Abstrak:

Metode
Sebuah survei etno-botani dilakukan di selatan Madagaskar dari Juli Agustus
2010. Bio-dipandu uji fraksinasi dilakukan pada kulit akar Diospyros quercina,
menggunakan sitotoksisitas uji bioassay pada P388 garis sel murine leukemia
sebagai model. Struktur senyawa sitotoksik telah ditetapkan oleh 1D dan 2D
NMR spektroskopi dan spektrometri massa.
Hasil
Biological experiments resulted in the isolation of three bioactive pure
compounds (named TR-21, TR-22, and TR-23) which exhibited very good in
vitro cytotoxic activities with the IC50 values of (0.017 50.0060) g/mL,
(0.0890.005) g/mL and (1.0270.070) g/mL respectively. Thus, they
support the claims of traditional healers and suggest the possible correlation
between the chemical composition of this plant and its wide use in Malagasy
folk medicine to treat cancer.
Kesimpulan:
Kemampuan senyawa terisolasi dalam penelitian ini untuk menghambat
pertumbuhan sel mungkin merupakan penjelasan yang rasional untuk
penggunaan Diospyros kulit akar quercina dalam mengobati kanker dengan
pengobat tradisional Malagasi. Penelitian lebih lanjut, oleh karena itu,
diperlukan untuk mengevaluasi in vivo aktivitas anti-neoplastik dari senyawa
sitotoksik sebagai obat antikanker yang efektif.

1. Introduction
Madagascar is reputed for the extraordinary richness of its flora
(biodiversity) and boasts a wide variety of indigenous species [1]. These
plants species represent an enormous reservoir of new molecules with
potential therapeutic activity waiting to be discovered.
In Madagascar as well as in the others parts of African continent, the
majority of people rely on traditional medicine for their health care needs
because the costs of conventional drugs are unaffordable [2-4]. In addition,
this big island is unique because of the endemicity of its flora which makes
85% of Malagasy biodiversity [5]. During an ethno-botanic survey conducted
in the south-west part of Madagascar, it was reported that the aerial part of
the plant known under the vernacular name Maintifototse (Malagasy name),
and scientifically named Diospyros quercina (Baill.) G.E. Schatz & Lowry
(Ebenaceae) is used by the local communauties to treat malaria and
incurable wounds. The genus Diospyros is represented in Madagascar by 21
endemic species and the majority of species are mostly distributed in the
south and southwest parts of Madagascar [6,7]. This plant is a well-known

and very important recipe in these regions because of its therapeutic values
in the Malagasy traditional medicine.
The aim of the present study was to isolate and characterize the
cytotoxic compounds from Diospyros quercina (Baill.) G.E. Schatz & Lowry
(Ebenaceae). Thus, if admitted that Diospyros
quercina contains such
cytotoxic compounds, the use of this plant as antimalarial or antidiabetic by
traditional healers should be carefully adjusted or discouraged because
recent findings have shown that many plants used in traditional medicine are
potentially toxic, mutagenic and carcinogenic[8]. Such information would be
useful in preventing the wide use of the plant by local communities and will
guide laboratory research in identifying new anticancer lead compounds.
In this present study, the crude extract of the root bark of Diospyros
quercina (Baill.) G.E. Schatz & Lowry (Ebenaceae) exhibited a good activity
with the IC50 value of 0.85 g/mL at 5 g/mL concentration. This interesting
preliminary result has conducted us to undertake further analyses in order to
purify and to elucidate the chemical structure(s) of the bioactive compounds
using a combination and spectroscopic techniques ( 1D and 2D NMR and
mass spectrometry in positive mode IES.SM-TOF 6.97Ev).
2. Materials and methods
2.1.
Selection and collection of plant material
Ethnobotanical information about plant species selected for this study
was obtained by interviewing traditional healers during field work which was
conducted in the south of Madagascar from July to August 2010. Diospyros
quercina (Baill.) G.E. Schatz & Lowry (Ebenaceae) was collected in the
Izombitse Sakaraha National Park at nearly 165 km from Toliara town, in the
south of Madagascar. The plant was identified by comparison with reference
specimen available at the Department
of Botany; Parc Botanique et
Zoologique de Tsimbazaza in Antananarivo, Madagascar. Voucher specimen
assigned sample number MQ-01 was deposited at the herbarium of the
Applied Chemistry Laboratory of the University of Toliara, Madagascar.
2.2.

Extraction and bioguided isolation

The air-dried and powdered root barks of Diospyros quercina (Baill.)
G.E. Schatz & Lowry (2 kg) were repeatedly extracted by cold percolation with
ethanol 90 (3 3 h, 15 L) at room temperature on a shake. Pooled organic
solvents were dried over Na2SO4
and evaporated until dryness at 40 C,
under reduced pressure to yield 30 g of crude extract. Twenty-five grams of
the ethanol crude extract was suspended in water and partitioned
successively with n-hexane, dichloromethane, ethyl acetate and n-butanol to
yield the corresponding soluble extract fractions. Bioassay-guided extraction
revealed interesting activity only with the dichloromethane extract fraction,
which exhibited an inhibition rate of leukemia P388 cell growth of 94.67% at
2 g/mL. Ten grams of the dichloromethane crude extract was first subjected
to fractionation, using a silica gel column chromatography eluted with nhexane and gradient of ethyl acetate (9:1 to 6:4) resulting into eight fractions
(F1F8). Two fractions, F5 and F6, showed strong cytotoxic activities with an
inhibition rate of 96.08% and 92.43% respectively at 0.5 g/mL. These

fractions were checked for purity by analytical TLC, and the zone was
detected with a UV lamp at 254 and 365 nm and by spraying with sulfuric
vanillin acid, followed by heating at 120 C for 1-5 min. F5 and F6 were
combined on the basis of TLC profile similarity and resubmitted to seperation
by silica gel column chromatography eluting with a mixture of diethyl
ether/chloroform/methanol (2/7.5/0.5); the column was in isocratic regime
and at the end, it resulted into seven fractions.
The fraction F63 showed cytotoxic activity with an inhibition rate of
99.012% at 0.25 g/mL. Then 100 mg of F63 was subjected to further
purification, using a silica gel column chromatography, eluting with n-hexane
and gradient of ethyl acetate furnished a pure compound (6 mg) and a
mixture of two compounds (40 mg) which were further separated by
preparative TLC using n-hexane/ethyl acetate/ acetone (2/6/2) as the solvent
system affording compounds 2 (4 mg) and 3 (5 mg). The three pure
compounds showed strong cytotoxic activities against murine P388 leukemia
cell lines after biological test. The purity of compounds was proved by HPLC
analysis. The mobile phase consisted of a CHCl3/MeOH (1/1) mixture of
chloroform and methanol; the chromatography was performed with isocratic
regime during 25 min. Eluted compounds were detected on the basis of their
UV absorption in the wavelength range from 190 nm to 315 nm. The purity
of each product was respectively 99.92% at =202 nm, 98.99% at =198
nm, and 99.42% at =245 nm.
2.3.
Cytotoxicity assay
The crude extract, the fractions (n-hxane, dichloromethane, ethyl acetate,
n-butanol and aqueous extracts) and the pure compounds were
systematically submitted to cytotoxicity test. In that way, murine P388
leukemia cells were grown in RPMI 1640 medium containing 0.01 nmol/L of
-mercapto-ethanol, 10 mmol/L of L-glutamine, 100IU/mL of G-penicillin ,
100 g/mL of streptomycin, 50 g/mL of gentamycin, and 50 g/mL of
nystatine, supplemented with 10% of fetal calf serum. Cells were maintained
at 37 C in a moisturized atmosphere with 5 % CO 2. The inoculums seeded
at 104 cells/mL at an optimal volume of 0.1 mL per well, were introduced into
flat-bottomed 95-well plates containing a serie of concentrations of
compounds (0.001, 0.01, 0.1 and 1 g/mL for compound/extract having
IC50<1 g/mL