Antioxidant and Antiproliferative Activities of the Essential Oils from Thymbra capitata and Thymus Species Grown in Portugal

The antioxidant and antiproliferative activities of the essential oils from Thymbra capitata and Thymus species grown in Portugal were evaluated. Thymbra and Thymus essential oils were grouped into two clusters: Cluster I in which carvacrol, thymol, p-cymene, α-terpineol, and γ-terpinene dominated and Cluster II in which thymol and carvacrol were absent and the main constituent was linalool. The ability for scavenging ABTS•+ and peroxyl free radicals as well as for preventing the growth of THP-1 leukemia cells was better in essential oils with the highest contents of thymol and carvacrol. These results show the importance of these two terpene-phenolic compounds as antioxidants and cytotoxic agents against THP-1 cells.


Introduction
Thymbra capitata and several Thymus species grown in Portugal produce essential oils (EOs) of interest for the food and fragrance industries and are also of medicinal value. Opposite to essential oils of T. capitata, characterized by a great chemical homogeneity with high carvacrol relative amounts, Thymus EOs show many chemotypes [1].
Earlier studies have shown the antioxidant potential of these EOs, but no previous report addressed the antiproliferative properties of the EOs from T. capitata and Thymus species grown in Portugal. For this reason, the main goal of the present work was to determine the antiproliferative activity of these EOs on the THP-1 leukemia cell line. Also, the in vitro antioxidant activity was evaluated with methodologies based on distinct mechanisms: one based on electron transfer and the other on hydrogen atom transfer (Trolox Equivalent Antioxidant Capacity (TEAC) and Oxygen Radical Antioxidant Capacity (ORAC), resp.).

Plant Material.
The aerial parts of Portuguese Thymbra and Thymus species, from collective or individual samples, were collected from wild-grown plants in the mainland of Portugal and in the Azores archipelago (Portugal). Plant material was stored at −20 ∘ C until extraction. In total, EOs isolated from 9 plant samples were evaluated for chemical composition and biological activity (Table 1). Certified   [13]. The absorbance was monitored spectrophotometrically at 735 nm for 6 min with a Shimadzu spectrophotometer 160-UV. The antioxidant activity of each sample was calculated as scavenging effect % (IA%) = (1 − / 0 ) × 100, where 0 is absorbance of the control and the absorbance in the presence of the sample. The values were compared with the curve for several Trolox concentrations and the values given as mM Trolox Equivalent Antioxidant Capacity.

Oxygen Radical Absorbance Capacity (ORAC) for EOs.
Fluorescein (FL) was the fluorescent probe used in the ORAC method, as described by Ou et al. [14]. EOs samples were diluted 1000 times in acetone before analysis. The equipment used was a Tecan Infinite M200 Microplate Reader. ORAC values were calculated according to [15]. Briefly, the net area under the curve (AUC) of the standards and samples was calculated. The standard curve was obtained by plotting Trolox concentrations against the average net AUC of the three measurements for each concentration. Final ORAC values were calculated using the regression equation between Trolox concentration and the net AUC and were expressed as mol Trolox/g EO. Tests were carried out in triplicate.

Antiproliferative Activity
Evidence-Based Complementary and Alternative Medicine 3 100 U/mL penicillin, and 100 g/mL streptomycin. Cells were incubated at 37 ∘ C in a humidified 5% CO 2 atmosphere.

Antiproliferative Activity Evaluation.
The growthinhibitory effect of EOs was measured using the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay adopted from Mosmann [16]. THP-1 cells were seeded in 96-well plate at 5 ⋅ 10 3 cells/well and exposed to different concentrations of EOs (10-500 g/mL) for 1 and 4 days. All test substances were dissolved in dimethyl-sulphoxide (DMSO). The solvent concentration in the incubation medium never exceeded 0.5%. Control cultures received the equivalent concentration of DMSO. After treatment, cells were incubated for 1 h in the usual culture conditions after addition of the same volume of medium containing MTT (2 mg/ mL). After this incubation, 150 L HCl (0.1 M) in isopropanol was added to dissolve the blue formazan crystals formed by reduction of MTT. Absorbance at 570 nm using a background reference wavelength of 630 nm was measured using a dual-wavelength Multiskan Spectrum (Thermo) plate reader. The mean absorbance values for the negative control (DMSO treated cells) were standardized as 100% absorbance (i.e., no growth inhibition) and results were displayed as absorbance (% of control) versus essential oil concentration. Tests were carried out in triplicate.

Statistical
Analysis. Data were analysed by one-way analysis of variance (ANOVA) using IBM SPSS Statistics version 20. Tukey's test was used to determine the difference at 5% significance level. Paired Student's -test was used in some tests to determine differences at 5% significance.

Chemical Composition of the EOs.
The identified components in the 9 EOs isolated from Thymbra and Thymus species, from mainland Portugal and Azores islands, are listed in Table 2 in their elution order on the DB-1 GC column, arranged according to the degree of correlation obtained after agglomerative cluster analysis based on the EOs chemical composition.
With variable amounts, these results are in accordance with previous studies on T. capitata as well as Thymus species grown in Portugal (for references, see Section 1).  Table 1.

Antioxidant
Activity. The EOs antioxidant activity was assessed using two methods, based on two distinct mechanisms: electron reaction-based method (TEAC) and hydrogen reaction-based method (ORAC).
Using TEAC method, the EO isolated from Th. caespititius collected in Terceira (Thc T) showed the highest antioxidant activity (27.3 mol TE/g EO) in contrast to the lowest antioxidant activity of Th. villosus EO (Thvl O: 3.7 mol TE/g EO). Large activity differences were observed among the 5 Th. caespititius EOs assessed (Table 2), with those isolated from plant material collected in mainland Portugal (Praia do Cortiço and Gerês) showing the lowest ability for scavenging ABTS radicals.
The lowest activities observed in Th. villosus and the two Th. caespititius EOs may be related with their main components: linalool and -terpineol, respectively (Tables 2 and 3), whereas the EOs with highest activity were dominated by thymol (Thc T) and carvacrol (Tc, Thc F, and Thc P). Although geraniol and 1,8-cineole predominated in Th. pulegioides (Thp SN) and Th. mastichina (Thm VC) EOs, they showed also relatively high percentages of thymol and carvacrol which may contribute to their scavenging capacity of ABTS (Table 2).
Dandlen et al. [3] did not observe correlation between Th. caespititius main EO component and the antioxidant activity after assaying the antioxidant activities of six Portuguese thyme EOs, by four methods: thiobarbituric acid reactive substances (TBARS), free radical scavenging activity through the capacity for scavenging DPPH (2,2-diphenyl-1-picrylhydrazyl), and the hydroxyl and superoxide anion radicals' scavenging. Indeed, in some cases, the same main component in different EOs of the same species but collected in different places of Portugal had different abilities for scavenging the free radicals and/or preventing lipid peroxidation. In the present work, in the group of Th. caespititius EOs, the highest Table 2: Percentage composition of the essential oils isolated from the aerial parts of Thymbra (Tc) and Thymus (Th) Portuguese species evaluated. Samples arranged according to the degree of correlation obtained after agglomerative cluster analysis based on the essential oils' chemical composition. For abbreviations and cluster analysis see Table 1 and Figure 1,    All components were identified based on a lab-made library created with reference essential oils, laboratory-synthesized components, laboratory isolated compounds, and commercial available standards. RI: in-lab obtained retention index relative to C 9 -C 21 -alkanes on the DB-1 column; t: traces (<0.05%). * Tentative identification based only on mass spectra. Evidence-Based Complementary and Alternative Medicine 7 activities were always in those in which thymol (Terceira) or carvacrol (Faial, Pico) prevailed (Tables 1 and 2).
As it was observed with the TEAC method, all thymol and carvacrol rich EOs (Thc T, Thc F, and Tc, Tables 2 and 3) showed also the highest scavenging peroxyl radicals capacity, by the ORAC method. Linalool and -terpineol rich EOs (Thc PC, Thc G, and Thvl O, Tables 2 and 3) showed the lowest activity.
Thymol and carvacrol's higher capacity for scavenging peroxyl radicals than linalool and 1,8-cineole was previously reported [17,18]. In contrast to the results obtained in the present work, -terpineol was considered by Bicas et al. [19] as possessing good capacity for scavenging peroxyl radicals. Since EOs are a complex mixture, this may reflect the presence of some other components that interfere with the capacity of this oxygenated monoterpene for scavenging peroxyl radicals.

Antiproliferative Activity.
The MTT assay is a sensitive, simple, and reliable method for evaluating antiproliferative activity of plant-based products. The cytotoxic activities of the essential oils of Thymbra and Thymus species from Portugal were studied with the THP-1 leukemia cell line by treating these cells with increasing amounts of the essential oils for 24 and 96 h (Figures 2 and 3). In both cases, essential oils decreased viability of THP-1 cells in a dose-dependent manner.
After one day (24 h), a great difference was observed between the cytotoxicities of the EOs from Th. mastichina and Th. caespititius from Gerês and even more from that of Pico ( Figure 2). These differences were detected even at low concentrations (<50 g/mL). At 10 g/mL, only 66% of THP-1 cells survived in the presence of the EO from Th. caespititius from Pico. At higher concentrations (>400 g/mL), EOs from Th. mastichina, Th. pulegioides, Th. caespititius from Praia do Cortiço, and Th. villosus showed the lowest cytotoxicity ( Figure 2). 1,8-Cineole, geraniol, -terpineol, and linalool were the main components of these EOs. Although thymol was present in low percentages in some samples (Thm VC and Thp SN), this was not enough for inhibiting the growth of THP-1 cells. Only EOs with higher thymol and carvacrol percentages were effective in preventing cell proliferation.
After four days (96 h), about 50% of THP-1 cells' survival was observed when exposed to 50 g/mL of Th. caespititius from Pico and T. capitata carvacrol rich EOs (Figure 3) These results support the importance of carvacrol and thymol among EOs components, since when present at low percentages the EOs did not inhibit the growth of THP-1 cells. The antiproliferative activity of thymol and carvacrol as well as Th. vulgaris EO against THP-1 cells was also reported by Aazza et al. [17]. Origanum onites carvacrol rich EO, between 62.5 and 125 g/mL, also presented toxicity against 5RP7 cancer cells (c-H-ras transformed rat embryonic fibroblasts) [20]. Also, Satureja sahendica thymol rich EO significantly reduced cell viability of the human colon adenocarcinoma (SW480), human breast adenocarcinoma (MCF7), choriocarcinoma (JET 3), and monkey kidney (Vero) cell lines [21].

Conclusions
In the Portuguese Thymbra and Thymus EOs studied, two main clusters were identified: one cluster grouping 8 samples 8 Evidence-Based Complementary and Alternative Medicine with diverse percentages of carvacrol, -terpineol, thymol, pcymene, and -terpinene and the other cluster with only one EO in which linalool predominated and thymol and carvacrol were absent. EOs with higher percentages of thymol and carvacrol showed the highest capacity for scavenging free radicals and preventing the growth of THP-1 cells.