A TLC-Direct Bioautography Method for Detection of Antiurolithiatic Metabolites.

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.

Efficient genomic DNA extraction protocol from medicinal rich Passiflora foetida containing high level of polysaccharide and polyphenol.

In Present work, the main objective is to develop less time consuming protocol for genomic DNA isolation from leaves of Passiflora foetida. Optimized protocol is cost effective, as it avoided use of expensive liquid nitrogen. The important parameters of CTAB buffer composition such as Polyvinylpyrrolidone PVP40000 (without PVP, 1%, 2%, 3.5%, 4.0%, 4.5%, 5.0%), CTAB (w, 1%, 2%, 3%, 4%, 5%), water bath temperature (30°C to 70°C) and duration on water bath for half hr and one and half hr has been optimized. CTAB (2%), PVP (1%), water bath temperature (70%), duration on water bath (1 hr) has efficiently yielded DNA quality of 200-1782 µg/0.5gm from leaf, stem, root, tendril and flower. However, 168 µg - 1782 µg of DNA has been obtained from 0.5 g of leaf of Passiflora foetida. Polyphenol contamination has been overcome using 5M NaCl and PVP. Acetate has been used for obtaining double-stranded DNA in stabilized form. Current DNA extraction protocol takes maximum of four hours for completion, which is many time savings. RAPD-PCR reaction parameters such as DNA concentration (100ng), Primer concentration (2 µM), Dream Taq polymerase (2 U), annealing temperature (29°C) and number of cycles for amplification of DNA has been optimized. Primer fragment Akansha 7 shows high polymorphism of 7 fragments ranges from 200bp - 2500 bp. Current optimized protocol of DNA isolation is specifically for Passiflora foetida, which can be used for downstream molecular techniques.