EVALUTION OF ANTI-UROLITHIATIC ACTIVITY OF RICINUS COMMUNIS L. LEAVES

EVALUTION OF ANTI-UROLITHIATIC ACTIVITY OF RICINUS COMMUNIS L. LEAVES

Rajshree Dahiya^*, Umesh K. Gilhotra and Anil K. Verma

Department of Pharmacology, G. D. Memorial College of Pharmacy, Jodhpur - 342001, Rajasthan India.

ABSTRACT: The present investigation was under taken to evaluate the anti-urolithiatic activity of ethanolic extract of Ricinus communis leaves in experimental animals. Ricinus communis Leaves were evaluated for anti-urolithiatic activity using Urolithiasis is a common disorder with higher recurrence rate in men. Super saturation of crystals with imbalance between levels of promoters and inhibitors of stone formation results in urolithiasis. Current medical management of urolithiasis is either costly or not without side effects. Therefore, traditionally reported more effective and safer anti-urolithiatic medicinal plants need to be studied. Thus, this study was aimed to evaluate the anti-urolithiatic activity of ethanolic extract of Ricinus communis leaves in male albino wistar rats. Ethylene glycol (0.75% v/v in drinking water; 28 days) induced urolithiasis preventive model were used to study the effect Ricinus communis leaves dose (200 mg/kg) and. Cystone (750 mg/kg) was used as a standard. At the end of the treatment changes in various physical parameters, promoters, inhibitors, renal function markers in urine and serum samples and histopathology of kidneys were observed. All the treatments significantly prevented the rise in promoters like calcium, oxalate, uric acid, and inorganic phosphate and increased the levels of magnesium and citrate like inhibitors in various biological samples. Thus ethanolic extract of Ricinus communis leaves have proved to be an effective drug in prevention of urolithiasis.

Urolithiasis, Ricinus communis leaves, Ethylene glycol, Hyperoxaluria, Histopathology

INTRODUCTION: Uretierolithasis is the condition where urinary stones are formed or located in the ureter ^{1, 2}. Urolithiasis is the most painful urologic disorder is calculi or stone formation in any location within the urinary tract due to imbalance between promoters (calcium, oxalate, uric acid, phosphate) and inhibitors (citrates, magnesium, potassium, pyrophosphate and urinary glycol-protein) of crystallization in urine.

Urolithiasis is one of the painful urologic disorders that occur in approximately 12% of the global population and its re-occurrence rate in males is 70 - 81% and 47 - 60% in female ^{3, 4}. Kidney stone formation is a complex that results from a succession of several physicochemical events including super-saturation, nucleation, growth, aggregation and retention within the kidneys ⁷.

Urinary stones can be classified according to stone composition as calcium stone, uric acid stone, Struvite stone, cystine stone and Miscellaneous types of stones-Protein matrix stones, Ammonium urate stones, Xanthine stones and Stones composed of drug. The lifetime risk is about 10 - 15% in the developed world, but can be as high as 20 - 25% in the Middle East.

Urolithiasis is a recurrent renal disease affecting 4 - 8% in UK, 15% in US, 20% in gulf countries and 11% population in India with a relapse rate of 50% in 5 - 10 years and 75% in 20 years ^{8, 9}. Presently, the available drug therapy for the treatment of urinary stone includes, antibiotics (for struvite stones), allopurinol (for uric acid stone), opiates and NSAID’s (for relieving pain), and diuretics (for renal stone removal). For kidney stones that do not pass on their own by pharmacological management, the most widely preferred technique is the lithotripsy. In this procedure, shock waves are used to break up a large stone into smaller pieces that can then pass through the urinary system. In case of failure with all other treatments, surgical invasive techniques have also been used like percutaneous nephrolithotomy or through ureteroscopy. Ricinus communis plant is common and quite wild in the jungles in India and it is cultivated throughout India, chiefly in the Madras, Bengal and Bombay presidencies. Leaves green or reddish-green, broad, palmately lobed, with 5 - 11 lobes, 30 - 60 cm ^{11, 14, 15}.

MATERIALS AND METHODS:

Plant Collection and Authentication: The collection of the leaves of Ricinus communis L. was done in the month of october from Jodhpur region, Rajasthan. Since the plants will be enriched with phytoconstituents during that time. The identification and authentication of the plant was carried out by Dr. S. L. Meena Scientict C and Botanical survey of India, Jodhpur (Rajasthan) (No.BSI/AZRC/I.12012/Tech./2014-15/PI.Id.)/713) was prepared and preserved Botanical survey of India, Jodhpur, Rajasthan, India.

Preparation of Plant Extract: About 75 gm of dried powder was properly kept in thimble and the soxhlet apparatus was set up. The extraction of powder was done with different solvents with solvents of increasing polarities like petroleum ether (60 -80 ºC), chloroform, benzene and ethanol. The flask with the given solvent is heated to a particular temperature. The vapour produced passes through the siphon tube into the thimble kept above where it is condensed and tickles down into the flask again through the thimble dissolving the active constituents in it. The method is described as the continuous extraction. The process is continued until all the soluble constituents get separated. The extract at the bottom was collected and the solvents were removed using reflex condenser and dried on water bath. Each time, before the extraction with other solvents, the powdered substance is air dried.  

Experimental Animals: Wistar rats weighing 180- 200 gm were brought from National Toxicological Center. They were housed in well ventilated cages (3 to 4 per cage),  under standard laboratory conditions of temperature (22 ± 3 °C) and 12/12 hr light/dark cell at least 1 week before experimentation on animals. Animals had free access to standard pellet diet and water ad libitum. Laboratory animal handling and experimental procedures were performed in accordance with the guidelines of CPCSEA.

Acute Toxicity Study: Dose was selected by using acute toxicity study (OECD, 423). The acute toxicity study for ethanolic extract of leaves of Ricinus communis was performed using rats. The animals were fasted overnight prior to the experiment and maintained under standard conditions. To find the LD₅₀ of ethanolic extract of leaves of Ricinus communis, four groups of rats, containing six in each group, were given Ricinus communis in the doses of 1000, 2000 and 4000, mg/kg orally. The animals were observed for 5 min every 30 min till 2 h and then at 4, 8 and 24 h after treatment for any behavioural changes / mortality. They were further observed daily for 7 days for mortality. No mortality up to 7 days after treatment was observed with the ethanolic extract of leaves of Ricinus communis and therefore was found safe up to dose of 2000 mg/kg ^{22, 23}. 

In vivo Anti-urolithiatic Activity:

Ethylene Glycol Induced Urolithiasis Model: ¹¹ Ethylene glycol induced urolithiasis was used to access the anti-urolithiatic activity in wistar albino rats. Animals shall have been divided into four groups containing six animals in each. Group 1 served as control and received regular rat food and drinking water ad libitum. Ethylene glycol (0.75%) in drinking water will fad to be group 2 to 4 for induction of renal calculi till 28 days. Group 3 will be received standard anti-urolithiatic drug (Cystone 750 mg/kg). Group 4 will be received ethanolic extract suspension in water and all extract will be given once daily by oral route.

Antiurolithiatic Activity: ^{12, 24}

Collection and Analysis of Urine: Animals will kept in separate metabolic cages and urine sample of 24 hrs will collected on day 14 and day 28. A drop of concentrated hydrochloric acid will add to urine before being store at 4 °C. Urine will be analysis for calcium, phosphate, oxalate, protein, creatinine, magnesium and uric acid content.

Serum Analysis: After the experimental period, blood will collected from the retro-orbital under anaesthetic conditions and the animals will be sacrifices by cervical decapitation. Serum will separate by centrifugation at 10,000 rpm for 10 min and analyzed for calcium, magnesium, phosphate, oxalate, creatinine, uric acid content.

Histopathological Studies: Kidney sample will be weight and fixed rapidly with 10% neutralized formalin (pH 7.4). Section of kidney fixed in paraffin wills prepared and stained with haematoxylin and eosin observed for histopathological changes.

RESULT: The results of the various experiments conducted so for reveal the following:

Phytochemical Investigation: Table 1.

TABLE 1: QUALITATIVE CHEMICAL ANALYSIS OF PETROLEUM ETHER, CHLOROFORM, BENZENE AND ETHANOLIC EXTRACT OF RICINUS COMMUNIS L.

(+): Present; (-): Absent.

Pharmacological Evaluation: Anti-urolithiatic effects of the ethanolic extract of the plant Ricinus communis L. under study was experimented by following studies.

Result of Biochemical Parameters: Table 2, Table 3, Table 4. The results of the anti-urolithiatic study showed that urine and serum levels of calcium, oxalate, phosphate, protine, uric acid and creatinine were increased from the 14^th day onwards following 0.75% ethylene glycol treatment in Urolithiatic control group (GB) contrary to this magnesium level was reduced followed the same treatment as revealed by the values in GB. Simultaneous treatment with ethanolic extract of Ricinus communis L. reduced the above mentioned elevated parameters and elevated the reduced parameter (i.e., magnesium). There is no significant difference between the normal and treatment with ethanolic extract indicating that they are equally potent (Fig. 1- 6).

TABLE 2: EFFECT OF RICINUS COMMUNIS LINN. EXTRACT ON URINE PARAMETERS IN 0.75% ETHYLENE GLYCOL TREATED RATS AFTER 14^th DAY

Group A: Normal control; Group-B: Disease control;

Group C: Treated with standard (Cystone);

Group D: Treated with test (Ricinus communis L.).

Values are expressed as Mean ± SEM for six animals in each group.

* = Values are significantly different from control.

** = Values are significantly different from lithiatic control, (p < 0.0001).

*** = Values are significantly different from lithiatic control, (p < 0.0001).

Values were finding out by using one way ANOVA followed by Newman – Keuls multiple range test.

TABLE 3: EFFECT OF RICINUS COMMUNIS LINN. EXTRACT ON URINE PARAMETERS IN 0.75% ETHYLENE GLYCOL TREATED RATS AFTER 28^th DAY

Group A: Normal control;

Group B: Disease control;

Group C: Treated with standard (Cystone);

Group D: Treated with test (Ricinus communis L.).

Values are expressed as Mean ± SEM for six animals in each group.

* = Values are significantly different from control.

** = Values are significantly different from lithiatic control, (p < 0.0001).

*** = Values are significantly different from lithiatic control, (p < 0.0001).

TABLE: 4 EFFECTS OF RICINUS COMMUNIS LINN. EXTRACT ON SERUM PARAMETERS IN 0.75% ETHYLENE GLYCOL TREATED RATS AFTER 28^th DAY

Group A: Normal control;

Group B: Disease control;

Group C: Treated with standard (Cystone);

Group D: Treated with test (Ricinus communis L.).

Values are expressed as Mean + SEM for six animals in each group.

* = Values are significantly different from control.

** = Values are significantly different from lithiatic control (p < 0.0001).

*** = Values are significantly different from lithiatic control (p < 0.0001).

Values were finding out by using one way ANOVA followed by Newman - Keuls multiple range test.

FIG. 1: EFFECT OF RICINUS COMMINUS L. ON URINE LEVEL OF CALCIUM (mg/dl) IN 0.75% ETHYLENE GLYCOL TREATED RATS AFTER 14^th DAYS

Group A:  Normal control;

Group B:  Disease control (0.75% Ethylene glycol);

Group C: Standard (0.75% Ethylene glycol + cystone);

Group D: Treatment (0.75% Ethylene glycol + Ricinus communis L.)

FIG. 2: EFFECT OF RICINUS COMMINUS L. ON URINE LEVEL OF OXALATE (mg/dl) IN 0.75% ETHYLENE GLYCOL TREATED RATS AFTER 14^th DAYS.

Group A: Normal control;

Group B: Disease control (0.75% Ethylene glycol);

Group C: Standard (0.75% Ethylene glycol +cystone);

Group D: Treatment (0.75% Ethylene glycol + Ricinus communis L.)

Histophathological Studies: There were no histopathological changes in tubules, glomeruli and blood vessels in normal group rats (GA). The histopathology of lithiatic group which are the treated with EG (0.75% v/v) sample of kidney showing crystals in animals. These groups also showed congestion and inflammation. On 28^th day of treatment the histopathology of kidney samples of rats treated with EG 0.75% v/v and standard drug cystone (750 mg/kg) were showing a mild congestion of the glomeruli. After treatment with Ricinus communis L. (200 mg/kg) upto 28 days. The histopathology of kidney sample showed with mild cloudy changes and inflammation (Fig. 7-10).

FIG. 3: EFFECT OF RICINUS COMMUNIS L. ON URINE LEVEL OF CALCIUM (mg/dl) IN 0.75% ETHYLENE GLYCOL TREATED RATS AFTER 28^th DAY

Group A: Normal control;

Group B: Disease control (0.75% Ethylene glycol);

Group C: Standard (0.75% Ethylene glycol + cystone);

Group D: Treatment (0.75% ethylene glycol + Ricinus communis L.)

FIG. 4: EFFECT OF RICINUS COMMUNIS L. ON URINE LEVEL OF OXALATE (mg/dl) IN 0.75% ETHYLENE GLYCOL TREATED RATS AFTER 28^th DAY

Group A: Normal control;

Group B: Disease control (0.75% Ethylene glycol);

Group C: Standard (0.75% Ethylene glycol + cystone);

Group D: Treatment (0.75% Ethylene glycol + Ricinus communis L.)

FIG. 5: EFFECT OF RICINUS COMMUNIS L. ON SERUM LEVEL OF CALCIUM (mg/dl) IN 0.75% ETHYLENE GLYCOL TREATED RATS AFTER 28^th DAYS

Group A: Normal control;

Group B: Disease control (0.75% Ethylene glycol);

Group C: Standard (0.75% Ethylene glycol + cystone);

Group D: Treatment (0.75% ethylene glycol + Ricinus communis L.)

FIG. 6: EFFECT OF RICINUS COMMUNIS L. ON SERUM LEVEL OF OXALATE (mg/dl) IN 0.75% ETHYLENE GLYCOL TREATED RATS AFTER 28^th DAYS

Group A: Normal control;

Group B: Disease control (0.75% Ethylene glycol);

Group C: Standard (0.75% Ethylene glycol + cystone);

Group D: Treatment (0.75% Ethylene glycol + Ricinus communis L.).

FIG. 7: KIDNEY SECTION OF THE GA (NORMAL CONTROL) RAT. NO HISTOPATHOLOGICAL CHANGES IN TUBULES, GLOMERULI AND BLOOD VESSELS

FIG. 8: KIDNEY SECTION OF THE GB (URO-LITHIATIC CONTROL) RAT FROM EG (0.75% v/v) SHOWING CRYSTALS, CONGESTION AND INFLAMMATION

FIG. 9: KIDNEY SECTION OF THE GC (TREATED WITH CYSTONE) RAT FROM EG 0.75% v/v AND   STANDARD DRUG CYSTONE (750 mg/kg) SHOWING MILD CONGESTION OF THE GLOMERULI

FIG. 10: KIDNEY SECTION OF THE GD (TREATED WITH R. COMMUNIS L.) RAT FROM R. COMMUNIS L. (200 mg/kg) SHOWING MILD CLOUDY CHANGES, CONGESTION OF INTERSTITUM AND INFLAMMATION

DISCUSSION: As traditional medicinal are usually taken by the oral route, same route of administration was used for evaluation of anti-urolithiatic effect of the Ricinus communis against ethylene glycol induced urolithiasis in rats. The discoveries of the clinical roles of these herbal remedies have made important contributions to the treatment of urinary stone disease as an alternative or adjunct therapy.

Sex hormones have substantial effect on crystal formation in the rat kidney through oxalate metabolism and oxidative cell damage. Testosterone is a promoter and estradiol an inhibitor of such crystal formation. Kidney stone formation or urolithiasis is a complex process that results from a succession of several physico-chemical events including super-saturation, nucleation, growth, aggregation and retention within renal tubules.

In the present study, chronic administration of 0.75% (v/v) ethylene glycol aqueous solution to male wistar rats resulted in Hyperoxaluria. Oxalate and calcium excretion were grossly increased in calculi induced animals. Since it is accepted that Hyperoxaluria is a for more significant risk factor in the pathogenesis of renal stones that hypercalciuria, the changes in urinary oxalate levels are relatively much more important than those of calcium. Increase urinary calcium is a factor favouring the nucleation and precipitation of calcium oxalate or apatite (calcium phosphate) from urine and subsequent crystal growth.

From the results of biochemical parameters and histopathological studies, it is obvious that the plant Ricinus communis L. has got inhibiting on the calcium oxalate crystal formation so it can be effectively used in animal modal to treat urolithiasis.

Chemical constituents of ethanolic extract of leaves of Ricinus communis L. reports as a diuretic in managment. Alkaloids, glycosides, tannins, phenolic compounds, triterpenoids etc. are responsible for diuretic activity. Mainly flavanoids and tannins are show the machanism of diuretic activity. These chemical constituents increase the urine output as well as urinary electrolyte concentration. Chemical constituents of leaves of Ricinus communis also show antioxidant activity. Mainly flavanoids are responsible for antioxidant activity. The evaluation of calcium and oxalate indicates that these two ions contribute significantly to stone formation. It has been reported, that 90% of all the stones analyzed contain calcium and that 50% to 65% contain mixture of both calcium oxalate and phosphate. Magnesium has an inhibitory action on stone formation. Magnesium complexes with oxalate, the reducing calcium oxalate super saturation in urine. As a result growth and nucleation rate of calcium oxalate crystals were reduced. The increase in urinary uric acid excretion was observed in urolithiatic rats. Increased excretion of uric acid has been reported in stone formers and hyperoxaluric rats.

Uric acid interferes with calcium oxalate solubility and it binds and reduces the inhibitory activity of glycosaminoglycans. The predominance of uric acid crystal in calcium oxalate stones and the observation that uric acid binding proteins are capable of binding to calcium oxalate and modulate it’s crystallization also suggests it primary role in stone formation. Treatment of Ricinus communis lowered the excretion of uric acid and reduced the risk of stone formation. In urolithiasis, the glomerular filtration rate (GFR) decrease due to the obstruction to the outflow of urine by stones in urinary system. Due to this, the waste products, particularly nitrogenous and uric acid get accumulated in blood.

CONCLUSION: The presented data indicate that administration of ethanolic extract of Ricinus communis L. Leaves to the rats with ethylene glycol induced urolithiasis reduced the formation of urinary stones and increase the total urinary output. Exact mechanism underlying this effect is not clear, but apparently related to diuretic, antioxidant effect and lowering of the stone forming constituents and suggested to explore the exact pharmacology of the drug and the study was closed with delight.

ACKNOWLEDGEMENT: I thankful to Mr. S. N. Kuchhawaha Sir, Chairman, G. D. Memorial College of Pharmacy, Jodhpur, for providing all necessary facilities for carry out research work. Special thanks to my respected guide Dr. Umesh Kumar Gilhotra Sir, Professor and Principal, G. D. Memorial College of Pharmacy, Jodhpur (Rajasthan) for her excellent guidance and dedicated efforts. I extend my sincere thanks towards Mr. Anil Kumar Verma Sir, Assistant Professor Department of Pharmacology, Mr. Parsant Mutta Sir for their help and constant support.

CONFLICT OF INTEREST: Nil.

REFERENCES:

1. Vijaya T, Sathish Kumar M and Ramarao NV: Urolithiatiasis and its causes – short review, The Journal of Phytopharmacology 2013; 2(3): 1-6.
2. Gilhotra UK, Govind M, Mohan AJM and Christina AJM: Antilithiatic activity of poly herbal formulation tablets by in vivo Journal of Applied Pharmaceutical Sciences 2013; 3(5): 43-48.
3. Joy Jyothi M, Prathyusha S, Mohanalakshmi S and Avs PK: Potent herbal wealth with litholytic activity:A Review. International Journal of Innovative Drug Discovery 2012; 2: 66-75.
4. Hossein H, Ali RK, Zahra KV and Motamed S: antiurolithiatic activity of Pinus eldarica fruits aqueous extract in rats urology Journal 2010; 7: 232-7.
5. Parmar RK, Kachchi NR, Tirgar PR, Desai TR and Bhalodiya PN: Preclinical evalution of anti-urolithiasis activity of Swertia chirata stems parmark. International Journal of Research of Pharmacy 2012; 3(8): 198-202.
6. Strand W, Jackson P and Ewalt D: Information for Parents About Urolithiasis: Kidney Stones. North Texas Pediatric Urology Associates 217-750.
7. Yadav RD, Alok S, Jain SK and Verma A: Herbal plants uses in the treatment of urolithiasis: A Review. International Journal of Pharmaceutical Sciences and Reaserch 2011; 2: 1412-1420.
8. Urologia SC and Ospedal AM: Lecco: Epidemiology of urolithiasis: an update. Clinical cases in mineral and bone metabolism 2008; 5(2): 101-106
9. Kachchhi NK, Parmar RK and Tirgar PR: Evaluation of the Antiurolithiatic Activity of Methanolic Extract of Celosia argentea root in Rats. International Journal of Phytopharmacology 2012; 3: 249-255.
10. Lopez M and Hoppe B: History, epimemiology and regional diversities of urolithiasis. Educational Review: Pediatr Nephrol 2010; 25: 49-59.
11. Philip MM and Hyacinth NM: Chapter 97, Urolithiasis 565-568.
12. Sharma AP and Filler G: Epidemiology of pediatric urolithiasis: Indian Journal of Urology 2010; 26(4): 516-522.
13. Stamatelou KK, Francis ME and Jones CA: Time Trends in Reported Prevalence of Kidney Stones in the United State. Kidney Int. 2003; 5: 1817-23.
14. Malan R, Walia A and Gupta S: New Frontiers Nephrolithiasis Pathophysiology and Managment of Kidney Stones: International Journal of Research in Ayurveda and Pharmacy 2011; 2: 775 -786.
15. Aggarwal KP, Narula S, Kakkar M and Tandon C: Nephrolithiasis: Molecular Mechanism of Renal Stone Formation and Critical Role Played by Modulators 2013.
16. Basavaraj DR, Biyani CS, Browning AJ and Cartledge JJ: The Role of Urinary Kidney Stone Inhibitors and Promoters in the Pathogenesis of Calcium Containing Renal Stones: European Association of Urology and European Board of Urology Update Series 2007; 5: 126-136.
17. Ricinus communis [Internet]. Wikipedia foundation, Inc; cited 2014 Aug. 18; Available from: http://en.wikipedia. org/ wiki/castor plants.
18. Ladda PL and Kamthane RB: Ricinus comminis (Castor) an overview, International Journal of Research in Pharmacology and Pharmacotherapeutics 2014; 3(2): 136-144.
19. Desu BSR, Supriya U and Lakshmi TNV: Evaluation of diuretic activity of Ricinus comminis leaves extract, An Internatonal Journal of Advances in Pharmaceutical Sciences 2013; 3: 405-409.
20. Jena J and Gupta AK: Ricinus comminis Linn: A Phytopharmacological Review, International Journal of Pharmaceutical Sciences 2012; 4: 975-1491.
21. Taha AK and Osman HE: Larvicidal Effects of Some Plant Extract Against Anopheles arabiensis Patton Larvae, Journal of Science and Technology 2011; 3: 473-1605.
22. Ladda PL: Screening of Ricinus communis Leaves for Anticonvulsant and Analgesic Activity, Asian Journal of Pharmaceutical and Clinical Research 2014; 3: 0974-2441.
23. OECD: Guideline for the Testing of Chemicals: Acute oral toxicity-Acute Toxic Class Method 2001; 423.
24. Gilhotra UK and Christina AJM: Effect of Roula aquatic on ethylene glycol induced urolothiasis in rats. International Journal of Drug Development and Research Jan-March 2011; 3: 0975-9344.
25. Richardson KE and Tolbert NK: Oxidation of Glyoxylic Acid to Oxalic Acid by Glycolic Acid Oxidase. The Journal of Biological Chemistry 1961; 236: 1280-1284.
26. Gilhotra UK, Christina AJM: Antilithiatic activity of kalanchoe pinnanta On 1% ethylene glycol induced lithiasis in rats. International Journal of Pharmaceutical Sciences Review and Research 2011; 187-192.
27. Shah BN, Raiyani KD and Modi DC: Antiurolithiasis activity studies of Momordica charantia Fruit. International Journal of Pharmaceutical and Chemical Sciences 2011; 1(1): 6-11.
28. Unnati A, Upadhyay S and Upadhyay U: Anti-Urolithiatic Activity of Dolichos biflorus Journal of Pharmacognocy and Phytochemistry 2013; 2(2): 209-213.
29. Indian Pharmacopoeia, 4^th Controller of Publications. India, New Delhi 1996; 2A: 54.
30. Khandelwal KR: The text book of Practical Pharmacognosy. Nirali Prakashan 25.1 - 25.9.
    

    ---

    How to cite this article:

    Dahiya R, Gilhotra UK and Verma AK: Evalution of anti-urolithiatic activity of Ricinus communis L. Leaves. Int J Pharm Sci Res 2017; 8(11): 4724-31.doi: 10.13040/IJPSR.0975-8232.8(11).4724-31.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---