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