A rapid, simple, sensitive and reproducible RP-HPLC method was developed for the determination of conessine (bioactive steroidal alkaloid) in Holarrhena pubescens, a highly valued medicinal plant extensively used in pharmaceuticals. Separation was achieved on Discovery (R) RP Amide C16 Supelco column (250 µm x 4.6 µm ID) using a mobile phase of acetonitrile: water (95: 5 v/v) with flow rate 0.6 ml/min detection at λ 210 nm. Assessments of six extraction techniques were also performed to evaluate the conessine content and further the efficiency of the extracts against bacterial strains was tested to address causative relationship between the both. The method was linear over a concentration range of 2.5-20 µg/ml (r2= 0.9991). Accuracy, precision and repeatability were all within the required limits. The mean recoveries measured at the three concentrations were higher than 98.8% with RSD< 2%. Extract obtained from ultrasonication method possessed highest conessine content (1.0647%) and inhibited most of the bacteria. The study confirms ultrasonic extraction to be an ideal method to obtain conessine enriched extract possessing enhanced antibacterial potential. The proposed RP-HPLC method is simple and may be applied for the quality assays of crude drug and phytopreparations.

INTRODUCTION: Holarrhena pubescens Wall. ex G. Don syn. H. antidysenterica Wall.(Apocynaceae) is a small deciduous tree distributed in Asia, tropical areas of Africa, Madagascar, Philippines, Malayan Peninsula and India ascending up to 1200 m 1 and mainly found in the sub-Himalayan tracts.