Seasonal Variation in Essential Oil Content, Chemical Composition and Antioxidant Activity of Teucrium polium L. Growing in Mascara (North West of Algeria)

Oxidative stress is involved in many human diseases ranging from inflammation to cancer.1 Thus, the development of new therapeutic agents proves to be indispensable in the fight against this phenomenon. Many researchers have indicated that essential oils (EOs) are considered as really active antioxidants.2,3 Recently, many antioxidant metabolites have been isolated from Mediterranean plants like Origanum vulgare L., Salvia officinalis L. and Thymus vulgaris L., of which several belong to the Lamiaceae family.4 The Teucrium genus (from the Lamiaceae family) includes 340 species distributed in the arid and rocky areas of the Mediterranean basin,5 of which 20 of them were reported in Algeria.6 Teucrium polium L. (germander) which is locally called Jâada (or Gattaba, khayatat lajrah) and is widely found in Mascara (North West of Algeria) has a small cluster of pink to white flowers. This plant has been used for over 2000 years in traditional medicine due to its hypoglycemic, antispasmodic, diuretic,7 anti-inflammatory, anti-rheumatoid,8 hypolipidemic,9 antioxidant,10,11 analgesic, antipyretic, wound healing, anti-microbial12 and cardioprotective properties.13 Thus, the biological activities of the genus Teucrium EOs depend on their chemical compounds which can be affected by geographic origin, environmental conditions (precipitation, temperature, etc) and stage of development.14,15 It has been seen that studies on the effect of abiotic and biotic factors on oil chemical compositions and biological activities of some medicinal and aromatic plants are available.16-19 However, no reports have been found concerning the EO of T. polium L. growing in Mascara. Therefore, the aim of the present study is to evaluate the variation of the chemical composition, oil yield and antioxidant activity of T. polium EO collected in North West of Algeria (Mascara province), Seasonal Variation in Essential Oil Content, Chemical Composition and Antioxidant Activity of Teucrium polium L. Growing in Mascara (North West of Algeria)

at different phenological stages (vegetative and flowering). This was done to select the best stage of harvest which would obtain the best quality and quantity of natural substances in EOs.

Plant Material Collection
Teucrium polium L. samples were collected at two phenological stages of 2015: vegetative in January-February and the flowering in May-June, from El Mamounia (Altitude: 658 m; Latitude: 35° 25′ 29″ N; Longitude: 0° 8′ 26″ E), situated in the North West of Algeria (Mascara province). About 800 g-100 0g of aerial parts of T. polium L. were isolated in laboratory. The authentication of plant species was carried out using the African flowering plants database and also by the intervention of different botanists of Biology department of the University of Mascara.
Essential Oil Extraction Samples of 60 g of the aerial part of the plant (stem and leaves) were extracted by hydrodistillation using a Clevengertype apparatus according to the method recommended in the European Pharmacopoeia. 20 Then, the obtained EO was dehydrated with anhydrous sodium sulphate and kept at +4°C in the dark until used. EO extractions were done in three replications and the extraction yield was expressed as the weight of EO volume on the weight of plant used (W/W).

Physicochemical Characteristics of Essential Oil
The physicochemical properties of the extracted EO were the organoleptic characteristics (appearance, color, smell), the chemical indexes such as pH value, acid and ester values and the physical indices for example refractive index and relative density. 21

GC-MS Analyses
The identification of different chemical compounds of EOs was carried by a gas chromatography-mass spectrometry (GC-MS) type Shimadzu QP2010 using a SUPELCO column (30 m × 0.25 mm fused silica capillary column) and 0.25 μm film thickness, the oven temperature was held at 60°C then programmed to 250°C at rate of 2°C min -1 during 110 minutes under the following conditions: helium was used as carrier gas at a flow rate of 1.13 mL/min; injector and detector temperatures: 250°C; injected volume: 1.0 μL by split less method, ionization energy 70 eV.
The oil chemical compounds were identified by comparison of their mass spectra, retention times and retention indices with those cited in the literature and given by the spectral library banks (NIST).

Antioxidant Activity DPPH Assay
The antioxidant activity of the EOs (S1, S2) and the standard antioxidant ascorbic acid were determined on the basis of the radical scavenging effect of the stable 2, 2-diphenyl-1picrylhydrazyl (DPPH) free radical activity according to the method described by Brand-William et al. 22 Fifty microliter of different concentrations of each sample were added to 1950 mL of methanol solution of DPPH (0.025 g/L). After incubation at room temperature for 30 minutes, the absorbance of these solutions was read at 517 nm against a control (containing only the DPPH solution) by UV-Visible Spectrophotometer (SHIMADZU, UVmini-1240) and the percentage inhibition (I%) was calculated using the following formula: I% = [(Abs 517 control-Abs 517 sample) / Abs 517 control] × 100 The concentration of sample necessary to scavenge 50% of the DPPH radical (IC 50 ) was calculated from the regression equations derived by the least-square method and prepared from the different concentrations of EOs. 23 A higher DPPH radical-scavenging activity is associated with a lower IC 50 values. All tests were carried out in triplicate.
The antioxidant activity was also expressed as ascorbic acid equivalent antioxidant capacity (AEAC) according to the equation: AEAC= [IC 50 (ascorbic acid) / IC 50 (sample)].10 5 . 24,25 Data Analysis The results are presented as mean ± SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) and P ≤ 0.05 were considered as significant.

Results
Yield and Physicochemical Analysis of Essential Oil Essential Oils extracted from aerial parts including stems, leaves of T. polium harvested during the vegetative (S1) and the flowering (S2) stages of growth showed a variable physicochemical characteristic (Table 1) accompanied by an oily liquid aspect, a light yellowish color with a strong odor. The yields of EO extracted from two stages (S1, S2) are variable (Table 1). This difference has been explained mainly by the influence of climatic conditions.
The meteorological data during 2006 to 2015 showed that the vegetative stage coincided with the winter period while the flowering stage coincided with the summer period ( Figure 1). The values are obtained from the National Office of Meteorology.
In addition, the average rainfall (2006 to 2015) was 408.1 mm fairly well distributed; the maximum rainfall received in autumn and winter were 58.2 mm and 565 mm respectively, 171.7 mm received in spring and 62.7 mm in summer. The maximum temperature recorded in August was 30.7°C. The coldest month was January with 10.8°C while August was the hottest. The average relative humidity varied from 78.9% in January to 45.9% in July. It was quite high in winter and spring with values greater than 60%. According to the Köppen-Geiger classification, Mascara has a warm Mediterranean climate with dry summer (Csa) (http://koeppen-geiger.vuwien.ac.at/).

Chemical Composition
Qualitative and quantitative analysis of the EOs from the vegetative period (winter leaves) and the flowering (summer leaves) of T. polium (Table 2) revealed that both types of oils contain limonene (29.87%-26.39% respectively) as major components. To the best of the researcher's knowledge, EOs from T. polium L. growing in Mascara (North West of Algeria) has been never reported in literature.
Monoterpene hydrocarbons, oxygenated monoterpenes and oxygenated sesquiterpene were the most represented in the flowering period (summer leaves), while the vegetative period (winter leaves) was characterized by a higher percentage of oxygenated monoterpenes, oxygenated sesquiterpene and low amount of monoterpene hydrocarbons.

Antioxidant Activity
The results of the present study showed that the two tested EOs exhibited moderate antioxidant activity. However, the EO extracted from vegetative aerial parts (winter period) had the best activity with IC 50 values from 3.90 ± 0.05 mg/   (AEAC) per g extracts scavenging DPPH˙ radicals for EOs S1, S2, respectively (P < 0.001). Results are shown in Figure 3.

Discussion
The yield of EO of the aerial part of T. polium L. depends on the stages of the plant growth, where the vegetative stage (S1) was dominated significantly (0.82%) following with the flowering (S2) stage (0.56%). The EO yields obtained in the present study were comparable to what has been reported by Lianopoulou et al 26 who found that winter leaves have a higher EO yield (0.68%) compared to summer leaves (0.37%) of T. polium collected in Northern Greece. They have also reported that winter leaves of T. polium exhibit a series of morphological and anatomical traits (absent in summer leaves) which may explain the higher production of EOs in winter as one of the defensive strategies against the chilling stress. Thus, McCaskill et al 27 reported the high EO yield in the winter leaves which correspond to the higher number of peltate glandular hairs in winter leaves, since they are responsible for the biosynthesis of natural products such as EO, sucrose esters and phenolic compounds. However, winter chilling stress activates oxidative stress in cells, 28 and induces the production of antioxidant enzymes and non-enzymatic antioxidants such as phenolic compounds, etc. 29 The winter leaves of T. polium have a higher photosynthetic rate compared to summer leaves. Furthermore, their mesophyll cells are dense and their chloroplasts are larger and more numerous with a higher number of grana which reflect to a larger CO 2 absorption area, and induce an increase in the rate of photosynthesis. In this fact, cells produce larger amounts of photosynthetic carbon which are invested in the biosynthesis of secondary metabolites necessary for the protection against the stress of low temperature such as phenolic compounds, EO etc. 26 The decrease of the yield of EO in the phenological stages (vegetative to flowering stages) was explicated by the adaptation of T. polium to climatic variation. Thus, the metabolites of the terpenoid pathway (monoterpenes, sesquiterpenes, and homoterpenes) were involved in many plant physiology and ecology. 30 According to the physico-chemical properties, good quality EOs have higher density, higher ester value and lower value of refractive index. The acid value must be less than 2 (low amount of free acids). 31 Hassan et al 32 found that oil extracted from T. polium growing in Saudi Arabia is light yellow, fragrant and pungent characteristic odor with refractive value 1.4850.
In general, it appears that the qualitative and quantitative changes of the EO composition of T. polium are significantly related with its development (the presence/absence of some typical components and the amount of the main constituents). Therefore, the variability of the chemical composition of EOs affects their antioxidant activity. As a result, the EOs extracted from the vegetative aerial parts had the best activity (IC 50 = 3.90 ±0.05 mg/mL) (P < 0.001). This is while, Mahmoudi and Nosratpour 42 found that the EO of T. polium L. (collected during flowering stage) from the West of Iran (Kerman province) was able to reduce the stable free radical DPPH with an IC 50 = 9200 μg/mL, where the main constituents were Spathulenol (15.06%), β-pinene (11.02%), β-myrcene (10.05%), germacrene B (10.11%), germacrene D (8.15%), bicyclogermacrene (8.25%) and linalool (4.02%). Hammoudi et al 37 found that the main compounds in the EO of T. polium (collected in November) were dl-limonene (11.18%), δ-cadinene (10.02%) and trans β-caryophyllene (9.15%) with a potential activity from 79.02 ± 0.00 mg AEAC per g extracts scavenging DPPH˙ radicals. However, T. polium subsp. capitatum oil ( collected in July) with t-cadinol (18.3%), germacrene D (15.3%) and β-pinene (10.5%) as the major components represented less efficiency than that of positive control (BHA) reaching a maximum of 12.7% for a concentration of 1000 mg/L. 43 Lemos et al 44 also showed that the EOs of Thymus vulgaris L (Lamiaceae family, 125 samples from Brazil harvested in each season) had moderate antioxidant activities evaluated by the DPPH method, and justified by the presence of the monoterpenes.
Thus, in the present study, the antioxidant activities of T. polium EOs can be attributed to the high concentration of major components, the variation in chemical composition, the presence/absence the oxygenated monoterpenes, monoterpene hydrocarbons and sesquiterpene. Furthermore, defining the component (s) responsible for this activity is very difficult.
Generally, EOs contain phenolic and non-phenolic compounds while phenolic compounds such as carvacrol and thymol have strong antioxidant potentials. 45,46 Whereas, the phenolic compound is a source of n H atoms that are transferred to DPPH • free radical then DPPH • converted into a stable molecule DPPH. Therefore, the addition of an antioxidant results on a decrease of absorbance proportional to the concentration and antioxidant activity of the compound. 47 However, some researchers have revealed that some EOs rich in non-phenolic compounds also had antioxidant potentials. [48][49][50] Wei and Shibamoto 51 revealed that EOs contain high percentages of hydrocarbon monoterpenes such as limonene and α-pinene, which demonstrate a significant antioxidant potential. Miguel 52 reported a good antioxidant activity for the EOs of Citrus sinensis, where limonene was the major constituents. According to Bayala et al, 53 the major constituents were α-terpineol (59.78%) and β-caryophyllene (10.54%) for Ocimum basilicum (Lamiaceae family) with the best ability to scavenge DPPH radical created in vitro with a percentage of inhibition of 55.67% for a concentration of 8 mg/mL.
Zengin and Baysal 54 tested some compounds individually and they showed that the overall antioxidant activity of α-terpineol was stronger than linalool and eucalyptol. Although Bicas et al 55 evaluated the antioxidant and antiproliferative activities of α-terpineol, they reported that this component exhibited potent free radical-scavenging activity and also had a cytostatic effect against six human cancerous cell lines (breast, lung, prostate, ovarian, and leukemia).
Antioxidant activity of the oil extracted from the leaves of Ocimum canum Sims. (Lamiaceae family) showed dose dependent free radical scavenging activity against DPPH (IC 50 523.55 ± 0.001 μg/mL), where the major compound was camphor (39.77%). 56  It has been reported that the overall antioxidant activity of oxygenated monoterpenes (linalool, carvacrol, α-terpineol etc) was the strongest, 52,55,58,59 followed in a descending order by monoterpene hydrocarbons and sesquiterpene hydrocarbons and their oxygenated derivatives which have very low antioxidant activities. 60 According to Viuda-Martos et al, 61 the good antioxidant activity of EOs compared to individual components can be attributed to the synergistic interaction of the EO constituents, the high percentages of the main components or to the micro components acting as pro-oxidants.
Although, some researchers have revealed that most natural antioxidative compounds (such as EO constituents, phenolic compounds, etc) often work synergistically with each other to create an effective defense system against free radical attack. 62,63 Conclusions Teucrium polium L. is a seasonally dimorphic plant showing its ability to adaptation to the drought stress and the chilling stress (the climatic variation). However, this variation effects the chemical composition of EOs (vegetative stage/flowering stage) and therefore their antioxidant activity. This fact is actually of great interest because the ability of T. polium EOs collected from the Mascara province may play an important role in the prevention of some diseases such as cancer and inflammatory diseases.
According to the results of the present study, the vegetative stage is selected as the best stage for harvesting the EO of Teucrium polium L. with the best antioxidant activity compared to the flowering stage EO.