Identification of volatile compounds and antioxidant, antibacterial, and antifungal properties against drug-resistant microbes of essential oils from the leaves of Mentha rotundifolia var. apodysa Briq. (Lamiaceae)

Ahmad Mohammad Salamatullah

doi:10.1515/chem-2022-0161

Article Open Access

Identification of volatile compounds and antioxidant, antibacterial, and antifungal properties against drug-resistant microbes of essential oils from the leaves of Mentha rotundifolia var. apodysa Briq. (Lamiaceae)

Ahmad Mohammad Salamatullah

Published/Copyright: June 9, 2022

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From the journal Open Chemistry Volume 20 Issue 1

Abstract

The present research work investigated antioxidant, antibacterial, and antifungal properties of essential oils from the leaves of Mentha rotundifolia var. apodysa Briq. (EOR). Hydro-distillation was used to extract EOR before being subjected to the chemical characterization by the use of GC/MS. Antioxidant activity was assessed by the use of three bioassays namely 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and total antioxidant capacity (TAC). Antimicrobial potency was tested against gram-negative and gram-positive bacteria namely Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumonia, while antifungal activity was tested against Aspergillus niger, Candida albicans, Aspergillus flavus, and Fusarium oxysporum. EOR yield was determined to be 1.31%, with 20 compounds wherein Menthol (31.28%) and Isomenthol (14.28%) constituted the greatest amount. Regarding antioxidant activity, EOR exhibited potent antioxidant power: DPPH (IC₅₀ value of 0.36 ± 0.03 mg/mL), FRAP (EC₅₀ value of 0.35 ± 0.03 mg/mL), and TAC (697.45 ± 1.07 mg EAA/g). Antibacterial activity results showed that EOR had broad antibacterial activity on the tested strains. Eventually, EOR resulted in the greatest inhibition zone diameters vs S. aureus (18.20 ± 0.41 mm) followed by E. coli (17.02 ± 0.5 mm). Antifungal activity results showed that EOR exhibited potent antifungal activity and resulted in the greatest inhibition zone diameters up to 51.32 ± 1.32 mm against Aspergillus flavus, and 34.51 ± 1.07 mm against Aspergillus niger.

Keywords: natural product; bioactive compounds; menthol; isomenthol; antimicrobial

1 Introduction

People have always been preoccupied with their state of health, seeking the best way to combat anything that may threaten their existence. Nowadays, many pathologies constitute a great danger, even more than that war brings [1]. It is thus fitting that the search for effective agents to control such pathologies is appreciated now more than ever [2]. At an earlier time, people have developed strategies to gather and domesticate edible plants, and use others as remedies for therapeutic purposes [3,4].

The use of plants for therapeutic properties is a very old practice that goes back many centuries ago. Egyptians, Chinese, and Indians are among the oldest people who have used natural remedies for therapeutic goals [5,6]. Medicinal herbs and extracts have extensively been studied for their efficacy in medication and have received particular attention as growth and health promoters [7]. Plant derivative substances have multiple interests, particularly essential oils have received special attention due to their important application as both medication and food agents. Phytochemicals, particularly those with anticarcinogenic and antibacterial capabilities, are increasingly being used in foods, as well as in preventative and therapeutic medicine [8]. Because of their richness in phenolic functional groups, essential oils are well known for their antimicrobial and antioxidant properties [9].

Antimicrobial resistance is a phenomenon that occurs when bacteria develop strategies to combat antibiotics designed to kill them, resulting in infections difficult to treat and increasing the risk of disease spread [10]. For a few decades, scientists have paid more attention to antimicrobial resistance worldwide since it has become one of the most concerning issues plaguing the healthcare system. In addition, the World Health Organization has stated that antimicrobial resistance is the most growing issue in 2019. The excessive use of drugs in human medicine, animal husbandry, and hygiene can contribute to the rise of antimicrobial resistance [7,11]. Nowadays, antimicrobial resistance is a significant worrying phenomenon, which can be involved in killing up to 10 million people by 2050 if no alternative drugs are developed to fight causative agents with huge economic impact [12].

Lamiaceae is the sixth-largest Angiosperm family with 12 subfamilies, 16 tribes, 9 subtribes, 236 genus, and more than 7,000 species [13]. This family may be found all over the world, although some species are indigenous. Tropical and temperate climes are typical habitats for these species [13].

The essential oil of Lamiaceae species is notably rich in volatile monoterpenes, sesquiterpenes, and diterpenes, which are composed of 10, 15, and 20 carbon atoms, respectively. The most important monoterpenes are α-pinene, β-pinene, 1,8-cineole, menthol, limonene, and γ-terpinene. The essential oil of Lamiaceae species has a high concentration of fatty acids as well [13,14,15]. Due to their richness in phenolic chemicals, including carvacrol, eugenol, and thymol, essential oils from Lamiaceae species possess antibacterial properties, even against clinically important drug-resistant pathogenic microorganisms [16].

Essential oils from Lamiaceae species were found to possess promising antifungal effects, even at low concentration (minimum inhibitory concentrations [MICs] < 1 mg/mL). Notably, Clinopodium, Thymbra, and Thymus oils showed the highest antifungal activity (MICs < 0.1 mg/mL) [17].

There are more than 30 species in the genus Mentha (family Lamiaceae), which are largely cultivated worldwide for the production of essential oils [18]. In this context, many species showed changes in metabolic pathways, resulting in differences in oil composition patterns, e.g., Mentha spicata L. and Mentha piperita L. [19].

In addition to their culinary usage, Mentha spp. is widely used in traditional medicine to treat gastrointestinal issues. Species in Mentha were originally used as natural remedies to cure stomachaches and chest pains, and it is commonly used as a natural preparation, particularly in the form of tea to stimulate digestion and treat biliary disorders including dyspepsia, flatulence, enteritis, gastritis, aerophagia, gastric acidities, intestinal colic, and spasms of the bile duct, gallbladder, and gastrointestinal tract [20]. Mentha rotundifolia var. apodysa Briq. investigated in this work was also used as antiemetic, antidiarrhea, anti-haemorrhoidal, and analgesic agents [3]. Mentha spp. extracts exhibited many pharmacological properties including analgesic, anti-inflammatory, antipyretic, DNA damage protecting activity, anti-androgenic, cytotoxic, antiviral, analgesic, antiparasitic, sedative, antichlamydial, antiviral, anticancer, antiemetic, radioprotection, anticholinesterase, hepatoprotective, antispasmodic, antimutagenic, antimutagenic, anti-allergic, and cardiovascular effects [21].

Given the diversity of essential oil profiles among Mentha species, we investigated the chemical composition, antibacterial and antifungal properties of essential oils derived from the aerial parts of Mentha rotundifolia var. apodysa Briq. (M. rotundifolia) as a first step in determining the potential benefits of this species.

2 Materials and methods

2.1 Chemicals

Trichloroacetic acid (TCA), triphenyltetrazolium chloride (TTC), 2,2-diphenyl-1-picryl hydrazyl (DPPH), iron trichloride (FeCl₃), potassium ferricyanide (K₃Fe(CN)₆), sulfuric acid, sodium phosphate, ammonium molybdate, ascorbic acid, and sodium chloride were of analytical grade and purchased from Sigma-Aldrich Company (Munich, Germany). Mueller-Hinton Broth, Potato Dextrose Agar (PDA), Mueller-Hinton Agar, Nutrient Agar, medium, Oxacillin, Ceftizoxime, and Streptomycin were purchased from Biokar Diagnostics company (Allonne, France).

2.2 Plant material and EOR extraction

M. rotundifolia was collected at the end of March 2021 (period of maximum flowering) from the Northeast Maghreb region (34.225101; −4.070307). Afterward, the plant was identified by a botanist before being deposited at the University Herbarium with MT/18042021. Next, the leaves were washed and dried in the shade in a dry and ventilated place for 12 days prior to extraction by use of a Clevenger (letslab-Glassco) for 4 h. Briefly, 200 g of M. rotundifolia previously ground into small pieces was soaked into a flask with 750 mL of distilled water. Next, the whole mixture was boiled for 3 h until full extraction to obtain essential oils, which were dehydrated on anhydrous sodium sulfate before being stored at 4°C until further use. The Mentha rotundifolia var. apodysa Briq. (EOR) yield was calculated and presented as a percent based on the mass of dried M. rotundifolia [2].

2.3 Identification of EOR volatile compounds

Identification of phytochemical compounds in EOR was carried out by use of gas chromatography-mass spectrometry (GC-MS; Shimadzu, Kyoto, Japan) [22]. To achieve this goal, GC-MS with a nonpolar silica capillary column type (Wcot Fused Silica), stationary phase (CP-SIL5CB), 50 m in length, 0.320 mm diameter with a film thickness of 0.250 mm was used to perform analysis. The column temperature was programmed to be 40–280°C at 3°C/min, while injector temperature was 240°C and detector temperature was 200°C. One milliliter/min was the flow rate for helium (carrier gas). The volume of the injected sample was 1 µL of oil previously diluted in hexane [23]. The chemical compounds in the essential oils were identified by comparing their calculated retention index (RI) to RI documented in the literature relative to the Adams database [24].

2.4 Antioxidant activity of EOR

The antioxidant effect of EOR was evaluated in vitro by use of three bioassays namely free radicals of DPPH, ferric reducing antioxidant power (FRAP), and total antioxidant capacity (TAC).

2.4.1 Radical scavenging activity of EOR

In the current study, the DPPH test was used to assess the antioxidant potency of EOR as described in an earlier work [25]. Briefly, 100 µL of EOR were mixed with 750 µL of DPPH solution (4.00 mg/100 mL) and a series of dilutions were performed to obtain concentrations ranging from 0.04 to 5.0 mg/mL. Next, the mixture was kept in the dark at ambient temperature for 30 min prior to reading at 517 nm. Methanol solution without EOR was used as a negative control. Results presented here were expressed as a percentage of DPPH inhibition by use of the following formula:

Inhibition = T 0 − T x T 0 × 100 ,

where T ₀ is the negative control absorbance and T _x is the sample absorbance.

2.4.2 Ferric reducing power of EOR

The reducing power of EOR was carried out following the method of Moattar et al. [26]. Briefly, 0.5 mL of a phosphate buffer solution and 0.5 mL of potassium ferricyanide [K₃Fe(CN)₆] (1%) were mixed with 0.1 mL of EOR and a series of dilutions were performed to obtain concentrations ranging from 0.04 to 5 mg/mL. After incubation for 20 min in a water bath at 50°C, 0.5 mL of TCA (10%), 0.50 mL of distilled water, and 0.10 mL of FeCl₃ (1%) were added to the reaction medium prior to reading the absorbance at a wavelength of 700 nm against a blank with reagents. Results were expressed as 50% effective concentration (EC₅₀).

2.4.3 Evaluation of the total antioxidant capacity of EOR

Total antioxidant power was also used to evaluate the antioxidant activity of EOR. Briefly, 25 µL of EOR was mixed with 1 µL of reagent solution (0.6 M of sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) and the mixtures were incubated in a water bath at 95°C/90 min prior to measuring the absorbance at 695 nm by use of a spectrophotometer. Twenty-five microliters of reagents solubilized in methanol were used as a negative control. Results of total antioxidant potency were expressed in mg EAA/g EOs. Ascorbic acid calibration was used as a positive control [27].

2.5 Antibacterial activity

2.5.1 Bacterial strains

In the current work, antibacterial potency of EOR was tested vs five bacterial strains; Staphylococcus aureus ATCC 6633, Escherichia coli K12, A. baumannii FMP 69, Klebsiella pneumonia FMP 97, and S. pneumoniae. All bacteria strains have been reported to be involved in nosocomial infections which constitute a major public health problem [28].

2.5.2 Disk diffusion method on solid medium

In the current study, the disk diffusion method on solid medium was used for antibacterial testing. Briefly, Petri dishes containing nutrient agar were inoculated with bacteria in 10⁷ to 10⁸ CFU/mL before being incubated at 37°C for 24 h. Subsequently, 6 mm sterile discs were soaked in 10 µL of EOR before being placed on Petri dishes prior to further inculcation at 37°C for 18 h. Results were expressed as an inhibition zone in millimeters [29]. Oxacillin, Ceftizoxime, and Streptomycin were used as positive control.

2.5.3 Determination of EOR MIC

Due to its high activity vs Bacteria as revealed by the disk diffusion method on solid medium, EOR was tested for its MIC. To achieve this goal, 200 µL from EOR prepared with 0.2% (0.088 to 90 mg/mL) were poured into 96-well microplates. Later, 50 µL of the earlier prepared inoculum with a density of 10⁷ to 10⁸ CFU/mL was added to the microplate wells, except for controls. Thereafter, the plates were enclosed with sterile parafilm and then incubated at 37°C for 18 h. Next, 10 µL of TTC (5 mg/mL) was added to each well to indicate bacterial growth [30].

2.6 Antifungal activity

Antifungal activity was performed on four fungal strains namely Candida albicans ATCC 10231, Aspergillus niger MTCC 282, Aspergillus flavus MTCC 9606, and Fusarium oxysporum MTCC 9913 as described in an earlier work [31]. Briefly, an emulsion was made with a 0.20% agar to assist germ/compound contact. Next, sterilized PDA medium contained in test tubes was mixed with different EOR-agar (0.2%) dilutions to prepare concentrations: 1/100, 1/200, 1/800, 1/1,600, ½,000, and 1/3,200 (v/v). The tubes were meticulously shaken and then poured into Petri dishes. The culture medium prepared with 0.2% agar solution was served as a control. Consequently, discs measuring 6 mm diameter were soaked in 10 µL of EOR and deposited on the surface of Petri dishes before being incubated at 27°C for 6 days. Regarding MIC, Petri dishes with concentrations that demonstrated no mycelial development were used to determine MIC as reported in a previous work [16].

2.7 Statistical analysis

Findings presented in the current research work were expressed in mean values with standard deviations of triplicate tests. Analysis of variance was performed by use of ANOVA and Tukey’s HSD (honestly significant difference) was used as a post hoc test for multiple comparisons. A significant difference was considered at p < 0.05.

3 Results and discussion

3.1 Yield extraction and identification of EOR volatile compounds

The yield of the essential oils recovered from M. rotundifolia was 1.31%. The findings of this study are closely consistent with those reported by Mata et al. (2007), who found that the yield of essential oils extracted from the leaves of Mentha spicata L. was 0.9%. Similarly, the yield of essential oil extracted from Mentha spicata L. was 1.8% according to Kofidis and coauthors [33]. Notably, M. rotundifolia essential oil yield was also comparable to M. suaveolens and M. arvensis whose essential oil yields were reported to be 0.79 and 0.36–1.36%, respectively, by Asekun et al. [34] and Mimica-Dukic et al. [35]. The results of GC-MS analysis profiled 20 compounds representing 98.45% of the total essential oil extracted from M. longifolia. EOR majorly constituted of oxygenated monoterpenes like Menthol (31.28%), Isomenthol (14.28%), Pulegone (9.03%), 1.8 Cineole (3.56%), and sesquiterpene like Caryophyllene (10.21%) (Table 1, Figures 1 and 2). This chemical composition shares some compounds with that reported by El-Kashoury and coauthors [36], who showed that M. suaveolens essential oil possessed oxygenated monoterpenes including Caryophyllene and also comparable to the findings reported by Benali et al. [37], who revealed that M. suaveolens essential oil possessed Piperitenone oxide, β-elemene, Butanone, methyl-5-ethylthiazole, β-trans-caryophyllene, E-β-Farnesene, α-Humulene, Epi-bicyclosesuiphellandrene, and Germacrene D.

Table 1

Phytochemical compounds of EOR profiled by GC-MS

Peak	RT	Compounds identified	CC	RI		Area (%)
Peak	RT	Compounds identified	CC	Calculated	Literature	Area (%)
1	5.39	1.8 Cineole	MO	993	1,014	3.56
2	9.96	Acetylpyrrole	O	1,058	1,060	1.08
3	16.57	Pulegol	MO	1,225	1,229	9.03
4	21.76	α-Terpinene	MO	1,019	1,017	5.33
5	22.48	Menthol	MO	1,165	1,171	31.28
6	23.89	Isomenthol	MO	1,180	1,182	14.28
7	25.38	Pulegone	MO	1,228	1,237	4.70
8	31.42	Caryophyllene	ST	1,665	1,408	10.21
9	34.90	Bulnesol	ST	1,670	1,671	7.01
10	39.27	Bisabolol	ST	1,674	1,671	6.21
11	46.36	Agglomerone	O	1,698	1,698	2.50
12	51.39	Santalol	ST	1,710	1,716	3.26
Chemical classes
Monoterpene (MO)						68.18%
Sesquiterpene (ST)						26.69%
Others (O)						3.58%
Total						98.45%

RT, retention time in minutes; CC, chemical classes; MO, monoterpene; ST, sesquiterpene; O, others.

Figure 1

Chromatogram of volatile compounds profiled by GC-MS.

Figure 2

Molecular structure of the dominant phytochemicals in EOR.

3.2 Antioxidant activity

Evaluation of the antioxidant activity of EOR was done by use of three methods (DPPH, FRAP, and TAC) (Table 2). The DPPH test revealed that the 50% free radical inhibition concentration (IC₅₀) was 0.36 ± 0.03 mg/mL for EOR (Figure 3). This value is considered important when compared to some medicinal plants known for their antioxidant power [31], and even when compared to synthesized agents such as butylated hydroxytoluene (BHT) and Quercetin recording IC₅₀ values of 0.07 ± 0.01 mg/mL and 0.12 ± 0.01 mg/mL, respectively. Antioxidant results obtained with DPPH bioassay were comparable to those reported by Salhi et al. [38], who showed that aqueous extract from M. suaveolens possessed strong antioxidant power with a calculated IC₅₀ value of 0.023 mg/mL. Meanwhile, IC₅₀ values recorded for ascorbic acid and BHA used as positive controls were 0.002 and 0.006 mg/mL, respectively (p < 0.05).

Table 2

Results of antioxidant activity of EOR by DPPH, FRAP, and TAC assays

	DPPH (IC₅₀ in mg/mL)	FRAP (EC₅₀ in mg/mL)	TAC (mg AAE/g)
EOR	0.36 ± 0.03	0.35 ± 0.03	697.45 ± 1.07
Quercetin	0.07 ± 0.01	0.032 ± 0.001	523.65 ± 1.02
BHT	0.12 ± 0.01	0.041 ± 0.001	562.24 ± 1.12

Figure 3

Antioxidant activity by the DPPH assay for EOR along with controls (BHT and Quercetin).

The FRAP method results were expressed in half-maximal effective concentration (EC₅₀). In this aspect, findings showed that the EC₅₀ of EOR was 0.35 ± 0.03 mg/mL, which remains important when compared to BHT (0.041 ± 0.001 mg/mL) and Quercetin (0.032 ± 0.001 mg/mL) used as positive controls. Results of the antioxidant power of EOR examined with FRAP assay were higher than that reported by Bouyahya et al. [39], wherein it was stated that essential oil M. suaveolens possessed antioxidant power with IC₅₀ = 0.084 ± 0.003 mg/mL and, therefore, this difference can get back to differences in environmental and edaphic factors affecting the chemical composition is plants.

Results of TAC of EOR were expressed as ascorbic acid equivalent per gram of essential oil. In this context, findings showed that TAC of EOR was 697.45 ± 1.07 mg EAA/g. These results are in accordance with those reported in a previous work [40], which showed that many species among genus Mentha possess antioxidant effects such as Mentha suaveolens L., Mentha officinalis L., Mentha piperita L., Mentha pulegium L., and Mentha royleana L.

Profiling of EOR by GC-MS showed many major compounds such as Menthol, Isomenthol, Caryophyllene, and Pulegone, which can be the responsible compounds for its antioxidant power since many studies have shown that antioxidant potency of essential oils is relative to their chemical compositions, especially bioactive compounds with hydroxyl functions [22]. Thus, essential oils higher in terpene alcohols and phenolic compounds are expected to possess antioxidant capacity [41].

3.3 Antibacterial activity

The antibacterial property of EOR was qualitatively evaluated by use of the agar diffusion method and its results are presented as inhibition zone diameters around discs (Table 3). From Table 3, it can be seen that EOR had broad antibacterial activity on the tested strains. Eventually, EOR resulted in the greatest inhibition zone diameters against S. aureus (18.20 ± 0.41 mm) followed by E. coli (17.02 ± 0.5 mm). However, this bacteria showed powerful resistance to antibiotics used as positive controls such as Kanamycin (9.80 ± 0.5 mm for A. baumannii) and Oxacillin (9.11 ± 0.4 mm for S. pneumoniae). It is thus fitting that analysis of variance revealed significant differences among the antibacterial activities of EOR and drugs used as references (p < 0.05).

Table 3

Antibacterial activity of EOR in solid and liquid mediums

	Diameter of the inhibition zone on solid medium (mm)			MIC (mg/mL)
Strain	EOR	Kanamycin	Oxacillin	EOR	Kanamycin	Oxacillin
E. coli	17.02 ± 0.5	0	0	0.038 ± 0.003	0.012 ± 0.001	0.011 ± 0.001
K. pneumoniae	16.03 ± 0.27	0	0	0.042 ± 0.003	0.017 ± 0.001	0.014 ± 0.001
A. baumannii	16.24 ± 0.25	9.80 ± 0.5	0	0.051 ± 0.001	0.013 ± 0.001	0.012 ± 0.001
S. pneumoniae	15.89 ± 0.5	0	9.11 ± 0.4	0.068 ± 0.001	0.016 ± 0.001	0.013 ± 0.001
S. aureus	18.20 ± 0.41	0	0	0.032 ± 0.005	0.014 ± 0.001	0.015 ± 0.001

Results are expressed as means of triplicate assays ± standard deviation.

The results of MIC of bacterial growth in liquid medium evaluated by the microdilution method were in agreement with those obtained with the aromatogram assay. More particularly, EOR induced the greatest inhibition zone diameters against E. coli and S. aureus and lowest MIC of 0.038 ± 0.003 and 18.20 ± 0.41 mg/mL, respectively. In order to determine the potentially responsible compounds for the antibacterial activity investigated here, we need to figure out Pulegone (9.03%) and Menthol (31.28%) (Table 1), which were reported to possess antibacterial effects vs Bacillus cereus and Staphylococcus aureus, with calculated inhibition zone diameters of 15 and 17 mm, respectively [42]. In comparison, the inhibition zone diameters of cis-cis-p-menthenolide isolated from M. suaveolens reached 18 and 21 mm against Bacillus cereus and Staphylococcus aureus, respectively. Mintlactone from Mentha piperita L. essential oils resulted in inhibition zone diameters in 11 and 12 mm against Bacillus cereus and Staphylococcus aureus, respectively [43]. Results presented here are consistent with those reported elsewhere [44], wherein it was stated that Mentha spicata L. oil has a stronger antibacterial effect than reference drugs namely streptomycin and penicillin.

Due to their excessive use, antibiotics can lose their effectiveness against many growing resistant microbes [45]. In this context, phytochemicals found in medicinal plants are considered as a reservoir of bioactive compounds that can serve as a source of antimicrobial compounds [46]. Scientific reports have shown that essential oils can be considered powerful agents to combat bacterial resistance due to their broad-spectrum antimicrobial effects. Essential oils result in bacterial death by targeting key determinants of pathogenicity, membrane potential, efflux pumps, syntrophic consortium of microorganisms, and R-plasmids [45,47].

3.4 Antifungal activity

Antifungal activity of EOR was carried out by use of the direct contact method on solid medium and its results are presented as inhibition zone diameters around discs. Results showed that the inhibition zone diameters generated by EOR vs fungal strains exceeded 30 mm (Table 4). EOR powerfully inhibited the growth of the mycelium of all fungal strains used for testing. A. flavus and A. niger were the mostly abolished fungi by EOR recording inhibition zone diameters of 51.32 ± 1.32 and 34.51 ± 1.07 mm, respectively. Similarly, the antifungal activity of EOR was clearly shown by MIC assay wherein EOR recorded values ranging from 6.04 ± 0.26 to 25.26 ± 0.55 µg/mL vs the studied fungal strains (Table 4).

Table 4

Results of antifungal activity of EOR by use of disc and microdilution methods

	Inhibition diameter (mm)		MIC (µg/mL)
Strains	EOR	Fluconazole	EOR	Fluconazole
A. niger	34.51 ± 1.07	46.12 ± 2.61	25.26 ± 0.55	3.51 ± 0.04
C. albicans	39.00 ± 1.5	43.08 ± 1.05	6.04 ± 0.26	2.54 ± 0.08
A. flavus	51.32 ± 1.32	69.41 ± 1.71	7.41 ± 0.40	2.62 ± 0.03
F. oxysporum	36.63 ± 2.10	37.52 ± 1.61	11.05 ± 0.76	3.72 ± 0.04

Results are expressed as means of triplicate assays ± standard deviations.

These results are in good agreement with those presented by Sokovic and Griensven [48], who showed that essential oil obtained from M. spicata had a stronger antifungal effect than Bifonazole used as a drug reference. Our findings corroborated the inhibitory effects of many essential oils from medicinal plants against fungi [42,35,24]. Several studies have been carried out to understand the mechanism of action of phytochemicals in fungi. It was reported that compounds can interfere with biomembranes and cause cellular damage followed by leakage of cellular material and ultimately to the death of the microorganisms [45,49]. Mycelial growth can be reduced or completely inhibited by the effects of phytochemicals acting on the functionality and structure of the cell membrane [50]. Menthol profiled in EOR along with other plants derivative essential oils have a high antifungal effect vs pathogenic strains: A. fumigatus, A. niger, A. flavus, and F. oxysporum [51]. Pulegone contained in M. suaveolens essential oil is reported to have powerful antifungal activity [52]. Antifungal activity of this compound can be due to the disruption of the lipid fraction of the plasma membrane altering its permeability, which leads to leakage of intracellular material [53].

Overall, EOR along with its major components, particularly Menthol (31.28%), and Pulegone (14.28%), exhibited important antimicrobial activity in the present work. It is feasible to assume that EOR has a broader and stronger antibacterial spectrum.

4 Conclusion

The present work sheds light on antioxidant, antibacterial, and antifungal potencies of essential oils extracted from the leaves of M. rotundifolia. Results revealed that the studied oils exhibited potent antioxidant and antimicrobial properties, which could be attributed to their richness in specific bioactive such as Menthol and Isomenthol. The outcome of the present study reported on the benefits of M. rotundifolia essential oils as effective eco-friendly agents with antioxidant and antimicrobial properties. Although the oil’s mechanism of action is still being examined, it is well known that a complex mixture of components could have multiple biological potentials simultaneously. Therefore, future research aims to understand the mode of action of pure molecules is warranted. In addition, research on nonhuman primates and people to determine the oil’s effects on species other than the target will be necessary before it can be used as a natural treatment to control infections.

Acknowledgment

The author extends his appreciation to Researchers Supporting Project number (RSP-2022R437), King Saud University, Riyadh, Saudi Arabia.

Funding information: This study was funded by Researchers Supporting Project number (RSP-2022R437), King Saud University, Riyadh, Saudi Arabia.
Author contributions: This work was completely performed by AMS.
Conflict of interest: The author declares that there is no conflict of interest.
Ethical approval: The conducted research is not related to either human or animal use.
Data availability statement: All data reported here are available from the author upon request.

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Received: 2022-02-21

Revised: 2022-04-15

Accepted: 2022-04-22

Published Online: 2022-06-09

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/chem-2022-0161

Keywords for this article

natural product; bioactive compounds; menthol; isomenthol; antimicrobial

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