ANTISICKLING ACTIVITIES OF CASSIA SIEBERIANA DC (LEGUMINOSAE)

Abstract

Sickle Cell Disease (SCD) affects primarily black populations. There are few drugs available for the management of SCD with deleterious side effects complicating the condition. Although many plants including Cassia sieberiana have been documented in the management of SCD, there is paucity of scientific evidence for their effectiveness and bioactive constituents. This study was designed to investigate the antisickling activities of morphological parts of C. sieberiana, isolate and characterise their constituents. The powdered samples of C. sieberiana (FHI- 112359) root, seed, pericarp and whole fruit were extracted into 70% ethanol by Soxhlet extraction and water by reflux. The extracts were used to either inhibit or reverse sodium metabisuphite induced sickling of HbSS erythrocytes from SCD patients in steady state, in vitro. The ethanol extract of the most active plant part (whole fruit) was successively partitioned to give n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc) and aqueous fractions. The fractions were screened for inhibition and reversal of sickling and inhibition of haemoglobin polymerisation. The percentage of inhibition, reversal and rate of decrease in HbSS polymerisation were calculated. The DCM and EtOAc fractions were purified using column and vacuum liquid chromatographic (VLC) techniques. Chromatographic fractions were screened for inhibitory and reversal activities and the active fractions were further purified using column and preparative thin layer chromatography to isolate compounds. Structures of isolated compounds were identified using 1D and 2D NMR (1H and 13C). Data were analysed using one-way ANOVA followed by Student t-test at α0.05. The ethanol extract of C. sieberiana whole fruit (CSF) at 180 minutes, exhibited the highest inhibitory activity (83.7±1.3)% compared to seed (65.7±2.2)%, pericarp (10.9±1.2)% extracts and was significantly different from vanillic acid-reference standard (50.4 ± 0.4)% and water whole fruit (51.4±1.6)% extract, while the root ethanol extract lysed the erythrocytes. The root ethanol extract exhibited a reversal activity of (88.9±0.8)%, compared to whole fruit (82.3±0.8)%, seed (82.2±1.0)% and pericarp extracts (55.6±1.4)%; para hydroxy benzoic acid reference standard (86.0±0.6)% and water whole fruit extract (84.4±1.9)%. The CSF exhibited both iii inhibitory and reversal activities and also decreased the rate of HbSS polymerisation by (73.5±0.06)%. The DCM and EtOAc fractions exhibited comparable inhibitory (77.9±1.1)%; (77.3±2.9)% and reversal (73.5±1.4)%; (76.8±3.8)% activities, respectively. The EtOAc and aqueous partitioned fractions decreased the rate of HbSS polymerisation by (80.6±0.02)% and (84.2±0.03)%, respectively, which were significantly lower than that of ascorbic acid (92.3±0.002)%. The DCM column fractions B3 and D3 exhibited inhibitory activities of (87.9±0.1)% and (91.7±1.5)%, respectively, which were significantly higher than the activities of all other column fractions. Fractions B3 (70.7±1.6)% and D3 (86.2±1.4)% also exhibited reversal activities. Nine phenolic compounds were isolated from; EtOAc fraction (E2, E3 and E4) and DCM fraction (M2, M3, G2, G3 and G4). One of the compounds - E3 was characterised as 4′-methoxy epiafzelechin.