Phytochemical Analysis and Study of Antioxidant, Anticandidal, and Antibacterial Activities of Teucrium polium subsp. polium and Micromeria graeca (Lamiaceae) Essential Oils from Northern Morocco

The protection of agricultural crops and the preservation of the organoleptic and health qualities of food products represent a major challenge for the agricultural and agro-food industries. Essential oils have received greater attention as alternatives to replace the control strategies based on pesticides against phytopathogenic bacteria and synthetic compounds in food preservation. The aims of this work were to study the chemical composition of Teucrium polium subsp. polium and Micromeria graeca essential oils and to examine their antioxidant and antimicrobial effects. To carry out this work, the chemical composition of the essential oil was determined using gas chromatography (GC) with the detection feature of mass spectrometry (MS). Subsequently, the antioxidant activity was investigated by DPPH and FRAPS assays. The antimicrobial effect was studied against phytopathogenic and foodborne pathogenic bacteria using the disc and the microdilution methods. Our results showed that GC-MS analysis of EOs allowed the identification of 30 compounds in T. polium EO (TPpEO), while 5 compounds were identified in M. graeca EO (MGEO). TPpEO had as major compounds β-pinene (19.82%) and germacrene D (18.33%), while geranial (36.93%) and z-citral (18.25%) were the main components of MGEO. The most potent activity was obtained from MGEO (IC50 = 189.7 ± 2.62 µg/mL) compared to TPpEO (IC50 = 208.33 ± 3.51 µg/mL. For the FRAP test, the highest reducing power was obtained from 1.32 ± 0.1 mg AAE/g of TPpEO compared to MGEO 0.51 ± 0.13 mg AAE/g of EO. Both EOs exhibited varying degrees of antibacterial activities against all the tested strains with inhibition zones in the range of 9.33 ± 0.57 mm to >65 mm and MIC values from 0.19 to 12.5 mg/mL. However, MGEO exhibits an interesting anticandidal effect with inhibition zone 44.33 ± 0.57 mm. The findings of this research establish the riches of EOs on volatile compounds, their important antioxidant activity, and their antimicrobial effect against the bacteria tested.


Introduction
e security of agricultural crops and both organoleptic and health qualities of food products represent a main defiance for the agricultural and agro-food industries [1,2]. e control of the problems caused by phytopathogenic bacteria is based on use of pesticides and antibiotics with potential side effects on the environment and living beings. Such chemicals are not very biodegradable and represent a risk of developing antibiotic resistance, which inspired the European Union to limit their use [3,4]. On the other hand, the preservation of food products is assured by synthetic compounds called "food additives," presented potential side effects on the consumer [5][6][7]. e plant extracts and essential oils as antioxidant and antimicrobial agents are focused to overcome these problems and to satisfy the improved demand for more natural solutions. For a long time, different cultures and civilizations worldwide have been using plants as drugs to treat numerous diseases [8][9][10]. e essential oils (EOs) are among the natural products of great interest in food, cosmetic, and pharmaceutical industries due to their antioxidant and antibacterial [11][12][13][14][15][16][17][18], antifungal [19][20][21][22][23][24][25], antiparasitic [6,26], insecticidal [27][28][29][30][31], and anticancer activities [32][33][34].
Morocco by its biogeographical position is characterized, on the one hand, by ecological and floristic diversities and, on the other hand, by a long tradition and expertise in the use of plant medicines [35][36][37]. Previous works in some regions of Morocco have shown that the Moroccan pharmacopoeia is dominated mainly by Lamiaceae [38][39][40][41]. A great economic importance is given to many of their species due to their EO production [42] and their traditional use [40,43,44]. In recent decades, in the goal to valorize the Moroccan Lamiaceae species, previous researchers have evaluated the antioxidant and antimicrobial activities of essential oils of many plants [45][46][47][48][49]. In this order, our study focused on two species of Lamiaceae, Teucrium polium subsp. polium and Micromeria graeca, locally known as "Jaâda" and "Bakolt'nhal," respectively. ese species have been strongly used in Moroccan traditional medicine [38-40, 44, 50].
To the best of our knowledge, no reports on the variation of essential oil composition and biological activities of these plants collected from the Province of Taza, Northern Morocco, are available. erefore, the objectives of this study were the identification of volatile compounds of hydrodistilled EOs of T. polium and M. graeca and the investigation of their antioxidant and antimicrobial activities. en, the plant sample (100 g) was subjected to hydrodistillation using a Clevengertype apparatus for 4 h. e essential oil was stored at 4°C until use.
e chemical composition of EOs was analyzed according the conditions described in our previous works [51,52]. For each compound, the Kovats retention index (RI) was calculated relative to a standard mix of n-alkanes between C9 and C31 (Sigma-Aldrich Co.). Identification of constituents was performed by comparison of RI and MS spectra with those reported in the literature and by computer matching with standard reference databases (NIST98, Wiley275, and CNRS libraries).

Free Radical Scavenging Activity.
e radical effect of EOs was evaluated using the radical 2.2-diphenyl-1-picrylhydrazyl (DPPH) as reported by Benali et al. [52] and Huang et al. [53], with some modifications. In brief, the DPPH solution (0.2 mM in methanol) was prepared. en, 2.5 mL of test sample at different concentrations (2.5-100 μg/mL) was added to 0.5 mL of DPPH solution, and the absorbance of samples was measured at 517 nm after 30 min. Ascorbic acid and Trolox were used as positive controls. e calculation of the antioxidant activity was done according to the following formula: where A 0 is the absorbance of the negative control and A s is the absorbance of the test sample at 30 min. e test was carried out in triplicate, and the IC 50 values were reported as mean ± SD.

Reducing Power of Ferric
Ions. e reducing activity of EOs was determined according to Benali et al. [52] and Oyaizu [54]. e mixture of the sample (1 mL), the phosphate buffer (2.5 mL, 0.2 M, pH 6.6), and the potassium ferricyanide (2.5 mL) was prepared. After incubation for 20 min at 50°C (water bath), 2.5 mL of trichloroacetic acid (10%) was added to the mixture. en, the solution was centrifuged at 3000 Trs/ min for 10 min. Finally, 2.5 mL of the supernatant was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride. Absorbance was measured at 700 nm.

Antimicrobial Activity.
e antibacterial activity was evaluating using disc diffusion method as described by Benali et al. [52] and Rota et al. [55], with some modifications. In brief, sterile disks (6 mm diameter) containing 12.5 µL of pure essential oil were applied onto the surface of the agar medium which were previously spread by the test inoculum concentrations. Gentamicin (15 μg), vancomycin (30 µg), streptomycin (25 μg), and amphotericin (10 μg) were used as a positive control. Negative control consisted of 10% dimethylsulfoxide (DMSO). After incubation as described above, the antimicrobial activity was assessed by measuring the diameter of inhibition zones. Tests were performed in triplicate.

Determination of Minimum Inhibitory Concentration.
MIC was determined only for strains considered very sensitive and essential oils considered very active leading to diameters larger than 15 mm [52][53][54][55][56]. Minimum inhibitory concentrations (MICs) were realized in sterile 96-well microplate as described by Güllüce et al. [22], with some modifications. First, 100 μL of MHB was distributed in all test wells, except the first well in which a volume of 200 μL containing the essential oil at a concentration of 25 mg/mL in 10% DMSO. A series of concentrations ranging from 0.097 to 25 mg/mL were prepared by the transfer of 100 μL by scalar dilutions from the first to the ninth well. en, except the 10th well used as sterility control, 10 μL of the suspension from each well was removed and replaced by the test inoculum concentrations as described above. e eleventh well was considered as positive growth control containing only broth medium. e last well containing 10% DMSO (v/v), without oils, was used as negative control. en, the plates were incubated at conditions of growth as described above. After incubation, a volume of 25 μL of an indicator of microorganism's growth was added in each well, and tetrazolium (MTT: 3-(4,5-dimethythiazol)-2-yl-2, 5diphenyltetrazolium bromide (Sigma)) was prepared at a concentration of 0.5 mg/mL in sterile distilled water. e microplate was re-incubated for 30 min at temperature 25°C or 37°C. Where microbial growth was inhibited, the solution keeps the initial color of MTT. To determine the minimum bactericidal concentration (MBC) value, 10 μL of broth from the uncolored wells was inoculated and incubated at growth conditions.

Statistical Analysis.
All experiments were done in triplicates and values of each were expressed as mean-± standard deviation (SD) and were subjected to analysis of variance (one-way ANOVA). e statistical analysis was performed using GraphPad Prism version 6.00 (GraphPad Inc., San Diego, California). Differences (between groups) were considered as statistically significant at p < 0.05.

Antioxidant Activity.
e essential oils were evaluated for their antioxidant effect using two methods, the DPPH free radical scavenging and the ferric ion reduction assay (FRAP). For the DPPH assay, as summarized in Table 2, the most potent activity was obtained from M. graeca (IC 50 � 189.7 ± 2.62 µg/mL), followed by T. polium (IC 50 � 208.33 ± 3.51 µg/mL), but they were all less potent than the standards used as positive controls, namely, Trolox and ascorbic acid (IC 50

Antimicrobial Activity.
e in vitro antimicrobial activity of the essential oils against the tested microorganisms was qualitatively and quantitatively confirmed by diameter of inhibition zone and the MIC values. As shown in Tables 3 and 4, the essential oils exhibited varying degrees of antibacterial activity against all tested strains. For the essential oil of TPpEO, the inhibition zones were in the range from 7.33 to 52 mm, with MIC values of 0.19 mg/mL and 0.78 mg/mL. C. michiganensis was the most sensitive bacteria to TPpEO with inhibition
ese results indicated the possibility of the chemical composition difference in Micromeria EOs from one species to another. e qualitative and/or quantitative difference between the oil composition in our results and those noticed in e diameter of the inhibition zones (mm), including diameter of disc (6 mm), is given as mean ± SD of triplicate experiments. nt: not tested; n.e: no effect. e sensitivity to the different oils was classified by the diameter of the inhibition halos as follows: not sensitive (−) for diameters less than 8 mm; sensitive (+) for diameters 9-14 mm; very sensitive (++) for diameters 15-19 mm; and extremely sensitive (+++) for diameters larger than 20 mm. previous works may be attributed to the ecological factors, genetic differences, environment, geographical origins, and season of harvest [68][69][70][71][72].
As indicated above, β-pinene, germacrene D, and α-pinene were among the major compounds of TPpEO chemical composition and nerol and z-citral were for MGEO. Previous research studies showed the antioxidant effect of β-pinene, germacrene D, and α-pinene tested individually [73][74][75][76][77]. Also, nerol and citral are known for their antioxidant efficacy [78][79][80]. ese proprieties can explain the antioxidant activity of both essential oils. e small difference of antioxidant activity between TPpEO and MGEO may be associated to the variability in chemical composition since the antioxidant mechanisms of essential oils are generally caused by several compounds' functional groups and their structure [81]. However, the difference observed between testing methods could be explained by the correlation between the chemical composition and/or each compound and the used method [82][83][84].
For the antibacterial activity, it is known that the Gramnegative bacteria are less sensitive to plant extracts than Gram-positive ones [85][86][87]. However, the present findings showed that essential oils of plants studied do not have selective antibacterial effects against microorganisms tested.

Conclusion
To the best of our knowledge, this is the first report contributing details on chemical composition and antioxidant and antimicrobial activities of Teucrium polium subsp. polium and Micromeria graeca essential oils from Northern Morocco. Our findings have shown that both essential oils are rich by volatile compounds which could be responsible for the observed antibacterial and antioxidant effects. TPpEO and MGEO may be proposed as natural antioxidant and antibacterial product for application on food preservation and management against phytopathogenic bacteria. Further in vivo studies will be recommended to investigate their biological proprieties and negative effects before the practical applications.
Data Availability e data used in this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.