ANTIUROLITHIC EFFECT OF PHYTOCHEMICALS: AN EXPLANATION OF THE UNDERLYING MECHANISMS 2 November 2015 Department of Zoology, School of Sciences, Gujarat University, Ahmedabad , India Dr. Sarmistha Saha Urolithiasis is the formation of stones in the urinary tract Frequency of renal stones was more in : Asia Europe North America Saudi Arabia Recurrence rate of renal stones about 75% in 20 years span UROLITHIASIS : GLOBAL PICTURE Rao and Das, Epidemiology of Urolithiasis, 2006 Rao and Das, Epidemiology of urolithiasis, 2006 RATIONALE Medical approach using surgical and invasive procedures including percutaneous nephrolithotomy, extracorporeal shock wave lithotripsy and ureteroscopy are costly, painful and associated with side effects Recurrence rate is still over 50% Substances first isolated from plants account for approx. 25% of western pharmacopoeia with another 25% derived from modification of chemicals having botanical origins (WHO, 1990) It is necessary to explore extensively the potential usage of medicinal plants with traditional claims to be having activity against urolithiasis and subject them to systematic phytochemical and pharmacological study so as to use them either alone or in combination Solanum xanthocarpum Family: Solanaceae Genus: Solanum Botanical name: Solanum xanthocarpum Distribution: Throughout India Fruits: The berries are green and white strips when young but yellow when mature, surrounded by the enlarged calyx. Isolation of Diosgenin from Solanum xanthocarpum fruits was one by silica gel column chromatography Characterization of disogenin was done by mp, UV-Visible spectrophotometry, FT-IR, 1H NMR and 13C NMR analysis. Diosgenin CAS No Classification Steroid Pharmacology Anti-oxidant, estrogenic, Anti-diabetic, anti-inflammatory activity PropertyValue or Information Molecular weight ColourColourless Physical stateWhite to off-white crystalline powder OdourOdourless Melting point C Density: at 20 C (g/cm 3 )1.13 Vapor density2.14 (air=1) Solubility: Water at 20 CNon soluble in water Organic solvent(s)Soluble in organic solvents, acetic acid Log P (octanol-water)6.34 Gallic acid [3,4,5-trihydroxybenzoic acid] CAS No Classification Carbocyclic carboxylic acid Pharmacology Anti-oxidant, anti-inflammatory, cytotoxic and bactericidal activity, Anti-angiogenic activity PropertyValue or Information Molecular weight ColourColourless Physical stateWhite crystalline powder OdourOdourless Melting point C Density: at 20 C (g/cm 3 )1.694 Vapor density2.14 (air=1) Solubility: Water at 20 C1.1 (g/100mL) Organic solvent(s)Soluble in alcohol, ether, glycerol, acetone Log P (octanol-water)0.70 Quercetin CAS No Classification Flavonol Pharmacology Anti-oxidant, anti-inflammatory, antiulcer activity PropertyValue or Information Molecular weight ColourYellow Physical stateYellow crystalline powder OdourOdourless Melting point316 C Density: at 20 C (g/cm 3 )1.799 Vapor density2.14 (air=1) Solubility: Water at 20 C > 1 mg/mL Organic solvent(s)Soluble in organic solvents, acetic acid Log P (octanol-water)2.16 OBJECTIVES Antiurolithiatic evaluation of diosgenin, gallic acid and quercetin against ethylene glycol - induced urolithiatic model in rats Protective effect of compounds against Calcium oxalate monohydrate (COM)-induced apoptotic death and oxidative stress in MDCK (Madin Darby Canine Kidney) renal epithelial cells In silico molecular docking of compounds WHY ETHYLENE GLYCOL MODEL? MATERIALS AND METHODS CHEMICALS Ethyene glycol was purchased from Merck, Mumbai, India. MDCK cells were obtained from National Centre of Cell Sciences, Pune, Maharashtra, India. Biochemical parameters were analyzed using diagnostic kits by Siemens in Siemens dimension xpand fully autoanalyzer, USA. All other chemicals were procured from Himedia, Mumbai, India and were of analytical grade. EXPERIMENTAL ANIMALS Inbred 12 14 weeks old Wistar strain female rats (Rattus norvegicus ) used in the study were obtained from Torrent Research Centre, Ahmedabad, India and weighed approximately 200 225 g at the initiation of the study. The research protocols were reviewed and approved by The Committee for the Purpose of Control and Supervision of Experiment on Animals (Reg 167/1999/CPCSEA), New Delhi, India. Blood samples were collected on 28 th day of treatment by retro-orbital sinus under mild anaesthetic conditions and urinalysis were done on 28 th of treatment. GroupsTreatments Number of animals Duration of treatment (Days) Day of autopsy 1Untreated control62829 th 2 Gallic acid control (10 mg/animal/kg body weight/day of gallic acid ) th 3 Quercetin control (10 mg/animal/kg body weight/day of quercetin ) th 4 Diosgenin control (10 mg/animal/kg body weight/day of diosgenin) th 5 *Ethylene Glycol (urolithiatic group; EG) 0.75% in drinking water th 6 EG along with 5 mg/animal/kg body weight/day of gallic acid th 7 EG along with 10 mg/animal/kg body weight/day of gallic acid th 8 EG along with 5 mg/animal/kg body weight/day of quercetin th 9 EG along with 10 mg/animal/kg body weight/day of quercetin th 10 EG along with 5 mg/animal/kg body weight/day of diosgenin th 11 EG along with 10 mg/animal/kg body weight/day of diosgenin th EXPERIMENTAL PROTOCOL STATISTICAL ANALYSIS The data were statistically analyzed using GraphPad Instat, software, version 3.0. The results were expressed as means S.E.M. Hypothesis testing methods included one-way Analysis of Variance (ANOVA) followed by Tukey multiple comparison test at a significance level of p < Results were expressed as mean SEM; n = 6. a as compared between untreated control (Group 1) and EG-treated (Group 5), b as compared between EG-treated (Group 5) and EG + antidote-treated (Groups 6-11). RESULTS Untreated Control Ethylene glycol Gallic acid 5 mg/kg Gallic acid 10 mg/kg Diosgenin 5 mg/kg Diosgenin 10 mg/kg Quercetin 5 mg/kg Quercetin 10 mg/kg Prevention of calcium oxalate monohydrate-induced apoptotic death and oxidative stress in MDCK cells Madin Darby Canine Kidney (MDCK) renal epithelial cells were obtained from National Centre of Cell Sciences, Pune, Maharashtra, India. The cells were maintained in Dulbeccos Modified Eagles Medium (DMEM) with 2.0 mM L-glutamine adjusted to contain 3.7 g/L sodium bi carbonate, 4.5 g/L glucose with 10% FBS and antibiotics. MDCK cells were exposed simultaneously to COM crystals at concentration of 80 g/cm 2 in the presence or absence of phyto- chemicals (5 and 10 g/mL) for 24 h. RESULTS caspase-9caspase-3 Caspase-9 Caspase-3 Compound ID Binding energy (kcal/mol) caspase-9; 1NW9 caspase-3; 1CP3 Gallic acid Quercetin Diosgenin In Silico Molecular Docking using AutoDock 4.0 Increase in urinary pH which correlates to an increase of urinary magnesium because reduction of calcium oxalate supersaturation by formation of complexes with calcium and direct inhibition of crystal growth with aggregation and increasing urinary pH, thus reducing recurrences Antioxidant property of phytocompounds and/or Diuretic activity Diosgenin was also able to attenuate the COM-induced cytotoxicity, prevented apoptosis and was capable of binding to active site of on caspase-9 and -3. SUMMARY AND CONCLUSIONS