HPLC assisted chemobiological standardization of α-glucosidase-I enzyme inhibitory constituents from Piper longum Linn-An Indian medicinal plant

Abstract

Formulations of traditional medicines are usually made up of complex mixture of herbs. However, effective quality control methods in order to select right quality materials are lacking. Though Piper longum is a widely used herb in several Ayurvedic formulations prescribed for various diseases, there is no analytical method in the literature so far which can help in selecting the right quality material with proper proportions of the active ingredients (α-glucosidase-I enzyme inhibitory principles). We employed a systematic bioassay guided fractionation method and isolated pipataline, pellitorine, sesamin, brachystamide B and guineensine as active principles. A reversed-phase high-performance liquid chromatography method was developed to quantify these active principles in the plant material, which can serve as an effective quality control tool. The separation was carried out using a Discovery HS F5 C-18 (ODS) column and the solvent system used was a gradient comprising of (A) acetonitrile and (B) water with a flow rate of 1 ml/min. The detection was performed using a PDA detector. Regression equation pertaining to all the bioactive isolates revealed a linear relationship (r² > 0.9995). The detection limits (S/N = 3) ranged from 0.005 to 0.001 μg/ml. Of all the active isolates, sesamin was identified to be present in maximum quantities (0.91%) where as brachystamide B was found in minimum quantity (0.01%).

Introduction

Piper longum (Piperaceae) is a slender aromatic climber with perennial woody roots occurring in the hotter parts of India. The fruits commonly known as “pippali” are used as a spice and also as a preservative in pickles. It is also used as cattle feed. In traditional Indian Medicinal system, Piper longum fruits have been advocated to be beneficial in diseases of respiratory tract infections like cough, bronchitis, asthma, malarial fever, diarrhea and jaundice (Krishnamurthi, 1969). The α-glucosidase-I inhibitory activity of a compound is been linked beneficial in diseases of viral origin, like diarrhea (Durantel et al., 2001), dengue (Courageot et al., 2000), hepatitis-C (Zitzmann et al., 1999), hepadnavirus (Block et al., 1998), HIV and SIV (Ratner et al., 1991, Van den Brock et al., 1996), and hence, presents broad-spectrum therapeutic activities (Mehta et al., 1998). Drug Celgosivir, a α-glucosidase-I inhibitor, has become a reality in this class of compounds, for the treatment of hepatitis-C (Sorbera et al., 2005). In our screening program of identifying potent α-glucosidase-I inhibitors from natural medicines, we encountered potent activity in Piper longum and isolated various molecules with varying activity potentials (Madhusudana Rao et al., 2004). Though, Piper longum is prescribed in several traditional pharmaceutical preparations, lack of technological inputs to identify and define molecular landscape of potentially defined bioactive mars the standardization of preparations bearing this medicinal plant.

In this report, we present standardization of active extract by reversed-phase high-performance liquid chromatography technique that identifies, defines, and quantifies active molecular fingerprints. This approach may serve as a simple, genuine, and appropriate technique to standardize Piper longum for further therapeutic exploitation.