**3.2 Extractive from** *Salacia chinensis* **(ES)**

The stems of *Salacia* species plants are pulverized and extracted with methanol for 3 h at 80°C. After filtration, the extract is evaporated to obtain a powder. The powder of the methanol extract is dissolved and purified using Sephadex LH-20 column chromatography. ES used in this study is kindly provided by Kobayashi Pharmaceutical Co., Ltd. (Osaka, Japan). Two compounds isolated from *Salacia* extracts, salacinol and kotalanol, strongly inhibit sucrase. Their structures are quite unique, bearing thiosugar sulfonium sulfate inner salt comprising a 1-deoxy-4-thio-D-arabinofranosyl cation and 1-deoxyaldosyl-3-sulfate anion (Fig. 2) (Shimoda et al, 1998; Yosikawa et al, 2001). It has been clarified that this extraction is not associated with toxicity or with blood biochemical or pathologic abnormalities in rats and other animals.

Fig. 2. Chemical structure of components of the extractive from *Salacia chinencis* (ES)

#### **3.3 Partially decomposed alginate by** *Vibrio alginolyticus* **SUN53 (Alg53)**

Alginate, which is a copolymer of α-L-guluronate and β-D-mannuronate, is a gelling polysaccharide found in great abundance as part of the cell wall and intracellular material in brown seaweeds (Fig. 3) (Wong et al, 2000). We demonstrated that partially decomposed alginate by *Vibrio alginolyticus* SUN53 (Alg53) had a competitive inhibitory effect for sucrase of the vesicles of the intestinal brush border membrane of rats. The procedure for the preparation of Alg53 has been described (Nakamura S et al, 2008).

Alginate (0.5%) (mean M.W., 55,000) partially hydrolyzed by HCl is incubated with *Vibrio alginolyticus* SUN53 (106 CFU/mL) in culture medium (pH 7.0) containing 0.025% yeast extract, 0.05% peptone, 1% NaCl and 0.01% FePO4 for 5 days at 25°C. After incubation, the supernatant is treated with 3 times-volume (75%) of ethanol to obtain low-M.W. hydrolyzed alginate. It is dried by freezing after ethanol is evaporated with a rotary evaporator. The

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

The stems of *Salacia* species plants are pulverized and extracted with methanol for 3 h at 80°C. After filtration, the extract is evaporated to obtain a powder. The powder of the methanol extract is dissolved and purified using Sephadex LH-20 column chromatography. ES used in this study is kindly provided by Kobayashi Pharmaceutical Co., Ltd. (Osaka, Japan). Two compounds isolated from *Salacia* extracts, salacinol and kotalanol, strongly inhibit sucrase. Their structures are quite unique, bearing thiosugar sulfonium sulfate inner salt comprising a 1-deoxy-4-thio-D-arabinofranosyl cation and 1-deoxyaldosyl-3-sulfate anion (Fig. 2) (Shimoda et al, 1998; Yosikawa et al, 2001). It has been clarified that this extraction is not associated with toxicity or with blood biochemical or pathologic

**Salacinol Kotalanol** Fig. 2. Chemical structure of components of the extractive from *Salacia chinencis* (ES)

Alginate, which is a copolymer of α-L-guluronate and β-D-mannuronate, is a gelling polysaccharide found in great abundance as part of the cell wall and intracellular material in brown seaweeds (Fig. 3) (Wong et al, 2000). We demonstrated that partially decomposed alginate by *Vibrio alginolyticus* SUN53 (Alg53) had a competitive inhibitory effect for sucrase of the vesicles of the intestinal brush border membrane of rats. The procedure for the

Alginate (0.5%) (mean M.W., 55,000) partially hydrolyzed by HCl is incubated with *Vibrio alginolyticus* SUN53 (106 CFU/mL) in culture medium (pH 7.0) containing 0.025% yeast extract, 0.05% peptone, 1% NaCl and 0.01% FePO4 for 5 days at 25°C. After incubation, the supernatant is treated with 3 times-volume (75%) of ethanol to obtain low-M.W. hydrolyzed alginate. It is dried by freezing after ethanol is evaporated with a rotary evaporator. The

**3.3 Partially decomposed alginate by** *Vibrio alginolyticus* **SUN53 (Alg53)** 

preparation of Alg53 has been described (Nakamura S et al, 2008).

al*,* 2003).

**3.2 Extractive from** *Salacia chinensis* **(ES)** 

abnormalities in rats and other animals.

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.

Fig. 3. Chemical structure of components of alginate
