PHYTOTHERAPY RESEARCH Phytother Res (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5175 α-Glucosidase Inhibitors from the Stems of Embelia ribes Phu Hoang Dang,1 Hai Xuan Nguyen,1 Nhan Trung Nguyen,1 Hanh Ngoc Thi Le2 and Mai Thanh Thi Nguyen1* Faculty of Chemistry, University of Science, Vietnam National University, Hochiminh City, Vietnam School of Education, An Giang University, An Giang, Vietnam From the ethyl acetate extract of the stems of Embelia ribes (Myrsinaceae), a new alkenylresorcinol, embeliphenol A (1), together with 11 known compounds have been isolated Their structures were elucidated on the basis of spectroscopic data All compounds possessed significant α-glucosidase inhibitory activity in a concentration-dependent manner, except for and Compounds 1, 3–6, 8, and 12 showed more potent inhibitory activity, with IC50 values ranging from 10.4 to 116.7 μM, than that of a positive control acarbose (IC50, 214.5 μM) Copyright © 2014 John Wiley & Sons, Ltd Keywords: Embelia ribes; embeliphenol A; α-glucosidase inhibition INTRODUCTION Diabetes mellitus (types I and II) is a most serious and chronic disease whose incidence rates are increasing with increasing levels of obesity and also with aging of the general population over the world Globally, type II diabetes (noninsulin-dependent diabetes mellitus) accounts for greater than 90% of the cases (Zimmet et al., 2001; Tewari et al., 2003) Postprandial hyperglycemia plays an important role in development of type II diabetes and complications associated with the diseases, such as microvascular (i.e., retinal, renal, and possibly neuropathic), macrovascular (i.e., coronary and peripheral vascular), and neuropathic (i.e., autonomic and peripheral) complications (Baron, 1998) One therapeutic approach to decrease postprandial hyperglycemia is to retard absorption of glucose via inhibition of carbohydrate-hydrolyzing enzymes, such as α-glucosidase, in the digestive organs (Holman et al., 1999) α-Glucosidase (EC 3.2.1.20, α-D-glucoside glucohydrolase) is widely distributed in microorganisms, plants, and animal tissues that catalyze the liberation of α-glucose from the nonreducing end of the substrate In type II diabetes, delaying glucose absorption after meals by inhibition of α-glucosidase is known to be beneficial in therapy (Tewari et al., 2003) Embelia ribes Burm f is a woody shrub that belongs to the family Myrsinaceae, which is sparsely distributed in the moist deciduous forests of India, Malaysia, South China, and Vietnam In the southern part of Vietnam, E ribes is known as ‘Ngu Linh Chi’ and has been used as Vietnamese traditional medicines for treatment of diabetes, inflammatory, intestinal worms, dental, oral, throat troubles, and skin diseases (Do, 2001) In addition, the ethanol solution extract of E ribes has been reported with the ability to protect pancreatic β-cell * Correspondence to: Prof Mai Thanh Thi Nguyen, Faculty of Chemistry, University of Science, Vietnam National University, Hochiminh City, Vietnam E-mail: nttmai@hcmus.edu.vn Copyright © 2014 John Wiley & Sons, Ltd (Bhandari et al., 2007) Some studies on the chemical constituents of E ribes have been reported a number of benzoquinones, benzofurans, resorcinol derivatives, and some phenolic compounds have been isolated from this plant (Lin et al., 2006; Haq et al., 2005) Our preliminary screening study revealed that the methanolic extract of the stems of E ribes exhibited significant α-glucosidase inhibitory activity with IC50 value of 0.13 μg/mL (Nguyen and Nguyen, 2012) Therefore, bioactivity-guided fractionation of MeOH extract was carried out, and a new alkenylresorcinol, embeliphenol A (1) was isolated together with eleven known compounds (2–12) In this article, we report the isolation and structure elucidation of the new compound by spectroscopic methods together with the α-glucosidase inhibitory activity of the isolated compounds MATERIAL AND METHOD General experimental procedures The infrared (IR) spectra were measured with a Shimadzu IR-408 spectrophotometer (SHIMADZU (ASIA PACIFIC) PTE LTD., Singapore) in CHCl3 solutions The nuclear magnetic resonance (NMR) spectra were taken on a Bruker Avance III 500 spectrometer (Bruker Biospin, BRUKER BIOSPIN AG, Bangkok, Thailand) with tetramethylsilane as an internal standard, and chemical shifts are expressed in δ values The high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on a Micro O-QIITOF mass spectrometer (Bruker Daltonics, BRUKER DALTONICS PTE LTD., Singapore) Analytical and preparative thin-layer chromatography (TLC) was carried out on precoated Merck Kieselgel 60 F254 or RP-18 F254 plates (0.25 or 0.5 mm thickness) Chemicals α-Glucosidase (EC 3.2.1.20) from Saccharomyces cerevisiae (750 UN) and p-nitrophenyl-α-D-glucopyranoside were obtained from Sigma Chemical Co (St Louis, MO, USA) Acarbose and dimethyl sulfoxide were purchased Received January 2014 Revised 13 March 2014 Accepted 24 April 2014 P H DANG ET AL from Merck (Darmstadt, Germany) Other chemicals were of the highest grade available Plant material The stems of E ribes was collected at An Giang province, Vietnam, in May 2011 and was identified by Ms Hoang Viet, Faculty of Biology, University of Science, Vietnam National University, Hochiminh City A voucher sample of the stem (AN1039) has been deposited at the Department of Analytical Chemistry of the University of Science, Vietnam National University, Hochiminh City Extraction and isolation The dried powdered stems of E ribes (5.3 kg) was extracted with MeOH (15 L, reflux, h × 3) to yield a MeOH extract (1000 g; IC50, 0.1 μg/mL) The MeOH extract was suspended in H2O and partitioned successively with hexane and ethyl acetate (EtOAc) to yield hexane (4.7 g; IC50, 1.0 μg/mL), EtOAc (200 g; IC50, 0.9 μg/mL), and H2O (780 g; IC50, 0.08 μg/mL) fractions, respectively The hexane and water fractions were not further investigated because of small amount (hexane fr.) as well as polar fraction (H2O fr.) The EtOAc fraction (200 g) was subjected to silica gel column (10 × 150 cm) chromatography eluted with MeOH-CHCl3 (0–30% MeOH) to give five fractions: fr.1 (4.7 g; IC50, 30.5 μg/mL), fr.2 (90.7 g; IC50 8.8 μg/mL), fr.3 (13.5 g; IC50, 2.7 μg/mL), fr.4 (28.4 g; IC50, 0.3 μg/mL), fr.5 (6.9 g; IC50, 1.0 μg/mL), and fr.6 (45 g; IC50, >100 μg/mL) Fraction fr.1 was rechromatographed on silica gel with acetone:hexane and MeOH:CHCl3 to gain three subfractions (fr.1.1–1.3); these fractions were applied to preparative TLC with acetone : hexane (1–3% acetone) and MeOH: CHCl3 (3% MeOH) to give compound 10 (10.2 mg), 11 (11.0 mg), and 12 (3.4 mg) Fraction fr.2 was separated by silica gel column chromatography with EtOAc : hexane to gain five subfractions (fr.2.1–2.5); fractions fr.2.1 and fr.2.2 were applied to reversed-phase preparative TLC with H2O : MeOH (20% H2O) and acetone : MeOH:H2O (8:1:1) to give compound (10.6 mg) and (10.1 mg); fractions fr.2.3 and fr.2.4 were subjected to silica gel column chromatography, eluted with acetone : hexane (1:5) to afford compound (13.4 mg) and (5.7 mg) Fraction fr.3 was chromatographed over silica gel eluting with EtOAc : hexane and purified by preparative TLC with EtOAc : CHCl3 : hexane (1:4:5) to obtain compound (5.1 mg) and (3.5 mg) Fraction fr.4 was subjected to silica gel column chromatography eluted with MeOH : CHCl3 to give three subfractions (fr.4.1–4.3), followed by ODS column chromatography and purified by reversed-phase preparative TLC with H2O : MeOH (3:7) to afford compound (10.2 mg), (7.2 mg), and (8.6 mg) Embeliphenol A (1) IR (CHCl3) cmÀ1: 3398, 2925, 2855, 1750, 1240, 1185 1H NMR (CDCl3, 500 MHz) and 13 C NMR (CDCl3, 125 MHz) (Table 1) HR-ESI-MS m/z: 415.2816 [M À H]- (Calcd for C26H39O4: 415.2848) (For further information, see supplementary data) α-Glucosidase inhibitory assay The inhibitory activity of α-glucosidase was determined according to the modified method of Kim et al (Kim et al., 2008) A 3-mM p-nitrophenyl-α-D-glucopyranoside (25 μL) and 0.2-U/ mL α-glucosidase (25 μL) in 0.01-M phosphate buffer (pH 7) were added to the sample solution (625 μL) to start the reaction Each reaction was carried out at 37 °C for 30 and stopped by adding 0.1 M Na2CO3 (375 μL) Enzymatic activity was quantified by measuring absorbance at 401 nm One unit of α-glucosidase activity was defined as amount of enzyme liberating p-nitrophenol (1.0 μM) per minute The IC50 value was defined as the concentration of α-glucosidase inhibitor that inhibited 50% of α-glucosidase activity Acarbose, a known α-glucosidase inhibitor, was used as positive control RESULT AND DISCUSSION The dried powdered stems of E ribes was extracted with reluxing MeOH, and the MeOH extract was suspended in H2O and then successively partitioned with hexane and EtOAc to yield hexane, EtOAc, and H2O fractions The hexane and EtOAc soluble fractions showed α-glucosidase inhibitory activity with IC50 values of 1.0 and 0.9 μg/mL, respectively Further separation and purification of EtOAc soluble fraction led to the isolation of a new alkenylresorcinol named embeliphenol A (1), together with 11 known compounds (2–12) These known compounds were identified as Table 1H and 13C NMR data for embeliphenol A (1) in CDCl3 (J values in parentheses) Position 1′ 2′ 7′, 13′ 8′, 9′, 11′, 12′ 10′ 17′ 1′′ 2′′ –O(C═O)O– –CH2– Copyright © 2014 John Wiley & Sons, Ltd δH δC — 6.51 (1H, t, J = 1.5 Hz) — 6.53 (1H, t, J = 1.5 Hz) — 6.57 (1H, t, J = 1.5 Hz) 2.54 (2H, t, J = 7.8 Hz) 1.57 (2H, m) 2.04 (4H, m) 5.35 (4H, m) 2.79 (2H, m) 0.89 (3H, t, J = 7.0 Hz) 4.30 (2H, q, J = 7.1 Hz) 1.38 (3H, t, J = 7.1 Hz) — 1.27 (brm) 152.0 106.1 156.4 113.3 145.9 113.5 35.9 31.1 27.4 128.1, 128.2, 130.3, and 130.4 25.8 14.4 64.9 14.4 153.7 22.5–32.0 Phytother Res (2014) α-GLUCOSIDASE INHIBITORS FROM THE STEMS OF EMBELIA RIBES Figure Structures of the isolated compounds from Embelia ribes resorcinol (2) (Poumale et al., 2008), 5-(8′Z-heptadecenyl)resorcinol (3) (Yoshikatsu et al., 1996), 1-(3,5-dihydroxyphenyl)nonan-1-one (4) (Hsun-Shuo et al., 2009), 1-(3,5-dihydroxyphenyl)heptan-1-one (5) (Hsun-Shuo et al., 2009), 3-methoxyl-5-pentylphenol (6) (McClanahan and Robertso, 1985), 5-O-methylrapanone (7) (McErlean and Moody, 2007), 5,6-dihydroxy-7-tridecyl-3-[4-tridecyl-3hydroxy-5-oxo-2(5H)-furylidene]benzo-2-oxo-3(2H)furan (8) (Lin et al., 2006), 5-methoxyeupomatenoid-8 (9) (Isogai et al., 1973), eupomatenoid-8 (10) (Fernandes et al., 1993), myristicin (11) (Chen et al., 2010), and 3, 4-methylenedioxy-5-methoxy cinnamyl alcohol (12) (Chen et al., 2010) (Fig 1) based on the spectroscopic analysis and comparison with literature data Compound exhibited an [M À H]À peak at m/z 415.2816 in the negative HR-ESI-MS, corresponding to the molecular formula C26H39O4 In the 1H NMR spectrum, the meta-coupled aromatic protons due to a 1,3,5-trisubstituted benzene ring appeared as three triplet [δH 6.51 (t, J = 1.5 Hz, H-2); 6.53 (t, J = 1.5 Hz, H-4), and 6.57 (t, J = 1.5 Hz, H-6)] The multiplet signals of two double bonds at δH 5.35 (m, H-8′, H-9′, H-11′, and H-12′) together with an ethoxyl group at δH 4.30 (q, J = 7.1 Hz, H-1′′); 1.38 (t, J = 7.1 Hz, H-2′′) and signals due to a long alkenyl side chain at δH 0.89 (t, J = 7.0 Hz, H-17′); 1.27 (overlapped methylene); 1.57 (homobenzyl 2H, m, H-2′); and 2.54 (benzyl 2H, t, J = 7.8 Hz, H-1′) Two multiplets at δH 2.04 (4H, H-7′, and H-13′) and 2.79 (2H, H-10′) identified as two allylic methylenes and a bisallylic methylene Moreover, the 13 C NMR spectrum exhibited signals for trisubstituted benzene [δC 152.0, 106.1, 156.4, 113.3, 145.9, and 113.5], four olefin carbons [δC 128.1, 128.2, 130.3, and 130.4], and one carbon of carbonate group (O(C═O) 1 Figure Connectivity (bold line) deduced by the H- H Correlation Spectroscopy (COSY) spectrum and significant HMBC correlations (arrow) observed for Copyright © 2014 John Wiley & Sons, Ltd O) at δC 153.7, together with long alkenyl side chain, which were assigned as shown in Fig by 2D-NMR spectra Signals of ethoxyl group were also observed at δC 64.9 (OCH2) and 14.4 (CH3) On the analysis of these spectra, compound was suggested to be an alkenylresorcinol The location of ethoxyl group was deduced to be at carbonate group, based on the heteronuclear multiple bond correlation (HMBC) between oxygenated methylene protons H-1′′ and carbonate group The chemical shifts and splitting patterns of aromatic protons indicated that carbonate group was attached to C-1 The benzylic and homobenzylic methylene protons exhibited simultaneously HMBC correlations with aromatic carbons C-5; this permitted assignment of the position of the long alkenyl side chain Remarkably, the bisallylic methylene signal at δ 2.79 (2H, H-10′) and 25.8 (C-10′) together with the HMBC correlations of two allylic methylenes and a bisallylic methylene further confirmed that the two double bonds were methylene-interrupted The stereochemistry of two double bonds were determined to be Z-configuration from the upfield shifted allylic carbon signals (δC 27.4, C-7′, C-13′, and δC 25.8, C10′) (Melvyn and Sirichai, 1989), and the position of two double bonds were confirmed by comparing these NMR data with those of 5-(8′Z,11′Z-heptadecadienyl)-1, 3-benzenediol (Barrow and Capon, 1991), which was isolated from the roots of E ribes (Lin et al., 2006) Thus, the aforementioned data led to propose the structure of compound to be 5-(8′Z,11′Z-heptadecadienyl)3-hydroxylphenyl ethyl carbonate (embeliphenol A) The isolated compounds were tested for their α-glucosidase inhibitory activity (Table 2) The assay was carried out at five different concentrations ranging from 10 to 250 μM, and all compounds possessed significant α-glucosidase inhibitory activity in a concentrationdependent manner, except for and Compounds 1, 3–6, 8, and 12 showed more potent inhibitory activity, with IC50 values ranging from 10.4 to 116.7 μM, than that of a positive control acarbose (IC50, 214.5 μM) Among the isolated compounds, the resorcinol derivatives possessed strong α-glucosidase inhibitory activity (1, 3–6) (Fig 3) The activity of these compounds greatly depended upon the nature of the substitution in resorcinol derivatives On the basis of the structure of resorcinol (2), it was observed that the compounds having Phytother Res (2014) P H DANG ET AL Table α-Glucosidase inhibitory activity of the isolated compounds Inhibition (I%) Compound 250 (μM) 100 (μM) 50 (μM) 25 (μM) 10 (μM) IC50 (μM) 10 11 12 Acarbose * — * * * * 54.4 ± 6.9 * * 55.7 ± 3.9 53.7 ± 3.0 87.3 ± 1.9 59.8 ± 1.2 88.6 ± 1.5 — 98.6 ± 1.3 99.9 ± 1.5 96.5 ± 2.0 74.0 ± 1.7 27.3 ± 3.8 66.7 ± 3.8 14.7 ± 2.1 24.4 ± 2.1 30.7 ± 2.2 48.2 ± 3.6 21.2 ± 2.2 65.1 ± 1.1 — 67.9 ± 1.8 93.9 ± 2.1 70.5 ± 2.0 43.2 ± 1.3 18.4 ± 1.3 43.1 ± 2.9 9.0 ± 3.4 16.0 ± 1.8 20.4 ± 3.9 31.0 ± 2.8 9.8 ± 1.4 32.1 ± 1.9 — 37.9 ± 3.2 72.8 ± 1.2 42.3 ± 2.6 14.2 ± 3.7 14.0 ± 3.1 30.4 ± 2.1 6.8 ± 1.7 13.1 ± 2.9 14.3 ± 4.7 21.8 ± 2.8 3.2 ± 1.7 10.1 ± 1.3 — 16.8 ± 2.1 45.9 ± 2.1 25.3 ± 1.5 1.8 ± 0.3 — 20.3 ± 1.3 — — — — — 47.4 >250 41.2 10.4 35.2 66.9 225.6 65.7 >250 222.6 224.4 116.7 214.5 * not tested due to unessential results (IC50 values can be identified without these results) —, not identified replacement of the hydroxyl group at C-1 by either an ethyl carbonate or a methoxyl group led to the slightly decrease of the activity (3 > > 6) In conclusion, we have reported a new alkenylresorcinol together with 11 known compounds isolated from E ribes Twelve compounds possessed α-glucosidase inhibitory activity This is the first report on α-glucosidase inhibitory activity of the stem of this plant These results suggested that the traditional use of E ribes for the treatment of diabetes in Vietnam may be attributable to the α-glucosidase inhibitory activity of its resorcinol derivatives, benzoquinone, neolignan, and phenylpropanoid constituents 100 Inhibition (%) 80 60 40 20 0 20 40 60 80 100 Figure Dose-dependent inhibition of α-glucosidase by 1, 3, 4, and This figure is available in colour online at wileyonlinelibrary com/journal/ptr the long chain ketone group at C-5 (4 and 5) displayed more stronger α-glucosidase inhibitory activity than those of the other compounds (1–3, 6), and the longer the carbon chain, the stronger the inhibition of α-glucosidase (4 > > > > 2) In the case of compounds possessing an alkyl or an alkenyl side chain at C-5, the Acknowledgements This work was supported by grant 104.01-2011.02 from Vietnam’s National Foundation for Science and Technology Development (NAFOSTED) Conflict of Interest The authors have declared that there is no conflict of interest REFERENCES Baron AD 1998 Postprandial hyperglycemia and α-glucosidase inhibitors Diabetes Res Clin Pr 40: 51À55 Barrow RA, Capon RJ 1991 Alkyl and alkenyl resorcinols from an Australian Marine Sponge, Haliclona Sp (Haplosclerida: Haliclonidae) Aust J Chem 44: 1393À1405 Bhandari U, Jain N, Pillai KK 2007 Further studies on antioxidant potential and protection of pancreatic β-cells by Embelia ribes in experimental diabetes Exp Diabetes Res 2007: 1À6 Chen CY, Wang HM, Chung SH, Lo WL, Yang WL, Yang SC 2010 Chemical constituents from the roots of Cinnamomum subavenium Chem Nat Compd 46: 474–477 Do TL 2001 Vietnamese Medicinal Plant Medicine Publisher: Hanoi Fernandes AMAP, Barata LES, Ferri PH 1993 Lignans and a neolignan from Virola oleifera leaves Phytochemistry 32: 1567À1572 Haq K, Ali M, Siddiqui AW 2005 New compounds from the seeds of Embelia ribes Burm Pharmazie 60: 69–71 Holman RR, Cull CA, Turner RC 1999 A randomized double-blind trial of acarbose in type diabetes shows improved glycemic control over years Diabetes Care 22: 960À964 Copyright © 2014 John Wiley & Sons, Ltd Hsun-Shuo C, Yi-Ju L, Shiow-Ju L et al 2009 Cytotoxic alkyl benzoquinones and alkyl phenols from Ardisia virens Phytochemistry 70: 2064–2071 Isogai A, Murakoshi S, Suzuki A, Tamura S 1973 Structures of new dimeric phenylpropanoids from Myristica fragrans Houtt Agr Biol Chem 37: 1479À1486 Lin P, Li S, Wang S, Yang Y, Shi J 2006 A nitrogen-containing 3-Alkyl-1,4-benzoquinone and a gomphilactone derivative from Embelia ribes J Nat Prod 69: 1629–1632 McClanahan RH, Robertso LW 1985 Microbial transformation of olivetol by Fusarium Roseum J Nat Prod 48: 660À663 McErlean CSP, Moody CJ 2007 First Synthesis of N-(3Carboxylpropyl)-5-amino-2-hydroxy-3-tridecyl-1,4-benzoquinone, an Unusual Quinone Isolated from Embelia ribes J Org Chem 72: 10298–10301 Melvyn VS, Sirichai W 1989 The Synthesis and identification of alkenyl and alkadienyl catechols of Burmese lac J Chem Soc Perkin Trans I 3: 431–439 Phytother Res (2014) α-GLUCOSIDASE INHIBITORS FROM THE STEMS OF EMBELIA RIBES Nguyen HX, Nguyen MTT 2012 Study on α-glucosidase inhibitory activity of An Giang Medicinal Plants Vietnam J Chem 50: 351À355 Poumale HM, Randrianasolo R, Rakotoarimanga JV et al 2008 Flavonoid glycosides and other constituents of Psorospermum and rosaemifolium BAKER (Clusiaceae) Chem Pharm Bull 56: 1428À1430 Tewari N, Tiwari VK, Mishra RC, et al 2003 Synthesis and bioevaluation glycosyl ureas as α-glucosidase inhibitors and their effect on mycobacterium Bioorgan Med Chem 11: 2911À2922 Yoshikatsu S, Yasuaki E, Hiroshi H, Yoshiki K, Akira S 1996 Isolation of 5-(8′Z-heptadecenyl)-resorcinol from etiolated Copyright © 2014 John Wiley & Sons, Ltd rice seedlings as an antifungal agent Phytochemistry 41: 1485–1489 Zimmet P, Alberti K, Shaw J 2001 Global and societal implications of the diabetes epidemic Nature 414: 782–787 SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publishers’s web site Phytother Res (2014) ... (Fig 3) The activity of these compounds greatly depended upon the nature of the substitution in resorcinol derivatives On the basis of the structure of resorcinol (2), it was observed that the compounds... 64.9 14.4 153.7 22.5–32.0 Phytother Res (2014) α-GLUCOSIDASE INHIBITORS FROM THE STEMS OF EMBELIA RIBES Figure Structures of the isolated compounds from Embelia ribes resorcinol (2) (Poumale et... plant These results suggested that the traditional use of E ribes for the treatment of diabetes in Vietnam may be attributable to the α-glucosidase inhibitory activity of its resorcinol derivatives,