Isolation and characterization of antibacterial compounds from Rhus Leptodictya.

Abstract

Rhus leptodictya is a member of the Anacardiaceae (mango family) and is used for treating bacteria related disease by indigenous cultures in South Africa. Domestic animals feed on the tree during times of drought and this apparently does not cause as much tainting of milk, as when stock feed on the related Rhus lancea. Beer is brewed from the fruits of R. leptodictya and various parts are used in traditional medicine. The Manyika people use powdered roots of R. leptodictya for acute pain in the chest and abdominal areas. The Xhosa people use roots for gall sickness in cattle. A lotion of branches and smoke from burning is used for eye complaints by Swati people. The main aim of the study was to evaluate the antibacterial activity of extracts of the leaves and twigs of R. leptodictya in order to confirm the traditional use and then to isolate and characterize antibacterial compounds. To determine the best extractants for the extraction of antibacterial compounds from the dried leaves and twigs of R. leptodictya, the efficacy of several extractants was determined. Seven different solvents of varying polarity hexane (Hex), chloroform (Chl), dichloromethane (DCM), ethyl acetate (Eth), acetone (Act), methanol (Met) and water (Wat) were used in serial extraction to extract compounds from the leaves and twigs. The mass extracted from 4g by different solvents ranged from 43-965 mg of dry weight. The plant extracts with the highest yield was the DCM extract followed by the methanol and acetone extracts. Thin layer chromatography (TLC) was used to analyze the chemical composition of the extracts using three eluent solvent systems of varying polarities i.e. CEF, BEA and EMW and sprayed with vanillin-sulphuric acid. The chemical composition of the different extracts was vii similar with the exception of methanol and water extracts, which had only one or two visible compounds after treating with vanillin spray reagent. Chromatograms developed in three different solvent systems (CEF, BEA and EMW) were sprayed with 2, 2-diphenylpicrylhydrazyl (DPPH) to evaluate antioxidant activity. There were few antioxidant compounds present. To evaluate the number of antibacterial compounds present in the fractions, bioautography was used against four most important nosocomial microorganisms. S. aureus, E. faecalis, P. aeruginosa and E. coli. Nearly all the crude serial extraction fractions contained compounds that inhibited the growth of S. aureus. The acetone fraction had the most lines of inhibition (6) followed by ethyl acetate (5). For quantitative evaluation of antibacterial activity, minimum inhibitory concentration (MIC) values were determined using a serial microplate dilution method. The MIC values for all the fractions against all the bacterial pathogens varied from 0.04-0.63 mg/ml. The acetone extract was the most active against four bacteria with the average MIC value of 0.36 mg/ml. Leaves and twigs were extracted in bulk with acetone and solvent-solvent extraction was employed which yielded seven fractions. Bioassay guided fractionation against S. aureus was used to isolate antibacterial compounds. The largest number of antibacterial compounds occurred in the carbon tetrachloride fraction. This fraction was subjected to silica gel column chromatography eluting with eluents of increasing polarity. Two pure compounds were isolated. These compounds were identified using NMR spectroscopy and mass spectroscopy, as 2, 3 dihydro-amentoflavone and lutein. The compounds had good activity against different bacteria with MIC values ranging from 20 to 60 μg/ml. Isolated compounds were investigated for cytotoxicity against Vero cells with an LC50 of 9.4 μg/ml for lutein and 9.8 μg/ml for 2.3- viii dihydro amentoflavone which indicated toxicity. The much higher toxicity against mammalian cells than again bacterial cells indicate that these compounds do not have a good therapeutic potential but support the external use for the treatment of wounds. This is the first report of these compounds from Rhus leptodictya. The results provide scientific support for the use of R. leptodictya in treating bacterial infection in humans and animals.