“ANTI-UROLITHIATIC ACTIVITY OF EXTRACTS OF PLANT

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“ANTI-UROLITHIATIC ACTIVITY OF EXTRACTS OF PLANT
“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.
1
2009-2010
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA.
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1.
Name of the candidate & Address.
Mr. PATEL SANJAY PARASOTAM BHAI
Post Graduate Student,
St. John’s Pharmacy College
No 6,9th cross,2nd Main, Vijaynagar 2nd Stage
(Hampinagar)
Bangalore- 560104
2.
Name of the Institution.
St. John’s Pharmacy College
#6, 9th cross, 2nd main, Vijayanagar
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.”
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6.
6.1
Brief resume of intended work
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.
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6.2
Review of Literature
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 3.
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.
4
Ethanolic extract of the fruits of Tribulus terrestris showed significant dose dependent protection
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
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effective in counteracting the free radical toxicity by bringing about a significant decrease in
peroxidase levels and increase in antioxidant status 14.
Schaftoside a flavonoid isolated from Desmodum styracifolium exhibited significant antiurolithiatic 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
4-OH Maysin and Cassiaoccidentalin B 20.Tannin 21.Mucilage 17. Tubulin 22.
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19
.
Biological activity:
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
activity in isolated guinea pig ileum 28.
Diuretic activity: Decocation of Mimosa pudica leaves exhibited Diuretic activity 28.
6.3
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.
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7.
Materials and methods
7.1
Source of data:
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
www.ijp-online.com
Will be used to obtain related information regarding this research protocol.
7.2
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,
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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.
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.
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)
ASSESSMENT OF ANTI-UROLITHIATIC ACTIVITY OF Mimosa pudica:
Induction of urolithiasis in rats by using sodium oxalate:
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.
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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:
 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.
Yes, the experimental models require usage of laboratory animals.
7.4
Has ethical clearance been obtained from your institution in case of 7.3?
Yes, ethical clearance has been obtained (copy enclosed)
10
8.
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.
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.
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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.
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.
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9.
Signature of the candidate
10. Remarks of the guide
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
13

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