“ Antiurolithic activity of Extracts of Roots of Ricinus Communis ”

1. PRESENTED BY JUHI TIWARI “Antiurolithic activity of Extracts of Roots of RicinusCommunis”

2. 1. Introduction • Herbal medicine : Medicinal plants are used by all the cultures across the world . According to W.H.O estimates around 80% of population in developing countries use herbal drugs for some aspects of primary health care. Herbs are the staple of medical treatment in many developing countries. • The urinary system: The urinary system consists of two kidneys, two ureters, the urinary bladder, and the urethra. Kidney: kidneys are the principal regulators of the internal environment of the body. The composition of all body fluids is either directly or indirectly regulated by the kidneys as they form urine from blood plasma. The kidneys which are two in number, lies on the posterior abdominal wall, one on each side of vertebral column, behind the peritoneal cavity and below the diaphragm. Kidneys are bean-shaped organs, about 11 cm long, 6 cm wide, 3 cm thick and weigh 150 g. They are embedded in, and held in position by, a mass of fat (Scanlon et al., 2007). • Introduction to Urolithiasis It is also called Nephrolithiasis or kidney stone. • Urolithiasis is the presence of calculi in the urinary tract. • Eighty percent of calculi are composed of calcium (either oxalate or phosphate). • Others composed of struvite, uric acid, xanthine or cystine.

4. UrolithiasisMartin L.J.A(2002) • Urolithiasis may be defined as the solid accumulation of the material that form in the urinary system which may be in the kidney, ureter, urinary bladder or other parts of system. • This process is also known as super saturation of the urine i.e greater number of solute in a solvent. • EpidemiologyJoual, A. (1997) • Urolithiasis is the third most common cause of urinary tract disease , predominantly in male gender in proportion of approximately 2:1 and is characterized by a recurrence rate of 50% and reaching 70% within 10 years. • Incidence of upper urinary tract stone was 2.4 fold greater in men than in women.

5. Process of stone formation (Atmaniaet al., 2004) Saturation Supersaturation Nucleation Crystal aggregation Stone formation Crystal growth • Etiology of Urolithiasis (Pfienet al., 1990) • Family History of Renal Stone Disease • Occupational or Situational Risk Factors • Systemic Diseases • Medications • Dietary Factors • Bacterial infection Symptoms of Kidney Stones (Parmaret al., 2004). • Renal pain • Hematuria • Pyuria • Dysuria • Oligouria • Abdominal distension • Nausea/vomiting • Fever and chills • Postrenalazotemia

6. Classification of Kidney StoneCoe FL (2005) • Calcium containing Stone • Calcium Oxalate Stone (60%) • Calcium Phosphate Stone (20%) • Struvite Stone (8%) • Uric Acid Stone (10%) • Cystine Stone(1.5%) • 2,8-di hydroxy adenine stone(0.5%) Causes for kidney stone Stones were formed when there is a decrease in urine volume and orexcess in stone forming substance in urine. Dehydration , different medical condition and also low level of dietary calcium intake may alter calcium oxalate balance thus resulting in increased excretion of oxalate and propensity to form oxalate stone.

7. Need of study • There are so many research has been already done in respect to urolithiasis but there is still need to research a safe and effective drug for the treatment of urolithiasis because there is no satisfactory drug in modern medicine which can dissolve stone therefore physician still remain to be depend on alternate system of medicine for their better relief , which is economically costly and produces major side effects . Objective of study • To conduct systematic phytochemical investigation of roots of RicinusCommunis . • To conduct acute toxicity . • To develop urolithiasis model in rats . • To evaluate anti urolithiatic activity of extract of roots of RicinusCommunis . • To evaluate antimicrobial activity of roots of RicinusCommunis .

8. Plant profile : RICINUS COMMUNIS :

9. RicinusCommunisTewari, D.D. (2012) RicinusCommunis : Castor Oil Plant . Herbaceous plant or semi woody large , dicot , shrub or small tree belonging to the family Euphorbiaceae . Reported activities Anti asthmatic , anti inflammatory, anti microbial , anti fungal , anti histaminic activities, Anti hepato toxicity, Antinociceptive, Antimicrobial activity, Antifungal activity, Antibacterial activity.

10. Animals And Experiments. Sprague Dawleyrats , weighing 100+5 gm from the animal house of Pinnacle Biomedical Research Institute, Bhopal. Animal maintenance and handling were in accordance to CPCSEA. • ExtractionPradnyaOnkar et.al (2012) • The powdered roots of Ricinuscommuniswas extracted by means of maceration process . powder was subjected to cold maceration with pet ether initially and afterward marc was treated with hydro-alcoholic solution in a jar, for about 7 days at room temperature. • Authentication Botansit : Dr. PradeepTiwari and Herbarium No. Bot./Her./B/1123 was deposited in the herbarium of the Hari Singh Gaur VishwaVidyalaya, Sagar (Madhya Pradesh). cont.

11. PRELIMINARY PHYTOCHEMICAL TESTING Khandelwal K.R., (2003) The crude extract of RicinusCommuniswas screened for the presence of different phyto chemical substance • Test for alkaloids : Dragendorff’s test, Mayer’s test, Hager’s test, Wagner’s test. • Test for saponins • Test for Glycosides : Legal’s test, Baljet’s test, Keller killani test and Borntrager’s test. • Test for carbohydrates : Molisch test, Fehling’s test, Benedict’s test 5) Test for tannins and phenolic compound 6) Test for flavonoids

12. Acute oral toxicityOECD 2001- guideline • Acute oral toxicity study of hydro alcoholic and petroleum ether extract was carried out according to OECD 423 guidelines.

13. Anti microbial activity • Well Diffusion Method (El-Mahmood et al., 2008 & Bauer et al.,1966): Zone of inhibition was measured. • Anti oxidant activity • DPPH Activity (Patil et al, 2009) : percentage of inhibition was measured • Hydrogen Peroxide Scavanging Assay (Patil et al, 2009) : percentage of inhibition was measured • Reducing Power Assay (Jayanthi and Lalitha 2011) : Increase in absorbance of the reaction mixture indicates the reducing power of the Samples. • Total Phenolic Content ( Maurya S , Singh D, 2010) : Line of regression from Gallic acid was used for estimation . • Total flavonoid Content ( Maurya S , Singh D, 2010) : Line of regression from rutin was used for estimation

14. Procedure of dpph and hydrogen peroxide assay • Preparation of Standard Ascorbic acid solutions • Preparation of Test solutions • Preparation of Control solution • Percentage of antioxidant activity of plant extract and Ascorbic acid was calculated by using formula:

15. Procedure of reducing power assay • Preparation of Standard Ascorbic acid solutions • Preparation of Test solutions. • Increase in absorbance of the reaction mixture indicates the reducing power of the Samples. • Procedure of total phenolic content • Different concentration of gallic acid and plant extract was prepared in methanol. • 0.5ml of each sample was introduced into test tube and mixed with 2.5ml of a 10 fold dilute folinCiocalteu reagent and 2ml of 7.5% sodium carbonate was prepared in methanol.

16. Calculation of tpc • Line of regression from Gallic acid was used for estimation of unknown phenol content.From standard curve of gallic acid line of regression was found to be : Thus the goodness of fit was found to be good for selected standard curve. By putting the absorbance of test sample (y = absorbance) in line of regression of above mentioned GA. Total flavonoid content : Total flavonoids were measured by a colorimetric method . standard solution of rutin was added to a 75 μl of NaNO2 solution, and mixed for 6 min, before adding 0.15 mL AlCl3 (100 g/L). After 5 min, 0.5 mL of NaOH was added. The final volume was adjusted to 2.5 ml with distilled water and thoroughly mixed. Absorbance of the mixture was determined at 510 nm against the same mixture, without the sample, as a blank. Total flavonoid content was expressed as mg rutin/g dry weight (mg rutine/g DW), through the calibration curve of Rutin. All samples were analysed in three replications.

17. Calculation of tfc Line of regression from rutin was used for estimation of unknown flavonoid content. From standard curve of rutin, line of regression was found to be : Thus the goodness of fit was found to be good for selected standard curve. By putting the absorbance of test sample (y = absorbance) in line of regression of above mentioned rutin.

18. InVitroAssesement of Anti urolithic Activity • Calcium Oxalate crystallization (Beghalia M., GhalemS.,Allali H., Belouatek A., Marouf A.2008 ,2009) : By determining the crystal size. • Procedure • Preparation of synthetic urine. • Simulation of the sedimentary crystal formation • The crystal size development by microscope was carried out at different time intervals of formation of crystals . Calculated percentage of Inhibition was based on the formula:

19. In vivo assesement of anti urolithic activityAnil T. Pawar (2012) • Ethylene glycol and ammonium chloride induced urolithiasis model was used to asses the antiurolithiatic activity in rats. • Cystone was taken to serve as a standard for anti urolithic activity. • Preparation of doses : All the doses were prepared in hydro alcoholic and DMSO solvent . In all cases, the concentration was prepared according to body weight of animal. • Evaluation of Antiurolithic activity : Animals were divided into 7 groups containing six animals in each group and kept in a metabolic cages individually for entire duration of experiment. All animals had free access to regular rat chow and drinking water ad libitum .

20. Experimental design for Antiurolithiatic activity • Group I- Control group • Group II- Lithiatic Control • Group III- Preventive Regimen • Group IV- Preventive Regimen • Group V- Preventive Regimen • Group VI- Preventive Regimen • Group VII- Cystone treated • Description of groups • Group I- Control group: Normal saline was given to the rats. • Group II- Control group: Lithiatic induction was done by ethylene glycol and ammonium chloride.This group serve as control for gp. III to gp. VI. • Group III- Hydroalcoholic extract of RicinusCommunis200 mg / kg bwt.was given with inducing agent to determine the antiurolithic ability of extract. Cont…

21. Group IV- Hydroalcoholic extract of RicinusCommunis400 mg / kg bwt.was given with inducing agent to determine the antiurolithic ability of extract. • Group V- Petroleum ether extract of RicinusCommunis200 mg / kg bwt.was given with inducing agent to determine the antiurolithic ability of extract. • Group VI- Petroleum ether extract of RicinusCommunis400 mg / kg bwt.was given with inducing agent to determine the antiurolithic ability of extract. • Group VII- Animals was treated with standard drug cystone 750 mg/kg bwt. To compare the result of extracts. All rats were housed in metabolic cages individually for entire duration of the experiment. On 22nd day all the groups was sacrificed. After the termination of experiment blood was collected by cardiac puncture and serum was separated by centrifugation at 15,000 rpm for 10 min.

22. HistopathologyDivakar K et .al • The abdomen was cut open to remove either kidney from each animal. Isolated kidneys were cleaned off extraneous tissue and preserved in 10% formalin. One of the isolated kidneys was then embedded in paraffin using conventional method and cut into 5 µm thick sections and stained using hematoxylin-eosin dye and finally mounted in diphenylxylene. Then the sections were observed under microscope for histopathological changes in kidney architecture and their photomicrographs was taken. Measurement of Biochemical Marker • Creatinine , BUN , Urea , Uric Acid , Phosphate , Calcium was determined by the method given in the protocol of Erba Diagnostic Ltd.. • Creatinine : Creatinine react with alkaline picrate to produce an orange-yellow color. The absorbance of the orange-yellow color formed is directly proportional to creatinine concentration . Creatinine was calculated by determining the ratio of change in absorbance of test and standard multiplied by concentration of standard. Cont….

23. Blood urea nitrogen and urea :The estimation of urea in serum involves the enzyme catalysed reactions in which the urea is react with water to form ammonia, which in turn reacts with α-ketoglutarate and NADH to form glutamate. The rate of decrease in absorbance is monitored at 340 nm and is directly proportional to urea concentration in the sample. • Uric Acid : Estimation is done with a modified Trinderperoxidase method using TBHB. Estimation of uric acid requires reaction with water and oxygen forming hydrogen peroxide which reacts with 2, 4, 6 Tribromo 3- hydroxyl benzoic acid and 4 Aminoantipyrine to form Quinoneimine. This is proportion to uric acid concentration. • Calcium : Calcium has numerous function within the body , not only as a structural factor in bones and teeth , but also in normal neuro muscular function and the clotting of the blood .OCPC reacts with calcium in alkaline solution to form a purple coloured complex . The intensity of the purple colour formed is proportional to the calcium concentration . • Phosphorus : The principle of the method is based on the reaction in which phosphorus reacts with ammonium molybdate to form phosphomolybdate. This unreduced Complex (phosphomolybdate) is directly proportional to inorganic phosphorus present in sample.

24. Kidney Homogenate AnalysisSpakal VD (2008) • Another isolated left kidney was then homogenized. The homogenate was centrifuged at 2,000 rpm for 10 min and supernatant separated. The enzymatic estimation of kidney homogenate was determined by SOD , GSH and LPO. Preparation of homogenate • Organ was rinsed with ice cold normal saline followed by 0.15 M trisHCl (pH 7.4). • Then the organ was divide and 10% w/v tissue was homogenize with 0.15 M trisHCl and 0.1 M Phosphate buffer . Then this homogenate was centrifuged at 15,000 rpm ,15 min , 4 °C, and take supernatant as sample. Supernatant was preserved in deep freeze at 2 °C.

25. Analytical Parameter of Kidney homogenate • Super oxide Dismutase, (Spakal VD 2008) : Percentage of inhibition was measured. • Lipid Peroxidase, (Spakal VD 2008) : Obtained result are equivalent to MDA. • Glutathione (Spakal VD 2008) : 5,5’-dithio bis-2-nitrobenzoic acid is reduced in presence of GSH to produce a yellow compound. The reduced chromogen is directly proportional to GSH conc.

26. SUPEROXIDE DISMUTASE Chemicals Used : sodium pyrophosphate buffer, phenazinemethosulphate ,Nitroblutetrazolium , NADH. Take OD at 560 nm (take butanol as blank) • LIPID PEROXIDE : Chemicals Used : SDS , acetic acid ,TBA, butanol , pyridine , Take OD at 532 nm (blank – butanol : pyridine / 15:1) (this absorbance will be of total MDA formed). • Glutathione : Chemicals Used : TCA, EDTA , Ellman’s reagent, sodium citrate solution. Take OD at 412 nm (water as blank)

27. Result • Physical Characters of Extracts : Hydroalcoholic Petroleum Ether • ColorBrown Greenish Brown • OdourSweet Aromatic • AppearanceSticky Sticky • %age yeild6.82% 1.45% • Phytochemical testing :Carbohydrate , reducing sugar, flavonoids, glycosides, tannin and phenolic compound are present in hydroalcoholic extract whereas tannin and phenolic and triterpenoids and steroids are present in petroleum ether extract.

28. Graphs showing antioxidant activity DPPH Assay • % Inhibition of DPPH by HA Extract at different concentration • % Inhibition of DPPH by PE Extract at different concentration Hydrogen peroxide scavenging assay Inhibition of Hydrogen peroxide by PE Extract at different concentration Inhibition of Hydrogen peroxide by HA Extract at different concentration

29. Graphs showing Antimicrobial activity • Zone of inhibition due to RicinuscommunisHA extract in different microorganism • Zone of inhibition due to RicinuscommunisPEEextract in different microorganism

30. Graph showing invitroantiurolithicactivty % inhibition of calcium oxalate crystal at different time interval due to HAE % inhibition of calcium oxalate crystal at different time interval due to PEE

31. Acute Oral Toxicity • No mortality was observed up to 2000 mg/kg, hence 2000 mg/kg was considered as NOAEL.1/10th and 1/5th of NOAEL, i.e.200 mg/kg and 400 mg/kg were selected as dose for further in vivo investigation. InVivoAntiurolithic Activity (Serological Analysis) CREATININE BUN Graph showing serum creatinine concentration in different groups. Graph showing blood urea nitrogen concentration in different groups.

32. URIC ACID UREA Graph showing urea concentration in different groups. Graph showing uric acid concentration in different groups. CALCIUM PHOSPHATE Graph showing serum calcium concentration in different groups. Graph showing phosphate concentration in different groups.

33. Graph showing effect on enzyme involved in oxidative stress SUPEROXIDE DISMUTASE LIPID PEROXIDASE Graph showing effect on LPOenzyme due to both extract in various groups. Graph showing effect on SOD enzyme due to both extract in various groups GLUTATHIONE Graph showing effect on glutathione enzyme due to both extract in various groups

34. Conclusion • Recently, there is increasing evidence that many healthy natural food and medicinal herbal and supplements have the potential to become valuable complementary therapy in the treatment of various renal disorders. In spite of tremendous advances in the field of medicine, there is no truly satisfactory drug for the treatment of urolithiasis. • In the present study, dried powder of RicinusCommuniswas subjected to extraction using petrolium ether and hydro-alcohol (70% Dis.water+30% Methanol). Some part of both extracts was reserved for preliminary phytochemical investigation and rest was utilized for pharmacological screening. • The preliminary phytochemical investigation of H A extract showed presence of carbohydrate , reducing sugar, flavanoids , glycoside , tannin and phenolic compound and in PE extract tannin and phenolic compounds and triterpenoids and steroids are present.

35. The present study indicates that the administration of RicinusCommunisextract to rats with ethylene glycol (0.75 %) and ammonium chloride (1 %) induced urolithiasis, reduced and prevented the growth of kidney stones and renal impairment. The hydroalcoholic extract was having significant protective effect against ethylene glycol with ammonium chloride induced urolithiasis than petrolium ether extract. Accordingly, it can be concluded that the supplementation of RicinusCommunishas a beneficial effect on urolithiasis induced by ethylene glycol with ammonium chloride solution and may be also by other chemical factors. • Whenever ,a patient is suffering from urolithiasis the chances of microbial infection in the kidney or urinary tract is much higher. This infection may be due to any particular pathogenesis or due to the histological damage that can occur due to sharp edges of the crystals. Some time the urinary tract itself gets injury and further microbial infection due to movement of small stone through the tract. The RicinusCommunisshowed significant antimicrobial potential against S.aureus, Kleibsiella , B. subtillusand E.Coli. These microorganisms are common for urinary tract infection. Some time these microorganism themselves create an environmental condition in the kidney or urinary tract that favours the formation of kidney stone. Thus being antimicrobial against these microorganisms the RicinusCommunis extract will decrease the chances of microbial infection as well as the chances of favourism for the condition in support of crystal growth.

36. This study also includes the in vitro antiurolithic study and it was observed that extract significantly inhibited formation of crystals. Hence extract was having some component that is avoiding formation of calcium oxalate crystals. To ascertain safety of any component intended to used inside the body, it needs to be confirmed. In present study extracts acute oral toxicity was confirmed on the basis of OECD 423 guidelines. It was observed that extract was not toxic upto the dose of 2000 mg/kg and hence it was considered as Not Observed Adverse Effect Limit (NOAEL). As mentioned above for assessment of antiurolithic activity of HA and PE extract urolithiasis was induced by using ethylene glycol and ammonium chloride. Ist group was not having any treatment and hence was called as vehicle treated. IInd group was having only induced with vehicle and rest groups were having treatments including standard drug Cystone in last group. Assessment was done on three bases, first on the basis of kidney function test (including Creatinine and BUN), estimation of factor involved in stone formation (Uric acid, Urea, Phosphate, and calcium), and effect of enzymes involved in oxidative stress (LPO, SOD, and GSH).

37. It was observed that in induced group (group 2) level of Creatinine, BUN was significantly higher as compared to vehicle treated animals. In chronic renal failure and uremia, an eventual reduction occurs in the excretion of creatinine. The most frequently determined clinical indices for estimating renal function depends upon concentration of urea in the serum. It is useful in differential diagnosis of acute renal failure and pre renal condition where BUN–creatinine ratio is increased.This confirmed that by administration of EG and AC malfunctioning in kidney occurred. In extract treated animals with HA extract has significant decrease in Creatinine and BUN level as compared to inducer group . Effect of PEextract was not significant. • Uric acid, Urea , Phosphate , and calcium level in kidney were also elevated in inducer group as compared to vehicle treated group. In extract treated animals with HA extract significant decrease in Uric acid, Urea, Phosphate, and calcium level as compared to inducer group . Effect of PE extract was not significant. Level of SOD and Glutathione was significantly less in inducer treated group as compared to vehicle treated animals and LPO level was significantly higher in induced treated animals this confirmed that in inducer group oxidative stress was much higher. In extract treated animals level of GSH and SOD was significantly higher as compared to inducer group and LPO was significantly less as compared to inducer group. • Thus from present investigation it can be concluded that hydroalcoholic extract of roots of Ricinuscommunispossess significant antiurolithic activity.

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