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

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.

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

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

Inhibitory Effects of the Phytochemicals Partially

investigated carefully before carrying out animal experiments.

Alg53. Each of phytochemicals has unique properties and structure.

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

**CH2OH**

**Fagomine (1,2-dideoxynojirimycin)**

**OH**

**<sup>H</sup> <sup>N</sup>**

**HO**

deoxynojirimycin and its derivatives

life for pre-diabetic and diabetic patients.

that of lactose intolerance.

1992; Yasuda et al, 2003).

**<sup>H</sup> <sup>N</sup>**

**OH**

**1-deoxynojirimycin**

**CH2OH**

**OH**

**HO**

Hydrolyzed Alginate, Leaf Extracts of *Morus alba* and *Salacia* Extracts on Dental Caries 227

α-glucosidase) provides many beneficial effects. However, if a large amount of sucrose and α-glucosidase are ingested simultaneously, transient diarrhea is caused because of an increase in osmotic pressure in the large intestine. This mechanism is thought to identical to

When a diet containing ELM or ES is added to Diet #2000 and given to rats, most of the rats suffer osmotic diarrhea during the experimental period and growth is slightly suppressed. ELM and ES strongly inhibit sucrase activity. Hence, a lot of sucrose of Diet #2000 is transferred to the lower intestine and may cause osmotic diarrhea. Osmotic diarrhea may reduce the immune response, and disturb anticariogenic effect of phytochemicals in experimental animals. If experimental animals catch illness except for dental caries during the experiment, the risk of dental caries infection may increase. Therefore, the properties and functional effects of test substance apart from anticariogenic effects need to be examined, and the concentration and form of test substance added to diets should be

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

We have investigated the anticariogenic effects of phytochemicals such as ELM, ES and

*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,

**CH2OH**

**OH**

Fig. 1. Chemical structures of components of the extractive from the leaves of *Morus alba*, 1-

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-of-

**HN**

**HO**

**N**

**1,4-dideoxy-1,4 imino-D-ribitol**

**OH**

**HOCH2**

**N**

**OH**

**imino-D-arabinitol**

**HOCH2**

**Iso-fagomine 1,4-dideoxy-1,4-**

**H**

**HO**

**H**

**OH**

of sucrose is reduced, the prevalence of dental caries is also reduced. Phytochemicals are commonly added to the diet to evaluate anticariogenic effects. After breeding, the molar is removed and the degree of dental caries is scored. The details of the experimental protocol have been described (Ooshima et al, 1981; Tsunehiro et al, 1997). The typical procedure of caries scoring is the Keyes Caries Score (Keyes, 1958).

According to the established method, we have carried out animal experiments using phytochemicals that revealed anticariogenic effects *in vitro*. Young rats were fed with diet #2000 containing phytochemicals with anticariogenic effects for 60 days. However, our results were inconclusive. Therefore, we would like to describe some key points for the planning of animal experiments based on our experience.
