New minor spirostanol glycosides from Helleborus thibetanus

Article abstract of DOI:10.1080/14786419.2016.1255888

Phytochemical reinvestigation of the dried roots and rhizomes of Helleborus thibetanus afforded four new minor spirostanol glycosides (1–4) and four known spirostanol glycosides (5–8). Their structures were determined on the basis of spectroscopic analyse

Full text of DOI:10.1080/14786419.2016.1255888

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
New minor spirostanol glycosides from Helleborus
thibetanus
Hui Zhang, Yan-Fang Su, Feng-Ying Yang & Xiu-Mei Gao
To cite this article: Hui Zhang, Yan-Fang Su, Feng-Ying Yang & Xiu-Mei Gao (2016): New
minor spirostanol glycosides from Helleborus thibetanus, Natural Product Research, DOI:
10.1080/14786419.2016.1255888
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Date: 14 November 2016, At: 05:59

Natural Product research, 2016
http://dx.doi.org/10.1080/14786419.2016.1255888
New minor spirostanol glycosides from Helleborus thibetanus
Hui Zhanga, Yan-Fang Sua,b, Feng-Ying Yanga,c and Xiu-Mei Gaod
atianjin Key laboratory for Modern drug delivery and high-efficiency, school of Pharmaceutical science
and technology, tianjin university, tianjin, P.r. china; bschool of Pharmaceutical science and technology,
collaborative Innovation center of chemical science and engineering (tianjin), tianjin, P.r. china;
cPharmaceutical engineering department, school of Biological science and technology, university of Jinan,
shandong, P.r. china; dtianjin Key laboratory of tcM chemistry and analysis, tianjin university of traditional
chinese Medicine, tianjin, P.r. china
ABSTRACT
ARTICLE HISTORY
received 10 July 2016
accepted 18 october 2016
Phytochemical reinvestigation of the dried roots and rhizomes of
Helleborus thibetanus afforded four new minor spirostanol glycosides
(1–4) and four known spirostanol glycosides (5–8). Their structures
were determined on the basis of spectroscopic analyses, including
1D and 2D NMR experiments, together with HR-ESI-MS and IR
measurements and the results of acid hydrolysis.
KEYWORDS
Helleborus; ranunculaceae;
steroidal saponins;
spirostanol
1. Introduction
Helleborus thibetanus Franch., a plant endemic to China, is mainly distributed in Sichuan,
Gansu and Shaanxi (Guan 1979). The roots and rhizomes of H. thibetanus, commonly known
as ‘Xiao-Tao-Er-Qi’, have been used as a folk medicine for traumatic cystitis, injury and ure-
thritis (Guo et al. 2003). Extracts of several Helleborus species have immunostimulatory and
anti-inflammatory properties (Čakar et al. 2014). Previously, the structure determination of
several steroidal saponins, one pregnane, one spirostanol sulphate, several bufadienolides
and phytoecdystones from H. thibetanus have been reported (Cheng et al. 2014; Zhang
CONTACT Yan-Fang su
suyanfang@tju.edu.cn
supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2016.1255888.
© 2016 Informa uK limited, trading as taylor & Francis Group

2
H. ZHANG ET AL
Figure 1. the structures of compounds 1–4.
et al. 2014a, 2014b, 2016). In continuation of our search for new steroidal glycosides from
this plant, we herein report the isolation and structure elucidation of four new minor spi-
rostanol steroidal saponins (1–4) (Figure 1) and four known spirostanol glycosides from the
title plant.
2. Results and discussion
Compound 1 was obtained as a white amorphous solid with molecular formula of C56H86O28,
1
determined by its HR-ESI-MS at m/z 1229.5198 ([M + Na]+), as well as its H and 13C NMR
spectroscopic data. The IR spectrum of 1 displayed the characteristic absorptions of hydroxy
groups at 3400 cm−1 and carbonyl group at 1734 cm−1. The 1H NMR spectrum of 1 showed
signals for two angular methyl groups at δH 0.96 (3H, s), 1.37 (3H, s), and an olefinic proton
signal at δH 5.62 (1H, s), an exomethylene group at δH 5.17 (1H, s) and 5.00 (1H, s). Five ano-
meric protons at δH 6.50 (1H, br s), 5.94, 5.40 (1H, d, J = 8.0 Hz), 4.89 and 4.61 were observed
in the 1H NMR spectrum and five anomeric carbon resonances at δC 100.6, 112.2, 106.0, 106.8
and 100.8 were observed in the 13C NMR spectrum, respectively. Additionally, the methyl
group at δH 1.39 (3H, d, J = 6 Hz) in the 1H NMR spectrum and δC 18.3 in the 13C NMR spectrum
were indicative of a 6-deoxyhexose unit. The presence of an acetyl group in 1 was shown
by the signals at δH 2.19 (3H, s) and δC 170.6 (C=O) and 21.0 (methyl). The above data and
one characteristic acetal signal at δC 111.8 suggested that 1 was a spirostanol saponin with
five monosaccharides and one acetyl group. Detailed comparison of NMR data of the agly-
cone moiety of 1 with those of Clintonioside B (Mimaki & Watanabe 2008) indicated the
presence of a methyl (δH 1.04; δC 14.7) and the disappearance of a hydroxymethyl. The methyl
group (δC 14.7) was placed at C-20 based on the COSY correlation from δH 1.04 (3H, d,
J = 7.0 Hz) to δH 2.88 (H-20) and the HMBC correlations of δH 1.04 (3H, d, J = 7.0 Hz) with δC
37.3 (C-20), δC 61.4 (C-17), and δC 111.8 (C-22). Additionally, the NOESY correlations between
H-1 and H-3, between Me-19 and Me-18/H-2ax/H-4ax, indicated the β-configurations of the
oxygenated substituents at C-1 and C-3. Furthermore, the configurations of C-23 and C-24
were determined to be S by the NOESY correlations between H-23 and H-20, between H-23
and Me-21/H2–27, between H-24 and H2–27 (Watanabe et al. 2003; Mimaki et al. 2003; Mimaki
& Watanabe 2008; Hayes et al. 2009). All of the data support assignment of the structure of

NATURAL PRODUCT RESEARCH
3
the aglycone of 1 as (23S,24S)-1β,3β,23,24-tetrahydroxy-spirosta-5,25(27)-diene. Acid hydrol-
ysis of 1 with 1 M HCl in dioxane-H2O (1:1) followed by TLC analysis produced apiose (api),
arabinose (ara), rhamnose (rha), xylose (xyl) and glucose (glc). The sequence and linkage
positions of the sugars were verified by detailed 2D NMR spectroscopic analysis. In the HMBC
spectrum, the correlations of δH 4.61 (H-1 of ara) with δC 84.2 (C-1 of the aglycone), H-1 (δH
6.50) of rha with C-2 (δC 72.5) of ara, H-1 (δH 4.89) of xyl with C-3 (δC 85.2) of ara, H-1 (δH 5.94)
of api with C-3 (δC 77.7) of rha were observed, which indicated that rha and xyl attached at
C-2 and C-3 of ara, respectively, api linked at C-3 of rha, and ara was attached at C-1 of the
aglycone. HMBC correlations between H-24 (δH 4.82) of the aglycone and C-1 (δC 106.0) of
glc established the linkage of glc and the aglycone. The linkage of the sugars was further
supported by the NOESY correlations of signals at H-1 (δH 3.77) of aglycone with H-1 (δH
4.61) of ara, H-2 (δH 4.59) of ara with H-1 (δH 6.50) of rha, H-3 (δH 3.99) of ara with H-1 (δH
4.89) of xyl, H-3 (δH 4.73) of rha with H-1 (δH 5.94) of api, H-24 (δH 4.82) of aglycone and H-1
(δH 5.40) of glc. Full assignments of 1 were accomplished by a combined analysis of DEPT,
COSY, HSQC, NOESY and HMBC spectra. Based on the above evidence, the structure of the
new spirostanol glycoside 1 was fully elucidated as (23S,24S)-24-[(O-β-D-glucopyranosyl)
oxy]-3β,23-dihydroxyspirosta-5,25(27)-diene-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-
α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside named
thibetanoside A.
Compound 2 was isolated as a white amorphous solid. HR-ESI-MS analysis revealed the
molecular formula C51H78O25 (m/z 1113.4729 [M + Na]+). The IR absorptions at 3427 cm−1 and
1725 cm−1 indicated the presence of hydroxy groups and a carbonyl group. Comparing the
1H, 13C NMR spectra of 1 and 2, the chemical shift of methyl signal of rha of 1 at δH 1.39 (3H,
d, J = 6.0 Hz) moved downfield to δH 1.66 (3H, d, J = 6.0 Hz) in 2, indicating the acetyl linked
at C-4 of rha was absent in compound 2 (Takaashi et al. 1995). The 13C NMR of 2 showed an
upfield shift of C-3 (δC 72.4) of rha, suggesting threre was no sugar attached to the C-3 of
rha. The carbonyl group was placed at C-21 based on the HMBC correlation between C=O
(δC 170.7) and H2–21 (δH 4.31–4.34) of the aglycone. Acid hydrolysis of 2 with 1 M HCl in diox-
ane-H2O (1:1) followed by TLC analysis produced ara, rha, xyl and glc. Consequently, 2 was
established to be (23S,24S)-21-acetyloxy-24-[(O-β-D-glucopyranosyl)oxy]-3β,23-dihydrox-
yspirosta-5,25(27)-diene-1β-yl O-(α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-
α-L-arabinopyranoside named thibetanoside B.
Compound 3 was isolated as a white amorphous solid and its molecular formula was
determined as C50H78O23 by HR-ESI-MS at m/z 1069.4826 [M + Na]+. Comparison of the 1H and
13C NMR signal assignments of 3 with 2, the signals of xyl were absent, which was also deduced
by an upfield shift of 8.4 ppm of C-3 of ara (δC 75.8) compared with 2. Furthermore, resonances
of the acetyl group in 2 were replaced by a methyl signal at δC 14.7 in 3. A set of six additional
signals corresponding to a fuc unit in compound 3 could be observed at δC 105.9, 73.6, 76.1,
83.2, 70.6, 17.4. Correspondingly, the anomeric proton signal at δH 5.13 (1H, d, J = 8.0 Hz) was
observed in the 1H NMR spectrum of 3. The sequence of the sugar chain at C-1 was deduced
from the following HMBC correlations: H-1 (δH 4.68) of ara with C-1 (δC 83.3) of the aglycone,
H-1 (δH 6.33) of rha with C-2 (δC 75.0) of ara. An HMBC correlation from H-1 (δH 5.13) of fuc
and C-24 (δC 82.2) of the aglycone, H-1 (δH 5.12) of glc and C-4 (δC 83.2) of fuc, gave evidence
for sugar linkage to C-24 of the aglycone. Acid hydrolysis of 3 with 1 M HCl in dioxane-H2O
(1:1) followed by TLC analysis produced ara, rha, fuc and glc. From the above evidence, the
structure of 3 was defined as (23S,24S)-24-{[O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl]

4
H. ZHANG ET AL
oxy}-3β,23-dihydroxyspirosta-5,25(27)-diene-1β-yl
O-(α-L-rhamnopyranosyl)-(1→2)-α-L-
arabinopyranoside named thibetanoside C.
Compound 4 was isolated as a white amorphous solid with a molecular formula of
C49H76O23, as determined by HR-ESI-MS with the molecular ion peak at m/z 1055.4669
[M + Na]+, suggesting the loss of C2H2O2 from 2. Its IR spectrum revealed absorptions at
3427 cm−1 due to hydroxy groups. The NMR spectra of 4 were superimposable with those
of 2, except for loss of the acetyl group. In the HMBC spectrum, a correlation between the
resonances of H-1 of glc (δH 5.39, d, J = 8 Hz) and C-24 (δC 82.4) of the aglycone established
that glc unit was attached at C-24 of the aglycone. Acid hydrolysis of 4 with 1 M HCl in diox-
ane-H2O (1:1) produced ara, xyl, rha and glc. Thus, the structure of 4 was assigned as (23S,24S)-
24-[(O-β-D-glucopyranosyl)oxy]-3β,23-dihydroxyspirosta-525(27)-diene-1β-yl
O-(α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside named
thibetanoside D.
In addition to the new steroidal saponins, 1–4, four known compounds, 5–8, were also
isolated and identified by comparison with published spectroscopic and physical data as
Hellebosaponin B (5) (Mimaki et al. 2003), Recurvoside D (6) (Takaashi et al. 1995), (23S,24S)-
21-acetoxy-24-[(O-β-D-glucopyranosyl)oxy]-3β,23-dihydroxyspirosta-5,25(27)-dien-1β-yl
O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[-β-D-
xylopyranosyl-(1→3)]-α-L-arabinopyranoside (7) (Watanabe et al. 2003) and (23S,24S)-21-
hydroxymethyl-24-[(O-β-D-fucopyranosyl)oxy]-3β,23-dihydroxyspirosta-5,25(27)-diene-1β-yl
O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-
xylopyranosyl-(1→3)]-α-L-arabinopyranoside (8) (Duckstein & Stintzing 2015).
3. Experimental
3.1. General experimental procedures
IR spectra were measured on a Bruker Tensor 27 spectrometer (Bruker Co., Ettlingen,
Germany) using KBr pellets. Optical rotations were determined on a Rudolph Research
Analytical Autopol II automatic polarimeter (Rudolph Research Analytical, USA). UV spec-
tra were obtained using an Agilent Cary 60 UV–vis spectrophotometer (Agilent, CA, USA).
NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer (Bruker Daltonics
Inc., Switzerland). High-resolution electrospray ionisation mass spectrometry (HR-ESI-MS)
was measured on a Bruker microTOF-Q II mass spectrometer (Bruker Biospin Inc.,
Germany). All solvents used were of analytical grade (Tianjin Jiangtian Chemical
Technology Co., Ltd, Tianjin, China). Column chromatographic isolations were performed
using silica gel (100–200 mesh, Qingdao Haiyang Chemical Co., Ltd, Qingdao, China),
D101 macroporous resin (Tianjin Haiguang Chemical Co., Ltd, Tianjin, China) and ODS
(40–63 μm, LiChroprep RP-18, Merck KGaA, Darmastadt, Germany). ODS was also used
as a stationary phase for the medium-pressure liquid chromatography (MPLC) system.
Thin-layer chromatography was performed on precoated silica gel GF254 plates (Qingdao
Haiyang Chemical Co., Ltd, Qingdao, China). Spots were visualised with UV light and by
spraying with 5% H2SO4 in EtOH, followed by heating. Analytical HPLC was performed
using an ODS column (Agilent ZORBAX SB-C18, 4.6 × 250 mm, 5 μm, CA, USA). Preparative
HPLC was carried out using an ODS column (Agilent ZORBAX SB-C18, 21.2 × 250 mm,
7 μm, CA, USA).

NATURAL PRODUCT RESEARCH
5
3.2. Plant material
The roots and rhizomes of H. thibetanus were collected in September of 2007 from Mei
County, Shaanxi Province in the People’s Republic of China, in September 2007, and were
authenticated by Prof. Zhen-Hai Wu, College of Life Sciences, Northwest A&F University,
China. A voucher specimen (S200609002) was deposited in School of Pharmaceutical Science
and Technology, Tianjin University, Tianjin.
3.3. Extraction and isolation
The air-dried roots and rhizomes of H. thibetanus (8.0 kg) were extracted twice with 6 L of
95% ethanol under reflux for 2 h and then with 6 L of 60% ethanol for 2 h. After removal of
the solvent in vacuo, the residue (1.5 kg) was suspended in water to a final volume of 5 L and
then successively extracted at room temperature with petroleum ether (PE), CHCl3, EtOAc,
and n-BuOH to give four extracts. The n-BuOH extract (934 g) was subjected to adsorption
on a D101 macroporous resin column, successively eluted with EtOH-H2O (0:100, 30:70, 50:50,
70:30 and 95:5) to afford five fractions. Fraction A (378 g), eluted with 30% EtOH, was chro-
matographed on silica gel eluting with EtOAc-MeOH (9:1→6:4) affording 126 fractions.
Fractions A77–A92 (50 g) were further chromatographed on a silica gel column eluted with
CH2Cl2-MeOH (80:20→0:100), yielding fractions 20–22 (6 g), which were then rechromato-
graphed by MPLC on an ODS with a step gradient of MeOH-H2O (34:66→100:0). Sub-fractions
46–47 (180 mg) were pooled and purified by preparative HPLC on ODS with CH3CN-H2O
(18:82) to yield compound 1 (10 mg) and compound 4 (8 mg). Repeated separation of MPLC
sub-fractions 48–49 (90 mg) by preparative HPLC on ODS with CH3CN-H2O (20:80) yielded
compound 7 (7 mg) and compound 8 (15 mg). MPLC sub-fractions 50–52 (70 mg) were sub-
jected to preparative HPLC on ODS with CH3CN-H2O (20:80) as the isocratic eluent system to
give fractions 8–21, which were further rechromatographed by preparative HPLC with CH3CN-
H2O (19:81) to yield compound 2 (8 mg) and compound 3 (12 mg). MPLC sub-fractions 57–58
(100 mg) were purified by repetitive preparative HPLC with CH3CN-H2O (21:79) to afford com-
pound 5 (10 mg). Fraction B (110 g), which eluted from the D101 resin column with 50% EtOH,
was chromatographed over silica gel with EtOAc-MeOH (9:1→6:4) to give 78 fractions. Fractions
B56-B72 (20 g) were fractionated by MPLC on ODS with MeOH–H2O (35:65→100:0) and by
preparative HPLC on ODS using CH3CN–H2O (19:81) to afford compound 6 (6 mg).
3.3.1. (23S,24S)-24-[(O-β-D-glucopyranosyl)oxy]-3β,23-dihydroxyspirosta-5,25(27)-
diene-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-
O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside (1)
White amorphous solid;[ꢀ]2D5 − 65(c = 0.4, MeOH); UV (MeOH) λmax (log ε) 255 (3.63) nm; IR
(KBr) vmax: 3400, 2935, 1734, 1640, 1036 cm−1; HR-ESI-MS: m/z 1229.5198 [M + Na]+ (calcd. for
[C56H86O28Na]+, 1229.5198). 1H NMR and 13C NMR spectroscopic data are presented in Table
S1and Table S2.
3.3.2. (23S,24S)-21-acetyloxy-24-[(O-β-D-glucopyranosyl)oxy]-3β,23-dihydroxysp-
irosta-5,25(27)-diene-1β-yl O-(α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-
(1→3)]-α-L-arabinopyranoside (2)
White amorphous solid;[ꢀ]2D5 − 34.3(c = 0.12, MeOH); UV (MeOH) λmax (log ε) 255 (3.17) nm;
IR (KBr) vmax: 3427, 2926, 1725, 1641, 1043 cm−1; HR-ESI-MS: m/z 1113.4729 [M + Na]+ (Calcd

6
H. ZHANG ET AL
1
for [C51H78O25Na]+, 1113.4724). H NMR and 13C NMR spectroscopic data are presented in
Table S1 and Table S2.
3.3.3. (23S,24S)-24-{[O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranosyl]oxy}-3β,23-
dihydroxyspirosta-5,25(27)-diene-1β-yl O-(α-L-rhamnopyranosyl)-(1→2)-α-L-
arabinopyranoside (3)
White amorphous solid;[ꢀ]2D5 − 69.6 (c = 0.29, MeOH); UV (MeOH) λmax (log ε) 255 (2.71) nm;
IR (KBr) vmax: 3426, 2923, 1638, 1052 cm−1; HR-ESI-MS: m/z 1069.4826 [M + Na]+ (Calcd for
[C50H78O23Na]+, 1069.4826). 1H NMR and 13C NMR spectroscopic data are presented in Table
S1 and Table S2.
3.3.4. (23S,24S)-24-[(O-β-D-glucopyranosyl)oxy]-3β,23-dihydroxyspirosta-5,25(27)-
diene-1β-yl O-(α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-
arabinopyranoside (4)
White amorphous solid;[ꢀ]2D5 − 66.7(c = 0.15, MeOH); UV (MeOH) λmax (log ε) 255 (3.54) nm;
IR (KBr) vmax: 3427, 2927, 1647, 1062 cm−1; HR-ESI-MS: m/z 1055.4669 [M + Na]+ (Calcd for
[C49H76O23Na]+, 1055.4669). 1H NMR and 13C NMR spectroscopic data are presented in Table
S1 and Table S2.
3.4. Acid hydrolysis of 1–4
A solution of compound 1 (3 mg) was heated in 1 M HCl (dioxane-H2O, 1:1, 6 mL) at 80 °C
for 2 h. The dioxane was removed under reduced pressure, the residue allowed to cool to
ambient temperature then diluted to 8 mL with H2O, and extracted with EtOAc (4 × 6 mL).
The aqueous layer was neutralised with Ag2CO3 to pH 6–7, filtered through a microporous
membrane and concentrated to a minimal volume. Five sugars were identified as glc, xyl,
ara, api and rha by comparison with authentic samples (glucose, xylose, arabinose, apiose
and rhamnose) by TLC (silica gel) with the solvent system EtOAc: MeOH: HOAc: H2O (20:5:3:3)
and CHCl3: MeOH: HOAc: H2O (16:10:3:3). The same procedures were carried out for com-
pound 2 (3 mg). TLC analysis of the sugar fraction under the same condition as in the case
of 1 indicated the presence of glc, xyl, ara and rha. Compound 3 (4 mg) was subjected to
acid hydrolysis as described for 1 to produce a sugar fraction. TLC analysis of which showed
the presence of glc, fuc, ara and rha. Compound 4 (3 mg) was subjected to acid hydrolysis
as described for 1 to produce a sugar fraction. TLC analysis of which showed the presence
of glc, xyl, ara and rha.
4. Conclusion
Four new minor spirostanol steroidal saponins (1–4) and four known compounds (5–8) were
isolated from the roots and rhizomes of H. thibetanus. Compound 6 was not reported from
Helleborus before. Compounds 5, 7, 8 were isolated from the title plant for the first time. This
is the first report of NMR data for compound 8 (Table S3).
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1–S38 and Tables
S1–S3.

NATURAL PRODUCT RESEARCH
7
Acknowledgements
We thank Professor Robert P. Borris for revising our manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
5
Funding
This work was supported by Program for New Century ExcellentTalents in University [grant number NCET-
09-0589]; Tianjin Municipal Science and Technology Commission [grant number 11ZCGHHZ00800].
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