Patil Anita Surendra, Paikrao Hariprasad Madhukarrao, Kale Ankit Subhash, Manik Surendra Raghoba
Lab No. 106, Plant Secondary Metabolite Lab, Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati, 444602 Maharashtra, India.
Government Institute of Forensic Science, Aurangabad, 431004 Maharashtra, India.
J Chromatogr Sci. 2017 May 1;55(5):578-585. doi: 10.1093/chromsci/bmx002.
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.
高草酸尿症是困扰全球大部分人群的主要泌尿系统疾病，主要涉及草酸钙（CaOx）晶体。印度古老的阿育吠陀医学体系宣称在治疗尿石症方面有更好的选择。一种来自“破石草”组的植物是叶下珠，具有抗尿石症活性，需要进行筛选和验证。在本研究中，提出了一种在琼脂凝胶系统中类似于抗菌杯碟扩散法的快速、简便且高效的抑制CaOx晶体的方法。还提出了一种新型的薄层色谱（TLC）直接生物自显影法来检测抗尿石症代谢物。它有助于定位叶下珠中的活性代谢物，进一步通过高分辨率液相色谱-质谱联用（LC-HRMS）分析对部分结构进行了表征。琼脂杯碟扩散法显示，柠檬酸钠和叶下珠提取物的50%抑制浓度（IC50）值分别为228.55和493.529 mg/mL。两个样品中显示出可见晶体抑制的最低浓度（最低抑菌浓度，MIC）均为20 mg/mL。在本研究中，一种独特的琼脂凝胶杯碟扩散和TLC直接生物自显影方法成功地筛选、检测并确认了叶下珠中抑制CaOx晶体的代谢物。通过LC-HRMS表征在叶下珠的生物活性部分检测到了块茎酸。
