23
23
23
1
Department of Biotechnology, Mohi- ud-Din Islamic University, Nerian Sharif, AJ&K, Pakistan
2
Pakistan Council for Science and Technology, Ministry of Science and Technology Islamabad, 45320, Pakistan
3
Department of Life Sciences, Yeungnam University, Gyeongsan 38541, Gyeongbuk, Republic of Korea
4
State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan
430062, China
5
Department of Biotechnology, University of Kotli Azad Jammu and Kashmir-Pakistan
6
CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization and Ecological Restoration,
Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of
Science, P.O Box 416, Chengdu, Sichuan 610041, China
7
University of Chinese Academy of Sciences, Beijing 100049, China
8
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences,
Beijing 100093, China
Corresponding author’s email: shangla123@gmail.com; saddamsaqib.qau@gmail.com
ABSTRACT
Mentha arvensis L. is traditionally used in folk medicine, and pharmacological industry due to presence of active
chemical substances. It is also valuable for food industry as additives because of the presence of antioxidant, cytotoxic,
antidiabetic and antimicrobial constituents. This study is intended to examine reactive oxygen species (Cvetanović et al.)
generation, lipid oxidation, cytotoxicity and antimicrobial effect of aqueous extract from M. arvensis L. prepared in
various solvents i.e. fermented methanol extract (FM.E), distilled water extract (DW.E) and methanol extract (M.E).
Phytochemical screening of the extract was qualitatively investigated for the isolation of alkaloids, flavonoids, fats and
oils, menthol and quinones. To check the potential of extract as preservative, pH, lipid oxidation and Fourier transformed
infrared spectroscopy (FT-IR) analysis was performed. Our results showed FM.E induce ROS generation, cytotoxicity
and inhibit Staphylococcus aureus (4.20±0.90 mm) and Pseudomonas aeruginosa (3.23±0.32 mm) growth. In addition,
in vivo results showed FM. E and M.E efficiently maintained chicken meat pH and reduced lipid oxidation. The presence
of essential phytochemicals was responsible for inhibition of biofilm formation. FT-IR analysis revealed the presence of
free OH stretching vibrations at 3878.69 cm-1, free NH at 3459.56 cm-1 and H-NH bond stretching 3388.02 cm-1 groups
in chicken meat which belong to M. arvensis L. extracts. These results suggest that menthol from M. arvensis L. extract
is favorable food additive against resistant pathogens.
Keywords: Mentha arvensis L; Phytochemicals; ROS generation; Lipid oxidation; Antimicrobial
Published first online October 20. 2021 Published final May 30. 2022
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Asghar et al., J. Anim. Plant Sci., 32 (3) 2022
medicinal plants offer a variety of alternatives to reduce and maintain food duration. Therefore, in this study we
bacterial growth (Ashmawy et al., 2018). have determined antioxidant, cytotoxic, antidiabetic,
From last few decades, consistent endeavors are antifungal and antibacterial activities of M. arvensis
in progress to control metabolic issue through ingestion different extracts. Additionally, all prepared extracts were
of dietary sources. The chemical drugs are now applied on chicken meat to assess lipid oxidation and pH
accessible for antibacterial applications but exhibit to evaluate M. arvensis extract role on food borne
serious side effects (Cuadrado et al., 2019), featuring the pathogens and retain food quality.
need to discover effective natural compounds. Health
benefits can be result by taking in a hygienic food MATERIALS AND METHODS
containing natural ingredients. Natural products, for
example, plants extract, either as pure forms or as Cultivation and preparation of Extracts: Plants were
adulterated form, give boundless chances to new cultivated in home garden at Tahlian (33.62° N and
medicate discoveries due to the unmatched chemical 73.77° E), Azad Kashmir, Pakistan. The leaves of fully
diversity and biocompatible nature (Cláudio et al., 2018; mature plant were washed with tap water, subsequently
Sasidharan et al., 2011). Recently many of these plants rinsed in distilled water (DW) and then air dried,
have been recommended for their preservative and food eventually ground into fine powder. The material was
additive ability, playing double role i.e. food flavor and extracted with three different solvents, D.W extract (DW.
bioactive compounds (Salehi et al., 2018). Plants aqueous E), methanol extract (M.E) and fermented methanol
extracts can be used as basic material, additive or extract (FM. E) (Shoba Thomas, 2001). In brief 50 g of
preservative in food industry such as bakery, the powder was soaked in the above-mentioned solvents,
confectionary products, ice creams, meat products and while fermented extract was prepared by inoculating
desserts (Sun‐Waterhouse, 2011). Aqueous extracts Lactobacillus plantarum (L. plantarum) in De Man,
usually obtained from aqueous phase through physical Rogosa and Sharpe broth (MRS) at 37 °C for 24 h and
process that does not influence their composition (Salehi diluted to get the initial culture. The M. arvensis extract
et al., 2018). But prior use at mass scale thorough of 5 % containing subculture of fresh bacteria (4 % v/v)
investigation such as cytotoxicity, antioxidant, was incubated at 37 °C for 24 h. Eventually, the extract
antidiabetic activities and lipid oxidation potential are was sterilized and filter using Whatman’s filter paper.
necessary to ensure their efficacy and safety through The stock solution was prepared at concentration of
research proof-of-concept for potential health claims. 0.100, 0.250 and 0.500 mg/mL and stored at 4 ºC for
Genus Mentha belongs to family Lamiaceae further use.
(Labiatae) comprising around 30 species distributed in
temperate regions of Eastern Asia (EA), South Asia (SA), Phytochemical analysis: The phytochemical analysis of
Australia, North America and South Africa (Anwar et al., all (DW.E, M.E and FM.E) prepared extract was
2019; Ayaz et al., 2020a; Ayaz et al. 2020b). These are performed to confirm the presence of alkaloids, fats and
aromatic perennial herbs, cultivated for their essential oils oils, menthol, flavonoids and quinones by following the
and culinary purposes. Therefore, many hybrids and protocol of Suja , and Williams (2016).
numerous cultivars are available. Species of the genus Cytotoxic Analysis: To confirm cytotoxic effect of all
Mentha such as M. arvensis contain various derivatives extracts 3T3 L1 mouse preadipocytes were obtained from
such as flavonoids, alkaloids, phenol, terpenes and Sigma Aldrich, Germany. The cells were sub-cultured in
polysaccharides. Terpenes and quinines are used in Dulbecco’s modified Eagle’s medium enrich with fetal
eatables and medicine, cosmetics and pesticides bovine serum (10 %) and Penicillium (1 %) was
industries (Chitrakar et al., 2019). Phytochemical deposited in 96 well plate. Cells were treated with 200 μl
screening allows identification and presence of essential respective extract and incubated at 37 °C for 4 h in 5 %
phytochemicals that can specify the role of Co2. Toxicity was checked by adding 20 μl sterilized 3-
phytochemical in food industry (Salehi et al., 2018). (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
Various medicinal activities of M. arvensis are reported bromide (MTT) reagent in separate wells and incubated
such as, anti-inflammatory, antiallergic, antifungal and at 37º C for 2 h. Eventually, 0.100, 0.250 and 0.500
antibacterial (Malik et al., 2012). Majority of the food mg/mL (200 µl) of all extracts (DW. E, M.E and FM.E)
and beverages industries are utilizing artificial M. was added to respective wells and mixed with the cells
arvensis extract or flavors in dietary products (Joseph et for the thorough suspension of formazan crystal (Lee et
al., 2018). Widespread use of synthetic food additives al., 2015). Distilled water treated cells were considered as
(Carmoisine, Indigocrarmine, Alkaline phosphatase etc.) a control. The quantity of viable cells was studied at 490
and preservatives (Benzoic acid, Sodium nitrite, Vitamin nm in a microplate ELISA spectrophotometer reader. The
E, and Vitamin C etc.) has led to huge health problems. cell’s sustainability was measured as a percentage of the
These issues triggered the search for new and viability of the control.
biocompatible strategies to inhibit food borne pathogens
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Asghar et al., J. Anim. Plant Sci., 32 (3) 2022
ROS generation: Antioxidant activity was determined thiobarbituric acid (TBA) test. TBA reactive substances
by assessing free radical scavenging activity practicing (TBARS) values were expressed as mg
2,2-diphenyl-1picrylhydrazyl (DPPH) assay (Kang et al., malonaldehyde/Kg sample. The samples were observed
2011). Samples were prepared with 0.100, 0.250 and randomly after every 5 d.
0.500 mg/mL (200 µl) concentrations. For positive
FT-IR analysis: FT-IR spectroscopy was performed to
control Vitamin C was used. The samples were kept at 25
confirm the presence of organic compounds on post
ºC for half hour and determined free radical scavenging
treated chicken meat using FT-IR spectrometer
activity by adding 50 μM DPPH solution (1:1) and
(Shimadzu, Japan) coupled with attenuated total
incubated at dark for 30 min. Finally, absorbance was
reflectance (Amaral et al., 2018). A small piece of post
measured by spectrophotometer at 517 nm. The IC50
treated 30 d chicken from all treatments was placed
value of the extracts was analyzed by following the
individually on ATR cell and the spectra was measured
formula (MubarakAli et al., 2018):
as % treatment in the range of 4000-400 cm-1 with a
Scavenging effect (%) = [100- (AC-AS/AC)]
resolution of 4 cm-1 applying 16 scans per sample.
In this equation AC stands for absorbance of the
control reaction, whereas AS is the absorbance of the Statistical analysis: All experiments were repeated
tested samples. thrice. Average mean values for all replicates were
analyzed by ANOVA whereas, significant difference was
Antibacterial activity: Antibacterial activity of all
separated by applying Bonferroni test set at P<0.05.
extracts was performed against pure culture of food
bacterial strains Staphylococcus aureus (P. aureus) and
Pseudomonas aeruginosa (P. aeruginosa) received from RESULTS AND DISCUSSION
department of Biotechnology Kotli university, Azad
Jammu and Kashmir. Well diffusion method was used to Taxonomy of Mentha arvensis L.: The plant
check their antibacterial activity with some modifications morphology was carefully examined that shows stems
(Salehi et al., 2015). The inoculum of the selected strains erect or ascending, with retrorse hairs on surface. Leavers
was incubated for 24 h in nutrient broth at 37 ºC. To arranged in opposite pairs and simple, ovate to ovate
perform antibacterial activity entire agar surface was elliptic having 2.5 x 1.2-2 cm size and serrulate to serrate
inoculated with bacterial inoculum. Cork borer of 6 mm eglandular hairs on both surfaces (Figure 1A). The root
was punched on the solid agar surface to make wells and stock spread in creeping manner from which the plant
filled with DW. E, M.E and FM. E (200 µl) of all body arise and grow erect. The plant habit is herbaceous
concentrations (0.100, 0.250 and 0.500 mg/mL). As a with creeping rhizomes (Figure 1B) from which the plant
control potato dextrose agar (PDA) dishes were body arise.
supplemented with same amount of DW. The Phytochemicals identification: Phytochemicals test
experimental data was obtained from three independent showed the presence of alkaloids, flavonoids, fats and
experiments and average was calculated. oils, quinones and steroids in M.E and FM. E while DW.
In vivo effect of menthol: All extracts (DW. E, FM. E E lack quinones (Table 1). In FM. E the presence of
and M.E (250 mg/mL) were applied on chicken meat to quinones may be because of microbial metabolites
determine the potential of extracts in meat preservation. secretion. The phytochemical analysis showed the
To check the effect of all extracts, chicken meat (300 ± presence of essential compounds which can help to
20 g weight) was purchased and packed in polystyrene protect the food material. In addition, use of fermented
boxed with flaked ice. After that visible dark meat, skin plant extracts in dietary products will enable
and bones were removed and cut into 0.4 x 3-4 cm sized pharmaceutical and food industry to produce health-
pieces. Subsequently, chicken meat samples were given oriented food products (Kieliszek et al., 2018). Previous
dip treatment for 2 h separately in DW. E, FM. E and studies showed that addition of solvents like methanol,
M.E (250 mg/mL) concentration, D.W was used as a chloroform, ethanol, and hexane enhance the
control. all treatments were well drained and packed in antimicrobial potential due to easy release of
separate airtight beakers containing polyvinyl dichloride phytochemicals (Alam et al., 2016; Johnson et al., 2011).
at 4 ºC for 30 d to assess lipid oxidation and pH of the The presence of alkaloids, flavonoids, fats and oils,
samples. quinones and steroids indicate that M. arvensis can be use
as significant cytotoxic and antioxidant agent (Bouyahya
pH analysis: The pH of the samples was measured on et al., 2020).
every 5th d post treatment of all extracts (250 mg/mL).
Readings were taken by sticking the probe into the Cells viability: Cytotoxic effect of M. arvensis extracts
sample’s beaker. showed significant activity against 3T3 L1 mouse cell
lines (Figure 2). FM. E was more active and showed
Lipid oxidation assay: Lipid oxidation of all extracts potential inhibition against cell lines. Minimum viability
using 250 mg/mL extracts was analyzed by 2-
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Asghar et al., J. Anim. Plant Sci., 32 (3) 2022
(IC50) was showed by FM. E 0.21±0.02 followed by M.E releasing active biological compounds in the host cell
0.19±0.01 Mean±SD and DW. E 0.25±0.01 Mean±SD (Shen et al., 2018). Cell division is a complex mechanism
respectively. We found that FM. E was significantly and can be treated by targeting multiple signaling
different from DW. E and comparatively more active pathways by using beneficial plant products in our daily
against 3T3 L1 mouse cell lines then DW. E and M.E. dietary products (Tamang Kailasapathy, 2010). The use
The basic mechanism against cancerous cells toxicity was of plant extract in food production and preservation can
generated due to apoptosis by stimulating reactive help to maintain the quality of food. The benefit of
oxygen species (Cvetanović et al., 2015) resulting in beneficial microbes in plant extract neutralize free
DNA damage and causing cell death (Wang et al., 2019). radicals and prevent cell division (Shen et al., 2018).
Our results are consistent with the study of Cvetanović et Thus, fermented extract through beneficial microbes
al. (2015). These results indicated that fermentation effects the food quality and prolong the food life.
enhance the cytotoxic activity of phytoextract by
Figure 2. Effect of M. arvensis L. extract on 3T3-L Cell’s viability. M.E represent menthol extract, DW. E
represent distilled water extract and FM.E represent Fermented menthol extract
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Asghar et al., J. Anim. Plant Sci., 32 (3) 2022
Antioxidant potential: Antioxidant activity of different compounds is linked with cytotoxicity because many
concentrations on the reducing power of DW. E, M.E and compounds revealed both antioxidant and cytotoxic
FM. E showed in (Figure 3). Our results revealed that activities (Benabdallah et al., 2018). These results
reducing power of all extracts increased with increase in suggest that phytochemicals of M. arvensis has
concentration, but FM. E was found more active outstanding potential to donate electron to free radicals
(1.47±0.03) than DW. E (1.68±0.06) and M.E resulting in production of stable non-reactive species and
(1.64±0.13). Hence it is confirmed that DPPH activity of restricting the free radical chain reaction (Bardaweel et
fermented plant extract increased two times as compared al., 2018).
to other solvents. Usually antioxidant activity of different
Figure 3. DPPH radical scavenging activity of M. arvensis L. extracts. M.E represent menthol extract, DW. E
represent distilled water extract and FM.E represent Fermented menthol extract.
Microbial growth inhibition: We found that all M. of active constituents (Murugan et al., 2018). Apart from
arvensis extracts are active against bacterial strains S. this modifying plant extracts with fermentation showed
aureus and P. aeruginosa used in this study (Figure 4A synergetic effect in controlling microbial growth. It
and B). FM. E showed maximum inhibition against both releases cellulase enzyme which break down the plant
strains S. aureus and P. aeruginosa and significant cellulose and activate secondary metabolites more
difference as compared to control, M.E and DW. E efficiently (Kadhim et al., 2016). Therefore, the use of
(Table 2). M.E showed significant inhibition at all plant extracts specifically fermented plant extracts can
concentrations (0.1, 0.250 and 0.500 mg/mL) against help to prolong the preservation period of the food.
both strains S. aureus and P. aeruginosa whereas, DW. E Fermented plant extracts prevent oxidation in the food
revealed significant difference as compared to control material eventually preventing the food material from
(Table 2). Previous results indicated that bactericidal bacterial attack.
effects of plant extracts usually because of the presence
S. aureus P. aeruginosa
Extracts DW. E M.E FM. E Control FM. E M.E DW. E Control
0.100 mg/mL 2.15±0.47a 3.96±0.45b 4.20±0.90c 0.26±0.15d 3.23±0.32b 3.06±0.31ab 2.77±0.27e 0.12±0.06d
0.250 mg/mL 1.36±0.16b 3.47±0.49a 4.17±0.24ab 0.19±0.17c 2.86±0.39d 2.73±0.24d 2.72±0.24d 0.19±0.17c
0.500 mg/mL 1.47±0.23b 3.34±0.52a 3.26±0.66a 0.3±0.1c 3.08±0.29a 3.11±0.27a 2.85±0.07d 0.19±0.11c
Antibacterial activity was assessed by measuring the zone of inhibition (mm). Different letters in the same row indicate significant
(P<0.05) differences
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Asghar et al., J. Anim. Plant Sci., 32 (3) 2022
Figure 4. Antibacterial activity of M. arvensis L. extracts against bacterial strains. (A) Staphylococcus aureus
activity (B) Pseudomonas aeruginosa activity
Effect on chicken meat lipid oxidation and pH: showed decrease in pH as compared to DW. E and M.E
Application of M. arvensis showed significant potential which proved that presence of microbial enzymes in
to maintain chicken meat lipid oxidation and pH. extracts result in pH decrease. It is understood that pH
and antioxidant concentration strongly influence the
pH control: Initial pH of all treatments was observed
storage ability of the food. The decrease in the pH might
from 5.8-6.1 (Table 3), whereas all samples showed
be due to the release of antioxidant compounds present in
increase in pH values from day first of storage to the last
the extract or transformation into new compounds due to
day. Our results showed that control showed increase in
fermentation process (Arabshahi-D et al., 2007). Our
pH after day 10 to end of the storage significantly higher
results indicated that FM.E can be use as potential pH
than M. arvensis all extracts. It means that treatment of
control agent in the food industry.
food with phytogenic extracts can prevent pH increase in
food and prolong storage time. In addition, FM. E
Lipid metabolism: All tested extracts screened were chicken meat pH and lipid oxidation which provide an
effective as antioxidants yield lower percentage of avenue for the use of fermented plant extracts in food
TBARS values over the 30 d period as compared to industry to produce health-oriented food.
control (Figure 5). The TBARS values of F.ME and M.E
FT-IR spectroscopy: To verify the effect of all plant
were significantly lower than DW. E and control. The
extracts on chicken meat and variations within plant
phenomena involve plant extract slow down oxidation
extracts FT-IR spectra was obtained. The FT-IR spectra
and lipid free radicals are more stable due to the presence
measured from all treatments are shown in Figure 6. The
of organic substances, which prolong the reaction time
major and common spectra observed in all samples
(Amaral et al., 2018). Lipid oxidation is the main process
including control associated with the presence of free OH
responsible for declining nutritional values and taste and
stretching vibrations at 3878.69 cm-1, free NH at 3459.56
aroma of food (Font-i-Furnols Guerrero, 2014). In food
cm-1 and H-NH bond stretching 3388.02 cm-1 and these
industry, it is mainly controlled by synthetic antioxidants
peaks are attributed to water and principal organic
due to low price but cause serious health problems
compounds of these samples (Rodiles‐López et al.,
(Abootalebian et al., 2016). Therefore, M. arvensis
2019). Another common band (2892.69-2699.06 cm-1) in
extract used in this study provide can be used as a natural
all samples associated with O-CH3 asymmetrical
antioxidant in food industry. Application of plant extracts
stretching vibration (Figure 6 A-C) (Prakash et al., 2018).
and fermentation played synergistic role in controlling
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Asghar et al., J. Anim. Plant Sci., 32 (3) 2022
Figure 6. Comparative FT-IR spectra of various plant extracts (A) M.E treated chicken meat spectra (B) DW. E
treated chicken meat spectra (C) FM. E treated chicken meat spectra (D) Control Chicken meat treated
spectra
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Asghar et al., J. Anim. Plant Sci., 32 (3) 2022
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