Alpha-Amylase Inhibitory Compounds From Lannea Schweinfurthi Stem Bark and Their Modes of Inhibition

Abstract

Management of hyperglycemia is an essential strategy in control of diabetes mellitus which remains a major health problem despite several therapeutic interventions particularly inhibitors of -amylase. Use of ethnobotanical options has prominently been reported, for instance, Lannea schweinfurthii reported for management of hyperglycemia, although there is no scientific information to validate its anti-hyperglycemic potential. The study was to determine structures of compounds from Lannea schweinfurthii stem bark which had in-vitro -amylase inhibitory activities and their modes of inhibition. Lannea schweinfurthii stem bark was collected from Kisumu County, Kisumu Karateng (34°45’0’’, 0°6’0’’), air dried under shade, ground into fine powder and sequentially extracted using n-hexane, dichloromethane, ethyl acetate and methanol. Crude n-hexane/DCM, EtOAc and MeOH extracts were subjected to in-vitro α-amylase inhibition assay where high inhibitory activities (p>0.05) were observed for EtOAc extract (IC50 = 0.578 mg/mL) and MeOH extract (IC50 = 0.497 mg/mL) while low inhibitory activity (P≤0.05) for n-hexane/DCM extract (IC50 = 1.024 mg/mL) relative to metformin (IC50 = 0.468 mg/mL) on α-amylase. Crude extracts were subjected to chromatographic separation techniques and purification on TLC, CC over silica gel and sephadex LH-20, that led to identification of two alkenyl cyclohexenone derivatives; (4R,6S)-4,6-dihydroxy-6-((Z)-nonadec-14’-en-1-yl)cyclohex-2-en-1-one (28), (2S,4R,5S)-2,4,5-trihydroxy-2-((Z)-nonadec-14’-en-1-yl)cyclohexan-1-one (29), two prenylated flavonoids; 5-hydroxy-7,8-(2’’,2’’-dimethylchromene)-flavanone (31), 5-methoxy-7,8-(2’’,2’’-dimethylchromene)-flavanone (32) and two sterols; stigmasterol (30), 3-O-[β-glucopyranosyl-(1’’→2’)-O-β-xylopyranosyl]-β-stigmasterol (33). α-Amylase inhibition assay of isolated compounds showed high inhibitory activities (IC50 = 0.665 mM and 0.580 mM) for compounds 31 and 33, respectively, relative to (p>0.05) metformin (IC50 = 0.468 mM) while 32 showed moderate inhibitory activity (IC50 = 0.826 mM) and 28 showed (IC50 = 1.245 mM) low inhibitory activity (P≤0.05). Kinetic analysis through Lineweaver-Burk plots depicted compounds 31 and 32 as competitive inhibitors based on the inhibition constants of Ki = 24.29 mM and Ki = 37.9 mM, respectively, while 33 depicted both Ki = 1.186 mM and Ki' = 4.184 mM indicative of mixed inhibition. Compounds 31 and 32 bound to active sites of free enzyme while 33 bound on both free enzyme and enzyme-substrate complex though small Ki = 1.186 mM indicated stronger binding to free enzyme and most active. High inhibitory activity of 33 was attributed to hydroxyl groups that could have bound to α-amylase active sites. Compounds 31, 32 and 33 suppressed carbohydrates hydrolysis through modulating α-amylase activity could reduce post-prandial hyperglycemia. Potential of 31, 32 and 33 to inhibit α-amylase supports usage of Lannea schweinfurthii stem bark in management of post-prandial hyperglycemia.