Two New Coumarins from Micromelum falcatum with Cytotoxicity and Brine Shrimp Larvae Toxicity

Two new coumarins, 7-methoxy-8-(2-hydroxmethyl-1-O-isovaleryl-4-butenyl)-coumarin (1) and 7-methoxy-8-(1-hydroxy-2-O-β-glucopyranosyl-3-methyl-4-butene-1-yl)coumarin (2), and twelve known coumarins 3–14 were isolated from the stem bark of Micromelum falcatum. The structures of compounds 1–14 were elucidated by extensive spectroscopic data analyses. The toxicity of compounds 1–14 was tested using a brine shrimp assay and in vitro antiproliferative assay against mammary cancer (F10) and lung cancer (HvEvc) cell lines by the MTT method. Some compounds had moderate activities. All compounds were also tested against the microorganisms Bacillus subtilis, Bacillus thuringiensis and Escherichia coli, but no activity was observed.


General
Optical rotation was measured on Polaptronic-HNQW5 high-resolution polarimeter. NMR spectra were recorded on a Bruker DRX-500 spectrometer with SiMe 4 as internal standard. ESI-MS was measured with a API2000 LC/MS/MS mass spectrometer (Applied Biosystems). HREI-MS was recorded on a Thermo MAT95XP spectrophotometer. Silica gel (200-300 mesh, Qingdao Haiyang Chemical Plant, Qingdao, China) and Sephadex LH-20 (Pharmacia) were used for column chromatography. Thin layer chromatography (TLC) was carried out on precoated silica gel G plates (Qingdao Haiyang Chemical Plant, Qingdao, China) and spots were visualized by spraying the plates with 50% H 2 SO 4 solution, followed by heating. Semi-preparative RPHPLC was carried out on ODS columns (YMC-Pack ODS-5-A, 250  10 mm, 5 m, YMC) with the CH 3 OH-H 2 O solvent system as eluents. A Waters 600 HPLC system equipped with a Waters 996 photodiode array detector was used for HPLC analysis.

Plant Material
The stem bark of M. falcatum (Lour.) Tan. was collected in October 2007 in Sanya, Hainan Province, China, and identified by Prof. Si Zhang. A voucher specimen is deposited at the Herbarium of South China Sea Institute of Oceanology (accession number: Dajian 020).

The Brine Shrimp Larvae Lethality Bioassay
According to the method described by Wanyoike [20], brine shrimp eggs (Ocean Star International, Inc., USA) were hatched in a large beaker containing natural sea water (South China Sea) and they were cultured at room temperature for 48 h. With the help of a light source, the larvae grouped together on one side of the vessel and were easily collected for the assay. The compounds 1-14 were dissolved in dimethyl sulfoxide (DMSO) at the concentration of 50 mg/mL, and then diluted in 96 well plate with 200 µL sea water for testing at the final concentrations of 5, 50 and 500 g/mL. Each test was processed in triplicate with approximate ten larvae. Brine shrimps were counted under a magnifying glass after 24 h of incubation and maintaining the 96 well plates under illumination. The controls were prepared in the same manner except that the test samples were omitted. The lethality of dead larvae was recorded and used for calculating the LC 50 by the Lanyu LC 50 analysis program (Version 1.01).

Antiproliferative Assays
Antiproliferative activities of compounds were evaluated by the MTT method using mammary cancer (F10) and lung cancer (HvEvc) cell lines. In MTT assay, the cell suspensions (200 L) at a density of 1 × 10 5 cells mL −1 were plated in 96-well microtiter plates and incubated for 24 h at 37 °C in a humidified incubator at 5% CO 2 . The tested compound solution (2 L in DMSO) at different concentrations was added to each well and further incubated for 72 h in the same condition. Then, the MTT solution (50 L) was added to each well and incubated for 4 h. The old medium (150 L) containing MTT was then gently replaced by DMSO. Absorbance was then determined on a Spectra Max Plus plate reader at 490 nm.

Antibacterial Assay
Compounds were tested against the microorganisms Bacillus subtilis, Bacillus thuringiensis, Escherichia coli and rifampicin was used as positive control for the three microorganisms. Procedures for the antimicrobial susceptibility assays were performed using a modified method [21]. Compounds were dissolved in absolute DMSO to give the needed amounts of 0.1, 1.0, and 10 g per 6 mm diameter paper disk, respectively. Twelve cm diameter dishes, filled with LB medium, were set for each microbe species. The inhibition zones surrounding each filter paper disk were measured at the end of an incubation period of 24 or 48 h at 27 °C. The absolute DMSO alone showed no inhibition zone (control).