Brazilian Journal of

Article
Pharmaceutical Sciences
[Link]

Green synthesis of silver nanoparticles using leaf

extracts of Mentha arvensis Linn. and demonstration
of their in vitro antibacterial activities
Santhanamari Thiyagarajan1*, Subramanian Kanchana2

Department of Medical Laboratory, Technology, College of Applied Medical Sciences,

1

Northern Border University, Arar, Kingdom of Saudi Arabia, 2Department of Chemistry,

Quaid-E-Millath Government College for Women (Aut.), Chennai, India

A study was carried out to synthesize silver nanoparticles (Ag-NPs) using plant extracts and
to explore their pharmaceutical application as antibacterial agents. Dried leaves of Mentha
arvensis Linn. were powdered and extracts were prepared using three different organic solvents.
Preliminary screening on antibacterial activity by agar well diffusion method indicated that
the methanolic extract has superior potential than the other two. Phytochemical analysis of
the leaf extract revealed that it possesses tannins, steroids, terpenoids and flavonoids. Ag-NPs
prepared using this extract were of spherical to cuboidal shape ranging in size from 40 to 70 nm.
It retained essential chemical groups and had the required surface plasmon resonance. Further
to the antimicrobial assay, the Ag-NPs, in contrast to the whole methanolic leaf extract, showed
better (100% vs. 80%) bactericidal activity against the indicator organisms. The zones of growth
inhibition for gram positive bacteria ranged between 14±0.6 and 23±0.3, while it was between
12±0.6 and 22±0.2 for gram negative bacteria. The Ag-NPs presented the minimum inhibitory
concentration values of 250 µg/mL for gram positive bacteria and 250 – 500 µg/mL for gram
negative bacteria. These findings suggest that the natural compounds present in M. arvensis L.
possess the potency to facilitate the synthesis and antibacterial action of Ag-NPs.

Keywords: Mentha arvensis. Leaf extract. Silver nanoparticles. Characterization. Antibacterial.

INTRODUCTION the world. It is commonly known as field mint, corn mint,

podina and so on. It has long been known as a herb of choice
Herbalism or botanical medicine, studied under the for food seasoning and food decoration purposes.
broad category of Pharmacognosy, is a traditional or folk Wild mint is often used for domestic herbal remedy
medicine practice based on the use of plants and plant as it offers numerous health benefits. Studies have reported
extracts. Many of the herbs and spices used by humans to that this plant owns a variety of chemicals like menthol,
season the food encompass useful medicinal compounds. menthofuran, isomnethone, α pinene, carvone, piperitenone
Mentha arvensis Linn. is a small perennial herb with a oxide, linalyl acetate, tannin, phenols, flavonoids, terpenes,
branched stem bearing trifling blue flower clusters on a alkaloids, eugenol, steroids, α-tocopherol, glycosides,
spike of scented green leaves and growing up to 75 cm (Nair, sugars and so on. It also possesses the flavonoids such as
Chanda, 2007). It belongs to the family Lamiaceae which menthoside, quercetin and isorhoifolin. The oil yield is 5%
includes roughly of 200 genera and more than 4000 species by distillation of leaves, which contain 40-50% menthol
(Snoussi et al., 2015). It is believed that it was originated (Santos et al., 2010; Pandey, Dubey, Saini, 2010; Biswas,
in Eurasia and now available in different habitats around Saha, Ali, 2014). The leaves, in particular, have substantial
pharmacological properties. Studies in recent years have
*Correspondence: S. Thiyagarajan. Assistant Professor of Microbiology demonstrated the antiviral, antibacterial (Gardiner, 2000;
& Immunology. College of Applied Medical Sciences. Northern Border
University. Arar, P.O. Box: 01321. Zip code: 91431, Kingdom of Saudi
Coutinho et al., 2009), antifungal (Pramila et al., 2012)
Arabia. Phone: +966 - 559354527. E-mail: drsthiyagarajan@[Link], and anti-helminthic (Sugandhi, Bai, 2011) properties of
Thiyagarajan.S@[Link]. ORCID number: [Link]
7943-8707. Scopus Author ID: 57191414106 M. arvensis. The antimicrobial property of this plant is

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 Santhanamari Thiyagarajan, Subramanian Kanchana

attributable to its possession of essential oil (Snoussi Silver has long been utilized since ancient times as
et al., 2015). Some researchers have established that an important agent in biological sciences, particularly
the secondary metabolites such as tannins prevent the in medicine, for various purposes. It is considered as
growth of microorganisms by way of inhibiting the a safe antimicrobial agent as it causes high lethality to
enzymes required for protein synthesis and oxidative bacteria while exerting minimal toxicity to animal cells
phosphorylation (Satya Prasad et al., 2015; Sosnowska (Hembram et al., 2018; Banerjee et al., 2017; Ali et al.,
et al., 2017). 2015). Studies have reported that owing to their extreme
In recent years, the effectiveness of antimicrobial stability and unique catalytic and biological properties,
chemotherapy is being questioned due to increasing silver nanoparticles (Ag-NPs) display considerable
incidences of complications, development of resistance antibacterial and antioxidant activities (Hembram et
by bacterial pathogens and deposition of drug residues al., 2018; Abdel-Aziz et al., 2014). Manjari et al. (2018)
in feed and environment (Mehdi et al., 2018; Hembram suggested that by virtue of these properties, the Ag-NPs
et al., 2018). As the plant based natural products are could serve as potential candidates for catalytic and
endowed with antimicrobial properties, there has been recycling applications. Researchers in recent years have
growing interest in carrying out research in this area. explored the extracts of different plants for the synthesis of
Interestingly, these phytochemicals, besides exerting Ag-NPs. However, common edible plants are often much
direct lethality, have the potency to modify the natural sought after for significant research studies owing to their
antibiotic resistance of pathogenic microorganisms commonness and easy availability (Saikia et al., 2015).
through facilitating the elimination of resistance coding Emergence and eventual complications caused by
plasmids and inhibition of drug transport via plasma antibiotic resistant bacteria, such as vancomycin resistant
membrane (Coutinho et al., 2009). S. aureus, extended spectrum beta-lactamase producing
Nanoscience and nanotechnology, dealing with Escherichia coli and Pseudomonas aeruginosa in recent
the materials of 1-100 nm size, are the fast developing decades is considered as a global threat to health care
sciences of 21st century. Owing to the morphology, size system from public health view point. In order to combat
and distribution characteristics the nanoparticles (NPs) such precarious pathogens, safe drugs of natural origin
exhibit different physical and chemical properties. In that are prepared using environment friendly and cost
recent years, the NPs have become essential objects in effective methods should be the high priority need in
various emerging fields dealing with optoelectronics, the present scenario. Therefore, the present study was
biosensors, nanocatalysts and so on (Parashar et al., carried out to synthesize Ag-NPs using leaf extract of
2009; Masurkar et al., 2011). Since the introduction of M. arvensis L. and characterize their properties and also
nanotechnology phenomenal modernizations have been to evaluate their antibacterial potential.
taking place in the field of medical sciences (Banerjee
et al., 2017). Compared to bulk metals, the metal NPs MATERIAL AND METHODS
display extraordinary catalytic properties by virtue of
their higher surface area-to-volume ratio (MubarakAli Material
et al., 2011). As the usual physical and chemical methods
of synthesis of metal NPs are encountered with higher Mint plant (Mentha arvensis Linn.) was collected
production cost and environmental pollution, synthesis from a local vegetable market in Egmore area, Chennai,
using plants and microbes is gaining vested interest in India. Plant material was identified and authenticated by
recent years. In green synthesis, the phytochemicals the experts in the Department of Botany, Quaid-E-Millath
present in the plants parts such as leaves, fruit peels, Govt. College for Women (Aut.), Chennai. Leaves of the
bark, callus and root are explored as bio-reductants as plant were removed and cleaned twice in sterile water.
well as capping agents for the synthesis of NPs, thus The wet leaves were spread over a tissue paper sheet and
becoming advantageous (Hembram et al., 2018). left for drying under shade for about 2-3 weeks. Then,

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Green synthesis of silver nanoparticles using leaf extracts of Mentha arvensis Linn. and demonstration of their in vitro antibacterial activities

the dry leaves were ground using a mechanical blender cut using a sterile cork borer. These plates were seeded
and the fine powder was collected in a sterile container. respectively with fresh cultures (18-24 h) of indicator
Solvents such as methanol, petroleum ether and bacteria (0.5 McFarland’s turbidity standard) by swab
chloroform (each of >95% purity) were procured from inoculation. Stock solution (1 mg/mL) of the drug was
Sisco Research Laboratories (India). Silver nitrate prepared using DMSO and four different volumes viz.,
(AgNO3) of 99.5% purity was purchased from Sigma- 25 µL, 50 µL, 75 µL and 100 µL (corresponding to the
Aldrich (USA). Mueller Hinton broth and agar media concentrations of 250, 500, 750 and 1000 µg/mL) of this
required respectively for maintaining bacterial cultures solution were loaded into four wells of MHA plate. Fifth
and antibacterial assay were received from HiMedia Ltd. well was loaded with 100 µL of DMSO, which served as
(India). Dimethyl sulfoxide (DMSO) and other analytical the control. Plates were incubated at 37oC for 24 h and
grade chemicals used for phytochemical analysis were diameter of zone of growth inhibition around each well
obtained from Merck (India). was measured using calibrated scale. Zone diameter of
>10 mm was considered positive for antibacterial activity.
Preparation of leaf extracts
Analysis of phytochemicals of leaf extract
Extracts of the leaves of M. arvensis L. were
prepared using three different solvents namely, methanol, Crude solvent extract of M. arvensis L. leaves was
petroleum ether and chloroform. Twenty grams of dry leaf
tested by standard procedures to analyze and determine
powder was mixed with 100 mL of solvent in a conical
the presence of various phytochemical constituents
flask and kept in rotary shaker at 150 rpm for 24 h. Then,
(Pramila et al., 2012; Mojab et al., 2003; Ghani, 2003)
it was filtered in a glass beaker and evaporated to make
as discussed below:
a final volume of one-fourth of the original volume. It
was stored at 4°C until further studies.
Tannins: Fifty milligram of the extract was mixed with
5 mL of distilled water, heated in water bath and filtered.
Screening of antibacterial activity of leaf extracts
Ferric chloride was added to this filtrate slowly until it
turned dark green indicating the presence of tannins.
The extracts of M. arvensis L. leaves were tested
for antibacterial activity against five indicator bacteria
Saponins: The plant extract of about 0.2 g was mixed
including one gram positive (Staphylococcus aureus)
and four gram negative bacteria (Escherichia coli, with 5 mL of distilled water and boiled. Presence of
Klebsiella pneumoniae, Peudomonas aeruginosa and saponins was confirmed by persistent frothing.
Proteus mirabilis). These bacteria were obtained from the
laboratory of Department of Microbiology, Institute of Steroids: To 20 mg of the extract, 1 mL of methanol was
Basic Medical Sciences, University of Madras, Chennai, added and filtrated. Subsequent addition of 1 mL conc.
where they were isolated from clinical specimens of H2SO4 resulting in yellow green fluorescence indicated
wound, stool, sputum and urine. the presence of steroids.
In order to screen the antibacterial activity of
different leaf extracts of M. arvensis L. the method of Terpenoids: The extract measuring 0.5 g was mixed in 2
agar well diffusion (Sugandhi, Bai, 2011; Singh, Shushni, mL of chloroform. Conc. H2SO4 was carefully added over
Belkheir, 2015) was adopted with slight modifications. to this mixture to form a layer. Reddish brown coloration
Briefly, five plates of sterile Mueller Hinton Agar (MHA) of interface confirmed the existence of terpenoids.
were prepared and each plate was labelled for testing the
drug (leaf extract) against an indicator bacterium. In each Flavonoids: The plant extract (0.2 g) was dissolved in
plate five wells each measuring 5 mm diameter were diluted NaOH. A slow addition of HCl to this solution

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 Santhanamari Thiyagarajan, Subramanian Kanchana

caused change of color from yellow to colorless, thus composition was achieved through Energy Dispersive
indicating the presence of flavonoids. X-ray analysis.

Anthraquinone: To 5 mL of chloroform 0.5 g of the FT-IR analysis: Fourier transform infrared spectroscopy
extract was added and shaken for 5 min. and filtered. The (Perkin Elmer system, USA) was adopted for detection
filtrate was mixed with an equal volume of 10% ammonia of reducing agents from the plant extract and also
solution and shaken until the ammonia layer turned pink for determining the size distribution, identification
violet or red showing positive result. of chemical bonds and functional groups of Ag-NPs.
Spectroscopic grade potassium bromide (KBr) was
Synthesis and characterization of silver dissolved (1:100) in extract and the FTIR spectra of Ag-
nanoparticles (Ag-NPs) NPs were recorded at the diffuse mode of reflectance
before and after the addition of AgNO3.
In order to synthesis the silver nanoparticles the
cold extraction method (Kelkawi, Kajani, Bordbar, 2017) Assay of antibacterial property of Ag-NPs
was followed. First of all, crude leaf extract (0.5 g) of M.
arvensis L. was added to 100 mL of de-ionized water Toxicity of Ag-NPs on bacterial growth was
and kept in vigorous stirring for 1 h. Then, 100 mL of tested by following agar well diffusion method (Singh,
AgNO3 (1×10 −3 M) was added to this and incubated at Shushni, Belkheir, 2015) as described above. Results were
room temperature (25°C) in dark for 48 h. Formation expressed in terms of minimal inhibitory concentration
of Ag-NPs was confirmed by observing the change of (MIC), i.e., the lowest concentration of drug which can
color of solution into brown. The mixture thus obtained cause visible inhibition of bacterial growth.
in this process was lyophilized to obtain the powder
of Ag-NPs. Characterization of Ag-NPs was carried Statistical analysis
out using UV-visible Spectrophotometry, SEM-EDX
analysis and FT-IR analysis and by carefully following Experiments were carried out in triplicate and the
the protocols adopted by Hembram et al. (2018), Banerjee results of analyses were presented as mean values ±
et al. (2017), Ali et al. (2015), Elumalai et al. (2010), and standard deviations (SD; n = 3). The data were subjected
Gabriela et al. (2017). to one-way analysis (ANOVA) and compared using
student’s t-test. Differences in values at P ≤0.05 were
UV-visible Spectrophotometry: The optical properties considered statistically significant.
i.e., surface plasmon resonance (SPR) of Ag-NPs and the
bioreduction of Ag+ in aqueous solution were determined RESULTS
using double beam UV-Visible spectrophotometer
(Thermo Spector_IC ~ Visio_pro Software V1.06, USA). Antibacterial activity and phytochemical analysis of
The reaction time was calculated through periodical leaf extract
recording of absorbance values of whole extract and
reduced Ag-NPs at a resolution of 1.5 nm between the The antibacterial efficacy of the solvent extracts was
band width ranges of 400 and 800 nm. determined in relation to their ability of inhibiting the
number of indicator bacteria. Among the three solvent
SEM-EDX analysis: Scanning Electron Microscopy for extracts of leaves of M. arvensis L. tested against five
analysis of physical properties (size, shape and surface indicator bacteria, the chloroform leaf extract (CLE) did
morphology) of Ag-NPs was performed using Hitachi not show any inhibitory effect (0%). While the petroleum
S-4500 SEM machine (Japan). Parallel identification ether leaf extract (PELE) could inhibit only E. coli (20%),
of elements and quantitative information on chemical the methanolic leaf extract (MLE) exhibited lethality

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Green synthesis of silver nanoparticles using leaf extracts of Mentha arvensis Linn. and demonstration of their in vitro antibacterial activities

against all the bacteria except P. aeruginosa (80%) II. The analysis showed the abundant presence of
(Table I). The results of the analysis for phytochemical flavonoids and a moderate presence of tannins, steroids
constituents of the leaf extract are presented in Table and terpenoids.

TABLE I - Results of assay of antibacterial activity of Leaf extracts of M. arvensis Linn. against indicator bacteria

Antibacterial activity of leaf extract

S. no Indicator bacteria Leaf Extract (zone dia. in mm)*
250 µg/mL 500 µg/mL 750 µg/mL 1000 µg/mL
CLE 3 ± 0.9 4 ± 0.4 6 ± 0.3 9 ± 0.7
1. Staphylococcus aureus PELE 6 ± 0.3 7 ± 0.2 7 ± 0.6 8 ± 0.2
MLE 11 ± 0.4 12 ± 0.2 16 ± 0.5 18 ± 0.1
CLE 5 ± 0.9 6 ± 0.8 6 ± 0.7 8 ± 0.2
2. Escherichia coli PELE 5 ± 0.3 7 ± 0.7 10 ± 0.2 12 ± 0.4
MLE 9 ± 0.3 13 ± 0.6 15 ± 0.4 16 ± 0.1
CLE 3 ± 0.4 4 ± 0.5 6 ± 0.1 7 ± 0.6
3. Klebsiella pneumoniae PELE 2 ± 0.9 5 ± 0.4 6 ± 0.8 7 ± 0.8
MLE 4 ± 0.6 8 ± 0.2 12 ± 0.6 13 ± 0.5
CLE 5 ± 0.4 5 ± 0.9 7 ± 0.4 8 ± 0.2
4. Pseudomonas aeruginosa PELE 4 ± 0.3 6 ± 0.6 7 ± 0.2 9 ± 0.1
MLE 4 ± 0.5 6 ± 0.4 7 ± 0.9 9 ± 0.3
CLE 4 ± 0.1 6 ± 0.3 6 ± 0.8 7 ± 0.5
5. Proteus mirabilis PELE 5 ± 0.7 7 ± 0.2 7 ± 0.6 8 ± 0.4
MLE 7 ± 0.7 11 ± 0.5 13 ± 0.3 14 ± 0.8
CLE, Chloroform leaf extract; PELE, Petroleum Ether leaf extract; MLE, Methanol leaf extract. *Mean value of triplicate
tests; Standard deviation +0.953 mm

TABLE II - Phytochemical constituents of methanol extracts of M. arvensis Linn

S. no. Phytochemicals tested Results*

1. Tannins ++

2. Saponins -
3. Steroids ++
4. Anthraquinone -
5. Terpenoids ++
6. Flavonoids +++
*-, negative / absent; ++, moderate presence; +++, abundant presence

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 Santhanamari Thiyagarajan, Subramanian Kanchana

Synthesis and characterization of Ag-NPs of container after the 35th hour of incubation. The UV- Vis
spectroscopy enabled the detection of silver SPR bands
Synthesis of Ag-NPs from the MLE was recognized at 430 nm. Formation of Ag-NPs was recorded through
by the change of color of reaction mixture into dark development of absorption peaks between the ranges 330
brown and development of dense precipitate at the bottom and 500 nm (Figure 1).

FIGURE 1 - UV-Visible Spectroscopy of M. arvensis Linn. mediated silver nanoparticles.

[Absorbance recorded between 300 and 600 nm; Appearance of SPR band at 430 nm]

SEM analysis indicated that the Ag-NPs were small, The FTIR analysis of Ag-NPs revealed that
spherical to square shaped and found in agglomerated the particles were potentially active encompassing
form (Figure 2). Analysis using dynamic light scattering functional groups ranging from 720 to 3413 cm-1
indicated that these particles were of the sizes ranging (Figure 4). Subsequent comparison with standard
from 40-70 nm. While the particles with the sizes of 56 interferrogram showed the occurrence of functional
and 61 nm were abundantly present, the mean size of the groups such as O-H (Alcohols, Phenols), C-H (Alkenes),
particles determined was 60 ± 23 nm endowed with the zeta C-H (Phenyl ring), NO2 (Nitro compound), N-H and
potential values ranging between -18 and -22 mV. The EDX C-N (Amines). While the broad peak at 3003 cm-1
spectrum deciphered the signals corresponding to elemental corresponded to the N-H group of the peptide linkage
silver (2.5-5.4%) and other associated compounds such as occurring in the extract, there was a preponderance of
Carbon, Nitrogen, Oxygen, Sodium, Chlorine, Potassium, alcohol and amine groups.
etc. producing signals with less volume (Figure 3).

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Green synthesis of silver nanoparticles using leaf extracts of Mentha arvensis Linn. and demonstration of their in vitro antibacterial activities

FIGURE 2 - Scanning Electron Micrograph of M. arvensis Linn. mediated silver nanoparticles. A, Spherical nanoparticles; B,
cuboidal nanoparticles

[Particles with sizes ranging 40-70 nm]

FIGURE 3 - EDAX spectrum of M. arvensis Linn. mediated silver nanoparticles.

[Spectrum showing peaks corresponding to C, O, N and Ag compounds]

Braz. J. Pharm. Sci. 2022;58: e19898 Page 7/14

 Santhanamari Thiyagarajan, Subramanian Kanchana
100.0

95

90 1
85

80

75

70

65

60

55

50
%T
720
45

40

35

30

25

20 3003 1377
1456
15 1742 1240
3413 1628 1158
10 2923 1045

5 2853

0.0
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0
FIGURE 4 - FTIR Spectrum of M. arvensis Linn. mediated silvercm-1
nanoparticles.

[Spectrum showing peaks corresponding to different functional chemical groups of Ag-nps]

Antibacterial activity of Ag-NPs proportionate with the ascending concentrations of

drug for the bacteria E. coli and K. pneumoniae were
The Ag-NPs of MLE of M. arvensis L. exhibited 12+0.6 to 19+0.8 and 14+0.1 to 22+0.7 respectively. A
substantial antibacterial activity against all the five comparatively high MIC (500µg/mL) was required for
indicator organisms. Bacteria such as E. coli, S. aureus, the bacteria P. mirabilis (Table III; Figure 5) with zone
K. pneumoniae and P. aeruginosa were inhibited diameters ranging 5+0.8 to 14+0.7. However, higher
even at the lowest concentration (250 µg/mL) of the susceptibility (14+0.6 to 23+0.3) was observed with
drug. The diameters of zones of growth inhibition the gram positive bacteria S. aureus.

TABLE III - Results of assay of antibacterial activity of silver nanoparticles

Antibacterial activity of Ag-nps (zone dia. in mm)*
S. no Indicator bacteria
250 µg/mL 500 µg/mL 750 µg/mL 1000 µg/mL
1. Staphylococcus aureus 14 ± 0.6 18 ± 0.2 20 ± 0.9 23 ± 0.3
2. Escherichia coli 12 ± 0.6 15 ± 0.6 16 ± 0.4 19 ± 0.8
3. Klebsiella pneumoniae 14 ± 0.1 16 ± 0.5 19± 0.4 22 ± 0.7
4. Pseudomonas aeruginosa 13 ± 0.3 17 ± 0.7 18± 0.6 20 ± 0.4

5. Proteus mirabilis 5 ± 0.8 10 ± 0.4 13 ± 0.5 14 ± 0.7

*Mean value of triplicate tests; Standard deviation +0.953 mm

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Green synthesis of silver nanoparticles using leaf extracts of Mentha arvensis Linn. and demonstration of their in vitro antibacterial activities

FIGURE 5 – MHA plates showing antibacterial activity of M. arvensis Linn. mediated silver nanoparticles against indicator
bacteria.

I, S. aureus; II, E. coli; III, K. pneumoniae; IV, P. aeruginosa; V, P. mirabilis

Drug concentrations (µg/mL) A, 250; B, 500; C, 750; D, 1000

DISCUSSION who independently demonstrated the antibacterial activity

of MLE of M. arvensis L. against both gram positive and
Screening of leaf extracts for antibacterial efficacy gram negative bacteria.
A comparative study of antibacterial activities of
For the purpose of preparing leaf extract of M. M. arvensis revealed that the MLE caused more lethality
arvensis L. three solvents namely, chloroform, methanol, on gram positive bacteria compared to gram negative
and petroleum ether were employed in the present study. bacteria. While this extract required an MIC of 500 µg/
Pramila et al. (2012), Singh, Shushni and Belkheir (2015) mL for most of the gram negative bacteria, it inhibited
and Bupesh et al. (2007) in their individual studies with the gram positive bacteria S. aureus even at a lower
another plant Mentha piperita used other solvents such as concentration (250 µg/mL). The relative resistance of
ethyl acetate, butanol, ethanol petroleum ether, chloroform gram negative bacteria to the antibacterial activity of
and so on for extraction of leaf contents. However, the MLE could be due to the presence of lipopolysaccharides
methanolic extract has been advocated as a superior solvent on the cell wall (Singh, Shushni, Belkheir, 2015).
especially in studies pertaining to antimicrobial assays. Interestingly, the MLE of our study showed superior
This is in agreement with the findings of the present study inhibitory activity with a zone diameter of 18 ± 0.1 and
as the methanolic leaf extract (MLE) of M. arvensis L. MIC of 250 µg/mL (Table I) than that of Farah, Zabta and
showed comparatively better activity by inhibiting 80% of Imran (2012) (15.66 ± 0.351; 2.5 mg/mL). The differences
indicator organisms (Table I) including gram positive and in antibacterial activities between these two studies may
gram negative bacteria except P. aeruginosa. Concomitant be attributed to the environmental and soil conditions of
findings were obtained by Sugandhi and Bai (2011), Farah, the plant’s habitat, cultivar and age of the plant, method
Zabta and Imran (2012) and Saxena, Patil and Khan (2011), and extraction of constituents of the plant material.

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 Santhanamari Thiyagarajan, Subramanian Kanchana

Phytochemistry of methanol leaf extract reducing AgNO3 in the process of production of Ag-NPs.
During the reaction with Ag+, the menthol gets converted
Phytochemical analysis of the MLE prepared for into menthone with the release of Ag0 (Sosnowska et al.,
the present study revealed that it possessed constituents 2017; Kamatou et al., 2013).
such as tannins, steroids, terpenoids and flavonoids
(Table II). Similar report has been made from the Physico-chemical characterization of Ag-NPs
studies of Sugandhi and Bai (2011) and Do Nascimento
et al., (2009). It is noteworthy that the biological and The UV-Vis spectrophotometry confirmed the
therapeutic properties of any plant are influenced by presence of silver nanoparticles in the MLE of M.
its phytochemical components. Possession of these arvensis L. through recording the change in color of the
compounds in the leaves of M. arvensis L. could be reaction mixture followed by raise in OD value (> 0.7).
attributed to its various pharmacological properties. The absorption spectra depicted the appearance of surface
Pramila et al. (2012) proposed that the antibacterial plasmon band at 430 nm, which is the characteristic of
and antifungal activity of methanolic leaf extract of Ag-NPs (Figure 1). Similar result has been obtained by
M. piperita is attributable to the presence of tannins Tambe and Tumane (2017), who recorded characteristic
and flavanoids. Besides, the terpenoids, owing to their absorption band for Ag-NPs at 439 nm. As the SPR is
inherent affinity towards the cell membrane, could cause dependent on the morphology and size of nanoparticles
the bacteriolysis through lipolytic activity (Snoussi et al., (Saikia et al., 2015), red shifting of its band indicated
2015). Menthol, principal component of mentha plants, the formation of larger Ag-NPs during the reduction
is added now-a-days in many commercial tooth pastes to reaction caused by the phytochemicals of the extract.
offer protection against oral microbial infections (Biswas, As the phenolics present in the extract are considered
Saha, Ali, 2014). to possess strong antioxidant property (Hembram et
al., 2018; Abdel-Aziz et al., 2014), by way of donating
Green synthesis of silver nanoparticles electrons they could have caused the reduction of Ag+
ions to Ag-NPs.
In recent years, researchers in the field of Occurrence in the medium of dense, aggregated
nanotechnology are showing exhilarated interest in Ag-NPs with a mean size of 60 ± 23 nm was recorded
alternative methods of synthesis of metal nanoparticles using SEM (Figure 2). Some independent studies which
(m-NPs) using natural plants as an eco-friendly synthesized the Ag-NPs from the leaves of another plant
approach. As the plant components possess non-toxic of the genus, M. piperita reported that the particles were
phytochemicals, the green synthesis of m-NPs intends spherical and of varying sizes viz., 5-50 nm (Sosnowska
to employ them as sources of reducing and stabilizing et al., 2017), 12 nm (Manjari et al., 2018), 15-65 nm
agents (Sosnowska et al., 2017; Ali et al., 2015). As the (Parashar et al., 2009) and 90 nm (MubarakAli et al.,
plants belonging to the genus Mentha have been reported 2011) respectively. In contrast to these reports, Ag-NPs
to contain various phytoconstituents, the present study of much smaller sizes viz., 1.9-4.3 nm and 5-30 nm
chose leaf extract of M. arvensis as a natural reducing have been synthesized respectively from the leaves of
agent for synthesizing Ag-NPs. MubarakAli et al. (2011) Eucalyptus globus (Ali et al., 2015) and M. piperita
in their studies on the synthesis of silver and gold NPs (Reddy et al., 2015) in other studies. Occurrence of
using the leaf extract of M. piperita suggested that relatively larger particles may be attributed to the
phytochemicals such as steroids, flavones, alkaloids, influence of lower pH and temperature which tend to
polysaccharides, oximes amino acids, etc. contribute cause lesser nucleation during the reduction reaction
significantly in the reduction of Au+ into Au0. (Ali et al., 2015). The EDX spectrum showing high
Particularly, the menthol, a secondary alcohol present peaks of Ag ions in the synthesis medium (Figure
abundantly in these plants, could play a pivotal role in 3) further confirmed the efficacy of MLE in the

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Green synthesis of silver nanoparticles using leaf extracts of Mentha arvensis Linn. and demonstration of their in vitro antibacterial activities

reduction of AgNO3. Manifestation of large numbers (Sosnowska et al., 2017; Saikia et al., 2015). Gabriela et
of feeble signals revealed the possibility of adherence al. (2017) in their studies on M. piperita demonstrated the
of other chemical elements on the surface of Ag-NPs. antibacterial activity of leaf extract against S. aureus and
Occurrences in the plant extract of phytochemicals E. coli with the respective MIC values of 51.5 and 2.49 µg/
could contribute to the stability of nanoparticles thereby mL, which are much lesser than that of our study. While the
preventing their aggregation (Ali et al., 2015). The better activity of Ag-NPs in their study could be attributed
existence of discrete Ag-NPs, as witnessed in the SEM to smaller size of particles (mean, 50 nm), the reduced
of our study, could be due to the capping effect and performance of Ag-NPs in our study may be due to their
steric hindrance caused by the compounds such as comparatively larger (mean, 60 ± 23 nm) size. Smaller the
tannins, terpenoids, flavonoids, carbohydrates, proteins size of Ag-NPs, the easier would be the transportation of
and other metabolites in MLE. particles across the bacterial cell wall, thereby manifesting
The FTIR analysis revealed the chemical complexity better lethal effects inside the cell.
of Ag-NPs owing to the possession of various functional The overall analysis of antibacterial activity of Ag-
groups (Figure 4). The stability of Ag-NPs in reaction NPs in our study revealed that they are equally toxic to
milieu depends on its stereochemistry which in turn rests both gram positive and gram negative bacteria. However,
on presence of chemical groups. The phytochemicals in terms of producing sizes of inhibitory zone, a sort of
in green synthesis could act as reducing agents and higher lethality was observed with gram positive bacteria
contribute to the steric conformation of Ag-NPs. Tambe than its counterpart. It can be noted from table III that
and Tumane (2017) have suggested that while O-H groups the diameters of growth inhibition caused by Ag-NPs
are offered by the flavonoids, C-H, N and C-O groups are against the gram positive bacteria S. aureus vs. across the
donated by alkaloids, amines and alcohols of the plant gram negative bacteria (maximum zone for each dilution)
extract respectively. were 14 ± 0.6 vs. 14 ± 0.1, 18 ± 0.2 vs. 17 ± 0.7, 20 ± 0.9
vs. 19± 0.4 and 23 ± 0.3 vs. 22 ± 0.7 at concentrations
Evaluation of antibacterial activities of Ag-NPs respectively for 250, 500, 750 and 1000 µg/mL (Table III).
Higher susceptibility of gram positive bacteria could be
The main focus of this study, i.e., evaluation of due to the presence of only the peptidoglycan layer which
antibacterial activity of green synthesized Ag-NPs is not a strong barrier to the permeability of Ag-NPs in
revealed that their activities were much superior than their cell wall. The cell wall of gram negative bacteria has
that of whole leaf extract. While the whole leaf extract more complex structure by virtue of a rigid phospholipid
inhibited the growth of four out of five indicator bacteria membrane along with lipopolysaccharide compounds,
(80%), the Ag-NPs exhibited toxicity against all of them which makes it less susceptible to antibacterial agents
even at lower concentrations (100%). This finding inferred (Farah, Zabta, Imran, 2012).
that the utilization of Ag-NPs in antimicrobial therapy has Although the exact mechanism of antibacterial
substantial advantage over the use of whole or purified action of Ag-NPs is not understood yet, some studies have
herbal extract. This is in agreement with the reports of proposed possible ways by which they cause lethality.
Ali et al. (2015), who demonstrated that while the mixture The silver present in the forms of Ag0 and Ag+ interact
of ethanolic extract of E. globulus leaves and Ag-NPs negatively with the cytoplasmic contents and DNA thus
inhibited the bacterial growth at a concentration of 25 preventing the replication and division of bacterial cell
µL (zone dia. 19-21 mm), the lone extract could cause (SivaKumar et al., 2015). Studies have established that the
only insignificant cytotoxic effects (zone dia. 8-10 mm) bacteria are incompetent in developing resistance against
even at a concentration of 100 µL. silver as they do with antibiotics. So, nanoparticles made
Studies conducted in recent years suggest that efficacy out of this metal could be best employed for controlling
of Ag-NPs in exhibiting antibacterial activity is significantly antibiotic resistant bacteria. Contemporary studies
influenced by the plant extract utilized for their synthesis have demonstrated the efficacy of Ag-NPs against drug

Braz. J. Pharm. Sci. 2022;58: e19898 Page 11/14

 Santhanamari Thiyagarajan, Subramanian Kanchana

resisting bacteria such as MRSA (Methicillin-resistant S. work through the project number IF-2020-NBU-218. The
aureus) (Chung et al., 2016), ESBL (extended spectrum authors thank and acknowledge the technical support
beta lactamase) producing P. aeruginosa and E. coli rendered by the Departments of Microbiology and
[18] (Ali et al., 2015) and multidrug resisting (MDR) Nanoscience, University of Madras, Chennai, India.
Salmonella enterica (Das et al., 2017).
In recent years, there has been increasing REFERENCES
demand for ecofriendly and cost effective synthesis
of metal nanoparticles. Our study suggests that proper Abdel-Aziz MS, Shaheen MS, El-Nekeety AA, Abdel-
standardization of synthesis of silver nanoparticles using Wahhab MA. Antioxidant and antibacterial activity of silver
nanoparticles biosynthesized using Chenopodium murale
leaf extract of M. arvensis L. could help in developing
leaf extract. J Saudi Chem Soc. 2014;18(4):356-63.
a promising drug to overcome the infections caused by
antibiotic resistant bacteria. Ali K, Ahmed B, Dwivedi S, Saquib Q, Al-Khedhairy AA,
Musarrat J. Microwave accelerated green synthesis of stable
silver nanoparticles with Eucalyptus globulus leaf extract
CONCLUSION and their antibacterial and antibiofilm activity on clinical
isolates. PLoS ONE. 2015;10(7):e0131178.
Mentha arvensis L. plant, as a whole, is regarded
Banerjee PP, Bandyopadhyay A, Harsha SN, Policegoudra
as a source of multiple nutraceutical and pharmaceutical RS, Bhattacharya S, Karak N, et al. Mentha arvensis
chemicals which offer numerous health benefits. The (Linn.)-mediated green silver nanoparticles trigger caspase
methanolic extract of leaves of this plant is 9-dependent cell death in MCF7 and MDA-MB-231 cells.
comparatively a better source for exploring its Breast Cancer - Targets Ther. 2017;9:265-78.
antimicrobial properties than other extracts. The Biswas NN, Saha S, Ali MK. Antioxidant, antimicrobial,
phytochemical constituents of leaves, especially cytotoxic and analgesic activities of ethanolic extract of
menthol, tannins, flavonoids, terpenoids and steroids Mentha arvensis L. Asian Pac J Trop Biomed. 2014;4(10):792-
97.
could act as natural reductants in the synthesis of silver
nanoparticles. Besides, these compounds contribute to Bupesh G, Amutha C, Nandagopal S, Ganeshkumar A,
the stability and stearic conformation of synthesized Sureshkumar P, Murali K. Antibacterial activity of Mentha
piperita L.(peppermint) from leaf extracts-a medicinal plant.
nanoparticles by way of donating essential chemical
Acta Agric Slov. 2007;89(1):73-79.
groups. Antibacterial activity of nanoparticles is plant
extract dependent and is significantly influenced by Chung IM, Park I, Seung-Hyun K, Thiruvengadam
M, Rajakumar G. Plant-mediated synthesis of silver
its size. Smaller silver nanoparticles exhibit better
nanoparticles: their characteristic properties and therapeutic
antibacterial activity as they can be easily transported applications. Nanoscale Res Lett. 2016;11:1–14.
across the cell membrane. Owing to its higher
Coutinho HD, Costa JG, Lima EO, Falcao-Silva VS,
permeability characteristic, the gram bacteria are more
Siqueira-Junior JP. Potentiating effect of Mentha arvensis
susceptible to silver nanoparticles than gram negative and chlorpromazine in the resistance to aminoglycosides
bacteria. Since plant based natural compounds serve of methicillin-resistant Staphylococcus aureus. In vivo.
as non-toxic reducing and stabilizing agents, green 2009;23(2):287-89.
synthesis of silver nanoparticles could be considered Das B, Dash SK, Mandal D, Ghosh T, Chattopadhyay S,
as a safe and most advantageous advantageous method. Tripathy S, et al. Green synthesized silver nanoparticles
destroy multidrug resistant bacteria via reactive oxygen
ACKNOWLEDGEMENTS species mediated membrane damage. Arabian J Chem.
2017;10(6):862-76.

The authors extend their appreciation to the Do Nascimento EM, Rodrigues F, Campos A, Da Costa
Deputyship for Research & Innovation, Ministry JG. Phytochemical prospection, toxicity and antimicrobial
activity of Mentha arvensis (Labiatae) from northeast of
of Education in Saudi Arabia for funding this research
Brazil. J Young Pharm. 2009;1(3):210-12.

Page 12/14 Braz. J. Pharm. Sci. 2022;58: e19898

Green synthesis of silver nanoparticles using leaf extracts of Mentha arvensis Linn. and demonstration of their in vitro antibacterial activities

Elumalai EK, Prasad TN, Hemachandran J, Therasa SV, Nair R, Chanda SV. Antibacterial activities of some
Thirumalai T, David E. Extracellular synthesis of silver medicinal plants of the western region of India. Turk J Biol.
nanoparticles using leaves of Euphorbia hirta and their 2007;31:231-36.
antibacterial activities. J Pharm Sci Res. 2010;2(9):549-54.
Pandey RR, Dubey RC, Saini S. Phytochemical and
Farah G, Zabta KS, Imran A. Screening of indigenous antimicrobial studies on essential oils of some aromatic
knowledge of herbal remedies for skin diseases among local plants. Afr J Biotechnol. 2010;9(28):4364-68.
communities of north west Punjab, Pakistan. Pak J Bot.
2012;44(5):1609-16. Parashar V, Parashar R, Sharma B, Pandey AC. Parthenium
leaf extract mediated synthesis of silver nanoparticles: a
Gabriela ÁM, Gabriela MD, Luis AM, Reinaldo PR, Michael novel approach towards weed utilization. Digest J Nanomat
HM, Rodolfo GP, et al. Biosynthesis of silver nanoparticles Biostruct. 2009;4(1):45-50.
using mint leaf extract (Mentha piperita) and their
antibacterial activity. Adv Sci Engin Med. 2017;9(11):914-23. Pramila DM, Xavier R, Marimuthu K, Kathiresan S,
Khoo ML, Senthilkumar M, et al. Phytochemical analysis
Gardiner P. Peppermint (Mentha piperita). The Center for and antimicrobial potential of methanolic leaf extract of
Holistic Education and Research. Revised May. 2000;2:1-22. peppermint (Mentha piperita: Lamiaceae). J Med Plants Res.
2012;6(2):331-35.
Ghani A. Medicinal plants of Bangladesh: Chemical
constituents and uses. 2nd ed. Dhaka: Asiatic Society of Reddy MC, Murthy KR, Srilakshmi A, Rao KS, Pullaiah
Bangladesh; 2003, p. 1-16, 381p. T. Phytosynthesis of eco-friendly silver nanoparticles and
Hembram KC, Kumar R, Kandha L, Parhi PK, Kundu CN, biological applications–a novel concept in nanobiotechnology.
Bindhani BK. Therapeutic prospective of plant-induced silver Afr J Biotechnol. 2015;14(3):222-47.
nanoparticles: application as antimicrobial and anticancer Saikia D, Gogoi PK, Phukan P, Bhuyan N, Borchetia S,
agent. Artif cells Nanomed Biotechnol. 2018;46(Sup 3):1-4. Saikia J. Green synthesis of silver nanoparticles using
Kamatou GPP, Vermaak I, Vilioen AM, Lawrence BM. Asiatic Pennywort and Bryophyllum leaves extract and their
Menthol: A simple monoterpene with remarkable biological antimicrobial activity. Adv Mater Lett. 2015;6(3):260-64.
properties. Phytochemistry. 2013;96:15–25. Santos AK, Costa JG, Menezes IR, Cansanção IF, Santos
Kelkawi AH, Kajani AA, Bordbar AK. Green synthesis of KK, Matias EF, et al. Antioxidant activity of five Brazilian
silver nanoparticles using Mentha pulegium and investigation plants used as traditional medicines and food in Brazil.
of their antibacterial, antifungal and anticancer activity. IET Pharmacogn Mag. 2010;6(24):335-38.
Nanobiotechnol. 2017;11(4):370–76. Satya Prasad M, Suman Joshi DSD, Narenda K, Nadiya SK,
Manjari G, Saran S, Devipriya SP, Rao AVB. Novel Synthesis Masthani SK, Padmaja Phani N, et al. A comparative study
of Cu@ZnO and Ag@ZnO Nanocomposite via Green of phytochemical analysis and in vitro antimicrobial activity
Method: A Comparative Study for Ultra-Rapid Catalytic and of three important vegetables from Brassicaceae family. Int J
Recyclable Effects. Catalysis Lett. 2018;148(8):2561-71. Res Ayurveda Pharm. 2015;6(6):767–72.

Masurkar SA, Chaudhari PR, Shidore VB, Kamble SP. Rapid Saxena R, Patil P, Khan SS. In vitro antibacterial effect of
biosynthesis of silver nanoparticles using Cymbopogan some plant essential oils against Staphylococcus aureus.
citratus (lemongrass) and its antimicrobial activity. Nano- Plant Archives. 2011;11(1):563-65.
Micro Lett. 2011;3(3):189-94.
Singh R, Shushni MAM, Belkheir A. Antibacterial and
Mehdi Y, Montminy MPL, Gaucher ML, Chorfi Y, Suresh antioxidant activities of Mentha piperita. L. Arabian J Chem.
G, Rouissi T, et al. Use of antibiotics in broiler production: 2015;8(8):322–28.
Global impacts and alternatives. Anim Nut. 2018;4(2):170-78.
SivaKumar T, Rathimeena T, Thangapandian V, Shankar T.
Mojab F, Kamalinejad M, Ghaderi N, Vahidipour H. Silver nanoparticles synthesis of Mentha arvensis extracts
Phytochemical screening of some Iranian plants. Iran J and evaluation of antioxidant properties. Biosci Bioengin.
Pharm Res. 2003;9:77-82. 2015;1(2):22-28.

MubarakAli D, Thajuddin N, Jeganathan K, Gunasekaran Snoussi M, Noumi E, Trabelsi N, Flamini G, Papetti A, De

M. Plant extract mediated synthesis of silver and gold Feo V. Mentha spicata essential oil: chemical composition,
nanoparticles and its antibacterial activity against antioxidant and antibacterial activities against planktonic
clinically isolated pathogens. Colloid Surf B: Biointerface. and biofilm cultures of Vibrio spp. strains. Molecules.
2011;85(2):360–65. 2015;20(8):14402-24.

Braz. J. Pharm. Sci. 2022;58: e19898 Page 13/14

 This is an open-access article distributed under the terms of the Creative Commons Attribution License.

Santhanamari Thiyagarajan, Subramanian Kanchana

Sosnowska M, Kutwin M, Adamiak A, Gawin K, Bugajska

Z, Daniluk K. Green synthesis of silver nanoparticles by
using aqueous mint (Mentha piperita) and cabbage (Brassica
oleracea var. capitata) extracts and their antibacterial
activity. Annals Warsaw Univ of Life Sci-SGGW Anim Sci.
2017;56(1):137-45.
Sugandhi BRM, Bai GM. Anti-microbial activity of Mentha
arvensis L. Lamiaceae. J Adv Lab Res Bio. 2011;1(1):11-14.
Tambe NA, Tumane PM. Mentha piperita silver nanoparticles
controlling the antibiotic resistance in bacteria and fungi – A
study. J Pharm Res. 2017;11(5):531-37.S
Received for publication on 23rd November 2019
Accepted for publication on 04th October 2020

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