Chapter 33
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Synergistic Antibacterial Effect of 5-Methylthiopentyl Isothiocyanate and (-)-Limonene on Periodontal Pathogen 1
2
Hideki Masuda and Saori Hirooka 1
Ogawa and Company, Ltd., 4-1-11, Nihonbashi Honcho Chuo-ku, Tokyo 103-0023, Japan Ogawa and Company, Ltd., 15-7, Chidori Urayasushi, Chiba 279-0032, Japan 2
The combination of 5-methylthiopentyl isothiocyanate, the minor component in volatiles obtained by the hydrolysis of wasabi, and (-)-limonene, the minor component of peppermint or spearmint essential oil, was found to show a synergistic antibacterial effect on Porphyromonas gingivalis, the primary bacterium responsible for causing periodontal disease using an isobolographic analysis, a time-kill method and a bio -luminescence method. The synergistic effect was shown to be significantly bacteriocidal within a few minutes. The different antibacterial mechanism leading to the synergistic effect was clarified from the different concentration of extracellular -adenosine 5'-triphosphate leaked out by the addition of 5 -methylthiopentyl isothiocyanate and that of (-)-limonene.
Periodontal disease, which is composed of light and severe symptoms, that is, gingivitis and periodontitis, respectively, is an oral infection caused by a © 2008 American Chemical Society
In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
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390 variety of pathogens (7-3). Among them, Porphyromonas gingivalis (P. gingivalis), a kind of obligate anaerobe, is reported as the mostfrequentlyfound periodontal bacteria in adult periodontitis patients. P. gingivalis grows, produces colonization in the deep pocket between dentin and gingival, and makes an inflammatory and destructive lesion in the periodontal tissue. To prevent periodontal disease, the habit of tooth-brushing is thought to be the easiest method. Recently, the use some foods and crude drugs have gradually increased in addition to the synthetic antibacterial materials (4-8). As a popular and traditional food, we have been focusing on wasabi (Wasabia japonica matum). In some of the volatiles obtained by the hydrolysis of wasabi, allyl isothiocyanate is well-known because of its high quantity and high antibacterial activity (9, 10). However, its flavor is strongly pungent and stimulative, so it is difficult to practically use. On the other hand, the comethylthioalkyl isothiocyanates, the minor components in the wasabi hydrolysate, were reported to have a radish-like and sweet flavor, and higher thresholds compaired to the threshold of allyl isothiocyanate (77). Therefore, from the viewpoint of flavor characteristics, the practical use of co-methylthioalkyl isothiocyanates is considered to be more interesting. However, their antibacterial effects have hardly been studied (72, 75). In addition, as for their antibacterial effect on periodontal pathogens, no report has been found. Therefore, in this study, we focused on the co-methylthioalkyl isothiocyanates, especially 5methylthiopentyl isothiocyanate (5-MITC). In order to enhance the pharmacological action, a combination of chemicals may be used. The synergistic effect obtained by combination of different components provides not only a pharmacological merit, but also a good flavor. As for the peppermint or spearmint essential oil and its flavor components, such as (-)-menthol, (-)-carvone, or thymol, some studies of their effect on the periodontal pathogens have already been reported (14-16). However, the antibacterial effect of (-)-limonene, the minor component in peppermint or spearmint essential oil, on periodontal pathogens has never been studied. In this combination study on P. gingivalis, we used (-)-limonene in combination with 5MITC (77).
Experimental Chemicals 5-Methylthiopentyl isothiocyanate (5-MITC) was prepared from 4-pentenyl isothiocyanate according to a reported method (18, 19). (-)-Limonene and chlorhexidine were purchased from Sigma-Aldrich, Inc. (St. Louis, MO) and used as received.
In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
391
5-Methylthiopentyl isothiocyanate
(-)-Limonene
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Microorganism and Media P. gingivalis ATCC33277 was obtained from the American Type Culture Collection (Manassas, VA, USA). A bacterial suspension for inoculation was prepared from an overnight culture on modified G A M broth (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) to produce a concentration of 1-8x10 colony forming units (CFU)/mL. The composition of the sterile diluent was as follows: K H P 0 (4.5 g), Na HP0 (6.0 g), L-cysteinHClH 0 (0.5 g), Tween 80 (0.5 g), agar (1.0 g), and distilled water (1000 mL). Each compound mentioned above was mixed and sterilized at 115 °C for 20 min in an autoclave (20). 7
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4
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4
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MIC o Determination 9
The minimum inhibitory concentration (MIC) determinations were performed using a microdilution technique (serial 2-fold dilution) in 96-well microplates (27, 22). The test compound solutions (26 pL), which were diluted with 100% N,N'-dimethylformamide (DMF), were added to 96-well microplates with 234 pL of modified G A M broth inoculated with l-8>
