Chemical components from the leaves of Ardisia insularis and their cytotoxic activity

Article abstract of DOI:10.1007/s12272-015-0591-x

One new oleanane triterpene glycoside, ardinsuloside (1), and twelve known compounds, demethoxybergenin (2), norbergenin (3), bergenin (4), 4-O-galloylbergenin (5), quercitrin (6), myricitrin (7), myricetin 3-O-(3′′-O-galloyl)-α-l-rhamnopyranoside (8), desmanthine-2 (9), epicatechin 3-O-galloyl ester (10), 3′-methoxyepicatechin 3-O-galloyl ester (11), gallic acid (12), and methyl galloate (13) were isolated from the leaves of Ardisia insularis. Their structures were established on the basis of spectral and chemical evidence, which were in agreement with those reported in literature. The cytotoxic activities of these compounds were evaluated on three cancer cell lines namely A-549 (human lung cancer), HT-29 (Human colon adenocarcinoma), and OVCAR (human ovarian carcinoma). The results revealed that compound 1 inhibited A-549, HT-29, and OVCAR cell lines with IC₅₀ values of 8.5 ± 1.2, 16.4 ± 3.1, and 13.6 ± 2.4 μM, respectively. The remaining compound showed weak cytotoxic activity. This result indicated that compound 1 could be useful in the treatment of cancer disease.

Full text of DOI:10.1007/s12272-015-0591-x

Arch. Pharm. Res.
DOI 10.1007/s12272-015-0591-x
RESEARCH ARTICLE
Chemical components from the leaves of Ardisia insularis
and their cytotoxic activity
Nguyen Thi Hong Van1 Trinh Anh Vien1,4 Phan Van Kiem3 Chau Van Minh3
•
•
•
•
Nguyen Xuan Nhiem2,3 Pham Quoc Long1 Luu Tuan Anh1 Nanyoung Kim2
SeonJu Park2 Seung Hyun Kim2
•
•
•
•
•
Received: 30 September 2014 / Accepted: 17 March 2015
Ó The Pharmaceutical Society of Korea 2015
Abstract One new oleanane triterpene glycoside, ardinsu-
loside (1), and twelve known compounds, demethoxyber-
genin (2), norbergenin (3), bergenin (4), 4-O-galloylbergenin
(5), quercitrin (6), myricitrin (7), myricetin 3-O-(300-O-gal-
loyl)-a-L-rhamnopyranoside (8), desmanthine-2 (9), epi-
catechin 3-O-galloyl ester (10), 30-methoxyepicatechin 3-O-
galloyl ester (11), gallic acid (12), and methyl galloate (13)
were isolated from the leaves of Ardisia insularis. Their
structures were established on the basis of spectral and che-
mical evidence, which were in agreement with those reported
inliterature. The cytotoxic activities of thesecompounds were
evaluated on three cancer cell lines namely A-549 (human
lung cancer), HT-29 (Human colon adenocarcinoma), and
OVCAR (human ovarian carcinoma). The results revealed
that compound 1 inhibited A-549, HT-29, and OVCAR cell
lines with IC50 values of 8.5 1.2, 16.4 3.1, and
13.6 2.4 lM, respectively. The remaining compound
showed weak cytotoxic activity. This result indicated that
compound1 couldbeusefulinthetreatment ofcancerdisease.
Keywords Ardisia insularis ꢀ Myrsinaceae ꢀ
Ardinsuloside ꢀ Cytotoxic activity
Introduction
Traditional Chinese medicines have attracted increasing
interest as potential sources of novel drugs with a wide
range of biological and pharmacological activities, in-
cluding anticancer activities (Vickers 2002). In 2014, there
will be an estimated 1,665,540 new cancer cases diagnosed
and 585,720 cancer deaths in the United States. Cancer
remains the second most common cause of death in the US,
accounting for nearly 1 of every 4 death (Cancer Facts &
Figs. 2014). More than half of currently available drugs are
natural compounds or are related to them, and in the case of
cancer this proportion surpasses 60 % (Newman and Cragg
2007). To find new medications, more attention has been
focused on natural compounds in plants, marine organisms,
and microorganisms.
Electronic supplementary material The online version of this
article (doi:10.1007/s12272-015-0591-x) contains supplementary
material, which is available to authorized users.
The genus Ardisia is the largest in the family of
Myrsinaceae, and approximately 500 species of evergreen
shrubs and trees are found throughout the subtropical and
tropical regions of the world. The leaves of Ardisia insu-
laris Mez. have been used in the folk medicine to treat
rheumatism, diarrhea, and liver diseases (Chi 2012). A
number of phytochemical studies of genus Ardisia has re-
ported the isolation of oleanane saponins (Mu et al. 2012b),
bergenin derivatives (Mu et al. 2012a), and quinones
& Seung Hyun Kim
kimsh11@yonsei.ac.kr
1
Institute of Natural Products Chemistry, Vietnam Academy
of Science and Technology (VAST), 18 Hoang Quoc Viet,
Caugiay, Hanoi, Vietnam
2
Yonsei Institute of Pharmaceutical Sciences, College of
Pharmacy, Yonsei University, Incheon 406-840, Korea
3
Institute of Marine Biochemistry, Vietnam Academy of
Science and Technology (VAST), 18 Hoang Quoc Viet,
Caugiay, Hanoi, Vietnam
´
(Kobayashi and de Mejıa 2005). However, chemical
4
components of A. insularis have not been studied yet. We
Industrial University of Ho Chi Minh city, Quang Tam,
Thanh Hoa, Vietnam
report herein the isolation, structural elucidation, and
123

N. T. H. Van et al.
evaluation of the in vitro cytotoxic activities of one new
and twelve known compounds from the leaves of A.
insularis.
Table 1 The NMR spectral data for compound 1
Pos.
1
daC,b
daH,b mult. (J in Hz)
Aglycone
Materials and methods
1
2
39.6
0.96 (m)/1.62 (m)
26.3
83.5
43.8
48.2
18.9
33.3
41.0
49.0
37.6
24.7
123.4
145.7
42.9
26.6
22.9
38.1
43.9
47.8
31.8
35.3
32.3
65.2
13.4
16.6
17.4
26.6
69.8
33.8
24.0
1.73 (m)/1.81 (m)
General procedures
3
3.61 (br d, 10.4)
4
–
Optical rotations were determined on a Jasco DIP-370
1
5
1.22 (m)
digital polarimeter. H NMR (400 MHz) and 13C NMR
6
1.40 (m)/1.50 (m)
(100 MHz) spectra were recorded on an Agilent 400-MR
NMR spectrometer and TMS was used as an internal
standard. Data processing was carried out with the
MestReNova 6.0.2 program. HR-ESI–MS spectra were
obtained using an Agilent 6550 iFunnel Q-TOF LC/MS
system. GC was recorded on a Shimadzu GC-2010 plus.
Preparative HPLC was carried out using an Agilent 1290
HPLC system. Column chromatography was performed on
silica gel (Kieselgel 60, 70–230 mesh and 230–400 mesh,
Merck) and YMC RP-18 resins.
7
1.32 (m)/1.65 (m)
8
–
9
1.64 (m)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
3-O-Ara
10
–
1.89 (m)
5.17 (br s)
–
–
1.32 (m)/1.84 (m)
1.19 (m)
–
Plant material
1.97 (m)
1.04 (m)/1.77 (m)
The leaves of A. insularis were collected in Quang Khe,
Dakglong, Daknong, Taynguyen, Vietnam in March, 2012,
and identified by Dr. Nguyen Quoc Binh, Vietnam National
Museum of Nature, VAST, Vietnam. A voucher specimen
(BMN-B0001532) was deposited at the Herbarium of In-
stitute of Natural Products Chemistry.
–
1.12 (m)
1.35 (m)/1.52 (m)
3.33 (d, 10.4)/3.64 (d, 10.4)
0.73 (s)
1.00 (s)
0.99 (s)
Extraction and isolation
1.20 (s)
3.10 (d, 11.2)/3.51 (d, 11.2)
0.87 (s)
The dried leaves of A. insularis (2.0 kg) were extracted with
MeOH three times at room temperature to yield 150 g of a
dark solid extract, which was then suspended in water and
successively partitioned with n-hexane and ethyl acetate
(EtOAc) to obtain n-hexane (AI1, 40.0 g), EtOAc (AI2,
45.0 g), and water (AI3, 65.0 g) extracts after removal sol-
vent in vacuo. The AI2 extract (45.0 g) was chro-
matographed on a silica gel column and eluted with CHCl3–
MeOH gradient (50:1 ? 1:1, v/v) to obtain four sub-frac-
tions, AI2A (8.0 g), AI2B (7.5 g), AI2C (12.5 g), and AI2D
(10.0 g). The AI2B fraction was chromatographed on an
YMC RP-18 column eluting with acetone–water (1:1, v/v) to
yield compounds 10 (80.0 mg) and 11 (19.0 mg). The AI2C
fraction was chromatographed on an YMC RP-18 column
eluting with acetone–water (0.8:1, v/v) to yield compounds 6
(42.0 mg) and 7 (55.0 mg). The water soluble fraction (AI3,
65.0 g) was chromatographed on a Diaion HP-20P column
eluting with water containing increasing concentrations of
MeOH (0, 25, 50, 75, and 100 %) to obtain five fractions,
0.88 (s)
106.1
72.1
84.2
69.5
66.9
4.35 (d, 7.6)
20
30
40
50
3.68 (dd, 7.6, 8.0)
3.64 (m)
4.03 (br s)
3.56 (d, 12.0)/3.86 (d, 12.0)
30-O-Glc
100
200
300
400
500
600
a
105.5
75.3
77.9
71.1
77.6
62.3
4.54 (d, 7.6)
3.30 (m)
3.35 (m)
3.30 (m)
3.30 (m)
3.65 (d, 4.8, 12.0)/3.83 (d, 12.0)
b
c
Measured in CD3OD, 100 MHz, 400 MHz, Assignments were
done by HMQC, HMBC, 1H-1H COSY and ROESY experiments,
Ara, L-arabinopyranosyl, Glc, D-glucopyranosyl
123

Chemical components from the leaves of Ardisia insularis and their cytotoxic activity
AI3A (30.0 g), AI3B (9.0 g), AI3C (13.0 g), AI3D (7.0 g),
and AI3E (6.0 g). The AI3B fraction (9.0 g) was chro-
matographed on a silica gel column eluting with CHCl3–
MeOH–water (5:1:0.1, v/v/v) to give four fractions, AI3B1
(3.0 g), AI3B2 (2.4 g), AI3B3 (2.1 g), and AI3B4 (1.0 g).
The AI3B2 fraction was chromatographed on an YMC RP-
18 column eluting with MeOH–water (1:1, v/v) to yield
compounds 12 (8.0 mg) and 13 (9.0 mg). The AI3B3 frac-
tion was chromatographed on an YMC RP-18 column elut-
ing with MeOH–water (2:1, v/v) to yield compounds 2
(5.0 mg), 3 (6.0 mg), and 4 (9.0 mg). The AI3C fraction was
chromatographed on a silica gel column and eluted with
CHCl3–MeOH gradient (50:1 ? 1:1, v/v) to obtain four
fractions, AI3C1 (8.0 g), AI3C2 (7.5 g), AI3C3 (12.5 g),
and AI3C4 (10.0 g). The AI3C2 fraction was chro-
matographed on an YMC RP-18 column eluting with
MeOH–water (2:1, v/v) to obtain compounds 5 (10.0 mg), 8
(17.0 mg), and 9 (9.0 mg). The AI3C3 fraction was chro-
matographed on an YMC RP-18 column eluting with ace-
tone–water (0.7:1, v/v) to yield compound 1 (6.0 mg).
Ardinsuloside (1) A white amorphous powder, [a]2D5:
?58 (c = 0.2, MeOH), C41H68O12, HR-ESI–MS found
m/z 787.4394 [M ? Cl]- (Calcd C41H68O12Cl- for
diphenyltetrazolium bromide (MTT) assay (Nhiem et al.
2012). The A-549 (human lung cancer), HT-29 (human
colon cancer) and OVCAR (human ovarian cancer) cell
lines were obtained from the korea cell line bank (KCLB)
and were grown in RPMI 1640 medium supplemented with
10 % fetal bovine serum and penicillin/streptomycin
(100 U/Ml and 100 g/Ml, respectively) at 37 °C in a hu-
midified 5 % CO2 atmosphere. The MTT assays were
performed as follows: 1.5 * 2.5 9 105 cells/Ml of human
cancer cells were treated for 3 days with isolated com-
pounds at concentration of 100 lM. After incubation,
0.1 mg (50 Ll of a 2 mg/Ml solution) MTT (Sigma, Saint
Louis, MO, USA) was added to each well and the cells
were then incubated at 37 °C for 4 h. The plates were
centrifuged at 1000 rpm for 5 min at room temperature and
the media was then carefully aspirated. Dimethylsulfoxide
(DMSO) was then added to each well to dissolve the for-
mazan crystals. The plates were read immediately at
540 nm on a microplate reader (Amersham Pharmacia
Biotech., USA). The compounds which result in more than
50 % human cancer cells dead will be further tested using
dose concentration: 1, 10, 50 and 100 lM. Mitoxantrone
was used to final concentrations of 1, 3, 10 and 20 lM as a
reference and DMSO 0.1 % was used as negative control.
All the experiments were performed three times and the
mean absorbance values were calculated. The results are
expressed as the relative cell viability percentage presented
by a reduction in the absorbance after the treatment of the
tested compounds compared to the untreated controls. A
dose–response curve was generated and the IC50 was de-
termined for each compound as well as each cell line.
1
787.4405), H- and 13C-NMR: see Table 1.
Acid hydrolysis of 1
Compound 1 (2.0 mg) was dissolved in 1 N HCl (dioxane-
H2O, 1:1, 1 Ml) and then heated to 80 °C in a water bath for
3 h. The acidic solution was neutralized with silver carbonate
and the solvent thoroughly driven out under N2 gas overnight.
After extraction with CHCl3, the aqueous layer was concen-
trated to dryness using N2 gas. The residue was dissolved in
0.1 Ml of dry pyridine, and then L-cysteine methyl ester hy-
drochloride in pyridine (0.06, 0.1 Ml) was added to the so-
lution. The reaction mixture was heated at 60 °C for 2 h, and
0.1 Ml of trimethylsilylimidazole solution was added, fol-
lowed by heating at 60 °C for 1.5 h. The dried product was
partitioned with n-hexane and H2O (0.1 Ml each), and the
organic layer was analyzed by gas liquid chromatography
(GC): column SPB-1 (0.25 mm 9 30 m); detector FID, col-
umn temp 210 °C, injector temp 270 °C, detector temp
300 °C, carrier gas He (2.0 Ml/min). Under these conditions,
standard sugars gave peaks at tR (min) 8.55 and 9.25 for D- and
L-glucose, 4.72 and 9.16 for D- and L-arabinose, respectively.
Peaks at tR (min) 8.55 and 9.16 of D-glucose and L-arabinose
for compound 1, respectively, were observed.
Results and discussion
Using various chromatographic techniques, one new and
twelve known compounds were isolated from the methanol
extract of the leaves of A. insularis.
Compound 1 was obtained as a white amorphous pow-
der. The molecular formula, C41H68O12, was determined on
the HR-ESI–MS at m/z 787.4394 [M ? Cl]- (Calcd.
787.4405 for C41H68O12Cl). The 1H-NMR spectrum of
compound 1 showed the following signals: one olefinic
proton at dH 5.17, one oximethine proton at dH 3.61, and
six tertiary methyl groups at dH 0.73, 0.87, 0.88, 0.99, 1.00,
and 1.20 (each 3H, s), assigned to oleane-type triterpene
aglycone; two anomeric protons at dH 4.35 (d, J = 7.6 Hz)
and 4.54 (d, J = 7.6 Hz), assigned to two sugar moieties.
The 13C-NMR and DEPT spectra of compound 1 revealed
41 carbon signals including seven quaternary, fourteen
methine, fourteen methylene, and six methyl carbons. The
1H- and 13C-NMR data of compound 1 were similar to
those of assamicoside A except for the disappearance of
Cytotoxicity assay
The effect of compounds 1–13 on the growth of human
cancer cells was determined by measuring the cytotoxic
activity
using
a
3-[4,5-dimethylthiazol-2-yl]-2,5-
123

N. T. H. Van et al.
30
18
29
OH
3'
20
17
OH
4'
1'
O
28
HO
11
25
26
H
CH2OH
R
7
2
14
1
OH
10
8
H
4
OGal
5
27
O
3
5
HO
HO
O
O
O
OH
O
H
R
OH
OH
HO
23
10
11
H
24
OH
3'
OMe
OH
OH
R1
1
4'
OH
1'
HO
HO
HO
O
7
2
3
R
O
2
OH
HO
O
OH
O
H
5
4
4
R1
10b
10
4a
OR2
O
OH
Me
10a
6a
1"
H
O
R
OH
OMe
R3
O
O
8
12
13
HO
HO
R2
6
O
OH
O
R1
H
OH
OH
OH
R2
H
H
Gal
H
R3
H
H
H
Gal
R1
R2
6
2
H
H
H
H
Gal: HO
7
8
9
3
4
5
OH
OMe
OMe Gal
O
HO
Fig. 1 Structures of isolated compounds 1–13 from Ardisia insularis
Fig. 2 The important HMBC
and COSY correlations of
compound 1
CH2O
H
OH
O
HO
HO
O
HMBC
COSY
O
O
OH
OH
HO
OH
two hydroxyl groups at C-16 and C-21 of the aglycone
(Tian et al. 2013). The carbons were assigned to the rele-
vant protons by means of an HMQC experiment (Table 1).
The HMBC correlations between H-23 (dH 3.33 and 3.64)
and C-3 (dC 83.5)/C-4 (dC 43.8)/C-5 (dC 48.2)/C-24 (dC
13.4); H-24 (dH 0.73) and C-3 (dC 83.5)/C-4 (dC 43.8)/C-5
(dC 48.2)/C-23 (dC 65.2) suggested two hydroxyl groups at
C-3 and C-23 of aglycone (Figs. 1, 2). The configurations
of both the hydroxyl at C-3 and the methyl groups at C-4
were proved to be b-orientation by the observation of
ROESY correlations of H-3 (dH 3.61)/H-23 (dH 3.33 and
3.64)/H-5 (dH 1.22) (Fig. 3) as well as of large coupling
constant of Ha-2 and Ha-3, J = 10.4 Hz). The HMBC
correlations between H-28 (dH 3.10 and 3.51) and C-16 (dC
22.9), C-17 (dC 38.1), C-18 (dC 43.9), and C-22 (dC 32.3)
confirmed the position of hydroxyl group at C-28. Acid
123

Chemical components from the leaves of Ardisia insularis and their cytotoxic activity
galloylbergenin (5) (Yoshidaetal. 1982), quercitrin (6) (Kiem
et al. 2010), myricitrin (7) (Kiem et al. 2010), myricetin 3-O-
(300-O-galloyl)-a-L-rhamnopyranoside (8) (Sun et al. 1991),
desmanthine-2 (9) (Nicollier and Thompson 1983), epi-
catechin 3-O-galloyl ester (10) (Braca et al. 2003), 30-
methoxyepicatechin 3-O-galloyl ester (11) (Aihara et al.
2009), gallic acid (12), and methyl galloate (13) (see Fig. 1).
Their structures were established on the basis of spectral and
chemical evidences, which were in agreement with those of
reported in literature. This isthe first time all these compounds
have been isolated from A. insularis.
The isolates 1–13 were evaluated for cytotoxic activity
against three human cancer cell lines (A-549, HT-29, and
OVCAR). Compound 1 exhibited the strongest cytotoxic
activity with the relative cell viability of 14.6–21.7 % at
the concentration of 100 lM. The remaining compounds
showed weak cytotoxic activity (Table 2). Mitroxantrone
was used as a positive control and inhibited A-549, HT-29,
and OVCAR cell lines with the IC50 values of 7.2 0.5,
3.1 0.3, and 8.4 0.9 lM, respectively. Whereas,
compound 1 inhibited A-549, HT-29, and OVCAR cell
lines with IC50 values of 8.5 1.2, 16,4 3.1, and
13.6 2.4 lM, respectively. This result indicates com-
pound 1 could be useful in the treatment of cancer disease
and further study of the cytotoxic effects of these com-
pounds by in vivo assay may be required.
Fig. 3 The important ROESY correlations of compound 1
Table 2 The effects of compounds 1–13 on the growth of human
cancer cell lines at the concentration of 100 lM
Compound
Relative cell viability (%)
A-549
HT-29
OVCAR
1
14.6 3.1
94.4 8.2
94.4 7.4
99.3 12.4
65.6 10.4
71.5 5.7
76.2 4.5
53.1 5.6
66.4 8.8
99.2 5.3
43.3 2.6
79.7 6.6
91.5 7.5
21.7 5.3
92.7 5.9
87.5 6.2
97.9 9.9
88.9 7.3
73.4 5.6
86.4 7.2
69.3 3.9
76.0 5.4
98.8 8.9
56.2 6.4
65.9 7.1
83.7 5.8
16.2 2.8
97.0 6.7
99.9 6.6
80.8 5.5
76.7 4.5
93.6 3.6
89.0 2.1
59.4 9.7
69.7 12.1
91.4 6.1
48.8 4.6
81.8 8.7
82.3 4.3
2
3
4
5
6
7
8
9
10
11
12
13
Supporting information
The NMR and HR-ESI–MS spectra of compound 1 are
available as Supporting information.
hydrolysis of compound 1 gave D-glucose and L-arabinose
(see Experimental). The coupling constants of the anomeric
Acknowledgments This research was funded by Vietnam National
Foundation for Science and Technology Development (NAFOSTED)
under grant number 104.01-2011.20. This research was also supported
by Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education,
Science and Technology (2011-0025129).
0
0
protons in two sugar moieties, JH-1 -H-2 = 7.6 Hz and
00
00
JH-1 -H-2 = 7.6 Hz confirmed the presence of b-D-glu-
copyranoside and a-L-arabinopyranoside moieties. More-
over, the HMBC correlations between glc H-10 (dH 4.54)
and ara C-30 (dC 84.2) as well as between ara H-30 (dH 3.64)
and glc C-100 ((dC 105.5) confirmed the sugar linkage to be
b-D-glucopyranosyl-(1 ? 3)-a-L-arabinopyranoside. This
sugar linkage to C-3 of aglycone was confirmed by HMBC
correlations from H-3 (dH 3.61) to ara C-10 (dC 106.1);
from ara H-10 (dH 4.35) to C-3 (dC 83.5). The important
HMBC and COSY correlations were shown in Fig. 2.
Consequently, the structure of compound 1 was determined
to be 3b,23,28-trihydroxyolean-12-ene 3-O-[b-D-glucopy-
ranosyl-(1 ? 3)-a-L-arabinopyranoside], a new compound
named ardinsuloside.
Conflicts of interest The authors report no conflicts of interest.
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