Two new triterpenoid glycosides from leaves of Cyclocarya paliurus

Article abstract of DOI:10.1080/14786419.2021.1931182

Two dammarane glycosides (1-2) were isolated from the leaves of Cyclocarya paliurus. The structures of new compounds were established by application of spectroscopic methods, including one-dimensional and two-dimensional NMR, HRESIMS, and chemical hydroly

Full text of DOI:10.1080/14786419.2021.1931182

Natural Product Research
Formerly Natural Product Letters
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Two new triterpenoid glycosides from leaves of
Cyclocarya paliurus
Huihui Sun, Hui Zhu, Jianping Wu, Yuyan Wang, Guihua Li, Yiqian Liu, Xiwen
Chang, Saiyu Ou, Wenwen Zha, Huiyun Chen, Rui Gui, Xiaoai He, Shijuan Lu,
Dangang Shangguan & Kangping Xu
To cite this article: Huihui Sun, Hui Zhu, Jianping Wu, Yuyan Wang, Guihua Li, Yiqian Liu,
Xiwen Chang, Saiyu Ou, Wenwen Zha, Huiyun Chen, Rui Gui, Xiaoai He, Shijuan Lu, Dangang
Shangguan & Kangping Xu (2021): Two new triterpenoid glycosides from leaves of Cyclocarya
paliurus, Natural Product Research, DOI: 10.1080/14786419.2021.1931182
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2021.1931182
Two new triterpenoid glycosides from leaves of
Cyclocarya paliurus
a
ꢀ
a
ꢀ
a
a
a
a
Huihui Sun , Hui Zhu , Jianping Wu , Yuyan Wang , Guihua Li , Yiqian Liu ,
a
a
a
a
a
c
Xiwen Chang , Saiyu Ou , Wenwen Zha , Huiyun Chen , Rui Gui , Xiaoai He ,
b
c
a
Shijuan Lu , Dangang Shangguan and Kangping Xu
a
Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, People’s Republic
b
of China; Haikou Municipal People’s Hospital and Central, South University Xiangya School of
Medicine Affiliated Haikou Hospital, Haikou, People’s Republic of China; Hunan Cancer Hospital,
c
Changsha, People’s Republic of China
ABSTRACT
ARTICLE HISTORY
Two dammarane glycosides (1-2) were isolated from the leaves of
Cyclocarya paliurus. The structures of new compounds were estab-
lished by application of spectroscopic methods, including one-
dimensional and two-dimensional NMR, HRESIMS, and chemical
hydrolysis. When evaluated against seven human cancer cell lines,
the two compounds exhibited selective cytotoxicity to MCF-
Received 2 January 2021
Accepted 10 May 2021
KEYWORDS
Cyclocarya paliurus;
triterpenoid glycosides;
cytotoxic activities
7
cells.
CONTACT Kangping Xu
Huihui Sun and Hui Zhu contributed equally to this article.
xukp395@csu.edu.cn; Dangang Shangguan
shangguandg@163.com
ꢀ
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2021.1931182.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
H. SUN ET AL.
1. Introduction
Cyclocarya paliurus (Batal.) Iljinskaja, a Chinese indigenous plant, is the sole species in
the genus Cyclocarya Iljinskaja of the Juglandaceae family. They have been used in
folk medicine to treat diabetes, hypertension, coronary heart disease and neurasthenia
(
1
The Editorial Committee of the Administration Bureau of Traditional Chinese Medicine
999). Previous phytochemical investigations on the leaves of C. paliurus have led to
the isolation of various chemical constituents including triterpenoid (Kennelly et al.
995; Li et al. 2012; Cui & Li 2015; Chen et al. 2018), flavonoids (Xie et al. 2010) and
1
phenolic acids (Zhang et al. 2010), of which some exhibited hypoglycemic (Kurihara
et al. 2003; Shi et al. 2009; Zhang et al. 2010; Li et al. 2011), antihyperlipidemic (Ma
et al. 2015; Yao et al. 2015), antioxidant (Yang et al. 2018), antimicrobial (Xie et al.
2012), anti-inflammatory (Jiang et al. 2014) and cytotoxic effects (Liu et al. 2007;
Cheng et al. 2009).
And in our previous study, several novel triterpenoids(Xuan et al. 2019; Sun et al.
2020a, 2020b) and flavonoids (Ye et al. 2020a, 2020b) were found in this plant. To fur-
ther explore the chemical diversity of triterpenoids, two undescribed triterpenoiids,
cyclocarioside Z12-Z13 (1-2), were isolated and identified. Herein, the isolation, struc-
ture elucidation, and biological evaluations were reported.
2. Results and discussion
2.1. Chemistry
Compound 1 was isolated as colorless amorphous powder. Its molecular formula
þ
C H O was identified by the HRESIMS m/z 754.5123 [M þ NH ] (calcd. 754.5105)
4
1
68 11
4
1
3
and C NMR data (supplementary material Table S1). Full NMR data analysis indicated
that 1 was a triterpenoid glycoside which was similar to cyclocarioside Z5 (Sun et al.
1
13
2
020a). The H and C-NMR data (supplementary material Table S1) for the side chain
suggested two double bond between C-23, C-24, C-25 and C-26, and a methyl at C-27.
Analysis of the coupling constants for olefinic protons H-23 and H-24 (J23,24 ¼ 15.5 Hz)
in the side chain supported E stereochemistry for the double bond. And there were
two anomeric proton signals at d 4.29 (d, 1H, J ¼ 6.0 Hz) and dH 4.34 (d, 1H,
H
1
J ¼ 7.5 Hz) for sugar moieties in the H-NMR spectrum. The monosaccharides obtained
by acid hydrolysis were identified by comparison on GC-MS analysis with authentic
sample as L-arabinose and D-quinovose. Furthermore, the relative configurations of 1
were determined by NOESY spectrum. Key correlations (supplementary material Figure
S2) between H-3 (d 3.34), H-1 (d 1.47, b-orientation) and H-29 (d 0.90), as well as
H
b
H
H
correlations between H-12 (d 4.12), H-11 (d 2.36, A-orientation), and H-9 (d 1.80)
H
A
H
H
indicated the H-3, H-12 were b-orientation and a-orientation. Besides, the configur-
13
ation at C-20 was deduced as “S” on the basis of optical rotation and C-NMR chem-
ical shift data comparison with reported analogous dammaranes (Sun et al. 2020a).
Thus, the compound 1 was determined as (20S,23E)-3a,12b,20-trihydroxy-12-O-b-D-
qunovopyranosyldammara-23,25-diene 3-O-a-L-arabinopyranoside.
Compound 2 was isolated as colorless amorphous powder, which had a molecular
formula of C H O determined by the HRESIMS. Detailed analysis of NMR data
4
1 70 13

NATURAL PRODUCT RESEARCH
3
showed that 2 had a high similarity to cyclocarioside K (Cui & Li 2015) except for
replacement of arabinopyranosyl moiety at C-12 by a glucopyranosyl moiety at the
same position in 2. Moreover, glucopyranosyl moiety was identified by acid hydrolysis
and GC-MS analysis with standard. Therefore, the structure of 2 was deduced as
(20S,24R)-20,24-epoxy-3a,12b,25-trihydoxy-12-O-b-D-glucopyranosyldammarane
3-O-
a-L-arabinopyranoside.
2.2. Cytotoxicity assay
The inhibitory effects of compound 1 and compound 2 against seven human cancer
cells (MCF-7, PC-3, Du145, NCI-1975, PC-9, SKVO3 and HepG2) were evaluated by the
MTT assay with positive control of STS. As shown in supplementary material Table S2,
all compounds displayed inhibitory activities on MCF-7 cells with IC50 values <35 lM.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a JASCO model 1020 polarimeter (Horiba, Tokyo,
Japan) at room temperature. Gas chromatography-mass spectrometer (GC-MS) were
measured on GCMS-QP2010 Ultra (SHIMADZU, Hongkong, China). NMR spectra were
measured on a Bruker AV-500 MHz spectrometer (Bruker, Karlsruhe, Germany) using
CD OD as solvent and tetramethylsilane (TMS) as an internal standard. The HRESIMS
3
were measured on a LC-LTQ Orbitrap Velos Pro ETD (Thermo Fisher, MA, USA) in posi-
tive ion mode. Column chromatography (CC) was performed on silica gel (200–300 or
80–100 mesh; Qingdao Peremanent Sea Silica Ltd., Qingdao, China), polyamide
(80–100 or 30–60 mesh, Taizhou Luqiao Sijia Biochemical Plastics Factory, Taizhou,
China), C18 reverse-phased silica gel (40–75 mm, Fuji, Kasugai, Japan). Analytical HPLC
experiments were conducted with a YMC Pack ODS-A column (5 lm, 250 mm ꢁ
4.6 mm i.d.; Tokyo, Japan) in Agilent 1100 (Agilent Technologies, Ltd) equipped with a
diode array detector (DAD) under reversed-phase. And semipreparative HPLC separa-
tions were carried out on an Agilent 1200 with YMC Pack ODS-A column (5 lm,
2
50 ꢁ 10 mm, YMC Co. Ltd., Kyoto, Japan). UV spectra were detected with Waters
Acquity UPLC equipped 2998 PDA Detector (America). All the analytic reagents were
analytical grade and purchased from Cologne Chemical Co., Ltd., Chengdu, China.
3.2. Plant material
The leaves of C. paliurus (collected in Xinning County, Shaoyang City, Hunan Province)
were provided by Hunan Heran Biotechnology Development Company, Hunan
Province, People’s Republic of China, in May 2016, identified by Prof. Kangping Xu
(Xiangya School of Pharmaceutical Sciences, Central South University). And its voucher
specimen (No. 20160820) was deposited in the Xiangya School of Pharmaceutical
Sciences, Central South University.

4
H. SUN ET AL.
3.3. Extraction and isolation
Whole leaves of C. paliurus (45.0 kg) were exhaustively extracted with 75% EtOH twice
under reflux (450 L ꢁ 2 h), and were dried under reduced pressure to get crude extract.
The crude extract was suspended in water and successively partitioned with CH Cl ,
2
2
EtOAc and n-BuOH(50L ꢁ 4 times for each solvent). The CH Cl extract (400 g) was
2
2
subjected to column chromatography on silica gel and eluted with a gradient mixture
of CH Cl /MeOH (from 100:0 to 0:100) to obtain ten Fr. (I-X) according to TLC patterns.
2
2
The Fr. X (60 g) was gradient elution of H O/MeOH (v/v, 100:0 to 0:100) through
2
Polyamide column to yield 5 fractions (Fr. A-Fr. E) according to analytical HPLC. Fr. B
(
13.2 g) was further chromatographed by a silica gel column with CH Cl /MeOH (10:0
2 2
to 0:10), and followed by a C18 reversed-phase column (from 10% to 100% aqueous
MeOH, stepwise). One of the fraction was found to develop crystal and compound 1
(3.5 mg) was isolated from the crystal (supplementary material Figure S24). Fr. C was
separated by a C18 reversed-phase column with a gradient system of MeOH/H O to
2
get five fractions (Fr. C1–Fr. C5). Compound 2 (2.4 mg) was isolated from Fr. C2 by
semi-preparative HPLC (2.5 mL/min, 230 nm, ACN-H O, 7.5:2.5, V/V).
2
3.3.1. Cyclocarioside Z12 (1)
Colorless amorphous powder; [a]25 D ꢂ 7.6 (c 0.10, MeOH); HPLC-UV (ACN-H O) kmax:
2
1
2
30 nm; H NMR (500 MHz in CD OD) data see supplementary material Table S1, and
C NMR (125 MHz in CD OD) data see supplementary material Table S1. HRESIMS, m/z
3
1
3
3
þ
7
54.5123 [M þ NH ] (calcd for C H NO 754.5105).
4
41 72
11
3.3.2. Cyclocarioside Z13 (2)
Colorless amorphous powder; [a]25 D ꢂ 29.6 (c 0.10, MeOH); HPLC-UV (ACN-H O)
2
1
kmax: 230 nm; H NMR (500 MHz in CD OD) data see supplementary material Table S1,
and C NMR (125 MHz in CD OD) data see supplementary material Table S1. HRESIMS,
3
1
3
3
þ
m/z 788.5159 [M þ NH ] (calcd for C H NO 788.5160).
4
41 74
13
3.4. Acid hydrolysis
The configurations of sugar moieties were established according to the published
method(Gan et al. 2015) with some modifications. Compounds 1-2 (each 1 mg) were
ꢃ
refluxed with 2 M HCl in the oil bath at 100 C for 2 h. The reaction mixture was neu-
tralized with Na CO and extracted with CHCl for three times. The aqueous layer was
2
3
3
concentrated and dried to obtain monosaccharide fraction. The mixture was dissolved
in 1 mL pyridine followed by 2 mg of L-cysteine methyl ester hydrochloride and heated
ꢃ
ꢃ
at 60 C for 1 h. Then trimethylsilylimidazole (1 mL) was added and heated at 60 C for
another 0.5 h. The reaction mixture was analyzed by GC–MS under the conditions:
ꢃ
Column, Rxi-5Sil MS (0.25 mm ꢁ 30.0 mm, 0.32 mm);front inlet 300 C, column
ꢃ ꢃ
1
50–300 C at 15 C/min. The sugar configurations of compounds 1-2 were identified
by the comparison of the retention times with authentic standard treated in the
same means.

NATURAL PRODUCT RESEARCH
5
3.5. Cytotoxic activity assay
All human cancer cell lines were gained from ATCC (Manassas, VA, USA). Cytotoxic
activities of all isolates (1-2) was tested by MTT assay with positive control of stauro-
sporine (STS). And specific experimental operations refer to published methods(Sun
et al. 2020b).
4. Conclusion
Two previously undescribed dammarane glycosides from Cyclocarya paliurus. were iso-
lated in this study. The cytotoxic activity assay suggested that compounds 1 and 2
have no broad-spectrum cytotoxicity against human cancer cells but showed a select-
ive inhibitory activity on MCF-7.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was supported by the Hainan Key Research Project（ ZDYF2020164,
ZDYF2020206） , Hunan Graduate Research and Innovation Project (No. CX20190247), Central
South University postgraduate independent exploration and innovation project (No.
2
019zzts191), Central South University College Students Innovation and Entrepreneurship
Project (No. S2020105330484; No. S202010533242X; No. 202010533021X; No. 202010533004S) ,
Hunan Science and Technology College Students Innovation and Entrepreneurship Project
(2020RC1001), Hunan Province Ordinary Higher Education Teaching Reform Research Project,
Central South University Deepening Innovation and Entrepreneurship Education Reform
Research Project (No. 2019CG006), Open Sharing Fund for the Large-scale Instruments and
Equipments of Central South University. We are very grateful to Prof. Guogen Liu (Modern
Analysis and Testing Centre of Central South University) and Prof. Hongping Long (High
Resolution Mass Spectrometry Laboratory of Advanced Research Center in the First Hospital of
Hunan University of Chinese Medicine) for the NMR and HRESIMS tests.
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