New Coumarins from Clausena lansium Twigs

Clausena lansium (Lour.) Skeels is a distant relative of citrus fruit belonging to the Rutaceae family. Several parts of this plant have been used as a folk medicine in China and Taiwan. For example, the leaves have been used for the treatment of coughs, asthma and gastro-intestinal diseases, and the seeds for acute and chronic gastrointestinal inflammation and ulcers. In addition, the fruits are used for influenza, colds and abdominal colic pains in the Philippines. Recently, the seed extract of C. lansium was found to exhibit antifungal, antiproliferative, and HIV reverse transcriptase-inhibitory activities. Previous chemical investigations of this plant have revealed a number of alkaloids and coumarins. As parts of our continuing study on chemical constituents and biological activity of Thai medicinal plants, we report herein the isolation and structure elucidation of two new coumarins (5 and 9) along with seven known coumarins (1-4, 6-8) from the twigs of C. lansium as well as the evaluation of cytotoxicity against KB, MCF7 and NCI-H187 cancer cell lines. In addition, the H and C NMR spectral data of 7 are also reported herein for the first time. Results and Discussion


Introduction
Clausena lansium (Lour.)Skeels is a distant relative of citrus fruit belonging to the Rutaceae family.Several parts of this plant have been used as a folk medicine in China and Taiwan.For example, the leaves have been used for the treatment of coughs, asthma and gastro-intestinal diseases, and the seeds for acute and chronic gastrointestinal inflammation and ulcers. 1 In addition, the fruits are used for influenza, colds and abdominal colic pains in the Philippines. 2Recently, the seed extract of C. lansium was found to exhibit antifungal, antiproliferative, and HIV reverse transcriptase-inhibitory activities. 35][6] As parts of our continuing study on chemical constituents and biological activity of Thai medicinal plants, we report herein the isolation and structure elucidation of two new coumarins (5 and 9) along with seven known coumarins (1-4, 6-8) from the twigs of C. lansium as well as the evaluation of cytotoxicity against KB, MCF7 and NCI-H187 cancer cell lines.In addition, the 1 H and 13 C NMR spectral data of 7 are also reported herein for the first time.

Results and Discussion
The combination of CH 2 Cl 2 and acetone extracts of twigs of C. lansium was separated by chromatographic techniques to yield two new coumarins, clausenalansimin A (5) and B (9), together with seven known compounds (1-4, 6-8).All structures were elucidated using spectroscopic data and compared with those reported in the literature.
Only the stable compounds and sufficient quantity were evaluated for their cytotoxicity against three human cancer cell lines including oral cavity cancer (KB), breast cancer (MCF7) and small cell lung cancer (NCI-H187).The results of cytotoxicity of the tested compounds (1-3, 5 and 6) are summarized in Table 2.All these compounds showed weak activity with cytotoxicity against KB, MCF7 and NCI-H187 cell lines, except coumarins 3 and 6 which were found to be inactive with MCF7 cancer cell line.

General procedures
The optical rotation [a] D values were determined with a Bellingham & Stanley ADP440 polarimeter.UV-Vis spectra were recorded with a Perkin-Elmer UV-Vis spectrophotometer.The IR spectra were recorded with a Perkin-Elmer FTS FT-IR spectrophotometer.The NMR spectra were recorded using 400 MHz Bruker spectrometer.Chemical shifts were recorded in parts per million (d) in CDCl 3 with tetramethylsilane (TMS) as an internal reference.The HRMS was obtained from a MicroTOF, Bruker Daltonics or MAT 95 XL mass spectrometers.Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60 H (Merck, 5-40 μm) and silica gel 100 (Merck, 63-200 μm), respectively.Precoated plates of silica gel 60 F 254 were used for analytical purposes.

Plant material
The twigs of C. lansium were collected in April 2008 from Nan Province, northern part of Thailand.Botanical identification was achieved through comparison with a voucher specimen number QBG 25077 in the herbarium collection of Queen Sirikit Garden, Mae Rim District, Chiang Mai, Thailand.

Cytotoxicity assay
The procedures for cytotoxic assay were performed by sulphorhodamine B (SRB) assay (anti-KB and MCF7) and colorimetric method (anti-NCI-H187) as described by Skehan et al. 6 In this study, three cancer cell lines, MCF7 (breast cancer), NCI-H187 (human, small cell lung cancer) and KB (oral human epidermal carcinoma) were used.Doxorubicin was the reference substance in this study.