A Comparative Analysis of the Chemical Composition, Anti-Inflammatory, and Antinociceptive Effects of the Essential Oils from Three Species of Mentha Cultivated in Romania

This work was aimed at correlating the chemotype of three Mentha species cultivated in Romania with an in vivo study of the anti-inflammatory and antinociceptive effects of essential oils. The selected species were Mentha piperita L. var. pallescens (white peppermint), Mentha spicata L. subsp. crispata (spearmint), and Mentha suaveolens Ehrh. (pineapple mint). Qualitative and quantitative analysis of the essential oils isolated from the selected Mentha species was performed by gas chromatography coupled with mass spectrometry (GC-MS). The anti-inflammatory activity of the essential oils was determined by the rat paw edema test induced by λ-carrageenan. The antinociceptive effect of the essential oils was evaluated by the writhing test in mice, using 1% (v/v) acetic acid solution administered intraperitonealy and by the hot plate test in mice. The results showed a menthol chemotype for M. piperita pallescens, a carvone chemotype for M. spicata, and a piperitenone oxide chemotype for M. suaveolens. The essential oil from M. spicata L. (EOMSP) produced statistically significant and dose-dependent anti-inflammatory and antinociceptive effects.


Introduction
The genus Mentha (Lamiaceae), present in the temperate regions of all five continents, consists of 18 species and 11 named hybrids [1]. The members of the genus Mentha can easily produce many intermediary forms by hybridization, polyploidy playing also an important role in the speciation, making the number of taxonomically valid species a subject of controversy [2]. Mentha species are characterized by high morphological variability but also by a great chemical diversity with respect to their essential oils, the main chemical constituents, rarely encountered in other temperate zone species [3]. The differences in essential oil composition among the members of this genus offer a variety
In this study, the GC-MS analysis of the essential oils from the tested samples showed a menthol chemotype for M. piperita pallescens cultivated in Romania, confirming the study of Schmidt et al., which found a rather similar menthol content (40.7%) in M. piperita originating from the Balkans [20].
Although our data confirmed a carvone chemotype for M. spicata, the carvone content was lower compared to M. spicata from India, where it reached 76.65% [21], or Turkey, where it reached 80.65% [22].
Our study showed also that a low content of carvone (1.555%) coupled with a high content of piperitenone oxide (73.773%), suggest a piperitenone oxide chemotype for M. suaveolens, encountered also in Southeastern Europe [23]. A different study (El-Kashouri et al.) found a higher percentage of carvone (24.72%-55.74%) in M. suaveolens harvested from North Africa [24]. The variations in chemical composition of essential oils from Mentha species can be attributed to several factors such as temperature, humidity, climate, harvest season, or photoperiod [25].

Anti-Inflammatory Activity of the Essential Oils
The anti-inflammatory activity of the tested essential oils was evaluated in vivo by the rat paw edema test induced by λ-carrageenan. The results of the experiment are presented in Table 2.  A statistically significant anti-inflammatory effect was observed in the groups treated with essential oils from M. spicata (EOMSP) 500 mg/kg, 2 h, 3 h, and 4 h after the induction of inflammation and M. piperita pallescens (EOMPA) 500 mg/kg, 4 h after the induction of inflammation. The most active sample, EOMSP (500 mg/kg), was superior to the reference drug diclofenac, 2 h and 4 h after the induction of inflammation. The essential oil from M. suaveolens (EOMSU) produced an inferior anti-inflammatory effect, and only at the highest dose.
The development of edema in the rat hindpaw following an injection of λ-carrageenan has been characterized as a biphasic event. Initially, the inflammatory reaction to carrageenan (0-1 h) is caused by the release of histamine, serotonin, bradykinin, complement and reactive oxygen species. In the second, accelerating the phase of swelling (2-4 h), an increased production of prostaglandins in the inflammatory area has been demonstrated [26].
Our experimental data suggest that several peripheral mechanisms could be responsible for the anti-inflammatory effect of essential oils from Mentha. Besides a reduction of prostaglandin concentration in the affected tissue, it is possible that the essential oils were able to also influence the first phase of carrageenan-induced edema formation, probably by inhibiting the release of other pro-inflammatory mediators.

Acetic Acid Induced Writhing Test in Mice
The antinociceptive activity of the tested essential oils was firstly evaluated by the acetic acid induced writhing test in mice. The results of the experiment are presented in Table 3. The administration of the reference drug, diclofenac, significantly reduced the number of writhes induced by acetic acid, the percentage of inhibition being 60.49%. The essential oil from M. spicata (EOMSP) showed a significant and dose-dependent antinociceptive effect, reducing the number of writhes at all the three tested doses, although the results were slightly inferior to the reference drug, diclofenac. The essential oils from M. piperita pallescens (EOMPA) and M. suaveolens (EOMSU) showed inferior antinociceptive effects in this experimental model.
Acetic acid is known to trigger an irritative reaction in the peritoneum, which induces the writhing response due to the sensitization of nociceptors by prostaglandins, excessively formed in the peritoneal cavity. The nociceptive properties of acetic acid might also be due to the release of cytokines, such as TNF-α, interleukin-1β, and interleukin-8, by resident peritoneal macrophages and mast cells [27]. Thus, the abdominal constriction response induced by acetic acid is a sensitive procedure to establish peripherally acting antinociceptives [28].
The protective effect of essential oils against the chemical noxious stimulus may be an indication for a decreased production of prostaglandins, thereby causing a reduction in the number of writhes. Our results from this experimental model indicate that antinociceptive effect of EOMSP might be mediated by the peripheral inhibition of prostaglandins synthesis or actions.

Hot Plate Test in Mice
To evaluate whether the antinociceptive effect of essential oils from Mentha might posses also a central mechanism, the hot plate test was used, the results being presented in Table 4. The mice treated with EOMSP presented the most significant antinociceptive effects in the hot plate test, starting 60 min after the administration, with a peak after 120 min, at the dose of 500 mg/kg. For this group, the percent analgesic score (PAS) varied between 60.24% at 60 min and 65.40% at 120 min. The antinociceptive effects of EOMPA were slightly inferior but significant at 60, 90, and 120 min, at the dose of 500 mg/kg. EOMSU produced only modest results. The effects of morphine, the reference centrally acting antinociceptive drug, were evident 60 min after administration, peaked at 90 min and continued for 2 h. The results showed that the essential oils had a rapid effect, which peaked at 120 min, probably due to specific pharmacokinetic parameters.
In our tested essential oils samples, terpenoids were the most important molecules, being very diverse from a structural point of view. Thus, in the essential oil from M. piperita pallescens (EOMPA), menthol, a monocyclic alcohol was the predominant constituent. In the essential oil from M. spicata (EOMSP), carvone, a monoterpene with ketonic function, was the majoritary compound, while in M. suaveolens (EOMSU), piperitenone-oxide, also a monoterpene, but with an epoxy group, was the main active constituent. The functional groups of these terpenic essential oil constituents can influence not only their chemical properties but also the pharmacological interactions with their molecular targets [29].
According to Galeotti et al., the antinociceptive effect of the essential oils in the hot plate test can be partially attributed to the high content of menthol which can directly stimulate opioid receptors, (-)-menthol having superior analgesic effects [30]. Additionally, Gaudioso et al. found that menthol can also produce a use-dependent block of the Na + channels from the dorsal root ganglion neurons with a subsequent pain modulation [31]. Although these studies indicate a central mechanism of action, Sun et al. suggested also that menthol, present in high concentrations in the essential oil from M. piperita grown in China, may inhibit PGE2 production with potent anti-inflammatory effects in vitro and in vivo [32].
Carvone, another important component present in the essential oils extracted from Mentha species, act mainly by peripheral mechanisms with a reduction of prostaglandin synthesis and inhibition of Nf-κB intracellular signaling, with subsequent anti-inflammatory and antinociceptive effects [33]. De Sousa et al. found that carvone and pulegone showed superior antinociceptive effects in the acetic acid-induced writhing test in mice, compared to piperitenone-oxide (rotundifolone) [34]. In another study, piperitenone-oxide was tested for its antinociceptive effects at doses of 10, 100, and 200 mg/kg, in the hot plate, writhing and tail-flick test, producing moderate effects only at the highest doses [35]. The majority of the experimental models used to study the anti-inflammatory and antinociceptive effects of terpenoids focused on acute inflammation, further research being needed to ascertain the validity of these findings in chronic inflammatory processes.
Our experimental study showed that the most significant anti-inflammatory and antinociceptive effects were produced by the essential oil from M. spicata (EOMSP), followed by the essential oil from M. piperita (EOMPA) and the essential oil from M. suaveolens (EOMSU) which showed only modest effects due to a different chemical composition.
According to our data, a high content of carvone and menthol could be responsible for the anti-inflammatory and antinociceptive effects, the amplitude of these effects depending on their concentration in the essential oil samples. However, due to the fact that essential oils are complex mixtures containing also other classes of molecules, it is possible that the pharmacological activity can be modulated by other minor components. Further research is needed to clarify the molecular mechanism of action of essential oils. For the isolation of essential oils, fresh leaves from each of the selected Mentha species were placed in a Clevenger apparatus and submitted to a hydrodistillation for 3 h. The yield of essential oil extraction was 0.98 mL/100 g herbal product for Mentha piperita pallescens, 1.03 mL/100 g herbal product for Mentha spicata, and 0.87 mL/100 g herbal product for Mentha suaveolens. Anhydrous sodium sulfate was added to the resulting organic phase in order to eliminate water traces, and the essential oils were then stored in sealed dark glass recipients at 4 • C. The GC-MS analysis and biological tests were performed on samples from the same batch for each of the tested Mentha species.

GC-MS Analysis of the Essential Oils
Qualitative and quantitative analysis of the essential oils isolated from the selected Mentha species was performed by gas chromatography coupled with mass spectrometry [36]. The gas chromatograph (Agilent Technologies model 7890A, Santa Clara, CA, USA) was equipped with a HP-5MS capillary column (phenyl-methyl-siloxane30 m × 250 µm × 0.25 µm, Agilent 19091S-433). Each diluted essential oil sample (1/100 in n-hexane, v/v) was injected in 1 µL volume, in split mode. The flow rate was 16 mL/min and the split ratio was 1:15. The oven temperature was linearly programmed from 60 • C to 240 • C (at rate of 3 • C/min) and then held for 10 min at the last temperature. The carrier gas was helium with a head pressure of 8.2317 psi. The mass spectrometer (Agilent Technologies model 5975C) operated in electron impact (EI) mode at 70 eV with an ion source temperature set at 250 • C. Mass spectra were acquired with the detector operating in scan mode, in 50-550 m/z range.
The retention index (RI) was calculated for all the volatile constituents using an n-alkane homologous series, using a linear temperature programmed equation [37]. The identification of essential oil components was performed by comparison of retention indices and mass spectra fragmentation patterns with the Willey and NIST database (6th ed.) as previously described [38]. ChemStation software (Agilent Technologies) was used for data analysis. The relative proportion of each individual component (%) was expressed as percent peak area relative to total peak area from the GC-MS analysis of the whole sample. The relative proportions of the components were presented as mean ± SD of three GC-MS analysis of each sample.

Animals
For the pharmacological experiments, 11 groups of male Charles River Wistar (Crl:WI) rats (n = 6) with a mean weight of 200 g and 22 groups of male Swiss albino mice (n = 6) with a mean weight of 30 g were obtained from the Practical Skills and Experimental Medicine Centre of the Iuliu Haţieganu University of Medicine and Pharmacy Cluj-Napoca (Romania). The animals were housed in polycarbonate type IV-S open-top cages (Tecniplast, Italy) and maintained under standard conditions (22 ± 2 • C, a relative humidity of 45% ± 10%, 12:12-h light:dark cycle). The animals had access to a standard pelleted feed (Cantacuzino Institute, Bucharest, Romania) and filtered water ad libitum throughout the experiment, except for the day when the test substances were administered. All experimental protocols were approved by the Ethics Committee of the Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania, and were conducted in accordance with the EEC Directive 63/2010, which regulates the use of laboratory animals for scientific purposes.

Anti-Inflammatory Activity
The anti-inflammatory activity of the essential oils from the selected species of Mentha was determined by the rat paw edema test induced by λ-carrageenan, according to the method of Winter et al. modified by Griesbacher et al. after the introduction of a commercially available plethysmomether [39][40][41]. Thus, the essential oils from M. piperita pallescens (EOMPA), M. spicata (EOMSP), and M. suaveolens (EOMSU), after being solubilized in the vehicle (1% Tween 80 aqueous solution), were orally administered to 9 groups of Crl:WI rats (n = 6) in three different doses-125 mg/kg bw, 250 mg/kg bw, and 500 mg/kg bw-one hour before the induction of inflammation. Rats in the control group were orally treated with the vehicle (10 mL/kg), while rats in the reference group received, also orally, 20 mg/kg bw diclofenac (Gerot Lannach GmbH, Lannach, Austria), a non-steroidal anti-inflammatory drug. Inflammatory edema was induced by a single injection of 0.1 mL of 1% λ-carrageenan (Sigma Aldrich, St. Louis, MO, USA) into the subplantar region of the left hind paw of each rat. The paw volume of each animal was determined before carrageenan injection and at 1, 2, 3, and 4 h after the induction of inflammation with a digital plethysmometer (model 7140, Ugo Basile, Varese, Italy). Edema volume and the percentage of edema inhibition were calculated as follows: where Vo is the mean paw volume before carrageenan injection, Vt is the mean paw volume at "t" hours, Et is mean edema volume in treated animals, and Ec is mean edema volume in the control group.

Acetic Acid Induced Writhing Test
The antinociceptive effect of the essential oils from the selected species of Mentha was evaluated by the writhing test in mice, using 1% (v/v) acetic acid solution administered intraperitonealy to induce abdominal constrictions [42]. Initially, the essential oils from M. piperita pallescens (EOMPA), M. spicata (EOMSP), and M. suaveolens (EOMSU), after being solubilized in the vehicle (1% Tween 80 aqueous solution), were orally administered to 9 groups of male Swiss mice (n = 6) in three different doses: 125 mg/kg bw, 250 mg/kg bw, and 500 mg/kg bw, orally, by gastric intubation. The mice in the control group (n = 6) were treated orally with the vehicle (10 mL/kg). The animals from the reference group (n = 6) were treated orally with an anti-inflammatory drug, diclofenac 20 mg/kg bw. After 30 min, all mice were injected intraperitoneally with 0.1 mL of 1% acetic acid solution, in order to induce abdominal constrictions (writhes). The animals were placed in an observation box, the writhes being counted over a period of 20 min. For scoring purposes, a writhe was indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb. The analgesic activity was evaluated by calculating the percentage of inhibition of the writhes with the formula: % inhibition = (mean no. of writhes for control group − mean no. of writhes for treated group) × 100/mean no. of writhes for control group.

Hot Plate Test in Mice
The antinociceptive effect of the essential oils from the selected species of Mentha was also evaluated by the hot plate test in mice [43], using a digital Hot/Cold Plate (model 35100, Ugo Basile Varese, Italy). Each mouse was initially placed on the plate heated at 55 • C in order to observe its pain responses (hind paw licking or jumping). The time (in seconds) needed for the development of this pain response was recorded by the device for each individual animal. The mice exhibiting response times shorter than 5 s or longer than 30 s were excluded from the study. Afterwards, the essential oils from M. piperita pallescens (EOMPA), M. spicata (EOMSP), and M. suaveolens (EOMSU), after being solubilized in the vehicle (1% Tween 80 aqueous solution), were orally administered to 9 groups of male Swiss mice (n = 6) in three different doses: 125 mg/kg bw, 250 mg/kg bw, and 500 mg/kg bw, orally, by gastric intubation. The mice in the control group (n = 6) were treated orally with the vehicle (10 mL/kg). The animals from the reference group (n = 6) were treated orally with a centrally acting antinociceptive drug, morphine, at 10 mg/kg bw. The evaluation of the response times was repeated for each individual animal at 30 min, 60 min, 90 min, and 120 min from the substance administration.
Percent analgesic score (PAS) was calculated for each group at all time intervals as: PAS = (Tf − Ti)/Tf × 100 where Tf = response time (in seconds) after drug administration, and Ti = response time (in seconds) before drug administration.

Statistical Analysis
Data were expressed as mean values ± SD and were statistically analyzed by one-way ANOVA method. The differences between the treated groups and the control group were evaluated by Dunnett's t-test, p-values ≤ 0.05 being considered statistically significant.

Conclusions
This research showed a menthol chemotype for M. piperita pallescens, a carvone chemotype for M. spicata and a piperitenone oxide chemotype for M. suaveolens, cultivated in Romania.
Our experimental results showed that the essential oil from M. spicata L. (EOMSP) with the particular chemical composition presented in the study had significant and dose-dependent anti-inflammatory and antinociceptive properties. Since the essential oil composition can be influenced by a variety of factors, further research is necessary to demonstrate if these promising results can be extrapolated to a wider variety of M. spicata samples, under different environmental conditions.