In Vitro Antibacterial Activities Of Plant Essential Oils Against Oral Bacteria

Indian Journal of Multidisciplinary Research Ind. J. Multi. Res. 2008. Vol. 4 (4) : 507 - 516

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ISSN - 0973 - 2225

IN VITRO ANTIBACTERIAL ACTIVITIES OF PLANT ESSENTIAL OILS AGAINST ORAL BACTERIA KOTESWARA RAO P.*, VARAPRASAD BOBBARALA**, D. ARYAMITHRA**, P. SUBHASHINI DEVI* AND T. RAGHAVA RAO* *Department of Biochemistry, Andhra University, Visakhapatnam, A.P-530003, India. **For U Biosciences, A/4A, Park lane Residency, East point colony, Visakhapatnam, A. P-530 017, India., Email: [email protected] (Received 25 July 2008, Accepted 23 October 2008) SUMMARY Essential oils, namely Cinnamon, Clove, Lavender, Rose, Eucalyptus, Lemon, Menthol, Tea tree oil, Mustered and Rosemary oils were screened for potential antibacterial activity against medicinally important bacterial strains, Which are present in dental plaques, namely Streptococcus mutans (MTCC 890), Streptococcus mutans (MTCC 497), Streptococcus salivarius, Streptococcus mitis, Lactobacillus fermentum, Streptococcus anginosus, Streptococcus gordonii, Lactobacillus acidophilus, and Staphylococcus aureus. The antibacterial activity was determined by agar well diffusion method. Out of ten essential oils tested, nine oils showed antibacterial activity against one or more strains. Cinnamon oil, Clove oil, Lavender oil, Rose oil, Eucalyptus oil, Lemon oil, Menthol, Tea tree oil and Rosemary oils exhibited significant inhibitory effect. Cinnamon oil showed promising inhibitory activity even at low concentration; where as Mustered oil did not shown activity against the tested bacteria. Streptococcus mutans (MTCC 890) was the most resistant strain, while the most susceptible bacterial strains were Streptococcus anginosus and Lactobacillus fermentum. Cinnamon and Tea tree oil were showed strong activity against all the tested bacterial strains. Hence these can be used to discover bio active natural products, which are reduce the oral bacteria serve as leads in the development of new pharmaceuticals that address unmet therapeutic needs in oral medicine. Majority of the oils showed antibacterial activity against the tested strains. However Cinnamon oil, Tea tree oil, Clove oil and Lemon oils were found to be inhibiting gram-positive bacteria. Cinnamon oil can be a good source of antibacterial agents. Key words: Antibacterial activity, Plant essential oils, Oral bacteria INTRODUCTION Essential oils are aromatic oily liquids obtained from plant materials (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots). Essential oils are complex mixers IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008

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comprising many single compounds. Essential oils have been shown to possess antibacterial, antifungal, antiviral insecticidal and antioxidant properties (Burt, 2004 and Kordali et al. 2005) essential oils are a rich source of biologically active compounds. There has been an increased interest in looking at antimicrobial properties of extracts from aromatic plants particularly essential oils (Milhau et al. 1997). Chemically they are derived from terpenes and their oxygenated compounds. Each of these constituents contributes to the beneficial or adverse effects. Therefore, it is reasonable to expect a variety of plant compounds in these oils with specific as well as general antimicrobial activity and antibiotic potential (Darokar et al. 1998). Essential oils such as Cinnamon, Clove, Eucalyptus, Lavender, Lemon, Menthol, Tea tree, Mustard, Rosemary and Rose oil have been traditionally used by people for various purposes in different parts of the world. A few recent studies have demonstrated antimicrobial activity against selected oral pathogens from natural sources Cinnamon, Clove and Rosemary oils had shown antibacterial and antifungal activity (Ouattara et al. 1997). In spite of all the information available on the nine oils selected for this study, we were not able to find antibacterial activity for all those oils. Hence this study was undertaken with the intention of finding out the efficacy of these essential oils as antimicrobial agents for commercial purposes. Dental plaque, a film of microorganisms on the tooth surface, plays an important part in the development of caries and periodontal diseases (Marsh 1992). Mutans streptococci can colonize the tooth surface and initiate plaque formation by their ability to synthesize extracellular polysaccharides from sucrose, mainly water-insoluble glucan, using glucosyltransferase (Gibbons and Van Houte 1975; Hamada and Slade 1980; Jacquelin et al. 1995). De novo synthesis of water-insoluble glucan is essential for the adherence of Streptococcus mutans and other oral microorganisms to the tooth surface, forming a barrier that prevents the diffusion of acids produced by the bacteria. Many antimicrobial compounds from plants and fungi (Shouji, et al. 2000)] which inhibit oral bacteria, particularly S. mutans, and control plaque and thus prevent caries have been investigated (Morgan et al. 2001; Wongkham et al. 2001; Koo et al. 2000; Ooshima et al. 2000; Jagtap and Karkera 1999; Matsumoto et al. 1999; Park et al. 1998; Sato et al. 1996; Didry, Dubreuil, and Pinkas 1994; Murio and Kubo 1993; Kubo, Himejima, and Muroi 1992; Kubo 1992; Chen, Lin, and Namba 1989). Use of plant-based alternatives for oral health has been successfully promoted. This study evaluated the in vitro antibacterial activity of essential oils against some oral micro organisms, Streptococcus mutans, Streptococcus salivarius, Streptococcus mitis, Lactobacillus fermentum, Streptococcus anginosus, Streptococcus mutans, Streptococcus gordonii, Lactobacillus acidophilus and Staphylococcus aureus. Thus, oils would be a useful compound for development of antibacterial agents against oral pathogens and has great potential for use in food additives and mouthwash for preventing and treating dental caries. IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008

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Table 1: List of selected essential oils and their properties S. No

COMMON NAME

BOTANICAL NAME

PROPERTIES

(FAMILY) Cinnamomum zeylanicum

1

Cinnamon oil

2

Clove oil

3

Eucalyptus oil

Eucalyptus globulus (Myrtaceae)

4

Lavender oil

Lavandula angustifolia (Labiatae)

5

Lemon oil

6

Menthol

Mentha arvensis (Lamiaceae)

7

Rose oil

Rosa damascena (Rosaceae)

8

Rosemary oil

9

Tea tree oil

(Lauraceae) Eugenia caryophyllus (Myrtaceae)

Citrus limon (Rutaceae)

Rosmarinus officinalis (Labiatae) Melaleuca alternifolia (Myrtaceae)

Carminative, Stomachic, Astringent and Antiseptic. Dental analgesic, Carminative, Stimulant and Antiseptic. Counter-irritant, Antiseptic, Expectorant, Cough reliever. Stimulant and Flavouring agent. Carminative, Stimulant, perfuming and flavouring agent. Antibacterial, Stimulant and Flavoring agent. Perfuming and Antibacterial agent. Carminative, Stimulant and flavouring agent. Chicken pox, Flu, Migraine, Urethritis.

MATERIALS AND METHODS Nine essential oils (Table 1) were obtained from Tegraj & Co (P) Ltd, India. These oils were selected based on literature survey and their use in traditional medicine. Quality of the oils was ascertained to be more than 98% pure. The tested microorganisms of Streptococcus mutans (MTCC 890), Streptococcus salivarius (MTCC 1938), Streptococcus mitis (MTCC 2696), Lactobacillus fermentum (MTCC 903), Streptococcus anginosus (MTCC 1929), Streptococcus mutans (MTCC 497), Streptococcus gordonii (MTCC 2695), Lactobacillus acidophilus (MTCC 447) and Staphylococcus aureus (MTCC 96) were purchased from The Microbial Type Culture Collection & Gene Bank (MTCC), Institute of Microbial Technology Sector39-A, Chandigarh, India. Microorganisms were inoculated into brain heart infusion broth and grown at 37oC for 24hours. The 24 and 48hr cultures were used in the determination of microbial growth inhibition of oils. IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008

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The readymade Brain Heart Infusion Agar (52g) was suspended in distilled water (1000ml) and heated up to boiling until it dissolved completely. The medium and the petri dishes were autoclaved at pressure of 15 lb/inc for 20 min. Agar well diffusion bioassay was employed for testing antibacterial activity of essential oils following the standard procedure (Linday 1962). The medium was poured into petri dishes under aseptic conditions in a laminar flow chamber. When the medium in the plates solidified, 0.5ml of 24h old culture of test organism was inoculated. After inoculation, cups were scooped out with 6mm sterile cork borer and the lids of the dishes were replaced. The tested essential oil at 50µl was introduced into each well and the plates were incubated at 37oC for 24h. Microbial growth was determined by measuring the diameter of zone of inhibition. For each bacterial strain the experiment was done two times and the mean values are presented. RESULTS AND DISCUSSION

Zone of Inhibition(mm)

Figure 1. Antibacterial activity of some medicinal plant oils against Antibacterial activity against S. mutans Streptococcus mutans MTCC 890 25 20 The antibacterial activity of oils of the different plants against Streptococcus mutans 15 oil, Lavender oil, Rose oil, Eucalyptus is shown in Figure 1. From which Cinnamon oil, Clove oil, Lemon oil, Menthol, Tea tree oil and Rosemary oils10exhibited significant activity. Maximum 5

Antibacterial activity against S. salivarius

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Figure 2. Antibacterial activity of some medicinal plant oils against Streptococcus salivarius MTCC 1938. IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008

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activity was observed in Cinnamon oil followed by Lemon oil, Tea tree oil, Clove oil, Rosemary oil, Eucalyptus oil and Menthol where as minimum antibacterial activity was recorded in Menthol other oils demonstrated no activity against S. mutans. All oils exhibited significant activity against Streptococcus salivarius in which figure 2 represents Cinnamon oil, Clove oil, Lavender oil, Rose oil, Eucalyptus oil, Lemon oil, Menthol, Tea tree oil and Rosemary oils exhibited significant activity. Maximum activity was showed in Cinnamon oil followed by Lemon oil, Tea tree oil, Clove oil, Rosemary oil, eucalyptus and Menthol where as minimum antibacterial activity was observed in Lavender oil. Antibacterial activity against S.mitis

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Figure 3. Antibacterial activity of some medicinal plant oils against Streptococcus mitis MTCC 2696.

Z one of In hib itio n(m m )

Apart from the results obtained Cinnamon oil, Menthol Clove oil, Lavender oil, Rose oil, Eucalyptus oil, Lemon oil, Menthol, Tea tree oil and Rosemary oils exhibited significant Antibacterial activity against L. fermemtum activity. Maximum activity was shown by Cinnamon oil, followed by Lemon oil, Tea tree oil, 25 Clove oil, Rosemary oil, and eucalyptus. Minimum antibacterial activity was reported in 20 Lavender oil and Eucalyptus oil. 15 10 5

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Figure 4. Antibacterial activity of some medicinal plant oils against Lactobacillus fermemtum MTCC 1938. IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008

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The antibacterial activity of oils of the different plants against Lactobacillus fermemtum is shown in figure 4. All oils exhibited significant activity against Streptococcus mutans in this figure. Maximum activity was recorded in Cinnamon oil followed by Lemon oil, Tea tree oil, Clove oil, Rosemary oil, Eucalyptus oil, Menthol and Rose oil. Minimum antibacterial activity was observed in Menthol and Rose oil. Antibacterial activity against S.anginosus

Z o ne of Inhibition(m m )

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Figure 5. Antibacterial activity of some medicinal plant oils against Streptococcus anginosus MTCC 1929.

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The antibacterial activity of oils of the different plants against Streptococcus anginosus is shown in Figure 5. From thee oils tested eight oils namely Cinnamon oil, Clove oil, Lavender oil, Eucalyptus oil, Lemon oil, Menthol, Tea tree oil and Rosemary oils exhibited significant activity. Maximum activity was shown by Cinnamon oil, followed by Rosemary oil, Lemon antibacterial activity against s. aureus oil, Tea tree oil, Clove oil, eucalyptus and Menthol. Menthol shows only minimum antibacterial 25 activity. Rose oil has no activity against Streptococcus anginosus. 20 15 10 5

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Figure 6. Antibacterial activity of some medicinal plant oils against Staphyloccus aureus MTCC 96. IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008

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The antibacterial activity of oils of the different plants against Streptococcus mutans is shown in Figure 6. From the above observations Cinnamon oil, Clove oil, Eucalyptus oil, Lemon oil, Menthol, Tea tree oil and Rosemary oils exhibited significant activity. Maximum activity was shown by Rosemary oil and Tea tree oil. Minimum antibacterial activity was shown by Eucalyptus oil. Other oils demonstrated no activity against S. aureus. Antibacterial activity against l. acdophilua

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Figure 7. Antibacterial activity of some medicinal plant oils against Lactobacillus acidophilus MTCC 447. All oils exhibited significant activity against Lactobacillus acidophilus. Cinnamon oil shows maximum activity against L. acidophilus and Lavender oil showed minimum antibacterial activity. Antibacterial activity againsty s. mutans

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Figure 8.Antibacterial activity of some medicinal plant oils against Streptococcus mutans MTCC 497. Figure 8 represents Cinnamon oil, Clove oil, Lavender oil, Eucalyptus oil, Lemon oil, Menthol, Tea tree oil and Rosemary oils exhibited significant activity. Maximum activity was observed in Cinnamon oil followed by Rosemary oil, Lemon oil, Tea tree oil, Clove oil, eucalyptus and Menthol. Menthol shows minimum antibacterial activity and Rose oils demonstrated no activity against Streptococcus anginosus. IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008

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Figure 9. Antibacterial activity of some medicinal plant oils against Streptococcus gordonii MTCC 2695. Apart from screening Cinnamon oil, Clove oil, Lavender oil, Eucalyptus oil, Lemon oil, Menthol, Tea tree oil and Rosemary oils exhibited significant activity. Maximum activity was shown by Cinnamon oil, followed by Tea tree oil, Lemon oil, Rosemary oil, Clove oil, Eucalyptus oil, Lavender oil and Menthol. Minimum antibacterial activity was shown by Menthol. Rose oils demonstrated no activity against Streptococcus gordonii. Thus, Cinnamon oil would be useful oil for development of antibacterial agents against oral pathogens and has great potential for use in food additives and mouthwash for preventing and treating dental caries. Very little work has been done on the possible medicinal applications of these Cinnamon oil and hence extensive investigation is needed to exploit their therapeutic utility to combat human oral diseases diseases. REFERENCES Burt S. A. 2004. Essential oils: their antibacterial properties and potential applications in foods. a review. Inter J. Food Microbiol., 94:223-253. Chen, C. P., C. C. Lin, & T. Namba. 1989. Screening of Taiwanese crude drugs for antibacterial activity against Streptococcus mutans. Journal of Ethnopharmacology 27(3):285-295. Darokar MP, Mathur A, Dwivedi S, Bhalla R, Khanuja SPS, Kumar S: 1998. Detection of antibacterial activity in the floral petals of some higher plants. Curr Sci., 75:187. Didry, N., L. Dubreuil, & M. Pinkas. 1994. Activity of thymol, carvacrol, cinnamaldehyde and eugenol on oral bacteria. Pharmaceutica Acta Helvetica 69:25-28. Gibbons R J, van Houte J. 1975; Bacterial adherence in oral microbial ecology. Annu Rev Microbiol. 29:19-44. IND. J. MULTI. RES. Volume - 4 ( No. 4). 2008

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