Oleanane triterpene saponins with cardioprotective activity from Clinopodium polycephalum

Article abstract of DOI:10.1080/10286020.2016.1254199

Two new triterpene saponins, clinopodiside VI (1) and saikosaponin c (2), along with six known saikosaponins (3–8), were isolated from the plant of Clinopodium polycephalum. Compounds 1–3 showed moderate inhibition against H9c2 cell damage induced by H

Full text of DOI:10.1080/10286020.2016.1254199

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Oleanane triterpene saponins with
cardioprotective activity from Clinopodium
polycephalum
Yu-Xia Hu, Wei Zhang, Wen Zhang, Yin-Di Zhu, Guo-Xu Ma, Nai-Liang Zhu,
Wen Sun, Ze-Xin Ma, Shi-Chun Yu, Xu-Dong Xu, Kai-Tuo Chen & Jun-Shan
Yang
To cite this article: Yu-Xia Hu, Wei Zhang, Wen Zhang, Yin-Di Zhu, Guo-Xu Ma, Nai-Liang Zhu,
Wen Sun, Ze-Xin Ma, Shi-Chun Yu, Xu-Dong Xu, Kai-Tuo Chen & Jun-Shan Yang (2016): Oleanane
triterpene saponins with cardioprotective activity from Clinopodium polycephalum, Journal of
Asian Natural Products Research, DOI: 10.1080/10286020.2016.1254199
To link to this article: http://dx.doi.org/10.1080/10286020.2016.1254199
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Date: 27 November 2016, At: 21:22

Journal of asian natural Products research, 2016
ꢁꢁp://ꢂx.ꢂꢃꢄ.ꢃꢅg/10.1080/10286020.2016.1254199
ꢀ
Oleanane triterpene saponins with cardioprotective activity
from Clinopodium polycephalum
a,b1
a1
a
b
b
b
Yu-Xia Hu , Wei Zhang , Wen Zhang , Yin-Di Zhu , Guo-Xu Ma , Nai-Liang Zhu ,
c
d
a
b
a
b
Wen Sun , Ze-Xin Ma , Shi-Chun Yu , Xu-Dong Xu , Kai-Tuo Chen and Jun-Shan Yang
ꢆ
b
sꢇꢀꢃꢃꢈ ꢃꢉ Pꢀꢆꢅmꢆꢇy, aꢊꢀꢋꢄ uꢊꢄvꢌꢅꢍꢄꢁy ꢃꢉ cꢀꢄꢊꢌꢍꢌ Mꢌꢂꢄꢇꢄꢊꢌ, hꢌꢉꢌꢄ 230012, cꢀꢄꢊꢆ; Kꢌy lꢆbꢃꢅꢆꢁꢃꢅy ꢃꢉ Bꢄꢃꢆꢇꢁꢄvꢌ
sꢋbꢍꢁꢆꢊꢇꢌ ꢆꢊꢂ rꢌꢍꢃꢋꢅꢇꢌ uꢁꢄꢈꢄzꢆꢁꢄꢃꢊ ꢃꢉ cꢀꢄꢊꢌꢍꢌ hꢌꢅbꢆꢈ Mꢌꢂꢄꢇꢄꢊꢌ, Mꢄꢊꢄꢍꢁꢅy ꢃꢉ eꢂꢋꢇꢆꢁꢄꢃꢊ, iꢊꢍꢁꢄꢁꢋꢁꢌ ꢃꢉ Mꢌꢂꢄꢇꢄꢊꢆꢈ
Pꢈꢆꢊꢁ dꢌvꢌꢈꢃpmꢌꢊꢁ, Pꢌkꢄꢊg uꢊꢄꢃꢊ Mꢌꢂꢄꢇꢆꢈ cꢃꢈꢈꢌgꢌ ꢆꢊꢂ cꢀꢄꢊꢌꢍꢌ aꢇꢆꢂꢌmy ꢃꢉ Mꢌꢂꢄꢇꢆꢈ sꢇꢄꢌꢊꢇꢌꢍ, Bꢌꢄjꢄꢊg 100193,
ꢇ
cꢀꢄꢊꢆ; hꢌꢆꢈꢁꢀ-ꢇꢋꢈꢁꢄvꢆꢁꢄꢃꢊ lꢆbꢃꢅꢆꢁꢃꢅy ꢃꢉ ꢁꢀꢌ Mꢄꢊꢄꢍꢁꢅy eꢂꢋꢇꢆꢁꢄꢃꢊ, Bꢌꢄjꢄꢊg uꢊꢄvꢌꢅꢍꢄꢁy ꢃꢉ cꢀꢄꢊꢌꢍꢌ Mꢌꢂꢄꢇꢄꢊꢌ,
ꢂ
Bꢌꢄjꢄꢊg 100029, cꢀꢄꢊꢆ; tꢀꢌ Mꢋꢍꢌꢋm ꢃꢉ cꢀꢄꢊꢌꢍꢌ Mꢌꢂꢄꢇꢄꢊꢌ, Bꢌꢄjꢄꢊg uꢊꢄvꢌꢅꢍꢄꢁy ꢃꢉ cꢀꢄꢊꢌꢍꢌ Mꢌꢂꢄꢇꢄꢊꢌ, Bꢌꢄjꢄꢊg
1
00029, cꢀꢄꢊꢆ
ABSTRACT
ARTICLE HISTORY
Two new triterpene saponins, clinopodiside VI (1) and saikosaponin
c (2), along with six known saikosaponins (3–8), were isolated from
the plant of Clinopodium polycephalum. Compounds 1–3 showed
moderate inhibition against H9c2 cell damage induced by H O .
rꢌꢇꢌꢄvꢌꢂ 10 Mꢆꢅꢇꢀ 2016
aꢇꢇꢌpꢁꢌꢂ 25 oꢇꢁꢃbꢌꢅ 2016
KEYWORDS
Clinopodium polycephalum;
2
2
ꢁ
ꢅꢄꢁꢌꢅpꢌꢊꢌ ꢍꢆpꢃꢊꢄꢊꢍ;
ꢇꢆꢅꢂꢄꢃpꢅꢃꢁꢌꢇꢁꢄvꢌ ꢆꢇꢁꢄvꢄꢁy
1
. Introduction
e crude drug “Clinopodii” is the dried aerial part of Clinopodium polycephalum (Van.)

C. Y. Wu et Hsuan ex Hsuan, and is used as a folk remedy for treatment of various uterine
bleeding, hematuria, epistaxis, gingival bleeding and bleeding wounds [1]. Many phytochem-
ical researches on C. polycephalum have been reported that triterpene saponins and flavo-
noids are the main active ingredients, and they exhibited different pharmacological activities
including anti-inflammatory and immunity [2], anti-hyperglycemic [3] and anti-tumor [4].
Our previous chemical studies led to the isolation of triterpene saponins. Further inves-
tigations of the extracts from the aerial parts of C. polycephalum resulted in the isolation
of three new triterpene saponins. is paper deals with the isolation and identification of
three triterpene saponins with their moderate inhibition against H9c2 cell damage induced
by H O , and five known triterpene saponins.
2
2
2
. Results and discussion
e alcohol extracts of C. polycephalum were subjected to silica gel column chromatography,

ODS and Sephadex LH-20 gel chromatography, then semi-preparative HPLC, and yielded
three new triterpene saponins clinopodisideVI (1), saikosaponin c (2), along with six known
CONTACT Xꢋ-dꢃꢊg Xꢋ
Yꢋ-xꢄꢆ hꢋ ꢆꢊꢂ Wꢌꢄ Zꢀꢆꢊg ꢇꢃꢊꢁꢅꢄbꢋꢁꢌꢂ ꢌqꢋꢆꢈꢈy ꢁꢃ ꢁꢀꢄꢍ wꢃꢅk.
xꢂxꢋ@ꢄmpꢈꢆꢂ.ꢆꢇ.ꢇꢊ
1
tꢀꢌ ꢍꢋppꢈꢌmꢌꢊꢁꢆꢈ ꢂꢆꢁꢆ ꢉꢃꢅ ꢁꢀꢄꢍ ꢆꢅꢁꢄꢇꢈꢌ ꢄꢍ ꢆvꢆꢄꢈꢆbꢈꢌ ꢃꢊꢈꢄꢊꢌ ꢆꢁ ꢀꢁꢁp://ꢂx.ꢂꢃꢄ.ꢃꢅg/10.1080/10286020.2016.1254199.
©
2016 iꢊꢉꢃꢅmꢆ uK lꢄmꢄꢁꢌꢂ, ꢁꢅꢆꢂꢄꢊg ꢆꢍ tꢆyꢈꢃꢅ & fꢅꢆꢊꢇꢄꢍ Gꢅꢃꢋp

2
Y.ꢀX. HU ET AL.
Figure 1. sꢁꢅꢋꢇꢁꢋꢅꢌꢍ ꢃꢉ ꢇꢃmpꢃꢋꢊꢂꢍ 1–8.
triterpene saponins (3–8) (Figure 1). eir structures were elucidated by 1D and 2D NMR
and MS analysis. Compounds 3–8 were identified by comparing their spectroscopic data
with published data for arjunglucoside I (3) [5], clinopodiside I (4) [6], saikosaponin b1 (5)
[
7], clinopodiside II (6) [8], buddlejasaponin IVb (7) [9] and buddlejasaponin IV (8) [10].
Compound 1 was assigned the molecular formula C H O , determined on the basis
3
6
58
9
+
of its positive HR-ESI-MS at m/z 657.3981 [M + Na] . e type and absolute configuration
of the sugar was identified as D-glucose on the basis of the TLC method comparison with
authentic monosaccharides (CHCl : MeOH: H O = 3: 2: 0.2), followed by gas chromatog-
3
2
1
raphy. In the H NMR spectrum, the coupling constants between the anomeric proton
(
δ 6.37, d, J = 7.8 Hz, H-1′) and the adjacent proton suggested that the glucose residue
H
1
was β-orientation. In the H NMR spectrum, signals were observed for six methyl singlets

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Figure 2. Kꢌy cosY ꢆꢊꢂ hMBc ꢇꢃꢅꢅꢌꢈꢆꢁꢄꢃꢊꢍ ꢃꢉ 1.
Figure 3. Kꢌy noesY ꢇꢃꢅꢅꢌꢈꢆꢁꢄꢃꢊꢍ ꢃꢉ 1.
(
δ 0.86, 0.97, 1.04, 1.24, 1.25, and 1.28), one anomeric proton signal at δ 5.10 (d, J = 7.8 Hz,
H
H
H-1′), two olefinic protons at δ 5.62 (d, J = 6.0 Hz, H-12) and 5.70 (d, J = 6.0 Hz, H-11).

H
1
3
e C-ATP spectrum showed 36 carbons including six methyl carbons at δ 14.0, 21.5,
C
2
1.7, 24.4, 26.5, and 33.5, four olefinic carbons at δ 116.4, 123.5, 145.7, and 155.8. e data
C
above indicated that 1 was an oxygenated oleanolic-type triterpenoid saponin derivative
1
13
with one sugar moiety. Comparison of the H NMR and C-APT data between 1 [δ 5.62
H
(H-12), 5.70 (H-11), δ 116.4 (C-11), 123.5 (C-12), 145.7 (C-13), 155.8 (C-9)] and clinop-
C
odiside I [δ 5.67 (H-12), 6.50 (H-11), δ 127.0 (C-11), 125.6 (C-12), 136.4 (C-13), 133.2
H
C
(C-18)] (Compound 4) [6] indicated that the conjugated double bonds at C-11/13 in 4
were changed to C-9/12. e HMBC (Figure 2) correlations of H-3/C-4, C-23, and C-24,
of H-23/C-3 and C-4 suggested the locations of the hydroxyl groups at C-3 and C-23. e
HMBC correlations from H-18 to C-16, C-17, and C-28 confirmed the locations of oxygen-
ated groups at C-16. e NOESY (Figure 3) correlations of CH -24/CH -25, H-9/CH -27,
3
3
3
and H-16/CH -27 indicated that the hydroxymethylene group of C-23 was α-oriented and
3
the hydroxyl of C-16 was β-oriented. e cross-peaks of CH -24/CH -25, CH -25/CH -26,
3
3
3
3
and H-18/CH -30 indicated that 24-CH , 25-CH , 26-CH and 30-CH methyl groups were
3
3
3
3,
3
β orientation. e arrangement of sugar unit was determined by HMBC experiment. Based
on the above spectral evidence, the structure of 1, clinopodiside VI, was defined as 3β, 16β,
2
3, 28-tetrahydroxyoleana-9(11), 12-dien-3-O-β-D-glucopyranoside.

4
Y.ꢀX. HU ET AL.
Compound 2 possessed a molecular formula of C H O Na based on HR-ESI-MS (m/z
4
2
68 13
+
1
8
03.4167 [M + Na] , calcd for 803.4558). e H NMR spectrum displayed seven methyl
signals at δ 0.85, 0.86, 0.96, 0.97, 1.07, 1.07, 1.52, two coupled olefinic signals at δ 5.70
H
H
(
4
at ∆
1H, d, J = 10.8 Hz), 6.52 (1H, dd, J = 10.8, 3.0 Hz), and sugar anomeric proton signals at δH
.95 (d, J = 7.8 Hz, H-1′) and 5.22 (d, J = 7.8 Hz, H-1′). e positions of two double bonds
1
1,12
11,38
and ∆
were also secured by HMBC correlations of H-11 (δ 6.52, d, J = 10.8,
H
3
.0 Hz) with C-10 (δ 36.7), C-8 (δ 40.7) and C-13 (δ 136.6), H-12 (δ 5.70, d, J = 10.8 Hz)
C C C H
with C-9 (δ 54.7), C-14 (δ 44.5), and C-18 (δ 133.5). On hydrolysis, the sugar units of 2
C
C
C
were identified as D-glucose and D-fucose by TLC and GC analyses. e linkage positions
of sugars were determined by comparing its NMR spectral data with that of saikosaponin
b1 (5) [10], clinoposaponin XV [11] and were further established by HMBC cross-peaks
between H-1′ and C-3, H-1′′ and C-2′, respectively. Based on the above results, it was con-
cluded that 2 has a similar structure to that of saikosaponin b1, except for the different
linkage positions of sugars. us, the structure of 2 was assigned as 3β, 16β, 23, 28-tetrahy-
droxyoleana-11,13(18)-dien-3-O-[β-D-glucopyranosyl(1→2)]-β-D-fucopyranoside and
named saikosaponin c.
In the in vitro cardioprotective assays, 1–3 exhibited moderate inhibition against H9c2
cell damage induced by H O . Compared with the cell viability of 63.3 ± 2.4% in the model,
2
2
1
–3 showed increased viability of 77.8 ± 2.6%, 80.9 ± 4.4% and 79.8 ± 2.7% at 100.0 μg/ml,
respectively, using quercetin as a positive control (cell viability of 84.1 ± 2.7%).
3
. Experimental
3
.1. General experimental procedures
Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter (Perkin Elmer,
Norwalk, USA) in MeOH at 20 °C. IR data were measured on FTIR-8400S spectrome-
ter (Shimadzu, Kyoto, Japan). UV data were run on a Shimadzu UV2550 spectrometer
(
Shimadzu, Kyoto, Japan). HR-ESI-MS were performed on a LTQ-Obitrap XL spectrometer.
NMR spectra were recorded on a Bruker AVIII 600 NMR spectrometer (Bruker Spectrospin,
Fällanden, Switzerland, chemical shif values are presented as δ values with TMS as the
internal standard). e GC was run on an Agilent 6890 N Gas Chromatograph (Agilent
Technology, Santa Clara, USA) using a HP-5 capillary column (30 m × 0.25 mm, i.d.)
(Agilent, Santa Clara, USA), with detection by FID. e initial temperature was set to 150 °C
for 2 min and then increased to 210 °C at a speed of 5 °C/min and the carrier gas was N2.
ODS reversed phase silica gel (40–63 μm, Merk, Darmstadt, Germany), Sephadex LH-20
(
Pharmacia, Uppsala, Sweden) and silica gel (100–200 mesh, Qingdao Haiyang Chemical
Co., Ltd, Qingdao, China) were used for column chromatography. HPLC separation was per-
formed on a CXTH LC-3000 HPLC system with a CXTH LC-3000 UV spectrophotometric
detector (Beijing Chuangxintongheng Science and Technology Co., Ltd., Beijing, China) and
a YMC (250 × 10 mm) semi-preparative column packed with C18 (5 μm, YMC CO., Ltd.,
Kyoto, Japan). TLC was carried out on silica gel precoated plates (Yantai Chemical Co. Ltd,
Yantai, China), and spots were detected by spraying with 10% H SO in EtOH followed by
2
4
heating.All solvents used were of analytical grade (Beijing ChemicalWorks, Beijing, China).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
3
.2. Plant material

e aerial part of Clinopodium polycephalum was provided from Anhui Institute for Sciinno
Drug Research, Anhui Province, China, in October 2013. e specimen was identified by
Jianli Zhou (Anhui University of Chinese Medicine, Anhui, China). A voucher specimen
(No. 20131028) has been deposited in the Anhui Science Institute for Drug Research Co. Ltd.
3
.3. Extraction and isolation
e air-dried and aerial parts of C. polycephalum (11.6 kg) were cut into small pieces and then

extracted three times with 70% aqueous EtOH (870 g). e solution was evaporated under
reduced pressure to obtain an extract without alcohol. e crude extract was suspended in
H O and respectively partitioned with petroleum ether, ethyl acetate, and n-butanol. e
2
n-BuOH extract (204 g) was subjected to column chromatography over silica gel, and eluted
with a gradient of CHCl -MeOH (1:0 to 0:1) to give three fractions (A–C). Fraction B was
3
subjected to ODS column, which was eluted with MeOH–H O (30:70, 50:50, 75:25, 90:10,
2
1
00:0) to give twelve fractions (B1–B12). B3 (1.2 g) was repeatedly separated by Sephadex
LH-20 (MeOH) and further purified by semi-preparative HPLC (flow rate 2.0 ml/min; UV
detection at 210 nm; eluent MeOH–H O 60:40; t =24.6 min) to offer compound 3 (39.3 mg).
2
R
B8 (0.8 g) was separated by Sephadex LH-20 (MeOH) and then isolated by semi-preparative
HPLC (flow rate 2.0 ml/min; UV detection at 210 nm; eluent MeOH–H O 80:20) to yield
2
compounds 1 (9.2 mg; t =27.5 min), and 4 (35.2 mg; t =25.4 min). B9 (3.2 g) was sepa-
R
R
rated by Sephadex LH-20 (MeOH) and further purified by semi-preparative HPLC (flow
rate 2.0 ml/min; UV detection at 210 nm; eluent MeOH–H O 80:20) to get compounds 2
2
(
14.5 mg; t =21.7 min), and 5 (60.6 mg; t =23.1 min). Fraction C (15.6 g) was subjected to
R
R
ODS column, which was eluted with MeOH–H O (30:70, 50:50, 75:25, 90:10, 100:0) to give
2
fif een fractions (C1–C15). Fractions C8 (131.3 mg), C10 (178.8 mg), and C12 (21.5 mg)
precipitated solids af er one week. Removing the supernatant and repeatedly recrystallizing
solids with acetone and MeOH provided white solids and yielded compounds 6 (40.6 mg),
7
(90.4 mg), and 8 (7.5 mg), respectively.
3
.3.1. Clinopodiside VI
2
0
White amorphous powder; [ꢀ] + 78.5 (c 0.05, MeOH); UV (MeOH) λmax (logε) 256
D
−
1 1
(
3.71), 262 (3.70), 281 (3.72) nm; IR (film) v 3367, 2942, 2875, 1457, 1067 cm . H
max
1
3
+
and C-APT spectral data see Table 1; HR-ESI-MS: m/z 657.3981 [M + Na] (calcd for
C H O Na, 657.3979).
3
6
58
9
3
.3.2. Saikosaponin c
2
0
White amorphous powder;[ꢀ]D + 1.67 (c 0.05, MeOH); UV (MeOH) λmax (logε) 251 (3.48),
−
1
1
13
2
56 (3.53) nm; IR (film) vmax 3391, 2934, 2880, 1730, 1070 cm . H and C-APT spectral
+
data see Table 1; HR-ESI-MS: m/z 803.4167 [M + Na] (calcd for C H O Na, 803.4558).
4
2
68 13
3
.4. Acid hydrolysis
A solution of each saponin (3 mg) was heated in 3 M TFA (4 ml) on a water bath for
h, and then extracted with EtOAc. e aqueous layer was evaporated to dryness with
3

6
Y.ꢀX. HU ET AL.
1
13
Table 1. h ꢆꢊꢂ c nMr ꢍpꢌꢇꢁꢅꢆꢈ ꢂꢆꢁꢆ ꢉꢃꢅ 1 ꢆꢊꢂ 2 (600 Mhz, Pyꢅꢄꢂꢄꢊꢌ-d ).
5
1
2
No.
δC
δH (J in Hz)
δC
δH (J in Hz)
1
38.0
1.93–1.95, m
38.6
1.81–1.83, m
1.09–1.10, m
2.30–2.32, m
2.08–2.09, m
4.13–4.15, m
1
.47–1.48, m
2.38–2.42, m
.02–2.03, m
2
26.6
26.2
2
3
4
5
6
82.4
41.0
44.1
18.5
4.30, ꢂꢂ, (10.8, 3.6)
82.4
44.5
48.2
18.5
1.81, ꢂ, (11.4)
1.80–1.82, m
1.54–1.56, m
1.80–1.82, m
1.40–1.41, m
1.40–1.42, m
1.32, m
1
.78–1.79, m
1.95–1.97, m
.37–1.38, m
7
31.4
32.9
1
8
9
43.7
155.8
39.2
116.4
123.5
145.7
44.0
40.7
54.7
36.7
127.3
125.9
136.6
44.5
2.07, ꢍ
10
11
12
13
14
15
5.70, ꢂ, (6.0)
5.62, ꢂ, (6.0)
6.52, ꢂꢂ, (10.8, 3.0)
5.70, ꢂ, (10.8)
36.6
2.34, bꢅ ꢁ, (12.6)
35.1
2.19, bꢅ ꢁ, (12.6)
1.54–1.55, m
4.06–4.08, m
1
.35–1.36, m
1
1
1
1
6
7
8
9
67.2
43.6
43.1
47.3
4.57–4.59, m
75.7
44.6
133.5
38.5
2.53, ꢂꢂ, (13.8, 4.8)
1.78–1.81, m
2.49, ꢂ, (13.2)
1.81, ꢂ, (13.2)
1
.17–1.18, m
2
2
0
1
27.0
32.5
32.9
35.3
1.92–1.94. m
.34–1.35, m
2.83, bꢅ ꢂ, (12.0)
.59–1.60, m
4.38, ꢂ, (10.2)
1.71–1.73, m
1.41–1.43, m
2.93, bꢅ ꢂ, (13.2)
1.54–1.55, m
4.40, bꢅ ꢍ
1
2
2
3
34.5
65.3
30.2
65.0
1
2
3
.70, ꢂ, (10.2)
1.04, ꢍ
1.25, ꢍ
1.24, ꢍ
1.28, ꢍ
3.73–3.75, m
1.07, ꢍ
2
2
2
2
2
4
5
6
7
8
14.0
26.5
21.5
21.7
69.8
13.1
19.0
17.6
22.2
64.3
0.86 ꢍ
0.97, ꢍ
1.07, ꢍ
3.74, ꢂ, (10.2)
4.37–4.39, m
4.17–4.19, m
0.85, ꢍ
4
.51, ꢂ, (10.2)
0.86, ꢍ
2
3
1
2
3
4
5
6
9
0
′
′
′
′
′
′
33.5
24.4
106.3
76.3
79.1
72.1
78.6
63.3
32.5
25.0
104.3
82.6
77.8
72.6
71.2
17.3
0.97, ꢍ
0.96, ꢍ
5.10, ꢂ, (7.8)
4.04, bꢅ ꢁ, (7.8)
4.14, bꢅ ꢁ, (9.6)
4.24, bꢅ ꢁ, (9.6)
3.98, m
4.95, ꢂ, (7.8)
4.49, bꢅ ꢁ, (8.4)
4.16–4.19, m
3.98, bꢅ ꢍ
3.69, q, (6.0)
1.52, ꢂ, (6.0)
4.58, ꢂꢂ, (11.4, 5.4)
4
.39, ꢂꢂ, (11.4, 3.6)
1′′
2′′
3′′
4′′
5′′
6′′
106.3
76.9
78.4
71.5
78.3
62.7
5.22, ꢂ, (7.8)
4.06–4.08, m
4.17–4.19, m
4.26, bꢅ ꢁ, (9.0)
3.73–3.76, m
4.40, bꢅ ꢍ
4.40, bꢅ ꢍ
ethanol in vacuo at 50 °C until neutral. e residues were determined in comparison with
authentic monosaccharides using TLC (CHCl :MeOH:H O = 3:2:0.2). For the sugars of
3
2
1
–3, the Rf values of glucose and fucose by TLC were 0.25 and 0.34, respectively. Also, the

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
absolute configurations of the sugars were determined by gas chromatography [12]. By
this method, L-cysteine methyl ester hydrochloride (0.06 mol/L) and hexamethyldisila-
zane–trimethylchlorosilane (HMDS-TMCS, 3:1) were added to the aqueous residue for
derivatization. e solution was then centrifuged and the precipitate was removed. Af er
these processes, n-hexane was used to extract derivate and analyzed by GC. D-Glucose
(
t =12.163, 13.800 min) and D-fucose (t =8.250, 8.930 min) were detected by comparing
R
R
with authentic monosaccharides.
3
.5. H9c2 Cell protection assay
4
H9c2 cells at a density of 5 × 10 cells per well in 96-well plates were cultured in dulbecco
modified eagle medium media (Hyclone, Los Angeles, USA) supplemented with 10% fetal
bovine serum (FBS, Hyclone, Los Angeles, USA), and L-glutamine (2 mM). Cultures were
maintained at 37 °C in 5% CO in a humidified incubator for 24 h. Af er 12 h of treatment
2
with different concentrations of drugs, followed by incubation with 150 mM H O for 2 h,
2
2
2
4
0 μl of 5 mg/ml MTT solution was added to each well (0.1 mg/well), and incubated for
h. e supernates were aspirated, and the formazan crystals in each well were dissolved
in 150 ml of DMSO. e absorbance was measured at 570 nm on a microplate reader
BioTek, Vermont). e survival rate of H9c2 cells was evaluated and the inhibition (%)
was obtained by the following formula: Inhibition (%) = [(OD(sample) – OD(control))/
OD(normal) – OD(control))] × 100.
(
(
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding

is work was financially supported by grants (Nos. 81173511 and 81374010) from the National
Natural Sciences Foundation of China.
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