Inhibitory effect of zingiber officinale towards Streptococcus mutans virulence and caries development: in vitro and in vivo studies

Sadaf Hasan, Mohd Danishuddin, Asad U Khan, Sadaf Hasan, Mohd Danishuddin, Asad U Khan

Abstract

Background: Streptococcus mutans is known as a key causative agent of dental caries. It metabolizes dietary carbohydrate to produce acids which reduce the environmental pH leading to tooth demineralization. The ability of this bacterium to tolerate acids coupled with acid production, allows its effective colonization in the oral cavity leading to the establishment of highly cariogenic plaque. For this reason, S. mutans is the only bacterium found in significantly higher numbers than other bacteria in the dental plaque. The aim of this study was to evaluate the effect of crude extract and methanolic fraction of Z. officinale against S. mutans virulence properties.

Results: We investigated in vitro and in vivo activity of crude extract and methanolic fraction at sub- MIC levels against cariogenic properties of S. mutans. We found that these extracts strongly inhibited a variety of virulence properties which are critical for its pathogenesis. The biofilm formation in S. mutans was found to be reduced during critical growth phases. Furthermore, the glucan synthesis and adherence was also found to be inhibited. Nevertheless, the insoluble glucan synthesis and sucrose dependent adherence were apparently more reduced as compared to soluble glucan synthesis and sucrose- independent adherence. Biofilm architecture inspected with the help of confocal and scanning electron microscopy, showed dispersion of cells in the treated group as compared to the control. The Quantitative Real Time PCR (qRT-PCR) data had shown the down regulation of the virulence genes, which is believed to be one of the major reasons responsible for the observed reduction in the virulence properties. The incredible reduction of caries development was found in treated group of rats as compared to the untreated group which further validate our in vitro data.

Conclusion: The whole study concludes a prospective role of crude extract and methanolic fraction of Z. officinale in targeting complete array of cariogenic properties of S. mutans, thus reducing its pathogenesis. Hence, it may be strongly proposed as a putative anti- cariogenic agent.

Figures

Figure 1
Figure 1
Kinetic killing curve. The bacteriostatic activity of CR extract and ME fraction at different sub- MIC levels on S. mutans cell cultures over 24h of incubation.
Figure 2
Figure 2
Effect on adherence, glucan synthesis, hydrophobicity & biofilm formation. Inhibitory effect of CR extract and ME fraction on the glass-dependent adherence in the absence (sucrose-independent SI) and presence of 5% sucrose (sucrose-dependent SD), water- soluble (WSG) and insoluble glucan (WIG) synthesis and on surface hydrophobicity by S.mutans(a & b). Also their effect on biofilm formation at 3h, 6h, 12h and 24 h of incubation (c & d). Each value is an average of triplicate assays, and each bar indicates ± standard deviation (n = 3).
Figure 3
Figure 3
Inhibitory effect on acid production and adaptation. Effect of sub-MIC concentration (128 μg ml−1) of CR extract and ME fraction on (a) glycolytic pH-drop (the values enclosed in box corresponds to the initial rate of the pH drop) (b) F- ATPase activity.
Figure 4
Figure 4
Effect on surface protein antigen. Direct binding ELISA of total protein from untreated S. mutans (control) and S. mutans treated with sub- MIC concentration of CR extract and ME fraction against polyclonal antibodies of Ag I/II rose in rabbit. Data are means ± SD (n = 3).
Figure 5
Figure 5
Docking analysis. Binding pattern of the compounds from crude extract (a&b) and methanolic fraction (c&d) analysed by GC-MS showing best docking score within the active sites of Spa and Brp A.
Figure 6
Figure 6
Electron microscopy. SEM (A) and CLSM images (B) of S. mutans biofilm formed in the presence and absence of the sub- MIC levels of extracts after 24 h of incubation.
Figure 7
Figure 7
Relative quantification of selected virulent genes expression by quantitative RT-PCR. Expression profile by real time PCR of various virulence genes of S. mutans in response to the treatment with sub- MIC levels of extracts. Each value is an average of triplicate assays. Data are means ± SD (n = 3).
Figure 8
Figure 8
In vivo validation. Effect of sub- MIC level of crude extract and methanolic fraction of Z. officinale on dental caries development in rats; means (SE), Keyes’ score.
Figure 9
Figure 9
Scanning electron micrographs of aseptically removed rat teeth. SEM analysis of rats teeth to evaluate the effect of CR extract and ME fraction on development of caries and extent of demineralization in untreated (a) and groups treated with crude extract and methanolic fraction (b’ & b”). Panels c, c’ & c” show dental surface of the teeth to observe biofilm formation. Panel b, b’ and b” shows the zoomed area focussed in red square of panel a, a’ and a” respectively.
Figure 10
Figure 10
Digital radiographs of rat teeth. Digital radiographs of untreated (a & b) and treated rat teeth with CR extract (c & d) and methanolic fraction (e & f) rat teeth to evaluate the anticariogenic effect of CR extract and ME fraction.

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