Effect of Harvest Time on Yield, Chemical Composition, Antimicrobial and Antioxidant Activities of Thymus vulgaris and Mentha pulegium Essential Oils

Department of Food Technology, Research Unit of Nuclear Technology, Environment and Quality Laboratory of Food Technology, National Institute of Agronomic Research (INRA), Tangier, Morocco. Department of Animal Production and Food Science, Veterinary Faculty, Universidad de Zaragoza, Spain. 3 Department of Chemistry, Faculty of Science and Technology, Université Abdelmalek Esaâdi, Morocco. 4 Department of Biology, Faculty of Science and Technology, Université Abdelmalek Esaâdi, Morocco. Department of Biology, Polydisciplinary Faculty of Larache, Université Abdelmalek Esaâdi, Morocco.


INTRODUCTION
Essential oils of medicinal and aromatic plants are subjected to a high demand. This demand is justified by the biological effects exhibited by some active compounds contained in these oils and by their condition of natural products as an alternative for replacing chemical substances in the pharmaceutical, cosmetic and food industries [1].
Among biological properties investigated in EOs, antimicrobial and antioxidant activities have been widely demonstrated [2][3][4][5][6]. For this reason, they have been proposed as potential agents for food preservation.
Nowadays, the major concern for EOs producers is to obtain oils with better quality and quantity, and with known and stable biological properties. However, the quantity (yield) and the quality (chemical composition) of EOs are influenced by the interaction among several factors such as genotype, environment, plant age, season and time of harvest [7][8][9][10][11].
While the effect of the vegetative cycle on yield and chemical composition of EOs has been well demonstrated [7,8,[12][13][14][15], its implication on biological properties has scarcely been studied. Among the few published data in this sense, it can be deduced that the plant vegetative cycle is of great importance in determining EOs biological properties [16,17].
Due to its geographical position, Morocco offers a wide range of Mediterranean bioclimates allowing the growth of rich flora, which is constituted by more than 4200 species. Aromatic and medicinal plants are estimated by 500 to 600 species, and most of them are endemic [18].
Morocco is a traditional supplier of medicinal and aromatic plants to Europe. More than twenty plants are used for EOs or other aromatic extracts production to be used in perfumery and cosmetic industry, as hygiene products, etc.
T. vulgaris and M. pulegium are two spontaneous aromatic and medicinal plants belonging to the Lamiaceae family that grow in several regions of Morocco. Both aromatic plants have an undeniable commercial interest because of the diverse biological activities of their extracts which have been widely demonstrated [19][20][21][22].
Despite the great interest of both plants, to the best of our knowledge, there are no studies about the effect of the vegetative cycle (preflowering and full-flowering stages) on their antioxidant or antimicrobial activities, which makes difficult the adequate exploitation of these natural resources.
This study aims to investigate the effect of the vegetative cycle (pre-fowering and full flowering stages) on the yield, chemical composition, antimicrobial and antioxidant activity of the EOs of T. vulgaris and M. pulegium harvested in the north and north east of Morocco, respectively.

Plant Material
The studied plants (T. vulgaris and M. pulegium) were collected during the period of April 2011 (Pre-flowering) to June 2011 (Full-flowering). T. vulgaris plants were harvested in the region of Beni Idder (391m, 35°23'59"N, 5°30'35"W) in the Northeast of Morocco and M. pulegium plants from Bougedour in the North (35m, 35°39'35"N, 5°50'59"W). The taxonomic identification was performed following the procedure described by Fennane et al. [23]. Subsequently, plants were dried for 48 h at 40ºC under ventilation. Leaves and flowers were used for the extraction of EOs.

Essential Oils Extraction
The EOs were extracted via steam distillation for 2 h using a Clevenger-type apparatus. The supernatant was separated by decantation after adding 50% of NaCl. The EOs were stored in sealed glass vials at 4ºC prior to analysis. The yield was based on the dry weight of the samples (% w/w). EOs were collected and stored at 4ºC in dark bottles in order to protect them from heat and light radiation.

Gas Chromatography/Mass Spectrometry (GC/MS) Analysis
The GC/MS analyses were carried out using a gas chromatograph coupled with Mass Identification of the components was made by the determination of the their retention indices (KI) relative to those of a homologous series of nalkanes (C8-C26) (Fluka, Buchs/sg, Switzerland) and by matching their recorded mass spectra with those stored in the spectrometer data base (NIST MS Library v. 2.0) and the bibliography [24].
Quantification was computed as the percentage contribution of each compound to the total amount present.

Antimicrobial Activity
EO antimicrobial activity was determined by paper disc agar plates. Microbial strains were provided by the Spanish Collection of Type Cultures (CECT). Two Gram negative bacteria, Escherichia coli (SCTC 471) and Salmonella Senftenberg 775W (ATCC 43845), and two Gram-positive, Listeria monocytogenes (SCTC 4031) and Staphylococcus aureus (SCTC 976), were used. A volume of 10 mL of Muller Hinton Agar medium (Liofilchem, Roseto degli Abruzzi, Italy) (MHA) was poured into Petri dishes. One hundred μL of culture bacteria were plated at a final concentration of 10 6 cells/mL approximately.
After 15 min, a paper disc Whatman No. 1 of 6 mm (Whatman International L7d Maidstone, England), impregnated with 10 μL of the EO, was placed on the agar surface. The plates were incubated for 24 h at 37ºC (E. coli, Salmonella Senftenberg, S. aureus) or 48 h at 30ºC (L. monocytogenes). The diameters of the resulting zones of inhibition were measured including the diameter of the paper disc. An inhibition zone (disk diameter included) lower than 9 mm was considered as non inhibitory. An average zone of inhibition was calculated after carrying out three independent replicates.

Radical scavenging activity assay
Following the method described by Blois [25], for each EO, 1 mL from a 1 mM methanol solution of 2,2-diphenyl-1-picrylhydrazyl (Sigma-Aldrich) (DPPH) was added to 3 mL of diluted EO in ethanol (1,15,30 uL/mL), butylated hydroxytoluene (Sigma-Aldrich) (BHT) (reference) or ethanol (control). Mixtures were shaken and incubated (room temperature/dark). After 30 min, absorbance at 517 nm was measured. Scavenging activity of DPPH radical was calculated as follows: where A blank is the absorbance of the control reaction (containing all reagents except the test compound), and A sample is the absorbance in presence of the test compound.

Statistical Analysis
Analyses of variance were performed by Statgraphics Plus 4.0 statistical software. All experiments were performed in triplicate and the results were presented as the mean with its standard deviation (n = 3) in each case. Differences were considered significant at P <0.05.

Yield and Chemical Composition
The yields of EO extraction for T. vulgaris and M. pulegium plants, as a function of the vegetative cycle, are illustrated in Table 1. For T. vulgaris EO, average yield at pre-flowering period was 1.9% while it was 3.6% at full-flowering stage. These percentages are relatively high when compared to some published data that found values ranging from 0.5 to 1.2% [7,18,26]. Table  1 also shows that yield values for M. pulegium EO were 0.9% and 3.5% for pre and fullflowering period respectively. These percentages were similar to those obtained by Hmiri et al. [20]. Therefore, these results demonstrate that the highest yield was obtained during fullflowering period. Therefore, it seems advisable to control the plants vegetative period in order to optimize oil production and obtain the maximum yield per plant. Similar trend has been previously reported by other authors [27,28]. Verma et al. [29] reported that EOs high yield at this stage is probably due to its ecological role in attracting pollinators and in being an antifungal defense mechanism.
The EOs obtained at pre-and full-flowering period were analyzed by GC-MS and results are included in Tables 2 and 3 for T. vulgaris and M. pulegium respectively. Chromatographic analysis highlighted the occurrence of six main components for T. vulgaris EO at higher levels than 0.5% of the content. Carvacrol was the major component with a content ranging from 70.97 to 74.10% accompanied with other components at relatively low levels: δ-terpinene (1.05 to 1,09%), p-cymene (5.17 to 8.74%), terpinene (4.08 to 5.89%), β -humulene (2.40 to 3.04%). Our results are in accordance with those obtained by Bouhdid [30] who found that carvacrol was the major compound. However, they differ from those obtained by Imelouane et al. [26] in eastern Morocco and Jordan et al. [8] from Murcia region in Spain. For Imelouane et al [26], camphore and camphene were the major compounds, while 1,8-cineole (29.2 to 36.5 %) and terpenyl acetate (18.2 to 25%) were identified as major compounds for Jordan et al. [8]. Such variations between countries could be due to numerous factors, such as differences in climatic conditions, geographical location, season at the time of collection, stage of development, processing of plant materials before extraction of the oils, and occurrence of different chemotypes. Table 2 also shows that vegetative cycle affects significantly the chemical composition of T. vulgaris EO. The differences consist of the variation of carvacrol percentage that increased from 70.97 to 74.10% and the decrease in the content of p-cymene from 8.74 to 5.17 and terpinene from 5.89 to 4.08%. Our results are in accordance with those obtained by Hudaib et al. [7] who found that the chemical composition of EOs varied markedly with vegetative cycle. These authors concluded that the end of vegetative cycle was the best period to obtain EOs with better quality and quantity. We also noted that when the content of carvacrol increased, the content of p-cymene and terpinene decreased. This might be explained by the simultaneous bioconversion of p-cymene and -terpinene to carvacrol [31].
Our results are similar to those reported by Sutour [32] who found that pulegone was the major compound, but differ from those of Mahboubi and Haghi [33] and Verma et al. [29] who identified piperitenone and menthol as the major constituents. As in T. vulgaris EO, mostly quantitative rather than qualitative variations were observed with respect to chemical composition as affected by vegetative cycle stages. The differences were reflected in the variation of pepiritenone percentage that increased from 1.71 to 6.54%, and the decrease of menthone from 6.25 to 1.37%. Nevertheless, the content of the major component (pulegone) was not affected. Similarly, Verma et al. [29] described considerable variation in the chemical composition of Mentha arvensis and Mentha piperita EOs at different stages of plants growth.

Antimicrobial Activity
The average diameters of the inhibition zone observed around the disks impregnated with the EOs of both plants studied are summarized in Table 4. According to these results, T. vulgaris EO showed a strong activity on all tested bacterial strains based on the inhibition diameters obtained between 30.7 and 36.7 mm. The largest antimicrobial activity was observed against L. monocytogenes (36.7 mm) and the weakest against E. coli (30.7 mm). Our results also demonstrated that Gram-positive bacteria were more sensitive to T. vulgaris EO than Gram-negative bacteria. Similar trend was observed by Rota et al. [3]. The resistance of Gram-negative bacteria to EOs is partly due to the complexity of the cell wall of these microorganisms which contains an outer membrane, unlike the simpler cell wall structure of the Gram-positive ones [1].
Despite the chemical variation of T. vulgaris EO between the two vegetative stages investigated (pre-and full-flowering period), this variation did not statistically (P <0.05) affected its antimicrobial activity against E. coli, Salmonella Senftenberg and S. aureus. Only L. monocytogenes was more sensitive to the EO from the full-flowering period. The difference in sensitivity of L. monocytogenes between preand full-flowering periods might be due to the higher concentration of carvacrol at this stage, which is a very effective antimicrobial agent, especially against Gram positive bacteria [5,34,35].
M. pulegium EO showed a lower antibacterial activity compared to the EO of T. vulgaris (Table  4); the inhibition diameters ranged from 9 mm to 12.7 mm. The largest antimicrobial activity was obtained against E. coli (12.7 mm) and the weakest against Salmonella Senftenberg (9 mm). Our results differ from those described by Ait-Ouazzou et al. [36] and Teixeira et al. [37] that demonstrated a higher antimicrobial activity of M. pulegium EOs.
Like in T. vulgaris, the chemical variation of M. pulegium EO during the vegetative cycle did not affect its antimicrobial activity against E. coli, Salmonella Senftenberg and S. aureus. In fact, the concentration of the major compound (pulegone) did not vary with the vegetative cycle. Again, only L. monocytogenes was more sensitive to the EO obtained during the preflowering period. The difference in sensitivity of L. monocytogenes between pre-and fullflowering periods could be due to the main differences found: the concentrations of menthone that changes from 6.25 to 1.37% and pepiritenone from 1.71 to 6.54% Therefore, these results point out the fullflowering period as the most appropriate time to harvest T. vulgaris EO and the pre-flowering for the M. pulegium EO in order to guaranty their greatest antimicrobial effect.

Antioxidant Activity
The effect of the vegetative cycle on the scavenging activity of DPPH radical of M. pulegium and T. vulgaris EOs is given in Table 5. As we can see, T. vulgaris EO showed a greater antioxidant activity than M. pulegium EO. Antioxidant activity of T. vulgaris increased with increasing concentration and it was comparable to the antioxidant effect of BHT at a concentration of 15 µL/mL. EOs from fullflowering period exhibited significantly higher antioxidant activity (P<0.05). This reduction capacity of free radicals of T. vulgaris EO is mainly due to its chemical profile, rich in carvacrol that has already been proved to possess a strong antioxidant activity [38][39][40].
Opposed to the results obtained with T. vulgaris EO, M. pulegium EO exhibited a higher antioxidant activity at pre-flowering than fullflowering period. Probably, the difference between the antioxidant activities from the two periods studied was related to the proportion of the present active components, especially the  content of menthone that decreased from 6.25 (pre-flowering) to 1.37% (full-flowering). Our results are in accordance with those obtained by Zhen et al. [41] who observed that the antioxidant activity of Citrus medica EOs decreased with increasing maturity at harvest. Also, Hussain et al. [16] showed that the antioxidant activity of Ocimum basilicum EOs varied significantly with the season.
So, our results demonstrate that full-flowering is not always the best period to harvest aromatic and medicinal plants with better antioxidant activity, but that depends on the type of plant.

CONCLUSION
As far as we know, this is the first time that the effect of vegetative cycle on the yield, chemical composition, antimicrobial and antioxidant activity of T. vulgaris and M. pulegium EO from Morocco has been studied.
According to our results, in general, full-flowering period is the best period to harvest T. vulgaris plants in order to produce EOs with better yield, antimicrobial and antioxidant activity. In contrast, pre-flowering stage is recommended to obtain M. pulegium EO with higher antioxidant and antimicrobial activity. The optimization of the harvest time will facilitate the exploitation of these natural resources and will allow producers to obtain EOs with better quality and quantity.

CONSENT
It is not applicable.