J Bagh College Dentistry Vol. 23(2), 2011 The antibacterial activity

The antibacterial activity of aqueous extract of peppermint and Bay leaf against Staphylococcus aureus

Luma M. Al-Hadi, B.Sc., M.Sc. (1)

ABSTRACT Background: Microbial resistance to antibiotics especially among Staphylococcal strains is a major threat to public health. Therefore plant extract are alternatives now to treat resistant organisms. Materials and Method: The effect of different concentration of two a aqueous extracts (peppermint and bay leaf) against Staphylococcus aureus was evaluated using the agar diffusion method and determination of minimum bactericidal concentration (MBC) value of peppermint and bay leaf were also done in this study. Results: The results of this study demonstrated that peppermint extract exhibit more activity than bay leaf on Staphylococcus aureus but both of them were found to have inhibitory effect against Staphylococcus aureu. The minimum bactericidal concentration was shown that 25% was the MBC for peppermint and 35% was the MBC for bay leaf. Conclusion: This in vitro study suggests that peppermint and bay leaf may be used as antibacterial agent against Staphylococcus aureus. Key words: Antibacterial activity, peppermint, bay leaf, Staphylococcus aureus. (J Bagh Coll Dentistry 2011;23(146- 150).

INTRODUCTION The spread of drug resistant pathogens is one Peppermint (Mentha piperita) is the most of the most serious threats to successful treatment (1) popular herbal remedies all over the world that of microbial diseases . belong to the family Lamiaceae (7). In vitro, The wide use of antibiotics in the treatment peppermint has significant antimicrobial and of bacterial infections has led to the emergence antiviral activities, strong antioxidant, antiulcer and spread of resistant strains. Staphylococcus and antitumor actions, antispasmodic, anti- aureus is recognized as one of the major cause of (2) inflammatory, antiseptic and some antiallergenic nosocomial infections and food poisoning . potential (8). Bay leaf (Laurus nobilis) belongs to Microbial resistance to antibiotic especially the family Lauraceae has shown antimicrobial and among Staphylococcal strains is a major threat to antifungal characteristics, hypoglycaemic and public health. Therefore it is extremely important antiulcerogenic properties. Bay leaf has been used to find new antimicrobial agents that are effective to treat epilepsy, neuralgia and Parkinsonism (9,10). for the treatment of infectious diseases caused by (3) The present study was undertaken in order to Staphylococcus aureus . evaluate the in-vitro antibacterial activity of Down the ages essential oils and other peppermint and bay leaf aqueous extract against extracts of plants have evoked interest as sources Staphylococcus aureus. of natural products. They have been screened for their potential uses as alternative remedies for the treatment of many infectious diseases (4). World MATERIALS AND METHODS Health Organization (WHO) noted that majority Evaluation of antibacterial effects of two of the world's population depends on traditional aqueous extracts (peppermint and bay leaf) medicine for primary healthcare. Medicinal and against Staphylococcus aureus was done in this aromatic plants which are widely used as study. The two aqueous extracts were prepared medicine and constitute a major source of natural (11) organic compound (1). according to Cowans that 100g of dried Plants extracts shown to possess powder plants were infused in 1000 ml of boiled antibacterial, antifungal, and antioxidant distilled water, and left for one hour at room properties like peppermint and bay leaf, theses temperature. Agitation of the infusion with stirrer extracts have been traditionally used by people for had been done alternatively. Then the infusions (5,6) were filtered by filter paper and residue discarded. various proposes in different part of the world . The two extracts left to dry in glass petridishes at o the incubator (37 c) the resulted powder kept in tightly closed glass container in refrigerator until used to prepare different concentrations. Five diagnosed isolates of Staphylococcus

(1) Assist. Lecturer, dep. Of basic sciences, college of dentistry, aureus obtained from Baghdad teaching univ. of Baghdad laboratories were used in the present study. An 18

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J Bagh College Dentistry Vol. 23(2), 2011 The antibacterial activity

hours culture of Staphylococcus aureus at 37 oC Determination of the minimum bactericidal in 10 ml Mueller Hinton broth was used. The concentration, the lowest concentration of theْ cultures were adjusted to approximately 105 antimicrobial agent causing negative growth CFU/ml with sterile saline solution. (fewer than three colonies) was done by serial Agar diffusion technique was applied to dilution of Mueller Hinton broth and lineation of study the antibacterial effect of the previously Mueller Hinton agar (12),(13). mentioned extracts. Final concentration of 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60% and 70% RESULTS of the aqueous extract of peppermint and bay leaf Each extract was tested against the five in Mueller agar were obtained for each extract isolates of Staphylococcus aureus. The separately. antibacterial activity of the extracts was recorded Mueller Hinton agar plates were swabbed as the mean diameter of the resulting inhibition with a suspension of Staphylococcus aureus zones of growth measured in (mm). The prepared as mentioned earlier, using sterile cotton antibacterial activity of peppermint and bay leaf swab. Plugs were removed from each agar plate aqueous extract is in figure (1). producing holes. The results revealed that peppermint To each hole 100 μl from different exhibited antibacterial activity against concentration of each extract was added and Staphylococcus aureus. As the mean of the allowed to diffuse at room temperature for 20 diameter of inhibition zones were 7mm, 8.5mm, min, the plates were then incubated aerobically o 10mm, 11mm, 12.5mm, 13mm, 15mm 16mm for overnight 37 C. the concentrations of 10%, 15%, 20%, 30%, 40%, 50%, 60% and 70% respectively.

16 14

12 Diameter of 10 inhibition 8

zones (mm) Peppermint 6 4 Bay leaf

2

0 5% 10% 15% 20% 30% 40% 50% 60% 70% Concentration of Peppermint and Bay leaf (%)

Figure 1: The relationship between the concentrations of peppermint and the bay leaf in (%) with the diameter of inhibition zones against Staphylococcus aureus in (mm).

Figure 1 also showed that bay leaf extract for peppermint and bay leaf for most exhibited antibacterial activity against concentrations used in this study and this was Staphylococcus aureus and the mean of the demonstrated in table 1. diameter of inhibition zone were 8mm, 10mm, Table 2 showed the minimum bactericidal 11mm, 12mm, 13.5mm, 14.5mm for the concentration (MBC) of peppermint and bay leaf concentrations of 20%, 30%, 40%, 50%, 60% and aqueous extract. The results of this study 70% respectively while the concentrations of 5%, demonstrated that the minimum bactericidal 10%, 15% did not give any inhibition zone. concentration (MBC) of peppermint aqueous The results of the present study using t-test extract for Staphylococcus aureus was 25% the demonstrated that there are significant differences majority of Staphylococcus aureus isolates were

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sensitive at this concentration it seems to be toxic Staphylococcus aureus sensitive at this to these bacteria. concentration and it was considered the MBC. For bay leaf aqueous extract the concentration of 35% was able to make all isolates or strains of

Table 1: Students t-test between peppermint and bay leaf aqueous extract for each test concentration in relation to the sensitivity of Staphylococcus aureus. Percent Mean extracts Significant concentration (mm) Peppermint 0 5% 0 Bay leaf 0 Peppermint 7 10% S Bay leaf 0 Peppermint 8.5 15% S Bay leaf 0 Peppermint 10 20% S Bay leaf 8 Peppermint 11 30% NS Bay leaf 10 Peppermint 12.5 40% S Bay leaf 11 Peppermint 13 50% NS Bay leaf 12 Peppermint 15 60% S Bay leaf 13.5 Peppermint 16 70% S Bay leaf 14.5 S: Significant difference at level P<0.05. Mean: mean of diameter of inhibition zones

Table 2: Minimum bactericidal concentrations of peppermint and bay leaf aqueous extract for Staphylococcus aureus. type of No. of isolates within the MBC of extract extract frequency % concentrations 5% 10% 15% 20% 25% 30% 35% 40% Peppermint extract 5 0 0 0 0 5 0 0 0 Bay leaf extract 5 0 0 0 0 0 0 5 0

DISCUSSIONS related to it's ability to inactivate microbial and cell envelope proteins (14). The antimicrobial effect of medicinal plants (17) is well documented and the results of different Deans and Baratta , shows that the studies provide evidence that some medicinal compounds from peppermint possess potent plants might indeed be potential sources of new antimicrobial activity and suggesting that the antibacterial agent even against some antibiotic – peppermint extracts should contains the effective resistant strains of bacteria (14). active constituents responsible for eliminating the In this current study using the agar diffusion bacterial pathogens. Menthol is the active technique it was observed that extract of compound in peppermint in addition to many peppermint produce antibacterial activity against potent compounds such as menthone, menthyl Staphylococcus aureus these results confirmed the acetate, menthofuran, and limnone and the earlier observation of earlier studies of Botani et.al. (15). studies suggested that the antibacterial activity of Gupta et.al also (16) reported that peppermint was peppermint was probably due to this compound effective against Staphylococcus aureus, found in peppermint and Menthol is virucidal against influenza, herpes and other viruses (18). Staphylococcus epidermids, Bacillus cereus, the (19) mode of antibacterial action of this extract is In another hand Chamsai et.al. reported that the effect of peppermint on Staphylococcus

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aureus appears to have good preventive treatment important antimicrobial activity against with regard to pathogen when using it as hand Staphylococcus aureus. sanitizer on hand and shoed higher antimicrobial activity compared to water washes. Tassan et.al. (20) REFERENCES also demonstrated that a low concentration of 1. Prabuseenivasan S, Jayakumar M, Ignaciuthu S. In essential oil of peppermint (<0.1%), the addition vitro Antibacterial Activity of Some Plants Essential of glucose in the growth medium prevented the Oils. MBC Complementary and Alternative Medicine formation of staphylococcal enterotoxin B. 2006; 6 (11):147. The results of our study revealed that bay leaf 2. Patterson JE, New gram-positive agents in L. Ran, 2005. The synergistic activity of antibiotics extract also exhibited antibacterial activity against nosocomial infection. Curr Opin Dis 2000; 13: 593-8. Staphylococcus aureus. This was in agreement 3. Adwan G. and Mhanna M. Synergistic Effects of (21) with the results of Hammer et.al. who found Plant Extracts and Antibiotics on Staphylococcus that bay leaf extract inhibit the growth of aureus Strains Isolated from Clinical Specimens Staphylococcus aureus this was also reported by Middle-East J Scientific Res 2008; 3 (3): 134-9. Özkan et.al. ('22) Dadalioglu and Evrendilek (23). 4. Tepe B, Daferera D, Sokmen M, Polissiou M, Sokmen A.. In vitro antimicrobial and antioxidant The effect of bay leaf may be explained by ' activities of the essential oils and various extracts of the action of its main components like 1, 8- Thymus eigii M. Zohary et P.H. Davis. J Agric Food (9) cineole, eugenol , sabinene and 4-terpineol . Chem 2004; 52:1132-7. While Celikel and Kavas (24) found 5. Burt SA, Essential oils: their antibacterial properties Staphylococcus aureus showed low susceptibility and potential applications in foods: a review. Inter J to bay leaf extract. This may be due to the Food Microbiol 2004; 94:223-53. 6. Irkin R, Korukluoglu M. Growth inhibition of differences in bacterial strains or/ and differences pathogenic bacteria and some yeasts by selected in the preparation production of extract, in this essential oils and survival of L. monocytogenes and study we used crude aqueous of bay leaf. C. albicans in apple-carrot juice. Foodborne Pathog Sensitivity of Staphylococcus aureus to Dis 2009; 6(3):387-94. different concentrations of peppermint and bay 7. Hayes, IR., Stavanja MS, Lawrence BM. Mint. The leaf extracts were tested separetly. As mentioned Genus Mentha. Boca Raton, CRC Press Taylor and Francis 2007; p: 422. earlier both of them have antibacterial activity 8. Mckay D.L., Blumberg J.B., A review of the against Staphylococcus aureus and the inhibition Bioactivity and potential health benefits of effect of each extracts increases with increasing Peppermint tea (Mentha piperita L.). Phytother Res concentration (24). 2006; 20(8):619-633. Findings of the present study are similar to 9. Verdian-rizi M, Variation in the essential oil those reported by Liu and Nakano (25) who found composition of Laurus nobilis of different growth stages cultivated in Iran. Journal of Basic and that bay leaf showed potent and maximum Applied Sciences 2009; 5(1): 33-36. activity against Staphylococcus aureus. 10. Chaudhry NM and Tariq P. Bactericidal activity of There are a few informations on the black pepper. Bay leaf. Aniseed and coriander against mechanism of inhibition of bacteria by plants oral isolates. Pak J Pharm Sci 2006, 19 (3), 214-8. extracts, some potential modes of action 11. Cowan M. Plant products as antimicrobial agents. suggested that interference with the phospholipid Clinical Microbiology Reviews 1999; 12 (4):564-82. 12. Barry, A. L.:The Antimicrobic Susceptibility Test, bilayer of the membrane may cause increased Principles and Practices. Lea and Febiger, permeability and loss of cellular constituents or Philadelphia; 1976. impairment of a variety of enzyme system and 13. Al-Mizrakchi A. Adherence of Mutans streptococci may affect the production of cellular energy and on teeth surfaces, Microbiological and Biochemical synthesis and structural components (26). studies, Phd thesis, Univ. of Al-Mustansiriya; 1998. From our results we noticed that peppermint 14. Kone, WM, Atindehon KK, Terreaux C, Hostettmann K, Traore D, Dosso M. Traditional medicine in north extract had more effects than bay leaf extract for cote-dIvoire: screening of so plants for antibacterial most concentrations used on Staphylococcus activity. J E thnopharmacol 2004; 93:43-9. aureus. While another study found that bay leaf 15. Mohsenzadeh, M. Evaluation of antibacterial activity exhibited greater activity than peppermint on of selected Iranian essential oils against Staphylococcus aureus. This may be due to the Staphylococcus aureus and Escherichia coli in differences in mode of action of each extract and nutrient broth medium. Pakistan journal of biological sciences: PJBS (Pak J Biol Sci) 2007; 10: 3693-7. the differences of molecular weight of the active 16. Gupta C, Garg AP, Uniyal RC, Kumari A. ingredient in each extract and the main factors Antimicrobial activity of some herbal oils against that determine antibacterial activity are type, common food-borne pathogens. African J of composition of the extract used, pH and Microbiology Res 2008; 2: 258-61. temperature of the environment (25). In conclusion 17. Deans, SG, Baratta, MT. Antimicrobial & peppermint and bay leaf were found to have Antioxidant properties some essential oils. Flau.Fragrance 1998; 235-44.

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J Bagh College Dentistry Vol. 23(2), 2011 The antibacterial activity

18. Bupesh G, Amutha C, Nandagopal S, Ganeshkumar 23. Dadalioglu I, Evrendilek GA. Chemical compositions A, Supereshkumar P. Antibacterial activity of Mentha and antibacterial effects of essential oils of Turkish piperita L. (peppermint) from leaf extracts – a oregano (Origanum minutiflorum), bay laurel (Laurus medicinal plant. Acta agriculturae Slovenica 2007; nobilis), Spanish lavender (Lavandula stoechas L.), 89:73-9. and fennel (Foeniculum vulgare) on common 19. Chamsai P, Tapnarong G, Junlapak D, Matan N. foodborne pathogens. J Agric Food Chem 2004; 52 Development of a hand sanitizing spray using (26): 8255-60. peppermint oil. As. J. Food Ag-Ind. 2009; 3(01): 178- 24. Celikel N and Kavas G. Antimicrobial properties of 83. some essential oils against some pathogenic 20. Tassou CC, Koutsoumanis K, Nychas GJE 2000. microorganisms. Czech J Food Sci 2008; 26 (3): 174- Inhibition of Salmonella enteridis and Staphyloccus 81. aureus on nutrient broth by mint essential oil. Food 25. Liu Z and Nakano H. Antibacterial activity of spice Res Internat. 2000; 48: 273-80. extract against food- related bacteria. J Fac. Appl 21. Hammer KA, Carson CF, Riley TV. Antimicrobial Biol sci 1996; 35: 181-90. activity of essential oils and other plasnt extracts. J 26. Kim J, Marshall MR, Wei C. Antibacterial activity of Appl Microbiol 1999; 86 (6): 985- 90. some essential oil components against five foodborne 22. Özkan G, Sağdiç O, Özcan M. Note: Inhibition of pathogens. J Agric Food Chem 1995; 43: 2839-45. Pathogenic Bacteria by Essential Oils at Different Concentrations Food Science and Technology International 2003; 9 (2): 85-8.

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