Chemical components from Metapanax delavayi leaves and their anti-BHP activities in vitro

Article abstract of DOI:10.1016/j.phytochem.2019.01.002

Two previously undescribed oleanane-type triterpene saponins named liangwanosides III–IV, and one undescribed eudesmane glycoside named liangwanoside A were obtained from the leaves of Metapanax delavayi, a Chinese folk medicine especially for tea used in Yunnan, together with four known compounds. The structures of the undescribed compounds were determined by detailed spectroscopic (1D/2D NMR), HR-ESI-MS data analysis and chemical evidence. The activity against human benign prostate hyperplasia was evaluated with BPH-1 cell line. Most of the isolated compounds showed moderate inhibitory activity against BPH-1 cells at 100 and 50 μM in vitro.

Full text of DOI:10.1016/j.phytochem.2019.01.002

Phytochemistry 160 (2019) 56–60
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Chemical components from Metapanax delavayi leaves and their anti-BHP
activities in vitro
a
a
b
a
a
c
a,∗
Chongzhi Sun , Yang Wu , Bei Jiang , Ying Peng , Mengyue Wang , Jiaxun Li , Xiaobo Li
a
School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, PR China
Institute of Materia Medica, College of Pharmacy and Chemistry, Dali University, Dali, 671000, PR China
Lanping County Bureau of Agriculture, Lanping, 671400, PR China
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Metapanax delavayi
Araliaceae
Anti-BPH activity
Oleanane-type triterpene saponin
Eudesmane glycoside
Two previously undescribed oleanane-type triterpene saponins named liangwanosides III–IV, and one un-
described eudesmane glycoside named liangwanoside A were obtained from the leaves of Metapanax delavayi, a
Chinese folk medicine especially for tea used in Yunnan, together with four known compounds. The structures of
the undescribed compounds were determined by detailed spectroscopic (1D/2D NMR), HR-ESI-MS data analysis
and chemical evidence. The activity against human benign prostate hyperplasia was evaluated with BPH-1 cell
line. Most of the isolated compounds showed moderate inhibitory activity against BPH-1 cells at 100 and 50 μM
in vitro.
1. Introduction
2. Results and discussion
Metapanax delavayi (Franch.) J. Wen & Frodin (Araliaceae), known
as “liang wang cha” in China, is mainly distributed in the northern
region of Vietnam, central and western regions of China (Committee,
007). According to local ethnic minorities lived in Yunnan Province of
China, the tender leaf and stem of M. delavayi has been used as food (Li
et al., 2015), which has an efficacy to treat prostatitis (Zhou et al.,
017). The stem bark of M. delavayi has been used in Chinese folk
medicine, which possesses the effects of clearing heat, anti-inflamma-
tion, expectorant, relieving cough and asthma (Zhou et al., 2017). The
phytochemical studies on M. delavayi led to the isolation and identifi-
cation of triterpenoid saponins (Kasai et al., 1987), polysaccharides
The aqueous extract from leaves of M. delavayi was firstly passed
through a D101 macroporous resin column, then subjected to silica gel,
Sephadex LH-20 column chromatography (CC) and RP-HPLC to yield
three undescribed compounds, liangwanoside III (1), liangwanoside IV
(2), liangwanoside A (3), and four known compounds, 3-O-α-L-arabi-
nopyranoside-3β-hydroxyolean-12-ene-28,29-dioic acid-28-O-[β-D-glu-
copyranosyl-(1 → 6)-β-D-glucopyranosyl] ester (4) (Zhang et al., 2018),
liangwanoside II (5) (Kasai et al., 1987), serratagenic acid-3-O-α-L-
2
2
arabinopyranoside (6) (Yu et al., 1995), byzantionoside
B (7)
(Matsunami et al., 2010). Serratagenic acid (8) (Kasai et al., 1987), the
aglycone of the oleanane-type triterpenoid saponin, was obtained by
acid hydrolysis method (See Fig. 1).
(
Miu, 2005), and several other components (William et al., 2008; Yang
et al., 2014), which exhibited diverse biological activities, such as an-
algesia (Ge et al., 2000), antimalarial (Liu et al., 2015) and anti-
oxidative activities (Miu, 2005). In order to clarify the effective che-
mical ingredients, the isolation and structural elucidation of aqueous
extract of M. delavayi leaves were carried out in the present study.
Three undescribed compounds (1-3) with four known compounds (4–7)
were obtained. The inhibitory activities against BPH-1 cells of com-
pounds 1–6 and the aglycone of the triterpene saponins named serra-
tagenic acid (8) were evaluated.
Compound 1 was obtained as a white amorphous powder. Its mo-
76
lecular formula was determined as C48H O19, evidenced from the HR-
–
1
ESI-MS data (m/z 955.49050 [M – H] , calcd. for 955.49026). The
NMR spectrum (Table 1) of 1 clearly indicated the presence of six
1.47, 1.23, 1.24, 1.15, 1.05, and 0.95, each 3H, s),
H
methyl protons (δ
H
which were labeled as methyls C-30, 23, 27, 26, 24, and 25, respec-
tively, and the characteristic proton signal of an olefinic proton (H-12,
δ
H
C
5.51) corresponded to the carbon signal at δ 124.0 (C-12) in the
HMQC spectrum (Supporting Information S3). A total of 48 carbon
resonances appeared in the 13C NMR spectrum (Table 1), of which 30
were ascribed to a triterpene skeleton, including the signals of two
carbonyl carbons (C-28, δc 176.8; C-29, δc 181.4), and the remaining
∗
Corresponding author.
E-mail address: xbli@sjtu.edu.cn (X. Li).
https://doi.org/10.1016/j.phytochem.2019.01.002
Received 28 September 2018; Received in revised form 3 December 2018; Accepted 7 January 2019
0031-9422/ © 2019 Elsevier Ltd. All rights reserved.

C. Sun et al.
Phytochemistry 160 (2019) 56–60
J = 14.4, 4.8 Hz, H-18), indicating that the relative stereochemistry of
this methyl group was β-orientation. Detailed analysis of the 1D and 2D
NMR data (Table 1 and Supporting Information S1–S6) revealed that
the aglycone of 1 was serratagenic acid which was reconfirmed by the
Table 1
1
H and 13C NMR data of compounds 1–2 in C
a
5
D
5
N (δ in ppm, J in Hz).
No.
1
2
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
NOSEY correlations of H-3/CH
-23, H-3/H-5, H-5/H-9, H-9/CH
3
-27,
3
CH -25/CH -26, H-18/CH -30 and H-18/CH
3
3
3
3
-26 (Kim et al., 2011).
b
b
1
2
1.57, m
39.4
28.6
1.56, m
1.01, m
1.87, m
1.83, m
3.44, m
39.4
28.6
b
b
b
b
b
b
b
The structure of 1 was similar to Liangwanoside II (Kasai et al., 1987),
with the difference of a reductional arabinopyranosyl group.
1
.01, m
1.87, m
.84, m
The 1H NMR spectrum showed three anomeric protons for three
1
3
4
5
6
3.44, dd (11.2, 5.0)
78.5
39.8
56.2
19.3
78.5
39.8
56.2
19.3
13
H
sugar moieties that resonated at δ 6.30, 4.99 and 5.90. The C NMR
spectrum of 1 displayed the presence of three sugar moieties including
two glucopyranosyls (δ 96.2, 74.5, 79.3, 71.3, 77.7, 69.8), (δ 105.5,
75.8, 77.0, 78.6, 78.5, 61.7) and one rhamnopyranosyl (δ 103.2, 73.3,
3.1, 74.3, 70.8, 19.0). After acid hydrolysis, the sugar units were
0.84, d (11.3)
0.84, d (11.6)
b
b
1.55, m
1.54, m
C
C
b
b
1
.40, m
1.37, m
C
b
b
b
7
1.45, m
.36, m
33.6
1.46, m
33.6
7
b
1
1.36, m
confirmed to be D-glucose and L-rhamnoside, which were identified by
GC-MS analysis. The orientation of the two glucopyranosyl residues
were deduced to be β, according to the large coupling constant of the
8
9
40.4
48.6
37.9
24.3
40.4
48.5
37.8
24.3
1.66, dd (10.8, 7.1)
1.66, dd (10.8, 7.1)
1
1
0
1
b
b
1.96, m
1.96, m
anomeric proton (Glc-1H, δ
shifts of Glc C-1′–C-6′ (δ
001), respectively. The anomeric proton (δ
showed a strong HMBC correlation with the carbons at positions Rha-3
(δ 73.1) and Rha-5 (δ 70.8), which indicated an α-orientation (Mair
et al., 2018).
As shown in Fig. 2, the HMBC correlation between Glc-1H (δ
and the carbonyl carbon at δ
group was posited at C-28. The HMBC cross-peaks of Glc-1′H (δ
to Glc-6C (δ 69.8) and Rha-1H (δ 5.90) to Glc-4′C (δ
6.30, d, J = 8.2 Hz) and the chemical
105.5, 75.8, 77.0, 78.6, 78.5, 61.7), (Ye et al.,
5.90) of the Rha unit
H
b
b
1
.95, m
1.95, m
C
12
13
14
15
5.51, s
124.0
144.1
42.8
5.52, s
124.0
144.1
42.8
2
H
2.35, t (12.1)
28.7
2.35, t (12.1)
1.17, m
28.7
C
C
1
.18, m
b
b
b
1
6
2.22, m
.03, m
24.0
2.22, m
23.9
6.30)
4.99)
b
H
2
2.03, m
C
176.8 indicated that the glucopyranosyl
1
1
1
7
8
9
47.5
41.3
41.3
47.4
41.3
41.3
3.34, dd (14.4, 4.8)
3.36, dd (14.1, 4.7)
2.56, t (13.9)
H
2.56, t (13.9)
78.6) confirmed
C
H
C
b
b
1
.92, m
1.92, m
the sequence of sugar moieties at C-28 was α-L-rhamnopyranosyl-(1 →
4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl. Thus, the structure
of 1 was elucidated as 3β-hydroxyolean-12-ene-28,29-dioic acid-28-O-
[α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-gluco-
pyranosyl] ester and named liangwanoside III.
Compound 2 was isolated as a white amorphous powder. The HR-
ESI-MS of 2 showed a major ion peak at m/z 809.43258 [M – H]
66
(calcd. 809.43235), supporting the molecular formula of C42H O15. A
comparison of the 1D and 2D NMR spectra (Table 1 and Supporting
Information S8–S14) similar to 1 revealed that the aglycone of 2 was
also serratagenic acid, with the difference of a reductional rhamno-
pyranosyl group. In the 1H NMR spectrum (Table 1), signals for two
2
2
0
1
42.6
29.6
42.6
29.5
2.23, mb
2.23, mb
1
.76, d (13.0)
1.77, d (13.3)
b
b
2
2
1.99, m
32.2
2.03, m
32.1
b
b
1
.91, m
1.94, m
2
2
2
2
2
2
2
3
2
2
3
4
5
6
3-CH
4-CH
5-CH
6-CH
7-CH
8
3
3
3
3
3
1.23, s
1.05, s
0.95, s
1.15, s
1.24, s
29.2
17.0
16.1
18.0
26.5
176.8
181.4
20.4
96.2
74.5
79.3
71.3
77.7
69.8
1.23, s
1.04, s
0.96, s
1.16, s
1.24, s
29.2
17.0
16.1
18.0
26.5
176.8
181.3
20.4
96.2
74.3
79.0
72.0
79.2
69.9
–
9
0-CH
8-Sugar Glc-1
3
1.47, s
1.47, s
6.30, d (8.2)
6.31, d (7.9)
4.17, m
b
4.17, m
4.26, m
4.38, m
4.18, m
anomeric protons at δ
J = 7.7 Hz) were diagnostic for the presence of two sugar residues. The
D NMR data confirmed the presence of two glucopyranosyl units (δ
96.2, 74.3, 79.0, 72.0, 79.2, 69.9; δ 105.8, 75.6, 78.9, 71.4, 78.5,
63.1). Acid hydrolysis of 2 afforded D-glucose by GC-MS analysis. The
coupling constants (Glc-1H, δ 6.31, d, J = 7.9 Hz; Glc-1′H, δ 5.07, d,
J = 7.7 Hz) confirmed the β-glycosidic linkages for two glucose units
Liang et al., 2011). The HMBC correlations (Fig. 2 and Supporting
Information S11) between the Glc-1H (δ 6.31) and the aglycone C-28
(δ 176.8), as well as the Glc-1′H (δ 5.07) and Glc-6C (δ 69.9), in-
H
6.31 (1H, J = 7.9 Hz) and δ
H
5.07 (1H,
b
b
4.24, m
b
b
4.25, m
2
C
b
b
4.24, m
b
C
4.72, m
4.76, d (10.7)
b
b
4
.37, m
4.39, m
b
Glc-1′
4.99, m
105.5
75.8
77.0
78.6
78.5
61.7
5.07, d (7.7)
4.04, t (8.0)
3.92, m
105.8
75.6
78.9
71.4
78.5
63.1
H
H
2′
3′
4′
5′
6′
3.98, t (8.4)
3.69, d (8.6)
4.45, t (9.4)
(
b
4.38, m
b
b
4.14, m
4.22, m
4.15, m
H
b
4.51, d (11.7)
C
H
C
b
b
4
.12, m
4.38, m
dicated the sugar residues at C-28 were determined to be β-D-gluco-
pyranosyl-(1 → 6)-β-D-glucopyranosyl. Consequently, 2 was identified
as 3β-hydroxyolean-12-ene-28,29-dioic acid-28-O-[β-D-glucopyranosyl-
(1 → 6)-β-D-glucopyranosyl] ester and named liangwanoside IV.
Compound 3 was obtained as a white amorphous powder. Its mo-
Rha-1
5.90, s
103.2
73.3
73.1
74.3
70.8
19.0
2
3
4
5
4.59, dd (9.3, 3.3)
b
4.71, m
4.36, m
5.01, m
b
b
6
-CH
3
1.72, d (6.2)
lecular formula was determined as C21
H + HCOOH] m/z 461.23970 (calcd. 461.23867). The IR spectrum
H
36
O
8
on the HR-ESI-MS [M –
–
a
b
1H NMR at 600 MHz and 13C NMR at 150 MHz in C
Signals overlapped.
5 5
D N.
−1
featured absorption bands at 3151 and 1762 cm
acteristic of hydroxy group and carbonyl group, respectively. The
NMR spectrum of 3 (Table 2) indicated the presence of three methyl
singlets (δ 0.98, 1.41, 1.15), two protons attached to oxygenated
carbons at δ 3.55 and δ 4.48, one anomeric proton at δ 4.97, and
one aldehyde proton at δ
9.73. In the 13C NMR and HSQC spectra of 3,
1 carbon signals were detected. These signals resulted from three
which were char-
1
H
1
8 were ascribed to three sugar moieties. The above spectroscopic data
1
2
H
indicated that 1 was a Δ -oleanane-type triterpenoid saponin (Zhao
et al., 2014). The HMBC correlations of H-18 (δ 3.34) to C-28 (δ
76.8) and CH 181.4) confirmed the
H
H
H
H
C
H
1
3
-30 (δ
H
1.47, 3H, s) to C-29 (δ
C
2
locations of carboxyl group at C-28 and C-29, respectively.
methyls, four methylenes, six methines (including two oxygenated), a
quaternary carbon, and an aldehyde group. Moreover, one set of proton
signals at δ 3.89–4.97, and their corresponding carbons resonating at
H
The NOESY spectrum of 1 (Supporting Information S6) showed NOE
enhancement between δ
H
1.47 (3H, s, CH
3
H
-30) and δ 3.34 (dd, 1H,
57

C. Sun et al.
Phytochemistry 160 (2019) 56–60
Fig. 1. Structures of compounds 1–7 isolated from the extract of M. delavayi, and serratagenic acid (8).
Table 2
1
H and 13C NMR data of compound 3 in C
a
5
D
5
N (δ in ppm, J in Hz).
No.
δ
H
(J in Hz)
δ
C
1
2
3.55, dd (11.5, 4.3)
1.88, m
78.2
29.7
1
.68, qd (13.2, 4.3)
3
1.54, m
25.9
b
1
.44, m
4
5
6
7
8
2.65, m
2.15, t (11.5)
48.1
45.7
78.4
43.0
23.8
4.48, dd (11.5, 4.4)
b
2.01, m
1.81, m
1
.64, tt (14.4, 4.4)
b
9
1.99, m
1
33.3
b
.46, m
1
11
0
42.0
26.0
23.3
24.5
12.1
203.7
102.3
74.9
79.1
71.6
78.2
63.3
Fig. 2. Key HMBC and NOESY correlations for compounds 1–2.
2.29, dq (13.9, 6.8)
0.98, d (6.8)
1.41, d (6.8)
1.15, s
9.73, d (4.5)
4.97, d (7.7)
4.04, t (8.4)
4.20, t (9.1)
4.30, t (9.1)
3.89, m
1
1
1
2-CH
3
3
3
3-CH
4-CH
C
δ 63.3, 71.6, 74.9, 78.2, 79.1 and 102.3, suggested the presence of a
hexose residue.
15
1′
The HMBC correlations of (Fig. 3) H-12, H-13 to a quaternary
2′
3′
4′
5′
C
carbon at δ 26.0 (C-11) and H-13 to C-12 showed that C-12 and C-13
were respectively connected with C-11. The HMBC correlation between
H-14 and C-10 supported that C-14 was attached to C-10, which was
further confirmed by the HMBC correlations from H-14 to C-1, C-5, and
6′
4.50, dd (11.5, 2.4)
1
1
4
.42, dd (11.5, 4.5)
C-9. H- H COSY correlation of H-15 to H-4 indicated that C-15 was
1
2
3
-OH
′-OH
′-OH
6.06, s
connected with C-4, which was confirmed by the HMBC correlations of
6.96, brs
7.13, brs
7.25, brs
5.53, brs
1
1
H-15 to C-3 and C-5. The H- H COSY correlations of H-5 to H-6, H-6 to
b
b
H-7 and the HMBC correlations of H-6 and H-7 to C-11 identified that C-
4′-OH
6′-OH
6
was connected between C-5 and C-7. The HMBC correlations of H-7 to
1
1
C-12 and C-13 and the H- H COSY correlation of H-11 to H-7 indicated
C-11 was attached to C-7. C-1 was attached to C-2 based on the 1H- H
COSY correlations from H-1 to H-2 then to H-3. It was also indicated
that the glycosidic site was attached to C-6 by the correlation of HMBC
from H-1′ to C-6. All the information mentioned above indicated that 3
had the same scaffold as 1α,6β-dihydroxy-5,10-bis-epi-eudesm-15-car-
boxaldehyde-6-O-β-D-glucopyranoside (Hao et al., 2015), but C-11 of 3
a
1H NMR at 600 MHz and 13C NMR at 150 MHz in C
Signals overlapped.
1
5 5
D N.
b
(
δ
C
26.0) was linked with a proton (δ
H
2.29), not a hydroxyl group.
The relative configurations of 3 were deduced from the analysis of
coupling constant and NOESY spectrum (Fig. 3). The coupling constant
of H-6 (J6,7 = 4.4 Hz, J5,6 = 11.5 Hz), and the interaction of H-6 with
H-7 and H-14 suggested that H-6, 7 and 14 were in α-orientations, H-5
was in β-orientation (Xu et al., 2010). H-14 interaction with H-4 sug-
gested α-orientation of H-4. H-5 interaction with H-1 and H-15 in-
dicated that H-1 and H-15 were in β-orientations. The coupling constant
of anomeric proton (δ
C-1′, δ 102.3; C-2′, δ
H
4.97, J = 7.7 Hz) and chemical shift of carbons
74.9; C-3′, δ 79.1; C-4′, δ 71.6; C-5′, δ 78.2;
Fig. 3. Key COSY, HMBC and NOESY correlations for compound 3.
(
C
C
C
C
C
58

C. Sun et al.
Phytochemistry 160 (2019) 56–60
600 MHz spectrometer (Bruker BioSpin Corp., MA, USA). HR-ESI-MS
data were obtained on a Waters ACQUITY™ UPLC-Q-TOF-MS (Waters
Corp., Milford, USA). Silica gel (200–300 mesh, Qingdao Haiyang
Chemical Co., Qingdao, China), D101-type macroporous resin (Baoen
Corp., Cangzhou, China), and Sephadex LH-20 (Pharmacia, Stockholm,
Sweden) were used for CC. RP-HPLC was performed on a Shimadzu LC
2010 AHT instrument, using a YMC ODS-AQ column (20 × 250 mm,
5 μm, YMC, Kyoto, Japan). GC-MS was conducted on an Agilent 7890A-
5975C instrument (Agilent Technologies, Inc., CA, USA). TLC was
carried out with GF254 plates (Qingdao Haiyang Chemical Co.,
Qingdao, China). Spots were visualized by spraying with 10% H SO in
2
4
EtOH followed by heating. The analytically pure reagents were from
Sinopharm Chemical Reagent Co., (Shanghai, China).
Fig. 4. The repressive effect of compounds 1–6 & 8 on the growth of BPH-1 cells
(n = 3).
4.2. Plant materials
C-6′, δ 63.3) indicated the sugar moiety was β-glucose (Gorin and
C
The leaves of Metapanax delavayi (Araliaceae) were collected from
Mazurek, 1975). The β-D-glucose of the sugar moiety was determined
by acid hydrolysis and comparison with an authentic sample by GC-MS.
Thus, the structure of 3 was assigned as 5-epi-eudesm-15-carbox-
aldehyde-6-O-β-D-glucopyranoside-1α,6β-diol, named liangwanoside A.
Their inhibitory activities against BPH-1 cells of compounds 1–6, 8
were studied in vitro (Fig. 4). In previous reports, compound 7 had ef-
fects of increasing alkaline phosphatase and inhibition of arachidonic
acid release activity (Bukhari et al., 2015; Mao et al., 2014). As 7 was
known compound and had a low content in M. delavayi leaves, we didn't
investigate its activity on anti-BPH. The result showed that compounds
Lanping County, Yunnan Province, China (GPS coordinates:
6°27′34.64″N, 99°19′1.25″E), in October 2014, autumn, and identified
by one of authors Prof. Xiaobo Li. A voucher specimen (20141001) was
deposited at School of Pharmacy, Shanghai Jiao Tong University.
2
4.3. Extraction and isolation
Dried and powdered leaves of M. delavayi (1 kg) were extracted
three times by water (3 × 10 L, 1 h each) under reflux. The combined
extraction filtrated with eight layers of gauze, and then concentrated
under reduced pressure to obtain a crude aqueous extract (380 g),
1
–6 inhibited cell proliferation for 48 h, and 8 didn't exhibit inhibitory
effect obviously. The inhibition rates of compound 1–6 were from 3.9%
to 10.9% at 50 μM, and from 9.9% to 21.0% at 100 μM, respectively. It
was reported that compound 4 possessed hepatoprotective effect
which was suspended in H
column. After eluting with H
eluted with 10%, 20%, 30%, 40% and 95% EtOH, to afford six fractions
F1–F6). F3 (13.6 g) was subjected to silica gel CC (CH Cl /MeOH,
5:1–3:1, v/v) to afford eight fractions (F3-1–F3-8). F3-2 was then se-
parated by silica gel CC eluted with CH Cl /MeOH (15:1–3:1), then
purified by RP-HPLC using MeCN/H O (10% v/v, 2 mL/min) as mobile
phase to yield compound 3 (95.1 mg, t = 17.9 min). F4 (20.7 g) was
chromatographed by silica gel CC using CH Cl /MeOH/H O (4:1:0.1,
v/v/v) as mobile phase, to yield 12 fractions. F4-1 to F4-12. F4-3
63.3 mg) was separated by repeated Sephadex LH-20 and purified by
2
O and chromatographed over a D101
2
O, the D101 column was successively
(
Zhang et al., 2018), and 5 showed the activity of IL-6 formation in-
(
2
2
hibition (Tsuji et al., 1997). In our present study, compounds 4–5 also
exhibited the anti-BPH activity in vivo, their inhibitory rates were
1
2
2
1
5.0 ± 1.4%, 16.0 ± 1.5% at 100 μM, respectively. But the un-
2
described compounds 1–3 showed stronger inhibitory effect with in-
R
hibitory rates of 18.5 ± 2.4%, 21.0 ± 2.4% and 18.4 ± 0.7% at
2
2
2
1
00 μM, respectively. The absorption of glycosides was often involved
metabolism such as deglycosylation to the aglycone after oral admin-
istration, and the prototype components as well as their aglycone could
be absorbed into plasma (Zhou et al., 2018). In our study, the glycosides
(
RP-HPLC MeCN/H
pound 7 (2.3 mg, t
CC with CH Cl
0 fractions, F5-1 to F5-30. F5-9 (72.6 mg) was separated via RP-HPLC
MeCN/H O, 20:80, v/v, 2 mL/min) to afford compound 4 (33.0 mg,
= 17.5 min). F5-8 (80.1 mg) was subsequently subjected to silica gel
CC eluting with CH Cl /MeOH/H O (4:1:0.1, v/v), and purified by RP-
HPLC using MeCN/H O (30% v/v, 2 mL/min, UV 210 nm) as mobile
phase to yield compound 1 (34.5 mg, t = 13.5 min). F5-7 (63.3 mg)
was repeated on a silica gel column, Sephadex LH-20 and RP-HPLC
MeCN/H O, 30:70, v/v, 2 mL/min) to yield compound 2 (27.0 mg,
= 23.5 min). F5-2 (47.6 mg) was subjected to passage over a silica
gel column eluted with CH Cl –MeOH (8:1, v/v) to give 13 fractions.
The eighth fraction (32.7 mg) was further purified over Sephadex LH-20
and RP-HPLC (MeCN/H O, 30:70, v/v, 2 mL/min, UV 210 nm) to give
2
O (10% v/v, 2 mL/min, UV 210 nm) to give com-
R
= 32.0 min). F5 (30.5 g) was separated by silica gel
(
1–2, 4–6) show stronger inhibitory activity of anti-proliferation on
2
2 2
/MeOH/H O (4:1:0.1, v/v/v) as mobile phase, to yield
BPH-1 cells than their aglycone (8). Their activities in vivo remain to be
further investigated.
3
(
2
t
R
3. Conclusion
2
2
2
2
A total of seven compounds were isolated from the aqueous extract
of M. delavayi leaves, including three undescribed and four known
compounds. Compounds 1–2, 4–6 were derivatives of serratagenic acid,
R
(
2
3
and 7 were sesquiterpenes. Compounds 1–6, which were demon-
t
R
strated to displayed moderate inhibitory activity against BPH-1 cells,
would be developed to the precursors of novel anti-BPH drugs.
Considering the large contents of the derivatives of serratagenic acid in
M. delavayi leaves, they might be the major bioactive components
against BPH in vivo and need to be further studied.
2
2
2
compound 6 (18.8 mg). F5-20 (11.3 g) was further separated by re-
peated Sephadex LH-20 eluted with MeOH to afford compound 5
(
10.2 g). Compound 5 (300 mg) was hydrolyzed by heating in 2 M HCl
(50 mL). The reaction mixture was partitioned between EtOAc (50 mL)
and H O each, and the EtOAc extract was subjected to silica gel CC
(CH Cl /MeOH/H O, 30:1:0.04, v/v/v) to afford compound 8
4. Experimental
2
4.1. General experimental procedures
2
2
2
(
63.2 mg), its identity was confirmed by comparison with published
Specific rotations were determined on a JASCO P-2000 polarimeter
NMR spectroscopic data.
(
Hachioji, Tokyo, Japan). UV spectra were recorded using a UV-1102
UV–vis spectrophotometer (Techcomp, Shanghai, China). IR spectra
were determined on an Equinox 55 FT-IR spectrometer (Bruker Optics
Inc., MA, USA). NMR spectra were recorded on a Bruker Avance
4.3.1. Liangwanoside III (1)
25
White powder; [α] –16.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
D
210 (3.61) nm; IR (KBr) νmax 3082, 2886, 1594, 1441, 1372, 1366,
59

C. Sun et al.
Phytochemistry 160 (2019) 56–60
181, 1054 cm 1; 1H NMR (C
50 MHz) see Table 1. HR-ESI-MS m/z 955.49050 [M – H] (calcd. for
−
D
5
N, 600 MHz) and 13C NMR (C
D
Funding
1
1
5
5
5
N,
–
C
48
H
75
O
19, 955.49026).
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
4
.3.2. Liangwanoside IV (2)
25
White powder; [α] –3.4 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
Acknowledgements
D
2
1
1
10 (3.51) nm; IR (KBr) νmax 3225, 2886, 1604, 1457, 1382, 1366,
−
1
1
13
187, 1048 cm
;
H NMR (C
5
D
5
N, 600 MHz) and C NMR (C
5
D
5
N,
The authors acknowledge the analysis support from Dr. Bona Dai
and Dr. Ruibin Wang at the Instrumental Analysis Center of Shanghai
Jiao Tong University for the measurement of NMR and optical rotation,
respectively.
–
50 MHz) see Table 1. HR-ESI-MS m/z 809.43258 [M – H] (calcd. for
C
42
H
65
O
15, 809.43235).
4
.3.3. Liangwanoside A (3)
25
White powder; [α] –5.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
Appendix A. Supplementary data
D
2
1
10 (3.23) nm; IR (KBr) νmax 3151, 2802, 1762, 1727, 1403, 1276,
−1
1
13
054, 985, 869 cm
;
H NMR (C
5
D
5
N, 600 MHz) and C NMR
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.01.002.
(
C
5
D
5
N, 150 MHz) see Table 2. HR-ESI-MS m/z 461.23970 [M –
–
37
H + HCOOH] (calcd. for C22H O10, 461.23867).
References
4
.4. Acid-hydrolysis and sugar analyses of the three undescribed
Bukhari, S., Feuerherm, A., Tunset, H., Isaksen, S., Sæther, M., Thvedt, T., Gonzalez, S.,
Schmid, R., Brunsvik, A., Fuglseth, E., 2015. A novel compound isolated from
Sclerochloa dura has anti-inflammatory effects. J. Serb. Chem. Soc. 81 55-55.
Committee, F. O. C. E., 2007. Flora of China, vol. 13 Science Press, Beijing.
Ge, F., Lai, X., Song, Z., Zhou, H., Qiu, Y., 2000. Analgesic effect of Nothopanax delavayi.
compounds (1–3)
The procedures of the acid-hydrolysis and sugar analyses of the
three undescribed compounds (1–3) were modified according to the
published literature (Zong et al., 2015). Compounds 1–3 (2 mg) were
dissolved in 2 M HCl (2 mL) and heated at 90 °C for 4 h, respectively.
The reaction mixture was extracted with EtOAc. The aqueous layer was
Chin. Wild Plant Resour. 19, 50–51.
Gorin, P.A.J., Mazurek, M., 1975. Further studies on the assignment of signals in 13C
magnetic resonance spectra of aldoses and derived methyl glycosides. Cheminform 6,
1212–1223.
Hao, X., Chen, J., Lai, Y., Sang, M., Yao, G., Xue, Y., Luo, Z., Zhang, G., Zhang, Y., 2015.
Chemical constituents from leaves of Cinnamomum subavenium. Biochem. Syst. Ecol.
2
evaporated under vacuum, diluted repeatedly with H O, and evapo-
rated in vacuo to furnish a neutral residue. The residue was dissolved in
pyridine (2 mL), to which 2 mg of L-cysteine methyl ester hydrochloride
was added. After heating at 60 °C for 2 h, the solvent was evaporated
6
1, 156–160.
Kasai, R., Oinaka, T., Yang, C., Zhou, J., Tanaka, O., 1987. Saponins from Chinese folk
medicine “liang wang cha,” leaves and stems of Nothopanax delavayi, Araliaceae.
Chem. Pharm. Bull. 35, 1486–1490.
Kim, K.H., Lee, I.K., Choi, S.U., Lee, J.H., Moon, E., Kim, S.Y., Lee, K.R., 2011. New
triterpenoids from the tubers of Corydalis ternata: structural elucidation and bioac-
tivity evaluation. Planta Med. 77, 1555–1558.
Li, X., Song, T., Zhang, Y., Xu, J., Zhang, Y., 2015. Research progress of Metapanax de-
lavayi. Beijing Agric. 17, 89–91.
Liang, D., Hao, Z., Zhang, G., Zhang, Q., Chen, R., Yu, D., 2011. Cytotoxic triterpenoid
saponins from Lysimachia clethroides. J. Nat. Prod. 74, 2128–2136.
Liu, Z., Xiao, C., Zhang, X., Xu, W., Peng, J., Jiang, B., 2015. Evaluation of antimalarial
activity of the ten herbs from West Yunnan. J. Dali Univ. 4, 1–4.
2
under N , and 0.2 mL trimethylsilylimidazole was added. The mixture
was kept at 60 °C for another 2 h and partitioned between n-hexane and
water. The organic layer centrifuged 20 minutes at 14000 rpm. The
supernatant (1 μL) was directly analyzed by GC-MS using a HP-5 ca-
pillary column. The acetate derivatives of D-glucose, L-rhamnose, and
L-arabinose had the following retention times, t
R
(min): D-glucose
(
21.84), L-rhamnose (20.06), and L-arabinose (19.09). Identification of
the sugar derivatives was based on a comparison of mass spectra with
those of authentic samples.
Mair, C.E., Grienke, U., Wilhelm, A., Urban, E., Zehl, M., Schmidtke, M., Rollinger, J.M.,
2
018. Anti-influenza triterpene saponins from the bark of Burkea africana. J. Nat.
Prod. 81, 515–523.
Mao, Y.-W., Lin, R.-D., Hung, H.-C., Lee, M.-H., 2014. Stimulation of osteogenic activity in
human osteoblast cells by edible Uraria crinita. J. Agric. Food Chem. 62, 5581–5588.
Matsunami, K., Otsuka, H., Takeda, Y., 2010. Structural revisions of blumenol C glucoside
and byzantionoside B. Chem. Pharm. Bull. 58, 438–441.
Miu, M., 2005. Studies on Active Polysacchrides from Metapanax davidii and M. Delavayi.
MA.Sc Thesis. China West Normal University.
Tsuji, T., Yada, Y., Yamaguchi, K., Yamazaki, K., Kasai, R., Otani, K., 1997. Interleukin 6
4
.5. BPH-1 cell survival test
The BPH-1 cells (Fenghui Biological Technology Co., Ltd., Hunan,
China) were cultured in a RPMI 1640 medium supplemented with 20%
FBS (Gibco, Melbourne, Australia), 100 U/mL penicillin, and 100 μg/
2
mL streptomycin. The cells were maintained in a humidified 5% CO ,
(IL-6) formation inhibitors containing saponins. Japan Patent JP09124489A.
William, S.N., Liu, Y., Chen, Y., 2008. Chemical composition of the essential oil of
Nothopanax delavayi leaves. Chem. Nat. Compd. 44, 539–540.
9
5% air atmosphere at 37 °C for different periods of time and were then
harvested for measurements. The BPH-1 cells were seeded in 96-well
plates (5 × 10 cells/well) and allowed to adapt overnight, then treated
Xu, X., Xie, H., Hao, J., Jiang, Y., Wei, X., 2010. Eudesmane sesquiterpene glucosides from
lychee seed and their cytotoxic activity. Food Chem. 123, 1123–1126.
Yang, Q., Zhang, J., Ouyang, S., Ye, W., Zhao, S., Yin, Z., 2014. Study on chemical
constituents from stem barks of Nothopanax delavayi. China J. Chin. Mater. Med. 39,
1858–1862.
Ye, W., Liu, X., Zhang, Q., Che, C., Zhao, S., 2001. Antisweet saponins from Gymnema
sylvestre. J. Nat. Prod. 64, 232–235.
Yu, S.S., Yu, D.Q., Liang, X.T., 1995. Triterpenoid saponins from the bark of Nothopanax
davidii. Phytochemistry 38, 695–698.
3
with 100 and 50 μM of compounds 1–6, 8 in the culture medium con-
taining 20% FBS for 48 h. Finasteride was used as a positive control.
The MTT solution (20 μL, 5 mg/mL) was added to each well, and the
cells were incubated for another 4 h. The formazan crystals were dis-
solved in 150 μL of DMSO. Cell viability was assessed by measuring the
absorbance at a 570 nm wavelength using a Thermo Varioskan micro-
plate photometer (Thermo, Waltham, USA). The inhibition rate
Zhang, Y., Zhu, H., Wei, X., Gao, D., Wang, D., Yang, C., 2018. C-3,28,29-trisubstituted
oleanane triterpenoid saponins, and its pharmaceutical composition for treatment of
liver diseases. China Patent CN108129542A.
(
%) = [(Acontrol – Asample)/(Acontrol – Ablank)] × 100%. Experiments
Zhao, Y., Wang, X., Wang, H., Liu, T., Xin, Z., 2014. Two new noroleanane-type triterpene
saponins from the methanol extract of Salicornia herbacea. Food Chem. 151, 101–109.
Zhou, G., Zhang, Y., Li, Y., Wang, M., Li, X., 2018. The metabolism of a natural product
mogroside V, in healthy and type 2 diabetic rats. J. Chromatogr. B 1079, 25–33.
Zhou, M., Zhou, W., Tian, X., 2017. Function and development prospect of food and
medicine in Nothopanax delavayi. Diet Health 4, 341–342.
Zong, J., Wang, R., Bao, G., Ling, T., Zhang, L., Zhang, X., Hou, R., 2015. Novel tri-
terpenoid saponins from residual seed cake of Camellia oleifera Abel. show anti-pro-
liferative activity against tumor cells. Fitoterapia 104, 7–13.
were performed in triplicate, and the values are the averages of three
independent experiments.
Conflicts of interest
There are no conflicts to declare.
60