Steroids from The Stem Bark of Dysoxylum nutans (Meliaceae) and Their Cytotoxic Effect Against MCF-7 Breast Cancer Cell Lines

Three steroids, 3α-hydroxystigmast-5(6), 22-diene-7-one (1), stigmasterol (2) and 3-hydroxy-7βmethoxystigmast-5(6)-ene (3), were isolated from the stem bark of Dysoxylum nutans. The chemical structures were identified by spectroscopic data, which includes IR, 1D-NMR, 2D-NMR, and HR-TOFMS as well as by comparing previously reported spectral data. Compounds 1-3 were tested for cytotoxic effect against MCF-7 breast cancer cell lines and compound 1 showed the strongest cytotoxic activity with an IC50 value of 20.13  0.06 μM.

As part of our investigation for anticancer substances from Indonesian Dysoxylum plants, methanol extract from dysoxyl nutans showed strong cytotoxic activity against MCF-7 breast cancer cell lines in vitro. D. nutans, which is a high plant and widely distributed in South East Asia (Luo et al., 2002;Cao et al., 2013). The plant is used in Indonesian for traditional medicine for fevers, infected wounds and skin diseases (Heyne, 1982). Although secondary metabolites of other Dysoxylum species have already been investigated, the phytochemical investigation of D. nutans has not yet been reported. The isolation, structure determination and cytotoxic effect of these isolated compounds are described.

General Experimental Procedure
Melting points were measured using an IA9000 electrothermal melting point apparatus (Bibby Scientific Limited, Staffordshire, UK). The optical rotations were recorded on a Perkin-Elmer 341 polarimeter (Waltham, MA, USA). The UV spectra was obtained using a TECAN Infinite M200 pro, with methanol (Switzerland). The IR was recorded on a SHIMADZU IR Prestige-21 in KBr (Kyoto, Japan). Mass spectra were measured using a Water QTOF HR-MS XEV otm mass spectrometer (Waters, Milford, MA, USA). The NMR data were recorded on Bruker 600 MHz (Billerica, MA, USA) and JEOL ECZ-600 spectrometer (Kyoto, Japan) at 600 MHz for 1 H and 150 MHz using tetramethylsilane as an internal standard. Column chromatography was conducted on silica gel 60 (70-230 mesh and 230-400 Mesh) (Merck, Darmstadt, Germany). TLC plates were precoated with silica gel GF 254 (Merck, Darmstadt, Germany 0.25 mm) and evidence was obtained by spraying with 10% sulphuric acid in ethanol, followed by heating.

Plant Material
The stem bark of D. nutans was obtained in Bogor Botanical Garden, West Java Province, Indonesia in August 2017. The plant specimen was deposited at Herbarium with collection number, III. F. 98.

Extraction and Isolation
The dried grounded stem bark (900.0 g) was extracted using methanol exhaustively (10 L) at room temperature for 5 days. Removal of the solvent on a rotary evaporator gives an extract of concentrated methanol (111.6 g). The concentrated methanol extract was first suspended in water and sequentially separated using n-hexane and ethyl acetate, and directly evaporated to give n-hexane (20.5 g) and ethyl acetate (10.5 g), respectively. The n-hexane soluble fraction (20.0 g) was fractionated by vacuum liquid chromatography (VLC) on silica gel using a gradient n-hexane-ethyl acetate to give 8 fractions (A-H). Fraction E (3.9 g) was separated by column chromatography on silica gel using 3% mixtures of n-hexane-ethyl acetate as eluting solvents (100:0-70:30) to give 8 subfractions (E1-E8). Sub-fraction E5 (1.1 g) was further separated by column chromatography on silica gel, with n-hexane-ethyl acetate (2% stepwise) as solvent system to give 7 subfractions (E5a-E5g). Similarly, sub-fraction E5e (0.1 g) was separated by column chromatography on silica gel, with 1% mixtures of n-hexane-ethyl acetate as a solvent (100:0-80:20) to give 1 (13.0 mg). Sub-fraction E5f was separated by column chromatography on silica gel, with n-hexane: ethyl acetate (8:1) as a solvent to give 2 (3.0 mg).

Bioassays of Cytotoxic Activity (Skehan et al., 1990)
MCF-7 cells were grown in 96-well plates with initial cell densities of approximately 3 x 10 4 cm -3 . After 24 hours of incubation for cell growth, various concentrations of the sample were added. Furthermore, the sample was first dissolved in DMSO at the required concentration.
The next six desired concentrations were prepared using PBS (phosphorus buffer solution, pH = 7.30 -7.65). The control wells only accept DMSO, and the test was stopped after an incubation period of 48 hours by adding PretoBlueTM Cell Viability Reagent and the incubation was further continued for 1-2 hours until the color change is observed. Optical density was read using a micro plate reader at 570 nm. IC 50 values were taken from cell charts of the percentage life plotted compared to the control (%), and the concentration of the tested compounds (µM). An IC 50 value is the concentration needed to inhibit 50% growth. Each test and analysis was carried out in triplicate and average.

RESULTS AND DISCUSSION
The concentrated methanol extract from the dried stem bark of D. nutans was extracted with n-hexane and ethyl acetate. The n-hexane extract was separated by vacuum-liquid chromatography (VLC) on silica gel 60 by gradient elution. The VLC fraction was separated by column chromatography on silica gel to give compounds 1-2. The ethyl acetate was prepared as described for compounds 1-2 and give compound 3 (Figure 1). were also observed in the 1 H-NMR spectrum. The 13 C-NMR together with DEPT spectra showed twenty nine carbon signals, which includes six methyls, eight methylenes, eight methines (including one oxygenated sp 3 carbons at δ C 70.5), three sp 2 methines (δ C 126.1, 138.0, 129.4), two sp 3 quaternary carbons, one sp 2 quaternary carbons (δ C 165.7) and 1 carbonyl at δ C 202.3. These unsaturation were calculated for eight out of the total seven degrees of unsaturation. All four degrees of unsaturation were consistent with the structure of tetracyclic stigmastane with additional carbonyl and olefin groups (Huang et al., 2009;Yan et al, 2014b).

Figure 2. Selected HMBC and 1 H-1 H COSY correlations for 1.
A detailed comparison of the NMR data of 1 with those of 3-hydroxystigmast-4,22diene-7-one, isolated from Hedyotis diffusa (Cayme & Ragasa, 2004), exhibited that the structures of the two compounds are very similar. The detail structure of 1 was supported from the 1 H-1 H COSY and HMBC experiments (Figure 2 , supporting the presence of stigmastane structure in compound 1. In the HMBC spectrum, the correlation of methyl protons to their neighboring carbons can influence the six methyls at C-10, C-13, C-20, C-25 (2 ), and C-29, respectively. The HMBC cross peak of the methylene protons at H-2 (δ H 1.48 and 1.80) and H-4 (δ H 2.10 and 2.13) on an oxygenated carbon at δ C 70.5 (C-3), indicated the hydroxyl group is located at C-3. Correlation from methine proton δ H 1.91 (H-8) and 1.20 (H-9) as well as an olefinic proton at δ H 5.55 to δ C 202.3 (C-7) were used to assign a carbonyl group is located at C-7. The stereochemistry of 1 was identified by a NOESY experiment (Figure 3), in which the NOESY correlations between Me-19 and H-3 indicated that the C-3 hydroxyl group is αoriented. Similar to the NOESY observations, the cross peak between Me-18 and H-20, indicated that Me-21 was α-oriented. Furthermore, the NOESY cross peak, which was also observed between Me-21 / H-17, showed that the side chain at C-17 was β-oriented. In addition, the correlation between H-24 and H-17, indicated that an ethyl chain was β-oriented. Therefore, the structure of compound 1 was determined to be 3α-hydroxystigmast-5(6),22diene-7-one.