Chemical constituents from the thorns of Gleditsia sinensis and their cytotoxic activities

Article abstract of DOI:10.1080/10286020.2020.1731799

A new aromatic glycoside (1) and a new natural product, neolignan (2), along with twenty-three known compounds (3-25), were isolated from the thorns of Gleditsia sinensis. According to the spectroscopic analyses (IR, UV, HRESIMS, NMR and ECD), the structures of isolates were elucidated. Herein, compounds 4, 6-8, 10-13, 15, 16, 18, 20, 23 were isolated from the plant of G. sinensis for the first time. Moreover, compounds 4, 6, 15 and 24 showed cytotoxic effects on human ovarian cancer (SKOV-3) cells with IC₅₀ values of 24.83 ± 4.90, 48.86 ± 9.11, 80.13 ± 5.62, 15.38 ± 2.21 μM, respectively. (Figure presented.).

Full text of DOI:10.1080/10286020.2020.1731799

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Chemical constituents from the thorns of Gleditsia
sinensis and their cytotoxic activities
Yu-Bo Zhang, Kar-Ho Lam, Li-Feng Chen, Hei Wan, Guo-Cai Wang, Kai-Fai Lee,
Chun-Wang Yip, Kwun-Hung Liu, Pak-Heng Leung, Hai-Yong Chan, Yi-Gang
Shi, Jia Zhao, Li-Xing Lao, Yao-Lan Li, Yan-Bo Zhang & Wei Meng
To cite this article: Yu-Bo Zhang, Kar-Ho Lam, Li-Feng Chen, Hei Wan, Guo-Cai Wang, Kai-Fai
Lee, Chun-Wang Yip, Kwun-Hung Liu, Pak-Heng Leung, Hai-Yong Chan, Yi-Gang Shi, Jia Zhao,
Li-Xing Lao, Yao-Lan Li, Yan-Bo Zhang & Wei Meng (2020): Chemical constituents from the thorns
of Gleditsiaꢀsinensis and their cytotoxic activities, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2020.1731799
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2020.1731799
Chemical constituents from the thorns of Gleditsia
sinensis and their cytotoxic activities
Yu-Bo Zhanga,b,†, Kar-Ho Lama,†, Li-Feng Chenb, Hei Wana, Guo-Cai Wangb,
Kai-Fai Leec, Chun-Wang Yipa, Kwun-Hung Liua, Pak-Heng Leungd, Hai-Yong
Chana, Yi-Gang Shia, Jia Zhaoa, Li-Xing Laoa, Yao-Lan Lib, Yan-Bo Zhanga and
Wei Menga
aYu Jin, Master of Gynaecology of Chinese Medicine & Integrative Medicine, Integrative Medicine,
Integrative Medicine Workstation for Training and Research (Hong Kong Branch), School of Chinese
b
Medicine, LKS Faculty of Medicine, The University of Hong Kong, Pok Fu Lam, Hong Kong; Institute
of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University,
c
Guangzhou 510632, China; Department of Obstetrics and Gynaecology, LKS Faculty of Medicine,
d
The University of Hong Kong, Pok Fu Lam, Hong Kong; Department of Pharmacology and
Pharmacy, LKS Faculty of Medicine, The University of Hong Kong, Pok Fu Lam, Hong Kong
ABSTRACT
ARTICLE HISTORY
Received 22 August 2019
Accepted 15 February 2020
A new aromatic glycoside (1) and a new natural product, neoli-
gnan (2), along with twenty-three known compounds (3-25),
were isolated from the thorns of Gleditsia sinensis. According to
the spectroscopic analyses (IR, UV, HRESIMS, NMR and ECD), the
structures of isolates were elucidated. Herein, compounds 4, 6-8,
10-13, 15, 16, 18, 20, 23 were isolated from the plant of G. sinen-
sis for the first time. Moreover, compounds 4, 6, 15 and 24
showed cytotoxic effects on human ovarian cancer (SKOV-3) cells
with IC50 values of 24.83 4.90, 48.86 9.11, 80.13 5.62,
15.38 2.21 lM, respectively.
KEYWORDS
Leguminosae; Gleditsia
sinensis; chemical
constituent; structural
identification; cytotoxic
activity
CONTACT Yan-Bo Zhang
ybzhang@hku.hk; Wei Meng
bmeng@hku.hk
†These authors contributed equally to this study.
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2020.1731799.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
Y.-B. ZHANG ET AL.
1. Introduction
The perennial plant Gleditsia sinensis Lam. (Leguminosae), is widely distributed in
China, such as Jiangsu, Hubei, Henan, Shandong and Sichuan Provinces [1]. The
thorns of G. sinensis, which is known as ‘Zao-Jiao-Ci’ in China, have been used for
the treatment of several illnesses, including cancer, carbuncle and skin diseases [2].
Moreover, the thorns of G. sinensis and seven other medicinal materials are mixed as
a Chinese medicine formula called “Kun Tai No. 1”, which is clinically used to treat
polycystic ovary syndrome [3]. Modern pharmacology researches have shown that the
plant of G. sinensis possesses anti-tumor [4, 5], anti-inflammatory [6], anti-allergic
[7], anti-angiogenic [8], and antibacterial [9] activities. In particular, the crude extract
and chemical constituents of the thorns of G. sinensis possessed a certain inhibitory
effect on various tumor cells, such as cervical, breast, leukemia, liver, oral, and gastric
cancer cells [5]. Previous phytochemical investigations on this plant have led to the
isolation of triterpenoid saponins [5], flavonoids [10], phenolic [11], and steroidal
[12] derivatives.
In our efforts to discover new anti-cancer metabolites from traditional medicinal
plants [13–15], we investigated the chemical constituents of G. sinensis. As a result, a
new aromatic glycoside (1) and a new natural product, neolignan (2), along with
twenty-three known compounds (3–25), were isolated from the thorns of G. sinensis
(Figure 1). Their chemical structures were determined by analysis of spectroscopic
and physical data. Then, all the isolates were evaluated for their cytotoxic activities
on human ovarian cancer cells by using the 3-(4,5-dimethylthiazol-2-yl)ꢀ2,5-diphe-
nyltetrazolium bromide (MTT) assay.
2. Results and discussion
25
Compound 1 was isolated as a white amorphous powder with ½aꢁD ꢀ 35.8 (c 1.0,
CH3OH), and has the molecular formula C25H28O13 as indicated by the HRESIMS at
m/z 559.1440 [M þ H]þ. The IR spectrum of 1 showed absorption bands for aromatic
ring (1609, 1513, 1455 cmꢀ1), carbonyl (1699 cmꢀ1), and hydroxyl group (3392 cmꢀ1).
1
The H NMR spectrum of 1 (Table 1), interpreted with the help of an HSQC experi-
ment, showed signals for a 1,2,4-trisubstituted aromatic group [dH 7.30 (1H, br s),
7.07 (1H, d, J ¼ 8.6 Hz), and 6.89 (1H, br d, J ¼ 8.6 Hz)], a symmetrically 1,3,4,5-tetra-
substituted aromatic group [dH 7.20 (2H, s)], a pair of mutually coupled aliphatic
protons [dH 7.41 (1H, d, J ¼ 15.6 Hz) and 6.40 (1H, d, J ¼ 15.6 Hz)], and three
methoxyls [dH 3.75 (6H, s) and 3.73 (3H, s)], and an anomeric proton [dH 5.08 (1H,
Figure 1. The chemical structures of 1-2.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. 1H and 13C NMR spectral data for compounds 1 and 2 (J in Hz).a
1
2
No.
dH
dC
No.
dH
dC
1
2
3
4
5
6
7
8
–
7.30 (br s)
–
128.4
111.1
149.1
148.0
114.7
121.7
143.3
118.2
168.2
99.3
73.1
76.6
70.3
74.0
1
2
3
4
5
6
7
8
9
–
134.1
111.9
148.7
147.0
115.7
121.0
74.1
7.02 (d, 1.8)
–
–
–
7.07 (d, 8.6)
6.89 (br d, 8.6)
7.41 (d, 15.6)
6.40 (15.6)
–
6.73 (d, 8.1)
6.84 (dd, 8.1, 1.8)
4.83 (d, 5.8)
4.36-4.38 (m)
3.82b
3.77 (dd, 12.2, 3.7)
–
86.1
62.2
9
10
20
30
40
50
60
5.08 (d, 6.7)
3.33b
10
20
30
40
50
60
70
80
90
132.7
111.4
151.9
149.2
118.8
120.8
133.8
125.2
74.2
3.33b
7.00 (br s)
–
3.26b
3.79b
–
4.62 (d, 11.2)
4.18 (dd, 11.2, 7.6)
64.1
6.88 (br s)
6.88 (br s)
6.54 (dt, 15.3, 1.1)
6.18 (dt, 15.3, 6.3)
4.05 (dd, 6.3, 1.1)
3.79 (s)
100
–
119.3
107.3
147.7
141.2
165.5
55.8
200,600
300,500
400
7.20 (s)
–
–
–
3-OMe
30-OMe
90-OMe
56.3
56.5
58.0
700
3.80 (s)
3.36 (s)
3-OMe
3.73 (s)
3.75 (s)
300,500-OMe
56.2
aMeasured at 500 (1H) and 125 (13C) MHz in DMSO-d6 for 1 and CD3OD for 2.
bOverlapped with other signals.
d, J ¼ 6.7 Hz)]. The 13C NMR spectrum of 1 (Table 1) in conjunction with the
DEPT-135 spectrum showed the existence of 25 carbon signals, including fourteen
aromatic or olefinic carbons (dC 149.1, 148.0, 147.7 ꢂ 2, 143.3, 141.2, 128.4, 121.7,
119.3, 118.2, 114.7, 111.1, and 107.3 ꢂ 2), two carbonyls (dC 168.2, 165.5), three
methoxyls (dC 55.8, 56.2 ꢂ 2), five oxygenated methines (dC 99.3, 76.6, 73.1, 74.0 and
70.3) and an oxygenated methylene (dC 64.1).
Detailed analysis of the 1D NMR data of 1 showed that compound 1 possessed
ferulic acid (dC 168.2, 149.1, 148.0, 143.3, 128.4, 121.7, 118.2, 114.7, 111.1, 55.8) [16],
4-hydroxy-3,5-dimethoxybenzoyl (dC 165.5, 147.7 ꢂ 2, 141.2, 119.3, 107.3 ꢂ 2,
56.2 ꢂ 2) [17], and glucose units (dC 99.3, 76.6, 74.0, 73.1, 70.3, 64.1) [17]. In the
HMBC spectrum of 1, the correlations between H-10 (dH 5.08) and C-4 (dC 148.0),
and between H2-60 (dH 4.62, 4.18) and C-700 (dC 165.5) indicated that the ferulic acid
and 4-hydroxy-3,5-dimethoxybenzoyl moieties were connected to C-10 and C-60 of
glucose unit, respectively. Moreover, the type of sugar residue was confirmed by the
3
acid hydrolysis of 1, which was detected as D-glucose by HPLC analysis. The JH1-H2
coupling constant demonstrated b-glycosidic linkages of D-glucose (J ¼ 6.7 Hz). Thus,
the structure of 1 was determined and named as 4-[6-O-(4-hydroxy-3,5-dimethoxy-
benzoyl)-b-D-glucopyranosyloxy]-ferulic acid.
Compound 2 was obtained as yellow oil, which was assigned the molecular for-
mula of C21H26O7 based upon an [M þ H]þ ion peak at m/z 413.1571 [M þ H]þ in
HRESIMS. The IR spectrum of 2 showed absorption bands for hydroxyl (3441 cmꢀ1).
1
The H NMR spectrum of 2 revealed the presence of the signals for eight olefinic
protons [dH 7.02 (1H, d, J ¼ 1.8 Hz), 7.00 (1H, br s), 6.88 (2H, br s), 6.84 (1H, dd,

4
Y.-B. ZHANG ET AL.
1
Figure 2. Key H-1H COSY and HMBC correlations of 1 and 2.
J ¼ 8.1, 1.8 Hz), 6.73 (1H, d, J ¼ 8.1 Hz), 6.54 (1H, dt, J ¼ 15.3, 1.1 Hz) and 6.18 (1H,
dt, J ¼ 15.3, 6.3 Hz)], six deshielded protons [dH 4.83 (1H, d, J ¼ 5.8 Hz), 4.37 (1H,
m), 4.05 (2H, dd, J ¼ 6.3, 1.1 Hz), 3.82 (1H, overlapped) and 3.77 (1H, dd, J ¼ 12.2,
3.7 Hz)], and three methoxyls [dH 3.80, 3.79 and 3.36 (each, 3H, s)]. The 13C NMR
and DEPT spectra exhibited twenty-one signals, including six quaternary carbons, ten
1
methines, two methylenes, and three methyls. Detailed analysis of the H and 13C
NMR data of 2 (Table 1) showed that 2 possessed two 1,3,4-trisubstituted benzene
rings [dC 148.7, 147.0, 134.1, 121.0, 115.7,111.9; dC 151.9, 149.2, 132.7, 120.8, 118.8,
111.4], a double bond [dC 133.8 and 125.2], two oxygenated methines [dC 86.1 and
74.1], two oxygenated methylenes [dC 74.2 and 62.2], and three methoxyls [dC 58.0,
56.5 and 56.3]. The NMR spectroscopic features of 2 were close to those of viburfor-
doside G [18]. The main difference was that the NMR signals of the glucopyranosyl
moiety in 2 disappeared. In the HMBC spectrum, the protons of three methoxyls
showed cross-peaks with the carbons of C-3/C-30/C-90, indicating that three methox-
yls were linked to C-3/C-30/C-90, respectively. The planar structure of 2 was further
1
confirmed by H-1H COSY and HBMC spectra (Figure 2). The erythro form of H-7
and H-8 in 2 was established by a small coupling constant (J7,8 ¼ 5.8 Hz) [18].
Furthermore, the DdC8ꢀC7 value of 2 (12.0) was identical to viburfordoside G [18],
which further verified that 2 should possess the same relative stereochemistry as that
of viburfordoside G (7,8-erythro). Finally, experimental ECD spectrum of 2 was in
good agreement with calculated ECD spectrum of 7S,8R-2 using a TDDFT [B3LYP/6-
31G(d)] (Figure 3), indicating that the absolute configurations of 2 were assigned to
be 7S, 8R. Thus, compound 2 was named as glensin A, which was previously
obtained (without detailed spectroscopic data) by a hydrolysis of viburfordoside G.
Herein it was reported for the first time as a molecule isolated from nature [18].
Twenty-three known compounds were identified as (7S,8R)ꢀ4,90-dihydroxyl-3,30-
dimethoxyl-7,8-dihydrobenzofuran-10-propylneolignan (3), (7S,8R)-threo-10-[30-hydroxy-
7-(4-hydroxy-3-methoxyphenyl)ꢀ8-hydroxymethyl-7,8-dihydrobenzofuran]acrylaldehyde
(4), (2R-trans)ꢀ2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)ꢀ3-(hydroxymethyl)ꢀ7-
methoxy-5-benzofuranpropanol acetate (5), (7R,8S)-dehydrodiconiferyl alcohol (6),
(-)-secoisolariciresinol (7), (þ)-pinoresinol (8), (þ)-medioresinol (9), syringaresinol
(10), garbanzol (11), 7,40-dihydroxy-5,30-dimethoxyflavanonol (12), 40-O-methyldihy-
droquercetin (13), (þ)-aromadendrin (14), (-)-eriodictyol (15), 5-hydroxy-7-methoxy-
flavanone (16), quercetin (17), scopoletin (18), 3-formylindole (19), indazole (20),
isovanillic acid (21), friedelin (22), lupenone (23), betulin (24), sitosterol (25) respect-
ively, by comparing their spectroscopic and physical data with those of related

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 3. Experimental and calculated ECD spectra of 2.
literatures. Herein, compounds 4, 6–8, 10–13, 15, 16, 18, 20, 23 were isolated from
the plant of G. sinensis for the first time. The references and chemical structures have
been listed in supporting material.
Then, the isolated compounds (1–25) were subjected to test their cytotoxic activ-
ities on human ovarian cancer SKOV-3 cells and human normal ovarian IOSE80 cells
using MTT assay. Cisplatin was adopted to be positive control in this experiment. As
a result (Table 2), compounds 4 and 24 showed moderate cytotoxic activities against
ovarian cancer cells SKOV3 with IC50 values of 24.83 4.90 and 15.38 2.21 lM, and
SI values of 15.20, 1.09, respectively. Compounds 6 and 15 showed weak cytotoxic
activities with IC50 values of 48.86 9.11 and 80.13 5.62 lM, respectively. However,
compounds 1–3, 5, 7–14 and 16–24 had not obvious activities (IC50>100 lM).
3. Experimental
3.1. General experimental procedures
IR and UV spectra were obtained on JASCO FI/IR-480 Plus Fourier transform spec-
trometer and JASCO V-550 UV/vis spectrophotometer (JASCO, Tokyo, Japan),
respectively. Optical rotations were measured on JASCO P-1020 polarimeter (JASCO,
Tokyo, Japan). NMR spectra were performed on Bruker AV-500 spectrometer
(Bruker, Faellanden, Switzerland). CD spectra were recorded on JASCO J-810 spec-
tropolarimeter (JASCO, Tokyo, Japan). HRESIMS data were tested on Agilent 6210
LC/Q TOF mass spectrometer (Agilent Technologies, CA, USA). Preparative HPLC
were performed on Varian Prostar system equipped with UV detector (Varian,
California, USA) and a preparative C18 column (Cosmosil, 5 mm, 20 ꢂ 250 mm,
Nacalai Tesque, Japan). Analytical HPLC were performed on Dionex chromatograph
(Dionex, California, USA) with an analytical column (Cosmosil, 5 mm, 4.6 ꢂ 250 mm,
Nacalai Tesque, Japan), a P680 pump, and a PDA-100 photodiode array detector.
Open column chromatography (CC) was performed using ODS (50 lm; YMC,

6
Y.-B. ZHANG ET AL.
Table 2. Cytotoxic activities of the active compounds.a
SKOV3
IOSE80
Compounds
IC50 (lM)b
CC50 (lM)c
SIc
4
6
15
24.83 4.90
48.86 9.11
80.13 5.62
15.38 2.21
11.08 0.18
73.42 13.24
15.20
>2.05
1.06
1.09
1.20
>100b
85.34 7.91
16.75 3.70
13.25 2.53
24
Cisplatind
aData are represented as the mean value SD.
bThe IC50 value of the sample was higher than 100 lM.
cSI value equals to IC50 (SKOV3 cells)/IC50 (IOSE80 cells).
dPositive control.
Tokyo, Japan), silica gel (200–300, 300–400 mesh, Qingdao Haiyang Chemical
Company, Ltd., Qingdao, China), and Sephadex LH-20 (25–100 lm, Amersham
Pharmacia Biotech AB, Uppsala, Sweden). Thin layer chromatography (TLC) was car-
ried out using precoated silica gel GF254 plates (Yantai Jiangyou Silica Gel
Technology Development Co., Ltd., Yantai, China). All the reagents were purchased
from Tianjin Damao Chemical Company (Damao, Tianjin, China).
3.2. Plant material
The dried thorns of Gleditsia sinensis were purchased in April 2018 from Luoyang
City, Henan Province of China, and identified by Prof. Guang-Xiong Zhou, Jinan
University, Guangzhou, China. A voucher specimen (No.20180318) was deposited at
the Institute of Traditional Chinese Medicine and Natural Products, Jinan University.
3.3. Extraction and isolation
The thorns of Gleditsia sinensis (30 kg) were powdered and extracted three times with
75% EtOH (50 L ꢂ 3, 24 h, each) at ambient temperature. The combined extracts were
evaporated in vacuo to afford a crude extract (1.8 kg), which was sequentially parti-
tioned into petroleum ether (PE), EtOAc, and n-BuOH. The PE part (126.5 g) was
chromatographed on silica gel column with a PE-EtOAc step gradient system (100:0
to 0:100) to give eight fractions (1-8). Fr. 3 (4.5 g) was purified with the aid of silica
gel column (PE-acetone, 100:0 to 1:1) to yield 18 (5.8 mg) and 24 (19.2 mg). Fr. 4
(278.0 mg) was purified by Sephadex LH-20 (CHCl3/CH3OH, 1:1) to obtain 22
(30.5 mg), and Fr. 6 (18.3 g) was separated by silica gel column (PE/EtOAc, 95:5 to
1:1) to obtain 23 (15.7 mg) and 25 (20.6 mg).
The EtOAc part (510.8 g) was subjected to silica gel column and eluted with
CHCl3/CH3OH (99:1 to 50:50) to afford five fractions (A-E). Fraction B (103.6 g) was
further separated with the aid of ODS column (CH3OH/H2O, 30:70 to 100:0) to give
six subfractions (B1-B6). Fr. B2 (8.7 g) was separated by silica gel column (PE/EtOAc,
90:10 to 0:100) and then purified by Sephadex LH-20 column (CHCl3/CH3OH, 1:1)
to obtain 19 (7.4 mg). Fr. B3 (2.2 g) was applied to Sephadex LH-20 column (CHCl3/
CH3OH, 1:1) to give 16 (10.0 mg) and 21 (15.9 mg). Fr. B5 (25.7 g) was separated by
silica gel column (CHCl3/CH3OH, 95:5 to 70:30) and further purified by preparative
HPLC (CH3OH/H2O, 85:15, 8 ml/min) to obtain 8 (9.3 mg), 9 (12.0 mg) and 10

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
(18.1 mg). Fr. B6 (1.5 g) was applied to silica gel column (CHCl3/CH3OH, 95:5 to
80:20) to yield 5 (17.7 mg). Fr. D (92.8g) was loaded onto a ODS column and eluted
with CH3OH/H2O (50:50 to 100:0) to give nine subfractions (D1-D9). Fr. D2 (3.6 g)
was chromatographed on a Sephadex LH-20 column (CHCl3/CH3OH, 1:1) and then
purified by preparative HPLC (CH3OH/H2O, 85:15, 8 ml/min) to yield 3 (15.7 mg), 4
(10.8 mg) and 6 (20.5 mg). Fr. D3 (2.1 g) was subjected to Sephadex LH-20 column
(CHCl3/CH3OH, 1:1) to yield 20 (14.6 mg), and Fr. D8 (4.7 g) was subjected to a silica
gel column (CHCl3/CH3OH, 95:5 to 70:30) to yield 7 (22.9 mg). Fr. D5 (15.8 g) was
separated by silica gel column (CHCl3/CH3OH, 100:0 to 80:20) and purified by
Sephadex LH-20 column (CHCl3/CH3OH, 1:1) to yield 11 (21.7 mg), 12 (18.0mg) and
13 (16.5 mg). Fr. D8 (1.5 g) was separated by ODS column (CH3OH/H2O, 20:80 to
90:10) to yield 1 (16.8 mg). Compounds 14 (20.5 mg) and 15 (13.2 mg) were isolated
from Fr. D9 (2.1 g) by Sephadex LH-20 column eluting with CHCl3-CH3OH (1:1).
Fraction E (39.4 g) was sequentially separated by ODS column (CH3OH/H2O, 30:70 to
90:10) and Sephadex LH-20CC (MeOH) to afford 2 (11.8 mg) and 17 (23.5 mg).
3.3.1. 4-[6-O-(4-Hydroxy-3,5-dimethoxybenzoyl)-b-D-glucopyranosyloxy]-ferulic acid (1)
25
Brown oil; ½aꢁD ꢀ 35.8 (c 1.0, CH3OH); UV (CH3OH) kmax 209 and 276 nm; IR (KBr)
1
ꢀmax 3392, 2941, 1699, 1609, 1513, 1455, 1423, 1336, 1269, 1228, 1071 cmꢀ1; H and
13C NMR spectroscopic data, see Table 1; HRESIMS: m/z 559.1440 [M þ H]þ (calcd
for C25H28O13Na, 559.1422).
3.3.2. Glensin A (2)
25
Yellow oil; ½aꢁD þ 42.4 (c 1.0, CH3OH); UV (CH3OH) kmax 205, 277 nm; IR (KBr)
1
ꢀmax 3441, 1630, 1510, 1382, 1267, 1115, 1027, 563 cmꢀ1; H and 13C NMR spectro-
scopic data, see Table 1; HRESIMS: m/z 413.1571 [M þ H]þ (calcd for
C21H26O7Na, 413.1571).
3.4. Acid hydrolysis of compound 1
Compound 1 (1 mg) was dissolved in 4.0 M HCl (10 ml) and refluxed at 90 ꢃC for
6 h. The reaction mixture was concentrated under reduced pressure and then parti-
tioned with equal volumes of chloroform and water. After the H2O layer was concen-
trated, the sugar residue was obtained. Then, the residue was dissolved in anhydrous
pyridine (1 ml), followed by addition of L-cysteine methyl ester hydrochloride (2 mg)
at 60 ꢃC for 1 h. The reaction solvent was dried under a stream of N2. Subsequently,
0.2 ml of trimethylsilyl imidazole was added at 60 ꢃC for another 1 h. The final prod-
uct was analyzed by HPLC and compared with standard sugar [flow rate: 0.8 ml/min;
injection volume: 10 ll; column: Cosmosil 5C18-MS-II column (4.6 ꢂ 250 mm);
mobile phase: 25% CH3CN-H2O (0.05% acetic acid); wavelength: 210 nm; column
temperature: 25 ꢃC)]. The same treatment was applied for standard sugar. The reten-
tion time of standard sugar was detected at 16.42 min (D-glucose).
3.5. Cytotoxicity assay
Compounds 1-25 were evaluated for their cytotoxic effects against human ovarian
cancer SKOV-3 cells and human normal ovarian IOSE80 cells by MTT assays. The

8
Y.-B. ZHANG ET AL.
cells were cultured in DMEM (dulbecco’s modified eagle medium) medium supple-
mented with 10% heat-inactivated fetal bovine serum. Then, cells were planted in
96-well plates with approximately 3000 cells per well and treated with 0-100 lM of
isolated compounds for 48 h at 37 ꢃC in 5% CO2. Subsequently, experiments were ter-
minated by adding 20 ml of MTT (10 mg/ml) into each well of the plates and then
incubated for 4 h. The supernatant was replaced by 100 ll of DMSO to dissolve the
formazan. MTT assay results were detected using a microplate reader spectrophotom-
eter (Thermo Labsystems Multiskan MK3) at 570 nm.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by grants from Innovation and Technology Support
Programme, Government of Hong Kong (Project code: UIM/321), National Key R&D
Program of China (No. 2017YFC1703800), and China Postdoctoral Science Foundation (No.
2019T120795).
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