Water-insoluble and water-soluble a-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 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. Low-molecular 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).
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