GC/MS Analysis and Antioxidant Activity of the Essential Oil leaves Thymus numidicus Poiret. growing in Mila

Essential oil components of the leaves of Thymus numidicus growing in Mila have been studied by gas chromatography mass spectrometry GC/MS to afford 60 Sixty compounds representing 94.0 % of the total oil and mainly represented by oxygenated monoterpenes,. The main constituents of the essential oil from the leaves were thymol (41.2%), β-cymene (12.9%), chlorocresol (11.2%), β-linalool (10.7%) and methyl thymol ether (3.3%). The antioxidant activity was evaluated by spectroscopic method, used for that free radical compound (DPPH) and in comparison with vitamin C as a standard. The essential oil showed a moderate activity against free radical compound (DPPH) 17.4% at 1M.


Introduction
The genus Thymus L., belonging to the Lamiaceae family represented by around 350 species of perennial, aromatic herbs and subshrubs predominantly found in Mediterranean region, Asia, Southern Europe and North Africa [1], and comprised in Algeria over 12 species [2].
Thymus species as well as many other aromatic plants biosynthesize variable amounts of volatile compounds known as essential oil; therefore, chemical classification of Thymus species was based on the main essential oil components and their chemical polymorphism, moreover, numerous chemotypes have been defined, such as carvacrol and thymol, γ-terpineol, thujone, geraniol, linalool and others [15].

Extraction
Essential oils were obtained by hydrodistillation of 100 g of dried leaves using a Clevenger-type apparatus for 3 h. The oil was stored in sealed vials protected from the light at +4°C before analyses. The oil sample was subsequently analyzed by GC-MS.

Gas chromatography/mass spectrometry (GC/MS)
Analyses were performed with a Varian CP-3800 gas chromatograph equipped with a DB-5 capillary column (30m × 0.25 mm; coating thickness 0.25 μm) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions: injector and transfer line temperatures 220 and 240°C, respectively; oven temperature programmed from 60°C to 240°C at 3°C/min; carrier gas helium at 1 mL/min; injection 0.2 μL (10% n-hexane solution); split ratio 1:30. Identification of the constituents was based on comparison of the retention times with those of authentic samples, comparing their linear retention indices relative to the series of nhydrocarbons, and by computer matching against commercial (NIST 98 and ADAMS) and homemade library mass spectra built up from pure substances and components of known oils and MS literature data [16].

Antioxidant activity
The capacity of essential oil extracted from Thymus numidicus Poiret. leaves to reduce the radical 2,2-diphenyl-1picrylhydrazyl (DPPH) was assessed using the method of Masuda et al. [17] modified in the laboratory. 15 μl of the essential oil at different concentrations was added to 1500 μl of a DPPH ethanolic solution. The mixture was shaken vigorously and left standing at room temperature for 05 min in the dark. The absorbance of the resulting solution was then measured at 517 nm. The normal purple color of DPPH will turn into yellow when its singlet electron is paired with a hydrogen atom coming from a potential antioxidant. The scavenging activity of essential oil was evaluated according to the formula: Where: A0 is the absorbance of the control at 05 min, and A1 is the absorbance of the sample at different times. All samples were analyzed in three replications.

Results and Discussion
The composition and percentage of the compounds are summarized in Table 1. They are listed by order of their retention times. The oil yield was 0.8 % (w/w) based on the dried weight which means that the organs are a potential oil source. Sixty compounds were identified in the essential oil, representing 94.0% of the total oil. The essential oils were dominated by a large amount of oxygenated monoterpenes (12.4%), monoterpene hydrocarbons (2.6%) and sesquiterpenes hydrocarbons (2.3%), while the oxygenated sesquiterpenes (0.9%) contents were very low. The main constituents of the essential oil were found to be, thymol (41.2%), βcymene (12.9%), chlorocresol (11.2%), βlinalool (10.7%) and methyl thymol ether (3.3%) and carvacrol (2.8%) and some other compounds were only present in minor amounts. In total, essential oil composition of Thymus numidicus Poiret. was considered as a rich source of oxygenated Monoterpenes The predominance of phenolic compounds is in agreement with previous results reported from Thymus numidicus Poiret. essential oil from Constantine (North-Eastern of Algeria) [18] which was mainly represented with thymol (68.2%), carvacrol (16.9%) and linalool (11.5%).
There are a few reports about the antioxidant activity of Thymus essential oils [21,22,23] showed that essential oils containing high amounts of thymol and carvacrol were reported to possess the highest antioxidant activity [24,25,26] and the principal active compounds of these oils are principally carvacrol, thymol, citral, eugenol, 1-8 cineole, limonene, pinene, linalool and their precursors [27].

Conclusion
Our results demonstrate the high chemical variability of constituents found in genus