α-Glucosidase Inhibitors from the Stems ofEmbelia ribes Phu Hoang Dang,1Hai Xuan Nguyen,1Nhan Trung Nguyen,1Hanh Ngoc Thi Le2 and Mai Thanh Thi Nguyen1* 1 Faculty of Chemistry, Universit
Trang 1α-Glucosidase Inhibitors from the Stems of
Embelia ribes
Phu Hoang Dang,1Hai Xuan Nguyen,1Nhan Trung Nguyen,1Hanh Ngoc Thi Le2
and Mai Thanh Thi Nguyen1*
1 Faculty of Chemistry, University of Science, Vietnam National University, Hochiminh City, Vietnam
2 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 2 and 9 Compounds 1, 3 –6, 8, and 12 showed more potent inhibitory
activity, with IC 50 values ranging from 10.4 to 116.7 μM, than that of a positive control acarbose (IC 50 , 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
micro-vascular (i.e., retinal, renal, and possibly neuropathic),
macrovascular (i.e., coronary and peripheral vascular),
and neuropathic (i.e., autonomic and peripheral)
compli-cations (Baron, 1998) One therapeutic approach to
de-crease postprandial hyperglycemia is to retard absorption
of glucose via inhibition of carbohydrate-hydrolyzing
enzymes, such asα-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
addi-tion, the ethanol solution extract of E ribes has been
reported with the ability to protect pancreatic β-cell
(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 prelim-inary screening study revealed that the methanolic extract of the stems of E ribes exhibited significant α-glucosidase inhibitory activity with IC50 value of 0.13μg/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 theα-glucosidase inhibitory activity
of the isolated compounds
MATERIAL AND METHOD General experimental procedures The infrared (IR) spectra were measured with a Shimadzu IR-408 spectropho-tometer (SHIMADZU (ASIA PACIFIC) PTE LTD., Singapore) in CHCl3solutions The nuclear magnetic reso-nance (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 pre-parative thin-layer chromatography (TLC) was carried out
on precoated Merck Kieselgel 60 F254or RP-18 F254plates (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
* 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
Phytother Res (2014)
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.5175
Received 2 January 2014 Revised 13 March 2014
Trang 2from 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
depos-ited 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,
3 h × 3) to yield a MeOH extract (1000 g; IC50, 0.1μg/mL)
The MeOH extract was suspended in H2O and
partitioned successively with hexane and ethyl acetate
(EtOAc) to yield hexane (4.7 g; IC50, 1.0μg/mL), EtOAc
(200 g; IC50, 0.9μg/mL), and H2O (780 g; IC50, 0.08μg/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,
30.5μg/mL), fr.2 (90.7 g; IC50 8.8μg/mL), fr.3 (13.5 g;
IC50, 2.7μg/mL), fr.4 (28.4 g; IC50, 0.3μg/mL), fr.5 (6.9 g;
IC50, 1.0μg/mL), and fr.6 (45 g; IC50, >100 μg/mL)
Fraction fr.1 was rechromatographed on silica gel with
ac-etone:hexane and MeOH:CHCl3to 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 6 (10.6 mg) and 7 (10.1 mg);
frac-tions fr.2.3 and fr.2.4 were subjected to silica gel column
chromatography, eluted with acetone : hexane (1:5) to
af-ford compound 8 (13.4 mg) and 9 (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 1 (5.1 mg) and 3 (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 col-umn chromatography and purified by reversed-phase pre-parative TLC with H2O : MeOH (3:7) to afford compound 2 (10.2 mg), 4 (7.2 mg), and 5 (8.6 mg)
Embeliphenol A (1) IR (CHCl3) cm 1: 3398, 2925,
2855, 1750, 1240, 1185.1H NMR (CDCl3, 500 MHz) and
13C NMR (CDCl3, 125 MHz) (Table 1) HR-ESI-MS m/z: 415.2816 [M 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 (625μL) to start the reaction Each reaction was carried out at 37 °C for
30 min and stopped by adding 0.1 M Na2CO3 (375μL) Enzymatic activity was quantified by measuring absor-bance at 401 nm One unit of α-glucosidase activity was defined as amount of enzyme liberating p-nitrophenol (1.0μM) per minute The IC50 value was defined as the concentration ofα-glucosidase inhibitor that inhibited 50%
ofα-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 showedα-glucosidase inhibitory activity with
IC50 values of 1.0 and 0.9μg/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 1. 1H and13C NMR data for embeliphenol A (1) in CDCl 3 ( J values in parentheses)
8 ′, 9′, 11′, 12′ 5.35 (4H, m) 128.1, 128.2, 130.3, and 130.4
Trang 3resorcinol (2) (Poumale et al., 2008), 5-(8
′Z-heptadecenyl)-resorcinol (3) (Yoshikatsu et al., 1996),
1-(3,5-dihydr-oxyphenyl)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-3-
hydroxy-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 1 exhibited an [M H] 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
trip-let [δH6.51 (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 atδH5.35 (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
sig-nals 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)
O) at δC 153.7, together with long alkenyl side chain, which were assigned as shown in Fig 2 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 1 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) be-tween oxygenated methylene protons H-1′′ and carbon-ate group The chemical shifts and splitting patterns of aromatic protons indicated that carbonate group was attached to C-1 The benzylic and homobenzylic methy-lene protons exhibited simultaneously HMBC correla-tions 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 deter-mined to be Z-configuration from the upfield shifted al-lylic carbon signals (δC27.4, C-7′, C-13′, and δC25.8,
C-10′) (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 struc-ture of compound 1 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 250μM, and all compounds possessed signifi-cantα-glucosidase inhibitory activity in a concentration-dependent manner, except for 2 and 9 Compounds
1, 3–6, 8, and 12 showed more potent inhibitory activity, with IC50values ranging from 10.4 to 116.7μM, than that
of a positive control acarbose (IC50, 214.5μM)
Among the isolated compounds, the resorcinol deriv-atives 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 cinol derivatives On the basis of the structure of resor-cinol (2), it was observed that the compounds having
Figure 1 Structures of the isolated compounds from Embelia ribes.
Figure 2 Connectivity (bold line) deduced by the 1
H-1H Correlation Spectroscopy (COSY) spectrum and significant HMBC correlations
(arrow) observed for 1.
Trang 4the 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-gluco-sidase (4> 5 > 3 > > 2) In the case of compounds
possessing an alkyl or an alkenyl side chain at C-5, the
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> 1 > 6)
In conclusion, we have reported a new alkenylr-esorcinol together with 11 known compounds isolated from
E ribes Twelve compounds possessedα-glucosidase in-hibitory activity This is the first report onα-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 phenylpro-panoid constituents
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.
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Table 2 α-Glucosidase inhibitory activity of the isolated compounds
Compound
Inhibition (I%)
IC 50 ( μM)
*
not tested due to unessential results (IC 50 values can be identified without these results).
—, not identified.
0
20
40
60
80
100
Figure 3 Dose-dependent inhibition of α-glucosidase by 1, 3, 4,
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