**4.1 Effects of Alg53 on acid production by** *S. sobrinus* **6715**

The production of organic acids by *S. sobrinus* 6715 is illustrated in Fig. 4. The positive control maintained the initial pH. The results indicated that Alg53 disturbed the conversion of the substrate to organic acids. In contrast, the absence of Alg53 resulted in an immediate decline in pH after addition of the substrate, with the pH finally reaching 4.1. The addition of Alg53 suppressed pH decline and maintained a pH of 5.0. This suppressive effect for the production of organic acids was dependent upon the concentrations of Alg53 in the reaction mixture (Fig. 5). The inhibitory effect on pH decline was also investigated using ELM, ES and oolong, but these phytochemicals did not inhibit the pH reduction by *S. sobrinus* 6715*.*

Inhibitory Effects of the Phytochemicals Partially

demonstrate considerable anticariogenic effects.

**1**

**0**

(Hashiguchi-Ishiguro et al, 2009)

**Rate of glucan**

**20**

**0**

**40**

**60**

**80**

**100**

**produced (%)**

pH was measured after incubation for 1 h. Control: water was added instead of Alg53

**2**

**3**

**p H**

**4**

**5**

**6**

Hydrolyzed Alginate, Leaf Extracts of *Morus alba* and *Salacia* Extracts on Dental Caries 231

insoluble (but not water-soluble) glucan by GTase from *S. sobrinus* 6715. Water-insoluble glucan is closely associated with the formation of biofilms on teeth surface. In addition, Alg53 has inhibitory effects on acid production and synthesis of glucan by mutans streptococci. That is, Alg53 has two types of anticariogenic effects. Accordingly, Alg53 may

> ×**1** ×**2** ×**5 Dilution factors of Alg53**

Fig. 5. Inhibitory effect by different concentrations of partially decomposed alginate by

**20**

**0**

**40**

**60**

**80**

**100**

**(A)** *S. sobrinus* **6715 (B)** *S. mutans* **MT8148**

*V. alginolyticus* SUN53 on acid production from glucose by *S. sobrinus* 6715

**Control ELM ES Oolong**

oolong on insoluble glucan produced by GTase

ELM, extractive from the leaves of *Morus alba*; ES, extractive from *Salacia chinensis* 

Reaction mixture [GTase, 0.2 mL; sucrose solution, 0.31 mL (includes 14C-sucrose, 20 μCi); test substance, 0.1 mL] incubated at 20° for 24 h at 37°C. The final concentration of sucrose in the reaction mixture was 1%. Glucan was expressed as the relative amount (%) of glucan produced as compared with the amount produced with the negative control (distilled water) (Hashiguchi et al, 2011).

Fig. 6. Inhibitory effects of the extractive from the leaves of *Morus alba*, *Salacia chinensis* and

**control**

**Control ELM ES Oolong**

#### **4.2 Effects of ELM, ES, Alg53 and oolong on glucan production by GTase from** *S. sobrinus* **6715 and** *S. mutans* **MT8148**

Oolong has been used as a functional food to prevent dental caries. Oolong was therefore used to compare the inhibitory effects of other phytochemicals on glucan production by GTase. The inhibitory effect of phytochemicals on water-insoluble glucan synthesis by GTase from *S.sobrinus* 6715 is illustrated in Fig. 6A. The original ELM solution reduced the production of water-insoluble glucan to 66% of that of the control (ELM-free). ES also significantly reduced the synthesis of water-insoluble glucan. The inhibitory effect of ES was remarkable compared with that of ELM. The inhibitory effect of oolong on the production of water-insoluble glucan by GTase was stronger than that of ELM and of a similar level to that of ES. Fig. 6B shows water-insoluble glucan synthesis by GTase from *S. mutans* MT8148. ELM significantly inhibited the glucan production by GTase from *S. mutans* MT8148, and the ratio of inhibition of production of water-insoluble glucan was 64% that of the control (ELM-free). The inhibitory effect of ES and oolong on glucan production by GTase from *S. sobrinus* 6715 was stronger than that by GTase from *S. mutans* MT8148. The inhibitory effect of ELM was of a similar level on glucan production by GTase from *S. sobrinus* 6715 and *S. mutans* MT8148.

Open circle, positive control (no production of acid); open square, negative control (no inhibition); closed triangle, with Alg53

In the positive control (no production of acid), Stephan's buffer (pH 7.0) was added instead of glucose. In the negative control (no inhibition), distilled water was added instead of Alg53. Data are mean values of duplicate assays (Hashiguchi-Ishiguro et al, 2009).

Fig. 4. Time-course of pH decrease with acid production by *S. sobrinus* 6715 from glucose with and without partially decomposed alginate by *V. alginolyticus* SUN53

The inhibitory effect of Alg53 on water-insoluble and water-soluble glucan synthesis by GTase from *S.sobrinus* 6715 is illustrated in Fig. 7. The original Alg53 solution and a ten-fold dilution of Alg53 solution reduced the production of water-insoluble glucan to 21% and 23%, respectively. However, Alg53 barely affected the production of water-soluble glucan by GTase. These results demonstrated that Alg53 clearly inhibits the synthesis of waterinsoluble (but not water-soluble) glucan by GTase from *S. sobrinus* 6715. Water-insoluble glucan is closely associated with the formation of biofilms on teeth surface. In addition, Alg53 has inhibitory effects on acid production and synthesis of glucan by mutans streptococci. That is, Alg53 has two types of anticariogenic effects. Accordingly, Alg53 may demonstrate considerable anticariogenic effects.

pH was measured after incubation for 1 h. Control: water was added instead of Alg53 (Hashiguchi-Ishiguro et al, 2009)

230 Contemporary Approach to Dental Caries

Oolong has been used as a functional food to prevent dental caries. Oolong was therefore used to compare the inhibitory effects of other phytochemicals on glucan production by GTase. The inhibitory effect of phytochemicals on water-insoluble glucan synthesis by GTase from *S.sobrinus* 6715 is illustrated in Fig. 6A. The original ELM solution reduced the production of water-insoluble glucan to 66% of that of the control (ELM-free). ES also significantly reduced the synthesis of water-insoluble glucan. The inhibitory effect of ES was remarkable compared with that of ELM. The inhibitory effect of oolong on the production of water-insoluble glucan by GTase was stronger than that of ELM and of a similar level to that of ES. Fig. 6B shows water-insoluble glucan synthesis by GTase from *S. mutans* MT8148. ELM significantly inhibited the glucan production by GTase from *S. mutans* MT8148, and the ratio of inhibition of production of water-insoluble glucan was 64% that of the control (ELM-free). The inhibitory effect of ES and oolong on glucan production by GTase from *S. sobrinus* 6715 was stronger than that by GTase from *S. mutans* MT8148. The inhibitory effect of ELM was of a similar level on glucan production by GTase from *S. sobrinus* 6715 and *S.* 

**Incubation time (min)**

**0 15 30 45 60**

Open circle, positive control (no production of acid); open square, negative control (no inhibition);

In the positive control (no production of acid), Stephan's buffer (pH 7.0) was added instead of glucose. In the negative control (no inhibition), distilled water was added instead of Alg53. Data are mean values

Fig. 4. Time-course of pH decrease with acid production by *S. sobrinus* 6715 from glucose

The inhibitory effect of Alg53 on water-insoluble and water-soluble glucan synthesis by GTase from *S.sobrinus* 6715 is illustrated in Fig. 7. The original Alg53 solution and a ten-fold dilution of Alg53 solution reduced the production of water-insoluble glucan to 21% and 23%, respectively. However, Alg53 barely affected the production of water-soluble glucan by GTase. These results demonstrated that Alg53 clearly inhibits the synthesis of water-

with and without partially decomposed alginate by *V. alginolyticus* SUN53

**4.2 Effects of ELM, ES, Alg53 and oolong on glucan production by GTase from** *S.* 

*sobrinus* **6715 and** *S. mutans* **MT8148** 

*mutans* MT8148.

**4**

of duplicate assays (Hashiguchi-Ishiguro et al, 2009).

**5**

**6**

**pH**

closed triangle, with Alg53

**7**

**8**

Fig. 5. Inhibitory effect by different concentrations of partially decomposed alginate by *V. alginolyticus* SUN53 on acid production from glucose by *S. sobrinus* 6715

ELM, extractive from the leaves of *Morus alba*; ES, extractive from *Salacia chinensis*  Reaction mixture [GTase, 0.2 mL; sucrose solution, 0.31 mL (includes 14C-sucrose, 20 μCi); test substance, 0.1 mL] incubated at 20° for 24 h at 37°C. The final concentration of sucrose in the reaction mixture was 1%. Glucan was expressed as the relative amount (%) of glucan produced as compared with the amount produced with the negative control (distilled water) (Hashiguchi et al, 2011).

Fig. 6. Inhibitory effects of the extractive from the leaves of *Morus alba*, *Salacia chinensis* and oolong on insoluble glucan produced by GTase

Inhibitory Effects of the Phytochemicals Partially

**Blank Control ELM ES Oolong**

2011).

with sucrose

**A: Right after incubation B: After washing**

ELM, extractive from the leaves of *Morus alba*; ES, extractive from *Salacia chinensis*

**100**

**0**

oligosaccharides to prevent dental caries.

**20**

**40**

**60**

**Rate of cell adhesion (%)**

**80**

**100**

Reaction mixture [cell solution, 0.5 mL; 2% sucrose (final concentration, 1%) in BHI, 0.8 mL; test substance 0.3 mL] incubated at 20° for 24 h at 37°C. In the blank, distilled water was added instead of sucrose. In the negative control, distilled water was added instead of test substance (Hashiguchi et al,

Fig. 8. Adhesion of *S. sobrinus* 6715 and glucan on smooth surfaces of glass after incubation

**60**

Hydrolyzed Alginate, Leaf Extracts of *Morus alba* and *Salacia* Extracts on Dental Caries 233

**Blank Control ELM ES Oolong**

**34**

**21**

**Control ELM ES Oolong**

Fig. 9. Inhibitory effects of the extractive from the leaves of *Morus alba* and *Salacia chinensis* 

In addition, Alg53 suppressed pH decline by the production of organic acids from glucose, whereas ELM and ES could not suppress pH decline as well as oolong. For the prevention of dental caries, Alg53 may be useful as a functional food that has two types of inhibitory effects on the synthesis of glucan by GTase and acid production. Alternative sweeteners for sucrose, such as sugar alcohols and oligosaccharides, are not used as substrates for acid production by mutans streptococci, so pH decline does not occur. However, alternative sweeteners cannot inhibit the production of organic acids from sugars. Therefore, we recommend that ELM and ES are used in a combination of sugar alcohols or

on the adhesion of *S. sobrinus* 6715 and glucan (Hashiguchi et al, 2011)

Reaction mixture [3% sucrose (final concentration, 1%) in 0.1 M phosphate buffer (pH 6.8), 1 mL; GTase from *S. sobrinus*, 0.3 mL; 0.1 M phosphate buffer (pH 6.8), 1.4 mL; Alg53, 0.3 mL] incubated at 20° for 24 h at 37°C. Glucan was expressed as the relative amount (%) of glucan produced as compared with the amount produced in the absence of Alg53. The amount of total carbohydrate was measured at 490 nm by the phenol-sulfuric acid method. Dates are expressed as mean values of duplicate assays (Hashiguchi-Ishiguro et al, 2009).

Fig. 7. Inhibitory effect of partially decomposed alginate by *V. alginolyticus* SUN53 on waterinsoluble and water-soluble glucan produced by GTase from *S. sobrinus*

#### **4.3 Effects of ELM and ES on sucrose-dependent cell adhesion on smooth surfaces**

The inhibitory effect of ELM on sucrose-dependent adherence of cells onto the surface of glass test tubes was examined using growing cells of *S. sobrinus* 6715. Fig. 8A shows that cells adhered to the surface of glass test tubes after incubation. The cells grew well and adhered to the glass surface of the control (no phytochemical), ELM, ES and oolong. However, cells and glucan did not adhere to the glass surface of blank test tubes (sucrose-free). Fig. 8B shows the conditions of test tubes in which the reaction mixture was removed by pipetting, and then washed gently with distilled water. As shown clearly in Fig. 8B, cell adhesion was very strong in control test tubes, but was feeble in ELM, ES and oolong tubes; cells were removed by washing. The results demonstrate that ELM and ES inhibit the adhesion of cells to the glass surface. Adhered cells that remained on the surface of glass test tubes after washing were suspended with 1 N NaOH and absorbance measured at 550 nm (Fig. 9). The cell number was 60% for ELM and 21% for ES compared with that of the control.

#### **5. Potential of phytochemicals as anticariogenic materials**

The main finding of this study is that three phytochemicals, partially decomposed alginate by SUN53 (Alg53), the extractive from the leaves of *Morus alba (*ELM) and the extractive from *Salacia chinencis* (ES), have inhibitory effects on glucan synthesis by GTase. It may become a key point that certain phytochemicals have inhibitory effects on α-glucosidase when we screen natural materials which inhibit Gtase activity. However, the degree of inhibitory effect is not always similar for sucrase and GTase. The inhibitory effect of ELM and ES on sucrase was very strong. The inhibitory constant (*K*i) of ELM and ES for sucrase was 2.1×10–4 mM and 6.7×10–4 mM, respectively (Oku et al, 2006).

**20**

**0**

Reaction mixture [3% sucrose (final concentration, 1%) in 0.1 M phosphate buffer (pH 6.8), 1 mL; GTase from *S. sobrinus*, 0.3 mL; 0.1 M phosphate buffer (pH 6.8), 1.4 mL; Alg53, 0.3 mL] incubated at 20° for 24 h at 37°C. Glucan was expressed as the relative amount (%) of glucan produced as compared with the amount produced in the absence of Alg53. The amount of total carbohydrate was measured at 490 nm

Fig. 7. Inhibitory effect of partially decomposed alginate by *V. alginolyticus* SUN53 on water-

**4.3 Effects of ELM and ES on sucrose-dependent cell adhesion on smooth surfaces**  The inhibitory effect of ELM on sucrose-dependent adherence of cells onto the surface of glass test tubes was examined using growing cells of *S. sobrinus* 6715. Fig. 8A shows that cells adhered to the surface of glass test tubes after incubation. The cells grew well and adhered to the glass surface of the control (no phytochemical), ELM, ES and oolong. However, cells and glucan did not adhere to the glass surface of blank test tubes (sucrose-free). Fig. 8B shows the conditions of test tubes in which the reaction mixture was removed by pipetting, and then washed gently with distilled water. As shown clearly in Fig. 8B, cell adhesion was very strong in control test tubes, but was feeble in ELM, ES and oolong tubes; cells were removed by washing. The results demonstrate that ELM and ES inhibit the adhesion of cells to the glass surface. Adhered cells that remained on the surface of glass test tubes after washing were suspended with 1 N NaOH and absorbance measured at 550 nm (Fig. 9). The cell number was

The main finding of this study is that three phytochemicals, partially decomposed alginate by SUN53 (Alg53), the extractive from the leaves of *Morus alba (*ELM) and the extractive from *Salacia chinencis* (ES), have inhibitory effects on glucan synthesis by GTase. It may become a key point that certain phytochemicals have inhibitory effects on α-glucosidase when we screen natural materials which inhibit Gtase activity. However, the degree of inhibitory effect is not always similar for sucrase and GTase. The inhibitory effect of ELM and ES on sucrase was very strong. The inhibitory constant (*K*i) of ELM and ES for sucrase

by the phenol-sulfuric acid method. Dates are expressed as mean values of duplicate assays

insoluble and water-soluble glucan produced by GTase from *S. sobrinus*

60% for ELM and 21% for ES compared with that of the control.

**5. Potential of phytochemicals as anticariogenic materials** 

was 2.1×10–4 mM and 6.7×10–4 mM, respectively (Oku et al, 2006).

**Dilution factors of Alg53 Dilution factors of Alg53**

**Control** ×**20** ×**10** ×**1**

**40**

**60**

**80**

**100**

**(A) Water-insoluble glucan (B) Water-soluble glucan**

**Control** ×**20** ×**10** ×**1**

**Rate of glucan**

**20**

**0**

(Hashiguchi-Ishiguro et al, 2009).

**40**

**60**

**80**

**100**

**produced (%)**

ELM, extractive from the leaves of *Morus alba*; ES, extractive from *Salacia chinensis* Reaction mixture [cell solution, 0.5 mL; 2% sucrose (final concentration, 1%) in BHI, 0.8 mL; test substance 0.3 mL] incubated at 20° for 24 h at 37°C. In the blank, distilled water was added instead of sucrose. In the negative control, distilled water was added instead of test substance (Hashiguchi et al, 2011).

Fig. 8. Adhesion of *S. sobrinus* 6715 and glucan on smooth surfaces of glass after incubation with sucrose

Fig. 9. Inhibitory effects of the extractive from the leaves of *Morus alba* and *Salacia chinensis*  on the adhesion of *S. sobrinus* 6715 and glucan (Hashiguchi et al, 2011)

In addition, Alg53 suppressed pH decline by the production of organic acids from glucose, whereas ELM and ES could not suppress pH decline as well as oolong. For the prevention of dental caries, Alg53 may be useful as a functional food that has two types of inhibitory effects on the synthesis of glucan by GTase and acid production. Alternative sweeteners for sucrose, such as sugar alcohols and oligosaccharides, are not used as substrates for acid production by mutans streptococci, so pH decline does not occur. However, alternative sweeteners cannot inhibit the production of organic acids from sugars. Therefore, we recommend that ELM and ES are used in a combination of sugar alcohols or oligosaccharides to prevent dental caries.

Inhibitory Effects of the Phytochemicals Partially

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