Chemical profiling study and antioxidant activity of wild Teucrium luteum subsp. flavovirens essential oil from Morocco

Plant material and essential oil isolation

The aerial parts of TLSF were collected in April 2016 (full bloom) in the area of Errachidia (Morocco) from 10 stations at least 5 km apart. Voucher specimens (R-2016) were deposited in the Herbarium of Sciences and Technologies Faculty, Moulay Ismail University, Errachidia, Morocco. The studied plant was curded at ambient temperature. For each sample, the dried plant (100 g) was water-distillated (3 hours) using a Clevenger-type apparatus as recommended by the European Pharmacopoeia (1997). The water is removed in the essential oil using anhydrous sodium sulfate, filtered, and saved at 4°C before analysis.

The collective essential oil representing the average of the 10 stations is obtained by mixing oils from each station with equal quantities.

GC-FID analysis

The GC-FID analysis was conducted with Perkin-Elmer Auto system XL GC apparatus equipped with a dual-flame ionization (FID) detection system and fused silica capillary columns (60 m × 0.22 mm inside diameter, layer thickness 0.25 μm), Rtx-1 (polydimethylsiloxane) and Rtx-wax (polyethylene glycol). The furnace temperature was programmed at 2°C/minute from 60°C to 230°C and maintained isothermally for 35 minutes at 230°C. The injector and detector temperature was kept at 280°C. A volume of studied oil (0.2 μl) was injected in fractional mode (1/50), with helium as a carrier gas (1 ml/minute). The determination of retention indices (RI) of the compounds was based on retention times. The peak areas of the GC allow us the calculation of the components relative concentrations without using correction factors.

GC-MS analysis

The essential oils were also analyzed using a Perkin-Elmer Turbo Mass quadrupole-detector, coupled to a Perkin-Elmer 88 Auto system XL, coupled with the two same fused-silica-cap described above. The GC conditions were the same as those detailed previously and the MS parameters were as follows: ion-source temperature, 150°; ionization energy, 70 eV; and the mass spectra by electron ionization acquired over a mass range of 35–350 Da during a scan-time of 1 second. The injection volumes for the oils were 0.1 μl.

Compound identification

The individual elements were determined using RI determined on polar and non-polar columns compared to those of authentic compounds or literature data (Adams, 2007; König et al., 2011) or using the computer comparison of mass spectra with those of commercial or our internal library, built with data from authentic literature compounds (NIST, 1999).

Antioxidant activities

DPPH assay

The antioxidant capacities of essential oil obtained from TLSF were determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging test as described in our previous study (Ouknin et al., 2018). The butylated hydroxytoluene (BHT) and ascorbic acid were considered as positive controls. The radical-scavenging activity is calculated according to Equation (1) as follows:

A₀ and A₁ represent the control absorbance and the sample absorbance after 30 minutes, respectively.

β-Carotene bleaching test

The antioxidant capacity was also evaluated using the coupled autoxidation of β-carotene and linoleic acid test as described by Ouknin et al. (2018). TLSF antioxidant activity has been evaluated in terms of bleaching β-carotene according to Equation (2) as follows:

where A_{β-carotene after 2h} represent the values of samples absorbance after 2 hours, and A_{initial β-carotene} represent the absorbance at the beginning of the experiment. All tests were made in triplicate, and oil concentration producing 50% of inhibition (IC₅₀) is determined by plotting the percentage of inhibition as a function to the oil concentration used.

Reducing power determination (FRAP)

The iron reduction capacity was conducted using the Oyiazu method (1986). Test ranges of 150–1,500 μg/ml for TLSF oil were prepared by a series of essential oil dilution with pure ethanol. The same for the test range of 5–100 μg/ml for control substances. The various concentrations of the samples were mixed with 2.5 ml of phosphate buffer (0.2 M, pH = 6.6) and 2.5 ml of K₃Fe(CN)₆ (1%). After the incubation of the mixture for 20 minutes at 50°C, 2.5 ml of Cl₃CCOOH (10%) was added. Then, the blend was centrifuged at 3,000 rpm for 10 minutes. A volume of 2.5 ml of the top layer was mixed with 0.5 ml of FeCl₃ (0.1%), and the UV absorbance was detected using a spectrophotometer at 700 nm. The oil concentration giving an absorbance of 0.5 (CI₅₀) is determined by plotting the following values at 700 nm referred to the corresponding oil concentration.

Essential oil composition

The analysis of the chemical composition of TLSF essential oils, harvested in 10 stations, shows that the chromatographic profiles are qualitatively and quantitatively similar. Hence, we have mixed all the oils with equal quantities to get a collective essential oil representing the average for the 10 stations. The chromatographic profile of the collective oil is given in Figure 1.

The average yield of essential oils obtained from TLSF is about 0.75%. However, the yield of essential oils obtained from previous reports of different species of Teucrium varied between 0.07% and 0.35% (v/w) (Djabou et al., 2010; 2012a; 2012; 2013a; 2013b; Muselli et al., 2009).

GC-FID and GC-MS analysis allow us the determination of 63 compounds, representing 98.1% of the total oil. From the Table 1 representing the TLSF essential oil chemical profiling, we can conclude that oxygenated sesquiterpenes (48.4%), hydrocarbon sesquiterpenes (22.0%), and hydrocarbon monoterpenes (20.1%) represent the main groups of constituents followed by oxygenated monoterpenes (7.6%). The main compounds (%>5%) identified are elemol (16.4%), α-pinene (12.0%), trans-caryophyllene (7.0%), α-humulene (6.4%), β-pinene (5.7%), and γ-eudesmol (5.3%).

Figure 1. GC-MS chromatogram of T. luteum subsp. flavovirens essential oil. [Click here to view]

To the authors’ knowledge, no previous study concerning the chemical composition of TLSF essential oil was reported in the literature.

On the basis of its constituents having a percentage higher than 5%, the essential oil of TLSF differs from oils of other species of the genus Teucrium previously studied (Djabou et al., 2010; 2012a; 2012b; 2013a; 2013b; Muselli et al., 2009). In fact, no other species simultaneously contains all of the six main compounds listed above. Except α-humulene, each of these constituents is present with a very small percentage in other species and with a much lower content than in Teucrium luteum. So, this group of compounds is a marker of this essential oil.

Antioxidant activities

The in vitro antiradical activity of TLSF essential oil was evaluated by the DPPH, bleaching test of β-carotene, and FRAP method.

The experimental results (Table 2) obtained by the DPPH test show clearly that the studied essential oil is effective in reducing the free radical DPPH^., with a strong antiradical activity compared to BHT with IC₅₀ of 13.75 ± 1.15 and 89.50 ± 3.14 μg/ml, respectively. The results obtained with the essential oil is comparable to those of ascorbic acid (IC₅₀ = 11.25 ± 0.11 μg/ml). Regarding the bleaching test of β-carotene, the examination of the results obtained for TLSF (Table 2) shows that the studied essential oil exhibits a significant anti-free radical activity with an IC₅₀ = 275.45 ± 1.25 μg/ml. This essential oil is less powerful antioxidant than the reference substances, Ascorbic acid and BHT, and their IC₅₀ are in the order of 45.75 and 75.14 μg/ml, respectively. About the reduction of Fe³⁺ to Fe²⁺by FRAP method, the results obtained show that the studied essential oil has a significant antioxidant activity with an IC₅₀ = 235.45 ± 2.50 μg/ml.

In overall, TLSF oil showed an important antioxidant activity. The observed activity can be assigned to components of the studied essential oil, such as elemol, α-pinene, β-pinene, trans-caryophyllene, α-humulene, γ-eudesmol and valerianol, and/or synergistic effects between all the compounds. The observed difference in the antiradical activity of the different tests could be ascribed to the different methods used for the evaluation. The antiradical properties of essential oils depend on the structural characteristics of their components; this activity is essentially attributed to the high reactivity of hydroxyl groups (Viuda-Martos et al., 2010).

Table 1. Qualitative and quantitative composition of T. luteum essential oil. [Click here to view]

Table 2. Antiradical activity of T. luteum subsp. flavovirens essential oil. [Click here to view]