Physicochemical Characterization and Antioxidant Properties of Essential Oils of M. pulegium (L.), M. suaveolens (Ehrh.) and M. spicata (L.) from Moroccan Middle-Atlas

The cosmetics and food fields are based on the use of synthetic substances to protect their products against oxidation. However, synthetic antioxidants were reported to have negative effects on human health. The interest to develop natural antioxidants from plants has been growing in recent decades. The aim of this study was to determine the antioxidant properties of three essential oils (EOs) of M. pulegium (L.), M. suaveolens (Ehrh.) and M. spicata (L.) from the Azrou and Ifrane regions. The organoleptic characteristics, yields and physical properties were determined for the selected EOs. Their chemical compositions were identified using GC-MS; then, their antioxidant activities were evaluated using the DPPH• free radical scavenging activity and were compared with the ascorbic acid standard. The determined physicochemical parameters of dry matter and EOs demonstrated their good quality. The analysis of the EOs showed the dominance of pulegone (68.86–70.92%) and piperitenone (24.81%), piperitenone oxide (74.69–60.3%), and carvone (71.56–54.79%) and limonene (10.5–9.69%) for M. pulegium, M. suaveolens and M. spicata, respectively, from Azrou and Ifrane. Additionally, the antiradical tests demonstrated the remarkable power of these EOs, especially M. pulegium EO (IC50 = 15.93 mg/mL), which recorded the best activity compared with ascorbic acid (IC50 = 8.849 mg/mL). The obtained results indicated that these EOs could be applied as natural antioxidants in the food industry.


Introduction
Antioxidants are substances that the body produces as a reaction to environmental and other pressures in order to prevent or decelerate cell damage caused by free radicals [1]. Free radicals can also be naturally produced by an organism during biosynthetic processes, such as vitamins C (ascorbic acid) and E, carotenoids and phenolic compounds, or they can be synthetic [2]. Synthetic antioxidants, such as BHA and BHT, are widely used in the food and cosmetics industries because of their chemical stability and availability relative to natural ones [3]. However, they are now suspected to cause potential risks to human health, such as cardiovascular disease and cancer [4].
in the food and cosmetics industries because of their chemical stability and availability relative to natural ones [3]. However, they are now suspected to cause potential risks to human health, such as cardiovascular disease and cancer [4].
Consequently, great attention has been given to the natural antioxidants of plant extracts, especially to the essential oils that are known to possess potential as natural agents for food conservation. Recently, several EOs were qualified as natural antioxidants and proposed to replace synthetic ones in specific areas of food preservation [5,6]. The genus Mentha is characterized by the diversity of its secondary metabolites used as traditional medicines, food additives and household remedies for gastrointestinal disorders. They are also used as mouth fresheners, antibacterial agents, tonics, astringents, mild laxatives and culinary herbs [7].
M. pulegium (L.), M. suaveolens (Ehrh.) and M. spicata (L.) are species that are widely used by the Moroccan population in traditional medicine and for the richness of their EOs in active principles required in the food and pharmaceutical industries. In this context, we aimed in this research to study the physicochemical properties of dry matter and the EOs of selected mints due to the scarcity of previous works dealing with them to show the effect of the origin region on the yields and constituents of the EOs and to evaluate their capacity to scavenge the DPPH • free radical.

Plant Material
The

Phytochemical Study 2.2.1. Quality Control of the Dry Matter Moisture Content
The moisture content (MS) was obtained by heating 5 g of the test sample, previously placed in dried crucibles, in an oven at 103 ± 2 • C [8]. The solid crucibles were weighed before and after drying. After 24 h, the MS was calculated and expressed as a percentage according to the following formula:

Determination of the pH
The procedure was performed according to NF V 05-108(1970) [9]. It defines the acidity of the product considered, measured with a pH meter. Ten milliliters of hot distilled water was added to 2 g of the sample. The mixture was crushed, filtered and allowed to cool. The electrode was immersed in a large volume of this filtrate and the pH value was noted. The electrode should be rinsed with distilled water before and after each measurement.

Ash Content
The ash content was determined according to NF V 05-113, 1972 [10]. The sample (4 g) was calcined at 550 • C in porcelain capsules. The capsules were placed in a muffle furnace until whitish ash of constant weight was obtained. They were removed from the oven and left to cool in the desiccator, then weighed. The organic matter was calculated first to deduce the ash percentage according to the following equations: OM% = ((W 1 −W 2 )/Ts) × 100 OM%: organic matter; W 1 : weight of the capsule and sample before calcination; W 2 : weight of the capsule and the sample after calcination; T S : test sample.

Then:
Ash % = 100 − OM% Determination of the Acidity According to NF V 05- 101 [11], the acidity was obtained by adding 10 g of powder to 50 mL of boiling distilled water; the solution was then put in a water bath for 30 min. After cooling, the mixture was made up to 100 mL with distilled water and filtered; a few drops of phenolphthalein were added to 10 mL of the recovered filtrate. Thereafter, the titration was carried out with KOH until the persistent pink color changed. The acidity was determined using the following equation: A% = ((250 V 1 × 100)/(V 0 M × 10)) × 0.07 M: mass in grams of the product taken; V 0 : volume in milliliters of the test sample; V 1 : volume in milliliters of the potassium hydroxide solution at the 0.1 N used.

Quality Control of the Mint Species EOs Determination of the Brix Degree
The Brix degree principle was based on measuring the concentration (%) of all solids (salts, sugar, proteins, fatty acids, etc.) dissolved in the oil using a refractometer device.

Determination of the Acid Value (AV)
For measuring the acidity of an EO, 1 g of the EO was dissolved in 5 mL of 96% alcohol, and a few drops of 1% phenolphthalein solution were then added. The mixture obtained was titrated using 0.1 N KOH solution (diluted in 96% ethanol) until a pale pink color appeared for 30 sec [12]. The following equation was then used to calculate the acid value: AV = (VKOH 0.1 × 56.1)/Mass of essential oil The iodine value was determined as described by Manuranjani et al. [13], where 0.5 g of the oil was taken for the analysis. The final solution was titrated with 0.1 N sodium thiosulphate using a starch indicator. The iodine value was then calculated using the following equation: Determination of the Peroxide Index The peroxide index (PV) provides valuable information on the oxidation state of essential oils. For this test, about 1.0 g of the oil sample was taken and 1.0 g of KI and 20 mL of the solvent mixture (glacial acetic acid/chloroform, 2/1, v/v) were added to it. The mixture was then boiled for one minute. In a flask containing 20 mL of 5% KIO 3 solution, the hot mixture solution was poured. A few drops of the starch solution were added to the mixture and titrated with 0.025 M sodium thiosulphate solution [13]. The following equation was then used to calculate the peroxide index: PV (m Eq/kg) = (titre × N/100) × weight of the sample N: normality of sodium thiosulphate solution.

Extraction and GC-MS Analysis of the Mint EOs
The extraction of essential oils was conducted via hydrodistillation of the mint aerial parts (100 g) using a Clevenger apparatus over 3 h. The isolated EOs were dehydrated with anhydrous sodium sulfate and stored in a refrigerator at a temperature of 4 • C until use. For the calculation of the yields, three replicates were performed for each mint and the following formula was used: The chromatographic analyses were performed using a Hewlett Packard gas chromatograph (HP 6890 series) equipped with an HP-5 capillary column (30 m × 0.25 mm × 0.25 microns' film thickness) and an FID detector set at 250 • C; the setup was fed with a gas mixture of H 2 /air. The mode of injection was split; the carrier gas used was nitrogen with a flow rate of 1.7 mL/min. The column temperature was programmed at a mounted rate of 4 • C/min from 50 to 200 • C for 5 min. The unit was controlled by a computer system type "HP Chem-Station" to manage the operation of the device and monitor the chromatographic analyses. GC-MS was carried out using a chromatograph Hewlett Packard (HP 6890) coupled to a mass spectrometer (HP 5973 series). Fragmentation was performed via electron impact at 70 eV. The used column was a capillary-type HP 5SM (30 m × 0.25 mm × 0.25 mm). The column temperature was programmed at a mounted rate of 4 • C/min from 50 to 200 • C for 5 min. The carrier gas was helium with a flow rate set at 1.7 mL/min. The injection mode was split type.
For the compound identification, the Kovàts index of each compound was calculated in relation to the retention time of a series of linear alkanes (C 7 -C 40 ). The calculated index was then compared with those of the Adams reference. The mass spectra of compounds

In Vitro Tests of the Antioxidant Activity of the EOs Using a DPPH • Assay
The scavenging DPPH • free radical potential was determined in vitro according to the protocol of Nikhat et al. [14]. The experiment was carried out using a visible UV spectrophotometer at a wavelength of 515 nm. The solution of DPPH • (1,1-diphenyl-dipicrylhydrazyl) at 5 M was prepared by dissolving 2.4 mg of the DPPH • powder in 100 mL of ethanol. Then, 2.8 mL of the obtained solution of DPPH • was mixed with 60 µL/mL of each EO or standard antioxidant (ascorbic acid) at different concentrations from 0.04 to 1.6 mg/mL. After 30 min of incubation in the dark at room temperature, the absorbance was read at 515 nm against a blank, which contained only ethanol. The positive control contained the DPPH • without the extract and the obtained values were subsequently converted into inhibition percentages. The radical scavenging activity was quantified using the following equation: The graph of the absorbance variation according to the concentration of extract allowed for determining the IC 50 (concentration corresponding to the loss of 50% of free radical's activity).

Data Analysis
To conduct the statistical calculations, we used SPSS software with a level of significance of <0.05 for variance values and GraphPad Prism 5 for Windows for the means and standard deviations. Additionally, the values of IC 50 were obtained from the third-degree polynomial trend curves in the absorbance graphs using Excel 2016.

Phytochemical Study
The phytochemical study consisted of the determination of the physicochemical properties of selected Mentha species in the form of dried matter and the EOs. These parameters allowed for evaluating their quality. However, there are few published works on the physical properties of these three mints. Moreover, the yields and the chemical composition of EOs were also determined.

Physicochemical Properties of the Dry Matter
The main calculated parameters of Mentha dried aerial parts are summarized in Table 1. There was a significant variation between species (p ≤ 0.01) and between origin regions for the same species (p ≤ 0.05) but the obtained values remained in compliance with the standards. The main parameters were used to test the quality of all EOs from both regions. Most plant nutrients are optimally available to plants within this pH range (6.5 to 7.5), plus this range of pH is generally very compatible with plant root growth [15]. In addition, plants prefer mildly acidic substances. The obtained values of pH were found to range from 5.45 to 5.95. All values obtained for all species were interesting but the best ones were found for the mints from Azrou compared with those from Ifrane. Generally, they demonstrated that our samples presented good quality.
Regarding the moisture contents, all studied Mentha species showed significant rates, except M. suaveolens from Ifrane, whose MC reached approximately 46.68%. Moreover, the values of M. pulegium MC, either from Azrou or Ifrane, were found to be lower than those obtained by Tanavar et al. [16] (65.85 ± 0.43%), while those found by Tarasevičienė et al. [17] in M. suaveolens leaves (20.93 ± 0.12%) and stems (20.70 ± 0.34%) ranged between the higher MC of pennyroyal from Azrou and the lower MC of that from Ifrane. However, the MC of our spearmint samples from both regions seemed to be less than that of spearmint from Sudan (76.01 ± 0.03%) [18]. Moisture content is widely used in the testing of water activity of many foods. A high value implies that the plant parts may have a short shelf life since microorganisms, which cause spoilage, thrive in foods with high moisture content. The possibility of spoilage reduces with the decrease in moisture content [19]. Consequently, our species were found to be of good quality since they were dried under laboratory conditions.
Ash content is an index of mineral contents in biota. The obtained percentages were nearly similar for all studied mints, except for M. spicata from Ifrane (21.87%), which had the highest percentage. However, the ash content of the fresh spearmint leaves from Sudan was found to be less than ours (3.48%) [18]. As reported by Dairo et al. [20], the ash content is within the range of values (16.30-17.31%) for some vegetables [21]. Thus, in a young leaf, the ash may constitute approximately 5% of the dry weight, while in a mature leaf, it may be 15% [22]. Tarasevičienė et al. [17] concluded that the leaves of M. suaveolens (10.92 ± 0.018%) were richer in crude ash than the stems (8.17 ± 0.168%). Therefore, the variation in the amount of ash depends on the part of the plant, age, treatment, etc. [22].
Based on the found results, the acidity rates of three Mentha species from both regions ranged from 0.163% to 0.608%. The lower rates were observed for all mint species from Azrou, especially for M. spicata (0.163% and 0.185% for Azrou and Ifrane, respectively). However, a remarkable increase in the acidity percentage was found for fresh spearmint leaves from Sudan (2.203%) [18].

Physicochemical Properties of the Mentha EOs
The extracted EOs have specific organoleptic properties. The yellow color with a lemony and very strong aromatic odor characterized the M. pulegium EO. The M. suaveolens EO was pale yellow with a fragrant odor, while the M. spicata EO was observed to be light yellow with a strong pleasant and fresh perfume. Similarly, the color of M. suaveolens from Saudi Arabia was pale yellow with a pleasant and distinct odor [23]. Moreover, the color of the tested spearmint essential oils from Pakistan was noted to be varied between yellow-green and brownish-yellow depending on their growing habitat, while the odor was similar to mint [24]. The physicochemical properties in the present analysis of Mentha oils served to prove their quality and their ability to be used safely.
The Brix degree, acidity index, iodine index and peroxide value were measured and their values are summarized in Table 2. They complied with the standards, even though there were slight significant differences according to the type of species and region (p ≤ 0.01).
The values of the Brix index determined for all mints ranged between the lowest index 71.6 • B recorded in the M. spicata EO from Azrou and the highest one was observed in the M. suaveolens EO (81.5 • B) from Azrou. These indices were greater than those recorded for peppermint oil (24.57-27.32 • B) [25] and comparable to those measured by Radi et al. [26] for some thyme species, such as 76.62 ± 0.05 • B (T. willdenowii) and 85.44 ± 0.05 • B (T. zygis).
The acid values (AVs) of the tested oils were found to be 21.91, 22.03 and 26.1 mg KOH/g for M. pulegium, M. suaveolens and M. spicata from Azrou, respectively, and 27.1-28.05 mg KOH/g for the mints from Ifrane. It was noted that the AVs found for M. pulegium and M. suaveolens from Azrou seemed to be similar. These results were almost comparable with the results of Salim et al. [27] and 19.7-25.2 (mg KOH/g), while those reported by Sulieman et al. [18] showed a lower value for the spearmint oil (0.0610 mg KOH/g oil). The iodine values (the weight of iodine absorbed by 100 parts by weight of fat, where the higher the iodine value, the greater ability of oil or fat to become rancid) reached for three mint oils ranged from 103.759 to 110.160 and 106.596 to 111.122 g I/100 g from the Azrou and Ifrane regions, respectively. However, Sulieman et al. [18] found a value (0.5467 mg I/g oil) that was lower than that calculated for our spearmint oil. There is no official maximum to be expected for the PV of EOs (PV of vegetable oils <10-15 m Eq O 2 /kg) [28]. Furthermore, the PV of a fragile EO stored for a long time in a small bottle that is regularly opened with little or no protection from heat and light can easily be of the order of 2-300 m Eq O 2 /kg according to ISO 18,321 [16]. Therefore, our samples presented good PVs that oscillated between 12 and 21.6 m Eq O 2 /kg as the lower and higher amounts for the three mint species from both regions.
Furthermore, all obtained physicochemical parameters varied between species and among the same species from the same region; this can be explained by the fact that these properties are strongly and directly affected by the distillation techniques, climatic conditions, plant varieties, regions, harvest periods, genotype, type of material and chemical composition [29].

The Yield of the Mint EOs
The yields obtained from the three mints EOs varied with the region of origin. The total yield percentages of the M. pulegium (L.), M. suaveolens (Ehrh.) and M. spicata (L.) EOs from Azrou reached 5.9 ± 0.014%, 1.8 ± 0.009% and 2.4 ± 0.012%, respectively, whereas those from Ifrane recorded lower percentages: 2.26 ± 0.013%, 1.25 ± 0.007% and 1.18 ± 0.014%, respectively. These percentages were found to be higher than those already studied from other regions of the Middle Atlas: 5.29% for M. pulegium EO [30], 1.55% for M. suaveolens EO from M'rirt [31] and 1.54% for M. spicata EO from Meknes [32]. Such variation in the EO yields may be linked to the morphological and biochemical diversity of the plants attributed to different typographical conditions and environmental factors [24].

Chemical Compositions of the Mentha Species EOs
According to the chromatographic analyses of the EOs extracted from all studied mints, the chromatographic profiles were measured to allow for identifying different chemical compositions of the analyzed EOs. Some variations were observed between the three mint species and between the same species from different regions ( Figure 2). According to the chromatographic analyses of the EOs extracted from all studied mints, the chromatographic profiles were measured to allow for identifying different chemical compositions of the analyzed EOs. Some variations were observed between the three mint species and between the same species from different regions ( Figure 2).  The chromatographic analysis of M. pulegium essential oils allowed for identifying 26 compounds that represented approximately 99.10% of the total composition of the EO from Azrou against 33 compounds (100%) from Ifrane. Monoterpenes dominated the totality of compounds compared with sesquiterpenes in both EOs. Moreover, the EO from Azrou was richer in monoterpenes and lower in sesquiterpenes than that from Ifrane (Table 3).  The pennyroyal EO from Ifrane was dominated (70.92%) by pulegone as the main chemotype, whereas the same EO from Azrou was greatly dominated by pulegone (68.86%) and piperitenone (24.81%), accompanied by other constituents with smaller percentages, such as chrysanthenol (1.03%), thymol (1.01%), limonene (0.9%) and menth-2-en-1-ol (0.57%). However, the constituents were specific to the EO from Ifrane with significant amounts: menthone (5.03%), Cyclocitral<β-> (3.49%) and Humulene<α-> (2.17%). Moreover, some compounds were common in both EOs from Ifrane and Azrou, though presenting varying proportions, such as limonene (1.64-0.09%), caryophyllene<E> (1.19-0.04%) and caryphyllene oxide (0.47-0.09%).
The chemical compositions of EOs of the studied Mentha species were spread out across molecular families (Figure 3). Significant differences (p < 0.05) were found between the studied mints and between regions of origin for the same species in terms of the chemical families' proportions.
The chemical compositions of EOs of the studied Mentha species were spread out across molecular families (Figure 3). Significant differences (p < 0.05) were found between the studied mints and between regions of origin for the same species in terms of the chemical families' proportions. For the M. pulegium EOs, both were dominated by ketones, but the EO from Azrou recorded a higher percentage 93.72% than that from Ifrane (83.22%). The hydrocarbons came in second place, reaching 6% in the EO from Ifrane, while it did not exceed 1.65% in the EO from Azrou. Other chemical families were present in lower proportions, such as  For the M. pulegium EOs, both were dominated by ketones, but the EO from Azrou recorded a higher percentage 93.72% than that from Ifrane (83.22%). The hydrocarbons came in second place, reaching 6% in the EO from Ifrane, while it did not exceed 1.65% in the EO from Azrou. Other chemical families were present in lower proportions, such as oxides (1.23%) and phenols (1.17%) without esters in the EO from Azrou. However, the Ifrane EO was marked by the presence of alcohols (3.9%), aldehydes (3.49%) and epoxides (1.06%), and the absence of phenols and ethers. A similarity was observed in the dominance of the oxides, hydrocarbons and ketones for M. suaveolens EOs with higher percentages of the oxides (75.39%) in Azrou than that of Ifrane (61.72%), while the EO from Ifrane was the richest in ketones (10.71%) and hydrocarbon (15.84%). However, the phenols and epoxides were absent in the EO from Azrou. Different from the M. suaveolens EOs, the M. spicata and M. pulegium EOs were characterized by the dominance of ketones. However, the oxides were absent in Azrou, whereas the esters, phenols and epoxides were present exclusively in the EO from Ifrane.
Regarding the collected data about phytochemical study, we deduced that the yields and chemical compositions of the essential oils varied according to the region of origin and within the same species. This variation was due to several factors, such as the method used; the used plant parts; the products and reagents used in the extraction; the environment; the plant genotype; geographical origin; the harvest period of the plant; the degree of drying; the drying conditions; temperature and drying time; and the presence of parasites, viruses and weeds [86].

Antioxidant Activity of the Mint EOs
The activity of the various mint EOs toward the radical DPPH • was assessed. The three mints were able to reduce the stable DPPH • to the yellow-colored diphenylpicrylhydrazine. This reduction capacity was determined via a decrease in the absorbance induced by antiradical substances that occurred in the EOs. As shown below, the percentage of DPPH • inhibition increased, as well as the concentration of ascorbic acid and the EOs (Figure 4). Moreover, the DPPH • inhibition percentages of the EOs were found to be important but less than ascorbic acid for all the applied concentrations. They reached 82%, 73% and 70% for M. pulegium (L.), M. suaveolens (Ehrh.) and M. spicata (L.), respectively.  Based on the obtained IC50 values, the three mint EOs exhibited a significant antiradical activity compared with the ascorbic acid (IC50 = 8.849 mg/mL). The degree of this activity decreased as follows: ascorbic acid > M. pulegium > M. suaveolens > M. spicata ( Figure  5). Therefore, the strongest effect was found for M. pulegium EO (IC50 = 15.93 mg/mL) and the weakest was found for M. spicata EO with IC50 = 16.88 mg/mL.  Based on the obtained IC 50 values, the three mint EOs exhibited a significant antiradical activity compared with the ascorbic acid (IC 50 = 8.849 mg/mL). The degree of this activity decreased as follows: ascorbic acid > M. pulegium > M. suaveolens > M. spicata ( Figure 5). Therefore, the strongest effect was found for M. pulegium EO (IC 50 = 15.93 mg/mL) and the weakest was found for M. spicata EO with IC 50 = 16.88 mg/mL. Based on the obtained IC50 values, the three mint EOs exhibited a significant antiradical activity compared with the ascorbic acid (IC50 = 8.849 mg/mL). The degree of this activity decreased as follows: ascorbic acid > M. pulegium > M. suaveolens > M. spicata ( Figure  5). Therefore, the strongest effect was found for M. pulegium EO (IC50 = 15.93 mg/mL) and the weakest was found for M. spicata EO with IC50 = 16.88 mg/mL. There are many reports in the literature concerning the antiradical power of mint EOs. They revealed a variation in the reaction of these EOs toward DPPH • . Regarding M. pulegium (L.), EO from Algeria showed a higher DPPH • radical scavenging activity of 90.54 ± 1.5% at a concentration of 1000 µg/mL [87]. Similarly, for EOs from Tunisia and Greece, their antiradical capacity was also demonstrated, with IC 50 values reaching 10 and 13.5 ± 0.5 µg/mL, respectively [58,88]. However, EOs extracted from Indian and Italian pennyroyal exhibited low antioxidant activities, with EC 50 values of approximately 147.36 mg/mL and 6.2 ± 0.2 mg/mL (BHT = 0.0049 mg/mL), respectively [89,90]. This low power can be explained by a lack of antioxidants in this oil. For Mata et al. [52], this EO showed no potential for trapping the DPPH • , which was probably due to its weak solubility under test conditions.
Spearmint oil also presented an antiradical potency but this was lower than the M. pulegium and M. suaveolens EOs. Our results concorded with previous studies. The inhibition capacity of Tunisian spearmint oil was found to be higher (IC 50 = 3 µg/mL) than the positive control BHT (IC 50 = 11.5 µg/mL) [93]. This oil seemed to be a more powerful antioxidant than that reported by Dhifi et al. [94], which had an IC 50 equal to 10 µg/mL. As for Mkaddem et al. [95], they deduced that the investigated spearmint oil had a moderate reducing power (IC 50 = 3476.3 mg/L) compared with ascorbic acid (4.4 ± 0.2 mg/L). Similarly, spearmint EO from Portugal showed antiradical activity that reached 31.45% [96], while that from the same origin revealed no antioxidant activity [95]. Moreover, a spearmint EO from Algeria had a significant scavenging activity expressed by an IC 50 close to 208.495 ± 4.247 µg/mL [97]. Recently, Alsaraf et al. [98] found the promising antioxidant activity of a spearmint EO from Oman with IC 50 = 26.64 µg/mL.
We note that our investigated oils presented the important ability to eliminate the radical DPPH • . This interesting property might be related to the chemical composition of EOs and mainly to the phenolic compounds that may play an important role in inhibiting the auto-oxidation of oils [6]. Consequently, the tested mint EOs were rich in oxygenated monoterpenes and sesquiterpenes, such as pulegone (68.86%) and thymol (1.01%) for the M. pulegium EO, carvone (71.56%) and limonene (10.50%) for the spearmint EO, and, finally, piperitenone oxide for the M. suaveolens EO. Moreover, other compounds found in lower amounts also showed their contribution to the antioxidant activity: such as β-pinene, 7terpinene, α-terpineol and spathulenol [93,96,99]. On the other hand, the higher antioxidant effect of the M. pulegium EO compared with the other tested EOs, as indicated in many mint species [89,100], may be explained by the high content of pulegone and by the presence of phenolic compounds, such as thymol, which acted as potential antioxidant [4,99,101].

Conclusions
The physical properties and chemical compositions of the three studied mints showed considerable variations according to the harvesting region. The data obtained revealed that the region was a crucial factor in the bioactive composition. The M. pulegium EO from Azrou was dominated by pulegone (68.86%) and piperitenone (24.81%) but the same EO from Ifrane contained pulegone as the main component (70.92%). Piperitenone oxide (74.69-60.3%), carvone (71.56-54.79%) and limonene (10.5-9.69%) were the major constituents with different rates for M. suaveolens and M. spicata from Azrou and Ifrane, respectively. Moreover, the identification of the physicochemical parameters of these oils led to demonstrating the quality that could be useful in future studies for medicinal or pharmaceutical purposes. The results also indicated that the mint EOs exhibited remarkable antioxidant properties that were comparable to ascorbic acid, especially the EO of M. pulegium, which was the most active. This activity was attributed to the occurrence of phenolic content in these EOs. These promising results showed that they have the potential to be a natural alternative to synthetic antioxidants in order to enhance the shelf life and safety of food.

Data Availability Statement:
The data are available from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.