title: 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.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors: Division of Oral Diagnosis, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, Sakado City, Saitama, Japan ymura@dent.meikai.ac.jp.
authors: Yukio
authors: Y
authors: Murakami
authors: Division of Oral Diagnosis, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, Sakado City, Saitama, Japan.
authors: Akifumi
authors: A
authors: Kawata
authors: Division of Oral Diagnosis, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, Sakado City, Saitama, Japan.
authors: Tadashi
authors: T
authors: Katayama
authors: Division of Oral Diagnosis, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, Sakado City, Saitama, Japan.
authors: Seiichiro
authors: S
authors: Fujisawa
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000737:chemistry / Q000494:pharmacology*; D000975:Antioxidants / Q000737:chemistry / Q000494:pharmacology*; D002083:Butylated Hydroxyanisole / Q000737:chemistry / Q000494:pharmacology*; D002084:Butylated Hydroxytoluene / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D052246:Cyclooxygenase 2 Inhibitors / Q000737:chemistry / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D000893:Anti-Inflammatory Agents; D000975:Antioxidants; D052246:Cyclooxygenase 2 Inhibitors; D014409:Tumor Necrosis Factor-alpha; D002084:Butylated Hydroxytoluene; D002083:Butylated Hydroxyanisole; D051546:Cyclooxygenase 2
keywords: BHA; BHT; COX2; Phenolic antioxidants; RAW264.7 cells; TNFα; anti-inflammatory activity; antioxidant combination; cytotoxicity