Journal of Natural Products
Article
Plant Material. Stem bark of F. ramontchi was collected in August
2005 from Namakia Toliara in Madagascar and authenticated by
Armand Rakotozafy by comparison with an authentic specimen held in
the Department of Botany, Parc Botanique et Zoologique de
Tsimbazaza, Antananarivo. A voucher specimen (MAD-0055) was
flow: 4 mL/min, detection: ELSD. Identification of D-glucose present
in the sugar fraction was carried out by comparison of its retention
time with those of authentic samples of L-galactose, D-galactose, D-
glucose, and L-glucose.
Chikungunya Virus-Cell-Based Antiviral Assay. Serial dilutions
of extract, fractions, or compounds, as well as the reference compound
chloroquine, were prepared in assay medium [MEM Rega3 (cat. no.
19993013; Invitrogen), 2% FCS (Integro), 5 mL of 200 mM L-
glutamine, and 5 mL of 7.5% sodium bicarbonate] that was added to
empty wells of a 96-well microtiter plate (Falcon, BD). Subsequently,
50 μL of a 4× virus dilution in assay medium was added, followed by
50 μL of a cell suspension. This suspension, with a cell density of 25
000 cells/50 μL, was prepared from a Vero cell line subcultured in cell
growth medium (MEM Rega3 supplemented with 10% FCS, 5 mL of
L-glutamine, and 5 mL of sodium bicarbonate) at a ratio of 1:4 and
grown for 7 days in 150 cm2 tissue culture flasks (Techno Plastic
Products). The assay plates were returned to the incubator for 6−7
days (37 °C, 5% CO2, 95−99% relative humidity), a time at which
maximal virus-induced cell death or cytopathic effect (CPE) is
observed in untreated, infected controls.
Subsequently, the assay medium was aspirated, replaced with 75 μL
of a 5% MTS (Promega) solution in phenol red-free medium, and
incubated for 1.5 h. Absorbance was measured at a wavelength of 498
nm (Safire2, Tecan); optical densities (OD values) reached 0.6−0.8
for the untreated, uninfected controls. Raw data were converted to
percentage of controls, and the EC50 (50% effective concentration, or
concentration that is calculated to inhibit virus -induced cell death by
50%) and CC50 (50% antimetabolic concentration, or concentration
that is calculated to inhibit the overall cell metabolism by 50%) were
derived from the dose−response curves. All assay conditions
producing an antiviral effect exceeding 50% were checked microscopi-
cally for minor signs of CPE or adverse effects on the host cell (i.e.,
altered cell morphology, etc.). A compound is only considered to elicit
a selective antiviral effect on virus replication when, following
microscopic quality control, at least at one concentration of
compound, no CPE nor any adverse effect is observed (image
resembling untreated, uninfected cells). Multiple, independent experi-
ments were performed.
́
deposited at the Institut Malgache de Recherches Appliquees.
Extraction and Isolation. The stem bark (1.8 kg) was successively
extracted with EtOAc and MeOH. After concentration, the EtOAc
extract (17.4 g) was subjected to silica gel column chromatography
(CC) using a gradient of n-heptane−acetone−MeOH (1:0:0 to
0:90:10) of increasing polarity, leading to 25 fractions on the basis of
TLC. Fraction 18 (286 mg; heptane−acetone, 30:70) was subjected to
a semipreparative C18 column using MeOH−H2O (60:40 + 0.1%
formic acid) at 3 mL·min−1 to afford flacourtoside A (1, 3.1 mg),
flacourtoside E (5, 4.8 mg), flacourtoside F (6, 11.3 mg), and itoside H
(9.3 mg). Fraction 19 (1100 mg; heptane−acetone, 20:80) was
purified on a preparative C18 column using MeOH−H2O (70:30 +
0.1% formic acid) at 21 mL·min−1 to afford flacourtoside B (2, 7.3
mg), scolochinenoside D (4.2 mg), and xylosmin (11.1 mg). Fraction
20 (908 mg; heptane−acetone, 10:90) was subjected to preparative
C18 chromatography using MeOH−H2O (70:30 + 0.1% formic acid)
at 21 mL·min−1 to afford flacourtoside C (3, 1.3 mg) and flacourtoside
D (4, 3.5 mg). Fraction 21 (4241 mg, heptane−acetone, 0:100) was
purified on a preparative C18 column using MeOH−H2O (40:60 +
0.1% formic acid) at 21 mL·min−1 to afford poliothrysoside (21.4 mg).
Flacourtoside A (1): greenish-yellow, amorphous powder; [α]24
D
−6 [c 0.1, MeOH]; UV [MeOH] λmax (log ε) 229 (3.95), 272 (3.62)
nm; IR νmax 3325, 1720, 1453, 1274, 1072, 715 cm−1; 1H NMR
(MeOD, 500 MHz) and 13C NMR (MeOD, 125 MHz), see Table 1;
HRESIMS m/z 399.1064 [M + Na]+ (calcd for C19H20O8Na,
399.1056).
Flacourtoside B (2): brown, amorphous powder; [α]24D −21 [c 0.1,
MeOH]; UV [MeOH] λmax (log ε) 232 (4.16), 257 (4.08), 268 (3.78),
326 (3.58) nm; IR νmax 3442, 1720, 1675, 1450, 1276, 1065, 704 cm−1;
1H NMR (MeOD, 500 MHz) and 13C NMR (MeOD, 125 MHz), see
Table 1; HRESIMS m/z 427.1009 [M + Na]+ (calcd for C20H20O9Na,
427.1005).
Flacourtoside C (3): brownish-beige, amorphous powder; [α]24
D
Enzymatic Activity Assay of the Dengue Polymerase.
Polymerase activity was assayed by monitoring the incorporation of
radiolabeled guanosine into a homopolymeric cytosine RNA template,
as previously described.22 The enzymes were produced and purified as
previously described.22 The determination of the IC50 of the pure
compounds followed a detailed procedure previously described.25 IC50
was determined using the following equation: % of enzyme activity =
100/[(1 + I2)/IC50], where I is the concentration of inhibitor. IC50 was
determined from curve-fitting using Kaleidagraph (Synergy Software).
For each value, results were obtained using triplicates in a single
experiment. 3′-Deoxy-GTP was used as the reference.
−11 [c 0.1, MeOH]; UV [MeOH] λmax (log ε) 203 (4.29), 226 (4.37),
256 (3.68) nm; IR νmax 3444, 1724, 1675, 1451, 1276, 1064, 709 cm−1;
1H NMR (MeOD, 500 MHz) and 13C NMR (MeOD, 125 MHz), see
Table 1; HRESIMS m/z 693.1809 [M + Na]+ (calcd for C33H34O15Na,
693.1795).
Flacourtoside D (4): yellow, amorphous powder; [α]24 +9 [c 0.1,
D
MeOH]; UV [MeOH] λmax (log ε) 229 (4.49), 280 (4.11) nm; IR νmax
3381, 1701, 1457, 1276, 1070, 715 cm−1; 1H NMR (MeOD, 500
MHz) and 13C NMR (MeOD, 125 MHz), see Table 2; HRESIMS m/
z 721.1734 [M + Na]+ (calcd for C34H34O16Na, 721.1744).
Flacourtoside E (5): brownish-beige, amorphous powder; [α]24D +8
[c 0.1, MeOH]; UV [MeOH] λmax (log ε) 230 (4.41), 274 (3.95), 281
(3.95) nm; IR νmax 3393, 1713, 1455, 1276, 1070, 713 cm−1; 1H NMR
(MeOD, 500 MHz) and 13C NMR (MeOD, 125 MHz), see Table 2;
HRESIMS m/z 703.1641 [M + Na]+ (calcd for C34H32O15Na,
703.1639).
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR spectra for compounds 1−6 and chromatograms for
determining the sugar are available free of charge via the
Flacourtoside F (6): brownish-beige, amorphous powder; [α]24
D
−14 [c 0.1, MeOH]; UV [MeOH] λmax (log ε) 205 (4.57), 228 (4.71),
274 (4.03), 281 (4.03) nm; IR νmax 3444, 1736, 1724, 1453, 1272,
1068, 713 cm−1; 1H NMR (MeOD, 500 MHz) and 13C NMR
(MeOD, 125 MHz), see Table 2; HRESIMS m/z 807.1909 [M + Na]+
(calcd for C41H36O16Na, 807.1901).
Acid Hydrolysis. The EtOAc extract, 487 mg, was heated under
reflux in 50 mL of 2 N HCl at 80 °C for 6 h. After removing HCl by
evaporation under vacuum, the mixture was diluted with H2O and
EtOAc (3 × 50 mL). The aqueous layer was neutralized with 0.1 M
NaOH. The residue containing the sugar was analyzed by UPLC/MS
under the following conditions: solvents: (A) ACN−H2O (30/70) +
0.1% NH4OH; (B) ACN−H2O (80/20) + 0.1% NH4OH, A/B (20/
80), flow: 0.17 mL/min, detection: MS. The analysis by SFC was
performed under the following conditions: cosolvent MeOH (15%),
CO2 flow rate: 3.4 mL/min, cosolvent flow rate: 0.6 mL/min, total
AUTHOR INFORMATION
Corresponding Author
■
*Tel: 33 1 69 82 30 85. Fax: 33 1 69 07 72 47. E-mail:
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to Museum National d’Histoire Naturelle for a
fellowship (M.B.). Financial support from the Centre de
́
Recherche et de Veille des Maladies Emergentes dans l’Ocean
■
́
757
dx.doi.org/10.1021/np300059n | J. Nat. Prod. 2012, 75, 752−758