Characterization of antimicrobial compounds from Combretum paniculatum, a plant with proven anti-HIV replication activity

Abstract

There is an urgent need to discover new antimicrobial and antiviral compounds owing to increasing problems of resistance to drugs encountered in many pathogenic organisms. There are also problems with currently used drugs in terms of side effects and expense. Plants have been used for many generations for healing purposes, and screening of extracts of these plants has often yielded positive results. In particular, plants with antimicrobial properties are the subject of much investigation. This study focuses on isolating the compounds responsible for biological activity in one such medicinal plant, Combretum paniculatum, extracts of which have been shown to possess antimicrobial activity. Members of the genus Combretum are widely used for medicinal purposes by many groups in Africa, to treat various conditions. Other researchers have discovered antifungal, antibacterial, anti-inflammatory and molluscicidal effects of these plants. One species of this genus, C. paniculatum, has been reported in the literature to have antiviral activity against HIV-2 with a promising selectivity index. It is important to exclude highly toxic effects of potential antimicrobial preparations. C. paniculatum extracts also displayed good antibacterial activity and some anti-inflammatory activity in other studies. Although many active compounds, especially antibacterial and antifungal, have been isolated from other Combretum species, little is known about the identity of compounds responsible for activity in C. paniculatum. In the initial stages of this project, the crude extracts of leaves of C. paniculatum were investigated for antiviral and cytotoxic activity. It was found that the acetone and water extracts of C. paniculatum leaves reduced the cytopathic effect of feline herpesvirus type 1 by 3.0 log10, a very promising result. Investigations were carried out to determine the best solvent to use for extracting the active components. It was found that acetone was the best extractant in terms of the number of compounds extracted from the plant after analysis using thin layer chromatography (TLC) and the number of bioactive compounds using bioautography against bacteria. Water extracted a large quantity of material. Different plant parts, namely stem bark, root bark and leaves, were screened for antiviral and antibacterial activity and the leaves and stem bark showed good activity. The test organisms were feline herpesvirus type 1 (FHV-1) for antiviral testing, and a range of Gram-positive and Gram-negative bacteria for antibacterial activity. Cytotoxicity against African green monkey kidney (Vero) cells was observed only at a relatively high concentration of 0.28 mg/ml. Based on availability and sustainability, the leaves were chosen for further work especially since leaves were used in the published data. Isolation of active compounds from a 70% acetone extract of a large quantity of C. paniculatum leaf material was carried out using bioassay-guided fractionation. The bioassay used to select the active fractions for further fractionation was an antibacterial assay since it is easier and more rapid to detect antibacterial activity than antiviral activity. Various techniques including column chromatography and high performance liquid chromatography (HPLC) were used to fractionate the extract to result in pure compounds. The isolated compounds were structurally elucidated by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) analysis. Nine compounds were identified as cholest-5-en-3-ol, 2-phyten-1-ol, isoquercitrin, p-coumaric acid, 2, 3, 8-tri-O-methylellagic acid, beta-sitosterol, gallocatechin, apigenin and apigenin-7-glucoside. The compounds were subjected to various bioassays to evaluate their biological activity. The isolated compounds had a broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens, as well as some antifungal and antimycobacterial activity. Cholest-5-en-3-ol, 2-phyten-1-ol, gallocatechin and apigenin were active against Escherichia coli (Gram-negative) and Mycobacterium vaccae, and against the fungi Sporobolomyces salmonicolor and Penicillium notatum. Cholest-5-en-3-ol and 2-phyten-1-ol were also active against Bacillus subtilis (Gram-positive). None of the compounds showed substantial antiviral activity against coxsackievirus strain B3 Nancy, influenzavirus type A strain Hong Kong and herpes simplex virus type 1 strain K1. The compounds were generally moderately cytotoxic to the HeLa cell line but were less toxic to the Madin-Darby Canine Kidney (MDCK) and Vero cell lines. The results obtained confirm the ethnobotanical use of C. paniculatum. Nine compounds with various biological activities were isolated from the leaf extract. The constituents responsible for antiviral activity still remain to be isolated and further work should be carried out making use of antiviral assay-guided isolation. These compounds may be present in low concentrations in C. paniculatum. Synergistic effects of isolated compounds on biological activity, particularly antiviral activity, could be investigated. The results reported here confirm that the presence of antibacterial activity in plant extracts is not an indicator of antiviral activity. Although the crude extracts of C. paniculatum had both antibacterial and antiviral activity, different compounds are responsible for antibacterial and antiviral activity respectively.