UJMR, Volume 6 Number 2, December, 2021, pp 46 - 53 ISSN: 2616 - 0668

https://doi.org/10.47430/ujmr.2162.007

Received: 2nd November, 2021 Accepted: 29th November, 2021

Liquid chromatography mass spectrometry Profile and In vitro Antimicrobial

Potentials of Mentha piperita (Mint) Hexane Extract on some Food-borne
pathogens

Musa Bashir*1 and Ruqayyah A. Usman2

1. Centre for Dryland Agriculture, Bayero University, Kano-Nigeria.
2. Department of Microbiology, Faculty of Life Science, Bayero University, Kano-Nigeria.
*Corresponding Author: Musa Bashir; [email protected] +2348039728221
Abstract
Mint herbs have been reported to possess several biological effects, and the dried leaves are
traditionally used for herbal tea or medicine. Extraction of plant materials was by maceration
method. Both preliminary qualitative screening of phytochemicals and Liquid Chromatography
Mass Spectrometer (LCMS) profiling revealed many useful metabolites. The phytochemicals
include: Saponin, Carbohydrate, Alkaloids, Cardiac glycoside, and Steroid. Some LCMS compounds
among others were also evaluated such as Quassin, Epioxylubimin, furocoumarinic acid glycoside
and Dioctylamine. The antimicrobial activity of Mentha piperita extract against some food-borne
pathogens such as Escherichia coli, Salmonella spp., Enterobacter spp. and Staphylococcus aureus
were evaluated using agar well diffusion method at different concentrations ranging from 2000
µg/ml - 250µg/ml. The results revealed that hexane mint extracts had activity against the
bacterial isolates. The highest activity was against E. coli (11.50 ±1.50) while the least activity
was against Enterobacter spp (6.14 ± 0.13). The potential bioactive metabolites identified could
be responsible for the antimicrobial activities observed. These findings have thus proven that
Mentha piperita is more effective against E. coli. Therefore this extract could be used in the
control and prevention of many food-borne pathogens and can be of importance in developing
new biopreservatives.
Key words: Metabolite, phytochemical, Mint, Antimicrobial, Pathogen.

INTRODUCTION reported the use of mint extract as an

Plants have always been a good source of antioxidant and antimicrobial bioactive natural
medicine and have been used from time extract (Wang et al., 2018). Many studies have
immemorial to heal various ailments (Natarajan revealed the inhibitory ability of this plant
et al., 2003). The genus Mentha belongs to the depending on the type of bacteria and its
Lamiaceae family, which include many species strong antimicrobial ability against Gram-
that differ widely in characteristics and positive bacteria, especially S. aureus (Talei et
cytogenetics (Ali et al., 2002). The fresh leaves al., 2017). Phytochemicals generally
(Mentha piperita) are commonly used as a raw have biological activity in the plant host and
vegetable or flavoring herb, whereas the dried play a role in plant growth or defence against
leaves are traditionally used for herbal tea or competitors, pathogens or predators (Molyneux
medicine (Natarajan et al., 2003). Mint et al., 2007).
(Mentha piperita) is a perennial herb with a Due to failure of some certain antibiotics to
unique aroma and colourful history. Mentha deliver a therapeutic potential and high cost,
piperita has been reported to be used most especially in low income or developing
internally as tea, tincture, oil or extracts, and nations, there is need to search for an
externally as rub or liniment (Sujana et al., alternative source of therapeutics particularly
2013). Mint herbs have been documented to from plant and to evaluate its metabolite
possess several biological effects, including contents using sensitive tool like Liquid
antioxidant, anti-inflammatory, anticancer, and Chromatography Mass Spectrometer (
antimicrobial activities (Wang et al., 2018). LCMS).Therefore this study is aimed to
The antioxidant activity of this plant determine the antimicrobial activity of Mentha
exclusively relies on its chemical composition piperita leave Hexane extract onsome food
and can prevent oxidative stress at the cellular pathogens and identify its LCMS profile.
level or in a living organism. Other studies have

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MATERIALS AND METHODS procedures. The crude extract was diluted with
Collection of Plant Materials hexane to the concentration of 1 mg/ml. The
Fresh mint leaves were obtained commercially qualitative phytochemical analysis of crude
from Bakin dogo market, Kaduna, Nigeria. It hexane mint leave was conducted to determine
was taken to herbarium unit of Biological the presence of secondary metabolites
Sciences Department of Bayero University Kano, (Tannins, Saponins, Flavonoids, Alkaloids,
where it was identified and a voucher specimen Steriods, Cardiac glycosides and Anthraquinone)
no. BUKHAN 337 deposited. The leaves were (Modupe et al., 2017).
washed first under running tap water and dried Liquid Chromatography Mass Spectrometry
at room temperature in dark. It was then (LCMS) Profile Analysis
grinded into smooth powder and kept in clean The samples were analysed using liquid
polythene nylon as reported by Modupe et al. chromatography (LC) tandem mass
(2017). spectrophotometer (MS) as described by
Extraction of Mentha piperita leaves (Piovesana et al., 2018) with some
The Mentha piperita leaves were extracted modifications. The extracted samples were
using hexane as reported by Modupe et al. reconstituted in Methanol and filtered through
(2017). Forty grams (40g) of the plant material polytetrafluoroethylene (PTFE) membrane filter
was soaked in 200ml of solvent (Hexane). The with 0.45 μm size. After filtration, the filtrate
mixture was left for 48 hours, and then it was (10.0 μl) was injected into the LC system and
filtered using a nylon sieve. The extract was allowed to separate on Sunfire C18 5.0µm
evaporated and kept labelled as the hexane 4.6mm x 150 mm column. The run was carried
extract. out at a flow rate of 1.0 mL/min, Sample and
Phytochemical Analysis Column temperature at 25oC. The mobile phase
Mentha piperita leave extracts was used for consists of 0.1% formic acid in water (solvent A)
preliminary qualitative screening of and 0.1% formic acid in Acetonitrile (solvent B)
phytochemicals as per standard biochemical with a gradient as in Table 1:

Table 1: Solvent Gradient

Time %A %B
0 95 5
1 95 5
13 5 95
15 5 95
17 95 5
19 95 5
20 95 5
From ratio of A/B 95:5 this ratio was maintained for further 1 min, then A/B 5:95 for 13min, to
15min. then A/B 95:5 to 17min, 19min and finally 20min. the Photodiode Array (PDA) detector was
set at 210-400nm with resolution of 1.2nm and sampling rate at 10 points/sec. The mass spectra
were acquired with a scan range from m/z 100 – 1250 after ensuring the following settings: ESI
source in positive and negative ion modes; capillary voltage 0.8kv (positive) and 0.8kv (negative);
probe temperature 600oC; flow rate 10 mL/min; nebulizer gas, 45 psi. MS set in automatic mode
applying fragmentation voltage of 125 V. The data was processed with Empower 3. The compounds
were identified on the basis of the following information, elution order, and retention time (Rt),
fragmentation pattern, and Base peak m/z (Piovesana et al, 2018)

Isolation of Microorganisms from Gurasa Pour plate method was used for plating the
Food pathogens such as Salmonella spp., E. samples. One (1ml) from the dilution 10-5, 10-4
coli, Enterobacter spp. and Staphylococcus and 10-3 were taken using sterile syringe and
aureus were grown and isolated from gurasa as then it was introduced into sterile petri dish,
described by Jideani, (2003). Twenty-five (25g) these were done in duplicate for the food
of homogenized sample was aseptically sample. The prepared media were poured into
weighed and dissolved in sterile 225ml of the Petri dishes containing 1ml of diluted
buffered in a 500ml conical flask to obtain a culture. The plates were swirled to mix
ratio 1:10. One (1ml) of the homogenate was properly. All plates were allowed to solidify and
introduced into a test tube containing 9ml of incubated at 37°C for 24 hours as reported by
the buffered peptone water and labelled 10 -1 Mbajiuka et al. (2014).
dilution and serially diluted into four test tubes
labelled 10-2, 10-3, 10-4 and 10-5

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Viable plate count stock solution of the extracts at concentrations

Colony counting machine was used for counting (2000, 1000, 500, and 250 µg/ml) were
the total aerobic bacteria contained on each introduced into their respective wells. 0.1ml of
plates (Mbajiuka et al., 2014) 250 µg/ml of sodium benzoic acid was
Gram staining technique introduced into the fifth well to serve as a
Smear of each isolate was made on the slide positive control for the bacterial isolates. The
and heat fixed. Primary stain (crystal violet) inoculated plates were left to stand for about
was applied for 45 second and washed with 30 minutes to allow diffusion of extract before
gentle running water. Lugol's iodine was added incubating at 37ºC for 24 hours. The zones of
for 45 seconds and was decolorized with clearance produced around the wells after
acetone - alcohol and washed with clean water. incubation was observed and measured using a
The slides were counter stained with 30% Vernier calliper and recorded (mm). Each of
safranin for 30 second and washed. It was then the experiment was conducted twice and the
air dried and examined at under oil immersion mean result was taken for the test organisms.
lens of the microscope used (Cheesbrough, Determination of Minimum Inhibitory
2006). Concentration and Minimum Bactericidal
Biochemical Tests concentration:
Different biochemical tests such as catalase, The minimum inhibitory concentration (MIC) is
coagulase, oxidase, indole, urease, methyl red, defined as the lowest concentration of the
voges-proskauer and citrate utilization test antimicrobial agent that inhibited visible
were conducted in order to further growth of microorganisms after overnight
characterize the organisms as described by incubation (Andrews, 2002). The doubling micro
cheesbrough, (2006). dilution broth method was used to determine
Preparation of different Concentrations of the MIC. Two (2ml) millilitres of the
the plant extract reconstituted crude extract at a concentration
Stock solution of concentration 2000 µg/ml was of 1000 µg/ml was added to 2ml of sterile
prepared by dissolving 2g of the plant extracts Mueller Hinton broth for the bacterial isolates,
in 1ml of dimetyhyl sulphoxide (DMSO) in glass Two (2ml) millilitres of this extract
vial bottles as reported by Cheesbrough (2006). concentration was transferred serially into test
And then diluted to have varied concentrations tubes numbered 1-9 until the 10th test-tube
(2000µg/ml, 1000 µg/ml, 500µg/ml and was reached, giving extract concentrations
250µg/ml). ranging from 1000-65.2 µg/ml. 0.1ml of an 18h
Standardization of Inoculum culture of bacteria previously adjusted to 0.5
The isolates were adjusted to 0.5 McFarland McFarland standard was inoculated into each of
standard (1.5 X 108 CFU/ml) using sterile the test tubes and the contents were
normal saline. McFarland standards were used thoroughly mixed. A test tube containing the
as a reference to adjust the turbidity of broth and bacteria inoculum was used as
microbial suspension so that the number of negative control. The inoculated culture tubes
microorganisms will be within a given range. were incubated at 37ºC and observed for
For the preparation of the 0.5 McFarland growth after 24 hours. The lowest
standard, 0.05ml of barium chloride (BaCl 2) concentration of extract showing no visible
(1.17% w/v BaCl2.2H2O) was added to 9.95ml of growth when compared with the control was
0.18M H2SO4 (1.0% w/v) with constant stirring. considered as the MIC as demonstrated by
To aid comparison the standard was compared Andrews (2002).
against a white background with a contrasting The minimum bactericidal concentration:
black line (Kalpana et al., 2013). This is the lowest concentration of
Antimicrobial Assay: antimicrobial agent that prevented the growth
The bioassay was carried out using the agar of an organism. 0.1ml aliquot from the tubes
well diffusion method described by that showed no visible bacterial growth from
Cheesbrough (2006). 0.1ml of the standardized the determination of minimum inhibitory
inoculums (1.5 x 108 CFU/ml) of the isolated concentration was inoculated on a sterile
organisms was inoculated onto sterile prepared Mueller Hinton Agar for 24 hours at 37◦C for the
Mueller Hinton Agar and was spread with a bacterial isolate. The lowest concentration in
sterile swab. Five wells were made with a 6mm which no growth occurred was taken as the
sterile cork borer into the agar plates minimum bactericidal concentration (MBC) as
containing the bacterial inoculums and 0.lml of demonstrated (Andrews, 2002).
the four different concentrations from the

48
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RESULTS carbohydrates, alkaloids, cardiac glycosides and

The preliminary phytochemical screening of steroids while tannins and flavoniods were
Mentha piperita hexane extract (table 2) absent.
indicated the presence of saponins,

Table 2: Phytochemical Screening of Mentha Piperita Hexane leave extract

Test Hexane Mint Extract
Saponins +
Tannins _
Carbohydrates +
Flavonoids _
Alkaloids +
Cardiac glycosides +
Steroids +
Key: HME- Hexane mint extract, + = positive, - =negative

Compounds such as Epioxylubimin, Furocoumarinic acid glycoside, Quassin (Quassinoids) and

Dioctylamine (Dialkylamines) were identified from the LCMS profiling studies as reported in table 3.
Both the total chromatogram and fragmentations of the identified metabolites are also presented
in figures 1-5 respectively.

Table 3: Liquid chromatography mass spectrometry Profile of Mentha piperita Hexane Extract
Peak Tentative compounds molecular mass (M) MZ (M+H)
1 Epioxylubimin 269 270
2 Furocoumarinic acid glycoside 366 367
3 Quassin (Quassinoids) 388 389
4 Dioctylamine (Dialkylamines) 241 242
Key: LCMS- Liquid chromatography mass spectroscopy, MZ – Mass to charge ratio

The antimicrobial potential of hexane Mentha piperita leave extract was presented in table 4. The
results revealed the potency of this plant extract against some food pathogens.
Table 4: Antimicrobial Activity of Mentha piperita Extract on Food Pathogens
zone of inhibition (mm)
Organisms Hexane mint extract Control
Conc. (µg/ml) 2000 1000 500 250 250
Staph. aureus 10.5±0.5 8.5±0.5 6.5±0.5 6.0±0 7.5±0.5
Salmonella spp. 9.0±0 7.5±0.5 6.6±0.4 6.0±0 7.5±0.2
E. coli 11.5±1.5 8.0±1.0 6.5±0.5 6.0±0 11.0±3
Enterobacter spp. 6.14±0.13 6.00±0 6.00±0 6.00±0 8.0±0
Key: Control (sodium benzoic acid)
The Minimum Inhibitory Concentration and Minimum Bactericidal Concentration of the hexane mint
extract indicates that the MIC can reduce the growth of most of the microorganisms used in this
studies drastically or eliminate it completely except for Enterobacter spp.

Table 5: Minimum Inhibitory Concentration and Minimum Bactericidal Concentration of Hexane

Mentha piperitaExtract(µg/ml)
Organisms HME (MIC) HME (MBC)
Salmonella 125 125
Staph. aureus 62.5 0
E. coli 62.5 62.5
Key:HME- Hexane mint extract,

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Figure 1: Total Chromatogram of the Hexane extract of Mentha peperita leave

Figure 2: Mass fragmentation of Dioctylamine (Dialkylamines) (242.564 mz)

Figure 3: Mass fragmentation of Epioxylubimin (270.525 mz)

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Figure 4: Mass fragmentation of Furocoumarinic acid glycoside (367.524 mz)

Figure 5: Mass fragmentation of Quassin (Quassinoids) (389.493 mz)

DISCUSSION extract of mint indicate the presence of vital

The present study revealed the presence of metabolites: (Epioxylubimin, Furocoumarinic
phytochemicals such as saponins, acid glycoside, dioctylamine and Quassin
carbohydrates, alkaloids, cardiac glycosides and (Quassinoid) which were identified based on
steroids while tannins and flavonoids were their molecular fragmentation pattern and
absent. This is in agreement with the work of compared to a data base for organic compounds
Sujana et al (2013) who reported the presence (SDBS data base for organic compound). .Most
of steroids and absence of tannins the leave of these compounds were reported severally in
hexane extract of Mentha piperita. But in previous researches to be responsible for
contrast with the work of Sontakke and Shinde antibacterial activities against microorganisms
(2019) who reported the presence of saponins, such as Staphylococcus aureus (Matsuura et al.,
carbohydrates, alkaloids, terpenoids, steroids 2007; Shumaila et al., 2021).
,tannins, phenols and flavonoids in the leave The antimicrobial activity of Hexane mint
hexane extract. This could be due to the extract revealed that, the extract inhibited the
differences of geographical origin of Mentha pathogens with the highest zone of inhibition of
piperita, moreso, Farooq et al. (2007) reported 11.5 ± 0.5, 10.5±0.5 and 9.0±0 against E. coli,
that plants occuring in varying habitats will staphylococcus aureus and salmonella spp.
have variation in the concentration and respectively. However higher zones were
composition of phytochemicals in the different obtained with methanol mint extracts reported
parts of such plants. Previous literatures have by Sujana et al. (2013), which could be as a
emphasized on the contribution of result of polarity differences. Therefore the
phytochemicals in antimicrobial and antimicrobial activity observed in this study
therapeutic properties, so this plant is may be as a results of these phytochemical
expected to have many medicinal uses (Kaur et constituents as well as the vital LCMS
al., 2010). The LCMS profile of the Hexane metabolites.
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The MIC and MBC of the hexane Mentha steroids that could be responsible for the
piperita extract indicated that the MIC that antimicrobial activities observed, and it was
inhibited or retarded the growth of the supported with the results of the LCMS profile
pathogens also killed them completely except that revealed the presence of Epioxylubimin,
for Staphylococcus aureus which was inhibited Furocoumarinic acid glycoside, dioctylamine
at a concentration of 62.50 µg/ml only. This and Quassin (Quassinoid . Additionally some of
indicates that Mentha piperita could have both the metabolites identified have been previously
bacteriostatic and bactericidal properties on reported to be responsible for antimicrobial
pathogens. activity and other medicinal potentials.
Therefore it can be concluded that hexane
CONCLUSION extract of Mentha piperita can be useful in the
The findings of this work revealed that Mentha control and prevention of many food-borne
piperita leave extracthas potential bioactive pathogens and can be of importance in
phytochemicals such as saponins, developing biopreservatives.
carbohydrates, alkaloids, cardiac glycosides and

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