Radical-scavenging and Pro-/anti-inflammatory Activity of Tetracycline and Related Phenolic Compounds With or Without Visible Light Irradiation.

BACKGROUND/AIM: Microbial tetracycline (TC) pastes have been employed to treat oral bacterial infection. In the present study, we investigated the kinetic radical-scavenging and pro-/anti-inflammatory activity of TC with or without visible light irradiation (VLI). MATERIALS AND METHODS: The radical-scavenging activity of TC and minocycline (MC) was determined by differential scanning calorimetry (DSC). The stoichiometric factor (n) and the rate constant of inhibition and propagation (kinh/kp) were determined. The levels of cyclooxygenase-2 (Cox2), tumor necrosis factor-α (Tnfα) or nitric oxide synthase 2 (Nos2) mRNA in RAW264.7 cells stimulated with lipopolysaccharide (LPS) were investigated using real-time reverse transcriptase-polymerase chain reaction. RESULTS: The n and kinh/kp values for 1 mM TC in 2,2'-azobisisobutyronitrile and benzoyl peroxide systems were 0.1-0.2 and 119-250, respectively, whereas the corresponding values for quercetin (QU) and resveratrol (RE) were 2-4 and 7-15, respectively. In RAW264.7 cells stimulated with LPS, Cox2 and Tnfα mRNA were over-expressed in the presence of TC. MC down-regulated only the expression of Cox2 by about 50% in LPS-stimulated cells. The anti-inflammatory activity determined on the basis of Cox2 inhibition declined in the order QU>RE>MC>TC. Upon application of VLI, only TC down-regulated the expression of LPS-stimulated Cox2 and Tnfα mRNA. After exposure to VLI, TC, but not MC, markedly up-regulated hemoxygenase-1 (Ho-1) expression. CONCLUSION: TC is a chain-breaking antioxidant with a large kinh Upon activation by VLI, TC may undergo degradation and its degradation products affect pleiotropic mediators such as Cox2, Tnfα and Ho-1. TC may be useful as a local photodynamic therapy for periodontal diseases.

Cytotoxicity and Pro-inflammatory Properties of Aliphatic Alpha, Beta-unsaturated Acid and Ester Monomers in RAW264.7 Cells and Their Chemical Reactivity.

BACKGROUND/AIM: α,ß-Unsaturated ester monomers such as methyl methacrylates (MMA), 2-hydroxyethyl methacrylates (2-HEMA), ethyleneglycol dimethacrylate (EGDMA) and triethyleneglycol dimetacrylate (TEGDMA) have been widely used in dentistry as dental materials. The present study was designed to clarify the proinflammatory activity of monomers. MATERIALS AND METHODS: The cytotoxicity of the monomers and their effects on the expression of cyclooxygenase-2 (Cox2), nitric oxide synthase 2 (Nos2) and heme oxygenase 1 (Ho-1) mRNAs in RAW264.7 cells were determined using a cell counting kit and real-time reverse transcriptase-polymerase chain reaction, respectively. RESULTS: The cytotoxicity declined in the order n-butyl acrylate (nBA) > acrylic acid > TEGDMA > EGDMA > methacrylic acid ≈ 2-HEMA > lauryl methacrylate > nBMA > MMA. nBA and EGDMA at 1 mM up-regulated the expression of Cox2 mRNA. In contrast, 1 mM nBA and 10 mM 2-HEMA up-regulated the expression of Nos2 mRNA. Up-regulation of Ho-1 mRNA expression was found for 0.1 mM nBA, 1 mM EGDMA and 2 mM TEGDMA. The electrophilicity, ω was calculated on the basis of the density function theory BLYP/6-31G*. CONCLUSION: nBA and EGDMA with high ω values exerted potent pro-inflammatory activities. nBA, EGDMA and TEGDMA upregulated Ho-1 gene expression. Ho-1 gene activation of monomers may promote resistance of chemical carcinogenesis in biological systems.

Cytotoxicity and Pro-/Anti-inflammatory Properties of Cinnamates, Acrylates and Methacrylates Against RAW264.7 Cells.

BACKGROUND/AIM: Periodontitis is a chronic inflammatory disease linked to various systemic age-related conditions. It is known that α,ß-unsaturated carbonyl compounds such as dietary cinnamates (ß-phenyl acrylates) and related (meth)acrylates can have various positive and negative health effects, including cytotoxicity, allergic activity, pro-and anti-inflammatory activity, and anticancer activity. To clarify the anti-inflammatory properties of α,ß-unsaturated carbonyl compounds without a phenolic group in the context of periodontal tissue inflammation and alveolar bone loss, we investigated the cytotoxicity and up-regulatory/down-regulatory effect of three trans-cinnamates (trans-cinnamic acid, methyl cinnamate, trans-cinnamaldehyde), two acrylates (ethyl acrylate, 2-hydroxyethyl acrylate), and three methacrylates (methyl methacrylate, 2-hydroxyethyl methacrylate, and triethyleneglycol dimethacrylate) using RAW264.7 cells. MATERIALS AND METHODS: Cytotoxicity was determined using a cell counting kit (CCK-8) and mRNA expression was determined using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Pro-inflammatory and anti-inflammatory properties were assessed in terms of expression of mRNAs for cyclo-oxygenase-2 (Cox2), nitric oxide synthase 2 (Nos2), tumor necrosis factor-alpha (Tnfa) and heme oxygenase 1 (Ho1). RESULTS: The most cytotoxic compound was 2-hydroxyethyl acrylate, followed by ethyl acrylate and cinnamaldehyde (50% lethal cytotoxic concentration, LC50=0.2-0.5 mM). Cox2 mRNA expression was up-regulated by cinnamaldehyde and 2-hydroxyethyl acrylate, particularly by the former. In contrast, the up-regulatory effect on Nos2 mRNA expression was in the order: cinnamaldehyde >> ethyl acrylate ≈ triethyleneglycol dimethacrylate >> methyl methacrylate ≈ methyl cinnamate. On the other hand, cinnamic acid and 2-hydroxyethyl methacrylate had no effect on gene expression. The two acrylates, but not cinnamates and methacrylates, up-regulated the expression of Ho1 mRNA at a non-cytotoxic concentration of 0.1 mM. Expression of Cox2, Nos2 and Tnfa mRNAs induced by Porphyromonas gingivalis lipopolysaccharide was greatly suppressed by cinnamaldehyde, methyl cinnamate and the two acrylates at 0.1 mM (p<0.05), and slightly, but significantly suppressed by cinnamic acid and methacrylates at 0.1-1 mM (p<0.05). CONCLUSION: Cinnamaldehyde and acrylates exhibited both anti-inflammatory and pro-inflammatory properties, possibly due to their marked ability to act as Michael reaction acceptors, as estimated from the beta-carbon 13C-nuclear magnetic resonance spectra. Methyl cinnamate exhibited potent anti-inflammatory activity with less cytotoxicity and pro-inflammatory activity, suggesting that this compound may be useful for treatment of periodontal disease and related systemic diseases.

Anti-inflammatory Activity of ß-Carotene, Lycopene and Tri-n-butylborane, a Scavenger of Reactive Oxygen Species.

BACKGROUND/AIM: The polyene carotenoids ß-carotene and lycopene are antioxidants that not only quench singlet oxygen but also inhibit lipid peroxidation. Tri-n-butyl borane (TBB) is used as an initiator for dental resin materials and is extremely reactive with oxygen and reactive oxygen species (ROS). This reactionability of TBB may be analogous to that of carotenoids with ROS. To clarify the biological activity of such ROS scavengers, we investigated the anti-inflammatory activity of ß-carotene, lycopene and TBB in terms of the expression of RNA for lipopolysaccharide (LPS)-induced cyclooxygenase-2 (Cox2), nitric oxide synthase 2 (Nos2) and tumor necrosis factor-alpha (Tnfa), and mRNA expression and up-regulation of heme oxygenase 1 (Hmox1) mRNA in RAW264.7 cells. MATERIALS AND METHODS: mRNA expression was investigated using real-time reverse transcriptase-polymerase chain reaction (PCR). The antioxidant activity of carotenoids was evaluated using the induction period method in the azobisisobutyronitrile or benzoyl peroxide-methyl methacrylate system. RESULTS: Hmox1 mRNA, but not Cox2 and Nos2 mRNA, was up-regulated by 100 µM ß-carotene and lycopene, and by 0.125% TBB. LPS-stimulated Cox2, Nos2 and Tnfa gene expression was inhibited by 50 µM ß-carotene and lycopene, and by 0.5-1% TBB. Both ß-carotene and lycopene had weak antioxidant activity, but ß-carotene showed pro-oxidant activity at higher concentrations. CONCLUSION: The anti-inflammatory activity of ß-carotene, lycopene and TBB may be related to their ROS-scavenging activity. Additionally, the activity of carotenoids and TBB may be attributed to the electrophilicity of ROS-induced carotenoid intermediates and boranes, respectively. Their anti-inflammatory activity may be attributable to enhancement of the potency of the electrophile/antioxidant response element transcription system in view of their up-regulation of Hmox1 mRNA expression.

Expression of Cyclooxygenase-2, Nitric Oxide Synthase 2 and Heme Oxygenase-1 mRNA Induced by Bis-Eugenol in RAW264.7 Cells and their Antioxidant Activity Determined Using the Induction Period Method.

BACKGROUND/AIM: To clarify the mechanisms responsible for the anti-inflammatory/proinflammatory activities of eugenol-related compounds, we investigated the cytotoxicity and up-regulatory/down-refgulatory effects of the biphenols curcumin, bis-eugenol, magnolol and honokiol, and the monophenols eugenol and isoeugenol, on major regulators of cyclooxygenase-2 (Cox-2), nitric oxide synthase 2 (Nos2) and heme oxygenase-1 (HO-1) mRNA in RAW264.7 cells. MATERIALS AND METHODS: mRNA expression was investigated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and the theoretical parameters were calculated using the DFT/B3LYP/6-31* method. Also, the antioxidant activity of eugenol-related compounds in combination with 2-mercapto-1-methylimidazole (MMI, as a model for glutathione (GSH)) was investigated using the induction period method for polymerization of methyl methacrylate initiated by benzoyl peroxide (BPO). RESULTS: The cytotoxicity of eugenol-related compounds showed a linear relationship with their softness (σ) and electrophilicity (ω). At a concentration of 50 µM, biphenols except for bis-eugenol elicited the expression of mRNA for both Cox-2 and Nos2, but monophenols did not. In contrast, bis-eugenol elicited Cox-2 gene expression, but down-regulated Nos2 gene expression. bis-Eugenol alone induced the expression of HO-1 mRNA, and when combined with MMI it showed a potent antagonistic effect on BPO-induced antioxidant activity. The ability of methoxyphenols to inhibit LPS-stimulated Cox-2 gene expression declined in the order curcumin >> isoeugenol > bis-eugenol >> eugenol, and the rank of ability was related to their ω value. CONCLUSION: Most eugenol-related compounds had proinflammatory activity at high concentrations. However, they had also anti-inflammatory activity at lower concentrations. Eugenol-related compounds may exert antioxidant and anti-inflammatory activity in LPS-stimulated RAW264.7 cells possibly by inhibiting the activation of nuclear factor-kappa B (Nf-ĸB), whereas bis-eugenol requires induction of HO-1 expression. bis-Eugenol as well as curcumin, may have anti-inflammatory and anticancer therapeutic applications.

Radical-scavenging and Anti-inflammatory Activity of Quercetin and Related Compounds and Their Combinations Against RAW264.7 Cells Stimulated with Porphyromonas gingivalis Fimbriae. Relationships between Anti-inflammatory Activity and Quantum Chemical Parameters.

BACKGROUND/AIM: The flavonoid quercetin exerts significant anti-inflammatory activity against chronic infections, including periodontal disease. However, it is unclear whether combination of quercetin with other flavonoids enhances antioxidant and anti-inflammatory activity. To clarify the molecular mechanism responsible for the anti-inflammatory activity of quercetin, we investigated the antioxidant, cytotoxicity and anti-inflammatory activity of quercetin and its related compounds, catechin and epicatechin, and their combinations. MATERIALS AND METHODS: Radical-scavenging activities were determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, and cytotoxicity against RAW264.7 cells was determined using a cell counting kit (CCK-8). The inhibitory effects of these compounds on the mRNA expression of cyclooxygenase-2 (Cox2), tumor necrosis factor-alpha (Tnfα) and nitric oxide synthase 2 (Nos2), in RAW264.7 cells stimulated with Porphyromonas gingivalis (Pg) fimbriae, was also determined using real-time polymerase chain reaction analysis. The phenolic O-H bond dissociation enthalpy (BDE) and quantum chemical parameters were calculated on the basis of density function theory (DFT) BLYP/6-31G*. RESULTS: The DPPH(•) radical-scavenging activity (EC50) of quercetin, catechin and epicatechin was 5.5, 7.7 and 6.2 µM, respectively, whereas the cytotoxicity (LC50) was 4.45, 4.80 and 4.95 mM, respectively. Quercetin had slightly higher cytotoxicity and anti-DPPH(•) activity than catechin and epicatechin. The BDE for the three flavonoids at the 4'-OH in the B ring, which is the initial active site, was about 75 kcal/mol. Furthermore, various combinations of quercetin with catechin or epicatechin exerted an antagonistic effect on anti-DPPH(•) activity. Gene expression of Cox2, Tnfα and Nos2 stimulated by exposure to Pg-fimbriae was markedly suppressed by quercetin, but was not modulated by its combination with epicatechin. The 50% inhibitory concentration of quercetin for Cox2 expression was approximately 10 µM, while that of catechin and epicatechin was approximately 500 µM. Values of the quantum chemical parameters softness (σ) and electronegativity (χ) were highest for quercetin among the three flavonoids tested. CONCLUSION: The potent anti-inflammatory activity of quercetin appears to be attributable to its high σ and χ values. Quercetin may be applicable as a preventive agent against inflammatory periodontal disease as a manifestation of systemic disease.

The Radical Scavenging Activity and Cytotoxicity of Resveratrol, Orcinol and 4-Allylphenol and their Inhibitory Effects on Cox-2 Gene Expression and Nf-κb Activation in RAW264.7 Cells Stimulated with Porphyromonas gingivalis-fimbriae.

BACKGROUND/AIM: Resveratrol is a polyphenol with efficient anti-oxidative and anti-inflammatory activity. To clarify the molecular mechanism responsible for its anti-inflammatory action, we investigated the radical scavenging activity, cytotoxicity and anti-inflammatory activity of resveratrol and its related compounds, orcinol and 4-allylphenol. MATERIALS AND METHODS: The radical scavenging activities of these compounds were determined by the DPPH (2,2'-diphenyl-1-picrylhydrazyl) assay and their cytotoxicities against RAW264.7 cells were determined using a cell-counting kit (CCK-8). The inhibitory effects of these compounds on cyclooxygenase-2 (Cox2) expression in RAW264.7 cells stimulated with Porphyromonas gingivalis (Pg) fimbriae were also determined using real-time polymerase chain reaction and western blot analysis, while inhibition of the fimbria-stimulated activation of nuclear factor-kappa B (Nf-κb) was evaluated using western blot analysis and enzyme-linked immunosorbent assay-like microwell colorimetric transcription factor activity assay, respectively. The quantum chemical parameters were calculated on the basis of the density function theory (DFT) BLYP/6-31G*. RESULTS: DPPH radical scavenging activity declined in the order resveratrol > orcinol > 4-allylphenol. The cytotoxicity of the compounds was in the order 4-allylphenol > resveratrol > orcinol. The inhibitory effect on Pg fimbria-stimulated Cox2 expression and Nf-κb activation was enhanced by resveratrol-alone. Resveratrol showed high electronegativity (χ) and softness (σ) values, as determined by quantum chemical calculations. CONCLUSION: Resveratrol exerts potent anti-inflammatory activity in RAW264.7 cells stimulated with Pg-fimbriae and may be applicable as a therapeutic agent for inflammatory periodontal disease as a manifestation of systemic disease.

Anti-inflammatory activity of the artificial antioxidants 2-tert-butyl-4-methoxyphenol (BHA), 2,6-di-tert-butyl-4-methylphenol (BHT) and 2,4,6-tri-tert-butylphenol (TBP), and their various combinations.

BACKGROUND/AIM: The artificial complex phenols, 2-tert-butyl-4-methoxyphenol (BHA), 2,6-di-tert-butyl-4-methylphenol (BHT) and 2,4,6-tri-tert-butylphenol (TBP) exert efficient antioxidant activity; however, they are considerable toxic and potentially tumor-promoting. These phenols, particularly in combinations, have enhanced antioxidant activity due to synergistic interactions and produce bioactive intermediates such as quinone methide. We investigated the anti-inflammatory activity of BHA, BHT and TBP, and combinations of BHT/BHA (in molar ratios of 1:1, 1:2, 1:3 and 2:1), BHT/TBP (1:1), and BHA/TBP (1:1), using gene-expression systems for cyclooxygenase-2 (Cox2) and tumor necrosis facto-alpha (Tnfa) in RAW264.7 cells. MATERIALS AND METHODS: The inhibitory effects of BHA, BHT and TBP on expression of Cox2 and Tnfa genes upon stimulation with Escherichia coli lipopolysaccharide (LPS) or Porphyomonas gingivalis (Pg) fimbriae were determined using real-time polymerase chain reaction. RESULTS: The inhibitory effect on expression of Cox2 and Tnfa genes upon stimulation with LPS and fimbriae was greatly enhanced by the combination of two antioxidants (molar ratio 1:1), BHT/BHA. In addition, that of the Cox2 gene, but not of Tnfa gene was slightly enhanced by a combination of equimolar BHT/TBP and BHA/TBP. None of the antioxidants alone exerted any anti-inflammatory activity upon stimulation with LPS, but a slight anti-inflammatory activity was observed upon stimulation with Pg fimbriae. The inhibitory effect of the BHT/BHA combination on expression of Cox2 mRNA upon stimulation with LPS was investigated at afferent molar ratios, and a molar ratio of 1:1 was found to have considerably less effect than a molar ratio of 1:2 or 2:1. The 1:3 combination had no effect. CONCLUSION: The combination of BHT and BHA at a molar ratio of 0.5-2 exerts potent anti-inflammatory activity. This anti-inflammatory activity on the generation of inflammatory mediators in LPS-activated RAW264.7 cells may be attributable to complex synergistic antioxidant activity of the combination of BHT and BHA. Our results suggest the potential usefulness of the BHT/BHA combination at an appropriate molar ratio as an antioxidant in foods and pharmaceuticals, whereas either antioxidant alone is unlikely to be effective.

Inhibitory effects of p-cresol and p-hydroxy anisole dimers on expression of the cyclooxygenase-2 gene and lipopolysaccharide-stimulated activation of nuclear factor-κB in RAW264.7 cells.

BACKGROUND/AIM: Phenolic compounds, particularly dihydroxybiphenyl-related compounds, possess efficient anti-oxidative and anti-inflammatory activity. We investigated the anti-inflammatory activity of 2,2'-dihydroxy-5,5'-dimethylbiphenol (p-cresol dimer), 2,2'-dihydroxy-5,5'-dimethoxybiphenol (pHA dimer), p-cresol, p-hydroxyanisole (pHA) and 2-t-butyl-4-hydroxyanisole (BHA). MATERIALS AND METHODS: The cytotoxicity of the investigated compounds against RAW264.7 cells was determined using a cell counting kit (CCK-8). Their inhibitory effects on cyclooxygenase-2 (Cox2) mRNA expression stimulated by lipopolysaccharide (LPS) were determined using northern blot analysis, and their inhibition of LPS-stimulated nuclear factor-kappa B (Nf-κb) activation was evaluated using enzyme-linked immunosorbent assay-like microwell colorimetric transcription factor activity assay. The molecular orbital energy was calculated on the basis of density function theory BLYP/6-31G*. RESULTS: The cytotoxicity of the compounds declined in the order pHA dimer > p-cresol dimer > BHA > p-cresol > pHA. The inhibitory effect on Cox2 expression and Nf-κb activation was enhanced by p-cresol dimer and pHA dimer, particularly the former, suggesting potent anti-inflammatory activity, whereas p-cresol and pHA showed weak activity, and BHA no activity. Both p-cresol dimer and pHA dimer were highly electronegative, as determined by quantum chemical calculations. CONCLUSION: Dimerization of p-cresol and pHA enhances their anti-inflammatory activity. p-Cresol dimer and pHA dimer, particularly the former, are potent anti-inflammatory agents.

Inhibitory effects of tocopherols on expression of the cyclooxygenase-2 gene in RAW264.7 cells stimulated by lipopolysaccharide, tumor necrosis factor-α or Porphyromonas gingivalis fimbriae.

BACKGROUND: Tocopherols, which include α-, ß-, γ-, and δ-tocopherol, protect cells against harmful free radicals and play an important role in preventing many human diseases such as cancer, inflammatory disorders, and ageing itself. However, the causal relationships between periodontal or oral chronic diseases and tocopherols have not been sufficiently studied. The present study investigated the inhibitory effects of these compounds on the expression of cyclooxygenase-2 (COX2) mRNA in RAW264.7 cells stimulated with lipopolysaccharide (LPS), tumor necrosis factor-α (TNFα) or fimbriae of Poryphyromonas gingivalis (Pg), an oral anaerobe. MATERIALS AND METHODS: The cytotoxicity (EC50) of tocopherols toward RAW cells was determined using a cell counting kit (CCK-8). The regulatory effect of these compounds on the expression of COX2 mRNA stimulated with LPS, TNFα or Pg fimbriae was investigated using real-time polymerase chain reaction (PCR). RESULTS: Each tocopherol had similarly low cytotoxicity. COX2 gene expression in RAW cells after exposure to the three different macrophage activators was inhibited by the tocopherols (p<0.01). Compared to α-tocopherol, ß-, γ- and δ-tocopherol exhibited greater inhibitory effects (p<0.05). CONCLUSION: Tocopherols exhibit anti-inflammatory activity, and ß-, γ- and δ-tocopherol have particularly more potent anti-inflammatory activity than α-tocopherol. Tocopherols may have potential utility for prevention of periodontal and chronic oral diseases.

Comparative inhibitory effects of magnolol, honokiol, eugenol and bis-eugenol on cyclooxygenase-2 expression and nuclear factor-kappa B activation in RAW264.7 macrophage-like cells stimulated with fimbriae of Porphyromonas gingivalis.

BACKGROUND: The anti-inflammatory activity of magnolol and related compounds is currently a focus of interest. In the present study, the inhibitory effects of these compounds on cyclooxygenase (COX-2) expression and nuclear factor-kappa B (NF-κB) activation were investigated in RAW264.7 macrophage-like cells stimulated with the fimbriae of Porphyromonas gingivalis, an oral anaerobe. MATERIALS AND METHODS: The cytotoxicity of magnolol, honokiol, eugenol and bis-eugenol against RAW264.7 cells was determined using a cell counting kit (CCK-8). The regulatory effect of these compounds on the expression of COX-2 mRNA, stimulated by exposure to the fimbriae was investigated by real-time polymerase chain reaction (PCR). NF-κB activation was evaluated by enzyme-linked immunosorbent assay (ELISA)-like microwell colorimetric transcription factor activity assay (Trans-AM) and western blot analysis. The radical-scavenging activity was determined using the induction period method in the methyl methacrylate-azobisisobutyronitrile (AIBN) polymerization system under nearly anaerobic conditions. The phenolic bond dissociation enthalpy (BDE) and orbital energy were calculated at the density functional theory (DFT) B3LYP/6-31G* level. RESULTS: The cytotoxicity against RAW264.7 cells declined in the order bis-eugenol>eugenol> honokiol>magnolol, whereas the radical-scavenging activity declined in the order honokiol, bis-eugenol>magnolol> eugenol. Magnolol and honokiol significantly inhibited the fimbria-induced expression of COX-2 at non-cytotoxic concentrations. Both the fimbria-stimulated binding of NF-κB to its consensus sequence and phosphorylation-dependent proteolysis of inhibitor κB-α were markedly inhibited by magnilol and honokiol, whereas eugenol and bis-eugenol did not inhibit COX-2 expression and NF-κB activation. Magnolol and honokiol possessed a high electronegativity (χ) value. CONCLUSION: Magnolol and honokiol exhibit antioxidative activity, low cytotoxicity, and anti-inflammatory activity. These compounds may be capable of preventing chronic inflammatory diseases induced by oral bacteria.

Effects of curcumin and capsaicin irradiated with visible light on murine oral mucosa.

The purpose of this study was to evaluate the histopathological effects of curcumin and capsaicin, with or without visible light (VL) irradiation for 5 min, on the oral mucous membrane in mice. Capsaicin-treated, but not curcumin-treated, buccal epithelium exhibited slight tissue damage; VL irradiation caused excessive tissue damage, particularly when combined with the former treatment. The TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method demonstrated that both capsaicin and curcumin induced apoptosis, with the apoptotic effect of capsaicin appearing at an early stage of application. VL irradiation increased the number of apoptotic cells, particularly those upon in the capsaicin-treated area. Capsaicin and curcumin acted as photosensitizers exposure to VL, in the presence of oxygen. Curcumin and capsaicin with VL irradiation could thus be used for photodynamic therapy in the clinical setting, especially in precancerous oral diseases.

Relationships between base-catalyzed hydrolysis rates or glutathione reactivity for acrylates and methacrylates and their NMR spectra or heat of formation.

The NMR chemical shift, i.e., the π-electron density of the double bond, of acrylates and methacrylates is related to the reactivity of their monomers. We investigated quantitative structure-property relationships (QSPRs) between the base-catalyzed hydrolysis rate constants (k1) or the rate constant with glutathione (GSH) (log k(GSH)) for acrylates and methacrylates and the (13)C NMR chemical shifts of their α,ß-unsaturated carbonyl groups (δC(α) and δC(ß)) or heat of formation (Hf) calculated by the semi-empirical MO method. Reported data for the independent variables were employed. A significant linear relationship between k1 and δC(ß), but not δC(α), was obtained for methacrylates (r(2) = 0.93), but not for acrylates. Also, a significant relationship between k1 and Hf was obtained for both acrylates and methacrylates (r(2) = 0.89). By contrast, log k(GSH) for acrylates and methacrylates was linearly related to their δC(ß) (r(2) = 0.99), but not to Hf. These findings indicate that the (13)C NMR chemical shifts and calculated Hf values for acrylates and methacrylates could be valuable for estimating the hydrolysis rate constants and GSH reactivity of these compounds. Also, these data for monomers may be an important tool for examining mechanisms of reactivity.

Porphyromonas gingivalis Fimbria-Induced Expression of Inflammatory Cytokines and Cyclooxygenase-2 in Mouse Macrophages and Its Inhibition by the Bioactive Compounds Fibronectin and Melatonin.

Porphyromonas gingivalis (Pg) fimbriae, in addition to lipopolysaccharide, are involved in the pathogenesis of periodontal disease. At the same time, bioactive compounds such as fibronectin (FN) and melatonin in saliva and gingival crevicular fluid have been reported to exert a preventive effect against periodontitis. Here, we review current knowledge regarding the potent inhibitory effects of FN and melatonin against Pg fimbria-induced induction of proinflammatory cytokines, cyclooxygenase-2 (COX-2) expression, and NF-kappa B activation in mouse macrophages and discuss their possible clinical application for prevention of periodontal diseases induced by oral bacteria.

Mechanisms of action of (meth)acrylates in hemolytic activity, in vivo toxicity and dipalmitoylphosphatidylcholine (DPPC) liposomes determined using NMR spectroscopy.

We investigated the quantitative structure-activity relationships between hemolytic activity (log 1/H(50)) or in vivo mouse intraperitoneal (ip) LD(50) using reported data for α,ß-unsaturated carbonyl compounds such as (meth)acrylate monomers and their (13)C-NMR ß-carbon chemical shift (δ). The log 1/H(50) value for methacrylates was linearly correlated with the δC(ß) value. That for (meth)acrylates was linearly correlated with log P, an index of lipophilicity. The ipLD(50) for (meth)acrylates was linearly correlated with δC(ß) but not with log P. For (meth)acrylates, the δC(ß) value, which is dependent on the π-electron density on the ß-carbon, was linearly correlated with PM3-based theoretical parameters (chemical hardness, η; electronegativity, χ; electrophilicity, ω), whereas log P was linearly correlated with heat of formation (HF). Also, the interaction between (meth)acrylates and DPPC liposomes in cell membrane molecular models was investigated using (1)H-NMR spectroscopy and differential scanning calorimetry (DSC). The log 1/H(50) value was related to the difference in chemical shift (ΔδHa) (Ha: H (trans) attached to the ß-carbon) between the free monomer and the DPPC liposome-bound monomer. Monomer-induced DSC phase transition properties were related to HF for monomers. NMR chemical shifts may represent a valuable parameter for investigating the biological mechanisms of action of (meth)acrylates.

Radical-scavenging activity of dietary phytophenols in combination with co-antioxidants using the induction period method.

The radical-scavenging activity of dietary phytophenols has been investigated by many researches due to their antioxidant, anti-inflammatory and anticancer property but the radical-scavenging effect of 2-phytophenol and the phytophenol:co-antioxidants, vitamin C and thiol combination under nearly anaerobic conditions still remains unknown. The radical-scavenging activity for seventeen phytophenols and for six synthetic phenols (positive controls) was investigated using the induction period method in the polymerization of methyl methacrylates (MMA) initiated by thermal decomposition of benzoyl peroxide (BPO) by monitoring differential scanning calorimetery (DSC). The k(inh) for the phytophenols was likely with the range 0.5 × 10³ M⁻¹s⁻¹-2.2 × 10³ M⁻¹s⁻¹, whereas that for synthetic phenols, hydroquinone and galvinoxyl, was with the range 7 × 10³ M⁻¹s⁻¹-8 × 10³ M⁻¹s⁻¹. Also, the additive scavenging effect of the (-)-epigallocatechin (EGC):(-)-epicatechin (EC) and the (+)-catechin:epicatechin (EC) combination was observed at 1:1 molar ratio, whereas that of the EC:quercetin combination showed the cancel (prooxidative) effect. Furthermore, the EGC:ASDB (L-ascorbyl 2,6-dibutylate) or 2-ME (2-mercaptoethanol) combination showed the prooxidative effect. Such enhancement of prooxidation in the combination may increase their toxic effects due to their cooxidation. Also, the synergic, additive or cancel effects of the flavonoid:vitamins E combination on the induction period in the BPO (a PhCOO* radical) and 2,2'-azobisisobutyronitrile (AIBN, an R* radical) systems are discussed.

Effect of melatonin on cyclooxygenase-2 expression and nuclear factor-kappa B activation in RAW264.7 macrophage-like cells stimulated with fimbriae of Porphyromonas gingivalis.

BACKGROUND: The possible link between melatonin and anti-inflammatory activity is currently a focus of interest. In the present study, COX-2 expression and NF-κB activation in RAW264.7 macrophage-like cells stimulated with the fimbriae of Porphyromonas gingivalis, an oral anaerobe, in the absence and presence of melatonin were investigated. MATERIALS AND METHODS: The cytotoxicity of melatonin and indole against RAW264.7 cells was determined using a cell counting kit. The regulatory effect of melatonin, and of indole on the expression of COX-2 mRNA stimulated by exposure to the fimbriae was investigated by Northern blot analysis. NF-κB activation was evaluated by both electrophoretic mobility-shift assay and Western blot analysis. RESULTS: The half maximal (50%) effective concentration (EC(50)) values for melatonin and indole were 3300 µM and 130 µM, respectively. Melatonin at non-cytotoxic concentrations significantly inhibited the fimbria-induced expression of COX-2. The fimbria-stimulated binding of NF-κB to its consensus sequence and phosphorylation-dependent proteolysis of inhibitor κB-α were markedly inhibited by melatonin. However, indole did not inhibit COX-2 expression and NF-κB activation. CONCLUSION: Melatonin may be able to prevent diseases induced by oral bacteria.

Radical-scavenging activity of melatonin, either alone or in combination with vitamin E, ascorbate or 2-mercaptoethanol as co-antioxidants, using the induction period method.

BACKGROUND: Melatonin shows antioxidant/prooxidant activity but its mechanism of action remains unknown. MATERIALS AND METHODS: The radical-scavenging activity of melatonin and various melatonin/co-antioxidant mixtures in a 1:1 molar ratio was evaluated in terms of the length of the induction period (IP) for polymerization of methyl methacrylate (MMA), initiated by thermal decomposition of 2,2'-azobisisobutyronitrile (AIBN) or by benzoyl peroxide (BPO) under nearly anaerobic conditions, which was monitored by differential scanning calorimetry (DSC). RESULTS: The observed IP (A) for a pinoline, L-ascorbyl 2,6-dibutyrate (ASDB), vitamin E (alpha-, beta-, gamma- or delta-T) or 2-mercaptoethanol (2ME) mixture was compared with the calculated total sum of IP (melatonin+each co-antioxidant) (B). For both the AIBN and BPO systems, the A/B for the melatonin/ASDB, beta-T, gamma-T or delta-T mixture was 0.3-0.7, whereas that for the melatonin/2ME mixture was approximately 1. For the AIBN system, the A/B for the melatonin/alpha-T or pinoline mixture was 0.7-0.8. By contrast, for the BPO system, that for the melatonin/alpha-T or pinoline mixture was approximately 1. CONCLUSION: The prooxidant effect of the melatonin/ascorbate or vitamin E mixtures induced by radical-oxidizing activity may help to explain the anticancer activity of melatonin in biological systems.

Radical-scavenging activity and cytotoxicity of p-methoxyphenol and p-cresol dimers.

Compoundswith two phenolic OH groups like curcumin possess efficient antioxidant and anti-inflammatory activity. We synthesized p-cresol dimer (2,2'-dihydroxy-5,5'-dimethylbiphenol, 2a) and p-methoxyphenol dimer (2,2'-dihydroxy-5,5'-dimethoxybiphenol, 2b) by ortho-ortho coupling reactions of the parent monomers, p-cresol (1a) and p-methoxyphenol (1b), respectively. Their antioxidant activity was determined using the induction period method, and their cytotoxicity towards RAW 264.7 cells was also investigated using a cell counting kit. The stoichiometric factors n (number of free radicals trapped by one mole of antioxidant moiety) for 2a and 2b were 3 and 2.8, respectively, being greater than those for 1a and 1b. The ratio of the rate constant of inhibition to that of propagation (k(inh)/k(p)) for 2a and 2b was similar to that for 2-t-butyl-4-methoxyphenol (BHA), a conventional food antioxidant. The 50% inhibitory dose (ID50) declined in the order 1b > 1a >> 2b > 2a > BHA. The cytotoxicity for 2a and 2b was significantly greater than that for the parent monomers (p < 0.001), but smaller than that for BHA (p < 0.01). Compounds 2a and 2b may be useful as food antioxidants.

Prediction of the reduced glutathione (GSH) reactivity of dental methacrylate monomers using NMR spectra - Relationship between toxicity and GSH reactivity.

It has been established that the toxicity of acrylate and methacrylate monomers is driven by their reactivity towards glutathione (GSH). With this relationship, the objective of this study was to predict the GSH reactivity of dental methacrylate monomers, and hence their toxicity, using the (13)C-NMR chemical shifts of beta-carbon (delta(Cbeta)) and the (1)H-NMR shifts of the protons attached to beta-carbon (delta(Ha), delta(Hb)). The different nucleophiles were chosen to compare the different nucleophilic reactions involving acrylate and methacrylate monomers. In conjunction with the use of literature data for monomer/GSH reactivity, significant linear relationships between GSH reactivity (log K) and delta(Cbeta )or delta(Ha )were observed (p<0.001). As for the oral LD(50 )values of some dental dimethacrylates in mice, they were estimated using linear regression curve fitting of GSH reactivity-toxicity response data. Results revealed an acceptable correlation between the oral LD(50 )values of acrylates and methacrylates and GSH reactivity (p<0.05, outlier: HEMA). In conclusion, the present findings suggested that NMR spectra might be useful for predicting the toxicity of dental methacrylates.