J Nat Med (2008) 62:349–353
DOI 10.1007/s11418-008-0244-1

NOTE

Antioxidant constituents in the dayflower (Commelina
communis L.) and their a-glucosidase-inhibitory activity
Makio Shibano Æ Koji Kakutani ÆMasahiko Taniguchi Æ Masahide Yasuda Æ
Kimiye Baba

Received: 5 February 2008 / Accepted: 17 March 2008 / Published online: 15 April 2008
Ó The Japanese Society of Pharmacognosy and Springer 2008

Abstract The dayflower, Commelina communis L., contains 1-deoxynojirimycin (DNJ) and (2R,3R,4R,5R)2,5bis(hydroxymethyl)-3,4-dihydroxypyrrolidine(DMDP),
potent a-glucosidase inhibitors. The extracts and powder ofthis herb are important food materials for prophylaxis
against type 2 diabetes. Eleven flavonoid glycosides as
antioxidants, isoquercitrin, isorhamnetin-3-O-rutinoside,isorhamnetin-3-O-b-D-glucoside, glucoluteolin, chrysoriol7-O-b-D-glucoside, orientin, vitexin, isoorientin, isovitexin,
swertisin, and flavocommelin, were identified from theaerial parts of C. communis. Their antioxidant activities
were measured using in vitro assays employing the 1,1diphenyl-2-picrylhydrazyl radical- and superoxide radicalscavenging assays. The results showed that glucoluteolin,orientin, isoorientin, and isoquercitrin are the predominantantioxidants in this herb. Moreover, isoquercitrin, isorhamnetine-3-O-rutinoside, vitexin, and swertisin inhibitedthe activity of a-glucosidase from rat intestine.
Keywords Dayflower, Commelina communis Commelina communis var. hortensis  Antioxidant 
Flavonoid glycoside  a-glucosidase inhibitor

M. Shibano (&)  M. Taniguchi  M. Yasuda  K. Baba
Osaka University of Pharmaceutical Sciences,
4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japane-mail: shibano@gly.oups.ac.jp
K. Kakutani
Pharmaceutical Research and Technology Institute,
Kinki University, 3-4-1 Kowakae Higashiosaka,
Osaka 577-8502, Japan

Introduction
Postprandial hyperglycemia is one of the most important
health issues in the 21st century, because it can develop
into type 2 diabetes, hypertension, and cardiovasculardisease [1]. The International Diabetes Federation has
estimated that approximately 500 million people worldwide experience some degree of dysglycemia [2]. This
uptake of excess sugar causes an imbalance between oxidants and antioxidants in the human body. Several
management strategies have been proposed for the earlystage of dysglycemia with the aim of preventing further
development. A key strategy is ‘‘lifestyle modification,’’involving changes in diet and exercise, which was shown to
reduce the incidence of type 2 diabetes by 58% [3].
It is widely accepted that dietary supplements can contribute significantly to health. In particular, in postprandial
hyperglycemia, herbs with a-glucosidase-inhibitory activity will become more important as food material. The
dayflower, Commelina communis L., is distributed widely
throughout the world. The whole plants have been used as a
febrifuge or a diuretic in Japanese folk medicine. This herb
contains 1-deoxynojirimycin (DNJ) and (2R,3R,4R,5R)2,5bis (hydroxymethyl)-3,4-dihydroxypyrrolidine (DMDP),
which are potent a-glucosidase inhibitors (Fig. 1), but noinvestigation of their antioxidant activity has been reported[4–6]. However, major phytochemicals, phenolic acid,flavonoids, coumarin derivatives, etc., are known to combat oxidative stress in the human body by helping tomaintain a balance between oxidants and antioxidants.
Moreover, the combination of a-glucosidase inhibitors andantioxidants will become more effective for the prophylaxis of type 2 diabetes with the use of dietary supplements.
In this paper, we report the isolation of 11 flavonoid
glycosides, isoquercitrin, isorhamnetin-3-O-rutinoside,

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J Nat Med (2008) 62:349–353

BIO-RAD Model 680XR plate readers (for radical-scavenging assays).

isorhamnetin-3-O-b-D-glucoside, glucoluteolin, chrysoriol7-O-b-D-glucoside, orientin, vitexin, isoorientin, isovitexin,
swertisin, and flavocommelin (Fig. 2), as antioxidants fromthe aerial parts of C. communis and C. communis var.
hortensis [7]. These compounds were also assayed todetermine whether they inhibit the activity of a-glucosidasefrom rat intestine.

Reagents and materials
HPLC-grade methanol and acetonitrile (for preparativeHPLC), reagent-grade dichloromethane, methanol, ethanol,and ethyl acetate (for extraction and column chromatography), 1,1-diphenyl-2-picrylhydrazyl (DPPH), sodium
acetate, acetic acid, and epigallocatechin gallate werepurchased from Nacalai Tesque Co., Ltd. (Kyoto, Japan).
Wakogel C-200 (for column chromatography) was purchased from Wako Pure Chemical Industries Ltd. (Osaka,
Japan). Silica gel 60F254-precoated TLC plates were purchased from Merck (Darmstadt, Germany). Ultrapure water
was prepared using a Millipore Milli-Q purification system(Bedford, MA). C. communis L. was collected in Takatsuki
ward (Osaka, Japan). C. communis var. hortensis wascultivated in Kusatsu (Shiga, Japan).

Materials and methods
General experimental procedures
The instruments used in this study were Shimadzu LC10AT, SPD-10A, and SCL-10A LC instruments (for preparative HPLC), Varian Mercury 300 and Unity Inova-500
NMR spectrometers (for NMR spectra measured in
CD3OD or pyridine-d5 using tetramethylsilane as aninternal standard), JEOL JMS-MS700V mass spectrometer(for mass spectra), and Shimadzu UV mini 1240 and

Isolation of antioxidants
After drying, the aerial parts (400 g) of the dayflowers
were refluxed three times with 70% EtOH (3 L) for 3 h.
The solution was concentrated under reduced pressure to
give an extract (75.5 g). The extract was chromatographedon a Diaion HP-20 column (Mitsubishi Chemical Co.,
Ltd., Tokyo, Japan). After washing the column withwater, the adsorbed material was eluted with MeOH (2 l).
The eluted fraction was concentrated in vacuo to give aflavonoid fraction (51.7 g). This fraction was chromatographed on a silica gel (400 g) column (5.3 i.d. 9 56 cm)

OH

NH

H
N

HOH2C

OH

HO
HO

CH2OH
OH

OH
DMDP

DNJ

Fig. 1 Chemical structures of DNJ and DMDP
Fig. 2 Chemical structures offlavonoid glycosides

OH
Glc
O

HO

OGlc

OH

Glc
OH

O

MeO

O

HO

Glc
OH

O

OH

orientinflavocommelinvitexin
OH

O

HO

OH

Glc

OH
O

HO

O

O

OH

isovitexinOO

swertisin

OH
GlcO

O

Glc
OH

isoorientin

OH

MeO

Glc
OH

O

OH

O

OH
O

HO

OH
O

HO
OGlc

OH

O

R=Me: chrysoriol-7-O-β -D-glucoside
R=H: glucoluteolin

123

OMe

OH

OR

OH

O

isoquercitrin

OGlc ROHO

R=Rham: isorhamnetin-3-O-rutinoside
R=H: isothamnetin-3-O- β -D-glucosideJ Nat Med (2008) 62:349–353

351

with CH2Cl2–MeOH. Each fraction was subjected to
HPLC separation [Develosil ODS-5 10 i.d. 9 250 mm(Nomura Chemical Co., Ltd., Seto, Japan) or Nucleosil
C-18 AB 10 i.d. 9 250 mm (Macherey-Nagel, Duren,
Germany), CH3CN-1% AcOH (12:88 or 15:85)]. Fractions
19–27 yielded isoquercitrin (22 mg), isorhamnetin-3O-rutinoside (64 mg), isorhamnetin-3-O-b-D-glucoside(13 mg), glucoluteolin (461 mg), chrysoriol-7-O-b-D-glucoside (32 mg), orientin (178 mg), vitexin (69 mg),isoorientin (22 mg), isovitexin (16 mg), swertisin (11 mg),and flavocommelin (20 mg).
Determination of free radical-scavenging activity
The effects of each fraction or flavonoid glycoside on
DPPH radicals were monitored according to the method of
Hatano et al. [8]. The sample was dissolved in 50% ethanol(concentration of 0.015–3 mg/ml). One milliliter of 0.1 M
acetate buffer (pH 5.5), 0.5 ml of 0.5 mM DPPH radical
ethanolic solution, and 1 ml of each of the prepared 50%
ethanol solutions were mixed. The mixture was shaken
vigorously and incubated at 30°C for 30 min in the dark,
and the absorbance was measured spectrophotometricallyat 517 nm. The solution without DPPH solution added
served as a blank. Lower absorbance of the reaction mixture indicated higher free radical-scavenging activity. The
percentage of DPPH-scavenging activity was expressed as{1-[(test sample absorbance - blank sample absorbance)/
blank sample absorbance]} 9 100 (%). The purification ofantioxidative compounds was done based on the antioxidative activity exhibited.
Determination of superoxide radical-scavenging
activity
Various concentrations (0.05–5 mg/ml) of 20% ethanol
solution of the flavonoid glycosides were prepared. The
superoxide anion-scavenging activity was determinedusing a xanthine/xanthine oxidase reduced WST-1 systemwith a SOD assay kit (Dojindo Molecular Technologies,
Inc., Tokyo, Japan) according to the manufacturer’s protocol. The water-soluble formazan dye was monitored atTable 1 Preliminary
experiments on dried dayflower
powder (5 g)aSample concentration
is 0.4 mg/mlb

Sample concentrationis 6.0 mg/ml

450 nm. Measurements were performed in duplicate, andthe concentration required for a 50% inhibition (IC50) of
WST-1 formazan formation was determined graphically.
Assay of a-glucosidase inhibition
The a-glucosidase-inhibitory activity was measured using
the modified method of Dahlqvist [9]. The reaction mixture
consisted of the above basic extract solution (25 ll), 200 ll
50 mM phosphate buffer (pH 7.0), 175 ll 100 mM sucrosein 10 mM phosphate buffer (pH 7.0), and 100 ll a-glucosidase from rat intestine (Sigma-Aldrich Co., St. Louis,MO) solution (a stock solution of 1.0 mg/ml in 10 mM
phosphate buffer, pH 7.0, was diluted 40-fold with thesame buffer). The reaction mixture was incubated for
30 min at 37°C. Then, 500 ll of an aqueous solution
containing 1% 3,5-dinitrosalicylic acid, 5% sodium potassium tartate, 1% NaOH, 0.2% phenol, and 0.05% sodium
sulfite was added to the incubated solution, and the mixture
was heated at 100°C for 10 min to stop the reaction. Thissolution was diluted with 2 ml of water, and the opticaldensity at 540 nm was measured (OD test). The control
sample was prepared by adding water instead of the extract
and by treating in the same way as test samples to give an
OD blank. The inhibition rates (%) were calculated using
the formula 100 - 100 9 (OD test - OD blank)/(control
OD test - control OD blank).

Results and discussion
In this study, preliminary experiments were carried out
using dried dayflower powder (5 g). The hot-water extracts(0.97 g) were chromatographed on Sep-Pak C18 cartridges(Waters, Milford, MA) using H2O, H2O–MeOH (1:1), and
MeOH. The yield and antioxidant activities of each fraction are shown in Table 1. Thin-layer chromatography(TLC) of the H2O–MeOH (1:1) fraction showed numerous
spots under 254-nm UV irradiation. These results suggested that the antioxidant components of the hot-water
extracts are flavonoid glycosides. In addition, the inhibitory
activities of these fractions were assayed for their ability to

FractionYield (g)DPPH radical-scavenging
capacity (%)aa-glucosidase-inhibitory
activity (%)b

Hot-water extracts(sep-pak C18 cartridge)

0.97

98.6

85.7

H2O

0.53

75.2

95.0

H2O–MeOH (1:1)

0.21

95.3

75.7

MeOH

0.09

7.6

25.8

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J Nat Med (2008) 62:349–353

Table 2 DPPH radical-scavenging effects of flavonoid glycosides
and epigallocatechin gallate

Table 3 Superoxide radical-scavenging activity of flavonoid glycosides and epigallocatechin gallate

Flavonoid glycoside

Flavonoid glycoside

EC50(lg/ml)(lM)

Isoquercitrin

6.2

13.4

Isorhamnetin-3-O-rutinoside

150[

–

Isorhamnetin-3-O-b-D-glucoside
Glucoluteolin

150[
5.8

Chrysoriol-7-O-b-D-glucoside

IC50(lg/ml)(lM)

Isoquercitrin

25.0

53.9

Isorhamnetin-3-O -rutinoside

385[

–

–
12.9

Isorhamnetin-3-O-b-D-glucoside
Glucoluteolin

385[
18.3

–
40.8

150[

–

Chrysoriol-7-O-b-D-glucoside

385[

–

Orientin

6.7

15.0

Orientin

32.6

72.8

Vitexin

18.7

43.3

Vitexin385[–

Isoorientin

6.9

15.4

Isoorientin

39.7

88.6

Isovitexin

18.9

43.8

Isovitexin

385[

–

Swertisin

94.5

211.7

Swertisin

385[–

Flavocommelin

150[

–

Flavocommelin

385[

–

Epigallocatechin gallate

1.2

2.7

Epigallocatechin gallate

3.9

8.5

inhibit the activity of a-glucosidase from rat intestine. The
H2O and H2O–MeOH (1:1) fractions potently inhibited aglucosidase activity. The active constituents of the H2O
fraction are DNJ and DMDP, but these polyhydroxylated
alkaloids are not present in the H2O–MeOH (1:1) fraction.
Therefore, we investigated the active compounds of thisfraction.
The 70% EtOH extract of C. communis was chromatographed on a Diaion HP-20 column. After washing thecolumn with water, the adsorbed material was eluted with
MeOH. The eluted fraction was chromatographed on silica
gel using CH2Cl2 and MeOH to afford 32 fractions. The
free radical-scavenging activity of each fraction was
assayed against DPPH radicals. Fractions 19–27 exhibitedstronger antioxidant activity than the others. These active
fractions were further subjected to reversed-phase preparative HPLC to afford 11 flavonoid glycosides. Thus,
bioassay-guided fractionation of the 70% EtOH extracts
led to the isolation of the antioxidants isoquercitrin [10],isorhamnetin-3-O-rutinoside [10], isorhamnetin-3-O-b-Dglucoside [11], glucoluteolin [12], chrysoriol-7-O-b-Dglucoside [12], orientin [13], vitexin [14], isoorientin [13],isovitexin [15], swertisin [16], and flavocommelin [17].
These known compounds were identified by comparison of
their spectral (1H-NMR, 13C-NMR, and MS) data with thereported values. These compounds were isolated from thisherb for the first time, except for flavocommelin, which
comprises the blue pigment of the dayflower. Moreover,these 11 flavonoid glycosides were also isolated from
C. communis var. hortensis using the same methods.
Table 2 shows the DPPH radical-scavenging activitiesof the isolated flavonoid glycosides. Isoquercitrin, glucoluteolin, orientin, vitexin, isoorientin, isovitexin, andswertisin had EC50 values of 13.4, 12.9, 15.0, 43.3, 15.4,

123

Table 4 a-glucosidase-inhibitory activity of flavonoid glycosides,
DNJ, and DMDP
Flavonoid glycoside

IC50 (M)

Isoquercitrin

2.4 9 10-4

Isorhamnetin-3-O-rutinoside

5.1 9 10-4

Isorhamnetin-3-O-b-D-glucoside[1.0 9 10-3

Glucoluteolin[1.0 9 10-3

Chrysoriol-7-O-b-D-glucoside[1.0 9 10-3

Orientin[1.0 9 10-3

VitexinIsoorientin

4.2 9 10-4
[1.0 9 10-3

Isovitexin[1.0 9 10-3

Swertisin

3.7 9 10-4

Flavocommelin[1.0 9 10-3

DNJ

1.5 9 10-4

DMDP

5.8 9 10-5

43.8, and 211.7 lM, respectively, and the activity of epigallocatechin gallate was 2.7 lM. Table 3 shows thesuperoxide radical-scavenging activities of the isolated
compounds. Glucoluteolin had the strongest activity, andisoquercitrin, orientin, and isoorientin were also relatively
potent. Comparing these four flavonoid glucosides, itappears that the phenolic hydroxyl groups at the 3- and 4positions of the flavonoid B ring play a key role in radical
scavenging, although the hydroxyl group at the 7-positionof the flavonoid A ring appears to have no effect. In
principle, the structure–radical-scavenging relationships ofthese flavonoid glycosides were in agreement with datareported in the literature [18].
These isolated compounds, DMDP, and DNJ wereassayed to determine whether they inhibit the activity of

J Nat Med (2008) 62:349–353

a-glucosidase from rat intestine (Table 4). Isoquercitrin,
isorhamnetine-3-O-rutinoside, vitexin, and swertisin
inhibited a-glucosidase from rat intestine to the same
extent as DNJ, but the other constituents did not inhibit theactivity of this enzyme.
We propose that these herbs (C. communis andC. communis var. hortensis) may be a useful food material
for type 2 diabetes patients and for prophylaxis against
dysglycemia.
Acknowledgments This work was supported by a grant from Kusatsu
City, Shiga, Japan. We are grateful to Dr. K. Minoura for 500-MHz
NMR spectral measurements and to Mrs. M. Fujitake for mass spectral
measurements at the Osaka University of Pharmaceutical Sciences.

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