Hebecarposides A?K, antiproliferative lanostane-type triterpene glycosides from the leaves of Lyonia ovalifolia var. hebecarpa

Article abstract of DOI:10.1016/j.phytochem.2018.03.012

Eleven previously undescribed lanostane-type triterpene glycosides, hebecarposides A?K, were isolated from the leaves of Lyonia ovalifolia var. hebecarpa (Ericaceae), along with two known analogues, lyonifolosides L and O. The structures of hebecarposides

Full text of DOI:10.1016/j.phytochem.2018.03.012

Phytochemistry 151 (2018) 32e41
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Phytochemistry
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Hebecarposides AꢀK, antiproliferative lanostane-type triterpene
glycosides from the leaves of Lyonia ovalifolia var. hebecarpa
a
,
b
,
1, Hanqi Zhang a 1, Junfei Zhou , Guanqun Zhan , Guangmin Yao
,
a
a
a, *
Yang Teng
a
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of
Science and Technology, Wuhan 430030, People's Republic of China
College of Pharmacy, Jiamusi University, Jiamusi 154007, People's Republic of China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 January 2018
Received in revised form
Eleven previously undescribed lanostane-type triterpene glycosides, hebecarposides AꢀK, were isolated
from the leaves of Lyonia ovalifolia var. hebecarpa (Ericaceae), along with two known analogues, lyoni-
folosides L and O. The structures of hebecarposides AꢀK were established by extensive spectroscopic
analysis and chemical methods, and the absolute configuration of C-24 in hebecarposides A and E was
18 March 2018
Accepted 31 March 2018
determined to be S and R, respectively, by a Mo
2
(OAc)
4
ꢀinduced electronic circular dichroism method.
This is the first report of the presence of lanostane-type triterpene glycosides in L. ovalifolia var. hebe-
carpa. All compounds were evaluated for their antiproliferative activities against five cancer cell lines,
SMMC-7721, HL-60, SW480, MCF-7, and A-549, and a normal epithelial cell line BEAS-2B, and none of
them showed general cytotoxity to the normal cell line BEAS-2B. Interestingly, hebecarposides C, D, G,
and K selectively inhibited the proliferation of HL-60 and SMMC-7721 cell lines, and hebecarposides C
and D showed significant anti-proliferative activities against A-549 cell lines than the positive control,
cis-platin. In addition, hebecarposides C and H exhibited more potent anti-proliferative activities against
MCF-7 than cis-platin.
Keywords:
Lyonia ovalifolia var. hebecarpa
Ericaceae
Lanostane-type triterpene glycosides
Mo
2
(OAc)
4
ꢀinduced ECD
Absolute configuration
Antiproliferative activity
©
2018 Elsevier Ltd. All rights reserved.
1
. Introduction
However, studies of Ericaceae triterpenoids are not thorough, and
the main triterpene skeletons are ursane, oleanane, lupane
(Bukreyeva et al., 2013; Dai and Yu, 2005; Huang et al., 2007; Way
et al., 2014; Yao et al., 2006), and dammarane (Dai and Yu, 2005).
Recently, Lv et al. (2016) reported the isolation of lanostane and
cycloartane triterpene glycosides with potent antiviral activity from
the twigs and leaves of Lyonia ovalifolia.
Lyonia is a small genus of Ericaceae family, and is mainly
distributed in Eastern Asia and North America. There are about 35
species of Lyonia in the world, and only six species and five varieties
in China. Lyonia ovalifolia var. hebecarpa (Franch. ex F.B. Forbes &
Hemsl.) Chun (Ericaceae) is endemic to China, and is mainly
distributed in Jiangsu, Anhui, Zhejiang, Guangdong, Guangxi, and
Yunnan provinces (Editorial Committee of the Flora of China, 1991).
The branches and leaves are used as a folk medicine for an astrin-
gent agent. Phytochemical studies on L. ovalifolia var. hebecarpa are
rare, only a megastigmane sesquiterpene glycoside and two ste-
roids were reported (Wang et al., 2001). In a course of search for
bioactive compounds from Ericaceae plants (Zhang et al., 2013,
Lanostane (also known as 4,4,14-trimethylcholestane)-type tri-
terpenoids possessing a 6/6/6/5 tetracyclic carbon ring system and
eight methyls are widely distributed in mushrooms (mainly
Ganoderma) and plants (Duru and Tel, 2015; Hamid et al., 2015;
Isaka et al., 2017; Jin et al., 2014; Tohtahon et al., 2017). Although
lanostane triterpene glycosides are the main components of sea
cucumbers (Holothurioidea and Echinodermata) and mushrooms
(Aminin et al., 2014; Kalinin et al., 2015; Lee et al., 2012), they are
relatively rare in plants, and only reported from Liliaceae (Adinolfi
et al., 1993; Ono et al., 2011; Ori et al., 2003a), Hyacinthaceae (Ori
et al., 2003b), Verbenaceae (Okwu and Offiong, 2009), Legumino-
sae (Mamedova et al., 2003), and Ericaceae (Lv et al., 2016). Erica-
ceae plants are famous for their beautiful flowers and their
structurally intriguing and bioactive diterpenoids components
(Zhang et al., 2013, 2015; Zhou et al., 2017a, 2017b; 2018a, 2018b).
2015; Zhou et al., 2017a, 2017b; 2018a, 2018b), the leaves of
*
Corresponding author.
E-mail address: gyap@mail.hust.edu.cn (G. Yao).
Y. Teng and H. Zhang contributed equally.
L. ovalifolia var. hebecarpa were investigated, leading to the isolation
1
https://doi.org/10.1016/j.phytochem.2018.03.012
031-9422/© 2018 Elsevier Ltd. All rights reserved.
0

Y. Teng et al. / Phytochemistry 151 (2018) 32e41
33
of eleven previously undescribed lanostane triterpene glycosides
1e11) and two known analogues (12 and 13) (Fig. 1). This is the
carboxyl group at
d
C
180.5 (C-30), and an arabinopyranosyl unit at
0
0
0
0
0
(
d
C
102.1 (C-1 ), 72.6 (C-2 ), 74.5 (C-3 ), 69.9 (C-4 ), and 67.0 (C-5 ) (Lv
first report of lanostane triterpene glycosides from L. ovalifolia var.
hebecarpa. In this paper, the isolation, structure elucidation, and
antiproliferative activities of thirteen lanostane triterpene gluco-
sides (1e13) are described.
et al., 2016). Apart from three indices of hydrogen deficiency
occupied by a double bond, a carboxyl group, and an arabinopyr-
anosyl unit, the remaining four indices of hydrogen deficiency
suggested that compound 1 is a tetracyclic triterpene glycoside. The
NMR data of 1 resembled those of 12 (lyonifoloside L) (Lv et al.,
2
016), except for an arabinopyranosyl unit in 1, instead of a glu-
2
. Results and discussion
copyranosyl unit in 12. The glycosidation at C-3 was proved by the
cross-peaks from the anomeric proton H-1 to C-3 (
from H-3 to the anomeric carbon C-1' (
0
d
C
82.2) and
The air-dried leaves of L. ovalifolia var. hebecarpa were extracted
d
C
102.1) in the HMBC
with 95% aqueous EtOH. The crude extract was suspended in H
2
O
1
1
spectrum of 1. The planar structure of 1 was confirmed by Hꢀ H
and then partitioned excessively with petroleum ether and chlo-
roform. The chloroform fraction was repeatedly subjected to silica
gel, reversed phase (RP) C18 silica gel, and Sephadex LHꢀ20 column
chromatography, as well as HPLC on a semipreparative XBꢀC18
column to yield eleven previously undescribed lanostane-type tri-
terpene glycosides (1e11) and two known analogues (12 and 13).
Known lanostane-type triterpene glycosides were identified to be
lyonifolosides L (12) and O (13) (Lv et al., 2016), respectively, by
comparison of their spectroscopic data with those reported in the
literature.
COSY, HSQC, and HMBC analyses (Fig. 2). The broad single peak of
1
H-3 (
d
H
3.39, br s) in the H NMR established its equatorial position
in the chair conformation of the cylcohexane in ring A (Fig. 2), and
H-3 was in a -orientation (Lv et al., 2016). NOESY analysis (Fig. 3)
and comparison of NMR data with 12 suggested that the relative
configuration of 1 is same to 12, ignoring the sugar moieties.
To determine the absolute configuration of the arabinopyranose,
b
1
was hydrolyzed by 2 mM HCl to obtain the sugar, and then, the
trimethylsilylthiazolidine derivatives of the sugar and standards, D
and -arabinose, were prepared. By comparing the retention times
L
Hebecarposide A (1) was obtained as a white amorphous power.
of these three trimethylsilylthiazolidine derivatives obtained from
gas chromatography (GC), the absolute configuration of the arabi-
nopyranose in 1 was determined to be L. The coupling constant of
Its molecular formula was established as C35
H
58
O
9
by the HRESIMS
H O Na, 645.3979) and
þ
at m/z 645.3978 [M þ Na] (calcd for C35
58 9
1
3
C NMR data, indicating seven indices of hydrogen deficiency. The
H NMR data of 1 (Table 1) showed resonances for seven methyl
0
the anomeric hydrogen J ¼ 6.2 Hz (
d
H
4.20, d, H-1 ) established the
1
a
-arabinopyranosyl linkage in 1. Due to the existence of a vic-diols
groups at
d
H
0.82 (3H, s, CH
-21), 1.16 (3H, s, CH
-28), and 0.90 (3H, s, CH
3
-18), 1.07 (3H, s, CH
-26), 1.13 (3H, s, CH
-29), an oxymethine at d
H
3
-19), 0.96 (3H, d,
-27), 0.98
3.39
4.20 (1H, d,
unit in the side chain,
a
dimolybdenum tetraaccetate
J ¼ 6.2 Hz, CH
3
3
3
[Mo
2
(OAc)
4
] ꢀinduced electronic circular dichroism (IECD) exper-
(
(
3H, s, CH
1H, br s, H-3
3
3
iment was implemented to establish the absolute configuration of
C-24 in 1 (Frelek et al., 2003; Gorecki et al., 2006, 2007; Snatzke
et al., 1981). To eliminate the effects of the arabinose and carbox-
ylic acid on the IECD, 1 was hydrolyzed by 0.6 mM TsOH$H
instead of HCl, to afford the genuine aglycone 1a, and then, 1a was
methylated with CH I to yield an ester 1b. As shown in Fig. 4, the
Mo (OAc) -induced ECD spectrum of the ester 1b exhibited a
b
0
), and an arabinopyranosyl unit at
d
H
0
0
J ¼ 6.2 Hz, H-1 ), 3.53 (1H, m, H-2 ), 3.52 (1H, m, H-3 ), 3.81 (1H, dd,
0
0
J ¼ 4.0, 2.9 Hz, H-4 ), 3.48 (1H, dd, J ¼ 12.4, 1.2 Hz, H-5 a), and 3.85
2
O,
0
13
(
1H, dd, J ¼ 12.4, 2.9 Hz, H-5 b) (Lv et al., 2016). The C NMR and
DEPT data of 1 (Table 2) exhibited a total of 36 carbon resonances
3
corresponding to seven methyls, ten methylenes, three methines
2
4
including two oxymethines at
quaternary carbons, one oxygenated tertiary carbon at
5), two tertiary sp2 carbons at
128.9 (C-8) and 141.9 (C-9), a
d
C
82.2 (C-3) and 80.6 (C-24), four
positive Cotton effect at 316 nm, suggesting the S configuration of
C-24 (Frelek et al., 2003; Gorecki et al., 2006, 2007; Snatzke et al.,
d
C
74.1 (C-
2
d
C
́
́
Fig. 1. Chemical structures of lanostane-type triterpene glycosides 1e13.

34
Y. Teng et al. / Phytochemistry 151 (2018) 32e41
Table 1
1
H NMR spectroscopic data for compounds 1ꢀ5b (
d in ppm, J in Hz, and 400 MHz).
No. 1a
1aa
1ba
2a
3b
4a
5b
5aa
5ba
1
1
2
2
3
5
6
6
7
7
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
a
b
a
b
b
a
a
b
a
b
1
1
2
2
5
5
6
6
7
8
9
0
1
1.41, m
2.10, m
1.95, m
1.61, m
3.39, br s
1.63, m
1.54, m
1.63, m
1.94, m
2.08, m
2.05, m
2.13, m
1.71, m
2.20, m
2.21, m
1.56, m
1.55, m
2.08, m
1.52, m
0.82, s
1.29, m
1.68, m
1.96, m
1.59, m
3.35, br s
1.62, m
1.56, m
1.62, m
2.01, m
2.07, m
2.15, m
2.25, m
1.69, m
2.32, m
2.07, m
1.57, m
1.59, m
2.09, m
1.51, m
0.81, s
1.30, m
1.67, m
1.90, m
1.56, m
1.38, m
2.09, m
1.95, m
1.55, m
3.35, overlap
1.62, m
1.59, m
1.65, m
2.01, m
2.09, m
2.07, m
2.17, m
1.69, m
2.21, m
2.20, m
1.55, m
1.56, m
2.09, m
1.52, m
0.81 s
1.46, m
2.07, m
1.97, m
1.57, m
3.42 br s
1.76, m
1.57, m
1.63, m
2.00, m
2.08, m
2.09, m
2.10, m
1.71, m
2.22, m
2.11, m
1.55, m
1.62, m
2.05, m
1.52, m
0.74, s
1.39, m
2.08, m
1.74, m
1.62, m
3.52, br s
1.75, m
1.52, m
1.61, m
1.98, m
2.08, m
1.74, m
2.16, m
1.70, m
2.19, m
2.11, m
1.57, m
1.57, m
2.06, m
1.48, m
0.72, s
1.54, m
2.04, m
1.99, m
1.84, m
3.64, overlap
1.95, m
1.55, m
1.62, m
2.19, m
2.33, m
2.19, m
2.37, m
1.79, m
2.78, m
2.48, m
1.70, m
1.80, m
2.46, m
2.09, m
0.94, s
1.29, m
1.68, m
1.93, m
1.59, m
3.35, overlap
1.62, m
1.54, m
1.61, m
2.00, m
2.08, m
2.15, m
2.26, m
1.72, m
2.32, m
2.09, m
1.49, m
1.33, m
2.09, m
1.55, m
0.82, s
1.46, m
1.63, m
1.96, m
1.58, m
3.35, overlap
1.57, m
1.56, m
1.62, m
1.92, m
2.07, m
2.17, m
2.22, m
1,73, m
2.28, m
2.08, m
1.55, m
1.43, m
2.05, m
1.44, m
0.82, s
3.35, br s
1.60, m
1.58, m
1.65, m
1.90, m
2.07, m
2.15, m
2.25, m
1.74, m
2.17, m
2.07, m
1.56, m
1.60, m
2.07, m
1.44, m
0.82, s
a
b
a
b
a
b
a
b
a
b
b
b
a
1.07, s
1.46, m
0.96, d (6.2)
1.06, s
1.05, s
1.06 s
1.05, s
1.03, s
1.14, s
1.67, s
1.06, s
1.05, s
1.47, m
0.96, d (6.4)
1.49, m
1.80, m
1.46, m
1.68, m
1.46, m
0.95, d (6.4)
1.52, m
1.80, m
1.47, m
1.68, m
1.46, m
0.96, d (5.4)
1.47, m
1.80, m
1.51, m
1.69, m
1.42, m
0.94, d (6.3)
1.72, m
1.81, m
1.46, m
2.03, m
1.41, m
0.94, d (6.1)
1.70, m
1.80, m
1.46, m
1.99, m
1.49, m
0.95, d (5.5)
1.49, m
1.27, m
1.45, m
1.78, m
1.49, m
0.95, d (5.5)
1.45, m
1.26, m
1.49, m
1.80, m
1.06, d, (5.2)
1.65, m
1.81, m
1.69, m
2.49, m
2a 1.70, m
2b 1.79, m
3a 1.50, m
3b 2.09, m
4
3.16, dd (10.2, 3.15, dd (10.0, 3.15, dd (10.0, 3.10, d (8.0)
3.15, dd (10.0, 1.2) 3.14, dd (10.2, 3.74, dd (10.0, 3.21, dd (10.3, 3.21, d (10.0,
1.3)
1.2)
1.6)
1.2)
2.0)
0.9)
0.8)
2
2
2
2
3
3
3
1
2
3
6
7
8
9
0a
0b
1.16, s
1.13, s
0.98, s
0.90, s
1.16, s
1.13, s
0.94, s
0.88, s
1.16, s
1.12, s
0.94, s
0.88, s
1.16, s
1.14, s
0.94, s
0.88, s
1.16, s
1.14, s
0.94, s
0.88, s
3.50, overlap
3.33, overlap
1.16, s
1.13, s
0.99, s
0.91, s
3.49, overlap
3.32, overlap
1.50, s
1.53, s
1.15, s
0.94, s
1.16, s
1.12, s
0.94, s
0.88, s
1.16, s
1.12, s
0.94, s
0.88, s
a
b
1
3.59, s
3.59, s
0
4.20, d (6.2)
3.53, m
3.52, m
4.26, d (6.8)
3.56, m
3.53, m
4.26, d, (7.0)
3.54, m
3.55, m
4.30, d (7.6)
3.15, m
3.34, m
4.69, d (6.8)
4.3, m
4.14, dd (8.5,
0
0
3
.3)
0
4
5
5
6
6
3.81, dd (4.0,
3.88, ddd (3.9, 2.9, 3.80, ddd (4.0, 2.8, 3.23, m
4.34, m
3.75, m
4.26, m
2
.9)
3.48, dd (12.4,
.2)
3.85, dd (12.4,
.9)
1.3)
1.2)
0
a
3.55, m
3.50, m
3.21, m
1
0
b
3.88, dd (12.8, 2.9) 3.86, dd (12.4, 2.8)
2
0
a
3.65, dd (11.8,
5
.7)
3.86, dd (11.8,
.9)
0
b
1
a
4
Recorded in methanol-d .
Recorded in pyridine-d5.
b
1
981), which was the same as 12 (Lv et al., 2016). Thus, the struc-
The
constant (J ¼ 6.8 Hz) of the anomeric proton H-1'. Therefore, the
structure of compound 2 was determined to be 3 -[( -arabino-
a-arabinopyranosyl linkage was deduced from the coupling
ture of compound 1 was identified as 3 -[( -arabinopyranosyl)-
oxy]-24(S),25-dihydroxylanost-8-en-30-oic acid.
a
a-L
a
a-L
Hebecarposide B (2), a white amorphous power, shared a same
pyranosyl)-oxy]-24(S),25-dihydroxylanost-8-en-30-oic acid.
Hebecarposide C (3) was obtained as a white amorphous power.
molecular formula of C35
H
58
O
9
with 1 as determined by the HRE-
þ
SIMS at m/z 645.3966 [M þ Na] (calcd for C35
H
58
O
9
Na, 645.3979)
Its molecular formula was determined as C35
H
60
O
8
by the HRESIMS
13
þ
13
and C NMR data. The NMR data (Tables 1 and 2) of 2 were similar
to 1, and the main differences were that C-3 ( 76.8) in 2 was
shielded, compared to that ( 82.2) in 1, while C-24 ( 89.5) in 2
was deshielded, compared to that in 1 ( 80.6). Thus, the arabi-
nopyranosyloxyl group should be located at C-24 in 2, instead of C-
(m/z 631.4193 [M þ Na] , calcd for C35
H
60
O
8
Na, 631.4186) and
C
d
C
NMR data. The NMR data (Tables 1 and 2) of 3 were similar to those
of 1, except for the presence of an oxymethylene ( 3.50, 3.33, H
30; 68.7, C-30) in 3, replacing of the carboxyl group ( 180.5, C-
30) in 1. HMBC correlations from H -15 ( 2.11, 1.55) to C-30 (
68.7) and from H -30 ( 3.50, 3.33) to C-8 ( 133.0), C-13 ( 46.9),
C-14 ( 56.9), and C-15 ( 26.6) confirmed the position of the
oxygenated methylene C-30 (Fig. 2). Thus, compound 3 was iden-
tified as -[( -arabinopyranosyl)-oxy]-24(S),25,30-trihydrox-
ylanost-8-ene.
Hebecarposide D (4), a white amorphous power, gave a
d
C
d
C
d
H
2
-
d
C
d
C
d
C
2
d
H
d
C
3
4
in 1. The HMBC correlations from the anomeric proton H-1' (
.26, d, J ¼ 6.8 Hz) of the arabinopyranose to C-24 ( 89.5) and
3.10, dd, J ¼ 8.0 Hz) to the anomeric carbon C-1' (
05.5) of the arabinopyranose confirmed this conclusion. The ab-
d
H
2
d
H
d
C
d
C
d
C
d
C
d
C
from H-24
b
(d
H
d
C
1
3
a
a-L
solute configuration of the arabinopyranose unit in 2 was deter-
mined to be L by the same chemical method and GC analysis as 1.

Y. Teng et al. / Phytochemistry 151 (2018) 32e41
35
Table 2
was then methylated with CH
I to give the aglycone ester 5b. The
3
13
C NMR spectroscopic data for compounds 1ꢀ5b (
No. 1a
d in ppm and 100 MHz).
Mo (OAc) -induced ECD spectrum of 5b (Fig. 4) showed a negative
2
4
1aa
1ba
2a
3b
4a
5b
5aa
5ba
Cotton effect at 314 nm, which was opposite to that of 1b. Thus, the
absolute configuration of C-24 was established to be R. Accordingly,
the structure of compound 5 was defined as 3a-[(a-L-arabinopyr-
anosyl)-oxy]-24(R),25-dihydroxylanost-8-en-30-oic acid by 2D
NMR analysis and the same chemical methods as 1. Therefore, the
chemical shifts of C-24 and H-24 may be used to assign its absolute
configuration.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
1
2
3
4
5
6
30.5
31.2
26.8
76.8
38.8
45.1
19.5
28.6
31.1
26.9
76.7
38.8
45.2
19.5
28.6
30.4
26.9
76.8
38.8
45.2
19.5
28.7
31.5
21.9
81.8
38.4
45.8
19.5
29.7
31.5
21.9
81.5
38.4
45.8
19.5
29.7
31.0
22.5
82.3
38.0
45.6
19.0
28.1
31.1
26.9
76.8
38.8
45.1
19.5
28.6
31.1
26.9
76.7
38.8
45.2
19.5
28.6
22.2
82.2
38.4
45.8
19.5
28.6
128.9 128.9 128.3 128.9 133.0 133.0 128.5 128.8 128.3
141.9 141.8 142.2 141.9 138.6 138.6 140.6 141.9 142.1
The molecular formula of hebecarposide F (6) was assigned as
þ
C
35
H
60
O
10 based on the HRESIMS (m/z 675.4136 [M þ Na] , calcd
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
38.7
23.4
32.7
48.3
64.1
29.0
29.2
52.4
18.4
20.4
37.8
19.5
34.9
31.1
80.6
74.1
25.8
25.1
29.3
23.2
38.8
23.3
32.9
48.1
64.0
29.1
29.2
52.3
18.5
20.0
37.9
19.4
34.9
31.1
80.8
74.0
25.8
25.0
28.9
24.0
38.8
23.4
33.0
48.3
64.4
28.9
29.2
52.3
18.4
19.9
37.9
19.2
34.9
31.1
80.7
74.0
25.9
25.0
28.9
23.0
38.8
23.4
32.9
48.2
64.1
29.1
29.6
52.5
18.6
20.1
37.8
19.2
34.3
31.1
89.5
73.7
26.8
25.0
28.9
23.0
38.7
22.5
32.5
46.9
56.9
26.6
29.6
53.0
17.8
19.6
38.4
19.6
35.1
29.9
80.8
74.1
25.0
26.0
29.3
23.1
38.6
22.5
32.5
46.9
56.9
26.5
29.3
53.0
17.9
19.6
38.4
19.6
35.1
29.9
80.8
74.1
26.0
25.0
29.1
23.1
68.6
38.3
23.2
32.1
47.5
63.4
29.0
30.2
51.9
18.4
20.2
36.9
19.2
34.4
29.4
79.4
73.1
26.2
26.5
29.2
23.0
38.8
23.4
32.9
48.1
64.0
30.0
29.0
52.5
18.5
20.0
37.3
19.2
34.4
31.1
79.9
74.0
25.8
25.1
28.9
24.0
38.8
23.4
33.0
48.4
64.4
29.0
30.3
52.7
18.4
19.9
37.2
19.1
34.3
28.6
79.8
74.0
25.9
25.0
28.9
23.0
13
60
for C35H O10Na 675.4084) and C NMR data. The NMR data
(Tables 3 and 4) of 6 were similar to those of 5, except for the typical
0
resonances for a glucopyranosyl unit at
d
H
4.30 (d, J ¼ 7.7 Hz, H-1 ),
0
0
0
0
3.17 (m, H-2 ), 3.38 (m, H-3 ), 3.26 (m, H-4 ), 3.24 (m, H-5 ), 3.66 (dd,
0
0
J ¼ 11.8, 5.6 Hz, H-6 a), and 3.86 (dd, J ¼ 11.8, 2.0 Hz, H-6 b) and
d
C
0 0 0 0 0
1
(
01.6 (C-1 ), 75.2 (C-2 ), 78.2 (C-3 ), 72.1 (C-4 ), 78.0 (C-5 ), and 63.2
0
C-6 ) in 6, replacing the
the cross peaks from H-1' (
3.50, br s) to C-1' ( 101.6) in the HMBC spectrum, the glyco-
a-
L-arabinopyranosyl unit in 5. Based on
d
H
4.30, d) to C-3 ( 82.0) and H-3 (
d
C
d
H
d
C
sidation position was determined to be C-3 in 6 (Fig. 2). The ab-
solute configuration of the glucopyranose in 6 was assigned to be D
by the same chemical method and GC analysis as 1, and the
glucopyranosyl linkage was determined by the coupling constant of
H-1' (J ¼ 7.7 Hz). Thus, the structure of 6 was established as 3 -[(
-glucopyranosyl)-oxy]-24(R),25,30-trihydroxylanost-8-en-30-oic
acid.
Hebecarposide G (7) had a molecular formula of C36
b-
a
b-
D
180.5 180.2 178.3 180.2 68.7
52.5
178.9 180.0 178.2
52.3
H
62
O
9
as
þ
1
deduced by the HRESIMS at m/z 661.4316 [M þ Na] (calcd for
0
102.1
72.6
74.5
69.9
67.0
105.5 101.7 101.0 103.2
13
6
61.4292) and C NMR data, indicating that it had one less index of
0
0
0
0
0
72.9
74.5
70.0
67.5
72.8
74.6
70.0
67.1
75.4
78.4
72.2
78.1
63.2
72.5
75.1
69.7
67.0
hydrogen deficiency than 6. The NMR data (Tables 3 and 4) of 7
showed similarities to those of 6, and the major difference was the
presence of an oxymethylene group (
d
H
3.47, 3.36, H
0) in 7, instead of the carboxyl group ( 180.5, C-30) in 6. In the
-30 correlated to C-8 ( 132.9), C-13 (
26.5), proving the location of the
OH at C-14 in 7 (Fig. 2). Hence, com-
was defined as -[( -arabinopyranosyl)-oxy]-
24(R),25,30-trihydroxylanost-8-ene.
2 C
-30; d 68.6, C-
3
d
C
a
Recorded in methanol-d
Recorded in pyridine-d5.
4
.
HMBC spectrum of 7, H
2
d
C
d
C
b
46.9), C-14 (
oxymethylene group 30-CH
pound
C C
d 56.9), and C-15 (d
2
7
3
a
a-L
molecular formula of C35
H
60
O
8
as determined by HRESIMS (m/z
þ
13
6
61.4289 [M þ Na] , calcd for C36
H
62
O
9
Na, 661.4292) and C NMR
Hebecarposide H (8) was proved to have a molecular formula of
þ
data. Based on the NMR spectroscopic data analysis (Tables 1 and
), hebecarposide D (4) was inferred to be similar to 3, except for
C
36
H
58
O
10 by the HRESIMS peak at m/z 673.3913 [M þ Na] (calcd
13
2
for C36
(Tables 3 and 4) of compound 8 were similar to those of 7, except for
that a ketone carbonyl ( 218.0, C-24) in 8 replaced the oxy-
methylene group ( 3.47, 3.36, H -30; 68.6, C-30) in 7. HMBC
correlations from H -26/H -27 ( 1.29) to C-24 confirmed the
location of the ketone carbonyl at C-24 (Fig. 2). Thus, compound 8
was determined as 3 -[( -glucopyranosyl)-oxy]-25-hydroxy-24-
oxolanost-8-en-30-oic acid.
58
H O10Na, 673.3928) and C NMR data. The NMR data
their sugar moieties. NMR spectra of 4 showed resonances for a
0
glucopyranosyl unit at
d
H
4.30 (1H, d, J ¼ 7.6 Hz, H-1 ), 3.15 (1H, m,
d
C
0
0
0
0
H-2 ), 3.34 (1H, m, H-3 ), 3.23 (1H, m, H-4 ), 3.21 (1H, m, H-5 ), 3.65
d
H
2
d
C
0
(
6
1H, dd, J ¼ 11.8, 5.7 Hz, H-6 a), and 3.86 (1H, dd, J ¼ 11.8, 1.9 Hz, H-
3
3
d
H
0
0
0
0
0
0
b);
d
C
101.0 (C-1 ), 75.4 (C-2 ), 78.4 (C-3 ), 72.2 (C-4 ), 78.1 (C-5 ),
0
and 63.2 (C-6 ) (Lv et al., 2016). HMBC correlations of H-3 (
br s) to C-1' (
proved the glycosidation position at C-3. The absolute configuration
of the glucopyranose in 4 was determined to be D by the same
chemical method and GC analysis as 1, and the coupling constant of
d
H
3.52,
81.5)
a
b-D
d
C
101.0) and H-1' (
d
H
4.30, d, J ¼ 7.6 Hz) to C-3 (
d
C
Hebecarposide I (9) was isolated as a white amorphous power.
þ
The sodium adduct ion at m/z 805.4705 [M þ Na] in the HRESIMS
13
70
and C NMR data assigned a molecular formula of C42H O13 to 9,
0
anomeric proton J ¼ 7.6 Hz (
d
H
4.30, d, H-1 ) established the
b
-
suggesting nine indices of hydrogen deficiency. Comparison of their
NMR data (Tables 3 and 4) revealed that compound 9 differed from
glucopyranosyl linkage in 4. Hence, compound 4 was established as
-[( -glucopyranosyl)-oxy]-24(S),25,30-trihydroxylanost-8-
ene.
3
a
b
-D
8 in the presence of an oxygenated methylene group (
each d, J ¼ 10.2 Hz, H -30; 68.1, C-30) in 9, replacing the carboxyl
group ( 181.2, C-30) in 8. Another major difference was the
presence of an additional rhamnopyranosyl moiety ( 6.50, s, H-
1''; 4.80, dd, J ¼ 3.1, 1.2 Hz, H-2''; 4.60, dd, J ¼ 9.3, 3.1 Hz, H-3''; 4.26,
m, H-4''; 4.74, dd, J ¼ 9.3, 6.2 Hz, H-5''; 1.72, d, J ¼ 6.2 Hz, H-6'';
H
d 3.97, 3.76,
2
d
C
Hebecarposide E (5) was determined to possess a same molec-
d
C
þ
ular formula as 1 by the HRESIMS peak at m/z 645.4007 [M þ Na]
d
H
13
(
58 9
calcd for C35H O Na, 645.3979) and C NMR data. Comparison of
their NMR data (Tables 1 and 2) revealed the deshielding of H-24
3.74, dd, J ¼ 10.0, 2.0 Hz) and the shielding of C-24 ( 79.4) in 5
than that ( , H-24; 80.6, C-24) in 1. Thus,
a
d
C
00
(d
H
d
C
101.3, C-1''; 72.9, C-2''; 72.0, C-3''; 74.5, C-4''; 69.9, C-5''; 19.1, C-6 )
in 9. The HMBC correlations from the anomeric proton H-1'' (
6.50, s) of the rhamnopyranosyl to C-2' ( 76.6) of the glucopyr-
d
H
3.16, dd, J ¼ 10.2, 1.3 H
Z
d
C
d
H
C-24 in compound 5 should have a different configuration from 1.
To determine the absolute configuration of C-24, compound 5 was
hydrolyzed by 0.6 mM TsOH$H
d
C
anosyl unit suggested the location of the rhamnopyranosyl unit at
0
2
O to obtain the aglycone 5a, which
C-2 of the glucopyranosyl unit. The location of the oxymethylene

36
Y. Teng et al. / Phytochemistry 151 (2018) 32e41
Fig. 2. 1Hꢀ H COSY and key HMBC correlations of compounds 1e11.
1
Fig. 3. Key NOESY correlations of compound 1.
group 30-CH
from H -30 (
23.9), C-13 (
2
d
OH at C-14 was deduced from the HMBC correlations
3.97, 3.76, each d, J ¼ 10.2 Hz, H -30) to C-8 (
46.2), and C-14 ( 56.4) (Fig. 2). The absolute
2
H
2
d
C
1
d
C
d
C
configurations of the arabinopyranose and glucopyranose were
determined to be L and D, respectively, by the same chemical
methods and GC analysis as 1. Thus, compound 9 was defined as 3
a-
[
a-L
-arabinopyranosyl-(1 / 2)-( -glucopyranosyl)-oxy]-25,30-
b-D
dihydroxy-24-oxolanost-8-ene.
The HRESIMS peak at m/z 803.4526 [M þ Na] and 13C NMR data
þ
Fig. 4. Mo (OAc) -induced ECD spectra of compounds 1b and 5b in DMSO solution.
2
4
established the molecular formula of hebecarposide J (10) to be
C
(
42
H
68
O
13 (calcd for C42
Tables 3 and 4) of 10 were similar to those of 9, with the major
difference of an aldehyde group ( 10.16, s, H-30; 201.4, C-30) in
0, instead of an oxymethylene group ( 3.97, 3.76, each d,
J ¼ 10.2 Hz, H -30; 68.1, C-30) in 9. HMBC cross-peaks of H-30 (
0.16, s) to C-8 ( 122.3), C-13 ( 46.4), and C-14 ( 67.7) verified
68
H O13Na, 803.4558). The NMR data
that the aldehyde group 30-CHO was located at C-14 (Fig. 2). Thus,
compound 10 was determined as 3-O- -[( -arabinopyranosyl)-
1 / 2)- -glucopyranosyl]-25-hydroxy-24,30-dioxolanost-8-
ene.
Hebecarposide K (11) had a molecular formula of C42
a
a-L
d
H
d
C
(
b-D
1
d
H
2
d
C
d
H
70 12
H O
1
d
C
d
C
d
C

Y. Teng et al. / Phytochemistry 151 (2018) 32e41
37
Table 3
1
H NMR spectroscopic data for compounds 6e11 (
d
in ppm, J in Hz, and 400 MHz).
8a
No.
6a
7a
9b
10a
11a
1
1
2
2
3
5
6
6
7
7
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
3
3
1
2
3
4
5
6
6
1
2
3
4
5
6
a
b
a
b
a
a
a
b
a
b
1
1
2
2
5
5
6
6
7
8
9
0
1
1.58, m
2.10, m
1.78, m
1.73, m
3.50, br s
1.65, m
1.56, m
1.65, m
1.97, m
2.08, m
2.09, m
2.23, m
1.71, m
2.22, m
2.03, m
1.58, m
1.35, m
2.01, m
1.51, m
0.83, s
1.58, m
2.10, m
1.91, m
1.70, m
3.53, overlap
1.78, m
1.49, m
1.61, m
2.01, m
2.07, m
1.55, m
2.15, m
1.77, m
2.15, m
2.10, m
1.51, m
1.35, m
1.97, m
1.51, m
0.83, s
1.06, s
1.49, m
0.95, d (6.0)
1.23, m
1.50, m
1.79, m
1.49, m
3.21, m
1.16, s
1.13, s
0.98, s
1.42, m
2.11, m
1.80, m
1.75, m
3.49, br s
1.65, m
1.56, m
1.65, m
2.01, m
2.10, m
1.73, m
2.24, m
1.72, m
2.26, m
2.20, m
1.55, m
1.45, m
2.07, m
1.52, m
0.81, s
1.42, m
2.00, m
2.05, m
1.79, m
3.93, br s
1.80, m
1.55, m
1.65, m
2.00, m
2.13, m
1.95, m
2.12, m
1.78, m
1.96, m
2.30, m
1.58, m
2.13, m
2.42, m
1.61, m
0.81, s
1.38, m
1.98, m
2.02, m
1.74, m
3.84, br s
1.81, m
1.52, m
1.62, m
1.97, m
2.08, m
1.90, m
2.17, m
1.77, m
1.90, m
2.30, m
1.58, m
2.15, m
2.41, m
1.55, m
0.76, s
1.44, m
1.93, m
2.03, m
1.74, m
3.77, br s
1.77, m
1.50, m
1.66, m
1.94, m
2.08, m
1.93, m
2.14, m
1.78, m
2.10, m
2.30, m
1.61, m
2.14, m
2.40, m
1.52, m
0.78, s
a
b
a
b
a
b
a
b
a
b
b
b
a
1.06, s
1.06, s
1.10, s
1.09, s
1.04, s
1.49, m
0.95, d (5.9)
1.25, m
1.48, m
1.79, m
1.41, m
3.22, m
1.16, s
1.47, m
0.94, d (6.1)
1.41, m
1.77, m
2.64, m
2.62, m
1.47, m
0.95, d (5.9)
1.55, m
2.00, m
2.94, m
2.92, m
1.50, m
0.81, d (6.4)
1.60, m
2.01, m
2.92, m
2.85, m
1.47, m
0.90, d (6.8)
1.51, m
1.76, m
2.93, m
2.90, m
2a
2b
3a
3b
4
6
7
8
9
b
1.29, s
1.29, s
0.98, s
0.91, s
1.57, s
1.57, s
1.22, s
0.87, s
3.97, d (10.2)
3.76, d (10.2)
4.98, overlap
4.28, m
1.56, s
1.56, s
1.22, s
0.87, s
10.16, s
1.56, s
1.56, s
1.27, s
0.89, s
1.06, s
1.13, s
0.98, s
0.91, s
b
a
0.91, s
0a
3.47, overlap
3.36, overlap
4.30, d (7.7)
3.18, m
3.35, m
3.27, m
3.21, m
3.66, dd (11.8, 5.3)
3.86, dd (11.8, 2.1)
0b
0
4.30, d (7.7)
3.17, m
3.38, m
3.26, m
3.24, m
4.27, d (7.8)
3.18, m
3.36, m
3.26, m
3.21, m
4.85, d (7.1)
4.34, m
4.33, m
4.80, overlap
4.27, m
4.26 m
0
0
0
0
4.32, m
4.16, dd (9.0, 8.8)
3.89, ddd (9.0, 5.6, 2.5)
4.35, dd (12.1, 5.6)
4.56, dd (12.1, 2.5)
6.50, s
4.80, dd (3.1, 1.2)
4.60, dd (9.3, 3.1)
4.26, m
4.15, dd (9.0, 9.0)
3.84, ddd (9.0, 5.4, 2.5)
4.36, m
4.54, dd (12.1, 2.5)
6.66, s
4.78, dd (3.4, 0.9)
4.52, dd (9.6, 3.4)
4.28, m
4.75, dd (9.4, 6.1)
1.68, d (6.1)
4.15, dd (9.0, 9.0)
3.85, ddd (9.0, 5.2, 2.5)
4.33, dd (11.5, 5.2)
4.51, dd (11.5, 2.5)
6.54, s
4.80, m
4.53, dd (9.3, 3.4)
4.28, m
0
0
a
b
3.66, dd (11.8, 5.6)
3.86, dd (11.8, 2.0)
3.66, dd (11.8, 5.7)
3.87, dd (11.8, 1.9)
0
0
0
0
0
0
0
0
0
0
0
0
4.74, dd (9.3, 6.2)
1.72, d (6.2)
4.73, dd (9.4, 6.2)
1.70, d (6.2)
a
4
Recorded in methanol-d .
Recorded in pyridine-d5.
b
þ
based on the HRESIMS peak at m/z 789.4764 [M þ Na] (calcd. for
cytotoxity. Interestingly, hebecarposides C (3), D (4), G (7), and K
(11) selectively inhibited the proliferation of HL-60 and SMMC-
þ
13
C
42 70
H O
12Na , 789.4765) and C NMR data. The NMR spectro-
scopic data (Tables 3 and 4) of 11 exhibited some similarities to
7721 cell lines with IC50 values ranging from 13.52 to 35.89 mM.
those of 10, but differed in the presence of a methyl group (
s, H -30; 24.8, C-30) in 11, replacing the aldehyde group (
s, H-30;
The HMBC cross peaks of H
45.1), C-14 ( 50.6), and C-15 (
the methyl group 30-CH at C-14 (Fig. 2). Thus, hebecarposide K
11) was identified as 3 -[ -arabinopyranosyl-(1 / 2)-( -glu-
copyranosyl)-oxy]-25-hydroxy-24-oxolanost-8-ene.
d
H
1.04,
10.16,
Hebecarposides C (3) and D (4) showed more potent anti-
proliferative activities against A-549 cell lines with IC50 values of
3
d
C
d
H
d
C
201.4, C-30) in 10. Thus, 11 is a 30-deoxy derivative of 10.
-30 ( 1.06, s) to C-8 ( 133.6), C-13
31.6) confirmed the location of
18.64 ± 0.52 and 27.59 ± 3.21
control, cis-platin (IC50 ¼ 28.23 ± 2.95
C (3) and H (7) exhibited more potent inhibitory activities against
the proliferation of MCF-7 (IC50 ¼ 22.15 ± 1.02 and 27.74 ± 1.29 M,
M).
mM, respectively, than the positive
3
d
H
d
C
mM). While, hebecarposides
(
d
C
d
C
d
C
3
a
m
(
a-
L
b
-
D
respectively) than the positive control (IC50 ¼ 35.02 ± 2.17
m
Analysis of the structure-activity of 1ꢀ13 revealed that the oxida-
Hebecarposides AꢀK (1e13) were evaluated for their anti-
tion state of C-30 plays an important role in the anti-proliferative
proliferative activities against five human cancer cell lines SMMC-
2 3
activity of lanost-8-ene glycosides, and 30-CH OH and 30-CH
7
721, HL-60, SW480, MCF-7, and A-549, and a normal epithelial
may be the active groups. The above data may provide a basis for
the further investigation and optimization of lanost-8-ene type
triterpene glycosides as an anti-proliferative agent for tumors, and
extensive studies are required for their structure-activity
relationship.
cell line BEAS-2B, and cis-platin was used as a positive control. As
shown in Table 5, compounds 1e13 did not show significant anti-
proliferative activities against the normal epithelial cell line
BEAS-2B (IC50 > 40 mM), suggesting that all of them have no general

38
Y. Teng et al. / Phytochemistry 151 (2018) 32e41
Table 4
a normal cell line, and all of them did not show general cytotoxity to
1
3C NMR spectroscopic data for compounds 6e11 (100 MHz).
the normal cell line BEAS-2B. Hebecarposides C (3), D (4), G (7), and
K (11) selectively inhibited the proliferation of cancer cell lines.
Preliminary structure-activity-relationship analysis revealed that
the oxidation state of C-30 plays an important role in the anti-
No.
6a
7a
8a
9b
10a
11a
1
2
3
4
5
6
7
8
9
30.6
22.3
82.0
38.3
45.7
19.4
28.5
128.8
141.9
38.7
23.4
32.7
48.3
64.1
29.0
28.9
52.5
18.3
20.3
37.2
19.2
31.1
34.4
79.9
74.0
25.8
25.1
29.1
23.0
180.5
101.6
75.2
78.2
72.1
78.0
63.2
31.4
22.5
81.5
38.3
45.8
19.5
29.9
132.9
138.6
38.6
21.9
32.5
46.9
56.9
26.5
29.0
53.1
17.8
19.6
37.7
19.2
29.2
34.6
79.9
74.0
25.8
25.1
29.1
23.0
68.6
101.1
75.4
78.4
72.1
78.0
63.2
30.5
22.3
82.0
38.3
45.7
19.4
28.5
129.0
141.6
38.7
23.4
32.7
48.2
64.3
29.0
29.1
52.2
18.4
20.3
37.0
19.1
31.0
34.0
218.0
78.1
26.9
26.9
29.1
23.0
181.2
101.6
75.2
78.3
72.2
78.0
63.2
30.9
21.9
79.2
37.8
45.7
19.3
29.1
123.9
137.0
38.2
20.8
25.9
46.2
56.4
31.7
29.3
52.0
17.7
19.7
36.9
19.2
31.1
34.7
216.8
77.1
27.6
27.7
28.9
23.4
68.1
99.2
76.6
80.0
72.7
78.8
63.2
101.3
72.9
72.0
74.5
69.9
19.1
30.5
22.2
80.8
37.7
45.7
19.0
28.3
122.3
145.1
38.6
22.7
26.6
46.4
67.7
31.3
29.8
53.4
17.4
20.2
35.9
18.9
31.0
33.4
216.7
77.1
27.7
27.7
29.2
23.4
201.4
100.7
76.1
80.2
72.6
78.7
63.3
101.6
72.8
73.4
74.3
69.6
19.1
30.9
22.7
81.4
37.6
46.1
19.1
28.7
133.6
135.9
37.7
21.7
26.5
45.1
50.6
31.6
31.7
50.8
16.4
20.0
36.7
19.2
31.4
33.6
216.9
77.2
27.6
27.7
29.2
23.5
24.8
101.1
77.3
80.6
72.7
78.5
63.4
101.8
72.8
73.2
74.3
69.8
19.2
proliferative activity of lanost-8-ene glycosides, and 30-CH
2
OH
and 30-CH may be the active groups. This is the first report of
3
lanostane-type triterpene glycosides from L. ovalifolia var. hebe-
carpa, and lanostane triterpene glycosides may be served as a
chemotaxonomic marker for the genus of Lyonia.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1
2
3
4
5
6
1
2
3
4
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
4. Experimental
4.1. General experimental procedures
Optical rotations were measured with a Rudolph Autopol IV
automatic. The ECD spectra were obtained on a JASCO Jꢀ80 spec-
trometer. 1D and 2D NMR spectra were recorded on a Bruker
1
AMꢀ400 spectrometer with referencing to the residuals of H (
d
H
13
3
.31 for methanol-d
4
or
d
H
7.19 for pyridine-d
5 C
) or C (d 49.2 for
methanol-d or 123.9 for pyridine-d
4
d
C
5
). HRESIMS were acquired
on a Bruker micrOTOF II spectrometer. Semi-preparative HPLC was
accomplished on a Dionex 680 system with a UV detector using a
reversed-phased (RP) C18 column (5
timate XBꢀC18). GC was analyzed using an Agilent 7820A gas
m,
mm, 10 ꢁ 250 mm, Welch Ul-
chromatography and capillary column (30 m ꢁ 0.25 mm ꢁ 0.5
m
Welch WM-1). The cytotoxicity assay was performed using a
Multiskan FC multifunctional microplate reader.
0
0
0
0
0
0
0
4.2. Plant material
The leaves of Lyonia ovalifolia var. hebecarpa (Franch. ex F.B.
Forbes & Hemsl.) Chun (Ericaceae) were collected at altitude about
0
0
0
0
0
0
0
0
0
0
0
0
ꢂ
0
00
ꢂ
0
00
2000 m, latitude 28 26 29 North and longitude 120 8 59 East,
Jinyun County, Zhejiang Province, China, in June 2015, and
authenticated by Professor Bing-Yang Ding at Wenzhou University.
A voucher specimen (No. 2015-0616) has been deposited at School
of Pharmacy, Tongji Medical College, Huazhong University of Sci-
ence and Technology.
a
4
Recorded in methanol-d .
Recorded in pyridine-d .
5
b
4
.3. Extraction and isolation
3
. Conclusions
A phytochemical investigation on the leaves of L. ovalifolia var.
The air-dried leaves of L. ovalifolia var. hebecarpa (50 Kg) were
ꢂ
extracted with 95% aqueous EtOH (3 ꢁ 70 L, each 24 h) at 45 C.
hebecarpa led to the isolation of eleven previously undescribed
lanostane triterpene glucosides, hebecarposides AꢀK (1e11), and
two known analogues, lyonifolosides L (12) and O (13). The struc-
tures of hebecarposides AꢀK (1e11) were established by extensive
After the removal of the solvent under the reduced pressure, the
2
crude extract (9.2 Kg) was suspended in H O (65 L) and then par-
titioned with petroleum ether (6 ꢁ 10 L) and chloroform (6 ꢁ 10 L).
The chloroform extract (5.2 Kg) was subjected to a silica gel
spectroscopic analysis, Mo
2
(OAc)
4
ꢀinduced ECD, and chemical
(
16.5 Kg, 100e200 mesh) column chromatography (CC) and eluted
with CH Cl
ꢀMeOH (50:1 to 0:1, v/v) to afford nine fractions
Fr.1ꢀFr.9. Fr.4 (43.5 g) was fractionated by an RP-C18 MPLC eluting
methods. Thirteen lanostane triterpene glucosides were evaluated
for their antiproliferative activities against five cancer cell lines and
2
2
Table 5
Anti-proliferative activities of compounds 1e13 against five cancer cell lines and one normal cell line (IC50 in
m
M)a,b
.
Compound
HL-60
A-549
SMMC-7721
MCF-7
SW480
BEAS-2B
Highest index of selectivityc
3
4
7
16.93 ± 0.29
16.55 ± 0.30
13.52 ± 0.36
25.60 ± 0.25
3.42 ± 0.17
18.64 ± 0.52
27.59 ± 3.21
>40
>40
28.23 ± 2.95
17.79 ± 0.53
18.59 ± 1.14
35.89 ± 1.10
24.88 ± 1.64
13.23 ± 1.17
22.15 ± 1.02
>40
27.74 ± 1.29
>40
33.32 ± 1.74
>40
>40
>40
26.90 ± 1.93
>40
>40
>40
>40
>40
>2.4
>2.4
>3.0
>0.6
>11.7
11
d
cis-platin
35.02 ± 2.17
a
Other compounds: IC50 > 40 mM.
b
c
IC50 values are presented as the means ± SD (n ¼ 3).
The “Highest index of selectivity” is the ratio of the IC50 value for the Beas-2B cell line over the lowest cancer cell IC50 value.
d
Positive control.

Y. Teng et al. / Phytochemistry 151 (2018) 32e41
39
with MeOHꢀH
2
O (from 20:80 to 100:0, v/v) to generate a major
fraction Fr.4D (10.2 g). Fr.4D was applied to a Sephadex LHꢀ20
4.3.8. Hebecarposide H (8)
White amorphous power; ½
2
5
1
a
ꢃ
ꢀ23 (c 0.2, MeOH); H NMR and
D
13
þ
column (MeOH) to obtain three subfractions Fr.4D1ꢀFr.4D3. Com-
C NMR data, see Tables 3 and 4; HRESIMS m/z 673.3913 [M þ Na]
pounds 7 (7.6 mg, t
R
¼ 38.1 min) and 12 (127.5 mg, t
R
¼ 33.5 min)
58
(calcd for C36H O10Na, 673.3928).
were obtained from Fr.4D1 (200 mg) by a semipreparative HPLC
eluting with MeCNꢀH O (50:50, v/v 1.5 mL/min). Subfraction
Fr.4D2 (48.6 mg) was separated by a semipreparative HPLC
O, 80:30, v/v, 1.5 mL/min) to give compound 8 (12.2 mg,
¼ 26.6 min). Compounds 1 (12.0 mg, t ¼ 26.6 min), 2 (7.0 mg,
¼ 25.5 min), and 5 (120.6 mg, t ¼ 24.5 min) were isolated from
O, 70:30, v/
v, 1.5 mL/min). Fr.5 (129.5 g) was chromatographed over an RP-C18
O (from 20:80 to 100:0, v/v) to obtain
2
4
.3.9. Hebecarposide I (9)
25
1
White amorphous power; ½
a
ꢃ
ꢀ82 (c 0.2, MeOH); H NMR and
D
(
MeCNꢀH
2
13
þ
C NMR data, see Tables 3 and 4; HRESIMS m/z 805.4705 [M þ Na]
t
t
R
R
70
(calcd for C42H O13Na, 805.4714).
R
R
Fr.4D3 (142 mg) by a semipreparative HPLC (MeCNꢀH
2
4.3.10. Hebecarposide J (10)
2
5
1
White amorphous power; ½
a
ꢃ
ꢀ188 (c 0.6, MeOH); H NMR and
D
MPLC eluting with MeOHꢀH
2
13
þ
C NMR data, see Tables 3 and 4; HRESIMS m/z 803.4526[M þ Na]
a major fraction Fr.5C (2.7 g). Fr.5C was applied to a Sephadex LH-20
column (MeOH) to afford five subfractions (Fr.5C1ꢀFr.5C5). Com-
68
(calcd for C42H O13Na, 803.4558).
pound 13 (15.1 mg, t
R
¼ 33.3 min) was obtained from Fr.5C1
4.3.11. Hebecarposide K (11)
(
1
148.7 mg) by a semipreparative HPLC (MeCNꢀH
.5 mL/min). Compounds 10 (12.6 mg, t
¼ 24.3 min) and 11 (7.0 mg,
¼ 38.2 min) were isolated from Fr.5C2 (102.5 mg) by a semi-
preparative HPLC (MeCNꢀH O, 80:20, v/v, 1.5 mL/min). Purification
of Fr.5C3 (14.2 mg) by a semipreparative HPLC (MeCNꢀH O, 67:33,
v/v, 1.5 mL/min) yielded compound 9 (2.6 mg, t
¼ 35.3 min). Frac-
tion Fr.5C4 (102.3 mg) was separated by a semipreparative HPLC
MeCNꢀH O, 80:20, v/v, 1.5 mL/min) to give compound 3 (3.6 mg,
¼ 33.3 min). Further purification of fraction Fr.5C5 (248.6 mg) by
O, 80:20, v/v, 1.5 mL/min)
2
O, 80:20, v/v,
2
5
1
White amorphous power; ½
a
ꢃ
ꢀ27 (c 0.4, MeOH); H NMR and
D
R
13
þ
C NMR data, see Tables 3 and 4; HRESIMS m/z 789.4764[M þ Na]
t
R
70
(calcd for C42H O12Na, 789.4765).
2
2
R
4.4. Acid hydrolysis and determination of the absolute
configuration of sugar moieties of compounds 1ꢀ11
(
2
t
R
Compound 1 (0.5 mg) was added to 1.5 mL 2 mM HCl, and the
ꢂ
a semipreparative HPLC (MeCNꢀH
2
mixture was stirred for 6 h at 80 C. After cooling to the room
afforded compounds 4 (20.0 mg, t
R
¼ 22.8 min) and 6 (7.0 mg,
temperature, the reaction mixture was extracted with EtOAc
(3 ꢁ 2.0 mL), and the aqueous layer was evaporated to dryness. The
dried sugar residue was dissolved in 0.2 mL anhydrous pyridine,
t
R
¼ 20.6 min).
and then 0.2 mg L-cysteine methyl ester hydrochloride was added
to the solution. The mixture was stirred at 60 C for 1.5 h, subse-
4
.3.1. Hebecarposide A (1)
ꢂ
2
5
1
White amorphous power; ½
a
ꢃ
ꢀ85 (c 0.4, MeOH); H NMR and
D
13
þ
quently, 0.1 mL N-trimethylsilylimidazole was added, and stirred at
C NMR data, see Tables 1 and 2; HRESIMS m/z 645.3978 [M þ Na]
ꢂ
6
0 C for another 1.5 h. The reaction mixture was suspended in
(
58 9
calcd for C35H O Na, 645.3979).
1
.0 mL H
2
O and extracted with n-hexane (3 ꢁ 1.0 mL). The layer of
n-hexane was directly analyzed by GC with a WM-1 capacity col-
umn. Nitrogen was used as a carrier gas at a flow rate of 1.0 mL/min.
Flame ionization detector (FID) and injection were set at 250 C.
4.3.2. Hebecarposide B (2)
2
D
5
1
White amorphous power; ½
a
ꢃ
ꢀ22 (c 0.1, MeOH); H NMR and
ꢂ
13
þ
C NMR data, see Tables 1 and 2; HRESIMS m/z 645.3966 [M þ Na]
The split ratio was 10:1 and the injection volume was 1
mL. The oven
(
58 9
calcd for C35H O Na, 645.3979).
temperature was programmed as follows: initial temperature at
ꢂ
ꢂ
ꢂ
2
30 C for 5 min, increased to 270 C at 10 C/min, and held
3.0 min at the final temperature. In the same way, the trimethylsi-
lylthiazolidine derivatives of the standards D- and -arabinose were
prepared and analyzed by GC. The GC retention times of the tri-
methylsilylthiazolidine derivatives of the hydrate of 1, L- and
arabinose were 8.83, 8.82, and 9.13 min, respectively, indicating the
presence of -arabinose in 1.
4.3.3. Hebecarposide C (3)
2
D
5
1
White amorphous power; ½
a
ꢃ
ꢀ12 (c 0.4, MeOH); H NMR and
L
13
þ
C NMR data, see Tables 1 and 2; HRESIMS m/z 631.4193 [M þ Na]
(
60 8
calcd for C35H O Na, 631.4186).
D
-
4.3.4. Hebecarposide D (4)
L
2
D
5
1
In the same way, compounds 2e11 were hydrolyzed by 2 mM
HCl, and the trimethylsilylthiazolidine derivatives of the hydrates
White amorphous power; ½
a
ꢃ
ꢀ20 (c 1.1, MeOH); H NMR and
13
þ
C NMR data, see Tables 1 and 2; HRESIMS m/z 661.4289 [M þ Na]
of 2ꢀ11, standards D-,
L-glucose, and L-rhamnose were prepared.
The trimethylsilylthiazolidine derivatives of the hydrates of com-
(
62 9
calcd for C36H O Na, 661.4292).
pounds 2, 3, and 5 gave GC retention times at 8.88, 8.90, and
4
.3.5. Hebecarposide E (5)
2
5
1
8.84 min, respectively, indicating the presence of
Detection conditions of the derivatives of standards D-,
L
-arabinose.
-glucose, L-
White amorphous power; ½
a
ꢃ
ꢀ15 (c 0.3, MeOH); H NMR and
D
13
þ
L
C NMR data, see Tables 3 and 4; HRESIMS m/z 645.4007 [M þ Na]
rhamnose, and hydrates of compounds 4 and 6e11 were different
(
58 9
calcd for C35H O Na, 645.3979).
ꢂ
ꢂ
at column temperature: initially 220 C for 5 min, raised to 270 C at
5
ꢂ
C/min, and kept 10 min. The GC retention times of the derivatives
-glucose, and -rhamnose were 13.35, 13.85, and
4.3.6. Hebecarposide F (6)
of standards D-,
L
L
2
5
1
White amorphous power; ½
a
ꢃ
ꢀ7 (c 0.4, MeOH); H NMR and
D
13.56 min, respectively. The trimethylsilylthiazolidine derivatives
of the hydrates of compounds 4 and 6e8 showed GC retention
times at 13.30, 13.34, 13.35, and 13.31 min, respectively, indicating
13
þ
C NMR data, see Tables 3 and 4; HRESIMS m/z 675.4136 [M þ Na]
(
60
calcd for C36H O10Na, 675.4084).
the presence of
D-glucose. Two sugars in compounds 9e11 were
4
.3.7. Hebecarposide G (7)
determined to be
D
-glucose and -rhamnose by the GC retention
L
2
5
1
White amorphous power; ½
aꢃ
ꢀ65 (c 1.5, MeOH); H NMR and
times of their trimethylsilylthiazolidine derivatives at 13.44 and
13.66 min (9), 13.34 and 13.67 min (10), and 13.33 and 13.64 min
(11), respectively.
D
13
þ
C NMR data, see Tables 3 and 4; HRESIMS m/z 661.4316 [M þ Na]
(
62 9
calcd for C36H O Na, 661.4292).

40
Y. Teng et al. / Phytochemistry 151 (2018) 32e41
4.5. Preparation of the aglycone of compounds 1 and 5
the compounds were calculated by Reed and Muench method
Reed and Muench, 1938).
(
A solution of compound 1 (5.2 mg) in 0.5 mL MeOH was added
4
6
0
m
L 0.6 mM TsOH$H
2
O in MeOH, and the mixture was stirred at
Acknowledgements
ꢂ
5 C for 72 h. After the removal of MeOH by evaporation, the re-
action mixture was diluted with 3.0 mL H
EtOAc (3 ꢁ 1.0 mL). The EtOAc extract was subjected to a silica gel
CC (200e300 mesh) eluting with a CH Cl OH (from 50:1 to
ꢀCH
0:1, v/v) gradient system to yield the aglycone 1a (2.4 mg). In the
same way, the aglycone 5a (3.6 mg) was prepared by the hydrolysis
of compound 5 (5.0 mg).
2
O and extracted with
This work was supported financially by National Natural Science
Foundation of China (U1703109, 81001368, and 31170323) and the
Fundamental Funds for the Central Universities (HUST:
2
2
3
2
2016YXMS148). We thank the Analytical and Testing Center at
Huazhong University of Science and Technology for ECD data
analysis.
4
.5.1. Hebecarpolic acid A (1a)
Appendix A. Supplementary data
25
ꢀ62 (c 0.3, MeOH); 1H NMR and 13C NMR
White power; ½ ꢃ
a
D
þ
data, see Tables 1 and 2; HRESIMS m/z 513.3561 [M þ Na] (calcd.
Supplementary data associated with this article can be found in
the online version, at https://doi.org/10.1016/j.phytochem.2018.03.
for C30
50 5
H O Na, 513.3556).
0
12. These data include MOL files and InChiKeys of the most
4
.5.2. Hebecarpolic acid E (5a)
important compounds described in this article.
25
ꢀ41 (c 0.1, MeOH); 1H NMR and 13C NMR
White power; ½ ꢃ
a
D
þ
data, see Tables 1 and 2; HRESIMS m/z 513.3553 [M þ Na] (calcd.
References
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50 5
H O Na, 513.3556).
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A mixture of 1a (2.4 mg) and K
drous DMF was added 4.6 L MeI, and then the mixture was stirred
at room temperature for 2 h. The reaction mixture was quenched
with Na SO (0.5 M), diluted with 1.0 mL H O, and extracted with
2 3
CO (1.0 mg) in 0.5 mL anhy-
m
2
3
2
EtOAc (3 ꢁ 1.0 mL). After concentration, the EtOAc extract was
subjected to a silica gel CC (200e300 mesh) eluting with a
CH
methyl ester 1b (1.8 mg). In the say way, the methyl ester 5b
2.4 mg) was prepared by the methylation of compound 5a.
2
Cl
2
ꢀCH
3
OH (from 50:1 to 30:1, v/v) gradient system to yield the
(
4
.6.1. Hebecarpolic acid A methyl ester (1b)
25
ꢀ51 (c 0.1, MeOH); 1H NMR and 13C NMR
White power; ½
aꢃ
Gorecki, M., Kaminska, A., Ruskowska, P., Suszczynska, A., Frelek, J., 2006. Dimo-
lybdenum method for determination of the absolute configuration of vic-diols-
foundations and developments. Pol. J. Chem. 80, 523e534.
D
þ
data, see Tables 1 and 2; HRESIMS m/z 527.3729 [M þ Na] (calcd.
52 5
for C31H O Na, 527.3712).
G oꢀ recki, M., Jabło nꢀ ska, E., Kruszewska, A., Suszczy nꢀ ska, A., Urba nꢀ czyk-Lipkowska, Z.,
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2 4
4
.6.2. Hebecarpolic acid E methyl ester (5b)
25
ꢀ35 (c 0.1, MeOH); 1H NMR and 13C NMR
White power; ½ ꢃ
a
D
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þ
data, see Tables 1 and 2; HRESIMS m/z 527.3718 [M þ Na] (calcd.
for C31
52 5
H O Na, 527.3712).
4.7. Determination of the absolute configuration of the 24,25-diol
moieties in compounds 1b and 5b
According to the method previously described (Frelek et al.,
2
003; Gorecki et al., 2006, 2007; Snatzke et al., 1981), Mo
was dissolved in dry DMSO to prepare a stock solution (1.0 mg/mL),
and then the freshly prepared solution of Mo (OAc) were added to
compound 1b (0.9 mg) in a molar ratio of 1.2:1. The Mo (OAc)
2 4
(OAc)
2
4
2
4
-
induced ECD (IECD) spectrum was immediately recorded every ten
minutes until the IECD spectrum was constant. After subtracting
the inherent ECD spectrum of 1b, the Mo
spectrum of 1b was prepared. The correlation between the absolute
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-induced ECD
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