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The phylogenetic relationship of four dental caries causing bacterial pathogens has been studied using ITS1 sequences of the standard strains were aligned by using the ClustalW computer program. The essential oil obtained from the leaves of Micromeria biflora Benth., obtained by hydrodistillaton. The chemical compositions of the essential oil from Micromeria biflora Benth was analyzed by gas chromatography-mass spectrometry (GC-MS). The GC/MS analysis showed eight major active constituents in the leaf essential oil of Micromeria biflora Benth. The antibacterial activity of the oil was evaluated against four dental caries causing bacteria such as Streptococcus mutans (MTCC 890); Lactobacillus acidophilus (MTCC 447); Streptococcus mitis (MTCC 2695) and Streptococcus salivarius (MTCC 1938) using broth microdilution method recommended by Clinical Laboratory Standards Institute (CLSI) formely (NCCLS). It's showed excellent activity against Streptococcus mutans with their Minimum inhibition concentration (MIC) 0.15 mg/ml and (IC50) 0.10 mg/ml and less effective against Lactobacillus acidophilus. The essential oil of Micromeria biflora Benth from leaf has played a significant role against dental caries causing bacteria. Relationships of the dental caries causing pathogens to the toxicity of the oil vis-à-vis phylogeny using molecular data of pathogens have also been discussed.
Key words: Antibacterial activity; Dental caries; Essential oil; Micromeria biflora Benth; Phylogenetic Analysis; Streptococcus mutans.
Dental caries is a multifactorial infectious disease, usually associated with increased numbers of Streptococcus mutans at the site of the disease. In addition, other microflora like Lactobacillus acidophilus, Streptococcus salivarius, Streptococcus mitis and Streptococcus sanguis are also involved in the process of causing dental caries. Estimation of the salivary levels of this organism may be useful for assessing caries risk in patients and for monitoring their response to preventive measures (1). Streptococcus mutans is an important component of the biofilms on teeth (dental plaque) associated with many forms of dental caries. Steptococcus mutans adheres firmly to the smooth tooth surfaces and produces sticky water insoluble dextran from dietary sucrose, forming plaque, which facilitates the accumulation of microorganisms. Streptococcus mutans and other organisms in the plaque produce organic acids such as lactic acid that gradually destroy the enamel and form a cavity (2).
The Micromeria biflora Benth, known as Indian wild Thyme, belongs to family Lamiaceae found in tropical, temperate Himalayas and Western Ghats. It is hardy to zone 0, flowers from June to August, and the seeds ripened from August to September. The flowers are hermaphrodite (have both male and female organs) and are insect-pollinated. A paste of the root was pressed between the jaws to treat toothache (3). The plant was rubbed and the aroma inhaled to treat nose bleeds (3). A paste of the plant was used as a poultice to treat wounds (3). The juice of the plant is taken internally and also inhaled in the treatment of sinusitis. The objective of this study was to investigate toxicity of the Micromeria biflora Benth. leaf essential oil vis-à-vis phylogeny using molecular data of pathogens. In particular, 16S rDNA sequences have been widely used to construct bacterial phylogenetic relationships (4, 5).
Materials and Method
Collection of Plant materials and extraction of essential oil
The essential oil was extracted from the fresh leaves of Micromeria biflora Benth. collected from the Himanchal Pradesh, India by hydro-distillatation using Clevenger's apparatus (6). A clear dark reddish yellow coloured oily layer was separated and dried with anhydrous sodium sulphate.
The essential oils obtained from Micromeria biflora Benth was studied on various parameters of physio-chemical properties such as Plant height, Oil yield, Colour, Specific gravity, Optical rotation, Refractive index and Solubility in 90% alcohol. The results are given in the Table 2.
Gas Chromatography analysis of the oil was performed on a Perkin- Elmer GC 8500, using a fused capillary column (25m x 0.55 mm i.d., film thickness 0.25um), coated with dimethtyl siloxane (BP-1). The oven temperature was programmed at 60oC to 2200C at 50C/min. then held isothermal at 220oC for 15 min. injector temperature, 250oC, detector temperature, 300oC, carrier gas, nitrogen at a linear velocity of 10 psi: split, 1:80
GC-MS data were obtained on a Shimazdu QP-2000 mass spectrometer at 70 ev and 2500C. GC column: Ulbon HR-1 equivalent to OV-1, fused silica capillary column 0.25 mm x 50m, film thickness 0.25 um. The initial temperature was 100oC for 7 min. and heated at 5oC/min to 250oC. Carrier gas helium at a flow rate of 2ml/min.The percentage composition of Micromeria biflora Benth leaves oil is given in the Table1.
Dental caries causing bacterial pathogens
Four dental caries causing bacterial pathogens were selected for this study; Streptococcus mutans (MTCC 890); Lactobacillus acidophilus (MTCC 447); Streptococcus mitis (MTCC 2695) and Streptococcus salivarius (MTCC 1938). Cultures were obtained from Microbial Type Culture Collection (MTCC), Chandigarh, India. The cultures of bacteria were maintained on Nutrient agar slants at 4°C throughout the study used as stock cultures.
Determination of Minimum Inhibitory Concentration (MIC) and IC50 by Broth micro dilution method
The antimicrobial activity of compounds was determined by broth micro dilution method recommended by Clinical Laboratory Standards Institute (CLSI) formerly NCCLS (7) using Mueller Hinton Broth. All the standard water borne bacterial cultures were maintained on Nutrient agar at 37oC. The 96-well tissue culture plates were used for twofold serial dilution. The proper growth control, drug control and the blank was adjusted onto the plate. Essential oil of Micromeria biflora Benth was dissolved in 5-10% DMSO at a concentration of 50mg/ml stock solution in case of natural antimicrobials, 20 µl of drug was added into 4th well of 96-well tissue culture plate horizontally having 180µl Mueller Hinton Broth (Fig. 1). So the maximum concentration of the test essential oil was 2.5mg/ml. From here the solution was serially diluted upto 4th well to 11th well resulting into the half of the concentration of test essential oil. The bacterial inoculum was prepared at 0.5 McFarland standards; the absorbance was equal to the inoculum suspension containing 1x107 cells per ml for bacterial isolates. Then standard bacterial inoculums was added and kept for incubation at 37oC in a moist chamber. The Minimum Inhibitory Concentration (MIC) and Inhibitory Concentration at 50% (IC50) was recorded spectrophotometrically at 492 nm using SpectraMaxplus 384 after 24 hrs incubation.
Figure 1. Format of drug testing by broth micro dilution method
Determination of minimum bactericidal concentration (MBC)
100 µl aliquot of inoculum was taken aseptically from incubated 96 well plate those well did not show turbidity and poured on to Nutrient agar plates then incubated for 24 hours at 37°C. MBC was defined as the lowest concentration of the essential oil at which 99.99% or more of the initial inoculum was killed. If there was no growth, it means the concentration was cidal. The number of surviving organisms was determined by viability counts. All tests were performed in triplicate.
To find out the reason; why the essential oil is more effective against dental caries causing bacteria, phylogenetic relationship have been studied including Streptococcus mutans; Lactobacillus acidophilus; Streptococcus mitis and Streptococcus salivarius using the Clustal W computer program (8) and GENETYX-MAC 10.1 software (Software Development Co., Ltd., Tokyo, Japan). Phylogenetic trees were then constructed by the DNA maximum-likelihood (ML) method in the PHYLIP program (Phylogeny Inference Package), version 3.5c (9) and the neighbor-joining (NJ) (10), method in the NJPLOT program (11), Bootstrap analysis with the Clustal W program was performed.
Nucleotide sequence accession numbers
Data for the phylogenetic analysis were obtained from sequences contained in the GenBank nucleotide sequence database (12). The ITS1 sequences of the standard strains used in this study Streptococcus mutans (accession no. AF204255); Lactobacillus acidophilus (accession no. HM162411); Streptococcus mitis (accession no. NC013853) and Streptococcus salivarius (accession no. S41233) were aligned (4,5,13,14).
Results and Discussion
Plant essential oils and extracts have been used for many thousands of years (15) especially in food preservation, pharmaceuticals, alternative medicine and natural therapies (16).
In the present study, composition and relative percentages of essential oil of Micromeria biflora was determined. 4 major constituents were identified with high content of Thymol 54%, Iso thymol 9.9%, Gurjurene 3.3% and β-caryophyllene 6.6% with RetentionTime (RT) 8.06, 8.83, 10.20 and 12.86 respectively. However, earlier studies suggested that the Micromeria biflora sp. Arabica K. Walth, essential oil was analyzed by GC-MS (17), 30 components were identified representing 98.2% of the total oil. The major constituents were trans-caryophyllene (43.7%), caryophyllene oxide (18.0%), spathulenol (8.5%), α-humulene (4.6%), α-myrcene (3.1%), and germacrene-D (3.1%) The present investigation of GC-MS analysis of Micromeria biflora essential oil, chemical constituents were quite different due to different agroclimatic changes. The major components and their retention times are summarized in Table 1.
Table 1. Mass Spectoscopy analysis of Micromeria biflora Benth leaf essential oil
R. Time (Scan)
I. Scan-F. Scan
Physio-chemical properties of Micromeria biflora Benth leaf essential oil showed various parameters of such as Plant height, Oil yield, Colour, Specific gravity, Optical rotation, Refractive index and Solubility in 90% alcohol. The results are given in the Table 2.
Table 2. Physio-chemical properties of Micromeria biflora Benth leaf essential oil
dark reddish yellow
Specific gravity at 25°C
0.8913 to 0.91260
-3 to -25
Refractive index at 20°C
1.468 to 1.488
Solubility in 90% alcohal
Filoche et al. (18) reported that essentail oil of Cinnamon showed antimicrobial potency (1.25-2.5 mg/ml) against Streptococcus mutans and Lactobacillus plantarum. However, in the present antibacterial activity of Micromeria biflora Benth leaf essential oil were assayed in vitro by a broth micro-dilution method against four dental caries causing bacteria such as Streptococcus mutans (MTCC 890); Lactobacillus acidophilus (MTCC 447); Streptococcus mitis (MTCC 2695) and Streptococcus salivarius (MTCC 1938)
According to the results, Micromeria biflora Benth leaf essential oil was found to be active against all dental caries causing bacteria. The strongest antibacterial activity was seen against Streptococcus mutans with a Minimum inhibitory concentration (MIC) value 0.15 mg/ml and IC50 value 0.10 mg/ml. While Minimum inhibitory concentration (MIC) value of Lactobacillus acidophilus; Streptococcus mitis and Streptococcus salivarius was 0.35 mg/ml, 0.20 mg/ml and 0.19 mg/ml respectively. These results are shown in Table 3. Nascimento et al., (19) also reported that the Hyptis pectinata essential oil exhibited considerable inhibitory effect against either all the clinical isolates obtained from patients' saliva or the ATCC strains tested, with minimum inhibitory and bactericidal concentrations of 200μg/mL.
Table 3. Anticaries activity of Micromeria biflora Benth leaf essential oil
Caries causing bacteria
Antibacterial activity in mg/ml
The phylogenetic relationships of dental caries causing bacteria were demonstrated by using internal transcribed spacer 1 (ITS1) obtained from GenBank. Alignment of the 16S rRNA nucleotide sequence, adjusted to 1,435 bases, was performed by the computer program MegAlign (DNASTAR Inc.). On the Phylogenetic analysis a clear picture can be drawn as shown in Fig. 2 and 3 Streptococcus mutans and Streptococcus salvarius belong to same genetical stock so; they were close to each other. That is the reason the Minimum Inhibitory Concentration (MIC) of test essential oil against S. mutans (0.15mg/ml) and S. salvarius (0.19mg/ml) reflect almost similar toxicity.
Figure 2. Alignment of ITS1, 16S rDNA sequences of dental caries causing bacterial pathogens
Figure 3. Results of cladogram (Neigbour Joining Tree plot) of dental caries causing bacteria using standard ITS1 sequences.
However, Lactobacillus acidophilus and Streptococcus mitis found to be closer to each other with their closed MIC 0.35 mg/ml, 0.20mg/ml. Results are showed in Fig. 2 and 3. An important characteristic of essential oils and their components is their hydrophobicity, which enable them to partition the lipids of the bacterial cell membrane and mitochondria, disturbing the cell structures and rendering them more permeable (20,21). Extensive leakage from bacterial cells or the exit of critical molecules and ions will lead to death (22).
In the present study clearly demonstrates that the leaf essential oil of M. biflora Benth was exhibited potent bactericidal action and as a therapeutic remedy against dental caries causing bacteria. The effectiveness of the oil was equal to those caries causing bacteria which are close in phylogenetic tree. As such, in future the oil can be used as a potential source of effective and cheap herbal formulation after undergoing successful multicentral topical testing.