standards. Test solution: Extract 1 g of substance by refluxing with 50 ml of methanol for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 5 mg each of 6-gingerol RS and 6-shogaol RS in about 25 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : toluene : diethyl ether (3.0 : 6.0 :1.0). Dry the plate in air and examine under UV 254 nm. Spray the plate with anisaldehyde- sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of the test solution shows bands corresponding to that of the standard solution (Fig. 1).

Other requirements:  Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 2 g, accurately weighed, of the substance being examined and reflux with methanol (25 ml x 3) on water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and transfer 90

 

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to a 100-ml volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, capsaicin RS in 25-ml volumetric flask and dissolve in about 10 ml of methanol and make up the volume. Filter through 0.42 m membrane. Chromatographic system: High performance liquid chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 55 volumes of acetonitrile, 44 volumes of 0.1 per cent orthophosphoric acid and 1 volume of methanol. Injection volume: 20 l. Flow rate: 1 ml per minu. Detection: UV 282 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and identify the peaks using the relative retention times, as below. Analyte 6-Gingerol Capsaicin 8- Gingerol 10-Gingerol 6-Shogaol

áu¸¶h¢ water extract

Fig. 2: HPLC chromatogram of áu¸¶h¢ water extract with Capsaicin as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents.

Relative retention time 0.8 1.0 1.6 4.0 2.04

Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article. API reference standards: API 6-Gingerol RS, 6-Shogaol RS and Capsaicin RS

Sum the peak areas of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in the substance being examined. Calculate the content of total gingerols from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

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SVARÛAPATRÌ Svar¸apatr¢ consists of dried leaflets of Cassia senna L. syn. C. angustifolia L., (Fam. Caesalpiniaceae), a small shrub, 60-75 cm high, found throughout the year, cultivated largely in southern India, especially in districts of Tinnevelly, Madurai, Ramanathapuram and collected and dried in shade for 7-10 days, till they attain yellowish-green colour, graded and then packed into large bales. It contains not less than 0.2 per cent of sennoside A and 0.1 per cent of sennoside B when assayed.

layers with spongy layer in between, palisade cells of upper surface longer than those of lower surface the latter having wavy anticlinal walls, prismatic crystals of calcium oxalate present on larger veins and clusters of calcium oxalate crystals distributed throughout the palisade and spongy tissues, midrib biconvex, bundles of midrib and larger veins, incompletely surrounded by a zone of pericyclic fibres and a crystal sheath of parenchymatous cells containing prismatic crystals of calcium oxalate

Synonyms: M¡rka¸·ik¡

c) Powder:

Other/Regional Language Names: Assamese: Sonamukhi; Bengali: Svarnamukhi, Sonapata; English: Indian Senna, Tinnevelly Senna; Gujarati: Mindhiaval; Hindi: Sanaya; Kannada: Nelavarike, Sonamukhi; Kashmiri: Sna; Malayalam: Sunnamukhi, Connamukhi; Marathi: Sonamukhi; Oriya: Sunamukhi; Punjabi: Sannamakhi, Sanapati, Sarnapatta; Tamil: Nilavarai, Nelavakai; Telugu: Sunamukhi; Urdu: Sena, Barg-e-Sana

Powder shows covering type of trichomes which are short, thick, unicellular, warty and frequently curved near the base. Rubiaceous or paracytic type of stomata are characteristic. Calcium oxalate cluster crystals occur in the cells of the mesophyll and as prisms in a sheath of cells around the fibres and as well freely distributed in powder (Fig. 1.).

Description: a) Macroscopic: Leaflets 2.5-6 cm long and 7-15 mm wide at centre, pale yellowish-green, elongated lanceolate, slightly asymmetric at base, margins entire, flat, apex acute with a sharp spine; both surfaces smooth with sparse trichomes, odour, faint but distinct, taste mucilagenous and disagreeable b) Microscopic: Transverse section of a leaflet through midrib shows an isobilateral structure, epidermal cells on both surfaces straight walled, containing mucilage; both surfaces bear scattered, unicellular hair, often conical, curved near base, thick-walled, non-lignified, warty cuticle, stomata paracytic, numerous on both surfaces, mesophyll consists of upper and lower palisade

Fig. 1: Powdered drug of SVARÛAPATRÌ (Cassia senna L.)

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Identity, Purity and Strength: Identification:

than 14.0 per cent (Appendix 2.1.5); Acidinsoluble ash: not more than 2.0 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 3.0 per cent (Appendix 2.1.8); Watersoluble extractive: not less than 25.0 per cent (Appendix 2.1.9)

Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using sennoside A and sennoside B as reference standards. Test solution: Extract 0.2 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 5 mg each of sennoside A RS and sennoside B RS in about 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase ethyl acetate : methanol : water (7.7 : 1.7 : 1.0). Dry the plate in air and examine under UV 366 nm. The chromatographic profile of the test solution shows bands corresponding to that of the standard solution (Fig. 2).

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4) 

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.4 g, accurately weighed, of the substance being examined and reflux with mixture of 0.05 M potassium dihydrogen orthophosphate and water (5 ml : 10 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates and transfer to a 50-ml volumetric flask, make up the volume with water. Filter through 0.42 m membrane. Standard solution: Take about 4 mg,   accurately weighed, each of sennoside A RS and sennoside B RS in a 25-ml volumetric flask, dissolve in about 5 ml of 0.05 M Potassium dihydrogen orthophosphate and 10 ml of water and make up the volume with water. Filter through 0.42 m membrane. Chromatographic system: High performance liquid Chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 83 volumes of 1 per cent acetic acid in water and 17 volumes of acetonitrile. Injection volume: 20 l. Flow rate: 2 ml per min. Detection: UV 350 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peaks. Calculate the content of sennoside A and sennoside B in the substance being examined from the peak response

366 nm  Rf

1.0

(1)

0.5

(2) 0.0 RS (1) RS (2)

T

Fig. 2: Thin-Layer Chromatogram of Svar¸apatr¢ RS: (1) Sennoside A and (2) Sennoside B; T: Test solution  Quantitative parameters: Foreign matter:  not more than 1.0 per cent (Appendix 2.1.3); Loss on drying: not more than 8.0 per cent (Appendix 2.1.4); Total ash: not more 93  

 

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API reference standards:

of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

API Sennoside A RS and Sennoside B RS

Constituents:

Bioactive anthraquinone glycosides viz., sennoside A, B, C and D; total anthraquinone derivatives occur in the range of 2 to 3 per cent. Palmidin A, aloe-emodin dianthrone-diglycoside, rhein-anthrone-8glycoside, rhein-8-diglucoside, aloe-emodin-8glucoside, aloe-emodin-anthrone-diglucoside, a primary glycoside having greater potency than sennosides A and B, naphthalene glycoside tinnevellin glucoside, sennoside G; kaempferol, its glucoside kaempferin and isorhamnetin, water-soluble polysaccharides

Svar¸apatr¢

Fig. 3: HPLC chromatograms of Svar¸apatr¢ with Sennoside A and Sennoside B as RS

Properties and Action: Rasa: Ka¶u, Tikta,

 

KaÀ¡ya; Gu¸a: Laghu, R£kÀa, T¢kÀ¸a; V¢rya: UÀ¸a; Vip¡ka: Ka¶u; Karma: Recana, KuÀ¶haghna

Additional requirements:

Important formulations: Paμcasak¡ra

Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents.

c£r¸a, S¡riv¡dy¡sava

Therapeutic uses: Gulma (abdominal lump), Udararoga (disease of abdomen), Vibandha (constipation)

Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

Dose: C£r¸a (powder): 0.5 to 2 g

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SVARÛAPATRÌ HYDRO-ALCOHOLIC EXTRACT 366 nm

Svar¸apatr¢ Hydro-Alcoholic Extract is a dried and powdered extract prepared from Svar¸apatr¢ (appropriately powdered). The extract contains not less than 2.0 per cent of sum of sennoside A and sennoside B when assayed.

   Rf 1.0

Method of Preparation: Take Svar¸apatr¢ suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under a reflux at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 60-650 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 21 per cent.

(1)

(2) 0.0 RS (1) RS (2) T Fig. 1: Thin-Layer Chromatogram of Svar¸apatr¢ hydro-alcoholic extract RS: (1) Sennoside A and (2) Sennoside B T: Test solution

Quantitative parameters:

Identity, Purity and Strength:

Loss on drying: not more than 5.0 per cent (Appendix 2.1.4); Total ash: not more than 13.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.0 per cent (Appendix 2.1.7); pH: 3.5-5.5 (Appendix 2.1.10); Total soluble solids: not less than 80.0 per cent (Appendix 2.1.11) (Method-I)

Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using sennoside A and sennoside B as reference standards. Test solution: Extract 0.2 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 5 mg each of sennoside A RS and sennoside B RS in about 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : methanol : water (7.7 : 1.7 : 1.0). Dry the plate in air and examine under UV 366 nm. The chromatographic profile of the test solution shows bands corresponding to that of the standard solution (Fig. 1).

Other requirements: Heavy metals:  Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.4 g, 95

 

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accurately weighed, of the substance being examined and reflux with mixture of 0.05 M potassium dihydrogen orthophosphate and water (5 ml : 10 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates and transfer to a 50-ml volumetric flask, make up the volume with water. Filter through 0.42 m membrane. Standard solution: Take about 4 mg, accurately weighed, each of sennoside A RS and sennoside B RS in a 25-ml volumetric flask, dissolve in about 5 ml of 0.05 M Potassium dihydrogen orthophosphate and 10 ml of water and make up the volume with water. Filter through 0.42 m membrane. Chromatographic system: High performance liquid Chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 83 volumes of 1 per cent acetic acid in water and 17 volumes of acetonitrile. Injection volume: 20 l. Flow rate: 2 ml per min. Detection: UV 350 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peaks. Calculate the content of sennoside A and sennoside B in the substance being examined from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Svar¸apatr¢ hydro-alcoholic extract

Fig. 2: HPLC chromatograms of Svar¸apatr¢ hydro-alcoholic extract with Sennoside A and Sennoside B as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standards: API Sennoside A RS and Sennoside B RS

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SVARÛAPATRÌ WATER EXTRACT 366 nm

Svar¸apatr¢ water Extract is a dried and powdered extract prepared from Svar¸apatr¢. The extract contains not less than 1.0 per cent of sum of sennoside A and sennoside B when assayed.

 Rf

1.0

Method of Preparation:

(1)

Take Svar¸apatr¢ suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under a reflux at a temperature between 80-850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 60-650 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 25 per cent.

0.5

(2) 0.0 RS (1) RS (2) T Fig. 1: Thin-Layer Chromatogram of Svar¸apatr¢ water extract RS: (1) Sennoside A and (2) Sennoside B T: Test solution 

Identity, Purity and Strength:

Quantative parameters:

Thin-layer chromatography:

Loss on drying: not more than 5.0 per cent (Appendix 2.1.4); Total ash: not more than 10.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.0 per cent (Appendix 2.1.7); pH: 3.5-5.5 (Appendix 2.1.10); Total soluble solids: not less than 80.0 per cent (Appendix 2.1.11) (Method-II)

Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using sennoside A and sennoside B as reference standards. Test solution: Extract 0.2 g of substance by refluxing with 50 ml of methanol for a period of 10-15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 5 mg each of sennoside A RS and sennoside B RS in about 10 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: ethyl acetate : methanol : water (7.7 : 1.7 : 1.0). Dry the plate in air and examine under UV 366 nm. The chromatographic profile of the test solution shows bands corresponding to that of the standard solution (Fig. 1).

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.4 g, accurately weighed, of the substance being examined and reflux with mixture of 0.05 M 97

 

 

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potassium dihydrogen orthophosphate and water (5 ml : 10 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates and transfer to a 50-ml volumetric flask, make up the volume with water. Filter through 0.42 m membrane. Standard solution: Take about 4 mg, accurately weighed, each of sennoside A RS and sennoside B RS in a 25-ml volumetric flask, dissolve in about 5 ml of 0.05 M potassium dihydrogen orthophosphate and 10 ml of water and make up the volume with water. Filter through 0.42 m membrane. Chromatographic system: High performance liquid Chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm, 5 m). Mobile phase: Filtered and degassed mixture of 83 volumes of 1 per cent acetic acid in water and 17 volumes of acetonitrile. Injection volume: 20 l. Flow rate: 2 ml per min. Detection: UV 350 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peaks. Calculate the content of sennoside A and sennoside B in the substance being examined from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Svar¸apatr¢ water extract

Fig. 2: HPLC chromatograms of Svar¸apatr¢ water extract with Sennoside A and Sennoside B as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standards: API Sennoside A RS and Sennoside B RS

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TULASÌ Lamina - epidermis and trichomes similar to those of petiole on both surfaces; stomata anomocytic and diacytic present on both surfaces and slightly raised above the level of epidermis; palisade single layered followed by 4-6 layers of closely packed spongy parenchyma with chloroplasts and oleo-resin; stomatal index 10-13-15 on upper surface and 14-1516 on lower surface; palisade ratio 3.8; vein islet number 31-33 c) Powder:

Tulas¢ consists of dried leaf of Ocimum tenuiflorum L. syn. O. sanctum L. (Fam. Lamiaceae), an erect, 30-60 cm high, much branched annual herb, found throughout the country and cultivated in sacred groves. It contains not less than 0.4 per cent of sum of oleanolic acid and ursolic acid when assayed. Synonyms: Suras¡, Surasa Other/Regional Language Names: Assamese: Tulasi; Bengali: Tulasi; English: Sacred Basil, Holy Basil; Gujarati: Tulasi, Tulsi; Hindi: Tulasi; Kannada: Tulasi; Malayalam: Tulasi; Marathi: Tulas; Punjabi: Tulasi; Tamil: Thulasi, Tulasi; Telugu: Tulasi; Urdu: Raihan, Tulsi a) Macroscopic: Leaves 2.5-5 cm long, 1.6-3.2 cm wide, ellipticoblong, obtuse or acute, entire or serrate, pubescent on both surfaces, petiolate, thin, petiole 1.5-3 cm long, hairy; odour aromatic; taste, characteristic

Fragment of lower Fragments of simple epidermis with trichomes glandular trichome and spongy mesophyll cells  

b) Microscopic: Leaf- Petiole - shows cordate outline, consisting of single layered epidermis composed of thin walled, oval cells having a number of covering and glandular trichomes; covering trichomes multicellular, uniseriate 1-8 celled long, rarely slightly reflexed at tip; glandular trichomes short, sessile or with 1-2 celled stalk, and 2-8 celled, balloon-shaped head, enclosed in a cuticular bladder, measuring 22-27  in dia., upper epidermis, followed by 3-4 layers of collenchymatous and 1-2 layers of parenchymatous cells; lower epidermis followed by 1-3 layers of collenchymatous and 2-3 layers of parenchymatous cells; three vascular bundles situated centrally, middle one larger than the other two, consisting of xylem and phloem

Glandular trichomes with stalk 

Surface view of epidermis with trichomes from the midrib portion

0.01mm

Epidermis in surface view

Midrib - epidermis, trichomes and vascular bundles similar to those of petiole, except reduced in cortical layers towards apical region of midrib

Epidermis in surface view with diacytic stomata 

Upper epidermis in surface view with glandular trichomes

0.01mm

Fig. 1: Powdered drug of Tulas¢ (Ocimum tenuiflorum L.) 99

 

Glandular trichomes in surface view

Fragment of lamina showing upper epidermis with glandular trichome, palisade and spongy mesophyll cells

Description:-

 

API, Part-I, Vol.-IX (Extracts); Monographs 

Light-green; shows fragments of polygonal, wavy to sinuous walled upper and lower epidermal cells in surface view, covering and glandular trichomes as a whole or in pieces, palisade and spongy parenchyma, anomocytic and diacytic stomata (Fig 1.)

spots/bands appear without charring. The chromatographic profile of the test solution shows a band corresponding to that of the standard solution (Fig. 2). Quantitative parameters: Foreign matter: not more than 2.0 per cent (Appendix 2.1.3); Loss on drying: not more than 12.0 per cent (Appendix 2.1.4); Total ash: not more than 19.0 per cent (Appendix 2.1.5); Acidinsoluble ash: not more than 3.0 per cent (Appendix 2.1.7); Alcohol-soluble extractive: not less than 6.0 per cent (Appendix 2.1.8); Watersoluble extractive: not less than 13.0 per cent (Appendix 2.1.9).

Identity, Purity and Strength: Identification: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using ursolic acid as a reference standard. Test solution: Extract 1 g of substance by refluxing with 50 ml of methanol for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 5 mg of ursolic acid RS in about 25 ml of methanol.

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Visible after derivatisation Rf 1.0

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.4 g, accurately weighed, of the substance being examined and reflux with methanol (10 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 25-ml volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, each of oleanolic acid RS and ursolic acid RS in a 25-ml volumetric flask and dissolve in about 15 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid Chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm). Mobile phase: Filtered and degassed mixture of 1 volume of water and 9 volumes of methanol. Injection volume: 20 l. Flow rate: 1 ml per min. Detection: UV 210 nm. Procedure: Inject 20 l of

0.5

0.0 RS T Fig. 2: Thin-Layer Chromatogram of Tulas¢ RS: Ursolic acid, T: Test solution Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: toluene : ethyl acetate : acetic acid (8.0 : 2.0 : 0.1). Dry the plate in air. Spray the plate with anisaldehyde - sulphuric acid reagent and heat at 1050 till the colour of the 100  

 

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the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peaks. Calculate the content of oleanolic acid and ursolic acid in the substance being examined from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Constituents: Essential oil containing eugenol, -caryophyllene; apigenin, apigenin-7-Oglucuronide, vicenin, vicenin-2, luteolin, luteolin7-O-glucuronide, galuteolin, orientin, molludistin, cirsilineol; gallic acid and its methyl and ethyl esters; ursolic acid. -carotene; rosmarinic, protocatechuic, vanillic, 4hydroxybenzoic, caffeic, chlorogenic acids; fatty acids; vanillin, 4-hydroxybenzaldehyde, cholesterol, campesterol, stigmasterol, sitosterol

Properties and Action: Rasa: Ka¶u, Tikta; Gu¸a: Laghu, R£kÀa, T¢kÀ¸a; V¢rya: UÀ¸a; Vip¡ka: Ka¶u; Karma: D¢pana, H¤dya, Jvaraghna, K¡sahara, K¤mighna, Kaphahara, Ras¡yana, áv¡sahara, V¡tahara

Tulas¢

Important formulations: M¡nasamitra va¶aka, Mah¡jvar¡´ku¿a rasa, Mukt¡paμch¡m¤ta rasa, Tribhuvanak¢rti rasa

Therapeutic uses:

Fig. 3: HPLC chromatograms of Tulas¢ with Oleanolic acid and Ursolic acid as RS

Apasm¡ra (epilepsy), Aruci (tastelessness), Gulma (abdominal lump), KÀata (wound), Hikk¡ (hiccup), K¡sa (cough), K¤miroga (helminthiasis / worm infestation), KÀaya (pthisis), KuÀ¶ha (disease of skin), P¡r¿va¿£la (intercostal neuralgia and pleurodynia), Pl¢h¡ (splenic disease), Prati¿y¡ya (coryza), áv¡sa (dyspnoea), áoÀa (cachexia)

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

Dose:

API reference standards:

C£r¸a (powder): 1-3 g

API Oleanolic acid RS and Ursolic acid RS

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TULASÌ HYDRO-ALCOHOLIC EXTRACT Tulas¢ Hydro-Alcoholic Extract is a dried and powdered extract prepared from Tulas¢ (appropriately powdered). The extract contains not less than 0.2 per cent of sum of oleanolic acid and ursolic acid when assayed.

the test solution shows a band corresponding to that of the standard solution (Fig. 1). Visible after derivatisation 1.0 Rf

Method of Preparation: Take Tulas¢ suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under a reflux at a temperature between 80850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 12 per cent.

0.5

0.0 RS T Fig. 1: Thin-Layer Chromatogram of Tulas¢ hydro-alcoholic extract RS: Ursolic acid, T: Test solution

Quantative parameters: Loss on drying: not more than 5.0 per cent (Appendix 2.1.4); Total ash: not more than 12.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.5 per cent (Appendix 2.1.7); pH: 3.5-5.5 (Appendix 2.1.10); Total soluble solids: Not less than 90.0 per cent (Appendix 2.1.11) (Method-I)

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using ursolic acid as a reference standard. Test solution: Extract 1 g of substance by refluxing with 50 ml of methanol for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 5 mg of ursolic acid RS in about 25 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: toluene : ethyl acetate : acetic acid (8.0 : 2.0 : 0.1). Dry the plate in air. Spray the plate with anisaldehyde - sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Residual solvent: Complies with the prescribed limits, (Appendix 3.8); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.4 g, 102

 

 

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accurately weighed, of the substance being examined and reflux with methanol (10 ml x 3) on a water bath for 15 min each, cool and filter. Combine all the filtrates, concentrate and transfer to a 25-ml volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, each of oleanolic acid RS and ursolic acid RS in a 25-ml volumetric flask and dissolve in about 15 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid Chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm). Mobile phase: Filtered and degassed mixture of 1 volume of water and 9 volumes of methanol. Injection volume: 20 l. Flow rate: 1 ml per min. Detection: UV 210 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peaks. Calculate the content of oleanolic acid and ursolic acid in the substance being examined from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Tulas¢ hydro-alcoholic extract

Fig. 2: HPLC chromatograms of Tulas¢ hydro-alcoholic extract with Oleanolic acid and Ursolic acid as RS

Additional requirements: Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standards: API Oleanolic acid RS and Ursolic acid RS

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TULASÌ HYDRO-ALCOHOLIC EXTRACT the test solution shows a band corresponding to that of the standard solution (Fig. 1).

Tulas¢ Water Extract is a dried and powdered extract prepared from Tulas¢. The extract contains not less than 0.02 per cent of sum of oleanolic acid and ursolic acid when assayed.

Visible after derivatisation

Method of Preparation:

1.0 R f

Take Tulas¢ suitably sized (powder or pieces) in an extractor. Add 50 per cent aqueous alcohol, about 3 times the quantity of raw material and heat under a reflux at a temperature between 80850 for 3-4 hours. Filter the extract through a filter (preferably 10 m pore size) to a suitable sized vessel. The marc is extracted three times more, filtering the extract each time into the same vessel. Concentrate the combined filtrate to a syrupy consistency and dry under vacuum (between 400-600 mm of Hg) at a temperature not exceeding 800 till the moisture is below 5 per cent. Mill the mass and sieve the powder through 500 m mesh to obtain the extract and pack. The yield obtained is about 12 per cent.

0.5

0.0 RS T Fig. 1: Thin-Layer Chromatogram of water extract RS: Ursolic acid, T: Test solution

Quantitative parameters: Loss on drying: not more than 5.0 per cent (Appendix 2.1.4); Total ash: not more than 10.0 per cent (Appendix 2.1.5); Acid-insoluble ash: not more than 1.0 per cent (Appendix 2.1.7); pH: 3.5-5.5 (Appendix 2.1.10); Total soluble solids: not less than 90.0 per cent (Appendix 2.1.11) (Method-II)

Identity, Purity and Strength: Thin-layer chromatography: Carry out thin-layer chromatography on a precoated silica gel 60F254 plate (Appendix 3.5) using ursolic acid as a reference standard. Test solution: Extract 1 g of substance by refluxing with 50 ml of methanol for a period of 15 min. Filter and concentrate the extract to 25 ml. Standard solution: Dissolve 5 mg of ursolic acid RS in about 25 ml of methanol. Procedure: Apply 10 l each of the test and standard solutions as bands at a height of 10 mm from the base of a 10 x 5 cm TLC plate and develop up to 8 cm from the base of the plate using the mobile phase: toluene : ethyl acetate : acetic acid (8.0 : 2.0 : 0.1). Dry the plate in air. Spray the plate with anisaldehyde- sulphuric acid reagent and heat at 1050 till the colour of the spots/bands appear without charring. The chromatographic profile of

Other requirements: Heavy metals: Complies with the prescribed limits, (Appendix 3.1); Microbial contamination: Complies with the prescribed limits, (Appendix 3.2); Pesticide residues: Complies with the prescribed limits, (Appendix 3.3); Aflatoxins: Complies with the prescribed limits, (Appendix 3.4)

Assay: Carry out the assay by liquid chromatography (Appendix 3.6). Test solution: Take about 0.4 g, accurately weighed, of the substance being examined and reflux with methanol (10 ml x 3) on a water bath for 15 min each, cool and filter. 104

 

 

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Additional requirements:

Combine all the filtrates, concentrate and transfer to a 25-ml volumetric flask and make up the volume. Filter through 0.42 m membrane. Standard solution: Take about 5 mg, accurately weighed, each of oleanolic acid RS and ursolic acid RS in a 25-ml volumetric flask and dissolve in about 15 ml of methanol and make up the volume with methanol. Filter through 0.42 m membrane. Chromatographic system: High performance liquid Chromatography. Column and stationary phase: C18 (250 mm x 4.6 mm). Mobile phase: Filtered and degassed mixture of 1 volume of water and 9 volumes of methanol. Injection volume: 20 l. Flow rate: 1 ml per min. Detection: UV 210 nm. Procedure: Inject 20 l of the standard solution and record the chromatogram. Inject 20 l of the test solution, record the chromatogram and measure the response for the analyte peaks. Calculate the content of oleanolic acid and ursolic acid in the substance being examined from the peak response of analytes. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent.

Storage: Store in well closed container protected from heat, light, moisture and against attack by insects and rodents. Labelling: The label states the official name, followed by the Latin binominal name and the part of the plant contained in the article.

API reference standards: API Oleanolic acid RS and Ursolic acid RS 

Tulas¢ water extract

Fig. 2: HPLC chromatograms of Tulas¢ water extract with Oleanolic acid and Ursolic acid as RS

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APPENDIX - 1   

1.1. APPARATUS FOR TESTS AND ASSAYS

125 106 90 75 63 53 45

1.1.1 Nessler Cylinders Nessler cylinders which are used for comparative tests are matched tubes of clear colourless glass with a uniform internal diameter and flat, transparent base. These comply with Indian Standard 41611967 and are of transparent glass with a nominal capacity of 50 ml. The overall height is about 150 mm, the external height to the 50 ml mark 110 to 124 mm, the thickness of the wall 1.0 to 1.5 mm and the thickness of the base 1.5 to 3.0 mm. The external height to the 50 ml mark of the cylinder used for a test must not vary by more than 1 mm.

120 150 170 200 240 300 350

Designation Test sieves of metal wire cloth are designated by the nominal size of aperture of the wire cloth, followed by the inscription ‘IS Sieve’. Examples: a. 5.60 mm IS Sieve b. 425 m IS Sieve

1.1.2. Sieves Sieves for pharmacopoeial testing are constructed from wire cloth with square meshes, woven from wire of brass, bronze, stainless steel or any other suitable material. The wires should be of uniform circular cross-section and should not be coated or plated. There must be no reaction between the material of the sieve and the substance being sifted.

Nominal aperture sizes of 1 mm and above, as well as their associated tolerances and wire diameters, are expressed in millimeters (mm) and for aperture sizes smaller than 1 mm, these are expressed in micrometers (m).

Sieves conform to the following specifications ‐

Unless otherwise specified, thermometers suitable for pharmacopoeial tests conform to Indian Standard 4825-1968 and are standardised in accordance with the ‘Indian Standard Method of Calibrating Liquidin-Glass Thermometers’, 6274-1971.

IS 460 (Pt I) 1985 (Reaffirmed 1998) mm 4.0 2.8 2.0 1.7 1.4 1.0 m 710 600 500 425 355 250 180 150

1.1.3. Thermometers

IS 460-1978 -4 6 8 10 12 16 -22 25 30 36 44 60 85 100

The thermometers are of the mercury-in-glass type and are filled with a dried inert gas, preferably nitrogen. They may be standardised for total immersion or for partial immersion. Each thermometer should be employed according to the condition of immersion under which it was standardised. In the selection of the thermometer it is essential to consider the conditions under which it is to be used. 1.1.4 Ultraviolet Lamp (For general purposes and for chromatography work) An instrument consisting of mercury vapour lamp and a filter which gives an emission band with maximum intensity at about 254 nm (near UV

 

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1.1.6. Weights and Balances

rays) and 366 nm (far UV rays) is used. To ensure that the required emission is being given by the lamp, carry out the following test periodically. Apply to a plate coated with silica gel G, 5 l of a 0.04 per cent w/v solution of sodium salicylate in ethanol (95 per cent) for lamps of maximum output at 254 nm and 5 l of a 0.2 per cent w/v solution in ethanol (95 per cent) for lamps of maximum output at 365 nm. Examine the spot in a position normal to the radiation. The distance between the lamp and the plate under examination used in a pharmacopoeial test should not exceed the distance used to carry out the above test.

Pharmacopoeial tests and assays require the use of analytical balances that vary in capacity, sensitivity and reproducibility. The accuracy needed for a weighing should dictate the type of balance. Where substances are to be “accurately weighed”, the weighing is to be performed so as to limit the error to not more than 0.1 per cent. For example, a quantity of 50 mg is to be weighed to the nearest 0.05 mg; a quantity of 0.1 g is to be weighed to the nearest 0.1 mg; and quantity of 10 g is to be weighed to the nearest 10 mg. A balance should be chosen such that the value of three times the standard deviation of the reproducibility of the balance, divided by the amount to be weighed, does not exceed 0.001.

1.1.5. Volumetric Glassware Volumetric apparatus is normally calibrated at 27˚. However, the temperature generally specified for measurements of volume in the analytical operations of the pharmacopoeia, unless otherwise stated, is 25˚. The discrepancy is inconsequential as long as the room temperature in the laboratory is reasonably constant and is around 27˚. Pharmacopoeial assays involving volumetric measurements require the use of accurately calibrated glassware. Volumetric apparatus must be suitably designed to assure accuracy. The design, construction and capacity of volumetric glassware should be in accordance with those laid down by the Bureau of Indian Standards. The tolerances on capacity for volumetric flasks, pipettes and burettes, as laid down in the relevant Indian Standards, are permissible.

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APPENDIX - 2  2.1. TESTS AND DETERMINATIONS

Chlorinated Soda Solution (Bleaching Solution): Dissolve 75 g of sodium carbonate in 125 ml of distilled water; triturate 50 g of chlorinated lime (bleaching powder) in a mortar with 75 ml of distilled water, adding it little by little. Mix the two liquids and shake occasionally for three or four hours. Filter and store, protected from light. Used for lightening highly coloured material, by warming in it and washing the tissues thoroughly.

2.1.1 Microscopic Identification of Botanical Substances: Microscopic identification of the botanical ingredients is a standard for statutory purposes in several monographs. Appropriate processing for separation and isolation with suitable clearing reagents and stains, and finally mounting a little part on a slide that helps to show the unit structures is to be followed. Identification of the discrete, but disoriented units will not be possible without proper isolation and should not be attempted.

Breamer’s Reagent: Dissolve 1 g of sodium tungstate and 2 g of sodium acetate in sufficient quantity of water to make 10 ml. Yellowish to brown precipitates; indicate the presence of tannins.

Monographs where the test is prescribed give both a relevant method of isolation and diagnostic features specific to the expected ingredient. Only a brief method and a few of the characteristics for each ingredient are given, but an analyst may use other methods of isolation and choose more characteristics to draw a correct conclusion.

Canada Balsam (as a Mountant): Heat Canada balsam on a water bath until volatile matter is removed and the residue sets to a hard mass on cooling. Dissolve residue in xylene to form a thin syrupy liquid. Used for making permanent mounts of reference slides of selected debris. Chloral Hydrate Solution: Dissolve 50 g of chloral hydrate in 20 ml of distilled water. A valuable clarifying agent for rendering tissues transparent and clear, by freeing them from most of the ergastic substances, but leaving calcium oxalate crystals unaffected.

1. Stains and Reagents for Microchemical Reactions: For the purpose of identification and characterization of materials expected to be included in the prescribed standards, the following stains and reagents are recommended for use wherever relevant, in addition to those mentioned in the monograph.

Chloral Iodine: Saturate chloral hydrate solution with iodine, leaving a few crystals undissolved; useful for detecting min grains of starch otherwise undetectable.

Acetic Acid: Dilute 6 ml of glacial acetic acid with 100 ml of distilled water; used for identification of cystoliths, which dissolve with effervescence.

Chlorziniciodine (Iodinated Zinc Chloride Solution): Dissolve 20 g of zinc chloride and 6.5 g of potassium iodide in 10 ml of distilled water. Add 0.5 g of iodine and shake for about fifteen min before filtering. Dilute, if needed, prior to use. Renders cellulosic walls bluish violet and lignified walls yellowish brown to brown.

Aniline Chloride Solution: Dissolve 2 g in a mixture of 65 ml of 30 per cent ethyl alcohol and 15 ml distilled water and add 2 ml of conc. hydrochloric acid. Lignified tissues are stained bright yellow. Bismarck Brown: Dissolve 1 g in 100 ml of 95 per cent of ethyl alcohol; used as a general stain for macerated material (with Schultze’s).

Chromic Acid Solution: 10 g of potassium chromate dissolved in 90 ml of dilute sulphuric acid. A macerating agent similar to Schultze’s.

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water, add 2 g of iodine to the solution and dissolved it; stains lignified walls yellow and cellulosic walls blue.

Corallin Soda: Dissolve 5 g of corallin in 100 ml of 90 per cent ethyl alcohol. Dissolve 25 g of sodium carbonate in 100 ml distilled water; keep the solutions separate and mix when required, by adding 1 ml of the corallin solution to 20 ml of the aqueous sodium carbonate solution. Prepare fresh each time, as the mixture will not keep for long. Used for staining sieve plates and callus bright pink and imparts a reddish tinge to starch grains and lignified tissues.

Lactophenol (Amman’s Fluid): Phenol 20 g, lactic acid 20 g, glycerin 40 g, dissolved in distilled water 20 ml; reveals starch grains in polarised light with a well-marked cross at hilum, and also min crystals of calcium oxalate as brightly polarising points of light. Methylene Blue: A solution of 0.1 g of methylene blue in 25 ml of ethyl alcohol (95 per cent). A general stain for nucleus and bacteria.

Cuoxam (Ammoniacal Solution of Copper Oxide): Triturate 0.5 g of copper carbonate in a mortar with 10 ml of distilled water and gradually add 10 ml of strong solution of ammonia (sp. gr. 0.880) with continued stirring; used for dissolving cellulosic materials.

Millon’s Reagent: Dissolve one volume of mercury in 9 volumes of fuming nitric acid (sp. gr. 1.52), keeping the mixture well cooled during reaction. Add equal volume distilled water when cool. Stains proteins red.

Eosin: 1 per cent solution in 90 per cent ethyl alcohol; stains cellulose and aleurone grains red.

Naphthol Solution: Dissolve 10 g of naphthol in 100 ml of ethyl alcohol; a specific stain for detection of inulin; cells containing inulin turn deep reddish violet.

Ferric Chloride Solution: A 5 per cent solution of ferric chloride in distilled water. Taninns containing tissues coloured bluish or greenish black. Glycerin: Pure or diluted as required with one or two volumes of distilled water. Used as a general mountant.

Phloroglucinol: 1g of phloroglucinol dissolved in 100 ml of 90 per cent ethyl alcohol; mount debris in a few drops, allow to react for a min, draw off excess of reagent with a filter paper strip and add a drop of conc. hydrochloric acid to the slide; lignified tissues acquire a deep purplish red colour; very effective on water washed material but not in chloral hydrate washed debris, for which alcoholic solution of safranin is more effective (See Safranin).

Haematoxylin, Delafield’s: Prepare a saturated solution of ammonia alum. To 100 ml of this add a solution of one g of haematoxylin in 6 ml of ethyl alcohol (97 per cent). Leave the mixed solution exposed to air and light in an unstopped bottle for three or four days. Filter and add to the filtrate 25 ml of glycerin and 25 ml of methyl alcohol. Allow the solution to stand exposed to light, till it acquires a dark colour (about two months). Refilter and store as a stock solution. Dilute it 3 or 4 times volumes with distilled water. Stains cellulosic fibers blue; used only on water washed material.

Picric Acid Solution (Trinitrophenol Solution): A saturated aqueous solution made by dissolving 1 g of picric acid in 95 ml of distilled water; stains animal and insect tissues, a light to deep yellow; in a solution with ethyl alcohol, aleurone grains and fungal hyphae are stained yellow.

Iodine Water: Mix one volume of decinormal iodine with 4 volumes of distilled water. Stains starch blue, and reveals crystalloids and globoids when present in aleurone grains.

Potash, Caustic: A 5 per cent aqueous solution; used to separate tenacious tissues of epidermis and also laticiferous elements and vittae, both of which are stained brown.

Iodine in Potassium Iodide Solution: Dissolve 1 g of potassium iodide in 200 ml of distilled 112  

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Foreign matter consists of any organism, part or product of an organism, other than that named in the definition of the product and mineral admixtures, such as soils, stones, sand and dust. It shall also include other than official parts of organism beyond their specified limits.

Ruthenium Red: Dissolve 0.008 g of ruthenium red in 10 ml of a 10 per cent solution of lead acetate; (to be freshly prepared) used for identification of most kinds of mucilage containing tissues, which turn pink. A 0.0008 g ruthenium red dissolved in 10 ml of distilled water and used immediately stains cuticular tissues in debris to a light pink.

Take 100 g of sample (unless otherwise specified) and spread in a thin layer on a suitable platform. Examine in daylight with unawed eye or using 6 x or 10 x magnifying glass and separate the foreign matter. Appropriate sieve can also be used to separate the foreign matter. Dust regarded as mineral admixture is separated by sifting the sample through a 250 m sieve. Weigh the sorted foreign matter and calculate the foreign matter content in per cent with reference to drug sample.

Safranin: A one per cent solution in 50 per cent ethyl alcohol; used to stain lignified cell walls deep red, even after clearing with chloral hydrate. Schultze’s Maceration Fluid: Add isolated debris to 50 per cent conc. nitric acid in a test tube and warm over water bath: add a few crystals of potassium chlorate while warming, till tissues soften; cool, wash with water thoroughly and tease out for mounting hard tissues; isolated cell structures are clearly revealed, but the structures are not useful for measurement of dimensions.

2.1.4. Determination of Moisture Content (Loss on Drying): Dry the evaporating dish for 30 min under the same conditions to be employed in the determination. Place about 5 to 10 g of powder/drug accurately weighed in a tared evaporating dish. For unpowdereded drug, prepare about 10 g of the sample by cutting, shredding so that the parts are about 3 mm in thickness. Seeds and fruits, smaller than 3 mm should be cracked. Avoid the use of high speed mills in preparing the samples, and exercise care that no appreciable amount of moisture is lost during preparation and that the portion taken is representative of the official sample. By gentle, sidewise shaking, distribute the test specimen as evenly as practicable to a depth of about 5 mm generally, and not more than 10 mm in the case of bulky materials. Place the loaded bottle in the drying chamber. Dry the test specimen at 1050 for 3 hours and weigh. Continue the drying and weighing at half an hour interval until difference between two successive weighing corresponds to, not more than 0.25 per cent.

Schweitzer’s Reagent: Same as Ammoniacal Copper Oxide Solution (Cuoxam). Sudan Red II I: Dissolve 0.01 g of sudan red III in 5 ml of ethyl alcohol (90 per cent) and 5 ml of pure glycerin; suberised walls of cork cells, and fatty material in cells are stained bright red. Sulphovanadic Acid (Mandelin’s Reagent): Triturate one g of ammonium vandate with 100 ml conc. sulphuric acid. Allow the deposit to subside and use the clear liquid. This is to be prepared fresh; useful for identification of alkaloids, particularly strychnine which turns violet in the cells containing it. 2.1.2. Net Content: The content of the final or retail pack shall be not less than 98 per cent of the declared net content. 2.1.3. Determination of Foreign Matter A. Foreign Matter The sample shall be free from visible signs of mould growth, sliminess, contamination by insects and other animal and animal products including animal excreta or any other noxious foreign matter.

2.1.5. Determination of Total Ash: Incinerate about 2 to 3 g, accurately weighed, of the ground drug in a tared platinum or silica dish at a 113

 

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temperature not exceeding 6000 until free from carbon, cool in a desiccator for 30 min and weigh without delay. If carbon free ash cannot be obtained in this way, exhaust the charred mass with hot water, collect the residue on an ashless filter paper, incinerate the residue and filter paper, add the filtrate, evaporate to dryness, and ignite at a temperature not exceeding 6000. Calculate the percentage of ash with reference to the air-dried drug.

Calculate the percentage of alcohol-soluble extractive with reference to the air-dried drug. 2.1.9. Determination Extractive:

of

Water-soluble

Proceed as directed for the determination of Alcohol-soluble extractive, using chloroform water (2.5 ml chloroform in purified water to produce 1000 ml) instead of ethanol.

2.1.6. Determination of Water-Soluble Ash:

2.1.10. Determination of pH Value:

Boil the ash obtained in (2.1.5) for 5 min with 25 ml of water; collect insoluble matter in a Gooch crucible, or on an ashless filter paper (Whatman 41), wash with hot water, and ignite for 15 min at a temperature not exceeding 4500. Substract the weight of the insoluble matter from the weight of the ash; the difference in weight represents the water-soluble ash. Calculate the percentage of water-soluble ash with reference to the air-dried drug.

The pH value of an aqueous liquid may be defined as the common logarithm of the reciprocal of the hydrogen ion concentration expressed in g per litre. For the purpose of pharmacopoeia pH is defined as the value given by a suitable, properly standardized, pH meter capable of reproducing pH values to 0.05 pH unit using an indicator electrode sensitive to hydrogen ion activity, the glass electrode and a suitable reference electrode. The instrument should be capable of sensing the potential across the electrode pair and for pH standardization purposes, applying an adjustable potential to the circuit by manipulation of “standardization,” “zero,” “asymmetry,” or “calibration” control, and should be able to control the change in millivolts per unit change in pH reading through a “temperature” and/or “slope” control. Measurements are made at 25 ± 20, unless otherwise specified.

2.1.7. Determination of Acid-insoluble Ash: To the crucible containing total ash, add dropwise 25 ml of dilute hydrochloric acid. Collect the insoluble matter on an ashless filter paper (Whatman 41) and wash with hot water until the filtrate is neutral. Transfer the filter paper containing the insoluble matter to the original crucible, dry on a hot plate and ignite to constant weight. Allow the residue to cool in a suitable desiccator for 30 min and weigh without delay. Calculate the content of acid-insoluble ash with reference to the air-dried drug. 2.1.8. Determination Extractive:

of

To standardize the pH meter, select two Buffer Solutions whose difference in pH does not exceed 4 units such that the expected pH of the material under test falls between them. Commercially available buffer solutions for pH meter standardization, having traceability to the National Standards can be used.

Alcohol-soluble

Macerate 5 g of the air dried drug, coarsely powdered, with 100 ml of alcohol of specified strength in a closed flask for 24 hours, shaking frequently during 6 hours and allowing to stand for 18 hours. Filter rapidly, taking precautions against loss of solvent, evaporate 25 ml of the filtrate to dryness in a tared flat bottomed shallow dish and dry at 1050, to constant weight and weigh.

Fill the cell with one of the Buffer Solutions for Standardization at the temperature at which the test material is to be measured. Set the “temperature” control at the temperature of the solution, and adjust the calibration control to make the observed pH value identical with that of the declared pH. Rinse the electrodes and cell 114

 

API, Part-I, Vol.-IX (Extracts); Appendices

with several portions of the second Buffer Solution for Standardization, then fill the cell with it, at the same temperature as the material to be measured. The pH of the second buffer solution is within ±0.07 pH unit of the declared value. If a larger deviation is noted, examine the electrodes and if they are faulty, replace them. Repeat the standardization until both Buffer Solutions for Standardization give observed pH values within 0.05 pH unit of the declared value without further adjustment of the controls.

Replace the aqueous ethanol with water and follow the procedure as given in Method- I. 2.1.12. Determination of Volatile Oil in Drugs The determination of volatile oil in a drug is made by distilling the drug with a mixture of water and glycerin, collecting the distillate in a graduated tube in which the aqueous portion of the distillate is automatically separated and returned to the distilling flask and measuring the volume of the oil. The content of the volatile oil is expressed as a percentage v/w.

When the system is functioning satisfactorily, rinse the electrodes and cell several times with a few portions of the test material, fill the cell with the test material, and read the pH value. Use carbon dioxidefree water for solution or dilution of test material in pH determinations. In all pH measurements, allow a sufficient time for stabilization.

The apparatus consists of the following parts (See Fig. 1). The apparatus described below is recommended but any similar apparatus may be used provided that it permits complete distillation of the volatile oil. All glass parts of the apparatus should be made of good quality resistance glass.

Unless otherwise specified in the monograph prepare 5 per cent w/v of the sample. Filter if it is not soluble completely and use the filtrate to measure the pH. 2.1.11. Determination of Total Soluble Solids: Method-I Take about one g, accurately weighed, of the substance being examined in a 100-ml volumetric flask, dissolve in 50 ml of 50 per cent v/v aqueous ethanol, sonicate for 10 min, heat on water bath (avoiding evaporation), cool and dilute to 100 ml with 50 per cent v/v aqueous ethanol. Mix and quickly pipette out 25 ml solution to a tared glass dish and evaporate. Centrifuge the remaining liquid for 10 min at 3000 rpm. Pipette out 25 ml of the supernatant obtained after centrifugation to a tared glass dish and evaporate. After evaporation of solvent, place the glass dishes in oven at 1050 to dry to a constant weight. The weight of residue obtained after centrifugation is not less than 90 per cent of the weight of the residue obtained before centrifugation.

Fig. 1: Apparatus for Volatile oil Content determination (a) Distilling Flask - A spherical flask, 1,000 ml capacity with ground neck, taper of ground socket 1 in 10, internal dia. of larger end 34.35 to 34.65 mm (b) Still Head - Graduated measuring tube and return flow tube made in one piece, in accordance with the following specifications. External diameter of the smaller end 31.0 to 31.2 mm. Minimum length of the ground zone -34 mm.

Determination of Total Soluble Solids: Method-II

Tube AC, length -220 to 240 mm 115

 

API, Part-I, Vol.-IX (Extracts); Appendices

Internal diameter -13 to 15 mm

pieces of porous earthen ware and one filter paper 15 cm cut into small strips, 7 to 12 mm wide, are also put in the distilling flask, which is then connected to the still head. Before attaching the condenser, water is run into the graduated receiver, keeping the tap T open until the water overflows, at P. Any air bubbles in the rubber tubing a-b are carefully removed by pressing the tube. The tap is then closed and the condenser attached. The contents of the flask are now heated and stirred by frequent agitation until ebullition commences. The distillation is continued at a rate which keeps the lower end of the condenser cool. The flask is rotated occasionally to wash down any material that adheres to its sides.

Bulb CD, length -100 to 110 mm Internal diameter -13 to 15 mm Spiral Condenser - Ground joint accurately fitting in the ground neck of the tube EG, taper 1 in 10 Tube EG, length -80 to 90 mm Internal diameter -30 to 40 mm Bulb B - length 20 to 22 mm Internal diameter -15 to 20 mm The distance between B and P is 120 to 125 mm. Junction P and the centre of the bulb B must be in the same horizontal plane. Measuring tube JL - Length of the graduated portion 144 to 155 mm capacity 2 milliliters graduated into fifths and fiftieths of a milliliter.

At the end of the specified time (3 to 4 hours) heating is discontinued, the apparatus is allowed to cool for 10 min and the tap T is opened and the tube L1 lowered slowly; as soon as the layer of the oil completely enters into the graduated part of the receiver the tap is closed and the volume is read.

Tube PL - Return flow tube - Internal diameter 7 to 8 mm Levelling tube I, length -450 to 500 mm. Internal diameter 10 to 12 mm tapering at the lower end with a wide top (20 to 25 mm diameter).

The tube L1 is then raised till the level of water in it is above the level of B, when the tap T is slowly opened to return the oil to the bulb. The distillation is again continued for another hour and the volume of oil is again read, after cooling the apparatus as before. If necessary, the distillation is again continued until successive readings of the volatile oil do not differ.

Rubber tubing a-b length 450 to 500 mm. Internal diameter 5 to 8 mm. (c) Burner - A luminous Argand burner with chimney and sensitive regulative tap. (d) Stand - A retort stand with asbestos covered ring and clamp carrying a piece of metal tubing connected by a short length of rubber tubing with the water inlet tube of the condenser jacket.

The measured yield of volatile oil is taken to be the content of volatile oil in the drug. The dimensions of the apparatus may be suitably modified in case of necessity.

The whole of the apparatus is effectively screened from draught. The apparatus is cleaned before each distillation by washing successively with acetone and water, then inverting it, filling it with chromic sulphuric acid mixture, after closing the open end at G, and allowing to stand and finally rinsing with water. Method of determination A suitable quantity of the coarsely powdered drug together with 75 ml of glycerin and 175 ml of water in the one litre distilling flask and a few 116  

API, Part-I, Vol.-IX (Extracts); Appendices

APPENDIX - 3  3.1. TEST FOR HEAVY METALS

blank solution following the same procedure omitting the sample.

3.1.1. Limits for Heavy Metals:

Prepare not less than 3 standard solutions of the element being examined of different concentrations, covering the 25 to 200 percentage of the range that may be present in the sample solution. Add separately the corresponding reagents as that for the test solution and prepare the blank reference solution with the corresponding reagents.

Table 1: Permissible Limits of Heavy Metals S. No. 1 2 3 4

Heavy Metal contents Permissible limits Lead 10 ppm Arsenic 3 ppm Cadmium 0.3 ppm Mercury 1 ppm

3.1.2. Determination of Lead, Cadmium, Arsenic and Mercury by Atomic Absorption Spectrophotometry or by Inductively Coupled Plasma:

Calibrate, operate the instruments as per manufacturer’s recommendations and set the analytical condition suitable for the analysis of lead, cadmium, arsenic, and mercury.

Procedure: Prepare a test solution of the substance being examined as follows:

Measure the absorbances of the blank reference solution and each reference solution of different concentrations separately, record the readings and prepare a calibration curve with the average value of 3 readings of each concentration on the ordinate and the corresponding concentration on the abscissa.

Transfer 3 g of the test substance to a clean, dry, 300ml Kjeldahl flask. [Note - A 800-ml flask may be used if the reaction foams excessively]. Clamp the flask at an angle of 450 and add a sufficient quantity of a concentrated nitric acid to moisten the substance thoroughly. Warm gently until the reaction commences, allow the reaction to subside and add portions of the same acid mixture, heating after each addition, until a total of 18 ml of the acid has been added. Increase the amount of heat, and boil gently until the solution darkens. Cool, add 2 ml of nitric acid and heat again until the solution darkens. Continue the heating, followed by addition of nitric acid until no further darkening occurs, then heat strongly to the production of dense, white fumes. Cool cautiously add 5 ml of water, boil gently to the production of dense, white fumes, and continue heating until the volume is reduced to a few ml. Cool, cautiously add 5 ml of water, and examine the colour of the solution. If the colour is yellow, cautiously add 1 ml of 30 per cent hydrogen peroxide, and again evaporate to the production of dense, white fumes and a volume of 2 to 3 ml. If the solution is still yellow, repeat the addition of 5 ml of water and the peroxide treatment. Cool, dilute cautiously with a few ml of water, and rinse into a 50-ml colour-comparison tube, taking care that the combined volume does not exceed 25 ml. Prepare a

Interpolate the mean value of the readings obtained with the test solution on the calibration curve to determine the concentration of each heavy metal. For more information on Apparatus refer API, Part I, Volume-VI, Appendix, 2.3.7 & 2.3.8 3.2. MICROBIAL LIMIT TESTS: Table 2: Microbial Contamination Limits

Sl. No.

1 2 3 4 5 6

Staphylococcus aureus/g Salmonella sp./g Pseudomonas aeruginosa/g Escherichia coli Total microbial plate count (TPC) Total Yeast & Mould

Permissible limits for herbal extracts and Powders

Permissible limits for plant materials which will be treated before use

Absent

-

Absent

Absent

Absent

-

Absent

10

1 05*/g

107/g

103/g

105/g

*For topical use, the limits shall be 107/g 117

 

Parameters

API, Part-I, Vol.-IX (Extracts); Appendices

The following tests are designed for the estimation of the number of viable aerobic microorganisms present and for detecting the presence of designated microbial species in the extract. The term ‘growth’ is used to designate the presence and presumed proliferation of viable microorganisms.

cultures described above and where the article is not suitable for applying the membrane filtration method it can be assumed that the failure to isolate the inoculated organism may be due to the bactericidal activity of the product. This may indicate that the article is not likely to be contaminated with the given species of microorganisms. However, monitoring should be continued to establish the spectrum of inhibition and bactericidal activity of the article.

Preliminary Testing: The methods given here in are invalid unless it is demonstrated that the test specimens (extracts) to which they are applied do not, of themselves, inhibit the multiplication under the test conditions of microorganisms that can be present. Therefore, prior to doing the tests, inoculate diluted extracts being examined with separate viable cultures of Escherichia coli, Salmonella species, Pseudomonas aeruginosa and Staphylococcus aureus. This is done by adding 1 ml of 24 hours broth culture containing not less than 1000 microorganisms to the first dilution (in buffer solution pH 7.2, fluid soyabean-casein digest medium or fluid lactose medium) of the test material and following the test procedure. If the organisms fail to grow in the relevant medium the procedure should be modified by (a) increasing the volume of diluent with the quantity of test material remaining the same, or (b) incorporating a sufficient quantity of a suitable inactivating agent in the diluents, or (c) combining the afore mentioned modifications so as to permit growth of the organisms in the media. If inhibitory substances are present in the extracts, 0.5 per cent of soya lecithin and 4 per cent of polysorbate 20 may be added to the culture medium.

Media Culture media may be prepared as given below or dehydrated culture media may be used provided that, when reconstituted as directed by the manufacturer, they have similar ingredients and / or yield media comparable to those obtained from the formulae given below. Where agar is specified in a formula, use agar that has a moisture content of not more than 15 per cent. Where water is called for in a formula, use purified water. Unless otherwise indicated, the media should be sterilized by heating in an autoclave (15 psi) at 1210 for 15 min. In preparing media by the formulas given below, dissolve the soluble solids in the water, using heat if necessary, to effect complete solution, add solutions of 0.1N hydrochloric acid or 0.1N sodium hydroxide in quantities sufficient to yield the required pH in the medium when it is ready for use. Determine the pH at 250 ± 20. Baird-Parker Agar Medium Pancreatic digest of casein Beef extract Yeast extract Lithium chloride Agar Glycerin Sodium pyruvate Water to

Alternatively, repeat the test as described in the previous paragraph, using fluid casein digestsoya lecithin-polysorbate 20 medium, to demonstrate neutralization of preservatives or other antimicrobial agents in the test material. Where inhibitory substances are contained in the extracts and the latter is soluble, the membrane filtration method described under total aerobic microbial count may be used.

Heat with frequent agitation and boil for 1 min. Sterilize, cool in between 450-500, add 10 ml of a one per cent w/v solution of sterile potassium tellurite and 50 ml of egg yolk emulsion. Mix thoroughly, but gently and pour into plates. (Prepare the egg-yolk emulsion by disinfecting the surface of whole shell eggs, aseptically

If in spite of incorporation of suitable inactivating agents and a substantial increase in the volume of diluent it is still not possible to recover the viable 118  

10.0 g 5.0 g 1.0 g 5.0 g 20.0 g 12.0 g 10.0 g 1000 ml

API, Part-I, Vol.-IX (Extracts); Appendices

Buffered Sodium Chloride - Peptone Solution pH 7.0 Potassium dihydrogen phosphate 3.56 g Disodium hydrogen phosphate 7.23 g Sodium chloride 4.30 g Peptone (meat or casein) 1.0 g Water to 1000 ml

cracking the eggs, and separating out intact yolks into a sterile graduated cylinder. Add sterile saline solution, get a 3 to 7 ratio of egg-yolk to saline. Add to a sterile blender cup and mix at high speed for 5 seconds). Adjust the pH after sterilization to 6.8 ± 0.2. Bismuth Sulphite Agar Medium Solution (1) Beef extract Peptone Agar Ferric citrate Brilliant green Water to

0.1 to 1.0 per cent w/v Polysorbate 20 or polysorbate 80 may be added. Sterilize by heating in an autoclave at 1210 for 15 min.

6.0 g 10.0 g 24.0 g 0.4 g 10.0 mg 1000 ml

Casein Soyabean Digest Agar Medium Pancreatic digest of casein 15.0 g Papaic digest of soyabean meal 5.0 g Sodium chloride 5.0 g Agar 15.0 g Water to 1000 ml

Dissolve with the aid of heat and sterilize by maintaining at 1150 for 30 min. Solution (2) Ammonium bismuth citrate Sodium sulphite Anhydrous disodium hydrogen phosphate Dextrose monohydrate Water to

Adjust the pH after sterilization to 7.3 ± 0.2.* Cetrimide Agar Medium Pancreatic digest of gelatin Magnesium chloride Potassium sulphate Cetrimide Agar Glycerin Water to

3.0 g 10.0 g 5.0 g 5.0 g 100 ml

Mix, heat to boiling, cool to room temperature, add 1 volume of solution (2) to 10 volumes of solution (1) previously melted and cooled to a temperature of 550 and pour.

Heat to boiling for 1 min with shaking. Adjust the pH so that after sterilization it is 7.0 to 7.4.* Desoxycholate-Citrate Agar Medium Beef extract Peptone Lactose Trisodium citrate Sodium thiosulphate Ferric citrate Sodium desoxycholate Neutral red Agar Water to

Bismuth Sulphite Agar Medium should be stored at 20 to 80 for 5 days before use. Brilliant Green Agar Medium Peptone Yeast extract Lactose Sucrose Sodium chloride Phenol red Brilliant green Agar Sodium chloride Water to

10.0 g 3.0 g 10.0 g 10.0 g 5.0 g 80.0 g 12.5 mg 12.0 g 5.0 g 1000 ml

5.0 g 5.0 g 10.0 g 8.5 g 5.4 g 1.0 g 5.0 g 0.02 g 12.0 g 1000 ml

Mix and allow to stand for 15 min. Gently boil with continuous stirring and continue boiling until solution is complete. Cool to 800, mix, pour and cool rapidly.

Mix, allow to stand for 15 min, sterilize by maintaining at 1150 for 30 min and mix before pouring.

Care should be taken not to overheat Desoxycholate Citrate Agar during preparation. It 119

 

20.0 g 1.4 g 10.0 g 0.3 g 13.6 g 10.0 g 1000 ml

API, Part-I, Vol.-IX (Extracts); Appendices

should not be remelted and the surface of the plates should be dried before use.

cent w/v solution of methylene blue. The finished medium may not be clear. Adjust the pH after sterilization to 7.1±0.2.

Fluid Casein Digest - Soya Lecithin Polysorbate 20 Medium Pancreatic digest of casein Soya lecithin Polysorbate 20 Water to

MacConkey Agar Medium Pancreatic digest of gelatin Peptone (meat and casein, equal parts) Lactose Sodium chloride Bile salts Agar Neutral red Crystal violet Water to

20.0 g 5.0 g 40.0 ml 1000 ml

Dissolve the pancreatic digest of casein and soya lecithin in water, heating in a water-bath at 480 to 500 for about 30 min to effect solution. Add polysorbate 20, mix and dispense as desired*. Fluid Lactose Medium Beef extract Pancreatic digest of gelatin Lactose Water to

Boil the mixture of solids and water for 1 min to effect solution. Adjust the pH after sterilization to 7.1 ± 0.2.*

3.0 g 5.0 g 5.0 g 1000 ml

MacConkey Broth Medium Pancreatic digest of gelatin Lactose Dehydrated ox bile Bromocresol purple Water to

Cool as quickly as possible after sterilization. Adjust the pH after sterilization to 6.9 ± 0.2. Lactose Broth Medium Beef extract Pancreatic digest of gelatin Lactose Water to

Mannitol-Salt Agar Medium Pancreatic digest of gelatin Peptic digest of animal tissue Beef extract D-Mannitol Sodium chloride Agar Phenol red Water to

Levine Eosin - Methylene Blue Agar Medium Pancreatic digest of gelatin 10.0 g Dibasic potassium phosphate 2.0 g Agar 15.0 g Lactose 10.0 g Eosin Y 400 mg Methylene blue 65.0 mg Water to 1000 ml

5.0 g 5.0 g 1.0 g 10.0 g 75.0 g 15.0 g 25 mg 1000 ml

Mix, heat with frequent agitation and boil for 1 min to effect solution. Adjust the pH after sterilization to 7.4 ± 0.2*.

Dissolve the pancreatic digest of gelatin, dibasic potassium phosphate and agar in water with warming and allow to cool. Just prior to use, liquify the gelled agar solution and the remaining ingredients, as solutions, in the following amounts and mix. For each 100 ml of the liquified agar solution use 5 ml of a 20 per cent w/v solution of lactose, 2 ml of a 2 per cent w/v solution of eosin Y and 2 ml of a 0.33 per

Nutrient Agar Medium: Nutrient broth gelled by the addition of 1 to 2 per cent w/v of agar. Nutrient Broth Medium Beef extract Peptone Sodium chloride Water to

                                                             Sterilize at 121º for 15 minutes in an autoclave

120  

20.0 g 10.0 g 5.0 g 10.0 mg 1000 ml

Adjust the pH after sterilization to 7.3 ± 0.2*

3.0 g 5.0 g 5.0 g 1000 ml

Adjust the pH after sterilization to 6.9 ± 0.2.



17.0 g 3.0 g 10.0 g 5.0 g 1.5 g 13.5 g 30.0 mg 1.0 mg 1000 ml

10.0 g 10.0 g 5.0 mg 1000 ml

API, Part-I, Vol.-IX (Extracts); Appendices

Dissolve with the aid of heat. Adjust the pH to 8.0 to 8.4 with 5M sodium hydroxide and boil for 10 min. Filter and sterilize by maintaining at 1150 for 30 min and adjust the pH to 7.3 ± 0.1.

alternatively add 50 mg of chloramphenicol immediately before use. Selenite F Broth Peptone Lactose Disodium hydrogen phosphate Sodium hydrogen selenite Water to

Pseudomonas Agar Medium for Detection of Flourescein Pancreatic digest of casein 10.0 g Peptic digest of animal tissue 10.0 g Anhydrous dibasic potassium phosphate 1.5 g Magnesium sulphate hepta hydrate 1.5 g Glycerin 10.0 ml Agar 15.0 g Water to 1000 ml

Dissolve, distribute in sterile containers and sterilize by maintaining at 1000 for 30 min. Fluid Selenite - Cystine Medium Pancreatic digest of casein Lactose Sodium phosphate Sodium hydrogen selenite l-Cystine Water to

Dissolve the solid components in water before adding glycerin. Heat with frequent agitation and boil for 1 min to effect solution. Adjust the pH after sterilization to 7.2 ± 0.2.

Tetrathionate Broth Medium Beef extract Peptone Yeast extract Sodium chloride Calcium carbonate Sodium thiosulphate Water to

Dissolve the solid components in water before adding glycerin. Heat with frequent agitation and boil for 1 min to effect solution. Adjust the pH after sterilization to 7.2 ± 0.2*. 40.0 g 10.0 g

Tetrathionate-Bile-Brilliant Medium Peptone Dehydrated ox bile Sodium chloride Calcium carbonate Potassium tetrathionate Brilliant green Water to

15.0 g 1000 ml

Sabouraud Dextrose Agar Medium with Antibiotics: To 1 liter of Sabouraud Dextrose Agar Medium, add 0.1 g of benzylpenicillin sodium and 0.1 g of tetracycline HCL or

Green

Broth 8.6 g 8.0 g 6.4 g 20.0 g 20.0 g 70.0 mg 1000 ml

Heat just to boiling; do not reheat. Adjust the pH so that after heating it is 7.0 ± 0.2.

                                                             Sterilize at 121º for 15 minutes in an autoclave

121  

0.9 g 4.5 g 1.8 g 4.5 g 25.0 g 40.7 g 1000 ml

Dissolve the solids in water and heat the solution to boil. On the day of use, add a solution prepared by dissolving 5 g of potassium iodide and 6 g of iodine in 20 ml of water.

Mix, and boil to effect solution. Adjust the pH after sterilization to 5.6 ± 0.2*.



5.0 g 4.0 g 10.0 g 4.0 g 10.0 mg 1000 ml

Mix and heat in flowing steam for 15 min. Adjust the final pH to 7.0 ± 0.2. Do not sterilize.

Pseudomonas Agar Medium for Detection of Pyocyanin Pancreatic digest of gelatin 20.0 g Anhydrous magnesium chloride 1.4 g Anhydrous potassium sulphate 10.0 g Agar 15.0 g Glycerin 10.0 ml Water to 1000 ml

Sabouraud Dextrose Agar Medium Dextrose Peptic digest of animal tissue and pancreatic digest of casein (1:1) Agar Water to

5.0 g 4.0 g 10.0 g 4.0 g 1000 ml

API, Part-I, Vol.-IX (Extracts); Appendices

Triple Sugar - Iron Agar Medium Beef extract Yeast extract Peptone Lactose Sucrose Dextrose monohydrate Ferrous sulphate Sodium chloride Sodium thiosulphate Phenol red Water to

Xylose -Lysine-Desoxycholate Agar Medium Xylose 3.5 g l-Lysine 5.0 g Lactose 7.5 g Sucrose 7.5 g Sodium chloride 5.0 g Yeast extract 3.0 g Phenol red 80.0 mg Agar 13.5 g Sodium desoxycholate 2.5 g Sodium thiosulphate 6.8 g Ferric ammonium citrate 800 mg Water to 1000 ml

3.0 g 3.0 g 20.0 g 10.0 g 10.0 g 1.0 g 0.2 g 5.0 g 0.3 g 24.0 mg 1000 ml

Mix, allow standing for 15 min, bringing to boil and maintain at boiling point until solution is complete, mix, distributing in tubes and sterilising by maintaining at 1210 for 15 min. Allow to stand in a sloped form with a butt about 2.5 cm long. Urea Broth Medium Potassium dihydrogen orthophosphate Anhydrous disodium hydrogen phosphate Urea Yeast extract Phenol red Water to

Heat the mixture of solids and water, with swirling, just to the boiling point. Do not overheat or sterilize. Transfer at once to a water-bath maintained at about 500 and pour into plates as soon as the medium has cooled. Adjust the final pH to 7.4 ± 0.2.

9.1 g 9.5 g 20.0 g 0.1 g 10.0 mg 1000 ml

Sampling: Use 10 ml or 10 g specimens for each of the tests specified in the individual monograph. Precautions: The microbial limit tests should be carried out under conditions designed to avoid accidental contamination during the test. The precautions taken to avoid contamination must be such that they do not adversely affect any microorganisms that should be revealed in the test.

Mix, sterilize by filtration and distribute aseptically in sterile containers. Vogel-Johnson Agar Medium Pancreatic digest of casein Yeast extract Mannitol Dibasic potassium phosphate Lithium chloride Glycerin Agar Phenol red Water to

3.2.1. Total Aerobic Microbial Count:

10.0 g 5.0 g 10.0 g 5.0 g 5.0 g 10.0 g 16.0 g 25.0 mg 1000 ml

Pre-treat the extracts and raw materials being examined as described below. Note: The raw materials needs to be ground as a coarse powder before analysis. Water-soluble products: Dissolve 10 g or dilute 10 ml of the extract preparation being examined, unless otherwise specified, in buffered sodium chloride-peptone solution pH 7.0 or any other suitable medium shown to have no antimicrobial activity under the conditions of test and adjust the volume to 100 ml with the* same medium. If necessary, adjust the pH to about 7.

Boil the solution of solids for 1 min. Sterilize, cool to between 450-500 and add 20 ml of 1 per cent w/v sterile solution of potassium tellurite. Adjust the pH after sterilization to 7.0 ± 0.2.

                                                             

Sterilize at 121º for 15 minutes in an autoclave

122  

API, Part-I, Vol.-IX (Extracts); Appendices

Products insoluble in water (non-fatty): Suspend 10 g or 10 ml of the extract preparation being examined, unless otherwise specified, in buffered sodium chloride-peptone solution pH 7.0 or any other suitable medium shown not to have antimicrobial activity under the conditions of the test and dilute to 100 ml with the same medium. If necessary, divide the preparation being examined and homogenize the suspension mechanically. A suitable surface-active agent such as 0.1 per cent w/v of polysorbate 80 may be added to assist the suspension of poorly wettable substances. If necessary, adjust the pH of the suspension to about 7.

buffered sodium chloride-peptone solution pH 7.0. For fatty substances add to the liquid polysorbate 20 or polysorbate 80. Transfer one of the membrane filters, intended for the enumeration of bacteria, to the surface of a plate of casein soyabean digest agar and the other, intended for the enumeration of fungi, to the surface of a plate of Sabouraud dextrose agar with antibiotics. Incubate the plates for 5 days, unless a more reliable count is obtained in shorter time, at 300 to 350 in the test for bacteria and 200 to 250 in the test for fungi. Count the number of colonies that are formed. Calculate the number of microorganisms per g or per ml of the extract preparation being examined, if necessary count bacteria and fungi separately.

Fatty products: Homogenise 10 g or 10 ml of the extract preparation being examined, unless otherwise specified, with 5 g of polysorbate 20 or polysorbate 80. If necessary, heat to not more than 400. Mix carefully while maintaining the temperature in the water-bath or in an oven. Add 85 ml of buffered sodium chloride-peptone solution pH 7.0 or any other suitable medium shown to have no antimicrobial activity under the conditions of the test, heated to not more than 400 if necessary. Maintain this temperature for the shortest time necessary for formation of an emulsion and in any case for not more than 30 min. If necessary, adjust the pH to about 7.

Plate count for bacteria: Using petri dishes 9 to 10 cm in diameter, add to each dish a mixture of 1 ml of the pretreated extract preparation and about 15 ml of liquified casein soyabean digest agar at not more than 450. Alternatively, spread the pretreated extract preparation on the surface of the solidified medium in a Petri dish of the same diameter. If necessary, dilute the pretreated extract preparation as described above so that a colony count of not more than 300 may be expected. Prepare at least two such Petri dishes using the same dilution and incubate at 300 to 350 for 5 days, unless a more reliable count is obtained in a shorter time.

Examination of the sample: Determine the total aerobic microbial count in the extract being examined by any of the following methods.

Count the number of colonies that are formed. Calculate the results using plates with the greatest number of colonies but taking 300 colonies per plate as the maximum consistent with good evaluation.

Membrane filtration: Use membrane filters 50 mm in diameter and having a nominal pore size not greater than 0.45 m the effectiveness of which in retaining bacteria has been established for the type of preparation being examined. Transfer 10 ml or a quantity of each dilution containing 1 g of the preparation being examined to each of two membrane filters and filter immediately. If necessary, dilute the pretreated extract preparation so that a colony count of 10 to 100 may be expected. Wash each membrane by filtering through it three or more successive quantities, each of about 100 ml, of a suitable liquid such as

Plate count for fungi: Proceed as described in the test for bacteria but use Sabouraud dextrose agar with antibiotics in place of casein soyabean digest agar and incubate the plates at 200 to 250 for 5 days, unless a more reliable count is obtained in a shorter time. Calculate the results using plates with not more than 100 colonies. Multiple-tube or serial dilution method: In each of fourteen test tubes of similar size place 9.0 ml of sterile fluid soyabean casein digest medium. Arrange twelve of the tubes in four sets of three 123

 

API, Part-I, Vol.-IX (Extracts); Appendices

3.2.2. Tests for Specified Microorganisms:

tubes each. Put aside one set of three tubes to serve as controls. Into each of three tubes of one set (“100”) and into fourth tube (A) pipette 1 ml of the solution of suspension of the test specimen (extract) and mix. From tube A pipette 1 ml of its contents into the one remaining tube (B) not included in the set and mix. These two tubes contain 100 mg (or 100 l) and 10 mg (or 10 l) of the specimen respectively. Into each of the second set (“10”) of three tubes pipette 1 ml from tube A, and into each tube of the third set (“1”) pipette 1 ml from tube B. Discard the unused contents of tube A and B. Close well and incubate all of the tubes. Following the incubation period, examine the tubes for growth. The three control tubes remain clear. Observations in the tubes containing the test specimen, when interpreted by reference to Table 3, indicate the most probable number of microorganisms per g or per ml of the test specimen.

Pretreatment of the extract sample being examined: Proceed as described under the test for total aerobic microbial count but using lactose broth or any other suitable medium shown to have no antimicrobial activity under the conditions of test in place of buffered sodium chloride-peptone solution pH 7.0. Escherichia coli: Place the prescribed quantity in a sterile screw-capped container, add 50 ml of nutrient broth, shake, allow to stand for 1 hour (4 hours for gelatin) and shake again. Loosen the cap and incubate at 370 for 18-24 hours. Primary test: Add 1.0 ml of the enrichment culture to a tube containing 5 ml of MacConkey broth. Incubate in a water-bath at 36-380 for 48 hours. If the contents of the tube show acid and gas, carry out the secondary test.

Table 3: Most Probable Total Count by Multiple-Tube Or Serial Dilution Method Observed combination of numbers of tubes showing growth in each set Number of mg (or ml) of specimen per tube 10 1 100 (100 l) (10 l) (1 l) 3 3 3 3 3 3 3 3 2 3 3 1 3 3 0 3 2 3 3 2 2 3 2 1 3 2 0 3 1 3 3 1 2 3 1 1 3 1 0 3 0 2 3 0 1 3 0 0

Secondary test: Add 0.1 ml of the contents of the tubes containing (a) 5 ml of MacConkey broth and (b) 5 ml of peptone water. Incubate in a water-bath at 43.5 - 44.50 for 24 hours and examine tube (a) for acid and gas and tube (b) for indole. To test for indole, add 0.5 ml of Kovac’s reagent, shake well and allow to stand for 1 min; if a red colour is produced in the reagent layer indole is present. The presence of acid and gas and of indole in the secondary test indicates the presence of Escherichia coli.

Most probable number of microorganisms per g or per ml >1100 1100 500 200 290 210 150 90 160 120 70 40 95 60 40 23

Carry out a control test by repeating the primary and secondary tests, adding 1.0 ml of the enrichment culture and a volume of broth containing 10 to 50 Escherichia coli (NCTC 9002) organisms, prepared from a 24-hour culture in nutrient broth, to 5 ml of MacConkey broth. The test is not valid unless the results indicate that the control contains Escherichia coli. Alternative test: By means of an inoculating loop, streak a portion from the enrichment culture (obtained in the previous test) on the surface of MacConkey agar medium. Cover and invert the dishes and incubate. Upon examination, if none of the colonies are brick-red in colour and have a 124

 

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surrounding zone of precipitated bile the sample meets the requirements of the test for the absence of Escherichia coli.

the colonies conforms to the description given in Table 4, the sample meets the requirements of the test for the absence of the genus Salmonella. If any colonies conforming to the description in Table 4 are produced, carry out the secondary test.

If the colonies described above are found, transfer the suspect colonies individually to the surface of Levine eosin - methylene blue agar medium, plated on Petri dishes. Cover and invert the plates and incubate. Upon examination, if none of the colonies exhibits both a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light, the sample meets the requirements of the test for the absence of Escherichia coli. The presence of Escherichia coli may be confirmed by further suitable cultural and biochemical tests.

Secondary test: Subculture any colonies showing the characteristics given in Table 4 in triple sugariron agar by first inoculating the surface of the slope and then making a stab culture with the same inoculating needle, and at the same time inoculate a tube of urea broth. Incubate at 360 to 380 for 18 to 24 hours. The formation of acid and gas in the stab culture (with or without concomitant blackening) and the absence of acidity from the surface growth in the triple sugar iron agar, together with the absence of a red colour in urea broth indicates the presence of Salmonella. If acid but no gas is produced in the cultures, the identity of the organisms should be confirmed by agglutination tests.

Salmonela: Transfer a quantity of the pretreated extract preparation being examined containing 1 g or 1 ml of the product to 100 ml of nutrient broth in a sterile screw-capped jar, shake, allow to stand for 4 hours and shake again. Loosen the cap and incubate at 35-370 for 24 hours.

Carry out the control test by repeating the primary and secondary tests using 1.0 ml of the enrichment culture and a volume of broth containing 10 to 50 Salmonella abony (NCTC 6017) organisms, prepared from a 24-hour culture in nutrient broth, for the inoculation of the tubes (a) and (b). The test is not valid unless the results indicate that the control contains Salmonella.

Table 4: Test for Salmonella Medium Bismuth sulphite agar Brilliant green agar

Deoxycholatecitrate agar Xylose-lysinedesoxycholate agar

Description of colony Black or green Small, transparent and colourless, or opaque, pinkish or white (frequently surrounded by a pink or red zone) Colourless and opaque, with or without black centers Red with or without black centres

Pseudomonas aeruginosa: Pretreat the extract preparation being examined as described above and inoculate 100 ml of fluid soyabean-casein digest medium with a quantity of the solution, suspension or emulsion thus obtained containing 1 g or 1 ml of the preparation being examined. Mix and incubate at 350 to 370 for 24 to 48 hours. Examine the medium for growth and if growth is present, streak a portion of the medium on the surface of cetrimide agar medium, each plated on Petri dishes. Cover and incubate at 350 to 370 for 18 to 24 hours. If, upon examination, none of the plates contains colonies having the characteristics listed in Table 5 for the media used, the sample meets the requirement for freedom from Pseudomonas aeruginosa. If any colonies

Primary test: Add 1.0 ml of the enrichment culture to each of the two tubes containing (a) 10 ml of selenite F broth and (b) tetrathionate-bilebrilliant green broth and incubate at 36-380 for 48 hours. From each of these two cultures subculture on at least two of the following four agar media: bismuth sulphate agar, brilliant green agar, deoxycholate citrate agar and xylose-lysine deoxycholate agar. Incubate the plates at 36-380 for 18 to 24 hours. Upon examination, if none of 125  

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conforming to the description in Table 5 are produced, carry out the oxidase and pigment tests.

the media listed in Table 6 to individual tubes, each containing 0.5 ml of mammalian, preferably rabbit or horse, plasma with or without additives.

Streak representative suspect colonies from the agar surface of cetrimide agar on the surfaces of Pseudomonas agar medium for detection of fluorescein and Pseudomonas agar medium for detection of pyocyanin contained in Petri dishes. Cover and invert the inoculated media and incubate at 330 to 370 for not less than 3 days. Examine the streaked surfaces under ultra-violet light. Examine the plates to determine whether colonies conforming to the description in Table 5 are present. If growth of suspect colonies occurs, place 2 or 3 drops of a freshly prepared 1 per cent w/v solution of N,N,N´,N´-tetramethyl-4phenylenediamine dihydrochloride on filter paper and smear with the colony; if there is no development of a pink colour, changing to purple, the sample meets the requirements of the test for the absence of Pseudomonas aeruginosa.

Incubate in water-bath at 370 examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours. If no coagulation in any degree is observed, the sample meets the requirements of the test for the absence of Staphylococcus aureus. Table 6: Tests for Staphylococcus aureus Selective medium VogelJohnson agar Mannitolsalt agar BairdParker agar

Table 5: Tests for Pseudomonas aeruginosa Medium

Cetrimide agar Pseudomonas agar medium for detection of fluorescein Pseudomonas agar medium for detection of pyocyanin

Characteristic colonial morphology Generally greenish

Fluoresce nce in UV light Greenish Yellowish

Generally colourless to yellowish Generally greenish

Blue

Oxid ase test

Gram stain

Posit -ive Posit -ive

Negative rods Negative rods

Posit -ive

Gram stain Positive cocci (in clusters) Positive cocci (in clusters) Positive cocci (in clusters)

Validity of the tests for total aerobic microbial count:

Negative rods

Grow the following test strains separately in tubes containing fluid soyabean-casein digest medium at 300 to 350 for 18 to 24 hours or, for Candida albicans, at 200 for 48 hours. Staphylococcus aureus Bacillus subtilis Escherichia coli Candida albicans

(ATCC 6538; NCTC 10788) (ATCC 6633; NCIB 8054) (ATCC 8739; NCIB 8545) (ATCC 2091; ATCC 10231)

Dilute portions of each of the cultures using buffered sodium chloride-peptone solution pH 7.0 to make test suspensions containing about 100 viable microorganisms per ml. Use the suspension of each of the microorganisms separately as a control of the counting methods, in the presence and absence of the preparation being examined, if necessary.

Staphylococcus aureus: Proceed as described under Pseudomonas aeruginosa, if upon examination of the incubated plates, none of them contains colonies having the characteristics listed in Table 6 for the media used, the sample meets the requirements for the absence of Staphylococcus aureus. If growth occurs, carry out the coagulase test. Transfer representative suspect colonies from the agar surface of any of

A count for any of the test organisms differing by not more than a factor of 10 from the 126

 

Characteristic colonial morphology Black surrounded by yellow zones Yellow colonies with yellow zones Black, shiny, surrounded by clear zones of 2 to 5 mm

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calculated value for the inoculum should be obtained. To test the sterility of the medium and of the diluent and the aseptic performance of the test, carry out the total aerobic microbial count method using sterile buffered sodium chloridepeptone solution pH 7.0 as the test preparation. There should be no growth of microorganisms.

Cypermethrin (and isomers) DDT (sum of p,p´-DDT, o,p´-DDT, p,pDDE and p,p´-TDE) Deltamethrin Diazinon Dichlorvos Dithiocarbamates (as CS2) Endosulfan (sum of isomers and endosulfan sulphate) Endrin Ethion Fenitrothion Fenvalerate Fonofos Heptachlor (sum of heptachlor and heptachlor epoxide) Hexachlorobenzene Hexachlorocyclohexane isomers (other than γ) Lindane (γ-hexachlorocyclohexane) Malathion Methidathion Parathion Parathion-methyl Permethrin Phosalone Piperonyl butoxide Pirimiphos-methyl Pyrethrins (sum of) Quintozene (sum of quintozene, pentachloroaniline and methyl pentachlorophenyl sulphide)

Validity of the tests for specified microorganisms: Grow separately the test strains of Staphylococcus aureus and Pseudomonas aeruginosa in fluid soyabean-casein digest medium and Escherichia coli and Salmonella typhimurium at 300 to 350 for 18 to 24 hours. Dilute portions of each of the cultures using buffered sodium chloride-peptone solution pH 7.0 to make test suspensions containing about 103 viable microorganisms per ml. Mix equal volume of each suspension and use 0.4 ml (approximately 102 micro-organisms of each strain) as an inoculum in the test for E. coli, S. typhimurium, P. aeruginosa and S. aureus, in the presence and absence of the extract preparation being examined, if necessary. A positive result for the respective strain of microorganism should be obtained. 3.3. PESTICIDE RESIDUE: Definition: For the purposes of the Pharmacopoeia, a pesticide is any substance or mixture of substances intended for preventing, destroying or controlling any pest, unwanted species of plants or animals causing harm during or otherwise interfering with the production, processing, storage, transport or marketing of vegetable drugs.

0.05 2.0 0.5 1.5 0.05 0.05 0.1 0.3 0.6 1.0 0.2 0.5 0.2 1.0 0.1 3.0 4.0 3.0 1.0

ADI×M MDD×100

ADI= Acceptable daily intake as published by FAOWHO, in milligrams per kilogram of body mass, M= body mass in kilograms (60 kg),

Limit (mg/kg) 0.02 0.05 1.0 3.0 0.05

MDD= daily dose of the drug, in kilograms If the drug is intended for the preparation of extracts, tinctures or other pharmaceutical forms whose preparation method modifies the content of pesticides in the finished product, the limits are calculated using the following expression:

0.5 0.2 0.1

127  

0.5 0.5 1.0 2.0 3.0

Note: Apart from the above, if any pesticides applied to the herb before or after harvesting should also be tested. The limit should be calculated using the following formula.

Table 7: Permissible Limits for Pesticide Residue: Substance Alachlor Aldrin and Dieldrin (sum of) Azinphos-methyl Bromopropylate Chlordane (sum of cis-, trans - and Oxythlordane) Chlorfenvinphos Chlorpyrifos Chlorpyrifos-methyl

1.0 1.0

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ADI×M×E MDD×100

for 3 min. Filter and wash the filter cake with two quantities, each of 25 ml of acetone. Combine the filtrate and the washings and heat using a rotary evaporator at a temperature not exceeding 400 until the solvent has almost completely evaporated. To the residue add a few milliliters of toluene and heat again until the acetone is completely removed. Dissolve the residue in 8 ml of toluene. Filter through a membrane filter (45 m), rinse the flask and the filter with toluene and dilute to 10.0 ml with the same solvent (solution A).

E= Extraction factor for of the method of preparation, determined experimentally. Higher limits can also be authorised, in exceptional cases, especially when a plant requires a particular cultivation method or has a metabolism or a structure that gives rise to a higher than normal content of pesticides. Reagents: All reagents and solvents are free from any contaminants, especially pesticides, that might interfere with the analysis. It is often necessary to use special quality solvents or, if this is not possible, solvents that have recently been redistilled in an apparatus made entirely of glass. In any case, suitable blank tests must be carried out.

Purification: Examine by size-exclusion chromatography. The chromatographic procedure may be carried out using:

Apparatus: Clean the apparatus and especially glassware to ensure that they are free from pesticides, for example, soak for at least 16 hours in a solution of phosphate-free detergent, rinse with large quantities of distilled water and wash with acetone and hexane or heptane.

a stainless steel column 0.30 m long and 7.8 mm in internal diameter packed with styrenedivinylbenzene copolymer (5 m).



as mobile phase toluene at a flow rate of 1 ml/min.

Performance of the column: Inject 100 l of a solution containing 0.5 g/l of methyl red and 0.5 g/l of oracet blue in toluene and proceed with the chromatography. The column is not suitable unless the colour of the eluate changes from orange to blue at an elution volume of about 10.3 ml. If necessary calibrate the column, using a solution containing toluene, at a suitable concentration, the insecticide to be analysed with the lowest molecular mass (for example, dichlorvos) and that with the highest molecular mass (for example, deltamethrin). Determine which fraction of the eluate contains both insecticides.

3.3.1. Test for Pesticides: The following methods may be used depending on the substance being examined, it may be necessary to modify, sometimes extensively, the procedure described hereafter. In any case, it may be necessary to use, in addition, another column with a different polarity or another detection method (mass spectrometry) or a different method (immunochemical methods) to confirm the results obtained. This procedure is valid only for the analysis of samples of vegetable drugs containing less than 15 per cent of water. Samples with a higher content of water may be dried, provided it has been shown that the drying procedure does not affect significantly the pesticide content.

Purification of the test solution: Inject a suitable volume of solution A (100 μl to 500 μl) and proceed with the chromatography. Collect the fraction as determined above (solution B). Organophosphorus insecticides are usually eluted between 8.8 ml and 10.9 ml. Organochlorine and pyrethroid insecticides are usually eluted between 8.5 ml and 10.3 ml.

Extraction (Method-I): To 10 g of the substance being examined, add 100 ml of acetone and allow to stand for 20 min. Add 1 ml of a solution containing 1.8 g/ml of carbophenothion in toluene. Homogenise using a high-speed blender

In a chromatography column, 0.10 m long and 5 mm in internal diameter, introduce a piece of 128

 



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defatted cotton and 0.5 g of silica gel treated as follows: heat silica gel for chromatography in an oven at 1500 for at least 4 hours. Allow to cool and add dropwise a quantity of water corresponding to 1.5 per cent of the mass of silica gel used; shake vigorously until agglomerates have disappeared and continue shaking for 2 hours using a mechanical shaker. Condition the column using 1.5 ml of hexane. Prepacked columns containing about 0.50 g of a suitable silica gel may also be used, provided they are previously validated.

evaporate and dry using rotary evaporator. Dissolve in 0.2 ml of n-hexane containing 10 ng/ml of carbophenothion and sonicate. 3.3.2. Quantitative Analysis: Refer API, Part I, Volume VI, Section 2.5.1. page 282 to 286 3.4. TEST FOR AFLATOXINS: Table 8: Permissible Limit of Aflatoxins Aflatoxin B1 B1+B2+G1+G2

Caution: Aflatoxins are highly dangerous and extreme care should be exercised in handling aflatoxin materials. This test is provided to detect the possible presence of aflatoxins B 1, B2, G1 and G2 in any material of plant origin. Unless otherwise specified in the individual monograph use the following method.

Concentrate solution B in a current of helium for chromatography or oxygen-free nitrogen almost to dryness and dilute to a suitable volume with toluene (200 l to 1 ml according to the volume injected in the preparation of solution B). Transfer quantitatively onto the column and proceed with the chromatography using 1.8 ml of toluene as the mobile phase. Collect the eluate (solution C).

Zinc Acetate - Aluminum Chloride Reagent: Dissolve 20 g of zinc acetate and 5 g of aluminum chloride in sufficient water to make 100 ml.

Extraction (Method-II): To 25 g of the substance being examined, add 300 ml of acetonitrile : water (3 : 1) and homogenise using a high-speed blender for 5 min. Filter and wash the filter cake with two quantities, each of 25 ml of acetonitrile water mixture. Transfer filtrate and rinse to a separating funnel.

Sodium Chloride Solution: Dissolve 5 g of sodium chloride in 50 ml of purified water. Test Solution 1: Transfer about 5 g of the powdered material, accurately weighed, to a glass-stoppered flask. Add 200 ml of a mixture of methanol and water (17 : 3). Shake vigorously by mechanical means for not less than 30 min and filter. [Note - If the solution has interfering plant pigments, proceed as directed for Test Solution 2]. Discard the first 50 ml of the filtrate and collect the next 40 ml portion. Transfer the filtrate to a separating funnel. Add 40 ml of sodium chloride solution and 25 ml of hexane and shake for 1 min. Allow the layers to separate and transfer the lower aqueous layer to a second separating funnel. Extract the aqueous layer in the separating funnel twice, each time with 25 ml of methylene chloride, by shaking for 1 min. Allow the layers to separate each time, separate the lower organic layer and remove the solvent from the combined and evaporate layers on a water bath. Cool the residue. If interferences exist in the

Add 50 ml of saturated sodium chloride and mix vigorously for 30 seconds. Add 50 ml hexane to the separating funnel and extract. Repeat extraction with hexane for another two times. Collect the hexane layer and pass the combined hexane layer through sodium sulphate. Collect the hexane and evaporate to dryness. Dissolve the residue in 25 ml hexane. Florisil column clean up: Use florisil solid phase extraction cartridges. Using bulb pipet transfer 2 ml of the hexane solution containing the pesticide residue in to the florisil cartridge. Elute with 12 ml of 15 per cent diethyl ether in hexane. Further elute with 12 ml of 50 per cent diethyl ether in hexane. Collect the elutes separately and 129  

Permissible Limit < 2 ppb < 5 ppb

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residue, proceed as directed for Cleanup Procedure; otherwise, dissolve the residue obtained above in 0.2 ml of a mixture of chloroform and acetonitrile (9.8 : 0.2) and shake by mechanical means, if necessary.

each for aflatoxins B1 and G1, 0.2 g/ml each for aflatoxins B2 and G2. Procedure: Separately apply 2.5, 5, 7.5 and 10 l of the Aflatoxin Solution and three 10 l applications of either Test Solution 1 or Test Solution 2 to a suitable thin-layer chromatographic plate coated with a 0.25 mm layer of chromatographic silica gel. Superimpose 5 l of the Aflatoxin Solution on one of the three 10 l applications of the Test Solution. Allow the spots to dry and develop the chromatogram in an unsaturated chamber containing a solvent system consisting of a mixture of chloroform, acetone and isopropyl alcohol (85 :10 : 5) until the solvent front has moved not less than 8 cm from the origin. Remove the plate from the developing chamber, mark the solvent front and allow the plate to airdry. Locate the spots on the plate by examination under UV light at 366 nm: the four applications of the Aflatoxin Solution appear as four clearly separated blue fluorescent spots; the spot obtained from the Test Solution that was superimposed on the Aflatoxin Solution is no more intense than that of the corresponding Aflatoxin Solution; and no spot from any of the other Test Solutions corresponds to any of the spots obtained from the applications of the Aflatoxin Solution. If any spot of aflatoxins is obtained in the Test Solution, the colour match the position of each fluorescent spot of the Test Solution with those of the Aflatoxin Solution to identify the type of aflatoxin present. The intensity of the aflatoxins spot, if present in the Test Solution, when compared with that of the corresponding aflatoxin in the Aflatoxin Solution will give an approximate concentration of aflatoxin in the Test Solution.

Test Solution 2: Collect 100 ml of the filtrate from the start of the flow and transfer to a 250 ml beaker. Add 20 ml of zinc acetate-aluminum chloride reagent and 80 ml of water. Stir and allow to stand for 5 min. Add 5 g of a suitable filtering aid, such as diatomaceous earth, mix and filter. Discard the first 50 ml of the filtrate, and collect the next 80 ml portion. Proceed as directed for Test Solution 1, beginning with “Transfer the filtrate to a separating funnel”. Cleanup Procedure: Place a medium-porosity sintered-glass disk or a glass wool plug at the bottom of a 10 mm x 300 mm chromatographic tube. Prepare slurry of 2 g of silica gel with a mixture of diethyl ether and hexane (3 : 1), pour the slurry into the column and wash with 5 ml of the same solvent mixture. Allow the absorbent to settle and add to the top of the column a layer of 1.5 g of anhydrous sodium sulphate. Dissolve the residue obtained above in 3 ml of methylene chloride and transfer it to the column. Rinse the flask twice with 1 ml portions of methylene chloride, transfer the rinses to the column and elute at a rate not greater than 1 ml per min. Add successively to the column 3 ml of hexane, 3 ml of diethyl ether and 3 ml of methylene chloride; elute at a rate not greater than 3 ml per min; and discard the eluates. Add to the column 6 ml of a mixture of methylene chloride and acetone (9 : 1) and elute at a rate not greater than 1 ml per min, preferably without the aid of vacuum. Collect this eluate in a small vial, add a boiling chip if necessary and evaporate to dryness on a water bath. Dissolve the residue in 2 ml of a mixture of chloroform and acetonitrile (9.8 : 0.2) and shake by mechanical means if necessary.

3.5. THIN-LAYER CHROMATOGRAPHY (TLC): Thin-layer chromatography is a technique in which a solute undergoes distribution between two phases, stationary phase and a mobile phase. The stationary phase acts as an adsorbent in a relatively thin, uniform layer of dry finely powdered material applied to a glass, plastic or

Aflatoxin Solution: Dissolve accurately weighed quantities of aflatoxins B1, B2, G1 and G2 in a mixture of chloroform and acetonitrile (9.8 : 0.2) to obtain a solution having concentration of 1.0 g/ml 130  

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metal sheet. Precoated plates are most commonly used. Separation may also be achieved on the basis of partition or a combination of partition and adsorption, depending on the particular type of stationary phase, its preparation and its use with different solvents.

and sealed. The chamber is fitted with a plate support rack that supports the plates, back to back, with lid of the chamber in place. (g) Graduated micro-pipettes capable of delivering microlitre quantities say 10 l and less. (h) A reagent sprayer that will emit a fine spray and will not itself be attacked by the reagent.

Identification can be effected by comparison of spots of identical Rf value and colour in unknown sample to a reference sample chromatographed on the same plate. A visual comparison of the size and intensity of the spots usually serves for semiquantitative estimation.

(i) An ultra-violet light, suitable for observation at short (254 nm) and long (366 nm) ultraviolet wavelengths. Preparation of plates: Unless otherwise specified in the monograph, the plates are prepared in the following manner. Prepare a suspension of the coating substance in accordance with the instructions of the supplier and, using the spreading device designed for the purpose, spread a uniform layer of the suspension, 0.20 to 0.30 mm thick, on a flat glass plate 20 cm long. Allow the coated plates to dry in air, heat at 1000 to 1050 for at least 1 hour (except in the case of plates prepared with cellulose when heating for 10 min is normally sufficient) and allow to cool, protected from moisture. Store the plates protected from moisture and use within 3 days of preparation. At the time of use, dry the plates again, if necessary, as prescribed in the monographs. Now a days pre coated plates of silica gel on glass/aluminium/ plastic sheets are also available.

Apparatus: (a) Flat uniformly thick glass plates of appropriate dimensions coated with a layer of adsorbent that allow the application of the necessary number of the solutions being examined along with reference solutions. The plates are prepared as described below; alternatively, commercially prepared plates may be used. (b) An aligning tray or a flat surface on which the plates can be aligned and rested when the coating substance is applied. (c) The coating substance consists of finely divided adsorbent materials, normally between 5 to 40 m in diameter is suitable for chromatography. It can be applied directly to the plate or can be bonded to the plate by means of plaster of paris (hydrated calcium sulphate) or with any other suitable binder. The adsorbent may contain fluorescing material to help in visualising spots that absorb ultra-violet light.

Method: Unless unsaturated conditions are prescribed, prepare the tank by lining the walls with sheets of filter paper; pour into the tank, saturating the filter paper in the process, sufficient of the mobile phase to form a layer of solvent 5 to 10 mm deep, close the tank and allow to stand for 1 hour at room temperature. Remove a narrow strip of the coating substance, about 5 mm wide, from the vertical sides of the plate. Apply the solutions being examined in the form of circular spots about 2 to 6 mm in diameter, or in the form of bands (10 to 20 mm x 2 to 6 mm unless otherwise specified) on a line parallel with, and 20 mm from, one end of the plate, and not nearer than 20

(d) A spreader which, when moved over the glass plate, will apply a uniform layer of adsorbent of desired thickness over the entire surface of the plate. (e) A storage rack to support the plates during drying and transportation. (f) A developing chamber that can accommodate one or more plates and can be properly closed 131  

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mm to the sides; the spots should be 15 mm apart. If necessary, the solutions may be applied in portions, drying between applications. Mark the sides of the plate 15 cm, or the distance specified in the monograph, from the starting line. Allow the solvent to evaporate and place the plate in the tank, ensuring that it is as nearly vertical as possible and that the spots or bands are above the level of the mobile phase. Close the tank and allow to stand at room temperature, until the mobile phase has ascended to the marked line. Remove the plate and dry and visualize as directed in the monograph; where a spraying technique is prescribed it is essential that the reagent be evenly applied as a fine spray.

plate with suitable instrumentation. Measurement is of the reflectance of the incident light from the spots by moving the plate or the measuring device. Likewise, fluorescence may be measured using an appropriate optical system. Apparatus: The apparatus measurement consist of:

direct

 a device for exact positioning and reproducible application of the amount of solutions onto the plate,  a mechanical device for moving the plate or the measuring device along the x-axis or the y-axis, (Applicator)  a recorder and a suitable integrator or a computer, and

For two-dimensional chromatography dry the plate after the first development and carry out the second development in a direction perpendicular to the first.

 a photometer with a source of light, an optical device for generating monochromatic light and a photocell of adequate sensitivity; for measurement of fluorescence, a suitable filter to prevent light used for excitation from reaching the detector while permitting emitted light or a specific portion thereof to pass (Densitometer)

When the method prescribed in the monograph specifies ‘protected from light’ or ‘in subdued light’ it is intended that the entire procedure is carried out under these conditions. Visualization:

Method: Prepare the test solution and reference solution as prescribed in the individual monograph. Use the same solvent for all the solutions and apply the same volume of each and develop the plate. Prepare and apply new fewer than 3 reference solutions of the substance under examination, the concentrations of which span the expected value in the test solution (about 80 per cent, 100 per cent and 120 per cent). Treat with the prescribed reagent, if necessary, and record the reflectance, the transmittance or fluorescence in the chromatograms obtained with all the solutions. Use the measured results to calculate the amount of substance in the test solution.

The phrases ultra-violet light (254 nm) and ultraviolet light (366 nm) indicate that the plate should be examined under an ultra-violet light having a maximum output at about 254 or at about 365 nm, as the case may be. The term secondary spot means any spot other than the principal spot. Similarly, a secondary band is any band other than the principal band. Rf Value: Measure and record the distance of each spot from the point of its application and calculate the Rf value by dividing the distance travelled by the spots by the distance travelled by the front of the mobile phase.

The requirement for resolution and separation are prescribed in the individual monograph.

3.5.1. Quantitative measurement The substances that have been separated after development of the plate and that respond to UVVis irradiation can be estimated directly on the 132  

for

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3.6. LIQUID CHROMATOGRAPHY:

absorbance depends on the concentration of the absorbing substance in the solution and the thickness of the absorbing layer taken for measurement.

Liquid chromatography is one of the widely used methods for separation and quantitative estimation of marker compounds present in herbal drugs. It is a liquid chromatographic system that uses narrow columns (~ 5 mm in diameter), pumping system operating at pressures up to 200 atm and suitable detectors. Reversed phase silica columns are widely used. A guard column is recommended to be fitted before the column to prevent the entry of unwanted compounds of the sample solution into the column. Sample introduction is done by syringe and a loop injector may be fitted with a fixed volume loop between 1-200l to facilitate accurate sample injection. Detection of the compound of interest is by retention time, UV absorbance fluorescence and electrical conductions. For majority of analyses, variable wavelength UV or photodiode array UV and RI detectors are used. Details of chromatographic conditions, e.g., column type, mobile phases, flow rats, detectors, etc. are given in detail in individual monographs. For accurate analysis, high purity reagents and HPLC grade solvents must be used.

For convenience of reference and for ease in calculations, the specific absorbance of a 1 per cent w/v solutions is adopted in this Pharmacopoeia for several substances unless otherwise indicated, and it refers to the absorbance of a 1 per cent w/v solution in a 1 cm cell and measured at a defined wavelength. It is evaluated by the expression. A (1 per cent, 1 cm) = A/cl Where c is the concentration of the absorbing substance expressed as percentage w/v and l is the thickness of the absorbing layers in cm. The value of A (1 per cent, 1 cm) at a particular wavelength in a given solvent is a property of the absorbing substance. Unless otherwise stated, measure the absorbance at the prescribed wavelength using a path length of 1 cm and at 240 to 260. Unless otherwise stated, the measurements are carried out with reference to the same solvent or the same mixture of solvents.

Columns

Determination of absorbance: Unless otherwise directed, measure the absorbance at the prescribed wavelength using a path length of 1 cm at 240 and 260. If necessary, the path length may be varied provided that compliance with Beer’s Law has been shown over the range in question.

1. Silica C 18 - Octadecyl silane chemically bonded to porous silica or ceramic particles. 2. Silica Nitrile- Nitrile groups chemically bonded to porous silica microparticles 3.7. Spectrophotometry

A statement in assay or test of the wavelength at which maximum absorption occurs implies that the maximum occurs either precisely at or within ± 2 nm of the given wavelength.

Ultraviolet and visible absorption spectrophotometry is the measurement of the absorption of monochromatic radiation by solutions of chemical substances, in the range of 185 nm to 380 nm, and 380 nm to 780 nm of the spectrum, respectively.

Likewise, a statement in a test of the absorbance, A, at a given wavelength or at the maximum at about a specified wavelength implies that the measured absorbance is within ± 3 per cent of the stated value.

The magnitude of the absorption of a solution is expressed in terms of the absorbance, A, defined as the logarithm to base 10 of the reciprocal of transmittance (T) for monochromatic radiation:

When an assay or test prescribes the use of a reference substance, make the spectrophotometric measurements with the solution prepared from the reference substance by the official directions and

A=log10 (I0/I) Where I0 is the intensity of the incident radiation. I is the intensity of the transmitted radiation. The 133  

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Stationary phases

then with the corresponding solution prepared from the substance under examination. Carry out the second measurement as quickly as possible after the first, using the same cell and same experimental conditions.

Stationary phases are contained in columns which may be:  a capillary column of fused-silica whose wall is coated with the stationary phase,

Unless otherwise specified, the requirements in the monographs for light absorption in the tests and assay apply to the dried or anhydrous material, where a standard is given for solvent content. In calculating the result, the loss on drying or contents of water solvent, determined by the method specified in the monograph, are taken in to account.

 a column packed with inert particles impregnated with the stationary phase,  a column packed with solid stationary phase. Capillary columns are 0.1 mm to 0.53 mm in internal diameter and 5 m to 60 m in length. The liquid or stationary phase, which may be chemically bonded to the inner surface, is a film 0.1m to 5.0m thick.

3.8. TEST FOR RESIDUAL SOLVENT: Residual ethanol limits: Not more than 5000 ppm

Packed columns, made of glass or metal, are usually 1 m to 3 m in length with an internal diameter of 2 mm to 4 mm. Stationary phases usually consist of porous polymers or solid supports impregnated with liquid phase.

Quantitative analysis of residual solvents: Gas chromatography (GC) is a chromatographic separation technique based on the difference in the distribution of species between two non-miscible phases in which the mobile phase is a carrier gas moving through or passing the stationary phase contained in a column. It is applicable to substances or their derivatives which are volatilised under the temperatures employed.

Supports for analysis of polar compounds on columns packed with low-capacity, low-polarity stationary phase must be inert to avoid peak tailing. The reactivity of support materials can be reduced by silanising prior to coating with liquid phase. Acid-washed, flux-calcinated diatomaceous earth is often used. Materials are available in various particle sizes, the most commonly used particles are in the ranges of 150 m to 180 m and 125 m to 150 m.

GC is based on mechanisms of adsorption, mass distribution or size exclusion. Apparatus: The apparatus consists of an injector, a chromatographic column contained in an oven, a detector and a data acquisition system (or an integrator or a chart recorder). The carrier gas flows through the column at a controlled rate or pressure and then through the detector.

Mobile phases: Retention time and peak efficiency depend on the carrier gas flow rate; retention time is directly proportional to column length and resolution is proportional to the square root of the column length. For packed columns, the carrier gas flow rate is usually expressed in millilitres per min at atmospheric pressure and room temperature. Flow rate is measured at the detector outlet, either with a calibrated mechanical device or with a bubble tube, while the column is at operating temperature. The linear velocity of the carrier gas through a packed column is inversely proportional to the square root of the internal diameter of the column for a given flow volume.

The chromatography is carried out either at a constant temperature or according to a given temperature programme. Injectors: Direct injections of solutions are the usual mode of injection, unless otherwise prescribed in the monograph. Injection may be carried out either directly at the head of the column using a syringe or an injection valve, or into a vaporisation chamber which may be equipped with a stream splitter. 134  

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Flow rates of 60 ml/min in a 4 mm internal diameter column and 15 ml/min in a 2 mm internal diameter column, give identical linear velocities and thus similar retention times.

reference is made to a particular commercial brand that has been found to be suitable for the purpose, but such statements do not imply that a different but equivalent commercial brand may not be used.

Helium or nitrogen is usually employed as the carrier gas for packed columns, whereas commonly used carrier gases for capillary columns are nitrogen, helium and hydrogen.

The chromatography is carried out either at a constant temperature or according to a given temperature programme.

Detectors: Flame-ionisation detectors are usually employed but additional detectors which may be used include: electron-capture, nitrogenphosphorus, mass spectrometric, thermal conductivity, Fourier transform infrared spectrophotometric, and others, depending on the purpose of the analysis.

Test solution: Place in the round bottom flask, accurately weigh about 1 g, of the substance being examined, dissolve in 15 ml of dimethylformamide. Heat the flask and collect the exactly 10 ml of distillate in a graduated cylinder. Cooling by circulating water is essential. Measure and record the volume.

Method: Equilibrate the column, the injector and the detector at the temperatures and the gas flow rates specified in the monograph until a stable baseline is achieved. Prepare the test solution(s) and the reference solution(s) as prescribed. The solutions must be free from solid particles.

Standard preparation ethanol: Prepare 500ppm of ethanol in dimethylformamide separately.

Performance: Criteria for assessing the suitability of the system are described in the chapter on Chromatographic separation techniques. The extent to which adjustments of parameters of the chromatographic system can be made to satisfy the criteria of system suitability are also given in this chapter.

Column with stationary phase: A fused-silica capillary column 30 m long and 0.25 or 0.32 or 53 mm in internal diameter coated with cross-linked 6 per cent polycyanopropylphenylsiloxane and 94 per cent polydimethylsiloxane having film thickness: 1.4 m, 1.8 m or 3 m .

Analytical procedure:

Chromatographic condition: Detector: Flame ionization detector

Temperature: Coloumn 340 to 1000 @ 150/min., then increase to 1800 @ 250/min then increase to 2250 @ 400/min. Injection port temperature 2500; detector temperature 2750.

Reagents: Solvents and reagents used in the preparation of solutions for examination should be of a quality suitable for use in gas chromatography. A wide range of chemical substances is used as stationary phases, including polyethylene glycols, high-molecular weight esters and amides, hydrocarbons, silicone gums and fluids (polysiloxanes often substituted with methyl, phenyl, nitrilo, vinyl or fluoroalkyl groups or mixtures of these) and microporous cross-linked polyaromatic beads. A suitable stationary phase, its concentration and the nature and grade of a suitable solid support are stated in the monograph. The column should be conditioned in accordance with the manufacturer’s instructions. In most cases

Carrier Gas: Nitrogen for chromatography at an appropriate flow Procedure: Inject 1 l standard solution and record the chromatogram. In the chromatogram obtained with test solution. If there is any peak corresponding to ethanol the peak area is not greater than the peak area in the chromatogram obtained with standard solution for ethanol.

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3.9. Stability Testing and Shelf Life Determination for New and Existing Ayurvedic Drugs

store the samples of same batch material at standard storage and accelerated storage conditions and test them periodically. Based on the evaluation of the results, the expiry date or shelf life may be determined.

(This guideline is not limited only to ASU extracts covered under this volume of API. It shall be applicable to all the licensed ASU medicines)

The second approach is to select samples from batches manufactured over a period of last five years spanning six months and evaluate them simultaneously. Based on the result obtained the expiry date or shelf life may be determined. This approach is applicable for existing products which do not have yet a declared shelf life. This approach has been referred in scientific literature as the “cross sectional approach”.

3.9.1 Scope and Objective The objective of this guideline is to specify the method of arriving at shelf life by stability testing. The shelf life determined by the process mentioned in this guideline can be used to decide the expiry date, in case a manufacturer wishes to assign a shelf life longer than one specified by the notification GSR 764(E) dated October 15, 2009.

3.9.3 Selection of batches

The guideline can be used for all patented and proprietary Ayurvedic medicines, both new and existing products.

Formal stability studies should be conducted on at least three primary batches. The primary batches should be of the same formulation as proposed for marketing. For new products, the batches should be manufactured to a minimum of pilot scale by the same route as, and using a method of manufacture and procedure that simulates the final process to be used for production batches. Pilot batches which are at least 1/10 of the commercial batch size can be used. The overall quality of the batches of drug placed in formal stability studies should be representative of the quality of the material made on production scale. Where possible, batches of drug product should be manufactured by using different batches of drug substance. Stability to be performed on each individual strength and container size of the product unless bracketing and matrixing is applied.

3.9.2 General Information on Stability Information of shelf life (expiry date) is mandatory requirement for all licensed Ayurvedic medicines. The stability depends on various factors like the nature of the product, the ingredients of the products, the packaging material etc. Stability studies are carried out to demonstrate that the medicine will remain suitable for consumption during shelf period when stored under the condition(s) mentioned on the packaging. On the product label, if there is no mention about any specific storage condition, then it is assumed that the product can be stored at room temperature (below 30o). For a suitable drug substance, retest period is more appropriate than expiry date.

For cross sectional approach at least two batches per year to be selected. For example if stability to be evaluated for four years eight batches should be selected.

The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of variety of environmental factors such as temperature, humidity, and light, to establish a retest period for drug substance or a shelf life for drug products.

3.9.4 Container and closure system The stability studies should be conducted on the dosage form packaged in the container and closure system proposed for marketing (including as appropriate, any secondary packaging and container

Two approaches can be followed to monitor the stability of the product. The first approach is to 136  

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label). If the container is too large for drug substances the stability studies should be conducted in a container and closure system that is the same as or simulates the packaging proposed for storage and distribution.

formulation (except for preservative concentration) intended for marketing. 3.9.6 Testing frequency For long term studies frequency of testing should be sufficient to establish the stability profile of the drug. For drug with proposed shelf life of at least 12 months, the frequency of testing at long term storage condition should normally be every 6 months over first year, and the second year and annually thereafter through the proposed re-test period or shelf life.

3.9.5 Specification Specification is a list of tests, reference to analytical procedures and proposed acceptance criteria. Stability study should include testing of those attributes of the drug that are susceptible to change during storage and are likely to influence quality, safety, and/or efficacy. The testing should cover as appropriate, the physical, chemical, biological, and microbiological attributes. Validated stability-indicating analytical procedures should be applied. Whether and to what extent replication should be performed will depend on the results from validation studies.

At the accelerated storage condition, a minimum of three time points including the initial and final time points (e.g. 0, 3 and 6 months) from a 6 month study is recommended. Reduced designs i.e., matrixing or bracketing, where the testing frequency is reduced or certain factor combinations are not tested at all, can be applied if justified.

The physical parameters included in the specification need not be limited to colour, odour, appearance, shape and taste only. The chemical parameters should include colour reaction, pH value, weight variation, disintegration, bulk density, extractive values, estimation of active or marker or category compound by suitable methods and chromatographic profiling. A suitable bioassay may be employed wherever possible.

3.9.7 Storage condition The world can be divided in to four climatic zones I - IV. This guideline address zone IV. The choice of test conditions defined in this guideline is based on an analysis of the effects of climatic conditions in the zone. Recommended storage conditions are Study

The limits of acceptance for the products should be those specified in pharmacopoeia. If limits are not available these should be derived from release specification. Shelf life acceptance criteria should be derived from consideration of all available stability information. It may be appropriate to have justifiable differences between the shelf life and release acceptance criteria based on the stability evaluation and the changes observed on storage. Any differences between the release and shelf life acceptance criteria for antimicrobial preservative content should be supported by a validated correlation of chemical content and preservative effectiveness demonstrated during development of the product in its final

Accelerated Long term

Minimum time 6 months 12 months

Other storage conditions are allowable if suitably justified. For products which are temperature sensitive, to be stored in lower temperature which will then become the condition designated long term storage temperature. The accelerated testing should be then carried out at least 100 more than the long term storage condition along with appropriate relative humidity condition for that temperature. The reference samples for the above study should be stored in a temperature less than 10o. 137

 

Storage condition 40o ± 2o/ 75 % RH ± 5 % 30o ± 2o/ 60 % RH ± 5 %

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3.9.8 Evaluation The purpose of stability is to establish, based on testing a minimum of at least three batches of the drug, a retest period applicable to all future batches for the drug substance, or a shelf life and label storage instructions applicable for all future batches of the drug product manufactured and packed under similar circumstances. An Ayurvedic drug can be considered to be stable if “no significant change” occurs during at any time of testing at accelerated storage condition or at real time storage condition. “Significant change” for a drug is defined as 1. A + or - 20 per cent change from the initial assay value (If the drug is analyzed for its marker). A + or - 15 per cent change from the initial assay value (If the drug is analyzed for its active compound). 2. Appearance of new spots in Identification by TLC (when compared with the sample stored in less than 10o) or completely disappearance of existing spot. 3. The physico-chemical parameters (moisture, ash, particle size) shall not vary beyond 25 per cent of the initial value. 4. Failure to meet the acceptance criteria as per individual monographs or specification. Failure to meet acceptance criteria for appearance (Physical attributes, and functionality tests e.g., colour, phase separation, caking, hardness).

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APPENDIX - 4 REAGENTS AND CHEMICALS  Acetic Acid - Contains approximately 33 per cent w/v of C2H4O2. Dilute 315 ml of glacial acetic acid to 1000 ml with water.

liberated iodine with 0.1 N sodium thiosulphate, using starch solution as indicator. Not less than 1 ml of 0.1 N sodium thiosulphate is required.

Acetic Acid, Glacial - CH3COOH =60.05

Odorous impurities - Neutralise 1.5 ml with sodium hydroxide solution; the solution has no odour other than a faint acetous odour.

Contains not less than 99.0 per cent w/w of C2H4O2. About 17.5 N in strength

Readily oxidisable impurities - To 5 ml of the solution prepared for the test for formic acid and oxidisable impurities, add 20 ml of water and 0.5 ml of 0.1 N potassium permanganate; the pink colour does not entirely disappear within half a min.

Description - At temperature above its freezing point a clear colourless liquid, odour, pungent and characteristic; crystallises when cooled to about 100 and does not completely re-melt until warmed to about 150. Solubility - Miscible with water, with glycerin and most fixed and volatile oils.

Non-volatile matter - Leaves not more than 0.01 per cent w/w of residue when evaporated to dryness and dried to constant weight at 1050.

Boiling range - Between 1170 and 1190 Congealing temperature - Not lower than 14.80

Assay - Weigh accurately about 1 g into a stoppered flask containing 50 ml of water and titrate with N sodium hydroxide, using phenolphthalein solution as indicator. Each ml of sodium hydroxide is equivalent to 0.06005 g of C2H4O2.

Wt. per ml - At 250about 1.047 g Heavy metals - Evaporate 5 ml to dryness in a porcelain dish on water-bath, warm the residue with 2 ml of 0.1 N hydrochloric acid and water to make 25 ml; the limit of heavy metals is 10 parts per million, Appendix 2.3.3.

Acetic Acid, Lead-Free - Acetic acid which complies with following additional test, boil 25 ml until the volume is reduced to about 15 ml, cool make alkaline with lead-free ammonia solution, add 1 ml of lead free potassium cyanide solution, dilute to 50 ml with water, add 2 drops of sodium sulphide solution; no darkening is produced.

Chloride - 5 ml complies with the limit test for chlorides, Appendix 2.3.2. Sulphate - 5 ml complies with the limit test for sulphates, Certain aldehydic substances - To 5 ml add 10 ml of mercuric chloride solution and make alkaline with sodium hydroxide solution, allow to stand for five min and acidify with dilute sulphuric acid; the solution does not show more than a faint turbidity.

Acetone - Propan-2-one; C3H6O = 58.08 (67-64-1)

Analytical reagent grade of commerce. A volatile, flammable liquid; boiling point, about 560; weight per ml, about 0.79 g. Complies with the following test. Water Not more than 0.3 per cent w/w, Appendix IX C, using anhydrous pyridine as the solvent.

Formic acid and oxidisable impurities - Dilute 5 ml with 10 ml of water, to 5 ml of this solution add 2.0 ml of 0.1 N potassium dichromate and 6 ml of sulphuric acid, and allow to stand for one min, add 25 ml of water, cool to 150, and add 1 ml of freshly prepared potassium iodide solution and titrate the

Acetonitrile - Methyl Cyanide; CH3CN = 41.05 General laboratory reagent grade of commerce 139

 

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Colourless liquid; bp about 810; wt. per ml, about 0.78 g Acetonitrile intended for use spectrophotometry complies with following test:

Foreign organic substances - Clean a glassstoppered cylinder thoroughly with hydrochloric acid, rinse with water and finally rinse with the alcohol under examination. Put 20 ml in the cylinder, cool to about 150 and then add from a carefully cleaned pipette 0.1 ml of 0.1 N potassium permanganate. Mix at once by inverting the stoppered cylinder and allow to stand at 150 for five min; the pink colour does not entirely disappear.

in the

Transmittance: not less than 98 per cent in the range 255 to 420 nm using water as the blank Alcohol Description - Clear, colourless, mobile, volatile liquid, odour, characteristic and spirituous; taste, burning, readily volatilised even at low temperature, and boils at about 780, flammable. Alcohol containing not less than 94.85 per cent v/v and not more than 95.2 per cent v/v of C2H5OH at 15.560.

Isopropyl alcohol and t-butyl alcohol - To 1 ml add 2 ml of water and 10 ml of mercuric sulphate solution and heat in a boiling waterbath; no precipitate is formed within three min. Aldehydes and ketones - Heat 100 ml of hydroxylamine hydrochloride solution in a loosely stoppered flask on a water-bath for thirty min, cool, and if necessary, add sufficient 0.05 N sodium hydroxide to restore the green colour. To 50 ml of this solution add 25 ml of the alcohol and heat on a water bath for ten min in a loosely stoppered flask. Cool, transfer to a Nesseler cylinder, and titrate with 0.05 N sodium hydroxide until the colour matches that of the remainder of the hydroxylamine hydrochloride solution contained in a similar cylinder, both solutions being viewed down the axis of the cylinder. Not more than 0.9 ml of 0.05 N sodium hydroxide is required.

Solubility - Miscible in all proportions with water, with chloroform and with solvent ether Acidity or alkalinity - To 20 ml add five drops of phenolphthalein solution; the solution remains colourless and requires not more than 2.0 ml of 0. 1 N sodium hydroxide to produce a pink colour. Specific gravity - Between 0.8084 and 0.8104 at 250 Clarity of solution - Dilute 5 ml to 100 ml with water in glass cylinder; the solution remains clear when examined against a black background. Cool to 100 for thirty min; the solution remains clear.

Fusel oil constituents - Mix 10 ml with 5 ml of water and 1 ml of glycerin and allow the mixture to evaporate spontaneously from clean, odourless absorbent paper; no foreign odour is perceptible at any stage of the evaporation.

Methanol - To one drop, add one of water, one drop of dilute phosphoric acid, and one drop of potassium permanganate solution. Mix, allow to stand for one min and add sodium bisulphite solution dropwise, until the permanganate colour is discharged. If a brown colour remains, add one drop of dilute phosphoric acid. To the colourless solution add 5 ml of freshly prepared chromotropic acid solution and heat on a water-bath at 600 for ten min; no violet colour is produced.

Non-volatile matter - Evaporate 40 ml in a tared dish on a water-bath and dry the residue at 1050 for one hour; the weight of the residue does not exceed 1 mg. Storage - Store in tightly-closed containers, away from fire. Labelling - The label on the container states “Flammable”. 140

 

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Alcohol, Aldehyde-free - Alcohol which complies with the following additional test:

proportionately lesser amounts of reagents or by appropriate dilution.

Aldehyde - To 25 ml, contained in 300 ml flask, add 75 ml of dinitrophenyl hydrazine solution, heat on a water bath under a reflux condenser for twenty four hours, remove the alcohol by distillation, dilute to 200 ml with a 2 per cent v/v solution of sulphuric acid, and set aside for twenty four hours; no crystals are produced.

Butan-1-ol - n-Butyl alcohol; butanol; C4H10O = 74.12 (71-36-3). Analytical reagent grade of commerce. A colourless liquid; boiling point, 1160 to 1190; d20 20 , about 0.81 Butan-1-ol - n-Butyl alcohol; butanol; C4H10O = 74.12 (71-36-3)

Alcohol, Sulphate-free - Shake alcohol with an excess of anion exchange resin for thirty min and filter.

Analytical reagent grade of commerce. A colourless liquid; boiling point, 1160 to 1190; d20 20 , about 0.81

Ammonia solution Sp. - Strong ammonia solution which complies with the following additional test.

Chloroform - Trichloromethane; CHCl3 = 119.4 (67-66-3)

Evaporate 10 ml to dryness on a water-bath. To the residue add 1 ml of dilute hydrochloric acid Sp. and evaporate to dryness. Dissolve the residue in 2 ml of dilute acetic acid Sp., add sufficient water to produce 25 ml and add 10 ml of hydrogen sulphide solution. Any darkening produced is not greater than that of a blank solution containing 2 ml of dilute acetic acid Sp., 1.0 ml of standard lead solution and sufficient water to produce 25 ml.

Analytical reagent grade of commerce containing 0.4 to 1.0 per cent w/w of ethanol A colourless liquid with a sweet, penetrating odour; boiling point, about 600; d20 20 1.475 to 1.481 Citric Acid - C6H8O7,H2O = 210.1 (5949-29-1). Analytical reagent grade of commerce. When used in the limit test for iron, complies with the following requirement:

0.1 M ammonia - Solution of any molarity x M may be prepared by diluting 75 x ml of strong ammonia solution to 1000 ml with water

Dissolve 0.5 g in 10 ml of water, add 0.1 ml of mercaptoacetic acid, mix, make alkaline with 10 M ammonia and add sufficient water to produce 20 ml. No pink colour is produced.

Anisaldehyde Sulphuric Acid Reagents - Mix in the following order 0.5 ml of anisaldehyde, 10 ml of glacial acetic acid, 85 ml of methanol and 5 ml of sulphuric acid.

Ether - C4H10O = 74.12 (60-29-7)

Bismuth nitrate - Analytical reagent grade

Analytical reagent grade of commerce.

Bromine ‐ Br2 = 159.8 (7726-95-6).

A volatile, highly flammable, colourless liquid; boiling point, 340 to 350; d20 20 0.7 13 to 0.7 15. Do not distil unless the ether complies with the following test for peroxides.

Analytical reagent grade of commerce. A heavy, brownish-red, fuming liquid, highly corrosive to the skin; d20 20 about 3.1.

Peroxides: Place 8 ml of potassium iodide and starch solution in a 12 ml ground-glass stoppered cylinder about 1.5 cm in diameter. Fill completely with the reagent being

To prepare 0.05 M bromine dissolve 3 g of potassium bromate and 15 g of potassium bromide in sufficient water to produce 1000 ml. Weaker solutions should be prepared using 141  

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examined, shake vigorously and allow to stand in the dark for 30 min. No colour is produced.

Strength per cent v/v 90 85 80 70 65 60 50 45 25 20 10

Store protected from light at a temperature not exceeding 150. The name and concentration of any added stabiliser are stated on the label. 1,4-Dioxane - 1,4-Dioxan; Diethylene Dioxide: C4H8O2 = 88.11 Analytical reagent grade of commerce. Colourless liquid with an ethereal odour; bp, about 1010; wt. per ml, about 1.03 g. Do not distil unless the dioxan complies with the test for peroxides. Peroxides: Place 8 ml of starch iodide solution in a 12 ml glass-stopperred cylinder about 1.5 cm in diameter. Fill completely with the reagent under examination, shake vigorously and allow to stand protected from light for 30 min; no colour is produced.

Volume of Ethanol (96 per cent) (Approx.) ml 934 885 831 727 676 623 519 468 259 207 104

0.83 0.85 0.86 0.89 0.90 0.91 0.93 0.94 0.97 0.975 0.986

Ethyl Acetate - C4H8O2 = 88.1 (14 1-78-6) Analytical reagent grade of commerce A colourless liquid with a fruity odour; boiling point, about 760 to 780; d20 20 0.90 1 to 0.904

Dragendorff’s Reagent

Solution B - A solution is made of 8 g potassium iodide in 20 ml water.

Folin Ciocalteu Reagent - Dilute commercially available Folin-Ciocalteu reagent (2N) with an equal volume of distilled water. Transfer it in a brown bottle and store in a refrigerator (40). It should be golden in colour. Do not use it if it turns olive green.

Stock solution - Equal volumes of solution A and B are mixed (Can be stored for a long time in dark glasses vessels)

Phosphomolybdotungstic Reagent (Folins reagent) Folin Ciocalteau phenol reagent of commerce.

Spray reagent - 1ml Stock solution is mixed with 2 ml glacial acetic acid and 10 ml water before use.

Dissolve 100 g of sodium tungstate and 25 g of sodium molybdate in 700 ml of water, add 100 ml of hydrochloric acid and 50 ml of orthophosphoric acid and heat the mixture under a reflux condenser for 10 hours. Add 150 g of lithium sulphate, 50 ml of water and 0.2 ml of bromine and boil to remove excess bromine (about 15 min), cool, dilute to 1000 ml with water and filter. The reagent should be yellow in colour. If it acquires a greenish tint, it is unsatisfactory for use but may be regenerated by boiling with 0.2 ml of bromine. Care must be taken to remove excess bromine by boiling.

Solution A - 0.85 g Basic Bismuth nitrate is dissolved in a mixture of 10 ml glacial acetic acid and 40 ml water.

Ethanol (96 per cent) - Alcohol - C2H6O. Analytical reagent grade ethanol of commerce containing not less than 95.1 per cent v/v and not more than 96.9 per cent v/v of C2H6O. A colourless liquid; weight per ml, about 0.81 g Diluted ethanols may be prepared by diluting the volumes of ethanol (96 per cent) indicated in the following table to 1000 ml with water.

Store at 20 to 80. 142  

Weight per ml (g)

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Analytical reagent grade of commerce containing not less than 84 per cent w/w of H3 PO4 and about 15.7 M in strength.

Formic Acid, Anhydrous - CH2O2 = 46.03 Analytical reagent grade formic acid of commerce containing not less than 98.0 per cent w/w of CH2O2.

A corrosive liquid; wt. per ml, about 1.75 g Potassium Dihydrogen Orthophosphate Potassium dihydrogen phosphate; KH2PO4 = 136.1 (7778-77-0)

A colourless, corrosive liquid with a pungent odour; d20 20 about 1.22 Assay: Weigh accurately a conical flask containing 10 ml of water, quickly add about 1 ml of the reagent and weigh again. Add 50 ml of water and titrate with 1M sodium hydroxide vs using 0.5 ml of phenolphthalein solution as indicator. Each ml of 1M sodium hydroxide VS is equivalent to 46.03 mg of CH2O2.

Analytical reagent grade of commerce, Colourless crystals Potassium Iodide - KI = 166.0 (7681-11-0) Analytical reagent grade of commerce, A white, crystalline powder Sodium Carbonate - Na2CO3,10H2O = 286.2 (6132-02-1)

Hydrochloric Acid - HCl = 36.46 (7647-01-0) Where no molarity is indicated use analytical reagent grade of commerce with a relative density of about 1.18, containing not less than 35 per cent w/w and not more than 38 per cent w/w of HCl and about 11.5 M in strength.

Analytical reagent grade of commerce. Melting point, greater than 3000. Sodium Molybdate - Na2MoO4,2H2O = 242.0 (10 102-40-6)

A colourless, fuming liquid

Analytical reagent grade of commerce

Solutions of molarity x M should be prepared by diluting 85x ml of hydrochloric acid to 1000 ml with water. Store in a container of polyethylene or other non-reacting material at a temperature not exceeding 300.

Sodium Tungstate - Na2WO4,2H2O = 329.9 (10213-10-2)

Lithium Sulphate - Li2SO4,H2O = 128.0 (10102-25-7)

When no molarity is indicated use analytical reagent grade of commerce containing about 96 per cent w/w of sulphuric acid and about 18 M in strength; an oily, corrosive liquid; wt. per ml, about 1.84 g.

Analytical reagent grade of commerce Sulphuric Acid - H2SO4 = 98.08 (7664-93-9)

Analytical reagent grade of commerce. Methanol - Methyl alcohol; CH4O = 32.04 (67-56-1) Analytical reagent grade of commerce

When solutions of molarity xM are required, they should be prepared by carefully adding 54x ml of sulphuric acid to an equal volume of water and diluting to 1000 ml with water.

A colourless liquid; boiling point, 640 to 650; d20 20 0.79 1 to 0.793 When ‘methanol’ is followed by a percentage figure, an instruction to use methanol diluted with water to produce the specified percentage v/v of methanol is implied.

When ‘sulphuric acid’ is followed by a percentage figure, an instruction to add, carefully, sulphuric acid to water to produce the specified percentage v/v (or, if required, w/w) proportion of sulphuric acid is implied.

Orthophosphoric Acid - Phosphoric acid; H3PO4 = 98.00 (7664-38-2)

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API, Part-I, Vol.-IX (Extracts); Appendices

Methanolic Sulphuric Reagent

Vanillin- 4-Hydroxy-3 - methoxybenzaldehyde; C8H8O3 = 152.2 (121-33-5)

Add dropwise 10 ml of sulphuric acid to 90 ml of ice- cold methanol.

Analytical reagent grade of commerce.

Sodium Sulphate, Anhydrous - Na2SO4 = 142.0 (7757-82- 6)

White to yellowish white, needles or crystalline powder, with an odour of vanilla.

Analytical reagent grade of commerce complying with the following test.

Melting point, about 810, determined without previous drying

Loss on drying: When dried at 1300, loses not more than 0.5 per cent of its weight.

Vanillin Sulphuric Acid Reagent Dissolve 1g of vanillin in 90 ml of ethanol; add drop wise 10 ml of sulphuric acid to ice- cold ethanol.

Sodium Carbonate Solution 30 g of sodium carbonate is dissolved in water and make up to 100 ml.

Water HPLC Grade Ultra-pure water

Tetrahydrofuran - Tetramethylene oxide; C4H8O = 72.11 (109-99-9)

Water, Carbon Dioxide-free

Analytical reagent grade of commerce.

Water, carbon dioxide-free: Water that has been boiled vigorously for a few min and protected from the atmosphere during cooling and storage

A clear, colourless, flammable liquid; boiling point, about 660; d20 20 about 0.89 Do not distil unless it complies with the following test: Peroxides: Place 8 ml of potassium iodide and starch solution in a ground-glass-stoppered cylinder with a capacity of 12 ml and about 1.5 cm in diameter and add sufficient of the substance being examined to fill the cylinder completely, shake vigorously and allow to stand for 30 min protected from light. No colour is produced. Tetrahydrofuran used in spectrophotometry complies with the following additional requirement. Transmittance not less than 20 per cent at 255 nm, 80 per cent at 270 nm and 98 per cent at 310 nm determined using water in the reference cell. Toluene - Methylbenzene; C7H8 = 92.14 (108-88-3) Analytical reagent grade of commerce A colourless liquid with a characteristic odour; wt. per ml, 0.865 to 0.870 g; boiling point, about 1100 144  

API, Part-I, Vol.-IX (Extracts); Appendices

APPENDIX - 5 WEIGHTS AND MEASURES  5.1.

Metric Equivalents of Classical Weights and Measures

The following table of metric equivalents of weights and measures, linear measures and measurement of time used in Classical Unit the Ayurvedic classics have been approved by the Ayurvedic Pharmacopoeia committee in consultation with Indian Yavodara Standards Institution. A´gula I. WEIGHTS AND MEASURES Classical Unit Metric Equivalent 1 Ratt¢ or Guμj¡ = 125 mg 8 Ratt¢ or Guμj¡ = 1 M¡Àa =1g 12 MaÀas = 1 KarÀa (Tol¡) = 12 g 2 KarÀas = 1 áukti = 24 g 2 áukti = 1 Palam = 48 g 2 Palas = 1 Pras¤ti = 96 g 2 Pras¤tis = 1 Ku·ava = 192 g 2 Ku·avas = 1 M¡nik¡ = 384 g 2 M¡nik¡s = 1 Prastha = 768 g 4 Prasthas = 1 Ë·haka = 3 kg 72 g 4 Ë·hakas = 1 Dro¸a = 12 kg 288 g 2 Dro¸as = 1 á£rpa = 24 kg 576 g 2 á£rpas =1 Dro¸¢ (V¡h¢) = 49 kg 152 g 4 Dro¸¢s = 1 Kh¡r¢ = 196 kg 608 g 100 Palas = 1 Tul¡ = 4 kg 800 g 20 Tul¡s = 1 Bh¡ra = 96 kg In case of liquids, the metric equivalents would be the corresponding litre and milliliter.

II. LINEAR MEASURES

Vitasti Aratni Hasta N¤pahasta (R¡jahasta) Vy¡ma

Metric Equivalent

1/8 of 3/4" 3/4"

0.24 cm 1.95 cm

9" 10 1/2" 18" 22"

22.86 cm 41.91 cm 45.72 cm 55.88 cm

72"

182.88 cm

III. MEASUREMENT OF TIME Unit

Equivalent (in hours, min & seconds)

2 KÀa¸as 2 Lavas 3 NimeÀas 1 Gha¶¢s 30 K¡À¶h¡s

= 1 Lava = 1 NimeÀa = 1 K¡À¶h¡

20 Kal¡s + 3 K¡À¶h¡s 30 Muh£rtas 15 Ahor¡tras 2 PakÀas 2 M¡sas

= 1 Muh£rta

3 Îtus 2 Ayanas 5 SaÆvatsara 1 Ahor¡tra of Devas 1 Ahor¡tra of Pitaras

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Inches

= 1 Kal¡

= 1 Ahor¡tra = 1 PakÀa = 1 M¡sa = 1 Îtu = 1 Ayana = 1 SaÆvatsara = 1Yuga

= 4.66 seconds = 24 min = 2 min 20 seconds = 48 min = 24 hours = 15 days = 30 days/1 month = 60 days/ Two Months = 6 Months = 12 months/1 Year = 5 Years = 1 Year = 1 Month

API, Part-I, Vol.-IX (Extracts); Appendices

5.2.

Metric System

Measures of Mass (Weights) 1 Kilogram (Kg) 1 Gram (g) 1Milligram (mg) 1 Microgram (g)

‐ ‐ ‐

is the mass of the International Prototype Kilogram the 1000th part of 1 Kilogram the 1000th part of 1 gramme the 1000th part of 1 milligram

Measures of capacity (Volumes) 1 Litre (1) is the volume occupied at its temperature of maximum density by a quantity of water having a mass of 1 Kilogram. 1 Millilitre (ml) is the 1000th part of 1 litre. The accepted relation between the litre and the cubic centimetre is 1 litre - 1000.027 cubic centimeters. Relation of capacity of Weight (Metric) One litre of water at 200 weighs 997.18 grams when weighed in air of density 0.0012 gram per millilitre against brass weights of density 84 grams per millilitre. Measures of Length 1 Metre (m) is the length of the International Prototype Metre at 0. ‐ 1 Centimetre (cm) the 100th part of 1 metre ‐ 1 Millimetre (mm) the 1000th part of 1 metre ‐ the 1000th part of 1 millimetre 1 Micron () the 1000th part of micron 1 Milliimicron (m) ‐  

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