Dry sap samples were obtained in Ethiopia from the stem bark of Boswellia carterii and Commiphora pyracanthoides Engler in August 2009. The plant materials were identified by a botanist at Harbin Medicine University-Daqing (Daqing, China) and a voucher specimen was stored at the Department of Pharmacology (School of Pharmacy, Harbin Medicine University-Daqing).

Subsequent to being frozen for 24 h, 30 g of the air-dried frankincense and myrrh samples were crushed into a powder. The essential oils from each sample were obtained through hydrodistillation for 3 h, according to the AB method described previously (12). Subsequently, the essential oils were diluted with 1% Tween 80 for a bioactivity analysis. The solution was prepared by mixing the myrrh and frankincense essential oils in a 1:1 ratio.

Analyses of the constituents of the essential oils were performed using gas chromatography mass spectrometry (GC-MS; Agilent Technologies, Santa Clara, CA, USA) and the GCMS-QP2010S mass spectrometer (Shimadzu Corp., Kyoto, Japan) with Rtx^®-50 elastic quartz capillary column (30×0.25 mm, 0.25 μm) and helium carrier gas (Beijing AP BAIF Gases Industry Co., Ltd., Beijing, China). The injector temperature was 230°C and the interface and ion-source heating temperatures were 300°C and 230°C, respectively. The temperature program consisted of 60°C for 1 min and 220°C for 15 min, with a heating rate of 5°C/min. The column head pressure was 70 kPa, the EI-mode was 70 eV and the scan-range was 20–500 amu with a cycle time of 0.65 sec. Mass spectral correlations were performed using NIST05.

Human cell lines (American Type Culture Collection, Rockville, MD, USA) obtained from breast (MCF-7) and hepatocellular (HepG2) carcinomas and cervical (HeLa), skin (HS-1) and small cell lung (A549) cancers, were maintained in monolayer tissue culture Petri dishes prior to examination. RPMI-1640 medium was supplemented with 10% fetal bovine serum (both Sigma-Aldrich, St. Louis, MO, USA), 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mM/l glutamine and cultures were maintained in a humidified atmosphere at 37°C in 5% CO₂.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to determine the effects of frankincense and/or myrrh essential oils on cell proliferation in the MCF-7, HepG2, HeLa, HS-1 and A549 cell lines. Briefly, 5×10³ cells/well were evenly distributed and incubated on 96-well plates (Iwaki, Tokyo, Japan) overnight. The cells were then treated with frankincense, myrrh and a mixture of the essential oils at concentrations of 0, 5, 10, 20, 40, 60, 80, 160, 180, 320 and 640 μg/ml, and incubated for 24 and 48 h. Subsequently, the medium in each well was replaced with 20 μl MTT (5 mg/ml in PBS) and incubated at 37°C for 4 h. The purple-blue formazan precipitate was dissolved in 100 μl dimethyl sulfoxide and the optical density was measured at a wavelength of 570 nm on a 96-well plate reader (Thermo Labsystems, Franklin, MA USA). The IC₅₀ was calculated as the concentration of compounds that achieved a 50% inhibition of cell viability. Data were analyzed using a SlideWrite program (Advanced Graphics Software, Inc., Rancho Santa Fe, CA, USA) to determine the IC₅₀ of each drug independently.

Isobologram curves were derived as described previously (13): IC₅₀ A and B = D_A / IC_X,A + D_B / IC_X,B; where IC₅₀ A and B indicates the combination concentration of drugs A and B at 50% inhibition, IC_X,A and IC_X,B indicates the concentration of the drugs that result in 50% inhibition independently and D_A and D_B indicates the concentrations of the two drugs as a mixture to achieve 50% inhibition. The isobologram curve was generated by plotting doses of drugs A vs. B predicted to simultaneously achieve 50% cell growth inhibition. A standard line of Loewe additivity was included to indicate a lack of interaction, and points below and above the line indicated synergy and antagonism, respectively.

Flow cytometry was used for the quantitative measurement of apoptosis. Briefly, 1×10⁶ MCF-7 cells were treated with 0, 10, 20 and 40 μg/ml frankincense and/or myrrh essential oils for 24 and 48 h, respectively. The cells were then collected by trypsinization and washed once with cold PBS. BD tubes were used and 100 μl suspension was added to each labeled tube followed by 10 μl Annexin V-FITC and 10 μl PI (20 μg/ml). Following incubation for ≥20 min at room temperature in the dark, 400 μl PBS binding buffer was added to each tube without washing. Within 30 min, the mixtures were analyzed using flow cytometry (BD FACSAria; BD Biosciences, San Jose, USA).

The content of the extracted oil of myrrh and frankincense was ~0.41 ml (2.05%, ml/g) and 0.62ml (2.06%, ml/g), respectively, and the total ion figures of the constituents were obtained by GC-MS analysis. The area normalization method was adopted to integrate the total ion peaks and the minimum area of the comparatively small peaks was set. Using a standard mass spectrum, 76 components were identified that accounted for 87.54% of the total myrrh essential oil (Table I). In addition, 99 components were identified that accounted for 91.26% of the total frankincense essential oil (Table II).

Myrrh and frankincense essential oils exhibited an inhibitory effect on the cell lines and a dose-dependent inhibition effect was noted. Among the five cell lines, MCF-7 and HS-1 were sensitive to the myrrh and frankincense essential oils (Table III).

All points were identified above the standard line of Loewe additivity, therefore, no synergistic effects were identified in the isobologram and combination index curves (Fig. 1).

The flow cytometry results showed that the myrrh, frankincense and the mixture of essential oils were capable of inducing apoptosis in the MCF-7 cells in a concentration-dependent manner (Fig. 2). A dose-dependent induction of the apoptotic cells was performed to investigate the apoptosis rate. The early- and late-stage apoptosis rates of the MCF-7 cells induced by 40 μg/ml myrrh, frankincense and the mixture of essential oils were 36.0, 77.3 and 45.8%, respectively (P<0.01).