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

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. 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

Inhibitory Effects of the Phytochemicals Partially

at 550 nm.

indicates the possibility to reveal anticariogenic effects.

the details of the procedure (Koo et al, 2002, 2010).

**2.2** *In-vivo* **experiments to evaluate anticariogenic effects** 

**2.2.1 Animals and diets to evaluate anticariogenic effects** 

Hydrolyzed Alginate, Leaf Extracts of *Morus alba* and *Salacia* Extracts on Dental Caries 225

between glucan and oral bacteria. Therefore, the measurement of sucrose-dependent cell adhesion is used to evaluate the formation of biofilms on teeth surface. If sucrose-dependent cell adhesion on the smooth surface is inhibited by phytochemicals, the test substance

This procedure has been described by Hamada and Torii (Hamada and Torii, 1978). After mutans streptococci (*S. sobrinus* or *S. mutans*) are grown in the medium, the collected cells are resuspended in the medium. The cell suspension (0.5 mL), sucrose solution (final concentration, 1%) and 0.3 mL of phytochemical solution are mixed in a new glass test tube, and incubated at angle 20° for 24 h at 37°C. After incubation, the reaction mixture containing nonadherent cell is gently removed by a Pasteur pipette. The glass test tube upon which mutans streptococci adhere to the surface is gently washed with distilled water at angle 20°. In the control, cell and glucan are not peeled by washing because of those are tightly adhere on test tube. Then, the cell and glucan are suspended in 1 N NaOH to measure absorbance

We have tried to evaluate the inhibitory effect of phytochemicals using this method, described below (Hashiguchi-Ishiguro et al, 2011). The degree of inhibitory effects on cell adhesion correlated roughly with inhibition of glucan production by phytochemicals. Anticariogenic effects of extractives from red wine, apple polyphenols and propolis are evaluated using this method (Furiga et al, 2008; Hayacibara et al, 2005; Yanagida et al, 2000). In this method, the surface of glass test tube is used for smooth surface model of teeth. The glass test tube used in this method must be new one that has very smooth surface with no flaw. Human saliva-coated hydroxyapatite beads are used as teeth surface model in other method (Venkitaraman et al, 1995), because teeth consists mainly of hydroxyapatite. The model of latter method reflects the situation of oral environment. Koo et al have reported

If anticariogenic effects of phytochemicals are demonstrated by some *in vitro* experiments as described above, an *in vivo* experiment using experimental animals (e.g., rats) is carried out to confirm that the test material has anticariogenic effects in the systemic body. The animal experiment to evaluate anticariogenic effects consumes much expense and time. In other method, human plaque is also used the *in vivo* experiment. "Touch electrode method" and "plaque sampling method" are used for the purpose of measuring human plaque pH (Frostell, 1970; Stephan, 1940). Mühlemann et al develop " indwelling plaque pH telemetry method" (Graf H, et al 1966). In this section, we show an outline on animal experiment.

Fifteen-day-old specific pathogen-free Sprague–Dawley (SD) rats are suitable for caries studies. The first and second molars are coming through at this age. Mutans streptococci are inoculated to animals during this period. If inoculation lags behind, the prevalence of dental caries is reduced (Ooshima et al, 1994). The number of mutans streptococci that must be inoculated to definitely cause dental caries is very important. The breeding period after inoculation with mutans streptococci is about 55 days. Diet #2000 is a popular diet in animal experiments on caries (Keyes and Jordan, 1964) and contains 56% sucrose. If the percentage

for the storage and administration of mutans streptococci. Hence, we are using a partially purified GTase from *S. mutans* and *S. sobrinus* to evaluate the inhibitory effects on glucan production. *S. sobrinus* GTase which synthesizes the water-insoluble glucan is released into the reaction medium during culture and that of *S. mutans* is localized mainly on the cell surface (Furuta et al, 1985). The properties and preparation method of GTase have been described (Baba et al, 1986; Furuta et al, 1985; Hamada et al, 1989).

An outline of our procedure to evaluate the inhibitory effect of phytochemicals on glucan production by GTase is given here. To measure the inhibition by phytochemicals for the synthesis of glucan from sucrose, 1 mL of 3% sucrose solution, 0.3 mL of GTase solution, 0.3 mL of test solution, and 1.4 mL of 0.1 M phosphate buffer are mixed and incubated at an angle of 20° for 24 h at 37°C. After the reaction is stopped in boiling water, the reaction mixture is centrifuged to separate water-insoluble and water-soluble glucans. The amount of total carbohydrate is measured using the phenol-sulfuric acid method (Dubois et al, 1956).

The phenol-sulfuric acid method is a popular and simple method for the determination of glucan produced by GTase (Koo et al, 2002). However, this method randomly determines the amount of whole carbohydrate in the sample without a clear difference between glucan production and the structure of phytochemicals in the reaction medium. Therefore, if the structure of test phytochemicals is similar to glucose polymer, alternative method is recommended to evaluate the inhibitory effect on glucan production. One method is the determination of radioactive carbon (14C) transferred to glucan from 14C-sucrose by GTase. Briefly, 14C-sucrose is added to the reaction mixture mentioned above and incubated under identical conditions. After incubation, the amount of 14C incorporated into glucan is measured by a liquid scintillation counter. This method requires specific facilities, but can specifically determine glucose incorporated to glucan fraction by GTase.

The preparation of GTase to evaluate inhibitory effects upon glucan production was carried out in our experiments. Briefly, to prepare partially purified GTase, after *S. sobrinus* 6715 was grown for 24 h at 37°C in 2 L of Brain Heart Infusion (Difco, Franklin Lakes, NJ, USA), the supernatant containing GTase was precipitated with 60% saturated (NH4)2SO4 for 24 h at 4°C. Low-molecular weight (<30,000) proteins contained in the precipitate were removed using an ultrafiltration system (Millipore, Billerica, MA, USA). The crude GTase obtained was further purified by chromatography using a Bio-gel hydroxyapatite (BioRad, Hercules, CA, USA) column. GTase fractions eluted to 0.5 M with a linear gradient from 0.1 M to 0.6 M phosphate buffer (pH 6.8) were used for the assay of inhibitory effects of phytochemicals (Venkitaraman et al, 1995; Yanagida et al, 2000).

After *S. mutans* MT8148 was grown for 24 h at 37°C in 2 L of Brain Heart Infusion (Difco), the collected cells were suspended with 8 M urea to obtain the cell-extract solution. Lowmolecular weight (<30,000) proteins contained in the solution were removed using an ultrafiltration system (Millipore). The resulting solution was used to evaluate the inhibitory effects on glucan production (Yanagida et al, 2000).

#### **2.1.3 Evaluation of inhibitory effects on sucrose-dependent cell adhesion on smooth surfaces by mutans streptococci**

When mutans streptococci are cultured in a medium containing sucrose, they strongly adhere on smooth surfaces. Dental biofilms are formed on teeth surface by interaction

for the storage and administration of mutans streptococci. Hence, we are using a partially purified GTase from *S. mutans* and *S. sobrinus* to evaluate the inhibitory effects on glucan production. *S. sobrinus* GTase which synthesizes the water-insoluble glucan is released into the reaction medium during culture and that of *S. mutans* is localized mainly on the cell surface (Furuta et al, 1985). The properties and preparation method of GTase have been described

An outline of our procedure to evaluate the inhibitory effect of phytochemicals on glucan production by GTase is given here. To measure the inhibition by phytochemicals for the synthesis of glucan from sucrose, 1 mL of 3% sucrose solution, 0.3 mL of GTase solution, 0.3 mL of test solution, and 1.4 mL of 0.1 M phosphate buffer are mixed and incubated at an angle of 20° for 24 h at 37°C. After the reaction is stopped in boiling water, the reaction mixture is centrifuged to separate water-insoluble and water-soluble glucans. The amount of total

The phenol-sulfuric acid method is a popular and simple method for the determination of glucan produced by GTase (Koo et al, 2002). However, this method randomly determines the amount of whole carbohydrate in the sample without a clear difference between glucan production and the structure of phytochemicals in the reaction medium. Therefore, if the structure of test phytochemicals is similar to glucose polymer, alternative method is recommended to evaluate the inhibitory effect on glucan production. One method is the determination of radioactive carbon (14C) transferred to glucan from 14C-sucrose by GTase. Briefly, 14C-sucrose is added to the reaction mixture mentioned above and incubated under identical conditions. After incubation, the amount of 14C incorporated into glucan is measured by a liquid scintillation counter. This method requires specific facilities, but can

The preparation of GTase to evaluate inhibitory effects upon glucan production was carried out in our experiments. Briefly, to prepare partially purified GTase, after *S. sobrinus* 6715 was grown for 24 h at 37°C in 2 L of Brain Heart Infusion (Difco, Franklin Lakes, NJ, USA), the supernatant containing GTase was precipitated with 60% saturated (NH4)2SO4 for 24 h at 4°C. Low-molecular weight (<30,000) proteins contained in the precipitate were removed using an ultrafiltration system (Millipore, Billerica, MA, USA). The crude GTase obtained was further purified by chromatography using a Bio-gel hydroxyapatite (BioRad, Hercules, CA, USA) column. GTase fractions eluted to 0.5 M with a linear gradient from 0.1 M to 0.6 M phosphate buffer (pH 6.8) were used for the assay of inhibitory effects of phytochemicals

After *S. mutans* MT8148 was grown for 24 h at 37°C in 2 L of Brain Heart Infusion (Difco), the collected cells were suspended with 8 M urea to obtain the cell-extract solution. Lowmolecular weight (<30,000) proteins contained in the solution were removed using an ultrafiltration system (Millipore). The resulting solution was used to evaluate the inhibitory

**2.1.3 Evaluation of inhibitory effects on sucrose-dependent cell adhesion on smooth** 

When mutans streptococci are cultured in a medium containing sucrose, they strongly adhere on smooth surfaces. Dental biofilms are formed on teeth surface by interaction

carbohydrate is measured using the phenol-sulfuric acid method (Dubois et al, 1956).

specifically determine glucose incorporated to glucan fraction by GTase.

(Venkitaraman et al, 1995; Yanagida et al, 2000).

effects on glucan production (Yanagida et al, 2000).

**surfaces by mutans streptococci** 

(Baba et al, 1986; Furuta et al, 1985; Hamada et al, 1989).

between glucan and oral bacteria. Therefore, the measurement of sucrose-dependent cell adhesion is used to evaluate the formation of biofilms on teeth surface. If sucrose-dependent cell adhesion on the smooth surface is inhibited by phytochemicals, the test substance indicates the possibility to reveal anticariogenic effects.

This procedure has been described by Hamada and Torii (Hamada and Torii, 1978). After mutans streptococci (*S. sobrinus* or *S. mutans*) are grown in the medium, the collected cells are resuspended in the medium. The cell suspension (0.5 mL), sucrose solution (final concentration, 1%) and 0.3 mL of phytochemical solution are mixed in a new glass test tube, and incubated at angle 20° for 24 h at 37°C. After incubation, the reaction mixture containing nonadherent cell is gently removed by a Pasteur pipette. The glass test tube upon which mutans streptococci adhere to the surface is gently washed with distilled water at angle 20°. In the control, cell and glucan are not peeled by washing because of those are tightly adhere on test tube. Then, the cell and glucan are suspended in 1 N NaOH to measure absorbance at 550 nm.

We have tried to evaluate the inhibitory effect of phytochemicals using this method, described below (Hashiguchi-Ishiguro et al, 2011). The degree of inhibitory effects on cell adhesion correlated roughly with inhibition of glucan production by phytochemicals. Anticariogenic effects of extractives from red wine, apple polyphenols and propolis are evaluated using this method (Furiga et al, 2008; Hayacibara et al, 2005; Yanagida et al, 2000). In this method, the surface of glass test tube is used for smooth surface model of teeth. The glass test tube used in this method must be new one that has very smooth surface with no flaw. Human saliva-coated hydroxyapatite beads are used as teeth surface model in other method (Venkitaraman et al, 1995), because teeth consists mainly of hydroxyapatite. The model of latter method reflects the situation of oral environment. Koo et al have reported the details of the procedure (Koo et al, 2002, 2010).
