Two new pregnane steroidal glycosides from Cynanchum taihangense

Article abstract of DOI:10.1080/14786419.2019.1672682

As our ongoing chemical investigation, two new pregnane steroidal glycosides, cynataihosides G (1), with a new aglycone, and H (2) were isolated from the 95% ethanol extract of Cynanchum taihangense. Their structures were elucidated on the basis of 1 D and 2 D NMR spectral data, HR-ESI-MS analysis and qualitative chemical methods. The compounds were subjected to detect the cytotoxicity against three human tumor cell lines (HL-60, THP-1 and PC-3). The compounds displayed no significant cytotoxicity. Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1672682.

Full text of DOI:10.1080/14786419.2019.1672682

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Two new pregnane steroidal glycosides from
Cynanchum taihangense
Yu-Bo Wang, Shan-Shan Su, Ming-Xu Tang, Dan Zhao, Gang Chen, Shao-Fei
Chen, Hai-Feng Wang & Yue-Hu Pei
To cite this article: Yu-Bo Wang, Shan-Shan Su, Ming-Xu Tang, Dan Zhao, Gang Chen, Shao-
Fei Chen, Hai-Feng Wang & Yue-Hu Pei (2019): Two new pregnane steroidal glycosides from
Cynanchumꢀtaihangense, Natural Product Research, DOI: 10.1080/14786419.2019.1672682
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1672682
Two new pregnane steroidal glycosides from Cynanchum
taihangense
a,b,c
d
b,c
e
b,c
Yu-Bo Wang , Shan-Shan Su , Ming-Xu Tang , Dan Zhao , Gang Chen ,
b,c
b,c
b,c
Shao-Fei Chen , Hai-Feng Wang and Yue-Hu Pei
a
b
School of Pharmacy, Jinzhou Medical University, Jinzhou, China; School of Traditional Chinese
c
Materia Medica, Shenyang Pharmaceutical University, Shenyang, China; Key Laboratory of Structure-
Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University,
d
Shenyang, China; Key Laboratory of Food Safety Research in Qinghai Province, Xining Customs
e
District, Xining, China; Research & Development Center, Zhejiang Xianju Pharmaceutical Co., Ltd,
Taizhou, China
ABSTRACT
ARTICLE HISTORY
As our ongoing chemical investigation, two new pregnane ster-
oidal glycosides, cynataihosides G (1), with a new aglycone, and
H (2) were isolated from the 95% ethanol extract of Cynanchum
taihangense. Their structures were elucidated on the basis of
Received 6 April 2019
Accepted 14 September 2019
KEYWORDS
Cynanchum taihangense;
steroidal glycosides; NMR
data; cytotoxicity;
1
D and 2 D NMR spectral data, HR-ESI-MS analysis and qualita-
tive chemical methods. The compounds were subjected to
detect the cytotoxicity against three human tumor cell lines
cynataihosides G, H
(
HL-60, THP-1 and PC-3). The compounds displayed no signifi-
cant cytotoxicity.
1. Introduction
Cynanchum taihangense (Tsiang and Li 1974; Li et al. 1995) belongs to Sectio
Vincetoxicum in Cynanchum (Asclepiadaceae). It is herbaceous vine and chiefly distrib-
uted in Shanxi Province, China. Cynanchum plants have been reported to be rich in
steroidal glycosides, which possess antitumor and cytotoxic activities (Chen et al. 2010;
Yin et al. 2016; Zhao et al. 2016; Liu et al. 2018). Cynanchum taihangense is a plant as
a Chinese herb with the function of inducing diuresis for treating strangurtia.
CONTACT Yue-Hu Pei
peiyueh@vip.163.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1672682.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
Y.-B. WANG ET AL.
Figure 1. Structures of compounds 1 and 2.
According to its function and the literatures, three human cancer (HL-60, THP-1 and
PC-3) cell lines were selected to detect the cytotoxicity. We have explored the 95%
EtOH extract of C. taihangense previously in order to find out the potential seco-preg-
nane glycosides (Wang et al. 2017, 2018). Continued phytochemical research obtained
two new seco-pregnane glycosides, which were named cynataihoside G (1), with a
new aglycone, and cynataihoside H (2) (Figure 1). This paper describes the structural
elucidation of new compounds and their cytotoxicity for human cancer cell lines (HL-
60, THP-1 and PC-3).
2. Results and discussion
2
0
Cynataihoside G (1) was obtained as white amorphous powder, [a] -73.3 (c 0.1,
D
þ
MeOH). Its positive HR-ESI-MS showed an ion peak at m/z 843.3738 [M þ Na] (calcd
1
for C H NaO , 843.3779), indicating a molecular formula of C H O . The H NMR
4
2
60
16
42 60 16
spectrum of 1 showed signals for two tertiary methyl groups at [d 1.12 (3H, s, H-19)
H
and 1.62 (3H, s, H-21)], two olefinic protons at [d 6.12 (1H, dd, J ¼ 11.5, 3.0 Hz, H-12)
H
and d 5.42 (1H, m, H-6)], two oxygen-substituted methine protons [d 4.74 (1H, dd,
H
H
J ¼ 12.8, 7.3 Hz, H-3) and 5.76 (1H, ddd, J ¼ 8.0, 7.4, 4.6 Hz, H-16)] and two oxygen-sub-
stituted methylene protons [d 4.44 (1H, dd, J ¼ 10.4, 7.4 Hz, H-15a) and 4.04 (1H, dd,
H
13
J ¼ 10.4, 4.6 Hz, H-15b)]. The C NMR data showed the presence of two groups of
double bond carbons [d 136.8 (C-5), 121.6 (C-6), 145.8 (C-12) and 130.5 (C-13)] and
C
three carbonyl groups [d 204.8 (C-2), 178.6 (C-14) and 167.2 (C-18)]. After analysis of
C
all the above data and comparing them with those of cynataihoside B (Wang et al.
2017), it was suggested that 1 had similar aglycone to that of cynataihoside B but sig-
nificant differences in the substitued situation of C-2. Further, in HMBC spectrum, the

NATURAL PRODUCT RESEARCH
3
correlation from H-4a at d 2.91, H-1a at d 2.67, H-1b at d 2.37 and H-3 at d 4.74
H
H
H
H
to C-2 at d 204.8 (Figure 1S) implied the aglycone of 1 was 2-carbonylstauntogenin.
C
Thus, the structure was determined to be 3b-hydroxy-18-oxo-15,20a:18,20b-diepoxy-
13,14:14,15-disecopregna-5,12-dien-14-oic acid 16-oxylactone-2-one. It was a new
seco-pregnane-type steroidal aglycone, and named as cynataihogenin G. The sugar
1
chain was linked at its C-3 hydroxyl group. For the sugar part, H NMR (600 MHz,
0
C D N) showed three anomeric proton signals at [d 5.26 (1H, br d, J ¼ 9.3 Hz, H-1 ),
5
5
H
0
0
000
5
.06 (1H, br d, J ¼ 9.6 Hz, H-1 ) and 4.97 (1H, br d, J ¼ 3.8 Hz, H-1 )], indicating the
existence of three sugars whose anomeric configurations were deduced by J values as
b-, b- and a-form, respectively. Meanwhile, there were three methyl of deoxysugars
signals at [d 1.31 (3H, d, J ¼ 6.2 Hz), 1.34 (3H, d, J ¼ 6.4 Hz) and 1.52 (3H, d, J ¼ 6.2 Hz)]
H
and three methoxy group of deoxy sugars peaks at [d 3.56 (3H, s), 3.54 (3H, s) and
H
3.36 (3H, s)], indicating 1 contained three deoxysugars. Comparing with the NMR data
of cynataihoside F (Wang et al. 2018), obviously, the sugar moiety of 1 had one less
13
glucopyranose unit, and the corresponding C NMR data were coincident except for a
0
00
000
000
slight change in the carbons C-3 , C-4 and C-5 of the terminal sugar. Therefore, it
assumed to be a-L-cymaropyranosyl-(1!4)-b-D-cymaropyranosyl-(1!4)-b-D-cymaro-
pyranosyl. Hydrolysis of 1 gave cymaroses, which were identified by comparison of its
R values with the reference standard. Moreover, in HMBC spectrum, the correlations
f
0
00
00
00
of d 4.97 (H-1 of a-cymarose) with d 81.8 (C-4 of b-cymarose), of d 5.06 (H-1 of
H
c
H
0
b-cymarose) with d 82.6 (C-4 of b-cymarose), and of d 4.74 (H-3 of 1) with d 95.5
C-1 of b-cymarose), confirmed the linkage of the sugar chain. In NOESY spectrum,
the NOEs of H-1 -Cym (d 5.06) and H-4 -Cym (dH 3.47), and of H-1 -Cym (dH 4.97)
and H-4 -Cym (d 3.43) further established its linkage. In addition, the relative stereo-
C
H
c
0
(
0
0
0
000
H
0
0
H
chemistry of 1 was elucidated through the NOESY experiments: H-19/H-8, H-11b (b-ori-
0
0
0
entated); H-17/H-12, H-21 (a-orientated); H-12/H-9 (a-orientated); H-5 /H-1 , C-3 -OCH
(
3
00 00 000 000
a-orientated); H-1 /H-5 (a-orientated); H-2 b/H-4 (a-orientated). Therefore, the
structure of 1 was elucidated as 3b-hydroxy-18-oxo-15,20a:18,20b-diepoxy-13,14:14,15-
disecopregna-5,12-dien-14-oic acid 16-oxylactone-2-one 3-O-a-L-cymaropyranosyl-(1!4)-
b-D-cymaropyranosyl-(1!4)-b-D-cymaropyranoside, and named cynataihoside G.
Cynataihoside H (2) was isolated as light yellow amorphous gum. Its molecular was
calculated as C H O based on a quasi-molecular ion peak at m/z 933.4664 (Calcd,
4
8 74 20
þ
1
13
9
33.4671) for [M þ Na] ion in the HR-ESI-MS data. Detailed analysis of H and
C
NMR spectrum indicated that compound 2 contains similar structure to sublanceoside
H2 (Warashina and Noro 2006) except for the replacement of the inner sugar. A com-
parison of the NMR data of 2 with those of glaucoside D (Nakagawa et al. 1983)
revealed that they have the same aglycone and the first connected deoxysugar. So 2
was proposed to consist of glaucogenin A and a sugar chain, b-D-glycopyranosyl-a-L-
cymaropyranosyl-(1!4)-b-D-cymaropyranosyl-(1!4)-b-D-oleandropyranosyl, which was
linked at C-3 hydroxyl group. Moreover, the coupling constants of the anomeric pro-
0
ton signals at [d 4.77 (1H, dd, J ¼ 9.7, 1.6 Hz, H-1 of b-D-Ole), 5.23 (1H, dd, J ¼ 9.7,
H
0
0
000
1
.6 Hz, H-1 of b-D-Cym), 4.93 (1H, br d, J ¼ 2.8 Hz, H-1 of a-L-Cym) and 5.00 (1H, d,
0
000
J ¼ 7.7 Hz, H-1 of b-D-Glc)] deduced the relative configuration of the sugars. The
0
HMBC spectrum obtained long-range correlations between H-1 -Ole (d 4.77) and C-3
H
00 0 000 00
d 84.7); H-1 -Cym (d 5.23) and C-4 -Ole (d 82.2); H-1 -Cym (d 4.93) and C-4 -Cym
C H C H
(

4
Y.-B. WANG ET AL.
0000 000
dC 82.1); H-1 -Glc (d 5.00) and C-4 -Cym (d 78.7) (Figure 1S). Besides, the NOESY
H C
(
0
0
0
000
correlations for H-1 -Cym (d 5.23) and H-4 -Ole (d 3.47), for H-1 -Cym (d 4.93) and
H
H
H
0
0
0000
000
H-4 -Cym (d 3.40), and for H-1 -Glc (dH 5.00) and H-4 -Cym (dH 3.94) were
H
observed. These correlations confirmed the sequence of the sugars and the linkage
sites to the aglycone moiety. On acid hydrolysis, 2 yielded oleandrose, cymarose and
glucose. In the NOESY spectrum, correlations of H-19 to H-2 and H-8 (b-orientated), of
0
0
0
00
00
H-17 to H-21 (a-orientated), of H-1 to H-3 and H-5 (a-orientated), of H-1 to H-5 (a-
0
00
000
0000
0000 0000
orientated), of H-5 to C-3 -OCH3 (b-orientated), and of H-1
a-orientated) indicated the orientation of the aglycone and sugars. Thus, the above
mentioned evidences with the corresponding 2D spectrum (HSQC, HMBC and NOESY)
See Table 1S and Figure 1S) assigned the structure of 2 as 15,20a:18,20b-diepoxy-
a,3b-dihydroxy-13,14:14,15-disecopregna-5,13(18)-dieno-14oic acid-16-oxy-lactone 3-
to H-3
and 5
(
(
2
O-b-D-glycopyranosyl-a-L-cymaropyranosyl-(1!4)-b-D-cymaropyranosyl-(1!4)-b-D-
oleandropyranoside, and named cynataihoside H.
The compounds for their cytotoxic activities against HL-60 (human leukemic pro-
myelocytic cell), THP-1 (human acute monocytic leukemia cell line) and PC-3 (prostate
cancer cell lines) were evaluated. 5-Fluorouracil was used as a positive control and the
IC50 values against HL-60, THP-1 and PC-3 were 6.38, 5.82 and 7.77 lM, respectively.
Compounds 1 showed cytotoxic activity on HL-60, THP-1 and PC-3 cell lines with the
IC50 values of 32.68, 40.91 and 32.64 lM, respectively, while compound 2 displayed
weaker activity than 1 against aforementioned three cell lines with IC50 values of
43.31, 54.77 and over 80 lM.
3. Experimental
3.1. General experiment procedure
The spectrometers for optical rotations, IR, NMR, HR-ESI-MS, as well as sugars analytical
HPLC and the separation instruments and materials such as analytical HPLC, and pre-
parative HPLC were the same as in our previous report (Wang et al. 2017).
3.2. Plant material
The fresh whole plants (4.7 kg) of C. panticulatum were collected in August 2014 at
Wangmang Ridge in Shanxi Province, China. A voucher specimen was identified by
Prof. Jing-Ming Jia of Shenyang Pharmaceutical University and has been deposited in
the School of Traditional Chinese Materia Medica of Shenyang Pharmaceutical
University (NO. SYPC201408316).
3.3. Extraction and isolation
The fresh whole plants (4.7 kg) of C. taihangense were extracted, partitioned and pre-
fractioned by the same procedure as that in our previous report (Wang et al. 2017) to
obtain thirteen fractions (Fr. 1 - 13).
Fr. Three was separated into eight fractions (Fr. 3-1 - Fr. 3-8) by silica gel column
using PE/acetone (100:1 to 0:100 v/v) as the eluent. Fr. 3-6 was further fractionated by

NATURAL PRODUCT RESEARCH
5
Sephadex LH-20 with CH Cl /MeOH (1:1 v/v) to afford four fractions (Fr. 3-6-1 - Fr. 3-6-
2
2
4
), and then Fr. 3-6-1 was submitted to another silica gel column with CH Cl /MeOH
2 2
(100:0 to 10:1, v/v) as the eluent to obtain seven fractions (Fr. 3-6-1-1 - Fr. 3-6-1-7). Fr.
3
-6-1-2 was sent to a semi-preparative HPLC in MeOH/H O (64:36 v/v, flowrate 2 mL/
2
min) to get Fr. 3-6-1-2-5, which were purified by another semi-preparative HPLC elut-
ing with CH CN/H O (52:48 v/v, flowrate 3.8 mL/min) to yield compound 1 (5 mg, t
R
3
2
3
0 min). Fr. 6-5 was chromatographed over D101 macroporous resin column with gra-
dient elution of MeOH/H O (20:100 - 100:0, v/v) to obtain five fractions (Fr. 6-5-1 - Fr.
2
6-5-5). Fr. 6-5-4 was subjected to ODS column, and then further isolated by semi-pre-
parative HPLC with the elution of CH CN/H O (49:51 v/v, flowrate 4 mL/min) to afford
3
2
compound 2 (21 mg, t 51 min).
R
3
.3.1. Cynataihoside G (1)
2
0
ꢀ1
White amorphous powder, [a] -73.3 (c 0.1, MeOH); IR (KBr) ꢀ
cm : 3438, 2932,
D
max
1
13
1
756, 1728, 1631, 1404, 1384,1313, 1165, 1089, 1058; H NMR and C NMR (C D N)
5 5
data see Table 1S; HR-ESI-MS m/z: 843.3738 [M þ Na]þ (calcd for
C H NaO , 843.3779).
4
2
60
16
3
.3.2. Cynataihoside H (2)
2
0
ꢀ1
Yellow amorphous gum, [a] -62.6 (c 0.4, MeOH); IR (KBr) ꢀ
cm : 3440, 2933, 1740,
max
D
1
13
1631, 1384, 1273, 1163, 1105; H NMR and C NMR (C D N) data see Table 1S; HR-ESI-
5 5
þ
MS m/z: 933.4664 [M þ Na] (Calcd for C H NaO 933.4671).
48
74
20
3.3. Acid hydrolysis of compounds 1 and 2
Acid hydrolysis was prepared by following the methods described in our previous
report (Wang et al. 2017, 2018). Solution of 1 (2 mg) in MeOH (1 mL) were heated
ꢁ
with 0.1 N H SO4 (1 mL) at 50 C for 30 min. Then the mixture was diluted with
2
2
ꢁ
mL of water and concentrated to 2 mL. After that, the solution was kept at 60 C
for further 30 min, followed by neutralizing with aqueous saturated Ba(OH)2
and the precipitates were filtered off. The filtrate was concentrated and analyzed
by TLC.
Compound 2 (6 mg) was hydrolyzed with 0.5 N H SO (3 mL) in 50% 1,4-dioxane
2
4
ꢁ
(
3 mL) at 95 C for 3 h. Reaction mixture was cooled, neutralized with aqueous satu-
rated Ba(OH)2 and the precipitates were filtered off. The filtrate was partitioned
between CH Cl and H O (Pawar et al. 2007). The aqueous layer was concentrated and
2
2
2
further subjected to TLC analyzation.
The known steroidal glycoside was hydrolyzed and purified by silica gel column to
give cymarose and oleandrose as the authentic samples of the deoxysugars. Cymarose
and oleandrose was identified by TLC comparison with authentic sample with three
solvent systems: R 0.51 and 0.45 with CHCl /CH OH (9:1 v/v), R 0.58 and 0.49 with
f
3
3
f
CH Cl /C H OH (9:1 v/v), and R 0.41 and 0.36 with PE/acetone (3:2 v/v) (Chen et al.
2
2
2
5
f
2008). The configurations of the deoxysugars were identified by the comparison of
those reported spectral data with those in the literatures.

6
Y.-B. WANG ET AL.
3.4. Determination of absolute configuration of glucose of 2
The above-mentioned concentrated aqueous layer of 2 was further subjected to HPLC
analysis under the following conditions: column, Shodex Asahipak NH2P-50 4E column
(
4.6 mm ꢂ 250 mm, 5 lm); flowrate, 0.8 mL/min; solvent, MeCN/H O (3:1 v/v); detec-
2
tion, OR (Jasco OR-4090) detector. D-glucose (t 10.8 min, positive polarity) in com-
R
pound 2 was identified by a comparison of the retention time and polarity.
3.5. Cytotoxicity assay
A MTT assay was used to determine the cytotoxicity effect of the compounds on three
cultured human cancer cell lines, including HL-60 (human leukemic promyelocytic
cell), THP-1 (human acute monocytic leukemia cell line) and PC-3 (prostate cancer cell
line). All the cell lines (American Type Culture Collection, Shanghai, China) was main-
tained in RPMI-1640 medium (Bioind, Israel) with 10% heat-inactivated fetal bovine
serum (FBS), 100 IU/ml penicillin, 100 lg/mL streptomycin and 1 mmol/L ʟ-glutamine in
ꢁ
a humidified atmosphere of 5% CO at 37.0 C. Logarithmic phase cells were used for
2
experiments. Appropriate dilutions of the test samples were added to the cultures.
The growth inhibition was calculated by comparing with control cells. 5-Fluorouracil
was used as a positive control.
4. Conclusions
Two new seco-pregnane steroidal glycoside, cynataihosides G (1), with a new agly-
cone, and H (2) were isolated from whole fresh plant of Cynanchum taihangense by
multiple separation methods. They are both new in nature. Meanwhile, the new com-
pounds were evaluated for their cytotoxicity for human cancer cell lines (HL-60, THP-1
and PC-3), but there is no obvious cytotoxicity.
Disclosure statement
The authors declare no conflict of interest.
Funding
This work was supported by the National Natural Science Foundation of China (NO. 31370375).
References
Chen G, Xu N, Li ZF, Zhang QH, Wu HH, Pei YH. 2010. Steroidal glycosides with anti-tumor activ-
ity from the roots of Cynanchum wallichii Wight. J Asian Nat Prod Res. 12(6):453–457.
Chen H, Xu N, Zhou YZ, Qiao L, Cao JQ, Yao Y, Hua HM, Pei YH. 2008. Steroidal glycosides from
the roots of Cynanchum amplexicaule Sieb. et Zucc. Steroids. 73(6):629–636.
Li PT, Gilbert MG, Stevens WD. 1995. Asclepiadaceae In: Flora of China Editorial Committee, editors.
Flora of China. Vol. 16. Beijing: Science Press; St. Louis: Missouri Botanical Garden, p. 205–223.
Liu JC, Yu LL, Chen SF, Lu XJ, Zhao D, Wang HF, Chen G, Pei YH. 2018. Two new 14, 15-seco-
pregnane-type steroidal glycosides from the roots of Cynanchum limprichtii. Nat Prod Res.
32(3):261–267.

NATURAL PRODUCT RESEARCH
7
Nakagawa T, Hayashi K, Wada K, Mitsuhashi H. 1983. Studies on the constituents of asclepiada-
ceae plants-LII: The structures of five glycosides glaucoside-A, -B, -C, -D, and -E from Chinese
drug “Pai-ch’ien” Cynanchum glaucescens HAND-MAZZ. Tetrahedron. 39(4):606–612.
Pawar RS, Shukla YJ, Khan SI, Avula B, Khan IA. 2007. New oxypregnane glycosides from appetite
suppressant herbal supplement Hoodia gordonii. Steroids. 72(6–7):524–534.
Tsiang Y, Li PT. 1974. Praecursores florae Asclepiadacearum sinensium. Acta Phytotaxonomica
Sin. 12:102.
Wang YB, Su SS, Chen SF, Tang MX, Chen G, Zhao D, Sang XN, Si YY, Wang HF, Pei YH. 2017.
C21 steroidal glycosides with cytotoxic activity from Cynanchum taihangense. Phytochem Lett.
6(20):218–223.
Wang YB, Su SS, Chen SF, Tang MX, Chen G, Zhao D, Sang XN, Si YY, Wang HF, Pei YH. 2018.
C21 steroidal glycosides from Cynanchum taihangense. J Asian Nat Prod Res. 20(3):263–269.
Warashina T, Noro T. 2006. Glycosides of 14,15-Seco and 13,14:14,15-disecopregnanes from the
Roots of Cynanchum sublanceolatum. Chem Pharm Bull. 54(11):1551–1560.
Yin ZQ, Yu SL, Wei YJ, Ma L, Wu ZF, Wang L, Zhang QW, Zhao M, Ye WC, Che CT, Zhang J.
2016. C21 steroidal glycosides from Cynanchum stauntonii induce apoptosis in HepG2 cells.
Steroids. 106:55–61.
Zhao D, Feng BM, Chen SF, Chen G, Li ZF, Lu XJ, Sang XN, An X, Wang HF, Pei YH. 2016. C21
steroidal glycosides from the roots of Cynanchum paniculatum. Fitoterapia. 113:51–57.