INTERNATIONAL JOURNAL OF AYUSH

albumin and total protein. Collection of blood and separation of serum Blood was collected from the retro-orbital plexus under mild Diethyl ether ... ...

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140

PANACEA INTERNATIONAL JOURNAL

International

IJAYUSH

Journal of AYUSH

Journal Homepage: www.prlpublisher.com/ijayush

ISSN: 2349 7025

Original Research Article

Volume 4 Issue 2

AN EVALUATION OF TREATMENT MODALITY OF MANDALIDAMSA WITH AJITAGADA IN EXPERIMENTAL LEVEL NITIN URMALIYA1, *MANOJ KUMAR GUPTA2, DINESH SINGH GAUR3, KRISNA KUMAR MISRA4, AJIT PAL SINGH CHAUHAN5 1. Assistant Professor, Department of Agad Tantra, government Ashtang Ayurvedic College and Hospital, Lokmanya Nagar, Indore, Madhyapradesh, India. 2. Assistant Professor, Department of Roga Nidan and Vikriti Vijnana Governme.nt Ayurvedic College and Hospital, Atarra Banda,Uttar pradesh, India. Email [email protected] 3. Associate Professor, Department of Shalakya, Government Ashtang Ayurvedic College and Hospital, Lokmanya Nagar, Indore, Madhyapradesh, India. 4. Assistant Professor, Department of Sharir Kriya Vijnana,Government Ayurvedic College and Hospital, Atarra Banda,Uttar pradesh, India 5. Professor Department of Sharir Kriya Vijnana, Government Ashtang Ayurvedic College and Hospital, Lokmanya Nagar, Indore, Madhyapradesh, India.

Article history: Received: 23th July 2015 Received in revised form: 28th July 2015 Accepted: 30th Aug. 2015 Available online: 25th Sept 2015

Abstract It is proposed to study the Renal-protective effect of Ajitagada yoga in Spague-dawley rats. All the animals were sacrified and

*Corresponding author: Dr Dr. Manoj Kumar E-mail: [email protected] Keywords: Visha Agada etc

kidney was used for the histopathological studies. Serum

These authors have no conflict of interest to declare. Copyright © 2012, International Journal of AYUSH Ayurveda, Yoga, Unani, Siddha and Homeopathy) All rights reserved

Ajitagada against gentamicin induced nephrotoxicity in Sprague-

creatinine level is used as an index of nephrotoxicity. The present study was undertaken to assess the nephroprotective effect of

dawley rats. Eight adult female Sprague-dawley rats weighing 160300g, the renal protective effect of Ajitagada’ was conducted at Veterinary college & sciences, Mannuthi, Thrissur.

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 INTRODUCTION: The fatal cases of poisoning are poisonous snakebites. In India it is believed that snakes bite about 2 million people annually, of which 15,00030,000 cases prove fatal. There are around 3000 species of snakes in the world of which 200 are found in India. Colubridae and Viperidae families of venomous snakes are of medical importance in India and in Kerala. Numbers of incidence of bites are more from Viper and Pit viper bites are not rare. No antivenom is being made against Indian Pit vipers. Systemic involvement has been hardly reported and symptomatic treatment is the existing mode in Modern system of medicine.Research is a scientific study to establish and analyse facts to contribute to the present knowledge.

MATERIALS AND METHODS: Experimental animals: The study was conducted in 40 adult female Sprague Dawley rats weighing 200 – 300 g.The rats were purchased from Small animal breeding station, Mannuthy. The animals were housed in appropriate cages in a well ventilate room with a 12-h light; 12-h dark cycle. They were maintained under identical feeding and management practices in the laboratory. An acclimatization period of four days was allowed before the commencement of the experiment. The experiment was conducted for a period of 30 days. Plant material/Drug: The whole plant of Boerhavia diffusa was collected from the campus of college of Veterinary and animal sciences, Mannuthy and identified. Preparation of aqueous extract of Boerhavia diffusa: The whole plant of Boerhavia diffusa was air dried at room temperature and coarsely powdered using an electrical pulverizer. 100g of the powder was mixed with 1 liter of distilled water and kept undisturbed for 24 hours. Then they were subjected to boiling for 30 minute with constant stirring. The extracts

were filtered through a muslin cloth and then kept in boiling water bath for the complete evaporation of water. Gentamicin sulphate: Gentamicin sulphate (procured from TTK pharma limited, Raja Annamalaipuram, Chennai, India) was administered at a dose rate of 80mg/kg intra peritoneal for eight days to induce Nephrotoxicity. Experimental design: The animals were randomly divided into 5 groups comprising eight animals each. The experiment was conducted for a period of 30 days (Actually 15 days are correct time for this study but I extended upto 30 days for observations).Eight rats were retained as healthy control. Rests of the rats were treated with Gentamicin sulphate at a dose rate of 80 mg/kg intra peritoneal for 8 days. Group 1 – Healthy control Group 2 – Gentamicin sulphate was given at a dose rate of 80mg/kg intra peritoneal for eight consecutive days and administered with vehicle Group 3 - Aqueous extract of Ajitagada was administered at a dose rate of 200 mg/kg p.o. from 9th day to day 30. Group 4 - Aqueous extract of Ajitagada was administered at a dose rate of 400 mg/kg p.o. from 9th day to day 30. Group 5 - Aqueous extract of Boerhavia Diffusa was administered at a dose rate of 400 mg/kg p.o. from 9th day to day 30. The blood was collected from all the animals on 0th, 9th, 15th, and 30th Day and serum was separated and used for the estimation of creatinine, urea, albumin and total protein. On 30th day, the rats were sacrified and both the kidneys were located and dissected out. The kidneys were

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 immediately weighed and used for conducting histopathological studies. Collection of biological samples- The blood was collected from all the animals on 0th, 9th, 15th and 30th day and serum was used for the estimation of creatinine, urea, albumin and total protein. Collection of blood and separation of serum Blood was collected from the retro-orbital plexus under mild Diethyl ether anaesthesia with heparinized capillary tubes, into sterile centrifuge tubes without adding any anticoagulant. It was kept at refrigeration temperature for half an hour, taken out and kept at room temperature for another half an hour. It was then centrifuged at 3200 rpm for 10 minutes and the clear serum obtained was pipette out. Kidney: The animals were euthanized and dissected upon and the kidney was collected. It was washed in running tap water to remove the blood clots and kept in chilled 0.9 percent sodium chloride. OBSERVATIONS Estimation of Serum parameters Creatinine: Creatinine in serum was determined based on jaffe kinetic method without deprotienisation in ‘BloodWorking Reagent Standard Sample

analyzer’ using Creatinine test kit from Agappe diagnostics limited, india. Clinical Significance: It is formed in muscles from phosphor creatinine. It is important form of energy being a stor of high energy phosphate. Creatinine determinations have one advantage over urea determination that it is not affected by a high protein diet. Serum creatinine is more specific and sensitive indicator of renal function simultaneous estimation of serum Urea and Creatinine provides better information. Serum urea nitrogen, creatinine ratio is > 15 in pre renal failure and < 10 in renal failure. Principle: Creatinine reacts with picric acid to produce a coloured compound, Creatinine alkaline picrate. The change in absorbance is proportional to the Creatinine concentration. Reagents: Reagents 1: Creatinine dye reagent Picric acid - 8.73 mmol/L Surfactant Reagent 2: Creatinine base reagent Sodium hydroxide- 300 mmol/L Sodium phosphate- 25 mmol/L Reagent 3: Creatinine standard Creatinine standard concentration 2 mg/dL Procedure Standard Sample 1000µL 1000 µL 100 µL 100 µL

Mix and read the value of sample through the Blood-analyzer give ammonia. This is toxic and so Normal value of Serum Creatinine in through a series of chemical reactions Sprague-dawley rat –0.2 – 0.8 mg/dl (urea cycle) non toxic urea is produced UREA Clinical significance: Proteins cannot be which is released into the blood excreted stored in human body, so excess should be in the urine. broken down. Amino acids which form the Elevated levels are seen during increased components of proteins break down to protein breakdown, dehydration, vomiting,

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 and diarrhoea. Also seen in any kind of renal disorder like Glomerular nephritis, chronic nephritis and nephritic syndrome. Principle Enzymatic determination of urea according to the following reaction. Urea + H2O urease= 2NH3 + CO2 NH3 + Salicylate = nitroprusside hypo chloride 2.2 – Dicarboxy Indophenols Reagents: Reagents 1: Urea – B Colour reagent R1 Blank

Sodium Salicylate 80 nmol/L Sodium nitroprusside 4 mmol/L Sodium hypochloride 45 MG/Dl Reagent 2: Urea – B R2 Phosphate buffer, (Ph 6.9) 60 mmol/L Urease- 25 KU/L Reagent 3 : Urea – B standard Urea – B standard concentration 40 mg/dL Procedure: Standard

1000µL 1000µL Working Reagent 10 µL Standard Sample Mix and incubate 5 min. at 37C then add 1000µL 1000µL Colour reagent Mix and incubate 5 min. at 37C then add 1000µL 1000µL DI Water

Sample 1000µL 10 µL 1000µL 1000µL

Mix well and measure the absorbance of sample and the standard against the reagent blank After that with the help of this formula, we disorders, Nephrotic syndrome, malnutrion can convert the Urea value into BUN and protein losses due to haemorrhage. value. So I got BUN value for observation. Principle: Colorimetric determination of total protein based on the principle of the BUN = UREA VALUE x 0.46 Biuret reaction (copper salt in alkaline Normal value of Serum BUN in medium). Protien in plasma or serum Sprague-dawley rat –15 – 21 mg/dl sample forms a blue coloured complex TOTAL PROTIEN Clinical significance: Protien forms the when treated with cupric ions in alkaline major portion of dissolved substancesm,in solution. The intensity of the blue colour is the plasma. They form the basic structural proportional to the protein concentration. components of the body. They constitutes REAGENT COMPOSITION the enzymes present in our body and also Total protein reagent act as source of energy. The other Potassium iodide 6 mmol/L functions include distribution of water, Potassium sodium tartarate 21 mmol/L buffering, transport of various Copper sulphate 6 mmol/L components, defence and coagulation of Sodium hydroxide -58 mmol/L blood in our body. Increased levels are Total protein standard found in dehydration and myeloma. Standard concentration 6 gm/dl Decreased levels are found in liver Procedure Blank Standard Sample

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 Reagent Standard Sample

1000µL -

1000µL 20 µL -

1000µL 20 µL

Mix and incubate for 10 min. at 37C. Measure the absorbance of sample and the standard against the reagent blank Principle: The reaction between albumin Normal value of Serum Total protien in from serum or plasma and the dye Sprague-dawley rat –5.6 – 7.6 mg/dl bromocresol –green produces a change in ALBUMIN Clinical significance: Albumin which is colour that is proportional to the albumin synthesized in the liver constitutes a Major concentration. part of the total proteins in the body, the REAGENT COMPOSITION other part being globulin; they form the Albumin reagent major portion of the dissolved substances Succinate buffer, (Ph 4.20) 75 mmol/L in the plasma. Functions of Albumin Bromocresol green 0.14 g/L includes distribution of extracellular fluid, Copper sulphate 6 mmol/L regulation of osmotic pressure, acts as a Sodium hydroxide 58 mmol/L transport agent for a wide variety of Albumin standard substances such as hormones lipids, Standard concentration 3 gm/dl vitamins etc. Increased levels are seen in Procedure dehydration. Decreased levels are seen in liver diseases (Hepatitis, Cirrhosis), malnutrition, kidney disorders, and increased fluid loss during extensive burn and malabsorption. Blank Standard Sample 1000µL 1000µL Reagent 1000µL 10 µL Standard Sample 10 µL Mix and incubate for 1 min. at 37C. Measure the absorbance of sample and the standard against the reagent blank Statistical analysis of data: The results Normal value of Serum Albumin in obtained were analysed using Analysis of Sprague-dawley rat –3.8 – 4.8 mg/dl covariance followed by Duncan’s multiple Histopathological examination of kidney Representative samples of kidney obtained range test for comparison between the from the dissected animals were fixed in groups as described by Snedecor and 10% formalin. They were then processed Cochran (1985). The cytoprotective and paraffin embedded as described by enzymes were analysed using Analysis of Sheehan and Hrapchak, 1980. The sections variance (ANOVA). The best treatment were stained with haematoxylin and eosin from each group was selected and they as per the technique followed by Bancroft were tested for significance using and cook, 1984. The sections were Student’s t test. examined in detail under light microscope. Results

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 Creatinine;

Table No.1 the Anova table for the serum Creatinine values of Degree of Mean square F-ratio 5% F limit freedom (MS) (d.f.)

Source of variation

Sum squares (SS)

Between columns (i.e. between days) Between rows (i.e. between groups) Interaction Within samples (Error)

41.5

2

20.75

71.81

3.07 (2,105)

12.67

4

3.1675

10.96

2.45 (4,105)

11.92 30.34

8 105

1.49 0.288952

5.15

2.02 (8,105)

Total

96.43

119

80

71.81

70 60 50 40

F-ratio

30

5% F limit

20

10.96

10

3.07

2.45

0 Between columns (i.e. between days)

Between rows (i.e. between groups)

The above table shows that all the three Frations are significant of 5% level. This analysis does not support the null hypothesis which means there is too much difference in Creatinine values in different Mean SD Group Group II 0.75 0.3 Group III 0.71 .39 On comparing the values of the serum Creatinine on 15th day, in between two groups, it was found that the mean score was 0.75 in Group II with SD + 0.3, and 0.71 with SD of + 0.39 in Group III. t Mean SD Group Group II

0.75

0.3

days (0th, 9th & 15th). Also different groups have too much difference. Table No. 2 Comparison on Serum Creatinine values of 15th day in between two group II & III.(Unpaired t - test) P values t - values 0.22

P>0.05

values was found 0.22 which was statistically insignificant, P>0.05. Table No. 3 Comparison on Serum Creatinine values of 15th day in between two group II & IV. (Unpaired t - test) P values t - values 1.36

P>0.05

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 Group IV 0.6 0.13 On comparing the values of the serum Creatinine on 15th day, in between two groups, it was found that the mean score was 0.75 in Group II with SD + 0.3, and 0.6 with SD of + 0.13 in Group IV. t Mean SD Group Group III 0.71 .39 Group IV 0.6 .13 On comparing the values of the serum Creatinine on 15th day, in between two groups, it was found that the mean score was 0.71 in Group III with SD + 0.39, and 0.6 with SD of + 0.13 in Group IV. t Mean SD Group Group III 0.71 .39 Group V 0.56 .25 On comparing the values of the serum Creatinine on 15th day, in between two groups, it was found that the mean score was 0.71 in Group III with SD + 0.39, and 0.56 with SD of + 0.25 in Group V. t Mean SD Group Group IV 0.6 .13 Group V 0.56 .25 On comparing the values of the serum Creatinine on 15th day, in between two groups, it was found that the mean score was 0.6 in Group IV with SD + 0.13, and 0.56 with SD of + 0.25 in Group V. t GROUP 0TH day (mean+SE)

values was found 1.36 which was statistically insignificant, P>0.05. Table No.4 Comparison on Serum Creatinine values of 15th day in between two groups III & IV. (Unpaired t - test) P values t - values 0.77 P>0.05 values was found 0.77 which was statistically insignificant, P>0.05. Table No. 5 Comparison on Serum Creatinine values of 15th day in between two groups III & V. (Unpaired t - test) P values t - values 0.93 P>0.05 values was found 0.93 which was statistically insignificant, P>0.05. Table No. 6 Comparison on Serum Creatinine values of 15th day in between two group IV & V. (Unpaired t - test) P values t - values 0.4 P>0.05 values was found 0.4 which was statistically insignificant, P>0.05. Table No. 7 Comparison on Serum Creatinine values of all days in between all groups. 9th day 15th day (mean+SE) (mean+SE)

I

0.41+0.06

0.41+0.04

0.41+0.04

II

0.42+0.05

2.14+0.37

0.75+0.10

III

0.47+0.05

3.06+0.51

0.71+0.14

IV

0.58+0.09

1.66+0.21

0.6+0.05

V

0.54+0.08

1.64+0.13

0.56+0.09

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 Creatinine values 0n 0th,9th & 15th day in all Groups 3.5 3 2.5 Creatnine value 0th day

2

Creatnine value 9th day

1.5

Creatnine value 15th day

1 0.5 0 Gr. -1

Gr. -2

Gr. -3

Gr. -4

Gr. -5

The results of the effect of Ajitagada on 0.71+0.14, 0.6+0.05 and 0.56+0.09 mg/dl serum creatinine levels were presented in respectively for groups I, II, III, IV and V. table. On Oth day, the mean serum The result indicated that Ajitagada at a creatinine values of groups I to V were dose rate of 200 mg/kg showed 0.41+0.06, 0.42+0.05, 0.47+0.05, considerable reduction in serum creatinine 0.58+0.09 and 0.54+0.08 mg/dl value than the other treatment groups. respectively. BUN th On 9 day, all the groups except the Table No.8 the Anova test table for the normal group showed an increase in serum serum BUN values creatine values with mean values of 0.41+0.04, 2.14+0.37, 3.06+0.51, 1.66+0.21 and 1.64+0.13 mg/dl respectively for groups I, II, III, IV and V. On 15th day, there was a significant reduction in serum creatinine values in groups III to V when compared with group II. The serum creatine values with mean values of 0.41+0.04, 0.75+0.10, Source of variation Sum of Degree of Mean F-ratio 5% F limit squares freedom square (SS) (d.f.) (MS) 2 116299.6 144.021 3.07 (2,105) Between columns 232599.2 (i.e. between days) 4 24805.13 30.71 Between rows (i.e. 99220.51 2.45 (4,105) between groups) 142834.2 8 17854.28 22.11 Interaction 2.02 (8,105) 84789.19 105 807.5161 Within samples (Error) Total 559443.2 119

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 160

144.021

140 120 100 80

F-ratio

60

5% F limit 30.71

40 20

3.07

2.45

0 Between columns (i.e. between days)

Between rows (i.e. between groups)

The above table shows that all the three Frations are significant of 5% level. This analysis does not support the null hypothesis which means there is too much difference in BUN values in different days Mean SD Group Group II 115.73 30.78 Group III 21..48 4.84 On comparing the values of the serum BUN on 15th day, in between two groups, it was found that the mean score was 115.73 in Group II with SD + 30.78, and 21.48 with SD of + 4.84 in Group III. t

Mean SD Group Group II 115.73 30.78 Group IV 32.54 14.55 On comparing the values of the serum BUN on 15th day, in between two groups, it was found that the mean score was 115.73 in Group II with SD + 30.78, and 32.54 with SD of + 14.55 in Group IV. t Mean SD Group Group III 21..48 4.84 Group IV 32.54 14.55 On comparing the values of the serum BUN on 15th day, in between two groups,

(0th, 9th & 15th). Also different groups have too much difference. Table No. 9 Comparison on Serum BUN values of 15th day in between two groups II & III. (Unpaired t - test) P values t - values 8.56 P>0.05 values was found 8.56 which was statistically significant, P>0.05. Table No. 10 Comparison on Serum BUN values of 15th day in between two groups II & IV. (Unpaired t - test) t - values 6.9

P values

P>0.05

values was found 6.9 which was statistically significant, P>0.05. Table No. 11 Comparison on Serum BUN values of 15th day in between two groups III & IV. (Unpaired t - test) P values t - values 2.04 P>0.05 it was found that the mean score was 21.48 in Group III with SD + 4.84, and 32.54

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 with SD of + 14.55 in Group IV. t values was found 2.04 which was statistically significant, P>0.05.

Table No. 12 Comparison on Serum BUN values of 15th day in between two groups III & V. (Unpaired t - test) P values t - values 2.57 P>0.05

Mean SD Group Group III 21..48 4.84 Group V 52.89 14.89 On comparing the values of the serum BUN on 15th day, in between two groups, it was found that the mean score was 21.48 in Group III with SD + 4.84, and 52.89 with SD of + 14.89 in Group V. t values

was found 2.57 which was statistically significant, P>0.05. Table No. 13 Comparison on Serum BUN values of 15th day in between two groups IV & V. (Unpaired t - test) P values t - values 2.76 P>0.05

Mean SD Group Group IV 32.54 14.55 Group V 52.89 14.89 On comparing the values of the serum BUN on 15th day, in between two groups, it was found that the mean score was 32.54 in Group IV with SD + 14.55, and 52.89 with SD of + 14.89 in Group V. t values GROUP 0TH day (mean+SE)

was found 0.22 which was statistically significant, P>0.05. Table No. 14 Comparison on Serum BUN values of all days in between all groups. 9th day 15th day (mean+SE) (mean+SE)

I

24.67+1.42

31.87+2.19

32.02+1.96

II III

36.09+2.49 23.11+2.37

114.99+11.69 87.33+11.44

115.73+10.77 21.47+1.69

IV

34.36+3.802

203.11+27.59

32.54+5.09

V

31.83+3.104

223.17+7.12

52.89+5.21

Comparison on serum BUN values 250

Values

200 0TH day

150

9th day 100

15th day

(mean) (mean) (mean)

50 0 I

II

III

IV

V

Different Groups

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 The results of the effect of Ajitagada on 32.54+5.09 and 52.89+5.21mg/dl serum BUN levels were presented in table. respectively for groups I, II, III, IV and V. On Oth day, the mean serum BUN values The result indicated that Ajitagada at a of groups I to V were 24.67+1.42, dose rate of 400 mg/kg showed 36.09+2.49, 23.11+2.37, 34.36+3.802 and considerable reduction in serum BUN 31.83+3.104 mg/dl respectively. value than the other treatment groups. th On 9 day, all the groups except the ALBUMIN normal group showed an increase in serum Table No.15 the Anova table for the BUN values with mean values of serum Albumin values 31.87+2.19, 114.99+11.69, 87.33+11.44, 203.11+27.59 and 223.17+7.12 mg/dl respectively for groups I, II, III, IV and V. On 15th day, there was a significant reduction in serum BUN values in groups III to V when compared with group II. The serum BUN values with mean values of 32.02+1.96, 115.73+10.77, 21.47+1.69, Source of variation Sum of Degree of Mean square F-ratio 5% F limit squares freedom (MS) (SS) (d.f.) Between columns (i.e. between days) Between rows (i.e. between groups) Interaction Within samples (Error)

0.247

2

0.123

0.753

3.07 (2,105)

19.87

4

4.96

30.39

2.45 (4,105)

.38 17.2

8 105

0.0475 0.163

.2289

2.02 (8,105)

Total

37.69

119

35 30.39 30 25 20 F-ratio 15

5% F limit

10 5

3.07 0.753

2.45

0 Between columns (i.e. between days)

Between rows (i.e. between groups)

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 in Abumin values in different days (0th, 9th & 15th) but different groups have differences due to chance. Table No. 16 Comparison on Serum Albumin values of all days in between all groups. 9th day 15th day (mean+SE) (mean+SE)

The above table shows that two F-rations are insignificant & F-rations are significant on between rows of 5% level. This analysis supports the null hypothesis in two places & does a not support in one place which means there is no difference GROUP 0TH day (mean+SE) I

4.01+0.14

4.08+0.13

3.96+0.17

II

3.46+0.13

3.46+0.08

3.42+0.09

III

2.81+0.18

3.18+0.09

2.46+0.12

IV

3.06+0.08

3.01+0.10

3.32+0.14

V

3.05+0.10

3.12+0.10

3.21+0.19

Means bearing the same superscript do not differ significantly at P<0.05

Values

Comparison on serum Albumin values 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

0TH day

(mean)

9th day

(mean)

15th day

I

II

III

IV

(mean)

V

Different groups

The results of the effect of Ajitagada on serum Albumin levels were presented in table. On Oth day, the mean serum Albumin values of groups I to V Were 4.01+0.14, 3.46+0.13, 2.81+0.18, 3.06+0.08 and 3.05+0.10 mg/dl respectively. On 9th day, all the groups except the normal group showed an Increase in serum Albumin values with mean values of 4.08+0.13, 3.46+0.08, 3.18+0.09, 3.01+0.10 and 3.12+0.10 mg/dl respectively for groups I, II, III, IV and V.

On 15th day, there was a significant reduction in serum Albumin values in groups III to V when compared with group II. The serum Albumin values with mean values of 3.96+0.17, 3.42+0.09, 2.46+0.12, 3.32+0.14 and 3.21+0.19 mg/dl respectively for groups I, II, III, IV and V. The result indicated that Ajitagada at a dose rate of 200 & 400mg/kg showed no considerable reduction in serum Albumin value than the other groups. TOTAL PROTIEN Table No.17 the Anova test table for the serum Total protien values

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 Source of variation

Sum squares (SS)

of Degree of Mean freedom square (d.f.) (MS)

F-ratio

5% F limit

Between columns (i.e. between days) Between rows (i.e. between groups) Interaction Within samples (Error)

0.729

2

0.365

1.79

3.07 (2,105)

5.95

4

1.49

7.31

2.45 (4,105)

4025 21.35

8 105

503.13 0.203

2474.38 2.02 (8,105)

Total

29.66

119

8

7.31

7 6 5 4

F-ratio

3.07

3

5% F limit

2.45 1.79

2 1 0

Between columns (i.e. between days)

Between rows (i.e. between groups)

The above table shows that two F-rations are significant & F-rations are insignificant on between columns of 5% level. This analysis does not support the null hypothesis in two places & supports in one place which means there is no difference GROUP 0TH day (mean+SE)

in Total protien values in different days (0th, 9th & 15th) but different groups have differences due to chance. Table No. 18 Comparison on Serum Total protein values of all days in between all groups. 9th day 15th day (mean+SE) (mean+SE)

I

6.06+0.16

6.06+0.15

6.11+0.17

II

6.05+0.05

6.11+0.09

5.66+0.07

III

5.77+0.11

5.96+0.06

5.98+0.23

IV

5.41+0.09

5.48+0.08

6.16+0.32

V

5.24+0.06

5.55+0.14

5.55+0.12

Means bearing the same superscript do not differ significantly at P<0.05

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140

Values

Comparison on serum Total protien values 6.4 6.2 6 5.8 5.6 5.4 5.2 5 4.8 4.6 I

II

III

IV

0TH day

(mean)

9th day

(mean)

15th day

(mean)

V

Different groups

The results of the effect of Ajitagada on serum Total protein Levels were presented in table.On Oth day, the mean serum Total protien values of groups I toV were 6.06+0.16, 6.05+0.05, 5.77+0.11, 5.41+0.09 and 5.24+0.06 mg/dl respectively. On 9th day, all the groups except the normal group showed an increase in serum Total protien values with mean values of 6.06+0.15, 6.11+0.09, 5.96+0.06, 5.48+0.08 and 5.55+0.14 mg/dl respectively for groups I, II, III, IV and V. On 15th day, there was a significant reduction in serum Total protien values in groups III to V when compared with group II. The serum Total protien values with mean values of 6.11+0.17, 5.66+0.07, 5.98+0.23, 6.16+0.32 and 5.55+0.12 mg/dl respectively for groups I, II, III, IV and V. The result indicated that Ajitagada at a dose rate of 200 & 400mg/kg showed no considerable reduction in serum Total protien value than the other groups. In other blood parameters like RBC, WBC, Neutrophil, Lymphocyte, Monocyte, Basophil, Eosinophil, there is no

significant change in different days in all groups. Histopathological examination of kidney In normal group (group 1), the microscopic examination of the kidney revealed the usual histological parameters. The tubular structures were largely intact without the presence of any mononuclear infiltrates in the interstitial and congestion in the vessels. In Gentamicin group (group 2), there were extensive proximal tubular necrosis and loss of the lining epithelium and regeneration features were predominantly sub capsular. Besides, there were interstitial oedema, perivascular oedema and multiple focal collections of mononuclear cells in the interstitial. The glomerular changes were quite marked (deffuse degenerative tubules). In ajitagada 200mg/kg treated group (group 3), the Proximal tubular epithelial cells showed varying degrees of regeneration but slight degenerative changes. Besides this, there were scattered small foci of mononuclear cell infiltration confined to sub scapular area. The epithelial cells of the proximal convoluted

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 tubules were more or less intact. However, a few cells showed mild degenerative changes characterized by vacuolar cytoplasm. So kidney looks like healthy. In Ajitagada 400mg/kg treated group (group 4), there were areas of tubular degeneration and necrosis along with areas of perivascular oedema and tubulo-interstitial mononuclear cell infiltrates at different foci throughout the cortex. Also Glomerular cells damage and tubular cells appear haemorrhagic, hyalinizing, basophilic and regenerating. In standard drug 400 mg/kg treated group (group 5), the Renal tubular cells showed varying degrees of dilatation with hyaline cast formation in the lumen and the lining tubular epithelial cells showed varying levels of vacuolar degeneration and necrosis. Areas of tubular degeneration and necrosis were observed at a few foci in the cortex along with varying degrees of regenerative changes. The results of histopathological examination of the Kidney revealed significant changes between ajitagada 200mg/kg and standard drug 400mg/kg. Ajitagada 400mg/kg also produced significant histological changes. Ajitagada 200mg/kg produced more regenerative changes in the kidney. Summary In experimental study ‘The renal protective effect of Ajitagada’ was conducted at Veterinary college & sciences, Mannuthi,Thrissur. The present study was undertaken to assess the nephroprotective effect of Ajitagada against gentamicin induced nephrotoxicity in Sprague-dawley rats.Eight adult female Sprague-dawley rats weighing 160300g,divided into 5 groups comprising

eight animals in each group, were used for the study. The experiment was conducted for a period of 30 days for observation although research design period was 15 days. Group 1 served as healthy control group. Gentamicin sulphate was administered to group 2, 3, 4 and 5 at a dose rate of 80mg/kg i.p. for eight days. Group 2 was retained as such without any treatment but administered with 2% Gumacacia till the completion of experiment. Group3 and 4 were administered with Ajitagada at a dose rate of 200mg/kg and 400mg/kg respectively for 22 days following gentamicin administration. Group 5 was administered with aqueous extract of Ideal drug at a dose rate of 400mg/kg for 22 days for 9th to 30th day following gentamicin administration for eight days.The blood was collected from all the animals on 0th, 9th, 15th and 30th day and serum was used for the estimation of creatinine, urea, albumin and total protein. On 30th day, all the animals were sacrified and kidney was used for the histopathological studies.Serum creatinine level is used as an index of nephrotoxicity. Serum creatinine level which was elevated following gentamicin administration was lowered on 15th day itself by Ajitagada, aqueous extract of ideal drug and without drug. There is no significant change between groups. Higher value of Creatinine naturally recovered to normal value. Serum urea levels were significantly lowered on 15th day by the Ajitagada and the mean values indicated that significant reduction was found with at 200mg/kg and aqueous extract of ideal drug at 400mg/kg. The serum albumin and total protein showed no significant reduction and they were found to be within normal range throughout the experiment.The

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 results substantiated by histopathological studies confirmed that treatment with Ajitagada and ideal drug alleviated the gentamicin induced proximal tubular necrosis, interstitial oedema, perivascular oedema and mononuclear cells infiltration. The regenerative changes were predominant with Ajitagada at rate of 200mg/kg.In the present study, Ajitagada and ideal drug showed a striking nephroprotective action in a dosedependent manner by decreasing the urea levels and lessened the negative effects of Gentamicin induced Nephrotoxicity possibly, by inhibiting free radical mediated process. The diuretic property of Ajitagada may also be one of the contributing factors for the nephroprotection than treatment with the Boerhavia diffusa.From the experiment, it is concluded that Ajitagada possess nephroprotective action and hence this drug can be recommended for the treatment of various Nephrotoxicosis in animals and man. DISCUSSION Nephrotoxicity occurs as a disturbance in renal function due tovarious drug Interaction, snake poisoning, inadequate elimination of radioactive contrast materials and chemicals. It is of great concern in patients with renal failure. Nephrotoxicity may limit the clinical usefulness of many diagnostics and therapeutic agents; recognition of factors associated with higher risk for renal injury is of great importance. However the end point of nephrotoxicity is always cell death; therefore it is important to identify the mechanism in addition to the site of action, in order to formulate a strategy for damage prevention. The strategies aimed

at ameliorating the nephrotoxicity are of clinical interest. A standard drug which provides nephroprotection is still a major question yet to be answered. Many herbal drugs with action on the urinary system are widely used in human. These herbal drugs are used traditionally but their efficacy and beneficial effects are not scientifically documented. Hence the present study was undertaken to assess the nephroprotective effect of Ajitagada in gentamicin –induced nephrotoxicity. Serum parameters: Serum creatinine and serum urea (serum markers of kidney function) have been consider the most important manifestations of severe tubular necrosis of kidney. (Ali et al.,2001, Afjal et al.,2004). Effect on creatinine: Serum creatinine levels were significantly increased in gentamicin group. These results are in accordance with the findings of Ramsammy et al. (1989) that the Serum creatinine Concentration was elevated significantly in gentamicin nephrotoxicity. Elevation of Serum creatinine was marked on 9th and 15th day of the experiment in gentamicin group. The Ajitagada and Boerhavia deffusa showed a reduction in Serum creatinine level on 15th day and by the end of experiment, the Serum creatinine levels of all the treatment groups were comparable with that of normal group. The studies conducted by Shirwaikar et al., (2004) proved that Aerva lanata provided nephroprotection indicated by reduction in Serum creatinine Level which was elevated by gentamicin administration. Kotin’s et al., (2004) reported that Hemidesmus indicus, a herbal drug, ameliorated the increased Serum creatinine level thereby providing nephroprotection. Similar results are also

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 shown by the Ajitagada under study where this herbal Formulation caused a reduction in the creatinine values thereby providing a striking nephroprotective effect.The significant tested between the groups respectively revealed that all the groups were equally effective (statistically insignificant). Thus it is concluded that the Ajitagada at the dose rate of 200mg/kg and 400mg/kg not more effective in providing nephroprotection. Effect on urea: Gentamicin administration caused an elevation in the level of serum urea.This is in accordance with the findings of Kozat et al. (2007) where elevated serum urea levels were found in gentamicin group. By the end of the experiment, the serum urea levels were significantly reduced in the treatment groups when compared with the control group.Maximum reduction in serum urea levels were shown by Ajitagada at a dose rate of 200mg/kg.Ali et al. (2005) observed that elevated serum urea level was decreades by curcumin at a dose rate of 200mg/kg in gentamicin induced renal damage. The results of the present study are also in accordance with the above findings and the Ajitagada showed significant reduction in serum urea levels thereby contributing towards the nephroprotective effect.The Insignificant tested between the Ajitagada and ideal drug treated group at 200, 400 mg/kg and 400 mg/kg respectively revealed that both the treatment groups were unequally effective (p=0.08).Ajitagada is more effective in compare to ideal drug. But, the Ajitagada produced the significant reduction in serum urea value at the lower dose. Thus it is concluded that the Ajitagada at the dose rate of 200mg/kg is

more effective in providing nephroprotection. Effect on albumin and total proteinThe results of serum parameters like albumin and total Protein revealed no Significant variation. All these parameters were within the normal range (Hrapkiewicz et al., 1998).The study conducted by Kotnis et al., (2004) to assess the nephroprotective effect of Hemidesmus indicus in gentamicin induced renal toxicity in rats revealed no difference in the levels of serum albumin and total protein as compared with the gentamicin and normal control groups.These findings are in accordance with the present study that no significant variation was observed in serum albumin and total protein and gentamicin induced nephrotoxicity had no influence on these parameters. Histopathological examination of kidney Histologically, gentamicin group showed severe proximal tubular necrosis and loss of lining epithelium. Besides, there were mononuclear cell infiltrations. In Ajitagada 200mg/kg treated group, varying degrees of regeneration and only small foci of mononuclear infiltration could be seen. Studies conducted by Vardi et al. (2005) found that gentamicin administration showed marked tubular necrosis and desquamation of the cortical epithelial cells and these changes were ameliorated by caffiec acid phenethyl ester.Gentamicin nephrotoxicity occurs as a result of binding of the cationic aminoglycosides with anionic phosphatidyl inositol which are present in the kidney. Recently, it is proposed that aminoglycoside nephrotoxicity results from endocytic retrieval of the drug by megalin, an endocytic-mediated receptor,

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 present in the apical membrane of proximal convoluted tubules. However, the generation of reactive oxygen species (ROS) has been one of the major contributing factors towardsnephrotoxicity. The mechanism of action through which these plant extracts provide nephroprotection is not clearly understood, but may be due to the scavenging of the free radicals that were generated by gentamicin administration mediated by the action of phytoconstituents present in the plant extracts.the more nephroprotective effect of Ajitagada may be attributed to the excretion of toxic substances alongwith increases diuresis. Further investigations are required to elucidate the exact mechanism underlying this possible beneficial effect.Thus, the findings of the present study validate the nephroprotective effect of Ajitagada for the management of renal disorders. CONCLUSION The present study was undertaken to assess the nephroprotective effect of Ajitagada against Gentamicin induced Nephrotoxicity in Sprague-Dawley rats. The results substantiated by histopathological studies confirmed that treatment with Ajitagada and ideal drug alleviated the Gentamicin induced proximal tubular necrosis, interstitial oedema, perivascular oedema and mononuclear cells infiltration. The regenerative changes were predominant with Ajitagada at rate of 200mg/kg. In the present study, Ajitagada showed a striking nephroprotective action in a dosedependent manner by decreasing the serum urea levels, and lessened the negative effects of Gentamicin induced

Nephrotoxicity possibly, by inhibiting free radical mediated process. The diuretic property of Ajitagada may also be one of the contributing factors for the nephroprotection. From the experiment, it is concluded that Ajitagada possess nephroprotective action and hence this drug can be recommended for the treatment of various Nephrotoxicosis in animals and man. REFERENCE 1. Agnives C.R.Dr., Unnikrishnan P.Dr., George M.J. Dr. (Editors) - (2002), Toxicology, Ayurvedic Perspective, 1st edition, Published by Dept. of Agadatantra, Vaidyaratnam P.S. Varier Ayurveda College, Kottakkal. 2. Aldo Castellani & Albert J. Chalmers – (1919), Manual of Tropical Medicine, 3rd edition, published by Bailliere, Tindall & Cox, London. 3. Anonymous - (1968), Ayurvediya Shabda kosha –Maharashtra Rajya Sahitya & Samskriti Mandal, Mumbai. 4. Anonymous - (1965), Visha Vaidya Jyotsnika, 1st Edition, Published by Reddiar Press and Book Depot, Thiruvananthappuram. 5. Anonymous - (1973), Visha Vaidya Sara Samgraham, 1st Edition, Published by Bharathavilasam Press, Thrissur 6. Anonymous - (1988), Yogaratnaakara, 4th Edition, Edited by Vaidya Lakshmipati Shastry, Choukhambha Sanskrit Sansthan, Varanasi. 7. Besson J. M. - (1999), Neurobiology of Pain, The Lancet, Vol. 353: Page No.1610-15, May 9, 1999. 8. Bhavamishra – (2003), Bhaavprakaash’a Samhita II Vol, Pandit Brahma Sh’ankar Mish’ra, 8th

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 Edition, Chaukhambha Sanskrit Sansthan, Varanasi. 9. Bhel – (1999), Bhel Samhita, Edt. Girijadayalu Sukla, Reprint, Chaukhamba Bharati Academy, Varanasi. 10. Chakrapan’idatta – (1992), Ayurveda Dipika, Commentary on Charaka Samhita, 5th Edition, Munshiram Manoharalal Publishers Pvt. Ltd., New Delhi. 11. Chakrapan’idatta – (2002), th Chakradatta, 4 Edition, VyakhyaIndradev Tripathi, Edited by Ramanath Dwivedi, Published by Chaukhambha Sanskrit Samsthan, Varanasi. 12. Charaka – (2004), Charaka Samhita, English translation by R.K. Sharma & Bhagwan Dash, Published byChowkhambha Sanskrit Series, Varanasi. 13. Dalhanacharya – (1997), Commentary on Sush’ruta Samhita, Vaidya Yadavji Trikamji Acharya, 6th Edition, Chaukhambha Sanskrit Sansthan, Varanasi. 14. Daniel B. Carr, Leonidas C. Goudas – (1999), Acute Pain, Lancet, Volume 353: Page No. 2051-58, June 12, 1999. 15. Govind Das – (1999), Bhaishajya Ratnavali- Vyakhya Ambikadatta Shastri, Edited by- Rajeshwardatta Shastri, 13th Edition, PubChaukhambha Sanskrit Samsthan, Varanasi. 16. Harrison – (2005), Harrison’s Principles of Internal Medicine, 16th Edition, Volume II, Edited by Dennis L. Kasper & others, Published by McGraw Hill Medical Publishing Division, New Delhi.

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 Published by-Chaukhambha Bharati Academy, Varanasi. 27. Priyavrata Sharma Aacharya th Aayurved ka Vaigyanik Ithihaasa, 4 Edition (Scientific History of Ayurveda) Choukhambha Orientalia, Varanasi. 28. Raghunatha Pandita Manohara (1959), Chikitsamanjaree, 1st edition, Published by- Scindia Oriental Institute, Ujjain. 29. Reddy K.S.N. - (2005), Essentials of Forensic Medicine And Toxicology, 24th Edition, Published by K. Suguna Devi, Hyderabad. 30. Sastry J.L.N. Dr. – (2005), nd Dravyaguna Vignyana, 2 edition, Published by Chaukhambha Orientalia, Varanasi. 31. Savnur H.V. - (1984), Ayurvedic Materia Medica, 1st Edition reprint, Sri Satguru Publications, Delhi. 32. Sharangadhara – (1994), Sharangadhara Samhita, 4th Edition, Edited by Radhakrishna Parashar, Published by shree Baidyanath Ayurved Bhavan Pvt Ltd, Nagpur. 33. Singhal S.K - (2005), Toxicology at Glance, 6th Edition, Published by The National Book Depot, Mumbai. 34. Sreekrishnan C.M. Dr. et al. – (1985), A clinical and experimental study on Darweekara visha with special reference to Swetarka (Calotropis procera) 35. Stanley L. Robbins & others – (2003), Robbin’s Basic Pathology, 7th Edition, Published by Reed Elsevier India Pvt. Ltd, New Delhi. 36. Sugumaran M. & Vetrichelvan T.– (2005), Antivenom Activity of Medicinal Plants, The Antiseptic, Vol. 102, No.12, Page No. 739-40.

37. Unnikrishnan P. Dr. et al. – (1981), Ayurvedic management of Echis carinatus poisoning with special reference to the action of Aristolochia indica. 38. Ushakumari P.K. et al. – (2005), An open clinical trial on the mode of administration of Shigrupunarnavadi churna in the treatment of Sashopha Kit’a Visha (Insect bite with swelling), Kottakkal. 39. Sirosha M. et al. – (2006), A comparative study on efficacy of Kottamtagaradi yoga with Sigrupunarnavadi yoga in Trimeresurus gramineus bite, Kottakkal. 40. Vagbhata (1991), Ashtanga Samgraha- Comm. Indu Sasilekha vyakhya, Pub. Central Council for Research in Ayurveda and Siddha, New Delhi. 41. Vagbhata – (1997), Ashtang Hridaya English Translation by Prof. K.R. Srikantha Murthy, 2nd Edition, Krishnadas Academy, Varanasi. 42. Vagbhata – (2002), Ashtang Hridaya – Sarvaangasundara & Ayurveda Rasaayana, Collated by Anna th Moreshwara Kunte, 9 Edition Chaukhamba Orientalia Publication, Varanasi. 43. Vagbhata – (2002), Ashtanga Sangraha – English Translation by Prof. K.R. Srikantha Murthy, 5th Edition, Choukhambha Orientalia, Varanasi. 44. G.D. Singhal – Ancient Indian surgery (vol. vii), 1st edition, Published by Chaukhamba Sanskrit Pratisthan, Delhi 45. Susruta – (2003), Susruta samhita – English Translation by Kaviraj Kunjalal Bhishagratna, 3rd Edition,

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INTERNATIONAL JOURNAL OF AYUSH (Ayurveda, Yoga, Unani, Siddha and Homeopathy) Volume 4 Issue 2, 2015; 120-140 Chaukhamba Orientalia Publication, Varanasi. 46. Susruta – (2005), Susruta samhita – Comm. Nibandhasangraha vyakhya by Vaidya Javaji Trikamji Acharya and Prof. P.V.Sharma,Chaukhamba Orientalia Publication, Varanasi. 47. Vaman Shivram Apte- (2002), Student’s Sanskrit – English Dictionary, Published by Nag publishers, Delhi. 48. Vinay kumar et al–(2003), Robbins Basic pathology, 7th Edition, Saunders publisher. 49. Whitaker Romulus – (2002) Common Indian snakes- A field guide, Published by Orient Longman Pvt. Ltd. Chennai. 50. Abdel-Naim, A.B., Abdel-wahab, M.H. and Atria, F.F.1999. Renalprotective effects of vitamin E and probucol against gentamicin induced nephrotoxicity in rats. Pharmacol. Res.40:183-187 51. Afzal, M.,Khan, N.A.,Ghufran, A., Iqbal, A. and Inamuddin, M.2004. Diuretic and nephroprotective effect of jawarish zaroonisada – a poly herbal formulation. J. Ethnopharmacol.91:219-223 52. Baliga, R.,Ueda, N., Walker, P.D. and Shah, S.V.1997. Oxidant mechanisms in toxic acute renal failure.Am.J.Kidney dis. 29:465 -477 53. El-Ashmawy, I., EI-Nahas, A.F. and salama, O.M.2006. Grape seed extract prevents gentamicin-induced nephrotoxicity and genotoxicity in bone marrow cells of mice. Basic clin.Pharmacol. Toxicol.99:230-236 54. Frame, P., Bannister, T., Tan, J. and Phair, J. 1973. Gentamicin kinetics and Nephrotoxicity in rabbits.Clin.Res.21:842

55. Ali, B.H.2002. The effect of treatment with the medicinal plant Rhazya stricta decne on gentamicin nephrotoxicity in rats. Hum.Exp.Toxicol20:199-203. CORRESPONDING AUTHOR Dr. Manoj Kumar Gupta Assistant Professor, Department of Roga Nidan and Vikriti Vijnana Government Ayurvedic College and Hospital, Atarra Banda,Uttar pradesh, India. Email : [email protected]

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