Design and study of anticaries effect of different medicinal plants against S.mutans glucosyltransferase

Fermentation of carbohydrates presents in a food by oral bacteria results in a decrease in the pH of plaque and demineralization of enamel and finally formation of dental caries [1‐4]. Streptococcus mutans (S.mutans) is considered to be the principal cariogenic bacterium in humans [5, 6]. It possesses several virulence factors that are associated with its cariogenicity [7]. An essential factor of S.mutans is its sucrose-dependent and glucan-mediated colonization of tooth surfaces. Glucans are glucose polymers synthesized by extracellular glucosyltransferases (gtfs) [1]. There are three S.mutans gtf’s: gtf B and gtf C synthesize primarily water-insoluble glucans (WIG) [8, 9] and gtf D, synthesize water-soluble glucans (WSG) [10]. Gtfs are involved in many biological processes such as cell-cell communications, signal transduction, immune response, microbial adhesion and infection [11]. Biofilm or dental plaque is formed through two different sequential steps: initial and reversible cell-to-surface attachment and subsequent sucrose-dependent adhesion of the microorganisms, which is firm and irreversible, gtfs are strongly involved in the latter step [12]. There are multiple ways that compounds can exert anticaries action in addition to gtf inhibition. In view of essential roles played by gtfs, intervention of gtfs has attracted remarkable interest among other ways for drug development since inhibitors of gtfs can potentially interfere with the pathological processes [13, 14]. Targeting gtf enzymes prevents the synthesis of extracellular polysaccharides (glucans) and is an attractive strategy for the development of anti-biofilm compounds, as the glucans are extremely important in the processes of biofilm formation and stabilization [15].

The use of medicinal plants or natural products has been one of the most successful strategies for the discovery of new medicines [16]. It already been reported that plant extracts and their components have significant antibacterial activity on oral bacteria, especially on S.mutans [17‐21]. Phytochemical rich extracts and their associated compounds have repeatedly shown inhibitory effects against adhesion, plaque and biofilm formation of S.mutans [22‐27]. As inhibition of essential virulence factor (gtf) is the primary goal for the prevention of dental carries and possibly other plaque related diseases, present study is aimed to report the anti-caries effects of four plant extracts (Gallnut, Guava, Neem, Indian beech) and two essential oils (Clove oil, Peppermint oil) against S.mutans gtfs.

Brain Heart Infusion broth (BHI), sucrose, ethanol, sodium dodecyl sulfate, acetic acid, sodium acetate, tween 80, dimethyl formamide (DMF) and ammonium sulfate (all are of analytical grade) were used in the present study and purchased from Merck, Mumbai.

Dental caries is a biofilm-mediated, sugar driven, multifactorial, dynamic disease that results in the phases of demineralization and remineralization of dental hard tissues. The synthesis of extracellular polysaccharide, including WIG & WSG is one of the most important virulence factors of S.mutans [39]. The WIG promotes the adhesive interactions of bacteria with the tooth surfaces and contributes the formation of dental biofilm [40]. Accordingly, in the present study, we examined whether selected plant extracts inhibits the synthesis of WIG and whether clove and peppermint oils inhibits the synthesis of WSG & WIG by crude gtfs as there is no previous molecular level mechanistic report on these samples. Our results were in correlation with the literature reports of the anti S.mutans activity of the test samples.

The hydroalcoholic extracts of T.chebula, P.guajava, A.indica and P.pinnata inhibit WIG synthesis by inhibiting S.mutans gtf activity in a dose dependent manner (Table 2). A.indica extract has shown highest percentage inhibition of 91.647 ± 0.445% at 10 mg/ml concentration. Order of gtfs inhibitory activity of the four plant extracts at their highest concentration of 10 mg/ml was A.indica > T.chebula > P.pinnata > P.guajava. T.chebula extract has shown predominant inhibitory action among tested extracts from 0.312 to 5 mg/ml (Fig. 1). It means gtf synthesizing WIG from sucrose was almost suppressed upon addition of 10 mg/ml concentration of A.indica extract. Highest correlation coefficient (R²) value is observed with A.indica when compared to other extracts. However, according to the 50 % inhibitory doses (IC₅₀) T.chebula would be considered as an effective gtf inhibitor as it has shown effect at lower concentration (1.091 mg/ml) when compared to other three plant extracts. It has been reported that gtf B and C are important factor for synthesis of WIG by S.mutans, gtf B synthesize primary WIG where as gtf C synthesizes WIG & WSG [41]. Although we identified that T.chebula, P.guajava, A.indica and P.pinnata extracts may inhibits gtf B by inhibiting WIG synthesis where as clove and peppermint oil inhibits both gtf B and gtf C by inhibiting WIG & WSG synthesis.

To study the molecular level mechanism responsible for the anticaries activity of clove oil and peppermint oil, the effect of these on the WIG & WSG synthesis by cell free extracellular gtf of S.mutans were examined. Various concentrations ranging from 3.125-100 mg/ml were used for both the samples and their concentrations are different from that of concentrations used for plant extracts (based on previously reported MIC against S.mutans). Dose dependent inhibition of gtf was observed in both the cases (Table 3) and especially from 25 to 100 mg/ml there was a drastic increase in % inhibition. IC₅₀ values of peppermint oil (44.693%) was significant when compared to clove oil (63.697%) (Fig. 2). Out of six test samples used in the present study, T.chebula extract has shown the low IC₅₀ value (Fig. 3).

R² value represents square root of the correlation coeffecient and A.indica has R² value of 0.9700 indicates 0.9700² × 100 (97%), the higher the percentage better the correlation between results. F-value is a numerical indicator of whether or not the result expressed by the method is coincidental, the higher the value, the less likely the result is due to chance (A.indica has highest F-value of 248 among tested extracts).

From kinetic analysis it was observed that T.chebula, P.guajava, A.indica and P.pinnata have shown the exponential curves (Fig. 4) indicative of deviation from normal response (hyperbolic) of the enzyme. From their Michael Menton constant (K_M) and Vmax values, it was identified that T.chebula, P.guajava and P.pinnata have shown uncompetitive inhibition as their K_M and Vmax values have been decreased when compared to control (without inhibitor). In case of A.indica it was found that K_M unchanged and Vmax decreased indicated that the pattern was of non-competitive inhibition (Table 4).

Similar analyses of clove and peppermint oils revealed the fact that response was sigmoidal and the pattern was of allosteric inhibition (Fig. 5). Sigmoid response curve suggests the cooperativity between the inhibitor and the enzyme and hill coefficient provides a way to quantify the degree of binding between inhibitor and enzyme. Both clove and peppermint oil have shown hill coefficient > 1 represents positive, cooperative binding (Table 4). Hill coefficient value is more (4.60 ± 0.59) for clove oil when compared to peppermint oil (1.92 ± 0.46) indicates that clove oil has more affinity for gtf. The present study mainly focused on the effect of plant extracts and essential oils on gtf inhibition and comparison of the same with commercial anti-carries agent. Here the criteria is to compare molecular level gtf inhibitory mechanism of tested samples so that whether they possess significant anti carries property or not.

The bilologically active phytoconstituents or secondary metabolites present in all four plant extracts have been responsible for their gtf inhibitory activity. Moreover gtf activity of bioactive compounds like tannins [42], flavonoids [43], terpenoids [44] were reported previously. The tannins in T.chebula fruits, terpenoids in A.indica twigs, isoflavones in twigs of P.pinnata and flavonoids and tannins present in leaves of P.guajava may be the attributing factors for S.mutans gtf inhibition.

The antibacterial properties of plant essential oils against bacteria found in the oral cavity are also well documented [45]. While the commercial mouthwashes containing essential oils are useful in the long-term control of plaque and mild to moderate gingivitis and are preferred to use in chlorhexidine containing mouth washes for long-term daily use [46]. Therefore, essential oils may be suitable additives in pharmaceutical products.

There has been reports about the menthol present in Mentha piperata (peppermint oil) and eugenol in Syzygium aromaticum (clove oil) and are considered as outstanding compounds exhibiting an antibacterial potential [47] which could be responsible for S.mutans gtf inhibition.

The present study also focused on the formulation of the polyherbal mouth rinse and its IC₅₀ value was found to be 7.938%w/v. The % inhibition at its maximum concentration of 15%w/v was found to be 95.936 ± 1.518 and it was very high when compared to 53.80 ± 1.83% inhibition of chlorohexidine commercial mouthwash. Dose dependent gtf inhibitory effect was observed in case of polyherbal mouth wash and chlorhexidine did not follow this pattern. From the results it was observed that gtf inhibitory potential of the polyherbal mouth rinse was significant (p < 0.05) compared to chlorhexidine mouthwash at tested concentrations (Fig. 6). From the shape of the exponential curve (concentration of inhibitor vs enzyme velocity) it was evident that polyherbal mouth rinse competitively inhibits gtf where as chlorhexidine shows non-competitive inhibition (Fig. 7).

From the studies it was concluded that among the four plant extracts and two essential oils tested against gtf activity A.indica extract has shown maximum percentage inhibition and T.chebula extract has shown the significant IC₅₀ value when compared to others. The results of kinetic analysis proposed that T.chebula, P.guajava, P.pinnata have shown uncompetitive gtf inhibition where as A.indica has shown non-competitive inhibition. Clove oil and Peppermint oil have proposed allosteric inhibition and the standard chlorhexidine has shown non-competitive inhibition. Moreover the polyherbal mouth rinse prepared from all four plant extracts and two essential oils has shown predominant gtf inhibition (95.936%) when compared to 54% shown by chlorohexidine mouthwash. Further clinical investigations are recommended for this polyherbal mouth rinse to utilize as anticaries agent.