Essential oil composition and cytotoxic activity in two species of the plant genus Vismia (Hypericaceae) from the Venezuelan Andes

Introduction: The Vismia genus belongs to the Hypericaceae family and comprises around 57 species of which 17 have been located in Venezuela. Previous investigations have been carried out in extracts as well as pure isolated compounds, revealing antimicrobial, antioxidant and anti-HIV, among other, biological activities. Objective: This investigation aims to determine the cytotoxic activity of essential oils from leaves of Vismia baccifera Triana & Planch (VBJ and VBV) and Vismia macrophylla Kunth (VM) collected in three different locations of the Venezuelan Andean region. Methods: Essential oils obtained by hydrodistillation were analyzed using gas chromatography-mass spectrometry (GC-MS) and their cytotoxic activity was analyzed following the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Human tumor cell lines from SKBr3, MCF-7 and PANC-1, two breast carcinomas and one pancreatic adenocarcinoma of ductal type, were tested with the oil samples and human dermis fibroblasts were used as non-tumor cells. Results: β-caryophyllene and trans-caryophyllene were present as major components in VBJ and VBV, respectively, while γ-bisabolene was the main component in the VM sample. Anticancer activity was observed on V. baccifera essential oil against SKBr3, MCF-7 and PANC-1. The selectivity index showed that VBV is highly selective against the SKBr3 cell line and has no activity against non-tumor cells. Conclusions: These results are considered a contribution to natural products research and may provide supportive data for future studies on cancer.


MATERIALS AND METHODS
Gas chromatography (GC): GC analyses were performed on a Perkin-Elmer AutoSystem gas chromatograph equipped with flame ionization detectors. Two capillary columns of different polarities were used: a 5 % phenylmethyl polysiloxane fused-silica column (AT-5, Alltech Associates Inc., Deerfield, IL) (60 m × 0.25 mm, film thickness 0.25 μm) and a polyethylene glycol fused-silica column (AT-WAX, Alltech Associates Inc., Deerfield, IL) of the same dimensions. The initial oven temperature was 60 °C; it was then heated to 260 °C at 4 °C/ min and the final temperature was maintained for 20 min. The injector and detector temperatures were 200 °C and 250 °C, respectively. The carrier gas was helium at 1.0 ml/min and the sample was injected using a split ratio of 1:100. Retention indices were calculated relative to C 8 -C 24 n-alkanes, using only the AT-5 capillary column and comparing values reported in the literature (Davies, 1990;Adams, 2007). The following conditions were applied: source temperature 230 °C; quadrupole temperature 150 °C; carrier gas helium, adjusted to a linear velocity of 34 m/s; ionization energy, 70 eV; scan range 40-500 amu; 3.9 scans/s. The injected volume was 1.0 μl of a 2 % dilution of oil in n-heptane. A Hewlett-Packard ALS injector was used with a split ratio of 1:100. The identification of the oil components was based on the Wiley Registry of Mass Spectral Data (6 th ed.) and NIST 05 data base library, followed by comparisons of mass spectral (MS) data with published literature (Adams, 2007) and the retention index calculation.
Cytotoxic activity assay: This analysis was carried out following the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. This method is based on the conversion of MTT (yellow tetrazolium salt) to purple formazan crystals by living cells that determines mitochondrial reductases activity (Mosmann, 1983;van Meerloo, Kaspers, & Cloos, 2011). Cell lines were used to determine the cytotoxic activity of the essential oils of the VM, VBJ and VBV samples. Samples at concentrations of 0.001, 0.01, 0.1, 1, 5, 10, 15, 25 and 100 μg/ml were placed along with a positive control (Taxol ® ) in 96-well plates (5 × 10 3 cells/well) and incubated at 37 °C with 5 % CO 2 for 24 h, to allow cells adhesion. After incubation time, 50 μl of sample dilutions and reference drug were added and re-incubated at 37 °C with 5 % CO 2 for 3 days. After 72 h of incubation, the culture medium was removed and cells were treated with 50 μl of MTT at a concentration of 0.4 mg/ml, allowing the formation of formazan crystals during 3 hours. These solid crystals were dissolved in 50 μl of DMSO and optical density was measured at 570 nm (DO 570 ) by using an ELISA spectrophotometer (Sunrise™ TECAN, Switzerland) (Raybaudi-Massilia et al., 2015;Suárez et al., 2016). Assays were conducted in triplicate and results were expressed as cytotoxicity percentages (% C), calculated using the following equation: Selectivity index: Selectivity index (SI) was expressed as the IC 50 (control cells) / IC 50 (tumor cell line) ratio. A selectivity index > 1 indicates that cytotoxicity on tumor cell lines surpasses that on healthy non-tumor cells (Nugroho et al., 2013).
Statistical analysis: Mean inhibitory concentration (IC 50 ) was calculated with 95 % confidence intervals by a linear regression equation and statistical analysis was performed using Prism 5.0 software (GraphPad, CA, USA). The global comparison was performed using two-way analysis of variance (ANOVA) followed by a Bonferroni's test for multiple comparisons. Significance levels of α= 0.05 and α= 0.001 were used.
In vitro cytotoxic activity was assessed in the three essential oil samples by using the MTT assay. VBJ inhibited the growth of PANC-1 (12.26 μg/ml), SKBr3 (4.99 μg/ml) and MCF-7 (20.49 μg/ml); while the essential oil of VBV only showed activity against SKBr3 (10.54 μg/ml); and the VM essential oil did not present any activity against the tumor cells evaluated in this investigation (Table 2).
According to the statistical analysis, VBJ essential oil exhibited significant differences (P < 0.001) for the tumor lines PANC-1 and MCF-7 with respect to the non-tumor cells, and significant differences (P < 0.001) compared to the VBV and VM groups for each tumor line. Furthermore, VBV is statistically significant (P < 0.001) for the tumor line SKBr3 with respect to the non-tumor cells, and also differs from the essential oil groups VBJ and VM in the SKBr3 tumor line (Table 2). On the other hand, VBV essential oil proved to be highly selective against SKBr3 cell line (9.5) and has no activity against non-tumor cells; showing a higher selectivity than Taxol ® (0.74), here used as a reference drug. VBJ essential oil, despite presenting cytotoxic activity for all tumor lines, showed the same activity against non-tumor cells, which indicates its low selectivity.
Despite the differences observed in the chemical composition of these oils, compounds like β-caryophyllene, germacrene D, β-elemene and α-humulene are present in all samples studied. Several investigations have explained that essential oil composition may vary considerably depending on the time of plant collection and are also influenced by environmental conditions at the harvesting date. In addition, the location where the plant is growing, either sun or shade, affects the composition and yield of the oil (Petinatti, Petinatti, Niehues, & Peporine, 2012) With respect to cytotoxic activity, VBJ essential oil proved to be active against the entirely cell lines assessed, with values between 6.16 to 20.49 µg/ml, being selective only to SKBr3 (SI 1.23), however, it showed activity against Hdf (Table 2). On the other hand, VBV showed a highly selective cytotoxic activity against SKBr3 cell line at 10.54 µg/ml, SI 9.49, and had no activity against Hdf. It is important to highlight that according to the selectivity index (SI 0.74), Taxol ® , used as reference drug, has no selectivity to this cell line. SKBr3 is considered a more aggressive and fast growing type of cancer than other carcinomas (Suárez et al., 2016). Thus, the results observed in the present investigation are considered a contribution to natural products research. As far as we know, there are no published works regarding the cytotoxic activity of essential oils from any Vismia species, however, antiproliferative activity was evaluated on essential oils obtained from 15 plants collected in Colombia by MTT assay on MCF-7, HeLa and HepG-2. Four species out of the 15 showed activity against MCF-7 and HeLa cell lines (IC 50 50 μg/ml). GC-MS analysis revealed that β-caryophyllene was a major component in those essential oils (Velandia, Quintero, Stashenko, & Ocazionez, 2018).
Ethical statement: authors declare that they all agree with this publication and made significant contributions; that there is no conflict of interest of any kind; and that we followed all pertinent ethical and legal procedures and requirements. All financial sources are fully and clearly stated in the acknowledgements section. A signed document has been filed in the journal archives.