Heliyon 10 (2024) e28125

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Research article

Quality criteria, chemical composition and antimicrobial activity

of the essential oil of Mentha suaveolens Ehrh
Moha Afrokh a, b, Khadija El Mehrach b, Khalid Chatoui a, Mohamed Ait Bihi b,
Hajar Sadki b, Abdelkader Zarrouk a, Mohamed Tabyaoui a, Saida Tahrouch b, *
a
Laboratory of Materials, Nanotechnology and Environment, Faculty of Sciences, Mohammed V University in Rabat, Av. Ibn Battouta, P. O. Box
1014 Agdal-Rabat, Morocco
b
Laboratoryof Plant Biotechnology, Faculty of Sciences, Ibn Zohr University, Box.8106, Agadir, Morocco

A R T I C L E I N F O A B S T R A C T

Keywords: The aim of the present study is the valorization of the essential oil of Mentha suaveolens Ehrh. The
Mentha suaveolens Ehrh research plan and methods included 3-axis: the first axis consists of studying the organoleptic and
Essential oil physicochemical characterization of the essential oil, the second is the chemical analysis carried
Organoleptic and physicochemical
out by Gas Chromatography/Mass Spectrometry (GC/MS) and the third consists of evaluating its
characterization
Antimicrobial activity
antimicrobial activity against selected microorganisms. The results obtained for the organoleptic
Oxygenated monoterpenes and physicochemical properties are as follows: appearance: Liquid, mobile and clear, odor: Strong
GC/MS analysis odor characteristic of Mentha suaveolens Ehrh, color: Pale yellow; relative density (0.92), misci­
bility with ethanol (1V/2V), freezing point (Tfreezing < − 10 ◦ C), refractive index (1.5256), rotating
power (+0.825), acid index (1.68), ester index (68.44), saponification index (70.13) and iodine
index (12.05).Chemical analysis identified 69 compounds which are mostly oxygenated mono­
terpenes such as piperitenone oxide (32.55%), pulegone (10.14%), piperitone oxide (8.34%), etc.
The microbiological tests were carried out by an agar diffusion test using the essential oil of
Mentha suaveolens Ehrh. The microbiological tests were carried out by a diffusion test on agar,
these tests are carried out on six microbial strains (five bacteria and one yeast).The inhibitory
effect of our oil is well marked against bacteria: Proteus mirabilis (17.50 ± 0.70 mm at 50 μL/mL),
Enterococcus faecalis (17.00 ± 1.00 mm at 50 μL/mL) and Staphylococcus coagulase negative (16.33
± 0.57 mm at 50 μL/mL) while it was moderate against Escherichia coli (14.33 ± 1.15 mm at 50
μL/mL) and Streptococcus spp (13.00 ± 0.00 mm at 50 μL/mL) as well as against yeast, Candida
albicans (15.33 ± 1.52 mm at 50 μL/mL). It appears from these results that our oil is of high
quality and can be used in several areas. The results obtained are therefore promising and thus
open the way for manufacturers to use this essential oil of Mentha suaveolens Ehrh in the phar­
maceutical, cosmetic, agricultural and food industries.

1. Introduction

Aromatic and medicinal plants have been known and used since the dawn of humanity for their cosmetic virtues and their various

* Corresponding author.
E-mail addresses: afrokhmoha@[Link] (M. Afrokh), [Link]@[Link] (K. El Mehrach), chatouikhalid@[Link] (K. Chatoui),
hajarsadki38@[Link] (H. Sadki), azarrouk@[Link] (A. Zarrouk), tabyaouihamid@[Link] (M. Tabyaoui), [Link]@[Link]
(S. Tahrouch).

[Link]
Received 29 April 2023; Received in revised form 11 March 2024; Accepted 12 March 2024
Available online 19 March 2024
2405-8440/© 2024 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
([Link]
M. Afrokh et al. Heliyon 10 (2024) e28125

therapeutic properties [1]. The family Lamiaceae (Labiatae) from the Latin labia meaning that the flowers have a characteristic
two-lipped shape, it includes nearly 700 species divided into 250 genera. The majority of these plants have medicinal properties and
constitute an important source of essential oils (EO) [2]. The essential oils of these plants all have a common characteristic: rich in
terpene constituents [3]. These species contain major groups of economically important secondary metabolites and can be used as an
ingredient in many formulations [4], Flavonoids, polyphenols, alkaloids, glycosides, terpenoids, and sesquiterpenes are the primary
metabolic chemicals that possess diverse antimicrobial, antifungal, cytotoxic, and antioxidant characteristics [5].
The genus Mentha is an important member of this family, and it is represented in Morocco by five main species: Mentha pulegium L.,
Mentha aquatica L., Mentha longifolia L., Mentha arvensis L. and Mentha suaveolens Ehrh [6]. Mentha suaveolens Ehrh, formerly named
Mentha rotundifolia (L) Hudson [7]is native to Africa, temperate Asia and Europe [8], it is known in Morocco under the name of
“Merssita” or “Timijja” [6]. It is a perennial herb that finds habitats in semi-arid and sub-humid zones along rivers [9]. These plants
have several therapeutic benefits, including analgesic, antispasmodic, sedative, appetizing, stimulant, tonic, antiviral, anticonvulsant
[10], anti common digestive and respiratory disorders [2]. Furthermore, the pharmacological and biological characteristics of this
variety’s essential oil include anti-inflammatory, antibacterial, antioxidant, anticancer, and antidiabetic actions [11]. In view of these
benefits of this plant, it is necessary to find solutions to the changing environmental conditions that generate abiotic stresses such as
exposure to chemicals and biotic stress [12].Thus, to increase the sources of benefits for human health and obtain significant results
[13].The chemical composition of the essential oil of African MS [14], has revealed its richness in menthone, pulegone, germacrene D,
borneoland piperitenone endowed with insecticidal, antibacterial and antifungal properties [15].
The objective of this study is the valorization of this plant, which grows in the region of Er-rich, south eastern of Morocco, by the
organoleptic and physicochemical characterization of its essential oil obtained by hydrodistillation. The chemical analysis carried out
by Gas Chromatography/Mass Spectrometry and the antimicrobial activity of this essential oil has been investigated against the
bacteria Enterococcus faecalis, Proteus mirabilis, streptococcus spp, Escherichia coli, Staphylococcus coagulase negative and the fungi:
Candida albicans.

2. Material and methods

2.1. Plant material

Mentha suaveolens Ehrh (MS) was collected near the town of Er-rich, located 65 km from the province of Errachidia (Latitude: N: 32◦
15′33.691″, Longitude: O: 4◦ 29′43.544″ and Altitude: 1321 m) in full bloom during September.

2.2. Kinetics study of the extraction

The distillation was carried out with a recycling commonly called cohesion as described in the European Pharmacopoeia. Samples
were taken every hour on the aqueous and organic phases and the hydrodistillation kinetics were measured. The hydrodistillation of
MS was performed using a Clevenger-type device. The distillation was carried out with a recycling commonly called cohesion as
described in the European Pharmacopoeia. This system makes possible to take hourly samples at regular intervals on the aqueous and
organic phases, which are intended for measuring hydrodistillation kinetics. The essential oil (EO) obtained was dehydrated using
anhydrous sodium sulphate before yield calculating. Afterward, it was stored in brown glass bottles sealed to protect it from air and
light at a temperature of 4 ◦ C.

2.3. Chromatographic analysis

2.3.1. Gas chromatographic analysis (GC-FID)

The PerkinElmer Autosystem (Whaltam, MA, USA) gas chromatograph (GC) with dual flame ionization (FID) detection was utilized
to analyze the samples. It was fitted with fused silica capillary columns (60 m × 0.22 mm internal diameter, 0.25 μm film thickness)
and various stationary phases. Rtx-Wax (polyethylene glycol) and Rtx-1 (polydimethylsiloxane). The oven was set to increase the
temperature by 2 ◦ C each minute from 60 to 230 ◦ C, and it was then maintained at that temperature isothermally for 35 min. At 280 ◦ C,
the temperatures of the injector and detector remained unchanged. Using helium as the carrier gas at a flow rate of 1 mL/min and an
injection volume of 0.2 μL of pure oil, samples were injected fractionally (1/50). By utilizing PerkinElmer software, linear interpolation
based on the Van den Dool and Kratz equation, and comparing the retention periods with those of a series of linear alkanes (C5–C30)
(Restek, Lisses, France), the retention indices (RIs) of the compounds were ascertained.

2.3.2. Gas chromatography-mass spectrometry (GC-MS)

Rtx-1 and Rtx-wax fused silica capillary columns from the PerkinElmer Autosystem XL system were used in conjunction with a
PerkinElmer turbo (quadrupole) mass detector to analyze the samples. The carrier gas, helium, was employed at a flow rate of 1 mL/
min. The oven temperature was planned to rise from 60 ◦ C to 230 ◦ C at a rate of 2 ◦ C/min and then be isothermally maintained at
230 ◦ C for 35 min, while the ion source temperature was kept at 150 ◦ C. The injector was maintained at 280 ◦ C and 70 eV of ionization
energy. Electron ionization mass spectra were obtained using a fractionation of 1/80 and an injection volume of 0.2 μL of pure oil,
covering the mass range of 35–350 Da.

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M. Afrokh et al. Heliyon 10 (2024) e28125

2.3.3. Components identification

Two primary approaches were used to identify the components of essential oils: (i) comparing the mass spectra of genuine reference
compounds, if feasible, with those of mass spectra libraries like WILEY275, NIST 02, and Adams; and (ii) assessing their retention
indices (RIs), which were computed using linear interpolation with respect to the retention times of a series of C-5 to C-30 alkanes, and
comparing these data with our own library of genuine compounds or with reference data from literary works [16,17].

2.4. Quality control of the essential oil

2.4.1. Organoleptic properties

In this study the organoleptic characteristics such as color, physical appearance and odor of the essential oil of Mentha suaveolens
Ehrh (EOMS) were determined and evaluated.

2.4.2. Physical properties

[Link]. Relative density (NFT-75 111, 2000). The determination of the density of extracted oils was carried out using an Eppendorf
tube with a capacity of 1 mL. The volume taken was 0.20 mL for the essential oil, as well as for the distilled water. The relative density
is given by the following formula(1) [18]:
m1 − m0
d20 = (1)
m − m0

With:
m1: mass in grams of the Eppendorf containing 0.20 mL of the [Link]://[Link]/fr-xl/feed
m0: mass in grams of the empty Eppendorf.
m: mass in grams of the Eppendorf containing 0.20 mL of distilled water.

[Link]. Miscibility with ethanol (NFT-75 101, 2000). Ethanol is added, in 0.5 mL portionsto 0.5 mL of essential oil. After each
addition, the mixture is stirred. When the solution becomes clear, the volume of ethanol is noted [18].The miscibility with ethanol was
calculated.

[Link]. Freezing point (NFT-75 102, 2000). The essential oil was placed, in a freezer, with a thermometer, which allowed following
slowly and gradually the temperature accompanying the solidification of the oil [18].The freezing point was recorded by the
researcher.

[Link]. Rotatory power (NFT-75 113, 2000). Certain chemical compounds exhibit specific rotation [α]tD , which results in a deviation
of the polarization plane of polarized light. This deviation suggests the presence of an asymmetric carbon. The Rotatory power [α ]tD
measured at a temperature t, using the D-line of Sodium (Na) λ = 589 nm as a light source is expressed by Biot’s law(2):
α
[α ]tD = (2)
L.C

With:
α: Value of the deflection angle of the polarized light read on the polarimeter expressed in degrees.
L: Length of the cell expressed in dm.
C: Concentration of the test solution in g/100 mL.
In order to evaluate the rotation angles a branded polarimeter was used: ATAGO AP-300, equipped with a cell filled with an
ethanolic solution of essential oil at a rate of 0.20 g for the sample in 100 mL of solvent.
The instrument measures the observed degree of rotation directly, allowing us to calculate the rotatory power of our essence [18].

[Link]. Refractive index (NFT-75 112, 2000). The refractive index is the ratio between the sine of the angles of incidence and
refraction of a light ray of determined wavelength, passing air through the maintained essential oil at a constant temperature. It is
measured by means of a refractometer, into which a few drops of distilled water, which is considered as a standard on the prism, are
introduced. The device is set at 1.333. These drops are wiped and replaced by a few drops of EO, then read. The refractive index at
temperature t is given by the formula(3) [18]:

(3)
′
[n]tD = ntD + 0, 00045(t’ − t)

With:
ntD : is the reference refractive index
ntD : is the measured refractive index.
′

t: reference temperature which is at 20 ◦ C.

t’: temperature at the time of measurement.

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M. Afrokh et al. Heliyon 10 (2024) e28125

2.4.3. Chemical properties

[Link]. Acid index (NFT-75 103, 2000). The acid index is defined as the number of milligrams (mg) of potassium hydroxide (KOH)
required to neutralize the free acids contained in 1 g of oil.
One gram of essential oil was added in an Erlenmeyer flask with 5 mL of 95% ethanol and about 5 drops of phenolphthalein. The
mixture was then titrated with an alcoholic solution of 0.1 N potassium hydroxide (KOH) until the solution turns pink. The acid index
(Ia) is determined by the following formula(4) [18]:
56, 11
Ia = V.C. (4)
m

With:
56.11: is the molar mass, expressed in grams per mole, of potassium hydroxide.
V: volume in mL of the KOH solution used for the titration.
C: concentration in mol/L of the KOH solution.
m: mass in grams of the test sample.

[Link]. Ester index (NFT-75 104, 2000). The ester indexis the number of mg of potassium hydroxide KOH necessary to neutralize the
acids released by hydrolysis in a basic medium of the esters contained in 1 g of oil.
One gram of essential oil and 25 mL of an alcoholic solution of 0.5 M KOH potassium hydroxide were introduced into a 100 mL flask
using a burette, as well as some pumice stones. The mixture was refluxed for 1 h. After cooling the solution, 20 mL of distilled water and
3drops of phenolphthalein are added.
Excess KOH was titrated with a solution of 0.5 N hydrochloric acid HCl until the pink color disappears. A blank operation was
carried out under the same conditions with the same reagents, replacing the EO with 1 mL of distilled water. The ester index Ie was
calculated by the following formula(5) [18]:
28.05
Ie = (V0 − V1 ) − Ia (5)
m

With:
V0: volume in mL of the HCl solution (0.5 N), measured for the blank test.
V1: volume in mL of the HCl solution (0.5 N), measured for the calculation of Ie.
m: mass in grams of the test sample.
Ia: value of acid index.

[Link]. Saponification index (NFT 60–206, 1984). The saponification indexis the number of mg of potassium hydroxide (KOH)
needed to neutralize the free acids and saponify the esters contained in 1 g of the substance.
A quantity of 1 g of essential oil was placed in a flask equipped with a condenser, and 25 mL of 0.5 N ethanolic KOH were then
added. The mixture was first boiled with stirring for 1 h, then, 3drops of phenolphthalein were added. The soapy solution was titrated
with HCl (0.5 N). At the same time, a blank test was carried out under the same conditions. The saponification index was given by the
following formula (6) [19]:
56.11.N.(V0 − V)
Is = (mg KOH / g EO) (6)
m

With:
56.11: molar mass of KOH.
V0: volume in mL of the hydrochloric acid solution for the blank test.
V: volume in mLof the hydrochloric acid solution used for the test portion.
N: exact normality of the hydrochloric solution.
m: massof the test sample in grams.

[Link]. Iodine index (NFT 60–203, 1984). The iodine index is the mass of iodine in grams likely to be fixed by 100g of the substance.
Onegramof essential oil, 20 mL of carbon tetrachloride (CCl4) and 25 mL of a solution of iodine (1 N) prepared in carbon tetra­
chloride were put in an Erlenmeyer flask. The mixture was kept in darkness for 2 h, then 20 mL of a solution of potassium iodide (KI) at
50% and 20 mL of distilled water were added. Excess iodine is titrated with 0.1 N sodium thiosulfate pentahydrate solution in the
presence of starch paste. Under the same conditions as above, a blank test was performed. The iodine index was calculated according to
the following relation (7) [19]:
12.69.N.( V0 − V1 )
Ii = (7)
m

With:

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M. Afrokh et al. Heliyon 10 (2024) e28125

Table 1
Chemical constituents of Mentha suaveolens Ehrh essential oil, in per cent (%).
R Ta(min) Calc. Area % Name of compound

10,17 58433904 0,04 ethyl 2-methylbutyrate

13,76 925 59128192 0,04 α-thujene
14,22 932 1166624000 0,82 α-pinene
15,03 944 897449920 0,63 camphene
16,33 966 607782208 0,43 verbenene
16,44 967 142679184 0,10 octen-3-ol
16,62 968 1234428416 0,87 sabinene
17,21 976 839217920 0,59 β-pinene
17,78 999 56475908 0,04 myrcene
18,05 1004 65311732 0,05 α-phellandrene
18,83 1015 54050572 0,04 α-terpinene
19,34 1024 133822360 0,09 p-cymene
19,78 1030 4015885056 2,83 limonene
19,87 1032 267900944 0,19 1,8-cineole (eucalyptol)
20,00 1033 76219888 0,05 (Z)-ocimene
20,49 1042 67859096 0,05 phenyl acetaldehyde
20,66 1044 47633020 0,03 (E)-ocimene
21,45 1056 140807808 0,10 γ-terpinene
22,18 1066 298938816 0,21 p-mentha-1,8-diene
23,39 1084 254868448 0,18 p-mentha-2,4(8)-diene
24,22 1098 2481846016 1,75 linalool
24,34 1101 86026296 0,06 nonanal
26,59 1134 491053504 0,35 dihydrolinalool
27,83 1151 3722602752 2,62 isopulegol
28,10 1155 3512162304 2,48 menthone
28,19 1156 205595632 0,14 isopulegol
28,80 1166 3890161920 2,74 neomenthol
28,94 1168 2587858176 1,82 menthol
29,19 1172 2643717376 1,86 borneol
29,79 1180 5722162176 4,03 terpinen-4-ol + isopulegol
30,04 1184 1007960128 0,71 isomenthol
30,42 1188 1388312064 0,98 cryptone
30,66 1192 966480960 0,68 α-terpineol
32,80 1223 742011328 0,52 coahuilensol, methylether
34,32 1246 1,4383E+10 10,14 pulegone
35,38 1262 1,1826E+10 8,34 piperitone oxide
35,56 1264 472312736 0,33 carveol oxide cis
35,67 1266 227414352 0,16 geranial
36,11 1273 1384287232 0,98 neomenthyl acetate
36,86 1284 1042828800 0,74 isobornyl acetate
37,03 1286 119624336 0,08 N Ia
37,30 1291 580543104 0,41 bornyl acetate
37,75 1296 426778624 0,30 2,4,6-trimethyl-3-methoxycyclohex2-enone
38,08 1303 361723840 0,26 NI
38,27 1306 920536064 0,65 isomenthyl acetate
38,60 1310 1630003072 1,15 dihydrocarvyl acetate
38,69 1312 444966656 0,31 carvacrol
39,05 1332 260792704 0,18 3-hexenyl butyrate
40,67 1343 4339761664 3,06 piperitenone
43,72 1389 4,6163E+10 32,55 piperitenone oxide
44,04 1394 292077856 0,21 β-elemene
44,72 1404 1559553408 1,10 methyl eugenol
44,80 1406 1006666496 0,71 NI
44,94 1407 2117318912 1,49 NI
45,07 1410 198096160 0,14 α-gurjunene
45,37 1415 1308774272 0,92 NI
45,77 1420 764911040 0,54 (E)-caryophyllene
46,22 1428 121632616 0,09 β-copaene
46,99 1439 152734336 0,11 NI
47,24 1444 266732064 0,19 muurola-3,5-diene cis
47,50 1454 165154592 0,12 spirolechepinene
47,72 1457 119235400 0,08 α-humulene
48,14 1464 100236728 0,07 alloaromadendrene
48,25 1466 185453088 0,13 γ-muurolene
49,45 1484 2147368448 1,51 germacrene D
50,27 1497 475189024 0,34 bicyclogermacrene
51,24 1513 510703200 0,36 γ-cadinene
51,52 1519 158842432 0,11 NI
(continued on next page)

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M. Afrokh et al. Heliyon 10 (2024) e28125

Table 1 (continued )
R Ta(min) Calc. Area % Name of compound

51,68 1521 641318272 0,45 δ-cadinene

55,02 1577 397151840 0,28 spathulenol
55,19 1580 383340768 0,27 2-phenethyl tiglate
55,37 1581 213837168 0,15 caryophyllene oxide
57,07 1610 280433024 0,20 α-epi-cadinol
58,60 1636 2045257856 1,44 α-epi-muurolol
58,77 1639 200641952 0,14 α-muurolol (torreyol)
59,28 1658 626716480 0,44 α-cadinol
60,99 1675 98215776 0,07 NI
61,31 1693 115422128 0,08 NI
a
Rt: Retention time, N I: Not Identified.

N: normality of the solution of sodium thiosulfate pentahydrate.

V0: volume in mL of the sodium thiosulfate pentahydrate solution used for the blank test.
V1: volume in mL of the sodium thiosulfate solution used for the determination of Ii.
m: mass in grams of the test sample.

2.5. Assessment of antimicrobial activities

Microorganisms used in this study are pathogenic speciesobtained from Oued Noune Laboratory for Medical Biology Analysis,
Guelmim (Morocco) and consisted of five bacteria species namelyEnterococcus faecalis, Proteus mirabilis, Streptococcus spp, Escherichia
coli, Staphylococcus coagulase negative and yeast: Candida albicans. Purity, viability and identification of the organisms were checked by
plating, gram staining, and the Vitek 2 System (Biomerieux vitek).

2.5.1. Antibacterial activity by agar disc diffusion method (DDM)

The antimicrobial activity was performed using the disc diffusion method on Muller-Hinton (MH) and Sabouraud (SB) agar,
following the CLSI recommendations [20]. Thus, the saline suspensions of microorganisms (NaCl 0.9%) were first adjusted to match
the 0.5 McFarland standard (equivalent to 1 × 108 cfu/mL), and then inoculated onto plate surfaces with a sterile cotton swab.
Whatman paper discs (about 5 mm in diameter) sterilized by autoclaving (121 ◦ C for 15 min) were soaked with the test oil at different
concentrations (30,40 and 50 μL/mL) and placed on the MH and SB agar surface. After 24 h of incubation at 37 ◦ C and 48 h at 25 ◦ C for
bacteria and yeasts respectively, the diameter of inhibition growth zones was measured using a ruler. Each trial was repeated three
times.

2.5.2. Determination of minimum inhibitory concentration (MIC)

The Minimum Inhibitory Concentrations (MIC) of tested oils were determined using the 96-well plate microdilution method [21].
Two-fold serial dilutions of the essential oil were made with BHI sterile broth. 25 μL of diluted oil were dispensed in each well of
columns 1 to 10, while columns 11 and 12 were reserved respectively for microbial inoculums and BHI medium broth (as a control to
monitor sterility).5 μL of microbial suspension previously adjusted to 0.5 Mac Farland (108 CFU/mL) was then added to all wells except
the column 12 which serves as a negative [Link] medium of 70 μL was distributed in the 96 wells and the microplate was
incubated at 37 ◦ C for 24 h. After incubation, the visual turbidity was observed and recorded. The MIC was determined visually as the
lowest concentration that led to growth inhibition.

2.5.3. Determination of minimum bactericidal concentration (MBC)

The Minimum Bactericidal Concentration (MBC) was determined by plating directly 100 μL of the content of wells with concen­
trations higher than the MIC value. The MBC was determined after incubation for 24 h at 37 ◦ C and it was considered as the lowest
concentration that totally inhibits growth. The antibacterial effect was judged to be bactericidal or bacteriostatic depending on the
ratio: MBC/MIC. Indeed, if MBC/MIC ≤4, the effect is bactericidal and if MBC/MIC> 4, the effect is bacteriostatic [22].

3. Results and discussion

3.1. Determination of the essential oil yield

According to the results, the evolution of the EO yield has a progressive growth. Thus, from 5 h of extraction; the curve is bent and
marks a plateau, which corresponds, the maximum yield that can be achieved, under the conditions considered. It corresponds to a
plateau that marks the end of the extraction process. The essential oil obtained gives a yield ranging from 0.53 to 1.93% during one to
6 h.
The output and content of the essential oil are influenced by several factors. Seasonal changes, the plant portion utilized, its
maturity level, its place of origin, and its genetic traits are a few of these.

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M. Afrokh et al. Heliyon 10 (2024) e28125

3.2. Chromatographic analysis

The essential oil of Mentha suaveolens Ehrh is characterized by the presence of piperitenone oxide as the main constituent with a
content of 32.55%. The latter is followed by pulegone (10.14%), piperitone oxide (8.34%), terpinen-4-ol + isopulegol (4.03%),
piperitenone (3.06%), limonene (2.83%), neo menthol (2.74%), isopulegol (2.62%), etc. Other compounds are identified but at
relatively low percentages such as borneol (1.86%), menthol (1.82%), linalool (1.75%), etc. (Table 1).
Numerous studies have been devoted to the chemical composition of the essential oils of MS. For example, a study carried out on the
essential oil of the fresh aerial parts of MS collected in the Al Hoceima National Park region (North of Morocco) confirmed the presence
of high quantities of Piperitenone oxide (44.3%), followed by Z-piperitone oxide (19.1%), terpinen-4-ol (3.8%), 1,2-epoxymenthyl
acetate (3.5%) and trans-hydrate Sabinene (3.1%) [23]. In addition, a study carried out on the EO of this species collected in the
North-West, confirmed the presence of the piperitenone oxide (56.28%), piperitenone (11,64%) and pulegone (6.16%) [24].While a
study recently carried out by Ref. [25], shows that Piperitenone oxide was the major constituent of the Azrou EO (74.69%), followed
by low percentages of γ-muurolene (5.53%), pulegone (2.34%), limonene (1.85%), [Link] general, previous research on the chemical
composition of the essential oil of Mentha suaveolens collected in various regions in Morocco has shown in the majority of cases, high
percentages for constituents belonging to the class of oxygenated monoterpenes, such as pulegone 85.47% in Beni-Mellal [26];
menthol 40.50% in Boulemane [27]; piperitenone oxide 81.69% in M’rirt [28]; piperitone 33.03% in Oulmes [29].

3.3. Quality control of the essential oil of Mentha suaveolens Ehrh

3.3.1. Determination of organoleptic properties

Organoleptic and physico-chemical properties are a means of verifying and controlling the quality of essential oil. Thus, the results
of the determination of organoleptic characteristics (appearance, color and odor) of the EOMS, grouped in Table 2, are close to those of
Mentha piperita L [18].

3.3.2. Determination of physical properties

The quality control verification results of the Mentha suaveolens Ehrh performed by the determination of the physical properties are
grouped in Table 3.
The miscibility with ethanol of the EOMS is a volume of essential oil for every two volumes of ethanol (1V/2V), we found that the
miscibility with ethanol of our oil is inagreement with those of Benbouali [30].This does not allow us to conclude that our essential oil
has a good miscibility with ethanol.
The freezing temperature of the EOMSis below − 10 ◦ C (Tfreezing. < − 10 ◦ C).
Comparing the value of the relative density mentioned previously, we found that this value is close to that of the EO of Mentha
piperita L which varies between 0.820 and 0.9900, according to AFNOR [18].
According to Ref. [31], the refractive index gives information on the quality of the EO; however, it varies with the content of
monoterpenes and oxygenated derivatives, a high content of monoterpenes would give a high index. For some authors, the low
refractive index of EO indicates its low refraction of light, which could favor its use in cosmetics [18]. Based on the value of the
refractive index of 1.5256, our EO has good quality and contains a large amount of monoterpenes and oxygenated derivatives. Thus,
the value found for our study is close to that of the oil of Mentha piperita L which varies between 1.4600 and 1.4670 according to
AFNOR [18] and in good agreement with those of Benbouali [30].
The rotary power is an important criterion of purity of the EO, which makes it possible to determine if it is optically active [32].
Thus, if the direction of rotation of the analyzer is in the clockwise direction, the oil studied is dextrorotatory (+). If the rotation of the
analyzer is in the counterclockwise direction, the oil studied is levorotatory (− ) [33].
According to the angle of rotation obtained α = + 0.33◦ , the rotational power value of the essential oil of Mentha suaveolens Ehrh is
+0.825. This oil is therefore dextrorotatory (+).

3.3.3. Determination of chemical properties

The chemical properties of the essential oil of Mentha suaveolens Ehrh are shown in Table 4.
The amount of free acids present is indicated by the acidity index. Our study’s index was less than 2, indicating that the essence was
well preserved and that there were few free acids present [18]. In addition, an acid index of less than 2 serves as an indicator of oil
quality. Our obtained results for the acid index agree with those given by Ref. [18] for the species Mentha piperita L. and are in
agreement with those of Benbouali [30].
In general, the higher the ester index, the better the quality of the EO [34]. From the chemical properties obtained, it appeared that

Table 2
Organoleptic properties of the essential oil of Mentha suaveolens Ehrh.
Organoleptic properties Mentha suaveolens (our results) Mentha suaveolens [18] Mentha
Piperita [18]

Aspect Liquid, mobile and crystal clear Liquid, mobile and crystal clear Liquid
Odor Strong characteristic smell at Mentha suaveolens Ehrh Spicy. Penetrating The strong smell, mint very refreshing.
Color Pale yellow Pale yellow Transparent to pale yellow

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M. Afrokh et al. Heliyon 10 (2024) e28125

Table 3
Physical properties of the essential oil of Mentha suaveolens Ehrh.
Physical properties Mentha suaveolens (our results) Mentha suaveolens [30] Mentha piperita [18]

Density at 20 ◦ C 0.920 0.930 0.820–0.9900

Miscibility with ethanol 1V/2V 90% 2V –
Freezing point Tfreezing < − 10 ◦ C – –
Refractive index 1.5256 1.5416 1.4600–1.4670
Rotary power +0.825 – –

Table 4
Chemical properties of the essential oil of Mentha suaveolens Ehrh.
Chemical properties Mentha suaveolens (our results) Mentha suaveolens [30] Mentha piperita [18]

Acid index 1.68 1.78 <5

Ester index 68.44 71.12 53–76
Saponification index 70.13 – –
Iodine index 12.05 – –

our essential oil is of good quality.

According to the results obtained, the ester index is in good agreement with that given by AFNOR [18]for the species Mentha
piperita L. which varies between 53 and 76 and in good agreement with that of Benbouali [30].
The saponification index is related to the length of the constituent fatty acids of the oil. Indeed, a low saponification index therefore
corresponds to fatty acids having a longer carbon chain. A high saponification index results in a high level of short chain fatty acids and
a higher glycerol content [35].
From the saponification index value obtained, it appears that our EO does not contain free fatty acids having a high molecular
weight.
The iodine index is the generally accepted parameter expressing the degree of unsaturation, ie the number of carbon-carbon double
bonds in fats or oils [36] also reported that the higher the amount of unsaturation, the greater the uptake of iodine; therefore, the
higher the iodine index, the higher the degree of unsaturation. In this regard, examination of Table 4 shows that the iodine index value
of the oil is low, that shows that our essential oil is less saturated. While a study recently carried out by Ref. [25], shows that the iodine
values (gram of iodine absorbed/100g of fat) obtained for Mentha suavenlens oil varied from 107.354 ± 0.018 to 106.61 ± 0.019.
g/100 g from the Azrou and Ifrane regions respectively [25]. The production, concentration, physical-chemical characteristics, and
composition of essential oils can all be influenced by a variety of circumstances. These include the plant’s age, its genotype, its parts
utilized, its surroundings, its drying process, its extraction technique, its harvesting period, its farming practices, and its drying
methods and length [37].

3.3.4. Antimicrobial activity

The results of the antimicrobial activity of the EOMS are summarized in Table 5.
The EO of round-leaved mint, evaluated in this study, is characterized by the variability of its activity with respect to the mi­
croorganisms tested Round-leaf mint EO, evaluated in this study, is characterized by the variability of its activity with respect to the
microorganisms tested. Its strong inhibitory effect is marked against bacteria: Proteus mirabilis (17.50 ± 0.70 mm at 50 μL/mL),
Enterococcus faecalis (17.00 ± 1.00 mm at 50 μL/mL) and Staphylococcus coagulase negative (16.33 ± 0.57 mm at 50 μL/mL) while it
was moderate against Escherichia coli (14.33 ± 1.15 mm at 50 μL/mL) and Streptococcus spp (13 ± 0.00 mm at 50 μL/mL) and our
results are closer to those obtained by Ref. [39] on Escherichiacoli (wild) (14.00 mm) and Proteus mirabilis (9.00 mm).Recently, the
results of the diffusion test conducted by Ref. [40] showed an inhibition of bacterial growth proportional to the diameter zone. In
addition, the diameters of inhibition produced by the EO vary according to the bacterial strain. The highest inhibition zone was
observed in Staphylococcus aureus (21.00 ± 1.00 mm), followed by Escherichia coli (17.00 ± 0.50 mm), Klebsiella pneumoniae (16.00 ±
0.50 mm), Streptococcus faecalis (14.00 ± 0.50 mm), Salmonella spp. (14.00 ± 0.40 mm) and Pseudomonas aeruginosa (13.00 ± 0.30

Table 5
Sensitivity of the studied strains to the essential oil of Mentha suaveolens Ehrh.
Strains Essential oil of Mentha suaveolens Nitrofurantoïne (cystitis) [38]

Inhibition zones (mm) Critical diameter (mm)

Bacteria Concentration (μL/mL) 30 40 50 S≥ R<

Staphylococcus coagulase négative 11.66 ± 0.57 13 ± 1.00 16.33 ± 0.57 13 13
Streptococcus spp 10.50 ± 0.70 12.00 ± 0.00 13.00 ± 0.00 15 15
Enterococcus faecalis 12.00 ± 0.57 14.66 ± 0.57 17.00 ± 1.00 15 15
Escherichia coli 11.50 ± 1.41 13.33 ± 0.57 14.33 ± 1.15 11 11
Proteus mirabilis 11.50 ± 0.70 14.50 ± 0.70 17.50 ± 0.70 11 11
Yeasts Candida albicans 10.66 ± 1.15 12.00 ± 1.15 15.33 ± 1.52 – –

8
M. Afrokh et al. Heliyon 10 (2024) e28125

mm).
Using the solid-phase and microtiter analysis method, essential oil extracts from plants grown in different parts of Morocco were
analyzed and their effectiveness against 19 bacterial strains, both Gram-positive and Gram-negative, as well as three species of fungi,
was assessed [41]. The essential oil containing a high concentration of pulegone showed significant inhibition of all bacterial strains,
while that containing a high proportion of piperitone oxide (PO) showed weaker activity. It appears that the activity of oils rich in PO
and piperitone oxide is less pronounced. These results suggest that the efficacy of essential oils is closely linked to their specific
chemical composition, an observation corroborated by another analysis in which the main aromatic components of these oils were
tested against the same organisms. It seems that oxidized piperitone has around half the potency of piperitone oxide against a variety of
microorganisms, including yeasts. These findings imply that the most fragrant component of wild mint essential oil extract (EOMS)
that may be the most active is pulegone [41]. The presence of significant concentrations of oxygenated monoterpenes with possible
antibacterial and antifungal characteristics, pulegone and menthone, may account for our essential oil’s antimicrobial action [42,43].
The measured MIC values are consistent with the zones of inhibition achieved, since the MIC is inversely proportional to the
diameter of the inhibition zones (Table 6). The highest MBC/MIC value was observed in Enterococcus faecalis. On the other hand, the
lowest values were observed in Escherichia coli and Proteus mirabilis. The minimum inhibitory concentration (MIC) for Staphylococcus
aureus was found to be 0.48 mg/mL, whereas Salmonella spp. and Pseudomonas aeruginosa showed a MIC of 7.81 mg/mL, as reported by
Ref. [40]. Moreover, the minimum bactericidal concentration (MBC) varied from 15.62 mg/mL in Pseudomonas aeruginosa to 0.48
mg/ml in Staphylococcus aureus.
Additionally, reference data [44] indicates that when M. suaveolens is compared to the reference antibiotics utilized, its MIC
(minimum inhibitory concentration), MBC (minimum bactericidal concentration), and MFC (minimum fungicidal concentration)
values are extremely low. This implies that, subject to thorough in vitro and in vivo toxicological and pharmacological evaluations, this
essential oil (EO) is a very strong antibacterial agent that may find use in the food industry and pharmaceutical formulations. Ac­
cording to reference data [44], the EOs examined had bactericidal properties since their BMC/MIC and MFC/MIC ratios were less than
or equal to four. Chemicals are classified as bacteriostatic or fungistatic if their CMB/CMI and CMF/CMI ratios are larger than four, and
as bactericidal or fungicidal if their ratios are less than or equal to four [45].
The precise mechanism of action of several EO constituents is still not known, although pioneering studies in the past have provided
some insights [46].However, before investigating the effect of EO on microbes, we should have a closer look at the cell-wall structure of
Gram-negative and Gram-positive bacteria (Fig. 1(a,b)).
In light of our results obtained for the essential oil on the microbial strains tested, we note that Gram-positive bacteria are more
sensitive to antimicrobial action than Gram-negative ones. The structural distinctions between Gram-positive and Gram-negative
bacteria explain this greater reactivity. In addition, antibacterial action against Gram-negative bacteria, namely Escherichia coli, is
relatively weak. The lipopolysaccharide-based outer membrane of this bacterium, which envelops the cell wall in peptidoglycan and
limits access to antimicrobial substances, could explain this discovery [48]. There is no discernible difference in minimum inhibitory
concentrations (MICs) between Gram-positive and Gram-negative bacteria, according to other researchers who have studied the
antibacterial action of essential oils [49–53]. The phospholipid bilayer or outer membrane of bacteria, changes in fatty acid
composition, increased membrane flexibility leading to leakage of potassium ions and protons, disruption of glucose uptake, inhibition
of enzymes or disruption of cellular processes are some of the possible mechanisms for the antimicrobial action of essential oils [54].
Studies have shown that interactions between terpenes and membrane proteins and phospholipids result in metabolite deficiency,
disruption of oxidative phosphorylation, suppression of nucleic acid synthesis and inhibition of the cellular respiratory chain. Ac­
cording to other studies, bioactive substances can significantly target the cell membrane, rendering microbial cells inactive [55]. In
fact, the thickening of the cell walls of Gram-positive strains due to interactions between bioactive chemicals and bacteria leads to cell
lysis. The negatively charged outer layer of Gram-negative bacteria can interact with other substances, making it easier for the
molecule to enter the intracellular space and causing disruption [56]. The chemical composition of essential oils, the surrounding
environment and the structures of the targeted bacteria influence their effectiveness as antimicrobials (Gram-positive or
Gram-negative) [57].

4. Conclusions and future prospects

The present study focused on the valorization of the essential oil of Mentha suaveolens Ehrh for its use in the industrial and medicinal
fields (pharmaceutical, cosmetic, agri-food, etc.). It turns out from the results obtained for the organoleptic properties (appearance:

Table 6
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of the essential oil of Mentha suaveolens Ehrh.
Strains Essential oil of Mentha suaveolens Nitrofurantoïne (cystitis) [38]

Bacteria Escherichia coli MIC (μL/mL) MBC (μL/mL) MBC/CMI Critical concentration mg/L
S≤ R>
20 20 1 64 64
Staphylococcus coagulase négative 20 40 2 64 64
Enterococcus faecalis 2,5 10 4 64 64
Proteus mirabilis 10 10 1 64 64
Streptococcus spp 10 20 2 64 64
Yeasts Candida albicans 10 – – – –

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M. Afrokh et al. Heliyon 10 (2024) e28125

Fig. 1. (a,b): The bacterial cell wall: (a) The Gram-positive envelope and (b) The Gram-negative envelope [47].

Liquid, mobile and clear, odor: Strong odor characteristic of Mentha suaveolens Ehrh and color: Pale yellow) and physicochemical
(relative density (0.92), miscibility to ethanol (1V/2V), freezing point (Tfreezing < − 10 ◦ C), refractive index (1.5256), turning power
(+0.825), acid index (1.68), d index ester (68.44), saponification index (70.13) and iodine index (12.05)) that our essential oil turns
out to be of good quality. Thus, chemical analysis shows that our oil is made up of several oxygenated monoterpene compounds such as
piperitenone oxide with a high percentage (32.55%), pulegone (10.14%), piperitone oxide (8.34%), etc. In addition, microbiological
tests carried out on six microbial strains (five bacteria and one yeast) show a well-marked inhibitory effect of our oil against bacteria:
Proteus mirabilis (17.50 ± 0.70 mm at 50 μL/mL), Enterococcus faecalis (17 ± 1.00 mm at 50 μL/mL) and Staphylococcus coagulase
negative (16.33 ± 0.57 mm at 50 μL/mL) while it was moderate against Escherichia coli (14.33 ± 1.15 mm at 50 μL/mL) and Strep­
tococcus spp (13 ± 0.00 mm at 50 μL/mL) as well as against a yeast, Candida albicans (15.33 ± 1.52 mm at 50 μL/mL. The results
obtained are therefore promising and thus open the way for manufacturers to use the essential oil of Mentha suaveolens in the phar­
maceutical, cosmetic, agricultural and particularly food industries. In addition, these properties could make them viable alternatives to
conventional preservatives, thus increasingthe shelf life of various food products. However, its application on an industrial scale as an
ingredient in perfumes, cosmetics and aromatic scents must be linked to its chemical composition. Indeed, dose management is
necessary and is defined as a series of interventions aimed at optimizing good antimicrobial activity in foods. In addition to playing an
antimicrobial and preservative role in food preservation, it also helps protect or improve certain physicochemical properties of foods in
order to maintain overall food quality.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

the authors confirm that data supporting the findings of this study are available within the article.

CRediT authorship contribution statement

Moha Afrokh: Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Conceptualization. Khadija El
Mehrach: Writing – review & editing, Writing – original draft, Supervision. Khalid Chatoui: Methodology, Formal analysis.
Mohamed Ait Bihi: Methodology, Conceptualization. Hajar Sadki: Formal analysis. Mohamed Tabyaoui: Writing – original draft,
Supervision, Conceptualization. Saida Tahrouch: Writing – review & editing, Writing – original draft, Supervision, Methodology,
Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.

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M. Afrokh et al. Heliyon 10 (2024) e28125

Acknowledgements

The authors would like to warmly thank Professor Jean-Marie BESSIÈRE (Montpellier, France) for his important and valuable help.

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