Two New Coumarins from the Leaves of Phellodendronamurense

Two new coumarins, PhellodenolsI (1) and J (2), were isolated from the leaves of Phellodendronamurense. The structures of these compounds were elucidated by extensive spectroscopic analysis. The two new compounds were tested against Hela, AGS, and MGC-803 human cancer cell lines and exhibited cytotoxic activities with IC50 values between 18.71± 5.23 μM and 36.92± 4.72 μM.


Introduction
Phellodendronamurense Rupr is an important member of the genus Phellodendron belonging to the family Rutaceae [1].It mainly distributes in Japan, Korea, and northeast of China, and has been used as a kind of traditional medicine for the treatment of intestinal function controlling and stomach aches for years [2].Modern pharmacological studies have established the anti-inflammatory, anti-psychic, and antioxidant activities of this plant [3].Previous phytochemical studies reveal the presence of coumarins, alkaloids, limonoidal triterpenes as well as phytosterols and their fatty acid estersin this plant [4].In our study, two new coumarins, phellodenolsI (1) and J (2), were isolated from the leaves of P.amurense (Figure 1).In this paper, we describe the structure elucidation of the two new compounds and their cytotoxicity against any human cancer cell lines.

General experimental procedure
Optical rotations were determined using a Perkin-Elmer 341 digital polarimeter.IR spectra were recorded on a FTIR-8400S spectrum and UV spectra were obtained on a Shimadzu UV2550 spectrometer.Melting points were determined without correction on a Fisher-Johns melting point apparatus (Fisher-Johns Scientific Company).High resolution mass spectra were measured on a Thermo Scientific LTQ-Obitrap XL (Thermo Scientific, Bremen, Germany).The NMR spectra were performed on Bruker AV Ш 600 spectrometers with TMS as an internal standard.Column chromatography was performed on silica gel (100-200 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, P. R. China) and Sephadex LH-20 (Pharmacia Biotech, Sweden).Precoated silica gel plates (F254, 0.25 mm, Qingdao Haiyang Chemical Co., Ltd., Qingdao, P. R. China) were used for TLC analysis.All solvents used for HPLC were HPLC grade (Fisher Scientific).

Plant material
The leaves of P. amurense were collected in November 2008 from Beijing, China and authenticated by Z Zhang.A voucher specimen of the plant (DLC-20101108) has been deposited at the herbarium of Institute of Medicinal Plant Development, Beijing, China.

Cytotoxicity assay
Cytotoxic testing was performed as the following previously described protocols [5,6].The cytotoxicity of the two new compounds were assessed using the MTT method against AGS, MGC-803 and Hela human cancer cell lines.AGS cells were cultured in Ham's F12 medium supplemented with 10% fetal bovine serum, while MGC-803 and Hela cells were cultured in DMEM medium.Cells were cultured in a 96-well plate and cultured 24 h.Dimethyl sulfoxide (DMSO) was used to dissolve the tested compounds and Taxol was used as a positive control.Each concentration was tested in triplicate.After incubation at 37 °C in 5% CO2 for 48 h, 10 μL of MTT (4 mg/mL) was added to each well and cultured for another 4 h.And then the supernatant was discarded and 150 μL DMSO was added to each well.The absorbance was detected on a microplate reader at a wavelength of 570 nm.

Results and Discussion
After successive stages of chromatography, the leaves of P. amurense extract yielded two new coumarins, phellodenolsI (1) and J (2).Their structures were elucidated on the basis of detailed analysis of the spectroscopic data.
Compound 1 was obtained as a white powder from in MeOH.Its molecular formula was deduced as C20H24O9 on the basis of 13CNMR data and the [M+Cl]-ion peak at m/z 443.1112 (calcd for C20H24ClO9, 443.1109) in the HRESIMS.The IR absorption bands indicated the presence of hydroxyl groups (3430 cm-1), a carbonyl group (1726 cm-1) and a double bond (1622 cm-1).The UV spectrum exhibited an absorption maximum for coumarin moiety (324 nm) [5].The planar structure of 1 was elucidated by analysis of the NMR spectra data.The 13CNMR and heteronuclear singlequantum coherence (HSQC) spectra displayed 20 carbon signals.

Figure 1 :
Figure 1: The structures of 1 and 2