CYTOTOXIC STEROIDS FROM THE STEM BARK OF Chisocheton cumingianus ( Meliaceae ) STEROID DENGAN AKTIVITAS SITOTOKSIK DARI KULIT BATANG Chisocheton cumingianus ( Meliaceae ) Dewa

Three cytotoxic steroids, stigmasterol (1), stigmast-5-en-3-ol (2) and -sitosterol-3-O-acetate (3) were isolated from the stem bark of Chisocheton cumingianus. The chemical structures of those compounds were identified based on spectroscopic data and by comparison with those data previously reported. All of the compounds isolated were evaluated for their cytotoxic effects against P-388 murine leukemia cells in vitro. Compounds 1-3 showed cytotoxicity activity against P-388 murine leukemia cells with IC50 values of 12.4, 60.8, and ˃ 100 g/mL, respectively.

As a part of our studies on anticancer candidate compounds from Indonesia Chisocheton plants, we already isolated a 7hydroxy coumarin from the stem bark of C. celibicus (Katja et al., 2015), and a 30-nor trijugin-type limonoid, chisotrijugin and lanostan-type triterpenoid, 3β-hydroxy-25ethyl-lanost-9(11),24(24′)-diene from the stem bark of C. cumingianus (Katja et al., 2016a, Katja et al., 2016b).In further search of cytotoxic compounds from Indonesia Chisocheton species, we found that n-hexane and ethyl acetate extracts of the stem bark of C. cumingianus exhibited a moderate cytotoxic activity against P-388 murine leukemia cells with IC50 value of 16.9 and 19.9 g/mL, respectively.We report herein the isolation and structural identification of the steroids 1-3, together with the cytotoxic activity against P-388 murine leukemia cells.

General
Melting points were measured on an electrothermal melting point apparatus IA9000.The IR spectra were recorded on a Perkin-Elmer 1760X FT-IR in KBr.Mass spectra were obtained with a Water Qtof HR-MS XEV otm mass spectrometer. 1H-and 13 C-NMR spectra were obtained with a JEOL JNM A-500 spectrometer using TMS as an internal standard.Chromatographic separations were carried out on silica gel 60 and ODS.TLC plates were precoated with silica gel and Octa desyl silane GF254 (ODS), detection was achieved by spraying with 10% H2SO4 in ethanol, followed by heating and under ultra-violet light with wavelength at 254 and 367 nm.

Plant material
The stem bark of C. cumingianus was collected in Bogor Botanical Garden, Bogor, West Java Province, Indonesia in April 2014.The plant was identified by the staff of the Bogoriense Herbarium, Bogor, Indonesia and a voucher specimen (No.Bo-1305316) was deposited at the herbarium.

Determination of cytotoxic activities
The cytotoxicity assay was conducted according to the method described previously (Sahidin et al., 2005;Alley et al., 1998).P-388 cells were seeded into 96-well plates at an initial cell density of approximately 3 x 10 4 cells cm -3 .After 24 h of incubation for cell attachment and growth, varying concentrations of samples were added.The compounds added were first dissolved in DMSO at the required concentration.Subsequent six desirable concentrations were prepared using PBS (phosphoric buffer solution, pH = 7.30 -7.65).Control wells received only DMSO.The assay was terminated after a 48 h incubation period by adding MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; also named as thiazol blue] and the incubation was continued for another 4 h, in which the MTTstop solution containing SDS (sodium dodecyl sulphate) was added and another 24 h incubation was conducted.Optical density was read by using a microplate reader at 550 nm.IC50 values were taken from the plotted graph of percentage live cells compared to control (%), receiving only PBS and DMSO, versus the tested concentration of compounds (g/mL).The IC50 value is the concentration required for 50% growth inhibition.Each assay and analysis was run in triplicate and averaged.

RESULTS AND DISCUSSION
The stem bark of C. cumingianus was grounded and successively extracted with nhexane, ethyl acetate, and methanol.The nhexane and ethyl acetate extract were chromatographed over a vacuum-liquid chromatographed (VLC) column packed with silica gel 60 by gradient elution.The fractions were repeatedly subjected to normal-phase and reverse-phase column chromatography to afford compounds 1-3 (Figure 1).
-sitosterol-3-O-acetate (3), was obtained as a colorless needle crystals, with m.p. 133-136 o C. The molecular formula was established to be C31H52O2 by HR-TOFMS m/z 457.7234 [M+H] + , calculated for C31H52O2, together with NMR spectral data (Table 1), thus requiring six degrees of unsaturation.The IR spectrum of 3 suggested the presence of saturated aliphatics (max 2875 cm -1 ), carbonyl (max 1728 cm -1 ) and ether group (max 1172 cm -1 ).The NMR spectra of 3 was similar to compound 1, except the absence of hydroxyl group and appearance of acetyl signals at [H 1.60 (3H, s), C 20.0, 173.5], suggested that 3 was 3-O-acetyl derivative of 1.A detailed comparison of the NMR data of 3 to those of -sitosterol-3-Oacetate (Elkader et al., 2013), revealed that the structure of both compounds were similar, therefore compound 3 was identified as a sitosterol-3-O-acetate (Figure 1) and was shown for the first time in this species.The stereochemistry of 3 was determined in line with -sitosterol-3-O-acetate based on the chemical shift in 13 C NMR spectrum, protonproton coupling constant values in 1 H NMR spectrum and biogenetic point of view the occurrences of steroid compounds in Chisocheton genus (Harneti et al., 2014;Yang et al., 2009).
The cytotoxic effects of the three isolated compounds 1-3 against P-388 murine leukemia cells were conducted according to the method described previous paper (Harneti et al., 2014;Sahidin et al., 2005;Alley et al., 1988) and were used an artonin E (IC50 0.3 g/mL) as a positive control (Hakim et al., 2007).Compounds 1-3 showed cytotoxic activity with IC50 values of 12.4, 60.8 and ˃ 100 g/mL, respectively.Among these steroid structures, compound 1 having two olefinic moieties showed the strongest activity, whereas compound 2 lacking one olefinic and compound 3 adding one acetyl groups showed decrease cytotoxic activity.These results suggested that the olefinic and acetyl moieties were important structural components for for cytotoxic activity.

CONCLUSION
Three steroid compounds, stigmasterol (1), stigmast-5-en-3-ol (2) dan -sitosterol-3-O-acetate (3) have been isolated from the stembark of Chisocheton cumingianus and was shown for the first time in this species.The presence of olefinic and acetyl moieties in steroid structure play important role for cytotoxic activity against P-388 murine leukemia cells.