Phenolic constituents from Brainea insignis

Article abstract of DOI:10.1248/cpb.58.868

Phytochemical investigation on the methanol extract of Brainea insignis led to the isolation of 16 phenolic compounds, including an unusual flavanol coupled with phenylpropyl and shikimic acid units, brainicin (1), and a new flavonol acylglycosides brainoside B (2). The structures of new compounds were elucidated on the basis of extensive spectroscopic analysis. Compounds 1-5 were evaluated for their cytotoxic effects. However, none of the compounds showed significant cytotoxic activity against the HL-60, A549, SMMC-7721, MCF-7, SW480 cell lines (IC₅₀>40 μM).

Full text of DOI:10.1248/cpb.58.868

8
68
Note
Chem. Pharm. Bull. 58(6) 868—871 (2010)
Vol. 58, No. 6
Phenolic Constituents from Brainea insignis
a,b
a,b
a,b
a
a
a
Kou WANG, Ming-Ming LI, Xuan-Qin CHEN, Li-Yan PENG, Xiao CHENG, Yan LI, and
,a
Qin-Shi ZHAO*
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese
b
Academy of Sciences; Kunming 650204, People’s Republic of China: and Graduate School of Chinese Academy of
Sciences; Beijing 100049, People’s Republic of China.
Received February 27, 2010; accepted March 23, 2010; published online March 31, 2010
Phytochemical investigation on the methanol extract of Brainea insignis led to the isolation of 16 phenolic
compounds, including an unusual flavanol coupled with phenylpropyl and shikimic acid units, brainicin (1), and
a new flavonol acylglycosides brainoside B (2). The structures of new compounds were elucidated on the basis of
extensive spectroscopic analysis. Compounds 1—5 were evaluated for their cytotoxic effects. However, none of
the compounds showed significant cytotoxic activity against the HL-60, A549, SMMC-7721, MCF-7, SW480 cell
lines (IC ꢅ40 mM).
5
0
Key words Brainea insignis; flavonol; brainicin; brainoside B; brainoside C
As the only species of the genus Brainea (Blechnaceae),
d
6.67 (overlapped, H-2ꢄ), 4.25 (s, br, H-3ꢄ), 3.75 (dd,
H
Brainea insignis (HOOK.) J. SMITH a kind of fern is widely Jꢁ7.0, 4.0 Hz, H-4ꢄ), 4.79 (dt, Jꢁ7.0, 5.0 Hz, H-5ꢄ), 1.71
distributed in south China. It is used as herbal medicine to (d, br, Jꢁ18.6 Hz, H-6ꢄa), and 2.40 (dt, br, Jꢁ18.6, 2.4 Hz,
treat cold, burn, bleeding wound, and ascariasis, etc., as well H-6ꢄb) were suggested to be a shikimic acid moiety. The
1
3
as occasionally planted for ornament because of its beautiful
C-NMR spectrum showed 31 carbon signals and supported
1,2)
shape. Previous chemical investigations of B. insignis have the above assignments (Table 1). In combination with the
been shown the presence of lignans, flavonoids, pyrone observed heteronuclear multiple bond connectivity (HMBC)
glucosides, stilbene, steroids, and a series of volatile correlations (Fig. 1) from H-2 to C-1ꢂ, C-2ꢂ, and C-6ꢂ, from
compounds. As a part of our search for naturally occurring H-4 to C-4a and C-5, from H-7ꢃ to C-1ꢃ, C-2ꢃ, and C-6ꢃ,
3)
4)
5)
5)
5)
6)
7—9)
bioactive metabolites from ferns of China,
we investi- from H-8ꢃ to C-9ꢃ, and from H-2ꢄ to C-7ꢄ, 1 was suspected
gated the chemical constituents of B. insignis, which led to to be composed of a flavan-3-ol unit, a phenylpropyl unit,
the isolation of 16 phenolic compounds, including an un- and shikimic acid unit and to be formed by coupling of these
usual flavanol coupled with phenylpropyl and shikimic acid units. The HMBC correlations of H-5ꢄ to C-9ꢃ, revealed the
units, brainicin (1), and a new flavonol acylglycosides braino
-
two oxygenated carbons C-5ꢄꢃ and C-9ꢃ were connected with
side B (2). Herein, we describe the isolation and structure
elucidation of new compounds.
Results and Discussion
Compound 1 was isolated as a brown amorphous solid.
The molecular formula was established as C H O by a
3
1
28 14
ꢀ
quasimolecular ion peak at m/z 623.1406 [MꢀH] in the
negative HR-electrospray ionization (ESI)-MS analysis (Calcd
for 623.1400), indicating 18 degrees of unsaturation. The IR
spectrum showed absorption bands at 3426, 1710, 1627,
ꢀ1
1
606, 1527 and 1449 cm , revealing the presence of hy-
droxyl, carbonyl and aromatic rings groups, respectively. The
1
aromatic region of the H-NMR spectrum of 1 showed the
presence of three sets of aromatic protons (Table 1). One set
corresponded to a tetrasubstituted aromatic ring with two
meta-coupling protons and appeared at d 5.88 (d, Jꢁ2.0 Hz,
H
H-6) and 5.98 (d, Jꢁ2.0 Hz, H-8), and two sets corresponded
to 3,4-dihydroxyphenyl groups at d 6.82 (d, Jꢁ1.5 Hz, H-
H
2
ꢂ), 6.71 (d, Jꢁ7.5 Hz, H-5ꢂ), and 6.67 (dd, Jꢁ7.5, 1.5 Hz,
H-6ꢂ), and at d 6.77 (d, Jꢁ2.0 Hz, H-2ꢃ), 6.71 (d, Jꢁ8.0 Hz,
H
H-5ꢃ), and 6.64 (dd, Jꢁ8.0, 2.0 Hz, H-6ꢃ), respectively. Reso-
nances due to a coupling system at d 5.14 (d, Jꢁ5.0 Hz, H-
H
2
2
), 3.97 (dd, Jꢁ10.7, 5.0 Hz, H-3), 3.05 (t, Jꢁ10.8 Hz, H-4),
.61 (t, Jꢁ10.6 Hz, H-8ꢃ), and 4.81 (d, Jꢁ10.0 Hz, H-7ꢃ)
suggested the presence of a –CH(O)–CH(O)–CH–CH–CH(O)–
1
1
unit. This unit was confirmed by the H– H correlation spec-
troscopy (COSY) experiment from the coupling of H-2/H-3,
H-3/H-4, H-4/H-8ꢃ, and H-8ꢃ/H-7ꢃ. The additional signals at
Chart 1.
∗
To whom correspondence should be addressed. e-mail: qinshizhao@mail.kib.ac.cn
© 2010 Pharmaceutical Society of Japan

June 2010
869
1
13
Table 1. H-NMR (500 MHz) and C-NMR (100 MHz) Data of 1—3 in CD OD
3
1
2
3
No.
No.
dH
dC
dH
dC
dH
dC
2
3
4
5.14 (d, 5.0)
3.97 (dd, 10.7, 5.0)
3.05 (t, 10.8)
80.2 d
71.3 d
36.9 d
98.1 s
155.9 s
96.1 d
2
3
4
4a
5
6
7
8
8a
1ꢂ
2ꢂ
3ꢂ
4ꢂ
5ꢂ
6ꢂ
1ꢃ
2ꢃ
3ꢃ
4ꢃ
5ꢃ
6ꢃ
1ꢄ
2ꢄ
3ꢄ
4ꢄ
5ꢄ
6ꢄ
158.2 s
135.1 s
179.8 s
105.7 s
162.9 s
99.8 d
165.6 s
94.7 d
158.2 s
122.9 s
117.4 d
145.9 s
149.7 s
115.0 d
123.7 d
100.9 d
84.4 d
77.7 d
70.9 d
77.7 d
62.2 t
158.2 s
135.0 s
180.1 s
105.6 s
162.9 s
99.9 d
165.6 s
94.8 d
158.4 s
122.2 s
132.7 d
116.3 d
161.6 s
116.3 d
132.7 d
101.6 d
82.7 d
76.6 d
70.1 d
76.1 d
61.7 t
4
5
6
7
8
8
1
2
3
4
5
6
1
2
3
4
5
6
7
8
9
1
2
3
a
5.88 (d, 2.0)
5.98 (d, 2.0)
6.17 (br s)
6.31 (br s)
6.13 (br s)
159.4 s
96.1 d
6.19 (br s)
a
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢄ
ꢄ
ꢄ
155.7 s
130.9 s
115.5 d
145.9 s
145.7 s
116.0 d
120.3 d
130.7 s
115.6 d
147.0 s
146.2 s
115.8 d
120.5 d
81.2 d
6.82 (d, 1.5)
7.69 (d, 2.0)
6.91 (d, 8.4)
7.66 (dd, 8.4, 2.0)
5.42 (d, 7.5)
3.76 (t, 8.8)
3.51 (m)
3.45 (m)
3.64 (m)
3.64 (m), 3.53 (m)
4.82 (d, 7.7)
3.44 (overlapped)
3.22 (m)
3.43 (overlapped)
3.70 (m)
8.09 (d, 8.5)
6.87 (d, 8.5)
6.71 (d, 7.5)
6.67 (dd, 7.5, 1.5)
6.87 (d, 8.5)
8.09 (d, 8.5)
5.12 (d, 7.5)
4.01 (t, 8.5)
3.48 (overlapped)
3.38 (m)
3.57 (m)
3.80 (m), 3.65 (m)
4.74 (d, 7.4)
3.43 (overlapped)
3.13 (m)
3.40 (m)
3.65 (overlapped)
4.44 (dd, 1.5, 11.8)
4.35 (dd, 6.3, 11.8)
6.77 (d, 2.0)
6.71 (d, 8.0)
6.64 (dd, 8.0, 2.0)
4.81 (d, 10.0)
2.61 (t, 10.6)
105.8 d
76.0 d
78.2 d
71.8 d
75.7 d
64.6 t
106.2 d
75.5 d
77.7 d
71.8 d
74.8 d
64.6 t
50.4 d
173.9 s
130.4 s
138.4 d
67.0 d
6.67 (overlapped)
4.25 (br s)
4.44 (dd, 2.0, 12.0)
4
.35 (dd, 6.0, 12.0)
4ꢄ
5ꢄ
6ꢄ
3.75 (dd, 7.0, 4.0)
4.79 (dt, 7.0, 5.0)
1.71 (br d, 18.6)
69.1 d
71.8 d
28.0 t
1ꢃꢃ
2ꢃꢃ
3ꢃꢃ
127.4 s
116.3 d
146.5 s
126.7 s
130.8 d
116.6 d
6.90 (d. 1.6)
7.08 (d, 8.3)
6.63 (d, 8.3)
2
.40 (br dt, 18.6, 2.4)
7
ꢄ
170.0 s
4ꢃꢃ
149.3 s
116.3 d
122.8 d
147.0 d
114.5 d
169.1 s
161.0 s
116.6 d
130.8 d
146.5 d
114.5 d
169.0 s
5
6
7
8
9
ꢃꢃ
ꢃꢃ
ꢃꢃ
ꢃꢃ
ꢃꢃ
6.68 (d, 8.1)
6.63 (d, 8.3)
7.08 (d, 8.3)
7.33 (d, 15.9)
6.01 (d, 15.9)
6.72 (dd, 1.6, 8.1)
7.38 (d, 15.9)
6.07 (d, 15.9)
d in ppm, J in Hz.
1
feature of the H-2 resonance in the H-NMR spectrum: d
H
1
0,11)
5
.14 (d, Jꢁ5.0 Hz).
And the C-2 aryl substituent was
suggested to be the equatorial orientation according to the
thermodynamically favoured conformation existing in natu-
1
2,13)
ral flavanones.
The 2,3-cis configuration was determined
by a small H-2/H-3 coupling constant of 5.0 Hz. The spatial
relationships were deduced from the nuclear Overhauser
enhancement spectroscopy (NOESY) spectra. The correla-
tions observed for H-4/H-2ꢂ (-6ꢂ), H-3/H-8ꢃ, H-8ꢃ/H-2ꢃ (-6ꢃ),
and H-4/H-7ꢃ indicated that H-2, H-3, and H-8 were cofacial,
while H-4 and H-7ꢃ were on opposite sides of the molecule.
The large coupling constants J ꢁ10.7 Hz, J4,8ꢃꢁ10.6 Hz,
Fig. 1. Key COSY, HMBC (from H to C) and ROESY Correlations of 1
3,4
and J7ꢃ,8ꢃꢁ10.0 Hz were also in agreement with the trans-ori-
entations of H-3/H-4, H-4/H-8ꢃ, and H-8ꢃ/H-7ꢃ. On the basis
a 5ꢄ,9ꢃ-ester linkage. Moreover, NMR experiments were of the above discussion and referring to published epicate-
1
0—12,14)
reperformed in DMSO-d , and crucial HMBC correlation chin derivatives,
the structure of 1 was finally as-
6
from the hydrogen of 3-OH to C-3 and C-4 were observed, signed and named brainicin.
which suggested that an ether linkage should be be-
Compound 2 was obtained as a yellow gummy solid, and
tween C-7ꢃ and C-5 but not between C-7ꢃ and C-3. Therefore, assigned the molecular formula C H O by the negative
3
6
36 20
ꢀ
the structure of 1 was confirmed as shown in Fig. 1.
HR-ESI-MS analysis ([MꢀH] at m/z 787.1706, Calcd for
The relative configuration of C-2 and C-3 in 1 was con- 787.1721). The UV and IR spectra of 2 showed absorption
cluded to be of the epicatechin type from the characteristic bands at 210, 268, 303 and 374 nm, and 3427 br, 1689, 1649,

8
70
Vol. 58, No. 6
ꢀ1
1
630, 1608, 1501, and 1444 cm , respectively, which are
indicative of the presence of aromatic rings, carbonyl and
hydroxyl groups.
The anomeric signals at d 100.9 (d) and 105.8 (d), as well
C
1
3
as the other characteristic signals of the C-NMR and distor-
tionless enhancement by polarization transfer (DEPT) spec-
tra (Table 1), displayed the presence of two glycopyranosyl
units. Besides, the spectra showed 15 carbon signals for 3-O-
15)
glycosylated quercetin and 9 carbon signals for a caffeoyl
16)
1
moiety. They were further supported by H-NMR spec-
Fig. 2. Key HMBC (from H to C) Correlations of 2
acquired with a Bruker AV-400 and DRX-500 spectrometers in CD OD at
trum: two broad singlets at d 6.17 and 6.31, typical of the
H
H-6 and H-8 protons of ring A of a flavonoid unit, one set
3
aromatic ABX system protons corresponded to 3,4- room temperature (d in ppm, J in Hz). ESI-MS (including HR-ESI-MS) and
FAB-MS were carried out on API QSTAR Pulsar i and VG Autospec-3000
mass spectrometers, respectively. Silica gel (200—300 mesh), Silica gel H
dihydroxyphenyl group of ring B [d 7.69 (d, Jꢁ2.0 Hz, H-
H
2
ꢂ), 7.66 (dd, Jꢁ2.0, 8.4 Hz, H-6ꢂ), and 6.91 (d, Jꢁ8.4 Hz,
(Qingdao Marine Chemical Ltd., China), LiChroprep RP-18 silica gel (40—
H-5ꢂ)], and a set protons of the caffeoyl moiety at d 7.38 (d,
H
63 mm, Merck, Dramstadt, Germany), and Sephadex LH-20 (Amersham
Pharmacia biotech, Sweden) were used for column chromatography. Prepar-
Jꢁ15.9 Hz, H-7ꢃꢃ), 6.90 (d, Jꢁ1.6 Hz, H-2ꢃꢃ) , 6.72 (dd, Jꢁ
1
.6, 8.1 Hz, H-6ꢃꢃ), 6.68 (d, Jꢁ8.1 Hz, H-5ꢃꢃ), and 6.07 (d, ative and semipreparative HPLC were performed on Shimadzu LC-8A
preparative liquid chromatograph with a Shimadzu PRC-ODS (K) column
and Agilent 1100 liquid chromatograph with a Zorbax SB-C18 (9.4 mmꢆ25
cm) column, respectively. Fractions were monitored by TLC and spots were
visualized by heating silica gel plates immersed with 15% H SO in ethanol.
Jꢁ15.9 Hz, H-8ꢃꢃ). In addition, the double bond was sug-
gested as trans-due to the coupling constant. Acidic hydroly-
sis of 2 gave D-glucose as the sole sugar moiety, which was
determined to have a b configuration on the basis of the large Solvents were distilled prior to use.
2
4
coupling constants of the anomeric protons [d 5.42 (Jꢁ7.5
Plant Material The aerial parts of B. insignis were collected in Jinping
County, Yunnan Province, PRC, in July 2007 and identified by professor
Xiao Cheng of Kunming Institute of Botany, Chinese Academy of Sciences.
A voucher specimen (No. 200707F03) has been deposited in the State Key
Laboratory of Phytochemistry and Plant Resources in West China, Kunming
H
Hz) and 4.82 (Jꢁ7.7 Hz)]. Assignment of glucosidic protons
system was achieved by analysis of H– H COSY and het-
eronuclear single quantum coherence (HSQC) experiments.
1
1
Connectivities of glucosyl, quercetin, and caffeoyl moi- Institute of Botany, Chinese Academy of Sciences.
eties were confirmed by the HMBC experiments, in which
Extraction and Isolation The dried and powdered plant materials
4.5 kg) were extracted with methanol (15.0 lꢆ3, each 2 d) at room tempera-
ture. After evaporation of the solvent in vacuo, the concentrate was sus-
(
correlations of H-1ꢃ (d 5.42) with C-3 (d 135.1), H-1ꢄ (d
H
C
H
4
.82) with C-2ꢃ (d 84.4), and H-6ꢄ (d 4.44, 4.35) with Cꢁ
C
H
pended into H O and partitioned successively with petroleum ether (PE) and
2
O (d 169.1) were observed. On the basis of all these results,
C
ethyl acetate (EtOAc). The EtOAc extract (150 g) was subjected to column
the structure of compound 2 was established as quercetin chromotography (CC) over silica gel (100—200 mesh) and eluted with gra-
dient PE–Me CO (1 : 0 to 0 : 1) to give five fractions 1—5. Fraction 2 was
3
-O-b-D-glucopyranosyl (1→2)-[(6ꢄ-O-trans-caffeoyl)-b-D-
2
further subjected to column chromatograph over silica gel to obtain three
subfractions 2.1—2.3. Subfraction 2.3 was further purified by silica gel CC
and Sephadex LH-20 (MeOH) to yield 6 (5 g) and 7 (10 mg). Fraction 3 was
glucopyranoside], named brainoside B (Fig. 2).
Compound 3 was initially determinated from immature
leaves of Brassica rapa L. ssp. chinensis L. (HANELT.) by
eluted with CHCl –MeOH (20 : 1 to 8 : 2) over silica gel CC to afford four
3
ROCHFORT and co-workers, but its spectroscopic data were subfractions 3.1—3.4. Subfraction 3.2 and 3.3 were separated repeatedly on
1
7)
silica gel and RP-18 (MeOH–H O gradient elution of increasing concentra-
lacking. In this paper, compound 3 was isolated as a yellow
2
1
13
tion) to obtain 5 (22 mg), 8 (7 mg), and 9 (116 mg). Fraction 4 was subjected
to silica gel CC eluting with EtOAc–MeOH (9 : 1 to 6 : 4) to yield five sub-
fractions 4.1—4.5. Subfractions 4.1 and 4.2 were chromatographed on silica
gummy solid and named brainoside C. The H- and C-
NMR data of 3 were showed in Table 1.
The structures of the known compounds 4—16 isolated gel and finally purified by semipreparative HPLC using MeOH–H O (30 :
were identified as camsibriside A, 3,3ꢂ,4-tri-O-methyl-4ꢂ- 70) and MeOH–H
O-rutinosylellagic acid,
vanillic acid, shikimic acid, icariside E , icariside E ,
brainic acid, 7-epiblechnic acid, (Z)-rosmarinic acid, ros-
marinic acid,
2
18)
O (32 : 68, plus 0.5% formic acid) as eluents respectively,
2
19)
20)
21)
25)
to afford 10 (7 mg), 11 (7 mg), 13 (3.5 mg), 14 (2.7 mg), and 15 (21 mg).
Subfraction 4.4 was further separated by silica gel CC to give 4 (2 g). Subse-
hyperin,
pectolinarigenin,
22)
23)
24)
3
5
quently, preparative HPLC (MeOH–H O, 30 : 70, plus 0.5% formic acid for
3)
3)
26)
2
subfraction 4.3 and MeOH–H O, 33 : 67, plus 0.5% formic acid for subfrac-
2
2
7,28)
29)
and 5-O-caffeoylshikimic acid, respec- tion 4.5) was used to purify 1 (21 mg), 12 (155 mg), 16 (11 mg), 2 (14 mg),
tively, by comparison of their spectroscopic data with litera- and 3 (35 mg) from subfractions 4.3 and 4.5.
23.9
Brainicin (1): Brown amorphous powder. mp 207—209 °C. [a]D ꢇ69.8
ture values.
(
1
1
cꢁ0.29, MeOH). UV lmax (MeOH) nm (log e): 291.8 (4.19), 216.4 (4.74),
Compounds 1—5 were evaluated for their cytotoxicity
against five human cancer cell lines using the 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (neg.) m/z: 623 [MꢀH] , 1247 [2MꢀH] . HR-ESI-MS (neg.) m/z:
method as reported previously. However, none of the com-
pounds showed significant cytotoxic activity against the HL-
ꢀ1
97.8 (4.52). IR (KBr) cm : 3426, 2925, 1710, 1627, 1606, 1527, 1449,
1
13
383, 1285, 1147, 1114, 1059. H- and C-NMR: see Table 1. ESI-MS
ꢀ
ꢀ
30)
ꢀ
6
23.1406 (Calcd for C H O , 623.1400).
31 27 14
2
5.8
Brainoside B (2): Yellow gummy solid. [a]D ꢀ67.2 (cꢁ0.12, MeOH).
UV lmax (MeOH) nm (log e): 374 (4.55), 303 (4.34), 268 (4.56), 210.0
(4.82). IR (KBr) cm : 3427, 2923, 1689, 1649, 1630, 1608, 1501, 1444,
1
7
60, A549, SMMC-7721, MCF-7, SW480 cell lines (IC ꢅ
ꢀ1
5
0
4
0 mM).
1
13
272, 1169, 1076. H- and C-NMR: see Table 1. HR-ESI-MS (neg.) m/z:
£
≠
87.1706 (Calcd for C H O , 787.1721).
3
6
35 20
2
5.8
Experimental
Brainoside C (3): Yellow gummy solid. [a]D ꢀ36.2 (cꢁ0.25, MeOH).
General Experimental Procedures Melting point was obtained on an UV lmax (MeOH) nm (log e): 315 (4.47), 268 (4.36), 206 (4.55), 196 (4.42).
ꢀ1
X-4 micro melting point apparatus. Optical rotations were measured on a IR (KBr) cm : 3427, 2921, 1688, 1655, 1606, 1512, 1444, 1360, 1278,
1
13
Horiba SEPA-300 polarimeter. IR spectra were obtained with a Tensor 27 1261, 1172, 1077. H- and C-NMR: see Table 1. HR-ESI-MS (neg.) m/z:
ꢀ
FT-IR spectrometer with KBr pellets. UV spectra were recorded on a Shi- 755.1840 (Calcd for C H O , 755.1823).
3
6
35 18
1
13
madzu UV-2401PC spectrophotometer. The H- and C-NMR spectra were
Acidic Hydrolysis of Compound 2 Compound 2 (6 mg) was hydrolyzed

June 2010
871
with 2 M HCl–dioxane (1 : 1, 4 ml) under reflux for 6 h. The reaction mixture
X., Zhao Q. S., Chem. Pharm. Bull., 57, 1123—1125 (2009).
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3
tralized with 2 M NaHCO3 then dried to give a monosaccharide mixture.
Then, a solution of the sugar mixture in pyridine (2 ml) was added to L-cys-
quet E., Dufourc E. J., de Freitas V., Laguerre M., Pianet I., Magn.
Reson. Chem., 44, 868—880 (2006).
teine methyl ester hydrochloride (about 1.5 mg) and kept at 60 °C for 1 h. 11) Cheng W., Zhu C. G., Xu W. D., Fan X. N., Yang Y. C., Li Y., Chen X.
Next, trimethylsilylimidazole (about 1.5 ml) was added to the reaction mix- G., Wang W. J., Shi J. G., J. Nat. Prod., 72, 2145—2152 (2009).
ture in ice water and kept at 60 °C for 30 min. The mixture was subjected to 12) Chokchaisiri R., Suaisom C., Sriphota S., Chindaduang A., Chuprajob
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1
80—280 °C; programmed increase, 3 °C/min; carrier gas, N2 (1 ml/min);
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Acknowledgments The work was supported financially by the NSFC-
Agric. Food Chem., 54, 4855—4860 (2006).
Joint Foundation of Yunnan Province (No. U0932602 to Q.-S. Z.) and the 18) Li N., Li X., Meng D. L., Guo Y. Q., Wang J. H., J. Asian Nat. Prod.
National Basic Research Program of China (973 Program, 2009CB522300).
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