Composition, antibacterial, antioxidant and antiproliferative activities of essential oils from three Origanum species growing wild in Lebanon and Greece

The essential oils from Origanum dictamnus, Origanum libanoticum and Origanum microphyllum were analysed by GC-MS, finding carvacrol, p-cymene, linalool, γ-terpinene and terpinen-4-ol as major components. The antioxidant activity by the DPPH and FRAP tests and the antiproliferative activity against two human cancer cell lines, LoVo and HepG2, were investigated, showing that the essential oil of O. dictamnus was statistically the most inhibitory on both the cell lines, while all the oils exerted a weak antioxidant activity. Furthermore, the samples were tested against 10 Gram-negative and Gram-positive bacteria; all the oils were active on Gram-positive bacteria but O. dictamnus essential oil was the most effective (MIC = 25–50 μg/mL), showing also a good activity against the Gram-negative Escherichia coli (MIC = 50 μg/mL). Data suggest that these essential oils and particularly O. dictamnus oil could be used as valuable new flavours with functional properties for food or nutraceutical products.


Introduction
The genus Origanum (Lamiaceae) is characterised by a large morphological and chemical diversity (Ietswaart 1980). Due to their several biological effects such as being antispasmodic, antimicrobial, expectorant, carminative and aromatic for whooping and convulsive coughs, Origanum species have been used as ethnopharmacological drugs in traditional medicine to treat various ailments (Barros et al. 2010). Origanum dictamnus L. was considered as Panacea according to the ancient Greeks and was used in many cases for its healing effects (Strid & Tan q 2015Taylor & Francis 1991. A number of studies describe the biological activities of its extracts, such as antioxidant (Proestos et al. 2013), antimicrobial (Alexopoulos et al. 2011) and cytotoxic activity against different cell lines (Chinou et al. 2007). Origanum microphyllum (Bentham) T. Vogel is a dwarf shrub endemic to Lefka Ori and Dhikti Mountains in Crete. The shrub of Origanum libanoticum Boiss is instead endemic to Lebanon (Arnold et al. 2000). To the best of our knowledge, the studies on the essential oil of O. microphyllum species are limited, and there are no previous papers on the biological activity of the essential oil of O. libanoticum. Furthermore, there are no reports on the biological activity of O. dictamnus collected on Mavri summit, Mt. Ida (Crete). The aim of this work was to compare the chemical composition of the essential oils from these three Origanum sp. in order to characterise the species through the qualitative and quantitative evaluation of chemical compounds. In view of the potential pharmaceutical applications, the antioxidant activity by the DPPH and FRAP tests, the antiproliferative activity against the two human cancer cell lines LoVo and HepG2, and the antimicrobial activity against 10 Gramnegative and Gram-positive bacteria were evaluated taking into account the chemical composition of the oils.

Antibacterial activity
The in vitro antimicrobial activity of the essential oils D, M and L against 10 bacteria species was evaluated by determining the MIC and the MBC using the broth dilution method. The oils showed an interesting activity mainly against the Gram (þ ) pathogens, while among Gram (-) bacteria only Escherichia coli was affected by the two oils D and M from Greece but not by the Lebanese oil L (Table S2). On the whole, the most active essential oil was D, which was very effective (MIC ¼ 25 mg/mL, MBC ¼ 50 mg/mL) against the Gram-positive Bacillus cereus, Bacillus subtilis and Staphylococcus epidermidis. The oil also showed a very good activity against Staphylococcus aureus, Streptococcus faecalis and the Gram (-) E. coli (MIC ¼ 50 mg/mL). Also the other Greek oil M showed good MIC and MBC values against all the Gram (þ ) pathogens except S. faecalis and against the Gram (2 ) E. coli (MIC ¼ 50 mg/mL for all). The activity of the oils can be very likely attributed to the existence mostly of carvacrol (Ipek et al. 2005). As regards L, whose antimicrobial activity was never shown before, its activity is probably due to the presence of linalool, thymol methyl ether and(E)-b-caryophyllene (Burt 2004).

Antiproliferative effects against human tumour cell lines
The antiproliferative activity of the essential oils was determined after 24 and 48 h of treatment. Effects on proliferation of LoVo and HepG2 cell lines are shown in Figure 1, which report the percentages of inhibition of cell viability after 24 and 48 h of treatment, respectively. All samples reduced cell viability in a dose-dependent manner compared with control (0.5% dimethyl sulphoxide-treated control cells). On colon carcinoma cell line, the essential oil of O. dictamnus (D), at the highest concentration (100 mg/mL), was statistically the most inhibitory causing a 58.39% of inhibition after 24 h of incubation. At the same concentration (100 mg/mL), the essential oils of O. microphyllum (M) and O. libanoticum (L) caused 47.12% and 48.15% of inhibition, respectively. A similar trend was observed on HepG2 cell viability after 24 h of treatment. In order to verify possible changes in antiproliferative activity of Origanum essential oils, effects on cell viability were also assessed after 48 h of incubation. At the highest concentration, 100 mg/mL D was still the most active sample on colon carcinoma LoVo cancer cell, inducing an inhibition of 61.96%. Inhibition induced by essential oils M (47.13%) and L (48.60%) was significantly lower. A different trend was observed on hepatocarcinoma cell line HepG2 cells: at the highest concentration (100 mg/mL), both D and L induced the highest antiproliferative activity (49.83% and 48.50% inhibition, respectively) in comparison to M (37.68%). The raw data were fitted through nonlinear regression in order to obtain the IC 50 parameter, which was detectable only on LoVo cell line treated with sample D. On the whole, O. dictamnus essential oil showed the best antiproliferative activity with IC 50 values of 84.76^1.03 mg/mL after 24 h and 72.26^1.05 mg/mL after 48 h of treatment.

Antioxidant activity
The essential oils of the three Origanum species were subjected to screening for their possible antioxidant activity by means of two spectrophotometric methods (DPPH and FRAP tests) and expressed as Trolox equivalents (TEs). Table S3 shows that according to the DPPH test, the essential oils revealed poor antiradical activity, and only the oil from O. dictamnus exerted a weak capacity to scavenge free radicals (0.15^0.04 mmol TE/L), very scarce if compared to artificial antioxidants such as butylhydroxytoluene and known antioxidant compounds common in essential oils such as thymol and carvacrol used at the same dose (Table S3). Using the FRAP test, all the samples showed significant activity, demonstrating that they are not active in catching free radicals but they have a major ferric reducing/antioxidant power (Table S3). Our results are in accordance with previous findings that show that, in most cases, the antioxidant activity of Origanum species is due to their polar extracts rather than other extracts or essential oils, and this is true also for O. dictamnus (Liolios et al. 2009;Liolios et al. 2010;Proestos et al. 2013). No previous studies were done on the antioxidant activity of O. libanoticum and O. microphyllum essential oils.

Conclusion
The results described in this paper may suggest that the three Origanum species analysed, O. dictamnus and O. microphyllum both endemic in Greece and O. libanoticum endemic in Lebanon, possess compounds with antimicrobial and cytotoxic properties. This is particularly true for O. dictamnus that exerted the best activity in all the assays. In addition, the data in this study are supporting the use of Origanum plants as tea or additive in foods, and traditional remedies for the treatment of infectious diseases and for the prevention of cancer and gastrointestinal system diseases.