Antimicrobial Potential and Chemical Profiling of Leaves Essential Oil of Mentha Species Growing under North-West Himalaya Conditions

Mentha essential oil is one of the most utilized essential oil in the food and pharmaceutical industries. The present study reports the chemical composition and antibacterial properties of leaf essential oils of Mentha species. Further, the effect of the harvesting period on essential oil yield was also investigated. Firstly, the cultivated Mentha piperita and wild Mentha longifolia, revealed significant differences in their chemical profile. M. longifolia essential oil was characterized with endo-borneol (1.12-6.2%), caryophyllene (2.72-7.03%), isopipertenone (0.07-0.36%), germacrene D (0.98-3.22%), 3-cyclopentene1-one,2-hydroxy-3-(3-methyl-2-butenyl)(21.91-56.72%) and piperitone oxide (8.96-39.31%), whereas, M. piperita leaves essential oil was found rich in isomenthone (5.97-6.75%), 1-menthone (7.32-18.32%) and menthol (18.03-58.53%), etc. The essential oils of both Mentha species exhibited strong antimicrobial activity as evaluated using poisoned food technique, dry weight method, and disc diffusion method against Candida albicans, Fusarium oxysporum, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli. Secondly, the maximum essential oil yield was observed in July month, 0.63±0.01 and 0.56±0.01%, respectively for M. piperita and M. longifolia.


INTRODUCTION
a-Terpinene, carveol, etc. constitute the bioactive composition of M. piperita. 13 Secondary metabolites (alkaloids, phenols, flavonoids, and so on) have therapeutic characteristics; their production and accumulation are influenced by the genotype as well as the plant's interaction with its environment. The photoperiod, temperature, relative humidity, and other climatic conditions influence the chemical profile because some chemicals accumulate over time in response to environmental changes. 14 The plant secondary metabolite products are also affected by harvesting time, plant age, and crop density. 15 As a result, it is critical to understand the appropriate time and age for plants to have a rich chemical profile with robust biological activities, so they may be used for essential oil extraction and medicine development against deadly infections in the long run.
The researchers in India, 16 Saudi Arabia, 17 Turkey, 18 Serbia, 19 and others have previously identified seasonal change in the phytochemical content and biological properties of essential oils in both Mentha species. The motive of this study was to analyze the chemical profiles of essential oils from M. piperita and M. longifolia leaves harvested in the northwestern Himalaya, as well as to look into their antibacterial efficacy against pathogenic bacterial strains (Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, and Staphylococcus aureus) and fungi (Fusarium oxysporum and Candida albicans)

MATERIAL AND METHODS Plant collection
In April-July 2018, 100 g leaves of cultivated M. piperita and wild M. longifolia were harvested from Y.S. Parmar University, Nauni, Solan nursery (30°85ˈ70 ̎ N, 77°16ˈ74 ̎ E) and Giripul (Distt. Sirmour; 30°52ˈ53 ̎ N, 77°12ˈ37 ̎ E), Himachal Pradesh, India, respectively, for the extraction of essential oil. The leaves were randomly sampled from the growing plot/area using a stratified random sampling method in the morning hours before noon for each month (April, May, June, and July).

Identification of plants
The Mentha species in the field were identified based on morphological characters (M. logifolia is characterized by ovate-lanceolate, greyish-green leaves with white hairs from the above, and purplish-white flowers, whereas in M. piperita leaves are ovate serrate, greencolored with long petiole, and purple flowers). 20,21 Collected plants were further authenticated in the herbarium of Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan, H.P., India. (UHF-Herbarium No. 13571). The voucher specimens have also been deposited in the herbarium of the School of Biological and Environmental Sciences, Shoolini University, Solan, H.P., India.

Isolation of essential oil
The leaves were harvested from plants (90 plants/3 quadrat) of M. piperita and M. longifolia and utilized for essential oil extraction using the hydrodistillation method. 22 Briefly, fresh leaves (100 g) were chopped into bits and combined with distilled water (1 litre) in a flask. In continuation, Clevenger-type equipment was used to distil the mixture for 3 hours at 60°C. The essential oil was extracted and stored at 4°C for further use.

Essential oil yield
Using the formula below, the essential oil yield in the percentage of each sample was computed: Oil yield = X 100 w1 w2 Where, w1 = net weight of oil (g); w2 = total weight of fresh leaves (g) Gas chromatography-mass spectrometry analysis of leaves essential oil GC-MS analysis of M. piperita and M. longifolia leaves essential oil was performed as per the standard method of Okut et al. in the SERF laboratory of Punjab University, Chandigarh, India. 23 The analysis was done using a GC-MS instrument (Thermo Trace 1300 GC coupled with Thermo TSQ 8000 Triple Quadrupole MS) fitted with a TG 5MS capillary column (30 m x 0.25 mm i.d., 0.25 µm film thickness) for qualitative determination. The essential oil was diluted 1/10 in n-hexane (v/v) before analysis. The Autosampler was built for the injection of essential oils. The injector temperature was 250°C. The temperature of the column was programmed from 60°C to 250°C, held for 60°C for 2 minutes, moderately raised to 200°C, and kept for 5 minutes with an increase of 15°C /minute to 220°C. Helium was employed as a carrier gas with 1.5 mL/minutes flow rate, and mass spectra were taken in scan mode. The ionization voltage and split ratio were 70 eV and 1:20, respectively. The ion source and interface were both at 230°C and 250°C, respectively. The solvent cut time was 3 minutes. A total of 1.0 μl of the sample was used in the analysis. The percentage of constituents was also determined using the response factor, and retention indexes (RIs) were calculated using the retention duration of n-alkanes (C10-C20) injected at the same temperature and under the same conditions. The phytoconstituents were identified by comparing their RI to those described in the literature, and by comparing their mass spectra to those recorded in the NIST 2.0 electronic library and the Wiley 275 libraries, with Match Factor (SI) and Reverse Match Factor (RSI) greater than 900. Antimicrobial activity

Microbial strains and growth conditions
To evaluate the antimicrobial potential, four bacterial [Gram positive bacteria (Bacillus subtilis (MTCC 5521) and Staphylococcus aureus (MTCC73) and Gram-negative bacteria (Escherichia coli (MTCC739) and Klebsiella pneumoniae (MTCC109))] and two fungal strains [Fusarium oxysporum (SR266-9), Candida albicans (ATCC90028) were selected. All microbial strains were procured from the Shoolini University's Yeast Biology and Plant Pathology laboratory. The bacteria were maintained on Nutrient Agar (NA) at 37°C for 18 to 24 h. The fungal strain such as F. oxysporum was maintained on Potato Dextrose Agar (PDA) medium, while C. albicans was maintained on Yeast Peptone Dextrose Agar (YPD) media at 25°C for 48 to 72 h. Agar disc diffusion method NA and YPD agar were spread with bacterial strains and yeast (0.5 McFarland standard), respectively. 24,25 The 6 mm diameter discs of sterile filter paper soaked with various concentrations (1.5 -3.0 mg/mL) of diluted essential oil (1:10 in DMSO, v/v) were kept on the inoculated plates. For positive control Ampicillin and Fluconazole (50 µg/mL) were used against tested bacterial and fungal strains, respectively. After the incubation period of 18-24 h at 37°C (in case of bacteria) and 48-72 h at 25°C (for fungi), inhibition zones were observed in the plates around the paper discs. An inhibition zone diameter was measured using HiMedia Zone scale. The experiment was carried out in triplicates.

Poisoned food technique
The leaves essential oils of both Mentha species (1:10 diluted in DMSO, v/v) at varying concentrations (1.5-3.0 mg/mL) were transferred to conical flasks containing 25 mL of sterilized PDA and then poured into Petri plates.
On the other hand, in the center of the Petri dish, 6-7 days old culture of F. oxysporum was placed and incubated at 25°C. After seven days, the radial development of fungal mycelium was checked. The findings were compared with negative control (plates with DMSO only). 26 The inhibition percentage of fungi in treatments was estimated using the formula below: Percent inhibition = X100 C -T C Where C represents the colony radius in the control plate, and T represents the pathogen radial growth in the presence of essential oil.

Dry weight method
Briefly, five test tubes containing sterile potato dextrose broth (5 mL) were inoculated with actively growing F. oxysporum mycelium (20 µL containing 1x10 6 spores/mL) obtained from a 6-7 days old culture. In four test tubes, 20 µL (1.5-3.0 mg/mL) of essential oil (1:10 in DMSO, v/v) was added, while one test tube was left without essential oil as a negative control, and all tubes were incubated at 25°C for 7 days. In the tubes, growth of visible mycelia represents the degree of effectivity of the essential oil. The fungal mycelia were further separated by filtration using Whatman filter paper No. 1. To achieve constant weight, the filter paper was dried at 60°C.
The growth inhibition of fungi was calculated by observing the control and sample mycelial dry weight. 27 The percentage growth inhibition was calculated using the following formula: Control -Test Control

Minimum inhibitory concentration (MIC)
The broth dilution method described by the Clinical and Laboratory Standards Institute was used for evaluating MIC. Briefly, essential oil was diluted geometrically (5-0.0095 %) in a 96-well microtiter plate, having growth control (NB/ PDB/YPD broth containing DMSO) and positive control well (NB/PDB/YPD broth inoculated with each microbial culture and containing 10 mg/ mL of Ampicillin, Hygromycin B and Fluconazole separately). Further, an equal number of cells (2 × 10 6 CFU mL-1, 0.5 McFarland) were inoculated into each well. At 37°C the microtiter plates were incubated for 24 h in case of bacteria and 25°C for 48 h in case of fungal strains. 26,28 The 15 µl of the resazurin dye was added to each well and plate was further incubated for 2-4 hours at 37°C. Any color changes after incubation period were observed. The blue color indicated bacterial growth whereas pink color indicated bacterial growth. Following the incubation period, the MIC values were reported as the lowest concentration of oil that suppresses observable microorganism growth. NB was used for antibacterial activity, while PDB and YPD Broth were used for antifungal activity.

Data analysis
Wherever necessary, analysis of variance (ANOVA), average and standard deviation were evaluated using GraphPad Prism 5.0. A p value <0.05 was considered statistically significant.

RESULTS AND DISCUSSION Essential oil yield
In all harvesting months, there was a significant variation in the yield of peppermint and mint leaves essential oil with respect to the plant's harvesting age (Table 1). The results showed that the least essential oil yield was achieved in April (0.26±0.02% and 0.63±0.01%, respectively) and the maximum was achieved in July month (0.63±0.01 % and 0.56±0.01 %, respectively) in both M. piperita and M. longifolia leaves. The M. piperita essential oil yield was higher as compared to M. longifolia essential oil.
The monthly variation in essential oil yield could be related to the fact that as the plant ages, the number of leaves and their surface area grows, resulting in the creation of more oil glands and hence a rise in essential oil content. The plant density and harvesting time, according to Mansoori, have a major impact on dry biomass and oil yield production because higher density improves plant vegetative growth and the number of leaves per plant, which enhances light interception and so increases plant essential oil yield. 29

Essential oil chemical composition
The volatile and semi-volatile components in the essential oil collected in each month, i.e., from April to May, were investigated using the GC-MS technique that revealed a unique chemical profile of essential oil of both M. piperita and M. longifolia. A total of six prominent phytochemical compounds (1-menthone, isomenthone, menthol, menthyl acetate, cryophyllene, and germacrene-D) were observed in the essential oil of M. piperita from April to July month ( Table 2; Fig. 1).
In comparison to previous findings, the principal compounds observed in M. longifolia oil were menthone, pulegone, limonene, and terpinolenone, but the present investigation revealed that several of these compounds were absent in the M. longifolia essential oil. A chemical, 3-cyclopentene-1-one,2-hydroxy-3-(3-methyl-2butenyl)-, was also observed in the GC-MS profile of M. longifolia essential oil, which is a new compound with unknown action. Similarly, most researchers identified menthofuran, limonene, and 1,8-cineole as important components in M. piperita essential oil, however, our investigation found none of these chemicals. 35,36 This difference in the essential oil chemical composition from plant species can be attributed to variation in climatic, edaphic conditions, harvesting period and drying technique, etc. 37 Additionally, chemical profile is influenced by all such factors that can boost the biosynthesis of some molecules while inhibiting the biosynthesis of others. 38

Antibacterial activity
The study revealed that as the concentration of essential oil (M. piperita and M. longifolia) increased from 1.5 -3.0 mg/mL the inhibition zone diameter was also increased from May to July month, and fluctuation in the essential oil activity of both Mentha species in each month was extremely minute (Table 3). In addition, M. piperita essential oil showed the lowest MIC (0.62 mg/mL) against B. subtilis, K. pneumoniae (MIC -0.62 mg/mL), and E. coli (MIC -1.25 mg/mL) in May and July month harvest, and against S. aureus, (MIC -0.62 mg/mL) The present findings are consistent with previous research. [39][40][41][42][43][44][45] Although, the effect of season on the antibacterial property of M. longifolia and M. piperita is known, there is no report on the ideal harvesting time for M. longifolia and M. piperita in terms of antibacterial potential of essential oil within one growing season. 32,37,46 In contrary to previous reports, in this study almost similar activity was reported against Gram positive as well as Gram negative strains. As per existing  literature, Gram positive bacterial strains are more susceptible to antibacterial agents as compared to Gram negative ones as the latter has a lipid bilayer that protects them against antimicrobials. 47 The bioactive content of M. longifolia and M. piperita essential oils is considered to be responsible for their antibacterial properties. Balkrishna et al. 48 described antibiotic resistance mechanisms such as molecular target transformation, efflux pump hyperactivity, biofilm growth, enzyme induced destruction, and drug transformation. They demonstrated that, antibacterial activity of plants is linked to their bioactive composition, and can be mediated by inhibiting efflux pump expression level, protein and DNA synthesis, biofilm, and others as shown in Fig. 4.
The essential oils of Mentha spp. might work through the mechanisms described above due to diverse bioactive components.

Fungicidal activity
The analysis revealed that as the concentration of the essential oil (0.5 mg/mL-3.0 mg/mL) increased, inhibition of fungal growth also increased (Tables 5 and 6). The lowest MIC values were observed with essential oil of M. piperita against C. albicans in July and May months (MIC-0.31 mg/mL).
Whereas, the essential oil extracted in June and May months showed the same MIC value of 0.62 mg/mL against F. oxysporum (Table 7). On the other hand, the M. longifolia essential oilMIC values revealed the maximum fungicidal activity against C. albicans in July month (MIC-1.5 mg/ mL), whereas, essential oil of May month harvest showed maximum antifungal activity (MIC-1.5 mg/ mL) against F. oxysporum (Table 7). These findings proved that both Mentha species essential oil were more efficient against Candida albicans than F. oxysporum. Additionally, M. longifolia essential oil has stronger fungicidal action than M. piperita essential oil, similar to the antibacterial results. Hussain et al. observed the maximum antifungal activity of the M. longifolia oil in the summer season and M. piperita oil in the winter season against F. oxysporum. 33 Kizil et al. also observed the significant antibacterial effect of M. piperita essential oil against C. albicans, and E. coli. 49 In the light of existing literature, it was found that, the antifungal and antibacterial potential of essential oil is attributed to the presence of some phytoconstituents with various mechanisms including hydrophobicity. 50 In addition, the mode of action of plant bioactive content is still unknown; it is thought that they interfere with cell membrane organization, resulting in a drop in membrane potential and a lower amount of ATP synthesis. 48 The antibacterial effect of M. piperita oil is due to menthol, menthone, piperitenone oxide, and carvone, according to the available literature. 34 Subsequently, according to Zouari-Bouassida et al. monoterpenoids are responsible for the antimicrobial potential of M. piperita essential oil. 45 Similarly, in M. longifolia essential oil the antimicrobial activity is due to the existence of menthol, menthone, piperitone oxide, carvone, etc. 33 However, some of these components were missing from M. longifolia essential oil obtained in the northwestern Himalaya. The compounds observed during the present study were piperitone oxide, 3-cyclopentene-1-one,2-hydroxy-3-(3methyl-2-butenyl)-, borneol, caryophyllene, cis-afarnesene, germacrene D, etc. and most of these compounds have been reported in the literature with various biological activities. 19,31,39,41,[51][52][53][54] Hence, the variation in antimicrobial potential of the essential oils harvested in the examined months could be related to variations in the quantity of these phytoconstituents in each month from April to July.

CONCLUSION
In conclusion, M. piperita essential oil harvested in July month was more effective against Klebsiella pneumoniae, Staphylococcus aureus and Candida albicans, whereas May month harvest showed good activity against Bacillus subtilis, Escherichia coli, and Fusarium oxysporum. Similarly, M. longifolia essential oil from the July month harvest was more potent against K. pneumoniae, E. coli, and Candida albicans, as compared to May month harvest (moderately effective against B. subtilis, and F. oxysporum). This may be due to the cumulative effect of biologically active phytocompounds such as menthol, isomenthone, piperitone oxide, carvone oxide, and others, which accumulated in July, and germacrene D, caryophyllene oxide, caryophyllene, a-terpineol, and others in May. As a result, the harvesting time was found to be a key player for the antimicrobial potential of Mentha essential oils, like M. piperita, M. longifolia essential oil can be utilized in the pharmaceutical, perfumery, and food industries.