title: A TLC-Direct Bioautography Method for Detection of Antiurolithiatic Metabolites.
abstract: Hyperoxaluria is major urinary disorder troubling largest population throughout the world predominantly involving calcium oxalate (CaOx) crystals. Ancient Ayurvedic system of medicine in India claims better option in treatment of urolithiasis. A plant from "Pashanbheda" group is Phyllanthus niruri L., possessing antiurolithiatic activity, needed to be screened and validated. In the present study, a rapid, easy and efficient method for CaOx crystal inhibition in the agar gel system analogous to antimicrobial well diffusion assay is proposed. A novel thin-layer chromatography (TLC)-direct bioautography method was also proposed to detect the antilithiatic metabolites. It helps to localize the active metabolites in P. niruri, further the partial structure elucidation was characterized by High Resolution Liquid Chromatography by mass spectroscopy (LC-HRMS) analysis. The agar well diffusion method shows 50% inhibitory concentration (IC50) value at 228.55 and 493.529 mg/mL for tri-sodium citrate and P. niruri extract, respectively. The lowest concentration showing visible crystal inhibition (minimum inhibitory concentration, MIC) in both samples was found to be 20 mg/mL. In this study, a unique agar gel well diffusion and TLC-direct bioautography method successfully screened, detected and confirmed CaOx crystal inhibitory metabolites from P. niruri. The tuberonic acid was detected in bioactive fraction of P. niruri by LC-HRMS characterization.
authors.affiliation: Lab No. 106, Plant Secondary Metabolite Lab, Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati, 444602 Maharashtra, India.
authors.forename: Anita Surendra
authors.initials: AS
authors.lastname: Patil
authors.affiliation: Government Institute of Forensic Science, Aurangabad, 431004 Maharashtra, India.
authors.forename: Hariprasad Madhukarrao
authors.initials: HM
authors.lastname: Paikrao
authors.affiliation: Lab No. 106, Plant Secondary Metabolite Lab, Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati, 444602 Maharashtra, India.
authors.forename: Ankit Subhash
authors.initials: AS
authors.lastname: Kale
authors.affiliation: Department of Botany, Sant Gadge Baba Amravati University, Amravati, 444602 Maharashtra, India.
authors.forename: Surendra Raghoba
authors.initials: SR
authors.lastname: Manik
mesh_terms: D002129:Calcium Oxalate / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D002855:Chromatography, Thin Layer / Q000379:methods*; D053040:Nephrolithiasis; D029803:Phyllanthus / Q000737:chemistry*; D010936:Plant Extracts / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology*; D012685:Sepharose
chemical_list: D010936:Plant Extracts; D002129:Calcium Oxalate; D012685:Sepharose