Ursane-type triterpenoid saponins from Elsholtzia bodinieri

Article abstract of DOI:10.1080/14786419.2018.1477144

Investigation of the n-BuOH extract of the aerial parts of Elsholtzia bodinieri led to the isolation of two new ursane-type triterpenoid saponins, bodiniosides O (1) and P (2), along with five known saponins, rotungenoside (3), 3,28-O-bis-β-d-glucopyranosides of 19α-hydroxyarjunolic acid (4), oblonganosides I (5), rotungenic acid 28-O-α-L-rhamnopyranosyl-(1→2)-β-d-glucopyranoside (6), and bodinioside M (7) isolated from the species. The structures of compounds 1 and 2 were characterized by spectroscopic data as well as acid hydrolysis and GC analysis as 3-O-β-d-xylopyranosyl-23-acetoxy-urs-12(13)-en-28-oic acid 28-O-β-d-xylopyranosyl-(1→6)-[β-d-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)]-β-d-glucopyranoside and 3-O-β-d-xylopyranosyl-23-hydroxy-urs-12(13)-en-28-oic acid 28-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside. Compounds 1 and 2 exhibited potent anti-HCV activities in vitro with a selective index of 30.63 and 9.08, respectively.

Full text of DOI:10.1080/14786419.2018.1477144

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Ursane-type triterpenoid saponins from Elsholtzia
bodinieri
Lunli Xiang, Lu Zhang, Xuanqin Chen, Xueshan Xia, Rongtao Li & Jindong
Zhong
To cite this article: Lunli Xiang, Lu Zhang, Xuanqin Chen, Xueshan Xia, Rongtao Li & Jindong
Zhong (2018): Ursane-type triterpenoid saponins from Elsholtzia bodinieri, Natural Product
Research, DOI: 10.1080/14786419.2018.1477144
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Natural Product research, 2018
https://doi.org/10.1080/14786419.2018.1477144
Ursane-type triterpenoid saponins from Elsholtzia bodinieri
Lunli Xiang#, Lu Zhang#, Xuanqin Chen, Xueshan Xia, Rongtao Li and Jindong Zhong
Faculty of life science and technology, Kunming university of science and technology, Kunming, P. r. china
ABSTRACT
ARTICLE HISTORY
received 24 January 2018
accepted 9 May 2018
Investigation of the n-BuOH extract of the aerial parts of Elsholtzia
bodinieri led to the isolation of two new ursane-type triterpenoid saponins,
bodiniosidesO(1)andP(2),alongwithfiveknownsaponins,rotungenoside
(3), 3,28-O-bis-β-d-glucopyranosides of 19α-hydroxyarjunolic acid (4),
oblonganosides I (5), rotungenic acid 28-O-α-L-rhamnopyranosyl-(1→2)-
KEYWORDS
Elsholtzia bodinieri; ursane-
type triterpenoid; saponins;
anti-hcV activities
β-d-glucopyranoside (6), and bodinioside M (7) isolated from the species.
The structures of compounds 1and 2were characterized by spectroscopic
data as well as acid hydrolysis and GC analysis as 3-O-β-d-xylopyranosyl-
23-acetoxy-urs-12(13)-en-28-oic acid 28-O-β-d-xylopyranosyl-(1→6)-[β-d-
glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)]-β-d-glucopyranoside
and 3-O-β-d-xylopyranosyl-23-hydroxy-urs-12(13)-en-28-oic acid 28-O-
β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside. Compounds 1 and 2
exhibited potent anti-HCV activities in vitro with a selective index of 30.63
and 9.08, respectively.
1. Introduction
Elsholtzia bodinieri Van’t (Labiatae), an annual herbaceous plant, was widely distributed in the
northwest and southwest districts of China, especially inYunnan and Guizhou Provinces. The
aerial part of the species was usually used as herbal tea or traditional folk medicine for the
CONTACT Jindong Zhong
jindongzhongkm@163.com
#these authors contributed equally to this work.
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1477144.
© 2018 Informa uK limited, trading as taylor & Francis Group

2
L. XIANG ET AL.
prophylaxis and treatment of cough, headache, pharyngitis, fever and hepatitis (College Jiangsu
New Medical 1985). The chemical compositions of E. Bodinieri are previously reported to
mainly contain triterpenoid saponins (Zhu et al. 2002; Li et al. 2005; Hu et al. 2007a), sesquit-
erpene glycosides (Hu et al. 2007b), clerodane diterpenoid glycosides (Hu et al. 2008), and
phenolic constituents (Hu et al. 2007c). Some of the compounds from the species were
revealed to possess antiviral and antibacterial activity (Guo et al. 2012). In a continuation of
our study on the medicinal plants of Elsholtzia family (Li et al. 2005, 2008; Zhao et al. 2015;
Zhong et al. 2016a, 2016b, 2016c), we have investigated the n-BuOH soluble fraction of
E. bodinieri crude acetonic extract. In this paper, we describe the isolation, biological activity
to anti-HCV, and structure elucidation of two new ursane-type triterpenoid saponins desig-
nated bodinioside O (1) and bodinioside P (2), along with five known triterpenoid saponins:
rotungenoside (3), 3,28-O-bis-β-d-glucopyranosides of 19α-hydroxyarjunolic acid (4), oblon-
ganosides I (5), rotungenic acid 28-O-α-L-rhamnopyranosyl-(1→2)-β-d-glucopyranoside (6)
and bodinioside M (7) from the aerial parts of this plant.
2. Results and discussion
Compound 1 was obtained as a white amorphous powder. Its molecular formula was deter-
mined as C60H96O27 according to the positive HR-ESI-TOF-MS, which exhibited a pseudomo-
lecular ion peak [M − H]− at m/z 1247.6067 (calcd for C60H95O27 1247.6061), indicating thirteen
degrees of unsaturation. It exhibited UV maximum absorption at 202.8 nm. Its IR spectrum
revealed absorption typical of hydroyl (3441 cm−1) and carbonyl (1721 cm−1).
The 1H NMR spectrum showed four tertiary methyl and two secondary methyl signals at
δH 0.84 (3H, s), 0.95 (3H, s), 1.02 (3H, s), 1.17 (3H, s), 0.97 (3H, d, J = 6.1 Hz) and 1.19 (3H, d,
J = 8.9 Hz), correlating with carbons at δC 13.0 (C-24), 17.8 (C-26), 16.3 (C-25), 23.4 (C-27), 21.4
(C-30) and 17.1 (C-29) in the HSQC spectrum, respectively. The signal at δH 5.45 (1H, br. s),
corresponding to the carbon at δC 126.2 (C-12) which coupled with δC 138.1 (C-13) in the
13C NMR spectrum, indicated the existence of a double bond. On the basis of the above
spectroscopic data, compound 1 was suggested to possess an urs-12-ene skeleton.
1
Comparison of its H and 13C NMR spectroscopic data with those of cynarasaponins D
(Shimizu et al. 1988), suggested that they had the same aglycone, 3,23-dihydroxyurs-12-en-
28-oic acid, but differ in the sugar moiety and an acetoxy group due to signals of δC 170.8
and 20.7 and δH 2.12 (3H, s).
The 1H NMR spectrum also displayed five anomeric signals at 6.49 (1H, br. s), 6.03 (1H, d,
J = 8.2 Hz), 5.13 (1H, d, J = 7.6 Hz), 4.87 (1H, d, J = 7.5 Hz), and 4.84 (1H, d, J = 7.5 Hz), which
correlated with anomeric carbon signals at δC 100.8 (d), 94.4 (d), 107.0 (d), 107.2 (d) and 105.6
(d) in the HSQC spectrum, respectively, suggesting the presence of five sugar moieties. Acid
hydrolysis of 1 with 1 M HCl produced l-rhamnose, d-glucose and d-xylose units as sugar
residues as determined by GC analysis of their corresponding trimethylsilylated l-cysteine
derivatives. Since NMR signals of five sugar units have undesirable overlapped effects, the
1
HMQC-TOCSY experiment was successfully used to distinguish and assign the H and 13C
NMR signals of each sugar moiety. The correlations from the anomeric proton signal at δH
4.87 to five carbon signals at δC 107.2 (anomeric carbon), 75.3, 78.2, 71.0, and 67.0, as well
as from five proton signals at δH 4.87, 4.00, 3.79, 4.23, and 4.37 to the anomeric carbon,
suggested the presence of d-xylopyranose (Xyl). In a similar way, the 1H and 13C NMR signals
for d-glucopyranosyl (Glc) and l-rhamnopyranosyl (Rha) were assigned. The β anomeric

NATURAL PRODUCT RESEARCH
3
Figure 1. the structure of compounds 1 and 2.
configurations for the xylopyranosyl and glucopyranosyl were determined by means of their
3 JH1,H2 coupling constants (3J = 7.5, 8.2 and 7.6 Hz, respectively). Comparison of the 13C signals
and observation of a broad singlet for H-1 deduced the α anomeric configuration for -rham-
l
nopyranosyl (Li et al. 2012b).
The sequence of the glycoside chains connected to C-3 and C-28 was established by
analysis of the HMBC correlations. The absence of any glycosidation shift for Xyl suggested
that Xyl was a terminal sugar unit attached at C-3 of the aglycone, which was further con-
firmed by HMBC correlation of HXyl-1 (δH 4.87) of C-3 (Figure 1). A series of HMBC correlations
from HGlc-1 (δH 6.03) to C-28, from HRha-1 (δH 6.49) to CGlc-2 (δc 74.7), from HXyl’-1 (δH 4.84) to
CGlc-6 (δc 68.8), and from HGlc′-1 (δH 5.13) to CRha-4 (δc 85.7), unambiguously clarified the
sequence of the second osidic part at C-28. In addition, HMBC correlation between H-23 (δH
4.55, 4.45) and the carbonyl carbon (δC 170.8) suggested that the acetoxy group was attached
at C-23.
Based on the above evidence, the structure of compound 1 was established as 3-O-β-d-
xylopyranosyl-23-acetoxy-urs-12(13)-en-28-oic acid 28-O-β-d-xylopyranosyl-(1→6)-[β-d-
glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)]-β- -glucopyranoside, a new ursane-type
d
saponin, and named as bodinioside O.
Bodinioside P (2), a white amorphous powder, was assigned the molecular formula of
C47H76O18, deduced from the pseudomolecular ions [M − H]− and [M + COOH]− at m/z 927.5
and 973.5, respectively, in the negative ESI-MS, and further confirmed by the positive
HR-ESI-MS (m/z 973.5002, [M + COOH]+), requiring nine degrees of unsaturation. A compre-
hensive analysis of 1H and 13C NMR spectral data suggested that 2 was also a saponin, with
urs-12-ene as the aglycone. Detailed comparison of the 1H and 13C NMR spectral data of 2
with those of 1 indicated they are highly structurally similar, except for the signal absence
of acetyl group in 1 and the difference of monosaccharides attached to C-28. Acid hydrolysis
of 2 also yielded two
d
-glucose and a -xylose as sugar residues as determined by GC analysis.
d

4
L. XIANG ET AL.
Table 1 anti-hcV activities of compounds 1 and 2 from E. bodinieri.
CC50 (nM)
EC50 (nM)
SI (CC50/EC50)
IFNα-1b
ribavirin
1
2
n.d.
1.08a
9.57
0.41
1.58
–
95.94
12.56
14.35
10.03
30.63
9.08
athe unit of IFN is Iu/ml.
1
In the H NMR spectrum of 2, three anomeric protons at δH 5.03 (d, J = 6.2 Hz), 6.15 (d,
J = 5.8 Hz) and 4.99 (d, J = 5.8 Hz) also indicated the presence of three sugar residues: a
xylopyranosyl (Xyl) and two glucopyranosyl (Glc) units. The Xyl unit was still deduced to link
to C-3 of the aglycone based on the HMBC correlation between δH 5.03 (H-1) of Xyl and δC
82.0 (C-3). Whereas, HMBC correlations from δH 6.15 (H-1) of Glc to δC 176.4 (C-28) of the
aglycone and from δH 4.99 (H-1) of Glc′ to δC 69.2 (C-6) of Glc, enabled the sugar chain at
C-28 to be assigned as β-d-glucopyranosyl (1→6)-β-d-glucopyranosyl. Thus, the structure of
2 was adequately illustrated as 3-O-β-
28-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside, and named bodinioside P.
The five known ursane-type triterpenoid saponins were determined as β- -glucopyranosyl
d-xylopyranosyl-23-hydroxy -urs-12(13)-en-28-oic acid
d
3β,19α,23-trihydroxyurs-12-en-28-oate (rotungenoside, 3) (Nakatani et al. 1989), 3,28-O-bis-
β-d-glucopyranosides of 19α-hydroxyarjunolic acid (4) (Higuchi et al. 1982), 3-O-β-d-
xylopyranosyl-3β,19α,23-trihydroxyurs-12(13)-en-28-oic acid-28-O-β-d-glucopyranosyl ester
(oblonganosides I, 5) (Wu et al. 2007), rotungenic acid 28-O-α-L-rhamnopyranosyl-(1→2)-β-
d-glucopyranoside (6) (Taketa and Schenkel 1994) and 3-O-β-d-xylopyranosyl-19α-hydroxy-
23-acetoxy-urs-12(13)- en-28-oic acid 28-O-α-L-rhamnopyranosyl-(1→2)-β-d-glucopyranoside
(bodinioside M, 7) (Zhong et al. 2016b) by comparison of the spectroscopic data with those
reported, respectively.
The anti-HCV activities of compounds 1 and 2 were evaluated, and their cytotoxicity was
measured in parallel with the determination of antiviral activity, using ribavirin as positive
control (CC50 = 95.94 nM, EC50 = 9.57 nM). Among the tested compounds, compound 1 exhib-
ited potent anti-HCV activity, with an EC50 value of 0.41 nM, a CC50 value of 12.56 nM, and a
SI value of 30.63. Moreover, compound 2 showed moderate anti-HCV activity, with an EC50
value of 1.58 nM, a CC50 value of 14.35 nM, and a SI value of 9.08 (Table 1). The isolated
compounds (1–7) were also evaluated for anti-HIV-1 effects and anti-influenza A virus activ
-
ities, but they were found to be inactive.
3. Experimental
3.1. General experimental procedures
Optical rotations were obtained using a Jasco DIP-370 digital polarimeter. UV spectra were
run on a UV-210A spectrophotometer. IR spectra were recorded on a Bio-Rad FtS-135 spec-
trophotometer in a KBr disk. NMR spectra were recorded using Bruker AM-400 and DRX-600
instruments with tetramethylsilane (TMS) as an internal standard. EIMS were measured on
a VG Auto-Spec-3000 spectrometer. ESIMS and HRESIMS were obtained with an API-Qstar-
TOF instrument. GC analysis was run on Agilent Technologies HP5890 gas chromatograph
with flame ionization detector. Semi-preparative HPLC was performed on an Agilent 1200
liquid chromatograph with a ZORBAX SB-C18 (5, 9.4 × 250 mm) column. Column

NATURAL PRODUCT RESEARCH
5
chromatography (CC) was carried out on silica gel (200–300 mesh, 100–200 mesh, 80–100
mesh, Qingdao Marine Chemical Factory, Qingdao, China), Diaion HP-20SS (63–150 mm
Mitsubishi Fine Chemical Industries Co., Ltd., Tokyo, Japan), ODS-C18 (75 μm, YMC Co., Ltd.,
Japan) and Sephadex LH-20 (Amersham Biosciences AB, Uppsala, Sweden). Fractions were
monitored by TLC plates (Si gel G, Qingdao Marine Chemical Factory, Qingdao, China), and
spots were visualized by heating silica gel plates sprayed with 5% H2SO4–EtOH.
3.2. Materials
The aerial parts of E. bodinieri Van’t were collected from Yuxi, Yunnan Province, P. R. China,
in May 2016, and identified by Dr Xuanqing Chen. A voucher specimen (KMUST 20160005)
was deposited at the Laboratory of Phytochemistry, Faculty of Life Science and Technology,
Kunming University of Science and Technology. Ribavirin and IFNα-1b were purchased from
Sigma-Aldrich, St. Louis, MO, USA. Human hepatoma Huh7.5.1 cells were a gift from Prof.
Ping Zhao (Second Military Medical University). The purity (>95%) of compounds 1 and 2
used for biological assay was determined by HPLC.
3.3. Extraction and isolation
The dried aerial parts of E. bodinieri (15 kg) were powdered and extracted with 75% aq.
Me2CO (3 × 35 L, 24 h, each) at room temperature, then concentrated in vacuo to yield an
extract, which was suspended in H2O, and successively partitioned with CHCl3, EtOAc, and
n-BuOH.
The n-BuOH extract (300.0 g) was separated over macroporous resin CC (Diaion HP-20SS)
eluting with MeOH/H2O (gradient 10, 30, 60, 90, and 100%, each 2.5 L) to afford five fractions
(Fr. A~E). Fr. B (50 g) was subjected to Sephadex LH-20 gel CC, eluting with MeOH/H2O
(gradient 10, 30, 50, 70, 90, and 100%, each 1 L) to obtain subfractions B-1 ~ B-4. Fr. B-2 (7.9 g)
was chromatographed successively over silica gel CC (chloroform/MeOH/H2O, gradient
20:1:0.1→5:5:0.5) to obtain subfractions B-2–1 ~ B-2–4. Fr. B-2–2 (1.2 g) was separated by
ODS (MeOH/H2O, gradient 10→100%) to obtain subfractions B-2–2-1 ~ B-2–2-4. Fr. B-2–2-2
(166.0 mg) was purified over semi-preparative HPLC (26% CH3CN, 3 mL/min) to give 6
(tR = 17.5 min, 7.7 mg), 4 (tR = 20.3 min, 15.6 mg), and 3 (tR = 22.0 min, 13.7 mg). Fr. B-2–2-3
(125.0 mg) was further purified on semi-preparative HPLC (25% CH3CN, 3 mL/min) to obtain
compounds 2 (tR = 22.6 min, 6.9 mg) and 5 (tR = 24.3 min, 18.3 mg). 1 (tR = 20.8 min, 5.6 mg)
and 7 (tR = 22.7 min, 13.6 mg) were prepared from Fr. B-2–2-4 (185.0 mg) on semi-preparative
HPLC (23% CH3CN, 3 mL/min).
Bodinioside O (1): white amorphous powder. [ꢀ]2D6.6 = −9.33 (c = 0.10, MeOH). IR (KBr):
3441, 2931, 1721, 1632, 1386, 1255, 1074 cm−1; UV λmax (MeOH) nm (logε): 206.8 (2.16); ESI-MS
(pos.) m/z: 1271 [M + Na]+); HR-ESI-MS (pos.) m/z: 1247.6067 [M − H]− (Calcd for C60H95O27,
1247.6061). 1H NMR (600 MHz, pyridine-d5): δ 1.50 (1H, m, H-1a), 0.87 (1H, m, H-1b), 2.06 (1H,
m, H-2a), 1.29 (1H, m, H-2b), 3.93 (1H, m, H-3), 1.23 (1H, m, H-5), 1.56 (1H, m, H-6a), 1.39 (1H,
m, H-6b), 1.58 (2H, m, H-7), 1.60 (1H, m, H-9), 2.00 (2H, m, H-11), 5.45 (1H, br. s, H-12), 1.50
(2H, m, H-15), 2.08 (1H, m, H-16a), 1.89 (1H, m, H-16b), 2.56 (1H, d, J = 10.9 Hz, H-18), 1.30
(1H, m, H-19), 1.42 (1H, m, H-20), 2.16 (2H, m, H-21), 2.08 (1H, m, H-22a), 1.78 (1H, m, H-22b),
4.55 (1H, m, H-23a), 4.45 (1H, m, H-23b), 0.84 (3H, s, H-24), 1.02 (3H, s, H-25), 0.95 (3H, s, H-26),
1.17 (3H, s, H-27), 1.19 (3H, d, J = 8.9 Hz, H-29), 0.97 (3H, d, J = 6.1 Hz, H-30), 23-OAC: 2.12 (3H,

6
L. XIANG ET AL.
s); 3-O-sugar: Xyl 4.87 (1H, d, J = 7.5 Hz, H-1), 4.00 (1H, m, H-2), 3.79 (1H, m, H-3), 4.23 (1H, m,
H-4), 4.37 (1H, m, H-5a), 3.75 (1H, m, H-5b); 28-O-sugar: Glc 6.03 (1H, d, J = 8.2 Hz, H-1), 4.30
(1H, m, H-2), 4.19 (1H, m, H-3), 4.29 (1H, m, H-4), 3.96 (1H, m, H-5), 4.60 (1H, m, H-6a), 4.23
(1H, m, H-6b), Rha 6.49 (1H, br. s, H-1), 4.72 (1H, m, H-2), 4.61 (1H, m, H-3), 4.38 (1H, m, H-4),
4.59 (1H, m, H-5′), 1.87 (3H, d, J = 6.2 Hz, H-6), Xyl′ 4.84 (1H, d, J = 7.5 Hz, H-1), 3.98 (1H, m,
H-2), 3.76(1H, m, H-3), 4.20 (1H, m, H-4), 4.28 (1H, m, H-5a), 3.62(1H, m, H-5b), Glc′ 5.13 (1H,
d, J = 7.6 Hz, H-1), 4.02 (1H, m, H-2), 4.12 (1H, m, H-3), 4.13 (1H, m, H-4), 4.14 (1H, m, H-5), 4.47
(1H, m, H-6a), 4.30 (1H, m, H-6b). 13C NMR (150 MHz, pyridine-d5): 38.5 (C-1), 26.0 (C-2), 82.0
(C-3), 42.3 (C-4), 48.3 (C-5), 18.3 (C-6), 33.3 (C-7), 40.1 (C-8), 48.5 (C-9), 36.6 (C-10), 23.5 (C-11),
126.2 (C-12), 138.1 (C-13), 42.3 (C-14), 28.3 (C-15), 24.6 (C-16), 48.1 (C-17), 53.0 (C-18), 38.1
(C-19), 39.3 (C-20), 30.7 (C-21), 36.6 (C-22), 64.6 (C-23), 13.0 (C-24), 16.3 (C-25), 17.8 (C-26),
23.4 (C-27), 176.5 (C-28), 17.1 (C-29), 21.4 (C-30), 23-OAC 170.8 (–C=O), 20.7 (–CH3); 3-O-sugar:
Xyl 107.2 (C-1), 75.3 (C-2), 78.2 (C-3), 71.0 (C-4), 67.0 (C-5); 28-O-sugar: Glc 94.4 (C-1), 74.7
(C-2), 79.6 (C-3), 70.6 (C-4), 77.2 (C-5), 68.8 (C-6), Rha 100.8 (C-1), 71.5 (C-2), 72.4 (C-3), 85.7
(C-4), 68.0 (C-5), 18.8 (C-6), Xyl′ 105.6 (C-1), 74.6 (C-2) 78.0 (C-3), 70.9 (C-4), 67.0 (C-5), Glc′
107.0 (C-1), 76.2 (C-2), 78.5 (C-3), 71.8 (C-4), 78.4 (C-5), 63.0 (C-6).
Bodinioside P (2): white amorphous powder.[ꢀ]2D4.9 = −4.29 (c = 0.25, MeOH). ESI-MS (neg.)
m/z: 927.5 [M-H]-, 973.5 [M + COOH]-; HR-ESI-MS (pos.) m/z: 973.5002 [M + COOH]+ (Calcd for
C47H76O18, 928.5032). 1H NMR (400 MHz, pyridine-d5): δ 1.57 (1H, m, H-1a), 0.78 (1H, m, H-1b),
2.22 (2H, m, H-2), 4.24 (1H, m, H-3), 1.23 (1H, m, H-5), 1.63 (1H, m, H-6a), 1.31 (1H, m, H-6b),
1.63 (1H, m, H-7a), 1.31 (1H, m, H-7b), 1.65 (1H, m, H-9), 1.91 (2H, m, H-11), 5.42 (1H, br. s,
H-12), 2.37 (1H, m, H-15a), 1.04 (1H, m, H-15b), 2.00 (1H, m, H-16a), 1.89 (1H, m, H-16b), 2.47
(1H, d, J = 11.2 Hz, H-18), 1.30 (1H, m, H-19), 1.02 (1H, m, H-20), 1.30 (1H, m, H-21a), 1.18 (1H,
m, H-21b), 1.90 (1H, m, H-22a), 1.74 (1H, m, H-22b), 4.27 (1H, m, H-23a), 3.67 (1H, m, H-23b),
0.92 (3H, s, H-24), 0.97 (3H, s, H-25), 1.14 (3H, s, H-26), 1.11 (3H, s, H-27), 0.88 (3H, d, J = 5.9 Hz,
H-29), 0.82 (3H, d, J = 6.1 Hz, H-30); 3-O-sugar: Xyl 5.03 (1H, d, J = 6.2 Hz, H-1), 3.98 (1H, m,
H-2), 3.85 (1H, m, H-3), 4.19 (1H, m, H-4), 4.25 (1H, m, H-5a), 3.61 (1H, m, H-5b); 28-O-sugar:
Glc 6.15.(1H, d, J = 5.8 Hz, H-1), 3.99(1H, m, H-2), 4.18 (1H, m, H-3), 4.17 (1H, m, H-4), 4.06 (1H,
m, H-5), 4.67 (1H, m, H-6a), 4.27 (1H, m, H-6b), Glc′ 4.99 (1H, d, J = 5.8 Hz, H-1), 4.06 (1H, m,
H-2), 4.16 (1H, m, H-3), 4.15 (1H, m, H-4), 4.05 (1H, m, H-5), 4.45 (1H, m, H-6a), 4.30 (1H, m,
H-6b). 13C NMR (100 MHz, pyridine-d5): 38.7 (C-1), 26.0 (C-2), 82.0 (C-3), 42.3 (C-4), 47.2 (C-5),
17.9 (C-6), 32.9 (C-7), 39.9 (C-8), 47.9 (C-9), 36.6 (C-10), 23.5 (C-11), 126.2 (C-12), 138.6 (C-13),
43.2 (C-14), 28.5 (C-15), 24.3 (C-16), 48.2 (C-17), 53.0 (C-18), 38.8 (C-19), 39.1 (C-20), 30.5 (C-21),
36.5 (C-22), 64.1 (C-23), 13.5 (C-24), 16.2 (C-25), 17.5 (C-26), 23.5 (C-27), 176.4 (C-28), 27.2
(C-29), 21.1 (C-30); 3-O-sugar: Xyl 106.9 (C-1), 75.4 (C-2), 78.1 (C-3), 70.9 (C-4), 66.9 (C-5);
28-O-sugar: Glc 95.7 (C-1), 74.9 (C-2), 78.4 (C-3), 70.6 (C-4), 77.6 (C-5), 69.2 (C-6), Glc′ 105.4
(C-1), 73.5 (C-2), 78.3 (C-3), 71.2 (C-4), 78.2 (C-5), 62.3 (C-6).
3.4. Acid hydrolysis for sugar analysis
A solution of 1 and 2 (1.0 mg for each compound) in 1 M HCl (0.4 mL) was heated at 90–100 °C
in a screw-capped vial for 5 h. The mixture was neutralized by addition of Amberlite IRA400
(OH− form) and then filtered. The filtrate was dried in vacuo, dissolved in 0.2 mL of pyridine
containing l-cysteine methyl ester (10 mg/mL) and reacted at 60 °C for 1 h. A solution (0.2 mL)
of trimethylsilylimidazole in pyridine (10 mg/mL) was added to this mixture, and it was
heated at 60 °C for 1 h. The final mixture was directly analyzed by GC [30QC2/AC-5 quartz

NATURAL PRODUCT RESEARCH
7
capillary column (30 m × 0.32 mm) with the following conditions: column temperature:
180/280 °C; programmed increase 3 °C/min; carrier gas: N2 (1 mL/min); injection and detector
temperature: 250 °C; injection volume: 4 μL; split ratio: 1/50]. The authentic samples d- and
l-glucose, d- and l-xylose, and l-rhamnose were treated in the same manner. Under these
conditions, the retention times of authentic samples d- and l-glucose, d- and l-xylose, and
l-rhamnose were 18.29, 18.87, 13.35, 14.01, and 14.97 min, respectively. During our studies,
d-glucose, d-xylose and l-rhamnose were detected in 1. d-glucose and d-xylose were detected
in 2.
3.5. Anti-HCV activity
Inhibitor preparation. For the inhibitory activity assays, compounds 1 and 2 were dissolved
and then serially diluted with DMSO, using DMSO as blank, ribavirin and IFNα-1b as positive
controls.
Cell line and cell culture. Stocks of infectious hepatitis C virus (HCV) J6/JHH-1 viral particles
were generated as previously described (Li et al. 2012a) and aliquoted for storage at −80 °C.
The HCV load (RNA copies) in stocks was measured with real-time quantitative RT-PCR.
Anti-HCV assay in vitro. Anti-HCV assay was carried out in clear-bottomed 96-well plates.
Huh7.5.1 cells were seeded at a density of 1.5 × 104 cells/well in 100 μL of DMEM culture
medium and incubated overnight, and virus stocks were added into the wells and incubated
for 8 h. Before the samples were added, the supernatants were removed and cells were
washed with completed medium five times. The samples or controls were serially diluted in
DMEM and then added to the appropriate wells. After incubation for 3 days, the supernatants
were collected to determine their viral load with real-time quantitative RT-PCR.
Cytotoxicity assay. The toxicities of the compounds were assayed by a modified MTT
method (Li et al. 2012a). In brief, the test samples were prepared at different concentrations.
After Huh7.5.1 cells had been seeded in a 96-well microplate for 4 h, the samples (20 μL)
were placed in each well and incubated for 3 days at 37 °C; then, 0.1 mL MTT was added for
4 h. After removal of the MTT medium, DMSO (100 μL/well) was added onto the microplate
for 10 min. The formazan crystals were dissolved, and the absorbance was measured on a
microplate reader at 490 nm.
4. Conclusion
Our present work on the plants of E. bodinieri yielded two new ursane-type triterpenoid
saponins, along with five known saponins. Two new compounds 1 and 2 exhibited potent
anti-HCV activities in vitro with a selective index of 30.63 and 9.08, respectively. This inves-
tigation should provide valuable information for further understanding of the E. bodinieri.
Disclosure statement
No potential conflict of interest was reported by the authors.

8
L. XIANG ET AL.
Funding
This work was supported by the National Natural Science Foundation of China [grant numbers
31660100, U1602222]; Talent Cultivation Project Foundation of Yunnan Province [grant number
KKSY201426122]; Yunnan Province applied Basic research project [grant number 2017BF034].
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