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

Sadaf Hasan 0 Mohd Danishuddin 0 Asad U Khan 0 0 Medical Microbiology and Molecular Biology lab. Interdisciplinary Biotechnology Unit, Aligarh Muslim University , 202002 Aligarh , India 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. - Background Dental caries is a complex and multifactorial condition which causes demineralization and progressive destruction of the dental enamel [1]. Streptococcus mutans, a member of endogenous oral microflora has long been implicated to play a key role in the pathogenesis of this disease [2,3]. S. mutans survival depends strictly on a biofilm lifestyle in its natural ecosystem i.e., dental plaque [4]. Dental plaque formation is important for its persistence since biofilms does not allow easy penetration of chemotherapeutic agents, permitting to cause resistance against antibiotics, immune factors and host-derived antibacterial agents [5]. The ability of this bacterium to produce (acidogenic) and tolerate (aciduric) acids coupled with its property of synthesizing extracellular glucans allows its effective colonization in the oral cavity leading to the establishment of highly cariogenic dental biofilms [6]. Extracellular glucans which are synthesized from sucrose by glucosyltransferases (GTFs) play a critical role in the adhesive interactions of S. mutans and contributes to the structural integrity of dental plaque [7]. S. mutans expresses three different GTFs viz., GTF B, GTF C and GTF D. The insoluble and soluble glucan is mostly synthesized by GTF B and GTC C respectively. However, GTF C is known to synthesize a mixture of soluble and insoluble glucans [8]. These virulence properties thus provide a unique microenvironment for unobstructed survival of S. mutans in the oral cavity [9]. Therefore, approaches to inhibit various factors governing the virulence properties of S. mutans, could be an alternative to prevent dental caries. There are numerous anti-plaque agents that are known to reduce dental biofilm formation amongst which fluoride is a well-known cariostatic agent [10]. However, its excessive use results in side effects like fluorosis and hence its use is limited [11]. Moreover, chlorhexidine which is considered as a standard anti-plaque agent have also been reported as genotoxic [12]. Therefore, despite of the presence of a variety of anti- plaque agents, the quest for an effective agent still continues. Therefore, the development of alternative therapeutic agents with anticariogenic properties and minimal side effects is a promising approach. Chemotherapeutic agents from natural products have proved to be promising source for the development of new drugs throughout human history [13]. Recently, several studies have shown the feasibility of using medicinal plants as a source of chemotherapeutic agents for the prevention of oral diseases [14]. Zingiber officinale (or ginger), is one of the most extensively used herbs in the Indian system of traditional medicine. Many studies have revealed its numerous pharmacological activities, such as, antioxidant, antibacterial, anti-inflammatory, antinociceptive [15], antimutagenic [16] and hepatoprotective [17]. Furthermore, there are several other studies showing the antibacterial effect of Z. officinale against a number of micro-organisms including S. mutans [18-20]. However, there are hardly any reports evaluating its anticariogenic potential. Hence, in view of the current need of alternative therapeutic approach, we initiated our study to provide an innovative and comprehensive insight of the effect of Z. officinale to inhibit various virulence traits of S. mutans. Plant material and preparation of extracts Dried roots of Zingiber officinale were purchased from the local market of Aligarh and the species was identified and authenticated in Department of Botany, A.M.U., Aligarh, India. Crude and solvent fractions of the rhizome were prepared as described earlier [3]. Bacterial strain and culture medium Bacterial strain used in this study was S. mutans UA159, which was grown in Brain Heart Infusion (BHI) Broth (Himedia Labs, Mumbai, India) at 37C in a 5% CO2 anaerobic atmosphere. It was sub cultured regularly and stored at-80C as glycerol stock. Determination of bacteriostatic (MIC) and bactericidal (MBC) concentration The MIC and MBC of the crude (CR) extract and methanolic (ME) fraction against S. mutans was determined by microdilution method [21]. For the determination of MIC, twofold serial dilution of the compound was performed. The inoculum was prepared in BHI, and density was adjusted to 0.5 Mcfarland standards (108 CFU ml1) and diluted to 1: 100 for the broth microdilution procedure. Microtitre plates were incubated at 37C, and the MIC was recorded after 24 h. The concentrations ranged from 2.44 to 5000 g ml1 in a series of twofold dilutions. The MIC was determined as the lowest concentration that totally inhibits visib (...truncated)