Elsevier

Bioorganic & Medicinal Chemistry

Volume 23, Issue 24, 15 December 2015, Pages 7641-7649
Bioorganic & Medicinal Chemistry

Phenolic group on A-ring is key for dracoflavan B as a selective noncompetitive inhibitor of α-amylase

https://doi.org/10.1016/j.bmc.2015.11.008Get rights and content

Abstract

A high throughput assay was applied to guide the isolation of a new pancreatic α-amylase inhibitor, dracoflavan B, from the dragon’s blood resin from Daemonorops draco. Applying C18 column, we successfully isolated both diastereomers and their structures verified by 1H NMR spectra in comparison with the literature values. Their activity in inhibition of pancreatic α-amylase with comparable IC50 values of 23 μM (A type) and 27 μM (B type) that are similar to that of acarbose. Dracoflavan B shows much weaker activity in inhibiting bacterial α-amylase and no activity towards fungal α-amylase. Moreover, both isomers show no inhibitory activity towards mammalian α-glucosidase. Kinetic analysis revealed that using starch as the substrate, dracoflavan B was a non-competitive α-amylase inhibitor with a Ki value of 11.7 μM. Lack of α-amylase inhibition for proanthocyanidin A2 dimer demonstrated that dracoflavan B hydrophobic nature of the B, A′, C′ and B′ rings are important for its α-amylase inhibition. In addition, selective chemical modification studies revealed that the phenolic group is also vital to dracoflavan B for its pancreatic α-amylase inhibition activity. Without the A ring phenolic hydrogen bond donor, the derivatives of dracoflavan B showed detrimental α-amylase inhibition. On the contrary, galloylation on the A ring phenolic OH group enhanced the activity as shown by the low IC50 (12 μM) against α-amylase which is 56% more potent as compared to dracoflavan B.

Introduction

Type II diabetes is one of the most serious chronic diseases associated with alarmingly increased rate due to aging population, unhealthy diets, and lifestyle.1, 2 For diabetic patients, controlling postprandial hyperglycemia is one of the main therapeutic targets. One way to do so is to reduce starch digestion rates by consuming low glycemic index (GI) foods or intake of starch hydrolase inhibitors.3, 4 Extensive research efforts have been focused on discovery of plant based starch hydrolase inhibitor as active ingredients for low GI foods.5, 6 Pancreatic α-amylase is a key enzyme in the digestive system in catalyzing the initial step in hydrolysis of starch to maltodextrin and oligosaccharides, which are degraded to glucose by brush border α-glucosidase in the small intestine.7 Modulation of pancreatic α-amylase activity through the therapeutic use of inhibitors would be of considerable medical relevance in prevention of postprandial hyperglycemia. Depending on the chemical nature, there are two types of α-amylase inhibitors, proteinaceous8, 9 and nonproteinaceous. The former are polypeptides isolated from plant, microbes, and animal and normally have high inhibition potency with IC50 in nanomolar concentration (cf. μM range for non-proteinaceous ones).10 However, the application of the proteinaceous inhibitors in mitigating postprandial hyperglycemia is challenged by their sensitivity to conformational changes due to denaturalization and protease digestion in the gastrointestinal tract. On the other hand, nonproteinaceous inhibitors, particularly pseudosugars such as acarbose, voglibose and miglitol are stable in the gastrointestinal tract and have found clinical application as anti-diabetic drug.11 They are also potent inhibitors of brush border α-glucosidase and have side effects of flatulence and diarrhea.12, 13, 14 Mechanistically, pseudosugar based inhibitors bind to the active sites of α-amylase and α-glucosidase and thus are competitive or mixed inhibitors as revealed by Michaelis–Menten kinetics analysis.15

Rarely reported are selective α-amylase inhibitors of non-proteinaceous nature. We hypothesize that, selective inhibitor of α-amylase instead of α-glucosidase may reduce the accumulation of fermentable oligosaccharides, which would be a feed stock of gut microflora as it move down the gastrointestinal track and lead to flatulence. Botanicals, particularly traditional medicine, is rich sources of bioactive compounds including starch hydrolase inhibitors. During our search for novel inhibitors from a few hundreds of commercially available botanical materials for traditional medicine use, we found that the crude extract of dragon’s blood belonging to Daemonorops draco exhibited selective pancreatic amylase inhibitory activity. It is thus an objective of this work in discovering such inhibitors from botanical sources using high throughput screening assay-guided fractionation and isolation of active compounds.

Starch digestion rate by α-amylase are most commonly measured by the dinitrosalicylic acid (DNSA) assay, which relies on the reduction of one of the nitro groups by reducing sugar generated due to starch hydrolysis. The DNSA assay could not be conveniently adapted to a high throughput fashion because the assay requires high temperature (95 °C) and basic media.16 Therefore, samples of the starch hydrolysis reaction need to be taken during the time course of the kinetic study and measured separately. In addition, recent research work has shown that tea catechins (reducing agent or antioxidants) cause interference in the DNSA assay.17 Because plant extracts typically contain polyphenolic compound, DNSA assay is not suitable for quantitation of α-amylase inhibition activity of botanical samples. We recently developed a high throughput assay on a 96-well microplate by monitoring turbidity changes during corn starch hydrolysis and applied the assay in studying starch hydrolase inhibition activity of polyphenolic compounds.18, 19 In the process of screening large amount of botanical materials for their potentials as starch hydrolase inhibitors, we discovered a new class of highly selective α-amylase inhibitor isolated from a resin called dragon’s blood.

Dragon’s blood is a common name used for resins and saps obtained from four plant genera; Croton (Euphorbiaceae), Dracaena (Dracaenaceae) mainly found in China, Daemonorops (Palmaceae), and Pterocarpus (Fabaceae).20 In Southeast Asian countries, the dragon’s blood resin is from Daemonorops draco. The chemical constituents of dragon blood are very complex,21 yet there is no study on the activity in starch hydrolase inhibition.

Section snippets

Assayed-guided isolation of dracoflavan B

We identified the active compounds in Daemonorops draco as dracoflavan B (Fig. 1) by high throughput assay-guided fractionation and identification. By applying semi-preparative column chromatography, we were able to separate two isomers with baseline resolution using achiral C18 column (Supplementary Fig. 1). The structures of the compounds were identified by comparing their 1H and 13C NMR spectra with that of literature report.21

Activity and selectivity of dracoflavan B on starch hydrolase inhibition

Figure 2 showed the time courses of starch digestion in the

Materials and instruments

Dragon’s blood resin was purchased from the local medical hall. Porcine pancreatic α-amylase, α-glucosidase in the form of rat intestine acetone powder, corn starch, 3,5-dinitrosalicyclic acid, acarbose and maltose were obtained from Sigma–Aldrich Chemical Co. (St Louis, MO). 1H NMR was recorded using Advance 300 MHz Bruker spectrometer in (CD3)2CO. Column chromatography was performed on silica gel 60 (Merck 40–60 nm, 230–400 mesh) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). The

Acknowledgement

D.H. would like to acknowledge the financial support from the National University of Singapore (Suzhou) Research Institute under the Grant number R-2012-N-006.

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