Coumarins and Xanthones from the Seeds of Mammea siamensis

Uma cumarina inédita, mammea E/BB ciclo D (1), juntamente com cinco compostos conhecidos, mammea E/BA ciclo D (2), suragina C (3), terapina B (4), 1,7-dihidroxixantona (5) e 1-hidróxi-5-metoxyxantona (6), foram isolados de sementes de Mammea siamensis. Suas estruturas foram caracterizadas usando dados de RMN 1D e 2D. Suragina C e terapina B mostraram atividade citotóxica contra adenocarcinoma de mama (MCF-7), câncer cervical humano (HeLa), câncer de colon (HT-29) e câncer oral humano (KB).


Introduction
Mammea siamensis (Miq) T. Anders.(Guttiferae), known in Thai as "Sarapi", is a small evergreen tree distributed in Thailand, Laos, Cambodia, Vietnam and Myanmar.The flowers of this plant have been used in traditional Thai medicine as a heart tonic.2][3][4] We have previously reported the isolation and structure determination of phenolic compounds from the seeds of this species. 5In a continuation of our study on this plant, we now report herein the isolation and structure elucidation of a novel compound, mammea E/BB cyclo D (1), together with three known coumarins, mammea E/BC cyclo D (2), 3 suragin C (3), 6 therapin B (4) 7 and two known xanthones, 1,7-dihydroxyxanthone (5) 8 and 1-hydroxy-5methoxyxanthone (6) 9 from the CH 2 Cl 2 extract (Figure 1).The cytotoxic activity of all isolates is also reported.

General procedures
Melting points were determined using a Fisher-John melting point apparatus.The optical rotation [α] D values were determined with a JASCO P-1020 polarimeter.UV spectra were measured with a UV-160A spectrophotometer (Shimadzu).The IR spectra were measured with a Perkin-Elmer FTS FT-IR spectrophotometer.The 1 H and 13 C NMR spectra were recorded using 500 MHz Varian UNITY INOVA and 300 MHz Bruker FTNMR Ultra Shield TM spectrometers.Chemical shifts were recorded in parts per million (δ) in CDCl 3 with tetramethylsilane (TMS) as an internal reference.The EIMS was obtained from a MAT 95 XL mass spectrometer.Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60 F 254 (Merck, 230-400 Mesh ASTM) and silica gel 100 (Merck, 70-230 Mesh ASTM), respectively.Precoated plates of silica gel 60 F 254 and reversed-phase (RP-18 F 254S ) were used for analytical purposes.

Extraction and Isolation
The seeds (224.5 g) of M. siamensis were extracted successively with CH 2 Cl 2 (500 mL) at room temperature for 5 days.The filtered samples were combined and the solvents were evaporated under reduced pressure to provide the CH 2 Cl 2 extracts (44.4 g).

Cytotoxicity assay
The procedure for cytotoxic assay was performed by sulphorhodamine B (SRB) assay as described by Skehan et al. 10 In this study, four cancer cell lines, MCF-7 (breast adenocarcinoma), HeLa (human cervical cancer), HT-29 (colon cancer) and KB (human oral cancer) were used.Camptothecin, the reference substance, exhibited activity toward MCF-7, HeLa, HT-29 and KB cell lines, with IC 50 range of 0.2-2.0μg mL -1 (Table 1).
The reported compounds were tested for their cytotoxicity against MCF-7 (breast adenocarcinoma), HeLa (human cervical cancer), HT-29 (colon cancer) and KB human oral cancer) cell lines.The results are summarized in Table 1.Only two coumarins, 3 and 4, were found to be active in this study.Suragin C (3) showed cytotoxic activities against all four cancer cell lines better than therapin B (4) (Table 1).It should be noted that the structural difference between suragin C (3) and therapin B ( 4) is only at C-6 (3 possesses a geranyl group while 4 contains a prenyl group).The presence of a geranyl moiety seems to be important for enhancing the cytotoxic activity.The anticancer drug used as a standard in our cytotoxic assay is camptothecin, which has an IC 50 in the range of 0.2-2.0μg mL -1 .