Anti-coxsackie virus B3 norsesquiterpenoids from the roots of Phyllanthus emblica

Article abstract of DOI:10.1021/np800792d

Three new norsesquiterpenoid glycosides, 4′-hydroxyphyllaemblicin B (1) and phyllaemblicins E (2) and F (3), were isolated from the roots of Phyllanthus emblica, together with three known compounds, phyllaemblic acid (4), phyllaemblicin B (5), and phyllae

Full text of DOI:10.1021/np800792d

J. Nat. Prod. 2009, 72, 969–972
969
Anti-Coxsackie Virus B3 Norsesquiterpenoids from the Roots of Phyllanthus emblica
Qing Liu,†,‡ Ya-Feng Wang,§ Rong-Jie Chen,† Mei-Ying Zhang,§ Yi-Fei Wang,§ Chong-Ren Yang,† and Ying-Jun Zhang*,†
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming 650204, People’s Republic of China, Graduate School of the Chinese Academy of Sciences, Beijing 100049, People’s Republic of
China, and Guangzhou Jinan Biomedicine Research & DeVelopment Center, Guangzhou 510632, People’s Republic of China
ReceiVed December 15, 2008
Three new norsesquiterpenoid glycosides, 4′-hydroxyphyllaemblicin B (1) and phyllaemblicins E (2) and F (3), were
isolated from the roots of Phyllanthus emblica, together with three known compounds, phyllaemblic acid (4),
phyllaemblicin B (5), and phyllaemblicin C (6). Of these, 3 is a new norsesquiterpenoid dimer. The structures of 1-3
were established by spectroscopic data information and by acidic hydrolysis. The isolated compounds, together with
two other known analogues, phyllaemblic acid methyl ester (7) and phyllaemblicin A (8), were evaluated for their
antiviral activity toward coxsackie virus B3 (CVB3) by an in vitro cytopathic effect inhibitory assay. Compounds 5-7
exhibited strong anti-CVB3 activity.
Phyllanthus emblica L. (Euphorbiaceae) is a shrub or tree with
a greenish-gray bark and feathery leaves, growing in tropical and
subtropical areas of mainland China, India, Indonesia, and the Malay
Peninsula. It has been used extensively in many traditional medicinal
systems, such as Ayurvedic medicine, Chinese herbal medicine,
and those used by the Tibetan, Mongolian, Dai, and Uigur people.1
The roots, bark, and leaves have been used for treating eczema,
warts, diarrhea, and headache after a fever in the People’s Republic
of China, and the roots have been used as an astringent and
hematostatic agent in Nepal.1 The fruit and its juice have been used
widely due to their purported biological activities, including
antibacterial,2 antioxidant,3 anti-inflammatory,4 antitumor,1 and
hepatoprotective effects.5 The methanolic and water extracts of the
fruits were found to markedly inhibit the reverse transcription of
HIV.6
Compound 1 was obtained as a white, amorphous powder. Its
molecular formula, C33H44O20, was determined on the basis of the
HRESIMS ([M - H]-, m/z 759.2333) and 13C and DEPT NMR
data. The IR spectrum showed the presence of hydroxyl (3421 cm-1
)
and carbonyl (1692 cm-1) groups in 1. The 1H and 13C NMR
spectroscopic data of 1 were closely related to those of 5, which
showed two sets of signals for two hexoses [anomeric centers at
δH 5.60 (d, J ) 8.0 Hz) and 4.20 (d, J ) 7.7 Hz); δC 93.7 and
106.2, respectively] and 14 carbon resonances, including one ketone
(δ 213.8), one carbonyl (δ 175.9), and six oxygen-bearing carbons
(δ 100.6, 76.2, 75.9, 71.5, 70.4, and 63.5). However, instead of
the benzoyl group in 5, the downfield shift of C-4′ (δ 163.2) and
the appearance of two doublet aromatic proton signals at δ 8.03
and 6.91 (each 2H, d, J ) 8.7 Hz) revealed the presence of a
p-hydroxybenzoyl group in 1. Acidic hydrolysis of 1 gave D-glucose
as the sole sugar moiety, which was determined to have a ꢀ
configuration on the basis of the large coupling constants of the
anomeric protons [δH 5.60 (d, J ) 8.0 Hz) and 4.20 (d, J ) 7.7
Hz)]. The locations of the sugar units and the p-hydroxybenzoyl
group in 1 were confirmed by a HMBC experiment, in which
correlations of H-1′′′ (δ 4.20, terminal glucosyl unit) with C-2′′ (δ
83.2, inner glucosyl unit), H-1′′ (δ 5.60, inner glucosyl unit) with
C-13 (δ 175.9), and H-10 (δ 5.29) and H-2′,6′ (δ 8.03) with C-7′
(δ 168.0) were observed. Therefore, the structure of 1 was
determined as 4′-hydroxyphyllaemblicin B.
Our previous collaborative chemical studies of P. emblica have
led to the isolation of several norsesquiterpenoids from the roots,7,8
organic acid gallates and polyphenols from the fruit juice,9,10 and
ellagitannins and flavonoids from the branches and leaves.10,11 Of
these, phyllaemblic acid and its glycosides phyllaemblicins A-C
were found to be the major norsesquiterpenoids from the roots. In
the course of our study on antiviral compounds from natural sources,
the norsesquiterpenoid glycoside phyllaemblicin B (5) exhibited
stronger anti-CVB3 activity in vitro than a positive control,
ribovirin. In order to evaluate its anti-CVB3 activity in vivo, a large-
scale preparation of 5 was carried out. This led to the isolation of
three new analogues, namely, 4′-hydroxyphyllaemblicin B (1) and
phyllaemblicins E (2) and F (3), together with two known
compounds, phyllaemblic acid (4) and phyllaemblicin C (6), in
addition to 5. The isolated compounds 1-6, together with two
previously isolated analogues, phyllaemblic acid methyl ester (7)
and phyllaemblicin A (8), were evaluated for their anti-CVB3
activity in vitro.
Compound 2 was assigned the molecular formula C38H54O23 from
the positive HRESIMS ([M + Na]+, m/z 901.2929), which was 2
mass units larger than that of 6. This difference corresponded to
the presence of two additional protons. The 1H and 13C NMR spectra
of 2 were very similar to those of 6, except for the loss of the
ketone signal at δ 213 and the appearance of an additional oxygen-
bearing methine carbon signal at δ 84.2. This observation suggested
that the C-7 ketone in 6 was reduced to a secondary alcohol in 2.
Acidic hydrolysis of 2 gave D-glucose and L-arabinose as sugar
residues. The coupling constants (J ) 8.1, 7.8 Hz) of anomeric
protons of the two glucose moieties indicated these to be in the ꢀ
configuration, and the coupling constants (J1,2 ) 7.1 Hz, J2,3 ) 9.0
Hz, J3,4 ) 9.0 Hz, J4,5 ) 12.6 and 2.6 Hz) of the L-arabinose moiety
suggested it to be in the R-pyranose form. Connectivities of the
sugar and benzoyl moieties were further confirmed by the HMBC
correlations of H-1′′ of the inner glucose (δ 5.53), H-1′′′ of the
middle glucose (δ 4.19), and H-1′′′′ of the arabinose (δ 4.50) with
C-13 (δ 175.8), C-2′′ of inner glucose (δ 84.1), and C-2′′′ of middle
glucose (δ 85.0), respectively. In the ROESY spectrum of 2, H-7
was only correlated with H-9, but not with H-5, suggesting that
The methanolic extract of the fresh roots of P. emblica was
subjected to column chromatography to afford compounds 1-6.
Of these, compounds 4-6 were identified as the previously reported
norsesquiterpenoid phyllaemblic acid (4)7 and the glycosides
phyllaemblicins B (5) and C (6),8 respectively, by comparison with
authentic samples and of their spectroscopic and physical data with
previously reported values.
* Corresponding author. Tel: +86-871-5223235. Fax: +86-871-5150124.
E-mail: zhangyj@mail.kib.ac.cn.
† Kunming Institute of Botany.
‡ Graduate School of the Chinese Academy of Sciences.
§ Jinan Biomedicine Research & Development Center.
10.1021/np800792d CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/17/2009

970 Journal of Natural Products, 2009, Vol. 72, No. 5
Notes
Experimental Section
H-7 was R-oriented. Accordingly, the structure of phyllaemblicin
E was established as shown in 2.
General Experimental Procedures. [R]D and UV data were
measured on a JASCO-20 polarimeter and Shimadzu UV-2401PC
spectrometer, respectively. IR spectra were measured on a Bruker
Tensor 27 spectrometer with KBr pellets. 1D- and 2D-NMR spectra
were run on Bruker AM-400 and DRX-500 instruments operating at
400 and 500 MHz for 1H and 100 and 125 MHz for 13C, respectively.
Coupling constants are expressed in hertz, and chemical shifts are given
on a ppm scale with tetramethylsilane as internal standard. The MS
data were recorded on a VG Auto Spec-3000 spectrometer (VG,
Manchester, U.K.) with glycerol as the matrix. HRESIMS were recorded
on an API Qstar Pulsa LC/TOF spectrometer. GC analysis was run on
a Shimadzu GC-14C gas chromatograph. Evaluations of cytotoxicity
and antiviral effects were performed on an Emax precision microplate
reader (Bio-Rad Company, Hercules, CA).
Column chromatography was performed with Sephadex LH-20
(Pharmacia Fine Chemical Co., Ltd. Uppsala, Sweden), Diaion HP20SS
(Mitsubishi Chemical Co., Tokyo, Japan), silica gel (200-300 mesh,
Qingdao Makall Group Co., Ltd. Qingdao, People’s Republic of China),
Chromatorex ODS (100-200 mesh, Fuji Silysia Chemical Co., Ltd.
Tokyo, Japan), and MCI gel CHP 20P (Mitsubishi Chemical Co.). Thin-
layer chromatography (TLC) was carried out on silica gel H-precoated
plates (Qingdao Makall Group Co., Ltd.). Dulbecco’s modified Eagle’s
medium (DMEM) and ribovirin were purchased from HyClone
Company (Logan, UT) and Guangzhou Shiqiao Pharmaceutical Co.,
Ltd. (Guangzhou, People’s Republic of China), respectively.
Plant Material. Roots of P. emblica were collected from Simao
city, Yunnan Province, People’s Republic of China, in May 2007. The
plant was identified by Mr. Jiang Li from Yunnan Academy of Forestry.
A voucher specimen (KUN No. 0619422) has been deposited in the
herbarium of Kunming Institute of Botany, Chinese Academy of
Sciences.
Compound 3 was obtained as a white, amorphous powder. The
molecular formula C54H66O27 was deduced from the positive
HRESIMS ([M + Na]+, m/z 1169.3658) data. Acidic hydrolysis
of 3 gave D-glucose as the sole sugar residue. The 1H and 13C NMR
spectra of 3 were closely related to those of 5, which exhibited
signals due to two ꢀ-glucopyranosyl units [δ 5.67 (d, J ) 8.1) and
4.25 (d, J ) 6.5)] together with two sets of signals almost
superimposable on those of 4, suggesting that there was another
phyllaemblic acid (4) moiety attached to the phyllaemblicin B (5)
unit in 3. On comparison with those of 5 (δH 3.52 and 3.56, δC
62.0), the H-6′′′ and C-6′′′ signals of the second glucose moiety in
3 were shifted downfield to δΗ 4.53 and 4.31 and δC 63.5,
respectively, suggesting that the C-6′′′ of this glucose unit is acylated
with another phyllaemblic acid (4) unit, in which the C-13 signal
was shifted upfield to δ 175.6 relative to that of the free
phyllaemblic acid (4, δ 179.0). The connectivities of each unit in
3 were further determined by 2D NMR experiments, including
1H-1H COSY, HMQC, and HMBC. In the HMBC spectrum of 3,
H-1′′ (δ 5.67) of the first glucose moiety and H-6′′′ (δ 4.53, 4.31)
of the second glucose moiety were correlated with C-13 (δ 174.8,
175.6) of the two carboxyls. Moreover, HMBC correlations of H-1′′′
(δ 4.25) of the second glucose moiety with C-2′′ (δ 83.8) of the
first glucose moiety and of both H-10 (δ 5.33) and H-2′,6′ (δ 8.18,
8.10) with C-7′ (δ 166.6) of the benzoyl group were observed.
Accordingly, the structure of 3 was determined to be a dimer of 4
and 5 through an ester linkage between C-6′′′ of 5 and C-13 of 4
and was named phyllaemblicin F.
Extraction and Isolation. The fresh roots of P. emblica (21.7 kg)
were extracted with methanol three times (2 h × 3) under reflux. The
extract (490 g) was divided into two partions, and each partion was
subjected to column chromatography over Sephadex LH-20 (6.4 × 50
cm), eluted with MeOH-H2O (3:7-1:0), to afford four fractions in
all. Fraction 1 (152 g) was applied to a Diaion HP20SS column (6.8 ×
60 cm) and eluted with MeOH-H2O (0:1-1:0) to give 11 subfractions.
Subfraction 8 (26.3 g) was chromatographed sequentially over silica
gel (4.8 × 50 cm, CHCl3-MeOH-H2O, 8:2:0.2 and 7:3:0.5), Chro-
matorex ODS (MeOH-H2O, 1:9-6:4), and MCI gel CHP 20P
(MeOH-H2O, 7:3-9:1) to afford 1 (358 mg), 2 (38 mg), 5 (9.1 g),
and 6 (6.2 g). Subfraction 9 (2.5 g) was subjected to passage over
Sephadex LH-20 (MeOH-H2O, 4:6-7:3), silica gel (CHCl3-MeOH-
H2O, 8:2:0.2), and MCI gel CHP 20P (MeOH-H2O, 7:3-9:1) to afford
3 (50 mg) and 4 (63 mg).
The isolated compounds 1-6 were tested for their in vitro anti-
CVB3 activities, together with two previously isolated analogues,
phyllaemblic acid methyl ester (7) and phyllaemblicin A (8), and
their CC50 (concentration that reduced the viability of HeLa cell
cultures by 50%), IC50 [concentration required to reduce 50% of
cytopathic effect (CPE)], and TI (therapeutic index ) CC50/IC50)
values are shown in Table 3. Compounds 5-7 exhibited significant
anti-CVB3 activity, while other compounds (1-4, 8) showed no
antiviral activity. This suggests that the C-7 ketone, the benzoyl
group at C-10, and esterification at C-13 are essential for anti-CVB3
activity. Once the ketone at the C-7 position was reduced to be a
secondary alcohol or the benzene ring of a benzoyl group was
substituted with a hydroxyl group, the anti-CVB3 activity of the
molecule (1 and 2) was lost. In addition, at least two sugar units
attached at the C-13 position are necessary for anti-CVB3 activity.
Compounds 4 and 8, with no or only one sugar unit, showed a
lack of anti-CVB3 activity.
4′-Hydroxyphyllaemblicin B (1): white, amorphous powder; [R]23
D
0 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 258 (3.68), 203 (3.68),
195 (3.41), 191 (3.42) nm; IR (KBr) νmax 3421, 2934, 1778, 1744, 1692,
1
1609, 1281, 1169, 1097, 851, 773, 617 cm-1; H and 13C NMR data,

Notes
Journal of Natural Products, 2009, Vol. 72, No. 5 971
Table 2. 1H and 13C NMR Spectroscopic Data of 3a
Table 1. 1H and 13C NMR Spectroscopic Data of 1 and 2a
1
2
part A
part B
position
1H
13C
1H
13C
position
1H
13C
1H
13C
1
2
3.91 brs
71.5
32.2
3.84 brs
72.1
28.7
1
2
3.99 d (2.5)
71.6
32.1
4.04 d (3.5)
71.4
32.2
2.04 brd (13.6)
1.75 dt (13.6, 2.5)
2.90 tt (12.9, 2.5)
2.35 brd (14.0)
1.86 dt (14.0, 3.6)
4.28 brs
2.19-2.22 m
1.57-1.66 m
2.63-2.72 m
2.08 dt (13.9, 3,8)
1.90 dt (13.9, 3.8)
3.83 brs
2.11-2.16 m
1.74 dt (16.7, 3.0)
2.92 brs
2.27 brd (18.5)
1.94 dt (18.5, 4.5)
4.23 brs
1.98-2.03 m
1.83 dt (16.0, 3.0)
3.00 dt (16.0, 3.0)
2.54 brd (18.5)
2.02-2.04 m
4.32 brs
3
4
32.1
29.4
34.5
28.4
3
4
31.5
28.8
31.6
28.8
5
6
7
8
9
76.2
75.9
213.8
100.6
32.8
76.0
76.7
82.4
102.6
36.1
5
6
7
8
74.9
75.9
212.2
99.8
32.1
70.1
33.4
62.6
75.2
75.7
212.3
99.8
32.7
69.9
33.6
62.6
4.15 t (3.2)
2.25 dd (14.8, 3.1)
1.96 dd (14.8, 3.1)
5.29 brd (2.8)
2.15 m
4.02 t (11.7)
3.54 dd (11.7, 2.8)
2.18-2.23 m
2.12 dd (14.6, 2.6)
5.32 d (2.5)
2.16-2.24 m
4.07 t (11.3)
9
1.94 dt (14.9, 2.7)
5.33 q (2.7)
2.10-2.16 m
3.48 dd (14.0, 5.5)
3.38-3.42 m
2.20 dt (18.5, 2.7)
5.33 q (2.7)
2.10-2.16 m
3.85 t (14.3)
10
11
12
10
11
12
70.4
34.4
63.5
71.9
34.1
63.1
3.72-3.75 m
3.55-3.61 m
13
14
1′
174.8
13.0
175.6
13.0
13
14
1′
175.9
13.1
175.8
13.1
0.77 d (6.8)
0.81 d (6.9)
0.87 d (6.8)
0.89 d (6.8)
131.9
130.5
129.3
133.7
166.6
92.8
83.8
77.4
70.1
75.6
132.0
130.5
129.5
134.0
166.6
123.1
133.2
116.5
163.2
168.0
93.7
83.2
77.9
70.5
79.0
132.2
130.9
129.9
134.5
167.9
93.8
84.1
77.5
70.3
78.8
2′, 6′
3′, 5′
4′
8.10 d (7.5)
7.57-7.68 m
7.57-7.68 m
8.18 d (7.4)
7.57-7.68 m
7.57-7.68 m
2′, 6′
3′, 5′
4′
8.03 d (8.7)
6.91 d (8.7)
8.14 d (7.2)
7.55 t (7.7)
7.67 t (7.4)
7′
7′
Glc-1′′
2′′
5.67 d (8.1)
3.41 dd (11.0, 8.1)
3.60-3.64 m
3.57-3.60 m
3.54 t (6.8)
3.58-3.62 m
3.47 dd (14.0, 5.5)
4.25 d (6.5)
3.20-3.25 m
3.30 dt (10.8, 6.5)
3.20-3.25 m
3.69-3.71 m
4.53 dd (15.0, 5.0)
4.31 dd (15.0, 2.0)
Glc-1′′
2′′
5.60 d (8.0)
3.49 brd (9.2)
3.60 brd (9.2)
3.36-3.40 m
3.40-3.43 m
3.92 dd (12.0, 2.4)
3.78 dd (12.0, 5.6)
4.20 d (7.7)
3.11 dt (7.7, 2.5)
3.25 dd (7.0, 2.5)
3.22-3.29 m
2.72-2.76 m
3.69 dd (12.1, 5.0)
3.59 dd (12.1, 2.0)
5.53 d (8.1)
3.23 brd (8.1)
3.45 brd (8.8)
3.44 brd (8.8)
3.35-3.38 m
3.85 brd (11.6)
3.64-3.68 m
4.19 d (7.8)
3.27 brd (7.8)
3.54-3.59 m
3.83 brs
3.28-3.33 m
3.45-3.50 m
3.54-3.60 m
4.50 d (7.1)
3.63-3.68 m
3.56 dt (9.0, 3.0)
3.83 brs
3′′
3′′
4′′
4′′
5′′
5′′
6′′
62.0
6′′
62.3
62.3
Glc-1′′′
2′′′
106.1
75.5
77.3
70.5
77.8
63.5
Glc-1′′′
2′′′
106.2
75.5
77.8
70.6
77.6
61.8
104.4
85.0
77.3
70.1
77.7
61.9
3′′′
3′′′
4′′′
4′′′
5′′′
5′′′
6′′′
6′′′
a Measured in CD3COCD3, 500 and 125 MHz for 1H and 13C NMR,
respectively, δ in ppm, J values in Hz.
Ara-1′′′′
2′′′′
107.0
73.6
74.2
69.6
67.6
3′′′′
Table 3. Anti-Coxsackie Virus B3 Effects of Compounds 5-7a
4′′′′
5′′′′
3.56 m
3.99 dd (12.6, 2.6)
compound
CC50 (µg/mL)b
IC50 (µg/mL)c
TId
5
6
7
50.2
67.7
213.6
426.0
7.8
11.0
21.8
20.3
6.4
6.2
9.8
a Measured in CD3OD, 400 and 100 MHz for 1H and 13C NMR,
respectively, δ in ppm, J values in Hz.
ribovirin
21.0
see Table 1; FABMS m/z 759 [M - H]- (100), 597 [M - H - 162
(glucosyl)]- (2), 435 [M - H - 324 (2 × glucosyl)]- (25); HRESIMS
m/z 759.2333 [M - H]- (calcd for C33H43O20, 759.2347).
a All data represent mean values for three independent experiments.
b CC50 ) concentration that reduced the viability of HeLa cell cultures
by 50%. c IC50 ) concentration required to reduce 50% of cytopathic
effect. d TI ) CC50/IC50.
Phyllaemblicin E (2): white, amorphous powder; [R]23 +11.0 (c
D
0.24, MeOH); UV (MeOH) λmax (log ε) 272 (2.56), 228 (3.56), 202
(3.55), 194 (3.31) nm; IR (KBr) νmax 3405, 2932, 1747, 1705, 1283,
1078, 781, 720 cm-1; 1H and 13C NMR data, see Table 1; FABMS m/z
902 [M + Na]+ (7), 879 [M + H]+ (12); HRESIMS m/z 901.2929 [M
+ Na]+ (calcd for C38H54O23Na, 901.2953).
3 °C/min; carrier gas, N2 (1 mL/min); injector and detector temperature,
250 °C; injection volume, 4 µL; and split ratio, 1/50. The configuration
of the sugar moiety was determined by comparing the retention time
with the derivatives of authentic samples. The retention times of
D-/L-glucose were 19.21/14.94 min, and those of D-/L-arabinose were
14.60/13.65 min, respectively.
Virus and Cell Cultures. Coxsackie virus B3 Nacy strain, which
was generously provided by the Wuhan Institute of Virology, Chinese
Academy of Sciences, was propagated in HeLa cell monolayers and
stored at -80 °C until used. Viral titers were determined by tissue
culture infectious dose (TCID50) assays, and the concentration of the
CVB3 titer used for the infections was 100 TCID50. HeLa cells were
obtained from the American Type Culture Collection and grown
routinely in complete medium (DMEM medium supplemented with
10% heat-inactivated newborn calf serum, 0.1% L-glutamine, 100 U/mL
penicillin, and 0.1 mg/mL streptomycin) at 37 °C in an atmosphere of
5% CO2. The test medium used for the cytotoxic assay as well as the
antiviral assay contained 2% of the appropriate serum.
Phyllaemblicin F (3): white, amorphous powder; [R]23 +11.6 (c
D
0.26, MeOH); UV (MeOH) λmax (log ε) 280 (2.49), 273 (2.64), 229
(3.98), 202 (3.91) nm; IR (KBr) νmax 3435, 3422, 2935, 1779, 1716,
1
1283, 1176, 1081, 1005, 952, 719 cm-1; H and 13C NMR data, see
Table 2; FABMS m/z 1169 [M + Na]+ (30), 1147 [M + H]+ (20), 727
[M + H - 420 (phyllaemblic acid)]+ (15), 413 [M + H - 420 - 324
(2 × glucosyl)]+ (17); HRESIMS m/z 1169.3658 [M + Na]+ (calcd
for C54H66O27Na, 1169.3689).
Acidic Hydrolysis of Compounds 1-3. Compounds 1-3 (each
8-10 mg) were hydrolyzed with 2 M HCl-dioxane (1:1, 4 mL) under
reflux for 6 h. The reaction mixture was extracted with CHCl3 five
times (2 mL × 5). The aqueous layer was neutralized with 2 M NaOH
and then dried to give a monosaccharide mixture. Then, a solution of
the sugar mixture in pyridine (2 mL) was added to L-cysteine methyl
ester hydrochloride (about 1.5 mg) and kept at 60 °C for 1 h. Next,
trimethylsilylimidazole (about 1.5 mL) was added to the reaction
mixture in ice water and kept at 60 °C for 30 min. The mixture was
subjected to GC analysis, run on a Shimadzu GC-14C gas chromato-
graph equipped with a 30 m × 0.32 mm i.d. 30QC2/AC-5 quartz
capillary column and an H2 flame ionization detector with the following
conditions: column temperature, 180-280 °C; programmed increase,
Cytotoxicity. Cytotoxicity of compounds 1-8 was determined by
a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
method,12 as previously reported. Briefly, HeLa cells were seeded in
96-well plates and grown to monolayers. After removal of the growth
medium (DMEM maintenance with 10% newborn calf serum), the test
compound solutions at series of concentrations (200-6.25 µg/mL) in

972 Journal of Natural Products, 2009, Vol. 72, No. 5
Notes
the test medium were added (100 µL each well). Then, the plates were
incubated at 37 °C for 48 h, and 20 µL of MTT solution (0.5 mg/mL,
in phosphate-buffered saline) was added to each well followed by
reacting for 4 h. Subsequently, the supernatant was removed and 100
µL of DMSO was added. After incubation at room temperature for 30
min, the optical density (OD) values were read on a microplate
spectrophotometer at dual wavelengths of 570 and 630 nm. The CC50
values (the concentration of test compounds that reduced the viability
of HeLa cell cultures by 50%) were calculated from each mean
dose-response curve.
Anti-CVB3 Activity Assay. Anti-CVB3 activities of the test
compounds were evaluated by the cytopathic effect (CPE) inhibitory
assay described previously.13,14 In brief, HeLa cells were grown in 96-
well plates and were allowed to form monolayers. Then, 50 µL of 100
TCID50 viral suspensions and an equal volume of serial 2-fold dilutions
of the test compounds at doses below CC50 were added to each well.
Noninfected and infected cells without the test compounds served as
cell and virus control, respectively. The plates were incubated at 37
°C in a humidified CO2 atmosphere. When virus control showed a
maximum CPE, the virucidal effects were determined by the MTT assay
as described above, and the IC50 values (the concentration of test
compounds required to reduce 50% of CPE) were calculated. Thus,
the therapeutic index (TI) was calculated from the ratio CC50/IC50.
of Phytochemistry and Plant Resources in West China, Chinese
Academy of Sciences (O807B11211, O807E21211).
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Acknowledgment. The authors are grateful to the staff of the
analytical group at State Key Laboratory of Phytochemistry and Plant
Resources in West China, Kunming Institute of Botany, Chinese
Academy of Sciences, for measuring the spectroscopic data. This work
was supported by the NSFC U0632010 and the State Key Laboratory
NP800792D