Characterisation of the Compounds of the Ethylacetate Extract of the Leaves of Alstonia Boonei De Wild and their Antioxidant and Antimicrobial Potentials

Dried and pulverised leaves of A. boonei De Wild (Apocynacea) were extracted using methanol for 48 h. The methanol extract obtained was defatted using n-hexane and fractionated using ethylacetate. The ethyl acetate fraction of the extract was subjected to vacuum liquid chromatography (VLC) in silica gel using gradients of hexane-ethyl acetate. The VLC fractions were further separated on Sephadex LH-20. A total of 10 compounds were successfully isolated and purified using the reverse phase semi preparative HPLC (L-7100, Merck/Hitachi). The structures of these compounds were determined using UV, HPLC-MS, one-dimensional: 1H, 13C and DEPT NMR, and two-dimensional: 1H1H COSY, HMQC, and HMBC NMR. The antioxidant properties were assessed using the 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging test. The isolated compounds were also subjected to anti-microbial studies using Agar well diffusion technique against the organisms: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. Based on the spectral data, the isolated compounds were identified as: quercetin-3-O- [α-L-rhamnopyranosyl(1→6) β-D-glucopyranoside] (1); quercetin-3-O- [α-L-rhamnopyranosyl(1→6) β-D-galactopyranoside] (2); kaempferol-3-O-[α-L-rhamnopyranosyl(1→6) β-D-glucopyranoside] (3); kaempferol-3-O-[α -L-rhamnopyranosyl(1→6) β-D-galactopyranoside] (4); quercetin-3-O-[α -L-rhamnopyranosyl(1→4) β-D-glucoctopyranoside] (5); kaempferol-3-O-[α -L-rhamnopyranosyl(1→4) β-D-glucopyranoside] (6); quercetin-3-O-[α -L-rhamnopyranosyl(1→2) β-D-glucopyranoside] (7); quercetin-3-O-[α -L-rhamnopyranosyl(1→2) β-D-galactopyranoside] (8); chlorogenic acid (9) and 4,5-dicaffeoylcinnamic acid (10). Compounds 1, 2, 5, 7, 8 (derivatives of quercetin) and 9, a caffeic acid derivative, showed a dose dependent antioxidant activity on DPPH free radical scavenging model with IC50 values of 52, 48, 36, 66, 56 and 22 μg/mL respectively. The three kaempferol derivatives (3, 4 and 6) showed poor anti-oxidant activity (IC50 >100 μg/mL). This suggests that the presence of at least two ortho coupled OH groups in RING B of the flavonoid nucleus is necessary for a good antioxidant activity. Compounds 7 and 8 were active against Escherichia coli with MIC values of 1.77 μg/mL and 1.92 μg/mL respectively. The profound antioxidant activity of the isolated quercetin derivatives and chlorogenic acid may explain the ethnomedicinal use of the leaf extract in the management of inflammatory disorders. These groups of compounds isolated could be very useful for SAR studies as well as other studies on the effects of glycosyl substitution patterns on chemical shifts of the ring carbons of flavonoid nuclei.