Cytotoxic isovaleryl sucrose esters from Ainsliaea yunnanensis: reduction of mitochondrial membrane potential and increase of reactive oxygen species levels in A549 cells

Article abstract of DOI:10.1039/c7ra01986f

Eight isovaleryl sucrose esters, named ainslosides A-H (1-8), were isolated from Ainsliaea yunnanensis Franch. Their structures, including the absolute configurations of the sugar residues, were elucidated by extensive analysis of NMR spectra and acid hydrolysis. All compounds were tested in vitro for cytotoxicity against four human tumour cell lines, A549, HCT116, MDA-MB-231, and BEL7404. Among the compounds tested, ainsloside B (2) showed potent cytotoxicity against the A549 cell line with an IC₅₀ value of 3.3 μM. Flow cytometry analysis showed that compound 2 can arrest the cell cycle at the G₀/G₁ phase and induce cell apoptosis in A549 cells. Further studies indicated that the apoptosis-inducing effect of compound 2 may be involved in the reduction of mitochondrial membrane potential (MMP) and increase of reactive oxygen species (ROS) level in A549 cells.

Full text of DOI:10.1039/c7ra01986f

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Cytotoxic isovaleryl sucrose esters from Ainsliaea
yunnanensis: reduction of mitochondrial
membrane potential and increase of reactive
oxygen species levels in A549 cells†
Cite this: RSC Adv., 2017, 7, 20865
Xin Fang, Zhi-Guo Zhuo, Xi-Ke Xu, Ji Ye, Hui-Liang Li, Yun-Heng Shen *a
ab
a
a
a
a
abc
and Wei-Dong Zhang*
Eight isovaleryl sucrose esters, named ainslosides A–H (1–8), were isolated from Ainsliaea yunnanensis
Franch. Their structures, including the absolute configurations of the sugar residues, were elucidated by
extensive analysis of NMR spectra and acid hydrolysis. All compounds were tested in vitro for cytotoxicity
against four human tumour cell lines, A549, HCT116, MDA-MB-231, and BEL7404. Among the
compounds tested, ainsloside B (2) showed potent cytotoxicity against the A549 cell line with an IC50
Received 17th February 2017
Accepted 4th April 2017
0 1
value of 3.3 mM. Flow cytometry analysis showed that compound 2 can arrest the cell cycle at the G /G
phase and induce cell apoptosis in A549 cells. Further studies indicated that the apoptosis-inducing
effect of compound 2 may be involved in the reduction of mitochondrial membrane potential (MMP) and
increase of reactive oxygen species (ROS) level in A549 cells.
DOI: 10.1039/c7ra01986f
rsc.li/rsc-advances
chemical constituents and bioactivities of A. yunnanensis were
Introduction
reported except for the isolation of several sesquiterpenoids,
12,13
The genus Ainsliaea of Asteraceae comprises about 70 species, triterpenoids, and some phenoloids.
As part of our
which is mainly distributed in the southeast of Asia. Many continuing efforts to discover structurally interesting bioactive
Ainsliaea species have been long used for the treatment of compounds from Ainsliaea species, eight isovaleryl sucrose
various diseases, such as rheumatism, traumatic injury, enter- esters, ainslosides A–H (1–8), were isolated from the whole
itis dysentery, sore throat, and urological and gynecological plants of A. yunnanensis. Compounds 1–8 were in vitro tested for
1
diseases. The chemical constituents and crude extracts from cytotoxicity against four human tumour cell lines A549,
Ainsliaea species have been reported to have various biological HCT116, MDA-MB-231, and BEL7404, and cell cycle arresting
2
3
4
activities, such as anti-microbial, cytotoxic, antiviral, antiox- and apoptosis induction of ainsloside B (2) in A549 cell line.
5
6–8
idant, and anti-inammation activities. Our previous inves- Herein, we described the isolation, structural elucidation, and
tigations on Ainsliaea species have reported the isolation of in vitro antitumor evaluation (Fig. 1).
a series of sesquiterpenoids, triterpenoids, sesquiterpenoid
lactone dimers and trimers, of which the rst two guaianolide
9
trimers, ainsliatrimers A and B from A. fulvioides, and a new
guaianolide dimer with an unusual carbon skeleton, ain-
10
sliadimer A from A. macrocephala, showed potent cytotoxicity
and anti-inammatory activities. Ainsliaea yunnanensis Franch.,
a kind of perennial plant of the genus Ainsliaea, is distributed
exclusively in China. In Chinese folk medicine, the whole plants
of A. yunnanensis have been used for the treatment of traumatic
11
injury and rheumatism pain. However, few studies of the
a
Department of Phytochemistry, School of Pharmacy, Second Military Medical
University, Shanghai 200433, P. R. China. E-mail: shenyunheng@hotmail.com;
wdzhangy@hotmail.com
b
Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P. R. China
c
Shanghai Institute of Pharmaceutical Industry, Shanghai 200400, P. R. China
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c7ra01986f
1
Fig. 1 Structures of compounds 1–8.
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inference. In addition, the relative conguration of the
anomeric proton of glucopyranosyl residue was determined to
Results and discussion
Ainsloside A (1) was obtained as colourless oil. Its molecular be a-oriented based on the small coupling constant (J ¼ 3.5 Hz)
15
formula was determined to be C37
positive HR-ESI-MS (m/z 785.3942 [M + Na] , calcd 785.3936), fructose was assigned to be b-orientation through the NOESY
62
H O
16 as deduced from between H-1 and H-2, while the relative conguration of
+
0
0
0
indicating seven degrees of unsaturation. The IR spectrum of 1 correlations of H-3 with H-1 and H-5 (Fig. 2). This conclusion
1
3
showed characteristic absorption bands of hydroxyl group (3471 also can be conrmed by comparing their C NMR data with
cm ) and carbonyl group (1743 cm ). The C and DEPT NMR those of a-glucose and b-fructose previously reported in litera-
ꢀ
1
ꢀ1
13
16
spectra (Table 1) displayed 37 carbon signals, in which 12 tures. Moreover, compound 1 was submitted alkaline hydro-
oxygenated carbon resonances were observed in the region 60 < lysis, and the hydrolysate was extracted with CHCl to afford
d < 105 ppm (d 105.5, 90.1, 78.1, 77.5, 77.0, 72.4, 70.6, 70.0,
8.8, 64.0, 63.6, 62.1), implying the presence of a disaccharide
residue. Characteristic signals from the NMR spectra, including
an anomeric proton at d
5.64 (d, J ¼ 3.5 Hz) and the corre-
sponding anomeric carbon at d 90.1, a hydroxymethyl at d
2.1, an hemiketal anomeric carbon at dC 105.5, and two
hydroxymethyls at dC 63.6 and 64.0, exhibited quite similarity
3
C
6
H
C
C
6
14
with those of sucrose previously reported in literatures, indi-
cating that the disaccharide moiety should be a sucrose residue.
1
1
The H– H COSY correlations of H-1/H-2/H-3/H-4/H-5/H-6 and
0
0
0
0
H-3 /H-4 /H-5 /H-6 , in combination with the HMBC correlation
from the anomeric proton (d 5.64) of glucopyranosyl unit to the
hemiketal anomeric carbon at d 105.5, supported the above
H
1
1
Fig.
2 Key H– H COSY, HMBC, and NOESY correlations for
C
compound 1.
1
13
3
Table 1 H (500 MHz) and C (125 MHz) NMR data of anslosides A–D (1–4) (in ppm) (CDCl ) (J in Hz)
1
2
3
4
No.
d
H
d
C
d
H
d
C
d
H
d
C
d
H
d
C
1
2
3
4
5
6
5.64 d (3.5)
4.90 dd (10.0, 3.5)
4.05 m
4.93 t (10.0)
4.26 m
90.1
72.4
70.0
70.6
68.8
62.1
5.55 d (3.5)
3.66 m
3.88 t (9.5)
4.87 t (9.5)
4.19 m
92.2
72.3
72.2
70.2
68.9
62.1
5.52 d (3.5)
3.77 m
5.25 t (9.5)
5.07 t (10.0)
4.29 m
4.18 dd (12.5, 2.0)
4.15 m
3.76 m
91.9
70.9
73.4
67.3
68.8
61.8
5.57 d (3.5)
4.89 dd (10.0, 3.5)
5.40 t (10.0)
3.49 t (9.5)
4.22 m
89.7
69.8
72.2
69.4
70.9
63.0
4.20 dd (12.0, 2.0)
4.15 m
4.36 m
4.15 dd (12.0, 5.5)
0
1
3.66 m
63.6
4.32 m
63.0
64.1
3.58 q (14.5)
63.7
3
—
.68 m
0
0
0
0
2
3
4
5
—
105.5
77.0
77.5
78.1
64.0
—
104.3
77.2
77.4
78.0
64.1
105.0
78.0
77.5
78.6
64.3
—
105.3
76.8
77.3
78.4
64.2
4.34 t (7.0)
5.17 t (7.0)
4.12 m
4.30 m
4.25 d (7.0)
5.16 d (7.0)
4.08 m
4.31 m
5.20 t (6.5)
4.12 m
4.34 m
4.34 m
5.16 t (7.0)
4.08 m
4.43 m
0
6
4.31 m
4.27 dd (12.0, 3.5)
C]O
—
173.0
—
173.0
172.9
172.7
172.6
172.2
43.2
43.1
43.1
43.0
42.8
25.7
25.6
25.6
25.6
25.4
22.3 ꢁ 10
—
174.1
172.6
172.6
172.5
171.5
43.3
43.0
43.0
43.0
42.8
25.7
25.6
25.6
25.4
25.3
22.3 ꢁ 10
—
174.1
173.5
172.8
172.7
172.0
43.4
43.0
43.0
42.8
42.7
25.6
25.6
25.5
25.5
25.2
22.3 ꢁ 10
1
1
1
1
72.9
72.6
72.5
72.4
CH
CH
CH
2
2.22–2.28 m
2.06–2.14 m
0.93–0.98 m
43.2
2.21–2.26 m
2.06–2.14 m
0.92–0.98 m
2.15–2.26 m
2.02–2.13 m
0.92–0.98 m
2.16–2.26 m
2.01–2.12 m
0.90–0.99 m
4
4
4
4
3.0
3.0
2.9
2.8
25.7
2
2
2
2
5.7
5.6
5.5
5.4
3
22.3 ꢁ 10
20866 | RSC Adv., 2017, 7, 20865–20873
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0 0
H H H H
isovaleric acid. The subsequent acid hydrolysis yielded two 5.40), H-6 (d 4.36), H-6 (d 4.27), H-4 (d 5.16), H-2 (d 4.89)
sugar units, and the sugar units were identied as D-glucose and with the corresponding ester carbonyls of ve isovaleryls at dC
D-fructose through the HPLC analysis and comparing their 174.1, 173.5, 172.8, 172.7, and 172.0, respectively, the structure
0
0
corresponding specic optical rotations with those of authentic of compound 4 was deduced as 2,3,4 ,6,6 -pentakis-isovaleryl-b-
2
3
23
samples (glucose: [a]

D
+53; fructose: [a]
eld region of the H NMR spectrum, three groups of over- (Fig. 3).
lapping signals, corresponding to methylene, methine, and Ainsloside E (5) was obtained as colourless oil. The molec-
methyl protons, were observed occurring at d 2.22–2.28 (10H, ular formula of 5 was inferred as C32 15 based on the HR-
m), 2.06–2.14 (5H, m), and 0.93–0.98 (30H, m), respectively ESI-MS (m/z 701.3365 [M + Na] , calcd 701.3360), implying the
D
ꢀ149). In the high D-fructofuranosyl-a-D-glucopyranoside, and named ainsloside D
1
H
54
H O
+
1
3
(
Table 1). Combining with the C and DEPT NMR, empirical existence of six degrees of unsaturation. The IR spectrum of 5
calculations indicated the presence of a total of ten methyls, ve showed characteristic absorption bands of hydroxyl group (3467
1
3
ꢀ1
ꢀ1
13
methines and ve methylenes. Also, the C NMR spectrum of 1 cm ), and ester carbonyl group (1739 cm ). In the C and
exhibited ve ester carbonyl resonances at d 173.0, 172.9, DEPT NMR spectra of 5 (Table 2), 12 carbon resonances in the
72.6, 172.5 and 172.4. Therefore, it may be proposed that the region 60 < d < 105 ppm showed characteristic signals of sucrose
structure of 1 composed of a sucrose moiety and ve isovaleryl (d 104.9, 90.1, 78.0, 77.3, 76.8, 72.0, 70.7, 70.7, 70.6, 64.4, 63.4,
moieties. Compared with sucrose, the C chemical shis of 1 63.2), in agreement with those of previously reported for sucrose
C
1
C
14
13
0
0
14
at C-2, C-4, C-6, C-4 , and C-6 , were shied to downeld, while in literature, revealing the presence of sucrose moiety. The
their adjacent carbons were shied to upeld, implying that the
ve isovaleryl moieties may be substituted at C-2, C-4, C-6, C-4 , H-4 /H-5 /H-6 further supported the above deduction. Similar
and C-6 , respectively. This deduction was further evidenced on to compounds 1–4, in the high eld region of the H NMR
1
1
0
H– H COSY correlations of H-1/H-2/H-3/H-4/H-5/H-6 and H-3 /
0
0
0
0

0
1
the basis of analysis of the HMBC spectrum which showed spectrum of 5 (Table 2), three groups of overlapping proton
0
cross-peaks between H-4 (d
H
5.17), H-4 (d
4.90) and the corresponding ester observed occurring in the region d
173.0, 172.9, 172.6, 172.5, and 172.4, respectively (4H, m) and 0.93–0.97 (24H, m), respectively. Meanwhile, the
H
4.93), H-6 (d
H
4.20, signals for methylene, methine, and methyl signals were
0
4
.15), H-6 (d
H
4.30), H-2 (d
H
H
2.20–2.29 (8H, m), 2.06–2.12
carbonyls at d
Fig. 2). Thus, compound 1 was deduced as 2,4,4 ,6,6 -pentakis-
isovaleryl-b-D-fructofuranosyl-a-D-glucopyranoside, and given carbonyls at dC 173.9, 173.0, 172.8, and 172.6, eight methyls,
a trivial name ainsloside A. four methylenes, and four methines. The information implied
Ainslosides B–D (2–4) shared the same molecular formula that there were only four isovaleryls in the structure of 5.
16 as ainsloside A (1) by analysis of HR-ESI-MS and Therefore, it could be concluded that compound 5 was a sucrose
NMR spectroscopy, and their IR spectra suggested the presence derivative with four isovaleryl groups. The downeld shied C-
of hydroxyl group and ester carbonyl group based on charac- 2, C-6, C-4 , and C-6 and their upeld shied adjacent carbons,
teristic absorption bands. The C and DEPT NMR spectra disclosed that the four isovaleryl groups were attached to C-2, C-
Table 1) of all the compounds also showed twelve carbon 6, C-4 , and C-6 positions, respectively. In the HMBC spectrum,
C
0
0
13
(
C and DEPT NMR spectra of 5 only exhibited four ester
37 62
C H O
0
0
1
3
0 0
(
signals in the region 60 < d < 105 ppm which revealed the same the proton signals of the sucrose residue were observed the
sucrose moiety as compound 1. Deducting the sucrose moiety, correlations from H-6 (d 4.36) to the ester carbonyl at d 173.9,
H
C
1
0
0
in combination with the H NMR spectrum (Table 1), the le 25 from H-6 (d 4.31) to the ester carbonyl at d 173.0, from H-4
H
C
carbons were assigned as ten methyls, ve methylenes, ve
methines, and ve ester carbonyls, which belonged to ve iso-
valeryls. Comprehensive analyses of 1D and 2D NMR spectra of
ainslosides B–D (2–4) suggested that these compounds were
isovaleryl substituted derivatives of sucrose similar to
compound 1 with different substitution pattern. The linkage
positions of the isovaleryls were assigned on the basis of
interpretation of the HMBC spectrum. In the HMBC spectrum
0
of compound 2, it could be observed the cross-peaks of H-4 (d
H
0
0
5
.16), H-4 (d
H
4.87), H-6 (d
H
4.15), H-6 (d
H
4.31), H-1 (d
H
4.32)
with the corresponding ester carbonyls of ve isovaleryls at d
C
1
73.0, 172.9, 172.7, 172.6, and 172.2, respectively. So,
0
0
0
compound 2 was deduced as 1 ,4,4 ,6,6 -pentakis-isovaleryl-b-D-
fructofuranosyl-a-D-glucopyranoside, and named ainsloside B.
In the same way, compound 3 was determined as 3,3 ,4,4 ,6-
0
0
pentakis-isovaleryl-b-D-fructofuranosyl-a-D-glucopyranoside by
0
the long-range HMBC correlations between H-3 (d
H
5.25), H-3
0
(d
H
4.31), H-6 (d
H
4.18), H-4 (d
H
5.20), H-4 (d
H
5.07) and the
174.1,
72.6, 172.6, 172.5, and 171.5, respectively, and named ainslo-
corresponding ester carbonyls of ve isovaleryls at d
1
C
1
1
side C. Meanwhile, by the key HMBC correlations of H-3 (d
H
Fig. 3 Key H– H COSY, and HMBC correlations for compounds 2–4.
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1
13
Table 2 H (500 MHz) and C (125 MHz) NMR data of anslosides E-H (in ppm) (CDCl
3
) (J in Hz)
5
6
7
8
No.
d
H
d
C
d
H
d
C
d
H
d
C
d
H
d
C
1
2
3
4
5
6
5.53 d (3.5)
4.78 dd (10.0, 3.5)
4.02 t (9.5)
3.40 t (9.5)
4.08 m
90.1
72.0
70.6
70.7
70.7
63.2
5.54 d (3.5)
4.87 dd (10.0, 4.0)
4.04 dd (10.0, 4.0)
4.94 t (9.5)
4.21 m
4.21 m
89.5
72.4
70.0
70.7
68.7
62.1
5.44 d (3.5)
3.69 dd (9.5, 3.5)
3.94 t (9.5)
4.84 t (10.0)
4.20 m
91.7
72.2
71.8
70.1
69.2
62.2
5.67 d (3.5)
4.87 dd (10.5, 4.0)
4.05 t (10.0)
4.95 t (10.0)
4.26 m
90.1
72.6
69.8
70.4
68.9
61.7
4.36 m
4.16 m
4.25 m
4
.13 dd (13.0, 6.0)
4.14 dd (12.5, 5.0)
3.61 q (12.5)
0
1
3.58 q (12.0)
63.4
3.62 q (7.0)
64.2
3.76 d (12.5)
63.9
64.0
3
—
.61 d (13.0)
0
0
0
0
2
3
4
5
—
104.9
78.0
77.3
76.8
64.4
—
104.5
78.3
75.8
79.6
64.1
104.7
78.7
78.0
78.5
64.7
—
105.5
76.9
76.7
81.5
61.4
4.08 m
5.16 t (7.0)
4.31 m
4.31 m
4.18 m
4.02 m
3.94 m
4.31 m
4.25 d (7.0)
5.17 t (7.0)
4.12 m
4.36 d (7.0)
5.26 t (6.5)
3.97 m
0
6
4.34 dd (12.0, 5.0)
3.84 dd (13.0, 3.0)
3.67 dd (13.0, 4.0)
C]O
—
173.9
—
173.3
172.9
172.8
172.4
43.2
43.0
43.0
42.9
25.7
25.7
25.6
25.5
22.3 ꢁ 8
—
173.1
172.8
172.7
172.6
43.2
43.1
43.0
42.8
25.7
25.7
25.6
25.4
22.3 ꢁ 8
—
173.1
172.9
172.8
172.7
43.2
43.1
42.9
42.8
25.7
25.7
25.7
25.4
22.3 ꢁ 8
1
1
1
73.0
72.8
72.6
CH
2
ꢁ 4
2.20–2.29 m
2.06–2.12 m
0.93–0.97 m
43.1
2.23–2.29 m
2.06–2.14 m
0.94–0.98m
2.18–2.27 m
2.03–2.14 m
0.92–0.98 m
2.23–2.30 m
2.06–2.13 m
0.93–0.97 m
4
4
4
3.0
2.9
2.9
CH ꢁ 4
25.7
2
2
2
5.7
5.6
5.4
CH
3
ꢁ 8
22.3 ꢁ 8
0 0
d 5.16) to the ester carbonyl at d 172.8, and from H-2 (d 4.78) as 2,4 ,6,6 -tetrakis-isovaleryl-b-D-fructofuranosyl-a-D-glucopyr-
H C H
(
C
to ester carbonyl at d 172.6, revealing that four isovaleroxyls anoside, and named ainsloside E.
0
0
were substituted at C-2, C-4 , C-6, and C-6 positions of sucrose
Ainslosides F–H (6–8) shared the same molecular formula
32 54
C H O15 as that of compound 5 due to analysis of their HR-
residue, respectively (Fig. 4). Thus, compound 5 were deduced
ESI-MS spectra. The IR spectra indicated the presence of
hydroxyl and ester carbonyl based on their characteristic
1
13
absorption bands. The H and C NMR spectra (Table 2) of 6–8
exhibited the same functionalities as ainsloside E (5), including
1
1
2 carbon resonances for a sucrose in the region 60 < d <
05 ppm and four ester carbonyls, eight methyls, four methy-
1
3
lenes, and four methines in C NMR and DEPT spectra,
together with three groups of overlapping proton signals for
methylene, methine, and methyl in the H NMR spectrum
1
(Table 1). The above evidences indicated that compounds 6–8
were four isovaleryls substituted sucrose ester similar to
compound 5 with different substitution pattern.
The linkage positions of four isovaleryls were determined by
HMBC experiment. In the HMBC spectrum of 6, the sucrose
0
protons at d
H
4.94 (H-4), 4.31 (H-6 ), 4.21 (H-6), 4.87 (H-2) were
correlated with the corresponding ester carbonyls of four iso-
valeryls at d
C
173.3, 172.9, 172.8, and 172.4, respectively,
0
implying that four isovaleroyoxyls were linked to C-4, C-6 , C-6,
and C-2 positions of sucrose moiety, respectively. Therefore, the
structure of 6 was deduced as 2,4,6,6 -tetrakis-isovaleryl-b-D-
0
fructofuranosyl-a-D-glucopyranoside, and named ainsloside F.
1
1
H
Similarly, the HMBC correlations from H-4 (d 4.84) to the ester
Fig.
4 Key H– H COSY, HMBC, and NOESY correlations for
compound 5–8.
carbonyl at d 173.1, from H-6 (d 4.16) to the ester carbonyl at
C
H
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0
d
C
172.8, from H-4 (d
H
5.17) to the ester carbonyl at d
C
172.7, developing anticancer drugs and most clinically used anti-
0
25
and from H-6 (d 4.34) to the ester carbonyl at d 172.6, cancer medicines can induce cell apoptosis. Considering the
H
C
0
0
proposed the structure of 7 as 4,4 ,6,6 -tetrakis-isovaleryl-b-D- strong inhibition of compound 2 against the proliferation of
fructofuranosyl-a-D-glucopyranoside, given a trivial name ain- A549 cells, compound 2 were further studied for its cell cycle
sloside G. The structure of 8 was identied as 2,4,4 ,6-tetrakis- arresting and apoptosis induction in A549 cells.
0
isovaleryl-b-D-fructofuranosyl-a-D-glucopyranoside on the basis
To determine if compound 2 can arrest cell-cycle progres-
0
of the HMBC correlations between H-4 (d
H
5.26) with the ester sion, A549 cells were exposed to various concentrations of
carbonyl at d
carbonyl at d
C
173.1, between H-2 (d
172.9, between H-4 (d
H
4.87) with the ester compound 2 for 24 hours, and the distribution of cells in the
H
4.95) with the ester cycle was determined by ow-cytometric analysis. As shown in
C
carbonyl at dC 172.8, and between H-6 (dH 4.25) with the ester Fig. 5, A549 cells treated with compound 2 exhibited a dose
carbonyl at 172.7 (Fig. 4). dependent increase in the proportion of cells in the G /G phase
0
1
Sucrose esters are of interest in such applications as fat and a decrease of cells in the G /M phase compared to the
2
substitutes, bleaching boosters, and emulsiers in the food and untreated control. This indicated that the growth inhibition
1
7,18
cosmetic industries.
Meanwhile, they also possessed bioac- induced by 2 in a dose-dependent manner occurs through the
/G phase.
tivities such as antimicrobial, antitumor, and insecticidal arrest of A549 cells in G
activities. Their properties depend strongly on their compo-
sitions in terms of degree of substitution and regiochemistry.
0
1
19
In addition, we examined compound 2-induced apoptosis in
20
A549 cells by using annexin V-FITC/PI double staining method.
Previous studies showed the hydroxyl group at OH-2 is very The X- and Y-axis represented annexin V-FITC and PI staining,
21
reactive in water just as it is in organic solvents, while the ester respectively. The right upper and lower right quadrant of the
22
at OH-6 is much more stable in synthesis. In our studies, the gure represented late and early stages of cell apoptosis,
content of ainsloside A is much more than others', which respectively. As illustrated in Fig. 6, compound 2 induced
0
0
suggests that the esters at OH-2, OH-4, OH-4 , OH-6 and OH-6
might be more stable or important in plant, at least in this
species.
All isolates were evaluated for in vitro cytotoxic activity
against four human tumour cell lines (A549, HCT116, MDA-MB-
231, and BEL7404), using MTT assay with doxorubicin as posi-
tive control (Table 3). Among these isolates, compounds 1, 2,
and 5 showed strong inhibitory activity against A549 cell line
with IC50 values of 9.74, 3.30, and 10.75 mM, compounds 1, 2, 4,
and 5 exhibited weak inhibitory activity against HCT116 cell line
with IC50 values of 10.77, 10.25, 16.56, and 16.10 mM, In addi-
tion, compounds 1, 2, 4, and 5 also showed weak inhibitory
activity against BEL7404 cell line with IC50 values of 11.45,
11.23, 15.95, and 12.77 mM. Of all the isolates tested, compound
2
displayed the strongest inhibitory activity against the prolif-
eration of A549 cell line in a dose and time dependent manner
Fig. 5 Cell cycle assay of A549 cells exposed to different concen-
trations of compound 2 were examined by flow cytometry (A). The
effect of compound 2 on the cell cycle and the expression of cell cycle
(Fig. S84, see ESI†).
Cell cycle dysregulation contributes to the aberrant cell
2
3,24
proliferation and development of cancer.
Moreover, target- regulators in A549 cells (B). The results are represented as the mean ꢂ
ing apoptosis is considered as one of the major strategies for SEM for three independent experiments with similar results. *P < 0.05
vs. the control.
Table 3 In vitro cytotoxic activities of compounds 1–8 (mean ꢂ SD, n ¼ 3)
IC50 (mM)
No.
A549
HCT116
MDA-MB-231
BEL7404
1
2
3
4
5
6
7
8
9.74 ꢂ 0.71
3.30 ꢂ 0.22
>100
19.93 ꢂ 0.93
10.75 ꢂ 1.07
60.06 ꢂ 2.71
46.19 ꢂ 3.21
66.00 ꢂ 1.17
0.034 ꢂ 0.002
10.77 ꢂ 0.87
10.25 ꢂ 0.53
68.51 ꢂ 0.86
16.56 ꢂ 0.67
16.10 ꢂ 1.12
23.11 ꢂ 1.73
23.65 ꢂ 2.01
54.82 ꢂ 3.15
0.045 ꢂ 0.003
39.10 ꢂ 1.87
19.59 ꢂ 0.69
>100
38.23 ꢂ 1.23
25.54 ꢂ 2.13
95.19 ꢂ 3.57
>100
11.45 ꢂ 0.57
11.23 ꢂ 0.61
95.48 ꢂ 4.47
15.95 ꢂ 1.31
12.77 ꢂ 0.78
29.86 ꢂ 1.75
53.61 ꢂ 1.31
80.03 ꢂ 2.16
0.12 ꢂ 0.01
>100
0.11 ꢂ 0.01
Doxorubicin
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Fig. 6 Compound 2-induced apoptosis in A549 cells. (A) Apoptosis
was evaluated using an annexin V-FITC apoptosis detection kit and
flow cytometry. The representative pictures are from A549 cells
incubated with different concentrations. (B) Compound 2 induced
apoptosis in A549 cells in a dose dependent manner. The data are
expressed as the means ꢂ SEM of three independent experiments with
the similar results. *P < 0.05; **P < 0.01 vs. the control.
Fig. 7 The MMP of A549 cells treated with compound 2 (A). The loss of
the MMP in A549 cells following treatment of compound 2 (B). The
data are expressed as the means ꢂ SEM for three independent
experiments with the similar results. *P < 0.05 vs. the control.
a dose-dependent increase in A549 apoptosis when treated with
a Shimadzu UV-2550 spectrometer. IR spectra were recorded on
a Bruker FTIR Vector 22 spectrometer using KBr pellets.
Column chromatography (CC) was performed on silica gel (100–
10 mM of 2 for 24 h. These results conrmed that the cytotoxicity
of compound 2 to A549 cells is associated with arrest of cell-
cycle progression and induction of apoptosis.
200, 200–300 mesh, Yantai, China), Sephadex LH-20 (GE
Depolarization of mitochondrial membrane potential
Healthcare Bio-Sciences AB, Sweden) and YMC-Gel ODS-A (50
mm; YMC, Milford, MA). Preparative TLC (0.4–0.5 mm) was
conducted with glass plates pre-coated with silica gel GF254
26,27
(MMP) is one of the characteristic event of apoptosis.
To
investigate whether compound 2-induced apoptosis was asso-
ciated with mitochondrial dysfunction, we analyzed MMP
changes of the A549 cells by staining with mitochondria
sensitive Rhodamine 123, and the results showed the MMP of
the A549 cells was decreased dose-dependently aer treatment
with different concentration of compound 2 for 24 h. The
experiment indicated that compound 2-induced A549 cells
apoptosis may be associated with the disruption of MMP
(Yantai, China). A semi-preparative column (Agilent ZORBAX
SB-C18, 5 mm, 9.4 ꢁ 250 mm) was used for HPLC (Shimadzu LC-
2010A HT), and an analytical column (YMC-Pack NH
2
, 5 mm,
1
2 nm, 4.6 ꢁ 250 mm) was used for analysis of sugar units on
a HPLC (Agilent 1100) instrument with an ELSD detector
SEDEX 85).
(
(
Fig. 7).
Previous studies showed that ROS (reactive oxygen species)
can induce apoptotic cell death in various types of cancer cell
28–30
aer treatment with anticancer drugs.
So, the association
between compound 2-induced apoptosis with elevated levels of
ROS in A549 cells was studied. When A549 cells were treated
with compound 2 for 24 h, the DCFH-DA uorescence was
shied to a higher intensity, indicating an increase in ROS
levels compared to the untreated control (Fig. 8). These data
suggested that the level of intracellular ROS may be involved in
compound 2-induced apoptosis in A549 cells.
Experimental
General experimental procedures
1
5
D and 2D NMR spectra were determined with a Bruker Avance-
00 spectrometer in CDCl with TMS as internal standard. ESI-
3
Fig. 8 Effects of compound 2 on intracellular ROS accumulation in
A549 cells (A). The results are presented as the percentage distribu-
tions for specific phases (B). The data are expressed as the means ꢂ
SEM for three independent experiments with the similar results. *P <
MS were acquired on an Agilent LC/MSD Trap XCT mass spec-
trometer, whereas HR-ESI-MS were measured using a Waters Q-
TOF micro mass spectrometer. Optical rotations were obtained
with a JASCO P-2000 polarimeter. UV spectra were obtained on 0.05 vs. the control.
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ꢀ
1
1
13
HPLC grade acetonitrile was purchased from Merck (Darm- 1120, 1014 cm ; H and C NMR data, see Table 1; HR-ESI-MS
+
stadt, Germany). Water was puried by a Milli-Q system from (positive) m/z 785.3939 [M + Na] (calcd 785.3936).
Millipore (Bedford, MA, USA). All other reagents were of Ainsloside B (1 ,4,4 ,6,6 -pentakis-isovaleryl-b-D-fructofur-
analytical grade and obtained from conventional commercial anosyl-a-D-glucopyranoside, 2). C H O ; colorless oil;
0 0 0
3
7
62 16
2
0
sources.
Human cell lines A549 (lung adenocarcinoma cells), HCT116
colon carcinoma cells), MDA-MB-231 (breast cancer cells) and 1095, 1011 cm ; H and C NMR data, see Table 1; HR-ESI-MS
[a]
n
D
+14 (c 0.077, MeOH); UV (MeOH) lmax 200.5 nm; IR (KBr)
max 3446, 2960, 2929, 2871, 1741, 1468, 1371, 1296, 1252, 1188,
ꢀ1
1
13
(
+
BEL7404 (hepatoma cancer cells) were obtained from the Cell (positive) m/z 785.3952 [M + Na] (calcd 785.3936).
0 0
Bank of Shanghai Institute of Biochemistry & Cell Biology,
Ainsloside
C
(3,3 ,4,4 ,6-pentakis-isovaleryl-b-D-fructofur-
Shanghai Institute for Biological Sciences, Chinese Academy of anosyl-a-D-glucopyranoside, 3). C H O ; colorless oil;
3
7
62 16
2
0
Sciences.
[a] +54 (c 0.112, MeOH); UV (MeOH) lmax 198.5 nm; IR (KBr)
D
n
3485, 3259, 2960, 2873, 1741, 1468, 1369, 1296, 1188, 1115,
max
ꢀ1
1
13
1
005 cm
;
H and C NMR data, see Table 1; HR-ESI-MS
Plant material
+
(positive) m/z 785.3946 [M + Na] (calcd 785.3936).
The whole plants of Ainsliaea yunnanensis were collected in
Baoshan city, Yunnan province, in August 2013 and authenti-
cated by Prof. Bao-Kang Huang in the Department of Pharma-
0
0
Ainsloside
D
(2,3,4 ,6,6 -pentakis-isovaleryl-b-D-fructofur-
colorless oil;
anosyl-a-D-glucopyranoside, 4).
37 62 16
C H O ;
2
0
[a]
D
+59 (c 0.054, MeOH); UV (MeOH) lmax 202.0 nm; IR (KBr)
cognosy, Second Military Medical University.
A voucher
n
3477, 2960, 2933, 2873, 1741, 1468, 1369, 1296, 1254, 1188,
max
specimen (20130813001) was deposited at the Herbarium of
School of Pharmacy, Second Military Medical University,
Shanghai, the People's Republic of China.
ꢀ1
1
13
1
120, 1005 cm ; H and C NMR data, see Table 1; HR-ESI-MS
+
(positive) m/z 785.3942 [M + Na] (calcd 785.3936).
0
0
Ainsloside
E
(2,4 ,6,6 - tetrakis-isovaleryl-b-D-fructofur-
colorless oil;
+39 (c 0.091, MeOH); UV (MeOH) lmax 198.0 nm; IR (KBr)
max 3467, 2960, 2933, 2873, 1739, 1468, 1369, 1296, 1255, 1190,
anosyl-a-D-glucopyranoside, 5).
32 54 15
C H O ;
Extraction and isolation
20
[a]
D
The air-dried whole plants of A. yunnanensis (15.0 kg) were
reuxed with 95% ethanol and 80% ethanol for each twice. The 1120, 1063, 1005 cm ; H and C NMR data, see Table 2; HR-
combined extract was chromatographed by a silica gel (100–200 ESI-MS (positive) m/z 701.3365 [M + Na] (calcd 701.3360).
n
ꢀ1
1
13
+
0
mesh) column eluted successively with petroleum ether (PE),
CHCl
Ainsloside F (2,4,6,6 -tetrakis-isovaleryl-b-D-fructofuranosyl-
2
0
, EtOAc, and n-BuOH, respectively. The EtOAc extract (100 a-D-glucopyranoside, 6). C32H O15; colorless oil; [a] +30 (c
54 D
3
g) was subjected to silica gel column (100–200 mesh, 200 g) 0.073, MeOH); UV (MeOH) lmax 199.0 nm; IR (KBr) nmax 3460,
chromatography (CC) eluted with gradient CHCl /MeOH 2960, 2931, 2873, 1741, 1468, 1371, 1296, 1190, 1099, 1012
3
ꢀ
1
1
13
(
100 : 1 to 1 : 1) to give 9 fractions (Fr.1–Fr.9) based on TLC cm ; H and C NMR data, see Table 2; HR-ESI-MS (positive)
analysis. The Fr.1 (16.6 g) was subjected to column chroma- m/z 701.3362 [M + Na] (calcd 701.3360).
tography on reverse-phase silica gel (ODS) eluted successively
with gradient MeOH–H
O to afford 8 fractions (Fr.1-1 to Fr.1-8). a-D-glucopyranoside, 7). C32
Subfraction Fr.1-2 (0.4 g) was subjected to semi-preparative 0.089, MeOH); UV (MeOH) lmax 198.5 nm; IR (KBr) nmax 3408,
HPLC (MeOH/H
O, 70 : 30) to give ainsloside B (2, 26 mg) and 2960, 2933, 2873, 1743, 1468, 1369, 1296, 1254, 1188, 1093, 1003
+
0 0
Ainsloside G (4,4 ,6,6 -tetrakis-isovaleryl-b-D-fructofuranosyl-
2
0
H O15; colorless oil; [a] +28 (c
54 D
2
2
ꢀ
1
1
13
ainsloside C (3, 52 mg). Fr.1-3 (3.5 g) was subjected to a silica gel cm ; H and C NMR data, see Table 2; HR-ESI-MS (positive)
+
CC (f 2.5 ꢁ 30 cm; 200–300 mesh, 50 g) eluted with gradient PE/ m/z 701.3371 [M + Na] (calcd 701.3360).
0
EtOAc (20 : 1 to 1 : 1) to give ainsloside A (1, 610 mg). From
Ainsloside H (2,4,4 ,6-tetrakis-isovaleryl-b-D-fructofuranosyl-
2
0
54 D
subfraction Fr.1-3-4 (0.9 g), ainsloside D (4, 28 mg) and ainslo- a-D-glucopyranoside, 8). C32H O15; colorless oil; [a] +45 (c
side E (5, 21 mg) were isolated aer CC over Sephadex LH-20 (f 0.100, MeOH); UV (MeOH) lmax 202.9 nm; IR (KBr) nmax 3456,
.5 ꢁ 150 cm; MeOH) followed by semi-preparative HPLC 2960, 2929, 2873, 1743, 1468, 1369, 1296, 1254, 1188, 1101,
2
ꢀ1
1
13
(MeOH/H
2
O, 65 : 35). Fr.1-5 (3.3 g) was chromatographed on 1065, 1012 cm ; H and C NMR data, see Table 2; HR-ESI-MS
+
silica gel (f 2.5 ꢁ 30 cm; 50 g, 200–300 mesh) eluted with (positive) m/z 701.3357 [M + Na] (calcd 701.3360).
gradient PE/EtOAc (20 : 1 to 1 : 1) followed by semi-preparative
HPLC (MeOH/H O, 65 : 35) to give ainsloside F (6, 20 mg). Fr.1-7
2
Sugar analysis
(
3
3.0 g) was chromatographed on silica gel column (f 2.5 ꢁ
ꢃ
0 cm; 40 g, 200–300 mesh) eluted with gradient PE/EtOAc A solution of 1 (35 mg) in 5% KOH (5 mL) was heated at 95 C for
20 : 1 to 1 : 1) to give four subfractions (Fr.1-7a–Fr.1-7d). Fr.1- 3 hours. The reaction mixture was acidied to PH 4 with 1 M
b (0.2 g) was subjected to semi-preparative HPLC (MeOH/ HCl, then extracted with CHCl
(3 ꢁ 10 mL) and n-BuOH(3 ꢁ 10
O, 60 : 40) to give ainsloside G (7, 23.0 mg). Fr.1-7d (0.3 g) was mL) each three times. The CHCl layer was concentrated to
(
7
3
H
2
3
subjected to semi-preparative HPLC (MeOH/H
2
O, 60 : 40) to afford isovaleric acid, which was elucidated by extensive anal-
give ainsloside H (8, 25 mg).
ysis of 1D NMR spectra. The n-BuOH layer was hydrolyzed in
0
0
ꢃ
Ainsloside
A
(2,4,4 ,6,6 -pentakis-isovaleryl-b-D-fructofur- 2 M HCl–MeOH (1 : 1, v/v, 10 mL) at 90 C for 2 h by heating in
anosyl-a-D-glucopyranoside, 1). C H O ; colorless oil; water bath. The aqueous phase was extracted with n-BuOH (3 ꢁ
3
7
62 16
2
0
[
n
a]
D
+25 (c 0.068, MeOH); UV (MeOH) lmax 197.5 nm; IR (KBr) 10 mL) aer acid hydrolysis and concentrated to yield a col-
O and directly
max 3471, 2960, 2927, 2873, 1743, 1468, 1369, 1296, 1254, 1188, ourless solid. The residue was dissolved in H
2
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analyzed by HPLC with an ELSD detector with reference stan- Mitochondrial membrane potential measurement
dards (MeCN–H O, 75/25): D-glucose eluted at 16.308 min, and
D-fructose at 13.116 min. Each of these eluates was individually
2
The mitochondrial membrane potential (MMP) was measured
by ow cytometry using Rhodamine 123 (Rh-123) as uoro-
collected, concentrated, and dissolved in H O. The obtained
2
chrome. A549 cells were treated with compound 2 for 24 h, and
2
3
sugar residues were identied as D-glucose, [a]
D
+53 (c 0.113,
2
O), through
ꢀ1
then incubated with 5 mg mL of Rh-123 for 30 min at room
2
3
H
2
O), and D-fructose, [a]
D
ꢀ149 (c 0.118, H
temperature in the dark. Aer centrifuged and washed twice
with PBS, the cells were resuspended in 1000 mL of PBS and
analyzed using ow cytometry with excitation and emission
wavelength of 488 and 530 nm, respectively.
comparisons of their specic rotations with those of the corre-
sponding authentic samples.
Isovaleric acid
Intracellular ROS production measurement
1
H NMR (in CDCl , 500 MHz) d 2.23 (2H, d, J ¼ 7.5 Hz), 2.12 (1H,
3
1
3
ROS levels were detected using a ow cytometer and a micro-
plate spectrophotometer (Molecular Devices, Sunnyvale, CA,
USA). Aer treatment, A549 cells were harvested and washed
with PBS and suspended in DMEM containing 10 mM 5(6)-car-
m), 0.99 (6H, d, J ¼ 7.0 Hz); C NMR d 178.8 (–COOH), 43.0
2 3 3
(CH ), 25.5 (CH), 23.3 (CH ), 22.3 (CH ).
0
0
Cytotoxic activity assays
boxy-2 , 7 -dichlorodihydrouorescein diacetate (carboxy-
ꢃ
H
2
DCFDA; Invitrogen) at 37 C for 20 min. The cells were
The viability of cells was determined by MTT assay to detect
functional mitochondria in living cells (Mosmann, 1983) using
doxorubicin as positive control. Human cell lines A549 (lung
adenocarcinoma cells), HCT116 (colon carcinoma cells), MDA-
washed again, and ow cytometry analysis was performed.
Statistical analysis
MB-231 (breast cancer cells) and BEL7404 (hepatoma cancer Data were analyzed using GraphPad Prism soware (GraphPad
ꢃ
cells) were incubated at 37 C in a Dulbecco's minimum soware Inc., San Diego, CA, USA). The comparison between
essential medium (DMEM) containing 10% FBS and penicillin– two groups was analyzed by unpaired Student t-test, and
4
streptomycin solution. About 1 ꢁ 10 cells per wells were seeded multiple comparisons were compared by one-way ANOVA
into 96-well microtiter plates. Aer twenty-four hours post- analysis of variance followed by Tukey post hoc test. Statistical
seeding, cells were treated with vehicle control or various signicance was determined as P < 0.05.
concentrations of samples for 48 h. 20 mL of MTT solution (5 mg
ꢀ
1
mL , Sigma Aldrich, St. Louis, MO, USA) was added to each
Conclusions
ꢃ
well and the tumor cells were incubated at 37 C in a humidied
atmosphere of 5% CO
2
air for 4 h. Upon removal of MTT/ Isovaleryl sucrose esters are a kind of rare second metabolites,
31–33
medium, 150 mL of DMSO was added to each well and the which have been sporadically isolated from some plants.
In
plate was agitated at oscillator for 5 min to dissolve the MTT- current study, eight isovaleryl sucrose esters were obtained from
formazan. The assay plate was read at a wavelength of 570 nm the whole plants of A. yunnanensis. It is the rst report of poly
using a microplate reader (Bio-Tek, Winooski, Vermont, USA).
isovaleryl sucrose derivatives from Ainsliaea species. Interest-
ingly, ainsloside B (2), a poly isovaleryl sucrose derivative with
0
0
0
1
,4,4 ,6,6 -pentakis-isovaleryl substitution, exhibited potent
Cell cycle arresting assay
inhibition against the proliferation of A549 with IC50 value of
A549 cells were incubated with compound 2 for 24 h, and then 3.3 mM, while other compounds showed weak inhibition or
trypsinized, washed with PBS, and xed with 70% of cold being inactive. It could be speculated that the quantity and
ꢃ
absolute ethanol at 4 C overnight. Aer washed twice with PBS, substitution pattern of isovaleryl may affect cytotoxicities of
the cells were incubated with 100 mL of RNase A for 30 min and these compounds. Meanwhile, ainsloside B (2) can arrest cell
then stained with 1 mL PI staining solution (3.8 mM sodium cycle of A549 at G /G phase and induce A549 cell apoptosis in
0
1
ꢀ
1
citrate, 50 mg mL PI in PBS, Beyotime) for 10 min at room dose-dependent manner. Further investigation indicated that
temperature in the dark. The DNA content of cells and cell cycle the apoptosis-inducing effect of compound 2 may be involved in
distribution were analyzed by ow cytometry (BD FACS Calibur). reduction of mitochondrial membrane potential (MMP) and
increase of reactive oxygen species (ROS) levels in A549 cells.
Therefore, polyisovaleryl sucrose esters could be a type of
important bioactive constituents of Ainsliaea plants.
Cell apoptosis assay
The apoptotic cells were quantied by using the annexin V-FITC
and PI double staining kit (DOJINDO, Japan). Briey, A549 cells
were incubated with compound 2 for 24 h. Then the cells were
Acknowledgements
centrifuged, and washed with ice-cold PBS. The cultures were The work was supported by Professor of Chang Jiang Scholars
suspended in 400 mL of binding buffer containing 5 mL annexin Program, NSFC (81573318, 81230090, 81520108030, 81373301,
ꢀ
1
V-FITC (10 mg mL ) for 15 min in the dark, and then incubated 1302658), Shanghai Engineering Research Center for the Prep-
ꢀ1
with 10 mL of PI (20 mg mL ) for 5 min. The cells were imme- aration of Bioactive Natural Products (10DZ2251300), the
diately analyzed by ow cytometry (BD FACSC Calibur).
Scientic Foundation of Shanghai China (12401900801,
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Paper
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1
0
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