Anti-Urolithiatic Activity of Extracts Of

“ANTI-UROLITHIATIC ACTIVITY OF EXTRACTS OF PLANT - Mimosa pudica L. IN RATS”.

By Mr. Patel Sanjay B.Pharm M. Pharm Dissertation Protocol Submitted to the

Rajiv Gandhi University of Health Sciences, Bangalore – 560 041, Karnataka.

In partial fulfillment Of the requirement for the degree of

MASTER OF PHARMACY IN PHARMACOLOGY

Under the Guidance of

Dr. Kailasam Koumaravelou M.Pharm.Ph.D. sProfessor, Department of Pharmacology and Toxicology .

Dept. of Advanced Pharmacology and Toxicology St. John’s Pharmacy College St. John’s educational institutions Bangalore- 560104.

2009-2010

1 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA.

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

Mr. PATEL SANJAY PARASOTAM BHAI Post Graduate Student, 1. Name of the candidate & Address. St. John’s Pharmacy College No 6,9th cross,2nd Main, Vijaynagar 2nd Stage (Hampinagar) Bangalore- 560104

St. John’s Pharmacy College #6, 9th cross, 2nd main, Vijayanagar 2. Name of the Institution. 2nd stage, (Hampi nagar), Bangalore- 560040. Tel: 91-80-23300958/ 23300668 Email: [email protected]

3. Course of the study & subject. Master of Pharmacy in Pharmacology

4. Date of admission. 15th June-2008

5. Title of the Topic :-

“Anti-Urolithiatic activity of extracts of plant -Mimosa pudica L. in rats.”

6. Brief resume of intended work

2 6.1 Need of the work

Urolithiasis is the third most common disorder of the urinary tract. The worldwide incidence of

urolithiasis is quite high and in north India more than 80% of urinary calculi are calcium oxalate

stones alone or calcium oxalate mixed with calcium phosphate 1.

Hyperoxaluria is the main initiating factor of human idiopathic calcium oxalate (CaOx) stone

disease. Oxalate is a powerful crystallization-driving factor present in the urine. Retention of which

enhances cell injury and causes early stages of lithogenesis 2.

Current strategies in treatment :

Surgical and other procedures: surgery is recommended for patients with severe pain who does

not respond to medications, for those with serious bleeding, and persistent fever, nausea, or

significant urinary obstruction. If no medical treatment is provided after surgery, stones reoccur in

50% of patients with in five years.

Hence the present study is designed to evaluate the anti-urolithiatic activity of extracts of wood bark

of Mimosa pudica.

6.2 Review of Literature

3 Anti-urolithiatic agents are used to reduce / dissolve the kidney stone precipitates, caused by chemical, crystalline and amorphous substances. The deposits may originate from the blood cells or from the renal tract casts or accumulation of foreign substances in urinary tract, which are involved in formation of kidney and urinary bladder stones. The term calculus is synonymous with uroliths, stones, or crystals, which are painful urinary disorders that start as salt/chemical crystals that precipitate from urine. Under normal circumstances, the urine contains certain substances that prevent crystallization. When urine fails to dissolve deposits effectively in urinary tract, such deposits assume higher proportions which may affect the passage of urine and known as urolithiasis

Anti-urolithiatic activity from plant drugs :

The two Sidha drugs, Aerva lanata and vediuppu chunnam, tested for hyperoxaluria, calculi induced in rats using ethylene glycol in drinking water. increased the urine volume, and reduced calcium oxalate and other crystallizing salts 4.

The Actinidia chinesis(Plum), Vaccinium macrocarpon(Cranberry) and Ribes nigrum(Blackcurrant) juice decreased the urinary pH, whereas the excretion of oxalic acid and the relative supersaturation for uric acid increased. Cranbery juice acidifies urine, and found useful in the treatment of urinary tract infection 5,6.

The effects of seven plant drugs Verbena officinalis, Lithospermum officinale, Taraxacum officinale, Equisetum arvense, Arctostaphylos ura-ursi, Arctium lappa and Silene saxifraga dugs are considered that it prevents and treat the kidney stone formation due to the presence of saponins 7.

The leaf extract of Coleus aromaticus has shown in reduction in deposition of urinary calculi induced by glycolic acid in experimental rats 8.

Ethanolic extract of the fruits of Tribulus terrestris showed significant dose dependent protection

4 against uroliths of rats induced by glass bead implantation 9.

The effect of aqueous extract of Tribulus terrestris reduced oxalates in rats, where oxalates induced by sodium glycolate. The glycolate resulted in hyperoxalurea and increased the activities of oxalate synthesizing lever enzymes like glycolate oxidase, glycolate dehydrogenase and lactate dehydrogenase and decreased kidney LDH activity, where the extract has reversed the activity of above said enzymes 10.

The medication for urolithiasis to relieve the acute pain, combined tinctures of wild yam

(Dioscorea villosa), cramp bark (Viburnum opulus), kava (Piper methysticum), and Jamica dogwood

(Piscidia piscipula), Drinking the infusion of equal parts of gravel root (Eupatorium purpureum), corn silk (Zea mays), pipissewa (Chimaphila umbellate), and kava, found to be effective in uroilithiasis 11.

Anti-urolithiatic activity from isolated constituents:

The efficacy of the pentacyclic triterpene isolated from Crataeva nurvula, lupeol and its ester, lupeol linoleate, against calcium oxalate urolithiasis in rats was examined. Pyridoxine deficient diet containing glycolic acid lead to increased excretion of stone forming constituents such as calcium oxalate and uric acid. Crystal deposition and subsequent renal tubular damage resulted in increased excretion of the tubular enzymes alkaline phosphatase, lactate dehydrogenase. Treatment with lupeol and lupeol linoleate reduced the extent of tubular damage with the evidence from reduced enzymuria and minimized the excretion of stone forming constituents 12.

The pentacyclic triterpenes lupeol and its structural analogue betulin from the plant Crataeva nurvula have been used on hyperoxaluric rats that minimized the tubular damage and reduced the markers of crystal deposition in the kidneys. Lupeol was found to be more effective than betulin 13.

Lupeol isolated from the plant Crataeva nurvula decreased the kidney oxalate level and also effective in counteracting the free radical toxicity by bringing about a significant decrease in peroxidase levels and increase in antioxidant status 14.

5 Schaftoside a flavonoid isolated from Desmodum styracifolium exhibited significant anti- urolithiatic activity by reducing the urine content like calcium, citric acid and creatinine 15.

The inhibitory effect of Desmodium styracifolium-triterpenoid (Ds-t) which was isolated from

D.styracifolium has inhibited the formation of calculi in rat kidneys by increasing the output of urine and excretion of citrate 15.

Different extracts of Alisma orientalis on urinary oxalate stone formation on rats was induced by ethylene glycol and ammonium chloride. In this, the ethyl acetate elution of ethyl acetate fraction of drug significantly inhibited urinary stone formation in rat16.

PLANT DESCRIPTION :

A plant, Mimosa pudica L. is known by the very rapid movement of the leaves when it is stimulated by touch, heating, etc.

Characters:-

Plant: Small woody herbs or low-spreading undershrub with hairy and prickly branches, hairs glandular 17. Leaves: Bipinnate, sensitive to touch, pinnae 1-2 pairs, leaflets 10-20 pairs, linear, glabrous 17. Flowers: Heads small, peduncled, globose, axilalry, pink-purple, Calyx campanulate, Petals crenate towards base 17. Pods: 1.5-2.5 cm long, closely prickly on the sutures 17.

Flowering and Fruiting time: Sept.-March in Indian conditions 17.

Scientific Name: Mimosa pudica Linn 17.

Family: Mimosoideae 17.

Synonyms: Sensitive Plant, Bashful Mimosa, Humble Plant, Touch-me-not 17.

Chemical Constituent: Mimosine, a non-protein plant amino acid 18. Flavonoid glycosides 19. 4- OH Maysin and Cassiaoccidentalin B 20.Tannin 21.Mucilage 17. Tubulin 22.

Biological activity:

6 Antibacterial activity: Hydroalcholic extracts of Mimosa pudica has activity against all the species namely, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi 23. Hyperglycemic effect: Ethanolic extract of Mimosa pudica leaves showed a significant hyperglycemic effect 24. Anticonvulsant activity: The decoction of Mimosa pudica leaves showed protection against pentylentetrazol and strychnine-induced seizures 25. Use in poisoning: Aqueous extracts of Mimosa pudica root possess compound(s), which inhibit the activity of cobra venom 26. Contraceptive and Antifertility : Methanolic extract of Mimosa pudica root, prolonged the length of the estrous cycle with significant increase in the duration of the diestrous phase 27. Spasmogenic activity: Ethanol extract of Mimosa pudica whole plant exhibited Spasmogenic 6.3 activity in isolated guinea pig ileum 28. Diuretic activity: Decocation of Mimosa pudica leaves exhibited Diuretic activity 28.

Objective of the study

1. Preparation of petroleum ether extract, ethanolic extract, aqueous extract of bark of Mimosa pudica using maceration process.

2. To investigate preliminary phytochemical constituents of petroleum ether extract, ethanolic extract, aqueous extract of Mimosa pudica.

3. Determination of LD50 of the aqueous extract of Mimosa pudica as per OECD guideline.

4. To establish the pharmacological profile of prepared extract for its anti-urolithiatic activity in male Wistar rats.

7. 7.1

Materials and methods Source of data:

7 Whole work is planned to generate data from laboratory studies i.e.; experiments are performed as described in reference, experimental studies in journals and in textbooks available with college, IISc library, Bangalore, RGUHS digital library (Helinet) and various institutions. Websites: www.sciencedirect.com www.pubmed.gov www.google.com www.jhetchem.com 7.2 www.ijp-online.com Will be used to obtain related information regarding this research protocol.

Method Of Collection Of Data The whole study is divided in the following five phases

PHASE -1:

Preparation of extracts:- petroleum ether extract, ethanolic extract, will be obtained by successive soxhlet extraction. The marc obtained after alcoholic extraction was macerated with water to obtain an aqueous extract.

PHASE -2:

Preliminary phytochemical investigation has been done in this phase as described by Khandelwal 29.

PHASE -3:

Acute oral toxicity

Acute toxicity study for the ether, ethanolic, aqueous extract of Mimosa pudica L. will be done according to the OECD guidelines No: 423 and, medium and high dose will be selected for treatment. Method:- The overnight fasted rats will be divided into 04 groups, each group consisting of 3 female animals. The EBR will be given in various doses (5, 50,300,2000) by gastric incubation with a syringe. After administration of the extract, the animal will be observed continuously for the first 2 hours and at 24 hrs to detect changes in behavioral responses and also for tremors, convulsion, salivation, diarrhea, lethargy, sleep, and coma and also will be monitored up to 14 days for the toxic symptoms and mortality.After 14 days of acute oral toxicity the survival mice will be rehabilitated and reused in anti tumor screening.

8 PHASE -4:

Anti-urolithiatic activity: Sixty healthy adult wister rats of either sex weighing (180-250g) will be divided into 10 groups consisting of 6 animals in each group 30.

ASSESSMENT OF ANTI-UROLITHIATIC ACTIVITY OF Mimosa pudica: Induction of urolithiasis in rats by using sodium oxalate: GROUP TREATMENT Group-1 Receive Saline (1 ml/kg) Group-2 Receive Sodium oxalate (7mg/100g,in) Group-3 Receive Sodium oxalate+vehicle (7 mg/100g, ip) Group-4 Receive Sodium oxalate+Cystone (7 mg/100g, ip + 500mg/kg,p). Group-5 Receive Sodium oxalate+ Ether extract.(Medium dose) Group-6 Receive Sodium oxalate+Ether extract.(High dose) Group-7 Receive Sodium oxalate+ Ethanolic extract.( Medium dose) Group-8 Receive Sodium oxalate +Ethanolic extract.(High dose) Group-9 Receive Sodium oxalate +Aqueous extract.( Medium dose) Group-10 Receive Sodium oxalate +Aqueous extract.(High dose)

Sodium oxalate induced urolithiatic model in rat will be used to assess the effect of petroleum ether extract Mimosa pudica. The study is designed to find out the effect of extracts of Mimosa pudica on therapeutic usage against sodium oxalate induced urolithiasis.

All rats will be housed in metabolic cages individually for entire duration of the experiment. The urine of each rat will be collected on 7th day after 6 hrs of sodium oxlalate injection with Thymol as a (preservative) and serum of each rat will be collected. Estimation of bio-chemical parameters viz Urea, Uric acid, Creatinine, Sodium, Chlorides and Potassium in serum and urine will be done. Therapeutic groups will be sacrificed on 7th day. Their right kidney will be examined for the presence of calcium oxalate crystals and stone formation by histological techniques. Crystal deposition is graded as grade ‘0’ – no deposition of crystals, grade ‘1’ – mild deposition of crystals, grade ‘2’- moderate deposition of crystals and grade ‘3’- higher amount of calcium oxalate crystals in kidney. Diuretic activity for the extracts which will show good anti-urolithiatic will be done which is one of the possible mechanism of action for the anti- urolithiatic activity.

Parameters:

9  Serum and urine electrolytes viz. sodium, potassium, chloride will be determined by electrolyte analyzer or flame photometry.  Serum / urine creatinine and uric acid will be determined with the help of auto analyser.  Urine and kidney oxalate concentration will be determined 31.  The pH of the urine is determined by pH meter.

Statistical analysis:

Values will be expressed as mean ± SEM from 6 animals. Statistical difference mean will be analyzed using one way ANOVA (analysis of variance) followed by Dunnett’s test p<0.05 will be considerable significant. 7.3

Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly. 7.4 Yes, the experimental models require usage of laboratory animals.

Has ethical clearance been obtained from your institution in case of 7.3? Yes, ethical clearance has been obtained (copy enclosed)

List of references: 1. Mitra SK, Gopumodhavan S, Venkataranganna MV, Sundaran R. Effect of Cystone, A Herbal Formulation on Glycolic Acid –Induced Urolithiasis in Rats. Phytother Res 1998;12:372-4.

2. Kumar R, Mukherjee M, Bhandari M, Kumar A, Sidhu H, Mittal RD. Role of Oxalabacter formigenes in calcium oxalate stone disease. A study from north India. Eur Urol 2002;41:318-22.

10 3. Kasper DL, Braunwald E, Fauci AS, Hauser SL, Longo DL, Jameson JL, Editors. Harrison’s Principles of Internal Medicine. 15 th ed. Newyork:McGraw-Hill 2003:2:2268-73.

4. Selvam R, Kalaselvi P, Govindraj A, Bala murugan V, Sathish Kumar AS. The effect of Aerva lanata and vediuppu chunnam in hyperoxaluria, calculi induced in rats using ethylene glycol. Pharmacological Research 2001;43(1):89-93.

5. Kahn HD, PanarielloVA, Saeli J, Sampson JR. Schwartz E. Effct of Cranberry juice in urine. J Am Diet Assoc1967;51(3):251-4.

6. Ke Bler T, Jansen B, Hesse A. European J of Clinical Nutrition 2002;56:1020-3.

7. Grases F, Melero G, Costa-Bauza A, Prieto R, March JG. Urolithiasis and phytotherapy. Int Urol Nephrol 1994;26(5):507-11.

8. Baskar R, Varalakshmi P, Amasaveni R. Indian drugs 1992;29:254-8.

9.Anand R, Patnaik GK, Srivastava S, Kulshrestha DK, Dhawan BN. Activity of certain fractions of Tribulus terrestris fruits against experimentally induced urolithiasis in rats. Ind J Exp Biol 1994;32(8):548-52.

10. Anand R, Patnaik GK, Kulshrestha DK, Dhawan BN. Activity of certain fractions of Tribulus terrestris fruits against experimentally induced urolithiasis in rats. Ind J Exp Biol 1994;32(8):548-52.

11.Anne McClenon ND. Altrnative and Complimentary Medicine 1999. USA.

12. Varalakshmi P, Shamila Y, Latha E, Jayanthi S. Effect of Crataeva nurvala on the biochemistry of the small intestinal tract of normal and stone-forming rats. J Ethnopharmacol 1991;31(1):67-73.

13. Varalakshmi P, Shamila Y, Latha E. Effect of Crataeva nurvala in experimental urolithiasis. J ethnopharmacol 1990;28(3):313-21.

14. Malini MM, Baskar R, Varalakshmi P. Effect of lupeol, a pentacyclic triterpene, on urinary enzymes in hyperoxaluric rats. Jpn J Med Sci Biol 1995;48(5-6):211-20.

15. Hirayama H, Wang Z, Nishi k, Ogawa A, Ishimatu T,Ueda S, et al. Effect of Desmodium styracifolium-triterpenoid on calcium oxalate renal stones. Br J Urology. 1993;71(2):143-7.

16. Zheng-guo C, Ji-houng L, Radman AM, Ji-zhou Wu, Ying CP,Zho SW. The effects of the active constituents of Alisma orientalis on renal stone formation. Zhongguo Zhong Yao Za Zhi 2003;28:1072-5.

17. http:// www.bio.miami.edu / momosa / mimosa.html .(accessed on 19th may2009)

18. Lalitha K, Kulothungan SR.Mimosine mitigates oxidative stress in selenium deficient seenlings of vigna rediata Biol Trace Elem Res. 2007;118(1):84-96.

11 19. Umi KY, Noriha A, Baki B, Sukari MA, Faridah A.Flavonoid glycosides in the leaves of Miomosa species.Biochemical Systomatics and Ecology 2003;31(4):443-5.

20. Annelisa L, Bernard W, Byung HU, Marc S, Robert A.the 4”-hydroxymaysin and cassiaoccidentalin B,two unusual C-glycosylflavones from Mimosa pudica. Biochemical Systomatics and Ecology 2002;30(4):375-7.

21. Fleurat-Lessard P, Frangne N, Maeshima M, Ratajczak R, Bonnemain JL, Martinoia E. Increased Expressipn of Vacular Aquaporin and H+-ATPase Related to Motor Cell Function in Mimosa pudica Plant Physiol.1997;114(3):827-34.

22. Chaudhuri AR, Biswas S.Cold stability of microtubules of Mimosa pudica. Biochem Mol Biol Int.1993 ;29(3):421-8.

23. Balakrishnan N, Bhaskar VH,Jayakar B and Sangameswaran B. Short communication Antibacterial activity of Mimosa pudica, Aegle marmelos and Sida cordifolia. Phcog Mag 2006;2(7):198-9.

24. Amalraj T,Ignacimuthu S. The Hyperglycemic effect of leaves of Mimosa pudica Linn. Fitoterapia 2002;73(4):351-2.

25. Bum EN, Taiwe GS, Nkainsa LA, Hiana IR, Bailabar T, Rakotonirina A. Validation of anticonvulsant and sedative activity of six medicinal plants. Epilepsy & Behavior2009;14(3):454-8.

26. M.Mahanta, A. Mukherjee.The Neutralisation of lethality, myotoxicity and toxic enzymes of Naja kaouthia venom by Mimosa pudica root extracts. J Ethnopharmacol 2001;75(1):55-60.

27. Ganguly M, Devi N, Mahanta R, Borthakur MK. Effect of Mimosa pudica root extract on vaginal estrous and serum hormones for screening of antifertility activity in albino mice. Contraception. 2007 ;76(6):482-5.

28. Khare CP.Indian herbal remedies;springer:2003.

29. Hodgkinson A, William A. An Improved colorimetric procedure for urine oxalate. Clin Chim Acta 1972;36:127-32.

30. Pankaj G, Nimesh P, Lokesh B, Zambare GN, Jain BB. Anti-urolithiatic effect of petroleum ether extract stem bark of Crataeva adansonii in rats.Pharmaceutical biology 2006; 44(3): 160-5.

31. Sreejayan, Rao MNA. Curcuminoids as potent inhibitors of lipid peroxidation. J Pharm Pharmaco 1994;46:1013-6.

9. Signature of the candidate

10. Remarks of the guide

12 11. Name & Designation of

11.1 Guide Dr. Kailasam Koumaravelou M.Pharm,Ph.D, Professor, Department of Pharmacology and Toxicology. St. John’s Pharmacy College Bangalore- 560104

11.2 Signature of Guide

11.3 Co – Guide

11.4 Signature of Co Guide

11.5 Head of the Department Dr. E.P. Kumar M.Pharn,Ph.D. Professor & Principal, Head, Department of Pharmacology and Toxicology. St. John’s Pharmacy College Bangalore- 560104.

11.6 Signature of HOD

12. 12.1 Remarks of the Chairman & Principal

12.2 Signature