Anti-viral and cytotoxic norbisabolane sesquiterpenoid glycosides from Phyllanthus emblica and their absolute configurations

Article abstract of DOI:10.1016/j.phytochem.2015.06.001

In an effort to identify anti-viral and cytotoxic compounds from Phyllanthus spp., 14 highly oxygenated norbisabolane sesquiterpenoids, phyllaemblicins H1-H14, were isolated from the roots of Phyllanthus emblica Linn, along with phyllaemblicins B and C and glochicoccinoside D. Their structures were determined on the basis of detailed spectroscopic analysis and chemical methods. Determination of absolute configurations of these compounds was facilitated by theoretical calculations of electronic circular dichroism (ECD) spectra using time-dependent density functional theory (TDDFT) for the aglycone components, and pre-column derivative/chiral HPLC analysis for the monosaccharides. The known glochicoccinoside D displayed potent activity against influenza A virus strain H3N2 and hand, foot and mouth virus EV71, with IC₅₀ values of 4.5 ± 0.6 and 2.6 ± 0.7 μg/ml, respectively. Phyllaemblicin H1 showed moderate cytotoxicity against human cancer cell lines A-549 and SMMC-7721, with IC₅₀ values of 4.7 ± 0.7 and 9.9 ± 1.3 μM, respectively.

Full text of DOI:10.1016/j.phytochem.2015.06.001

Phytochemistry 117 (2015) 123–134
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Anti-viral and cytotoxic norbisabolane sesquiterpenoid glycosides from
Phyllanthus emblica and their absolute configurations
Jun-Jiang Lv a,c,1, Shan Yu a,b,1, Ying Xin a,b, Rong-Rong Cheng a, Hong-Tao Zhu a, Dong Wang a,
Chong-Ren Yang a, Min Xu a, , Ying-Jun Zhang a,
⇑
⇑
a State Key Laboratory of Phytochemistry & Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Lanhei Road 132#, Kunming 650201,
People’s Republic of China
b University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
c Chemistry and Chemical Engineering College, Chongqing University, Chongqing 400030, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to identify anti-viral and cytotoxic compounds from Phyllanthus spp., 14 highly oxygenated
norbisabolane sesquiterpenoids, phyllaemblicins H1–H14, were isolated from the roots of Phyllanthus
emblica Linn, along with phyllaemblicins B and C and glochicoccinoside D. Their structures were deter-
mined on the basis of detailed spectroscopic analysis and chemical methods. Determination of absolute
configurations of these compounds was facilitated by theoretical calculations of electronic circular
dichroism (ECD) spectra using time-dependent density functional theory (TDDFT) for the aglycone com-
ponents, and pre-column derivative/chiral HPLC analysis for the monosaccharides. The known glochicoc-
cinoside D displayed potent activity against influenza A virus strain H3N2 and hand, foot and mouth virus
Received 4 December 2014
Received in revised form 30 May 2015
Accepted 2 June 2015
Keywords:
Yu Gan Zi
Phyllanthus emblica
Euphorbiaceae
ECD calculation
Antiviral
EV71, with IC50 values of 4.5 0.6 and 2.6 0.7
cytotoxicity against human cancer cell lines A-549 and SMMC-7721, with IC50 values of 4.7 0.7 and
9.9 1.3 M, respectively.
lg/ml, respectively. Phyllaemblicin H1 showed moderate
l
Norbisabolane sesquiterpenoid glycosides
Phyllaemblicins
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Phyllanthus emblica Linn. is an important medicinal plant that
has been described in Chinese herbal medicine (called Yu Gan
Zi), Ayurvedic medicine, and Tibetan medicine. Its roots are used
for treatment of hypertension and enteritis in China (Xie, 1996).
Continued studies on the chemical composition of this plant led
herein to identification of 14 new sesquiterpenoid glycosides,
along with three known ones. Their structures featured phyllaem-
blic acid (1a) as the aglycone connected to different sugar moieties,
e.g., glucopyranosyl, xylopyranosyl, arabinopyranosyl, 6-deoxyglu-
copyranosyl, and/or myo-inositol units were determined on the
basis of detailed spectroscopic analysis. A pre-column deriva-
tive/chiral separation/DAD detection method was established and
applied to determine the absolute configurations of the sugar parts
of the new glycosides. Quantum chemical calculated electronic cir-
cular dichroism (ECD) using time-dependent density functional
theory (TDDFT) was used to elucidate the absolute configurations
of the aglycone. Moreover, the antiviral activities of the isolated
compounds against enterovirus 71 (EV71) for hand, foot and
mouth disease, influenza A virus strain H3N2, herpes simplex virus
(HSV-1), and Coxsackie virus B3 (CVB3), as well as their cytotoxic
activities against human cancer cell lines, were evaluated. Herein,
the results obtained in this study were presented.
Highly oxygenated bisabolane sesquiterpenoid glycosides are
primarily present in the roots of several Phyllanthus spp.
(Euphorbiaceae). Isolation of phyllanthocin from Phyllanthus
brasiliensis Muell (Kupchan et al., 1977), phyllaemblicins A–F and
G1–G8 from Phyllanthus emblica Linn (Lv et al., 2014a; Zhang
et al., 2000a,b, 2001), and phyllanthacidoids A–U from
Phyllanthus acidus Skeels (Lv et al., 2014b; Vongvanich et al.,
2000) have been previously reported. These compounds showed
potent antileukemic (Kupchan et al., 1977), cytotoxic
(Vongvanich et al., 2000) and anti-viral (Liu et al., 2009; Lv et al.,
2014a,b) activities, which attracted considerable attention by
pharmacologists and organic chemists (Moore and Powis, 1986;
Smith et al., 1991).
⇑
Corresponding authors.
E-mail addresses: xumin@mail.kib.ac.cn (M. Xu), zhangyj@mail.kib.ac.cn (Y.-J.
Zhang).
1
These authors contributed equally to this article.
http://dx.doi.org/10.1016/j.phytochem.2015.06.001
0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

124
J.-J. Lv et al. / Phytochemistry 117 (2015) 123–134
2. Results and discussion
Taking into account the high contents of tannins and sugars in
the 70% ethanolic extract of the air-dried P. emblica roots, Diaion
HP20SS column chromatography (CC) was first used to remove
sugars and to enrich the sesquiterpenoid glycoside (50–80%
CH3OH in H2O) fraction. Silica gel CC was then employed to remove
most of the tannins and to fractionate the sesquiterpenoid glyco-
side. Then, Sephadex LH-20 CC of the sesquiterpenoid glycoside
fraction eluted with
a CH3OH/H2O gradient system further
enriched the sesquiterpenoid glycosides (0–30% CH3OH in H2O).
The enriched target fractions were next further purified by passage
over MCI-gel CHP20P, Toyopearl HW-40C and RP-8, followed by
preparative HPLC (p-HPLC) to afford 17 compounds (1–17), respec-
tively, with purification guided by thin layer chromatography (TLC)
and HPLC-DAD analyses. The new compounds 1–14 were all nor-
bisabolane type sesquiterpenoid glycosides with phyllaemblic acid
as the aglycone (Fig. 1). Three known sesquiterpenoid glycosides
were determined to be phyllaemblicins B (15) (Zhang et al.,
2000a) and C (16) (Zhang et al., 2000a), and glochicoccinoside D
(17) (Xiao et al., 2007), respectively, by comparing with authentic
samples directly and their spectroscopic and physical data with
those previously reported in literatures.
2.1. Structure elucidations of new compounds
Phyllaemblicin H1 (1) was obtained as a white amorphous pow-
der, with a molecular formula of C35H46O20, as established from
HRESIMS m/z 831.2556 [M+HCOO]ꢀ. Its 1H NMR spectrum
(Table 1) showed characteristic signals arising from a benzoyl
group (dH 8.17, 7.54, and 7.64), an acetyl methyl group (dH 2.10,
s, 3H), one methyl group at dH 0.90 (3H, d, J = 6.8 Hz), and two
anomeric protons of two sugar units [dH 5.57 (1H, d, J = 8.2 Hz),
and dH 4.21 (1H, d, J = 7.6 Hz)]. In its 13C NMR spectrum
(Table 1), apart from the resonances of one benzoyl and two hexo-
syl groups, 14 carbon signals arising from one methyl (dC 13.3),
four methylenes including one oxygen-bearing one (dC 63.6), five
methines (three oxymethines), one ketal carbon (dC 100.6), one
carboxyl (dC 175.9), one oxygen bearing quaternary carbon (dC
75.7), and one carbonyl (dC 213.8) were observed. The aforemen-
tioned data indicated that 1 was a highly oxygenated bisabolane
sesquiterpenoid glycoside (Zhang et al., 2000b). These NMR fea-
tures were comparable to those of phyllaemblicin B, a known
highly oxygenated norbisabolane glycoside isolated from the title
plant (Zhang et al., 2000a), except for an additional acetyl group.
Based on the HMBC correlation (Fig. 2) of H-600 of the inner glucosyl
to the acetyl dC 172.8 (C-10000), it was determined that compound 1
was 600-acetyl phyllaemblicin B.
Phyllaemblicin H2 (2) had the same molecular formula as 1,
deduced from the HRESIMS. The NMR spectroscopic data
(Table 1) of 2 were similar to 1. Extensive analysis of the 2D
NMR spectra indicated that 2 was also an acetylated derivative of
phyllaemblicin B. The HMBC correlation between the terminal glu-
cosyl H-6000 (dH 4.17) to dC 173.1 allowed the connection of the
acetyl group to C-6000 of the terminal glucosyl. Accordingly, com-
pound 2 was determined to be 6000-acetyl phyllaemblicin B.
Both phyllaemblicins H3 (3) and H4 (4) had a disaccharide moi-
ety. One of the sugars was assigned as a glucopyranosyl [anomeric
proton at dH 5.58, d, J = 8.2 Hz for 3 and dH 4.20, d, J = 7.6 Hz for 4]
unit, on the basis of 1D and 2D NMR spectra. Another sugar moiety
was a xylopyranosyl (anomeric proton at dH 3.95, d, J = 7.7 Hz) for 3
and an arabinopyranosyl (anomeric proton at dH 5.50, d, J = 7.7 Hz)
for 4. The monosaccharides in 3 and 4 were further confirmed by
acid hydrolysis followed by pre-column derivatization/chiral
separation/DAD detection (Fig. 3). After acid hydrolysis, the
Fig. 1. Structures of compounds from P. emblica.
reaction of monosaccharides with 1-phenyl-3-methyl-5-pyra-
zolone (PMP) in 0.3 M NaOH solution at 70 °C was carried out
(Honda et al., 1989). After chiral HPLC separation, the monosaccha-
rides were determined to be
glucose and -arabinose for 4, by comparing their retention times
(tR) with the corresponding standards -glucose (tR, 15.0 min),
xylose (tR, 15.5 min) and -arabinose (tR, 11.3 min) (Fig. 3).
D-glucose and D-xylose for 3, and D-
L
D
D-
L
The sugar moiety of phyllaemblicin H5 (5) comprised of two hex-
osyl units, including one glucosyl unit. Two anomeric protons at dH
5.52 (1H, d, J = 8.1 Hz, H-100) and 4.19 (1H, d, J = 8.0 Hz, glucosyl H-
1) were observed in the 1H NMR spectrum (Table 2). All of the proton
and carbon signals from the two hexosyl units were unambiguously
assigned by HSQC-TOCSY experiments. Besides the proton spin sys-
tem of the glucosyl moiety, the proton spin system of H-100/H-200/H-
300/H-400/H-500/H-600 was constructed for another hexosyl unit. The
presence of the methyl signal [dC 18.2 and dH 1.33] and the large cou-
pling constants (Table 2) of H-100 to H-400 indicated that this
monosaccharide was a 6-deoxy b-glucopyranosyl unit. In the
HMBC spectrum of 5, the correlations from H-100 to the carboxyl car-
bon of the aglycone at dC 176.1, and H-1000 to C-200 confirmed the con-
nectivity of the disaccharide moiety. Using the same method as
described for 3 and 4, the glucosyl unit in 5 was determined to have
a D configuration (rt = 15.0 min), while the absolute configurations
of the 6-deoxy b-glucosyl unit (tR, 15.7 min) were not determined
due to the lack of an authentic sample.
The MS and 13C NMR spectra of phyllaemblicin H6 (6) indicated
that there was only one hexosyl unit. However, two anomeric pro-
ton signals [dH 5.11 (d, J = 3.8 Hz) and 4.52 (d, J = 7.8 Hz)] with one

J.-J. Lv et al. / Phytochemistry 117 (2015) 123–134
125
Table 1
13C and 1H NMR Spectroscopic data for compounds 1–4 in CD3OD (d in ppm).
No.
1
2
3
4
dC
dH
dC
dH
dC
dH
dC
dH
1
2
71.6, CH
3.92 brs
71.4, CH
3.91 brs
71.2, CH
3.91 brs
71.4, CH
3.94 brs
32.5, CH2 1.74 ddd (2.5, 13.0, 14.0) 32.7, CH2 1.77 ddd (2.5, 13.0 14.0) 32.5, CH2 1.76 ddd (2.5, 13.0 14.0) 32.3, CH2 1.77 ddd (2.5, 13.0, 14.0)
2.05a
1.95a
1.89a
2.04 brd (14.0)
3
4
32.3, CH
2.88 tt (3.0, 13.0)
32.5, CH
2.94 tt (3.0, 13.0)
32.3, CH
2.95 tt (3.0, 13.0)
32.2, CH
2.93 tt (3.0, 13.0)
29.4, CH2 1.88 ddd (3.7, 13.0, 14.6) 29.4, CH2 1.89 ddd (3.8, 13.0, 14.6) 29.4, CH2 1.87a
29.2, CH2 1.90 ddd (4.0, 13.0, 14.5)
2.34 brd (14.5)
76.4, CH
75.5, C
213.7, C
100.6, C
32.7, CH2 2.02 dd (3.4, 15.0)
2.32 dd (3.4, 15.0)
2.31 brd (3.9, 14.6)
2.36 brd (14.6)
4.28 brs
2.35 brd (14.4)
4.27 brs
5
6
7
8
9
76.3, CH
75.7, C
213.8, C
100.6, C
4.28a
76.4, CH
75.7, C
213.8, C
100.6, C
32.9, CH2 2.00 dd (3.0, 14.6)
2.30 dd (3.5, 14.6)
76.3, CH
75.7, C
214.0, C
100.6, C
32.8, CH2 1.98 dd (3.5, 15.0)
2.31 dd (3.5, 15.0)
4.31 brs
32.8, CH2 1.98 dd (3.0, 14.9)
2.31a
10
11
12
71.2, CH
34.4, CH
5.34 brs
2.18 m
71.2, CH
34.5, CH
63.5, CH2 3.56 dd (4.6, 11.5)
4.01 dd (11.5, 11.5)
176.3, C
13.3, CH3 0.89 d (6.9)
132.4, C
131.0, CH 8.17 d (8.0)
130.1, CH 7.57 t (8.0)
134.6, CH 7.66 t (8.0)
167.8, C
93.9, CH
82.6, CH
78.1, CH
70.8, CH
79.0, CH
5.36 brs
2.18 m
71.1, CH
34.3, CH
5.34 brs
2.17 m
71.0, CH
34.3, CH
63.4, CH2 3.60 dd (4.0, 11.5)
4.06 dd (11.5, 11.5)
175.8, C
13.1, CH3 0.92 d (6.9)
132.2, C
130.9, CH 8.20 d (8.0)
129.7, CH 7.57 t (8.0)
134.8, CH 7.65 t (8.0)
167.8, C
5.38 brs
2.20 m
63.6, CH2 3.58a
63.4, CH2 3.57a
4.05 dd (11.5, 11.5)
4.05 dd (11.5, 11.5)
13
14
10
175.9, C
13.3, CH3 0.90 d (6.8)
132.4, C
175.9, C
13.1, CH3 0.89 d (6.9)
132.2, C
20,60
30,50
40
131.0, CH 8.17 d (8.0)
130.0, CH 7.54 t (8.0)
134.5, CH 7.64 t (8.0)
168.0, C
93.7, CH
82.8, CH
77.9, CH
70.9, CH
76.3, CH
130.8, CH 8.17 d (8.0)
130.0, CH 7.57 t (8.0)
134.5, CH 7.65 t (8.0)
167.9, C
94.0, CH
84.0, CH
77.6, CH
70.8, CH
78.9, CH
70
100
5.57 d (8.2)
5.59 d (8.1)
5.58 d (8.2)
94.4, CH
82.7, CH
77.3, CH
70.6, CH
5.50 d (7.7)
3.44 dd (7.7, 8.4)
3.60 brd (8.4)
3.63 m
200
3.42 dd (8.2, 9.2)
3.62 dd (9.2, 9.2)
3.46 dd (9.2, 9.2)
3.60 m
3.45 dd (8.1, 9.0)
3.63 dd (9.0, 9.0)
3.45 dd (9.0, 9.0)
3.39 m
3.28 dd (8.2, 9.0)
3.57 dd (9.0, 9.0)
3.43 dd (9.0, 9.0)
3.38 m
300
400
500
67.5, CH2 3.34a
3.95 dd (3.9, 11.4)
600
64.5, CH2 4.28a
4.42 dd (2.0,12.3)
106.0, CH 4.21 d (7.6)
62.3, CH2 3.73 dd (5.2, 12.3)
3.87 dd (2.3,12.3)
105.7, CH 4.29 d (7.8)
62.2, CH2 3.74 dd (5.0, 12.0)
3.88 dd (2.0,12.0)
106.9, CH 3.95 d (7.7)
1000
2000
3000
4000
5000
105.8, CH 4.20 d (7.6)
76.0, CH
78.0, CH
71.2, CH
77.9, CH
3.11 dd (7.6, 9.1)
76.0, CH
77.6, CH
71.2, CH
75.4, CH
3.13 dd (7.8, 9.0)
3.27 dd (9.0, 9.0)
3.22 dd (9.0, 9.0)
3.09 m
75.9, CH
77.5, CH
71.0, CH
3.07 dd (7.7, 9.1)
3.18 dd (9.1, 9.1)
3.31a
75.8, CH
77.7, CH
70.7, CH
77.7, CH
3.14 dd (7.6, 9.2)
3.27 dd (9.2, 9.2)
3.27 m
3.24 dd (9.1, 9.1)
3.23 ma
2.82 ddd (2.1, 4.3, 9.2)
67.1, CH2 2.83 dd (11.1, 11.1)
3.70 dd (5.3, 11.1)
2.81 ddd (2.3, 4.2, 9.6)
6000
62.1, CH2 3.58 m
64.6, CH2 4.17 m
62.0, CH2 3.59 dd (5.3, 12.3)
3.65 dd (2.2, 12.3)
21.0, CH3 2.10 s
172.8, C
21.0, CH3 1.98 s
173.1, C
CH3CO
CH3CO
Data were recorded at 150 MHz for 13C and 600 MHz for 1H.
a
Overlapped.
HO
O
Compared to 6, phyllaemblicin H7 (7) had one more glucopyra-
nosyl unit (anomeric proton at dH 4.51, 1H, d, J = 7.5 Hz, H-1000)
attached to the inner glucosyl C-200. This was confirmed by the
HMBC correlation from H-1000 to C-200. As for 8, it had one more ara-
binopyranosyl (dC 107.5, dH 4.50, 1H, d, J = 8.2 Hz) unit than com-
pound 7, based on extensive analysis of HSQC-TOCSY and 1H-1H
COSY spectra. The additional arabinosyl group in 8 was connected
to C-2000 of the middle glucosyl, taking into account the HMBC cor-
relation from H-10000 to C-2000. The anomeric free glucosyl unit in
OH
O
O
O
O
O
O
H
O
O
O
O
HO
HO
HO
HO
HO
O
OH
both 7 and 8 mainly existed as
the substitution at the C-2 position.
a-form (Tables 2 and 3), due to
Fig. 2. 1H–1H COSY (—) and HMBC (?) correlations of 1.
Phyllaemblicin H9 (9) had the same molecular formula
C33H44O19 as phyllaemblicin B (15), on the basis of HRESIMS.
proton integral suggested that the hexosyl moiety existed as a mix-
ture of
system of dH 5.11 (H-100)/H-200/H-300/H-400/H-500/H-600 was con-
structed for the -isomer. The large coupling constants (Table 2)
for H-200, H-300, and H-500 suggested that it was an
-glucopyranosyl.
a
and b isomers. In the 1H–1H COSY spectrum, a proton spin
However, the 1H NMR spectrum (Table 3) of 9 displayed only one
anomeric proton for b-glucopyranosyl (dH 3.91, 1H, d, J = 7.8 Hz,
H-1000). H-100 was assigned at dH 4.83 based on its HMBC correlation
with C-13 of the aglycone. This oxymethine proton (dH 4.83) was
obviously involved in the proton spin system for (H-100)/H-200/H-
300/H-400, and H-100/H-600/H-500. Together with the HMBC correlations
from H-300 to C-400 and from H-400 to C-200, an inositol moiety could
be constructed, whose relative configuration was determined by its
coupling constants from H-100 to H-600 (Table 3) and it was assigned
as myo-inositol. The HMBC correlation from H-1000 to C-200
a
a
In the same way, the resonances for the b-isomer were also com-
pletely assigned. The down-field chemical shift (dC 65.0) suggested
that C-600 in the glucosyl group was substituted. This was further
confirmed by HMBC correlations from H-600 to C-13 of the aglycone.
Therefore, the glucosyl moiety in 6 was attached to C-13 through
the C-600 hydroxyl group.

126
J.-J. Lv et al. / Phytochemistry 117 (2015) 123–134
Fig. 3. HPLC chromatograms of chiral separation of monosaccharides from hydrolysis of compounds 3, 4, 7 and 10. (PMP: 1-phenyl-3-methyl-5-pyrazolone).
confirmed that the terminal glucosyl unit was connected to myo-
inositol C-200. Accordingly, the saccharide part of 9 was elucidated
as myo-inositol-2-O-b-glucopyranosyl.
400/H-300/H-200/H-100 was established, and the inner b-glucosyl was
constructed. The terminal glucopyranosyl unit with an anomeric
a
center was also constructed. On the basis of the HMBC correlation
The MS and NMR data (Table 3) of phyllaemblicin H10 (10) indi-
cated the presence of two hexosyl and one pentosyl units, includ-
ing two glucopyranosyl [anomeric protons at dH 5.63 and 4.53
(each 1H)] and one xylopyranosyl (anomeric proton at dH 4.63
(1H, d, J = 7.5 Hz)) moieties, which was confirmed by HSQC-
TOCSY and 1H–1H COSY experiments. The HMBC correlation from
one glucosyl anomeric proton at dH 5.63 to the aglycone C-13 (dC
175.7) allowed assignment of this glucosyl at the inner position.
The chemical shifts of the inner glucosyl C-200 and C-300 shifted to
dC 79.8 and 87.7, suggesting that both C-200 and C-300 in 10 were
substituted. In the HMBC spectrum, the signals of H-1000 (dH 4.53)
of the second glucosyl group and H-10000 (dH 4.63) of the xylosyl
group were correlated with the inner glucosyl C-200 (dC 79.8) and
C-300 (dC 87.7), respectively. Based on the above evidence, the sugar
moiety of 10 was elucidated as S12. Acid hydrolysis followed by
chiral separation analysis further determined the absolute config-
from H-100 to the C-2000 (dC 82.7), the sugar moiety of 11 was deter-
mined as S13
.
The 1D NMR spectra and HRMS of phyllaemblicin H12 (12)
indicted that the sugar moiety was comprised of three hexosyl
and one pentosyl units. Based on the HSQC-TOCSY and 1H–1H
COSY spectra measured on an 800 MHz NMR spectrometer, four
proton spin systems for three b-glucopyranosyl and one
a-ara-
binopyranosyl groups were successfully constructed (Fig. 4),
whose corresponding carbon signals were also unambiguously
assigned. In the HMBC spectrum of 12, H-100 (dH 5.60, d,
J = 8.0 Hz) of the inner glucosyl, H-1000 (dH 4.14, d, J = 8.0 Hz) of
the middle glucosyl, H-10000 (dH 4.64, d, J = 8.0 Hz) of the terminal
arabinosyl, and H-100000 (dH 4.62, d, J = 8.0 Hz) of the terminal gluco-
syl were correlated respectively with C-13 (dC 175.9) of the agly-
cone, C-200 (dC 84.0) of the inner glucosyl, C-2000 (dC 82.4) and C-3000
(dC 87.0) of the middle glucosyl. Thus, the sugar moiety in 12
uration of the monosaccharides as
D-glucose and
D-xylose, which
was constructed as S14.
were the same as those for 3 (Fig. 3).
Phyllaemblicins H1–H12 (1–12) had the same aglycone as phyl-
laemblic acid, whose relative configurations were established on
the basis of their proton coupling constants and ROESY correlations
(Zhang et al., 2000b). Taking compound 5 as an example, the small
coupling constants of H-1 and H-5 suggested both protons were in
an equatorial orientation. The coupling constants of H-3 (tt, J = 3.0,
13.0 Hz) indicated that H-3 was axially orientated. In the chair
Phyllaemblicin H11 (11) had the same molecular formula
33H44O19 as 7. The 1D NMR spectra were also comparable to those
C
of 7. In the HMBC spectrum, the signal of H-600 (dH 4.23 and 4.38) of
one glucosyl unit showed correlations with the aglycone C-13 (dC
175.9). According to the HSQC-TOCSY and 1H–1H COSY correla-
tions, the proton spin system of dH 4.23 and 4.38 (H-600)/H-500/H-

J.-J. Lv et al. / Phytochemistry 117 (2015) 123–134
127
Table 2
13C and 1H NMR Spectroscopic data for compounds 5–7 in CD3OD (d in ppm).
No.
5
6b
7
a
c
a
c
dC
dH
dC
dH
dC
dH
1
2
71.6, CH
32.5, CH2
3.92 brs
71.1, CH
33.1, CH2
3.92 brs
71.6, CH
33.1, CH2
3.92 brs
1.74 ddd (2.5, 12.5, 14.0)
1.95 m
2.86 tt (3.5, 12.5)
1.90 ddd (3.7, 13.0, 14.8)
2.33 m
1.74 ddd (2.5, 13.0 14.5)
1.99 dd (3.0, 14.5)
2.90 tt (3.0, 13.0)
1.89 ddd (4.0, 13.0, 14.5)
2.32d
1.74 ddd (2.5, 13.0, 14.5)
1.96 brd (14.5)
2.85 m
3
4
32.3, CH
29.4, CH2
32.1, CH
28.8, CH2
32.1, CH
28.8, CH2
1.90d
2.32d
5
6
7
8
9
76.4, CH
75.6, C
213.8, C
100.7, C
32.8, CH2
4.28 brs
76.3, CH
75.6, C
213.8, C
100.5, C
32.6, CH2
4.30 brs
76.4, CH
75.6, C
213.9, C
100.5, C
32.5, CH2
4.28 brs
1.97 dd (3.5, 14.5)
2.30 dd (3.5, 14.5)
5.34 brs
2.00 dd (1.5, 15.0)
2.31d
5.35 brs
2.21 m
3.62 dd (4.4, 11.3)
4.05 dd (11.3, 11.3)
1.99 dd (2.5, 14.5)
2.32 m
5.36 brs
10
11
12
71.2, CH
34.5, CH
63.6, CH2
71.1, CH
34.2, CH
63.4, CH2
71.1, CH
34.3, CH
63.4, CH2
2.17 m
2.19 m
3.57a
3.60d
4.03 dd (11.5, 11.5)
4.04 dd (11.4, 11.4)
13
14
10
176.1, C
13.3, CH3
132.2, C
131.0, CH
130.0, CH
134.5, CH
168.2, C
177.1, C
13.1, CH3
132.0, C
130.9, CH
129.6, CH
134.4, CH
168.0, C
a
177.2, C
13.1, CH3
132.0, C
130.8, CH
129.7, CH
134.4, CH
168.0, C
0.89 d (6.9)
0.94 d (6.9)
0.99 d (6.9)
20,60
30,50
40
8.16 d (8.0)
7.53 t (8.0)
7.63 t (8.0)
8.16 d (8.2)
7.60 t (8.2)
7.65 t (8.2)
8.17 d (8.0)
7.57 t (8.0)
7.65 t (8.0)
70
b
a
b
100
200
3000
400
500
600
93.8, CH
83.4, CH
77.8, CH
76.1, CH
74.5, CH
18.2, CH2
5.52 d (8.1)
93.9, CH
73.8, CH
74.7, CH
71.5, CH
70.7, CH
65.0, CH2
98.3, CH
76.1, CH
77.8, CH
71.3, CH
75.4, CH
65.0, CH2
5.11 d (3.8)
4.52 d (7.8)
93.6, CH
83.0, CH
73.6, CH
71.4, CH
70.7, CH
64.8, CH2
5.38 d (3.5)
3.40 dd (8.1, 9.0)
3.55 dd (9.0, 9.0)
3.11 dd (9.0, 9.0)
3.42 m
3.37 dd (3.8, 9.3)
3.71 dd (9.3, 9.3)
3.31d
3.99 ddd (2.0, 5.3, 9.3)
4.32 dd (2.0, 11.9)
4.25 dd (5.3, 11.9)
3.16 dd (7.8, 9.2)
3.37 dd (9.2, 9.2)
3.31d
3.48 ddd (2.1, 5.6, 9.2)
4.21 dd (2.1, 11.9)
4.36 dd (5.7, 11.9)
3.48 dd (3.5, 9.5)
3.88 dd (9.5, 9.5)
3.38d
4.00 ddd (2.0, 5.0, 9.7)
4.32 dd (2.0, 11.8)
4.26 dd (5.0, 11.8)
4.51 d (7.5)
1.33 d (6.3)
1000
2000
3000
4000
5000
6000
106.1, CH
76.5, CH
77.9, CH
70.9, CH
78.0, CH
63.6, CH2
4.19 d (8.0)
3.09 dd (8.0, 8.8)
3.23 m
106.0, CH
75.4, CH
77.9, CH
71.4, CH
77.9, CH
62.6, CH2
3.30 dd (7.5, 9.0)
3.31d
3.23 m
3.34 dd (9.0, 9.0)
3.31d
3.68 dd (5.0, 12.0)
3.87 dd (2.5, 12.0)
2.78 ddd (2.3, 4.3, 9.5)
3.56d
3.62d
a
b
c
Data were recorded at 100 MHz.
Data were recorded at 600 MHz for 1H NMR and 150 MHz for 13C NMR.
Data were recorded at 800 MHz.
d
Overlapped.
conformation of the A ring (Fig. 5), H-3 was located at the opposite
face of H-1 and H-5. This was confirmed by the ROESY correlations
of H-3 with H-2 eq (dH 1.99) and H-4 eq (dH 2.32), and strong
ROESY correlations of H-1 with H-2ax, and H-5 with H-4ax. The
large coupling constant (11.5 Hz) of JH-12,H-11 indicated an axial ori-
entation of H-11, and a broad singlet of H-10 suggested its equato-
rial orientation locating at the same side as H-11 in a chair shaped
tetrahydropyran ring C. Together with the ROESY correlations of H-
4 eq and H-3 with the benzoyl aromatic protons (dH 8.16), the rel-
ative configurations of the aglycone of 5 were established as shown
in Fig. 5.
by the configuration at C⁄-10. The strong negative Cotton effect at
320 nm was produced by the n–p⁄ excitation of the carbonyl (C-7)
group, which was adjacent to the stereo centers of C⁄-6 and C⁄-8.
The experimental ECD curves agreed well with the calculation
results. Thus, the absolute configurations of the aglycone moiety
of compounds 1–12 were determined as 1S, 3S, 5R, 6R, 8S, 10S, 11R.
Phyllaemblicin H13 (13) was obtained as a white powder, pos-
sessing a molecular formula of C38H52O24, as established from the
HRESIMS. Its NMR spectra (Table 4) resembled phyllaemblicin C
(16), except for signals arising from a benzoyl moiety. The mutu-
ally coupled four aromatic proton signals at dH 7.03 (1H, dd,
J = 1.0, 8.0 Hz, H-30), 7.59 (1H, ddd, J = 1.8, 8.0, 8.4 Hz, H-40), 7.06
(1H, ddd, J = 1.0, 8.0, 8.4 Hz, H-50), and 8.12 (1H, dd, J = 1.8,
8.0 Hz, H-60) established a 2-hydroxyl benzoyl moiety in 13,
instead of a benzoyl moiety in 16. The relative configuration of
13 was established by ROESY correlations and coupling constants
as for 5, and the absolute configuration was determined by ECD
calculations. Due to the different substitution in the benzoyl moi-
ety, the ECD curve of 13 differed from phyllaemblic acid. Though
the experimental ECD curve of 13 and the calculated ECD curve
of the aglycone of 13 did not match very well at 280–300 nm, there
was a weak peak at 287 nm in the experimental spectrum corre-
sponding to the peak between 280 and 300 nm in the calculated
ECD curve (Fig. 7 left). This was confirmed by comparing the
The absolute configurations of compounds 1–12 were deter-
mined by comparing their experimental ECD curves with the
experimental and calculated ECD curves of phyllaemblic acid
(Fig. 6). The conformer search was carried out using the mechanics
MMFF method. The resulting conformers (<20 kJ/mol) were re-op-
timized using DFT at the B3LYP-SCRF/6-311G (d,p) level. Ten of the
conformers with energy <2 kcal/mol were considered for ECD cal-
culation. These lower energy conformers differed in the orienta-
tions of the benzoyl moiety (Supplementary S-108 Fig. 4). The
calculated ECD spectra of the conformers were similar and compa-
rable to the averaged ECD spectrum (Supplementary S-108 Fig. 5).
The Cotton effects at 230 nm and 250 nm were produced by
excitation of the benzoate moiety, and their signs were determined
p–
p⁄

128
J.-J. Lv et al. / Phytochemistry 117 (2015) 123–134
Table 3
13C and 1H NMR Spectroscopic data for compounds 8–11 in CD3OD (d in ppm).
No.
8
9b
10a
11
a
c
b
c
dC
dH
dC
dH
dC
dH
dC
dH
1
2
71.5, CH
3.91 brs
71.8, CH
3.93 brs
71.6, CH
3.94 brs
71.2, CH
3.92 brs
33.1, CH2 1.73 ddd (1.9, 13.0, 14.6) 31.6, CH2 1.78 ddd (3.0, 13.0 15.2)
32.2, CH2 1.79 ddd (2.5, 13.1, 14.5) 33.0, CH2 1.76 ddd (2.7, 13.0, 14.0)
1.92d
2.84 m
2.00d
2.92 tt (2.4, 13.0)
2.06 dd (2.5, 14.5)
2.91 tt (3.0, 13.1)
1.98 dd (3.2, 14.0)
2.83 tt (3.3, 13.0)
3
4
32.2, CH
32.2, CH
32.3, CH
32.1, CH
28.8, CH2 1.90d
29.8, CH2 1.87 ddd (3.4, 13.0, 14.4) 29.3, CH2 1.92 ddd (3.6, 13.1, 14.5) 29.0, CH2 1.93 ddd (3.3, 13.0, 14.3)
2.30d
4.25 brs
2.62 brd (14.4)
4.33 brs
2.35 brd (14.5)
4.31 brs
2.28 brd (14.3)
4.29 brs
5
6
7
8
9
76.3, CH
75.6, C
213.7, C
76.7, CH
75.6, C
213.9, C
100.6, C
32.9, CH2 2.04 dd (2.4, 15.0)
2.26 dd (3.7, 15.0)
76.3, CH
75.5, C
213.9, C
100.6, C
32.7, CH2 2.03 dd (4.0, 14.6)
2.28 dd (3.0, 14.6)
76.4, CH
75.7, C
213.8, C
100.5, C
32.4, CH2 1.99 dd (3.4, 14.9)
2.35 dd (3.3, 14.9)
100.6, C
32.5, CH2 1.97d
2.30d
10
11
12
71.2, CH
34.3, CH
5.33 brs
2.18d
70.9, CH
34.4, CH
5.37 brs
2.20 m
71.0, CH
34.3, CH
5.39 brs
71.2, CH
34.3, CH
5.33 dd (3.3, 6.4)
2.20d
2.21d
63.5, CH2 3.57d
63.4, CH2 3.62d
63.5, CH2 3.59d
63.6, CH2 3.62d
4.01d
4.07 dd (11.4, 11.4)
4.06 dd (11.4, 11.4)
4.04 dd (11.1, 11.1)
13
14
10
177.3, C
176.9, C
13.1, CH3 0.89 d (7.1)
132.1, C
131.0, CH 8.21 d (8.3)
130.0, CH 7.61 t (8.3)
134.9, CH 7.86 t (8.3)
167.9, C
175.7, C
13.1, CH3 0.92 d (7.0)
132.1, C
130.8, CH 8.18 d (8.0)
129.9, CH 7.58 t (8.0)
134.5, CH 7.68 t (8.0)
167.9, C
175.9, C
13.1, CH3 0.95 d (7.1)
132.1, C
129.6, CH 8.17 d (8.2)
130.9, CH 7.55 t (8.2)
134.4, CH 7.68 t (8.2)
167.9, C
13.0, CH3 0.91 d (6.8)
132.1, C
20,60 130.7, CH 8.14 d (8.0)
30,50 129.6, CH 7.53 d (8.0)
40
70
134.4, CH 7.64 t (8.0)
168.1, C
100
200
300
400
500
600
93.5, CH
84.4, CH
73.4, CH
71.2, CH
70.8, CH
5.33 d (3.2)
75.1, CH
83.9, CH
73.6, CH
73.4, CH
73.5, CH
4.83 dd (9.6, 9.6)
93.9, CH
79.8, CH
87.7, CH
69.5, CH
78.4, CH
5.63 d (8.0)
3.67 dd (8.0, 9.0)
3.76d
105.9, CH 4.51 d (7.8)
3.37d
3.78 dd (9.6, 9.6)
3.61 dd (2.6, 9.6)
4.08 dd (2.6, 2.6)
3.47 dd (2.6, 9.6)
75.2, CH
77.7, CH
71.8, CH
75.5, CH
3.32 dd (7.8, 9.7)
3.41 dd (9.7, 9.7)
3.28 dd (9.7, 9.7)
3.48 ddd (2.1, 7.2, 9.7)
3.85 dd (9.4, 9.4)
3.39d
3.95 ddd (2.3, 5.4, 9.6)
3.52 dd (9.0, 9.0)
3.44 m
64.8, CH2 4.26 m
72.7, CH2 3.93 dd (9.6, 9.6)
62.2, CH2 3.77d
64.5, CH2 4.23 dd (7.2, 11.8)
4.38 dd (2.1, 11.8)
3.92 dd (1.5, 12.1)
104.3, CH 4.53 d (8.0)
1000
2000
3000
4000
500000
6000
105.1, CH 4.54 d (8.2)
106.5, CH 3.91 d (7.8)
93.8, CH
82.7, CH
73.6, CH
71.6, CH
72.9, CH
5.27 d (3.6)
85.4, CH
77.5, CH
71.2, CH
77.7, CH
3.42 dd (8.2, 9.1)
75.8, CH
78.2, CH
70.4, CH
77.3, CH
3.08 dd (7.8, 9.4)
3.16 dd (9.4, 9.4)
3.24 dd (9.4, 9.4)
2.36 m
75.6, CH
78.2, CH
71.0, CH
77.3, CH
3.04 dd (8.0, 9.0)
3.39 dd (3.6, 9.3)
3.86 dd (9.3, 9.3)
3.38d
3.55 dd (9.1, 9.1)
3.23 dd (9.0, 9.0)
3.38d
3.26d
3.29 dd (2.5, 5.5, 9.1)
2.84 ddd (2.4, 4.2, 9.0)
3.79 ddd (2.5, 6.9, 9.4)
62.5, CH2 3.66d
3.83d
61.6, CH2 3.39d
62.3, CH2 3.55d
62.5, CH2 3.71d
3.79d
3.50 dd (2.8, 12.7)
3.61d
10000
20000
30000
40000
50000
107.5, CH 4.50 d (8.2)
105.1, CH 4.63 d (7.5)
73.7, CH
74.3, CH
69.5, CH
3.65d
75.4, CH
78.4, CH
71.2, CH
3.29d
3.31d
3.28d
3.56 dd (4.8, 9.3)
3.81 brs
67.7, CH2 3.64d
3.98d
67.2, CH2 3.28d
3.94d
a
b
c
Data were recorded at 125 MHz for 13C or 500 MHz for 1H.
Data were recorded at 150 MHz for 13C or 600 MHz for 1H.
Data were recorded at 800 MHz.
d
Overlapped.
HO
Glc
O
HO
HO
Glc
HO
HO
HO
HO
O
O
O
HO
O
Glc
O
OH
O
HO
OH
Ara
HO
Fig. 4. Key 1H–1H COSY (—) and HMBC (?) correlations of S14.
Fig. 5. Key ROESY correlations in the lowest energy conformer of 5.
experimental CD curve of 13 with those of phyllaemblic acid, 7 and
10 with the benzoyl chromophore (Fig. 7 right). It is obvious that
the TDDFT computation overestimated the intensity of the peak
at 280–300 nm. However, it could still predict the tendency of
the experimental ECD curve between 280 and 300 nm. Thus, the
absolute configuration of 13 was determined (Fig. 7).
The molecular formula C33H44O19 of phyllaemblicin H14 (14)
was deduced from the HRESIMS. Its NMR spectra were similar to
those of 40-hydroxy phyllaemblicin B (15), except that a methine
at dC 48.6 in 14 replaced the oxygenated quaternary carbon of C-

J.-J. Lv et al. / Phytochemistry 117 (2015) 123–134
129
Fig. 6. Experimental ECD curves of compounds 1–12 and 14, and the calculated ECD curve (TD-DFT/B3LYP-SCRF/6-311G (d,p)) of their aglycones.
Table 4
13C and 1H NMR Spectroscopic data for compounds 12–14 in CD3OD (d in ppm).
No.
12b
13a
14a
dC
dH
dC
dH
dC
dH
1
2
71.6, CH
31.9, CH2
3.95 brs
71.5, CH
31.7, CH2
3.94 brs
65.9, CH
35.0, CH2
4.32 brs
1.81 ddd (2.7, 13.4, 15.1)
2.01 brd (15.1)
2.94 tt (3.2, 13.4)
1.90 ddd (4.6, 13.4, 14.6)
2.45 brd (14.6)
4.33 brs
1.80 ddd (3.1, 13.0, 14.3)
1.60 ddd (2.2, 13.0 14.5)
1.98c
2.16c
3
4
32.3, CH
29.9, CH2
32.2, CH
29.6, CH2
2.95 tt (2.4, 13.0)
1.86 dd (5.3, 13.0, 15.0)
2.49 brd (15.0)
4.31 brs
31.8, CH
31.2, CH2
2.96 tt (3.2, 13.0)
1.86 (ddd, 4.2, 13.0, 14.5)
2.45 brd (14.5)
4.59 brs
5
6
7
8
9
76.5, CH
75.5, C
213.6, C
100.6, C
33.0, CH2
76.6, CH
75.5, C
213.5, C
100.5, C
33.0, CH2
72.6, CH
48.6, CH
212.2, C
101.0, C
32.6, CH2
2.54 t (5.7)
2.04 dd (3.6, 14.5)
2.27 dd (3.0, 14.5)
5.41 brd (3.3, 6.4)
2.20 m
3.60 m
4.05 dd (11.3, 11.3)
2.05 dd (3.2, 15.0)
2.22 dd (3.1, 15.0)
5.54 brs
2.00 dd (3.0, 14.7)
2.16c
10
11
12
70.9, CH
34.5, CH
63.5, CH2
71.4, CH
34.4, CH
63.4, CH2
70.6, CH
34.5, CH
63.8, CH2
5.34 dd (3.0, 5.8)
2.21c
2.16c
3.61c
3.59 dd (4.8, 11.5)
4.02 dd (11.5, 11.5)
3.98c
13
14
10
175.9, C
13.3, CH3
132.2, C
176.2, C
13.2, CH3
114.7, C
161.9, C
119.0, CH
137.4, CH
121.2, CH
132.4, CH
170.3, C
93.6, CH
84.7, CH
77.5, CH
70.1, CH
79.1, CH
62.4, CH2
176.1, C
13.1, CH3
122.9, C
133.3, CH
116.5, CH
163.4, C
116.5, CH
133.3, CH
168.1, C
93.7, CH
83.3, CH
77.9, CH
70.6, CH
79.1, CH
62.3, CH2
0.89 d (6.9)
0.88 d (7.5)
0.90 d (7.0)
20
130.2, CH
131.0, CH
134.7, CH
131.0, CH
130.2, CH
167.8, C
93.8, CH
84.0, CH
77.5, CH
70.1, CH
79.1, CH
62.4, CH2
8.21 d (8.2)
7.62 t (8.2)
7.72 t (8.2)
7.62 t (8.2)
8.21 d (8.2)
8.08 d (8.0)
6.94 d (8.0)
30
7.03 dd (1.0, 8.0)
40
7.59 ddd (1.8, 8.0, 8.4)
7.06 ddd (1.0, 8.0, 8.4)
8.12 dd (1.8, 8.0)
50
6.94 d (8.0)
8.08 d (8.0)
60
70
100
200
300
400
500
600
5.60 d (8.0)
5.58 d (8.1)
5.62 d (8.0)
3.32c
3.31c
3.51 dd (8.0, 9.1)
3.63 dd (9.1, 9.1)
3.55 dd (9.1, 9.1)
3.42c
3.80 dd (5.8, 12.1)
3.93 dd (2.0,12.1)
4.22 d (7.8)
3.13 dd (7.8, 9.0)
3.27 dd (9.0, 9.0)
3.27 dd (9.0, 9.0)
2.73 ddd (3.4, 3.5, 9.0)
3.56c, 3.61c
3.57 dd (8.7, 8.7)
3.56 dd (9.5, 9.5)
3.50 dd (9.5, 9.5)
3.40 m
3.75 dd (5.1, 12.2)
3.90 dd (2.3, 12.2)
4.18 d (7.9)
3.53c
3.41 ddd (2.3, 5.6, 8.5)
3.78 dd (5.6, 12.3)
3.92 brd (12.3)
4.14 d (8.0)
3.45 dd (8.0, 9.3)
3.50 dd (9.3, 9.3)
3.37 dd (9.3, 9.3)
2.63 ddd (9.3, 2.5, 2.5)
3.51c, 3.56c
1000
2000
3000
4000
5000
6000
10000
20000
30000
40000
50000
104.9, CH
82.4, CH
87.0, CH
69.1, CH
76.9, CH
62.1, CH2
106.1, CH
73.4, CH
74.5, CH
69.9, CH
67.0, CH2
105.2, CH
85.4, CH
77.8, CH
70.1, CH
77.1, CH
61.5, CH2
107.4, CH
73.9, CH
74.2, CH
69.7, CH
67.8, CH2
106.3, CH
76.0, CH
77.6, CH
70.6, CH
77.6, CH
61.7, CH2
3.25 dd (7.9, 9.0)
3.33c
3.30 dd (9.5, 9.5)
2.55 dt (9.5, 3.3)
3.53 m
4.64 d (8.0)
4.48 d (7.2)
3.62 dd (8.0, 9.6)
3.54 dd (3.5, 9.6)
3.85 m
3.63 brd (12.3)
3.98 dd (2.5, 12.3)
4.62 d (8.0)
3.65 dd (7.2, 9.2)
3.56 dd (3.7, 9.2)
3.82 brs
3.63 dd (1.6, 12.7)
3.99c
100000
200000
300000
400000
500000
600000
104.5, CH
75.4, CH
78.4, CH
71.6, CH
78.3, CH
62.6, CH2
3.26 dd (8.0, 9.1)
3.39 dd (9.1, 9.1)
3.31 dd (9.1, 9.1)
3.40 m
3.66c, 3.92c
a
b
c
Data were recorded at 150 MHz for 13C or 600 MHz for 1H.
Data were recorded at 200 MHz for 13C or 800 MHz for 1H.
Overlapped.

130
J.-J. Lv et al. / Phytochemistry 117 (2015) 123–134
Fig. 7. Experimental ECD curves of phyllaemblic acid, compounds 7, 10, and 13, and the calculated ECD curve (TD-DFT/B3LYP-SCRF/6-311G (d,p)) of the aglycone of 13.
6 (dC 75.9) in 15. This led to up-field shifts of C-1 and C-5, and a
down-field shift of C-2 in 14. The methine at dC 48.6 was assigned
as C-6, based on the 1H–1H COSY correlations of its corresponding
proton (dH 2.54, 1H, t, J = 5.7 Hz, H-6) with H-1 and H-5. The cou-
pling constant of H-6 and its ROESY correlations with H-1 and H-
5 suggested that H-6 was b-orientated. Therefore, the relative con-
figurations of rings A and C were determined to be the same with
those of 5. Moreover, compound 14 had a similar ECD curve to that
of phyllaemblic acid. Thus, the absolute configuration of 14 was
established as 1S, 3S, 5R, 6R, 8S, 10S, 11R.
pre-column derivatization/chiral separation/DAD detection
method takes advantages of low sample consumption (0.5–
1.0 mg) and simple operation, by which the absolute configura-
tions of the monosaccharides can be determined rapidly. Using
the TDDFT calculated ECD method, the absolute configurations of
the aglycones were established. Moreover, the main constituents
of this plant, phyllaemblicin B (15) and glochicoccinoside D (17),
showed potential anti-viral activities against H3N2 and EV71.
Phyllaemblicin H1 (1) displayed cytotoxicity against human caner
cell lines A-549 and SMMC-7721.
2.2. Antiviral activities
4. Experimental
Isolated compounds (2–3, 6–8, 11–12, 15–17) with an amount
greater than 10 mg were evaluated for their antiviral activities
against enterovirus 71 (EV71), influenza A virus strain H3N2, her-
pes simplex virus (HSV-1), and coxsackie virus B3 (CVB3) (Table 5).
Compound 6 displayed inhibitory activity against HSV-1, whereas
8 and 12 showed anti CVB3 activities. The known compounds,
phyllaemblicin B (15) and glochicoccinoside D (17), displayed
anti-viral activities against influenza A virus strain H3N2, with
4.1. General procedures
IR and UV spectra were measured on a Bruker Tensor 27 spec-
trometer with KBr pellets and Shimadzu UV 2401PC, respectively.
1D and 2D NMR spectra were run on Bruker AM-400, DRX-500 and
AVANCE III-600 and AVANCE-800 NMR spectrometers operating at
400, 500 and 600 and 800 MHz for 1H, and 100, 125 and 150 and
200 MHz for 13C, respectively. Coupling constants are expressed
in Hertz and chemical shifts are given on a ppm scale with solvents
as internal standard. ESI-MS and HRESIMS were measured at
Bruker HCT/Esquire and Agilent G6230. ECD and optical rotations
were detected at Applied Photophysics and Jasco P-1020. The high
performance liquid chromatography apparatus was an Agilent
1260 with a DAD detector. Column chromatography (CC) was per-
formed with Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd.
Uppsala, Sweden), Diaion HP20SS (Mitsubishi Chemical Co.,
Tokyo, Japan), MCI-gel CHP20P (Mitsubishi Chemical Co., Tokyo,
IC50 values of 2.6 0.7 and 4.5 0.6
to the positive control ribavirin (IC50 = 25.0 0.5
l
g/ml, respectively, compared
g/ml).
l
Compounds 16 and 17 also exhibited inhibitory activities against
hand, foot and mouth virus EV71, with IC50s of 2.6 0.8 and
2.6 0.7
lg/ml, respectively, whereas the positive control ribavirin
gave an IC50 = 158.7 3.3
lg/ml. Compounds 2, 3, 7 and 11 had no
inhibitory activities towards the tested viruses with concentrations
above their CC50 values.
2.3. Cytotoxicity activities
Japan), RP-8 gel (40–60 lm, Merck, Darmstadt, Germany),
Toyopearl HW-40C (TOSOH, Japan), Chromatorex ODS (Fuji
Silysia Chemical Co. Ltd), silica gel (200–300 mesh, Qingdao
Hailang Group Co., Ltd. Qingdao, People’s Republic of China) and
All isolates were evaluated for their cytotoxicities against five
human cancer cell lines, i.e., lung cancer (A-549), human myeloid
leukemia (HL-60), breast cancer (MCF-7), hepatocellular carcinoma
(SMMC-7721), and colon cancer (SW480). Cis-platin was a broad-
spectrum first-line anti-tumor drug, and was utilized as a positive
control. Only phyllaemblicins H1–H2 (1–2) showed cytotoxicities
(Table 6). Especially 600-acetylated phyllaemblicin B (2) selectively
inhibited the growth of A-549 and SMMC-7721 cell lines, with IC50
a 250 ꢁ 9.4 mm, i.d., 5
l
m Sunfire C18 column (Waters), respec-
tively. A CHIRALPAK AD-H column (i.d., 5
lm, 4.6 ꢁ 250 mm,
Daicel corporation) was used for chiral separation. TLC was carried
out on precoated silica gel GF254 plates, which were visualized by
ultraviolet and spraying with 10% H2SO4 in EtOH solution.
Quantum chemical calculations were carried out at HPC Center,
Kunming Institute of Botany, CAS, China.
values of 4.7 0.7 and 9.9 1.3 lM, respectively. Acetylation of the
saccharide moiety of norbisabolane sesquiterpenoid glycosides can
thus significantly improve their cytotoxicities.
4.2. Plant materials
3. Conclusion
Roots of P. emblica were collected in Baoshan City, Yunnan
Province, People’s Republic of China, and identified by Prof. C. R.
Yang (Kunming Institute of Botany, Chinese Academy of
Sciences). A voucher specimen (KIB-ZL-0100020) is deposited in
the State Key Laboratory of Phytochemistry and Plant Resource
Fourteen new norbisabolane sesquiterpenoid glycosides, phyl-
laemblicins H1–H14, were discovered from the roots of P. emblica.
These compounds have the rare norbisabolane sesquiterpenoid
aglycone and various saccharide moieties (S1–S14). The established

J.-J. Lv et al. / Phytochemistry 117 (2015) 123–134
131
Table 5
Anti-viral activities of the isolated compounds (IC50 l
g/ml).a
Compounds
H3N2
IC50
EV71
CVB3
IC50
HSV-1
IC50
CC50
IC50
CC50
CC50
CC50
6
8
12
15
16
17
Ribavirin
GH
Acyclovir
ND
ND
ND
2.6 0.7
NA
4.5 0.6
25.0 0.5
ND
ND
ND
ND
6.9 0.9
13.4 1.5
13.4 2.1
>500
ND
ND
ND
ND
NA
2.6 0.8
2.6 0.7
158.7 3.3
ND
ND
ND
ND
6.9 0.9
10.0 1.9
9.9 1.7
>500
ND
NA
26.7 2.4
106.8 3.7
55.1 0.7
6.9 0.9
13.4 1.5
13.4 2.1
ND
10.5 1.1
NA
NA
NA
NA
NA
ND
ND
26.7 2.4
76.0 4.8
21.0 1.8
NA
NA
NA
ND
27.2 0.8
ND
106.8 3.7
55.1 0.7
6.9 0.9
9.0 1.5
13.4 2.1
ND
>200
ND
ND
>200
ND
ND
ND
ND
0.033 0.007
NA: not active above the concentration of CC50. ND: not detected. GH: Guanidine hydrochloride.
a
Compound concentration reducing the viability of MDCK or Vero cells culture by 50%.
Table 6
a Sephadex LH-20 column, eluting with CH3OH:H2O (0:100 to
80:20), to afford Fr.D1–D4. Fr.D2 was purified by Chromatorex
ODS CC, eluted with CH3OH:H2O (30:70 to 80:20), to give 15
(38 g). Fr.E (24.4 g) was applied to a Sephadex LH-20 column,
eluted with CH3OH:H2O (0:100 to 80:20), to afford Fr.E1–Fr.E3.
Cytotoxic activities of compounds 1 and 2 (IC50
lM).
Compounds A-549
HL-60
MCF-7
SMMC-7721 SW480
1
2
4.7 0.7 18.9 1.1 18.8 0.7
12.3 0.3 28.6 1.0 >40
9.9 1.3
17.5 1.0
3.3 0.4
18.4 0.6
32.9 1.5
16.3 1.1
Cisplatin
3.6 0.5
1.0 0.2 13.0 0.8
Fr.E1 was fractionated on
CH3OH:H2O (30:70 to 80:20), to afford Fr.E1.1–Fr.E1.8. Fr.E1.2
was applied to Toyopearl HW-40C column, eluted with
a
RP-8 column, eluted with
a
CH3OH:H2O (0:100 to 30:70), to give Fr.E1.2.1–1.2.3. Fr.E1.2.2
was separated using silica gel CC and preparative HPLC
(CH3CN:H2O, 15:85 to 30:70) to afford 14 (6 mg) and 3 (10 mg).
Fr.F (24.4 g) was passed over a Sephadex LH-20 column, eluted
with CH3OH:H2O (0:100 to 80:20), to afford Fr.F1–Fr.F3. Fr.F1
was fractionated by RP-8 CC, eluted with CH3OH:H2O (30:70 to
80:20), to produce Fr.F1.1–1.9. Fr.F1.2 was subjected to silica gel
CC to afford Fr.F1.2.1–1.2.4, and Fr.F1.2.2 was purified by prepara-
tive HPLC, eluted with CH3CN:H2O (15:85 to 30:70), to yield 5
(3 mg). Fr.F1.3 was passed through a Toyopearl HW-40C column,
eluted with CH3OH:H2O (0:100 to 30:70), to give Fr.F1.3.1–1.3.5.
Fr.F1.3.2 was purified by preparative HPLC, eluted with
CH3CN:H2O (15:85 to 30:70), to yield 2 (17 mg). Fr.F1.3.4 was sub-
jected to preparative HPLC, eluted with CH3CN:H2O (15:85 to
30:70), to afford 1 (3 mg), 4 (2 mg) and 6 (21 mg).
in West China of Kunming Institute of Botany, Chinese Academy of
Sciences.
4.3. Extraction and isolation
Air dried roots of P. emblica (109 kg) were extracted with
EtOH:H2O (500 L, 70:30, v/v) under conditions of reflux for three
times (each time 2 h) to give an extract (7.8 kg), which was sus-
pended in H2O (22.5 L) and partitioned with n-BuOH. The organic
layer was concentrated in vacuo and subjected to Diaion HP20SS
CC, eluted with CH3OH:H2O (0:100 to 100:0), to afford four major
fractions (Fr.1–4). Fr.3 (400 g) was applied to a silica gel column,
gradient eluted with CHCl3:CH3OH:H2O (50:1:0 to 7:3:0.5) to give
seven sub-fractions (Fr.A–Fr.G). Fr.A was subjected to Sephadex
LH-20 CC, eluted with CH3OH:H2O (0:100 to 80:20) to afford
Fr.A1–Fr.A2. Fr.A1 (3.2 g) was passed through a MCI-gel CHP20P
column, eluted with CH3OH:H2O (30:70 to 90:10) to give four frac-
tions (Fr.A1.1–Fr.A1.4). Fr.A1.4 was purified by Toyopearl HW-40C
CC, eluted with CH3OH:H2O (0:100 to 30:70) to give 12 (15 mg).
Fr.B was passed through Sephadex LH-20 column eluting with
CH3OH:H2O (0:100 to 30:70), to afford Fr.B1–Fr.B2. Fr.B1 was frac-
tionated by MCI-gel CHP20P CC, eluted with CH3OH:H2O (30:70 to
80:20), to afford Fr.B1.1–Fr.B1.6. Fr.B1.2 was fractionated by
Toyopearl HW-40C CC, eluted with CH3OH:H2O (0:100 to 30:70),
to give Fr.B1.2.1–1.2.2, and Fr.B1.2.1 was purified by RP-8 CC,
eluted with CH3OH:H2O (30:70 to 80:20), to afford 11 (15 mg).
Fr.B1.5 was purified by Toyopearl HW-40C CC, eluted with
CH3OH:H2O (0:100 to 30:70) and preparative HPLC (CH3CN:H2O,
15:85 to 30:70), to afford 9 (2 mg). Fr.B1.6 was passed through a
Toyopearl HW-40C column, eluted with CH3OH:H2O (0:100 to
30:70) to give Fr.B1.6.1–1.6.4, and Fr.B1.6.2 was purified by RP-8
CC, eluted with CH3OH:H2O (30:70 to 80:20) to afford 16 (5.0 g).
Fr.B1.6.3 was applied to a RP-8 column, eluted with CH3OH:H2O
(30:70 to 80:20), to afford 8 (24 mg) and 13 (3 mg). Fr.C was passed
through a Sephadex LH-20 column, eluted with CH3OH:H2O (0:100
to 80:20), to afford Fr.C1–Fr.C4. Fr.C2 was applied to a RP-8 col-
umn, eluted with CH3OH:H2O (30:70 to 80:20) to produce
Fr.C2.1–2.6. Fr.C2.5 was purified by Toyopearl HW-40C CC, eluted
with CH3OH:H2O (0:100 to 30:70) to afford 17 (4.1 g). Fr.C2.6
was subjected to preparative HPLC (CH3CN:H2O, 15:85 to 30:70)
to give 7 (28 mg), and 10 (6 mg). Fr.D (73 g) was passed through
4.3.1. Phyllaemblicin H1 (1)
White amorphous powder; [
(MeOH) kmax (log ) 200.6 (1.10), 228 (1.11), 272 (0.15) nm; ECD
a]
25 = +6.4 (c 0.8, CH3OH); UV
D
e
(in MeOH, kmax [nm], /[mdeg]) 205 (3.9), 227 (ꢀ4.3), 245 (3.4),
322 (ꢀ4.3); IR (KBr) mmax 3431, 2925, 1719, 1280, 1078 cmꢀ1; For
1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) spec-
troscopic data, Table 1; MS (ESI): m/z 821 [M+Cl]ꢀ; HRMS (ESI):
m/z 831.2556 [M+HCOO]ꢀ (calcd for C36H47O22, 831.2565).
4.3.2. Phyllaemblicin H2 (2)
White amorphous powder; [
(MeOH) kmax (log ) 200.6 (1.08), 228 (1.08), 272 (0.19) nm; ECD
a]
25 = +4.7 (c 1.2, CH3OH); UV
D
e
(in MeOH, kmax [nm], /[mdeg]) 204 (5.2), 226 (ꢀ4.0), 246 (4.2),
322 (ꢀ4.7); IR (KBr) mmax 3431, 2925, 1719, 1280, 1078 cmꢀ1; For
1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) spec-
troscopic data, see Table 1; MS (ESI): m/z 821 [M+Cl]ꢀ; HRMS (ESI):
m/z 831.2556 [M+HCOO]ꢀ (calcd for C36H47O22, 831.2565).
4.3.3. Phyllaemblicin H3 (3)
White amorphous powder; [
(MeOH) kmax (log ) 200.0 (1.06), 228.6 (1.05) nm; ECD (in MeOH,
a]
25 = +10.4 (c 1.2, CH3OH); UV
D
e
kmax [nm], /[mdeg]) 205 (5.2), 226 (ꢀ4.6), 246 (3.8), 321 (ꢀ4.5);
IR (KBr) mmax 3431, 2925, 1719, 1280, 1078 cmꢀ1; For 1H NMR
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) spectroscopic

132
J.-J. Lv et al. / Phytochemistry 117 (2015) 123–134
data, see Table 1; MS (ESI): m/z 749 [M+Cl]ꢀ; HRMS (ESI): m/z
1077, 1040 cmꢀ1; For 1H NMR (CD3OD, 500 MHz) and 13C NMR
(CD3OD, 125 MHz) spectroscopic data, see Table 3; MS (ESI): m/z
911 [M+Cl]ꢀ; HRMS (ESI): m/z 921.2867 [M+HCOO]ꢀ (calcd for
759.2335 [M+HCOO]ꢀ (calcd for C33H43O20, 759.2353).
4.3.4. Phyllaemblicin H4 (4)
C39H53O25, 921.2881).
White amorphous powder; [
(MeOH) kmax (log ) 200.2 (1.13), 228.4 (1.13), 272.6 (0.18) nm;
a]
25 = +2.4 (c 0.6, CH3OH); UV
D
e
4.3.11. Phyllaemblicin H11 (11)
ECD (in MeOH, kmax [nm], /[mdeg]) 208 (2.2), 226 (ꢀ3.9), 246
White amorphous powder; [
(MeOH) kmax (loge) 201.4 (1.28), 227.2 (1.06), 274.0 (0.49) nm;
a
]
25 = ꢀ7.9 (c 0.3, CH3OH); UV
D
(3.0), 321 (ꢀ4.0); IR (KBr) mmax 3424, 2928, 1719, 1280,
1080 cmꢀ1
;
For 1H NMR (CD3OD, 600 MHz) and 13C NMR
ECD (in MeOH, kmax [nm], /[mdeg]) 207 (1.4), 227 (ꢀ2.6), 247
(2.1), 322 (ꢀ3.2); For 1H NMR (CD3OD, 800 MHz) and 13C NMR
(CD3OD, 150 MHz) spectroscopic data, see Table 3; MS (ESI): m/z
767 [M+Na]+; HRMS (ESI): m/z 743.2384 [MꢀH]ꢀ (calcd for
(CD3OD, 150 MHz) spectroscopic data, see Table 1; MS (ESI): m/z
737 [M+Cl]ꢀ; HRMS (ESI): m/z 759.2346 [M+HCOO]ꢀ (calcd for
C33H43O20, 759.2353).
C
33H43O19, 743.2404).
4.3.5. Phyllaemblicin H5 (5)
White amorphous powder; [
a
]
D
25 = +13.2 (c 0.9, CH3OH); UV
4.3.12. Phyllaemblicin H12 (12)
(MeOH) kmax (log
e) 200.0 (1.05), 228.2 (1.02), 272.4 (0.03) nm;
White amorphous powder; [a]
25 = +7.1 (c 1.6, CH3OH); UV
D
ECD (in MeOH, kmax [nm], /[mdeg]) 202 (5.7), 226 (ꢀ4.4), 245
(MeOH) kmax (loge) 201.8 (1.24), 228.2 (1.12) nm; ECD (in MeOH,
(4.3), 323 (ꢀ4.9); IR (KBr) mmax 3425, 2930, 1719, 1280,
kmax [nm], /[mdeg]) 208 (3.2), 228 (ꢀ2.5), 245 (3.4), 322 (ꢀ3.6);
IR (KBr) mmax 3424, 2928, 1711, 1283, 1078 cmꢀ1; For 1H NMR
(CD3OD, 800 MHz) and 13C NMR (CD3OD, 200 MHz) spectroscopic
data, see Table 4; MS (ESI): m/z 1073 [M+Cl]ꢀ; HRMS (ESI): m/z
1037.3338 [MꢀH]ꢀ (calcd for C44H61O28, 1037.3355).
1076 cmꢀ1
;
For 1H NMR (CD3OD, 800 MHz) and 13C NMR
(CD3OD, 100 MHz) spectroscopic data, see Table 2; MS (ESI): m/z
751 [M+Na]+; HRMS (ESI): m/z 773.2486 [M+HCOO]ꢀ (calcd for
C34H45O20, 773.2510).
4.3.6. Phyllaemblicin H6 (6)
4.3.13. Phyllaemblicin H13 (13)
White amorphous powder; [
a
]
D
25 = +42.3 (c 0.9, CH3OH); UV
White amorphous powder; [
(MeOH) kmax (log
a]
25 = ꢀ31.9 (c 1.1, CH3OH); UV
D
(MeOH) kmax (log
e) 200.4 (1.13), 228.6 (1.15), 272.8 (0.22) nm;
e) 206.0 (1.39), 235.8 (0.96), 306.4 (0.55) nm;
ECD (in MeOH, kmax [nm], /[mdeg]) 204 (5.4), 228 (ꢀ3.4), 244
ECD (in MeOH, kmax [nm], /[mdeg]) 211 (ꢀ3.6), 220 (1.6), 233
(ꢀ1.4), 249 (2.7), 289 (ꢀ1.1), 324 (ꢀ6.5); IR (KBr) mmax 3419,
2927, 1717, 1615, 1081 cmꢀ1; For 1H NMR (CD3OD, 600 MHz)
and 13C NMR (CD3OD, 150 MHz) spectroscopic data, see Table 4;
MS (ESI): m/z 891 [MꢀH]ꢀ; HRMS (ESI): m/z 891.2765 [MꢀH]ꢀ
(calcd for C38H51O24, 891.2776).
(5.0), 321 (ꢀ4.9); IR (KBr) mmax 3429, 2927, 1716, 1280,
;
1114 cmꢀ1 For 1H NMR (CD3OD, 600 MHz) and 13C NMR
(CD3OD, 150 MHz) spectroscopic data, Table 2; MS (ESI): m/z 605
[M+Na]+; HRMS (ESI): m/z 627.1926 [M+HCOO]ꢀ (calcd for
C28H35O16, 627.1931).
4.3.7. Phyllaemblicin H7 (7)
4.3.14. Phyllaemblicin H14 (14)
White amorphous powder; [
a]
25 = +33.5 (c 1.1, CH3OH); UV
White amorphous powder; [
(MeOH) kmax (log
a]
D
25 = ꢀ9.4 (c 1.0, CH3OH); UV
D
(MeOH) kmax (log
e
) 199.8 (1.09), 228.8 (1.11), 271.4 (0.11) nm;
e
) 201.8 (1.24), 257.8 (1.12) nm; ECD (in MeOH,
ECD (in MeOH, kmax [nm], /[mdeg]) 206 (4.4), 227 (ꢀ5.6), 245
kmax [nm], /[mdeg]) 207 (8.1), 235 (ꢀ5.5), 261 (1.4), 310 (ꢀ7.0);
IR (KBr) mmax 3428, 2928, 1708, 1609,1280, 1078 cmꢀ1; For 1H
NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) spectro-
scopic data, see Table 4; MS (ESI): m/z 767 [M+Na]+; HRMS (ESI):
m/z 743.2401 [MꢀH]ꢀ (calcd for C33H43O19, 743.2404).
(4.6), 321 (ꢀ5.4); IR (KBr) mmax 3429, 2929, 1718, 1280,
1075 cmꢀ1
;
For 1H NMR (CD3OD, 800 MHz) and 13C NMR
(CD3OD, 100 MHz) spectroscopic data, see Table 2; MS (ESI): m/z
767 [M+Na]+; HRMS (ESI): m/z 789.2440 [M+HCOO]ꢀ (calcd for
C34H45O21, 789.2459).
4.4. Acid hydrolysis of compounds 3–5, 7, and 10
4.3.8. Phyllaemblicin H8 (8)
White amorphous powder; [
a]
25 = +35.0 (c 0.8, CH3OH); UV
Compounds 3–5, 7 and 10 (each 0.5–1.0 mg) were dissolved in
D
(MeOH) kmax (log
e) 200.0 (1.13), 229.0 (1.10), 271.8 (0.05) nm;
2 M HCl (400 lL) in a vial separately. Then, each vial was sealed
ECD (in MeOH, kmax [nm], /[mdeg]) 201 (4.9), 227 (ꢀ4.5), 246
and incubated at 80 °C in a water bath. After 6 h, each solution
was cooled to room temperature, and extracted with CHCl3
(3.9), 322 (ꢀ4.7); IR (KBr) mmax 3430, 2926, 1717, 1281,
1076 cmꢀ1
;
For 1H NMR (CD3OD, 800 MHz) and 13C NMR
(400
l
L ꢁ 2). Each aqueous layer was neutralized by passaging
(CD3OD, 125 MHz) spectroscopic data, see Table 3; MS (ESI): m/z
over a small column with Amberlite IRA-401. TLC was used to
911 [M+Cl]ꢀ; HRMS (ESI): m/z 875.2814 [MꢀH]ꢀ (calcd for
identify the type of monosaccharide by comparing with the
C38H51O23, 875.2827).
authentic
sugars,
with
elution
system
CHCl3:n-
BuOH:MeOH:HAc:H2O (17:10:6:2:3), and the Rfs of standard sug-
ars were 0.40 for arabinose, 0.53 for 6-deoxy b-glucose, 0.46 for
xylose and 0.35 for glucose.
4.3.9. Phyllaemblicin H9 (9)
White amorphous powder; [
(MeOH) kmax (loge) 200.6 (0.93), 228.6 (0.94) nm; ECD (in MeOH,
a
]
D
25 = ꢀ0.6 (c 0.4, CH3OH); UV
kmax [nm], /[mdeg]) 207 (3.7), 226 (ꢀ3.7), 245 (4.3), 322 (ꢀ4.2);
For 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz)
spectroscopic data, see Table 3; MS (ESI): m/z 751 [M+Na]+;
HRMS (ESI): m/z 743.2391 [MꢀH]ꢀ (calcd for C33H43O19, 743.2404).
4.5. Precolumn derivatization and chiral separation
The mixture of monosaccharides from compounds 3–5, 7 and
10 were added separately into
a
small sample tube.
Derivatization was carried out as reported by Honda et al.
(1989). Briefly, 1-phenyl-3-methyl-5-pyrazolone (PMP) dissolved
in MeOH (0.5 M, 50 lL) and NaOH solution (0.3 M, 50 lL) were
4.3.10. Phyllaemblicin H10 (10)
White amorphous powder; [
(MeOH) kmax (log
a
]
25 = ꢀ7.3 (c 0.6, CH3OH); UV
D
e
) 200.0 (1.01), 228.6 (1.07), 272.2 (0.06) nm;
added in each tube, respectively. The tubes were sealed and incu-
ECD (in MeOH, kmax [nm], /[mdeg]) 203 (5.9), 227 (ꢀ4.1), 246
bated at 70 °C for 30 min. After cooling to room temperature, HCl
solution (0.1 M, 50 lL) was added, and the solution was
(4.7), 322 (ꢀ5.1); IR (KBr) mmax 3424, 2926, 1717, 1605, 1280,

J.-J. Lv et al. / Phytochemistry 117 (2015) 123–134
133
evaporated to dryness. The residue was partitioned between H2O
(200 L) and CHCl3 (200 L), and the aqueous layer was evapo-
4.7.3. Anti-virus assay
l
l
MDCK and Vero cells were plated in 96-well plates and incu-
bated for 24 h (5% CO2, 37 °C) to form monolayers. Virus suspen-
sions were added to each well and to allow the adsorption for
2 h. Then the viruses were removed and a serial twofold dilution
rated to dryness. The resultant PMP-monosaccharide was dissolved
in EtOH/n-hexane solution (1:4, v/v) and analyzed by HPLC using
CHRIALPAK AD-H column, with an elution solvent system of n-hex-
ane/i-PrOH (87:13) at 25 °C, and detected by DAD detector. The
standard sugars were analyzed using the same methods. The reten-
of the tested compounds at dose below CC50 (50 lL per well) each
with equal volume were added, and followed incubation for 72 h.
The virus-induced CPE of the tests was expressed as a percentage
in comparison with the parallel virus control and cell control.
IC50 values (the concentration of test compounds required to
reduce 50% of CPE) were calculated by regression analysis of the
dose–response curves, and the therapeutic index (TI) was calcu-
lated from the ratio CC50/IC50. Ribavirin was tested as the positive
control for anti EV71 and H3N2, and Guanidine hydrochloride and
Acyclovir were tested as the positive control for anti CVB3 and
HSV-1, respectively.
tion time of standard sugars were
(17.5 min), -xylose (15.5 min), -xylose (16.8 min),
(11.3 min), and -arabinose (18.5 min), respectively.
D
-glucose (15.0 min),
L-glucose
D
L
L
-arabinose
D
4.6. Quantum chemical calculations
Conformational analyses were carried out using Monte Carlo
search with molecular mechanics MMFF in Spartan’06
(Wavefunction Inc. Irvine, CA). The resultant conformers were re-
optimized using DFT at the B3LYP-SCRF/6-311G (d, p) level using
the integral equation formalism variant of the polarizable contin-
uum model (IEF-PCM). The free energies and vibrational frequen-
cies were calculated at the same level to confirm their stability,
and no imaginary frequencies were found. The optimized low
energy conformers with energy <2 kcal/mol were considered for
ECD calculation. The TDDFT/B3LYP-SCRF/6-311G (d,p) method
was applied to calculate the excited energies, oscillator strength
and rotational strength. All the calculations were run with
Gaussian ’09 (Frisch et al., 2009). The excited energies and rota-
tional strength values were used to simulate ECD spectra of each
conformer by introducing the Gaussian Function. The final ECD
spectra of each compound were obtained by averaging all the sim-
ulated ECD spectra of all conformers according to their excited
energies and Boltzmann distribution (Lv et al., 2014a,b). The band
shape of the calculated ECD curves were all 0.3 eV if there is no
illustration.
4.8. Human cancer cell cytotoxicity assay
As previously reported (Lv et al., 2013).
Acknowledgment
The authors are grateful to the members 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. We
also thank Fudan University for antiviral assay. This work was
supported by the National Natural Science Foundation of China
(NSFC 81473121), the 973 Program of Ministry of Science and
Technology of P.R. China (2011CB915503), the National Science
& Technology Support Program of China (2013BAI11B02), the
12th 5-Year National Science & Technology Supporting Program
of China (2012BAI29B06), the Fourteenth Candidates of the
Young Academic Leaders of Yunnan Province (Min Xu,
2011CI044) and by West Light Foundation of the Chinese
Academy of Sciences.
4.7. Antiviral bioassay
4.7.1. Virus and cell culture
Dog kidney cells MDCK, were used as target cells for H3N2 virus
infection, which were cultured in M199 (Sigma, St. Louis, USA) sup-
plemented with 10% fetal bovine serum (FBS), 500 unit/ml ampi-
Appendix A. Supplementary data
cillin, 300 lg/ml streptomycin and 0.25 lg/ml amphotericin B.
Supplementary data associated with this article can be found.
The data include 1H and 13C NMR, HSQC, HMBC, HSQC-TOCSY,
1H–1H COSY, ROESY, HRESIMS spectra for compounds 1–14, low
energy conformers of calculated ECD, and chromatograms of stan-
dard sugars in chiral separation. These data are available free of
Maintenance medium (MM) was the same as M199 but containing
5% fetal bovine serum. Vero cells were used as target cells for CVB3,
EV71, H3N2, HSV-1 and virus infection, which were cultured in
MEM. Cell culture was maintained at 37 °C in a humidified 5%
CO2 atmosphere. Virus strains CVB3, EV71, H3N2, and HSV-1 were
provided by Shanghai Municipal Center for Disease Control and
Prevention. The viral titer was tested by the cytopathic end-point
assay (Schmidtke et al., 2001) and expressed as 50% tissue culture
infective dose (TCID50).
charge
via
internet
http://www.sciencedirect.com.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.
2015.06.001.
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