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.

Comparative study of the alkyl and peroxy radical-scavenging activity of 2-t-butyl-4-methoxyphenol (BHA) and its dimer, and their theoretical parameters.

BACKGROUND: 2-t-Butyl-4-methoxyphenol (BHA) has considerable toxicity and undesirable potential tumor-promoting activities. To clarify the free radical mechanism of BHA-induced toxicity, the comparative radical-scavenging activity of BHA and its dimer (bis-BHA, 3,3'-ditert-butyl-5,5'-dimethoxy-1,1'-biphenyl-2,2'-diol) with or without 2-mercapto-1-methylimidazole (MMI) was studied using the induction period method. MATERIALS AND METHODS: The induction period and propagation rate (Rp) were determined by differential scanning calorimetry (DSC) monitoring of polymerization of methyl methacrylate, initiated by the thermal decomposition of benzoyl peroxide (a source of the peroxy radical, PhCOO*) or 2,2'-azobisisobutyronitrile (a source of the alkyl radical, R*) under nearly anaerobic conditions. The anti-1,1'-diphenyl-2-picrylhydrazyl (DPPH) radical- and O2(-)-scavenging activities were also investigated. Furthermore, theoretical parameters were calculated from the DEFT/B3LYP and HF/6-31G*//B3LYP levels. RESULTS: For both PhCOO* and R* the inhibition rate constant (k(inh)) for BHA and bis-BHA was almost identical, but a marked decrease in the Rp(inh)/Rp(con) was found for the former. The BHA/MMI mixture (1:1 molar ratio) oxidized by R* reduced the total radical-scavenging activity by approximately 20% . BHA showed lower anti-DPPH radical- and higher O2(-)-scavenging activity. CONCLUSION: Upon PhCOO* or R* scavenging, BHA with a lower BDE, IP(koopman's), electronegativity, and electrophilicity value, but not bis-BHA with higher corresponding values, highly suppressed propagation. This may be due to the formation of highly reactive free-radical intermediates, which are potentially toxic.

Radical-scavenging activity of the reaction products of isoeugenol with thiol, thiophenol, mercaptothiazoline or mercaptomethylimidazole using the induction period method.

The reaction products in the presence of Lewis acid of isoeugenol (1) with ethanethiol, thiophenol, 2-mercaptothiazoline or 2-mercapto-1-methylimidazole (ISO-S1-ISO-S-4) were obtained. The radical-scavenging activity of these compounds was investigated using the induction period method for polymerization of methyl methacrylate (MMA) initiated by thermal decomposition of 2,2'-azobisisobutyronitrile (AIBN) and benzoyl peroxide (BPO) and monitored by differential scanning calorimetry (DSC). For BPO, the stoichiometric factor (number of free radicals trapped by one mole of antioxidant moiety, n) declined in the order isoeugenol (1.8) > ISO-S-1 (1.6) > ISO-S-2 (1.2) > ISOS- 3 (0.9) > ISO-S-4 (0.3), whereas for AIBN, their n values were about 1, except for ISOS- 3 (0.6). The ratio of the rate constant of inhibition to that of propagation (k(inh)/k(p)) for BPO declined in the order ISO-S-4 (56) > ISO-S-3 (15) > ISO-S-2 (11) >ISO-S-1 (9) > isoeugenol (8). Similarly, for AIBN the k(inh)/k(p) of the reaction products (33-57) was greater than that of isoeugenol (31). The reaction products of isoeugenol with a SH group showed greater inhibition rate constants (kinh) than the parent compound isoeugenol.

Radical-scavenging activity of natural methoxyphenols vs. synthetic ones using the induction period method.

The radical-scavenging activities of the synthetic antioxidants 2-allyl-4-X-phenol (X = NO2, Cl, Br, OCH3, COCH3, CH3, t-(CH3)3, C6H5) and 2,4-dimethoxyphenol, and the natural antioxidants eugenol and isoeugenol, were investigated using differential scanning calorimetry (DSC) by measuring their anti-1,1-diphenyl-2-picrylhydrazyl (DPPH) radical activity and the induction period for polymerization of methyl methacrylate (MMA) initiated by thermal decomposition of 2,2'-azobisisobutyronitrile (AIBN) and benzoyl peroxide (BPO). 2-Allyl-4-methoxyphenol and 2,4-dimethoxy-phenol scavenged not only oxygen-centered radicals (PhCOO*) derived from BPO, but also carbon-centered radicals (R*) derived from the AIBN and DPPH radical much more efficiently, in comparison with eugenol and isoeugenol. 2-Allyl-4-methoxyphenol may be useful for its lower prooxidative activity.

Kinetic radical-scavenging activity of colchicine and tropolone.

The kinetics of radical-scavenging activities for colchicine and tropolone remain unknown. Their antioxidant activities were determined by the induction period (IP) method in the polymerization of methyl methacrylate initiated by thermal decomposition of 2,2'-azobisisobutyronitrile (AIBN, R*) or benzoyl peroxide (BPO, PhCOO*) using differential scanning calorimetry (DSC) under nearly anaerobic conditions. The IPs for colchicine and tropolone were very short despite the addition of a high concentration of these compounds relative to initiators; the stoichiometric factor (n, the number of moles of PhCOO* trapped by the antioxidant) was approximately 0.03 and 0.04 for colchicine and tropolone, respectively. The n value of these compounds for R* was less than that for PhCOO*. The rate constant of inhibition to that of propagation (kinh/kp) for both compounds was 23-27, and the difference between them was considerably small. Both compounds had weak antioxidant properties at very high concentrations.

Predicting the biological activities of 2-methoxyphenol antioxidants: effects of dimers.

UNLABELLED: Selective cyclooxygenase (COX)-2 inhibitors have attracted much attention in relation to the design of non-steroidal anti-inflammatory agents (NSAIDs). The relationship between experimentally derived data on the antioxidant capacity, cytotoxicity and COX-2 inhibition for a range of 2-methoxyphenols and their calculated descriptors was investigated. MATERIALS AND METHODS: Quantitative structure-activity relationship (QSAR) studies were performed on a series of 2-methoxyphenols that act as COX-2 inhibitors using electronic descriptors, such as the highest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO), ionization potential (IP), chemical hardness (q), and electronegativity (chi), which were calculated by the CONFLEXIPM3 method. The antioxidant capacity of a range of 2-methoxyphenols was evaluated by 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity, and the anti-peroxy radical activity (stoichiometric factor, n) was determined by the induction period method in the polymerization of methyl methacrylate (MMA) initiated by thermal decomposition of benzoyl peroxide (BPO). The 50% cytotoxic concentration (CC50) against human submandibular gland tumor cell line (HSG) was determined by the MTT method. RESULTS: Cytotoxicity declined in the order of curcumin > dehydrodiisoeugenol > isoeugenol >bis-MMP > eugenol > ferulic acid > 2-methoxy-4-methylphenol (MMP) > bis-eugenol > bis-ferulic acid. The inhibitory effects on LPS-induced COX-2 gene expression in RAW 264.7 cells were determined by Northern blot assay. The majority of 2-methoxyphenols studied were COX-2 inhibitors. In particular, dehydrodiisoeugenol was a potent inhibitor, followed by bis-ferulic acid and curcumin. A linear relationship between anti-DPPH radical activity (log 1/IC50) and IP for 2-methoxyphenols except for dehydrodiisoeugenol was observed (r2=0.768.) The n for methoxyphenols was less than 2 in most cases. A linear relationship (r(2)=0.713) between the log (1/CC50) and the r1-term except for ferulic acid was observed. COX-2 inhibition, except for hesperetin, was related to the chi-term (r(2)=0.685). CONCLUSION: It may be possible to predict the mechanism responsible for the biological activities of 2-methoxyphenols.

Preventive effect of ortho dimer of butylated hydroxyanisole on activator protein-1 activation and cyclooxygenase-2 expression in macrophages stimulated by fimbriae of Porphyromonas gingivalis, an oral anaerobe.

Butylated hydroxyanisole (BHA; a mixture of 2- and 3-BHA) is widely used as a potent antioxidant, but is reported to have adverse effects, such as carcinogenesis and pro-inflammatory activity, possibly due to the pro-oxidant property of this compound. 2-Methoxyphenol dimers derived from ferulic acid were recently demonstrated to inhibit the expression of lipopolysaccharide-stimulated cyclooxygenase-2 (COX-2) via redox-sensitive transcription factors such as nuclear factor kappa B or activator protein-1 (AP-1), due to a weakening of its pro-oxidant property by dimerization. To develop anti-inflammatory and/or anticancer drugs for the prevention of oral diseases, such as leukoplakia and destructive chronic periodontitis, whether 2-BHA (2-tert-butyl-4-methoxyphenol) and its synthetic ortho dimer, bis-BHA (3,3'-di-tert-butyl-5,5'-dimethoxy-1,1'-biphenyl-2,2'-diol) can inhibit AP-1 transcriptional activity stimulated by Porphyromonas gingivalis fimbriae was examined. The fimbria-stimulated AP-1 activation of RAW 264.7 murine macrophages was markedly inhibited by bis-BHA. However, BHA showed slight inhibition. Furthermore, bis-BHA significantly inhibited fimbria-induced COX-2 gene expression, which is closely involved with inflammation and carcinogenesis. These findings suggest that bis-BHA may possess a potent anti-inflammatory effect against oral diseases.

Kinetic radical-scavenging activity of melatonin.

Carbon-centred free radicals can be involved in damage to biological systems under hypoxiclanoxic conditions as well as in ischaemia/reperfusion injury. The antioxidant activities of melatonin against carbon-centred radicals are poorly understood. The aim of this study was to investigate the antioxidant properties of melatonin against carbon-centred radicals in a biomimetic model system consisting of growing methyl methacrylate (MMA) radicals (poly-MMA radicals, PMMA*). The kinetics of the polymerization of MMA initiated by thermal decomposition of 2,2'-azobis(isobutyronitrile) (AIBN; R* radical) or benzoyl peroxide (BPO; PhCOO* radical) in the presence of melatonin were investigated by the induction period method under nearly anaerobic conditions. As melatonin concentrations increased, the length of the induction period (IP) increased, but for the BPO system the IP reached a plateau at a molar ratio of BPO to melatonin of 5:1, indicating that the oxidation of melatonin by PhCOO* was limited. At low concentrations of melatonin, the stoichiometric factor (n, the number of free radicals trapped by the antioxidant moiety) for melatonin was approximately 2, but as the melatonin concentration increased the n value decreased markedly to 0.1. These observations suggest that melatonin may possess catalytic activity contributing to radical avoidance. The initial rate of polymerization (Rp) in the BPO system was markedly suppressed by high concentrations of melatonin, suggesting a strong interaction between oxidative end-products formed from melatonin and PMMA*. Under conditions where n was about 2, the kinh values for melatonin in the BPO system and the AIBN system were 6.58 x 10(4) M(-1)S(-1) and 2.49 x 10(3) M(-1)s(-1), respectively. In the BPO system, the kinh of melatonin was of a similar magnitude to that of a-tocopherol, whereas in the AIBN system the kinh of melatonin was 100-fold greater than that of tocopherol. The present findings suggest that melatonin may be able to scavenge harmful carbon-centred radicals in vivo.

An ortho dimer of butylated hydroxyanisole inhibits nuclear factor kappa B activation and gene expression of inflammatory cytokines in macrophages stimulated by Porphyromonas gingivalis fimbriae.

Butylated hydroxyanisole, BHA, is widely used as a potent antioxidant, but its adverse effects such as carcinogenesis and proinflammatory activity have been reported, which are possibly due to the prooxidant property of this compound. We recently demonstrated that the dimer of 2-methoxyphenols exhibits cyclooxygenase-2 inhibition, because of lessening of its prooxidant property caused by the dimerization. In the present study, toward our goal of developing a chemopreventive agent for chronic periodontal diseases, we examined whether 2-BHA (2-tert-butyl-4-methoxyphenol) and its synthetic ortho dimer, bis-BHA (3,3'-di-tert-butyl-5,5'-dimethoxy-1,1'-biphenyl-2,2'-diol) could inhibit the Porphyromonas gingivalis fimbria-stimulated inflammatory reaction. The fimbria-induced expression of interleukin-1beta and neutrophil chemoattractant KC genes in RAW264.7 murine macrophages was strongly inhibited by bis-BHA. In contrast, 2-BHA showed only slight inhibition. bis-BHA also significantly inhibited the fimbria-stimulated phosphorylation-dependent degradation of the alpha inhibitor of nuclear factor-kappaB and the transcriptional activity of this factor in the cells. These findings suggest that bis-BHA possesses anti-inflammatory activity against chronic periodontal diseases.

Tumor-specific cytotoxicity and apoptosis-inducing activity of berberines.

Berberine iodide (IK-1) and acetoneberberine (IK-2) showed higher cytotoxicity against five human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4, NA, CA9-22) and one human promyelocytic leukemia (HL-60) cell lines, than against normal human oral tissue-derived cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), producing a tumor specificity index of 4.0 and 3.6, respectively. IK-1 was more potent than IK-2 in inducing the production of apoptotic cells, internucleosomal DNA fragmentation, the activation of caspases-3, -8 and -9, and the increased expression of proapoptotic BAD protein, with a corresponding decrease in the expression of anti-apoptotic Bcl-2 protein in HL-60 cells. These compounds did not induce internucleosomal DNA fragmentation (only producing larger DNA fragment), nor increased the Bad protein expression in HSC-2 cells. The present study demonstrated the tumor-specific cytotoxicity and apoptosis-inducing activity of berberines, suggesting their possible antitumor potentiaL

Cytotoxic activity of styrylchromones against human tumor cell lines.

A total of 6 newly-synthesized styrylchromones (SC-1 approximately SC-6) were compared for their cytotoxic activity against three normal oral human cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF) and four human tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, submandibular gland carcinoma HSG, promyelocytic leukemia HL-60). All compounds showed higher cytotoxic activity against tumor cell lines than against normal cells. Among the 6 compounds, SC-3, SC-4 and SC-5, which have one to three methoxy groups, showed higher tumor specificity and water solubility. The cytotoxic activity of SC-3 and SC-5 was slightly reduced by a lower concentration of NADH, a quinone reductase, but that of SC-3 was enhanced by higher concentrations of NADH, possibly due to demethylation of the methoxy groups. Agarose gel electrophoresis demonstrated that SC-3 and SC-5 induced intemucleosomal DNA fragmentation in HL-60 cells and production of large DNA fragment in HSC-2 cells. Both SC-3 and SC-5 enhanced the enzymatic activity to cleave the substrates for caspases 3, 8 and 9, suggesting the activation of both extrinsic and intrinsic apoptosis pathways. ESR spectroscopy showed that these compounds produced no detectable amount of radical and did not scavenge superoxide anion generated by the hypoxanthine-xanthine oxidase reaction. The highly tumor-specific cytotoxic action and apoptosis-inducing capability of SC-3 and SC-5 suggest their applicability for cancer chemotherapy.

Dehydrodiisoeugenol, an isoeugenol dimer, inhibits lipopolysaccharide-stimulated nuclear factor kappa B activation and cyclooxygenase-2 expression in macrophages.

o-Methoxyphenols such as eugenol and isoeugenol exhibit anti-oxidant and anti-inflammatory activities, but at higher concentrations act as oxidants and potent allergens. We recently demonstrated the eugenol dimer bis-eugenol to be an efficient inhibitor of lipopolysaccharide (LPS)-induced inflammatory cytokine expression in macrophages without cytotoxicity. This result suggested that dimer compound of o-methoxyphenols may possess anti-inflammatory activity. Thus, we further synthesized dehydrodiisoeugenol and alpha-diisoeugenol from isoeugenols, and investigated whether these dimers could inhibit LPS-stimulated nuclear factor kappa B (NF-kappaB) activation and cyclooxygenase (COX)-2 gene expression, both of which are closely involved in inflammation and mutagenesis. The expression of the COX-2 gene was strongly inhibited by dehydrodiisoeugenol in RAW264.7 murine macrophages stimulated with LPS. In contrast, isoeugenol and alpha-diisoeugenol did not inhibit it. Dehydrodiisoeugenol also significantly inhibited LPS-stimulated phosphorylation-dependent proteolysis of inhibitor kappaB-alpha and transcriptional activity of NF-kappaB in the cells. These findings suggest that dehydrodiisoeugenol acts as a potent anti-inflammatory agent.

Kinetic radical scavenging activity and cytotoxicity of 2-methoxy- and 2-t-butyl-substituted phenols and their dimers.

The dimers bis-EUG, bis-MMP, bis-BHA, bis-BMP and bis-DBP were synthesized from the monomers 4-allyl-2-methoxyphenol (EUG), 2-methoxy-4-methylphenol (MMP), 2-t-butyl-4-methoxyphenol (BHA), 2-t-butyl-4-methylphenol (BMP) and 2,4-di-t-butylphenol (DBP), respectively. The stoichiometric factors (n; number of free radicals trapped by one mole of phenolic moiety) of these compounds were determined by induction period methods with a kinetic approach in the 2'2-azobisisobutyronitrile (AIBN) and benzoyl peroxide (BPO) systems at 70 degrees C. The n values for bis-EUG, bis-MMP and bis-BHA were approximately two-fold greater than those for their monomers in both the AIBN and BPO systems, whereas the n values for bis-BMP and bis-DBP were identical to those of their monomers. bis-EUG, bis-MMP and bis-BHA, containing methoxy groups, were potent antioxidants. The n values (1.3-1.6) for EUG and MMP were considerably less than 2, as is commonly observed for the stoichiometric factors of phenolic compounds. The antiradical efficiencies against DPPH (diphenylpicrylhydrazyl) of the monomers and their dimers were also investigated, likewise indicating that bis-EUG, bis-MMP and bis-BHA were potent antioxidants. DBP and bis-DBP were less effective radical scavengers because of the steric factor of their bulky t-butyl groups. On the basis of cytotoxic activity against a human submandibular gland carcinoma cell line (HSG) and human gingival fibroblasts (HGF), these compounds could be classified into a high-activity group (DBP, bis-DBP and bis-BMP, with butylated hydroxytoluene (BHT) as a positive control) and a low-activity group (MMP, EUG, BHA, BMP, bis-BHA and bis-EUG). The cytotoxicity of EUG and BHA was markedly reduced by dimerization, whereas that of MMP was enhanced. The sensitivity index (ratio of 50% cytotoxic concentration for HGF cells to that for HSG cells) of EUG, MMP, bis-MMP and bis-BHA was approximately 9, 5, 7 and 2, respectively, whereas that of the other compounds was approximately 1. Potential mechanisms of cytotoxicity were assessed by PM3 semiempirical molecular orbital (MO) calculations. Tumor cells were highly sensitive to 2-methoxy-4-alkylphenols such as EUG and MMP, possibly due to the formation of cytotoxic quinone methide intermediates. In contrast, the high sensitivity index of bis-MMP may be related to the production of a highly reactive substance, CH3+, via oxidation. Structure-activity relationship (SAR ) models using PM3 calculations may be useful to predict biological activity during the development of potential anticancer drugs.

Dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol liposomes as predictors of the cytotoxicity of bis-GMA related compounds.

In light of recent development, dental materials such as 2, 2-bis [4-2(-hydroxy-3-methacryloyloxypropoxy)phenyl] propane, ( bis-GMA); 2, 2-bis [4-(1-hydroxymethyl-2-methacryloxy)phenyl] propane, (iso-bis-GMA); and triethyleneglycol dimethacrylate, (TEGDMA) were investigated to determine whether their phase transition properties (phase transition temperature, temperature width, cooperativity) could be induced in samples of DPPC or DPPC/cholesterol (CHOL) liposomes using differential scanning calorimetry (DSC). The changes in phase transition properties of DPPC liposomes caused by addition of TEGDMA were greater than those caused by addition of bis-GMA or iso-bis-GMA, but the extent of changes in the properties of DPPC/CHOL (10:1 or 4:1) liposomes declined in the order of bis-GMA > iso-bis-GMA > TEGDMA. The degree of alteration was related to the cytotoxicity of these compounds. DPPC/CHOL liposomes were found to be better predictors of cytotoxicity than DPPC liposomes. Whether the computational approach to studying the molecular mechanism of alteration is applicable using descriptors such as reactivity of energy of the highest occupied molecular orbital (HOMO) and/or lowest unoccupied molecular orbital (LUMO) was investigated, and the data suggested that these descriptors are useful for studying the interactive roles of dental materials.

Radical-scavenging activity of butylated hydroxytoluene (BHT) and its metabolites.

To clarify the radical-scavenging activity of butylated hydroxytoluene (BHT), a food additive, stoichiometric factors (n) and inhibition rate constants (kinh) were determined for 2,6-di-tert-butyl-4-methylphenol (BHT) and its metabolites 2,6-di-tert-butyl-p-benzoquinone (BHT-Q), 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHA-CHO) and 3,5-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadiene-1-one (BHT-OOH). Values of n and kinh were determined from differential scanning calorimetry (DSC) monitoring of the polymerization of methyl methacrylate (MMA) initiated by 2,2'-azobis(isobutyronitrile) (AIBN) or benzoyl peroxide (BPO) at 70 degrees C in the presence or absence of antioxidants (BHT-related compounds). The n values declined in the order BHT (1-2) > BHT-CHO, BHT-OOH (0.1-0.3) > BHT-Q ( approximately 0). The n value for BHT with AIBN was approximately 1.0, suggesting dimerization of BHT. The kinh values declined in the order BHT-Q ((3.5-4.6) x 10(4) M(-1)s(-1)) > BHT-OOH (0.7-1.9 x 10(4) M(-1)s(-1)) > BHT-CHO ((0.4-1.7 x 10(4) M(-1)s(-1)) > BHT ((0.1-0.2 x 10(4) M(-1)s(-1)). The kinh for metabolites was greater than that for the parent BHT. Growing MMA radicals initiated by BPO were suppressed much more efficiently by BHT or BHT-Q compared with those initiated by AIBN. BHT was effective as a chain-breaking antioxidant.