PHYTOCHEMICAL AND BIOLOGICAL STUDIES ON SOME CONSTITUENTS OF VERNONIA AMYGDALINA

Abstract

Some saponins and flavonoids occuring in the leaves of Vernonia amygdalina (Compositae) and the biological response they elicited were investigated. C18 fractionation of a 169 g methanolic extract of the leaves using 30-70% methanol (MeOH) as eluting solvents afforded three saponins, a natural stigmastane-steroid aglycone and three flavonoid compounds. The saponins were purified by a combination of normal and reverse phase column and high performance liquid chromatography. The flavonoids were purified by reverse phase HPLC isoeratically and then through either polyamide or cellulose columns. The chemical structures of the saponin compounds were elucidated by a combination of U.V. mass spectral and nuclear magnetic resonance (NMR) data. The three saponin compounds and a natural aglcone were identified as 3β-glucosyl-1,7,8,9,11,20,22-hexadehydro-16α,21,24-trihydroxy-5α-stigmastane 21-23,22-25,25-27.-tricarbolactone (Vemonioside D): 3β-glucosyl-7,8,9,11-tetradehydrol 16α, 25-dihydroxy-23-acetoxy-5α stigmastane-24,28-propanone (Vernonioside E): 3β-glucosyl-7,8,9,11-tetradehydro-16α-one-24,28-propanone, -5α stigmastane-21,23-carbolactone (Vernonioside A3) and 3β-hydroxy-7,8,9,11,20,22-hexadehdro-16α-one-24e.28e-dihydroxy-5α-stigmastane-21,23-carbolactone. Vernoniodside D was shown to be the most abundant glycoside in the plant material, occuring at a level of about 0.27% DM. The crude saponin content of the plant material was determined to be 2.8% on DM basis. The structures of the flavonoid compounds were established by a combination of UV and mass spectral data as: 3,4,5,7-tetrahy droxy-flavone (lutcolin): 3,4,5-trihydroxy-7-O-glucuronyl-flavone and 3,4,5-trihydroxy-7-O-glucosyl-flavone. The 7-O-glucuronyl derivative was found to be the most abundant in the plant tissue. Elemental analysis of the dry leaves for both macro-and micro-elements showed the total dietary fibre contents to be 19 and 20.13% DM, respectively. The fat content was approximately 6% DM. Iron(Fe), zinc (Zn) and molybdenum (Mo) occured at levels of 890,34.3 and 2.2 ppm, respectively. Feeding 2-week old growing mice of both sexes with standard diet amended with 25% powered leaves: equivalent amounts of the alcoholic extracts, crude or purified saponins for 14 days did not alter their feed performance, while the group fed fibre preparate showed an increased appetite towards food. These treatments caused severe body weight losses, increased fecal and urine output and even deaths, especially in groups fed high saponin diets, compared with the control group. At necropsy, the liver and plasma cholesterol levels were reduced and saponin-concentration dependence. The gastro-intestinal tract (GIT) appeared darker and enlarged and these toxic effects were attributed to perturbations in cholesterol and nutrient absorption along the gut, caused by saponins in the plant material. Phytotoxic evaluation of the two novel vernoniosides D and, the crude extracts and crude saponin mixtures on wheat seed germination and growth of germinated seedlings, showed that two vernonoisides caused the delay of wheat seed germination and inhibited the growth of root by 71.17% and 78.86% respectively. Both inhibited the growth of shoot by 40%. The extracts also exhibited similar effects but less significantly. The effects showed both concentration and structure-dependence. Vernonioside D inhibited the growth of Trichderma viride (a soil microbe) by 13.4% and two plant pathogens: Gaeumannomyces graminis v. tritici and Cephalosporium gramineton by 12.3% and 20% respectively at a drug concentration of 200 g/ml. At the same concentration the crude extracts and the partially purified mixture stimulated the growth of the two pathogens by over 100%. The antioxidant activity of the three flavones determined by measuring the coupled oxidation of β-carotene and linoleic acid at 50⁰C showed that at drug concentration of 15 mg/L, luteolin was significantly (P<0.05) more potent than the synthetic antioxidant (BHT) and the two flavone glycosides. The antioxidant activity of the three flavonoids showed both concentration and structure-dependence.