healthmedicinet

Collection of plant material

The Stem bark of Garcinia ovalifolia (Clusiaceae), was collected in Makenene (Centre region of Cameroon) in December 2010
and identified by Victor NANA of the National Herbarium Cameroon and a sample specimen
is deposited on the voucher no. 20854/SRFCam.

Extraction and isolation of compounds

Air-dried and powdered stem bark of G. ovalifolia (2.5 kg) were macerated in methanol (5 L) for 48 h at room temperature. The solution
obtained was then filtered through Whatman No. 1 filter paper. The filtrate solution
was concentrated under vacuum into a paste to give a dark brown crude extract (150 g).
The slurry was made of crude extract (100 g) by dissolving in MeOH, adsorbed on 120 g
of silica gel (60–120 mesh) which was subjected to Vacuum Liquid Chromatography (VLC)
column packed with 800 g of silica gel (120–200 mesh). Elution was carried out using
using hexane/ethyl acetate and ethyl acetate/methanol gradients as eluents at a flow
rate of 2 mL/min. Fractions (250 mL each) were collected as follows: pure hexane (fractions
1–5), hexane/ethyl acetate 75/25 (fractions 6–12), hexane/ethyl acetate 50/50 (fractions
13–25),ethyl acetate (fractions 26–33), acetate/methanol 90/10 (fractions 116–125)
and methanol (fractions 126–132). These fractions were pooled on the basis of the
thin layer chromatography analysis on seven sub-fractions from A to G respectively.

Further chemical investigation of sub- fractions B, C and D was carried out using
column chromatography, preparative tin layer chromatography and recrystallization
in different solvent yielded three compounds: 7-epigarcinol (250 mg); isogarcinol
(25 mg) manniflavanone (40 mg) respectively. All these structures were obtained by
the means of spectroscopic analysis including 1D and 2D NMR and mass spectra.

Cell culture

Human promyelocytic leukemia (HL-60 cells) and prostate cancer (PC-3 cells) were obtained
from European Collection of Cells Culture (ECCC), Sigma–Aldrich, India. They were
grown in RPMI-1640 medium containing 10 % Fetal bovine serum (FBS),penicillin (100 IU/mL)
and streptomycin (100 µg/mL medium).The cells suspension was kept in the incubator
(Thermocom Electron Corporation, USA) at 37 °C, 5 % CO
₂
; 98 % humidity. Cells were used for different assays during logarithmic growth phase
while the untreated control cultures received only the vehicle (DMSO  0.1 %).

Cells viability and treatments

The human promyelocytic leukemia (HL-60 cells) and prostate cancer (PC-3 cells) were
seeded in 96 different well plates containing 15 × 103 and 6 × 103 cells/100 µL/well,
respectively. The cultured cells were then treated (triplicate wells per condition)
by adding 100 µL of serial dilutions of the three molecules (7-epigarcinol, isogarcinol
and manniflavanone) in DMSO to give a final concentration of 100, 30, 10 and 1 µg/mL.
The HL-60 treated cells were incubated immediately while for PC-3 cells, the molecules
were added after 24 h of incubation. In addition, the DMSO alone was added to another
set of cells as the solvent control (DMSO  0.1 %). The cells were then incubated
for another 48 h prior to the addition of 20 µL of 2.5 mg/mL solution of 3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) into each well. The incubation was continued for
3 h before the media was removed. A mixture of DMSO (150 µL) was added to each well
and mixed to ensure dissolving of the crystal formazan before the absorbance at 570 nm
was measured. Three replications of each experiment were performed and fifty percent
of inhibitory concentration (IC
₅₀
) of each extract was calculated.

DNA content and cell cycle phase distribution

HL-60 cells (1 × 10
⁶
cells/2 mL/well) were treated with molecules at 20, 50, 100 µg/mL for 24 h. They were
harvested and washed with 1 mL of PBS, then centrifuged at 400 g for 5 min at 4 °C.
The pellet was suspended in 100 µL of PBS and 900 µL of hypertonic citrate buffer
(PI-25 µg/mL, RNAase- 40 µg/mL, sodium citrate-0.1 % and Triton-100X-0.03 %) and incubated
at 37 °C in dark for 20 min. Finally, cells were analyzed immediately on flow cytometer
FACS Calibur (Becton–Dickinson, USA). The data were collected in list mode on 10,000
events and illustrated in a histogram, where the number of cells (counts) is plotted
against the relative fluorescence intensity of PI (FL-2; ?em: 585 nm; red fluorescence).
The resulting DNA distributions were analyzed by Modfit (Verity Software House Inc.,
Topsham, ME) for the proportions of cells in G
₀
–G
₁
, S- phase, and G
₂
-M phases of the cell cycle 26].

Hoechst 33258 staining of cells for nuclear morphology

HL-60 cells (2 × 10
⁶
 cells/3 mL/well) were treated with isogarcinol and 7-epigarcinol at different concentrations
of extract for 24 h. They were collected, centrifuged at 400 g and washed once with
PBS. A solution of Hoechst (Hoechst, 10 µg/mL; citric 10 mM; Na
₂
HPO
₄
0.45 M; Tween-20 0.05 %) was added in each tube and kept in the dark at room temperature
for 30 min. The mixture was washed with PBS and the pellet suspended in 100 µL of
PBS/glycerol (1:1). The solution (10 µL) was poured into the slide and nuclear morphology
alterations observed under fluorescence microscope (Olympus ×70, magnification ×20)
26].

Mitochondrial membrane potential (MMP) assay

HL-60 cells (1 × 10
⁶
cells/2 mL/well) were treated with isogarcinol and 7-epigarcinol at different concentrations
for 24 h. Thirty minutes before the end of the experiment, the cell culture was treated
with Rhodamine-123 (200 nM) and kept in the dark for 30 mn. Cells were collected,
centrifuged (400 g; 4 °C; 5 min), the pellet was washed with 1 mL of PBS and centrifuged
as mentioned earlier. The fluorescence intensity of 10,000 events was analyzed in
FL-1 channel on BD FACSCalibur (Becton–Dickinson, USA) flow cytometer. The decrease
in fluorescence intensity because of membrane mitochondrial potential loss was analyzed
in FL-1 channel and the change of potential membrane (??m) was assessed by comparing
fluorescence.

Reactive oxygen species (ROS) assay

ROS production was monitored by flow cytometry using 2?,7?-dichlorodihydrofluorescin
diacetate (DCFH
₂
-DA). This dye is a stable non polar compound that readily diffuses into cells and
is hydrolyzed by intracellular esterase to yield 2?,7?-dichlorodihydrofluorescin (DCFH
₂
), which is trapped within the cells. Hydrogen peroxide or low molecular weight peroxides
produced by the cells oxidizes DCFH
₂
to a highly fluorescent compound 2?,7?-dichlorofluorescein (DCF). Thus, the fluorescence
intensity was proportional to the amount of hydrogen peroxide produced by the cells.
Briefly, HL-60 cells (1 × 10
⁶
cells/2 mL/well) were treated with isogarcinol and epigarcinol at different concentrations
for 24 h. Thirty minutes before the end of the experiment, the cell culture was treated
with DCFH
₂
-DA (50 µM) and kept in the dark. Cells were then collected, centrifuged (200 g; 4 °C;
5 min) and the pellet was washed with 1 mL of PBS and centrifuged as mentioned earlier.
The pellet was suspended in 500 µL of PBS and the fluorescence was assessed by comparing
two fluorescence emission 480 nm/530 nm using a flow-cytometer (BD-LSR).

Statistical analysis

The viability experiments were done in triplicates and each data point represents
the average of at least 3 independent experiments. For other assays, three independent
experiments were made and one of them had been chosen as result to post in this study.