A new coumarin from the roots of Micromelum minutum

Abstract A new coumarin, minutuminolate (1), together with eleven known coumarins (2–12), was isolated from the roots of Micromelum minutum. The structures of these compounds were established on the basis of their 1D and 2D NMR spectroscopic data. Compounds 2, 5, 10, 11 and 12 showed cytotoxicity against KB cell line. In addition, compounds 2, 3, 4, 7, 11 and 12 also showed weak cytotoxicity against NCI-H187 cell line.


Introduction
The genus Micromelum belongs to the flowering plants in the Rutaceae family. Micromelum minutum (Forst. F.) Wight & Arn (syn. M. pubescens Blume), or 'Hat-Sa Khun' in Thai, is a tropical small spineless tree that can reach up to 3 m in height. It is widely distributed in Southeast Asia and in Pacific islands. The stems, flowers, leaves and roots are used medicinally for a variety of indications (Pongboonrod 1958). A water decoction of the roots is traditionally used for treatment of fever, giddiness and haemorrhoids (Bunyapraphatsara & Chokechaijaroenporn 2000). Previous phytochemical studies on the genus Micromelum have revealed a number of coumarins, particularly the 6-and 8-prenylated 7-methoxycoumarins (Lamberton et al. 1967;Cassady et al. 1979;Das et al. 1984;Kamperdick et al. 1999), polyoxygenated flavonoids, triterpenoids (Tantishaiyakul et al. 1986;Rahmani et al. 2003;Susidarti et al. 2006), quinolone alkaloid (Tantivatana et al. 1983) and carbazole alkaloids (Siridechakorn © al. 2012). In addition, we have previously reported C-7-oxygenated coumarins from the fruits of M. minutum (Lekphrom et al. 2011). our continuous efforts to the phytochemistry research on the roots of this plant led to the isolation of a new coumarin (1) and eleven known coumarins (2-12) ( Figure 1). In this paper, we describe the isolation, structural elucidation and biological activities of these compounds.

General experimental procedures
Melting points were determined using an electrothermal IA9200 digital melting point apparatus (Bibby Scientific Limited, Staffordshire, UK). optical rotations were measured on a JASCoDIP-1000 digital polarimeter (JASCo Inc., USA), and UV spectra were recorded using an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). IR spectra were obtained using a Bruker Tenser 27 spectrophotometer (Bruker, Germany). NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer (Varian Inc., USA) using CDCl 3 , CD 3 oD and DMSo-d 6 as solvents. The internal standards were referenced from the residue of those solvents. The HR-eSI-ToF-MS were recorded on a Bruker microToF mass spectrometer (Brucker, Germany). Column chromatography (CC) was carried out on MeRCK silica gel 60 (230-400 mesh) (Merck, Darmstadt, Germany). Thin-layer chromatography was carried out with pre-coated MeRCK silica gel 60 PF254 (Merck, Darmstadt, Germany); the spots were visualised under UV light (254 and 365 nm) and further by spraying with anisaldehyde and then heating until charred.

Plant material
The roots of M. minutum were collected in Amphoe Muang, Khon Kaen Province, Thailand, in June 2011. The plant material was identified by Prof. Pranom Chantaranothai, Department of Biology, Khon Kaen University, Thailand, where a voucher specimen (S. Kanokmedhakul 10) was deposited.

Extraction and isolation
Air-dried roots of M. minutum (1.1 kg) were grounded into powder and then extracted successively with hexane, etoAc and MeoH. Removal of solvents from each extract under reduced pressure gave crude hexane (14.5 g, 1.32%), etoAc (123.2 g, 11.20%) and MeoH (66.2 g, 6.02%) extracts. The etoAc extract (100.0 g) was separated over silica gel CC, eluted with gradient systems of hexane-etoAc and etoAc-MeoH to give ten fractions, eF 1 -eF 10 . Fraction eF 2 was purified by silica gel CC, gradually eluted with CH 2 Cl 2 -etoAc and further purified by preparative TLC using CH 2 Cl 2 -etoAc (99 : 1) as developing solvent to give 2 as a colourless oil (65.0 mg, R f = 0.77). Fraction eF 7 was subjected to silica gel CC and gradually eluted with a gradient of hexane-etoAc to give five subfractions, eF 7.1 -eF 7.5 . Subfraction eF 7.2 was purified by silica gel CC, eluted with a gradient system of CH 2 Cl 2 -etoAc to afford 3 as a white solid (501.2 mg). The solid of fraction eF 7.4 was recrystallised with hexane-etoAc (30:70) to yield 4 as a white solid (175.4 mg). Fraction eF 8 was dissolved in etoAc, and the precipitate was filtered to obtain 5 as a white solid (2.7 g). The filtrate was evaporated in vacuo and a residue (10.5 g) was separated over silica gel flash column chromatography, eluted with gradient systems of hexane-etoAc and etoAc-MeoH to give nine fractions, eF 8.1 -eF 8.9 . Fraction eF 8.4 was dissolved in etoAc and the precipitate was filtered to yield 6 as a white solid (1.9 g). Fraction eF 8.5 was purified by preparative TLC using hexane-etoAc (60:40) as developing solvent to yield 1 as a colourless oil (25.7 mg, R f = 0.37). Fraction eF 9 was separated on silica gel CC, eluted with a gradient system of hexane-etoAc to give nine subfractions, eF 9.1 -eF 9.9 . Subfraction eF 9.2 was purified by preparative TLC using CH 2 Cl 2 -etoAc (40:60) as developing solvent to yield 7 as a colourless oil (25.7 mg, R f = 0.44). Subfraction eF 9.4 was subjected to silica gel CC and eluted with a gradient system of hexane-etoAc to give four subfractions, eF 9.4.1 -eF 9.4.4 . Subfraction eF 9.4.2 was further purified by preparative TLC using hexane-etoAc (40 : 60) as developing solvent to yield 8 as a white solid (591.7 mg, R f = 0.24). The solid in subfraction eF 9.4.3 was recrystallised from MeoH to yield 9 as a white solid (95.2 mg). The MeoH extract (60.2 g) was separated on silica gel CC, eluted with gradient systems of hexane-etoAc and etoAc-MeoH to give six fractions, MF 1 -MF 6 . Fraction MF 4 was separated by CC, eluted with a gradient system of hexane-etoAc to give ten subfractions, MF 4.1 -MF 4.10 . Subfraction MF 4.5 was purified by silica gel CC, eluted with a gradient system of hexane-CH 2 Cl 2 and further purified by preparative TLC using CH 2 Cl 2 -etoAc (95 : 5) as developing solvent to yield 12 as a white solid (8.8 mg, R f = 0.34). Fraction MF 4.8 was separated by silica gel CC, eluted with a gradient system of CH 2 Cl 2 -etoAc to give 11 as a white solid (4.6 mg). Fraction MF 5 was dissolved in etoAc and the precipitate was filtered to yield an additional amount of 5 (49.3 mg). Fraction MF 6 was purified by silica gel CC, eluted with a gradient system of hexane-etoAc and preparative TLC using hexane-etoAc (95:5) as developing solvent to yield 10 as a white solid (9.0 mg, R f = 0.33).

Cytotoxicity assay
Cytotoxic assays against human epidermoid carcinoma (KB), human small cell lung cancer (NCI-H187), and human breast cancer (MFC-7) cell lines were performed employing the colorimetric method as described by Skehan and co-workers (Skehan et al. 1990). The reference substances were ellipticine and doxorubicin.

Conclusion
Chemical investigation of the etoAc and MeoH extracts from the roots of M. minutum led to the isolation of twelve compounds with classical coumarin characteristic of the genus. In this report, one new coumarin, minutuminolate (1), and eleven known compounds (2-12) were isolated. This is the first report of coumarins 6 and 7 isolated from Micromelum species. It was found that compounds 2, 5, 8, 9 and 10 have previously been isolated from the fruits of M. minutum. In addition, compound 5 is the main chemical constituent isolated from both fruits and roots of this plant.