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Original Article

Antibacterial and Antibiofilm Properties of Azadirachta

indica (Neem), Aloe vera (Aloe vera), and Mentha
piperita (Peppermint) against Multidrug‑Resistant Clinical
Isolates
Priya Mehrishi1, Priti Agarwal2, Shobha Broor1, Amisha Sharma3
1
Department of Microbiology, SGT Medical College, Hospital and Research Institute, SGT University, Gurugram, 2Department of Microbiology, ESIC Medical College
and Hospital, Faridabad, Haryana, 3Department of Microbiology, Maharishi Markandeshwar Medical College and Hospital, Maharishi Markandeshwar University,
Solan, Himachal Pradesh, India

Abstract
Background: Misuse of antibiotics globally has resulted in the development of resistant bacterial strains. One of the sole reasons for bacteria
being resistant to antibiotics is the production of biofilm. Biofilms are microbial communities which get adhere to solid surfaces easily
and pose an important virulence factor for causing many chronic infections. Therefore, there is an urge to find out new potential sources
which can be used as an alternative to the existing antibiotics. Methods: The present study was conducted on three medicinal plant extracts
Azadirachta indica, Aloe vera, and Mentha piperita to assess their antibacterial and antibiofilm properties against 58 multidrug‑resistant
clinical isolates using agar well diffusion method, minimum bactericidal concentration (MBC), and crystal violet modified assay at 50, 25,
12.5, and 6.25 mg/ml concentration. Results: A. indica showed a maximum zone of inhibition of (17.8 ± 1.52 mm) and (18.1 ± 1.45 mm) at
50 and 25 mg/ml concentration. Biofilm inhibition was more than 80% for Staphylococcus aureus and Pseudomonas aeruginosa and MBC
came out to be 6.25 ± 2.96–6.25 ± 4.91 mg/ml (mean range). A. vera showed the highest zone of inhibition for S. aureus (18.2 ± 1.48 mm)
at 50 mg/ml concentration followed by Staphylococcus saprophyticus (17.8 ± 1.48 mm) and Staphylococcus epidermidis (18.0 ± 1.60 mm).
Biofilm inhibition was seen more than 50% and MBC was 50 ± 23.14–50 ± 25.72 mg/ml (mean range). Conclusion: All the three plant extracts
were effective, but A. indica and A. vera were found to be more potent than M. piperita.

Keywords: Antibacterial, antibiofilm activity, medicinal plant extracts, multidrug resistant

Introduction more on the development of such therapeutic activities which

Misuse of antibiotics globally has resulted in the development
of resistant bacterial strains. One of the sole reasons for
bacteria being resistant to antibiotics is the production of
biofilm. Biofilms are microbial communities which get adhere
to solid surfaces easily and pose an important virulence factor
for causing many chronic infections. Bacteria get protected
inside the biofilm exo‑polysaccharide layer due to which it
is resistant to the effect of antibiotics.[1,2] Therefore, there
is an urge to find out new potential sources which can be
used as an alternative to the existing antibiotics. Research
on medicinal plants as new antimicrobial sources has been
well‑documented.[3] New technological science is focusing
have no harmful side effects. Thus, the interest has shifted
toward the medicinal plants which have known bioactive
compounds such as flavanoids, tannins, alkaloids, steroids, and
Address for correspondence: Prof. Priti Agarwal,
Department of Microbiology, ESIC Medical College and Hospital,
Faridabad ‑ 121 001, Haryana, India.
E‑mail: pritidragarwal2@gmail.com
ORCID: 0000‑0002‑5774‑6837
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Quick Response Code: How to cite this article: Mehrishi P, Agarwal P, Broor S, Sharma A.
Website: Antibacterial and antibiofilm properties of Azadirachta indica (neem),
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Aloe vera (Aloe vera), and Mentha piperita (peppermint) against
multidrug‑resistant clinical isolates. Biomed Biotechnol Res J
2022;6:98-104.
DOI:
10.4103/bbrj.bbrj_178_21 Submitted: 06‑Aug‑2021; Revised: 27-Aug-2021;
Accepted: 13‑Oct‑2021; Published: 11-Mar-2022.

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Mehrishi, et al.: Antibacterial and antibiofilm properties of A. indica, A. vera, and M. piperita
phenol compounds.[4,5] Medicinal plants such as Azadirachta
indica (Neem) are known since ancient times. Activity of A.
indica with its different fractions such as root, leaves, bark, oil,
and stem has been reported to have antimicrobial properties.
Leaves of A. indica have some important antibaterial and
antifungal components such as B-sitosterol, quercetin and
polyphenolic flavonoids whereas seed part contains geduninin
component.[6,7] The active compounds present in Aloe vera are
anthraquinone aloin, aloe‑emodin, acemannan amino acids,
sterols, and vitamins.[8] Mentha piperita (Peppermint) contains
phenolic compounds such as α‑pinene, citronellol, and major
compounds such as menthol and carvone with minute amounts
of menthone and men‑thylacetate which possess antimicrobial
activity.[9] Therefore, the aim of our study is to demonstrate
the antibacterial and antibiofilm activity of methanolic extract
of A. indica (Neem), A. vera, and M. piperita (Peppermint)
against multidrug‑resistant clinical isolates.
Methods
The present study was conducted in the Microbiology
Department of SGT Medical College, Hospital and
Research Institute, Gurugram. All clinical specimens sent
to microbiology laboratory were screened for isolation
of Pseudomonas aeruginosa, Acinetobacter baumannii,
Staphylococcus  aureus, Staphylococcus saprophyticus, and
Staphylococcus epidermidis strains which were further
assessed for their multidrug resistance status. [10] All the
bacterial isolates which showed multidrug‑resistant patterns
were further checked for their biofilm production and
classified accordingly in three groups: strong, moderate, and
weak biofilm producers.[11] The present study was approved
by the Institutional Ethical Clearance Committee of SGT
University Reference Number SGTU/FMHS/MICRO/341/15
on August 22, 2016.
Collection of plants
A. indica (UHF herbarium: 13588), A. vera (UHF herbarium:
13589), and M. piperita (UHF herbarium: 13591) were
certified from Department of Forestry, Dr. Yashwant Singh
Parmar University of Horticulture and Forestry, Nauni, Solan,
Himachal Pradesh. Referral ATCC bacterial strains of the
similar isolates were simultaneously tested and single testing
for biofilm inhibition assay was performed in the laboratory
for antibacterial activity.
Plant extract preparation
The methanolic extracts of the above‑mentioned plants were
prepared. Dried leaves of A. vera, A. indica, and M. piperita
plants were crushed and soaked in methanol (50 ml). Further,
the methanolic mixtures of plants were boiled continuously
with an interval of few minutes between each boiling time.
Clear supernatant was obtained after centrifugation at
5000 rpm was done for 5 min. 0.2 um (Micropore filters) was
used to filter the supernatant and filtrate was further stored at
4°C.[12]
Biomedical and Biotechnology Research Journal ¦ Volume 6 ¦ Issue 1 ¦ January-March 2022
Antimicrobial activity by using agar well diffusion method
Sterile Petri plates containing Mueller–Hinton agar were prepared.
Fresh culture suspensions (0.5 McFarland) of isolated bacteria
were swabbed on the respective plates. Sterile gel puncher was
used to make wells over the agar plates into which plant extracts
were added at various concentrations of (50, 25, 12.5, and
6.25 mg/mL). These plates were further incubated for 24 h at
37°C. After incubation, the diameter of inhibitory zones around
each well was measured in mm and recorded.[12,13]
Detection of minimum bactericidal concentration
Minimum bactericidal concentration (MBC) is defined
as the concentration producing a 99.9% reduction in
colony‑forming units number in the initial inoculums. It was
determined by twofold dilutions of the plant extracts which
were prepared at different concentrations of 50, 25, 12.5, and
6.25 mg/ml. Microorganism suspension of 100 µL was added
to the above‑mentioned concentration of extracts making the
final density of the suspension to 105 cells/ml and was further
incubated at 37°C for 24 h after which it was sub‑cultured on
MuellerHinton agar. On the next day, bacterial growth was
observed. MBC was determined as the lowest concentration
of plant extract that failed to show any bacterial growth in the
subcultures.[14]
Detection of biofilm formation by bacterial isolate using
modified crystal violet assay
Tissue culture plate of 96 wells was used. Mueller–Hinton
broth (50 µl) was added to each well followed by 50 µl of
fresh bacterial suspensions (1.0 McFarland) and was kept for
incubation at 37°C for 48 h. To check for the formation of
biofilm, contents in the wells were firstly washed with normal
saline (200 µl) followed by 0.1% crystal violet stain (200 µl)
and incubated for 20 min. After the incubation, each well was
completely washed with the deionized water and fixed later
with 96% ethanol (200 µl). ELISA reader was used to check
for the optical density (OD) of bacterial adherence at 630 nm
and biofilm formation was assessed using the formula.
OD of bacteria= [(OD growth control – OD sample)/OD
growth control] × 100.
Strains were classified as follows:[15]
No biofilm producer: OD ucODc
Weak biofilm producer: ODc < OD ≤ 2 × ODc
Moderate biofilm producer: 2 × ODc < OD ≤ 4 × ODc
Strong biofilm producer: 4 × ODc < OD.
Determination of antibiofilm activity of plant extracts using
modified crystal violet assay
Sterile tissue culture plates of 96 wells were used. Mueller–Hinton
broth (50 µl) was added to each well. Twofold serial dilutions
of plant extract at concentrations of 50, 25, 12.5, and 6.25 mg/
ml were made in the tissue culture plates to which 50 µl of
fresh bacterial suspensions (1.0 McFarland turbidity standard
matched) was added. Growth control (bacteria without plant
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Mehrishi, et al.: Antibacterial and antibiofilm properties of A. indica, A. vera, and M. piperita

extract) was used. Modified crystal violet assay was performed

Table 1: Distribution of bacterial isolates into strong,
again after 24 h of incubation as described above. Percentage of
moderate, and weak biofilm producers
biofilm reduction was calculated using the following formula:
[(OD growth control – OD sample)/OD growth control] ×100. Bacterial Strong biofilm Moderate biofilm
isolate (58) producers (9) producers (49)
The biofilm inhibition concentration (BIC50) was defined as
S. aureus (18) 3 15
the lowest concentration of extracts that showed 50% inhibition
P. aeruginosa (20) 4 16
on the biofilm formation.[15] A. baumannii (7) 2 5
S. epidermidis (8) Nil 8
Results S. saprophyticus (5) Nil 5
S. aureus: Staphylococcus aureus, P. aeruginosa: Pseudomonas aeruginosa,
A total of 58 MDR bacterial isolates were obtained from A. baumannii: Acinetobacter baumannii, S. epidermidis: Staphylococcus
clinical specimens. Out of 58 MDR isolates, 49 moderate epidermidis, S. saprophyticus: Staphylococcus saprophyticus

Table 2: Antibacterial and antibiofilm activity of Azadirachta indica

Bacterial isolate Concentration Mean±SD
(mg/mL)
Zone of inhibition (mm) MBC (mg/ml) Biofilm reduction (%)
S. aureus (ATCC 50 18.3±0.58 6.25 85
25923) 25 18.0±1.00 81
12.5 16.7±0.58 69
6.25 14.7±1.53 70
S. aureus (18 isolates) 50 17.8±1.52 6.25±4.91 81±0.05
25 18.1±1.45 84±0.03
12.5 16.8±1.42 72±0.02
6.25 14.8±1.38 74±0.03
P. aeruginosa (ATCC 50 15.0±1.00 6.25 80
27853) 25 13.3±1.15 86
12.5 11.3±0.58 71
6.25 9.7±0.58 70
P. aeruginosa (20 50 14.1±1.41 6.25±3.04 84±0.03
isolates) 25 12.0±1.41 84±0.03
12.5 10.1±1.52 75±0.03
6.25 10.3±1.49 74±0.03
A. baumannii (ATCC 50 14.3±0.58 6.25 77
19606) 25 11.3±0.58 85
12.5 12.3±0.58 82
6.25 10.0±1.00 74
A. baumannii (07 50 14.6±2.07 6.25±2.96 73±0.02
isolates) 25 9.9±1.46 81±0.03
12.5 12.9±1.68 78±0.05
6.25 10.0±1.63 77±0.06
S. epidermidis (ATCC 50 18.3±1.15 6.25 55
12228) 25 17.0±0.00 50
12.5 16.3±1.53 55
6.25 15.7±0.58 52
S. epidermidis (08 50 18.4±1.51 6.25±2.89 51±0.02
isolates) 25 16.8±1.58 53±0.02
12.5 15.0±1.41 52±0.02
6.25 16.6±1.41 51±0.01
S. saprophyticus 50 13.3±0.58 6.25 55
(ATCC 15305) 25 13.0±1.00 73
12.5 12.7±1.15 75
6.25 12.0±1.00 70
S. saprophyticus (05 50 12.6±1.52 6.25±3.62 52±0.02
isolates) 25 12.2±1.48 69±0.05
12.5 13.4±1.52 71±0.03
6.25 12.8±1.48 69±0.04
S. aureus: Staphylococcus aureus, P. aeruginosa: Pseudomonas aeruginosa, A. baumannii: Acinetobacter baumannii, S. epidermidis: Staphylococcus
epidermidis, S. saprophyticus: Staphylococcus saprophyticus, MBC: Minimum bactericidal concentration, SD: Standard deviation

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and 9 were strong biofilm producers. Extracts of A. indica
(Neem), A. vera, and M. piperita (Peppermint) plants were
tested against these 58 MDR isolates at four different
concentrations of 50, 25, 12.5, and 6.25 mg/ml, as described
in Table 1.
Antibacterial and antibiofilm activity of Azadirachta
indica (neem)
A. indica showed its best activity against S. aureus with the
highest zone of inhibition of 17.8 ± 1.52 mm and 18.1 ± 1.45 mm
at 50 mg/ml and 25 mg/ml concentration, respectively. Biofilm
inhibition was more than 80% for S. aureus and P. aeruginosa
and MBC came out to be 6.25 ± 2.96–6.25 ± 4.91 mg/ml
(mean range), as described in Table 2.
Antibacterial and antibiofilm activity of Aloe vera
A. vera showed the highest zone of inhibition for
S. aureus (18.2 ± 1.48 mm) at 50 mg/ml concentration
followed by S. saprophyticus (17.8 ± 1.48 mm) and
S. epidermidis (18.0 ± 1.60 mm). Biofilm inhibition was seen

Table 3: Antibacterial and antibiofilm activity of Aloe vera

Bacterial isolate Concentration Mean±SD
(mg/mL)
Zone of inhibition (mm) MBC (mg/ml) Biofilm
reduction (%)
S. aureus (ATCC 25923) 50 18.3±1.53 50 52
25 16.3±0.6 50
12.5 19.6±0.58 54
6.25 12.3±0.58 55
S. aureus (18 isolates) 50 18.2±1.48 50±25.72 53±0.03
25 14.7±1.75 53±0.03
12.5 19.6±1.29 51±0.03
6.25 12.0±1.64 51±0.04
P. aeruginosa (ATCC 50 14.3±1.15 50 75
27853) 25 13.0±1.0 63
12.5 13.6±0.58 52
6.25 13.6±0.58 57
P. aeruginosa (20 isolates) 50 13.9±1.35 50±20.63 70±0.04
25 11.8±1.52 60±0.04
12.5 13.5±1.61 55±0.02
6.25 13.8±1.54 54±0.02
A. baumannii (ATCC 50 15.3±0.58 50 55
19606) 25 13.3±0.6 50
12.5 14.6±0.58 52
6.25 10.3±1.53 54
A. baumannii (7 isolates) 50 14.6±1.72 50±22.49 52±0.01
25 12.1±1.68 52±0.02
12.5 14.9±1.57 51±0.02
6.25 10.1±1.68 50±0.02
S. epidermidis (ATCC 50 13.3±0.58 50 57
12228) 25 16.7±0.6 54
12.5 17.6±0.58 52
6.25 19.6±0.58 51
S. epidermidis (8 isolates) 50 13.1±1.46 50±23.14 53±0.02
25 17.3±1.13 52±0.02
12.5 18.0±1.60 50±0.01
6.25 20.9±1.96 49±0.01
S. saprophyticus (ATCC 50 12.3±0.58 50 50
15305) 25 17.3±0.6 54
12.5 17.0±1.00 52
6.25 20.3±0.58 55
S. saprophyticus (5 isolates) 50 11.8±1.48 50±0.0 52±0.02
25 16.6±1.52 50±0.02
12.5 17.8±1.48 51±0.02
6.25 20.6±1.14 51±0.03
S. aureus: Staphylococcus aureus, P. aeruginosa: Pseudomonas aeruginosa, A. baumannii: Acinetobacter baumannii, S. epidermidis: Staphylococcus
epidermidis, S. saprophyticus: Staphylococcus saprophyticus, MBC: Minimum bactericidal concentration, SD: Standard deviation

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Mehrishi, et al.: Antibacterial and antibiofilm properties of A. indica, A. vera, and M. piperita

more than 50% and MBC was 50 ± 23.14–50 ± 25.72 mg/ml
(mean range), as described in Table 3.
Antibacterial and antibiofilm activity of Mentha
Piperita (peppermint)
M. piperita showed its best antibacterial activity for
S. saprophyticus with zone of inhibition of 19.8 ± 1.79 mm and
18.8 ± 1.18 mm at 50, 25 mg/ml concentration. It did not show
any antibacterial activity at 12.5 and 6.25 mg/ml concentration
with no zone of inhibition against P. aeruginosa, S. epidermidis,
and S. saprophyticus. Biofilm inhibition was <50% in case of
S. aureus but was more than 50% for other bacterial isolates.
MBC came out to be 50 ± 11.18–50 ± 25 mg/ml (mean range),
as described in Table 4.
Discussion
Increasing multidrug resistance status in the bacteria has
become a global concern due to the inadequate use of
antibiotics. Therefore, finding an alternative for antibiotics is

Table 4: Antibacterial and antibiofilm activity of Mentha piperita

Bacterial isolate Concentration Mean±SD
(mg/mL)
Zone of inhibition (mm) MBC (mg/ml) Biofilm reduction (%)
S. aureus (ATCC 25923) 50 14.6±0.58 50 30
25 14.3±0.6 25
12.5 11.3±0.58 22
6.25 12.6±0.15 24
S. aureus (18 isolates) 50 13.9±2.03 50±21 25±0.04
25 14.4±1.62 21±0.05
12.5 10.3±1.71 19±3.0
6.25 12.9±1.88 21±0.02
P. aeruginosa (ATCC 27853) 50 15.3±0.58 50 55
25 12.7±0.6 54
12.5 ± 53
6.25 ± 50
P. aeruginosa (20 isolates) 50 14.7±2.18 50±19.18 53±0.03
25 13.0±1.76 52±0.03
12.5 ‑ 50±0.02
6.25 ‑ 51±0.03
A. baumannii (ATCC 19606) 50 14.0±1.00 50 52
25 12.7±0.6 57
12.5 11.3±0.58 55
6.25 11.0±1.00 52
A. baumannii (7 isolates) 50 14.1±1.21 50±25 55±0.02
25 13.0±1.41 53±0.03
12.5 10.6±1.51 51±0.02
6.25 12.1±1.86 50±0.01
S. epidermidis (ATCC 12228) 50 15.3±0.58 50 67
25 14.7±1.5 65
12.5 ± 62
6.25 ± 60
S. epidermidis (8 isolates) 50 14.3±2.12 50±20.86 63±0.03
25 15.1±2.23 61±0.03
12.5 ‑ 59±0.03
6.25 ‑ 59±0.02
S. saprophyticus (ATCC 50 20.3±0.58 50 52
15305) 25 19.3±0.6 54
12.5 16.0±1.00 51
6.25 ‑± 55
S. saprophyticus (5 isolates) 50 19.8±1.79 50±11.18 50±0.02
25 18.8±1.18 50±0.01
12.5 16.6±1.52 50±0.02
6.25 ‑ 50±0.01
S. aureus: Staphylococcus aureus, P. aeruginosa: Pseudomonas aeruginosa, A. baumannii: Acinetobacter baumannii, S. epidermidis: Staphylococcus
epidermidis, S. saprophyticus: Staphylococcus saprophyticus, MBC: Minimum bactericidal concentration, SD: Standard deviation

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Mehrishi, et al.: Antibacterial and antibiofilm properties of A. indica, A. vera, and M. piperita
an urgent need which has focused attention on natural products.
In our study, methanolic extract of A. indica (Neem) has shown
great activity against biofilm‑producing bacteria. More than
70% and 80% reduction in biofilm was seen against strong
and moderate clinical isolates of S. aureus, P. aeruginosa,
S. epidermidis, S. saprophyticus, and A. baumannii at
all the concentrations (50, 25, 12.5, and 6.25 mg/ml). In
concordance to our study, Geethashri et  al.[16] have also
reported the suppression in biofilm of E. feacalis using
A. indica extract at 7.5 mg/ml concentration. Another study
done by Jahan et al.[17] revealed strong antibiofilm activity by
methanolic extract of A. indica at 2 mg/ml concentration against
biofilm‑producing P. aeruginosa. In our study, methanolic
extract of A. indica gave maximum zone of inhibition for
S. aureus (17.8 ± 1.52 mm), S. epidermidis (18.4 ± 1.51 mm),
A. baumannii (14.6 ± 2.07 mm), P. aeruginosa (14.1 ± 1.41 mm),
and S. saprophyticus (12.6 ± 1.52 mm) at 50 mg/ml
concentration. Almost similar results were given by
Tirumalasetty et  al.[18] in which 50 mg/ml concentration of
methanolic extract of A. indica has shown 20 mm zone of
inhibition for S. aureus and 14 mm for P. aeruginosa. MBC for
A. indica came out to be 6.25 ± 2.96 mg/ml–6.25 ± 4.91 mg/ml
(mean range) in our study, whereas Arévalo‑Híjar et  al.[19]
revealed 25ug/ml MBC for Enterococcus faecalis. Another
study done by Abalaka et al.[20] revealed MBC of 50 mg/ml
for S. aureus and P. aeruginosa which is quite high compared
to our study.
In the present study, methanolic extract of A. vera did not
reveal higher anti‑biofilm activity as inhibition in biofilm was
52%at 50 mg/ml concentration for S. aureus. Biofilm inhibition
ranged between 50% and 54% for isolates of S. saprophyticus,
S. epidermidis, and A. baumannii. In contrast to our study,
Abraham et al.[21] reported no effect of A. vera extract on biofilm
inhibition, whereas a study by Sasirekha et al.[22] reported <50%
reduction in biofilm using 200 µl/ml concentration of A. vera
extract. In our study, A. vera gave a maximum zone of inhibition
of 20.9 ± 1.96 mm for S. epidermidis and 20.6 ± 1.14 mm for
S. saprophyticus at the concentration of 6.25 mg/ml, whereas
Abakar et  al.[23] revealed 19 and 25 mm zone of inhibition
against P. aeruginosa ATCC 27853 and S. aureus ATCC
25923. Another study by Malar et al.[24] revealed a 10.5 mm
zone of inhibition for S. aureus. In our study, MBC was found
to be 50 ± 23.14–50 ± 25.72 mg/ml (mean range). In contrast
to this, Muhuha et al.[25] reported MBC of 180 mg/ml against
multidrug‑resistant S. aureus and P. aeruginosa using A. vera
extract.
M. Piperita (Peppermint) extracts did not show any effect on
biofilm formation as the reduction was <50%. In concordance
to our study, Abraham et al.[21] presented similar results with
M. piperita extract as did not have any effect on inhibiting
the biofilm formation. In the current study, the best activity
of M. piperita extract was seen for S. saprophyticus with the
highest zone of inhibition of 19.8 ± 1.79 mm at 50 mg/ml
concentration and 14.4 ± 1.62 mm zone of inhibition for
S. aureus at 25 mg/ml concentration. In concordance to our
Biomedical and Biotechnology Research Journal ¦ Volume 6 ¦ Issue 1 ¦ January-March 2022
study, 15 mm zone of inhibition for S. aureus and 24 mm zone of
inhibition for P. aeruginosa has been reported by Mathur et al.[26]
using 200 µg/ml extract of M. piperita. In our study, MBC
of M. piperita came out to be 50 ± 11.18–50 ± 25 mg/ml
(mean range), whereas Radaelli et al.[9] have mentioned MBC
of 10 mg/ml for S. aureus using M. piperita oil extract which
is comparatively lower to our study.
Conclusion
All the three plant extracts exhibited good activity, but
A. indica and A. vera plant extracts were found to be more
potent than M. piperita as antibacterial and antibiofilm agents.
Limitation of study
No limitation in the study samples.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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