C21 steroidal glycosides from Prunella Vulgaris

Article abstract of DOI:10.1080/07328303.2015.1069828

A new C₂₁ steroidal glycoside, qinyangshengenin-3-O-β-D-digitoxopyranoside (1), together with a known steroidal glycoside, qinyangshengenin-3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-digitoxopyranoside (2), was isolated from

Full text of DOI:10.1080/07328303.2015.1069828

This article was downloaded by: [New York University]
On: 02 August 2015, At: 10:36
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: 5 Howick Place, London, SW1P 1WG
Journal of Carbohydrate Chemistry
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lcar20
C21 Steroidal Glycosides from Prunella
vulgaris
Hua-Yong Loua, Jia-Ning Yaoa, Jie Pana, Shan Zhenga, Ya-Zhou Liua,
Guang-Yi Lianga & Wei-Dong Pana
a The Key Laboratory of Chemistry for Natural Products of Guizhou
Province and Chinese Academy of Sciences, Guiyang 550002, PR
China
Published online: 29 Jul 2015.
Click for updates
To cite this article: Hua-Yong Lou, Jia-Ning Yao, Jie Pan, Shan Zheng, Ya-Zhou Liu, Guang-Yi Liang
& Wei-Dong Pan (2015) C21 Steroidal Glycosides from Prunella vulgaris, Journal of Carbohydrate
Chemistry, 34:6, 358-364, DOI: 10.1080/07328303.2015.1069828
To link to this article: http://dx.doi.org/10.1080/07328303.2015.1069828
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Journal of Carbohydrate Chemistry, 34:358–364, 2015
ꢀ
C
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2015.1069828
C21 Steroidal Glycosides from
Prunella vulgaris
Hua-Yong Lou, Jia-Ning Yao, Jie Pan, Shan Zheng, Ya-Zhou Liu,
Guang-Yi Liang, and Wei-Dong Pan
The Key Laboratory of Chemistry for Natural Products of Guizhou Province and
Chinese Academy of Sciences, Guiyang 550002, PR China
GRAPHICAL ABSTRACT
A new C21 steroidal glycoside, qinyangshengenin-3-O-β-D-digitoxopyranoside
(1), together with
a
known steroidal glycoside, qinyangshengenin-3-O-β-D-
oleandropyranosyl-(1→4)-β-D- cymaropyranosyl-(1→4)-β-D-digitoxopyranoside (2),
was isolated from the medicinal plant Prunella vulgaris Linn. The structure of 1 was
elucidated on the basis of extensive spectroscopic analysis, including HR-MS, 1D and
2D NMR experiments, and chemical evidence.
Received September 13, 2014; accepted July 2, 2015.
Address correspondence to Guang-Yi Liang and Wei-Dong Pan, the Key Laboratory of
Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences,
Guiyang 550002, PR China. E-mail: guangyi liang@126.com; wdpan@163.com
Color versions of one or more of the figures in the article can be found online at
www.tandfonline.com/lcar.
358

C21 Steroidal Glycosides from Prunella vulgaris
359
Figure 1: The structures of compounds 1 and 2.
Keywords Prunella vulgaris Linn; C21 steroidal glycoside; Isolation; Qingyangshen-
genin
INTRODUCTION
Prunella vulgaris Linn., a traditional Chinese medicine belonging to the fam-
ily of Labiatae, is widely distributed in southern China. Modern pharmacolog-
ical studies on this herb medicine showed that it had extensive pharmacologi-
cal activities, such as hypoglycemic,[1] antimicrobial,[2] anticancer,[3] and other
biological activities.[4] Extensive studies on the chemical constituents in this
plant have resulted in the isolation of coumarins,[5] triterpenoids,[5] phenyl-
propanoids,[6] saccharide,[7] diterpenes,[8] and others.[9] In our recent study on
this plant collected in Guizhou Province, a new and a known C21 steroidal gly-
coside were isolated from the whole herb of P. vulgaris Linn. In this article, we
report the isolation and structural elucidation of these two compounds (Fig. 1).
The structure of the new compound 1 was elucidated on the basis of extensive
spectroscopic analysis, including HR-MS, 1D and 2D NMR experiments, and
chemical evidence, and the known compound 2 was determined by comparing
its spectroscopic data with that of the literature.
RESULTS AND DISCUSSION
Compound 1 was obtained as a white amorphous power, and its molecular
formula was determined to be C34H46O11 by HR-ESI-MS at m/z 631.3115
[M+H]+ (Calcd. 631.3118), indicating 12 degrees of unsaturation. The 1H
NMR spectrum of 1 (Table 1) showed two obvious steroidal methyl signals
at 1.64 (3H, s) and 1.15 (3H, s) ppm. The 13C NMR and DEPT spectra of 1
exhibited 34 carbon signals, including 4 methyl groups, 8 methylene groups,

H-Y. Lou et al.
360
Table 1: 1H (400 MHz) and 13C (100 MHz) NMR data of 1 and 2 in CD3OD
1
2
No.
δH, J/Hz
δC
δH, J/Hz
δC
1
1.15 (m)1.90 (m)
1.60 (m)
39.8
30.1
79.3
39.8
140.2
119.6
35.2
75.0
45.1
38.1
25.5
74.0
59.1
90.0
34.3
33.5
93.1
10.6
18.5
212.1
27.8
166.8
122.3
132.8
116.2
163.6
116.2
132.8
97.1
39.8
69.2
74.4
70.8
18.6
1.15 (m)1.89 (m)
1.60 (m)
39.8
30.2
79.3
39.0
140.2
119.7
35.2
75.0
45.1
38.1
25.5
74.1
59.1
90.0
34.3
33.5
93.1
10.6
18.5
212.1
27.8
166.8
122.3
132.8
116.2
163.6
116.2
132.8
97.0
39.8
69.5
83.8
70.0
18.6
100.7
36.4
76.9
83.6
64.7
19.4
58.6
102.9
37.4
81.6
76.9
73.3
18.5
57.4
2
3
3.55 (m)
3.55 (m)
4
2.42 (m)
2.39 (m)
5
6
5.36 (d, 4.8)
2.18 (m)
5.36 (m)
2.19 (m)
7
8
9
1.59 (m)
1.58 (m)
10
11
12
13
14
15
16
17
18
19
20
21
1’
1.76 (m)1.92 (m)
4.75 (dd, 11.6, 4.4)
1.76 (m)1.92 (m)
4.78 (dd, 9.6, 3.2)
1.89 (m)2.05 (m)
1.75 (m)
1.89 (m)2.08 (m)
1.73 (m)
1.64 (s)
1.15 (s)
1.65 (s)
1.15 (s)
2.07 (s)
2.08 (s)
2’
3’
7.80 (d, 8.8)
6.82 (d, 8.8)
7.80 (d, 6.8)
6.82 (d, 7.2)
4’
5’
6’
6.82 (d, 8.8)
7.81 (d, 8.8)
4.97 (dd, 9.6, 1.6)
1.59 (m)
6.82 (d, 6.8)
7.80 (d, 7.2)
4.76 (dd, 9.2, 2.8)
1.58 (m)
7’
1”
2”
3”
4”
5”
6ꢁꢁ
4.00 (m)
3.85 (m)
3.13 (dd, 3.2, 9.6)
3.75 (m)
3.18 (m)
4.23 (m)
1.23 (d, 6.4)
1.22 (d, 6.4)
4.83 (dd, 9.2, 2.8)
1.65 (m)
1ꢁꢁꢁ
2ꢁꢁꢁ
3ꢁꢁꢁ
4ꢁꢁꢁ
5ꢁꢁꢁ
6ꢁꢁꢁ
-OCH3
1ꢁꢁꢁꢁ
2ꢁꢁꢁꢁ
3ꢁꢁꢁꢁ
4ꢁꢁꢁꢁ
5ꢁꢁꢁꢁ
6ꢁꢁꢁꢁ
-OCH3
3.83 (m)
3.20 (m)
4.23 (m)
1.13 (d, 6.4)
3.44 (s)
4.59 (dd, 9.6, 1.6)
1.60 (m)
3.31 (m)
3.20 (m)
3.61 (m)
1.32 (d, 6.4)
3.35 (s)

C21 Steroidal Glycosides from Prunella vulgaris
361
Figure 2: The key HMBC correlations (
compound 1.
) and selected NOESY correlations (
) of
12 methane groups, and 10 quaternary carbons. The characteristic chemical
shift of a carbon signal at δC 97.1 was typical for anomeric carbon of saccha-
rides, and the aglycone moiety signals showed the presence of one typical car-
bonyl carbon (δC 212.1), one double bond (δC 119.7, 140.2), and one benzoyl
group (δC 166.8, 163.6, 122.3, 132.8×2, 116.2×2). All the signals assignable
to the aglycone moiety were similar to those of a C21 steroid, qingyangshen-
genin, with only a tiny difference for the chemical shifts of C-5 (−0.1), C-6
(+0.1), C-20 (+0.1), and C-1’ (-0.1) as compared to that of the reference.[10]
The co-TLC results of the acidic hydrolyzate of 1 with an authentic sam-
ple of qingyangshengenin further supported this conclusion. In addition, pro-
ton signals assigned to one methyl group [δH 1.23 (3H, d, J = 6.4 Hz)],
one methylene group [δH 1.59 (2H, m)], and one anomeric proton [δH 4.97
1
(dd, J = 9.6, 1.6 Hz)] in the H NMR spectrum indicated the presence of a
β-digitoxopyranose unit in 1 on the basis of the coupling constant.[11] From
the 2D NMR spectra, the HMBC correlation from H-1ꢁꢁ (4.97) to C-3 (79.3)
indicated that the monosaccharide unit was connected to C3-OH of qingyang-
shengenin. In the NOESY experiment, the NOE correlations of H-3ꢁꢁ/H-4ꢁꢁ and
H-4ꢁꢁ/6ꢁꢁ-CH3 showed that they were cofacial, and NOE correlation of H-1ꢁꢁ/H-
5ꢁꢁ indicated that they were a-oriented (Fig. 2). Therefore, compound 1 was
considered to be qingyangshengenin-3-O-β-digitoxopyranoside. The absolute
configuration (D or L) of the digitoxopyranose unit was further confirmed by
20
comparing its optical rotation value ( α +41.6 (c 0.2, H2O) with that of the
[ ]D
20
references ( α +48.4 (c 0.9, H2O) for D-digitoxopyranose) after acidic hydrol-
[ ]D
ysis of 1.[11,12] Therefore, the structure of 1 was unambiguously established as
qingyangshengenin-3-O-β-D-digitoxopyranoside (Fig. 1).
Compound 2 was isolated as a white powder. The molecular formula
was established as C48H70O17 by ESI-MS (m/z 919 [M+H]+, 941 [M+Na]+),

H-Y. Lou et al.
362
which was supported by 1D NMR spectroscopic data (Table 1). Comparison
of the NMR and MS data of 2 with those of the reference[13,14] indicated
that compound 2 was qinyangshengenin-3-O-β-D-oleandropyranosyl-(1→4)-β-
D-cymaropyranosyl-(1→4)-β-D- digitoxopyranoside (Fig. 1).
Compounds 1 and 2 were tested in vitro for cytotoxic activity against the
human leukemia cancer cell line HL60 and the human lung cancer cell line
A549.[15] The results, however, showed that the two compounds had no clear
inhibition on these cancer cells.
CONCLUSION
A
new C21 steroidal glycoside named qingyangshengenin-3-O-β-D-
digitoxopyranoside (1), along with a known steroidal glycoside (2), was
isolated from the whole herb of P. vulgaris Linn. The structure of compound
1 was elucidated on the basis of extensive spectroscopic analysis, including
HRMS, 1D and 2D NMR experiments, and chemical evidence. The known
compound 2 was determined by comparison of its spectroscopic data with that
reported in the literature.
EXPERIMENTAL SECTION
General Methods
Optical rotation was measured at 20◦C using a Rudolph-IV polarimeter
equipped with a 2.5-cm pathlength cell. All NMR data were collected on a
Varian INOVA 400-MHz spectrometer in CDCl3 or CD3OD (TMS as inter-
nal standard). MS data were recorded on Agilent 1100 LC/MS and STAR
Pulsar I system spectrometers. The semipreparative HPLC was performed
on a Waters-600 machine with a W2489 UV detector, column: ODS (5 μm,
10 × 150 mm, Waters Co, Ltd., USA). Silica gel (200–300 mesh and 300–400
mesh, Qingdao Ocean Chemical Factory, PR China) was used for TLC and col-
umn chromatography, respectively. Sephadex LH-20 (25–100 μm, Amersham
Biosciences, Sweden) was also used for chromatography.
Plant Material
The whole herb of P. vulgaris Linn. was collected in Longli, Guizhou
Province, of P.R. China, in June 2008, which was authenticated by Prof.
Deyuan Chen from Guiyang College of Traditional Chinese Medicine. The
voucher specimen (No. 20080624) was deposited at the Key Laboratory of
Chemistry for Natural Products of Guizhou Province and Chinese Academy
of Sciences.

C21 Steroidal Glycosides from Prunella vulgaris
363
Extraction and Isolation
The air-dried and powdered whole herb of P. vulgaris Linn. (10 kg) was
extracted with 75% EtOH under reflux for 5 h three times. Then the combined
EtOH extracts were concentrated under reduced pressure to yield a residue,
which was suspended in H2O and extracted successively with petroleum ether
and ethyl acetate. The ethyl acetate soluble extract (180 g) was subjected to
column chromatography on silica gel and eluted with CHCl3/MeOH (100:0-
0:100) to afford seven subfractions 1-7. Fr. 1 was further isolated on an MCI
column and purified repeatedly by silica gel to provide compound 2 (13 mg). Fr.
4 was purified by reverse-phase RP-18 column (MeOH-H2O) and semiprepar-
ative HPLC to yield compound 1 (29 mg).
Acidic hydrolysis: A solution of compound 1 (15.0 mg) in MeOH (5 mL)
and 2N HCl (10 mL) was heated at 60◦C for 3 h. After hydrolysis, the sol-
vent was filtered off and evaporated, and the residue (the mixture of agly-
cone and sugar) was subjected to CC (column chromatography, CHCl3:MeOH
= 50:1–0:1) to yield fractions of sugar and aglycone. The aglucone fraction of
1 showed the same TLC behavior as qingyangshengenin (Rf 0.4, CHCl3:MeOH
= 10:1). Sugar fraction was identified by TLC (Rf 0.4; CHCl3:MeOH = 8:1) as
20
well as optical rotation α +41.6; c 0.2, H2O), which indicated the presence of
[ ]D
20
a D-digitoxopyranoside unit ( α +48.4; c 0.9, H2O) according to the reference.
[ ]D
Qingyangshengenin-3-O-β-D-digitoxopyranoside (1), white amorphous
20
power ( α +1.7; c 0.7, CH3OH); H and 13C NMR, see Table 1; HR-ESI-MS
1
[ ]D
m/z 631.3115 [M+H]+ (Calcd 631.3118, C34H46O11). Qinyangshengenin-
3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-
(1→4)-β-D-
digitoxopyranoside (2), white amorphous powder; 1H and 13C NMR, see
Table 1; ESI-MS m/z 919 [M+H]+, 941 [M+Na]+.
ACKNOWLEDGMENTS
We thank Prof. Jian-Xin Zhang and Prof. Dao-Ping Wang from the Key Lab-
oratory of Chemistry for Natural Products of Guizhou Province and Chinese
Academy of Sciences, Guiyang, China, for NMR and mass spectrometric mea-
surements, respectively.
FUNDING
We are grateful for the financial support from the National Natural Science
Foundation of China (81160390) and the Science and Technology Department
of Guizhou Province (QKHRZ [2011]33, QSZHZ [2010]71, and QKHYSCN
[2009]4010).

H-Y. Lou et al.
364
SUPPLEMENTAL MATERIAL
Supplemental data for this article can be accessed on the publisher’s website.
REFERENCES
1. Liu, B.L.; Zhu, D.N.; Wang, G. Effects of the alcohol extract of Spica Prunellae on
blood glucose in mice. J. Chin. Pharm. Univ. 1995, 26, 44–46.
2. Yang, L.; Yang, Z.L.; Jia, G.Y. Study on antimicrobial activity of extract of Prunella
vulgaris L. J. Hainan Normal Univ. 2013, 26, 51–53.
3. Feng, L.; Jia, X.B.; Zhu, M.M.; Chen, Y.; Feng, S. Chemoprevention by Prunella
vulgaris L. extract of non-small cell lung cancer via promoting apoptosis and regulating
the cell cycle. Asia. Pac. J. Cancer Prev. 2010, 11, 1355–1358.
4. Liu, Y.; Song, S.J.; Xu, S.X. Advances in the study on the chemical constituents and
biological activities of Prunella vulgaris L. J. Shenyang Pharm. Univ. 2003, 20, 55–59.
5. Kojima, H.; Ogura, H. Triterpenoids from Prunella vulgaris. Phytochemistry 1986,
25, 729–733.
6. Wang, Z.J.; Zhao, Y.Y.; Wang, B.; Li, J.X.; Ai, T.M.; Chen, Y.Y. A new phenyl-
propanoid and triterpenoids from Prunella vulgaris. J. Chin. Pharm. Sci. 2000, 9,
128–130.
7. Zhang, X.; Nie, S.P.; Li, C.; Xie, M.Y. Ethanol fractionation precipitation and
physico-chemical properties of soluble polysaccharides from Prunella vulgaris Linn.
Sci. Technol. Food Ind. 2012, 33, 87–90.
8. Lou, H.Y.; Zheng, S.; Li, T.L.; Zhang, J.X.; Fei, Y.; Hao, X.J.; Liang, G.Y.; Pan,
W.D. Vulgarisin A, a new diterpenoid with a rare 5/6/4/5 ring skeleton from the chinese
medicinal plant Prunella vulgaris. Org. Lett. 2014, 16, 2696–2699.
9. Meng, Z.M.; He, L.W. Studies on constituents of Pruenlla vulgaris L. J. Chin.
Pharm. Univ. 1995, 25, 329–331.
10. Ma, X.X.; Jiang, F.T.; Yang, Q.X.; Liu, X.H.; Zhang, Y.J.; Yang, C.R. New pregnane
glycosides from the roots of Cynanchum otophyllum. Steroids 2007, 72, 778–786.
11. Ma, L.F.; Shan, W.G.; Zhan, Z.J. Polyhydroxypregnane glycosides from the roots of
Cynanchum otophyllum. Helv. Chim. Acta 2011, 94, 2272–2282.
12. Abe, F.; Mori, Y.; Okabe, H.; Yamauchi, T. Steroidal constituents from the roots
and stems of Asclepias fruticosa. Chem. Pharm. Bull. 1994, 42, 1777–1783.
13. Chen, G.; Xu, N.; Pei, Y.H. C21 steroidal glycosides from Cynanchum wallichii
Wight. J. Asian Nat. Prod. Res. 2009, 11, 177–182.
14. Chen, G.; Xu, N.; Li, Z.F.; Zhang, Q.H.; Wu, H.H.; Pei, Y.H. Steroidal glycosides
with anti-tumor activity from the roots of Cynanchum wallichii Wight. J. Asian Nat.
Prod. Res. 2010, 12, 453–457.
15. Zhang, M.S.; Yang, F.M.; Wang, D.P.; Zhang, J.X.; Sun, Q.Y.; Liang, G.Y.; Pan,
W.D. Novel N-oxide derivatives of benzyltetrahydroisoquinoline alkaloid from tubers
of Stephania viridiflavens H.S. Lo et M. Yang. Phytochem. Lett. 2012, 5, 96–99.