J Periodontol • January 2016
Antibacterial Activity of Curcumin Against Periodontopathic Bacteria Shusuke Izui,* Shinichi Sekine,* Kazuhiko Maeda,* Masae Kuboniwa,* Akihiko Takada,* Atsuo Amano,* and Hideki Nagata*
Background: Curcumin is a polyphenol extracted from root of turmeric and known to possess multifunctional properties, including antibacterial activity. Although previous studies have investigated the effects of curcumin on microorganisms, available knowledge on the effects of curcumin on periodontopathic bacteria is still limited. In this study, the antibacterial effect of curcumin on periodontopathic bacteria is investigated, particularly Porphyromonas gingivalis. Methods: Representative periodontopathic bacteria were cultured in media with and without various curcumin concentrations, and the optical density at 600 nm was measured for 60 hours. The inhibitory effect of curcumin on P. gingivalis Arg- and Lys-specific proteinase (RGP and KGP, respectively) activities were assessed using spectrofluorophotometric assay. Analysis of biofilm formation by P. gingivalis with or without Streptococcus gordonii was conducted using confocal laser-scanning microscopy (CLSM). Results: Curcumin inhibited the growth of P. gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Treponema denticola in a dose-dependent manner. Bacterial growth was suppressed almost completely at very low concentrations of curcumin. Conversely, 100 mg/mL curcumin did not suppress the growth of Aggregatibacter actinomycetemcomitans. It also demonstrated inhibitory effects against RGP and KGP activities in a dose-dependent manner. CLSM revealed that curcumin suppressed P. gingivalis homotypic and P. gingivalis– S. gordonii heterotypic biofilm formation in a dose-dependent manner. A concentration of 20 mg/mL curcumin inhibited these P. gingivalis biofilm formations by >80%. Conclusion: Curcumin possesses antibacterial activity against periodontopathic bacteria and may be a potent agent for preventing periodontal diseases. J Periodontol 2016;87:83-90. KEY WORDS Biofilms; curcumin; periodontal diseases; Porphyromonas gingivalis. * Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, Osaka, Japan.
C
hronic periodontitis (CP), one of the main causes of tooth loss, is an inflammatory disease caused by periodontopathic bacteria in periodontal tissues.1 Among them, the red complex consisting of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia is considered to be an important consortium in the progression of periodontitis.2 At present, plaque control, particularly, mechanical plaque control methods such as toothbrushing, is considered to be one of the most effective methods for preventing periodontal disease. Although antibacterial chemicals, such as chlorhexidine, are also used as chemical plaque control, their possible side effects are of concern. Recently, studies have investigated adjunctive methods that use nutrients and functional foods to maintain the health status of periodontal tissues; for example, extracts of plants, including mastic exudated from Pistacia lentiscus3 and macrocarpals from Eucalyptus globulus,4,5 have also been investigated for their effects on periodontal health, particularly because of their safety. Curcumin is a yellow, water-insoluble pigment extracted from root of turmeric, a commonly used spice and food-coloring agent in Southeast Asian cooking. It has also been used as a therapeutic agent in Ayurvedic medicine for thousands of years.6 Recently, its anti-inflammatory,7,8 antioxidant,9-12 and anticancer13-15 effects have been studied extensively. It is also reported to have antibacterial and antifungal properties.16 For example, curcumin doi: 10.1902/jop.2015.150260
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Curcumin Inhibits Virulence Factors of Porphyromonas gingivalis
was shown to be highly effective against Trichomonas vaginalis17 and to possess significant antibacterial effects on Helicobacter pylori.18 It also demonstrated strong antifungal activity against the Candida species, including Candida albicans19 and Paracoccidioides brasiliensis.20 Moreover, it also inhibits the formation of biofilm of Escherichia coli, Pseudomonas aeruginosa,21 Klebsiella pneumonia,22 and C. albicans.23 Among the oral bacteria, curcumin was reported to inhibit sortase A of Streptococcus mutans, a major cariogenic bacteria, resulting in reduced S. mutans biofilm formation.24 Regarding the effect of curcumin on periodontopathic bacteria, very recently, Shahzad et al.25 reported an inhibitory effect of curcumin on the planktonic growth of periodontopathic bacteria, such as Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and P. gingivalis. However, additional effects of curcumin on periodontopathic bacteria are still unclear. In this study, the effects of curcumin on the growth of periodontopathic bacteria, such as P. gingivalis and T. denticola, as well as the protease activity of P. gingivalis are investigated. Moreover, the inhibitory effects of curcumin on P. gingivalis homotypic biofilm formation and P. gingivalis–Streptococcus gordonii interbacterial biofilm formation are examined. MATERIALS AND METHODS Bacterial Strains and Culture Conditions P. gingivalis ATCC33277, Prevotella intermedia ATCC49046, F. nucleatum ATCC23726, A. actinomycetemcomitans ATCC29522 and 29523, T. denticola ATCC33520, and S. gordonii G9B were kept as frozen stocks in the laboratory of the authors. Clinical isolate P. gingivalis OMZ314 was a kind gift from Prof. van Winkelhoff (Academic Centre for Dentistry, Amsterdam, The Netherlands). Periodontopathic bacteria were grown in prereduced trypticase soy broth (TSB) † containing 1 mg/mL yeast extract, 5 mg/mL hemin, and 1 mg/mL menadione in an anaerobic system‡ in an atmosphere of 80% N2, 10% CO2, and 10% H2 at 35C. S. gordonii was grown in brain heart infusion broth§ at 35C for 16 hours. Growth Assay of Periodontopathic Bacteria The aforementioned periodontopathic bacteria were grown in 10 mL TSB, anaerobically for 48 hours. Bacterial culture (500 mL) was then inoculated into 10 mL fresh TSB containing 0, 5, 10, 15, and 20 mg/mL curcumin.i Growth was evaluated by measuring the optical density at 600 nm (OD600) using an ultravioletvisible spectrophotometer.¶ After 48 hours of incubation, the lowest concentration at which no growth (OD600 nm 100
ATCC29523
>100
F. nucleatum ATCC23726
10
P. gingivalis ATCC33277
15
OMZ314
10
P. intermedia ATCC49046
10
T. denticola ATCC33520
5
P. gingivalis was treated with various concentrations (0, 1, 5, 10, and 20 mg/mL) of curcumin for 30 minutes. After washing three times with PBS, the treated or untreated P. gingivalis was added to the well to form homotypic P. gingivalis biofilm. Biofilm Formation by P. gingivalis With S. gordonii To investigate the effects of curcumin on the early stage of biofilm formation, analysis of biofilm formed by P. gingivalis with S. gordonii was conducted in the same manner, with slight modifications.26 To evaluate the effect of curcumin on P. gingivalis colonization on S. gordonii biofilm, P. gingivalis was treated with curcumin, and curcumin was removed by thorough washing with PBS before adding to the well in which S. gordonii biofilm formed antecedently. Samples of 1.5 · 109 CFU/mL S. gordonii G9B were stained with 15 mg hexidium iodide¶¶ (HI) for 15 minutes and then washed three times with PBS. S. gordonii (5 · 107 CFU) was then inoculated into PBS in individual chambers coated with human whole saliva and cultured anaerobically in a well at 37C for 16 hours. P. gingivalis OMZ314, stained with FITC as mentioned above, was treated with various concentrations (0, 1, 5, 10, and 20 mg/mL) of curcumin for 30 minutes. After washing three times with PBS, 5 · 106 CFU of P. gingivalis were added to the well in which S. gordonii biofilm was formed. The mixtures were incubated anaerobically at 37C for 24 hours in the dark on a rotator, as mentioned above. Biofilm Analyses by Confocal Laser-Scanning Microscopy Analysis of biofilm formation was conducted with a confocal laser-scanning microscope (CLSM).## After the wells were washed with PBS, homotypic or heterotypic biofilm formed at the bottom of the wells were observed at a magnification of ·40, using a helium–neon
laser (543-nm wavelength) to visualize FITC-stained P. gingivalis and an argon laser (488-nm wavelength) to visualize HI-stained S. gordonii. Six fields from each confocal laser-scanning microscopy image were selected randomly, and the volumes of P. gingivalis and S. gordonii were calculated using a software program.*** The level of inhibition exerted by curcumin on the formation of biofilm was calculated as follows: percentage inhibition = (1 A/B) · 100, where A is volume ratio of P. gingivalis when curcumin was added, and B is volume ratio of P. gingivalis without addition of curcumin. Statistical Analyses Dose dependency and culture curve were analyzed using the Jonckheere-Terpstra test and Dunnett test, respectively, using statistical software.††† The planktonic culture and protease experiments were performed in triplicates, and the biofilm data were based on the average of six fields. RESULTS Antibacterial Activity of Curcumin Against Periodontopathic Bacteria Curcumin significantly inhibited the growth of the tested periodontopathic bacteria, with the exception of A. actinomycetemcomitans. The MIC of curcumin against tested periodontopathic bacteria is shown in Table 1. MIC of curcumin for P. gingivalis was 10 to 15 mg/mL. Similar results were obtained for P. intermedia, F. nucleatum, and T. denticola. Less than 10 mg/mL curcumin completely inhibited growth of these periodontopathic bacteria. Conversely, it failed to demonstrate marked effects against two strains of A. actinomycetemcomitans, ATCC29522 and 29523, even at a concentration of 100 mg/mL. Among the periodontopathic bacteria, P. gingivalis is considered to be one of the most influential microorganisms in CP. Moreover, P. gingivalis was classified based on the gene type of major fimbriae (FimA), and those with Type II FimA have been reported to be detected most frequently in patients with periodontitis.27 Therefore, P. gingivalis was targeted, and the effects of curcumin on it were investigated. Two strains of P. gingivalis were selected: 1) strain ATCC33277, which is a representative with Type I FimA; and 2) OMZ314 with Type II FimA. Growth curves of these P. gingivalis strains with and without curcumin are shown in Figure 1. Their growth was inhibited by curcumin, even at a concentration of 5 mg/mL, and, at concentrations >15 mg/mL, there was a significant inhibition of the bacterial growth. These results indicate that curcumin exerted strong antibacterial activity against ¶¶ ## *** †††
LSM 510 v.3.2, Carl Zeiss, Oberkochen, Germany. Molecular Probes. Imaris software v.5.0.1, Bitplane, Zurich, Switzerland. 2006 Excel statistics software, SSRI, Tokyo, Japan.
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Curcumin Inhibits Virulence Factors of Porphyromonas gingivalis
Volume 87 • Number 1
Figure 1.
Effect of curcumin on planktonic growth of P. gingivalis. P. gingivalis ATCC33277 with Type I FimA (A) and P. gingivalis OMZ314 with Type II FimA (B). Concentrations of curcumin: black diamonds = 0 mg/mL; black squares = 5 mg/mL; black triangles = 10 mg/mL; white squares = 15 mg/mL; white triangles = 20 mg/mL. Values are expressed as means – SDs derived from three experiments consisting of triplicate samples. Culture curve was analyzed using the Dunnett test. *P
Antibacterial Activity of Curcumin Against Periodontopathic Bacteria Shusuke Izui,* Shinichi Sekine,* Kazuhiko Maeda,* Masae Kuboniwa,* Akihiko Takada,* Atsuo Amano,* and Hideki Nagata*
Background: Curcumin is a polyphenol extracted from root of turmeric and known to possess multifunctional properties, including antibacterial activity. Although previous studies have investigated the effects of curcumin on microorganisms, available knowledge on the effects of curcumin on periodontopathic bacteria is still limited. In this study, the antibacterial effect of curcumin on periodontopathic bacteria is investigated, particularly Porphyromonas gingivalis. Methods: Representative periodontopathic bacteria were cultured in media with and without various curcumin concentrations, and the optical density at 600 nm was measured for 60 hours. The inhibitory effect of curcumin on P. gingivalis Arg- and Lys-specific proteinase (RGP and KGP, respectively) activities were assessed using spectrofluorophotometric assay. Analysis of biofilm formation by P. gingivalis with or without Streptococcus gordonii was conducted using confocal laser-scanning microscopy (CLSM). Results: Curcumin inhibited the growth of P. gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Treponema denticola in a dose-dependent manner. Bacterial growth was suppressed almost completely at very low concentrations of curcumin. Conversely, 100 mg/mL curcumin did not suppress the growth of Aggregatibacter actinomycetemcomitans. It also demonstrated inhibitory effects against RGP and KGP activities in a dose-dependent manner. CLSM revealed that curcumin suppressed P. gingivalis homotypic and P. gingivalis– S. gordonii heterotypic biofilm formation in a dose-dependent manner. A concentration of 20 mg/mL curcumin inhibited these P. gingivalis biofilm formations by >80%. Conclusion: Curcumin possesses antibacterial activity against periodontopathic bacteria and may be a potent agent for preventing periodontal diseases. J Periodontol 2016;87:83-90. KEY WORDS Biofilms; curcumin; periodontal diseases; Porphyromonas gingivalis. * Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, Osaka, Japan.
C
hronic periodontitis (CP), one of the main causes of tooth loss, is an inflammatory disease caused by periodontopathic bacteria in periodontal tissues.1 Among them, the red complex consisting of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia is considered to be an important consortium in the progression of periodontitis.2 At present, plaque control, particularly, mechanical plaque control methods such as toothbrushing, is considered to be one of the most effective methods for preventing periodontal disease. Although antibacterial chemicals, such as chlorhexidine, are also used as chemical plaque control, their possible side effects are of concern. Recently, studies have investigated adjunctive methods that use nutrients and functional foods to maintain the health status of periodontal tissues; for example, extracts of plants, including mastic exudated from Pistacia lentiscus3 and macrocarpals from Eucalyptus globulus,4,5 have also been investigated for their effects on periodontal health, particularly because of their safety. Curcumin is a yellow, water-insoluble pigment extracted from root of turmeric, a commonly used spice and food-coloring agent in Southeast Asian cooking. It has also been used as a therapeutic agent in Ayurvedic medicine for thousands of years.6 Recently, its anti-inflammatory,7,8 antioxidant,9-12 and anticancer13-15 effects have been studied extensively. It is also reported to have antibacterial and antifungal properties.16 For example, curcumin doi: 10.1902/jop.2015.150260
83
Curcumin Inhibits Virulence Factors of Porphyromonas gingivalis
was shown to be highly effective against Trichomonas vaginalis17 and to possess significant antibacterial effects on Helicobacter pylori.18 It also demonstrated strong antifungal activity against the Candida species, including Candida albicans19 and Paracoccidioides brasiliensis.20 Moreover, it also inhibits the formation of biofilm of Escherichia coli, Pseudomonas aeruginosa,21 Klebsiella pneumonia,22 and C. albicans.23 Among the oral bacteria, curcumin was reported to inhibit sortase A of Streptococcus mutans, a major cariogenic bacteria, resulting in reduced S. mutans biofilm formation.24 Regarding the effect of curcumin on periodontopathic bacteria, very recently, Shahzad et al.25 reported an inhibitory effect of curcumin on the planktonic growth of periodontopathic bacteria, such as Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and P. gingivalis. However, additional effects of curcumin on periodontopathic bacteria are still unclear. In this study, the effects of curcumin on the growth of periodontopathic bacteria, such as P. gingivalis and T. denticola, as well as the protease activity of P. gingivalis are investigated. Moreover, the inhibitory effects of curcumin on P. gingivalis homotypic biofilm formation and P. gingivalis–Streptococcus gordonii interbacterial biofilm formation are examined. MATERIALS AND METHODS Bacterial Strains and Culture Conditions P. gingivalis ATCC33277, Prevotella intermedia ATCC49046, F. nucleatum ATCC23726, A. actinomycetemcomitans ATCC29522 and 29523, T. denticola ATCC33520, and S. gordonii G9B were kept as frozen stocks in the laboratory of the authors. Clinical isolate P. gingivalis OMZ314 was a kind gift from Prof. van Winkelhoff (Academic Centre for Dentistry, Amsterdam, The Netherlands). Periodontopathic bacteria were grown in prereduced trypticase soy broth (TSB) † containing 1 mg/mL yeast extract, 5 mg/mL hemin, and 1 mg/mL menadione in an anaerobic system‡ in an atmosphere of 80% N2, 10% CO2, and 10% H2 at 35C. S. gordonii was grown in brain heart infusion broth§ at 35C for 16 hours. Growth Assay of Periodontopathic Bacteria The aforementioned periodontopathic bacteria were grown in 10 mL TSB, anaerobically for 48 hours. Bacterial culture (500 mL) was then inoculated into 10 mL fresh TSB containing 0, 5, 10, 15, and 20 mg/mL curcumin.i Growth was evaluated by measuring the optical density at 600 nm (OD600) using an ultravioletvisible spectrophotometer.¶ After 48 hours of incubation, the lowest concentration at which no growth (OD600 nm 100
ATCC29523
>100
F. nucleatum ATCC23726
10
P. gingivalis ATCC33277
15
OMZ314
10
P. intermedia ATCC49046
10
T. denticola ATCC33520
5
P. gingivalis was treated with various concentrations (0, 1, 5, 10, and 20 mg/mL) of curcumin for 30 minutes. After washing three times with PBS, the treated or untreated P. gingivalis was added to the well to form homotypic P. gingivalis biofilm. Biofilm Formation by P. gingivalis With S. gordonii To investigate the effects of curcumin on the early stage of biofilm formation, analysis of biofilm formed by P. gingivalis with S. gordonii was conducted in the same manner, with slight modifications.26 To evaluate the effect of curcumin on P. gingivalis colonization on S. gordonii biofilm, P. gingivalis was treated with curcumin, and curcumin was removed by thorough washing with PBS before adding to the well in which S. gordonii biofilm formed antecedently. Samples of 1.5 · 109 CFU/mL S. gordonii G9B were stained with 15 mg hexidium iodide¶¶ (HI) for 15 minutes and then washed three times with PBS. S. gordonii (5 · 107 CFU) was then inoculated into PBS in individual chambers coated with human whole saliva and cultured anaerobically in a well at 37C for 16 hours. P. gingivalis OMZ314, stained with FITC as mentioned above, was treated with various concentrations (0, 1, 5, 10, and 20 mg/mL) of curcumin for 30 minutes. After washing three times with PBS, 5 · 106 CFU of P. gingivalis were added to the well in which S. gordonii biofilm was formed. The mixtures were incubated anaerobically at 37C for 24 hours in the dark on a rotator, as mentioned above. Biofilm Analyses by Confocal Laser-Scanning Microscopy Analysis of biofilm formation was conducted with a confocal laser-scanning microscope (CLSM).## After the wells were washed with PBS, homotypic or heterotypic biofilm formed at the bottom of the wells were observed at a magnification of ·40, using a helium–neon
laser (543-nm wavelength) to visualize FITC-stained P. gingivalis and an argon laser (488-nm wavelength) to visualize HI-stained S. gordonii. Six fields from each confocal laser-scanning microscopy image were selected randomly, and the volumes of P. gingivalis and S. gordonii were calculated using a software program.*** The level of inhibition exerted by curcumin on the formation of biofilm was calculated as follows: percentage inhibition = (1 A/B) · 100, where A is volume ratio of P. gingivalis when curcumin was added, and B is volume ratio of P. gingivalis without addition of curcumin. Statistical Analyses Dose dependency and culture curve were analyzed using the Jonckheere-Terpstra test and Dunnett test, respectively, using statistical software.††† The planktonic culture and protease experiments were performed in triplicates, and the biofilm data were based on the average of six fields. RESULTS Antibacterial Activity of Curcumin Against Periodontopathic Bacteria Curcumin significantly inhibited the growth of the tested periodontopathic bacteria, with the exception of A. actinomycetemcomitans. The MIC of curcumin against tested periodontopathic bacteria is shown in Table 1. MIC of curcumin for P. gingivalis was 10 to 15 mg/mL. Similar results were obtained for P. intermedia, F. nucleatum, and T. denticola. Less than 10 mg/mL curcumin completely inhibited growth of these periodontopathic bacteria. Conversely, it failed to demonstrate marked effects against two strains of A. actinomycetemcomitans, ATCC29522 and 29523, even at a concentration of 100 mg/mL. Among the periodontopathic bacteria, P. gingivalis is considered to be one of the most influential microorganisms in CP. Moreover, P. gingivalis was classified based on the gene type of major fimbriae (FimA), and those with Type II FimA have been reported to be detected most frequently in patients with periodontitis.27 Therefore, P. gingivalis was targeted, and the effects of curcumin on it were investigated. Two strains of P. gingivalis were selected: 1) strain ATCC33277, which is a representative with Type I FimA; and 2) OMZ314 with Type II FimA. Growth curves of these P. gingivalis strains with and without curcumin are shown in Figure 1. Their growth was inhibited by curcumin, even at a concentration of 5 mg/mL, and, at concentrations >15 mg/mL, there was a significant inhibition of the bacterial growth. These results indicate that curcumin exerted strong antibacterial activity against ¶¶ ## *** †††
LSM 510 v.3.2, Carl Zeiss, Oberkochen, Germany. Molecular Probes. Imaris software v.5.0.1, Bitplane, Zurich, Switzerland. 2006 Excel statistics software, SSRI, Tokyo, Japan.
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Curcumin Inhibits Virulence Factors of Porphyromonas gingivalis
Volume 87 • Number 1
Figure 1.
Effect of curcumin on planktonic growth of P. gingivalis. P. gingivalis ATCC33277 with Type I FimA (A) and P. gingivalis OMZ314 with Type II FimA (B). Concentrations of curcumin: black diamonds = 0 mg/mL; black squares = 5 mg/mL; black triangles = 10 mg/mL; white squares = 15 mg/mL; white triangles = 20 mg/mL. Values are expressed as means – SDs derived from three experiments consisting of triplicate samples. Culture curve was analyzed using the Dunnett test. *P
