Three new steroidal saponins from the roots and rhizomes of Rohdea chinensis (Baker) N.Tanaka (synonym Tupistra chinensis Baker)

Article abstract of DOI:10.1080/14786419.2019.1616723

Three new steroidal saponins (1–3), together with four known compounds (4–7), were isolated from the roots and rhizomes of Rohdea chinensis Baker, and their structures were determined as (24S, 25 R)-1β-hydroxy-3β-[(β-D-glucopyranoside)oxy]-spirost-5-en-24-yl-β-D-glucopyranoside (1) and (24S)-spirost-25(27)-en-1β, 3β, 4β, 5β, 6β, 24β-hexahydroxy-24-O-β-D-glucopyranoside (2), (22S, 25S)-1β, 3β, 4β, 5β, 26, 27-hexanol-furospirost-5, 26-O-β-D-glucopyranoside (3), together with four known compounds 3-epi-diosgenin-3-β-D-glucopyranosid (4), 3-epiruscogenin (5), 25(R)-1β-hydroxy-spirost-5-en-3α-yl-O-β-D-glucopyranoside (6) and tupichinin A (7), on the basis of physico-chemical properties and spectral analysis. In this study, compounds 1–3 and 5–7 were evaluated for their cytotoxic activity against SW620, A549 and HepG2 tumor cell lines. Among them, compound 7 showed moderate cytotoxicity against two human cancer cell lines A549 and HepG 2 with IC₅₀ values of 25.3 ± 2.6 and 26.1 ± 2.5 μM, respectively.

Full text of DOI:10.1080/14786419.2019.1616723

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Three new steroidal saponins from the roots and
rhizomes of Rohdea chinensis (Baker) N.Tanaka
(synonym Tupistra chinensis Baker)
Bei Song, Wenli Huang, Yuze Li, Yangyang Zhang, Huawei Zhang, Yi Jiang,
Chong Deng, Xiaomei Song & Jianli Liu
To cite this article: Bei Song, Wenli Huang, Yuze Li, Yangyang Zhang, Huawei Zhang, Yi Jiang,
Chong Deng, Xiaomei Song & Jianli Liu (2019): Three new steroidal saponins from the roots and
rhizomes of Rohdeaꢀchinensis (Baker) N.Tanaka (synonym Tupistraꢀchinensis Baker), Natural
Product Research, DOI: 10.1080/14786419.2019.1616723
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1616723
Three new steroidal saponins from the roots and
rhizomes of Rohdea chinensis (Baker) N.Tanaka (synonym
Tupistra chinensis Baker)
c
a
c
c
Bei Songa,b , Wenli Huang , Yuze Li , Yangyang Zhang , Huawei Zhang , Yi
ꢀ
ꢀ
Jiangc, Chong Dengc, Xiaomei Songc and Jianli Liua
aKey Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education,
b
College of Life Science, Northwest University, Xi’an, China; The Second Affiliated Hospital, Shaanxi
c
University of Chinese Medicine, Xianyang, China; College of Pharmacy, Shaanxi University of
Chinese Medicine, Xianyang, China
ABSTRACT
ARTICLE HISTORY
Received 31 January 2019
Accepted 1 May 2019
Three new steroidal saponins (1–3), together with four known
compounds (4–7), were isolated from the roots and rhizomes of
Rohdea chinensis Baker, and their structures were determined as
(24S, 25 R)-1b-hydroxy-3b-[(b-D-glucopyranoside)oxy]-spirost-5-en-
24-yl-b-D-glucopyranoside (1) and (24S)-spirost-25(27)-en-1b, 3b,
4b, 5b, 6b, 24b-hexahydroxy-24-O-b-D-glucopyranoside (2), (22S,
25S)-1b, 3b, 4b, 5b, 26, 27-hexanol-furospirost-5, 26-O-b-D-gluco-
pyranoside (3), together with four known compounds 3-epi-dios-
genin-3-b-D-glucopyranosid (4), 3-epiruscogenin (5), 25(R)-1b-
hydroxy-spirost-5-en-3a-yl-O-b-D-glucopyranoside (6) and tupichi-
nin A (7), on the basis of physico-chemical properties and spectral
analysis. In this study, compounds 1–3 and 5–7 were evaluated
for their cytotoxic activity against SW620, A549 and HepG2 tumor
cell lines. Among them, compound 7 showed moderate cytotox-
icity against two human cancer cell lines A549 and HepG 2 with
IC50 values of 25.3 2.6 and 26.1 2.5 lM, respectively.
KEYWORDS
Rohdea chinensis; steroidal
saponins; cytotoxicity
CONTACT Xiaomei Song
Xianyang, 712046, China; Jianli Liu
Songxiaom@126.com
College of Pharmacy, Shaanxi University of Chinese Medicine,
Key Laboratory of Resource Biology and Biotechnology
jlliu@nwu.edu.cn
in Western China, Ministry of Education, College of Life Science, Northwest University, Xi’an, 710069, China.
ꢀ
Bei Song and Wenli Huang contributed equally to this work.
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1616723.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
B. SONG ET AL.
1. Introduction
Rohdea chinensis Baker belongs to family Liliaceae (Convallarieae), which is used as an
endemic herbal medicine, known as “Kai-Kou-Jian”, and it is mainly distributed in the
Qinba Mountains of Shaanxi Province in China (Song and Liu 2011). It has long been
used in Chinese folk medicine for the treatment of pharyngitis, rheumatic diseases
and snake bite (Pan et al. 2012; Wu et al. 2014). Previous phytochemical investigation
on this plant revealed the presence of spirostanol sapogenins, flavonoids, lignans, a
pregnane glycoside, a pregnane genin and other chemical constituents from R. chinen-
sis rhizomes (Pan et al. 2000; Pan et al. 2006; Liu et al. 2012; Xiao et al. 2015; Xiang
et al. 2016). The modern pharmacological experiments indicated that the extracts of
this species possessed significant antitumor activity (Pan et al. 2012). Moreover, ster-
oidal saponins isolated from this plant have exhibited potent bioactivities including
anticancer, anti-inflammatory, antifungal and antiviral (Liu et al. 2013; Wei et al. 2014;
Xiang et al. 2016). As part of our exploration of the diversity of bioactive saponins in
medicinal herbs growing in the Qinba Mountains (Li et al. 2015; Song, Li et al. 2015;
Song, Zhang et al. 2015; Cui et al. 2018), three new steroidal compounds (24S, 25 R)-
1b-hydroxy- 3b- [(b-D- glucopyranoside)oxy]spirost-5-en-24-yl-b-D-glucopyranoside (1)
and (24S)-spirost-25(27)-en-1b, 3b, 4b, 5b, 6b, 24b-hexahydroxy-24-O-b-D-glucopyrano-
side (2), (22S, 25S)-1b, 3b, 4b, 5b, 26, 27-hexanol-furospirost-5, 26-O-b-D-glucopyrano-
side (3), together with four known compounds 3-epi-diosgenin-3-b-D-glucopyranoside
(4) (Takahira et al. 1977), 3-epiruscogenin (5) (Pan et al. 2003), 25(R)-1b-hydroxy-spi-
rost-5-en-3a-yl-O-b-D-glucopyranoside (6) (Park et al. 2014) and tupichinin A (7) (Pan
et al. 2012) (Figure 1) were obtained from rhizomes of R. chinensis. This study reported
the isolation and structural elucidation of three new steroidal saponins by detailed
analysis of their 1D and 2D NMR spectra as well as enzymatic hydrolysis and their
cytotoxic evaluation against A549, SW620 and HepG 2 tumor cells. Among them, com-
pound 7 showed moderate cytotoxicity against two human cancer cell lines A549 and
HepG 2 with IC50 values of 25.3 2.6 and 26.1 2.5 lM, respectively.
2. Results and discussion
Compound 1 was isolated as an amorphous solid, its molecular formula was deter-
mined as C39H62O15 from the HR-ESI-MS at m/z 793.3972 [M þ Na]þ (calcd for
793.3986). The typical steroid methyl proton signals at dH 1.01 (d, J ¼ 6.8 Hz, Me-21),
1
1.30 (s, Me-19), 1.16 (d, J ¼ 6.4 Hz, Me-27) and 0.83 (s, Me-18) in the H NMR spectrum,
and olefinic carbon signals at dC 140.2 (C-5) and 124.8 (C-6) and a quaternary carbon
signal at dC 112.1 (C-22) in the 13C NMR spectrum, indicated that 1 was a D5(6)-spiro-
stanol (Pan et al. 2003). The assignments of the NMR signals associated with 1 were
1
derived from H–1H COSY, HSQC, HMBC and NOESY experiments. Three oxygenated
methane carbon signals at dC 76.2, 75.5 and 82.0 were attributed to C-1, C-3 and C-24,
which showed correlations with proton signals at dH 4.09 (1H, ca., H-1), 3.99 (1H, ca.,
H-3) and 4.07 (1H, ca., H-24) in the HSQC spectrum. In the HMBC spectrum of 1,
HMBC correlations of H-19/C-1, H-4/C-3, H-24/C-25 and H-26/C-24 revealed that three
hydroxyl groups were linked at C-1, C-3 and C-24 of the aglycone, respectively. HMBC
correlations of H-19/C-1, C-5, C-9 and C-10, H-18/C-12, C-13, C-14 and C-17, H-17/C-20

NATURAL PRODUCT RESEARCH
3
Figure 1. The structures of compounds 1–7.
and C-22, H-3/C-1 and C-5, H-6/C-5, C-8 and C-10, were assigned. Moreover, HMBC cor-
relations of H-26/C-22, C-24 and C-25, H-27/C-24, C-25 and C-26 were observed. Thus,
the structure of the aglycone moiety was determined as 1, 3, 24-triol-spirost-5-en. In
addition, the HMBC correlation signals of H-Glc-10/C-3 and H-Glc-100/C-24, indicated
that glucosyl groups were connected as (Glc-100-O-C-24) and (Glc-10-O-C-3) (Figure S1).
The two glucosyl moieties were identified as D-glucose by enzymatic hydrolysis of 1,
followed by TLC comparison with a reference compound and optical rotation deter-
mination (Hudson and Dale 1917), and judged to be in a b-configuration (Zhang et al.
2012) from the coupling constants of the anomeric protons (7.8 Hz and 7.8 Hz, respect-
ively). Meanwhile, in the NOESY spectrum, the correlation of H-2a/H-9, H-2b/H-4a, H-1/
H-4b, and H-3/H-4b supported the A/B cis ring junction pattern and also indicated
a-axial configurations of H-1 and H-3 and b-orientation of 1-OH and 3-OH. In addition,
the proton multiplicity of H-24, with the J values of 11.6 Hz (H-24/H-23a), 4.7 Hz (H-24/
H-23b), and 4.9 Hz (H-24/H-25), and NOE correlations of H-23ax/H-20/Me-27 and H-
26ax/H-16/H-24 in 1 were consistent with the known compound surculoside A with C-
22a, C-24S and C-25R configurations (Yokosuka et al. 2000). Four typical absorption
bands at 963 cmꢁ1(A), 922 cmꢁ1 (B), 901 cmꢁ1(C), and 878 cmꢁ1(D) were observed in
the IR spectra, and intensity of C band was stronger than B band, which suggested
that the absolute configuration of C-25 was R (Kravets et al. 1986). Accordingly, com-
pound 1 was identified as (24S, 25 R)-1b-hydroxy-3-[(-D-glucopyranoside) oxy]spirost
-5-en -24-yl-b-D-glucopyranoside.
Compound 2 was obtained as a white amorphous powder and showed a positive
reaction in the Liebermann–Burchard test. Its molecular formula was deduced as
C33H52O13 from the quasi-molecular ion peak at m/z 657.3471 [M þ H]þ (calcd for
1
C33H53O13, 657.3486) in the HR-ESI-MS. The H NMR spectrum showed three methyl
protons at dH 0.78 (3H, s, Me-18), 1.90 (3H, s, Me-19), and 1.07 (3H, d, J ¼ 6.9 Hz, Me-
21), the geminal protons at dH 5.13(1H, br.s, H-27a) and 5.14 (1H, br.s, H-27b) as two
singlets and the coupling constants of approximately 0 Hz, characteristic of an exo-
methylene, ascribable to the aglycone proton signals of this steroidal saponin.
Comparison of the NMR data of 2 and the known compound tupichigenin B (Pan et al.

4
B. SONG ET AL.
2000), indicated almost similar NMR spectroscopic features, suggested the existence of
the 1-OH, 3-OH, 4-OH, 5-OH and 6-OH. Therefore, the aglycone of 2 was identified as 1,
3, 4, 5, 6, 24-hexanol-spirost-25(27)-en. In addition, HMBC correlations of H-24/C-23 and
C-Glc-1, H-Glc-1 and C-24, indicated the glucopyranoside was linked at C-24 (Figure S2).
Similarly as compound 1, the results of the enzymatic hydrolysis procedure and analysis
of detail NOESY spectra data showed the structure of 2 was elucidated as (24S)-spirost-
25(27)-en-1b, 3b, 4b, 5b, 6b, 24b-hexahydroxy-24-O-b-D-glucopyranoside.
Compound 3 had a molecular formula C39H64O18, was deduced from the quasi-
1
molecular ion peak at m/z 819.4020 [M - H]- (calcd for C39H63O18, 819.4014). The H
NMR spectrum showed three typical steroidal methyl signals at dH 0.89 (3H, s, Me-18),
1.67 (3H, s, Me-19) and 1.15 (3H, d, J ¼ 6.5Hz, Me-21). The 13C NMR spectrum exhibited
the C-22 carbon of the aglycone at dC 121.1, characteristic of the furospirostanol skel-
eton (Yang et al. 2005). In addition, the observation of anomeric proton signal at dH
5.23 (1H, d, J ¼ 7.4 Hz) and 4.92 (1H, d, J ¼ 7.6 Hz), together with enzymatic hydrolysis
of 3 affording d-glucose, suggested that 3 were a furospirostanol glucoside.
Comparison the NMR data of 3 with the known compound (20S, 22 R)-spirost-25(27)-
en-1b, 3b, 4b, 5b-tetraol-5-O-b-D-glucopyranoside (Song, Li et al. 2015), their NMR
spectroscopic features of the ring A-E were similar, indicated the existence of the 1-
OH, 3-OH, 4-OH, and 5-OH. Their difference was mainly display on the ring F. The
chemical shifts of dC 74.9 (C-26) and dC 65.2 (C-27) were due to two oxygenated car-
bon groups. Moreover, HMBC correlations of H-27/C-24, C-25 and C-26 were observed
(Figure S3). In the NOESY spectrum of 3, the NOE correlations of H-2a/H-4/H-3/H-7a/H-
9/H-1 and H-1/Me-19, indicated b-orientation of 1-OH, 3-OH and 4-OH. The NOE corre-
lations of H-20/H-23b/Me-27 and the carbons in the F-ring (Table S1) verified that the
stereochemistry of C-22 and C-25 was in the S-configuration (Yang et al. 2005; Qin
et al. 2016). In addition, the HMBC correlation signals of H-Glc-10/C-5 and H-Glc-100/C-
26, indicated that glucosyl groups were connected as (Glc-100-O-C-26) and (Glc-10-O-C-
5). The two glucosyl moieties were identified as D-glucose by enzymatic hydrolysis of
3, followed by TLC comparison with a reference compound and optical rotation deter-
mination (Hudson and Dale 1917), and judged to be in a b-configuration (Zhang et al.
2012) from the coupling constants of the anomeric protons (7.6 Hz and 7.4 Hz, respect-
ively). Therefore, compound 3 was identified as (22S, 25S)-1b, 3b, 4b, 5b, 26, 27-hexa-
nol-furospirost-5, 26-O-b-D-glucopyranoside.
Compounds 1–3 and 5–7 were evaluated for their cytotoxic activity against SW620,
A549 and HepG2 tumor cell lines by MTT method. Among them, compound 7 showed
moderate cytotoxicity against two human cancer cell lines A549 and HepG 2 with IC50
values of 25.3 2.6 and 26.1 2.5 lM, respectively.
3. Experimental
3.1. General experimental procedures
Optical rotation indices were determined in methanol on a Rudolph Autopol II digital
polarimeter (Rudolph, Hackettstown, NJ, USA). The IR spectra were recorded on a
Bruker TENSOR-27 instrument. The HR-ESI–MS spectra was taken on an Agilent
Technologies 6550 Q-TOF. 1 D and 2 D NMR spectra were recorded on a Bruker-

NATURAL PRODUCT RESEARCH
5
AVANCE400 instrument with TMS as an internal standard. The analytical HPLC was per-
formed on a Waters 2695 Separations Module coupled with a 2996 Photodiode Array
Detector and a Accurasil C18 column (4.6 mm ꢂ 250 mm, 5 mm particles, Ameritech,
America). Semipreparative HPLC was performed on a system comprising a Shimadzu
LC-6AD pump equipped with an SPD-20A UV detector and a Ultimate XB-C18 (10 mm
ꢂ250 mm, 5 mm particles) or YMC-Pack-ODS-A (10 mm ꢂ250 mm, 5 mm particles).
D101 was from Sunresin New Materials Co. Ltd. (Xi’an, China). Silica gel was purchased
from Qingdao Haiyang Chemical Group Corporation (Qingdao, China).
3.2. Plant material
The roots and rhizomes of R. chinensis Baker were collected from Taibai region of
Qinba mountains in Shaanxi Province on August in 2010, and identified by Senior
experimentalist Wang Ji-tao. A voucher specimen (herbarium No.20100816) has been
deposited in the Medicinal Plants Herbarium (MPH), Shaanxi University of Chinese
Medicine, Xianyang, China.
3.3. Extraction and isolation
The air-dried and powdered underground parts of R. chinensis (1.5 kg) were extracted
with 65% EtOH three times at 80 ꢃC. The combined EtOH extracts was evaporated to
6 L, and applied to a resin D101 column, eluting with H2O, 20% EtOH, 60% EtOH, and
95% EtOH to give four fractions (Fr.1-4). Fr.3 (79 g) were separated by silica gel column
chromatography in elution with gradient solvent system (CHCl3-MeOH-H2O, 100:0:0-
0:50:50) producing ten fractions (Fr.3-1 – Fr.3-10). Fr.3-9 (3.5 g) was subjected to
reversed-phase ODS, using MeOH–H2O (0:100 – 70:30) as the eluent to afford six frac-
tions (Fr.3-9-1 – Fr.3-9-6). Fr.3-9-4 (0.8 g) was purified by HPLC (Ultimate XB-C18,
10 mm ꢂ 250 mm, 5 mm particles, flow rate: 1.0 ml/min) with CH3CN-H2O (33:67) as
mobile phase to afford compound 1 (15 mg) and compound 2 (10 mg). Fr.3-4 (6.8 g)
was subjected to rechromatography on silica gel elution with CHCl3-MeOH (15:1) to
afford compound 3 (14 mg), 4 (7 mg) and 5 (9 mg). Fr.3-6 (8 g) was rechromatographed
on silica gel elution with CHCl3-MeOH (10:1) to afford compound 6 (11 mg) and
7 (18 mg).
3.3.1. (24S, 25 R)-1-hydroxy-3-[(-D-glucopyranoside)oxy]spirost-5-en-24-yl--D-gluco-
pyranoside (1)
A white amorphous powder,½aꢄD15:5-80.0 (c 0.4, MeOH); IR (KBr) mmax: 3406, 2932, 1616,
1
1026, 963, 922, 901, 878, 836 cmꢁ1; H NMR and 13C NMR spectral data, see Table S1;
m/z 793.3972 [M þ Na]þ (calcd for C39H62O15Na 793.3986).
3.3.2. (24S)-spirost-25(27)-en-1, 3, 4, 5, 6, 24-hexahydroxy-24-O--D-glucopyrano-
side (2)
A white amorphous powder;½aꢄ1D5:0-128.0 (c 0.4, MeOH); IR (KBr) mmax: 3402, 2927, 1640,
920, 875 cmꢁ1 1H-NMR and 13C-NMR spectral data, see Table S1; m/z 657.3471
;
[M þ H]þ (calcd for C33H53O13, 657.3486),

6
B. SONG ET AL.
3.3.3. (22S, 25S) -1, 3, 4, 5, 26, 27-hexanol-furospirost-5, 26-O--D-glucopyrano-
side (3)
A white amorphous powder;½aꢄD15:2-84.6 (c 0.3, MeOH); IR (KBr) mmax: 3430, 2930, 1719,
1
1452, 1025, 913, 802 cmꢁ1. H NMR and 13C NMR spectral data, see Table S1; m/z 819.
4020 [M - H]- (calcd for C39H63O18, 819.4014)
3.4. Enzymatic hydrolysis of compounds 1 ꢅ 3 and absolute sugar configuration
determination
The solutions of compounds 1 (5 mg), 2 (6 mg) and 3 (4 mg) in H2O (3 ml) were indi-
vidually hydrolyzed with b-glucosidase (10 mg, Almonds Lot 1264252, Sigma-Aldrich,
St. Louis, MO, USA) at 37 ꢃC for 24 or 36 h. Each reaction mixture was extracted with
EtOAc for three times (3 ꢂ 3 ml) to yield the EtOAc extract and H2O phase after remov-
ing the solvents. The aqueous extracts were subjected to CC over silica gel eluted
with CH3CN/H2O (8:1) to yield glucose with positive optical rotation, and the value of
[a]2D0 : þ46.9 ꢅ þ49.2 (c 0.16, H2O). The solvent system CH3CN/H2O (6:1) was used for
the TLC analysis of glucose and authentic sugar samples.
3.5. Cytotoxicity experiments
The cytotoxic activity assay toward the A549, SW620 and HepG 2 tumor cell lines
were measured by the MTT method in vitro, using 5-fluorouracil as positive control.
Briefly, 1 ꢂ 104 mLꢁ1 cells were seeded into 96-well plates and allowed to adhere for
24 h. Compounds 1–3 and 5–7 were dissolved in DMSO and diluted with complete
medium to six degrees of concentration (from 0.001 mmolꢆLꢁ1 to 0.3 mmolꢆLꢁ1) for
inhibition rate determination. After incubation at 37 ꢃC for 4 h, the supernatant was
removed before adding DMSO (100 ml) to each well. 5-fluorouracil (5-Fu) was used as
positive control. The inhibition rate (IR) and IC50 were calculated. Values are
ꢀꢀ
mean SD, n ¼ 3,
p < 0.01 vs. DMEM control. The IC50 values are shown in
Table S2.
4. Conclusion
Three new steroidal saponins (24S, 25 R)-1b-hydroxy-3b-[(b-D-glucopyranoside)oxy]-spi-
rost-5-en-24-yl-b-D-glucopyranoside (1) and (24S)-spirost-25(27)-en-1b, 3b, 4b, 5b, 6b,
24b-hexahydroxy-24-O-b-D-glucopyranoside (2), (22S, 25S)-1b, 3b, 4b, 5b, 26, 27-hexa-
nol-furospirost-5, 26-O-b-D-glucopyranoside (3), together with four known compounds
(4–7), were isolated from the roots and rhizomes of R. chinensis Compound 7 showed
moderate cytotoxicity against two human cancer cell lines A549 and HepG 2 with IC50
values of 25.3 2.6 and 26.1 2.5 lM, respectively.
Disclosure statement
No potential conflict of interest was reported by the authors.

NATURAL PRODUCT RESEARCH
7
Funding
This project was financially supported by the Special Foundation of Shaanxi Provincial Education
Department (No.14JF005); the Open Research Fund of Key Laboratory of Basic and New Herbal
Medicament Research, Shaanxi university of Chinese medicine (No.17JS030, 2017KF02); the
Innovative Team of Pharmacodynamic Substances and Applications of “Qinba Characteristic Qi-
Medicines” in Taibai Mountains, Shaanxi university of Chinese medicine; the National Natural
Science Foundations of China (grant No.81503237); Key R&D Program of Shaanxi Province (grant
No.2017SF-360, 2015SF073, 2018SF-324).
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