Two new xanthones and cytotoxicity from the bark of Garcinia schomburgkiana.

Two new xanthone derivatives, named schomburgones A (1) and B (2), along with eight known compounds, including xanthones (3-8) and anthraquinones (9-10) were isolated from the bark of Garcinia schomburgkiana. Their structures were determined by spectroscopic analysis especially 1D and 2D NMR spectroscopies. All isolated compounds were evaluated for their cytotoxicity against five cancer cell lines (KB, HeLa S-3, HT-29, MCF-7 and HepG-2). Compounds 3-6 and 8 showed good cytotoxicity against all the five cancer cell lines with IC50 values in the range of 1.45-9.46 µM.


Introduction
Garcinia schomburgkiana Pierre (family Clusiaceae) is a medium-sized tree distributed in Thailand, Laos, Vietnam, and Cambodia. In folk medicine in these countries, its leaves, roots, and fruits are used for the treatment of cough, menstrual disturbances, expectorant, laxative and diabetes [1]. Previous chemical and biological studies on the chemical constituents of G. schomburgkiana showed the presence of xanthones, depsidones, biphenyls, flavonoids, triterpenoids, and phloroglucinols, some of which exhibited antimalarial activity and cytotoxicity [2], [3]. Here, we reported two new xanthone derivatives, named schomburgones A (1) and B (2), along with six known xanthones (3)(4)(5)(6)(7)(8) and two known anthraquinones (9-10) from the bark of this plant. The structures of all isolated compounds were elucidated using spectroscopic methods especially 1D and 2D NMR spectroscopies and compared with their 1 H and 13 C NMR spectroscopic data from the literature. The cytotoxicity of all isolated compounds was evaluated using the MTT method against five cancer cell lines.
Schomburgone B (2) was obtained as yellow oil. Its molecular formula was determined as C 19 (Table 1) were shown to be similar to those of the known xanthone, pancixanthone A [14], except that the hydroxyl group at C-3 of pancixanthone-A was substituted by a methoxy group. In the HMBC correlations of 2 (Fig. 2), the methoxy proton at δ H 3.91 showed a cross-peak with δ C 166.1 (C-3). Moreover, two methyl protons at δ H 1.61 showed cross-peaks with δ C 113.9 (C-4) and 156.3 (C-2′), confirming that a 1,1-dimethylallyl group was connected at C-4. Thus, the completed assignment of schomburgone B was determined as 2.
In previous research many xanthones showed cytotoxicity [15]. Therefore, all isolated compounds were evaluated in vitro for their cytotoxicity against five cancer cell lines (KB, HeLa S-3, HT-29, MCF-7 and HepG-2) ( Table 2).  Table 2) of xanthones suggest that the ortho hydroxy group at C-5 and C-6 and the 1,1-dimethylallyl group at C-4 might improve the cytotoxicity as inferred from the comparison of their cytotoxicity of compounds 1-10.

General experimental procedures
NMR spectra were recorded on Bruker 400 AVANCE spectrometer. HRESIMS spectra were obtained using a Bruker MICROTOF model mass spectrometer. The UV-visible absorption spectra were recorded on a UV-2550 UV-Vis spectrometer (Shimadzu, Kyoto, Japan). The IR spectra were measured on a Nicolet 6700 FT-IR spectrometer using KBr discs.

Cytotoxicity assay
All isolated compounds (1-10) were subjected to cytotoxic evaluation against KB (human epidermoid carcinoma), HeLa S-3 (human cervical carcinoma), HT-29 (human colon adenocarcinoma), MCF-7 (human breast adenocarcinoma) and HepG-2 (human liver carcinoma) cell lines employing the colorimetric method [16]. Doxorubicin was used as the reference substance which exhibits activity against five cancer cell lines. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (Sigma Chemical Co., USA) was dissolved in saline to make a 5 mg/mL stock solution. Cancer cells (3 × 10 3 cells) suspended in 100 μg/wells of MEM medium containing 10% fetal calf serum (Gibco BRL, Life Technologies, NY, USA) were seeded onto a 96-well culture plate (Costar, Corning Incorporated, NY, USA). After 24 h pre-incubation at 37 °C in a humidified atmosphere of 5% CO 2 /95% air to allow cellular attachment, various concentrations of test solution (0.1, 0.3, 1.0, 3.0, 10.0, 30.0, and 100.0 μM, each 10 μL/well) were added and these were then incubated for 48 h under the above conditions. At the end of the incubation, 10 μL of tetrazolium reagent was added into each well followed by further incubation at 37 °C for 4 h. The supernatant was decanted, and DMSO (100 μL/ well) was added to allow formosan solubilization. The optical density of each well was detected using a Microplate