Article pubs.acs.org/JAFC
Bactericidal Activity and Mechanism of Photoirradiated Polyphenols against Gram-Positive and -Negative Bacteria Keisuke Nakamura,*,† Kirika Ishiyama,† Hong Sheng,# Hiroyo Ikai,‡ Taro Kanno,‡ and Yoshimi Niwano† †
Laboratory for Redox Regulation, ‡Division of Molecular and Regenerative Prosthodontics, and #Division of Advanced Prosthetic Dentistry, Tohoku University Graduate School of Dentistry, 4-1 Seiryo, Aoba-ku, Sendai 980-8575, Japan ABSTRACT: The bactericidal effect of various types of photoirradiated polyphenols against Gram-positive and -negative bacteria was evaluated in relation to the mode of action. Gram-positive bacteria (Enterococcus faecalis, Staphylococcus aureus, and Streptococcus mutans) and Gram-negative bacteria (Aggregatibacter actinomycetemcomitans, Escherichia coli, and Pseudomonas aeruginosa) suspended in a 1 mg/mL polyphenol aqueous solution (caffeic acid, gallic acid, chlorogenic acid, epigallocatechin, epigallocatechin gallate, and proanthocyanidin) were exposed to LED light (wavelength, 400 nm; irradiance, 260 mW/cm2) for 5 or 10 min. Caffeic acid and chlorogenic acid exerted the highest bactericidal activity followed by gallic acid and proanthocyanidin against both Gram-positive and -negative bacteria. It was also demonstrated that the disinfection treatment induced oxidative damage of bacterial DNA, which suggests that polyphenols are incorporated into bacterial cells. The present study suggests that blue light irradiation of polyphenols could be a novel disinfection treatment. KEYWORDS: polyphenols, photoirradiation, bactericidal effect, Gram-positive bacteria, Gram-negative bacteria
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INTRODUCTION Polyphenols occurring in fruits, nuts, vegetables, and flowers have antioxidant activity.1−3 The phenolic hydroxyl group in their structures acts as a hydrogen donor, and they can effectively scavenge free radicals.4,5 The beneficial antioxidative activity of polyphenols has been well studied and applied to health promotion.6−8 Besides the antioxidative activity, some polyphenols are also antimicrobial and antiviral.9−13 In our previous study, it was demonstrated that the antimicrobial activity of polyphenols, such as gallic acid and proanthocyanidin, was considerably enhanced by blue light irradiation at a wavelength of around 400 nm.14,15 When Staphylococcus aureus suspended in an aqueous solution of the polyphenols was exposed to blue light, they were killed with a >5 log reduction of viable counts within 15−30 min. It was also demonstrated that exposing an aqueous solution of the polyphenols to blue light led to photo-oxidation of the polyphenolic hydroxyl group, resulting in the generation of reactive oxygen species (ROS) in the presence of dissolved oxygen.14 Hydrogen peroxide (H2O2) is produced via electron transfer from photo-oxidized polyphenols to dissolved oxygen. The H2O2 is subsequently photolyzed by the blue light, resulting in the generation of hydroxyl radicals (•OH),16 which would be a main contributor of the bactericidal activity.14,15 Because the reaction will begin with the photo-oxidation of the polyphenolic hydroxyl group, the bactericidal activity may be different for each polyphenol depending on the reactivity of the polyphenolic hydroxyl group related to the chemical structure. However, it has not been studied what kind of polyphenol exerts effective bactericidal activity when photoirradiated. To date, there is little available scientific evidence regarding the disinfection treatment based on the photoirradiation of polyphenols.14,15 It is still unclear whether the disinfection treatment is effective against other bacterial species besides S. aureus. It is speculated that the affinity of polyphenols to © XXXX American Chemical Society
bacterial cells is one of the factors that will affect the bactericidal activity of the disinfection treatment.14,15 Thus, it may be reasonable to assume that the effect of the disinfection treatment varies between Gram-positive and -negative bacteria because of the difference in their cell wall structures. The cell wall of Gram-positive bacteria consists of a thick peptidoglycan layer, whereas Gram-negative bacteria have an outer membrane composed of lipopolysaccharide and phospholipid in addition to a thin peptidoglycan layer.17 This difference in the cell wall structure may influence the affinity of polyphenols to bacterial cells. Furthermore, the target site where polyphenols show the affinity has not been fully understood. Because it was demonstrated that lipid peroxidation was induced by the photoirradiation of gallic acid in S. aureus,14 one of the targets would be the cytoplasmic membrane. Polyphenols may also penetrate into cytoplasm as reported in the case of mammalian cells,18 so that they may cause oxidative damage to the cellular components in the cytoplasm when photoirradiated. The aim of the present study was, therefore, to compare the bactericidal effects of various types of photoirradiated polyphenols against Gram-positive and -negative bacteria and to elucidate the mode of action.
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MATERIALS AND METHODS
Reagents. Reagents were purchased from the following sources: caffeic acid (CA) and gallic acid hydrate (GA) from Tokyo Chemical Industries (Tokyo, Japan); (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCg), catalase, ammonium ferrous sulfate, sulfuric acid, xylenol orange, and sorbitol from Wako Pure Chemical Industries Special Issue: 27th ICP and 8th Tannin Conference (Nagoya 2014) Received: December 3, 2014 Revised: February 1, 2015 Accepted: February 6, 2015
A
DOI: 10.1021/jf5058588 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Structures of polyphenols used in the present study. and Streptococcus mutans JCM5705, and three different species of Gram-negative bacteria, Aggregatibacter actinomycetemcomitans JCM2434, Escherichia coli JCM5491, and Pseudomonas aeruginosa JCM6119, were used. A bacterial suspension was prepared in sterile physiological saline from a culture grown on brain−heart infusion (BHI) agar (Becton Dickinson Labware, Franklin Lakes, NJ, USA). E. faecalis, S. aureus, E. coli, and P. aeruginosa were cultured aerobically at 37 °C for 20 h, whereas S. mutans and A. actinomycetemcomitans were cultured anaerobically using Anaero Pack (Mitsubishi Gas Chemical Co., Tokyo, Japan) at 37 °C for 48 h before the preparation of bacterial suspension. In a plastic cuvette, 450 μL of polyphenol solution (CA, GA, ChA, EGC, EGCg, or PA) or pure water (PW) was mixed with 50 μL of the bacterial suspension to reach final concentrations of 1 mg/mL for polyphenol and approximately 107 colony-forming units (CFU)/mL for the bacteria. Then, the samples were exposed to LED light for 5 or 10 min. After irradiation, 50 μL of the sample was mixed with an equal volume of sterile catalase solution (5000 U/mL) to terminate the bactericidal effect of H2O2 generated by photo-oxidation of the polyphenol. A 10-fold serial dilution of the mixture was prepared using sterile physiological saline, and 10 μL of the diluted solution was seeded onto an agar plate. P. aeruginosa was seeded onto deoxycholate hydrogen sulfide lactose agar (Becton Dickinson Labware) to obtain colonies with an entire edge for counting, whereas the others were seeded onto BHI agar. The agar plates with each bacterial species were cultured in the same way as described above, and the CFU/mL was determined. In addition, a bacterial suspension that was kept for 10 min in a light-shielding box instead of being exposed to LED light was subjected to the same procedure. The bacterial initial count (inoculum size) in each assay was also evaluated by the viable counting method. All tests were performed in triplicate. Bactericidal Effect of LED Light on Bacteria Pretreated with Polyphenols. An additional bactericidal assay was conducted to examine if polyphenols taken up by bacteria would exert bactericidal activity. S. aureus and E. coli were used as representative Gram-positive and -negative bacteria, respectively. In a microtube, 1800 μL of each
(Osaka, Japan); proanthocyanidin (PA; Leucoselect) from Indena (Milano, Italy); 5,5-dimethyl-1-pyrroline N-oxide (DMPO) from Labotec (Tokyo, Japan); chlorogenic acid (ChA) and 4-hydroxy2,2,6,6-tetramethylpiperidine N-oxyl (TEMPOL) from Sigma-Aldrich (St. Louis, MO, USA). All other reagents used were of analytical grade. Light Source. An experimental device equipped with a lightemitting diode (LED) with a wavelength of 400 nm (NHH105UV, Lustrous Technology, Shiji, Taiwan) was used according to our previous study.14 The output power of the LED measured using a power meter (FieldMate, Coherent, Santa Clara, CA, USA) was set to be 400 mW/LED corresponding to an irradiance of 130 mW/cm2 at a distance of 15 mm from the LED. A four-clear-sided methacrylate plastic cuvette containing the sample was placed in the experimental device. LED light irradiation was performed toward both sides of the plastic cuvette (total irradiance = 260 mW/cm2). Preparation of Polyphenol Solution. The structures of polyphenols used in the present study are shown in Figure 1. Each polyphenol was dissolved in pure water to be 1.1 mg/mL. On the preparation of CA, ChA, and EGC solutions, the tubes containing samples were put in hot water (90 °C) to heat them up and were agitated so that each polyphenol could be completely dissolved. CA, ChA, and EGC solutions were subjected to high-performance liquid chromatography (HPLC) using a Prominence HPLC system (Shimadzu, Kyoto, Japan) with a Shimpak-HRC ODS colum (5 μm, ⌀4.6 × 150 mm), and a Flex FX-15 HPLC system (PerkinElmer, Waltham, MA, USA) with a PerkinElmer Brownlee Supra column C18 (2.1 μm, ⌀2.1 × 100 mm). It was confirmed that the polyphenols were not decomposed by heating. The pH of each polyphenol solution measured using a pH meter (Seven Go SG2, Mettler Toledo, Greifensee, Switzerland) is also shown in Figure 1. The polyphenol solutions were used in the experiments without further pH adjustment. Comparison of Bactericidal Activity of Photoirradiated Polyphenols. All bacterial species used in the present study were purchased from the Japan Collection of Microorganisms, RIKEN BioResource Center (Wako, Japan). Three different species of Grampositive bacteria, Enterococcus faecalis JCM7783, S. aureus JCM 2413, B
DOI: 10.1021/jf5058588 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Table 1. Logarithmic Reduction of CFU/mL after Treatment with Each Polyphenol under Light-Shielding Condition for 10 mina CA
GA
ChA
EGC
EGCg
PA
PW
G(+)
E. faecalis S. aureus S. mutans
0.1 0.4 0.8**
0.1 0.2 1.0**
0.0 0.4 1.5**
0.0 0.0 0.0
0.0 0.1 0.0
2.2** 0.3 0.5**
0.0 0.0 0.0
G(−)
A. actinomycetemcomitans E. coli P. aeruginosa
1.5** 0.1 2.7**
0.7 0.2** 2.7**
0.9* 0.4** 2.8**
0.2 0.1 0.6**
3.5** 1.0** 1.0**
0.2 0.1 1.5**
0.0 0.0 0.6**
a
Significant differences from the initial bacterial count are shown, p < 0.05 (*) and p < 0.01 (**). G(+), Gram-positive; G(−), Gram-negative; CA, caffeic acid; GA, gallic acid; ChA, chlorogenic acid; EGC, epigallocatechin; EGCg, epigallocatechin gallate; PA, proanthocyanidin; PW, pure water. Each value represents the mean of triplicate assays.
Table 2. Logarithmic Reduction of CFU/mL after LED Irradiation of Each Polyphenol for 5 mina CA
GA
ChA
EGC
EGCg
G(+)
E. faecalis S. aureus S. mutans
2.6** >5** >5**
0.0 0.6** 1.9**
1.0** 1.0** >5**
0.0 0.1 0.3
0.0 0.0 0.4
3.0** 1.3** 1.5**
PA
0.0 0.0 0.4
PW
G(−)
A. actinomycetemcomitans E. coli P. aeruginosa
>5** >5** >5**
>5** 2.2** >5**
>5** 3.2** >5**
1.6** 0.2 2.1**
3.0** 1.1 3.1**
>5** 0.9 3.8**
2.4** 0.3 2.0**
a Significant differences from the initial bacterial count are shown, p < 0.05 (*) and p < 0.01 (**). G(+), Gram-positive; G(−), Gram-negative; CA, caffeic acid; GA, gallic acid; ChA, chlorogenic acid; EGC, epigallocatechin; EGCg, epigallocatechin gallate; PA, proanthocyanidin; PW, pure water. Each value represents the mean of triplicate assays.
polyphenol solution was mixed with 200 μL of the bacterial suspension to reach final concentrations of 1 mg/mL for polyphenol and approximately 109 CFU/mL for the bacteria. The mixture was incubated for 1 min at room temperature. Then, the bacteria were collected by centrifugation at 5000g at 4 °C for 10 min. Following the decantation of the supernatant, the pellet was resuspended in 1000 μL of sterile saline. The suspension was further diluted about 100 times with sterile saline to reach a final concentration of approximately 107 CFU/mL. An aliquot (500 μL) of the diluted suspension was put in a plastic cuvette and exposed to LED light for 10 min. After irradiation, determination of CFU/mL was performed in the same way as described above. Oxidative DNA Damage. To examine if the photoirradiated polyphenol causes oxidative DNA damage in bacteria, biochemical analysis was performed. CA was used as a representative polyphenol, and S. aureus and E. coli were used as representative Gram-positive and -negative bacteria, respectively. In a plastic cuvette, 1800 μL of CA solution was mixed with 200 μL of the bacterial suspension to reach final concentrations of 1 mg/mL for CA and approximately 109 CFU/ mL for the bacteria. The sample was exposed to LED light for 5 min. After irradiation, 200 μL of sterile catalase solution (5000 U/mL) was added to the sample. Then, the bacteria were collected by centrifugation, and DNA was extracted using a commercial kit (Isoplant, Nippon Gene, Tokyo, Japan). Concentration of DNA was adjusted to 200 μg/mL by reading an absorbance at 260 nm using a spectrophotometer (Gene Quant 1300, GE Healthcare, Bukinghamshire, UK). The formation of 8-hydroxydeoxyguanosine (8-OHdG), which is a marker of oxidative DNA damage,19 was analyzed using a commercial kit (OxiSelect Oxidative DNA Damage ELISA Kit, Cell Biolabs, San Diego, CA, USA). The 8-OHdG formation caused by photoirradiated CA [CA(+)L(+)] was compared to the treatment with CA alone [CA(+)L(−)], LED irradiation alone [CA(−)L(+)], and no treatment [CA(−)L(−)]. Under the CA(−) condition, pure water was added to the reaction system. Under the L(−) condition, the samples were kept in a light-shielding box. H2O2 Determination. The H2O2 content of each polyphenol solution and pure water with or without the 10 min exposure to LED light was determined by the colorimetric method based on the
peroxide-mediated oxidation of Fe2+ followed by the reaction of Fe3+ with xylenol orange.20 In brief, 500 μL of reaction mixture consisting of 500 μM ammonium ferrous sulfate, 50 mM sulfuric acid, 200 μM xylenol orange, and 200 mM sorbitol was added to 500 μL of polyphenol solution with or without LED irradiation. The polyphenol solution was diluted with pure water beforehand as appropriate. After the 45 min incubation at room temperature, absorbance at 560 nm was read using a spectrophotometer (Gene Quant 1300). The assay was calibrated using a standard curve, which was obtained in the range between 0.25 and 5 μM H2O2. Because the sample was mixed with an equal volume of the reaction agent, the detection limit was 0.5 μM. Electron Spin Resonance (ESR) Analysis of Hydroxyl Radicals. Qualitative and quantitative analyses of •OH generated by photoirradiation of polyphenol solution were performed using an ESR spin trapping technique according to our previous studies.14,21 In brief, polyphenol solution was mixed with DMPO, a spin trap agent, in a plastic cuvette to reach final concentrations of 1 mg/mL for polyphenol and 300 mM for DMPO. Then, the sample was exposed to LED light for 1 min. After irradiation, the sample was transferred to a quartz cell for ESR spectrometry. The ESR spectrum was recorded on an X-band ESR spectrometer (JES-FA-100; JEOL, Tokyo, Japan). The measurement conditions for ESR were as follows: field sweep, 331.41−341.41 mT; field modulation frequency, 100 kHz; field modulation width, 0.1 mT; amplitude, 200; sweep time, 2 min; time constant, 0.03 s; microwave frequency, 9.420 GHz; and microwave power, 4 mW. TEMPOL (10 μM) was used as a standard to calculate the concentration of spin-trapped radicals, and the ESR spectrum of manganese (Mn2+) held in the ESR cavity was used as an internal standard. The concentration of •OH was determined using digital data processing (JEOL). Statistical Analyses. The statistical significance (p < 0.05) in the CFU/mL obtained in the bactericidal test was assessed by Dunnett’s multiple-comparison test using the initial bacterial count as a control group. The analyses for the bactericidal test were performed following logarithmic conversion. When a colony was not detected, the value of the detection limit (102 CFU/mL) was used for the statistical analysis. Statistical significance (p < 0.05) in the 8-OHdG formation was assessed by using the Tukey−Kramer honest significant difference C
DOI: 10.1021/jf5058588 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 3. Logarithmic Reduction of CFU/mL after LED Irradiation of Each Polyphenol for 10 mina CA
GA
ChA
EGC
EGCg
PA
PW
G(+)
E. faecalis S. aureus S. mutans
4.0** >5** >5**
0.0 2.9** 3.2**
>5** >5** >5**
0.1 0.7 0.6**
0.1 0.2 0.6**
>5** 3.6** 4.6**
0.0 0.2 1.0**
G(−)
A. actinomycetemcomitans E. coli P. aeruginosa
>5** >5** >5**
>5** >5** >5**
>5** >5** >5**
2.7** 1.0** 3.8**
>5** 2.2** >5**
>5** 1.6** >5**
3.4** 0.9** 3.0**
a
Significant differences from the initial bacterial count are shown, p < 0.05 (*) and p < 0.01 (**). G(+), Gram-positive; G(−), Gram-negative; CA, caffeic acid; GA, gallic acid; ChA, chlorogenic acid; EGC, epigallocatechin; EGCg, epigallocatechin gallate; PA, proanthocyanidin; PW, pure water. Each value represents the mean of triplicate assays.
Figure 2. Bactericidal activity of a 1 min pretreatment with polyphenols against S. aureus (a) and E. coli (b). Each value represents the mean of triplicate assays with standard deviation. Significant differences from the CFU/mL of PW-L(−) are shown, p < 0.05 (∗) and p < 0.01 (∗∗). CA, caffeic acid; GA, gallic acid; ChA, chlorogenic acid; EGC, epigallocatechin; EGCg, epigallocatechin gallate; PA, proanthocyanidin; PW, pure water; L(+,−), with or without LED irradiation; ND, not detected.
Figure 3. Formation of 8-OHdG in S. aureus (a) and E. coli (b) suspended in caffeic acid (CA) aqueous solution with or without LED irradiation. Each value represents the mean of sextuplicate assays with standard deviation. Significant differences are shown, p < 0.05 (∗) and p < 0.01 (∗∗). CA(+,−), with or without CA; L(+,−), with or without LED irradiation. multiple-comparison test, and the yield of H2O2 and •OH in each polyphenol solution with or without LED irradiation was assessed by Student’s t test. When H2O2 was not detected, the value of the detection limit (0.5 μM) was used for the statistical analysis.
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most susceptible to the polyphenols and was killed with a ≥1 log reduction of CFU/mL by CA, GA, ChA, EGCg, and PA (Table 1). When exposed to LED light, the bactericidal activity of each polyphenol was enhanced in an irradiation time-dependent manner (Tables 2 and 3). Phenolic acids, such as CA, GA, and ChA, showed higher bactericidal activity than flavonoid, such as EGC and EGCg. In particular, photoirradiated CA and ChA killed all bacterial species with a ≥4 log reduction of CFU/mL within 10 min. Following CA and ChA, photoirradiated GA and PA showed relatively high bactericidal effect, although E. faecalis and E. coli were not effectively killed by GA and PA, respectively. LED irradiation alone (PW) also showed bactericidal activity, especially against A. actinomycetemcomitans and P. aeruginosa. Compared to PW, photoirradiated EGC showed no or only a little additional bactericidal effect.
RESULTS
Bactericidal Assay. The logarithmic reduction of CFU/mL from the initial bacterial counts after each treatment is summarized in Tables 1−3. Under the light-shielding condition, certain bacterial species showed susceptibility to the polyphenols (Table 1). E. faecalis was killed by PA with a 2.2 log reduction of CFU/mL in 10 min, whereas the other polyphenols hardly killed the bacteria (Table 1). A. actinomycetemcomitans showed high susceptibility to EGCg and was killed with a 3.5 log reduction of CFU/mL (Table 1). Of the bacterial species used in the present study, P. aeruginosa was D
DOI: 10.1021/jf5058588 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 4. Quantification of H2O2 (a) and DMPO-OH, a spin adduct of hydroxyl radical (b), generated in each polyphenol solution with or without LED irradiation. Each value represents the mean of triplicate assays with standard deviation. Significant differences are shown, p < 0.05 (∗) and p < 0.01 (∗∗). CA, caffeic acid; GA, gallic acid; ChA, chlorogenic acid; EGC, epigallocatechin; EGCg, epigallocatechin gallate; PA, proanthocyanidin; PW, pure water; L(+,−), with or without LED irradiation; ND, not detected.
light for 1 min, the ESR signal of DMPO-OH, a spin adduct of OH, was detected. The presence of the spin adduct was confirmed by hyperfine coupling constants of aN = aH = 1.49 mT.22 The yields of DMPO-OH generated in each polyphenol solution significantly increased by LED irradiation, whereas LED irradiation of PW did not increase the yield (Figure 4b). The highest yield of DMPO-OH (about 1 μM) was observed in photoirradiated PA, followed by EGC, ChA, CA, GA, and EGCg.
Although photoirradiated EGCg was effective against Gramnegative bacteria, it showed no additional bactericidal effect against Gram-positive bacteria. As a whole, Gram-negative bacteria tended to show higher susceptibility to the disinfection treatment than Gram-positive bacteria. Bactericidal Effect of LED Light on Bacteria Pretreated with Polyphenols. When S. aureus was pretreated with polyphenols for 1 min followed by being exposed to LED light for 10 min, pretreatment with CA, GA, ChA, and PA significantly reduced the CFU/mL from the initial bacterial count (Figure 2a). However, the logarithmic reduction of CFU/mL in all cases was 5 log reduction of CFU/mL when exposed to LED light for 10 min. Pretreatment with EGC, EGCg, and PA followed by LED irradiation for 10 min also significantly reduced the CFU/mL, although the reduction was 1−2 log of CFU/mL. Pretreatment with CA, GA, and ChA without LED irradiation also significantly reduced the CFU/mL from the initial bacterial count, although the reduction was
