https://doi.org/10.52973/rcfcv-e34431
Received: 30/03/2024 Accepted: 20/05/2024 Published: 25/07/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34431
ABSTRACT
Mentha rotundifolia is a member of the Lamiaceae family and
is distributed mainly in traditional medicine in Algeria and the
Mediterranean locality. This study aimed to demonstrate how the
extraction technique can determine the type of active ingredients
obtained, as well as their potential pharmacological and therapeutic
effects. The methanolic extracts were obtained by maceration and
Soxhlet apparatus. The study of the chemical composition was
determined by colourimetric methods and liquid chromatography–
mass spectrometry (LC–MS/MS); the antioxidant activity was
evaluated invitro by three tests: DPPH test, ferrous ion chelating
test and reducing power test. The antimicrobial effect of extracts was
evaluated by agar diffusion assay against six microbial strains. The
results demonstrate that the best yield of extraction, Total phenolic
and avonoid contents, and antioxidant activity were found to be
higher in the extract obtained by Soxhlet than those obtained by
maceration. The phytochemical screening tests identied polyphenols,
avonoids, tannins, terpenoids, and quinones, and anthraquinones and
saponins were absent in both extracts. The LC–MS/MS revealed the
presence of several phenolic compounds, the predominant in both
extracts was rosmarinic acid. The antimicrobial activity shows that
both extracts have no effect. In conclusion, this study reveals that
extraction by Soxhlet is more suitable than extraction by maceration
for this plant.
Key words: Antioxidant; extraction; LC/MS/MS; maceration; Mentha
rotundifolia; Soxhlet
RESUMEN
Mentha rotundifolia es miembro de la familia Lamiaceae y se
distribuye ampliamente en la medicina tradicional en Argelia y la
localidad mediterránea. El objetivo de este estudio fue demostrar
cómo la técnica de extracción puede determinar el tipo de
principios activos obtenidos, así como sus potenciales efectos
farmacológicos y terapéuticos. Los extractos metanólicos se
obtuvieron mediante maceración y aparato Soxhlet. Se determinó el
estudio de la composición química mediante métodos colorimétricos
y espectrometría de masas por cromatografía líquida (LC–MS/MS).
La actividad antioxidante se evaluó in vitro mediante tres pruebas:
prueba de DPPH, prueba de quelación de iones ferrosos y prueba
de poder reductor. El efecto antimicrobiano de los extractos se
evaluó mediante un ensayo de difusión en agar contra seis cepas
microbianas. Los resultados demuestran que el mejor rendimiento
de extracción, el contenido de fenólicos y avonoides totales, la
actividad antioxidante fue mayor en los extractos obtenidos por
Soxhlet que los obtenidos por maceración. Las pruebas de detección
toquímica permitieron identicar polifenoles, avonoides, taninos,
terpenoides, quinonas y la ausencia de antraquininas y saponinas
en ambos extractos. La LC–MS/MS reveló la presencia de varios
compuestos fenólicos, el predominante en ambos extractos fue el
ácido rosmarínico. La actividad antimicrobiana muestra que ambos
extractos no tienen ningún efecto. En conclusión, este estudio revela
que la extracción mediante Soxhlet es más adecuada que la extracción
por maceración para esta planta.
Palabras clave: Antioxidante; extracción; LC/EM/EM; maceración;
Mentha rotundifolia; Soxhlet
Comparative study of chemical composition Antioxidant and Antimicrobial
activity of Methanolic Extracts of Mentha rotundifolia
Estudio comparativo de la composición química, actividad antioxidante y
antimicrobiana del extracto metanólicos de Mentha rotundifolia
Siham Ferdjioui* , Rachid Belhattab
Ferhat Abbas University, Department of Biochemistry, Laboratory of Applied Microbiology. Setif, Algeria.
*Corresponding author: ferdjioui_89@yahoo.fr
Comparative study of Mentha rotundifolia methanolic extracts / Ferdjioui and Belhattab ___________________________________________
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INTRODUCTION
Due to its richness in secondary metabolites, plants can be considered
a rich source of therapeutic compounds. Variation in extraction methods
usually depends on the duration of the extraction period, the solvent
used, the pH of the solvent, temperature, the particle size of the plant,
and the solvent–to–sample ratio. The majority of extraction methods
involve the separation of medicinally active compounds of plants from
the inactive components by using selective solvents [1, 2].
The solubility of compounds in the solvent depends on their
chemical natures, which vary for compounds of others; this structural
diversity is responsible for diversity in molecules’ physicochemical
properties. For this reason, it is dicult to develop a universal method
for extracting all effective compounds from plants [3].
Oxidative stress can be dened as the state in which the free
radicals in our body outnumber our antioxidant defenses. An
antioxidant is a molecule capable of inhibiting the oxidation of another
molecule. Antioxidants break the free radical chain of reactions by
sacricing their own electrons to feed free radicals without becoming
free radicals themselves [4]. Thus, it is essential to develop effective
and natural antioxidants that can protect the body from free radicals
and retard the progress of many chronic diseases [5].
Mentha rotundifolia is one of the Lamiaceae species that is widely
distributed in Algeria. It is used as a condiment, and it has been applied
in traditional medicine for a wide range of actions: stimulative, tonic,
stomachic, carminative, analgesic, antispasmodic, anti–inammatory,
sedative, hypotensive and insecticidal [6]. This plant was also the subject
of several scientic studies, which made it possible to determine its
therapeutic effect as an antioxidant [7], anti–inammatory [8], and
antimicrobial [9]. The objective of this study was to investigate the
effect of temperature in the extraction of phenolic compounds from M.
rotundifolia and its antioxidant and antimicrobial activity.
MATERIAL AND METHODS
Plant materials
The plant M. rotundifolia was collected in the region of Djemila
Wilaya of Setif (Algeria) during the period of plain Florissant. Professor
Laouer H, a botanist in the laboratory of Botanical Sciences, Ferhat
ABBES Setif–1 University, Algeria, carried out the botanical identity
of the plant (family, genus, and species). The aerial parts were air–
dried in the shade at room temperature away from humidity and then
powdered by an electric grinder.
Extraction
Extraction by maceration
Twenty g of plant powder was put into a maceration bottle and
lled with 96% methanol for 48 hours (h), with the solvent renewed
after 24 h. The ratio between plant powder and ethanol was 1:10.
Afterwards, the ltrate separated from the residue using Watman
lter paper was collected and evaporated using a rotary evaporator
(Rotavator, Büchi; Swiss) at 45°C until a dry extract was obtained.
Soxhlet extraction
Twenty g of aerial parts powder was packed in a Watman lter paper
and placed in the Soxhlet extractor (Büchi; Swiss). Then, 200ml of
methanol was poured into the roundbottom ask. The solvent was
heated using the dismantle, which began to evaporate, moving through
the apparatus to the condenser. The condensate then dripped into the
reservoir containing the plant extract. The process was made to run
for a total of 6 h. Finally, the extract was collected, and the methanol
was evaporated using a rotary evaporator (BUCHI rotavap Suiss) at
45°C. The extract was stored at room temperature for further use.
Phytochemical screening
Phytochemical tests were performed on methanolic extracts to verify
the presence of some compounds (polyphenols, avonoids, tannins,
terpenoids, quinines, and saponins). Their detection is achieved
using the methods described by Bagre et al.[10], Khaldi etal.[11],
Vayalakshmi et al. [12].
Determination of total phenolic compounds
The polyphenols in extracts was quantied using Folin–Ciocalteu
reagent according to the method describedby Li et al.[13]. Briey,
an aliquot of 200 μL of the extract was mixed with 1 ml of Folin–
Ciocalteu reagent for 4 min, followed by the addition of 800 μL of
Na
2
CO
3
aqueous solution (7,5%). The absorbance was measured
(SECOMAN Spectrophotometer, French) at 765 nm after two hours
of incubation. The polyphenol content was expressed as mg gallic
acid equivalent (GAE)·g extract
-1
.
Determination of total avonoids
The total avonoid content of each extract was determined by a
colourimetric method as described by Kosalec et al. [14]. In brief, 1 ml of
each extract was added to 1 ml of aluminum chloride (AlCl
3
) methanolic
solution (2%) and allowed to stand for 30 min, the absorbance of the
mixture was measured at 430 nm. The total avonoid content was
reported as mg of quercetin equivalent (QE)·g extract
-1
.
Identication and quantication of phenolic compounds in extracts
using liquid Chromatography/ Mass Spectrometry (LC/MS/MS)
Methanol was used to dissolve the extracts and standard at a rate
of 1.0 mg·mL
-1
. For every analysis, a volume of 20 μL was continuously
injected at a ow rate of 1.0 mL·min
-1
onto an Agilent Zorbax 150 mm
× 4.6 mm C18 column. A gradient solvent system was used for the
study, with aqueous–formic acid (0.10%) serving as solvent (A) and
acetonitrile (100%) serving as solvent (B). A 35–min run time was
allocated to a ve–step linear gradient elution, wherein solvent A
was reduced to 10% and solvent B was increased to 90%.
A triple–quadrupole mass spectrometer (API 3200; MDS Sciex,
Concord, ON, Canada) received the full flow from the high–
performance liquid chromatography (HPLC). The mass spectrum
information was collected in negative ion mode with a capillary voltage
of 4500 V, an Electrospray Ionization (ESI) ion source, a cone voltage
of 70 V, a collision energy of 35 eV, a drying temperature of 650°C, N
2
as the drying gas at a ow rate of 4.0 L·min
-1
, and Analyst software
version 6. The eluted samples and standards were found at 280 nm [7].
Antioxidant activity
Diphenyl–1–picrylhydrazyl (DPPH•) radical scavenging assay
All substances that can donate a hydrogen atom or an electron
to DPPH can be considered antioxidants and, therefore, radical
TABLE I
Amounts of total phenolic compounds and
avonoids in Mentha rotundifolia extracts
Extract Polyphenols
(a)
Flavonoids
(b)
Methanolic extract obtained by maceration
(MEM)
141.571 ± 0.143 15.636 ± 0.030
Methanolic extract obtained by Soxhlet
(MES)
168.642 ± 1.642 33.045 ± 0.76
(a)
: Expressed as mg galic acid equivalent (GAE) per gram of extract,
(b)
: Expressed as
mg quercitin equivalent (QE) per gram of extract
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scavengers. The degree of discolouration of the violet colour of DPPH
as it gets reduced indicates the radical scavenging potential of the
antioxidant [15]. The capacity of extracts to scavenge DPPH was
determined following the method of Sarikurkcu et al. [16]. Briey: 0.1
mL of extracts of different concentrations was mixed with 1 ml DPPH
solution (0.008% in ethanol). The mixture was shaken and left to stand
for 2 h at room temperature. The absorbance was measured at 515
nm. The scavenging activity of extracts was compared with that of
Butylated hydroxytoluene (BHA) as standard. The capability to scavenge
the DPPH• radical was calculated using the following equation:
%I
A
AA
10
0
0
01
#
=
-
=
G
Where A
0
is the absorbance of the control, and A
1
the absorbance
of the sample.
Metal ion chelating assay
Transition metal species such as ferrous iron (Fe
2+
) can facilitate
the production of ROS within animal and human systems; the capacity
of compounds to chelate iron can provide a valuable antioxidant
capability [17]. The Metal ion chelating effect of the extract was
estimated using the method reported by Le et al. [17]. Briey, one
ml of each extract at a different concentration was mixed with 1 ml
of FeCl
2
(0.6 mM). After 5 min, the reaction was initiated by adding 1
ml ferrozine (5 mM). The tubes were vortexed and allowed to stand
for 10 min at room temperature. Absorbance was measured at 562
nm. The ratio of ferrozine–Fe
2+
complex formation inhibition was
calculated as follows:
%I
A
AA
100
control
controlsample
#
=
-
=
G
The EDTA was used as a positive control.
Reducing power assay
The plant extract components’ reducing power might be a signicant
indicator of its potential antioxidant activity [18]. The presence of
reductants such as antioxidant substances in the samples causes
the reduction of the Fe
3+
/ferricyanide complex to the ferrous form.
Therefore, Fe
2+
can be monitored by measuring the formation of Perl’s
Prussian blue at 700 nm [17]. The reducing power of extracts was
assessed by the method described by Beyhan et al. [17], 1 ml of extracts
was mixed with 2.5 ml phosphate buffer (pH=6.6) and 2.5 ml of 1%
potassium ferricyanide [K
3
Fe(CN)
6
] solution. The mixture was incubated
at 50°C for 20 min, and then 2.5 mL of 10% Trichloroacetic acid was
added. After vigorous agitation, 2.5 mL of this solution was mixed
with 2.5 mL of distilled water and 0.5 ml ferric chloride (FeCl
3
) (0.1%).
The absorbance was measured at 700 nm. The reducing power of the
extracts was compared with that of Butylated Hydroxyanisole (BHA) as
a positive control. Higher absorbance indicates higher reducing power.
Antimicrobial activity
The study of antimicrobial activity was performed by the method
of agar diffusion against two Gram–negative bacteria: Pseudomonas
aeruginosa ATCC 27853 and Escherichia coli ATCC 25922, one gram–
positive bacteria Staphylococcus aureus ATCC 25923 and three fungi
(Aspergillus niger 2CA936, Aspergillus avus NRRL391, Candida albicans
ATCC1024). Sterilized Whatman paper discs of 6 mm diameter were
impregnated with 20 µl of solution of extracts (200–500 mg·ml
-1
),
and the discs were then placed in Petri dishes previously inoculated
by Muller–Hinton agar for bacteria, Potato Dextrose Agar (PDA) for
fungus, and Sabouraud + Chloramphenicol for Candida albicans.
Different standard antibiotics (according to the type of bacteria)
(Gentamicin (GEN), Imipenem (IMP), Cefoxitin (CX), Pristinamycin (RP),
Vancomycin (VA), Piperacillin (PRL), Ciprooxacin (CIP), Clindamycin
(CD)) and antifungal (Amphotericin B (AM), Chloramphenicol (CTR),
Nystatin (NY)) were used as positive control.
The Petri dishes are incubated (Memmert ovens, Germany) at
37°C·24h
-1
for bacteria, at 37°C for 48 h for yeast, and at 27°C for 72 h
for fungi. The negatif control was an impregnated disk DMSO (Dimethyl
sulfoxide) alone. Different antibiotic and antifungal disks were used as
positive control. Three repetitions were performed for each test [19].
Statistical analysis
The results were presented as the mean ± standard deviation (SD).
Statistical data analysis was performed using the GraphPad Prism 5
program with the Dunnett test; the level of signicance was set at P<0.05.
RESULTS AND DISCUSSION
Phytochemical analyses
In the present study, methanolic extracts of M. rotundifolia were
evaluated for their phytochemical screening, polyphenolic contents,
antioxidant, and antimicrobial activity.The best extract yield was
recorded by the Soxhlet apparatus with 16.05 % against 9.2 % for the
maceration. The phytochemical screening revealed the presence of
polyphenols, avonoids, tannins, quinones, and terpenoids. However,
saponins and anthraquinones were absent in both extracts.
Amounts of total polyphenols and avonoids
The contents of total polyphenols, and total flavonoids were
determined in plant extracts by using Folin–Ciocalteu and AlCl
3
reagents respectively, the results are shown in TABLE I. As can be
seen, the Soxhlet apparatus seems to be signicantly (P<0.05) better
than the maceration to extract total polyphenols and avonoids.
Identication and quantication of phenolic compounds using
LC/MS/MS
The results of this study demonstrate that among the 24 standards
used, 17 phenolic compounds were identied in MEM and 15 in MES.
Among the phenolic compounds identied: phenolic acids (Vanilic
acid, Rosmarinic acid, Ellagic acid, Syringic acid…), avonols (Myrtillin,
Quercetin, Rutin, Isoquercitin), flavone (Apigenin, Luteolin) and
anthocyanins (Cyanin chloride).The predominant compound in both
TABLE II
Phenolic prole of methanolic extracts obtained by maceration and Soxhlet, identied by LC– MS/MS
Compounds C
1
(ng·ml
-1
) MEM C
2
(ng·ml
-1
) MES RT Q1 Q3 MWT
Apigenin 213.00 2.99 8.40 269.000 151.0 270.120
Isoquercitrin 57.60 nt 7.00 464.900 300.0 464.400
Catechol nt nt 5.50 109.000 109.0 110.110
Epicatechin nt nt 6.57 289.100 108.8 290.300
Gallic Acid 152.00 nt 1.53 169.000 124.6 170.120
Procyanidin B
2
nt nt 6.40 577.100 407.0 / 289.0 578.520
Quercetin 3–O–Galactoside 328.00 60.00 7.00 463.000 301.0 464.379
Luteolin 275.00 118.00 8.00 285.000 217.0 286.240
Chlorogenic Acid 357.00 96.50 5.94 353.155 190.4 /84.8 / 93.1 354.310
Epigallocatechin Gallate nt nt 6.66 456.579 168.2 / 168.5 / 124.8 458.372
Cyanin Chlorid 304.00 203.00 8.00 286.198 133.4 /132.8 / 150.6 287.100
Myrtillin 65.40 8.62 7.01 462.178 299.8 / 270.8 / 254.7 500.800
Quercetin nt nt 8.01 300.604 150.4 302.200
Rutin 337.00 168.00 6.84 609.419 299.0 / 299.9 / 270.9 610.520
Caec Acid 156.00 95.10 6.50 178.465 134.2 / 106.4 / 89.1 18.160
Ellagic Acid 7.98 nt 8.01 300.703 149.7 / 150.6 / 149.9 302.197
Ferulic Acid 109.00 56.00 7.20 192.807 133.9 / 133.4 / 177.8 194.180
Hydroxybenzoic Acid 198.00 84.70 6.48 134.779 88.4 / 106.8 / 89.1 135.120
P–Cumaric Acid 178.00 153.00 7.06 162.756 118.8 / 118.1 / 92.7 164.160
Rosmaric Acid 18900.00 7690.00 7.38 358.319 160.7 / 161.0 / 132.7 360.320
Syringic Acid 116.00 126.00 6.48 196.718 120.4 / 120.7 / 152.3 198.170
Transcinamaldehyde Acid nt 975.00 7.95 131.797 103.7 / 102.8 / 101.9 132.160
Vanilic Acid 206.00 221.00 6.38 166.660 107.8 / 151.2 / 151.7 168.150
Hypericin nt nt 10.53 503.000 405.0 504.450
Q1: compound molecular weight, Q3: fragment molecular weight, MWT: molecular weight, nt: not found
Comparative study of Mentha rotundifolia methanolic extracts / Ferdjioui and Belhattab ___________________________________________
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extracts was Rosmaric Acid (18900 and 7690 ng·mL
-1
for MEM and
MES respectively) (TABLEII). Furthermore, the study showed that the
amounts of the majority of phenolic compounds identied are higher
in MEM than in MES. However, the quantities of syringic acid, vanillic
acid, and trans–cinnamaldehyde were higher in MES.
The medicinal value of the plant is due to the phytochemical
constituents they produce, which exhibit certain physiological actions
on human body [20].The result of this study shows the presence of
polyphenols, avonoids, tannins, quinones, and terpenoidsM. rotundifolia
extracts. This diversity of compounds can lead to a diversity of plant
therapeutic properties. The determination of the amount of total
polyphenols and avonoids demonstrate the richness of both extracts
on this compounds with a predominance in the extract obtained by
Soxhlet. In a study carried out by Boussouf etal. [8] on the leaves of the
same plant from the northern Algeria, the methanolic extract obtained by
maceration gave respective contents of polyphenols and avonoids of
350.10 ± 0.96 mg galic acid equivalent (GAE·g
-1
) of extract and 79.44 ± 0.76
mg quercitin equivalent (EQ·g
-1
) of extract. These differences with our
study may be due to several factors that can inuence the contents
and the nature of phenolic compounds in extracts, such as the region
and period of the harvest of the plants, the part used, the time and
the extraction temperature, the polarity of the solvent [1]. In order to
identify the phenolic compounds of extracts, LC–MS/MS was carried
out. Based on comparing their chromatographic proles and retention
times with those of the standards used, several phenolic compounds
with therapeutic interest are identied. Rosmarinic acid was the major
compound and has been the subject of numerous scientic studies
demonstrating its therapeutic effects, such as antioxidant properties
[21], anticancer [22], anti–inammatory [23].
Antioxidant activity
DPPH radical scavenging assay
The IC50 for DPPH radical–scavenging activity reported in TABLEIII
demonstrates that the radical–scavenging activity of extracts
increased with increasing concentration of extract. The methanolic
extract obtained by the Soxhlet apparatus had shown better (P<0.05)
scavenging activities than those obtained by maceration. However,
the activity of BHA was much more marked (P<0.001) than extracts.
Determination of metal chelating activity
The results of this test also demonstrate that the extract
obtained by maceration has a significantly smaller chelating
TABLE III
IC
50
Values of Mentha rotundifolia Extracts and standard for DPPH Scavenging
Activity, Metal chelating activity and EC
50
for reducing power
Test MES (µg·ml
-1
) MEM (µg·ml
-1
) BHA (µg·ml
-1
) EDTA (µg·ml
-1
)
DPPH
scavenging
133.160 ± 11.346 265.491 ± 2.221 20.701± 0.065 NT
Metal chelating
activity
2194.000 ± 0.038 3417.000 ± 0.011 NT 11.100 ± 0.060
Reducing
power
468.000 ± 1.000 550.325 ± 3.613 89.000 ± 0.285 NT
NT: not tested
TABLE IV
Antimicrobial activities of Mentha rotundifolia extracts against the bacterial and fungal strains tested
Escherichia coli
ATCC 25922
Pseudomonas aeruginosa
ATCC 27853
Staphylococcus aureus
ATCC 25923
Aspergillus niger
2CA936
Aspergillus avus
NRRL391
Candida albicans
ATCC1024
MEM 200 and 500 (µg·ml
-1
) NI NI NI NI NI NI
MES 200 and 500 (µg·ml
-1
) NI NI NI NI NI NI
Antibiotics
27 (CX) 34 (PRL) 30 (RP) 9 (AM) 8 (AM) 19 (AM)
28 (GEN) 39 (CIP) 30.5 (CD) 11 (NY) 11 (NY) 21 (NY)
37 (IMP) 38 (IMP) 16 (VA) 20(CTR) 24 (CTR) 33 (CTR)
NI: No inhibition zone observed around the discs (6 mm) impregnated with 20 µl of methanolic extracts.
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power capacity (IC50=3417 ± 0.011 µg·ml
-1
) than the Soxhlet extract
(IC50=2194 ± 0.038µg·ml
-1
) (TABLE III).
Reducing power
In this assay, The EC50 values indicated that the BHA showed a good
reducing power (EC50= 89 ± 0.285 µg·ml
-1
); the EC50 values of extracts
demonstrate that the activity of the extract obtained by maceration
is signicantly lower (P<0.001) than of Soxhlet extract (TABLE III).
No correlation was noted between levels of some phenolic
compounds determined by HPLC/ MS–MS and antioxidant activity; this
may be, explained by the presence of other compounds not identied
in this study in soxhlet extract with a good antioxidant effect.
Antimicrobial activity
The antimicrobial activity of methanolic extracts was evaluated
against two Gram–negative bacteria: Pseudomonas aeruginosa ATCC