ANTIUROLITHIATIC AND NEPHROPROTECTIVE ACTIVITY OF GOKSHURADI GUGGUL AN AYURVEDIC PREPARATION IN RATS

Protocol of Dissertation Submitted

By

Mr. VIKAS. R. DHOLE

To

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA

Under the guidance of

Dr. NANU. R. RATHOD

Asst. Professor

Department of Pharmacology,

HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY,

BAGALKOT- 587101, KARNATAKA.

(2010-2011)


RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA-BANGALORE

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERATATION

1. / NAME OF THE CANDIDATE AND ADDRESS / Mr. VIKAS DHOLE
DEPARTMENT OF PHARMACOLOGY
H.S.K.COLLEGE OF PHARMACY
B.V.V.S. CAMPUS
BAGALKOT-587101, KARNATAKA.
2. / NAME OF THE INSTITUTION / H.S.K.COLLEGE OF PHARMACY
B.V.V.S. CAMPUS,
BAGALKOT- 587101, KARNATAKA.
3. / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN
PHARMACOLOGY
4. / DATE OF ADMISSION TO COURSE / 22nd June 2010
5. / TITLE OF THE TOPIC:
“Antiurolithiatic and Nephroprotective activity of Gokshuradi Guggul An Ayurvedic Preparation in Rats”
6. / Brief resume of the intended work
6.1 Need for the study:
Kidney stones are one of the most common urological problems in the United States, 13% of men and 7% of women will develop a kidney stone during their lifetime and the prevalence of Kidney stone formation or urolithiasis is a complex process that is a consequence of an imbalance between promoters and inhibitors in the kidneys1. The recurrence of urolithiasis represents a serious problem as patients who have formed one stone are more likely to form another. Not all standard Pharmaceutical drugs used to prevent urolithiasis are effective in all patients and many have adverse effects that compromise their long-term use.
Renal calculi can be broadly classified in two large groups: 1) Tissue attached and 2) unattached. Tissue attached calculi are mainly integrated by Calcium oxalate monohydrate (COM) renal calculi, with a detectable attachment site to the renal papilla and basically consisting of a core located near to the attachment site (concave zone) and radially striated concentrically laminated peripheral layers. Unattached calculi, with no detectable site of attachment to papilla, are developed in renal cavities of low or reduced urodynamic efficacy and can exhibit diverse composition and structures. Several reports have been published since Randall's first description of papillary calcifications and their possible active role in the genesis of COM papillary calculi At present, it seems clear that renal epithelial cell injuries play a decisive role in such a type of renal calculi development and in fact the lithogenic effect caused by ethylene glycol (EG) must be mainly attributed to the oxidative damage caused by the high level of oxalate generated by EG. Thus, although EG rat model can be questioned as a general model to study renal stone formation, it must be considered as an interesting model to evaluate renal papillary stone development, at least for those stones which genesis is linked to oxidative cell damage. Are most likely due to antioxidative effects. To further investigate the potential of gokshuradi guggul as a therapy for lithiasis, the present study2.
6.2 Review of literature:
Plant Profile:
Title of plant: Commiphora mukul, Linn Family: Burseraceae Synonyms:Guggula
Parts used: Gum Habitat: India
Chemical constituents: Gum-resin, bitter principle, carminative3.
Medicinal action and uses: Emulcent, carminative, antispasmodic, antiseptic, diuretics, demulcent3.
Pharmacological activity: Antibacterial activity, antiseptic4.
Title of plant: Fagonia Arabica, Linn Family: Zygophyllaceae Synonyms:Dhmasa
Parts used: Gum Habitat: India
Chemical constituent: Ca, Na, P, Cu, Fe, Mn Zn
Medicinal action and uses: Laxative, stimulant Bitter tonic, diuretic & astringent3.
Pharmacological activity: Antibacterial activity, myocardial infraction5.
Title of plant: Tribulus terrestris, Linn. Family: Zygophyllaceae Synonyms: Gokshuradi
Parts used: Root Habitat: India
Chemical constituents: Alkaloid, resin, fixed oil, essential oil3
Medicinal action and uses: Disorder of genitourinary tract, gout, impotence, Demulcent, diuretic, tonic3.
Pharmacological activity: antiurolithiatic, antimicrobial, antifungal activity6.
Title of plant: Cyperus rotundus,Linn. Family: Cyperaceae Synonyms : Musta
Parts used : Tuber Root, Habitat: India.
Chemical constituents: Fat, sugar, gum, carbohydrates, starch.
Medicinal action and uses: Fever, diarrhoea, anthelminitics, Astringent.
Pharmacological activity: Antidiabetic activity on rats7.
Title of plant: Embilica officinalis. Family: Euphorbiaceae. Synonyms: Amla
Parts used: Dried fruit Habitat: India
Chemical constituents: Fixed oil, volatile oil3.
Medicinal action and uses: Ageing & restore organ, diarrhea, Diuretic3.
Pharmacological activity: Antitumor, antimicrobial, antioxidant activity8.
Title of plant: Terminalia belerica. Family: Combretaceae Synonyms: Beleric
Parts used: Fruits Habitat: India
Chemical constituents: Tannins, anthraquinones, resins3.
Medicinal action and uses: Coughs, liver Gastrointestinal Tract diseases3.
Pharmacological activity: Antimicrobial, bronchial asthma, antidiarrea9.
Title of plant: Terminalia chebula Family: Combretaceae, Synonyms: Heritaki
Parts used: Fruits Habitat: India
Chemical constituent: Tannins, anthraquinones, resins.
Medicinal action and uses: Treatment of Diarrhea, dysentery, urinary diseases3.
Pharmacological activity: Antioxidant & probable radioprotector, anticancer activity10.
Title of plant: Piper nigrum, Linn. Family: Piperaceae, Synonyms-: Black pepper
Parts used: Dried unripe Fruit, Habitat: India
Chemical constituents: Alkaloid piperidin, volatile oil3.
Medicinal action and uses: Stimulant, Carminative, antipyretic, antiseptic3.
Pharmacological activity: antibacterial activity, CNS stimulant, analgesic & antipyretic
Activity12.
7. / Title of plant: Piper lognum,Linn. Family: Piperaceae Synonyms: Pippali,

Parts used: Dried unripe Fruit Habitat: India

Chemical constituents: Resins, volatile oil, starch gum3.

Medicinal action and uses: Carminative, diuretic, vermifuge3.

Pharmacological activity: Immunomodulatory & anticancer, antiamoebic, arthritis11.

Title of plant: Zingiber officinale, Family: Scitaminaceae Synonyms: Sunth

Parts used: Rhizomes Habitat: India
Chemical constituents: Volatile oil resins, starch3.

Medicinal action and uses: Diuretic, asthma, dyspepepsia3.

Pharmacological activity: Dyslipidemia in diabetic rats, inflammation, arthritis13.
6.3 Objectives of the study:
Traditionally well known Ayurvedic preparation called as Gokshuradi guggul will be used for the present study. The purpose of the study is to scientifically evaluate antilithiasis and Nephroprotective effect of Gokshuradi guggul on rat
1.Evaluation of Gokshuradi guggul on Ethylene glycolated water (E.G.W) 0.75% inducedLithiasis in rats
2. Evaluation of Gokshuradi guggul on Gentamicin induced Nephrotoxicity in rats
Methods and Materials
7.1 Source of data:
a) Literature survey, books, website- Pubmed, Medline, Scidirect, Scirus, Elesvier,etc.
b) Publication of Journals.
c) Lab based studies.
7.1.1 Ethylene glycolated water 0.75% (E.G.W ) induced Lithiasis in rats:
Group I : Normal
Group II : Control (E.G.W 0.75% ) Lithiasis.
Group III : Effect of Cystone 750 mg/kg on E. G.W induced lithiasis.
Group IV : Effect of Gokshuradi guggul low dose on E.G.W induced lithiasis.(C.R)
Group V : Effect of Gokshuradi guggul moderate dose on E.G.W induced lithiasis. (C.R)
Group VI : Effect of Gokshuradi guggul High dose on E.G.W induced Lithiasis. (C.R)
Group VII : Effect of Gokshuradi guggul low dose on E.G.W induced lithiasis. (P.R)
Group VIII : Effect of Gokshuradi guggul medium dose on E.G.W induced lithiasis. (P.R)
Group IX : Effect of Gokshuradi guggul high dose on E.G.W induced lithiasis. (P.R)
Abberivations: (C.R) = curative regimen, (P.R) = preventive regimen
7.1.2 Gentamicin Induced Nephrotoxicity in rats:
Group I : Normal
Group II : Control (Gentamicine 100 mg/kg)
Group III : Effect of Selenium (6 mg/kg) on Gentamicin induced nephrotoxicity
Group IV : Effect of Gokshuradi guggul low dose on Gentamicin induced nephrotoxicity.
GroupV : Effect of Gokshuradi guggul medium dose on Gentamicin induced nephrotoxicity.
GroupVI : Effect of Gokshuradi guggul high dose on Gentamicin induced
nephrotoxicity
7.3 Methods:
7.3.1. Preparation of Gokshuradi guggul
The Gokshuradi guggul an ayurvedic preparation prepared as per the method14. The Gokshuradi guggul containing the following plants preparation; Zingiber officinali, Piper nigrum, Piper lognum, Cyperus rotundus, Terminalia balerica, Terminalia chebula, Tribulus terrestris, Fagonia Arabica and Embillica officinalis, Commiphera mukul.
7.3.2 Ethylene glycolated water 0.75 % (E.G.W) induced Lithiasis in rats:
Lithiasis was induced in rats by using the method of Selvam et al. With modification the animal were selected from acclimatized crowed and randomly divided into 9 groups of 6 rats. The treatment was carried out for 28 days. The urine was collected on days 7, 14, 21 and 28th for 24 hr by keeping the animals in polypropylene metabolic cages. The collected urine was used for analysis of calcium (using calcium liquid kits), magnesium using standard methods. The volume of urine collected from all groups will be recorded. Finally, the prevalence of lithiasis will be confirmed by histopathological studies of the kidneys15.
7.3.3 Gentamicin induced nephrotoxicity in rats:
Wister albino rats were assigned 6 groups and each group contains 6 rats. The Nephrotoxicity was induced by intraperitoneal administration of 100 mg/kg/day for 8 days, along with Gentamicin the Gokshuradi guggul an Ayurvedic preparation was continued for 8 days. On the 8th day, rats were placed in separate metabolic cages for 24 hr for urine collection to determine urine output and urine creatinine content. The blood samples were collected via retro-orbital puncture for estimation of the serum creatinine and blood urea nitrogen (BUN). The changes in body weight were recorded and kidney were weighed and processed for histopathological examination16.
7.3.4 Evaluation of biochemical parameters: The urine parameters sodium, potassium, urea, creatinine, phosphate, oxalate17, body weight and tissue kidney homogenate glutathione (GSH)18, superoxide dismutase (SOD)19, lipid peroxidase (LPO)20, catalase (CAT)21, protein were estimated22.
7.3.5 Histopathological studies:
The isolated kidneys were weighed and transferred in to 10% formalin (pH 7.4) The pathological changes were observed in the section of kidney fixed in paraffin and stained with hematoxylin and eosin15.
7.3.6 Statistical analysis: The obtained data was analyzed using one-way ANOVA followed by Dunnette’s multiple comparison test P< 0.05 was consider significant.
7.4 Does the study require any investigation or to be conducted on patients or other humans/animal? If so please describe briefly
Yes, the above study required to be carried out in rat. The effects of preparation on antiurolithiatic and nephroprotectivity will be studied by considering physiological, pathological, biochemical parameters using animal models.
7.5 Has ethical clearance been obtained from your institution for performing various tests on animals?
Yes, the study is cleared institutional animal’s ethics committee & the copy is enclosed.
8. / REFERENCES:
1.  Kasper D., Braunwald E., Fauci., Hauser s., Longo D. Harrison’s principle of internal medicine. 16thed. McGraw-hill, USA 2005; pp 1710-1711.
2.  Touhami M., Amine L., Farouk L. Lemon juice has protective activity in a rat urolithiasis model. J BMC urol 2007; 1: 7-18.
3.  Nadkarni A. Indian materia medica 3rded. Popular prakashan India 2007; 1: pp.167, 428, 480, 533, 663, 665, 1202, 1205, 1229, 1308.
4.  Asif M., Sabir A. Antibacterial activities of some constituents from Oleo-gum resin of Commiphora mukul. Fitoterapia 2004; 75: 204-208.
5.  Sweta S., Prasad A., Singh R. Effect of Fagonia arabica on in vitro thrombolysis. J BMC 2007; 10:140-145.
6.  Anand A., Patnaik G., Shrivastava S. Evaluation of antiurolithiatic activity of Tribulus terrestris. Pharmaceutical Biology 1994; 32: 217-224.
7.  Nishicant A., Raut A., Gaikwad N. J. Antibacterial activity of hydro-ethanolic extract of Cyperus rotundus alloxan induced rat. Fitoterapia 2006; 77: 585-588.
8.  Jinak K., Girija K., Ramdasa K. Antitumor activity of Embilica officinalis
J Ethanopharmacol 2001; 75: 65-69.
9.  Elizabeth K. M. antimicrobial activity of Terminila bellerica. Indian J. Clin. Biochem 2005; 20: 150 -153.
10.  Naik G. H., Priydarshini K. I., Naik. D. B., Mohan H. Studies on the aqueous extract of Terminalia chebulla as a potent antioxidant and probable radio protector. International Journal of Phytotherapy & Phytopharmacology 2004; 25: 530-538.
11.  Sunila E. S., Khutan G m. Immuno modulator & Anti-tumor activity of Piper lognum fruits and Piperine. J Ethanopharmacol 2004; 90: 340-346.
12.  Pravin B.,Reddy V., Hora K., Chinna B. Antibacterial constituent from the berries of Piper nigrum. Phytomedicine 2004; 11: 697-700.
13.  Penna S. C., Medeiors M. V., Ambire F. S. Zingeber officinalis rhizomes on rat paw and skin edema. J Ethanophrmacol 2004; 90:310-315
14.  Sharangdhara., srikanta murthy K. R. A Treatise on Auyrveda Sharangdhara samitha 50thed Chaukhambha orientalia Varanasi India 2003; pp 109.
15.  Selvam R., Kalaiselvi P., Govindaraj A., Balamurugan V., Satish Kumar A. S. Effect of Arva lanata leaf extract and vediuppu chunnam on the urinary risk factors of calcium oxalate urolithiasis during experimental hyperoxaluria. Pharmacol Res 2001; 43: 89-93.
16.  Kalyan S. B., Christina A. J. M., Sundar B.S., Selvakumar S., Sundara K.S. Antilithiatic activity of Hibiscus sabdariffa linn. on ethylene glycol-induced lithiasis in rats. Natural Product radiance 2009; 8: 43-47.
17.  Hodgkinson A.,Willams A. An improved colorimetric procedure for urine oxalate. Clin Chim Acta 1972; 36: 127-132.
18.  Ellman G. L. Tissue sulfhydryl groups. Arch Biochem Biophys 1959; 82: 70-73.
19.  Beauchamp C., Fridovich I. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971; 44: 276-287.
20.  Ohkawa H., Ohishi N., Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acids reaction. Anal Biochem 1979; 95: 351-358.
21. Sinha A. Calorimetric assay of catalase. Anal Biochem 1972; 47: 389-394.
22.  Lowry O. H., Rosebrough N. J., Farr L., Randall R. J. Protein measurement with the
Folin-Phenol reagent. J Biol Chem 1951; 193: 265-272
9. / SIGNATURE OF CANDIDATE / (Mr. VIKAS DHOLE)
10. / REMARKS OF THE GUIDE / The present study has been proposed based on traditional claim. The results obtained from the study may validate its traditional therapeutic claim.
11. / NAME AND DESIGNATION OF
THE GUIDE / Dr. Nanu. R. Rathod
Asst. Professor
12. / SIGNATURE
13. / CO-GUIDE
14. / SIGNATURE
15. / HEAD OF THE DEPARTMENT / Dr. I. S. MUCHANDI
H.O.D. Dept of Pharmacology
H. S. K. College of Pharmacy,
BAGALKOT-587101.
16. / SIGNATURE
17. / REMARKS OF THE PRINCIPAL / The above mentioned information is correct and I recommend the same for approval.
18. / NAME OF THE PRINCIPAL / Dr. I. S.MUCHANDI
H.O.D. Dept. of Pharmacology
H. S. K. college of pharmacy,
BAGALKOT-587101.
19 / SIGNATURE


OFFICE OF THE INSTITUTIONAL ANIMAL ETHICS COMMITTEE (IAEC)

HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY,

BAGALKOT-587101, KARNATAKA.

REG NO.821/01/a/CPCSEA, Dated: 6th AUG 2004 UNDER THE RULES 5(a) OF THE

“BREEDING OF AND EXPERIMENTS ON ANIMALS (Control and Supervision)

RULES 1998”

Ref: HSKCP/IAEC, Clear / 2010-11 / 1-12

CERTIFICATE

This is to certify that Mr. Vikas Dhole a student of first year M. Pharm is permitted to carry out experiments on animals for the dissertation/thesis work entitled “ Antiurolithiatic and Nephroprotective activity of Gokshuradi Guggul An Ayurvedic Preparation in Rats” as per details mentioned and after observing the usual formalities laid down by IAEC as per provision made by CPCSEA.