3.1 OBJECTIVE

L-rhamnose, D-glucuronic acid ( Saponin A). Saponin B is the ß-D-galactopyranosyl ester of Saponin A (Hariharan and Rangaswami, 1970). Chapter-3 Gener...

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Chapter-3 3.1

General phytochemical analysis

OBJECTIVE

To perform proximate and phytochemical analysis for the collected plants Scoparia dulcis Linn. and Achyranthes aspera Linn. 3.2

INTRODUCTION

Phytochemicals can be defined as the chemicals that are produced by plants. Currently the term is being used only for those plant chemicals that may have healthrelated effects but are not considered essential nutrients (proteins, carbohydrates, fats, minerals, and vitamins). Plants have valuable source of natural products for maintaining human health, especially in the past, with more intensive studies for natural therapies. The use of plant compounds for pharmaceutical purposes has gradually increased in Brazil and other countries. According to World Health Organization (WHO) medicinal plants would be the best source to obtain a variety of drugs (Ellof, 1998). At the same time the usage of herbal drugs must and should be supported by analytical methods and techniques relevant to the extraction, separation, purification, identification, qualification and quantification of substances in the medicinal plants, which would give more justification for its intended usage. Medicinal plants contain components of therapeutic values, hence they are used as remedies for human diseases. Some of them are also used for prophylactic purposes. An increasing interest in herbal remedies has been observed in several parts of Nigeria and many of the herbal remedies have been incorporated into orthodox medicinal plant practice (Nostro et al, 2000). Many disease including malaria, epilepsy, infantile convulsion, diarrhea, dysentery, fungal and bacterial infections are being managed traditionally using medicinal plants for many years and now these systems require screening for their defined properties (Sofowora, 1996). 3.3

LITERATURE REVIEW

Achyranthes aspera Linn. : Achyranthes aspera Linn. seeds contain the compounds saponins A and B. They are glycosides of oleanolic acid. The carbohydrate components are the sugars D-glucose, L-rhamnose, D-glucuronic acid ( Saponin A). Saponin B is the ß-D-galactopyranosyl ester of Saponin A (Hariharan and Rangaswami, 1970).

25

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Figure-3.1: Photograph of plant Scoparia dulcis Linn.

Figure-3.2: Photograph of plant Achyranthes aspera Linn.

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The chemical constituents of Achyranthes aspera Linn. are triterpenoid saponins possessing oleanolic acid as aglycone, viz. A, B, C and D as major chemical constituents. Other constituents of the plant are ecdysterone, long chain alcohol, viz. 17-penta triacontanol, 27-cyclohexyl heptaeosan-7-ol, 16-hydroxyl 26-methyl heptacosan-2one and 36, 47-dihydroxy hen-pentacontan-4one. It also contains a water soluble base, betaine. Different chemical constituents reported by different scientist are Saponins from alcoholic extract of defatted seeds (Gopalanchari and Dhar, 1985), Oleanic acid from seeds (Khastgir et al, 1958), Saponins A and B (Hariharan and Rangaswami, 1970), Saponins C and D from unripe fruits (Sheshadri et al, 1981), protein, Fe, Ca, phosphorous (Satyanaryana et. al. 1964), Achyranthine, N-methyl pyrrolidine –3 carboxylic acid (Basu, 1957), Water soluble base, betaine (Kapoor and Singh, 1967), Vitamin C (Hasan, 1962), Ecdysterone (Banerjee and Chandha, 1970), Inokosterone ecdysterone in callus and tissue culture (Hiroshi et al, 1971), Enzyme level (Purohit et al, 1980). Achyranthes aspera Linn. plant roots reported to contain oleanolic acid, saponins, amino acids and hentriacontane and a long chained carbohydrate is also found. In the shoots an aliphatic dihydroxyketone 36, 37-dihydroxyhenpentacontan-4on and triacontanol were found (Batta & Rangaswami, 1973). Two long chain compounds isolated from the shoots of Achyranthes aspera have been characterized as 27-cyclohexylheptacosan-7-ol and 16-hydroxy.26-methylheptacosan-2- on by chemical and spectral investigations (Misra et al, 1993). Simple color reaction was performed for Achyranthes aspera Linn. stems as an investigation for alkaloids and reported to contain alkaloids. The shoots extracted with petrol forms yellow semi-solid mass. From this a pink colored essential oil 17pentatriacontanol was separated (Gariballa et al, 1983). The whole plant was extracted with methanol, after the removal of the solvent the residue was extracted successively with different solvents and isolated in butanol through column chromatography. Ecdysterone, a phytoecdysone was isolated and characterized by its colour and special chemical reactions. Contents (g/kg) reported as 0.25 (seeds), 0.09 (roots), 0.04 (stem, leaves) (Banerji et al, 1971). Anti viral activity and anticancer activity was reported for methanolic extract of leaves (Chakraborty et al, 2002). Leaves extract reported to contain antimicrobial 27

Chapter-3

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activity against Staphylococcus aureus, Bacillus subtilis, E. coli (Bashir et al, 1992). Cardiac toxicity was also reported for Achyranthes aspera Linn. (Han & Un, 2003). Scoparia dulcis Linn.: Scoparia dulcis Linn. was qualitatively reported to contain alkaloids, tannins, saponins, terpinoids, flavonoids, phenols and cardiac glycosides. Some of them are reported as alkaloids 0.81%, phenols 0.04%, tannins 6.23%, flavonoids 0.88% and saponins 0.00% approximately (Edeoga, Okwu and Mbaebie, 2005). Scoparia dulcis found to posses antiviral, inhibitory and antitumor activity (Hayashi et al, 1993). Scoparia dulcis Linn. was reported to contain antidiabetic activity, the plant identified to contain some chemical compounds like Scopadulcic Acid A, Scopadulcic Acid B, Scopadulciol, Scopadiol, Scopadulin, Scoparic Acid A, Scoparic Acid B, Scoparic Acid C, 4-epi-scopadulcic Acid B, scoparic acid D, Dulcidiol, Acacetin, Apigenin, Betulinic acid, cirsitakaoside, diacetyl scopadol, iso-dulcinol, Sitosterol, vitexin, Scutellarein, hispidulin, aphidicolin, eugenyl beta-D-glucopyranoside, Daucosterol (Rupjyoti Saikia et al, 2011). Preliminary phytochemical screening concluded on various successive extracts of Scoparia dulcis Linn. powder indicated the presence of carbohydrates, steroids, Triterpenoid glycosides, flavonoid glycoside, phytosterols, steroids, mucilage and saponins (Vaishali Parekh et al, 2011) Alpha amyrin is also quantified in Scoparia dulcis Linn. whole plant powder extract by high performance liquid chromatography (Lakshmu naidu P.V et al, 2012) 3.4

MATERIALS AND METHODS

Scoparia dulcis Linn. was collected during the flowering seasons i.e., monsoon, winter and summer from different geographical regions namely Visakhapatnam, Srikakulam and Mumbai (India). Sample collection dates were mentioned as described below for Scoparia dulcis Linn. Visakhaptanam on 17January2006, Srikakulam on 15January2006 and Mumbai on 22January2006, Visakhaptanam on 11April2006,

Srikakulam

on

10April2006

and

Mumbai

on

7April2006,

Visakhaptanam on 5June2006, Srikakulam on 4June2006 and Mumbai on 10June2006. Sample collection dates for Achyranthes aspera Linn. Visakhaptanam on

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17January2006, Srikakulam on 15January2006 and Mumbai on 22January2006 and in the other season it was dried and not obtainable. The plant material was thoroughly washed to remove soil particles, dust, etc. and properly drained to remove excess of water by spreading over newspaper for 6 hours in shade, away from sunlight. The plant material was then placed in a preset oven at 45 ± 50C. The plant material was allowed to dry for 4 days, after drying, it was powdered using an electric mixer-grinder and sieved through a BSS mesh No.85 sieve. The sieved powder was stored in commercially available airtight plastic (Amber PET) containers and this powdered plant material was used for research work in the present thesis. The plant material collected during monsoon, from above different regions was used for the study. Proximate analysis: Crude drugs before use in formulation was subjected to proximate analysis, after which stored in quarantine store and they remained there for long time. During storage proper ventilation, humidity controls, suitable temperature and light conditions should be ensured to maintain their original pharmacological action; however, it is observed that crude plant materials before processing, if are not analyzed can lead to changes in their original characteristics. To avoid this crude drugs should be tested for the following tests as per the USP and Indian Herbal Pharmacopoeia (IHP). 1. Foreign organic matter 2. Ethanol soluble extractives 3. Water soluble extractives 4. Total ash content 5. Acid insoluble ash 6. Water soluble ash 7. Loss on drying 8. Percentage moisture content

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Foreign organic matter: Medicinal plant materials should be entirely free from visible signs of contamination i.e. moulds, insects and other animal contamination, including animal excreta. Any soil, stones, sand, dust and other foreign organic matter must be removed before medicinal plant materials were cut or ground for testing. Macroscopic examination can conveniently be employed for the determination of foreign matter in whole or cut plant materials. Procedure: The whole plant material was washed thoroughly with water to remove the dust particles on the surface of the plant and the soil particles adhering to the roots. Excess water was allowed to drain off by spreading the plant material on filter paper. Then 500g of the washed and drained whole plant material was taken and spread as a thin layer on a white, clean muslin cloth. Foreign matter was sorted by visual inspection and by using magnifying lens (6x). The portions of the sorted foreign matter were weighed and the contents of foreign matter in grams per 100g of the sample were calculated. The procedure was carried out for a total of three sets. Extractable matter: This method determines the amount of phytoconstituents extracted with solvents from a given amount of medicinal plant material. Here according to Indian Herbal Pharmacopoeia, ethanol and water were used as solvents to determine the extractable matter. Procedure: Accurately weighed 4g each whole plant material of Scoparia dulcis Linn. and Achyranthes aspera Linn. were placed in glass-stoppered conical flasks separately. To this 100 cm3 of water was added. The flask was shaken frequently for six hours, and then allowed to stand for eighteen hours. The contents were filtered. The filtrates were transferred to previously weighed clean beakers and evaporated to dryness on a water-bath. After evaporation the extracts were dried at 105º C for six hours and kept in desiccators for cooling. The beakers were weighed and percent extractable matter in water was calculated. The above procedure was repeated twice. Ethanol soluble extractable matter was determined by following the above procedure except that ethanol was used instead of water, as extracting solvent. The experiment was repeated three times. Ash content: The ash remaining after the ignition of medicinal plant materials is determined by three different methods, which measure total ash, Acid-insoluble ash and Water-soluble ash. The total ash method is designed to measure the total amount of material remaining after ignition. This includes both ‘physiological ash’ which is 30

Chapter-3

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derived from the plant tissue itself, and ‘non-physiological ash’ which is the residue of the extraneous matter (e. g. sand and soil) adhering to the plant surface. Acidinsoluble ash is the residue obtained after boiling the total ash with dilute hydrochloric acid and igniting the remaining insoluble matter. This measures the amount of silica present as sand and siliceous earth. Water-soluble ash is the difference in weight between the total ash and the residue after treatment of the total ash with water. Total ash content: Apparatus: Silica dish, dessicator, air oven and muffle furnace. Procedure: Accurately weighed 2g of the dried material was taken in a tarred silica dish and was ignited with a flame of Bunsen burner for about 1 hr. The ignition was completed by keeping it in a muffle furnace at 5500C ± 200C till grey ash was formed. It was then cooled in desiccators and weighed. The process was repeated (ignition, cooling and weighing) till the difference in the weight between two successive weighing was less than 1 mg. Acid insoluble ash content: Chemicals: Dilute HCl , 5 N HCl, and AgNO3 solution. Apparatus: Silica dish, dessicator, air oven and muffle furnace. Procedure: Accurately weighed 2g of the dried material was taken in a porcelain/silica dish and was ignited with a bunsen burner for about 1 hr. The porcelain dish was kept in a muffle furnace at 5500C ± 200C till grey ash was obtained. The ash was moistened with concentrated HCl and evaporated to dryness after which it was kept in an electric air oven maintained at 1350C ± 20C for 3 hr. After cooling, 25 cm3 of dilute HCl was added, and was kept covered with watch glass and heated on a water bath for 10 minutes. It was then allowed to cool, filtered through Whatman filter paper no. 41. The residue was then washed with hot water till washings were free from chloride (as tested with AgNO3 solution). The filter paper and the residue were put in a dish and ignited in a muffle furnace at 5500C ± 200C for 1 hr. The dish was removed and cooled in desiccators and weighed. The process was repeated till the difference between two successive weights was found to be less than 1 mg.

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Water-soluble ash content: Apparatus: Silica dish, dessicator, air oven and muffle furnace. Procedure: 25 cm3 of distilled water was added in a silica dish containing the 0.5g of ash and boiled for ten minutes. The insoluble matter was collected on an ashless filter paper. The residue was washed with hot water and ignited in a crucible for fifteen minutes at a temperature not exceeding 450ºC. The water-soluble ash was calculated; by subtracting the weight of this residue from the weight of the total ash. Loss on drying: Apparatus: Wide mouthed Stoppard weighing bottle, dessicator and air oven. Procedure: 5g each of Scoparia dulcis Linn and Achyranthes aspera Linn. whole plant powdered samples were weighed in wide mouthed stopper weighing bottle. The bottle was then placed with lid open in an air oven maintained at 1050 C ± 20 C. The sample was kept in an oven for 2 hr. The bottle was then removed, covered and placed in a desiccators. The bottle was weighed after cooling to room temperature. The bottle was again kept in the oven for 2 hrs and the above procedure was repeated (heating, cooling and weighing) till the difference in the weight between two successive weighing was less than 5 mg. Three readings for each sample were recorded. Moisture content (Karl-Fischer titrimetric method): Instrument: Digital automatic karl fischer titrator. Model : Veego / Matic – MD. Reagents: Karl Fischer (K/F) reagent, methanol K/F grade, commercial grade methanol (only for cleaning the dispensing system) and distilled water. Procedure: The instrument was turned on and the speed of magnetic stirrer was adjusted. Methanol was neutralized and the titre factor was determined by calibrating the K/F reagent. This was done by adding 10 µl of distilled water with the help of a microlitre syringe in the reaction vessel and completing the titration. The calibration of the reagent was done in triplicate. The readings were noted and the titre factor was calculated. The data for determination of titre factor reported in Table and it was calculated using the following formula Titer factor =

mg of water added (wt.) / Reading in cm3 (vol)

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Sample analysis: Exactly 100 mg of the plant powder was weighed and added to the titration vessel and the titration was allowed to complete. The results obtained are given in result tables. Percentage of moisture = Titre factor x reading x 100 / Weight of sample (in mg) Selection of solvent for extraction and optimization studies: Extraction the term used pharmaceutically, involves the separation of medicinally active constituents from complex matrix of plant or animal tissues by the use of selective solvents in standard extraction procedures. The products so obtained from plants are relatively impure liquids, semisolids or powders intended only for oral or external use. These include classes of preparations such as decoctions, infusions, fluid extracts, tinctures, extracts (semisolid) and powdered extracts. Such preparations are popularly known as galenicals. The present research work concentrates primarily on basic extraction procedures for crude drugs to obtain the therapeutically desirable portion and eliminate the unwanted material by treatment with a selective solvent, known as the menstrum. The principal methods of extraction are maceration, percolation, digestion, infusion and decoction. The quality of the finished product can be enhanced by optimization of primary extracts. The United State Pharmacopoeia (USP) provides general guidelines for maceration and percolation under the heading of tinctures. Choice of extracting solvent: To obtain complete extraction of a given active principle from a solid material, the ideal solvent is obviously one that has maximum selectivity, best capacity for extraction in terms of coefficient of saturation of the product in the medium and its compatibility with the properties of the material to be extracted. In the present work different solvents, from non-polar to polar, were used to optimize the extractive values of Scoparia dulcis Linn and Achyranthes aspera Linn. Procedure: Optimization of amount of solvent: In this experiment, the amount of Scoparia dulcis Linn. and Achyranthes aspera Linn. whole plant powder taken was kept constant throughout the experiment. In different sets of volumetric flasks, accurately weighed Scoparia dulcis Linn. and Achyranthes aspera Linn. whole plant powder was taken. In these different sets of volumetric 33

Chapter-3

General phytochemical analysis

flasks, different amounts of various solvents were added and kept for 1.0 hr. Then these solvents were filtered through Whatman filter paper no. 41 in pre-weighed dry beakers separately and solvents were evaporated on a water-bath to dryness. The dried residue was then weighed and the percentage extractions were calculated. From the percentage extraction values, the amount of solvent was optimized. From the graph of volume of solvent (in cm3) versus percentage extraction as shown in figure optimization of time of extraction, it was observed that the percentage extraction levels remain constant after certain volume of a solvent used for extraction. More or less Methanol and Ethanol had same % extraction. Hence methanol is selected as solvent for extractions. The solvent volume and time of extraction were optimized using the same. Optimization of time: For optimization of time of contact between powder and solvent, the optimized volume of solvent was added to the sample and the contents in the flasks were filtered after different time intervals. The above procedure was repeated and the percentage extractive values were calculated. From the percentage extractive values, the optimization time was determined. For optimization of the time, methanol was added to the samples kept in different flasks. The contents in the flasks were filtered after different time intervals and the percentage extractive values were calculated. Optimization of number of extractions: For optimization of the number of extractions, the optimized amount of selected solvent was added to the sample in different sets of flasks and these flasks were kept aside for the optimized time. Then the contents of the flasks were filtered separately through Whatman filter paper no. 41 in pre-weighed dry beakers. The residues were again taken in a flask and extracted repeatedly using optimized solvent and time. The above procedure was repeated and the percentage extraction values were calculated. From these values number of extractions was optimized. Phytochemical analysis: Plant constituents of medicinal importance form an extensively diverse group of chemical compounds showing greater variation in solubility and stability. They can be broadly classified as follows: (Camille et al, 1996).

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 Fixed oils, fats and waxes (lipids)  Phenols  Tannins  Proteins  Alkaloids  Carbohydrates  Glycosides  Volatile oils  Resin and resin combinations Extraction of phytochemicals: The precise mode of extraction depends on the texture and type of the substances to be isolated. The classical chemical procedure for obtaining constituents from dried plant tissues is to continuously extract powered material in a ‘Soxhlet apparatus’ with a range of solvents. When investigating the complete phytochemical profile of a given plant species, fractionation of crude extract is desirable, in order to separate the main classes of constituents from each other prior to chromatographic analysis. Procedure: Following is the procedure based on varying polarity of solvents, which was employed for determination of phytochemical profile of Scoparia dulcis Linn. and Achyranthes aspera Linn. (Harbone, 1998). a) 2g of the dried whole plant powder of Scoparia dulcis Linn and Achyranthes aspera Linn. was separately soxhlet extracted with a mixture of methanol and distilled water (50 cm3) in the volume ratio 4:1. The extract was cooled and filtered through Whatman filter paper No. 41 into a dry and pre-weighed beaker. b) The residue was extracted with 125 cm3 of (5 x 25 cm3) of ethyl acetate and filtered into a dry, pre-weighed beaker. The residue obtained after filtration comprised plant fibers. Weight of the extract was noted down and the plant fibers were dried in an oven at 60 ±50C and percent crude fiber was calculated. c) The filtrate obtained from step (b) was evaporated to dryness on a water bath maintained at 45±50C. After evaporation of ethyl acetate, the beaker was allowed to cool to room temperature in a desiccator. After cooling, the weight of beaker

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containing the residue was noted down. The residue obtained was the neutral extract and consisting of fats and waxes. d) The filtrate obtained from step (a) was evaporated to approximately 1/10th of its volume by heating in a water bath maintained at a temperature less than 700C. It was acidified with 2M H2SO4. The acidified filtrate was extracted using 75 cm3 (3x25 cm3) chloroform in a separating funnel. It was then transferred to a dry preweighed beaker. Chloroform layer was evaporated to dryness on a water bath maintained at 45±50C. e) After evaporation of chloroform the beaker was allowed to cool to room temperature in a dessicator. After cooling the weight of this beaker containing the residue was noted down, the residue obtained was moderately polar extract and consisting of terpenoids and phenolics. f) The aqueous acid layer obtained from step (d) was basified (pH was adjusted to10 with 2M NaOH). It was then extracted with 60 cm3 (2x30 cm3) of mixture of chloroform and methanol in the volume ratio 3:1, followed by extraction with 40 cm3 (2x20 cm3) chloroform in a separating funnel. The aqueous basic layer was transferred to a dry pre-weighed beaker. The aqueous basic layer was evaporated to dryness on a water bath maintained at 700C. After evaporation of the solvent, the beaker was allowed to cool to room temperature in a dessicator. g) The weight of the beaker containing the residue was noted down. The residue obtained was polar extract consisting of quaternary alkaloids and N-oxides. h) The organic layer (chloroform and methanol) was transferred to a dry, preweighed beaker, placed on a water bath maintained at 45±50C. After evaporation of the solvent, the beaker was allowed to cool to room temperature in a desecrator. After cooling, the weight of this beaker containing the residue was noted down. The residue obtained was the basic extract consisting of alkaloids. i) 2g of the dried whole plant powder of Scoparia dulcis Linn. and Achyranthes aspera Linn. was extracted with 100 cm3 methanol (4x25 cm3) in a dry, stoppered conical flask. The extract was filtered into a dry, pre-weighed beaker. The filtrate obtained was evaporated to dryness on a water bath maintained at 700C. After evaporation, the beaker was allowed to cool to room temperature in a dessicator. After cooling, the weight of beaker containing the residue was noted down. The residue obtained was reconstituted in methanol to obtain a final concentration of 36

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100 mg/cm3. This extract was filtered through Whatman filter paper No. 41. The filtrate obtained was the plant extract. Each of the extracts obtained was spotted on HPTLC plate. The plate was developed in previously optimized mobile phase and the chromatograms of different extracts were compared with plant extract under same experimental conditions. Flow chart for extraction of phytochemicals: Plant powder Methanol + Water (4:1) (50 cm3) Soxhlet extraction

The filtrate evaporated below 700C, acidified H2SO4. extracted 75 cm3 (3x25 cm3) chloroform evaporated at 45±50C to get terpenoids and phenols

Residue 5 x25ml ethyl acetate extraction

Ethyl acetate evaporate to get neutral extract and consisting of fats and waxes

Plant fibers dried in an oven at 60 ±50C.

The aqueous acid layer pH 10 with NaOH extracted 60 cm3 (2x30 cm3) chloroform and methanol 3:1,extraction 40 cm3 (2x20 cm3) chloroform

The aqueous basic layer evaporated at 700C quaternary alkaloids and N-oxides

Chloroform and methanol layer evaporated at 45±50C basic extract consisting of alkaloids

HPTLC Analysis of phytochemical extracts: Sample application: The HPTLC separation of plant extract and extracts of separated phytochemicals was performed on pre-coated silica gel 60 F254 HPTLC plates. Ten micro liters of the extracts comprising alkaloids, fats and waxes, phenolics and terpenoids, basic extracts and polar extract and plant extract were applied at a distance of 10 mm/ from the base of chromatographic plate and 7mm width on HPTLC plate using Camag Linomat-IV sample applicator. Methodology: The methodology followed for High Performance Thin Layered Chromatography was mentioned in the Chapter-8. 37

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The individual chromatograms of all separated phytochemicals i. e. alkaloids, fats and waxes, phenolics and terpenoids, basic extracts and polar extract are compared with plant extract by overlay, for Scoparia dulcis Linn. and Achyranthes aspera Linn. were shown in figure 3.9 and 3.10 respectively. Thus each peak in the chromatogram of plant extract decides which phytochemicals they belong to. 3.5

RESULTS OBTAINED

Foreign organic matter observations and results: It is observed that the percentage foreign organic matter in Scoparia dulcis Linn. and Achyranthes aspera Linn. whole plant is between 0.032 % to 0.040%. The results are given in Table 3.1. and Table 3.2. Calculations: % Foreign organic matter = (M1 - M) × 100 M2 Where M = Weight of empty dish in g M1 = Weight of dish with foreign matter in g M2 = Weight of sample (whole plant material) in g For Scoparia dulcis Linn. (29.8148

-

29.6822)

500

x 100

= 0.026 %

x 100

= 0.028 %

For Achyranthes aspera Linn. (29.9342

-

29.7921)

500

Table 3.1. Percentage foreign organic matter in Scoparia dulcis Linn. % Foreign organic matter Mumbai

Visakhapatnam

Srikakulam

% Mean

Scoparia dulcis Linn.

0.031

0.026

0.028

0.028

% RSD

5.6

3.8

10.7

8.9

Sample

Note: Each observation is mean of three readings.

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Table 3.2. Percentage Foreign organic matter in Achyranthes aspera Linn. % Foreign organic matter

Sample

% Mean

Mumbai

Srikakulam

Visakhapatnam

Achyranthes aspera Linn.

0.03

0.022

0.028

0.027

% RSD

3.8

7.1

5.4

15.6

Note: Each observation is mean of three readings. Extractable matter observations and results: It is observed that the percentage ethanol extractable matter in Scoparia dulcis Linn. and Achyranthes aspera Linn. whole plant powder is between 21.00 % to 25.00 % and percentage water extractable matter is between 28.00 % to 30.00 %. The results are given in Table 3.3, 3.4, 3.5 and 3.6. Table 3.3. Percentage of ethanol extractable matter in Scoparia dulcis linn. Extractive value (%) Visakhapatnam Srikakulam

% Mean

Sample

Mumbai

Scoparia dulcis Linn.

21.29

23.42

22.95

22.55

% RSD

2.9

2.6

2.5

5.0

Note: Each observation is mean of three readings. Table 3.4. Percentage of ethanol extractable matter in Achyranthes aspera Linn. Sample

Extractive value (%) Mumbai Visakhapatnam Srikakulam

% Mean

Achyranthes aspera Linn.

23.2

23.95

24.42

23.86

% RSD

2.6

2.0

2.1

2.6

Note: Each observation is mean of three readings. Table 3.5. Percentage of water extractable matter in Scoparia dulcis Linn. Extractive value (%) Visakhapatnam Srikakulam

% Mean

Sample

Mumbai

Scoparia dulcis Linn

27.66

29.13

28.93

28.57

% RSD

2.7

3.7

4.6

2.8

Note: Each observation is mean of three readings.

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Table 3.6. Percentage of water extractable matter in Achyranthes aspera Linn. Extractive value (%) Visakhapatnam Srikakulam

% Mean

Sample

Mumbai

Achyranthes aspera Linn.

29.33

29.93

29.24

29.50

% RSD

2.1

3.7

2.2

1.3

Note: Each observation is mean of three readings. Ash content observations and results: It is observed that the percentage total ash content of Scoparia dulcis Linn and Achyranthes aspera Linn whole plant powder is between 3.17% to 3.23% and 4.76% to 4.98% respectively. Table 3.7. Total ash content of Scoparia dulcis Linn. % Total ash content

Sample

% Mean

Mumbai

Visakhapatnam

Srikakulam

Scoparia dulcis Linn.

3.23

3.17

3.17

3.19

% RSD

3.4

2.4

6.5

1.1

Note: Each observation is mean of three readings. Table 3.8. Total ash content of Achyranthes aspera Linn. % Total ash content

Sample

% Mean

Mumbai

Visakhapatnam

Srikakulam

Achyranthes aspera Linn.

4.76

4.98

4.89

4.88

% RSD

3.7

1.5

5.1

2.3

Note: Each observation is mean of three readings. Acid insoluble ash content observations and results: It is observed that the percentage acid insoluble ash content of Scoparia dulcis Linn. and Achyranthes aspera Linn. whole plant powder is between 0.85 % to 0.95 %.and 0.255% to 0.265% respectively.

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Table 3.9 Acid insoluble ash content of Scoparia dulcis Linn. % Acid insoluble ash Mumbai

Visakhapatnam

Srikakulam

% Mean

Scoparia dulcis Linn.

0.85

0.91

0.95

0.90

% RSD

2.4

5.7

3.2

5.6

Sample

Note: Each observation is mean of three readings. Table 3.10. Acid insoluble ash content of Achyranthes aspera Linn. % Acid insoluble ash Mumbai

Visakhapatnam

Srikakulam

% Mean

Achyranthes aspera Linn.

0.265

0.255

0.2599

0.2599

% RSD

7.5

8.1

7.3

1.9

Sample

Note: Each observation is mean of three readings.

Water-soluble ash content observations and results: It is observed that the percentage water-soluble ash content of Scoparia dulcis Linn. and Achyranthes aspera Linn. whole plant powder is found to be between 2.28 % to 2.34 %. and 4.14% to 4.17 % respectively. Table 3.11. Water soluble ash content of Scoparia dulcis Linn. % Water soluble ash Mumbai

Visakhapatnam

Srikakulam

% Mean

Scoparia dulcis Linn.

2.34

2.30

2.28

2.31

% RSD

4.3

5.8

2.5

1.3

Sample

Note: Each observation is mean of three readings.

Table 3.12. Water soluble ash content of Achyranthes aspera Linn. % Water soluble ash Mumbai

Visakhapatnam

Srikakulam

% Mean

Achyranthes aspera Linn.

4.14

4.14

4.17

4.15

% RSD

2.1

0.6

1.4

0.4

Sample

Note: Each observation is mean of three readings. 41

Chapter-3

General phytochemical analysis

Loss on drying observations and results: It is observed that the percentage of loss on drying of Scoparia dulcis Linn. and Achyranthes aspera Linn. whole plant powder is between 8.00% to 9.00% Table 3.13. Percentage loss on drying of Scoparia dulcis Linn. % Loss on drying Visakhapatnam Srikakulam

% Mean

Sample

Mumbai

Scoparia dulcis Linn.

8.24

8.86

8.16

8.42

% RSD

1.8

3.0

1.

4.6

Note: Each observation is mean of three readings.

Table 3.14. Percentage loss on drying of Achyranthes aspera Linn. % Loss on drying Visakhapatnam Srikakulam

% Mean

Sample

Mumbai

Achyranthes aspera Linn.

8.58

8.59

8.59

8.59

% RSD

1.2

3.9

3.5

0.1

Moisture content observations and results: Table 3.15. Determination of moisture content (Titre Factor) of Scoparia dulcis Linn.

Sr. No.

Weight of water added in mg

Volume of reagent added in cm3

Titer factor

1.

10

1.85

5.41

2.

10

1.83

5.46

3.

10

1.86

5.38

Mean

5.42

Table 3.16. Determination of moisture content (Titre Factor) of Achyranthes aspera Linn. Sr. No.

Weight of water added in mg

Volume of reagent added in cm3

Titer factor

1.

10

1.73

5.78

2.

10

1.78

5.61

3.

10

1.80

5.55

Mean

5.55 42

Chapter-3

General phytochemical analysis

Table 3.17. Moisture content of Scoparia dulcis Linn. Sr. No.

Weight of powder in mg

Volume of reagent added in cm3

% Moisture

1.

100

1.23

6.66

2.

100

1.18

6.39

3.

100

1.15

6.23

Mean

6.43

Table 3.18. Moisture content of Achyranthes aspera Linn. Sr. No.

Weight of powder in mg

Volume of reagent added in cm3

% Moisture

1.

100

1.08

6.24

2.

100

1.08

6.85

3.

100

1.07

5.93

Mean 6.34 Following are the parameters (Table 3.19) required for checking the quality of raw material of whole plant powder of Scoparia dulcis Linn. and Achyranthes aspera Linn. before going for processing. These quality control parameters depend on cultivation, handling of the raw material and storage condition. Table 3.19. Proximate analysis of Scoparia dulcis Linn. and Achyranthes aspera Linn. Sr. No.

Parameter

1

Foreign organic matter

2

Ethanol soluble extractive

% Content Scoparia dulcis Achyranthes aspera Linn. Linn. 0.028 0.027 22.55

23.86

3

Water soluble extractive

28.57

29.50

4

Total ash

3.19

4.88

5

Acid-insoluble ash

0.90

0.26

6

Water soluble ash

2.31

4.15

7

Loss on drying

8.42

8.59

8

Moisture content

6.43

6.34 43

Chapter-3

General phytochemical analysis

Solvent optimization for extraction and acceptability: Table 3.20. Optimization of extraction conditions for selecting suitable solvent Scoparia dulcis Linn.

S. No.

Solvent

Volume (cm3)

Time (Min)

No. of Extractions (n)

Extractive value (%)

SD

% RSD

1.

Water

100

60

3

27.96

1.47

5.3

2.

Methanol

100

60

3

11.91

1.4

11.8

3.

Ethanol

100

60

3

12.51

0.99

7.9

4.

Acetonitrile

100

60

3

8.75

0.67

7.7

5

Acetone

100

60

3

7.35

0.83

11.3

6

Chloroform

100

60

3

6.85

0.33

4.8

Note: All values are mean of three extractions. Table 3.21. Optimization of extraction conditions for selecting suitable solvent Achyranthes aspera Linn. %

S. No.

Solvent

Volume (cm3)

Time (min.)

No. of Extractions (n)

Extractive value (%)

SD

1.

Water

100

30

3

29.0

1.32

4.6

2.

Methanol

100

60

3

14.40

0.9

6.3

3.

Ethanol

100

60

3

15.13

1.18

7.8

4.

Acetonitrile

100

60

2

10.24

0.91

8.9

5

Acetone

100

60

3

9.88

0.83

8.4

6

Chloroform

100

60

3

12.12

0.93

7.6

RSD

44

Chapter-3

General phytochemical analysis

Table 3.22. Optimization of amount of solvent for extraction Scoparia dulcis Linn.

Set No.

Weight of powder

1

0.5

Amount of solvent in cm3 25

2

0.5

50

8.03

0.95

11.8

3

0.5

75

10.32

0.99

9.6

4

0.5

100

11.91

1.1

9.2

5

0.5

150

12.02

1.48

12.3

6

0.5

200

12.12

0.53

4.4

Extractive value (%)

SD

% RSD

7.50

0.91

12.2

%Extraction

15.0 10.0 5.0 Amt of solvent

0.0 0

100 200 Amt. in ml

300

Figure 3.3. Optimization of amount of solvent for extraction of Scoparia dulcis Linn.

45

Chapter-3

General phytochemical analysis

Table 3.23. Optimization of amount of solvent for extraction Achyranthes aspera Linn

Set No. 1

Weight of powder in g 0.25

2

Amount of solvent in cm3

Extractive value (%)

SD

% RSD

25

9.01

0.49

5.4

0.25

50

10.42

0.88

8.4

3

0.25

75

12.00

1.5

12.5

4

0.25

100

13.00

0.92

7.1

5

0.25

125

13.04

0.56

4.3

6

0.25

150

13.00

1.44

11.1

Figure 3.4. Optimization of amount of solvent for extraction of Achyranthes aspera Linn.

46

Chapter-3

General phytochemical analysis

Optimization of time: Table 3.24. Optimization of time of extraction for Scoparia dulcis Linn.

1

Weight of powder in g 0.5

Time of extraction in min 30

2

0.5

3

%

Extractive value (%)

SD

7.74

0.66

8.5

60

11.77

1.63

13.8

0.5

90

12.42

1.04

8.4

4

0.5

120

13.11

0.85

6.4

5

0.5

180

13.71

0.75

5.5

6

0.5

240

13.76

0.46

3.4

Set No.

RSD

%Extraction

15 10 5 Tim e of Extraction

0 0

100

200

300

Time in min

Figure 3.5. Optimization of time of extraction for Scoparia dulcis Linn.

47

Chapter-3

General phytochemical analysis

Table 3.25. Optimization of time of extraction for Achyranthes aspera Linn.

1

Weight of powder in g. 0.25

2

0.25

60

12.16

0.81

6.7

3

0.25

90

12.44

1.08

8.7

4

0.25

120

12.36

1.23

9.9

5

0.25

150

12.48

1.03

8.2

Set No.

Time in minutes

Extractive value (%)

SD

% RSD

30

7.44

0.64

8.6

% Extraction

15 10 5 Time of Extraction 0 0

50

100

150

200

Time in min

Figure 3.6. Optimisation of time of extraction for Achyranthes aspera Linn.

48

Chapter-3

General phytochemical analysis

Optimization of number of extractions: Table 3.26. Optimization of number of extractions for Scoparia dulcis Linn.

1

Weight of powder in g 0.5

2

0.5

2

15.08

1.35

9.0

3

0.5

3

16.85

0.57

3.4

4

0.5

4

17.51

0.31

1.7

5

0.5

5

17.62

0.15

0.9

6

0.5

6

17.68

0.19

1.1

Set No.

No. of Extraction

Extractive value (%)

SD

% RSD

1

13.74

0.67

4.9

% extraction

20 15 10 nos of extraction

5 0 0

2

4

6

8

nos of extraction

Figure 3.7. Optimization of number of extraction for Scoparia dulcis Linn

49

Chapter-3

General phytochemical analysis

Table 3.27. Optimization of number of extractions for Achyranthes aspera Linn.

Set No.

Weight of powder in g

Number of extractions

Extractive value (%)

SD

% RSD

1

0.2500

1

12.16

0.35

2.9

2

0.2500

2

13.72

0.42

3.0

3

0.2500

3

14.40

0.69

4.8

4

0.2500

4

14.60

0.56

3.8

5

0.2500

5

14.66

0.58

3.9

% of Extraction

20 15 10 5 nos of extraction

0 0

2 4 nos of Extraction

6

Figure 3.8. Optimisation of number of extraction for Achyranthes aspera Linn.

50

Chapter-3

General phytochemical analysis

Phytochemical analysis: Table 3.28. Percent phytochemicals from Scoparia dulcis Linn. Extractive

SD

% RSD

1.7

0.1

5.9

Moderately polar extract (Terpenoids and phenolics)

1.8

0.06

3.1

3

Basic extract (Most alkaloids)

0.96

0.02

2.2

4

Polar extract (Quaternary alkaloids and N-oxides)

19.34

0.67

3.4

5

Fibers

76

1.32

1.7

Total

99.8

Sr. No.

Phytochemical extract

1

Neutral extract (Fats and waxes)

2

value (%)*

Table 3.29. Percent phytochemicals from Achyranthes aspera Linn.

Sr. No.

Extractive Phytochemical extract

value (%)

SD

% RSD

*

1

Neutral extract (Fats and waxes)

1.3

0.1

7.7

2

Moderately polar extract (Terpenoids and phenolics)

1.13

0.12

10.2

3

Basic extract (Most alkaloids)

0.70

0.1

14.3

4

Polar extract (Quaternary alkaloids and N-oxides)

21.24

0.9

4.2

5

Fibers

75.5

1.0

1.3

99.87 Total * Each observation is mean of three readings.

51

Chapter-3

General phytochemical analysis

Results: Separate extracts of the isolated phytochemical constituents and the plant extract in methanol were prepared as discussed earlier. The comparison of phytochemical constituents in isolated extracts and in plants extract was required to study their equivalence. The data obtained after chromatographic scan under the same chromatographic conditions was studied carefully. The Rf values were compared for the data treatment. The plant extract of Scoparia dulcis Linn. in methanol showed nine peaks at Rf values 0.22, 0.28, 0.33, 0.41, 0.45, 0.56, 0.65, 0.71 and 0.77 which are representative of individual phytochemical type. The alkaloid extract showed three peaks at Rf-values 0.34, 0.56 and 0.71 representing the presence of different alkaloids. The chromatogram of the basic extract showed eight peaks with Rf values 0.23, 0.28, 0.34, 0.46, 0.57, 0.66, 0.72 and 0.78. The fats and waxes showed two peaks with Rf values 0.64 and 0.71. Mid polar extract showed six peaks with Rf values 0.27, 0.33, 0.40, 0.51, 0.56 and 0.71 representing the presence of terpenoids.

52

Chapter-3

General phytochemical analysis

Fats and waxes Terpenoids Plant extract Alkaloids Basic extracts

Figure 3.9: The overlay of densitometric chromatogram of Scoparia dulcis Linn.

Fats and waxes Terpenoids Plant extract Alkaloids Basic extracts

Figure 3.10: The overlay of densitometric chromatogram of Achyranthes aspera Linn.

53

Chapter-3

General phytochemical analysis

Table 3.30. Phytochemical analysis comparative results of characteristic spots Scoparia dulcis Linn. S No.

Rf value

1

0.22



2

0.23

3

Whole plant Alkaloid

Fats & Waxes

Phenolics & Terpenoids

Basic extract



















0.27











4

0.28











5

0.33











6

0.34











7

0.40











8

0.41





9

0.45



10

0.46

11

0.51

12

0.56

13

0.57

14

0.64

15

0.65

16

0.66

17

0.71

18

0.72

19

0.77

20

0.78

√ √ √



√ √ √

√ √ √





√ √

√ √

54

Chapter-3

General phytochemical analysis

The plant extract of Achyranthes aspera Linn. in methanol showed seven peaks at Rf values 0.23, 0.33, 0.45, 0.49, 0.60, 0.66 and 0.73 which are representative of individual phytochemical type. The alkaloid extract showed four peaks at Rf values 0.20, 0.30, 0.33 and 0.43 representing the presence of different alkaloids. The chromatogram of the basic extract showed seven peaks with Rf values 0.25, 0.29, 0.35, 0.46, 0.61, 0.68 and 0.73. The fats and waxes showed five peaks with Rf values 0.33, 0.59, 0.66, 0.71 and 0.78. Mid polar extract showed ten peaks with Rf values 0.21, 0.24, 0.29, 0.35, 0.41, 0.49, 0.59, 0.61, 0.73 and 0.80 representing the presence of terpenoids.

55

Chapter-3

General phytochemical analysis

Table 3.31. Phytochemical analysis comparative results of characteristic spots in Achyranthes aspera Linn.

SNo .

Rf value

1

0.20

2

0.21

3

0.23

4

0.24

5

0.25

6

0.29

7

0.30

8

0.33

9

Whole plant

Alkaloid

Fats & Waxes

Phenolics & Terpenoids

Basic extract

√ √ √ √ √ √



0.35





10

0.41



11

0.43

12

0.45

13

0.46

14

0.49

15

0.59

16

0.60

17

0.61

18

0.66

19

0.68

20

0.71

21 22

0.73 0.78

√ √





√ √ √ √

√ √



√ √ √



√ √ √









56

Chapter-3 3.6

General phytochemical analysis

DISCUSSION OF RESULTS IN LIGHT OF OTHERS WORK

No literature reported for the content of foreign organic matter, ethanol soluble extractives, water soluble extractives, total ash content, acid insoluble ash, water soluble ash for the plants Scoparia dulcis and Achyranthes aspera. No HPTLC method reported to identify the chemical constituents present in the whole plant. Several methods reported that methanolic, ethanolic extracts were having pharmacologically active. In accordance with the present data, the ethanol extracts have more percentage of extraction. The above data shows that the plants have more mid polar components that are extractable in ethanol. 3.7

CONCLUSION

Type of chemical constituents present in the above two plants are identified. Qualitative HPTLC method developed to identify the type of chemical constituents present in the whole plant. The solvent required for extraction selected. The maximum extraction of the whole plant powder of Scoparia dulcis Linn. and Achyranthes aspera Linn. found in water are 27.96 and 29.0 respectively. The optimum extraction conditions of different solvents are given in Table 3.20 and 3.21. The % extraction for the solvent methanol for Scoparia dulcis and Achyranthes aspera are 11.91 and 14.40 respectively. The % extraction for the solvent ethanol for Scoparia dulcis and Achyranthes aspera are 12.51 and 15.13 respectively. Comparatively Ethanol is having more % extraction capacity rather than methanol. Hence ethanol used for extraction as ethanol extracts can be used directly for oral administration. Even though water posses more extraction capacity it is not used because of its susceptibility for microbial contamination. These optimized parameters can be used for bulk production of whole plant powder extracts of Scoparia dulcis Linn. and Achyranthes aspera Linn. 3.8

FUTURE PROSPECTS

The ethanol and mid polar extracts should be further separated using different techniques such as flash chromatography followed by preparative column chromatography. 57

Chapter-3

General phytochemical analysis

The separated components have to be purified, characterized

and identified by

different techniques such as Mass, NMR and IR. The purified components are to be screened for the anti diabetic and anti HIV activity.

3.9

REFERENCES

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Chapter-3

General phytochemical analysis

Hayashi T, Okamuka K, Kawasaki M, Morita N. Phytochemistry., 1993; 35 (2): 353356. Hiroshi H, Hisanon J, Takemota T. Chem. Pharm. Bull., 1971; 19(12): 438. Indian Herbal Pharmacopoeia, A joint publication of Regional Research Laboratory(CSIR) Jammu tawi and Indian Drugs Manufacturer’s Association., 1998; 2. Kapoor VK, Singh HK. Ind. J. Pharm., 1967; 29(10): 281. Khastgir H, Sengupta SK, Sengupta P. J Ind. Che. Soc., 1958; 35: 693. Lakshmu Naidu PV, Kishore Kumar K, Sujatha S, Narasimha Raoa M., Journal of Pharmacy Research., 2012; 5 (4) : 1970. Misra TG, Singh RS, Pandey HS. Phytochemistry., 1993; 33(1): 221-3. Nostro A, Germano MP, D'Angelo V, Marino A, Cannatelli MA. Lett. Appl. Microbiol., 2000; 30(5): 379. Purohit S, Bhattacharya I C. J. Sci. Ind. Res., 1980; 20C (8): 246. Rupjyoti Saikia, M. Dutta Choudhury, A. Das Talukdar and Pankaj Chetia. Assam University Jour. of Science & Technol., 2011; 7 (I): 173-180. Satyanarayana MS, Sushila BA, Rao, AN, Vijaraghwan, PK. J. Food sci. tech., 1964; 1(12): 26. Sheshadri V, Batt AK, Rangaswami S. Ind. J. Chem., 1981; 20B (9) : 773. Sofowora A. J. Altern. Complement. Med., 1996; 2 (3): 365-372. United States Pharmacopoeia, 24 NF (19).

The United States Pharmacopoeia

Convention Inc. USA, 2000. Vaishali Parekh, Saurabh Parmar, Urvashi Shah., International Journal of Universal Pharmacy and Life Sciences., 2011: 1(2).

59