in-vitro antibacterial activity of mentha piperita leaf extracts to some selective pathogenic...

International Standard Serial Number (ISSN): 2321-7049

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International Journal of Medical & Pharmaceutical Sciences Research and Review Vol. 1 (2)

INTERNATIONAL JOURNAL OF MEDICAL &

PHARMACEUTICAL SCIENCES RESEARCH & REVIEW Research Article……

Received: 08-08 -2013; Accepted:16-08-2013

IN-VITRO ANTIBACTERIAL ACTIVITY OF MENTHA PIPERITA LEAF

EXTRACTS TO SOME SELECTIVE PATHOGENIC BACTERIAL

STRAINS

Imran Zamin*, Abdul Majid, Izhar Ali, Tahir Ali Khan, Muhammad Ibrar, Junaid Ali

shah, Zakir Ullah, Imran Aman, Muhammad Naveed, Ijaz Khan, Malik Mujaddad Ur

Rahman

Department of Microbiology, Hazara University, Mansehra Pakistan

Corresponding Author: [email protected]

ABSTRACT

The peppermint, Mentha × piperitta is a plant that represents the oldest and

traditional medicinal herbs used in both Eastern and Western traditions until

recent time. The peppermint has a history of use in herbal medicine dating back

to the ancient Egyptian, Greek and Roman times and also important for microbial

management program. Pakistan has a great wealth of M. piperita which has

valuable use as food not only in Pakistan but all over the world. The present

study was carried out in vitro to determine antibacterial activity of M. piperita

leaf extracts against Pathogenic bacterial strain by using leaf extract.

Antimicrobial activity of M. piperita was evaluated by disk diffusion methods.

The zone of inhibition against the tested bacteria was found ranging from 20.00

to 24.31mm. The highest zone of inhibition produced by Cold water extract S.

typhi was (29.21 mm), E. coli (27.31 mm) and S. aureus was (24 mm)

respectively. The extracts of M. piperita leaf were found to be valuable

antibacterial agent. This study gives the way for further deliberation and

investigates to find out the active compounds accountable for the plant biological

mailto:[email protected]


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activity with the necessary minimum inhibitory concentration. Further research

should accomplish to detect the precise system of action by which extracts exert

their antimicrobial effect to recognize which can be used in drug development for

safe health care services.

KEYWORD: Medicinal Plant, Bacteria, ATCC, Disk diffusion method,

Antimicrobial activity, M. piperita

INTRODUCTION

Plants are the key to life on the earth as they directly or indirectly supply

approximately 80% of human caloric and protein intake, the remainder being

derived from animal products. They are economically important to man due to

their multiple applications, such as pharmaceuticals, flavors, fragrance,

insecticides, dyes, food additives, toxins etc (Mehta et al., 2012).

Mentha piperita L. (Peppermint) is a perennial, glabrous and strongly scented

herb belonging to family Lamiaceae. It thrives well in humid and temperate

climate and most widely cultivated in temperate region of Europe, Asia, United

States, India and Mediterranean countries (Mehta et al., 2012). The peppermint

(Mentha × piperitta L.), as a representative of the large family Lamiaceae,

belongs to the oldest and traditional medicinal and culinary herbs (Sustrikova and

Salamon, 2004). Peppermint is a non-native herbaceous plant, it is a perennial,

which can reach 100 cm in height (40 inches) has four-sided stem. It is

commonly known as Peppermint, Brandy mint, Candy mint, Lamb mint, Balm

mint, Vilayati pudina or Paparaminta (Punit and Mello, 2012).

Peppermint oil is used in daily life. It is used in Pepperment candy, Chewing

gum, Candy care, Ments chocolate, Shampoo, Insects repellants and used for

flavoring pharmaceuticals and oral preparations, such as toothpastes, dental

creams, and mouth washes. Higher and aromatic plants have traditionally been


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used in folk medicine as well as to extend the shelf life of food, showing

inhibition against bacteria, fungi and yeast (Ebenezer et al., 2011). Mint essential

oils are generally used externally for antipruritic, astringent, rubefacient,

antiseptic, and antimicrobial purposes, and for treating neuralgia, myalgia,

headaches, and migraines (Fatiuh et al., 2002). Peppermint oil or peppermint tea

is often used to treat gas and indigestion; it may also increase the flow of bile

from the gall bladder. Peppermint oils relaxing action also extended to tropical

use, when applied tropically it acts as counterirritant and analgesic with the

ability to reduce pain and improve blood flow to the affected area. (Sureshkumar

et al., 2007).

Peppermint oil and menthol have moderate antibacterial effects against both

gram-positive and gram negative bacteria. Peppermint extracts are bacteriostatic

against Streptococcus pyrogens, Streptococcus aureus, Streptococcus pyrogens,

Serratia marcescens, E.coli and Mycobacterium avium. Peppermint is also found

to have antiviral and fungicidal activity. Menthol is virucidal against influenza,

herpes and other viruses. Aqueous extracts of peppermint leaves were antiviral

against influenza A, Newcastle disease virus in egg and cell culture system.

Menthol and peppermint oil are fungicidal against Candida albicans, Aspergillus

albus and Dermatophytic fungi and verity of parasites (Sureshkumar et al., 2007;

Alexander and Heinz, 2004; Katikia et al., 2011). This essential oil dilates blood

vessels and inhibits bacteria; especially menthol has a broad spectrum

antibacterial activity. Peppermint oil is found to be strongly effective against

Staphylococcus aureus (S. aureus), Bacillus cereus (B. cereus), Bacillus subtilis

(B. subtilis), Enterococcus faecium (E. faecium), Klebsiella pneumoniae (K.

pneumoniae) and (E. coli). Peppermint is also found to have antiviral and

fungicidal activity. It is virucidal against influenza, herpes and other viruses.


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Antifungal activity of the essential oil of M. piperita was also reported

(Ebenezer, et al., 2011; Beata, 2007). The study confirms that both aqueous as

well as organic solvent leaf extracts possess strong antibacterial properties

against various pathogens viz., Bacillus substillus, Pseudomonas aureus,

Pseudomonas aerogenosa, Serratia marcesens and Streptococcus aureus

(Sureshkumar et al., 2007: Pramila et al., 2012).

The present study was conducted to investigate the antibacterial activity of M.

piperita leaf extracts against gram positive ATCC (American type cell Culture)

bacteria S. aerious ATCC®6538, and gram negative bacteria Escherichia coli

ATCC®25922, and Salmonella typhimurium ATCC

®14028.

MATERIALS AND METHODS

This research work was conducted at the Microbiology research Laboratory,

Department of Microbiology, Hazara University Mansehra, Pakistan.

Plant Materials

Healthy, disease free, mature M. piperita leaf was collected directly from

Department garden Hazara University Mansehra Pakistan and brought to

Department of Microbiology, Hazara University, Mansehra Research Laboratory.

The leaves were cleaned with tap water. After cutting the leaf into small pieces,

they were air dried in room temperature for 7 days, and then dried leaves were

crushed into a fine powder by blender machine.

Cold Water Used For Leaf Extraction

The Adebayo and Ishola (2009) method of extraction was used. Three grams

powdered samples of leaf and root was soaked in 30 ml cold water in 250ml

sterile flask and rotated on shaker at 150 rpm for 24 hours at room temperature.

The extract was filtered through a muslin cloth and then centrifuged at 4400 rpm


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for 7 minutes. The supernatant were collected and the pellet was discarded. These

steps were repeated three times. The coming supernatant was considered as 100%

concentration of extract. The cold water extracts were evaporated to dryness

using a rotary evaporator (Stuart, Barloworld and Model RE 300). Their crude

extracts were evaporated in a water bath to give gummy solid residue. The

obtained crude extracts were 0.54 g leaf and 0.72g root.

Media Preparation

Nutrient Agar

Nutrient Agar was enrichment medium for the growth of microorganisms.

Medium was prepared by adding 13g of dehydrated powder using electrical

balance into 1 liter of distilled water. PH was adjusted by electrical pH meter at

7.4 and was boiled to dissolve completely.

MEDIA STERILIZATION

All Media were sterilized by using automatic autoclave (SANYO) at 121°C for

15 minutes.

Media Pouring and Drying

Media was poured in pre-sterilized glass Petri plates of 90mm in Laminar Flow

Hood which was sterilized by overnight exposure of UV light and disinfected

with 70% ethanol solution. Media plates were kept open for half an hour in the

Laminar Flow Hood for drying and solidifying media.

Test Microorganisms

The in-vitro activity of the extracts was assayed against the bacterial strains. All

the ATCC (MicroBioLogics) against gram positive bacteria S. aerious

ATCC®6538, and gram negative bacteria Escherichia coli ATCC

®25922, and

Salmonella typhimurium ATCC®14028. Which were kindly provided by Dr.

Malik Mujaddad Ur Rehman, Assistant Professor, and Head of Department of

Microbiology, Hazara University, Mansehra, Strains were maintained on Nutrient


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Agar Tubes at 4 oC. The antibiotic efficacy of the plant extracts was evaluated

against given strains.

Standardization of Inoculums

After the incubation time, single selective colony of each bacterium from their

respective selective agar medium was inoculated into 5ml NB and incubated for

4-6 hours at 37°C in incubator (NAPCO). 1% v/v solution of sulphuric acid

(H2So4) was prepared by adding 1 ml of concentrated sulphuric acid to 99 ml of

distilled water. 1.175% w/v solution of barium chloride was also prepared by

dissolving 2.35 g of dehydrated barium chloride in 200 ml of distilled water.

McFarland Standard No.0.5 was prepared by mixing 0.1ml of 1.175% w/v of

barium chloride with 9.9 ml of 1% v/v sulphuric acid. Nutrient agar plates were

prepared and pure bacterial cultures were swabbed in these plates and incubated

for 370C for 24 hours. With the help of sterile wire loop, three to four well

isolated colonies from each plate were transferred to test tubes containing fresh

nutrient broth to make bacterial suspension. These test tubes were incubated at

370C for six hours. Turbidity of these suspensions was adjusted by using nutrient

broth to McFarland Standard No. 0.5 by visually comparing the turbidity of

bacterial suspension with McFarland standard.

INOCULATION OF TEST ORGANISMS

100µl of 1McFarland bacterial suspensions were aseptically introduced and

spread using pre-sterilized cotton swabs on surface of MHA plates.

Disk Diffusion Method

The disc diffusion method was used to determine the growth inhibition of

bacteria by plant extracts (Nostro et al., 2000). In the disc diffusion method, the

discs were placed aseptically over the bacterial culture on nutrients agar plates

and incubated at 37oC for 24hrs. After inoculation for 24 hrs, the zones of


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inhibition around the discs were measured by Digital Vernier Caliper (Mitutoyo).

The experiment replicated three times to confirm the reproducible results.

Evaluation of Antimicrobial Activity

Antimicrobial activity of M. piperita leaf extract was tested using agar well

diffusion method. With the help of sterile micropipette tips M. piperita leaf

extract (cold water) 100µl were poured into the wells. The plates were incubated

at 370C for 24 hours. After incubation, the diameter of the resulting zone of

inhibition was measured with the help of Digital Vernier Caliper (Mitutoyo) and

the average values were recorded. Each antimicrobial assay was performed three

times. Mean values were reported in this report.

Data Analysis

All data were measured average value of three replicates and standard error (±).

Results were subjected to Microsoft excel 2007.

RESULTS

In the present study, the antimicrobial activity of the Cold water extracts against

two gram negative and one gram positive bacterial strains and their potential

activity were qualitatively and quantitatively assessed by the presence or absence

of inhibition zones and MIC values.

Antibacterial activity

The extracts of the investigated plant species showed antimicrobial activities

against all tested bacterial strains. Results of the antimicrobial activity obtained

using the well diffusion assay is summarized in Table 1 and 2 and Figure 1.


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Table 1: Activity of Cold water extract of M. piperita leaf against Gram negative

bacterial strains.

In table 1 showed that Cold water extract of M. piperita leaf make a zone of

inhibition on gram negative bacterial strain including E. coli (29.31 mm) and S.

typhimurium (27.21 mm) were determined.

Table 1: Activity of Cold water extract of M. piperita leaf against Gram positive

bacterial strains.

In table 2 showed that Cold water extract of M. piperita leaf make a zone of

inhibition on gram positive S. areues (24 mm) were determined

Bacterial strains Zone of inhibition on bacterial strain of M. piperitta extract

Disk diffusion methods

E. coli 29.31 mm

S. typhimurium 27.21 mm

Bacterial strains Zone of inhibition on bacterial strain of M. piperitta

extract

Disk diffusion methods

S. areues 24 mm


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Figure 1: Zone of inhibition of M. piperita leaf extract on gram negative and

positive bacterial strains

DISCUSSION

The aim of this research was to find out M. piperita plant extract against Gram

positive and Gram negative bacteria. M. piperita plant with several other

compounds that have antimicrobial activity (Sureshkumar et al., 2007). The

antimicrobial activities of medicinal plants are qualified due to the presence of

alkaloids, and flavonoids (Burapedjo and Bunchoo 1995; Fewell and Roddick,

1993). These reports and presence of flavonoids, alkaloids in different extract of

M. piperita confirm it’s prospective against all selected pathogens bacterial

strain.

The current study suggests that the Cold water leaf extract of M. piperita have a

board spectrum of antimicrobial activity, although the degree of vulnerability

could different between different microorganisms. The antimicrobial activity

found in this present conducted study may be ascribed to the presence of


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secondary metabolites either individually or in combination of various types of

chemical composition present in the plant material.

The plant active substances were soluble in organic solvents so plant extracts

obtained more activity than commercial antibiotics (Boer et al., 2005). Results of

this study showed that the potential usefulness M. piperita in the treatment of

various pathogenic diseases or infection as it may help in the innovation of new

chemical classes of antibiotics or drugs.

Antimicrobial agents now days available in the market are inadequate due to their

low effectiveness, toxicity, and prove costly in case of long-drawn-out treatment.

The discovery of a potent medication from plant origin will be a great

development in microbial infection therapies. Therefore, there is needed to

develop new antimicrobial agents which can satisfy the current demand.

CONCLUSION

This study gives the way for further deliberation and investigates to find out the

active compounds accountable for the plant biological activity with the necessary

minimum inhibitory concentration. Further research should accomplish to detect

the precise system of action by which extracts exert their antimicrobial effect to

recognize which can be used in drug development for safe health care services.


www.ijmpsrr.com

ACKNOWLEDGEMENTS

We are grateful to the Department of Microbiology Hazara University, Mansehra

for approving, funding and facilitating this study. We also appreciate Dr. Kashif

Haleem, Dr. Azam Hayyat, Dr. Muhammad Farooq and Dr. Isfahan Tuseef

(Assistant professor Microbiology Department, Hazara University, Mansehra) for

helpful discussions and advice during development of the manuscript and special

thanks to Muhammad shabeer Lab assistant Microbiology Research Laboratory.

COMPETING INTERESTS

The authors declare that they have no competing interests.

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in-vitro antibacterial activity of mentha piperita leaf extracts to some selective pathogenic...

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