J. Korean Soc. Appl. Biol. Chem. 52(5), 555-559 (2009)
Short Communication
Antibacterial Activities of Phenethyl Isothiocyanate and Its Derivatives against Human Oral Pathogens Hyung Wook Kim, Chi Hoon Lee, Min-Gi Kim and Hoi-Seon Lee* Department of Bioenvironmental Chemistry, College of Agriculture & Life Sciences, Chonbuk National University, Chonju 561-756, Republic of Korea Received December 24, 2008; Accepted June 19, 2009
The antibacterial activity of Sinapis alba seed oils was tested against oral bacteria. The active constituent of S. alba oil was identified as phenethyl isothiocyanate. Phenethyl isothiocyanate showed strong and moderate inhibition at 0.75 and 0.5 mg/disc, respectively, against Porphyromonas asaccharolytica, P. gingivalis and Streptococcus sobrinus. Similarly, benzoyl isothiocyanate and benzyl isothiocyanate had moderate inhibition at 0.5 mg/disc against P. asaccharolytica, P. gingivalis and S. sobrinus. These results indicate that the effectiveness of isothiocyanate derivatives against oral pathogens is related to their aromatic structure. Key words: Sinapis alba antibacterial activity, oral bacteria,
Major oral health problems include dental caries, periodontal diseases, gingivalis inflammation and tooth loss [Tsai et al., 2006]. These problems are caused by oral microorganisms. A sugar-rich diet together with smoking may cause increasing oral bacterial diseases [Zambom et al., 1996; Philip, 2006]. These diseases cause suffering, pain and their psychosocial impact significantly diminishes the quality of life of an individual. According to the Ministry of Health and Social Welfare in Korea, the prevalence rate of dental caries was 23.5% and the prevalence rate of periodontal diseases was 27.7% in 2006. The prevalence rate among OECD nations like Korea is 1.1-2.2 times higher than those of other countries [MHSW, 2007]. The effective control of major oral pathogens such as Actinomyces, Actinobacillus, Bacteroides, Streptococcus and Porphyromonas species that cause dental caries and periodontal diseases is thus an important issue in Korean society [Van Oosten et al., 1987; Song et al., 2006; Rajasuo et al., 2007]. Although numerous chemicals and antibiotics have been widely used for the *Corresponding author Phone: +82-63-270-2544; Fax: +82-63-270-2550 E-mail:
[email protected]
Abbreviations: BHI, brain heart infusion; EI-MS, electron
impact mass spectrometry; HPLC, high performance liquid chromatography; PEITC, phenethyl isothiocyanate; TLC, thin layer chromatography doi:10.3839/jksabc.2009.093
, phenethyl isothiocyanate derivatives
purposes of prevention and disease therapy against oral bacterial diseases, their excessive use can cause an increases in resistance in the target pathogens and serious side effects such as the development of bacterial vomiting, diarrhea and tolerance teeth strain [Jarvinen et al., 1993; Song et al., 2006]. Further research is needed to find alternative antibacterial agents that are safe for humans and specific for oral pathogens. Studies designed to identify solutions to antibiotics resistance and undesirable side effects have often focused on novel agents against oral pathogens from diverse sources including edible plant extracts and the essential oils of herbal plants [Shin et al., 2002; Kyung et al., 2007; Choi et al., 2008]. In this regard, Sinapis alba seeds are used as a spice and herbal supplement in medicinal practice [Court, 1986]. S. alba seeds are known to induce hydroxyl radical scavengers and have antioxidant activity, antimicrobial activity, anticancer and antineoplastic properties [Amarowicz et al., 1996; Chung and Osawa, 1998; Tesaki et al., 1998; Eskin et al., 2007]. However, relatively few studies have been conducted to evaluate the activity of S. alba seeds against oral pathogens despite its excellent pharmacological effects. Therefore, we conducted this study to isolate and identify the active antibacterial constituents from S. alba seeds. Moreover, its commercially available derivatives were investigated to evaluate structure-activity relationships. The oral bacteria used in this study were Porphyromonas asaccharolytica ATCC 25260, P. gingivalis ATCC 33277, Streptococcus mutans ATCC 25175 and S.
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sobrinus ATCC 27607. Stock cultures of these strains
were routinely stored on an Eggerth-Gagnon (EG) liver extract-Field’s slant at −80oC and subcultured on an EG agar (Eiken chemical, Tokyo, Japan) plate when required. The plates were incubated anaerobically at 37oC for 2 days in an atmosphere of 80% N2, 15% CO2 and 5% H2 in an anaerobic chamber (Hirayama, Tokyo, Japan). The bacteria were then grown in a brain heart infusion (BHI) broth (pH 7.6). Only P. gingivalis ATCC 33277 was cultured in Porphyromonas gingivalis broth (PGB) medium (BHI 1.85%, yeast extract 0.5%, L-cystein 0.05%, hemin solution 1%, menadione solution 0.1%). Acetyl isothiocyanate, allyl isothiocyanate, benzoyl isothiocyanate, benzyl isothiocyanate, butyl isothiocyanate, ethyl isothiocyanate and methyl isothiocyanate were purchased from Fluka (Buchs, Switzerland) or Aldrich (Milwaukee, WI). Tetracycline was purchased from Sigma (St. Louis, MO). All other chemicals were of reagent grade. S. alba seeds were purchased from a local market in Chonju, Korea. The air-dried seeds (1.2 kg) were ground to a fine powder and then subjected to supercritical fluid extraction using an accelerated solvent extractor system (SFX 3560, ISCO Inc., Lincoln, Nebraska, U.S.A.) to obtain essential oil (yield 0.63%). The essential oil (5 g) was loaded on a silica gel (70-230 mesh, Merck, Darmstadt, Germany) column (550 mm i.d.×660 mm) and eluted stepwise with hexane and mixtures of hexane: ethyl acetate (9:1, 6:1, 3:1, 1:1, v/v) resulting in six fractions (D1-D6). The D4 fraction exhibited growthinhibiting activity against oral bacteria. Therefore, this fraction was rechromatographed on another column (300 mm i.d.×780 mm). In this step, the D4 fraction was eluted with hexane:ethyl acetate (4:1, v/v) resulting in the active D42 fraction. The active fraction was purified by preparative HPLC (Japan Analytical Industry Co., Ltd., Tokyo, Japan). The D42 was then purified by a JAI gel W Series column (W-252 500 mm+W-253 500 mm) for separation of the active constituent. The conditions used were chloroform at a flow rate of 3.0 mL/min and detection at 215 nm. The active D423 fraction was further rechromatographed using a JAI gel GS Series Column (W-253 500 mm+W-252 500 mm) and a mixture of
hexane:chloroform (1:3, v/v) under the same conditions. All fractions were analyzed by thin layer chromatography (TLC, hexane:ethyl acetate, 6:1, v/v). Fractions with similar patterns were combined and bioassayed at 5.0 mg/ disc. Finally, the potent active compound was isolated as D4233 (503 mg). The structure of the active principle was measured by instrumental analyses. 1H and 13C-NMR spectra were obtained by a JNM-ECA 600 spectrometer (JEOL Ltd., Tokyo, Japan; 1H-600 MHz; 13C-150 MHz) in CDCl3 as an internal standard. A UV-visible absorption spectrum was obtained by a UV spectrometer (DR 4000 spectrophotometer, HACH, Japan). To assay the growth inhibitory activity of the S. alba seed-derived compounds on the test microorganisms, one loopful of each test oral bacteria was suspended in 1 mL of sterilized physiological saline. An aliquot (0.1 mL) of the bacterial suspension was seeded on an EG agar plate. A 0.1 mL test compound in acetone solution was applied to a paper disc (Advantec diameter 8 mm and thickness 1 mm, Toyo Roshi, Tokyo, Japan) using a Drummond glass microcapillary. After evaporation of the solvents, the discs were placed on the agar surface inoculated with the test oral bacteria. All plates were incubated anaerobically at 37oC for 2 days. The control discs received 0.1 mL of acetone, which exhibited no adverse effect against the microorganisms used. All tests of growth inhibition were replicated three times. The inhibitory responses were classified as described previously [Lim et al., 2007]: potent response, ++++, zone diameter >30 mm; strong response, +++, zone diameter 21-30 mm; moderate response, ++, zone diameter 16-20 mm; weak response, +, zone diameter 1015 mm; and little or no response, -, zone diameter 30 mm, ++++; 21-30 mm, +++; 16-20 mm, ++; 10-15 mm, +; +++) against P. asaccharolytica, P. gingivalis and S. sobrinus. At a low dose of 0.25 mg/ disc, benzyl isothiocyanate weakly (+) inhibited the growth of P. asaccharolytica, P. gingivalis and S. sobrinus. However, growth-inhibitory activity of acetyl isothiocyanate, allyl isothiocyanate, butyl isothiocyanate, ethyl isothiocyanate and methyl isothiocyanate was not detected against the four oral pathogens. Antibiotics are known to cause disturbance of oral microbiota which result in various diseases or abnormal physiological states.
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Table 2. Growth-inhibitory activities of the isolated compound and its derivatives against oral bacteria Bacterial straina Dose (mg/disc) P. asaccharolytica P. gingivalis S.mutans S. sobrinus b 2.0 Acetyl isothiocyanate 1.0 2.0 Allyl isothiocyanate 1.0 2.0 ++++ ++++ +++ ++++ 1.0 +++ +++ ++ +++ 0.75 +++ +++ + +++ 0.50 +++ +++ +++ Benzoyl isothiocyanate 0.25 ++ ++ ++ 0.10 + + + 0.05 + + + 2.0 ++++ ++++ +++ ++++ 1.0 ++++ ++++ ++ +++ 0.75 +++ ++++ + +++ Benzyl isothiocyanate 0.50 ++ ++ ++ 0.25 + + + 0.10 2.0 Butyl isothiocyanate 1.0 2.0 Ethyl isothiocyanate 1.0 2.0 Methyl isothiocyanate 1.0 2.0 ++++ ++++ ++ +++ 1.0 +++ +++ ++ +++ 0.75 +++ +++ + +++ 0.50 ++ ++ ++ Phenethyl isothiocyanate 0.25 + ++ + 0.10 + 0.05 + 0.50 ++++ ++++ ++++ ++++ 0.25 ++++ ++++ ++++ ++++ Tetracycline 0.10 +++ ++++ +++ +++ 0.05 +++ ++++ ++ +++ a o b Cultured on Eggerth-Gagnon agar at 37 C for 2 days in an atmosphere of 80% N2, 15% CO2 and 5% H2. Inhibitory zone diameter >30 mm, ++++; 21-30 mm, +++; 16-20 mm, ++; 10-15 mm, +;
