Brenes ” in Costa Rica and their Cytotoxic Activity on Tumor Cell Lines

A composição química dos óleos essenciais obtidos de folhas, cascas e troncos de Ocotea gomezii e O. morae de populações silvestres da Costa Rica, está sendo descrita pela primeira vez. Os óleos de O. gomezii são constituídos principalmente por sesquiterpenóides enquanto os de O. morae apresentaram monoe sesquiterpenóides na mesma proporção. A análise da composição química por CG/EM e CG/DIC resultou na identificação de 166 componentes, correspondente a 89,4-98,1% dos óleos totais. Quando comparada a atividade de todos os óleos obtidos sobre linhagens de células CCF-STTG1, Hep3B, HepG2, H-460, AGS, N-87, SW-620, MCF-7 e VERO, observou-se que as células de astrocitoma foram as mais resistentes aos mesmos. Concluiu-se que os óleos essenciais de folhas, cascas e tronco de Ocotea gomezii e Ocotea morae podem conter alguns compostos tóxicos, mas o uso potencial dos mesmos contra as células tumorais foi muito baixo, pois são tóxicos na mesma extensão, para as linhagens de células tumorais e não-tumorais.


Introduction
The genus Ocotea (Lauraceae) is widely represented in the American Tropics with 300-400 species, being the largest genus of this family in Mesoamerica, with 102 species. 1 Lauraceae is a family with about 2500-3000 species of mostly tropical trees. 2 This family is an important component of cloud forests in Costa Rica where the individuals occur in high abundance and diversity. 3,4t can be recognized by the simple, alternate, stiff and aromatic elliptic to obovate leaves and fruits often borne in a cup.Worldwide, this family has a considerable economic importance because it is used as a source of timber for construction and furniture (Nectandra, Ocotea, Persea spp.), as a crop (Persea americana Mill., avocado), and to obtain flavors for food industry, perfumery and medicines (Cinnamomum zeylanicum Bl., C. cassia Pressl.).
Ocotea gomezii W.C. Burger is an unusual species distinguished by its ferruginous puberulence and broadly rounded leaves.It is a tree of about 6-10 m tall and endemic of Costa Rica.The geographic distribution of the species includes the Central Volcanic Mountain and extends from near Volcán Rincón de la Vieja in the West, to Moravia de Chirripó in the East of Costa Rica. 3 O. morae Gómez-Laur. is a tree of 18-22 m tall, with large fruits, readily recognized by their large 65 mm broad cupules, and 58 mm long and 60 mm wide fruits. 5This tree is also endemic of Costa Rica and its geographic distribution is restricted to the humid Cordillera de Tilarán slopes at ca. 850 m of elevation at the "Reserva Biológica Alberto M. Brenes", managed and administered by the Universidad de Costa Rica. 6everal phytochemical investigations have been performed on plants of the genus Ocotea.These plants are well known as a source of aporphine alkaloids, 7,8 lignans and neolignans 9,10 and phenylpropanoids. 11The chemical composition of the volatile oils of Ocotea species has been the subject of several studies.  Theswo endemic plants, Ocotea gomezii and O. morae, are barely studied from the chemical point of view.One report indicates that the aporphine alkaloid (+)-preocoteine is present in the bark of O. gomezii. 38everal Ocotea essential oils have been studied for their biological activities.For instance, oils from flower calyces and leaves of O. quixos ("flor de canela", American cinnamon) presented in vitro antioxidant, antibacterial and antifungal activities 24,39 and also anti-inflammatory 40 and antiplatelet properties. 41,42Oil from the calyces of O. bofo also presented antimicrobial and antioxidant activities. 28ssential oil from the stem bark of O. bracteosa presented molluscicidal activity, 33 and oils of O. duckei showed significant cardiovascular effects. 37Setzer and co-workers 29 also determined the activity of leaf essential oils of ten Ocotea species from Monteverde, Costa Rica, against cruzain (Chagas disease).
Regarding the cytotoxic effect against human cells, there are several studies with Ocotea species that include: O. endresiana, 36 O. floribunda, 35 O. meziana, 43 O. praetermissa, 36 O. tonduzii, 34 O. veraguensis, 44 O. whitei 44 and some unidentified ones (Ocotea new species "los llanos" and Ocotea new species "small leaf"). 43Basically, it has been demonstrated that some of them showed some toxicity against breast cancer cells. 34,36,43,44There is also one study with essential oil from O. floribunda that showed cytotoxicity against hepatoma cells HepG2. 35n this paper, we report the chemical composition and cytotoxic properties of six essential oils obtained from O. gomezii and O. morae from three different parts of the plants (leaves, bark and wood) and we show their complex composition and inespecific toxicities.
Identification of the oil components was performed using the retention indices on a DB-5 type column, 45 and by comparison of their mass spectra with either those published in the literature 46 or those from our own database.Integration of the total chromatogram, expressed as area percent, has been used to obtain quantitative compositional data.

Cytotoxicity assay
Various concentrations of essential oils, previously dissolved in 95% ethanol, were added to the plates in 100 µL of fresh medium and incubated for 48 h.After incubation, [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] (MTT) was added to each well to a final concentration 0.5 mg mL -1 and after 2 h at 37 °C medium was carefully removed from the plates and 95% ethanol was added to the wells with the purpose of dissolving formazan crystals. 47Absorbances were read at 570 nm and viability percentages were calculated, using samples incubated with 95% ethanol dissolved in culture medium as 100% viability values.(R)-(+) Limonene (Sigma Aldrich) was used as a standard to assure its values were always constant and the cells remained equally resistant to its effects in every performed experiment.LD 50 values were calculated from concentration versus viability plots using SlideWrite ® Plus 6.1 (Advanced Graphics Software, Inc., Carlsbad, CA), to obtain the concentrations able to induce 50% of cytotoxicity.

Statistical analysis
Cytotoxicity values were analyzed by ANOVA followed by Tukey's test and p < 0.05 were considered statistically significant.

Results and Discussion
From the hydrodistilled oils, a total of 166 compounds were identified, accounting for 89.4-98.1% of the total composition of the essential oils.The chemical composition of the volatiles is listed in Table S1 (see Supplementary Information, SI).
The oils were predominantly terpenoid in nature like other studied Ocotea oils from Costa Rica. 26,30,32f the six oils analyzed in this work only the leaf oil from O. morae contained a very small quantity of the benzenoid compounds benzaldehyde (0.1%) and the esters benzyl benzoate (0.4%) and benzyl salicylate (0.1%).Oils from Costa Rica Ocotea spp.][14][15][16][17][18][19][20][21][22][23][24][25] We determined the cytotoxicity of essential oils on eight different tumor cell lines, and non-tumoral cells (Vero).Cell lines were derived from tumors from lung, liver, colon, breast, stomach (primary tumor and liver metastasis) and an astrocytoma (Table 1).Except for bark oils, all the other volatiles showed statistically significant differences in toxicity between astrocytoma cells and the other ones, but the effect was not observed among the other cell lines, or between tumor cell lines and non-tumor Vero cells.When we compared the effect of the oils taking all the cell lines together, we only observed statistically significant differences between samples OgB and OgW; OgB and OmL and OgW and OgL.The effect of limonene was very low compared to the volatiles tested, but worked well as an internal standard for the experiments, to assure the cells were kept under the same degree of sensitivity along the time they were in culture.
One of the few Ocotea essential oils reported in the literature for its biological activities is O. quixos, which shows antifungical and antibacterial activities. 39This leaf oil presents as main identified compounds: caryophyllene, humulene and eremophyllene.The first compound is present in significant amounts in O. morae oils too, whereas humulene is present in small amounts in the oils of both plants analyzed here.
43,44 We showed here that these species have some toxicity, but due to their complex chemical composition, no assumptions can be made about the compounds responsible for these activities.Compounds such as germacrene D, β-caryophyllene, α-cadinene and αand β-pinene have been shown to be toxic on cell lines such as MCF-7, a breast carcinoma cell line. 43ll these compounds are present in the Ocotea species tested in this article, so they could be responsible for the relative toxicity observed here.Another compound found in these volatiles, 1,8-cineole, has been shown to induce apoptosis on KB cells (human oral epidermoid carcinoma), indicating that could play a role in the cytotoxicity observed here. 48Some antagonistic effects between some of the compounds present in these oils have been also reported in the literature. 43ompound

Table 1 .
LD 50 of six essential oils obtained from different parts of the plants O. gomezii and O. morae Superscript letters represent statistically significant differences in the cytotoxicity observed between some of the oils on the different cell lines (comparison is made in each column).p < 0.05 is considered statistically significant.Limonene is used as an internal standard.

Table S1 .
Percentage composition of the essential oils from O. gomezii and O. morae from Costa Rica Chemical Analysis of Essential Oils from Ocotea gomezii W.C. Burger and Ocotea morae Gómez-Laur.

Table S1 .
Percentage composition of the essential oils from O. gomezii and O. morae from Costa Rica (cont.)