Cytotoxic steroidal saponins from the roots and rhizomes of Maianthemum henryi

Article abstract of DOI:10.1080/14786419.2019.1641812

Henryiosides F and G (1 and 2), two new steroidal saponins along with two known analogues (3 and 4) were obtained from the roots and rhizomes of Maianthemum henryi. Their structures were determined by physicochemical properties and spectroscopic methods including 1D, 2D-NMR, IR and HR-ESI-MS data analysis. Cytotoxic activity in human HepG2 and SW620 tumour cells were evaluated by the MTT method and all of the saponins exhibited cytotoxicity with IC₅₀ values ranging from 15.33 μM to 57.85 μM.

Full text of DOI:10.1080/14786419.2019.1641812

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Cytotoxic steroidal saponins from the roots and
rhizomes of Maianthemum henryi
Xin Zhang, Jing Sun, Xiaofei Zhang, Shuo Zhang, Xuan Zhang, Xuanji Xue &
Zengjun Guo
To cite this article: Xin Zhang, Jing Sun, Xiaofei Zhang, Shuo Zhang, Xuan Zhang, Xuanji Xue &
Zengjun Guo (2019): Cytotoxic steroidal saponins from the roots and rhizomes of Maianthemum
henryi, Natural Product Research, DOI: 10.1080/14786419.2019.1641812
To link to this article: https://doi.org/10.1080/14786419.2019.1641812
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1641812
Cytotoxic steroidal saponins from the roots and rhizomes
of Maianthemum henryi
a,b
ꢀ
b
ꢀ
b
c
d
Xin Zhang , Jing Sun , Xiaofei Zhang , Shuo Zhang , Xuan Zhang ,
a,e
a,e
Xuanji Xue and Zengjun Guo
a
b
School of Pharmacy, Xi’an Jiaotong Univeristy, Xi’an, China; School of Pharmacy, Shaanxi University
c
of Chinese Medicine, Xianyang, China; School of Traditional Chinese Medicine, Beijing University of
Chinese Medicine, Beijing, China; The Second Affiliated Hospital of Shaanxi University of Chinese
Medicine, Xianyang, PR China; Shaanxi key laboratory of “Qiyao” resources and anti-tumor
d
e
acitivities, Xi’an, China
ABSTRACT
ARTICLE HISTORY
Received 23 May 2019
Accepted 1 July 2019
Henryiosides F and G (1 and 2), two new steroidal saponins along
with two known analogues (3 and 4) were obtained from the
roots and rhizomes of Maianthemum henryi. Their structures were
determined by physicochemical properties and spectroscopic
methods including 1D, 2D-NMR, IR and HR-ESI-MS data analysis.
Cytotoxic activity in human HepG2 and SW620 tumour cells were
evaluated by the MTT method and all of the saponins exhibited
cytotoxicity with IC50 values ranging from 15.33 lM to 57.85 lM.
KEYWORDS
Maianthemum henryi;
henryioside F; henryioside
G; structure determination;
cytotoxicity
1. Introduction
Maianthemum henryi (Baker) LaFrankie (synonym Smilacina henryi (bekev) Wang et
Tang) is a perennial herb in the genus Maianthemum (Liliaceae) and is mainly distrib-
uted in Shaanxi, Hebei and Yunnan province in china (Qiao et al. 2019). Its roots,
named Pian Tou Qi in the region of Qinba Mountains of Shaanxi province, are usually
used as traditional Chinese medicines (TCMs) for the treatment of rheumatism,
CONTACT Jing Sun
Xin Zhang and Jing Sun contribute equally to this work.
ph.175@163.com; Zengjun Guo
guozj@mail.xjtu.edu.cn
ꢀ
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1641812.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
X. ZHANG ET AL.
traumatic injury and impotence (Lin et al. 2018; Zhang et al. 2013). Steroidal saponins,
flavoids and nucleosides have been reported from the previous studies (Lin et al.
2018; Qiao et al. 2019; Zhang et al. 2013). In our research project of searching for the
bioactive constituents from TCMs (Guo et al. 2014; Hui et al. 2012; Xu et al. 2017), an
investigation of secondary metabolites of M. henryi was carried out and two new ster-
oidal saponins (henryiosides F and G) along with two known analogues, henryioside A
(3) (Zhang et al. 2013) and (25S)-5a-spirostan-9(11)-en-3b, 17a-diol-3-O-b-D-glucopyra-
nosyl-(1!2)-[b-D-glucopyranosyl-(1!3)]-b-D-galactopyranoside (4) (Cui et al. 2018),
were obtained. In addition, their cytotoxic activities in human HepG2 and SW620
tumour cells were also evaluated. Details of the isolation, structural elucidation and
cytotoxic activities of the saponins are presented in this paper.
2. Results and discussion
2
0
ꢂ
Compound 1 was obtained as a white amorphous powder, with [a] ꢁ29.6 (c 0.3,
D
MeOH). The HR-ESI-MS spectrum showed a negative molecular ion peak at m/z
－
1
1
047.5007 [M－ H] corresponding to a molecular formula of C H O (calculated
5
0 80 23
－
1
047.5018 [M－H] ). In the H NMR spectrum of 1, the characteristic proton signals
were observed, such as four steroid methyl groups at d 1.03 (3H, s, H-18), 0.78 (3H, s,
H
H-19), 1.25 (3H, d, J ¼ 6.8 Hz, H-21) and 0.70 (3H, d, J ¼ 7.1 Hz, H-27), an olefinic proton
at d 5.40 (1H, d, J ¼ 5.3 Hz, H-11) and four anomeric protons at d 4.86 (1H, d,
H
H
J ¼ 7.6 Hz, Gal-H1), 5.21 (1H, d, J ¼ 7.8 Hz, Glc-H1), 5.26 (1H, d, J ¼ 7.7 Hz, Glc’-H1) and
1
3
5
.59 (1H, d, J ¼ 7.5 Hz, Xyl-H1) were observed. The C-NMR spectrum of 1 showed the
signals a distinctive quaternary carbon signal of spirostane type steroids at d 110.8
C
(
C-22) (Song et al. 2015), a double bond carbons at d 146.3 (C-9) and 116.8 (C-11) as
C
well as four methyl carbons at d 17.6 (C-18), 18.0 (C-19), 10.0 (C-21) and 17.4 (C-27)
C
and four anomeric carbons at 102.5 (Gal-C1), 105.1 (Glc-C1), 104.9 (Glc’-C1) and 105.0
(
(
Xyl-C1). These NMR data compared with the reported compound henryioside A (3)
Zhang et al. 2013) indicated the different spectroscopic features in the F ring.
1
Detailed analysis of the H-NMR spectrum of 1 showed the chemical shifts of the H-27
signals which transferred to upfield at d 0.70 (<1.0) compared with that in henryio-
H
side A (d 1.02 > 1.0, 25S absolutely configuration) (Zhang et al. 2011). This differences
H
ꢁ
1
along with the IR absorptions observed at 979, 912, 894, 842 (912 < 894) cm con-
firmed the 25R absolutely configuration of 1 (Cui et al. 2018; Song et al. 2015). Acid
hydrolysis of 1 resulted in the products of D-galactose, D-glucose and D-xylose which
2
0
ꢂ
were established by their optical rotation data (Gal: [a] þ29.0 in MeOH, Glc:
D
2
0
ꢂ
20
D
ꢂ
[
4
a] þ48.8 in MeOH and Xyl: [a] þ33.6 in MeOH) and R values (BuOH-AcOH-H O,
D
f
2
:1:5 upper layer, Gal: 0.39, Glc: 0.36 and Xyl: 0.54) with the authentic sugar samples
(Chai et al. 2014). Coupling constants of the anomeric proton signals (J1,2 > 7.0 Hz)
suggested the b-configurations for the D-galactose, D-glucose and D-xylose (Zhang
et al. 2016). Accordingly, the structure of 1 was determined as (25R)-5a-spirostan-
9
(11)-en-3b, 17a-diol 3-O-b-D-glucopyranosyl-(1!2)-O-[b-D-xylopyranosyl-(1!3)]-O-
b-D-glucopyranosyl-(1!4)-b-D-galactopyranoside and named as henryioside
Figure 1).
F
(

NATURAL PRODUCT RESEARCH
3
Figure 1. Structures of compounds 1–4.
2
0
ꢂ
Compound 2 was obtained as a white amorphous powder, with [a] ꢁ26.1 (c 0.3,
D
MeOH). The HR-ESI-MS spectrum showed a negative molecular ion peak at m/z
－
1
1
047.5006 [M－ H] corresponding to a molecular formula of C H O (calculated
5
0 80 23
－
047.5018 [M－H] ). Four steroid methyl groups at d 1.05 (3H, s, H-18), 0.82 (3H, s,
H
H-19), 1.40 (3H, d, J ¼ 6.8 Hz, H-21) and 1.07 (3H, d, J ¼ 7.1 Hz, H-27) along with an ole-
1
13
finic proton 5.43 (1H, s, H-11) were observed in the H-NMR spectrum of 1. The C-
NMR spectrum displayed 50 carbon signals, in which a double bond carbons at dC
1
48.3 (C-9) and 123.1(C-11) and four methyl groups at d 10.9 (C-18), 17.8 (C-19), 14.0
C
(
(
C-21) and 16.3 (C-27) were observed. These NMR data compared with the literature
Qiao et al. 2019) supported the (25S)-5a-spirostan-9(11)-en-3b, 12b-diol aglycone of 1.
This inference was deduced from 2D-NMR data analysis including HSQC, HMBC,
1
1
NEOSY and H- H COSY experiments (Figure S16). The 3-O-b-D-glucopyranosyl-(1!2)-
O-[b-D-xylopyranosyl-(1!3)]-O-b-D-glucopyranosyl-(1!4)-b-D-galactopyranoside sugar
chain in 2 was determined by the HMBC correlations observed from Gal-H1/C-3, from
Glc-H1/Gal-C4, from Glc’-H1/Glc-C2 and from Xyl-H1/Glc-C3 and their NMR data which
was in agreement with the literature (Liu et al. 2012; Zhang et al. 2016; Zhang et al.
2013). The b-configurations for the D-galactose, D-glucose and D-xylose in 2 were
identified as the same method as 1. Compound 2 was thus determined as (25S)-5a-
spirostan-9(11)-en-3b, 12b-diol 3-O-b-D-glucopyranosyl-(1!2)-O-[b-D-xylopyranosyl-
(
1!3)]-O-b-D-glucopyranosyl-(1!4)-b-D-galactopyranoside and named as henryioside
G (Figure 1).
To the best of our knowledge, more than 30 cytotoxic steroidal compounds have
been isolated from the genus Maianthemum (Lin et al. 2018; Liu et al. 2012; Qiao et al.
2019; Yang et al. 2009; Zhang et al. 2013; Zhang et al. 2006). In this paper, compounds
1
ꢁ4 were assayed for their regarding cytotoxic activities in human HepG2 and SW620
tumour cells, the results (Table 1) showed that all of the saponins exhibitated inhibi-
tory effects with IC50 values ranging from 15.33 lM to 57.85 lM. The comparison with
the structures of 1ꢁ4 indicated that the 3-O-b-D-glucopyranosyl-(1!2)-O-[b-D-

4
X. ZHANG ET AL.
a
Table 1. Cytotoxic activities of compounds 1–4 in HepG2 and SW620 tumour cells (IC50 lM).
b
Compounds
1
2
3
4
5-FU
HepG2
SW620
a
19.60 ± 1.76
27.41 ± 2.41
24.06 ± 1.52
22.59 ± 0.85
15.33 ± 1.09
16.06 ± 1.28
57.85 ± 3.50
48.77 ± 4.68
12.07 ± 0.82
15.84 ± 1.21
IC50 values were expressed as mean ± SD (n ¼ 3).
b
5-FU ¼ 5-fluorouracil was used as the positive control.
xylopyranosyl-(1!3)]-O-b-D-glucopyranosyl-(1!4)-b-D-galactopyranoside sugar chain
is important to the cytotoxic activities.
3. Experimental
3.1. General experimental procedures
Optical rotation was measured using a Rudolph Autopol VI polarimeter (Rudolph,
USA); IR spectra were recorded on a Nicolet iS10 instrument (Thermo Fisher Scientific,
USA); 1D and 2D NMR spectra were recorded on a Bruker-Avance 400 instrument
(
Bruker Corp. Karlsruhe, Germany); Semipreparative HPLC was performed on Agilent
infinity II system equipped with a UV detector and a YMC-Pack-ODS-A (10 mm ꢃ
50 mm, 5lm particles) column. The HR-ESI-MS spectra were taken on an Agilent
2
Technologies 6650 Q-TOF (Agilent Technologies). Sephadex LH-20 gel and ODS C18 (5
lm) silica gel was purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden).
Silica gel was purchased from Qingdao Haiyang Chemical Group Corporation
(Qingdao, China).
3.2. Plant material
The roots and rhizomes of Maianthemum henryi (Baker) LaFrankie (synonym Smilacina
henryi (bekev) Wang et Tang) were collected on August in 2017 from Qinba
Mountains in Shaanxi Province of China, and were authenticated by one of our co-
authors Prof. Jing Sun (Shaanxi University of Chinese Medicine). A voucher specimen
(herbarium No. SH-201708) is deposited in School of Pharmacy, Xi’an Jiaotong
University, Xi’an 710061, China.
3.3. Extraction and isolation
The air-dried roots and rhizomes of M. henryi (6.6 kg) were extracted with 80% EtOH
under reflux for three times (2h, 2h, 1h, successively). The concentrated residue was
partitioned with petroleum ether (PE) and n-BuOH. The n-BuOH extract (130.2 g) was
subjected to column chromatography (CC) on silica gel (1 kg), eluting with gradient
solvent system (CH Cl -MeOH-H O, 100:0:0 ꢁ 60:40:10) to give six fractions (Fr.1 – Fr.6).
2
2
2
Fr.4 (19.1 g) was subjected to CC on silica gel (200 g), eluting with (CH Cl -MeOH-H O,
2
2
2
1
00:10:0 ꢁ 80:20:5) to give six subfractions (Fr.4-1 ꢁ Fr.4-6). Fr.4-3 (3.1 g) was subjected
to CC on Sephadex LH-20 gel (100 g) eluting with (CH Cl -MeOH 100:100) to give ten
2
2
subfractions (Fr.4-3-1 ꢁ Fr.4-3-10). Fr.4-3-3 (61.1 mg) was purified by HPLC (YMC-Pack-
ODS-A, 10 mm ꢃ 250 mm, 5 lm particles, flow rate: 1.5 mL/min) with MeCN-H O (70:30)
2
as mobile phase to afford compound 4 (9.0 mg; t ¼ 28 min); Fr.5 (23.8 g) was
R

NATURAL PRODUCT RESEARCH
5
subjected to CC on silica gel (200 g), eluting with (CH Cl -MeOH-H O,
2
2
2
1
00:10:0 ꢁ 70:30:5) to give five subfractions (Fr.5-1 ꢁ Fr.5-5). Fr.5-2 (2.6 g) was sub-
jected to CC on Sephadex LH-20 gel (100 g) eluting with (CH Cl -MeOH 100:100) to
2
2
give nine subfractions (Fr.5-2-1 ꢁ Fr.5-2-9). Fr.5-2-2 (45.2 mg) was purified by HPLC
YMC-Pack-ODS-A, 10 mm ꢃ 250 mm, 5 lm particles, flow rate: 1.0 mL/min) with MeCN-
(
H O (74:26) as mobile phase to afford compound 2 (11.4 mg; t ¼ 47 min); Fr.5-3
2
R
(
4.8 g) was subjected to CC on Sephadex LH-20 gel (100 g) eluting with (CH Cl -MeOH
2 2
1
00:100) to give twelve subfractions (Fr.5-2-1 ꢁ Fr.5-3-12). Fr.5-3-3 (113.5 mg) was puri-
fied by HPLC (YMC-Pack-ODS-A, 10 mm ꢃ 250 mm, 5 lm particles, flow rate: 1.5 mL/
min) with MeCN-H O (75:25) as mobile phase to afford compound 1 (14.3 mg, t ¼
2
R
5
8 min) and compound 3 (38.2 mg, t ¼ 55 min).
R
3.4. Identification of structures
2
0
ꢂ
ꢁ
Henryioside F (1), a white amorphous powder; [a] ꢁ29.6 (c 0.3, MeOH); IR (KBr)ꢀ
:
D
max
－
1
3
407, 2926, 1626, 1378, 1070, 1041, 979, 912, 894, 842 cm ; m/z 1047.5007 [M－H]
corresponding to a molecular formula of C H O
0 79 23
H-NMR (pyridine-d , 400 MHz, d in ppm) : 1.19 (1H, m, H-1a), 1.52 (1H, m, H-1b), 1.63
－
－
(calculated 1047.5018 [M－H] ).
5
1
5
(1H, m, H-2a), 2.07 (1H, m, H-2b), 3.94 (1H, m, H-3), 1.29 (1H, m, H-4a), 1.73 (1H, m, H-
4b), 0.96 (1H, m, H-5), 1.11 (1H, m, H-6a), 1.15 (1H, m, H-6b), 0.84 (1H, m, H-7a), 1.63
(
(
4
1
1
2
1H, m, H-7b), 2.03 (1H, m, H-8), 5.40 (1H, d, J ¼ 5.3 Hz, H-11), 1.68 (1H, m, H-12a), 2.99
1H, d, J ¼ 17.0 Hz, H-12b), 2.01 (1H, m, H-14), 1.53 (1H, m, H-15a), 2.34 (1H, m, H-15b),
.34 (1H, d, J ¼ 7.3 Hz, H-16), 1.03 (3H, s, H-18), 0.78 (3H, s, H-19), 2.73 (1H, dd, J ¼ 6.8,
4.1 Hz, H-20), 1.25 (3H, d, J ¼ 6.8 Hz, H-21), 1.61 (1H, m, H-23a), 1.64 (1H, m, H-23b),
.55 (1H, m, H-24a), 2.21 (1H, m, H-24b), 1.53 (1H, m, H-25), 3.71 (1H, d, J ¼ 11.0 Hz, H-
6a), 3.84 (1H, dd, J ¼ 2.4, 11.0 Hz, H-26b), 0.70 (3H, d, J ¼ 7.1 Hz, H-27), 4.86 (1H, d,
J ¼ 7.6 Hz, H-Gal-1), 4.38 (1H, m, H-Gal-2), 4.04 (1H, m, H-Gal-3), 4.57 (1H, m, H-Gal-4),
3
.97 (1H, m, H-Gal-5), 4.14 (1H, m, H-Gal-6a), 4.62 (1H, m, H-Gal-6b), 5.21 (1H, d,
J ¼ 7.8 Hz, H-Glc-1), 4.38 (1H, m, H-Glc-2), 4.12 (1H, m, H-Glc-3), 3.78 (1H, m, H-Glc-4),
.05 (1H, m, H-Glc-5), 4.01 (1H, m, H-Glc-6a), 4.48 (1H, d, J ¼ 10.4 Hz, H-Glc-6b), 5.26
1H, d, J ¼ 7.7 Hz, H-Glc-1’), 4.06 (1H, m, H-Glc-2’), 3.88 (1H, m, H-Glc-3’), 4.15 (1H, m, H-
Glc-4’), 4.10 (1H, m, H-Glc-5’), 4.33 (1H, m, H-Glc-6’a), 4.57 (1H, d, J ¼ 10.3 Hz, H-Glc-
4
(
6
’b), 5.59 (1H, d, J ¼ 7.5 Hz, H-Xyl-1), 4.01 (1H, m, H-Xyl-2), 4.07 (1H, m, H-Xyl-3), 4.08
13
(
(
1H, m, H-Xyl-4), 3.65 (1H, t, J ¼ 10.7 Hz, H-Xyl-5a), 4.19 (1H, m, H-Xyl-5b). C-NMR
pyridine-d , 100 MHz, d in ppm) : 35.8 (C-1), 29.9 (C-2), 77.3 (C-3), 34.9 (C-4), 43.3 (C-5),
5
28.8 (C-6), 33.6 (C-7), 36.7 (C-8), 146.3 (C-9), 38.2 (C-10), 116.8 (C-11), 34.2 (C-12), 44.4
(C-13), 50.5 (C-14), 33.6 (C-15), 90.1 (C-16), 89.0 (C-17), 17.6 (C-18), 18.0 (C-19), 46.1 (C-
2
2
0), 10.0 (C-21), 110.8 (C-22), 27.2 (C-23), 27.1 (C-24), 30.7 (C-25), 70.0 (C-26), 17.4 (C-
7), 102.5 (Gal-C1), 73.2 (Gal-C2), 75.6 (Gal-C3), 79.9 (Gal-C4), 75.3 (Gal-C5), 60.6 (Gal-
C6), 105.1 (Glc-C1), 81.4 (Glc-C2), 86.8 (Glc-C3), 70.5 (Glc-C4), 77.6 (Glc-C5), 63.0 (Glc-
C6), 104.9 (Glc-C1’), 76.2 (Glc-C2’), 77.7 (Glc-C3’), 71.0 (Glc-C4’), 78.8 (Glc-C5’), 62.5 (Glc-
C6’), 105.0 (Xyl-C1), 75.1 (Xyl-C2), 78.7 (Xyl-C3), 70.8 (Xyl-C4), 67.4 (Xyl-C5), or see table
S1 (Supplementary material).
2
0
ꢂ
Henryioside G (2), a white amorphous powder; [a] ꢁ26.1 (c 0.3, MeOH); IR (KBr)
D
ꢁ
1
－
ꢀ
: 3414, 2928, 1642, 1373, 1067, 988, 919, 893, 849 cm ; m/z 1047.5006 [M－H]
max

6
X. ZHANG ET AL.
－
－
corresponding to a molecular formula of C H O
(calculated 1047.5018 [M－H] ).
H-NMR (pyridine-d , 400 MHz, d in ppm) : 1.17 (1H, m, H-1a), 1.51 (1H, m, H-1b), 1.64
50 79 23
1
5
(
4
(
1H, m, H-2a), 2.06 (1H, m, H-2b), 3.92 (1H, m, H-3), 1.29 (1H, m, H-4a), 1.73 (1H, m, H-
b), 0.96 (1H, m, H-5), 1.11 (1H, m, H-6a), 1.13 (1H, m, H-6b), 0.86 (1H, m, H-7a), 1.64
1H, m, H-7b), 2.02 (1H, m, H-8), 5.43 (1H, s, H-11), 4.14 (1H, s, H-12), 3.31 (1H, m, H-
1
4), 1.57 (1H, m, H-15a), 2.04 (1H, m, H-15b), 4.43 (1H, d, J ¼ 7.3 Hz, H-16), 2.31(1H, t,
J ¼ 7.3 Hz, H-17), 1.05 (3H, s, H-18), 0.82 (3H, s, H-19), 2.13 (1H, m, H-20), 1.40 (3H, d,
J ¼ 6.8 Hz, H-21), 1.41 (1H, m, H-23a), 1.94 (1H, m, H-23b), 1.53 (1H, m, H-24a), 2.04 (1H,
m, H-24b), 1.57 (1H, m, H-25), 3.38 (1H, d, J ¼ 11.0 Hz, H-26a), 4.11 (1H, m, H-26b), 1.07
(
3H, d, J ¼ 7.1 Hz, H-27), 4.86 (1H, d, J ¼ 7.6 Hz, H-Gal-1), 4.39 (1H, m, H-Gal-2), 4.04 (1H,
m, H-Gal-3), 4.56 (1H, m, H-Gal-4), 3.99 (1H, m, H-Gal-5), 4.14 (1H, m, H-Gal-6a), 4.61
(
1H, m, H-Gal-6b), 5.21 (1H, d, J ¼ 7.7 Hz, H-Glc-1), 4.37 (1H, m, H-Glc-2), 4.13 (1H, m, H-
Glc-3), 3.79 (1H, m, H-Glc-4), 4.04 (1H, m, H-Glc-5), 4.01 (1H, m, H-Glc-6a), 4.46 (1H, d,
J ¼ 10.4 Hz, H-Glc-6b), 5.26 (1H, d, J ¼ 7.7 Hz, H-Glc-1’), 4.07 (1H, m, H-Glc-2’), 3.88 (1H,
m, H-Glc-3’), 4.15 (1H, m, H-Glc-4’), 4.10 (1H, m, H-Glc-5’), 4.33 (1H, m, H-Glc-6’a), 4.57
(
4
1H, d, J ¼ 10.3 Hz, H-Glc-6’b), 5.58 (1H, d, J ¼ 7,5 Hz, H-Xyl-1), 4.02 (1H, m, H-Xyl-2),
.07 (1H, m, H-Xyl-3), 4.09 (1H, m, H-Xyl-4), 3.65 (1H, t, J ¼ 10.5 Hz, H-Xyl-5a), 4.16 (1H,
1
3
m, H-Xyl-5b). C-NMR (pyridine-d , 100 MHz, d in ppm) : 35.7 (C-1), 29.9 (C-2), 77.3 (C-
5
3), 34.9 (C-4), 43.2 (C-5), 28.7 (C-6), 33.5 (C-7), 36.2 (C-8), 148.3 (C-9), 38.1 (C-10), 123.1
(C-11), 78.5 (C-12), 45.2 (C-13), 53.1 (C-14), 32.9 (C-15), 81.4 (C-16), 61.8 (C-17), 10.9 (C-
18), 17.8 (C-19), 43.8 (C-20), 14.0 (C-21), 110.1 (C-22), 26.4 (C-23), 26.2 (C-24), 27.6 (C-
25), 65.2 (C-26), 16.3 (C-27), 102.5 (Gal-C1), 73.2 (Gal-C2), 75.6 (Gal-C3), 79.9 (Gal-C4),
75.3 (Gal-C5), 60.6 (Gal-C6), 105.2 (Glc-C1), 81.4 (Glc-C2), 86.8 (Glc-C3), 70.5 (Glc-C4),
77.6 (Glc-C5), 63.0 (Glc-C6), 104.9 (Glc-C1’), 76.2 (Glc-C2’), 77.8 (Glc-C3’), 71.1 (Glc-C4’),
78.8 (Glc-C5’), 62.5 (Glc-C6’), 105.0 (Xyl-C1), 75.1 (Xyl-C2), 78.7 (Xyl-C3), 70.8 (Xyl-C4),
67.4 (Xyl-C5), or see table S2 (Supplementary material).
3.5. Acid hydrolysis assay
Solutions of 1 and 2 (5 mg each) were hydrolysed in 2 M hydrochloric acid (5 mL) at
ꢂ
8
0 C for 2 h. After cooling, each solution was concentrated under vacuum, dissolved
with water, and extracted twice with dichloromethane (CH Cl ). The aqueous parts
2
2
were subjected to CC on ODS C18 silica gel (10 g), eluting with (MeCN-H O, 5:95) to
2
give three products. The D configurations of the galactose, glucose and the xylose
moieties in 1 and 2 were confirmed through their optical rotation data (Gal:
2
0
ꢂ
20
ꢂ
20
ꢂ
[
(
a] þ29.0 , MeOH, Glc: [a] þ48.8 , MeOH and Xyl: [a] þ33.6 , MeOH) and R values
D
D
D
f
BuOH-AcOH-H O, 4:1:5 upper layer, Gal: 0.39, Glc: 0.36 and Xyl: 0.54) with the authen-
2
tic sugar samples.
3.6. Cytotoxicity assay
The cytotoxic activity assay in the human HepG2 and SW620 cell lines were measured
4
by the MTT method (positive control: 5-fluorouracil, IC 12.07 lM). Briefly, 1 ꢃ 10
50
ꢁ
1
mL
cells were seeded into 96-well plates and allowed to adhere for 24 h.
Compounds 1 and 2 were dissolved in DMSO and diluted with complete medium to 6

NATURAL PRODUCT RESEARCH
7
ꢂ
degrees of concentrations for inhibition rate determination. After incubation at 37.8 C
for 4 h, the supernatant was removed before adding DMSO (100 lL) to each well, the
IC50 values were calculated (Table 1).
4. Conclusions
A total of four steroidal saponins were obtained from the roots and rhizomes of M.
henryi, of which, henryiosides F and G (1 and 2) were determined as two new ones.
Moreover, the isolated saponins (1ꢁ4) were assayed for their regarding cytotoxic
activities in human HepG2 and SW620 tumour cells, the results (Table 1) showed that
all of the saponins exhibited inhibitory effects with IC50 values ranging from 15.33 lM
to 57.85 lM. This study enriched the chemical and pharmacological diversity of
M. henryi.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was financially supported by the Key Laboratory Project of Shannxi Education
Department (Study on the antitumor pharmacodynamic substance basis of Smilacina henryi).
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