Bio-assay guided isolation and identification of α-glucosidase inhibitors from the leaves of Aquilaria sinensis

Article abstract of DOI:10.1016/j.phytochem.2010.11.025

Eight α-glucosidase inhibitors including four new compounds were isolated from the 70% aqueous ethanolic extract of leaves of Aquilaria sinensis (Lour.) Gilg by activity-directed fractionation and purification processes. The ethanolic extract was first separated into petroleum ether, ethyl acetate, n-butanol and water soluble fractions and screened for inhibitory activity against α-glucosidase. Further activity-directed investigation lead to the isolation of four new compounds with moderate inhibitory activity, viz, aquilarisinin (1), aquilarisin (2), hypolaetin 5-O-β-d-glucuronopyranoside (3) and aquilarixanthone (4) from the n-butanol fraction, and four known compounds showing potent activity including mangiferin (5), iriflophenone 2-O-α-l-rhamnopyranoside (6), iriflophenone 3-C-β-d-glucoside (7) and iriflophenone 3,5-C-β-d-diglucopyranoside (8) from the most potent ethyl acetate fraction. The structures of these compounds were determined by extensive spectroscopic analyses, including IR, UV, ESIMS, HRESIMS, 1D and 2D NMR.

Full text of DOI:10.1016/j.phytochem.2010.11.025

Phytochemistry 72 (2011) 242–247
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Bio-assay guided isolation and identification of
a-glucosidase inhibitors
from the leaves of Aquilaria sinensis
b
Jie Feng a, Xiu-Wei Yang a, , Ru-Feng Wang
⇑
a State Key Laboratory of Natural and Biomimetic Drugs, Department of Natural Medicine, School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China
b School of Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100102, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 April 2009
Received in revised form 2 August 2010
Available online 6 January 2011
Eight a-glucosidase inhibitors including four new compounds were isolated from the 70% aqueous eth-
anolic extract of leaves of Aquilaria sinensis (Lour.) Gilg by activity-directed fractionation and purification
processes. The ethanolic extract was first separated into petroleum ether, ethyl acetate, n-butanol and
water soluble fractions and screened for inhibitory activity against
ted investigation lead to the isolation of four new compounds with moderate inhibitory activity, viz, aqui-
larisinin (1), aquilarisin (2), hypolaetin 5-O-b- -glucuronopyranoside (3) and aquilarixanthone (4) from
the n-butanol fraction, and four known compounds showing potent activity including mangiferin (5),
iriflophenone 2-O- -rhamnopyranoside (6), iriflophenone 3-C-b- -glucoside (7) and iriflophenone
3,5-C-b- -diglucopyranoside (8) from the most potent ethyl acetate fraction. The structures of these com-
a-glucosidase. Further activity-direc-
Keywords:
Aquilaria sinensis
Thymelaeaceae
D
a
-
L
D
a-Glucosidase inhibitors
D
Benzophenone glycoside
Flavonoid glucuronide
Xanthone glycoside
pounds were determined by extensive spectroscopic analyses, including IR, UV, ESIMS, HRESIMS, 1D and
2D NMR.
Ó 2011 Published by Elsevier Ltd.
1. Introduction
an incense as well as a traditional sedative, analgesic, and digestive
medicine in East Asia. Phytochemical investigations of agarwood
Diabetes mellitus is a chronic metabolic disorder which is char-
acterized by hyperglycemia and accompanied by various chronic
vascular complications; thus, the control of postprandial blood glu-
cose surges is critical for the treatment of diabetes. One of the ther-
apeutic approaches for postprandial hyperglycemia is to reduce
carbohydrate uptake after meal. It is well-known that complex
polysaccharides are hydrolyzed by amylases to dextrins which
and its oil have led to isolation of sesquiterpenes and chromone
derivatives (Nakanishi et al., 1984; Ishihara et al., 1991; Yagura
et al., 2005; Liu et al., 2008). The leaves of A. sinensis have been
used traditionally in China for treatments of inflammation and
anaphylaxis and their extracts have exhibited notable analgesic,
anti-inflammatory (Zhou et al., 2008) and mild cathartic (Iinuma
et al., 2007) activities. As for the chemical constituents of the
leaves, several flavonoids and benzophenone glycosides have been
reported previously (Iinuma et al., 2007; Qi et al., 2009). In the
are further hydrolyzed to glucose by intestinal
a-glucosidase be-
fore entering blood circulation through intestinal epithelium
absorption. For this reason, postprandial hyperglycemia may be
course of our characterization studies on
a-glucosidase inhibitors
treated by amylase and
a-glucosidase inhibitors via delaying glu-
in natural medicines, the ethyl acetate (EtOAc) soluble fraction of
cose absorption. Synthetic amylase and
a-glucosidase inhibitors,
the 70% aqueous ethanol extract from the leaves of A. sinensis
for example, acarbose (chemical structure shown in Fig. 1), are
widely used for the treatment of patients with type II diabetes,
but they are also reported to cause various side-effects; therefore,
was found to have inhibitory effects against
Consequently, the chemical constituents of Aquilaria leaves were
thoroughly investigated in this paper guided by bio-assay of
glucosidase inhibitors.
a-glucosidase activity.
a-
safer natural amylase and a-glucosidase inhibitors are desired and
many compounds have been reported from plant sources (Hiroyuki
et al., 2001; Matsui et al., 2001; Wansi et al., 2007; Kim et al., 2008;
Lee et al., 2008). Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae),
distributed in South China such as Hainan, Guangxi, Guangdong,
Fujian and Taiwan provinces, is the principal source of the expen-
sive agarwood. The latter is a rich in resin which has been used as
2. Results and discussion
2.1. Chemistry
Dried leaves of A. sinensis were extracted with 70% aqueous eth-
anol, and the extract was concentrated in vacuo and kept overnight
for precipitation. The resulting suspension was filtered and succes-
sively fractionated with petroleum ether (PE), EtOAc and n-butanol
⇑
Corresponding author. Tel.: +86 10 82805106; fax: +86 10 62070317.
E-mail addresses: xwyang@bjmu.edu.cn, xwyang@hsc.pku.edu.cn (X.-W. Yang).
0031-9422/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.phytochem.2010.11.025

J. Feng et al. / Phytochemistry 72 (2011) 242–247
243
Table 1
1H and 13C NMR spectroscopic data (DMSO-d6) of 1, 6 and 6a.a
Position
1
6
6a
dH
dC, mult.
dC, mult.
dC, mult.
1
2
3
4
5
6
109.7, qC
155.6, qC
94.0, CH
159.5, qC
96.8, CH
156.5, qC
193.1, qC
130.4, qC
131.6, CH
115.3, CH
161.9, qC
115.3, CH
131.6, CH
98.6, CH
70.0, CH
70.0, CH
82.0, CH
68.1, CH
17.9, CH3
104.6, CH
74.5, CH
76.6, CH
69.7, CH
77.1, CH
109.5, qC
155.5, qC
93.9, CH
159.4, qC
96.6, CH
156.4, qC
192.8, qC
130.1, qC
131.4, CH
115.1, CH
161.8, qC
115.1, CH
131.4, CH
98.9, CH
70.1, CH
70.1, CH
71.5, CH
69.5, CH
17.9, CH3
106.6, qC
157.9, qC
94.3, CH
160.4, qC
94.3, CH
157.9, qC
194.4, qC
130.6, qC
131.5, CH
114.7, CH
161.5, qC
114.7, CH
131.5, CH
6.14 d, 2.0
6.05 d, 2.0
7
10
20
7.56 d, 8.7
6.82 d, 8.7
30
40
50
6.82 d, 8.7
7.56 d, 8.7
5.14 br s
60
Rha-100
Rha-200
Rha-300
Rha-400
Rha-500
Rha-600
Glc-1000
Glc-2000
Glc-3000
Glc-4000
Glc-5000
Glc-6000
3.05 br d, 7.8
3.35 dd, 5.4, 7.8
3.34 dd, 5.4, 8.0
3.34 dd, 5.4, 8.0
1.14 d, 5.4
4.32 d, 7.8
2.97, dd, 7.8, 8.4
3.11 t, 8.4
3.36 t, 8.4
3.06 t, 8.7
3.45 dd, 8.0, 12.0
3.62 d, 12.0
61.2, CH2
a
C-multiplicities were established by a HSQC experiment.
mode and 245 [Mꢀ162–146ꢀH]ꢀ in the negative mode ESIMS
spectrum demonstrated the presence of a disaccharide chain con-
sisting of a rhamnosyl–glycosyl moiety (Ding et al., 2008). Finally,
the linkage pattern of the disaccharide chain was further con-
firmed by 2D NMR spectroscopy such as 1H–1H COSY, HSQC
and HMBC analyses. The HMBC data showed correlations from
H-1000 [d 4.32 (1H, d, J = 7.8 Hz, terminal H-1 of glucopyranosyl)]
to C-400 [d 82.0 (inner C-4 of rhamnopyranosyl)] and from H-100 [d
5.14 (1H, br s, inner H-1 of rhamnopyranosyl)] to C-2 (d 155.6),
corroborating the linkage pattern of glucopyranosyl-(1?4)-
rhamnopyranosyl and its attachment to C-2 of the aglycone. Thus,
Fig. 1. Chemical structures of compounds 1–8, 2a, 6a, acarbose and Key HMBC
(H?C) correlations of 4.
(n-BuOH). The EtOAc and n-BuOH extracts were successively sub-
jected to silica gel, polyamide and Sephadex LH-20 column chro-
matography in combination with bioassays with the purpose of
tracking
a-glucosidase inhibitors. As a result, four new (1–4) and
four known (5–8) compounds were obtained.
Compound 1 was obtained as a yellowish amorphous powder.
Its molecular formula was determined as C25H30O14 on the basis
of ESIMS and HRESIMS data. In the ESIMS spectrum, two quasi-
molecular ion peaks appeared at m/z 577 [M+Na]+ and 555
[M+H]+, suggesting a molecular weight of 554. The positive HRE-
SIMS analysis also showed quasi-molecular ion peaks at m/z
555.1711 [M+H]+ (calcd. for C25H31O14, 555.1708) and 577.1516
[M+Na]+ (calcd. for C25H30NaO14, 577.1528), which confirmed
the molecular formula mentioned above. Compound 1 was char-
acterized as chalcone, this being partially supported by the
stretching vibration signals at 3383 and 1725 cmꢀ1 indicative of
an associated hydroxyl and a carbonyl group, respectively, in its
IR spectrum. The signals observed in 1H and 13C NMR (Table 1)
spectra of 1 were similar to those of the known compound 6 (Ii-
numa et al., 2007), except for a set of signals representative of a
1 was identified as iriflophenone 2-O-b-
-rhamnopyranoside, named as aquilar- isinin.
Compound 2, obtained as a yellowish amorphous powder, was a
flavone (Fig. 1) as evidenced by comparison of its spectroscopic
data with a known flavone, i.e. isoscutellarein 8-O-b- -glucurono-
D-glucopyranosyl-(1?4)-O-
a-L
D
pyranoside (2a) (Billeter et al., 1991). In its IR spectrum, the strong
IR bands at 3381 and 1726 cmꢀ1 were closely similar to those of 1,
indicating an associated hydroxyl and a carbonyl group, respec-
tively. This carbonyl group was confirmed by its 13C NMR reso-
nance signal at d 182.0. The 1H NMR spectroscopic data [d 8.14
(2H, d, J = 8.4 Hz, H-2, 6); 6.88 (2H, d, J = 8.4 Hz, H-3, 5)] disclosed
the characteristic of a 1,4-disubstituted B-ring, while six quater-
nary 13C NMR carbon signals (d 124.9, 129.2, 148.1, 149.6, 150.7
and 161.2) corresponding to O-linked aromatic carbons in combi-
nation with a 1H NMR resonance at d 6.68 (1H, s) that demon-
b-D-glucopyranosyl group (Breitmayer and Voelter, 1989). This
glucopyranosyl group was linked to C-400 of the rhamnopyranosyl
moiety through an 1 ? 4 glycosidic bond as evidenced by the
downfield chemical shift of this carbon (+10.5 ppm). The entire
structure of 10 as shown in Fig. 1, was definitely determined
based on the evidence below. Firstly, acid hydrolysis of 1 yielded
an aglycone 6a (iriflophenone), which was identical to that ob-
tained by acidic hydrolysis of 6, and two monosaccharides, i.e.
glucose and rhamnose were identified by comparison with the
respective authentic samples by paper chromatography (PC). Sec-
ondly, both ion peaks at m/z 247 [Mꢀ162ꢀ146+H]+ in the positive
strated the 5,6,7,8,40-pentaoxygenated flavone skeleton of
2
(Agrawal, 1989; Breitmayer and Voelter, 1989; Ponce et al.,
2004). A sharp three-proton singlet at dH 3.88 was assigned to a
methoxyl group which was proven to be located at C-5, since this
singlet showed a HMBC correlation with one of the oxygenated
quaternary carbons at dC 149.6 (C-5). Compound 2 was thus
established to be a monoglycosided flavone from the characteristic
b-
troscopic data: see Table 2]. This b-
connected the skeleton at C-8 via an oxygen atom, since the
D
-glucopyranosiduronyl signals of NMR [1H and 13C NMR spec-
-glucopyranosiduronyl group
D

244
J. Feng et al. / Phytochemistry 72 (2011) 242–247
(C-200), 76.2 (C-300), 71.9 (C-400), 76.7 (C-500) and 168.5 (C-600) (Billeter
Table 2
1H and 13C NMR spectroscopic data (DMSO-d6) of 2, 2a and 3.a
et al., 1991). Acid hydrolysis (see Section 4) together with fragmen-
tation ion peaks at m/z 325 [Mꢀ176+Na]+ and 303 [Mꢀ176+H]+ in
the positive ESIMS spectrum established the existence of glucu-
ronic acidyl group. This group connected C-5 (d 157.5) via an oxy-
gen atom, this being supported by a correlation noted between a
proton signal at d 4.37 (1H, d, J = 7.0 Hz, H-100) and C-5 in the HMBC
spectrum. This glucosidic bond was also b-oriented as judged from
the coupling constant of H-100 (J = 7.0 Hz) (Billeter et al., 1991).
According to above description, the molecular formula of 3 was
Position
2
2ab
3
dH
dC, mult.
dC
dH
dC, mult.
2
3
4
5
6
7
8
9
164.1, qC
102.7, CH
182.0, qC
149.6, qC
129.2, qC
148.1, qC
124.9, qC
150.7, qC
103.1, qC
121.4, qC
129.2, CH
116.2, CH
161.2, qC
116.2, CH
129.2, CH
106.4, CH
73.7, CH
76.2, CH
71.8, CH
75.5, CH
170.5, qC
56.1, CH3
163.8
102.3
181.7
157.2
99.0
157.2
125.2
149.2
103.3
121.0
128.9
116.0
161.1
116.0
128.9
106.3
73.6
163.5, qC
102.4, CH
180.5, qC
157.5, qC
99.0, CH
163.5, qC
128.3, qC
149.8, qC
102.0, qC
122.7, qC
114.3, CH
146.5, qC
149.3, qC
116.0, CH
118.5, CH
108.6, CH
74.3, CH
76.2, CH
71.9, CH
76.7, CH
168.5, qC
6.68 s
6.43 s
8.36 s
C
21H18O13, this also being confirmed by an quasi-molecular ion
peak at m/z 479.0822 [M+H]+ (calcd. for C21H19O13, 479.0820). Fi-
10
10
nally, 3 was elucidated as hypolaetin 5-O-b-D-glucuronopyranoside
(Fig. 1).
20
8.14 d, 8.4
6.88 d, 8.4
7.95 br s
30
Compound 4 also was isolated in the form of a yellowish amor-
phous powder. Its IR spectrum differed slightly from the former 3
compounds in that the bands at 3394–3380 cmꢀ1 were representa-
tive of aliphatic and aromatic hydroxyl groups, and bands at 1647
and 1587 cmꢀ1 were characteristic signals of a chelated carbonyl
group. Its 1H and 13C NMR signals, including five phenolic hydroxyl
resonances at dH 13.59 (1H, s, 1-OH), 10.50 (1H, s, 6-OH), 9.75 (1H,
s, 7-OH), 9.54 (1H, s, 3-OH) and 5.59 (1H, s, 4-OH) (Gómez-Zaleta
et al., 2006), two shielded aromatic methine signals at dH 7.31
(1H, s, H-8) and 6.73 (1H, s, H-5), as well as a 13C NMR resonance
at dC 179.2 for a carbonyl group (Agrawal, 1989; Breitmayer and
Voelter, 1989) (Table 3) closely resembled those of the known
compound 5 (mangiferin), indicating that compound 4 also had a
similar xanthone structure (Fig 1). Compared to mangiferin, com-
pound 4 had an additional hydroxyl group at d 5.59 (1H, s, 4-OH)
and a set of NMR signals for a xylopyranosyl instead of a glucopyr-
anosyl group. This additional hydroxyl group in 4 was located at
C-4, inasmuch as the 13C NMR signal of this carbon (d 153.8) was
shifted downfield about 60 ppm and the 1H NMR signal for H-4
was absent in comparison to mangiferin. The pentosyl group was
40
50
6.88 d, 8.4
8.14 d, 8.4
4.66 d, 7.8
3.52, m
3.57 t, 9.0
3.26, m
6.80 br d, 8.1
7.29 br d, 8.1
4.37 d, 7.0
3.65–3.17 m
3.65–3.17 m
3.65–3.17 m
3.65–3.17 m
60
GlcA-100
GlcA-200
GlcA-300
GlcA-400
GlcA-500
GlcA-600
5-OMe
OH
76.1
71.5
75.3
170.1
3.55 d, 9.0
3.88 s
8.39 br s
a
C-multiplicities were established by a HSQC experiment.
b
2a was isoscutellarein 8-O-b-D
-glucuronopyranoside and the 13C NMR spec-
troscopic data was reported in Billeter et al. (1991).
anomeric proton signal at dH 4.66 (H-100) showed a HMBC correla-
tion with another oxygenated quaternary carbon at dC 124.9 (C-8)
(Yang et al., 2003). Acid hydrolysis of 2 confirmed the presence of
this glucuronic acidyl group. At first, a quasi-molecular ion peak at
m/z 217.0318 [C6H10NaO7]+ in the positive ESIMS spectrum of the
hydrolysate established the presence of glucuronic acid. Secondly,
comparison of the hydrolysate with an authentic glucuronic acid
on PC (n-BuOH–H2O–HOAc, 4:2:1, v/v/v) provided the same con-
clusion. The entire molecule of 2 was deduced to have a formula
of C22H20O13 from the quasi-molecular ion peaks at m/z 515
[M+Na]+ and 493 [M+H]+ in the positive mode, and at m/z 983
[2MꢀH]ꢀ and 491 [MꢀH]ꢀ in the negative mode ESIMS spectra.
It was further confirmed by HRESIMS with this having an ion peak
at m/z 493.0964 [M+H]+ (calcd. for C22H21O13, 493.0976). Hence,
compound 2 was elucidated as 5-methoxy-6-hydroxy-isoscutella-
identified as b-D
-xylopyranosyl on the basis of its 1H and 13C
NMR spectroscopic data (Agrawal, 1989, 1992; Rancon et al.,
1999). This xylopyranosyl group connected C-2 through the C–C
Table 3
1H and 13C NMR spectroscopic data (DMSO-d6) of 4 and 5.a
Position
4
5
dH
dC, mult.
dC, mult.
rein 8-O-b-D-glucuronopyranoside (Fig. 1), trivially named as
aquilarisin.
1
2
3
4
4a
4b
5
6
7
158.3, qC
105.4, qC
160.7, qC
153.8, qC
154.2, qC
150.9, qC
102.4, CH
154.2, qC
143.6, qC
107.7, CH
111.6, qC
101.4, qC
179.2, qC
74.3, CH
71.9, CH
81.0, CH
71.9, CH
69.2, CH2
161.8, qC
108.1, qC
163.9, qC
93.3, CH
156.3, qC
150.8, qC
102.7, CH
154.1, qC
143.8, qC
107.7, CH
111.8, qC
101.3, qC
179.2, qC
73.1, CH
70.2, CH
79.0, CH
70.7, CH
81.7, CH
61.6, CH2
Compound 3 was isolated as a yellowish powder. The strong IR
bands at 3385 and 1725 cmꢀ1 were indicative of an associated hy-
droxyl and a carbonyl group, respectively. The carbonyl group was
confirmed by the resonance at dC 180.5 (C-4) in its 13C NMR spec-
trum (Table 2) (Agrawal, 1989; Breitmayer and Voelter, 1989).
Compound 3 was demonstrated to be a derivative of 5,7,8,30,40-
pentahydroxyflavone (Fig. 1) by comparison of its NMR spectro-
6.73 s
7.31 s
8
8a
8b
C@O
10
scopic data with those published for hypolaetin 8-O-b-D-glucuron-
opyranoside (Billeter et al., 1991; Bilia et al., 1996). In the 1H NMR
spectra, the aromatic methine signals at d 7.95 (1H, br s), 7.29 (1H,
br d, J = 8.1 Hz) and 6.80 (1H, d, J = 8.1 Hz) were assigned to H-20,
H-60 and H-50 of the B ring, respectively, and the resonance at d
6.43 (1H, s) was assigned to the H-3 of the C ring. Thus, another
aromatic methine proton signal at d 8.36 (1H, s) must be assignable
to A ring, and its exact position was H-6 because of its HMBC cor-
relation with a carbon signal at d 102.0 (C-10) which was assigned
based on its HMBC correlation with H-3. Similar to compound 2 it
also possessed a glucuronosyl group as indicated from its 1H and
13C NMR spectroscopic data including dH 4.37 (1H, d, J = 7.0 Hz,
H-100), 3.65–3.17 (4H, m, H-200 – H-500), and dC 108.6 (C-100), 74.3
4.86 d, 9.5
3.92 dd, 8.0, 9.5
3.30 t, 8.0
20
30
40
3.43 m
50
3.30 d, 10.0 3.84 dd, 6.0, 10.0
60
1-OH
3-OH
4-OH
6-OH
7-OH
13.59 s
9.54 s
5.59 s
10.50 s
9.75 s
a
C-multiplicities were established by a HSQC experiment.

J. Feng et al. / Phytochemistry 72 (2011) 242–247
245
bond due to the HMBC correlations of H-10 (d 4.86) and C-1 (d
158.3), C-2 (d 105.4) and C-3 (d 160.7) which not only established
the linking position, but also showed the characteristic chemical
shift pattern of a C-glycoside. The nature of the C-glycoside was
confirmed by acid hydrolysis results of compound 4, in which none
of the usual fragmentation patterns for O-glycosides in the mass
spectrum were observed (Rancon et al., 1999). The large coupling
constant of H-1 (J = 9.5 Hz) indicated a b-orientation of this C–C
glycosidic linkage. On the basis of both NMR spectroscopic data,
in combination with the quasi-molecular ion peaks at m/z 855
[2M+K]+ and 431 [M+Na]+ detected in positive ESIMS spectrum,
the molecular formula C18H16O11 was established. Thus was fur-
ther confirmed by the positive HRESIMS with an ion peak at m/z
431.0592 [M+Na]+ (calcd. for C18H16NaO11, 431.0590). Finally,
with that of the positive control acarbose with an IC50 value of
372.0 37.8 g/mL, which was comparable to the reported val-
ues (Li et al., 2005). In addition to 5 and 6, 1, 3, 7 and 6a also
exhibited inhibitory effect against -glucosidase which was
about twofold of that of acarbose, while 2, 4 and 8 showed
inhibitory effect almost the same as that of acarbose (chemical
structure shown in Fig. 1). Therefore, they may have potential
l
a
as lead compounds for development of potent
a-glucosidase
inhibitors. This is the first report of
activity of these compounds.
a-glucosidase inhibitory
3. Conclusion
In summary, compounds 5 and 6 are the main constituents of
the leaves of A. sinensis, compounds 1–4 were new compounds,
and compound 7 was isolated from the leaves of A. sinensis for
the first time, whereas its pharmacokinetic profile had been re-
ported by us previously (Feng et al., 2009). Compounds 1–8 and
the structure of
4 was elucidated as 2-C-b-D-xylopyranosyl-
1,3,4,6,7-pentahydroxyxanthone (Fig. 1), which has been named
as aquilarixanthone.
Known compounds were identified by various spectroscopic
methods including MS, 1D and 2D NMR spectroscopic analysis
and by comparing experimentally obtained data with those de-
scribed in the literature. In this way, mangiferin (5) (Catalano
6a exhibited inhibitory activity in vitro against
a-glucosidase, of
which 5 was the most potent one. In addition, it is found that
the chemical constituents of the leaves differ significantly from
the resin-deposited part of the trunk of A. sinensis based on our
results together with previous reports (Nakanishi et al., 1984;
Ishihara et al., 1991; Yagura et al., 2005; Iinuma et al., 2007; Liu
et al., 2008; Qi et al., 2009), and accordingly support their different
folklore use for some diseases.
et al., 1996; Mishra et al., 2006), iriflophenone 2-O-
anoside (6) (Iinuma et al., 2007), iriflophenone 3-C-b-
(7) (Murakami et al., 1986), and iriflophenone 3,5-C-b-
pyra-noside (8) (Iinuma et al., 2007) were identified.
a
-
L
-rhamnopyr-
-glucoside
-digluco-
D
D
2.2.
a-Glucosidase inhibitory activity
4. Experimental
The bio-assay of
a-glucosidase inhibitor was employed in this
investigation to direct the discovery of bioactive compounds. All
of the chromatographic fractions, and compounds (1–8) isolated
from leaves of A. sinensis, as well as an acidic hydrolysate (6a) of
4.1. General experimental procedures
Optical rotations were measured on an Autopol III polarimeter
with MeOH as solvent at 22°. IR spectra were taken on a Thermo
Nicolet Nexus 470 FT-IR spectrophotometer. UV spectra were ob-
tained on a Varian Cary-300 ultraviolet–visible photometer in
MeOH solution. Mass spectra were recorded on a MDS SCIEX API
ASTAR spectrometer (for ESIMS) and a Bruker DALTONICS APEX
IV Fourier transform ICR high-resolution mass spectrometer (for
HRESIMS). 1D and 2D NMR spectra were recorded on a JEOL JNM
300 (300 MHz for 1H NMR and 75 MHz for 13C NMR) using
DMSO-d6 as solvents, with TMS as int. standard. Open column
chromatography (CC) was carried out using silica gel (200–300
mesh, Qingdao Marine Chemical Co., Qingdao, PR China), polyam-
ide C-100 (Wako Pure Chemical Industries Ltd., Osaka, Japan) and
Sephadex LH-20 (Pharmacia, Fine Chemicals, Inc., Piscataway, NJ,
USA) as stationary phase. TLC was conducted on silica gel GF254
plates (Merck) and reversed-phase C18 silica gel plates (Merck).
6 were evaluated for their
lowing preliminary screening, the EtOAc fraction showed very
strong inhibitory effects against -glucosidase, leading to the iso-
a-glucosidase inhibitory activity. Fol-
a
lation of compounds 5–8, whereas four new compounds 1–4
were obtained from the less potent n-BuOH fraction. Compounds
1–8 and 6a were further studied for their concentration
dependent activity in order to calculate IC50 values (Table 4).
Compounds 5 and 6, the major components in the leaves of A.
sinensis, showed very strong inhibition with IC50 values of
126.5 17.8 and 143.7 10.6 lg/mL, respectively, when compared
Table 4
Inhibitory effects of the fractions, compounds 1–8, 6a, and acarbose against
a
-glucosidase.
Samples
IC50 valuesa
g/mL
p-Nitrophenyl-
a
-
D
-glucopyranoside (PNPG), p-nitrophenol (PNP),
-glucose, -rhamnose, -glucuronic
l
lM
a-glucosidase, and authentic
D
L
D
Total extract
Petroleum ether fraction
EtOAc fraction
n-BuOH fraction
H2O-soluble fraction
1
2
3
4
5
6
7
8
6a
1056.0 28.6
1046.0 42.1
366.0 45.1
990.1 59.1
993.2 68.2
151.6 22.1
312.3 22.5
142.9 13.3
276.7 56.1
126.5 17.8
143.7 10.6
165.1 11.3
273.6 14.5
138.3 7.3
acid were obtained from Sigma Chemical Co. (St. Louis, Mo, USA).
Bovine serum albumin (BSA) and acarbose were ordered from
Amersco Inc. (USA) and Bayer Healthcare Company Ltd. (Germany),
respectively. All other chemicals were analytical-grade commercial
preparations.
273.7 39.8
634.7 45.7
298.9 27.9
678.1 137.4
299.7 42.3
366.7 27.0
404.7 27.7
454.4 24.0
562.1 29.6
576.2 58.5
4.2. Plant material
Plant material was collected from ‘‘The National GAP Base of
Chinese Materia Medica for Aquilaria sinensis (Lour.) Gilg’’ at
Dianbai County, Guangdong Province of China in July 2005 and
identified by Prof. Xiu-Wei Yang as the leaves of A. sinensi. A
voucher specimen (No. 20050720) has been deposited in the State
Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, China.
acarbose
372.0 37.8
a
IC50: concentration of inhibitor to inhibit 50% of its activity, which were
obtained by interpolation of concentration–inhibition curves. All values were
expressed as means standard deviation (n = 3).

246
J. Feng et al. / Phytochemistry 72 (2011) 242–247
4.3. Exaction and isolation
TOFMS m/z 983 [2MꢀH]ꢀ, 491 [MꢀH]ꢀ; positive HRESIMS m/z
493.0964 [M+H]+ (calcd. for C22H21O13, 493.0976).
Dried leaves of A. sinensis (10 kg) were powdered and extracted
with EtOH–H2O (7:3, v/v) (100 l ꢁ 3 times, 3 h for the first time,
and 2 h for the 2nd and 3rd times) under conditions of reflux.
The combined EtOH–H2O solubles were evaporated under reduced
pressure to afford a extract (2 kg, yield 20%, w/w). This was dis-
solved in H2O (6 l) and partitioned successively with petroleum
ether (18 l ꢁ 5 times), EtOAc (18 l ꢁ 5 times) and n-BuOH (18 l ꢁ
5 times) to yield the corresponding extracts of (150 g, 1.5%, w/w),
(80 g, 0.8%, w/w), (165 g, 1.65%, w/w), and the residual H2O layer
of (705 g, 7.05%, w/w). All extracts were screened for inhibitory
4.3.3. Hypolaetin 5-O-b- -glucuronopyranoside (3)
D
Yellowish powder; ½a D22
ꢀ 53:5 (c 0.69, MeOH); IR (KBr) mmax
ꢃ
3385, 1725, 1656, 1606, 1510, 1437, 1357, 1260, 1055, 1073,
1007, 945, 840, 715 cmꢀ1
; UV (MeOH) kmax 224, 259, 271,
355 nm; For 1H and 13C NMR spectroscopic data, see Table 2; posi-
tive ESITOFMS m/z 501 [M+Na]+, 479 [M+H]+, 325 [Mꢀ176+Na]+,
303 [Mꢀ176+H]+; negative ESITOFMS m/z 955 [2MꢀH]ꢀ, 477
[MꢀH]ꢀ, 301 [Mꢀ176ꢀH]ꢀ; positive HRESIMS [M+H]+ m/z
479.0822 (calcd. for C21H19O13, 479.0820).
activity against
a-glucosidase according to a slightly modified
method described in previous papers (Krakenaite and Glemzha,
1983; Cremonesi et al., 2003; Li et al., 2005). As a result, only the
EtOAc extract showed strong activity relative to the reference com-
pound, acarbose.
4.3.4. Aquilarixanthone (2-C-b-
D-xylopyranosyl-1,3,4,6,7-
pentahydroxyxanthone) (4)
Yellowish amorphous powder; ½a D22
ꢀ 10:3 (c 0.80, MeOH); IR
ꢃ
(KBr) mmax 3394–3380, 1647, 1621, 1587, 1478, 1430, 1357, 1291,
1173, 1077, 1036, 878, and 826 cmꢀ1; UV (MeOH) kmax 241, 261,
317, 368 nm; For 1H and 13C NMR spectroscopic data, see Table 3;
positive ESITOFMS m/z 855 [2M+K]+, 431 [M+Na]+, 331, 313, 301;
The active EtOAc extract (70 g) was separated by silica gel CC
and eluted with CHCl3–MeOH mixtures of increasing polarity. A to-
tal of seven fractions (Fr.1: 1.1 g; Fr.2: 10.1 g; Fr.3: 1.7 g; Fr.4:
3.3 g; Fr.5: 4.5 g; Fr.6: 6.1 g, and Fr.7: 45 g) were collected and
combined on the basis of TLC analysis. Fr. 7 (45 g) was fractionated
using polyamide CC eluted with H2O:MeOH gradient mixtures
(55:45 ? 0:100) to give four fractions (ca. 250 ml each) as fr.
7–1 ꢂ fr. 7–4. Fr. 7–1 was successively purified by CC on Sephadex
LH-20 (MeOH–H2O = 7:3, v/v) and RP-18 (MeOH–H2O = 45:55, v/v)
to afford compounds 5 (1.0 g), 6 (4.0 g), 7 (50.0 mg), and 8
(20.0 mg), respectively.
The n-BuOH extract (155 g) was dissolved in H2O (465 l) and
kept overnight at room temperature (for at least 8 h). The resulting
precipitate was collected by filtration, and compound 5 (22.2 g) was
obtained from it after repeated recrystallization with mixture of
MeOH and CHCl3. The filtrate was subjected to D-101 macroreticu-
lar resin CC eluted successively with H2O and EtOH–H2O (15:85,
30:70, 40:60, and 95:5, v/v) to give five fractions: B1 (43.5 g), B2
(30.8 g), B3 (30.0 g), B4 (33.0 g), and B5 (5.8 g), respectively. B3
(33.0 g) was fractionated by silica gel CC eluted with CHCl3–MeOH
gradient mixtures (9:1 ? 6:1) to give five fractions (ca. 250 ml
each) as fr. B3–1 – fr. B3–5. Fr. B3–2 was successively purified by
CC on Sephadex LH-20 (MeOH:H2O = 70:30, v/v) and RP-18
(MeOH:H2O = 45:55, v/v), and preparation TLC (CHCl3:MeOH:-
H2O:HCOOH = 8:2:0.1:0.1, v/v/v/v), respectively, to provide com-
positive HRESIMS m/z 431.0592 [M+Na]+ (calcd. for C18H16NaO11
,
431.0590).
4.3.5. Acid hydrolysis of 6
Compound 6 (200 mg) was hydrolyzed in 3 M HCl/MeOH
(40 mL) at 70 °C for 3 h. After being neutralized with 4 M NaOH/
H2O and extracted with EtOAc (400 ml), the EtOAc solubles were
evaporated in vacuo to give a residue. The latter was further puri-
fied by preparative silica gel TLC (CH2Cl2–EtOAc = 7:3, v/v) to yield
iriflophenone (6a) (156 mg). Yellowish amorphous powder; 1H
NMR spectroscopic data (300 MHz, DMSO-d6) d 9.80 (4H, br s,
OH ꢁ 4), 5.81 (2H, s, H-3, H-5), 7.53 (2H, d, J = 8.7 Hz, H-20, H-60),
6.76 (2H, d, J = 8.7 Hz, H-30, H-50); 13C NMR spectral data
(75 MHz, DMSO-d6), see Table 1; EIMS m/z 246 [M]+.
4.3.6. Acid hydrolysis of 1, 2 and 3
Compound 1 (1 mg) was dissolved in 10% H2SO4 (1 ml) and
heated at 95 °C for 1 h. After cooling to room temperature, the reac-
tion solution was neutralized with satd. NaHCO3 and extracted with
EtOAc (2 ml ꢁ 3). The combined EtOAc phase was evaporated, and
iriflophenone was identified by direct comparison with 6a on silica
gel TLC. The aqueous layer was condensed and subjected to paper
chromatography (PC) (n-BuOH–H2O–HOAc, 4:2:1, v/v) together
pound
1 (20.5 mg). Fr. B3–3 was successively purified by
Sephadex LH-20 CC (MeOH:H2O = 70:30, v/v) and RP-18 (MeOH:-
H2O = 45:55, v/v), respectively, to afford compounds 2 (8.3 mg), 3
(20.4 mg), and 4 (10.1 mg).
with authentic D-glucose and L-rhamnose. Consequently, the
hydrolysates were coincident with authentic samples. Acid hydro-
lysis of compounds 2 and 3 were performed in the same manner
as that of 1. The aqueous layer was concentrated and subjected to
PC (n-BuOH–H2O–HOAc, 4:2:1, v/v) together with authentic
4.3.1. Aquilarisinin (iriflophenone 2-O-b-
D-glucopyranosyl-(1?4)-
D-glucuronic acid, as a result, the hydrolysate was coincident with
O-a-L-rhamnopyranoside) (1)
authentic sample.
Yellowish amorphous powder; ½a D22
ꢃ
ꢀ 25:2 (c 1.02, MeOH); IR
(KBr) mmax 3383, 1725, 1626, 1597, 1509, 1458, 1383, 1322, 1280,
1170, 1073, 1032, 973, 925, and 821 cmꢀ1; UV (MeOH) kmax 224,
290 nm; For 1H and 13C NMR spectroscopic data, see Table 1;
positive ESITOFMS m/z 577 [M+Na]+, 555 [M+H]+, 247 [Mꢀ162–
146+H]+; negative ESITOFMS m/z553 [MꢀH]ꢀ, 245[Mꢀ162ꢀ146ꢀH]ꢀ;
4.4. Biological assays
Inhibitory
photometrically in a 96-well microtiter plate (Corning Costar,
Cambridge, MA, USA) based on p-nitrophenyl- -glucopyranoside
a-glucosidase activities were determined spectro-
a-D
positive HRESIMS m/z 555.1711 [M+H]+ (calcd. for C25H31O14
,
(PNPG) as a substrate following the slightly modified method de-
scribed in the papers (Krakenaite and Glemzha, 1983; Cremonesi
555.1708); 577.1516 (calcd. for C25H30NaO14, 577.1528).
et al., 2003; Li et al., 2005). In brief, 20
[0.8 U/ml -glucosidase in 0.01 M potassium phosphate buffer
(pH 6.8) containing 0.2% of BSA] and 120 l of the test compound
or the extract in 0.5% DMSO of 0.01 M potassium phosphate buffer
were mixed, and was preincubated at 37 °C prior to initiation of the
reaction by adding the substrate. After 15 min of preincubation,
ll of enzyme solution
4.3.2. Aquilarisin (5-methoxy-6-hydroxy-isoscutellarein 8-O-b-
a
D
-glucuronopyranoside) (2)
l
Yellowish amorphous powder; ½a D22
ꢃ
ꢀ 62:1 (c 0.65, MeOH); IR
(KBr) mmax 3381, 2926, 1726, 1656, 1610, 1583, 1509, 1433, 1358,
1254, 1174, 1059, 1021, and 839 cmꢀ1; UV (MeOH) kmax 225,
373, 344 nm; For 1H and 13C NMR spectroscopic data, see Table 2;
positive ESITOFMS m/z 515 [M+Na]+, 493 [M+H]+; negative ESI-
PNPG solution (20
ll) [5.0 mM PNPG in 0.1 M potassium phos-
phate buffer (pH 6.8)] was added and then incubated together at

J. Feng et al. / Phytochemistry 72 (2011) 242–247
Kim, K.Y., Nama, K.A., Kurihara, H., Kim, S.M., 2008. Potent
247
a
-glucosidase inhibitors
37 °C. After 15 min of incubation, 0.2 M Na2CO3 (80 ll) in 0.1 M
purified from the red alga Grateloupia elliptica. Phytochemistry 69, 2820–2825.
Krakenaite, R.P., Glemzha, A.A., 1983. Some properties of two forms of alpha-
glucosidase from Saccharomyces cerevisiae-II. Biokhimiia 48, 62–68.
potassium phosphate buffer was added to the test tube to stop
the reaction. The amount of PNP released was quantified using a
UVmax Kinetic Microplate Reader (Molecular Dynamics, Inc., Sun-
nyvale, California, CA, USA) at 405 nm.
Lee, S.S., Lin, H.C., Chen, C.K., 2008. Acylated flavonol monorhamnosides,
a
-glucosidase inhibitors, from Machilus philippinensis. Phytochemistry 69,
2347–2353.
Li, T., Zhang, X.D., Song, Y.W., Liu, J.W., 2005. A microplate-based screening method
for alpha-glucosidase inhibitors. Chin. J. Clin. Pharmacol. Ther. 10, 1128–1134.
Liu, J., Wu, J., Zhao, Y.X., Deng, Y.Y., Mei, W.L., Dai, H.F., 2008. A new cytotoxic 2-(2-
phenylethyl)chromone from Chinese eaglewood. Chin. Chem. Lett. 19, 934–936.
Acknowledgements
This research was partly supported by National High Technology
Research and Development Program of China (2006BAI 06A01-02).
We thank Prof. Norman G. Lewis of Institute of Biological Chemis-
try, Washington State University for editing the manuscript.
Matsui, T., Ueda, T., Oki, T., Sugita, K., Terhara, N., Matsumoto, K., 2001.
a-
Glucosidase inhibitory action of natural acylated anthocyanins: 1. Survey of
natural pigments with potent inhibitory activity. J. Agric. Food Chem. 49, 1948–
1951.
Mishra, B., Indira Priyadarshini, K., Sudheerkumar, M., Unnikrishhnan, M.K., Mohan,
H., 2006. Pulse radiolysis studies of mangiferin: a C-glycosyl xanthone isolated
from Mangifera indica. Radiat. Phys. Chem. 75, 70–77.
Murakami, T., Tanaka, N., Wada, H., Saiki, Y., Chen, C.M., 1986. Chemical and
chemotaxonomical studies on Filices LXIII. Xanthone derivatives of
Hypodematium fauriei Tagawa, H. crenatum Kuhn and Gymnocarpium robertianum
Newm. (G. jessoense Koidz.). Yakugaku Zasshi 106, 378–382.
Nakanishi, T., Yamagata, E., Yoneda, K., Nagashima, T., Kawasaki, I., Yoshida, T., Mori,
H., Miura, I., 1984. Three fragrant sesquiterpenes of agarwood. Phytochemistry
23, 2066–2067.
Ponce, M.A., Scervino, J.M., Erra-Balsells, R., Ocampo, J.A., Godeas, A.M., 2004.
Flavonoids from shoots and roots of Trifolium repens (white clover) grown in
presence or absence of the arbuscular mycorrhizal fungus Glomus intraradices.
Phytochemistry 65, 1925–1930.
References
Agrawal, P.K., 1989. Carbon-13 NMR of Flavonoids. Elsevier, Amsterdam.
Agrawal, P.K., 1992. NMR spectroscopy in the structural elucidation of
oligosaccharides and glycosides. Phytochemistry 31, 3307–3330.
Bilia, A.R., Ciampi, L., Mendez, J., Morelli, I., 1996. Phytochemical investigations of
Licania genus. Flavonoids from Licania pyrifolia. Pharm. Acta Helv. 71, 199–204.
Billeter, M., Meier, B., Sticher, O., 1991. 8-Hydroxyflavonoid glucuronides from
Malva sylvestris. Phytochemistry 30, 987–990.
Breitmayer, E., Voelter, W., 1989. Carbon-13 NMR Spectroscopy. VCH, Weinheim.
Catalano, S., Luschi, S., Flamini, G., Cioni, P.L., Nieri, E.M., Morelli, I., 1996. A
xanthone from Senecio mikanioides leaves. Phytochemistry 42, 1605–1607.
Cremonesi, F., Torti, E., Pecile, A., Groppetti, D., Biondi, P., 2003. Evaluation of alpha-
glucosidase activity in dog semen and its use in fertility diagnosis. Vet. Res.
Commun. 27, 587–589.
Qi, J., Lu, J.J., Liu, J.H., Yu, B.Y., 2009. Flavonoid and a rare benzophenone glycoside
from the leaves of Aquilaria sinensis. Chem. Pharm. Bull. 57, 134–137.
Rancon, S., Chaboud, A., Darbour, N., Comte, G., Barron, D., Raynaud, J., Cabalion, P.,
1999. A new C-glycosyl xanthone isolated from Davallia solida. Phytochemistry
52, 1677–1679.
Ding, S., Dudley, E., Plummer, S., Tang, J., Newton, R.P., Brenton, A.G., 2008.
Fingerprint profile of Ginkgo biloba nutritional supplements by LC/ESI–MS/MS.
Phytochemistry 69, 1555–1564.
Wansi, J.D., Lallemand, M.C., Chiozem, D.D., Toze, F.A.A., Mbaze, L.M., Naharkhan, S.,
Iqbal, M.C., Tillequin, F., Wandji, J., Fomum, Z.T., 2007. a-Glucosidase inhibitory
constituents from stem bark of Terminalia superba (Combretaceae).
Phytochemistry 68, 2096–2100.
Feng, J., Yang, X.W., Liu, T.H., 2009. RP-LC Quantification and pharmacokinetic study
of iriflophenone 2-O-a-rhamnopyranoside in rat plasma. Chromatographia 70,
1227–1231.
Gómez-Zaleta, B., Ramírez-Silva, M.T., Gutiérrez, A., González-Vergara, E., Güizado-
Rodríguez, M., Rojas-Hernández, A., 2006. UV/vis, 1H, and 13C NMR
spectroscopic studies to determine mangiferin pKa values. Spectrochim. Acta
A 64, 1002–1009.
Yagura, T., Shibayama, N., Ito, M., Kiuchi, F., Honda, G., 2005. Three novel diepoxy
tetrahydrochromones from agarwood artificially produced by intentional
wounding. Tetrahedron Lett. 46, 4395–4398.
Yang, H., Protiva, P., Cui, B.L., Ma, C.Y., Baggett, S., Hequet, V., Mori, S., Weinstein, I.B.,
Kennelly, E.J., 2003. New bioactive polyphenols from Theobroma grandiflorum
(‘‘Cupuacu’’). J. Nat. Prod. 66, 1501–1504.
Zhou, M.H., Wang, H.G., Suolangjiba, Kou, J.P., Yu, B.Y., 2008. Antinociceptive and
anti-inflammatory activities of Aquilaria sinensis (Lour.) Gilg. Leaves extract. J.
Ethnopharmacol. 117, 345–350.
Hiroyuki, F., Tomohide, Y., Kazunori, O., 2001. Efficacy and safety of Touchi extract,
an
a-glucosidase inhibitor derived from fermented soybeans, in non-insulin-
dependent diabetic mellitus. J. Nutr. Biochem. 12, 351–356.
Iinuma, M., Hara, H., Oyama, M., 2007. Laxative and food containing the same. PCT
Int. Appl. WO2007083594, A1 20070726, p. 29.
Ishihara, M., Tsuneya, T., Uneyama, K., 1991. Guaiane sesquiterpenes from
agarwood. Phytochemistry 30, 3343–3347.