Inhibition of Cariogenic Factors of Mutans Streptococci

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1 Biocontrol Science, 2005, Vol.10, No.4, Note Inhibition of Cariogenic Factors of Mutans Streptococci by Persimmon Tannin SACHIKO YOSHIOKA*, HIROAKI TOYAMA, NORIAKI KISHIMOTO, AND TOKIO FUJITA Department of Agricultural Chmistry, Faculty of Agriculture, Kinki University, Nakamachi, Nara , Japan Received 8 February 2005/Accepted 11 August 2005 Persimmon tannin is especially known well as a fruit polyphenol. It inhibited the growth of streptococci of the mutans group. It also exhibited a strong inhibitory effect on glucosyltransferase-i (GTase-I) activity. The sucrose-dependent adherence of the bacterial cells was examined with persimmon tannin in vitro. The tannin inhibited the production of adherent materials at the lowest concentration among the polyphenols tested. Persimmon tannin exhibited an inhibitory effect on cariogenic factors at the steps of tooth-decaying bacterial growth, insoluble glucan synthesis, and dental plaque formation, and is expected to be a used for the prevention of dental caries. Key words : Persimmon tannin/polyphenol/mutans streptococci/glucosyltransferase The scientific name of persimmons is Diospyros kaki Thunb. There are more than 190 species in the world although they are especially well-known as a Japanese fruit. Persimmons are divided into sweet and astringent persimmons, but there are almost no sweet persimmons in China or the Korean Peninsula; sweet persimmons may be unique to Japan (Imai, 1990). Sweet persimmons are edible as they are, while astringent persimmons are eaten after being dried or after being soaked in alcohol or hot water to remove their astringency. In addition to their use as food, astringent persimmons have been used as the raw material for Kakishibu. Although the manufacturing method of Kakishibu slightly varies among regions, compressed persimmon juice is fermented, filtered, and matured to prepare the product. Kakishibu is prepared nationwide in Japan, and applied to woods, bamboo, cloth, and paper as a protective agent, or these materials are immersed in the varnish. Kakishibu has also been used by people as an effective for humans as a folk remedy for burns, chilblains, and stomach ulcers (Yoshimura, 1999 and *Corresponding author. Tel: , Fax: ). In addition, Kakishibu is known to have antibacterial activity (Inoue et al., 1980; Nishiyama and Kozaki, 1984). Its activity against gram-negative bacteria is particularly strong because Kakishibu contains 5% or more persimmon tannin with a molecular weight of about 15,000, possessing many phenolic OH groups (Matsuo and Ito, 1977). Persimmon tannin is a condensed form of catechin gallate, gallocatechin gallate, and catechin, and is considered to have strong antibacterial action. It has recently been reported that apple polyphenol, a condensed polyphenol as is persimmon tannin, has an action against dental caries (Yanagida et al.,2000). It is well known that polyphenols have inhibitory effects on cariogenic factors. However, there have been only a few reports about the condensed polyphenols abundantly contained in fruit despite the report on the effect of apple polyphenols on dental caries. Thus, we investigated the inhibitory effect of persimmon tannin on cariogenic factors. Dental caries are tooth decay, and many etiological bacteria are present in the mouth. The main pathogenic factor accounting for the greatest ratio is collectively named mutans streptococcus. The species isolated from humans are almost entirely limited to

164 S. YOSHIOKA ET AL. Streptococcus mutans and Streptococcus sobrinus. These bacteria produce glucosyltransferase TABLE 1. MIC of various polyphenolic compounds against S. mutans and S. sobrinus. (GTase), and synthesize adhesive insoluble glucan in the mouth using sucrose as the substrate.

2 164 S. YOSHIOKA ET AL. Streptococcus mutans and Streptococcus sobrinus. These bacteria produce glucosyltransferase TABLE 1. MIC of various polyphenolic compounds against S. mutans and S. sobrinus. (GTase), and synthesize adhesive insoluble glucan in the mouth using sucrose as the substrate. Toothdecaying bacteria adhere to the tooth surface with the insoluble glucan produced, and form dental plaque. The bacteria produce organic acid by glucose metabolism in the dental plaque, and decrease the ph on the tooth surface, causing decalcification of enamel, and forming dental caries (Jason, 1979). GTase produced by mutans streptococcus has been classified by enzymological, immunological, and molecular genetic analyses, and the presence of many types has been clarified. However, the enzymes have been arbitrarily divided into GTase-S and GTase-I based on whether the glucan produced by the enzyme is soluble or insoluble. Regarding S. mutans, GTase-I has not been recovered from the culture supernatant, regardless of the presence or absence of sucrose (Baba et al., 1986). The enzyme of S. mutans is called cell-associated (CA) GTase, and the presence of multiple types has been suggested (Fujiwara et al., 1992 and 1996). As for S. sobrinus, most activities of GTase-S and GTase-I were recovered from the culture supernatant in the absence of sucrose, and at least 3 types of GTase are present (Furuta et al., 1985). We used persimmon tannin manufactured by Maruzen Pharmaceutical Co., Ltd. and examined its inhibitory effects on cariogenic factors. Commercial tannic acid, epigallocatechin gallate (EGCg), which is most abundantly contained among green tea polyphenolic compounds (Sakanaka et al., 1989) were used to compare the inhibitory effects. The test bacterial strains were S. mutans JCM 5175, S. mutans JCM 5176, and S. sobrinus ATTC All test bacteria were precultured in BHI at 37 Ž for h with shaking. The minimum growth inhibitory concentrations were measured by the broth microdilution method. Since persimmon tannin precipitates when mixed with medium components, modified Czapek-Dox medium was used. Modified Czapek-Dox medium consisted of 36.0g of glucouse, 2.0g of ammonium nitrate, 1.0g of dipotassium hydrogenphosphate, 0.5g of magnesium sulfate heptahydrate, 0.5g of potassium chlorede, 0.37g of peptone, and 0.25g of yeast extract, which were dissolved in 1000ml of distilled water and the ph adjusted to 6.0. The MIC of persimmon tannin, epigallocatechin gallate, and tannic acid against the test bacteria are shown in Table 1. The growth of S. mutans was judged after culture for 24 h, but the judgment of S. sobrinus was made after culture for 48 h because no difference in the S. mutans : judged after culture for 24 h. S. sobrinus : judged after culture for 48 h. turbidity was observed after culture for 20 h. The MIC of persimmon tannin was 62.5ƒÊg/ml for S. mutans and 125ƒÊg/ml for S. sobrinus. The MIC of epigallocatechin gallate and tannic acid were 500-1,000g/ml and ƒÊg/ml, respectively. It has been reported that among green tea polyphenols, the growth inhibition of tooth-decaying bacteria by epigallocatechin gallate was the 3rd strongest after gallocatechin and epigallocatechin, and the MIC was 250-1,000ƒÊg/ml (Sakanaka et al., 1989). The results for epigallocatechin gallate obtained in this study were similar. However, the MIC of tannic acid was 15ƒÊg/ml for S. mutans and 31.3ƒÊg/ml for S. sobrinus, and these were the lowest among the polyphenols tested in this study. These MIC values were almost the same as those in the study report by Teraoka (1987), in which the antibacterial action of a combined agent of tannic acid, zinc acetate, and strontium acetate was mainly due to tannic acid. It has been reported that the galloyl group had affinity for the bacterial lipid membrane, suggesting its involvement in the strong antibacterial activity (Kajiya et al., 2001 and 2002). Persimmon tannin contains galloyl groups (Matsuo, 1977). The residues may have been involved in the strong antibacterial effect on S. mutans and S. sobrinus in this study. It has been suggested that injury and decreased fluidity of the bacterial membrane caused by adsorption by catechins are involved in the exertion of the antibacterial activity (Ikigai et al., 1993 and 1998), but the antibacterial action mechanism of polyphenols has not been elucidated, and remains to be solved. The following study was on the inhibition of GTase activity by polyphenolic compounds. Crude GTase was produced in the culture fluid by S. sobrinus. We had to use the medium in an amount large enough for the mass production of the crude enzyme. Then TTY substitute medium was employed because it was a cheap and effective medium for the growth of the bacteria. After being cultured with shaking in TTY substitute medium at 37 C for 20 h, the culture was centrifuged (6,000 ~g, 10 min) to collect the supernatant. The supernatant was combined with 50%

3 ANTI-CARIES ACTIVITY OF PERSIMMON TANNIN 165 saturated ammonium sulfate and kept at 4 Ž for 30 min. The precipitate was recovered by centrifugation (6,000 ~g, 20 min), dissolved in 10mM phosphate buffer (ph 6.8), and dialyzed against the same buffer for 24 h, and the obtained solution was regarded as the crude enzyme solution. The prepared crude GTase was subjected to SDS-PAGE, and a band at a position corresponding to 156 kda was confirmed. We used the crude enzyme for the measurement of insoluble glucan synthesis activity. The obtained crude GTase solution at 0.3 ml, 3.4% sucrose solution (containing dextran) at 0.5 ml, and distilled water or the sample polyphenol (final concentration: 10-1,000ƒÊg/ml) at 0.2ml, were mixed for reaction at 37 Ž for 18 h. After the reaction, the mixture was centrifuged (14,000 ~g, 10min), and the supernatant was removed. The precipitate was washed with phosphate-ethanol buffer (phosphate buffer: ethanol=1:1) to remove residual polyphenol. Insoluble glucan was dissolved with 1N NaOH, and the production of insoluble glucan was measured by the phenol-sulfuric acid method. The results regarding the inhibition of insoluble glucan synthesis by the prepared crude GTase, presented by the concentration of polyphenol added, are shown in Fig. 1. The insoluble glucan synthesis rate was presented by designating the amount of glucan synthesized when distilled water was added instead of polyphenol as 100%. Persimmon tannin inhibited the enzyme activity to a degree similar to that by tannic acid at a polyphenol concentration of 500ƒÊg/ml, and completely inhibited insoluble glucan synthesis at 1,000ƒÊg/ml. However, the inhibition was stronger at a low concentration of ƒÊg/ml than that by tannic acid. The concentration for 50% inhibition of the synthesis rate was lowest in the graph, showing about a 65% inhibition rate at 100ƒÊg/ml. This finding clarified that persimmon tannin strongly inhibits GTase-I activity produced by S. sobrinus. Inhibition of GTase-I by persimmon tannin may not be due to competition for the substrate, unlike palatinose (Ooshima et al. 1983; Topitsoglow et al. 1984), and the binding of tannin with protein may play an important role. Persimmon tannin is the main component of persimmon astringency used in the process of 'orisage' in the production of Japanese Sake. Persimmon tannin is a polymer with a molecular weight of about 15,000, and contains many hydroxyl groups (Matsuo, 2000), exhibiting a strong protein-precipitating effect. Ito (1991) and Kakiuchi et al. (1986) suggested that the presence and the number of galloyl groups are important factors the for inhibition of GTase activity. Koga et al. (1993) suggested that the strong GTase-inhibiting activity of FIG. 1. Inhibitory effect of polyphenolic compounds on insoluble glucan synthesis by cell-free glucosyltransferase (GTE) of S.sobrinus. Symbols: œ,persimmon-tannin;, epigallocatechin gallate (EGCg);,Tannic acid. The glucan synthesis means the relative amount (%) of insoluble glucan produced at a certain polyphenol concentration as compared to the amount produced in the absence of any polyphenol. oolong tea polyphenol is derived from the action on protein molecules, and the inhibitory activity is dependent on the molecular weight of polyphenol, that is the degree of polymerization of catechins. This study clarified a strong inhibitory effect of a polyphenol polymer, persimmon tannin, on GTase-I produced by S. sobrinus. We examined whether the persimmon tannin that showed a strong inhibition activity against the GTase- I also showed an inhibitory effect on the dental plaque formation. For the bacterial adherence test, glass tubes of which the inner surface was very smooth like that of tooth were used. After culture in BHI, the test bacteria were centrifuged (3,500 ~g, 15min), washed, and resuspended with 10 mm phosphate buffer, adjusting to OD600=6.0, and used as a cell suspension. Sucrose (3%) dissolved in phosphate buffer as the substrate solution and phosphate buffer or various polyphenol solutions dissolved in the same buffer (final concentration: ƒÊg/ml) as samples were mixed in test tubes (7 ~70mm). These were incubated at 37 Ž for 18h with the test tubes tilted at 30 degrees. After the reaction, the supernatant containing the soluble glucan and nonadherent cells was removed from the glass tube. The adherent materials were freeze-dried and weighed. The effect on the adherence rate to the glass surface is presented as the concentration of polyphenol added in Fig. 2. The adherence rate was calculated

166 S. YOSHIOKA ET AL. by designating the amount of adherence when phosphate buffer was added instead of polyphenol as 100%. Because S. mutans JCM 5175 and S.

4 166 S. YOSHIOKA ET AL. by designating the amount of adherence when phosphate buffer was added instead of polyphenol as 100%. Because S. mutans JCM 5175 and S. mutans JCM 5176 had shown results, only the results for S. mutans 5175 are shown here. Particularly, persimmon tannin inhibited synthesis of adherent materials to 10% or lower at 80ƒÊg/ml for S. mutans and at 160ƒÊg/ml for S. sobrinus. Yanagida et al. (2000) reported that the apple polyphenol had shown the deposit rate of 20% by 200ƒÊg/ml. However, persimmon tannin suppressed the deposit rate up to 6.9% by 160ƒÊg/ml. Koga et al. (1993) reported that the inhibition of bacterial adherence increased with an increase in the degree of polymerization of catechins as in the inhibition of GTase. Ito (1991) reported that tannic acid markedly inhibited adherence in a bacterial adherence test using a disc of hydroxyapatite, which comprises more than 95% of tooth enamel, and Aoki et al. (1992) suggested that the galloyl group in the molecular structure is important for this inhibitory action. In this study, a polyphenol polymer containing galloyl A B FIG. 2. Inhibitory effects of polyphenols on the extent of sucrose-dependent cell adherence of S. mutans 5175 (A) and S. sobrinus (B). Symbols: œ, Persimmon-tannin;, epigallocatechin gallate (EGCg);,Tannic acid. The cell adherence means the relative amount (%) of adherent cells at a certain polyphenol concentration as compared to the amount detected in the absence of any polyphenol. groups, persimmon tannin, exhibited the strongest inhibitory effect on bacterial adherence. Based on the above findings, persimmon tannin containing the pyrogallol and galloyl groups in the molecular structure may be useful as a dental cariespreventive agent. ACKNOWLEDGMENT Our deepest thanks go to Mr. Y. Nakamura, Maruzen Pharmaceutical Co., Ltd., for providing us with the persimmon tannin used in this study. REFERENCES Aoki, T., Katoh, H., Taguchi, T., Fukukmoto, M., Ito, M., Uehara, S., Makimura, M., and Otake, S. (1992) Inhibition of cariogenic factors of Streptococcus mutans by green tea catechins (in Japanese). Nihon Univ. J. Oral Sci., 18, Baba,T., Ogawa, T., Okahashi, N., Yakushiji, T., Koga, T., Morimoto, M., and Hamada, S., (1986) Purification and characterisation of the extracellular D- glucosyltransferase from serotype c Streprococcus mutans. Carbohydrate Res., 158, Fujiwara, T., Kawabata, S., and Hamada, S. (1992) Molecular characterization and expression of the cellassociated glucosyltransferase gene from Streptococcus mutans. Biochem. Biophys. Res. Commun., 187, Fujiwara, T., Tamesada, M., Zhuan B., Kawabata, S., Kimura, S., and Hamada, S. (1996) Deletion and reintroduction of glucosyltransferase genes of Streptococcus mutans and role of their gene products in sucrose dependent cellular adherence Microb. Pathog., 20, 225- Furuta, T., Koga, T., Nishizawa, T., Okahashi, N., and Hamada, S. (1985) Purification and characterization of glucosyltransferases from Streptococcus mutans J. Gen. Microbiol., 131, lkigai, H., Nakae, T., Hara, Y., and Shimamura, T.(1993) Bactericidal catechins damage the lipid bilayer. Biochim. Biophys, Acta, 1147, lkigai, H., Hara, Y., Otsuru, H., and Shimamura, T.(1998) Mechanism of membrane damage by (-)- epigallocatechin gallate: comparison with polymyxin B (in Japanese). Jpn, J. Chemother., 46, lmai, K., (1990) History of Folk Customs of Persimmon. Gendaisouzousya (in Japanese), Osaka. Inoue S., Mura K., and Tanimura W.,(1981) Growth inhibition of acetic acid bacteria, lactic acid bacteria and yeast by a Kakishibu (in Japanese). Showa Women's University Gakuen, 502, Jason M. Tanzer.(1979) Essential dependence of smooth surface caries on, and augmentation of fissure caries by, sucrose and Streptococcus mutans infection. Infect. Immun., 25, Kakiuchi, N., Hattori, M., Nishizawa, M., Yamagishi, T., Okuda, T., and Namba, T.(1986) Studies on dental caries prevention by traditional medicines. VIII. lnhiitory effect of various tannins on glucan synthesis by glucosyltransferase from Streptococcus Pharm. Bull., 34, mutans. Chem.

5 ANTI-CARIES ACTIVITY OF PERSIMMON TANNIN 167 Koga, K., Nakahara, K., and Ono, H. (1993) Anti-caries activity of catechin oligomer synthesized by peroxidase. Foods Food Ingred. J., 157, Matsuo, T., Ito, S.(1977) On persimmon tannin (in Japanese). Kagaku to seibutsu. 15, Matsuo, T. (2000) Chemistry and utilization of tannin and its related compounds in fruits (in Japanese). 1. Agric. Horticul., 75, Nishiyama R., and Kozaki M.,(1984) Inhibitory function of Kakishibu (persimmon tannin) toward cell growth of microorganisms (in Japanese). Hakkokogaku, 62, Ooshima, T., Izumitani, A., Sobue, S., Okahashi, N., and Hamada, S.(1983) Non-Cariogenicity of the disaccharide palatinose in experimental dental caries of rats. Infect. lmmun., 39, Teraoka, K.,(1990) An approach to caries prevention by a zinc acetate-strontium acetate-tannic acid mixture: biological and physicochemical actions of the mixture on enamel, oral streptococci and dental plaque (in Japanese). J. Osaka Univ. Dental Soc., 23, Topitsoglow, V., Sasaki, N., Takazoe, I., Frostell, G..(1984) Effect of frequent rinses with isomaltulose (PalatinoseR) solution on acid production in human dental plaque. Caries Res., 18, Yanagida, A., Kanda, T., Tanabe, M., Matsudaira, F., and Jose, G. O. C.(2000) Inhibitory effects of apple polyphenols and related compounds on cariogenic factors of mutans streptococci. J. Agric. Food Chem., 48, Yoshimura, K.(1999) Persimmon tannin paints friendly to natural environment (in Japanese). Toso-to-toryo. 587, Yoshimura, K.(2002) Natural material persimmon tannin (in Japanese). Architecture Community, 83,