Inhibition of α-amylase and α-glucosidase by alkaloids and phenolics from stem bark of Zanthoxylum chalybeum and Zanthoxylum gilletii

Abstract

Diabetes prevalence is on the rise with conventional drugs either being unaffordable, unavailable or having undesirable side effects. The plants Zanthoxylum chalybeum (ZC) and Zanthoxylum giletii (ZG) are reported for management of diabetes by local communities. Root bark and stem bark extract from two plants has shown in vivo anti-diabetic activity against alloxan streptozotocin–induced diabetic rats. Two benzophenanthridine alkaloids and a phenolic were established to inhibit the α-amylase and α-glucosidase activities. Based on the molecular variations of benzophenanthridine alkaloids and phenolics, the inhibitory effects of only three compounds were not predictive enough for the trends of inhibition against α-amylase and α-glucosidase. Molecular structural requirement including enzymatic inhibitory modes of alkaloids and phenolics from Zanthoxylum plants was also unknown. Further research to establish the structural characteristics exhibiting the enzymatic inhibition of these compounds is thus necessary to provide insight into their potential in hyperglycemic management. Therefore, the study intends to determine the structures of these compounds, their relative α-amylase and α-glucosidase inhibitory activities, and kinetic modes of inhibition of the active molecule. The stem bark of ZC was collected from Homa Hills, Kenya and stem bark of ZG from Kakamega forest, Kenya. Both the samples were air dried, ground into fine powder followed by acid-base alkaloid extraction comprising of 25% v/v of aq.NH4OH: ethyl acetate-petroleum ether: 2% v/v H2SO4; chloroform fractionation into alkaloid and non-alkaloids extracts. The fractions were subjected to chromatographic separations then characterization of the pure isolates using mass spectrometry, NMR, and UV-vis spectroscopic techniques. Eleven compounds were isolated: six alkaloids {Norchelerythrine (9), Phenanthridine A (42), chalybemide A (39), chalybemide B (40), chalybemide C (41) and skimmianine (21); two phenolics {2,3-epoxy-6,7-methylenedioxy coniferyl alcohol (10) and sesamine(46})} from Z. chalybeum. Whereas Z. gilettii yielded six compounds: four alkaloids {9, 42, Zanthocapensine (43), Zanthoamide D (45)}; and two phenolics {Zanthocapensol (44) and sesamine (46)}. Ten compounds showed inhibitory activities against α-amylase and α-glycosidase in the range of IC50 = 43.22-49.36 μM which had no significant difference (P > 0.05) relative to acarbose (IC50 = 42.67, 44.88) following in vitro bioassay by Worthington Enzyme assay protocols except sesamine (IC50 = 54.67, 54.77) which showed lower activity. The kinetic analysis based on Lineweaver-Burk plots {1/velocity vs 1/[Substrate] at constant [Inhibitor] (sample compounds)} established modes of inhibition and on each kinetic mode, Dixon plots {1/velocity vs [Inhibitor] at constant [Substrate]} were used to establish the respective enzymatic dissociation constants (Ki). Compound 10 showed competitive inhibition with Ki of 5.54 and 17.21 µM as acarbose (Ki = 6.14 and 22.40 µM) against α-amylase and α-glucosidase, respectively. Alkaloids 9, 21, 42, and 43 showed mixed modes of inhibitions (Ki ranges 2.74-3.10 µM and 4.73-9.17 µM) in α-amylase and α-glucosidase enzymes, respectively. Compounds with amides functionality 39, 40, 41, 45 showed non-competitive inhibition (Ki = 11.05-26.69 and 17.56-44.58) against α-amylase and α-glucosidase, respectively, except compound 40 which showed uncompetitive inhibition (Ki = 44.58 µM) against α-glucosidase same as 44 (Ki = 26.28 and 20.62 µM). Compounds showing competitive inhibition similarly exhibited stronger binding (lower Ki values) to the enzymes followed by mixed inhibitors and noncompetitive modes while uncompetitive inhibitors showed the highest Ki. Compounds 10, 9, 21, 42 and 43 exhibited good activity against α-amylase and α-glucosidase thus were considered as lead compounds for management of hyperglycemia. The study offers guidance for future investigations into drug discovery using these kinds of molecular templates.