http://informahealthcare.com/phbISSN 1388-0209 print/ISSN 1744-5116 online

Editor-in-Chief: John M. PezzutoPharm Biol, Early Online: 1–4

! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.814692

ORIGINAL ARTICLE

Inhibitors of a-glucosidase and a-amylase from Cyperus rotundus

Hong Hanh Thi Tran1, Minh Chau Nguyen1,2, Hoang Tram Le1, Thi Luyen Nguyen1, Thanh Binh Pham1,Van Minh Chau1, Hoai Nam Nguyen1, and Tien Dat Nguyen1

1Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam and2School of Chemical Engineering, Hanoi University of Science and Technology, 1-Dai Co Viet, Hanoi, Vietnam

Abstract

Context: A methanol extract of Cyperus rotundus L. (Cyperaceae) rhizomes showed inhibitoryactivity against a-glucosidase and a-amylase, two enzymes involve in carbohydrate digestion.Objective: Identification of compounds from C. rotundus rhizomes responsible for the inhibitionof a-glucosidase and a-amylase.Materials and methods: Compounds were identified by a phytochemical investigationusing combined chromatographic and spectroscopic methods. a-glucosidase and a-amylaseinhibitory activities were evaluated by in vitro enzyme inhibition assays.Results: A new (2RS,3SR)-3,40,5,6,7,8-hexahydroxyflavane (1), together with three known stilbenedimers cassigarol E (2), scirpusin A (3) and B (4) were isolated. Compound 2 inhibited botha-glucosidase and a-amylase activities while the flavane 1 only showed effect on a-amylase,and compounds 3 and 4 were active on a-glucosidase. All four compounds showed significant2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity.Discussion: The inhibitory activities against a-amylase and a-glucosidase of the C. rotundusrhizomes were reported for the first time. Stilbene dimers are considered as potent inhibitorsof a-glucosidase and promising antihyperglycemic agents.Conclusion: The isolated compounds may contribute to the antidiabetic property of C. rotundus.

Keywords

Antihyperglycemia, cyperaceae, flavane,stilbene dimmers

History

Received 29 March 2013Revised 20 May 2013Accepted 10 June 2013Published online 16 September 2013

Introduction

Cyperus rotundus L. (Cyperaceae) is distributed world-

wide and has been used in many traditional remedies for

treatment of menstrual disorders, dysmenorrhea, stomach-

ache and inflammation (Tang & Eisenbrand, 2011;

Venkatasubramanian et al., 2010; Vo, 2004). This plant has

recently attracted a great deal of attention due to the variety of

chemical compositions and broad range of biological

activities. The strong antioxidant properties of C. rotundus

have been shown to be due to its polyphenol, terpene and

essential oil contents (Kilani et al., 2008; Priya-Rani &

Padmakumari, 2012; Yazdanparast & Ardestani, 2007). It has

also been reported that C. rotundus showed cytotoxic and

apoptosis-inducing effects against various tumor cells

(Kilani et al., 2008; Kilani-Jaziri et al., 2009; Sayed et al.,

2007). Jin et al. (2011) reported that sesquiterpenes prepared

from a 70% ethanol extract of the rhizomes of C. rotundus

exerted significant anti-allergic activity in vitro and in vivo.

Nootkatone, a sesquiterpene isolated from C. rotundus,

was found to have potent inhibitory effects on collagen-,

thrombin- and arachidonic acid-induced platelet aggregation

(Seo et al., 2011). The antidiabetic activity of C. rotundus has

also been evaluated in animal models. Oral administration

of 200 and 500 mg/kg of 70% ethanol extract of C. rotundus

rhizomes significantly lowered blood glucose levels in

alloxan-induced hyperglycemic rats (Raut & Gaikwad,

2006). The aerial parts of C. rotundus showed antihypergly-

cemic effects via inhibition of protein glycation in a fructose-

mediated model (Ardestani & Yazdanparast, 2007). Several

flavonoids isolated from C. rotundus aerial parts inhibited

a-amylase (Sayed et al., 2008).

Diabetes is a group of metabolic diseases characterized by

chronic hyperglycemia resulting from deficiency in insulin

secretion or action. One therapeutic approach for treating

diabetes is to decrease postprandial glycemia by inhibition

of the enzymes responsible for carbohydrate hydrolysis, such

as a-glucosidase and a-amylase (Souza et al., 2012). In our

search for antidiabetic agents of natural origins, a methanol

extract of C. rotundus rhizomes was found to show significant

inhibitory activity against a-glucosidase and a-amylase.

Phytochemical investigation of the methanol extract of

C. rotundus rhizomes led to the isolation of a new flavan-3-

ol (1) and three stilbene dimers, cassigarol E (2), scirpusin

A (3) and scirpusin B (4) (Morikawa et al., 2010) (Figure 1).

These compounds showed strong a-glucosidase and a-amy-

lase inhibitory effects as well as 2,2-diphenyl-1-picrylhydra-

zyl (DPPH) radical scavenging activity.

Correspondence: Nguyen Tien Dat, Department of Bioactive Products,Institute of Marine Biochemistry, Vietnam Academy of Science andTechnology, 18-Hoang Quoc Viet, Hanoi, Vietnam. Tel: 84-4-37917049.Fax: 84-4-37917054. E-mail: ngtiend@imbc.vast.vn

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Materials and methods

General experimental procedures

Optical rotation values were recorded using a JASCO P-2000

digital polarimeter (JASCO, Tokyo, Japan). The infrared (IR)

spectrum was obtained from a Tensor 37 FT-IR spectrometer

(Bruker, Ettlingen, Germany). Nuclear magnetic resonance

(NMR) experiments were carried out on a Bruker AM500 FT-

NMR spectrometer (Bruker, Rheinstetten, Germany) using

tetramethylsilane as internal standard. The electrospray

ionization mass spectrometry were recorded on an Agilent

1200 series liquid chromatography-mass selective detector

Ion Trap (Agilent Technologies, Waldbronn, Germany). The

high resolution electrospray ionization mass spectrometry

(HR-ESI-MS) were recorded on an Fourier transform ion

cyclotron resonance mass spectrometer (Bruker Dal-tonics,

Bremen, Germany).

Plant material

The rhizomes of C. rotundus were collected in Dong Anh,

Hanoi, Vietnam, in September 2011 and identified by

Dr. Tran Huy Thai, Institute of Ecology and Biological

Resources, Vietnam Academy of Science and Technology.

The voucher specimens were deposited at the herbarium of

the Institute of Ecology and Biological Resources.

Extraction and isolation

The air-dried and powdered rhizomes of C. rotundus (4.0 kg)

were extracted with methanol (10 L� 3 times) at room

temperature. The combined extracts were concentrated to

give 300.0 g of crude extract, which was then resuspended

in water (1.5 L) and successively partitioned with hexane and

ethyl acetate (each 0.5 L� 3 times) to obtain 71.3 and 179.0 g

of hexane and ethyl acetate residues, respectively. The ethyl

acetate residue was chromatographed on a silica gel column

eluted with a gradient of 1–100% methanol in chloroform

to afford three fractions E1–3. The E1 was fractionated

on a silica gel column eluted with hexane-ethyl acetate (10:1,

1:1 and 1:10 v/v) to give three fractions E1.1–3. Compound 2

(20.0 mg) was purified from E2.2 by using a reverse phase

C18 column eluted with methanol–water (1:1 v/v). The fraction

E2.3 was divided into three fractions E2.3.1–3 by silica gel

column (chloroform–acetone–water 5:1:0.05 v/v). Compounds

3 (30.5 mg) and 4 (17.5 mg) were isolated from E2.3.2 by silica

gel column eluted with chloroform–acetone (2:1 v/v). The

fraction E2.3.3 was passed through a Sephadex LH-20 column

(methanol–water 1:1 v/v) to obtain 1 (11.2 mg).

(2RS,3SR)-3,40,5,6,7,8-hexahydroxyflavane (1): white

solid, optically inactive. IR �max(KBr): 3400, 1620, 1530,

1470 and 1150 cm�1. 1H NMR (500 MHz, CD3OD): d 2.53

(1H, dd, J¼ 6.5, 16.5 Hz, H-4a), 2.90 (1H, dd, J¼ 5.5,

16.5 Hz, H-4b), 4.00 (1H, dd, J¼ 8.0, 14.0 Hz, H-3), 4.62

(1H, d, J¼ 8.0 Hz, H-2), 6.80 (2H, d, J¼ 8.0 Hz, H-30,50)and 7.23 (2H, d, J¼ 8.0 Hz, H-20,60). 13C NMR (125 MHz,

CD3OD): d 82.8 (C-2), 68.8 (C-3), 28.8 (C-4), 158.3 (C-5),

157.7 (C-6), 158.3 (C-7), 157.4 (C-8), 156.9 (C-9), 100.9

(C-10), 131.5 (C-10), 129.6 (C-20), 116.0 (C-30), 156.9 (C-40),116.0 (C-50) and 129.6 (C-60). HR-ESI-MS: m/z 307.0810

[MþH]þ (calcd. 307.0818 for C15H15O7).

Figure 1. Structure of compounds 1–4 isolated from C. rotundus.

2 H. H. T. Tran et al. Pharm Biol, Early Online: 1–4

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Assay for a-glucosidase inhibition

The a-glucosidase (G0660-750UN, Sigma-Aldrich, St. Louis,

MO) enzyme inhibition assay was performed according to the

previously described method (Ali et al., 2002). The sample

solution (2 ml dissolved in dimethyl sulfoxide; DMSO) and

0.5 U/ml a-glucosidase (40ml) were mixed in 120 ml of 0.1 M

phosphate buffer (pH 7.0). After 5 min pre-incubation, 5 mM

p-nitrophenyl-a-D-glucopyranoside solution (40 ml) was

added, and the solution was incubated at 37 �C for 30 min.

The absorbance of released 4-nitrophenol was measured at

405 nm by using a microplate reader (Molecular Devices,

Sunnyvale, CA). Acarbose was used as positive control.

Assay for a-amylase inhibition

The a-amylase (A8220, Sigma-Aldrich, St. Louis, MO)

enzyme inhibitory activity was measured using the method

reported by Kusano et al. (2011) with slight modifications.

Substrate was prepared by boiling 100 mg potato starch in

5 ml phosphate buffer (pH 7.0) for 5 min, then cooling to

room temperature. The sample (2 mL dissolved in DMSO) and

substrate (50mL) were mixed in 30 mL of 0.1 M phosphate

buffer (pH 7.0). After 5 min pre-incubation, 5 mg/mL

a-amylase solution (20 mL) was added, and the solution was

incubated at 37 �C for 15 min. The reaction was stopped by

adding 50 mL 1 M HCl and then 50 mL iodine solution was

added. The absorbances were measured at 650 nm by a

microplate reader. Acarbose was used as positive control.

DPPH radical scavenging activity

The antioxidant activity of the isolated compound was

evaluated by its scavenging capacity of the DPPH radical.

Briefly, tested sample (10 mL) at various concentrations was

mixed with 150 mM DPPH solution (190mL) 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.

(þ)-Catechin was used as positive control.

Results and discussion

Compound 1 was obtained as a yellow solid. HR-ESI-MS

showed the peak at m/z 307.0810 [M þ H]þ corresponding to

the molecular formula C15H14O7.

The NMR data of 1 suggested a flavan-3-ol skeleton based

on signals characteristic for the C ring with two oxymethine

protons at dH 4.62 (1H, d, J ¼ 8.0 Hz, H-2) and 4.00 (1H, dd,

J ¼ 8.0, 14.0 Hz, H-3) and a pair of methylene protons at dH

2.53 (1H, dd, J ¼ 6.5, 16.5 Hz, H-4a) and 2.90 (1H, dd,

J ¼ 5.5, 16.5 Hz, H-4b). These protons gave correlations with

carbon resonances at dC 82.8 (C-2), 68.8 (C-3) and 28.8 (C-4)

in the heteronuclear multiple quantum coherence spectrum,

respectively. An A2B2 spin coupling system at dH 6.80 (2H, d,

J ¼ 8.0 Hz, H-30, H-50) and 7.23 (2H, d, J ¼ 8.0 Hz, H-20, H-

60) was characteristic of the 40-substituted pattern of the B

ring. The remaining NMR signals indicated that ring A was

fully oxygenated. These data were very similar to those of

2R,3R-3,5,6,7,8,40-hexahydroxyflavane (Zeng et al., 2011)

except for the signals of the C ring. The large coupling

constant of H-2 and H-3 was indicative of the 2,3-trans

relative configuration of 1 (Sang et al., 2002). The null optical

rotation suggested the racemic mixture of this flavane-3-ol.

Thus, 1 was determined as (2RS,3SR)-3,40,5,6,7,8-

hexahydroxyflavane.

The inhibitory effects of the isolated compounds against

a-glucosidase and a-amylase were evaluated in comparison

with the antidiabetic acarbose. As shown in Table 1, the most

active compound was cassigarol E (2), which inhibited both

a-amylase and a-glucosidase with IC50 values of 21.7 and

210.5 mM, respectively. The flavanol 1 inhibited a-amylase

at a dose similar to 2 but had no effect on a-glucosidase.

In contrast, 3 and 4 were only active against a-glucosidase.

In addition, all compounds exhibited DPPH radical scaven-

ging activity.

Stilbene dimers derived from resveratrol, including

scirpusin A (3), have been shown to be potent inhibitors

of a-glucosidase (Lam et al., 2008; Wan et al., 2011). The

stilbene cassigarol E (2) has been shown to exhibit antioxi-

dant, antiallergic and antitumor activities (Morikawa et al.,

2010; Wada et al, 2009; Xiang et al., 2005). In this study, this

compound showed strong inhibitory effects against a-gluco-

sidase and a-amylase. Previous studies have indicated that the

addition of a hydroxyl group increases the biological activities

of stilbene (Lam et al., 2008; Morikawa et al., 2010; Richard

et al., 2011). Consistent with these reports, our results

indicated that the inhibitory effects of scirpusin B (4) against

a-glucosidase were stronger than those of the less hydro-

xylated derivative, scirpusin A. The a-glucosidase inhib-

ition may contribute to the blood glucose-lowering effect

of scirpusin B in the glycogen-loaded mouse model

reported previously by Kobayashi et al. (2006). Although

the a-amylase inhibitory activity of the aerial parts of

C. rotundus has been reported (Sayed et al., 2008), this

is the first study of the effects of C. rotundus rhizomes

on a-amylase and a-glucosidase activities. These results may

contribute to characterization of the antidiabetic properties

of C. rotundus extract (Ardestani & Yazdanparast, 2007;

Raut & Gaikwad, 2006).

It has been reported that oxidative stress, through the

production of reactive oxygen species, is an important factor

for the development of diabetes mellitus, and a high blood

sugar level in diabetics can cause the overproduction of free

radicals (Johansen et al., 2005; Psaltopoulou et al., 2011;

Sabu & Kuttan, 2002). Antioxidants act as free radical

scavengers due to their redox properties and therefore prevent

and repair free radical-induced damage (Karunakaran & Park,

2013; Williams et al., 2013). Consistent with these reports,

our study demonstrated that the C. rotundus rhizomes

Table 1. Inhibitory effects of 1–4 against a-glucosidase, a-amylaseand DPPHa.

Compounds a-amylase a-glucosidase DPPH

1 24.2� 1.1 na 163.0� 12.62 21.7� 1.4 210.5� 17.3 78.6� 3.73 na 168.1� 12.5 108.3� 7.24 na 94.3� 6.8 55.1� 3.8Acarbose 776.1� 36.7 2060� 97.5 –(þ)-Catechin – – 43.2� 2.4

aValues (IC50 in mM) are means� SD from at least three experiments.na: not active.

DOI: 10.3109/13880209.2013.814692 Antidiabetic constituents from Cyperus rotundus 3

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contained polyphenols as both inhibitors of carbohydrate

digestive enzymes and scavengers of free radicals and hence

can be used as a complementary therapeutic medicine for the

management of diabetic complications (Golbidi et al., 2011;

Johansen et al., 2005).

Conclusion

Phytochemical fractionation of the methanol extract of

C. rotundus rhizomes led to the isolation of (2RS,3SR)-

3,40,5,6,7,8-hexahydroxyflavane (1), together with three

known stilbene dimers, cassigarol E (2), scirpusin A (3) and

scirpusin B (4). Compound 2 showed inhibitory effects

against both a-glucosidase and a-amylase activities. The

flavan-3-ol 1 inhibited a-amylase, while 3 and 4 were

inhibitors of a-glucosidase. All four compounds showed

significant DPPH scavenging activity.

Acknowledgements

We thank the Institute of Chemistry, Vietnam Academy

of Science and Technology, for the NMR and HRMS

measurements.

Declaration of interest

Authors declare no conflicts of interest.

This work is supported, in part, by the Ministry of Science

and Technology (NCCBDHUD/2011-2014) and the National

Foundation for Science and Technological Development

(NAFOSTED 104.01-2011.54).

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