Two new glycosides isolated from Sapindus mukorossi fruits: effects on cell apoptosis and caspase-3 activation in human lung carcinoma cells

Article abstract of DOI:10.1080/14786419.2015.1054283

Two new glycosides (1, 2) and two saponins (3, 4) were isolated from the fruits of Sapindus mukorossi Gaertn. The two glycosides were designated as sapindoside G (1) and 4′′,4′′′′′-O-diacetylmukurozioside IIa (2). All four compounds exhibited inhibitory effects against A549 human lung adenocarcinoma cells with inhibition rates up to 69.2–83.3% at a concentration of 100 μg/mL. Flow cytometric analysis revealed that compounds 1–4 could suppress A549 cell growth by promoting cell apoptosis, which was related to the activation of caspase-3.

Full text of DOI:10.1080/14786419.2015.1054283

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Two new glycosides isolated from
Sapindus mukorossi fruits: effects on
cell apoptosis and caspase-3 activation
in human lung carcinoma cells
Xuan-Ming Zhanga, De-Po Yangab, Zhi-Yong Xiea, Qing Lia, Long-Ping
Zhub & Zhi-Min Zhaoab
a School of Pharmaceutical Sciences, Sun Yat-sen University
(Higher Education Mega Center), 132 Waihuan Rd East, Panyu
District, Guangzhou 510006, China
b Guangdong Technology Research Center for Advanced Chinese
Medicine, Guangzhou 510006, China
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Published online: 09 Jul 2015.
To cite this article: Xuan-Ming Zhang, De-Po Yang, Zhi-Yong Xie, Qing Li, Long-Ping Zhu & Zhi-Min
Zhao (2015): Two new glycosides isolated from Sapindus mukorossi fruits: effects on cell apoptosis
and caspase-3 activation in human lung carcinoma cells, Natural Product Research: Formerly
Natural Product Letters, DOI: 10.1080/14786419.2015.1054283
To link to this article: http://dx.doi.org/10.1080/14786419.2015.1054283
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NATURAL PRODUCT RESEARCH, 2015
http://dx.doi.org/10.1080/14786419.2015.1054283
Two new glycosides isolated from Sapindus mukorossi fruits:
effects on cell apoptosis and caspase-3 activation in human
lung carcinoma cells
Xuan-Ming Zhanga, De-Po Yangab, Zhi-Yong Xiea, Qing Lia, Long-Ping Zhub
and Zhi-Min Zhaoab
aSchool of Pharmaceutical Sciences, Sun Yat-sen University (Higher Education Mega Center), 132 Waihuan
b
Rd East, Panyu District, Guangzhou 510006, China; Guangdong Technology Research Center for Advanced
Chinese Medicine, Guangzhou 510006, China
ABSTRACT
ARTICLE HISTORY
Received 10 February 2015
Accepted 17 May 2015
Two new glycosides (1, 2) and two saponins (3, 4) were isolated from
the fruits of Sapindus mukorossi Gaertn. The two glycosides were
designatedassapindosideG(1)and4′′,4′′′′′-O-diacetylmukurozioside
IIa (2). All four compounds exhibited inhibitory effects against A549
human lung adenocarcinoma cells with inhibition rates up to 69.2–
83.3% at a concentration of 100 μg/mL. Flow cytometric analysis
revealed that compounds 1–4 could suppress A549 cell growth by
promoting cell apoptosis, which was related to the activation of
caspase-3.
KEYWORDS
Sapindus mukorossi;
glycosides; spectroscopic
analysis; flow cytometric;
apoptosis; caspase-3
GRAPHICAL ABSTRACT
1. Introduction
The genus Sapindus belongs to the Sapindaceae family, which consists of 2000 species (Rao
etal.2012).Sapindusmukorossi Gaertn,commonlyknownasWu-huan-zi,isprevalentinsouthern
China and has been used in the treatment of asthma, dermatological disorders and hepatic
disorders (Sharma et al. 2011). According to the literature, there are several phytochemicals
present in the pericarp, seeds, leaves, roots, stems and galls. The major constituents in the
fruit are saponins (10.0–11.5%) and sugars (10%) (Verma 2012). The glycosides isolated
CONTACT Zhi-Min Zhao
zhaozhm2@mail.sysu.edu.cn
© 2015 Taylor & Francis

2
X.-M. ZHANG ET AL
from S. mukorossi are mainly sesquiterpene oligoglycosides and triterpenoidal saponins
of hederagenin, dammarane and tirucullane (Upadhyay & Singh 2012). These glycosides
have antimicrobial (Ibrahim et al. 2006), cytotoxic (Chen et al. 2010), molluscicidal (Huang
et al. 2003; Upadhyay & Singh 2011), insecticidal (Rahman et al. 2007), fungicidal (Supradip
et al. 2010) and hepatoprotective (Peng et al. 2014) properties. In this study, we isolated and
characterized two new glycosides and evaluated the in vitro antiproliferative activity of all
compounds against A549 cells.
2. Results and discussion
2.1. Structural elucidation
Compound 1 was a white amorphous powder. Its molecular formula was C63H102O28 based
on HR-ESI-MS and MS/MS data (Figure 1). The 1H NMR spectrum had signals characteristic
of seven tertiary singlet methyl groups [δH 0.87, 1.02, 1.04, 1.15, 1.33, 1.33, and 1.34], two
secondary methyl groups [δH 1.57 (3H, d, J = 5.4 Hz) and 1.57 (3H, d, J = 5.4 Hz)], an olefinic
proton [δH 5.50 (1H, br s)] and an oxygen-bearing methine proton [δH 3.32 (1H, overlapped
‘os’)]. The 1H NMR spectrum of 1 revealed the presence of six anomeric proton signals at δH
4.88 (1H, d, J = 7.8 Hz, Ara H-1′), 6.23 (1H, br s, Rha H-1′′), 5.36 (1H, d, J = 7.4 Hz, Xyl H-1′′′),
4.90 (1H, d, J = 8.0 Hz, Glc H-1′′′′), 5.30(1H, d, J = 6.8 Hz, Xyl H-1′′′′′) and 6.25 (1H, br s, Rha
H-1′′′′′′), which were correlated with the 13C NMR signals for anomeric carbons at δC 105.6
(C-1′), 102.0 (C-1′′), 108.0 (C-1′′′), 105.7 (C-1′′′′), 107.8 (C-1′′′′′) and 102.1 (C-1′′′′′′), respec-
tively. The corresponding seven angular methyl groups [δC 16.1, 17.7, 17.7, 24.3, 26.7, 28.7,
and 28.7], olefinic signal [δC 123.0, 145.3] and carboxyl group [δC 180.7] were detected. These
findings implied that compound 1 had an oleanane-type triterpene and six sugar moieties.
The linkage points of the sugar units to each other and to the aglycone were determined
by the following HMBC correlations (see supplementary material, Figure S1): δH 4.88 (Ara-1′)
with δC 89.3 (aglycon C-3), δH 6.23 (Rha-1′′) with δ 75.9 (Ara C-2′), δ 5.36 (Xyl-1′′′) with δC 83.5
(Rha C-3′′), δH 4.90 (Glc-1′′′′) with δC 89.3 (Xyl C-3′C′′), δH 5.30 (Xyl-1′′H′′′) with δC 67.9 (Glc C-6′′′′)
and δH 6.25 (Rha-1′′′′′′) with δC 83.3 (Xyl C-2′′′′′). The relative configuration of the aglucone
and the sugar linkages were confirmed by ROESY spectrum (Figure S2). The relatively large
coupling constants for the anomeric protons of compound 1 revealed an α-configuration
of the arabinose unit and a β-configuration of the glucose and xylose units. Even though
the anomeric protons of two rhamnose moieties were observed as singlets in the 1H NMR
spectrum, the 13C NMR shifts of Rha C-5 at δC 69.7 and 70.0 indicated an α-configuration
(Wang et al. 2013). Acid hydrolysis of compound 1 yielded L-arabinose, d-glucose, d-xylose
and l-rhamnose, which were detected by GC-MS analysis of their derivatives. Accordingly,
compound 1 was identified as 3-O-α-l-rhamnopyranosyl-(1→2)-β-d-xylopyranosyl-(1→6)-
β-d-glucopyranosyl-(1→3)-β-d-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arab-
inopyranosyl oleanolic acid, and was named sapindoside G.
Compound 2 was a white powder. The negative ion HR-ESI-MS spectra of compound
2 showed quasimolecular ion peaks at m/z 1231.56188 [M−H]−; its molecular formula was
C55H92O30 (Figure 1). The 1H and 13C NMR spectra of compound 2 had signals characteristic
of three methyl groups [δH 1.01 (3H, d, J = 7.2 Hz, Me-15), 1.60 (3H, s, Me-14) and 1.63 (3H,
s, Me-13)]; two methylene groups bearing an oxygen function [δH 3.34 (1H, m, H-12α), 3.84
(1H, os, H-12β), 4.28 (1H, os, H-1α) and 4.56 (1H, os, H-1β)], two tri-substituted olefins [δH 5.21
(1H, m, H-6), 5.60 (1H, os, H-2)]; two β-d-glucopyranosyl moieties [δH 4.68 (1H, d, J = 7.2 Hz,
Glc H-1′′′′) and 4.73 (1H, d, J = 7.2 Hz, Glc H-1′)]; four α-l-rhamnopyranosyl moieties [δH 1.61

NATURAL PRODUCT RESEARCH
3
Figure 1. Structure of compounds 1–4.
(3H, Rha Me-6′′), 1.61 (3H, Rha Me-6′′′′′), 1.67 (3H, Rha Me-6′′′), 1.70 (3H, Rha Me-6′′′′′′),
5.62 (1H, br s, Rha-1′′′′′′), 5.63 (1H, br s, Rha-1′′′), 5.69 (1H, br s, Rha-1′′′′′) and 5.74 (1H, br
s, Rha-1′′)]; and two acetyl groups [δH 2.00 (3H, s) and 2.09 (3H, s)]. Detailed analysis of 1D
1
NMR, HSQC, H-1H COSY and HMBC spectra (Figure S3) established the assignment of an
alkyl glycoside similar to mukurozioside IIa (Morikawa et al. 2010), except for the additional
signals attributed to the acetyl groups. The binding sites for the acetyl groups of compound
2 were elucidated by HMBC and acetylation shifts. The presence of a correlation between
δH 5.71 (Rha-4′′)/δ 171.3 and δH 5.71 (Rha-4′′′′′)/δC 171.4 revealed that the acetyl groups
were located at 4′′Cand 4′′′′′ position in the rhamnopyranosyl moiety. The acetylation shifts
[δC 71.1 (Rha-3′′), 76.2 (Rha-4′′), 67.8 (Rha-5′′); δC 71.1 (Rha-3′′′′′), 76.3 (Rha-4′′′′′) and 67.8
(Rha-5′′′′′)] confirmed the assignment of the acetate groups. In the ROESY spectrum, corre-
lations at H2-1/H3-13 and H2-5/H3-14 and the absence of any correlations at H-2/H3-13 and
H-6/H3-14 indicated that the olefinic bonds in the aglucone were both trans relationships.
The linkages between sugar units were also concluded from ROESY spectrum (Figure S4).
Consequently, compound 2 was identified as 4′′,4′′′′′-O-diacetylmukurozioside IIa.
By comparing the physical and spectroscopic data with those reported in the literature
(Chirva et al. 1970; Nakayamak et al. 1986), compounds 3 and 4 were identified as Hishoushi-
saponin Ee and Sapindoside A, respectively.
2.2. Cell viability assay
MTT assay results revealed that A549 cell growth was inhibited in a dose-dependent man-
ner by compounds 1–4. All four compounds exhibited inhibitory effects with inhibition
rates up to 69.2–83.3% at a concentration of 100 μg/mL (Table S1). Compounds 1, 3 and
4 had better inhibitory effects than compound 2 against A549 cells, with IC50 values of
33.61 0.24, 46.75 0.07 and 33.19 0.13 μg/mL, respectively. Compound 2 had an IC50
value of 98.35 0.18 μg/mL.
2.3. Cell cycle and cell apoptosis
To further confirm the induction of cell apoptosis, cells were stained with annexin V/PI for
flow cytometry. As shown in Figure S5, the early apoptotic rates of A549 cells were 52.4%

4
X.-M. ZHANG ET AL
(1), 4.9% (2), 52.4% (3) and 56.6% (4). Compared to untreated A549 cells, the percentage of
late apoptotic cells increased by 1.8% (1), 3.3% (2), 13.9% (3) and 8.1% (4) following 24-h
treatment. Compound 2 had a weaker effect on A549 cells apoptosis than the other three
compounds. Late apoptosis was induced in the test concentration of compound 2. Therefore,
the inhibition of A549 cell growth by glycosides is attributed to an induction in cell apop-
tosis. Compound 2 has different inhibitory mechanisms than compounds 1, 3 or 4. For cell
cycle analysis, the percentages of cells in G0/G1, S and G2/M phases were determined by
flow cytometry. The effect of compounds 1–4 on the cell cycle distribution of A549 cells is
shown (Figure S6). As a result, the compounds had little influence in the A549 cell cycle arrest.
2.4. Induction of apoptosis by activating caspase-3
Caspase family proteins play crucial roles in cell apoptosis. To assess whether compounds
1–4 activate the caspase-dependent cell death pathway, we studied the activation of
caspase-3 using colorigenic tetrapeptide substrates such as Ac-DEVD-pNA, which is selec-
tive for caspase-3 enzymatic activities. Compared to the control group, compound-treated
groups had higher absorbance measurements at 405 nm. After a 2-h treatment with
compound 1, caspase-3 activity increased from 1.44 0.26 μM pNA/2 h of protein to
4.33 0.48 μM pNA/2 h of protein in a concentration-dependent manner (Figure S7). The
activity of the enzyme increased in the presence 6.25–75 μg/mL of compounds 2–4 and
decreased in the presence of 100 μg/mL of compounds 2–4. These results indicated that com-
pounds 1–4 from S. mukorossi activated caspase-3. A high caspase-3 activity was observed
during the early apoptotic stage. The highest caspase-3 enzymatic activities obtained were
4.33 0.48 μM pNA/2 h (with 100 μg/mL of 1), 5.09 0.17 μM pNA/2 h (with 75 μg/mL
of 3) and 3.47 0.40 μM pNA/2 h (with 75 μg/mL of 4). Compound 2 had a lower induction of
caspase-3 activity, consistent with the apoptotic rate described in the flow cytometry results.
3. Conclusions
Two new glycosides (1, 2) and two saponins (3, 4) were isolated from the fruits of S. mukorossi
Gaertn. Compounds 1, 3 and 4 showed inhibition of A549 cell growth in a dose-dependent
manner by promoting cell apoptosis via activation of caspase-3. Studies are in progress to
elucidate the possible mechanism of action of these glycosides.
Funding
The authors acknowledge the financial support obtained from the National Natural Science Foundation
of China [grant number 81102782], the Postdoctoral Science Foundation of China [grant number
2011M501368], and the Special Project on the Integration of Industry, Education, and Research of
Guangdong Province [grant number 2011B090400140].
Supplemental data and research materials
Supplemental data for this article can be accessed at http://dx.doi.10.1080/14786419.
2015.1054283.
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