Chemical composition of Mentha suaveolens and Pinus halepensis essential oils and their antibacterial and antioxidant activities

Abdelhakim Bouyahya, Omar Belmehdi, Jamal Abrini, Nadia Dakka, Youssef Bakri

1Laboratory of Human Pathologies Biology, Department of Biology, Faculty of Sciences, and Genomic Center of Human Pathologies, Faculty of Medicine and Pharmacy, Mohammed V University in Rabat, Morocco

2Biology and Health Laboratory, Department of Biology, Faculty of Sciences, Abdelmalek Essaadi University, Tetouan, Morocco

Keywords：Mentha suaveolens Pinus halepensis Essential oil Antioxidant effect Antibacterial activity

A BSTRACT Objective: To determin the chemical compounds of Mentha suaveolens (M. suaveolens)and Pinus halepensis (P. halepensis) essential oils (Eos) and evaluate their antioxidant and antibacterial activities.Methods: The chemical composition of P. halepensis and M. suaveolens EOs was determined by GC-MS analysis. The antioxidant activity was evaluated using DPPH, ABTS and FRAP assays. The antibacterial effect was tested against 6 bacterial strains using the well diffusion method and micro-dilution assay.Results: The major components of P. halepensis EOs were β-caryophyllene (28.04%), myrcene(23.81%) and α-pinene (12.02%). However, piperitenone oxid (56.28%), piperitenone (11.64%)and pulegone (6.16%) were the major components of M. suaveolens EOs. M. suaveolens EOs showed remarkable antioxidant activities compared with P. halepensis EOs, showing antioxidant capacity values of IC50=(64.76±2.24) µg/mL, IC50=(82.73±3.34) µg/mL, and IC50=(93.35±4.45) µg/mL, revealed by DPPH, ABTS and FRAP assays, respectively. However,P. halepensis EOs showed interesting antibacterial effects against all bacterial strains. The most sensible strains to P. halepensis EOs were Staphylococcus aureus [(34.00±0.50) mm], Listeria monocytogenes [(31.00±1.50)] mm and Proteus mirabilis [(29.00±2.25)mm]. Furthermore, the lowest minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)values were revealed by P. halepensis EOs against Staphylococcus aureus [MIC=MBC=0.125%(v/v)] and Listeria monocytogenes [MIC=MBC=0.25% (v/v)].Conclusions: P. halepensis and M. suaveolens EOs contain bioactive compounds that could have potential applications against bacterial infections and oxidative stress related diseases as well as for food conservation. However, further investigations are necessary to isolate and investigate the action mechanisms of these bioactive compounds.

1. Introduction

Oxidative stress results from unbalancing between generation and degradation of free radicals in cells. Currently, several data showed that the oxidative stress is a major risk factor of genesis for several diseases such as inflammation, diabetes, and cancer[1]. Synthetic antioxidant drugs have shown several side effects that require the screening of natural antioxidants. In this context, several studies currently focused on the secondary metabolites of medicinal plants such as essential oils( EOs) as source of novel antioxidants.

On the other hand, the bacteria continue to develop sophisticated mechanisms to resist against antibiotic actions[2]. The resistance developed by some bacteria towards a large number of antibiotics challenges the search of new alternative molecules, especially those extracted from natural sources including medicinal plants.

Among the bioactive compounds containing in medicinal plants,we found EOs, which exhibited various biological activities such as antimicrobial, antioxidant, antitumor and anti-inflammatory effects[3,4]. These effects are certainly attributed to terpenoids bioactive compounds containing in EOs.

Mentha suaveolens (M. suaveolens) and Pinus halepensis (P.halepensis) are two medicinal plants used traditionally for the treatment of some diseases in several countries including Morocco.In fact, M. suaveolens is used by Moroccan population to treat pathologies of digestive system, allergy and diabetes, while P.halepensis is used against stomach disorders[5].

M. suaveolens and P. halepensis EOs showed some biological properties such as antimicrobial and antioxidant effects[6]. However,in Morocco, EOs of these two species have not been largely studied.This study aimed to determine the chemical composition of P.halepensis and M. suaveolens EOs, and evaluate their antibacterial and antioxidant properties.

2. Materials and methods

2.1. Plant material and EOs extraction

The leaves of P. halepensis (voucher specimen: RAB16) and the flowery tops of M. suaveolens (voucher specimen: RAB08) were collected during the summer months (June) from the North-West of Morocco. The samples were air dried at room temperature under dark condition. The extraction of EOs was carried out by hydrodistillation method using clevenger-type apparatus. The obtained oils were dried, weighed and stored at 4 ℃ until use.

2.2. Gas Chromatography-mass spectrometry(GC-MS)analysis EOs

The volatile components of P. halepensis EOs and M. suaveolens EOs were analyzed by GC-MS using the same parameters previously published[6]. The identification of volatile compounds was carried based on the literature and the retention index. The confirmation of each compound was made by comparison of its mass spectra with those of NIST02 library data.

2.3. Antioxidant activity

2.3.1. The stable 2,2-diphenyl-1-picrylhydrazyl radical(DPPH) free radical-scavenging assay

DPPH was used for the determination of free radical-scavenging activities of P. halepensis and M. suaveolens EOs[6,7]. Briefly, 0.2 mL aliquots of various concentrations of methanol dissolved EO’s samples were added to 1.8 mL of a 0.004% DPPH methanolic solution. After 30 min incubation time at room temperature, the absorbance was measured at 517 nm as optical density[6]. The EOs antioxidant activities expressed as the percent of scavenging DPPH radical was calculated by the following formula:

Acis the absorbance of the control (without oil), and Atis the absorbance of the sample. Trolox and ascorbic acid were used as positive controls,and EOs concentration providing 50% inhibition (IC50) was calculated by plotting the inhibition degrees against the sample concentrations. The test was carried out in triplicate, and the IC50values were reported as means±standard deviation (SD).

2.3.2. Reducing ferric power determination

Reductive ability was investigated by the Fe3+to Fe2+transformations in presence of P. halepensis and M. suaveolens EOs[6,7]. EOs and control(ascorbic acid and trolox) were mixed separately and successively with 2.5 mL volumes of phosphate buffer (0.2 M, pH 6.6) and potassium ferricyanide [K3Fe (CN)6] (1%). After incubating the mixture at 50 ℃ for 20 min, 2.5 mL of trichloroacetic acid (10%) was added to the mixture and centrifuged for 10 min at 3 000 rpm. Finally, a volume of 2.5 mL was collected from the upper layer of the obtained solution and mixed with an equal volume of distilled water and 0.5 mL FeCl3(0.1%). The absorbencies were measured at 700 nm by a spectrophotometer and plotted against the corresponding sample concentrations to deduce the EO concentration providing 50% of absorbance decrease (IC50). Trolox and ascorbic acid were used as positive controls. The test was carried out in triplicate and IC50values were reported as means±SD.

2.3.3. 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging activity

ABTS radical-scavenging activity of EO was determined according to the method described by El Bouzidi et al.[6] with some modifications.The ABTS radical cation was produced by the reaction between 5 mL of ABTS stock solution and 5 mL of 2.45 mM potassium persulfate(K2S2O8) solution, stored in the dark at room temperature for 16 h.Before use, this solution was diluted with water to get an absorbance of (0.700±0.015) at 734 nm and equilibrated at 30 ℃. EOs at various concentrations were diluted with dimethyl sulfoxide to get sample solution. A volume of 5 mL sample solution was homogenized with 195 mL of ABTS solution, the mixture was incubated at room temperature for 6 min and its absorbance was recorded at 734 nm. Blanks were run in each assay. ABTS scavenging ability was expressed as IC50(µg/mL) and the inhibition percentage was calculated using the following formula:

Where: A0is the absorbance of the control, and A1is the absorbance of the sample.

2.4. Antibacterial activity

2.4.1. Bacteria strains

The antibacterial effects of M. suaveolens and P. halepensis EOs were evaluated against Escherichia coli (E. coli) K12 (Laboratory of Food Microbiology, UCL, Belgium: MBLA), Staphylococcus aureus CECT 994(S. aureus), Listeria monocytogenes serovar 4b CECT 4032 (L.monocytogenes) and Proteus mirabilis (Spanish type culture collection:CECT)(P. mirabilis), Pseudomonas aeruginosa IH (P. aeruginosa)(Institute of hygiene, rabat, Morocco: IH), and Bacillus subtilis 6633 (B. subtilis)(German collection of microorganisms: DSM). Strains were stored in inclined agar medium at 4 ℃. Before use, the bacteria were revived by two subcultures in an appropriate culture medium; lysogeny broth (LB) (Biokar Diagnostics, Beauvais, France) at 37 ℃ for (18-24) h. For the test, final inoculum concentrations of 106CFU/mL bacteria were used according to the National Committee for Clinical Laboratory Standards, USA.

2.4.2. Determination of inhibition diameters

To determine the inhibition diameters of P. halepensis and M.suaveolens EOs against the tested bacteria, we have used agar-well diffusion assay as described by Bouhdid et al.[8]. This method is mostly used as preliminary screening assay to further quantitative investigations. Briefly, a basal layer medium was made by cooling Muller-Hinton agar on Petri dishes to solidify. An 8 mm diameter cylinder was deposited under aseptic conditions on Muller-Hinton agar layer. A total of 6 mL LB medium containing 0.8% agar and inoculated by a fresh culture of the tested bacterial strain was used as a top medium layer. The wells formed were filled with 50 µL of P. halepensis and M. suaveolens EOs after enriched LB medium solidification. After 24 h incubation time at an appropriate temperature for each tested strain, all cultures were examined and the diameters of the appeared inhibition zones were measured in millimeters. All the bacterial growth inhibition assays were performed in triplicate.

2.4.3. Determination of minimal inhibitory concentration (MIC)and minimal bactericidal concentration (MBC)

The MIC of M. suaveolens and P. halepensis EOs against the tested bacteria were determined according to the broth micro-dilution assay described by Bouhdid et al.[9] and Bouyahya et al.[6]. The test was carried out on liquid medium using a 96-wells polypropylene microtitre plate. After deposing a 50 µL (different concentrations) of the tested oil in each well, 50 µL ( final concentration of 106CFU/mL) of a bacterial suspension was added to each well. After incubation at 37 ℃ for 18 h,the resazurin (10 µL) was added to each well to assess bacterial growth.Bacterial growth was detected after incubation at 37 ℃ for 2 h by reduction the blue dye resazurin to pink resoru fin, and then the MIC was determined as the lowest EO concentration that prevented resazurin color changes. To determine the MBC, 10 µL were transferred from negative subcultures to on plat count agar medium and then the MBC were determined after 24 h incubation time at 37 ℃[8].

2.5. Statistical analysis

The comparison of means between groups was performed with oneway analysis of variance (ANOVA) followed by Tukey test. Differences were considered significant when P ＜ 0.05.

3. Results

3.1. Chemical composition

The chemical composition of P. halepensis and M. suaveolens EOs was identified using GC-MS (Figure 1 and 2). The obtained results are listed in Table 1. As shown, 20 compounds were identified in M. suaveolens EOs, while P. halepensis EOs contain 23 volatile compounds. The main compounds of P. halepensis EOs were β-caryophyllene (28.04%),myrcene (23.81%), α-pinene (12.02%), and caryophyllene oxide(6.78%). However, for M. suaveolens EOs, the main compounds were piperitenone oxide (56.28%), piperitenone (11.64%), and pulegone(6.16%).

Figure 1. GC-MS analysis of P. halepensis essential oils.

Figure 2. GC-MS analysis of M. suaveolens essential oils.

3.2. Antioxidant activity

To evaluate the antioxidant effects of P. halepensis and M. suaveolens EOs, 3 complementarily assays were used: DPPH scavenging activity assay, ABTS and FRAP tests, and in order to compare the obtained data, the results were expressed as IC50values (Table 2). As listed in the table, M. suaveolens EOs revealed higher antioxidant capacities than P. halepensis EOs. Indeed, M. suaveolens EOs showed antioxidant capacity values at IC50=(64.76±2.24) µg/mL, IC50=(82.73±3.34) µg/mL, and IC50=(93.35±4.45) µg/mL using DPPH, FRAP and ABTS assays, respectively. Moreover, it has been noticed that the DPPH assay was more sensitive than FRAP and ABTS tests.

Table 1. Chemical composition of P. halepensis and M. suaveolens EOs.

Table 2. Antioxidant activities of P. halepensis and M. suaveolens EOs (µg/mL).

3.3. Antibacterial activity

The antibacterial activity of P. halepensis and M. suaveolens EOs was evaluated against 6 bacterial strains using agar well diffusion assay.The obtained results were expressed as the diameters of inhibition around the wells (Table 3). As summarized in the table, P. halepensis EOs showed a higher antibacterial effect than M. suaveolens EOs against some strains. Indeed, P. halepensis EOs exhibited remarkable inhibition of S. aureus, L. monocytogenes and P. mirabilis with inhibition zones of (34.00±0.50) mm, (31.00±1.50) mm and(29.00±2.25) mm, respectively. These effects were higher than those obtained with chloramphenicol. For the determination of the MIC and MBC, the micro-dilution assay was used. The obtained results were listed in Table 4. As shown, P. halepensis EOs demonstrated remarkable inhibition of S. aureus [MIC=MBC=0.125% (v/v)] and L. monocytogenes [MIC=MBC=0.25% (v/v)]. However, M. suaveolens EOs showed an antibacterial inhibition at moderate concentrations.

Table 3. Antibacterial activity of P. halepensis and M. suaveolens EOs using agar-well diffusion assay (mm).

Table 4. P. halepensis, M. suaveolens EOs, and chloramphenicol (µg/mL) against 6 human pathogen bacteria strains tested by microdilution assay.

4. Discussion

Several studies have shown that EOs possess remarkable biological properties such as anticancer, anti-inflammatory, antioxidant and antimicrobial effects[3,4]. Morocco, as a Mediterranean country, is rich in vegetation, in particularly medicinal plants. The chemical and biological valorization of Moroccan medicinal plant secondary metabolites may be an important strategy to develop alternative drugs. Our study focused on the characterization of P. halepensis and M. suaveolens EOs and the investigation of their antibacterial and antioxidant activities.

P. halepensis is a woody plant, so for the EOs extraction the leaves were used. However, M. suaveolens is an herbaceous plant,and in this case the flowery tops were used to extract the EOs. It has been noticed that the major compounds of M. suaveolens EOs(pulegone, piperitenone and piperitenone oxide) were totally absent in P. halepensis EOs. On the other hand, the major compounds of P. halepensis EOs existed in M. suaveolens EOs but in low concentrations.

Several studies have reported the chemical composition of P.halepensis and M. suaveolens EOs[10-13]. Our findings are not completely in accordance with the literatures. These differences are attributed certainly to the variability of ecological conditions between the areas of collection. Indeed, Llorens-Molina et al.[14]showed that the chemical composition of M. suaveolens EOs is variable between several regions. Moreover, the chemical variability between P. halepensis EOs was also proved by Dob et al.[15],which determined the volatile compounds of P. halepensis EOs in several areas from Algeria and revealed different results[15]. Other several studies currently showed that the chemical composition of same species is influenced by various factors particularly climatic conditions[14,16]. For this reason, the comparison of the essential oil composition described in different studies is sometimes difficult,if not impossible, not only because different methodologies of isolation and analysis may have been used, but also when different plant parts and various developmental stages are involved. Recently,some studies have reported that chemical flexibility of secondary metabolites of P. halepensis is due essentially to response against induced stress by climatic changes[17,18].

Generally, the synthesis of secondary metabolites in medicinal plants is affected by various internal and external factors, which in fluence the regulation of these metabolites in space and time via several epigenetic pathways such as DNA methylation, histones modifications and remodelage of chromatin in order to respond against specific physiological functions[19,20].

Piperitenone oxide, the major compound of M. suaveolens EOs, is known for its cardiovascular and anti-nociceptive effects[21,22] and as an insect repellent. However, pulegone, one of the main components of this plant, is known for its antimicrobial activity[23], as well as for its hepatoprotective effect[24]. On the other hand, β-caryophyllene the main component of P. halepensis EOs, is known for its various biological activities such as anticancer and analgesic properties[25],local anesthesic activity[26], anti-arthritic and anti inflammatory effects[27]. Moreover, α-pinene and myrcene also possess numerous biological activities such as antimicrobial[28]. These various activities of these biomolecules explain its importance as alternative for medical purposes. In addition, as it has been shown that the major components vary between these two studied plants; these EOs can be complementary as bioactive compounds sources.

The evaluation of antioxidant activity revealed that the IC50values obtained with the DPPH test were lower than those obtained with the FRAP and ABTS tests. The differences between the two results can be explained by the mechanism of the reaction involved. The major compounds of P. halepensis EOs are β-caryophyllene, α-pinene,myrcene and p-cymene, while the major compounds of M. suaveolens EOs are piperitenone oxid and piperitenone, which could explain the differences in the antioxidant activity between them. In addition, the oxygenated monoterpenes found in these EOs may act as antioxidant agents.

The antiradical activity of our P. halepensis EOs using DPPH test [IC50=(113.25±7.18) µg/mL] was more important than that of Algerian P. halepensis EOs evaluated at different vegetative stages[IC50values ranging from (201.28±2.75) to (236.18±3.73) µg/mL][13].Comparatively, this DPPH-radical scavenging activity was lower than that of the synthetics antioxidants ascorbic acid and trolox[IC50=(22.61±1.08) µg/mL and (34.12±2.13) µg/mL, respectively].The antioxidant properties of P. halepensis EOs and MSEO are certainly attributed to the phenolic compounds present in these EOs, which are able to neutralize the free radicals via different mechanisms. However, the use of three experimental methods to estimate the antioxidant activity showed that the antioxidant effect of P. halepensis and M. suaveolens EOs does not only depend on the chemical composition, but also on the medium where reaction takes place. Our results are in accordance with other studies carried out on the antioxidant activities of P. halepensis and M. suaveolens EOs[13,29].

The antibacterial activity of M. suaveolens and P. halepensis EOs was determined against 6 strains of reference using agar well diffusion assay and microtitration assays. The results revealed that P. halepensis EOs showed remarkable inhibitory effects on all tested microorganisms. It would be related to their major components specially β-caryophyllene which is known by its antibacterial effect[30]. However, it is difficult to attribute the activity of a complex mixture to a single or particular constituent. In fact, there is some evidence that minor components have a critical part to play in antibacterial activity, possibly by producing a synergistic effect between other components. The variation in chemical composition of essential oil might be responsible for the different antibacterial effects.

The mechanisms of action of plant EOs have been mostly studied.Nevertheless, the studies about the antimicrobial action of essential oil and their components still remains. It has been reported that the antimicrobial actions of essential oil are linked to one of the most important EOs characteristics, its hydrophobicity resulting in increased cell permeability and consequent leaking of cell constituents. The most known targets of EOs are: cell wall and membrane disturbance, alteration of ATP production and protein synthesis, pH disturbance, intra-cytoplasmic changes, DNA damage,and quorum sensing inhibition[31]. Moreover, this study revealed that Gram-negative bacteria are more resistant to P. halepensis and M. suaveolens EOs than the Gram-positive ones. The resistance of Gram-negative bacteria can be attributed to the presence of outer membrane surrounding the cell wall[32].

In this study, the antioxidant and antibacterial activities of M.suaveolens and P. halepensis EOs were evaluated. The analysis of the chemical composition showed considerable qualitative variability in the composition of P. halepensis and M. suaveolens EOs. The variability in essential oil composition is the results of an adaptive process to particular ecologic conditions (geographical regions,climate conditions, altitude), period of collection of the plant, the studied part (leaves or flowers), state of plant (fresh or dry) and method of extraction of the essential oil. The antioxidant effects were showed mostly by M. suaveolens EOs, while P. halepensis EOs showed interesting antibacterial properties. The results revealed the eventually possibilities to use of M. suaveolens EOs as source of antioxidant compounds and P. halepensis EOs as source of antibacterial compounds. However, further studies are required to isolate bioactive components and to elucidate their antioxidant and antibacterial mechanisms.

Conflict of interest statement

The authors declare that there is no conflict of interest.

Acknowledgement

The authors would like to thank le Centre National pour la Recherche Scientifique et Technique et l’Agence Nationale des Plantes Médicinales et Aromatiques for supporting the funding of this study.