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molecules
Article
A Comparative Analysis of the Chemical
Composition, Anti-Inflammatory, and
Antinociceptive Effects of the Essential Oils from
Three Species of Mentha Cultivated in Romania
Cristina Mogosan 1, Oliviu Vostinaru 1 ,*, Radu Oprean 2, Codruta Heghes 3, Lorena Filip 4,
Georgeta Balica 5and Radu Ioan Moldovan 6
1Department of Pharmacology, Physiology and Physiopathology, Iuliu Hatieganu University of Medicine
and Pharmacy, Cluj-Napoca 400349, Romania; cmogosan@umfcluj.ro
2Department of Analytical Chemistry, Iuliu Hatieganu University of Medicine and Pharmacy,
Cluj-Napoca 400349, Romania; roprean@umfcluj.ro
3
Department of Drug Analysis, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca 400349,
Romania; cmaier@umfcluj.ro
4Department of Bromatology, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca 400349,
Romania; lorenafilip@yahoo.com
5Department of Pharmaceutical Botany, Iuliu Hatieganu University of Medicine and Pharmacy,
Cluj-Napoca 400337, Romania; bgeorgeta@umfcluj.ro
6SC Fares Biovital Laboratories SRL, Orastie 335700, Romania; cercetare@fares.ro
*Correspondence: oliviu_vostinaru@yahoo.com; Tel.: +40-74-118-5163
Academic Editor: Thomas J. Schmidt
Received: 15 November 2016; Accepted: 6 February 2017; Published: 10 February 2017
Abstract:
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.
Keywords: Mentha spp.; menthol; carvone; piperitenone oxide; anti-inflammatory; antinociceptive
1. 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
Molecules 2017,22, 263; doi:10.3390/molecules22020263 www.mdpi.com/journal/molecules
Molecules 2017,22, 263 2 of 11
of strains with high contents of menthol, menthone, carvone, linalool, or other valuable terpenoid
components synthesized by the mevalonic acid pathway [
4
]. Essential oils from Mentha species
are widely used in food and beverage or cosmetic industries due to their flavoring properties [
5
].
Since ancient times, Mentha species have been used in traditional medicine for their carminative and
antispasmodic properties in a variety of disorders of the gastro-intestinal tract or cholecystopathies [
6
].
More recently, several pre-clinical studies which investigated the pharmacological effects of the
active constituents from Mentha species, demonstrated significant antimicrobial, antifungal, and
antiviral activities [
7
–
9
], strong antioxidant and anticancer actions [
10
,
11
], but also antinociceptive,
anti-inflammatory, and antiallergic properties [
12
–
14
]. Thus, evidence-based research demonstrated
that Mentha species can be used as complementary or alternative remedies in a variety of pathologic
conditions [
15
]. Due to their economic importance, extended cultures of Mentha species can be found
in various climatic regions, in Europe, North-America or Asia [16].
In Romania, three species of Mentha are frequently cultivated. Mentha piperita L. var. pallescens
(white peppermint) was among the first cultivated medicinal and aromatic plant, an experimental
culture being created in 1908 [
17
]. Mentha spicata L. subsp. crispata (spearmint) is a creeping
rhizomatous and perennial herb present in the spontaneous flora from the Balkan Peninsula but
also under cultivation. Mentha suaveolens Ehrh. var. variegata (pineapple mint) is used as an ornamental
plant but also in traditional medicine of Mediterranean areas, being extensively cultivated in Southern
Europe [18]. It has an extremely variable chemotype, creating confusion between species.
Although some Mentha species were extensively studied, there is little information regarding
the anti-inflammatory and antinociceptive properties of their essential oils, the major constituents.
This original work is aimed at correlating the chemotype of three Mentha species from Romania with
an
in vivo
study of the anti-inflammatory and antinociceptive effects, with the purpose of exploring
potential benefits of essential oils in the treatment of various inflammatory conditions.
2. Results and Discussion
2.1. GC-MS Analysis of the Essential Oils
The GC-MS analysis of the essential oils isolated from the selected species of Mentha revealed the
presence of over 30 compounds, mainly with terpenoid structures. Their retention indices and relative
proportions in the studied samples are listed in Table 1.
Table 1. Chemical composition of the essential oils from the selected Mentha species.
No. Compound RIlit aRIcal b% in EOMPA c% in EOMSP d% in EOMSU e
1 alpha-pinene 939 950 0.133 ±0.03 0.220 ±0.05 0.791 ±0.12
2 sabinene 971 980 0.071 ±0.00 0.136 ±0.03 0.342 ±0.08
3 beta-pinene 976 983 0.232 ±0.04 0.471 ±0.19 1.497 ±0.42
4 myrcene 990 994 0.119 ±0.02 0.095 ±0.00 0.462 ±0.11
5 2-octanol 997 997 0.617 ±0.18 0.201 ±0.06 0.305 ±0.07
6 para-cymene 1022 1025 0.292 ±0.11 0.140 ±0.02 0.230 ±0.05
7 limonene 1027 1028 0.346 ±0.09 1.569 ±0.48 2.969 ±1.02
8 1,8-cineole 1029 1031 1.587 ±0.64 2.567 ±0.69 0.118 ±0.04
9 gamma-terpinene 1057 1058 0.407 ±0.05 0.106 ±0.00 -
10 menthone 1151 1155 15.742 ±2.73 7.225 ±1.81 -
11 isomenthone 1162 1165 7.735 ±2.03 3.325 ±0.82 -
12 menthol 1170 1177 39.695 ±3.26 12.774 ±2.48 0.128 ±0.03
13 terpineol 4 1175 1179 2.182 ±0.55 1.221 ±0.33 0.679 ±0.19
14 isomenthol 1181 1184 0.493 ±0.14 0.192 ±0.04 -
15 alpha-terpineol 1188 1191 0.449 ±0.17 0.622 ±0.21 0.246 ±0.04
16 dihydrocarveol 1192 1195 0.120 ±0.02 1.120 ±0.24 -
17 estragole 1195 1199 0.929 ±0.18 - -
18 trans-carveol 1216 1219 0.085 ±0.00 1.113 ±0.47 -
19 cis-carveol 1228 1232 - 1.221 ±0.31 -
20 pulegone 1235 1239 2.140 ±0.80 3.763 ±1.04 -
21 carvone 1240 1244 2.377 ±0.73 41.215 ±4.18 1.555 ±0.44
22 piperitone 1250 1254 2.096 ±0.75 0.647 ±0.13 0.340 ±0.08
23 neomenthyl acetate 1274 1276 0.135 ±0.02 0.101 ±0.00 -
24 trans-anethole 1282 1286 5.374 ±0.94 0.113 ±0.02 -
Molecules 2017,22, 263 3 of 11
Table 1. Cont.
No. Compound RIlit aRIcal b% in EOMPA c% in EOMSP d% in EOMSU e
25 menthyl-acetate 1294 1295 3.022 ±1.21 1.912 ±0.89 -
26 menthylcamphor - 1300 0.478 ±0.17 0.497 ±0.12 0.498 ±0.15
27 eugenol 1354 1357 0.214 ±0.05 0.184 ±0.03 -
28 piperitenone oxide 1366 1366 - - 73.773 ±6.41
29 beta-bourbonene 1383 1384 0.222 ±0.04 0.951 ±0.27 0.293 ±0.05
30 cis-jasmone 1395 1399 - - 2.124 ±0.65
31 caryophyllene 1418 1418 0.112 ±0.03 2.289 ±0.99 0.604 ±0.23
32 germacrene-d 1479 1480 - - 3.309 ±1.19
33 viridoflorol 1592 1590 - - 1.455 ±0.68
a
Retention indices from literature.
b
Calculated retention indices.
c
EOMPA: essential oil from M. piperita pallescens.
d
EOMSP: essential oil from M. spicata.
e
EOMSU: essential oil from M. suaveolens. Relative proportions are expressed
as mean ±SD of three GC-MS analysis of each sample.
Thus, in the essential oil from M. piperita pallescens (EOMPA), the major compounds were menthol
(39.695%
±
3.26%), menthone (15.742%
±
2.73%), and isomenthone (7.735%
±
2.03%). Our data
also showed the presence of estragole (0.929%
±
0.18%) in EOMPA, but the low content complies
with the European Medicines Agency recommendations regarding the use of herbal medicinal products
containing estragole [
19
]. In the essential oil from M. spicata (EOMSP) carvone (41.215%
±
4.18%) was the
major compound, followed by menthol (12.774%
±
2.48%), menthone (7.225%
±
1.81%), and pulegone
(
3.763% ±1.04%
). In the essential oil from M. suaveolens (EOMSU) piperitenone oxide (73.773%
±
6.41%)
was the major compound followed by germacrene-d (3.309% ±1.19%) and limonene (2.969% ±1.02%).
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].
2.2. 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.
Table 2.
Effect of the essential oils from the selected Mentha species on carrageenan-induced rat
paw edema.
Group Dose
Edema 1 h (mL) Edema 2 h (mL) Edema 3 h (mL) Edema 4 h (mL)
(% inhib.) (% inhib.) (% inhib.) (% inhib.)
Control (vehicle) - 0.56 ±0.11 1.30 ±0.13 2.00 ±0.20 2.34 ±0.27
EOMPA 500 mg/kg 0.36 ±0.20 0.96 ±0.40 1.40 ±0.64 1.12 ±0.84 *
(35.71%) (25.15%) (30.00%) (52.13)
EOMPA 250 mg/kg 0.42 ±0.04 1.11 ±0.53 1.69 ±0.73 1.80 ±0.46
(25.00%) (14.61%) (15.50%) (23.07%)
EOMPA 125 mg/kg 0.50 ±0.15 1.21 ±0.66 1.81 ±0.98 2.03 ±1.18
(10.71%) (6.92%) (9.50%) (13.24%)
EOMSP 500 mg/kg 0.36 ±0.17 0.52 ±0.28 * 0.74 ±0.26 * 0.88 ±0.26 *
(35.71%) (60.00%) (63.00%) (62.39%)
EOMSP 250 mg/kg 0.44 ±0.27 0.78 ±0.19 * 1.23 ±0.46 1.54 ±0.86
(21.42%) (40.00%) (38.50%) (34.18%)
Molecules 2017,22, 263 4 of 11
Table 2. Cont.
Group Dose
Edema 1 h (mL) Edema 2 h (mL) Edema 3 h (mL) Edema 4 h (mL)
(% inhib.) (% inhib.) (% inhib.) (% inhib.)
EOMSP 125 mg/kg 0.50 ±0.37 1.03 ±0.69 1.68 ±1.13 2.01 ±0.64
(10.71%) (20.76%) (16.00%) (14.10%)
EOMSU 500 mg/kg 0.54 ±0.35 0.98 ±0.64 1.23 ±0.89 1.41 ±0.78
(3.57%) (24.61%) (38.50%) (39.74%)
EOMSU 250 mg/kg 0.63 ±0.22 1.20 ±0.80 1.56 ±0.73 1.84 ±0.98
(-) (7.69%) (22.00%) (21.36%)
EOMSU 125 mg/kg 0.70 ±0.31 1.42 ±0.69 1.79 ±0.46 2.13 ±1.36
(-) (-) (10.5%) (8.9%)
Diclofenac 20 mg/kg 0.36 ±0.06 * 0.55 ±0.11 * 0.68 ±0.08 * 1.06 ±0.17 *
(35.71%) (57.69%) (66.00%) (54.70%)
* Statistically significant, p≤0.05. Values are expressed as Mean ±SD.
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.
2.3. Antinociceptive Activity of the Essential Oils
2.3.1. 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.
Table 3.
Effect of the essential oils from the selected Mentha species in the acetic acid induced writhing
test in mice.
Group Dose (mg/kg) No. of Writhes (X ±SD) Percentage of Inhibition (%)
Control (vehicle) - 32.4 ±10.19 -
EOMPA 500 mg/kg 20.8 ±4.25 35.80
EOMPA 250 mg/kg 23.6 ±7.22 27.16
EOMPA 125 mg/kg 25.6 ±5.15 20.98
EOMSP 500 mg/kg 17 ±7.87 * 47.53
EOMSP 250 mg/kg 18.8 ±9.23 * 41.97
EOMSP 125 mg/kg 19.8 ±6.60 * 38.88
EOMSU 500 mg/kg 24.2 ±2.56 25.30
EOMSU 250 mg/kg 28 ±2.89 13.58
EOMSU 125 mg/kg 30.6 ±2.05 5.55
Diclofenac 20 mg/kg 12.8 ±4.12 * 60.49
* Statistically significant, p≤0.05. Values are expressed as Mean ±SD.
Molecules 2017,22, 263 5 of 11
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-1
β
, 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.
2.3.2. 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.
Table 4. Effect of the essential oils from the selected Mentha species in the hot plate test in mice.
Group Response (s) at
0 min (PAS)
Response (s) at
30 min (PAS)
Response (s) at
60 min (PAS)
Response (s) at
90 min (PAS)
Response (s) at
120 min (PAS)
Control (Vehicle) 8.78 ±2.18 9.12 ±1.94 8.19 ±1.53 7.85 ±1.80 7.13 ±1.96
(-) (-) (-) (-) (-)
EOMPA 500 mg/kg 9.05 ±1.76 15.89 ±2.27 20.98 ±6.17 * 23.44 ±4.72 * 25.12 ±8.47 *
(-) (43.04%) (56.86%) (61.39%) (63.97%)
EOMPA 250 mg/kg 10.34 ±2.20 13.44 ±3.67 17.39 ±4.99 * 19.89 ±4.63 21.27 ±5.46 *
(-) (23.06%) (40.54%) (48.01%) (51.38%)
EOMPA 125 mg/kg 9.42 ±1.96 11.29 ±2.16 14.52 ±4.21 17.11 ±3.92 18.41 ±4.52
(-) (16.56%) (35.12%) (44.94%) (48.83%)
EOMSP 500 mg/kg 9.84 ±1.98 18.23 ±3.76 24.75 ±6.71 * 27.42 ±6.06 * 28.44 ±4.77 *
(-) (46.02%) (60.24%) (64.11%) (65.40%)
EOMSP 250 mg/kg 10.31 ±0.84 14.97 ±3.70 19.42 ±4.63 22.93 ±6.35 * 24.03 ±6.15 *
(-) (31.12%) (46.91%) (55.03%) (57.09%)
EOMSP 125 mg/kg 9.24 ±1.87 12.19 ±3.90 15.55 ±3.09 * 18.32 ±4.57 * 20.84 ±5.82 *
(-) (24.20%) (40.57%) (49.56%) (55.66%)
EOMSU 500 mg/kg 10.11 ±3.09 13.42 ±3.23 18.11 ±2.04 20.75 ±4.54 * 21.04 ±6.64 *
(-) (24.66%) (44.17%) (51.27%) (51.94%)
EOMSU 250 mg/kg 9.19 ±1.96 11.41 ±2.74 15.14 ±4.41 17.02 ±5.15 16.14 ±3.90
(-) (19.45%) (39.29%) (46.00%) (43.06%)
EOMSU 125 mg/kg 10.04 ±2.11 10.11 ±1.76 13.45 ±2.98 14.25 ±4.50 13.89 ±3.70
(-) (6.9%) (25.35%) (29.54%) (27.71%)
Morphine 10 mg/kg 10.23 ±2.92 11.50 ±4.21 35.63 ±7.58 * 38.72 ±6.64 * 37.45 ±6.77 *
(-) (11.04%) (71.28%) (73.57%) (72.68)
* Statistically significant, p≤0.05. Values are expressed as Mean ±SD.
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,
Molecules 2017,22, 263 6 of 11
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-
κ
B 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.
3. Materials and Methods
3.1. Plant Material and Essential Oil Isolation
The selected Mint species were Mentha x piperita L. var. officinalis Sole f. pallescens Camus (white
peppermint), Mentha spicata L. subsp. crispata (spearmint), and Mentha suaveolens Ehrh. var. variegata
(pineapple mint).
The plants were harvested in flowering phase from experimental fields of the Fares BioVital
Laboratories Orastie (Hunedoara County, Romania, Latitude: 45
◦
49.9998
0
N Longitude: 23
◦
12
0
E) in
July 2015. The experimental cultures of the three Mentha species were created in a randomized complete
block design with four replications. The plants were planted 10 cm apart in 70 cm rows to rows, in
Molecules 2017,22, 263 7 of 11
a clay sandy soil. The plants received normal inter-cultural operations and irrigation. The average
day temperature during summer season was 20
◦
C. After harvesting, voucher specimens (No. 1516,
No. 1517 and No. 1518) were deposited in the Herbarium of the Department of Pharmaceutical Botany
from the Faculty of Pharmacy, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca,
Romania. 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.
3.2. 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
µ
m
×
0.25
µ
m, Agilent 19091S-433). Each diluted essential
oil sample (1/100 in n-hexane, v/v) was injected in 1
µ
L 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/zrange.
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.
3.3. 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 Ha¸tieganu 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.
3.4. 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
–
41
].
Thus, the essential oils from M. piperita pallescens (EOMPA), M. spicata (EOMSP), and M. suaveolens
Molecules 2017,22, 263 8 of 11
(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:
Edema volume (mL) = Vt −Vo
Inhibition of edema (%) = [1−(Et/Ec) ×100]
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.
3.5. Antinociceptive Activity
3.5.1. 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.
3.5.2. 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
Molecules 2017,22, 263 9 of 11
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.
3.6. 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.
4. 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.
Author Contributions:
C.M., O.V., and R.I.M. conceived and designed the experiments; C.M. and O.V. performed
the pharmacological experiments, analyzed the data and wrote the paper; G.B. identified and prepared the vegetal
material; R.O., C.H., L.F., and R.I.M. performed the GC-MS analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the essential oils are available from the authors.
©
2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
