Labdane and Abietane Diterpenoids from Juniperus oblonga and Their Cytotoxic Activity

A phytochemical investigation of the whole plant of Juniperus oblonga led to the isolation of one previously undescribed labdane diterpenoid, (4R,5S,9S,10R)-13-des-ethyl-13-oxolabda-8(17),11E-dien-19-oic acid (1), together with nine known diterpenoids (2–3, 6–12), two lignans (4, 5), and a coumarin (13). The structures of all the compounds were elucidated on the basis of spectrometric data, primarily one-dimensional (1D)- and two-dimensional (2D)-NMR and mass spectrometry. Electronic circular dichroism (ECD) calculations determined the absolute configuration of 1. In addition, the isolated compounds were evaluated for their cytotoxic activity against three human tumor cell lines (HepG2, MCF-7, and HeLa). 6,12-Dihydroxyabieta-5,8,11,13-tetraen-7-one (6) showed moderate cytotoxicity against all three cell lines with IC50 values ranging from 24.41 μM to 58.39 μM and trilobinone (10) showed weaker activity with IC50 values ranging from 56.93 μM to 79.98 μM. None of the isolated diterpenoids have been previously reported from Juniperus oblonga, and five compounds are here reported from the genus Juniperus for the first time.


Introduction
Juniperus oblonga M. Bieb. belongs to the family Cupressaceae (Cypress family). The genus Juniperus is one of the largest conifer genera and it is widely distributed in the temperate regions of the Northern Hemisphere [1]. Juniperus is a well-known source of folk medicines in several parts of the world [2]. For traditional medicine, some species represent drugs with several properties, such as antitussive and haemostatic activities [3], antifertility effect [4], and antitumor activity [5]. The berries also have antimicrobial activity and anti-hypercholesterolemic activity [6,7]. The Juniperus species are used as an insect repellent and in the treatment of fever and dysuria in Bhutan [8]. Juniperus oblonga belongs to the subgenus Oxycedrus of the genus Juniperus. Its ripe berries have been found to exert diuretic and antiscorbutic effects [9], and the essential oils that were obtained from the fruits and branchlets of this plant possess antioxidant and anti-glycation properties [10].
Our continuing phytochemical investigation on Juniperus oblonga has led to the discovery of one undescribed labdane diterpene, nine known diterpenes, two known ligans, and a coumarin. Herein we report the isolation and structural elucidation of the undescribed diterpenoid by extensive spectroscopic techniques and chemical means. The CD exciton chirality method and calculated ECD spectra determined the absolute configuration of the new compound. X-ray diffraction analysis determined the crystal structures of compound 3 and 6. This is the initial report of the crystal structure of 6. The cytotoxicity of the isolated compounds was evaluated against three human tumor cell lines-HepG2, MCF-7, and HeLa.
When isolated quantities permitted, the isolated compounds were evaluated for cytotoxicity against three cancer cell lines, including a human hepatocellular carcinoma cell line (HepG2),a human breast cancer cell line (MCF-7) and a human cervical carcinoma cancer cell line (HeLa), as well as a normal human liver cell line (LO2), at an initial concentration of 4 mg/mL (Table 2) using a standard MTT assay [30]. The IC50 values were determined for compounds that showed greater than 50% inhibition at this concentration. Compound 6 exhibited moderate cytotoxicity against all three cell lines, with IC50 values ranging from 24.41 to 58.39 μM, while 10 showed weaker activity, with IC50 values that ranged from 56.93 to 79.98 μM ( Table 3). The remaining compounds did not exhibit cytotoxicity. The absence of cytotoxicity against the normal human liver cell line (LO2) may have some importance. While none of the compounds that were isolated in this study have the potential for development into clinically useful anticancer agents, other labdane and abietane diterpenoids have shown antibacterial, antifungal, and anti-inflammatory activities [17,[31][32][33]. Members of these diterpene families not being cytotoxic to normal human cells may enhance their potential for development for these other bioactivities.  When isolated quantities permitted, the isolated compounds were evaluated for cytotoxicity against three cancer cell lines, including a human hepatocellular carcinoma cell line (HepG2),a human breast cancer cell line (MCF-7) and a human cervical carcinoma cancer cell line (HeLa), as well as a normal human liver cell line (LO2), at an initial concentration of 4 mg/mL (Table 2) using a standard MTT assay [30]. The IC 50 values were determined for compounds that showed greater than 50% inhibition at this concentration. Compound 6 exhibited moderate cytotoxicity against all three cell lines, with IC 50 values ranging from 24.41 to 58.39 µM, while 10 showed weaker activity, with IC 50 values that ranged from 56.93 to 79.98 µM ( Table 3). The remaining compounds did not exhibit cytotoxicity. The absence of cytotoxicity against the normal human liver cell line (LO2) may have some importance. While none of the compounds that were isolated in this study have the potential for development into clinically useful anticancer agents, other labdane and abietane diterpenoids have shown antibacterial, antifungal, and anti-inflammatory activities [17,[31][32][33]. Members of these diterpene families not being cytotoxic to normal human cells may enhance their potential for development for these other bioactivities.

X-ray Diffraction Analyses
The single crystal X-ray diffraction data were collected on a ROD, Synergy Custom system, HyPix diffractometer (Rigaku, Japan). The crystal was maintained at 159.99(10) K during data collection. The structures were solved using Olex2 with the ShelXT structure solution program using intrinsic phasing and refined with the ShelXL refinement package while using least squares minimization.

Extraction and Isolation
Fresh plant samples were freed of extraneous matter, air-dried, and then milled to a coarse powder. A 1 kg portion of each dried sample was extracted with methanol (3 × 4 L). After the removal of solvent, the resulting viscous oil was dispersed in 1 L of methanol: water (9:1) and extracted with n-hexane (3 × 1 L). The hexane-depleted hydroalcoholic phase was freed of methanol, dispersed in distilled water (1 L), and then sequentially extracted with dichloromethane and water saturated n-butanol (each 3 × 1 L). The resulting solvent-soluble fractions were each evaporated to dryness in vacuo, while the residual aqueous fraction was freed of solvent and lyophilized. The dichloromethane part (7.46 g) was fractionated by column chromatography on silica gel, eluting with a step-gradient of CH 2 Cl 2 /MeOH (100:0, 99:1, 98:2, 97:3, 95:5, 90:10, 80:20, 50:50, 100:0) to give twenty-seven fractions (Fr. .
Compound 3 was crystallized by the slow evaporation of the CH 2 Cl 2 /MeOH mixture from Fr. 5.

Cytotoxicity Assay
MTT assay was used to measure the in vitro cytotoxicity of the isolated compounds. Human breast adenocarcinoma cell line (MCF-7), human liver hepatocellular carcinoma cell line (HepG2), and human cervical cancer cell line (HeLa) were cultured in Dulbecco's modified Eagle's medium (DMEM) and Roswell Park Memorial Institute medium (RPMI 1640), which was supplemented with 10% fetal bovine serum (FBS) at 37 • C in a humidified atmosphere of 5% CO 2 . The cells were cultured in 96-well plates for 24 h and then treated with test compounds at various concentrations (8-125 µM) for 72 h. After incubation for another 4 h with a 20 µL aliquot of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] solution (5 mg/mL in PBS), the medium was discarded, and 150 µL of DMSO was added to dissolve the produced formazan. The absorbance was measured at 490 nm and 570 nm using a microplate reader. Doxorubicin was used as a positive control. Each experiment was carried out in triplicate. The IC 50 values were calculated using Graphpad Prism 5 software.

Calculations of the CD Spectra
The theoretical calculations of the model molecules were carried out using Gaussian 09. MMFF94 was used to initially perform conformational analysis. The conformers were optimized at the B3LYP/6-31 G (d) level. Room temperature equilibrium populations were calculated according to the Boltzmann distribution law.

Conclusions
Thirteen compounds, including one previously undescribed labdane diterpene, nine known diterpenoids, two lignans, and a coumarin were isolated from Juniperus oblonga, and their structures were primarily elucidated on the basis of NMR and MS studies. The absolute configuration of the new compound was determined by CD exciton chirality and calculated ECD methods. The crystal structures were determined for two of the diterpenes (3 and 6) by single crystal X-ray diffraction analyses. The isolated compounds were tested for cytotoxicity against three cell lines, with 6 showing moderate cytotoxicity against all three cell lines and 10 exhibiting weaker activity.
Supplementary Materials: The following are available online: NMR, MS data of compound 1, data of ECD calculation for compound 1, single crystal X-ray diffraction data and refinement parameters with bond lengths and bond angles of compound 3 and 6. IC 50 valves of selected compounds.
Author Contributions: M.K., V.A. and D.A. collected and identified the plant material, prepared and submitted voucher specimens and reviewed this manuscript. Y.Q. and R.P.B. conceived and performed the isolation, structure determination and cytotoxicity testing of the isolated compounds, prepared and reviewed this manuscript.