**1. Introduction**

220 Contemporary Approach to Dental Caries

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Many studies on natural materials with anticariogenic effects have been carried out. Anticariogenic materials, such as polyphenols from oolong tea (Nakahara et al, 1993) and polyphenols from cacao (Ito et al, 2003), are known to be inhibitors of glucosyltransferases (GTases). These compounds have been used in foods to prevent or reduce dental caries. Sugar alcohols and oligosaccharides, which are not utilized as the substrate of GTase, are known as alternative sweeteners to sucrose (Kawanabe et al, 1992; Makinen et al, 1995; Ooshima et al, 1992; Van Loveren, 2004).

We investigated the potentiality of inhibitory effects of some phytochemicals on dental caries, because it is very interesting that phytochemical components inhibit the activity of not only GTase but also α-glucosidase. We have clarified that some phytochemicals such as partially decomposed alginate (Alg53), extractives from the leaves of *Morus alba* (ELM) and extractives from *Salacia chinensis* (ES) have inhibitory effects on disaccharidases such as maltase, sucrase and trehalase.

Alginate is a polyuronic saccharide that is isolated from the cell walls of a number of brown seaweed species around the world, and produced as an extracellular matrix by certain bacteria (Draget et al, 2003). It has a gelling ability, stabilizing properties and high viscosity. Alginate and its decomposed derivatives are widely used in foods, cosmetics and pharmaceutical industries (Ci et al, 1999; Johnson et al, 1997). Alginate hydrolysates exhibit many bioactivities, such as stimulating human keratinocytes, accelerating plant root growth and enhancing penicillin production from cultures of *Penicillium chrysogenum* (Ariyo et al, 1998; Kawada et al, 1997; Natsume et al, 1994). We have clarified that alginic acid with lowered molecule (mean molecular weight about 55,000) has suppressive effects on the elevation of blood glucose and insulin secretion (Oku et al, 1997) and improves defecation ant the fecal conditions (Oku et al, 1998).

*Morus alba* has traditionally been cultivated in China, Korea and Japan to use its leaves to feed silkworms. Recently, health benefits of *Morus alba* have been clarified and naturally occurring 1-deoxynojirimycin (DNJ) was isolated from its roots (Yagi et al, 1976). DNJ is glucose analogue with a secondary amine group instead of an oxygen atom in the pyranose

Inhibitory Effects of the Phytochemicals Partially

Several typical methods are described below.

dependent cell adhesion.

**2.1** *In-vitro* **experiments to evaluate anticariogenic effects** 

**2.1.1 Evaluation of inhibitory effects on pH decline by acid production** 

Therefore, the rapid and reliable method is suitable for *in vitro* assay.

**2.1.2 Evaluation of inhibitory effects on glucan production by GTase** 

Water-insoluble and water-soluble α-linked glucans produced from sucrose due to the action of GTases adhere to the surfaces of teeth and promote the development of dental caries. GTase inhibitors disturb the production of these glucans and prevent the development of dental caries. The inhibitory effect of test substances on GTase has been evaluated by partially purified GTase from mutans streptococci, particularly *Streptococcus mutans* and *Streptococcus sobrinus,* which are considered to be the primary causative agents of dental caries in humans. Partially purified GTase can be conveniently used to evaluate the inhibitory effects of test substances on glucan production because it is stable and readily administered after preparation. If *S. mutans* and *S. sobrinus* are directly used to evaluate the inhibitory effects of test substances on glucan production, the assay is complicated and additional effort is required

Hydrolyzed Alginate, Leaf Extracts of *Morus alba* and *Salacia* Extracts on Dental Caries 223

Because the inducing factor of dental caries is complicate, some types of *in vitro* experiments have to be carried out and judged carefully to recognize characteristics of phytochemicals.

*In vitro* experiments to evaluate the anticariogenic effects of phytochemicals include observations of pH decline by acid production, inhibitory effects on glucan production by GTase from mutans streptococci and sucrose-dependent cell adhesion on smooth surfaces by mutans streptococci*.* In addition, antibacterial effect and the evaluation of plaque accumulation or enamel demineralization by using artificial mouth also have been investigated (Hinoide et al, 1984; Pigman et al, 1952). We have evaluated the anticariogenic effects of phytochemicals based on pH decline, glucan production by GTase and sucrose-

Oral bacteria produce organic acids from sugars. If oral pH declines to approximately 5.5 due to organic acids produced, enamel demineralization of teeth begins. If organic acids produced from sugars by mutans streptococci are omitted or decreased and oral pH does not decline to 5.5, enamel demineralization is prevented by buffering action of saliva. Therefore, the degree of pH decline of the culture medium is measured during the incubation with oral bacteria (especially mutans streptococci) in the presence and absence of test substance with anticariogenic effects. Our procedure to measure pH decline is rapid and reliable. Cell suspension and phytochemical solution are added to 20 mM glucose solution in Stephan's buffer (pH 7.0) in a test tube (total volume, 1.0–2.0 mL), and incubated for 10– 60 min at 37°C under anaerobic conditions after mixing. During incubation, the pH of the reaction medium is periodically measured with a portable pH meter (Hashiguchi-Ishiguro et al, 2009). Another method uses an incubation period of 24-48 h. But oral pH declines to about 4 immediately after the ingestion of sugars as glucose and sucrose (Lingström et al, 2000). Actually, the pH of reaction medium resulted in an immediate decline within 30 min in our study. In practical eating, if we intake sugars from meals or snacks, the food is masticated and swallowed within a few minutes and the pH in oral cavity declines.

ring of glucose. Then, DNJ has also been found in the leaves and fruits of *Morus alba* (Asano et al, 1994, 2001). Ever since, preventive effect of *Morus alba* on diabetes by α-glucosidase inhibitor has been extensively studied. Furthermore, *Morus alba* has been clarified multiple biological and physiological effects, as well as hypoglycemic, anti-oxidant and decrease in serum triacylglycerol (TG) level (Kojima et al, 2010). In a long term treatment study, intake of *Morus alba* does not cause harmful effects (Kimura et al, 2007).

The roots and stems of *Salacia* species plants have been used in the Ayurvedic system of Indian medicine to treat diabetes mellitus (DM) (Li et al, 2008). *Salacia* is a woody climbing plant belonging to the Celastrceae family that is found in limited regions of India and Sri Lanka. Currently, extracts of *Salacia* are consumed in commercial foods and food supplements in Japan for the treatment of diabetes and obesity. The water soluble portion of the methanolic extract inhibits α-glucosidase. Moreover, Beppu et al have reported that *Salacia reticulata* has improvement effect of fasting blood glucose and HbA1c levels in human including mild type 2 diabetics and has no toxicity (Beppu et al, 2006). The potential genotoxicity of *Salacia oblonga* extract was evaluated and it was determined not to be genotoxic (Flammang et al, 2006).

These phytochemicals, Alg53, ELM and ES competitively inhibit sucrase, maltase and trehalase of vesicles of the brush border membrane of rat intestine. The activity of GTase is inhibited by some polyphenols. We hypothesized that phytochemicals that inhibit αglucosidases such as sucrase may also inhibit the synthesis of glucan from sucrose by GTase, because the latter is also a type of enzyme related to carbohydrate metabolism. Conversely, acarbose and DNJ are known to competitively inhibit sucrase and GTase, and suppress the postprandial elevation of blood levels of glucose and insulin (Newbrun et al, 1983). These chemicals are used as medicine for the treatment of DM.

We have investigated the anticariogenic effect of some phytochemicals using simple *in vitro*  methods. The inhibitory effect of phytochemicals on the production of glucan from sucrose by GTase can be used for screening of the anticariogenic effects of natural materials. Surveys of natural materials with anticariogenic effects are important for reduction of the development of dental caries. Discovering new materials to prevent dental caries could expand the repertoire of the development of functional foods for oral health. These functional foods for oral health should be used in combination with different types of materials because the development of dental caries is related to multiple factors.

In this chapter, we introduce the procedures employed to evaluate the anticariogenic effects of phytochemicals. We also discuss the properties of three phytochemicals, Alg53, ELM and ES. Although many natural materials have been studied for anti-cariogenic effect, in our knowledge, this is the first and unique report that α-glucosidase inhibitor also inhibit GTase activity in natural materials. That is to say, Alg53, ELM and ES are expected as multiple functional food materials which have the effects of prevention to dental caries, diabetes and obesity. Moreover, if we search for natural materials that inhibit GTase, it might be a key point that certain materials have inhibition of α-glucosidase activity.
