Bioactivity-guided isolation of new antiproliferative compounds from Juniperus foetidissima Willd.

Abstract Based on a literature survey on cytotoxic medicinal plants, Juniper species were identified as interesting source of antitumor compounds. Using bioassay-guided fractionation against Caov-4 cancer cells on acetone extract of leaves and branchlets of Juniperus foetidissima led to the isolation of a new 3H-benzofuaran-2-one: 4-methyl-3-methoxy-3H-benzofuaran-2-one (1), a new sesquiterpene: 4,9(α)-dihydroxy-nardosin-6-en (2) and an already known labdane-type diterpene: 15-hydroxy-8(17),13(E)-labdadiene-19-carboxilic acid (3). Compounds 1–3 exhibited cytotoxic effects, with moderate cytotoxicity against the EJ-138 bladder and CAOV-4 ovary cancer cell lines.


Introduction
Bladder and ovary cancers are common cancers in women. Discovery of safe and potent anticancer compounds is thus important to treat these cancers. Ethnobotanical knowledge and biological evaluation of medicinal plants can provide investigational new drugs to develop effective cancer therapies. Cupressus semipervirens, Juniperus communis, Juniperus excelsa, Juniperus foetidissima, Juniperus oblonga, Juniperus sabina, Platycladus orientalis and Taxus baccata are Iranian conifers. A few of them have exhibited interesting cytotoxic activities against a panel of cancer cells (Emami et al. 2005(Emami et al. , 2007Sadeghi-aliabadi et al. 2010). Among them, J. foetidissima (Cupressaceae), locally known as 'Ardush' , is a tree with 5-15 m high, crown slender conical, branched to the ground. J. foetidissima has leaves of 2.0-3.4 mm, with branchlets of 1.2-2.0 mm diameter and 1-3 seeds per cone which differentiate it from J. excelsa with 0.6-1.4 mm leaves, 0.7-1.5 mm in diameter branchlets and 3-6 seeds per cone. It is grown in the mountains of Greece, Albania, Yugoslavia, Asia Minor to transcaucasia and north parts of Iran (Riedl 1968;Emami et al. 2007;Marcysiak et al. 2007). In the Iranian traditional medicine, Rhazes (854 CE -925 CE) in an old pharmacopoeias named Alhavi have suggested fruits and leaves of Juniper in combination with figs can be used for the treatment of a type of hyperplasia inside the nose (Al-rhazes & Al Havi 2005). In recent studies, a wide range of cytotoxic and antibacterial activities of the compounds of Juniper species stimulated our interest to study the chemical composition of J. foetidissima (El Sawi & Motawe 2008;Muto et al. 2008;Bahri et al. 2013;Robichaud et al. 2013). previous studies on this plant described β-thujone, cedrol, sabinene and abietal as the main components of essential oil of its leaves and fruits (tunalier et al. 2002), as well as cedrane-type sesquiterpene oxides such as 8, 14-cedranozide, 8,14-cedranoxide, 8,14-cedranediol, 8-cedren-13-ol and cedrolic acid in its nonpolar chloroform extract (Baggaley et al. 1968). We report here a cytotoxicity-guided fractionation and isolation of a new 3H-benzofuaran-2-one, a new sesquiterpene and an known labdane-type diterpene from the acetone extract of J. foetidissima leaves and branchlets. these compounds exhibited cytotoxic activities against EJ-138 and Caov-4 Human bladder and ovary cancer cells.

General
Isolation of compounds were carried out with open column chromatography using silica gel (60-200 μm, Merck, Germany) and normal preparative HplC using a YMC-pack-Sil column (250 × 20 mm i.d., YMC, Japan). the structures of the compounds were elucidated by 1 H-nMR, BB 13 C-nMR, DEpt, COSY, HMBC, nOESY, ft-IR and HREI-MS. the nMR spectra were acquired with Bruker AV-400 using CDCl 3 as solvent. the Infrared spectra were acquired by Rayleigh WQf-510 ftIR spectrophotometer, with naCl discs. the HREI-MS spectra were obtained with Waters Q-tOf Micro YA019 mass spectrometer in m/z and EI-MS spectra with Varian MAt 312 spectrometer.

Plant material
the leaves and branchlets of J. foetidissima were collected from Azadshahr (Golestan province, north of Iran), in July 2011. the plant was authenticated by Dr Iraj Mehregan and a voucher specimen (no.1419) deposited at the Herbarium of the pharmacognosy Department, School of pharmacy, Isfahan University of Medical Sciences, Isfahanl, I.R. Iran.

Cytotoxicity MTT assay
EJ-138 and CAOV-4 cancer cell lines from pasteur Institute, Iran, were grown adherently in RpMI-1640 media, supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ ml streptomycin at 37 °C in 5% CO 2 /95% air. Cells were seeded at 5000 cells per well in 5% CO 2 at 37 °C in RpMI medium containing 10% fBS, 100 units/ml penicillin and 100 μg/ml streptomycin, in 96-well plates. After overnight incubation, breast cancer cells were treated with different concentrations (1, 10, 20, 40, 80 and 100 μM) of compounds 1-3 for 48 h. Doxorubicin hydrochloride (Ebewe pharma, Unterach, Austria) as standard drug was applied as a positive control at concentrations of 0.01, 0.10, 1.00 and 10.00 μM. Mtt was added to the wells following with incubation for another 4 h at 37 °C. Each experiment was independently carried out in triplicates and the absorbance was read by the microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm. Cell viability percentages using the formula: (mean OD of treated cells /mean OD of control cells) × 100 were expressed as percent of control cells which were not treated (Zarei et al. 2013).

Statistical analysis
All data are reported as mean ± SD of the mean and the IC 50 values were calculated using Excel based program. One-way AnOVA: post-hoc Dunnett test was used and p < 0.05 was considered to indicate a statistically significant difference.