Comparative study of chemical compositions and antimicrobial effect of different genius of Thymus harvested during two period of development

1 Laboratory of Organic Synthesis-Modeling and Optimization of Chemical Processes, Badji Mokhtar University, P.O. Box 12, 23000 Annaba, Algeria. 2 EPST-Annaba, P.O. Box 218, 23000 Annaba, Algeria. 3 Faculty of natural science and life, University of Mohamed Cherif Messaadia, P.O. Box 12, 41000 Souk-Ahras, Algeria. 4 Laboratory Development and Control of Hospital Pharmaceutical Preparations. Medical Science Faculty, University of Badji MokhtarAnnaba, LP 205 Ezzafrania, Annaba (23000) Algeria. 5 Department of Chemistry, Faculty of Science, Badji Mokhtar University, P.O. Box 12, 23000 Annaba, Algeria. 6 Department of Process Engineering, Faculty of Engineering, Badji Mokhtar University, P.O. Box 12, 23000 Annaba, Algeria.


INTRODUCTION
Essential oils, called usually essences are volatile substances contained in vegetation.Their volatility opposes them from fixed oils which are lipids.Essential oils are mixtures of numerous constituents called chemotype.The Thymus species being a part of the botanical family of Lamiaceae, include several developing around the Mediterranean sea, especially in North Africa it consist in 300-400 species (Evans, 1989;Pedersen, 2000).All species of the Thymus genus produce essential oils .
They are aromatic plants very used in galenic pharmacy and beauty care.This is due to their wide biological activity: antibacterian (Essawi et al., 2000;Marina, 1999), anti tumoral (Jaafar et al., 2007), anti oxydant (Miura et al., 2002) and antifungal (Giordani et al., 2008).Our work consists on the study of the chemical composition of the essential oil extracted from species of Thymus (T.nimidicus, T. ciliatus and T. algeriensis) collected before and during flowering periods from different regions in eastern Algeria.Antimicrobial activity of Thymus essential oils in two phase of growth were determined by agar disc diffusion method (Malabadi et al., 2012).

Plant material
The different species of Thymus were collected: T. numidicus from Berrahal a western region of the department of Annaba, Thymus algeriensis from the region of Selaoua Anouna and the Thymus ciliatus from Ain Makhlouf in the southwest of the city of Guelma.The gathered Thymus was dried until the stabilization of their weight.
The extraction of the essential oil was made by hydrodistallation (Clevenger-type apparatus).The distillations were realized by boiling, during 3 hours, 50 g of the plant dried with 500 ml of water in a ball of 1L overcome a column 60 cm of length and connected with a refrigerant.The yields in essential oil were determined with a regard to the dried material.These oils were stored and maintained optimally at 4° C protected from light according to literature protocol (Moumni et al., 2013a).

Essential oils analysis
The qualifications of the constituents of essential oil was realized by a chromatogram type SHIMATZU QP2010, provided with an FID type detector, equipped with a capillary column OV 17, of 25 m length and 0.25 mm diameter, with a thickness of the movie of 0.25 μm.The temperature of the oven was maintained at 60°C during 1 minute, and then increased at 200°C with a speed of 3°C/min.Later we had maintained in isotherm during 16 min.The temperature of injections is 250°C, the volume of each of the samples 0.1μl.After 1/10 dilution in the ethanol grad chromatography each sample, was injected in Split Less mode.
The fragmentation is made by electronic impact in 70 Ev, the carrier gas is the helium with a constant pressure of 25.6 kPa.The identification of the compounds was made by comparison of the mass spectrum of with those contained in the NIST bookshop.

Antimicrobial activity
The antimicrobial activity of essential oils of Thymus taxa was qualitatively determined using the method aromatogram solid environment.This technique involves depositing sterile discs, impregnated with essential oils of the Thymus taxa to the surface of Petri dishes in a solid environment carrier " Muller Hinton agar and nutritive blood" of the bacteria studied.DMSO aprotic organic solvent chosen for its inactivity bacteria.A range of 6 dilutions 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 was prepared.The bacterial strains used are gram-positive: Escherichia coli 22 and gram-negative: Staphyllococcus 23, Pseudomonas 53, they have been obtained from microbiological laboratory, Faculty of Medicinal Science, University of Badji Mokhtar-Annaba.Algeria.

RESULTS AND DISCUSSION
The yields of essential oils of the various species of Thymus of several regions in eastern Algeria were calculated with regard to the dray vegetable material for two periods: pre and during flowering.Table 1 summarizes the obtained results.

Chemical composition of the essential oils
The chromatographic analysis of the essential oil has allowed us to identify their constituents.The obtained results are mentioned in (table 2).
Fig1: Yields of essential oils during the two periods.

T. numidicus
The essential oil yield in before flowering period was (1.28%) and became (2.85%) during the flowering period, this increase may explain the change of the rate of the components.the result obtained during flowering is better compared to (Zeghib et al., 2013), which was (2.00%).In period before flowering, we found 24 components; that number climbed to 43 components during the flowering period.The major component is linalool in before flowering period (31.14%) and thymol (39.66%) during flowering period.The percentage of thymol increased (20.28%) before flowering period to (39.66%) during flowering, the same remark is attributed to γ terpinene, the rate increased from (0.16%) to (6.12%).We also note the appearance of new components during the flowering period: carvacrol (23.52%), eucalyptol (1.88%) and α phyllandrene (0.26%).The essential oil of T. numidicus was dominated by oxygenated monoterpenes in the two periods with 54.48% and 76.96% (Figure 2).The essential oil composition of Thymus numidicus obtained in flowering period is in agreement with the result found by (Zeghib et al., 2013) where thymol is the most important compound (23.92%)followed by linalool (17%).

T. algeriensis
The essential oil yields of T. algeriensis obtained in two periods are slightly different: (1.155%) in period before flowering and (1.176%) during flowering period.The number of components fell for 30 to 19 during flowering period.Camphor is the predominant compound in before flowering period (33.30%), but it completely fells during the flowering period where we found only (3.30%).
During the flowering period, we have the P-cymen-7-ol with a important percentage (26.18%)while this compound did not exist in before flowering period.The major components obtained during the flowering period found by (Amarti et al., 2010) and(Chemat et al., 2012) were respectively: camphor (27.70%) and thymol (71.45%).
The isolimonene and limonene were (5.5%) and (5.13%) in before the flowering period have completely disappeared in flowering period.We also noticed the appearance of new components during flowering period such as Methyl tert-butyl ether (19.29%), verbenone (12.84%), and γ terpinene (5.30%).The oxygenated monoterpenes represent a significant rate: (61.86%) before the flowering period and (77.56%) during the flowering period.

Microbial activity
The discs diffusions tested with 10 μl of the essential oils of different taxa of Thymus collected before and during flowering period have proved the good results with reference bacteria microorganisms with (7.3 to 50.8 mm) inhibition zones.The results obtained are mentioned in table 3. The tests of antibacterial activities on the three referenced strains have shown that the majority of essential oils of three taxa (T.numidicus, T. ciliatus.and T. algeriensis) have high inhibition values in particular for dilutions 1 / 4, 1/8 and 1/16.
The bacterial strain Escherichia coli 22 showed sensitivity to essential oils of three taxa.Essential oils of Thymus algeriensis obtained during flowering period with 1/4 dilution gave a very significant inhibition diameter (42.2mm), followed by the essential oil of Thymus numidicus in pre flowering and flowering periods with inhibition diameters (34.25mm) and (35mm).The results obtained during the flowering period is better than found by (Zeghib et al., 2013), which was (20.4 ± 0.5 mm).With the 1/8 dilution inhibition the important result was observed with the essential oil of Thymus ciliatus harvested before flowering period (30.2mm).The germ Staphyllococcus 23 showed a high sensitivity to essential oil Thymus ciliatus harvested before flowering period with diameter between 24.3mm and 50 mm depending on the dilution, and the essential oil of Thymus numidicus obtained during flowering with (43.3 mm) for a dilution 1/4.Pseudomonas 53 showed resistance for the full range of dillution of essential oils except for Thymus numidicus in two periods of development with dilutions 1/4 and 1/8.
The same result was found by (Zeghib et al., 2013) for the T. numidicus harvested during flowering with (16.4 ± 0.5 mm) of inhibition against Pseudomonas 53.(Kouch et al., 2014) found (38.5 mm) of inhibition with the essential oil obtained during flowering.The results obtained show that the three essential oils are effective against Escherichia.coli 22, while essential oil of T. ciliatus obtained before flowering and essential oil of T. numidicus in flowering period are most effective against Staphyllococcus 23.The importance of antimicrobial activity for the species thymus is related to their high content of phenolic compounds; in fact several authors (Pellecuer et al., 1980), (Gergis et al., 1990), (Panizzi et al., 1993), (Sivropoulou et al., 1996;Trombetta et al., 2002;Satrani et al., 2008;Chemat et al., 2012) showed that the essential oils rich in phenolic derivatives possess strong antimicrobial activity.(Dorman et al., 2000) showed that thymol is the compound that has the broadest spectrum of antibacterial activity, which explains our results found with the essential oils of T. numidicus and T. cilatus before and in flowering periods, where the percentage of thymol was between (20, 28% and 54, 04%), the latter is totally absent in the essential oil composition of T. algeriensis.Camphor is the main component for T. algeriensis, it is also known for his anti-microbial activity (Aligrannis et al., 2000) and (prudent et al., 1993).

CONCLUSION
In this work we studied the chemical composition of essential oils of different genius of Thymus (T.numidicus, T. ciliatus and T. algeriensis) collected before and during the flowering periods in different regions of Eastern Algeria.The chemical study of essential oil of the collected Thymus, revealed that essential oil yield of the three Thymus species in two stages were ranged from (1.002%) to (2.850%).
The chemotype of T. algeriensis is camphor before flowering stage with (33.30%) and p-cymen-7-ol in flowering period with (26.98%).We concluded that the chemical compositions of different geniuses of Thymus are mostly constituted by oxygenated monoterpens.The essential oils were tested by 3 bacterial strains and gave good results, we explain this by the presence of phenol derivatives such as thymol , linalool and γ terpineol.

Table 1 :
Yields in essential oil of various studied species.

Table 2 :
Chemical compositions of the Thymus essential oils in different parts of Eastern Algeria before and during flowering periods.
Yield of oxygenated monoterpens of the Thymus during two period of development.

Table 3 :
Antimicrobial activity of the different genius of Thymus collected before and during the flowering period in different regions of Eastern Algeria.