Steroidal glycosides from the underground parts of Hosta ventricosa and their anti-inflammatory activities in mice

Article abstract of DOI:10.1080/14786419.2019.1638379

Two new pregnane glycosides, 2α, 3β-dihydroxy-5α-pregn-16-en-20-one-3-O-{α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-galactopyranoside} (1) and 2α, 3β-dihydroxy-5α-pregn-16-en-20-one-3-O-{β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside}(2), have been isolated along with two known spirostanol saponins from the underground parts of Hosta ventricosa. Their structures were elucidated on the basis of chemical and spectral evidence. The anti-inflammatory activities of these steroidal glycosides were evaluated using a xylene-induced ear edema model. Our results indicated that the compounds exhibited promising anti-inflammatory activities.

Full text of DOI:10.1080/14786419.2019.1638379

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Steroidal glycosides from the underground parts
of Hosta ventricosa and their anti-inflammatory
activities in mice
Hong-Biao Chu, Nan-Nan Li, Zong-Ping Zhang, Xiao-Yue Hu, Cai-Yun Yu & Lei
Hua
To cite this article: Hong-Biao Chu, Nan-Nan Li, Zong-Ping Zhang, Xiao-Yue Hu, Cai-Yun Yu &
Lei Hua (2019): Steroidal glycosides from the underground parts of Hostaꢀventricosa and their anti-
inflammatory activities in mice, Natural Product Research, DOI: 10.1080/14786419.2019.1638379
To link to this article: https://doi.org/10.1080/14786419.2019.1638379
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1638379
Steroidal glycosides from the underground parts of Hosta
ventricosa and their anti-inflammatory activities in mice
Hong-Biao Chu, Nan-Nan Li, Zong-Ping Zhang, Xiao-Yue Hu, Cai-Yun Yu and
Lei Hua
Department of Pharmacy, School of Medicine, Jinggangshan University, Ji’an, Jiangxi, China
ABSTRACT
ARTICLE HISTORY
Received 31 March 2019
Accepted 25 June 2019
Two new pregnane glycosides, 2a, 3b-dihydroxy-5a-pregn-16-en-
20-one-3-O-fa-L-rhamnopyranosyl-(1!2)-[b-D-glucopyranosyl-
(1!4)]-b-D-galactopyranosideg (1) and 2a, 3b-dihydroxy-5a-
pregn-16-en-20-one-3-O-fb-D-glucopyranosyl-(1!2)-[b-D-
xylopyranosyl-(1!3)]-b-D-glucopyranosyl-(1!4)-b-D-gal-
actopyranosideg(2), have been isolated along with two
known spirostanol saponins from the underground parts of
Hosta ventricosa. Their structures were elucidated on the
basis of chemical and spectral evidence. The anti-inflammatory
activities of these steroidal glycosides were evaluated using a
xylene-induced ear edema model. Our results indicated that the
compounds exhibited promising anti-inflammatory activities.
KEYWORDS
Hosta ventricosa; steroidal
glycosides; pregnane
glycosides; anti-
inflammatory
CONTACT Hongbiao Chu
hongbiaochu@163.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1638379.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
H.-B. CHU ET AL.
1. Introduction
The genus Hosta belongs to the family Liliaceae, with approximately 40 species distrib-
uted in the temperate and subtropical zones of Asia (Liu et al. 2011). Previous phyto-
chemical studies on Hosta have afforded structurally-diverse and biologically-active
compounds, such as steroids, alkaloids, flavonoids, and monoterpenes, with some of
them showing potent anti-inflammatory, cytotoxic, antibacterial, antiviral, and antioxi-
dant activities (Budzianowski 1990; Mimaki et al. 1996, 1997, 1998; Wang et al. 2007a,
2007b; Yada et al. 2010; Wang et al. 2017; Yang et al. 2017; He et al. 2018). The ethno-
pharmacological significance of the genus Hosta led us to investigate the chemical
constituents of one of its species, Hosta ventricosa. This plant is widely cultivated in
China and Western countries not only for its ornamental value but also for its use as a
medicinal herb and economic value as a food source (Schmid 1991). This paper
describes the isolation and structural elucidation of two new pregnane compounds
along with two known steroidal compounds isolated from the underground parts of
H. ventricosa. In addition, the anti-inflammatory activities of these compounds were
determined using a xylene-induced ear edema model.
2. Results and discussion
A 95% ethanol extract obtained from the underground parts of H. ventricosa was con-
centrated with a rotary evaporator, suspended in water, and then successively parti-
tioned with petroleum ether and n-butanol. The n-butanol extract was subjected to
column chromatography using silica-gel, Sephadex LH-20, MCI gel, and ODS-C18 on a
reversed-phase HPLC system, which afforded four compounds including two
new pregnane glycosides 1 and 2 (Figure 1). The known compounds were identified
as
(25 R)-2a,
3b-dihydroxy-5a-spirost-9-en-12-one-3-O-fb-D-glucopyranosyl(1!2)-
[b-D-xylopyranosyl(1!3)]-b-D-glucopyranosyl(1!4)-b-D-galactopyranosideg (3) and
(25 R)-3b-hydroxy-5a-spirost-9-en-12-one-3-O-fb-D-glucopyranosyl(1!2)-[b-D-xylopyra-
nosyl (1!3)]-b-D-glucopyranosyl(1!4)-b-D-galactopyranosideg (4) by comparing their
MS and NMR data with those reported in the literature (Mimaki et al. 1995).
Compound 1 was obtained as a white amorphous powder. The ESI-MS spectrum
gave a quasi-molecular ion peak at m/z 825.4348 [M þ Na]þ and HR-TOF-MS suggested
a molecular formula of C39H62O17 (m/z 801.3914, calcd. 801.3909, [M–H]-). The IR spec-
trum (KBr) showed characteristic absorption bands at 3418 and 1661 cmꢀ1 correspond-
ing to hydroxyl and a, b-unsaturated carbonyl groups, respectively. The 1H-NMR
spectrum (Table S1) of 1 revealed the presence of three methyl groups at dH 0.89 (3H,
s, CH3-18), 0.87 (3H, s, CH3-19), and 2.21 (3H, s, CH3-21), two oxymethines at dH 4.05
(1H, overlapped, H-2) and 3.82 (1H, m, H-3), one olefin at dH 6.57 (1H, dd, J ¼ 3.1, 1.
7 Hz, H-16), and three anomeric proton doublets at dH 4.92 (1H, d, J ¼ 7.6 Hz, Gal, H-1ꢀ),
5.15 (1H, d, J ¼ 7.8 Hz, Glc, H-100), and 6.22 (1H, d, J ¼ 1.2 Hz, Rha, H-1000). This informa-
tion was supported by the 13C-NMR data obtained for compound 1. The 13C-NMR
spectrum of 1 (Table S2) showed signals for three methyl groups (dC 13.2, 15.9, and
26.9), two carbons bearing a hydroxyl group (dC 70.3 and 84.9), one olefin carbon (dC
144.5), and three anomeric carbons (dC 100.8, 107.0, and 102.1). Acid hydrolysis of 1
using 2 M hydrochloric acid gave aglycone 1a (C21H32O3), D-galactose, D-glucose, and

NATURAL PRODUCT RESEARCH
3
Figure 1. Structures of compounds 1–4.
L-rhamnose in a 1:1:1 ratio as the carbohydrate compounds. The absolute configura-
tions of the sugar units were assigned using HPLC analysis of the chiral derivatives of
the sugars in the acid hydrolysate. An a-anomeric configuration for the rhamnose unit
1
was concluded from the multiplicity of the anomeric H NMR signal (d, J ¼ 1.2 Hz) of
rha and the chemical shift of rha C-5000 (dC 69.2). The b-anomeric configuration for
both galactose and glucose was assigned from their coupling constants (J1,2 >7.0 Hz)
(Agrawal et al. 1985; Agrawal 1992). In the HMBC spectrum (Figure S7), cross-peaks
due to long-range correlations between gal H-1ꢀ(dH 4.92) of galactose and C-3 (dC 84.
9) of the aglycone, rha H-1000 (dH 6.22) of rhamnose and C-2ꢀ(dC 76.6) of galactose, glc
H-100 (dH 5.15) of glucose and C-4ꢀ (dC 81.1) of galactose indicated that a trisaccharide
moiety, -3-O-a-L-rhamnopyranosyl-(1!2)-[b-D-glucopyranosyl-(1!4)]-b-D-galactopyra-
noside was linked to the aglycone at C-3. In addition, we concluded that the trisac-
charide was linked to the C-3 hydroxyl position of the aglycone because in the 13C
NMR spectrum of 1, the signal due to C-3 was shifted down-field by 8.5 ppm, whereas
the signals due to C-2 and C-4 were shifted up-field by 2.5 and 1.8 ppm, respectively,
when compared to those of 1a.
Upon comparison of the 13C NMR data with those of steroidal saponins (Mimaki
et al. 1997; Yada et al. 2010), the 13C signals due to parts A and B, including C-19,

4
H.-B. CHU ET AL.
were superimposable on those of steroidal saponins, indicating the presence of a 2a,
3b - hydroxyl group bearing a triglycoside at C-3. The 16-en-20-one structure of 1 was
in good agreement between the NMR data obtained for the D-ring, C-18 methyl and
acetylmethyl groups, and those previously reported for pregnane glycoside (Mimaki
et al. 1997). This data implies that compound 1 was a triglycoside of a pregnane with
a 16-en-20-one structure derived from a spirostanol via Marker’s degradation (Gould
et al. 1952). The relative configuration of compound 1 was determined from the
NOESY spectrum (Figure S9). The correlation of d 0.87 (H-19) to d 4.05 (H-2) suggested
the relative configuration at H-19 and H-2 to be b orientation, while, d 3.82 (H-3) to d
0.97 (H-5), suggested the relative configuration at H-3 and H-5 to be a orientation. On
the basis of the above-mentioned evidence, compound 1 was identified to be 2a, 3b-
dihydroxy-5a-pregn-16-en-20-one-3-O-fa-L-rhamnopyranosyl-(1!2)-[b-D-glucopyranosyl-
(1!4)]-b-D-galactopyranosideg.
Compound 2 was obtained as a white amorphous powder. The ESI-MS spectrum
gave a quasi-molecular ion peak at m/z 973.4245 [M þ Na]þ and HR-TOF-MS suggested
a molecular formula of C44H70O22 (m/z 949.4286, calcd. 949.4280, [M–H]-). The IR spec-
trum (KBr) showed characteristic absorption bands at 3407 and 1649 cmꢀ1 correspond-
1
ing to hydroxyl and a, b-unsaturated carbonyl groups. The 13C and H NMR spectra
(Table S1 and Table S2) of 2 were closely related to those of 1 except for the sugar
moieties, indicating that 2 was also a pregnane-type glycoside with an identical agly-
1
cone to that found in compound 1. The 13C and H NMR spectra showed the presence
of galactose, two glucose, and xylose moieties in 2, instead of galactose, glucose, and
rhamnose found in 1. This was confirmed by acid hydrolysis of 2 which gave 1a, D-
galactose, D-glucose, and L-xylose in a 1:2:1 ratio. The absolute configurations of the
sugar units were assigned using HPLC analysis of the chiral derivatives of the sugars in
the acid hydrolysate. The HMBC cross-peaks (Figure S19) due to the long-range corre-
lations between gal H-1ꢀ (dH 4.91) of galactose and C-3 (dC 84.0) of the aglycone, glc
H-100 (dH 5.58) of glucose and C-4ꢀ(dC 79.5) of galactose, glc H-1000 (dH 5.21) of glucose
and C-200 (dC 81.2) of glucose, and xyl H-10000 (dH 5.23) of xylose and C-300 (dC 87.1) of
glucose indicate that a tetrasaccharide moiety, -3-O-b-D-glucopyranosyl-(1!2)-[b-D-
xylopyranosyl-(1!3)]-b-D-glucopyranosyl-(1!4)-b-D-galactopyranoside, was linked to
the aglycone at C-3. The relative configuration of compound 2 was determined from
the NOESY spectrum (Figure S21). The correlation of d 0.69 (H-19) to d 4.08 (H-2)
suggested the relative configuration at H-19 and H-2 to be b orientation, while, d 3.86
(H-3) to d 1.02 (H-5), suggested the relative configuration at H-3 and H-5 to be a
orientation. Consequently, 2 was determined to be 2a, 3b-dihydroxy-5a-pregn-16-en-
20-one-3-O-fb-D-glucopyranosyl-(1!2)-[b-D-xylopyranosyl-(1!3)]-b-D-glucopyranosyl-
(1!4)-b-D-galactopyranosideg.
The effects of the four compounds identified in the present study on edema forma-
tion in mice were investigated using a xylene-induced ear edema model. The applica-
tion of xylene in mouse ears brought about a significant increase in ear weight in the
xylene control group (Table S3). In contrast, the control group mice treated with
aspirin (500 mg/kg) showed significant (p < 0.01) inhibition (60.38%) compared to that
of the saline-control group. The animals in the groups treated with compound 1
(25 mg/kg), 2 (50 mg/kg), 3 (12.5 mg/kg), and 4 (25 mg/kg) showed significant

NATURAL PRODUCT RESEARCH
5
inhibition of ear edema of 66.97%, 70.74%, 60.66%, and 79.24%, respectively (for all,
p < 0.01 versus the saline control group). The results suggest that compounds 1–4
have anti-edematogenic effects.
3. Experimental
3.1. General experimental procedures
Column chromatography was performed using silica gel (200–300 mesh, Qingdao
Marine Chemical Inc., China), Sephadex LH-20 (25–100 lm, Pharmacia Fine Chemical
Co., Ltd., Sweden), and MCI–gel CHP 20P (75–150 mm, Mitsubishi Chemical, Tokyo,
Japan). 1 D NMR [1H (600 MHz) and 13C (150 MHz)] and 2 D NMR [1H-1H COSY, HSQC,
HMBC, and NOESY] spectroscopy was recorded on a Bruker Avance600 instrument
(Bruker, Switzerland) at the Centre for Analysis Facilities at Nanchang University.
Optical rotations were measured on a WZZ-2S polarimeter (Shanghai Physics and
Optics Instrument Inc., China). IR spectroscopy was obtained on a Nicolet 5700 spec-
trophotometer using KBr pellets (Nicolet, USA). HR-TOF-MS was recorded on a Trip
TOFTM 5600 spectrometer (Sciex, USA).
3.2. Plant material
Plant material was collected from Wugongshan, Jiangxi Province of China in October
2014 and identified by Prof. Zhaochang Liang (School of Medicine, Jinggangshan
University, Ji’an, China). A voucher specimen (JU 0810021) was deposited in the herb-
arium of Jinggangshan University.
3.3. Animals
Kunming mice (18–22 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd,
and fed a commercial diet with water ad libitum. The animals were housed in cages
maintained on a natural 12 h of light and dark cycle. Ethical clearance for performing
the experiments on animals was obtained from Institutional Animal Ethics Committee,
School of Medicine, Jinggangshan University.
3.4. Extraction and isolation
Dried and powdered plant material (9 kg) obtained from the underground parts of H.
ventricosa was extracted three times with 95% ethanol heated under reflux. The resi-
due was suspended in water and partitioned with petroleum ether and n-butanol,
respectively. The n-butanol portion (800 g) was separated into 6 fractions (Frs A–F)
using a silica gel column eluted with EtOAc–MeOH (30:1 to 6:1). Fr. D (17.9 g) was sub-
jected to MCI-gel column chromatography using 20, 40, 60, 80 and 100% MeOH as
the eluent (each concentration gradient for 1.5 L). The 80% MeOH fraction (10.4 g) was
further purified using a Sephadex LH-20 column chromatography to afford a crude
mixture (6.2 g). Compounds 1 (645 mg, tR 26.5 min), 2 (897 mg, tR 32.5 min), 3

6
H.-B. CHU ET AL.
(1690 mg, tR 51.3 min), and 4 (1587 mg, tR 60.0 min) were obtained from the crude mix-
ture using semi-preparative HPLC (Zorbax ODS-C18, MeOH–H2O ¼ 7:3).
3.4.1. 2a, 3b-dihydroxy-5a-pregn-16-en-20-one-3-O-fa-L-rhamnopyranosyl-
(1fi2)-[b-D-glucopyranosyl-(1fi4)]-b-D-galactopyranosideg (1)
White amorphous powder. [a]2D0¼ ꢀ29.5ꢁ (c ¼ 0.1, CH3OH). IR (KBr): 3418, 2933, 1661,
1372, and 1073 cmꢀ1. HR-TOF-MS: m/z 801.3914 (calcd. 801.3909 for C39H61O17,
1
[M–H]-). H NMR (600 MHz, pyridine-d5) d: 6.57 (1H, dd, J ¼ 3.1, 1.7, H-16), 6.22 (1H, d,
J ¼ 1.2, H-1000 of Rha), 5.15 (1H, d, J ¼ 7.8, H-100 of Glc), 4.92 (1H, d, J ¼ 7.6, H-1ꢀof Gal),
4.81 (1H, m, H-5000), 4.76 (1H, m, H-2000), 4.55 (1H, overlapped, H-3000), 4.53 (1H, d,
J ¼ 6.0, H-6ꢀb), 4.52 (1H, overlapped, H-4ꢀ), 4.51 (1H, m, H-600b), 4.50 (1H, m, H-2ꢀ), 4.26
(1H, m, H-4000), 4.25 (1H, m, H-6ꢀb), 4.24 (1H, m, H-3ꢀ), 4.19 (1H, overlapped, H-300), 4.17
(1H, overlapped, H-600a), 4.07 (1H, overlapped, H-200), 4.05 (1H, overlapped, H-2), 4.04
(1H, m, H-400), 3.96 (1H, m, H-5ꢀ), 3.92 (1H, m, H-500), 3.82 (1H, m, H-3), 2.51 (1H, m, H-
12b), 2.21 (3H, s, H-21), 2.18 (1H, m, H-1b), 2.10 (1H, m, H-15b), 1.98 (1H, m, H-4b),
1.81 (1H, m, H-15a), 1.78 (1H, m, H-4a), 1.58 (3H, d, J ¼ 6.0, H-6000), 1.51 (1H, m, H-11b),
1.49 (1H, m, H-7b), 1.35 (1H, m, H-8), 1.29 (1H, m, H-11a), 1.28 (1H, m, H-12a), 1.24 (1H,
m, H-6b), 1.24 (1H, m, H-14), 1.19 (1H, m, H-6a), 1.09 (1H, m, H-1a), 0.97 (1H, m, H-5),
0.89 (3H, s, H-18), 0.87 (3H, s, H-19), 0.83 (1H, m, H-7a), 0.63 (1H, m, H-9). 13C NMR
(150 MHz, pyridine-d5) d: 196.1 (C-20), 155.2 (C-17), 144.5 (C-16), 107.0 (C-100 of Glc),
102.1 (C-1000 of Rha), 100.8 (C-1ꢀ of Gal), 84.9 (C-3), 81.1 (C-4ꢀ) , 78.7 (C-300), 78.4 (C-500),
76.6 (C-2ꢀ) , 76.2 (C-3ꢀ) , 75.4 (C-200), 75.3 (C-5ꢀ), 73.9 (C-4000), 72.6 (C-3000), 72.2 (C-2000), 71.9
(C-400), 70.3 (C-2), 69.2 (C-5000), 62.8 (C-600), 60.7 (C-6ꢀ) , 56.1 (C-14), 54.7 (C-9), 46.3 (C-13),
45.3 (C-1), 44.7 (C-5), 36.7 (C-10), 35.0 (C-12), 33.2 (C-4), 33.0 (C-8), 32.0 (C-15), 31.7 (C-
7), 27.9 (C-6), 26.9 (C-21), 21.2 (C-11), 18.3 (C-6000), 15.9 (C-19), 13.2 (C-18).
3.4.2. 2a, 3b-dihydroxy-5a-pregn-16-en-20-one (1a)
1
White amorphous powder. ESI-MS: m/z 333 ([M þ H]þ). H NMR (600 MHz, pyridine-d5)
d: 6.58 (1H, dd, J ¼ 3.1, 1.7, H-16), 4.01 (1H, m, H-2), 3.82 (1H, m, H-3), 2.54 (1H, m, H-
12b), 2.21 (3H, s, H-21), 2.18 (1H, m, H-1b), 2.10 (1H, m, H-15b), 1.88 (1H, m, H-4b),
1.85 (1H, m, H-15a), 1.83 (1H, m, H-4a), 1.67 (1H, m, H-11b), 1.57 (1H, m, H-7b), 1.54
(1H, m, H-8), 1.34 (1H, m, H-11a), 1.31 (1H, m, H-12a), 1.28 (1H, overlapped, H-14), 1.27
(1H, overlapped, H-6b), 1.22 (1H, m, H-6a), 1.19 (1H, m, H-1a), 1.19 (1H, m, H-5), 0.88
(3H, s, H-18), 0.87 (1H, m, H-7a), 0.83 (3H, s, H-19), 0.70 (1H, m, H-9). 13C NMR
(150 MHz, pyridine-d5) d: 196.1 (C-20), 155.2 (C-17), 144.5 (C-16), 76.4 (C-3), 72.8 (C-2),
56.1 (C-14), 54.9 (C-9), 46.3 (C-13), 46.0 (C-1), 45.2 (C-5), 37.4 (C-10), 37.0 (C-12), 35.0
(C-4), 33.0 (C-8), 32.0 (C-15), 31.8 (C-7), 28.1 (C-6), 26.9 (C-21), 21.3 (C-11), 16.0 (C-19),
13.4 (C-18).
3.4.3. 2a, 3b-dihydroxy-5a-pregn-16-en-20-one-3-O-fb-D-glucopyranosyl-(1fi2)-
[b-D-xylopyranosyl-(1fi3)]-b-D-glucopyranosyl-(1fi4)-b-D-galactopyranosideg (2)
White amorphous powder. [a]2D0¼ ꢀ39.0ꢁ (c ¼ 0.1, CH3OH). IR (KBr): 3407, 2929, 1649,
1
and 1069 cmꢀ1. HR-TOF-MS: m/z 949.4286 (calcd. 949.4280 for C44H69O22, [M–H]-). H
NMR (600 MHz, pyridine-d5) d: 6.62 (1H, dd, J ¼ 3.1, 1.7, H-16), 5.58 (1H, d, J ¼ 7.8, H-100
of Glc), 5.23 (1H, m, H-10000 of Xyl), 5.21 (1H, m, H-1000 of Glc), 4.91 (1H, d, J ¼ 7.8, H-1ꢀof

NATURAL PRODUCT RESEARCH
7
Gal), 4.60 (1H, overlapped, H-4ꢀ), 4.59 (1H, overlapped, H-6ꢀb), 4.56 (1H, m, H-6000b), 4.48
(1H, m, H-2ꢀ), 4.48 (1H, m, H-600b), 4.35 (1H, m, H-200), 4.33 (1H, m, H-6000a), 4.22 (1H,
overlapped, H-6ꢀa), 4.22 (1H, overlapped, H-3000), 4.22 (1H, overlapped, H-50000b), 4.20
(1H, m, H-4000), 4.15 (1H, m, H-3ꢀ), 4.10 (1H, overlapped, H-300), 4.09 (1H, overlapped, H-
4
0000), 4.08 (1H, m, H-2), 4.06 (1H, m, H-600a), 4.04 (1H, m, H-30000), 3.97 (1H, m, H-5ꢀ), 3.94
(1H, m, H-2000), 3.91 (1H, m, H-20000), 3.86 (1H, overlapped, H-3), 3.85 (1H, m, H-500), 3.85
(1H, m, H-5000), 3.82 (1H, m, H-400), 3.66 (1H, m, H-50000a), 2.51 (1H, m, H-12b), 2.25 (3H, s,
H-21), 2.15 (1H, m, 1 b), 2.09 (1H, m, H-15b), 1.90 (1H, m, H-4b), 1.84 (1H, m, H-15a),
1.69 (1H, m, H-4a), 1.51 (1H, m, H-11b), 1.49 (1H, m, H-7b), 1.35 (1H, m, H-8), 1.31 (1H,
m, H-11a), 1.27 (1H, m, H-6b), 1.26 (1H, overlapped, H-12a), 1.25 (1H, overlapped, H-
14), 1.16 (1H, m, H-6a), 1.09 (1H, m, H-1a), 1.02 (1H, m, H-5), 0.88 (3H, s, H-18), 0.87
(1H, m, H-7a), 0.69 (3H, s, H-19), 0.63 (1H, m, H-9). 13C NMR (150 MHz, pyridine-d5) d:
196.5 (C-20), 155.4 (C-17), 144.9 (C-16), 104.9 (C-100 of Glc), 104.8 (C-1000 of Glc), 104.7
(C-10000 of Xyl), 103.2 (C-10 of Gal), 87.1 (C-300), 84.0 (C-3), 81.2 (C-200), 79.5 (C-4ꢀ), 78.7
(C-5000), 78.5 (C-30000), 78.2 (C-3000), 77.6 (C-500), 76.1 (C-3ꢀ), 75.8 (C-5ꢀ), 75.5 (C-2000), 75.2 (C-
2
0000), 72.5 (C-2ꢀ), 71.4 (C-4000), 70.8 (C-400), 70.5 (C-2), 70.5 (C-40000), 67.3 (C-50000), 62.9
(C-6000), 62.7 (C-600), 60.7 (C-6ꢀ), 56.3 (C-14), 54.9 (C-9), 46.5 (C-13), 45.4 (C-1), 44.8 (C-5),
37.0 (C-10), 35.2 (C-12), 34.1 (C-4), 33.2 (C-8), 32.3 (C-15), 31.9 (C-7), 28.1 (C-6), 27.2
(C-21), 21.4 (C-11), 16.2 (C-19), 13.3 (C-18).
3.5. Acid hydrolysis of compounds 1 and 2
A solution of 1 (50 mg) in 2 M HCl (dioxane–H2O ¼ 1:1, 8 mL) was heated at 100 ꢁC for
2 h. After cooling, the reaction mixture was neutralized upon the addition of NH4OH
and extracted with EtOAc. The combined organic layers were evaporated and isolated
using semi-preparative HPLC to afford aglycone 1a (21 mg), which was identified using
MS and NMR spectroscopy. The aqueous layer was evaporated to dryness to obtain
the sugar fractions. A 2- mg portion of the sugar residue was dissolved in H2O (1 mL)
and mixed with EtOH (1 mL) containing (s)-a-methylbenzylamine (17 lL) and NaBH3CN
(8 mg), respectively. The mixture was agitated at 40 ꢁC for 4 h, glacial acetic acid
(0.2 mL) added, and then concentrated in vacuo. Acetic anhydride (0.3 mL) and pyri-
dine (0.3 mL) were added to the resulting residue at room temperature for 24 h. After
the pyridine was removed, an aqueous solution of the reaction mixture was subjected
to column chromatography on a Cleanert C18-N column (Agela) using H2O, 20 and
50% CH3CN (15, 15, and 10 mL) as the eluent. The 50% CH3CN fraction was analyzed
using HPLC under the following conditions: Agilent SB-C18 column (4.6 ꢂ 250 mm,
5 lm); mobile phase, 40% CH3CN; flow rate, 0.8 mL/min; and DAD detection, 230 nm.
The absolute configuration of the monosaccharides was determined by comparing the
retention time tR (min) of their derivatives with those of authentic samples: 19.51 min
(D-galactose derivative), 22.77 min (D-glucose derivative), and 24.93 min (L-rham-
nose derivative).
A solution of 2 (10 mg) in 2 M HC1 (dioxane–H2O ¼ 1:1, 2 mL) was treated identi-
cally to that of compound 1 to give la (2.4 mg) and the saccharide fraction. D-galact-
ose, D-glucose, and D-xylose were identified in a 1: 2: 1 ratio in the saccharide fraction
of 2 upon conversion into their 1-[(S)-N-acetyl-a-methylbenzylamino]-1-deoxyalditol

8
H.-B. CHU ET AL.
acetate derivatives followed by HPLC analysis: 19.48 min (D-galactose derivative),
22.82 min (D-glucose derivative), and 18.12 min (D-xylose derivative).
3.6. Xylene-induced ear edema
The xylene-induced ear edema test was performed as previously described (Tang et al.
1984). Briefly, a total 140 mice were randomly divided into fourteen groups consisting
of 5 males and 5 females in each group. The different groups were treated with saline,
compounds 1–4 (HPLC purity ꢃ 98%, 12.5, 25, and 50 mg/kg), and aspirin (500 mg/kg).
The mice were orally administrated once a day for 5 successive days. On the fifth day,
after administration for 1 h, the mice in each group were daubed with 0.05 mL xylene
on two sides of the right ear and the left ear was used as the control. After the appli-
cation of xylene for 0.5 h, all mice were euthanized under ether anesthesia and both
ears were cut down along the auricle baseline. The round pieces of the ears were
sampled using a punch (diameter ¼ 8 mm) and weighed on an electronic balance.
3.7. Statistical analysis
All data were expressed as the mean standard error of mean (per group n ¼ 10). The
normally distributed data were subjected to two-way ANOVA followed by Dunnett’s
test. p < 0.05 was considered statistically significant.
4. Conclusions
In conclusion, two new pregnane glycosides, 2a, 3b-dihydroxy-5a-pregn-16-en-20-one-
3-O-fa-L-rhamnopyranosyl-(1!2)-[b-D-glucopyranosyl-(1!4)]-b-D-galactopyranosideg
(1) and 2a, 3b-dihydroxy-5a-pregn-16-en-20-one-3-O-fb-D-glucopyranosyl-(1!2)-
[b-D-xylopyranosyl-(1!3)]-b-D-glucopyranosyl-(1!4)-b-D-galactopyranosideg
(2)
along with two known spirostanol saponins (3 and 4) were isolated and character-
ized on the basis of chemical and spectral evidence (1H-1H COSY, HSQC, HMBC, and
NOESY). All the compounds exhibited promising anti-inflammatory activities. Our
present investigation demonstrates that H. ventricosa is a potential source of anti-
inflammatory natural products.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was supported by the National Natural Science Foundation of China (31260084) and
the Scientific Research Fund of Jiangxi Provincial Education Department (GJJ150769).

NATURAL PRODUCT RESEARCH
9
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