Scanning electron microscopy of Streptococcus mutans biofilm grown in BHI supplemented with 0.25% sucrose; (a) control, (b) with sub-MIC concentration of crude extract of Trachyspermum ammi and (c) with sub-MIC concentration of petroleum ether fraction of Trachyspermum ammi . 

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... each sequence was performed to search the suitable template. Best templates for modeling the structures were extracted from protein data bank. The three dimensional structures of four genes brpA, gtfB, gtfC and spaP were constructed by using MODELLER 9v7 (Sali and Blundell 1993). Energy minimization of selected structures was performed by using CHARMm package (Brooks et al. 1983) in Discovery studio 2.5. Energy minimizations were carried out in 2000 steps with conjugate minimization of the designed side chains and solvent to remove bad contacts. Energy minimized structures were evaluated for its stereochemical quality by using PROCHEK (Laskowski et al. 1993). The binding pockets and active sites of selected structures (brpA, gtfB, gtfC and spaP) were identified by using POCKET finder programme (www.modelling.leeds.ac.uk/pocketfinder). The compound was obtained from Pubchem database having PubChem id 6989. Molecule, 2-isopropyl-5-methyl-phenol was docked to the binding sites of each structure by GOLD 5.0 (Genetic Optimization for Ligand Docking) (Jones et al. 1995) with default parameters. The scoring function GoldScore implemented in GOLD was used to rank the docking positions of the molecule. All the experiments were performed in triplicates. For each outcome, data were summarized as mean ± standard deviation. Statistical analysis was performed using SPSS (version 11.5, Chicago). For quantitative real time RT-PCR, one tail Student’s t -test was used to calculate the significance of the difference between the mean expression of a given experimental samples and the control samples. A p value of <0.05 was considered significant. The minimum inhibitory concentration (MIC) of the crude extract (CR) was found to be 320 ␮ g/ml while that of the petroleum ether (PE) fraction was 40 ␮ g/ml. The major active constituents of Trachyspermum ammi extract and its active fraction were determined by GC–MS (Table 2). The predominant com- ponent was 2 isopropyl-5-methyl-phenol in the crude (52.05%) as well as in the petroleum ether fraction (83.64%). Other constituents present in the crude extract were oleic acid (8.37%), octadecanoic acid (5.86%), (3 ␤ ,24S)-stigmast-5-en-3-ol (3.88%), lupeol acetate (3.18%), hexadecanoic acid (3.02%), stigmasta- 5,22-dien-3 ␤ -ol (2.80%) and lup-20(29)-en-3-yl acetate (2.61). The petroleum ether fraction contained constituents such as lup-20(29)-en-3 ␤ -ol (2.62%), lup-20(29)-en-3yl acetate (1.86), n- hexatriacontane (1.21%), oleic acid (1.16%), n-hexacosane (0.90%), ␤ -stigmasterol (0.85%) and cholest-5-en-3 ␤ -ol (0.78%). Moreover, the MIC of thymol was also determined and was found to be 156.25 ␮ g/ml. The adherence assay was performed at concentrations below MIC to rule out the reduction in biofilm due to antimicrobial activity of the extracts tested (Fig. 1). It was found that at 20 ␮ g/ml (sub-MIC), the PE fraction exhibited strong anti adherence effect by 84.25% reduction, while the CR fraction at this concentration was found to reduce by 11.57%. The effective reduction by CR was observed at 40 ␮ g/ml. Whereas, the reduction in adherence in the presence of thymol at its sub-MIC was observed as 14.59%. The inhibitory effect of the CR and PE fraction on biofilm formation is shown in Fig. 2. Biofilm formation by S. mutans was significantly inhibited by CR and PE fractions at concentrations 80 ␮ g/ml and 10 ␮ g/ml compared to control ( p < 0.01). The reduction in the ability to form biofilm was in a dose dependent manner. As these fractions did not affect the growth of S. mutans , as evaluated through determination of the minimum inhibitory concentration, the extracts affected the ability of S. mutans to form biofilm rather than its growth. Both the samples were efficient in reducing the production of acids (Table 3). The decrease of pH was significantly inhibited in the presence of CR (160 ␮ g/ml) and PE (20 ␮ g/ml) fractions, showing at the onset pH 7.0 and 7.2 while after 12 h of incubation pH 6.5, and 6.8, respectively. However, thymol had no effect on the production of acids by S. mutans . On the other hand, untreated cells (control) showed pH 7.3 at the onset and pH 5.3 after 12 h of incubation. This is significant, because to the extent that the process of acidifi- cation of the medium between the bacterial plaque and the outer surface of the tooth is decreased, the possibility of demineralization of this surface, an essential factor in the formation of dental caries, is also decreased. Fig. 3 shows the inhibition of insoluble glucan synthesis (%) by crude GTF with increasing extract concentrations (2.5–160 ␮ g/ml). The effectiveness in inhibition of insoluble glucan synthesis was found to be dependent on the extract concentration in the reaction mixture. The inhibitory effect of PE fraction was stronger than CR fraction. Scanning electron microscopy revealed the influence of the plant extracts on the ability of Streptococcus mutans to synthesize extracellular polysaccharides (Fig. 4). As supported by the results of the effect of the extracts on glucan synthesis, there is reduced amount of polysaccharides produced at sub-MIC concentrations of CR as well as PE fraction. The control sample shows clumping of cells in the polysaccharide matrix while the cells in the treated samples shows scattered cells indicating reduced interaction between cells and thus reduced biofilm formation. Table 4 shows the weekly recovery of Streptococcus mutans for 5 weeks. It was found there was no significant reduction in the recovery of S. mutans in either the CR treated or PE treated rats. However the caries score were found to be reduced in the treated groups, reducing the total smooth surface as well as the sulcal surface caries (Table 5). The percentage of total smooth surface caries and sulcal surface caries was 84.83% and 87.61% for crude extract and 53.93% and 73.11% for petroleum ether fraction of T. ammi , respectively. The expression profile of gtfB , gtfC , gtfD , ftf , relA , spa P , vic R , smu0160 and gbpB in S. mutans biofilms treated with the crude and petroleum ether fraction of T. ammi was determined (Fig. 5). Analysis of our data shows that the selected extracts modulated the expression of specific virulence genes by S. mutans , and also disrupted the accumulation and structural organization of extracellular polysaccharides (EPS) and bacterial cells in the matrix, which affected the biochemical and physiological properties of the biofilms in vitro . The addition of sub-MIC concentration of CR and PE fractions of T. ammi , markedly reduced the mRNA levels of the selected genes to different extents in S. mutans biofilms. The CR maximally reduced the expression of the rel A gene and spa P genes by 56.66% and 58.52% respectively, while the most effected gene by PE fraction were ftf and gbpB with 69.57% and 91.4% reduction in their expression, respectively. In this study, the concentrations of the extract selected for the study were devoid of any significant bactericidal activity against biofilms under experimental conditions. The structure geometry of selected models checked by Ramachandran plot from PROCHECK program shows that 88–90% residues located in the most favorable region which indicates the good quality of the modeled structures. Docking studies of 2-isopropyl-5-methyl-phenol compound with selected structures indicate that this compound was able to dock with best 62.82 GOLD score in binding sites of N-terminal region of gtfC (Fig. 6a). However, the C-terminal regions of this protein was able to make interaction with 57.71 GOLD score, suggesting that compound 2-isopropyl-5- methyl-phenol favor the gtfC for making the interaction complex (Fig. 6b). Another protein brpA was able to make interaction with 2-isopropyl-5-methyl-phenol with 59.57 GOLD score, which is the second highest score among the complexes selected in the present work (Fig. 6c). However, 2-isopropyl-5-methyl-phenol was found to dock in the active site of spaP with the lowest binding affinity of 47.78 (Fig. 6d and e). The importance of Streptococcus mutans in the development of caries has been well documented. Studies on oral microbiota have shown a high number of this microbe in biofilm and saliva, and this can predispose to a significant increase in caries activity (Russell 2009). For the pathogenicity of this bacterium, S. mutans optimizes the expression of a variety of virulence factors, including extracellular polysaccharide synthesis systems, biofilm formation, and cell–cell signaling. Numerous drugs have been tested for their effect on dental biofilm formation and maturation. The most common of them contain antibacterial agents, which reduce the number of viable microorganisms in the biofilm. Although effective, such antibacterial applications have several undesirable side effects (Chung et al. 2006). Development of novel chemotherapeutic approaches, other than microbiocides, that disrupt the establishment, structure and virulence of dental biofilms could be a promising route to prevent or reduce the pathogenesis of oral infectious diseases such as dental caries. In recent times, focus on plant research has increased all over the world and a large body of evidence has collected to show immense potential of medicinal plants used in various traditional systems. More than 13,000 plants have been studied. Use of natural plant extracts for the manipulation and altering bacterial adhesion in biofilm, without affecting their viability, represents a targeting approach that is fast gaining popularity (Clarkson 2000). The compounds present in these plant extracts may act by reducing the total mass of causative microorganisms but do not affect the viability of the oral bacteria, thereby decimating the development of resistant strains or secondary infections. Thus, application of natural agents appears to be a promising approach in oral hygiene. The purpose of this study was to evaluate the cariostatic effect of ...

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... electron microscopy revealed the influence of the plant extracts on the ability of Streptococcus mutans to synthesize extracellular polysaccharides (Fig. 4). As supported by the results of the effect of the extracts on glucan synthesis, there is reduced amount of polysaccharides produced at sub-MIC concentrations of CR as well as PE fraction. The control sample shows clumping of cells in the polysaccharide matrix while the cells in the treated samples shows scattered cells indicating ...