**3. Preparation and property of phytochemicals with anticariogenic effect**

We have investigated the anticariogenic effects of phytochemicals such as ELM, ES and Alg53. Each of phytochemicals has unique properties and structure.

### **3.1 Extractive from the leaves of** *Morus alba* **(ELM)**

*Morus alba* has been used for centuries in Japan as a tea infusion. *Morus alba* contains DNJ and some of its derivatives, which are well known as α-glucosidase inhibitors, as shown in Fig. 1 (Asano et al, 1994). D-glucose analogs such as voglibose, miglitol and acarbose, with nitrogen-in-rings, have been used for the treatment of DM (Drent et al, 2002; Raimbaud et al, 1992; Yasuda et al, 2003).

Fig. 1. Chemical structures of components of the extractive from the leaves of *Morus alba*, 1 deoxynojirimycin and its derivatives

We have clarified that ELM competitively inhibits the activity of sucrase, maltase, and isomaltase using human and rat intestinal homogenates, and significantly suppresses the increment in blood glucose levels, when ELM is administered with sucrose to rats (Oku et al, 2006). In addition, we found that confections with ELM effectively suppress the postprandial blood levels of glucose and insulin in healthy humans (Nakamura M et al, 2009). We suppose that confections with ELM can contribute to the prevention and the quality-oflife for pre-diabetic and diabetic patients.

Inhibitory Effects of the Phytochemicals Partially

**D-mannuronic acid block** 

**L-guluronic acid block**

Fig. 3. Chemical structure of components of alginate

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

ES, and oolong as the positive control.

not inhibit the pH reduction by *S. sobrinus* 6715*.*

**D-mannuronic acid and L-guluronic acid block** 

**4.** *In vitro* **evaluation of the anticariogenic effects of phytochemicals** 

In this section, we introduce the anticariogenic effects evaluated using the three *in vitro* methods described above. The phytochemicals used in our experiments were Alg53, ELM,

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

Hydrolyzed Alginate, Leaf Extracts of *Morus alba* and *Salacia* Extracts on Dental Caries 229

mean M.W. of partially decomposed alginate is approximately 1,000 by column chromatography using a Sephadex G-15 column. Tseng et al reported that alginate lyase isolated from *Vibrio alginolyticus* (ATCC17749) has specificity for polymannuronic blocks, and Haug et al reported that depolymerizing alginate by lyase yields a product containing deoxyuronic acid (Tseng et al, 1992; Haug et al, 1967). Therefore, it is considered that Alg53 also comprises penta- or hexa-mannuronic acid with deoxymannuronic acid as the nonreducing terminal moiety. The conversion ratio of Alg53 by *Vibrio alginolyticus* SUN53 is very low, so we could not obtain a sufficient amount of Alg53 for *in vivo* experiments using animals. We have to develop a culture condition in which Alg53 is produced effectively.

To prepare the ELM solution, the leaves are extracted with 50% ethanol, and ethanol is removed with a rotary evaporator. ELM used in this study is kindly provided by Toyotama Healthy Food Co., Ltd. (Tokyo, Japan). The original extract solution contains 0.24% DNJ. A small amount of several types of DNJ derivative is measured using liquid chromatographymass spectrometry (LC-MS). It has been clarified that this extraction is not associated with toxicity or hematologic, blood biochemical, or pathologic abnormalities in rats (Miyazawa et al*,* 2003).
