Synergistic Antioxidant Activity and Chemical Composition of Essential Oils From Thymus fontanesii , Artemisia herba-alba and Rosmarinus officinalis

In an attempt to preserve human health and avoid autooxidation affecting both the sensory and nutritional quality of foods, synthetic preservatives such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole were introduced in the food industry. However, with the current resurgence of interest in the application of safe organic materials instead of synthetic materials, which are suspected for their carcinogenicity, essential oils are increasingly sought as natural alternatives.1,2 Essential oils represent a “green” alternative in the nutritional and pharmaceutical fields due to their incredible biological properties.3,4 Essential oils are not only used in monotherapy but also have been used in combinations for many years.5 They are used to act synergistically to further enhance their effects. The possible synergistic effect produced by the combination of plant essential oils was referred as an efficient strategy to inhibit or reduce the natural oxidation process of foods.6 The combination of essential oils with antioxidant effects approach may lead to new naturel preservatives. A Few research have studied the synergistic effect of plant essential oils. Grosso et al suggested that the combination of thymol, carvacrol and thymoquinone in the volatile oil of Satureja montana may be responsible for the increase in antioxidant activity, using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging and rancimat methods.7 Thymus fontanesii and Rosmarinus officinalis belong to the Lamiaceae family. While, Artemisia herba-alba belongs to the Asteraceae family. These species are especially known for their important biological properties such as antibacterial and antioxidant activities. To our knowledge, no study has investigated the antioxidant properties of T. fontanesii, A. herba-alba and R. officinalis essential oils blend. The purpose of the present study was (i) Synergistic Antioxidant Activity and Chemical Composition of Essential Oils From Thymus fontanesii, Artemisia herbaalba and Rosmarinus officinalis

to determine the chemical composition of the essential oil of these plants, (ii) to evaluate the antioxidant power of each essential oil by DPPH and FRAP assays and (iii) to investigate the possible synergistic impacts of the combination of three essential oils.

Materials and Methods
Plant Material and Extraction of the Oils Aerial parts of T. fontanesii, A. herba-alba and R. officinalis were collected at the flowering stage on May to June, 2017.The plant materials were botanically identified by the Laboratory of Ecology and Ecosystem Management of the University of Tlemcen, Algeria.Voucher specimens were deposited with the Herbarium of the University of Tlemcen.T. fontanesii (T.on.04/2018), A. herba-alba (A.h.a.05.2018) and R. officinalis (R.of 04/2018).
Essential oils were obtained from fresh material (300-400 g) by hydrodistillation for 5 hours using a Clevenger-type apparatus with yields (w/w) of 3.7% for T. fontanesii, 0,7% for A. herba-alba and 0.4% for R. officinalis.

Gas Chromatography
Gas chromatography (GC) analyses were carried out using a Perkin Elmer Clarus 600 GC apparatus (Walhton, MA, USA) equipped with a single injector and two flame ionization detectors (FIDs).The apparatus was used for simultaneous sampling of 2 fused-silica capillary columns (60 m × 0.22 mm, film thickness 0.25 μm) with different stationary phases: Rtx-1 (polydimethylsiloxane) and Rtx-Wax (polyethylene glycol).Temperature was programed 60 to 230°C at 2°C min −1 and then held isothermal 230°C (30 minutes).Carrier gas was hydrogen (0.7 mL min −1 ).Injector and detector temperatures were held at 280°C.Split injection was conducted with a split ratio of 1:80.Injected volume was 0.1 μL.

Gas Chromatography-Mass Spectrometry (GC/MS)
The oils and the fractions obtained by GC were investigated using a Perkin Elmer TurboMass quadrupole analyzer, directly coupled with a Perkin Elmer Autosystem XL equipped with 2 fused-silica capillary columns (60 m × 0.22 mm, film thickness 0.25 μm), Rtx-1 (polydimethylsiloxane) and Rtx-Wax (polyethylene glycol).Other GC conditions were the same as described above.Ion source temperature was 150°C and energy ionization 70 eV; electron ionization mass spectra were acquired with a mass range of 35-350 Da and scan mass of 1 s.Oil injected volume was 0.1 μL and fraction injected volume was 0.2 μL.

Component Identification
The identification of each compound was carried out by comparison: (i) retention indices calculated respectively on polar and apolar columns with those of standard compounds of the laboratory library 8,9 or those reported in the literature; (ii) mass spectra (electronic impact) with those of standard compounds or those present in computerized banks. 10,11

Determination of Antioxidant Activity of Essential Oils DPPH Free Radical Scavenging Assay
The free radical-scavenging activity of essential oils and combinations were measured using DPPH, as described in the literature. 12At first, 500 mg/L of each essential oil stock solution was prepared.A series of dilution with varying concentrations (0.1 to 100 mg/L) was prepared by dissolving various masses of essential oil in ethanol.In regards to essential oils blends, a ratio of 1: 1 by volume was mixed for each combination and a series of concentrations ranging from 0.1 to 50 mg/L were prepared.After on, 100 µL of each concentration was then mixed with 25 µL of 0.5 mM DPPH.After a 30 minutes incubation period at room temperature, the absorbance was measured at 517 nm using spectrophotometer.Ascorbic acid was used as standard and DPPH mixture without any sample served as blank.Inhibition of free radical DPPH in percent (I%) was calculated as follows: As presented above, A blank is the absorbance of the control reaction (without oils), and A sample was the absorbance in the presence of essential oils.From the obtained RSC values, the IC 50 values, which represented the concentrations of the extracts caused 50% neutralization, were determined by linear regression analysis.

Ferric-Reducing Antioxidant Power Assay
The ferric reducing antioxidant power (FRAP) of the essential oils was tested as described earlier 13 .Different concentrations of the essential oils dissolved in ethanol (80%) were mixed with 2.5 mL of phosphate buffer (pH = 6.6) and 2.5 mL of potassium ferricyanide.Later, the mixture was incubated at 50°C for 20 minutes, and then trichloroacetic acid (10%, 1,25 mL) was added.After shaking the mixture vigorously, it was mixed with distilled water (5 mL) and ferric chloride (0.1%, 5 mL).After 30 minutes of incubation, absorbance was read at 700 nm against ethanol (80%) as blank.Analyses were performed in triplicate.Increased absorbance of the reaction meant increased reducing power and compared to the synthetic antioxidant BHT as reference.

Statistical Analysis
The data are presented as the means ± standard deviations from the three replicates.Calculations were performed using the SAS v. 9.1.3program.

Chemical Composition of Essential Oils
GC-FID and GC-MS analysis of T. fontanesii, A. herbaalba and R. officinalis oils accounted for 99.3%, 93.4% and 91.1% of oils, respectively and allowed the identification of 31, 20 and 19 components, respectively (Table 1).All components were identified by comparison of their mass spectrum and retention indices with those of our laboratoryproduced "Arômes" library (Table 1).The essential oil of T. fontanesii obtained from the aerial parts was dominated principally by monoterpenoid phenol (84.7%).The main components were thymol (76.6%) and p-cymene (7.4%).2).Essential oils blends were very effective compared to the individual essential oils tested.The combination of T. fontanesii, A. herba-alba and R. officinalis essential oils showed a significant increase in antioxidant activity with an IC 50 of 2.6 mg/L, almost equal to the synthetic antioxidant used as reference (IC 50 = 2,3 mg/L) (Table 3).The combination of essential oils of T. fontanesii and R. officinalis showed also good antioxidant activity with an IC 50 of 7.2 mg/L.However, the essential oils blends of T. fontanesii and A. herba-alba, and A. herba-alba and R. officinalis had promising antioxidant activities with IC 50 s of 23.9 and 39.2 mg/L, but were still lower than the synthetic antioxidant (BHT) (Table 3).
Ferric Reducing Antioxidant Power Assay FRAP assay is a widely used method that uses antioxidants as reductants in a redox-linked colorimetric reaction, wherein Fe 3+ is reduced to Fe 2+ .Figure 1 depicts the reducing power of individual and blend essential oils.All oils showed the presence the reductive effects, which increased with an increase in concentration.However, the combination of T. fontanesii, A. herba-alba and R. officinalis essential oils has been as effective on reducing power, compared to the synthetic antioxidant BHT.The individual and blends essential oils displayed chelating effects on ferrous ions, suggesting that it can sequestrate Fe-ions or reduce the concentration of metal.

Discussion
As a result, T. fontanesii presented the highest antioxidant effect, probably related to its chemical profile, especially the relatively high percentage of phenolic compounds.Thymol and p-cymene are the most frequently occurring constituents of essential oils obtained from thyme species, with many biological activities.5][16] The moderated antioxidant activity obtained from R. officinalis essential oil could be the consequence of appreciable content of camphene, borneol, verbenone, 1,8-cineole and α-pinene, that represented more than 71% of the total oil.The lowest antioxidant activity was recorded for A. herba-alba oil where chrysanthenone, camphor, camphene and 1,8-cineole represented more than 66.3% of the total oil.The antioxidant activity obtained from A. herba-alba and R. officinalis essential oils could be the consequence of appreciable contents of oxygenated monoterpenes (80.2 and 65.9%, respectively)  17 showed that camphor, one of the main components in essential oil of Salvia hispanica, has lower antioxidant activity.This is while, 1,8 cineole and borneol were naturally found in many aromatic plants and showed weak antioxidant activity. 18,19However, no information was found in the available literature about the biological activities of chrysanthenone and verbenone.Furthermore, monoterpene hydrocarbons are known to have noticeable antioxidant activities. 20The obtained findings showed that essential oil combinations applied a promising synergistic antioxidant effect by decreasing the half maximal inhibitory concentration.The strong synergistic effect was found by combining T. fontanesii, A. herba-alba and R. officinalis essential oils (Table 3).It seems that the association of thymol and p-cymene with 1,8-cineole, chrysanthenone, camphor, borneol and verbenone increases the antioxidant activity.T. fontanesii and R. officinalis essential oils blend also showed a good antioxidant capacity when compared to the value of the synthetic antioxidant.It is evident that this synergistic effect was found when thymol and p-cymene was paired with 1,8-cineole, borneol and verbenone.However, the blends of T. fontanesii and A. herba-alba, A. herba-alba and R. officinalis essential oils showed the lowest antioxidant activities.It seems that the combination of thymol and p-cymene with chrysanthenone, camphor and 1,8-cineole produce low antioxidant power.While, the absence of thymol and p-cymene of blend significantly decreases this power.
In conclusion, the results showed that essential oil of T. fontanesii is a good source of monoterpenoid phenols as thymol and p-cymene.The essential oil had the best antioxidant activity using DPPH and FRAP methods.While, R. officinalis and A. herba-alba essential oils rich with oxygenated monoterpenes showed the lowest antioxidant activities by means of highest IC 50 values.On the other hand, blends of essential oils showed an increase of antioxidant capacity.It seems that the monoterpenoid phenols (i.e.thymol and p-cymene) associated with oxygenated monoterpenes such as 1.8-cineole, chrysanthenone, camphor, borneol and verbenone play a pivotal role in this activity and produce stronger synergistic effect.These results, therefore, represent a basis for further studies that could lead to the development of a new treatment based on the combination of these essential oils as natural antioxidant agents, both in food and pharmaceuticals fields.

Table 1 .
Chemical Composition of the Essential Oils a Order of elution is given on apolar column (Rtx-1).b Retention indices of literature on the apolar column (lRIa) reported from Konig. 9 c Retention indices on the apolar Rtx-1 column (RIa).d Retention indices on the polar Rtx-Wax column (RIp).e Identification mode; RI. retention indices; MS. mass spectrometry in electron impact mode.