A new megastigmane sulphoglycoside and polyphenolic constituents from pericarps of Garcinia mangostana

Abstract A megastigmane sulphoglycoside together with three phenolic compounds were isolated from the water-soluble fraction of the pericarps of Garcinia mangostana. The structure of the new compound was determined as 4-O-sulpho-β-d-glucopyranosyl abscisate (1) by spectroscopic data. Proanthocyanidin A2 (2) showed potent α-glucosidase inhibitory and DPPH scavenging activities with IC50 values of 3.46 and 11.6 μM, respectively.


Introduction
The plants in the genus Garcinia (Guttiferae) are well known to be rich in biologically active compounds including xanthones, benzophenones, proanthocyanidins and biphenyls (Mungmee et al. 2013;Feng et al. 2014;Fouotsa et al. 2014;Jiang et al. 2014;Anu Aravind et al. 2015). Mangosteen (Garcinia mangostana Linn.) is a plant that is widely cultivated in Vietnam and other Southeast Asian countries. The pericarp of this plant has been used as a remedy in traditional medicine for the treatment of diarrhoea, inflammation, skin infections and wounds (Vo 2012). Previous studies have shown that it contains high amounts of polyphenolic constituents, in which xanthones have been most frequently reported (Obolskiy et al. 2009;Dharmaratne et al. 2013;Morelli et al. 2015;Zhou et al. 2015). Mangosteen pericarp has exhibited a variety of interesting biological activities including antibacterial, antifungal, anti-inflammatory, antimalarial and antidiabetic (Pedraza-Chaverri et al. 2008;Obolskiy et al. 2009;Dharmaratne et al. 2013). α-Glucosidase, a membrane-bound enzyme at the epithelium of the small intestine, hydrolyses starch oligosaccharides to monosaccharides. The inhibitors of this enzyme delay carbohydrate digestion and thus cause a reduction in the rate of glucose absorption and lower the postprandial hyperglycaemia. Therefore, the inhibition of α-glucosidase plays a major role in preventing the rise of the postprandial glucose level in diabetics (Kumar et al. 2011).

Results and discussion
All isolated compounds were tested for their α-glucosidase inhibitory activity. Most of the isolated compounds are phenolic and may possess antioxidant activity, thus the compounds were evaluated for their DPPH scavenging capacity. As shown in Table 1, proanthocyanidin A2 (2) was most active with the IC 50 values of 3.46 and 11.6 μM for α-glucosidase inhibitory and DPPH scavenging activities, respectively. The known antioxidant compound (-)-epicatechin (3) and the benzofuran glycoside (4) also showed moderated α-glucosidase inhibitory effect. The new megastigmane sulphoglycoside (1) exhibited significant DPPH scavenging capacity in comparison with ascorbic acid. A previous study reported that the prenylated xanthones in the pericarps of mangosteen potently inhibited α-glucosidase (ryu et al. 2011). In the present work, proanthocyanidin A2, a polar compound isolated from the water-soluble fraction of mangosteen pericarps showed potent α-glucosidase inhibitory activity in comparison with the antidiabetic drug acarbose. This result coincided with the previous study assuming that proanthocyanidin components in the pericarps of mangosteen are potent inhibitors of α-amylase, another enzyme involving in the metabolism of carbohydrates and hyperglycaemia (Loo & Huang 2007). Although previous study showed that total proanthocyanidins from Cinnamomum cassia strongly inhibited α-glucosidase (Kang et al. 2014), the present paper is the first report for the α-glucosidase inhibitory activity of proanthocyanidin A2. A lot of studies have shown that the prenylated xanthones in mangosteen are potent inhibitors of α-glucosidase. However, these compounds are mostly soluble in organic solvents of moderate polarity. Our result showed that the water-soluble fraction of mangosteen pericarp contains attractive α-glucosidase inhibitors. Thus, these data reinforce the health benefit of mangosteen as an alternative medicine to help lower postprandial glucose absorption.

General procedures
Optical rotation values were recorded on a JASCO P-2000 digital polarimeter (JASCO, Tokyo, Japan). The uV spectra were recorded on a JASCO V-630 spectrophotometer (JASCO, Tokyo, Japan). The Ir spectrum was obtained from a Tensor 37 FT-Ir spectrometer (Bruker, ettlingen, Germany). NMr experiments were carried out on a Bruker AM500 FT-NMr spectrometer (Bruker, rheinstetten, Germany) using tetramethylsilane (TMS) as internal standard. The Hr-eSI-MS were recorded on an FT-ICr mass spectrometer (Bruker Dal-tonics, Bremen, Germany).

Plant material
The fruits of G. mangostana were purchased from markets in Hanoi in June, 2011 and identified by Prof Tran Huy Thai, Institute of ecology and Biological resources, Vietnam Academy of Science and Technology. The voucher specimens (BK-10) were deposited at School of Chemical engineering, Hanoi university of Science and Technology. The fruit pericarps were collected, air dried and powdered.

Extraction and isolation
The air-dried and powdered materials (2.0 kg) were extracted with methanol (4 L × 3 times) at room temperature for 24 h. The combined extracts were concentrated under vacuum to obtain a crude residue (152.0 g, 81% α-glucosidase inhibition at 100 μg/mL), which was then resuspended in water (1 L), and extracted by chloroform (1 L × 3 times). The organic layers were combined and concentrated to give 48.5 g of chloroform residue. The water layer (87% α-glucosidase inhibition at 100 μg/mL) was passed through a dianion HP-20 column and washed with 1.5 L water, then eluted with 50 and 100% methanol (1 L each). The eluant from 100% methanol was concentrated and chromatographed on a rP-18 column using methanol-water (1:4 v/v) as the mobile phase to afford five fractions of FA1-5. Compound 2 (60.3 mg) was purified from FA2 by using a silica gel column eluted by chloroform:methanol:water (45:10:1 v/v). Fraction FA3 was passed through a C-18 reverse-phase column using methanol:water (1:2 v/v) as the mobile phase to obtain compounds 3 (98.5 mg) and 4 (8.0 mg). FA5 was chromatographed on a silica gel column eluted with ethyl acetate:methanol:water (60:10:1 v/v) to give 1 (6.1 mg).

Acid hydrolysis of 1
Compound 1 (2 mg) was heated in 1 N HCl (1 mL) at 80 °C for 2 h, then the solution was cooled and extracted with ethyl acetate (1 mL × 3). The organic and aqueous layers were concentrated to 1 mL and their optical rotations were checked. (S)-abscisic acid was obtained from the organic layer, [ ] 25 D = +107.6 (c 0.03, MeOH). The sugar product in the aqueous layer was identified as d-glucose by silica gel TLC (rf 0.55 developed with acetone-methanol-water 100:10:1 (v/v) and was sprayed with a 10% sulphuric acid solution containing 2% vanillin), and by the positive value of optical rotation ([ ] 25 D = +3.2 (c 0.03, H 2 O) in comparison with d-glucose standard. The addition of BaCl 2 to the aqueous solution produced a white precipitate indicating the presence of SO 2− 4 anions.

Assay for α-glucosidase inhibition
The α-glucosidase (G0660-750uN, Sigma) enzyme inhibition assay was performed according to a previously described method (Luyen et al. 2013). The sample solution (2 μL dissolved in DMSO) and 0.5 u/mL α-glucosidase (40 μL) were mixed in 120 μL of 0.1 M phosphate buffer (pH 7.0). After 5 min pre-incubation, 5 mM p-nitrophenyl-α-d-glucopyranoside solution (40 μL) was added and the solution was incubated at 37 °C for 30 min. The absorbance of released 4-nitrophenol was measured at 405 nm using a microplate reader (Molecular Devices, CA). The IC 50 value was graphically measured using a plot of the per cent inhibition vs. log of the concentration of the test compound.

DPPH radical scavenging activity
The antioxidant activity of the isolated compounds was evaluated by its scavenging capacity of the DPPH radical. Briefly, the tested samples (10 μL) at various concentrations were mixed with 150 μM DPPH solution (190 μL) in 96 well plates. The plate was incubated in the dark at room temperature for 30 min. Then the absorbance of the reaction mixture was measured at 520 nm on a microplate reader. The IC 50 value is defined as the concentration of a sample required to scavenge 50% of the DPPH radical.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work is supported by a grant from the Ministry of Industry and Trade [grant number CNHD.ĐT 054/14-16].