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Introduction
There has been a significant decline in the prevalence of dental caries in some developed countries due to the increasing awareness of common dental hygiene techniques among the general population. However, no significant reduction has been seen in other parts of the world, including South Korea, and dental caries continues to be an important public health issue here1). There are more than 700 species of bacteria that exist in the mouth, occupying various oral surfaces. Some of them adhere to teeth, damaging tooth structures, and resulting in the development of caries. Among these, is a group of streptococcal species called mutans streptococci (MS) and two important caries-causing bacteria in the group are Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus). These bacteria have excellent tooth adhesion properties, thereby increasing cariogenicity with acidogenicity, and are thus, an important cause of dental caries2).
MS synthesize extracellular polysaccharides such as glucans and fructans from sucrose through the action of glucosyltransferase (GTFase) and fructosyltransferase (FTFase), respectively3). They serve as efficient energy sources for MS within the oral cavity4).
Glucans are sticky glucose polymers that trap oral bacteria, leading to the formation of a biofilm. This serves as a first step in the pathogenesis of dental caries2,3). Insoluble glucans help S. mutans and S. sobrinus to adhere to the tooth surface and maintain the dental biofilm. S. mutans and S. sobrinus generate organic acids from glucan in addition to dietary sugars. Of these, lactic acid dissolves hydroxyapatite, a chemical component of tooth enamel5). Besides, glucans make the biofilm more condense and insoluble, and thus, act as a barrier that prevents penetration of saliva into the biofilm leading to a reduced salivary buffering capacity. This then causes the acid to stagnate within the biofilm, finally resulting in dental caries6). Therefore, suppressing the growth of S. mutans and S. sobrinus is important to lower the occurrence of dental caries.
Many medicinal plants targeting streptococci for the prevention of dental caries have been studied to date. Camellia sinensis7) or Magnolia officinalis8) have been reported to have antibacterial effects. However, toxicities and many side effects such as digestive disorders and hypersensitivity reactions have also been reported with the use of these plant extracts9). Thus, there was a need to identify an effective agent to control dental caries with fewer side effects.
Rheum palmatum is a species of flowering plant of the knotweed family Polygonaceae. It is commonly called Chinese rhubarb10), Turkey rhubarb11), or East Indian rhubarb11). Rheum palmatum is a herbaceous perennial related to the edible rhubarb and is primarily used in traditional medicine. In Chinese medicine, it has been used as a laxative and an anti-inflammatory drug in the treatment of various disorders. It has also been reported to possess antibacterial and antiproliferative properties12). Also, recent in vivo studies have shown that Rheum palmatum could play a beneficial role in the treatment of liver injuries and hepatitis13). However, the effects of Rheum palmatum on oral streptococci including S. mutans and S. sobrinus have not yet been investigated.
Previously, in the course of the development of preventive agents for dental caries, we had tested the antibacterial activities of several plant extracts which have been reported to be effective in the treatment of oral diseases in oriental medicine. Among them, the Rheum palmatum extract showed the best antibacterial effects. Thus, this study aimed to study the anticariogenic activity of Rheum palmatum including its inhibitory effects on the growth, glucan synthesis, and biofilm formation of the cariogenic bacteria, S. mutans, and S. sobrinus.
Materials and Methods
1. Bacterial culture
S. mutans and S. sobrinus were successively cultured twice in a brain heart infusion (BHI) broth (BD, MO, USA) in a 5% CO2 incubator at 37℃ before use.
2. Preparation of Rheum palmatum extract and other natural extracts
Rheum palmatum extract and other natural extracts used in the study were taken from the Okcheondang located in Yeongcheon. The extraction was done with hydrothermal water using a reflux cooling extractor with 10 times (w/v) distilled water per 100 g. The extracts were filtered using a filter paper (Whatman No. 2) and then concentrated with a rotary vacuum evaporator (Buchi rotavapor R-100, Germany) before freeze drying (TFD5505, ilShin BioBase Co. Ltd., Korea). The concentration of the extracts was adjusted from 0.1% (1 mg/ml) to 10% (100 mg/ml) by diluting them in a phosphate-buffered saline solution (PBS).
3. Screening experiments of herbal extracts against S. mutans and S. sobrinus
5 ml of Mentha piperascens, Ulmus macrocarpa Hance, Syzygium aromaticum, Polygonum tinctorium, Coptis chinesis, and Rheum palmatum extracts suspended in PBS at concentrations of 0 to 5% were added into a 96-well plate and the bacteria were inoculated at 1×105 colony forming units (CFU)/well. After incubation at 37℃ for 24 h, the absorbance was measured at 650 nm using a spectrophotometer (Tecan, Männedorf, Switzerland). No herbal extracts were added to the control group.
4. Measurement of the growth of S. mutans and S. sobrinus
Equal quantities of BHI broth and Rheum palmatum extract were added to the 96-well plate and each bacterial species was inoculated at 1×105 CFU/well. After incubating at 37℃ for 24 h, absorbance was measured at 650 nm using a spectrophotometer. Rheum palmatum extract was not added to the control group.
5. Estimation of viable cell count post-treatment
5 ml of Rheum palmatum extract was added into each tube at concentrations varying from 0 to 2.5%. S. mutans and S. sobrinus (1×104 CFU/ml) grown and diluted in the BHI broth were inoculated into the tube containing the Rheum palmatum extract. After 18 h of incubation in a CO2 incubator, the mixture was diluted with PBS and inoculated with BHI agar. The number of viable cells was then measured after 48 h of incubation.
6. Measurement of biofilm formation
The BHI broth with 5% sucrose in a 24-well plate was inoculated with S. mutans and S. sobrinus, respectively, and mixed with the Rheum palmatum extract in concentrations ranging from 0 to 10%. After incubation at 37℃ for 72 h to induce biofilm production, the biofilm obtained was washed three times with PBS and then stained with 0.1% crystal violet solution for 10 min. After washing three times with PBS and drying in a hood for 15 min, the biofilm was dissolved by adding 100% ethanol and the absorbance was measured at 570 nm using a spectrophotometer.
7. Glucan synthesis test
S. mutans and S. sobrinus were inoculated to 1×105 CFU/ml into glass test tubes containing BHI broth with or without 5% sucrose and then the Rheum palmatum extract was added in concentrations varying from 0 to 2.5%. After incubation at 37℃ for 24 h, 1 ml of the culture supernatant was centrifuged and 200 μl of supernatant was transferred into the 96-well plate, and then the absorbance was measured at 650 nm by a spectrophotometer. No Rheum palmatum extract was added to the control group.
Results
1. Effect of Rheum palmatum extract on the growth of oral streptococci
In our previous study, we screened various natural extracts for their antibacterial properties. Based on this screening, Rheum palmatum was selected as the most effective. To examine its antibacterial activity, S. mutans, S. sobrinus, S. oralis, S. mitis, and S. salivarius were treated with the Rheum palmatum extract at 37℃ for 24 h and the optical density was then measured. All concentrations of the Rheum palmatum extract inhibited the growth of all the Streptococcal species used in the experiment, and the growth of all the test strains was inhibited at concentrations above 0.6% (Fig. 1).
Among the streptococci, S. mutans and S. sobrinus are the major causative agents of dental caries. A viable cell count assay was carried out to examine in detail, the effect of the Rheum palmatum extract on the growth of S. mutans and S. sobrinus. Rheum palmatum extract at concentrations of 1.25% and 2.5% significantly decreased the number of viable cells of S. mutans and S. sobrinus, compared to the control group. The Rheum palmatum extract thus inhibited viable cells in a concentration-dependent manner (Fig. 2).
2. Effect of Rheum palmatum extract on biofilm formation
We examined whether Rheum palmatum extract inhibited biofilm formation which is an early step in the development of dental caries. As shown in Fig. 3, the biofilm formation by S. mutans and S. sobrinus was decreased 20-fold or more at concentrations of 1.25 and 2.5% of Rheum palmatum, compared to the control.
3. Effect of Rheum palmatum extract on glucan synthesis
We examined the effect of Rheum palmatum on glucan synthesis which is essential for bacterial adherence and biofilm formation. The Rheum palmatum extract was observed to inhibit glucan synthesis (Fig. 4). Specifically, the 1.25% and 2.5% concentration levels of the Rheum palmatum extract significantly inhibited glucan synthesis by both S. mutans and S. sobrinus, compared to the control.
Discussion
We screened the various candidate oriental medicines including Rheum palmatum for their antibacterial activity, inhibition of glucan synthesis and formation of biofilms. In accordance with our previous study, we found that Rheum palmatum extract most effectively inhibited the growth of S. mutans and S. sobrinus, synthesis of glucan, and formation of biofilms.
According to traditional Chinese medicine, Rheum palmatum is one of the most ancient and medically useful herbs14). Rheum palmatum has thick roots, hollow and erect stems, and small white-green or purple-red flowers clustered on its branches and belongs to the Rheum L. genus from the Polygonaceae family14). Anthraquinones are a major group of the polyphenol constituents of Rheum palmatum, and these include aloe-emodin, rhein, emodin, chrysophanol, and their glycosides15). Recent studies have reported anticancer16), antiviral17), antioxidant18), and antidiabetic activities of anthraquinones15). Also, Rheum palmatum has long been used as an anti-inflammatory, anti-fibrotic and anticancer medicine in China19). In the ancient Chinese book “Shen Nong Ben Cao Jing”, the rhizome of Rheum palmatum was classified as an important medicinal plant20). Furthermore, there is clinical evidence of the efficacy of Rheum palmatum in treating chronic hepatic diseases20). Although studies have been conducted to evaluate the role of Rheum palmatum in various diseases, its effect on dental caries has not been examined to date.
In this study, the Rheum palmatum extract showed a strong inhibitory effect on the growth of S. mutans and S. sobrinus in a dose-dependent manner. Specifically, the number of viable bacteria was significantly reduced when S. mutans and S. sobrinus were exposed to Rheum palmatum extract at a concentration of 2.5% for 18 h. To date, Rheum palmatum has been found to have antibacterial effects only on Staphylococcus aureus and Pseudomonas aerginosa21). The antibacterial effect of Rheum palmatum extract on S. mutans and S. sobrinus can make it a useful agent for use in the prevention of dental caries.
Next, we investigated the effects of Rheum palmatum extract on the formation of biofilms by S. mutans and S. sobrinus. The microbial population, including streptococci, forms a dental biofilm in the oral cavity that is composed of bacteria and bacterial products including polysaccharides enclosed in a matrix of extracellular material derived both from the cells themselves and the environment2). Compared to floating bacteria, the biofilm can withstand a variety of environmental stresses such as nutrient depletion, dehydration, pH shifts, and osmotic shock. Biofilms adversely affect oral health and are resistant to commonly used antibacterial agents, making treatment of dental caries a significant challenge22). Therefore, it is important to inhibit the biofilm formation by streptococci to prevent dental caries. In this study, the Rheum palmatum extract significantly reduced the biofilm formation by S. mutans and S. sobrinus.
Finally, we examined the effects of Rheum palmatum extract on glucan synthesis by streptococci. Glucans are a major factor contributing to the ability of streptococci to adhere to the tooth surface and for bacterial cells to aggregate within a biofilm. The presence of glucan, allows the bacteria to remain adhered to the teeth, despite the action of physical forces such as mastication23). For the prevention of dental caries, it is not only necessary to inhibit the biofilm formation, but also essential to inhibit the synthesis of glucans. S. mutans synthesizes glucans from sucrose using GTFase enzyme. Both soluble and insoluble glucans have strong adhesive properties23). Specifically, the insoluble glucans synthesized by the oral bacteria play an important role in the formation of biofilm by promoting their adhesion and accumulation24). Various Chinese herbal medicines25), ribocitrin26), and polyphenols of cocoa bean husk27) have been studied to assess their GTFase inhibitory activity28). Of these, extracts of two Chinese herbal medicines, the Coptis chinensis extract29) and Radix pulsatillae extract30) have been shown to simultaneously inhibit the formation of the biofilm and the synthesis of glucans. The present results with Rheum palmatum extract are similar, as the extract inhibited not only the formation of biofilm but also glucan synthesis.
Conclusions
This study showed that the Rheum palmatum extract inhibited the growth, biofilm formation, and glucan synthesis of S. mutans and S. sobrinus. Therefore, Rheum palmatum extract could be suggested as a potential candidate for the control of dental caries by virtue of its antibacterial effects.
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