Investigation of Chemical Compositions and Biological Activities of Mentha suaveolens L. from Saudi Arabia

Mentha is an aromatic plant used since antiquity for its pharmaceutical virtues. The climate of Saudi Arabia favors the growth of aromatic plants including Mentha suaveolens L. The aim of this study is to analyze the volatile oils of different parts of fresh and dried Mentha suaveolens L. grown in Saudi Arabia (Aljouf area) using Gas Chromatography/Mass Spectrometry (GC/MS) and Gas Chromatography Flame Ionization Detector (GC/FID) techniques, to recognize the effect of drying on chemical composition, then to evaluate the antioxidant and antifungal activities of different extracts. In total, 118 compounds were identified via GC/MS and GC/FID, in which carvone is the main volatile constituent (stems, leaves, whole plant 45–64%). This investigation deduces that Mentha belonged to the carvone chemotype. Then, the analysis of non-volatile constituents of fresh and dried Mentha was performed by HPLC. The main phenolic compound of fresh and dried Mentha for different parts was rosmarinic acid (ranging from 28,002.5 to 6558 µg/g). The ethanolic extract of fresh stem showed the highest antifungal activity (53% inhibition) compared with miconazole (60% inhibition) but the ethanoic extract of dry stem showed no activity. Additionally, all ethanolic extracts, whether for fresh or dry Mentha, have antioxidant activity more than 90% while the antioxidant activity of whole plant volatile oil is equal to 53.33%. This research shows that M. suaveolens L. could be applied to manufacture natural antioxidants, antifungal, and flavoring agents.


Introduction
M. suaveolens L. is common plant in Saudi Arabia known for its strong antifungal, anti-aflatoxigenic, and antioxidant potential [1]. Mentha species have been well recognized for their aromatic and medicinal purposes since ancient times [2]. Recently, several Mentha species gained increasing interest for their potential use as natural food preservatives because of their strong antifungal, anti-aflatoxigenic, and antioxidant properties [1,3]. These biochemical properties are mainly due to the presence of several aromatic and phenolic compounds in different parts of the Mentha species. The chemical composition of Mentha species differs according to the growing season [4,5]. Additionally, the volatile oil composition of Mentha species from various populations and geographical regions indicated that the plants relate to either carvone or menthol or linalool chemotypes [6]. Several Mentha species were investigated in the Kingdom of Saudi Arabia. Burham et al. [7] analyzed the chemical composition of the volatile oils extracted from the leaves of M. longifolia harvested from tive compounds and other acids such as cinnamic acid, gentisic acid, protocatechuic acid, hydroxybenzoic acid, and vanillic acid [17]. Antioxidants are of major interest to scientists, however, many carcinogenic and toxic effects were reported for synthetic antioxidants, making natural antioxidants promising alternatives [18]. The volatile oils of M. suaveolens have several proven and beneficial biological activities, especially antioxidant activity [4].
Other studies also showed that the volatile oils extracted from M. suaveolens exhibited potent antifungal activity against Aspergillus niger and Candida albicans [5].
To our knowledge, there is no report on the chemical composition and biological activities of the volatile oils of M. suaveolens L. native to Saudi Arabia (Aljouf area). Therefore, the objective of this study was to extract non-volatile and volatile oils from M. suaveolens L. and to identify the chemical constituents using GC, GC-MS and high-performance liquid chromatography (HPLC). Moreover, biological activities (antioxidant and antifungal activities) are presented in this study.

Analysis of Volatile Oils of Fresh and Dried M. suaveolens L. by GC/MS and GC/FID
The volatile constituents obtained from M. suaveolens L. under study were pale yellow with a pleasant and distinct odor. The total yield percentages of volatile constituents from M. suaveolens L. of fresh and dried whole plant extracts were 0.57 ± 0.03% and 0.225 ± 0.01%, respectively. The total yield percentages of fresh and dried leaf extracts were 0.235 ± 0.012% and 0.24 ± 0.016%, respectively. The total yields of fresh and dried stem extracts were 0.24 ± 0.016% and 0.9 ± 0.05%, respectively.

Drying Effect on Volatile Oil Constituents of Mentha
A variation in chemical composition concentration for some compounds was found due to the drying effect as shown in Figure 1. As shown in the figure, the LOC is the highest concentration in all plant extracts. While the S and the HOC concentration increases after the drying process in all plant extracts, no significant changes were found in the concentration of the M after the drying process.

Chemical Composition of Non-Volatile Constituents of Fresh and Dried Mentha by Using HPLC
The analysis of fresh Mentha non-volatile constituents was performed by HPLC. The major phenolic derivatives identified in the ethanolic extract from the fresh whole plant were rosmarinic acid (2223.3µg/g), rutin (676.7 µg/g), and ferulic acid (226.7 µg/g). Additionally, the main phenolic derivatives found in fresh leaves of Mentha were rosmarinic acid (28,002.5 µg/g), rutin (3383.8 µg/g), and ferulic acid (1520 µg/g). In fresh stems of Mentha, the main phenolic derivatives identified were rosmarinic acid (6558µg/g), catechin (1340.4 µg/g), and naringenin (371.8 µg/g). The other components are listed in Table 2. In addition, the analysis of dried Mentha non-volatile constituents was performed by HPLC. The major phenolic derivatives identified in the ethanolic extract from the dried whole plant of Mentha were rosmarinic acid (21,191.9 µg/g), rutin (252.16 µg/g), and quercetin (153.8 µg/g). The main phenolic derivatives found in dried leaves of Mentha were rosmarinic acid (15,165.1 µg/g), rutin (194.7 µg/g), and quercetin (156.98 µg/g). Moreover, the main phenolic derivatives found in dried stems of Mentha were rosmarinic acid (8378.4µg/g), naringenin (130.3 µg/g), and catechin (68.1 µg/g). The other components are recorded in Table 2. Antioxidant activities of Mentha were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. DPPH radical scavenging activity of the positive standard Butylated hydroxytoluene (BHT) and plant extracts were expressed by inhibition percentage (%). The ability of the tested extract to act as an electron donor in the conversion of DPPH• into DPPH-H was studied.
Antioxidant Activity of Volatile Oils from Mentha suaveolens L.
The percentages of inhibition were measured for all the samples presented in Figure 2. The results showed that the antioxidant activity of samples ranged from 31% to 53%. Antioxidant Activity Non-Volatile Constituents from Mentha DPPH• (purple-colored) was reduced to DPPH-H (yellow colored) by using tested samples. The obtained results showed that all the ethanolic samples have good antioxidant activity with values exceeding 90%. The inhibition% measured for all samples is presented in Figure 3. Isolated fungi of the studied white cheese samples show that a total of 13 fungal isolates were recovered on PDA media. The isolated fungi were identified as seven species belonging to four genera. The sequence of the most dominant species was closely related to Penicillium glabrum (Genbank accession number, MW534476.1) with 100% similarity https://www.ncbi.nlm.nih.gov/nuccore/MW534476.1 (2 February 2021).
Penicillium sp. was the most predominant genus encountered in 37.4% of the total fungi recovered on PDA media. Two species of Penicillia were most common, P. glabrum was the most prevalent (22.4% of the total fungi) and P. aurantiogriseum (14.9%). Three species of Aspergilla were identified, A. niger was the most prevalent (11.21% of the total fungi), followed by A. ustus (9.3%), and A. fumigatus (7.47%). Geotrichum was isolated from one sample only cultivated on PDA medium. Antifungal Activity of Volatile Oils from M. suaveolens L.
Antifungal activity was estimated by determining the capacity of inhibition of mycelial growth of the fungi species under study. Leaves of fresh Mentha presented the highest biological activity compared with the other extracts from Mentha, as shown in Figure 4. In total, 20 mg\ml of fresh and dried Mentha leaf extracts prevented the growth of the fungi by 51.18 ± 0.15 and 47.06 ± 0%, respectively. Additionally, the fungi can be inhibited by using fresh and dried stems of Mentha extracts, and the percentage is 48.53 ± 0.15% and 45.44 ± 0.23%, respectively, followed by inhibition by the whole plant, fresh and dried Mentha extracts, with the percentages of 41.53 ± 0.23% and 35.29 ± 0%, respectively.

Antifungal Activity of Non-Volatile Constituents from Mentha
Antifungal activity was estimated by determining the capacity of inhibition of mycelial growth of the species under study. Fresh stem extracts of Mentha had the highest biological activity compared with the other extracts from Mentha. A concentration of 20 mg/mL of fresh stems, leaves, and whole plant extracts of Mentha can prevent the growth of the fungi by 52.94 ± 0.24%, 41.47 ± 0.05%, and 39.12 ± 0.096%, respectively, as shown in Figure 5. The other extracts, including dried (whole plant, leaves, and stems), of Mentha did not affect the growth and sporulation of the tested fungi. Antifungal activity of volatile oils and non-volatile constituents of Mentha, examined using analysis of variance and presented in Table 3, shows that mean squares were highly significant for treatments.

Discussion
The volatile oils obtained from Mentha under study were pale yellow with a pleasant and distinct odor. The total yield percentages of volatile oils from different parts of fresh and dried Mentha varied from 0.225% to 0.9%. Our results were in close agreement with Petretto et al. [29] and Kasrati et al. [14], who reported that the volatile oil yields of the aerial parts of M. suaveolens ssp. insularis from Sardinia was 0.2%. The non-volatile constituents obtained from Mentha under study were yellow to deep green with a pleasant and distinct odor. The total yield percentages of non-volatile constituents from different parts of fresh and dried Mentha varied from 0.6% to 3.4%. Our results agree with Barchan et al. [30] who found that the content of non-volatile constituents of M. pulegium and M. piperita from Morocco to be 3.4% and 4%, respectively. In contrast, non-volatile constituents were found in high levels (5%) in the extracts of three Algerian mints [31]. Moreover, El-Ghorab [32] reported that non-volatile constituents of Egyptian Mentha were 4.5%.
The volatile constituents of fresh and dried Mentha from Sakaka belonged to the carvone chemotype. Our results agree with El-Kashoury et al. [4], who reported that the carvone was the main compound in Mentha in different seasons during the year (56% to 31%). Other studies are in agreement with our results where Mentha was classified by the carvone pathway [6,33]. Hussain et al. [34] and Boukhebti et al. [35] analyzed the chemical composition of Mentha from Pakistan and Algeria, in which the main compound in both studies was carvone (59.5% and 59.4%, respectively). Although the carvone percentage in leaves was higher (64.31%) than that reported by Hussain et al. [34], the percentage was lower in whole plant (43.65%) compared to Boukhebti et al. [35]. These differences in carvone concentration might be due to different environmental conditions. In another study of fresh Mentha in Morocco, pulegone was the major compound, accounting for 17.61% [13], while in our study, the percentage was 3.8% in the whole plant, 2.1% in leaves, and 2.2% in stems. Additionally, Sutour et al. [36] reported a higher level of pulegone in their samples (44.4%) compared to our results. In another study of dried Mentha in Morocco (Iguer region), pulegone was absent, agreeing with our results [14]. These changes in the concentration of pulegone might be due to the different habitats from which plants were collected. In our results, α-Terpineol concentration in fresh Mentha whole plant (0.15%) was lower than that reported by Salhi et al. (0.4%) [37]. In another study of M. suaveolens ssp. insularis in Sardinia, the percentage of the linalool was 1.37%, slightly lower than what was observed in this study for fresh whole plants (1.54%) [29]. In an recent study, α-Humulene was found in a high concentration (1.09%) in dried Mentha (Morocco) whole-plant extract compared to our results (0.04% in dried whole plant) [15]. In the same study, terpinene (γ) was among the major compounds accounting for 5.33% in dried whole-plant extract, which is a much higher percentage than the one we observed (0.03% in dried whole-plant extract).
The chemical compounds in fresh and dried Mentha analyzed by GC/MS can be categorized into four chemical classes: M, LOC, HOC, and S as shown in Table 2. The chemical classes were classified in the following decreasing order: high percentage of LOC due to its high content of carvone in all plant extracts, followed by S, HOC, and M in all different extracts of fresh and dried Mentha.
The above results (Table 2) were similar to those reported by Petretto et al. [29] during their work on fresh M. suaveolens ssp. insularis in Sardinia, especially high percentages were reported for oxygenated compounds (87.1%) in the whole plant extract. Moreover, the percentage of sesquiterpenes (14.2%) was similar to the percentage reported in the fresh whole plant extract (15.83%) in our study [29]. El-Kashoury et al. [4] analyzed the chemical composition of fresh M. suaveolens Ehrh. in Egypt. The highest percentage of oxygenated compounds was 62.9% in their whole plant extract, which was much lower than the percentages reported in our study. Our results were in close agreement with Hussain et al. [34], who identified oxygenated compounds in dried leaves of Mentha from Pakistan occurring at 81.5%. In the same study, sesquiterpenes were found at 6.1% in dried leaf extract compared to 12.90% in our analysis. However, the monoterpenes percentage was (1.15%) lower than reported in their study (9.09%) [34]. In another study of dried Mentha in Taif from KSA, the percentage of the oxygenated compounds was 91.9%, which was slightly higher than our results [8]. In contrast, Burham et al. [7] reported a much lower percentage of oxygenated compounds in dried leaves (30.4%) of Mentha from Albaha Area southern KSA. They also reported higher percentages of monoterpenes and sesquiterpenes (54.3% and 26.08%, respectively) compared to our observations in leaf extracts (1.15% and 12.90%, respectively) [7].
Based on the above results, significant change in the chemical composition of volatile oils might be due to different plant parts, environmental factors, seasons, geographical location, and plant age of the Mentha plant.
Previous investigation of the chemical composition of the volatile oils from Mentha in Senegal showed that the pulegone rate dropped in the oils of the dried plants [38]. Similarly, our results showed a sharp drop in pulegone concentration to very low or zero levels in dried M. suaveolens L. Kohari et al. [9] reported the drop in α-Terpineol percentage in the dried Japanese peppermint (0.27% in fresh to 0.18% in dried). This result was in close agreement with our results. The 1,8-cineole concentrations in Mentha from Senegal were lower in the dried plants, which was similar to our observations [38]. Moreover, in our results, the percentages of monoterpenes after drying the whole plant and leaves decreased. Similar results were previously reported [9,39]. In a study of Mentha from Senegal, monoterpene levels increased from 3.4% to 4.7% after drying [38]. Sesquiterpenes were also increased after drying M. suaveolens L. in our analysis, which also agrees with the studies of Diop et al. [38] and Ahmed et al. [39]. Different factors could contribute to the observed variation in volatile constituents. One of these factors is the chemical properties of volatile oils, mainly their structure and volatility. The type of plant is also a significant factor in this regard. There is also a chance for new compounds to form based on the chemical reactions involved, such as glycoside hydrolysis, oxidation, esterification, etc. [39]. Changes in volatile compound concentrations after drying have been reported and explained by the continuous biosynthesis after harvest [40].
Non-volatile components of fresh and dried Mentha were in close agreement with those reported by Elansary et al. [16] and Mišan et al. [44]. They investigated the phenolic compounds of Mentha species and reported, rosmarinic acid as a main compound. In comparison, Kulig et al. [45] reported that rutin was the primary phenolic acid in Mentha from the Slovak Republic, which was the case in our study for the fresh and dried whole plant and fresh and dried leaf extracts. Additionally, Farnad et al. [17] analyzed Mentha from West Azerbaijan and found that the main compounds were chlorogenic acid and rutin. Previous studies on Mentha species revealed some phenolic derivatives in the genus, such as rosmarinic acid, caffeic acid, ferulic acid, and catechin [31,43,44]. The highest antioxidant activity (53.33%) was observed in the whole-plant extract of dried Mentha, which can be explained by its high carvone content (45%), and which is known for its antioxidant activity.
Indeed, Elmastaş et al. [46] reported that the antioxidant activity of carvone is equal to 95%. Additionally, it contains a high concentration of copaene (β) (11%), which has high antioxidant activity [47]. The mild antioxidant activity of the volatile oils of Mentha may be due to its weak phenolic compounds. Approximate results were obtained by Bardaweel et al. [48], who showed that volatile oils have weak antioxidant activity.
Good antioxidant activity of non-volatile constituents of Mentha was observed (94.23 ± 0.007% for the fresh whole plant, 94.50 ± 0.005% for fresh leaves, 94.27 ± 0.01% for fresh stems, 92.83 ± 0.02% for the whole dried plant, 90 ± 0.014% for dried leaves, and 93.33 ± 0.007% for dried stems). These results agree with Mata et al. [49], who reported that ethanol extracts have good antioxidant activity.
The high antifungal activity of volatile oils of Mentha can be explained by its high content of carvone, which showed high antifungal activity [55]. The volatile oils of fresh Mentha contain pulegone, which also exhibited high antifungal activity [56]. Additionally, it contains 1,8-cineole, which showed good antifungal activity but was lower than carvone [57].
The high antifungal activity was observed for the non-volatile constituents of fresh extracts of Mentha due to the high concentration of rosmarinic acid, catechin, and luteolin, known for their high antifungal activity [58][59][60]. Moreover, the fresh stem extracts of Mentha contained a high concentration of eugenol, which showed high antifungal activity [61].

HPLC Analysis of Non-Volatile Compounds
The non-volatile extracts were analyzed using HPLC, instrumental details and condition according to the reported method [63]. All phenolic standards were prepared in methanol within the range from 0.5 to 100 µg/mL and these standards were used for quantitative and qualitative identification of phenolic compounds in M. suaveolens L. extract chemical composition depending on the standard calibration curve for each standard and retention time, respectively (see Supplementary Materials).

Antioxidant Activity
The scavenging DPPH free radical ability was determined in vitro according to the procedure of Yue and Xu [64]. DPPH solution (0.1 mM, 1.8 mL) was mixed with 0.2 mL of each diluted Mentha extract stock solution (5 mg/mL) and incubated in the dark for 30 min at 25 • C. In this assay, the percentage of DPPH reduction by samples was compared to BHT. The experiment was repeated three times. The following formula was used to quantify radical scavenging activity: A 0 is the absorbance of the control reaction (t = 0 min). A 1 is the absorbance of the tested extract solution (t = 30 min).

Antifungal Activity
Ten samples of locally fabricated cheese were gathered arbitrarily from farmer's houses of Al-Jouf-Sakaka in KSA. Samples were collected in (sterile, clean, and dry) containers and shipped to test centre within 1-2 h of collection (at 4 + 2 • C).

Detection and Isolation of Fungi
The dilution-plate method [22] was used to isolate and identify fungi from the collected cheese samples. Samples were inoculated on potato dextrose agar (PDA) obtained from Oxoid [24]. Rose Bengal (30 ppm) was used as an antibacterial agent.
One gram of white cheese was suspended in 90 mL sterilized distilled H 2 O using a rotating shaker to homogenate the suspension. Then, 10 serial dilutions were prepared, and 1 mL of each dilution was inoculated into a petri dish. Then, melted medium was poured, mixed well, and left to solidify. After solidification, Petri dishes were incubated at 27 ± 2 • C for 5-7 days. Colonies were counted and isolated for purification and identification.

Identification of the Isolated Fungi
Morphological identification of the isolated fungi was carried out based on their macro and microscopic characteristics using the taxonomic methods [28,63]. Additionally, the most dominant species were identified using molecular analysis of the ITS1-5.8S rRNA-ITS2 region (animal health research institute, Dokki, Giza, Egypt).

Effect of Volatile Oils and Non-Volatile Extract on Mycelium Growth of Isolated Fungi
In total, 15 mL of PDA medium were placed in each Petri dish (9 cm diam.), and after solidification, a circular hole (2 cm, diam.) was formed. Then, 1 mL of each different concentration of the tested extract was added to each well. Each Petri dish was inoculated with four fungal species and incubated at 28 • C for seven days. PDA medium without additives was used as a negative control, whereas plates containing 20 mg/mL Miconazole Nitrate were used as a positive control. Each treatment was repeated three times and subjected to statistical analysis. The diameter of inhibition zones was measured for each well, and the results were statistically analyzed. In total, 1 mL of plant extract (20 mg/mL) was added to each well to determine the inhibition zone and inhibition percentage (%).
The diameter of inhibition zones was measured, and the percentage of antifungal activity was calculated according to Equation (2): DC = the diameter of fungal mycelium growth in the control Petri dish. DT = the diameter of fungal mycelium growth in the treated Petri dish, containing Mentha extracts.

Statistical Analysis
Separated completely randomized designs as one-way ANOVA were conducted for statistical analysis procedure of the obtained data. Both antioxidant and antifungal activities of volatile oils and non-volatile extracts of Mentha were carried out in triplicate experiments. Each experiment included seven treatments (whole plant of fresh Mentha, whole plant of dried Mentha, leaves of fresh Mentha, leaves of dried Mentha, stems of fresh Mentha and stems of dried Mentha as well as the control treatment with BHT). Duncan's multiple range test [65] was used to estimate the performance of mean treatments depending on significance of mean of squares according to the ANOVA table at a significance level of p = 0.05, where a different superscript letter refers to a significant difference among those treatments. MSTAT-Cv.2.10 software program package processed all data [66].

Conclusions
M. suaveolens L. from the Sakaka region was investigated for its chemical components and biological activities for the first time. Regarding the main chemical components of volatile oils, carvone dominated in all samples. The volatile oil M. suaveolens L. was carvne chemo type. Additionally, the main phenolic compounds in ethanolic extract were rosmarinic acid, rutin, catechin, and naringenin which are responsible for the antioxidant and antifungal activities of Mentha. It was found that copaene had the highest concentration in M. suaveolens L. in comparison with the same species in another locality. Fungi (Penicillium glabrum) isolated from white cheese was inhibited by Mentha extracts for the first time as well as fungi (Penicillium glabrum) isolated from white cheese that was inhibited by Mentha extracts for the first time.