New ecdysteroid and ecdysteroid glycosides from the roots of Serratula chinensis

Article abstract of DOI:10.1080/10286020.2016.1209492

Three new ecdysteroid glycosides (1–3) and one new ecdysteroid (4), were isolated from the roots of Serratula chinensis. Their structures were established on the basis of extensive spectroscopic analysis and chemical methods.

Full text of DOI:10.1080/10286020.2016.1209492

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
New ecdysteroid and ecdysteroid glycosides from
the roots of Serratula chinensis
Zi-Yue Zhang, Wei-Qun Yang, Chun-Lin Fan, Hui-Nan Zhao, Xiao-Jun Huang,
Ying Wang & Wen-Cai Ye
To cite this article: Zi-Yue Zhang, Wei-Qun Yang, Chun-Lin Fan, Hui-Nan Zhao, Xiao-Jun
Huang, Ying Wang & Wen-Cai Ye (2016): New ecdysteroid and ecdysteroid glycosides
from the roots of Serratula chinensis, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2016.1209492
To link to this article: http://dx.doi.org/10.1080/10286020.2016.1209492
Published online: 18 Jul 2016.
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Date: 23 July 2016, At: 00:39

Journal of asian natural Products research, 2016
http://dx.doi.org/10.1080/10286020.2016.1209492
New ecdysteroid and ecdysteroid glycosides from the roots of
Serratula chinensis
Zi-Yue Zhanga,b,c, Wei-Qun Yanga,b,c, Chun-Lin Fanb,c, Hui-Nan Zhaob, Xiao-Jun Huangb,c,
Ying Wangb,c and Wen-Cai Yea,b,c
adepartment of Phytochemistry, china Pharmaceutical university, nanjing 210009, china; binstitute of
traditional chinese Medicine & natural Products, college of Pharmacy, Jinan university, Guangzhou 510632,
china; cJnu-hKust Joint laboratory for neuroscience & innovative drug research, college of Pharmacy, Jinan
university, Guangzhou 510632, china
ABSTRACT
ARTICLE HISTORY
received 22 april 2016
accepted 1 July 2016
Three new ecdysteroid glycosides (1–3) and one new ecdysteroid (4),
were isolated from the roots of Serratula chinensis. Their structures
were established on the basis of extensive spectroscopic analysis and
chemical methods.
KEYWORDS
compositae; Serratula
chinensis; ecdysteroid;
ecdysteroid glycoside
1. Introduction
e plant Serratula chinensis S. Mooe (Compositae) is a perennial herb widely distributed
in southern China [1]. e roots of this plant, called “Guang Sheng Ma” in Chinese, are
widely used as an alternative to the traditional Chinese medicine Cimicifugae foetida L.
(“Sheng Ma”) (Ranunculaceae) for the treatment of pharyngitis and morbilli in southern
areas of China, such as Guangdong, Guangxi, and Fujian provinces [2]. Previous phyto-
chemical investigations of this plant had led to the isolation of ecdysteroids, sphingolip-
ids, and cerebrosides [3–5]. Among them, ecdysteroids are the major and characteristic
components of the roots of S. chinensis, some of which have been reported to show potent
antioxidant, hepatoprotective, immunomodulatory, antitumor, and erythropoietic activ-
ities [6–11]. During the course of our ongoing program to assess the chemical and bio-
logical diversities of medicinal plants growing in southern China [12–17], we carried out
extensive phytochemical investigation on the roots of S. chinensis and had reported the
isolation of several known ecdysteroids [18]. More recently, our further study of this plant
led to the finding of three new ecdysteroid glycosides, 20-hydroxyecdysone-2-O-β-D-ga-
lactopyranoside (1), 3-O-acetyl-20-hydroxyecdysone-2-O-β-D-galactopyranoside (2),
3-O-acetyl-20-hydroxyecdysone-2-O-β-D-glucopyranoside (3) and a new ecdysteroid,
24-O-acetyl-epi-abutasterone (4) (Figure 1). To the best of our knowledge, there are only
six ecdysteroid glycosides isolated from the genus Serratula [11,18], three of which were
reported here. In this paper, we report the isolation and structural elucidation of these new
compounds.
CONTACT Ying Wang
wangying_cpu@163.com; Wen-cai Ye
chywc@aliyun.com
© 2016 informa uK limited, trading as taylor & francis Group

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Z.-Y. ZHaNg eT aL.
Figure 1. chemical structures of 1–4.
2. Results and discussion
Compound 1 was obtained as amorphous powder. e molecular formula of 1 was estab-
lished as C33H54O12 on the basis of the quasi-molecular ion at m/z 665.3532 [M + Na]+ by its
HR-ESI-MS. e IR spectrum showed the presence of hydroxyl (3406 cm−1) and carbonyl
1
(1660 cm−1) groups. e H NMR spectrum of 1 showed signals for five angular methyl
groups (δH 0.91, 0.98, 1.20, 1.20, and 1.22, each 3H, s), three oxygenated methines [δH 3.34
(1H, m), 4.05 (1H, dt, J = 12.5, 3.5 Hz), and 4.16 (1H, d, J = 3.5 Hz)], as well as one olefinic
proton [δH 5.82 (1H, d, J = 2.0 Hz)]. In the 13C NMR spectrum of 1, thirty-three carbon sig-
nals were observed. Among them, twenty-seven of which could be assigned to the aglycone
moiety including five methyls (δC 18.0, 21.0, 24.2, 28.9, and 29.7), six oxygenated methines
or quaternary carbons (δC 66.0, 71.3, 76.4, 77.9, 78.4, and 85.2), two olefinic carbons (δC
122.6 and 168.3), as well as a carbonyl (δC 206.5). Detailed interpretation of the 2D NMR
(1H-1H COSY, HSQC and HMBC) spectra of 1 allowed the assignment of all proton and
carbon resonances (Table 1). e aglycone of 1 was identified as 20-hydroxyecdysone by
comparison of the NMR data of 1 with those published in literature [3]. In the NOESY
spectrum of 1, correlations between H-9 (δH 3.18) and H-2 (δH 4.05), as well as between
H3-19 (δH 0.98) and H-5 (δH 2.39) confirmed the presence of cis junction of rings A and B.
Chemical shif values of C-12 (δC 32.5) and C-15 (δC 31.7) confirmed the trans junction of
rings C and D [19]. e α-configurations of H-2 and H-3 could be confirmed by the large
3JH1β-H2 coupling constant (12.5 Hz) and small 3JH2-H3 coupling constant (3.5 Hz), as well as
by the NOE correlation between H-2 (δH 4.05) and H-9 (δH 3.18). Furthermore, the NOE
correlations between H-9 (δH 3.18) and H-12α (δH 1.76), between H-12α (δH 1.76) and H-17
(δH 2.41) suggested the presence of α-configuration of H-17 (Figure 2). e configurations
of H-21 and H-22 were determined by comparison of the NMR data of C-17, C-20, C-21,
C-22, and C-25 [3,20,21] among 1, 20-hydroxyecdysone and 22-epi-20-hydroxyecdysone
both in CD3OD and C5D5 N [3,20,21].
In addition, the 1H and 13C NMR spectra of 1 showed the presence of a sugar anomeric
proton signal at δH 4.41 (1H, d, J = 7.5 Hz, H-1′) and the corresponding carbon signal at δC
103.3 (C-1′). Acid hydrolysis of 1 yielded the D-galactose, which was identified by HPLC
analysis [22]. e relative anomeric configuration of the D-galactose was determined to be
β based on the 3JH1-H2 coupling constant of the anomeric proton (J = 7.5 Hz). e linkage
position of the monosaccharide could be deduced by the HMBC correlation between the
anomeric proton H-1′ (δH 4.41) and C-2 (δC 76.4) (Figure 2). erefore, the structure of 1
was determined as 20-hydroxyecdysone-2-O-β-D-galactopyranoside.

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Z.-Y. ZHaNg eT aL.
Figure 2. Key hMBc and noesY correlations of 1.
Compound 2 was isolated as amorphous powder. e molecular formula of 2was assigned
as C35H56O13 by its HR-ESI-MS with a quasi-molecular ion peak at m/z 707.3609 [M + Na]+.
Comparison of the 1H and 13C NMR data of 2 with those of 1 (Table 1) revealed that they
were very similar, except for the presence of signals assigned to an acetyl group [δH 1.96 (3H,
s); δC 21.5 and 171.2] in 2, and the significant downfield shif of C-3 and H-3 signals of 2. In
the HMBC spectrum, the correlation between H-3 (δH 5.65) and C-1′′ (δC 171.4) indicated
that the additional acetyl group was located at C-3 in 2. Acid hydrolysis of 2 also afforded
D-galactose, which was identified by HPLC analysis [22]. Furthermore, the 3JH1-H2 coupling
constant of the anomeric proton (J = 7.5 Hz) established the β-configuration of the sugar
unit. e HMBC correlation between H-1′ (δH 4.84) and C-2 (δC 73.9) further confirmed
that the C-2 position of the aglycone was glycosylated with D-galactose. us, compound
2 was deduced to be 3-O-acetyl-20-hydroxyecdysone-2-O-β-D-galactopyranoside.
Compound 3 showed the same molecular formula as 2 by its HR-ESI-MS at m/z 707.3609
1
[M + Na]+. e H and 13C NMR spectral data of the aglycone part of 3 were in accord-
ance with those of 2 (Table 1), suggesting that the aglycone of 3 was 20-hydroxyecdysone.
Besides, the 1H and 13C NMR spectra displayed the signals due to one sugar moiety. Acid
hydrolysis of 3 afforded D-glucose. e relative anomeric configuration of D-glucose was
3
determined to be β based on the JH1-H2 coupling constant (J = 7.5 Hz). In the HMBC
spectrum, correlations between H-1′ (δH 4.95) and C-2 (δC 74.1), as well as between H-3
(δH 5.70) and C-1′′ (δC 171.4) suggested that the D-glucose and acetyl group were attached
to the C-2 and C-3 positions, respectively. us, the structure of 3 was determined as
3-O-acetyl-20-hydroxyecdysone-2-O-β-D-glucopyranoside.
Compound 4 gave a molecular formula C29H46O9 by its HR-ESI-MS at m/z 561.3045
1
[M + Na]+. e H NMR spectrum of 4 indicated the presence of signals for five methyl
groups (δH 0.90, 0.98, 1.19, 1.20 and 1.21, each 3H, s), four oxygenated methines [δH 3.31(1H,
m), 3.84 (1H, m), 3.96 (1H, br s), and 5.09 (1H, dd, J = 10.5, 1.5 Hz)], as well as one ole-
finic proton [δH 5.82 (1H, d, J = 2.5 Hz)]. e 13C NMR and DEPT spectra of 4 showed the
signals for twenty-nine carbons, including five methyls (δC 18.1, 21.0, 24.4, and 26.0 × 2),
seven oxygenated carbon signals (δC 68.5, 68.7, 72.7, 73.8, 77.6, 78.7, and 85.2), two olefinic
carbons (δC 121.8 and 167.9), as well as a carbonyl (δC 206.2). e NMR spectral data of 4
were similar to those of 24-epi-abutasterone [23], except for the presence of signals assigned
to an acetyl group [δH 2.11 (3H, s); δC 21.0 and 173.0] (Table 1). e HMBC correlations
between H-24 (δH 5.09) and C-1′ (δC 173.0)/C-22 (δC 73.8)/C-23 (δC 33.1)/C-26, 27 (δC
26.0 × 2) suggested that the additional acetyl group was located at C-24 in 4. e relative
configuration of 4 could be further determined on the basis of NOESY experiment, which
was identical to 24-epi-abutasterone. However, the stereochemistry of C-24 in the side-chain

JourNaL oF asiaN NaTuraL ProduCTs researCH
5
could not be assigned based on current evidence. us, the structure of 4 was established
as 24-O-acetyl-epi-abutasterone.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on Jasco P-1020 digital polarimeter at room temperature
(Jasco, Tokyo, Japan). UV data were obtained on a JASCO V-550 UV/vis spectrophotometer
(Jasco, Tokyo, Japan). Jasco FT/IR-480 plus Fourier Transform Spectrometer was used for
scanning IR spectra with KBr pellets (Jasco, Tokyo, Japan). NMR spectra were acquired with
Bruker AV-500 Spectrometer (500 MHz for 1H NMR and 125 MHz for 13C NMR, Bruker,
Fällanden, Switzerland). HR-ESI-MS analyses were recorded with Agilent 6210 ESI/TOF
mass spectrometer (Agilent, Pala Alto, CA, USA). Silica gel (200–300 mesh or 300–400
mesh, Qingdao Haiyang Chemical Corporation, Qingdao, China) and Sephadex LH-20
(Pharmacia, Uppsala, Sweden) were used for column chromatography.
3.2. Plant material
e roots of Serratula chinensis were purchased from Qingping market of traditional
Chinese medicine, Guangzhou city, Guangdong province of China, in October of 2012,
and authenticated by Professor Guang-Xiong Zhou (College of Pharmacy, Jinan University).
A voucher specimen (No. 20,121,015) was deposited in the Institute of Traditional Chinese
Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou, China.
3.3. Extraction and isolation
Air-dried and powdered roots (20.0 kg) of S. chinensis were extracted with 95% (v/v) ethanol
for three times. e solvent was removed under vacuum to obtain a crude extract (370 g),
which was then suspended in H2O and successively extracted with petroleum ether, EtOAc
and n-BuOH. e n-BuOH fraction (190 g) was subjected to silica gel column chromatog-
raphy eluted gradually with CHCl3-MeOH (100:0–0:100, v/v) to yield seven fractions (Fr.
1-Fr. 7). Fr. 4 (14 g) was further subjected to silica gel column chromatography eluted by
CHCl3-MeOH (100:0–0:100, v/v) to yield seven subfractions (Fr. 4a-Fr. 4 g). Fr. 4d (1.6 g)
was further purified with Sephadex LH-20 column chromatography (MeOH:H2O, 70:30,
v/v) to yield 1 (10 mg), 2 (16 mg) and 4 (13 mg), respectively. Fr. 4e (0.8 g) was further
separated by preparative HPLC using MeOH-H2O (65:35, v/v, 6 ml/min) to afford 3 (RT:
25.2 min, 6 mg).
3.3.1. Compound 1
Amorphous powder;[ꢀ]D25 + 13.1 (c 0.01, MeOH); UV (MeOH) λmax: 242 nm; IR (KBr) vmax
:
3406, 2954, 2928, 1660, 1384 cm−1; 1H and 13C NMR spectral data see Table 1; HR-ESI-MS
m/z: 665.3532 [M + Na]+ (calcd for C33H54O12Na, 665.3507).

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3.3.2. Compound 2
Amorphous powder; [ꢀ]2D5 + 21.0 (c 0.01, MeOH)；UV (MeOH) λmax: 242 nm; IR (KBr)
v
max: 3420, 2925, 2857, 1647, 1382, 1057 cm−1; 1H and 13C NMR spectral data see Table 1;
HR-ESI-MS m/z: 707.3609 [M + Na]+ (calcd for C35H56O13Na, 707.3613).
3.3.3. Compound 3
Amorphous powder;[ꢀ]D25 + 13.4 (c 0.01, MeOH); UV (MeOH) λmax: 242 nm; IR (KBr) vmax
:
3423, 2962, 2923, 1728, 1660, 1382, 1051 cm−1; 1H and 13C NMR spectral data see Table 1;
HR-ESI-MS m/z: 707.3609 [M + Na]+ (calcd for C35H56O13Na, 707.3613).
3.3.4. Compound 4
Amorphous powder;[ꢀ]D25 + 46.4 (c 0.01，MeOH); UV (MeOH) λmax: 242 nm; IR (KBr) vmax
:
3420, 2964, 2929, 1713, 1648, 1383, 1054 cm−1; 1H and 13C NMR spectral data see Table 1;
HR-ESI-MS m/z: 561.3045 [M + Na]+ (calcd for C29H46O9Na, 561.3034).
3.4. Acid hydrolysis and sugar analysis
Compounds 1–3 (each 2 mg) were dissolved in 2 M HCl (10 ml) and stirred at 80 °C for 3 h,
respectively [22]. e solution was extracted with chloroform to yield an aglycone fraction
and an aqueous fraction. e aqueous layer was evaporated to give a residue, followed by
adding with anhydrous pyridine (0.5 ml) and L-cysteine methyl ester (1 mg). e reaction
mixture was heated at 60 °C for 1 h. Afer that, O-aryl isocyanate ester (5 μl) was added
to the solution, which was then heated at 60 °C for another 1 h. Filtered with membrane
(0.45 μm) afer reaction, the solution was analyzed with HPLC under the following condi-
tions: Cosmosil 5C18-MS-II (4.6 × 250 mm, i.d. 5.0 μm), mobile phase: acetonitrile-0.05%
formic acid (25:75), detector wavelength: 250 nm, flow rate: 0.8 ml/min, acquisition time:
40 min. e derivatives of authentic D-glucose, L-glucose, D-galactose and L-galactose were
prepared by the same method, and showed the retention times of 20.65, 18.69, 16.78 and
17.71 min. As a result, D-galactose [tR (min): 16.72, 16.77] from the hydrolyzates of 1 and
2, and D-glucose [tR (min): 20.53] from the hydrolyzate of 3 were detected.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was supported by the National Natural Science Foundation of China [grant number
81473117], [grant number 81503210], [grant number 81502939]; the Joint Fund of NSFC-Guangdong
Province [grant number U1401225].
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