ABSTRACT
Hinokitiol is found in the heartwood of several cupressaceous plants and is frequently added to cosmetic products such as hair restorers, skin lotions, and body soaps because of its potent and broad-spectrum antibacterial activity. In this study, we established a simple method of hinokitiol determination by high-performance liquid chromatography (HPLC) with dual-wavelength ultraviolet detection at 240 and 345 nm, using a reversed-phase C4 column (RP-4). The retention time of hinokitiol was 7.1 min at both wavelengths. The value of the symmetry coefficient of the hinokitiol peak was close to 1 when the RP-4 column, not an RP-8 or RP-18 column, was used. With the RP-4 column, the regression equation for hinokitiol showed good linearity in the range of 0.05-5 microg/ml, with a detection limit (signal-to-noise ratio of 3) of 0.005 microg/ml at 240 nm and 0.01 microg/ml at 345 nm. The coefficients of variation at 240 and 345 nm were less than 8.2% and 8.7%, respectively, and the recovery was good. The proposed method was used for the determination of hinokitiol in commercial hair restorers, skin lotions, and body soaps.
Subject(s)
Chromatography, High Pressure Liquid/methods , Cosmetics , Monoterpenes/analysis , Spectrophotometry, Ultraviolet/methods , Tropolone/analogs & derivatives , Tropolone/analysisABSTRACT
Transforming growth factor-beta(1) (TGF-beta(1)) increases synthesis of secreted protein, acidic and rich in cysteine (SPARC), as well as fibronectin (FN) and type I collagen. However, little is known about the regulatory mechanism of SPARC expression. We examined the effect of FN on SPARC expression by TGF-beta(1) in cultures of human periodontal ligament cells (HPL cells). TGF-beta(1) increased the SPARC and SPARC mRNA levels in HPL cells. Extracellular matrix (ECM) produced by HPL cells in the presence of TGF-beta(1) also increased the SPARC levels. Contents of FN and type I collagen in the ECM were increased by TGF-beta(1). HPL cells cultured on FN-coated plates secreted more SPARC than those on non-coated plates. However, type I collagen had little effect on SPARC levels. The addition of anti-alpha5 antibody to the cultures abolished the increase in SPARC mRNA expression by TGF-beta(1). This study demonstrated that FN may be partly involved in the increase in SPARC expression by TGF-beta(1) in HPL cells.
Subject(s)
Fibronectins/pharmacology , Osteonectin/biosynthesis , Periodontal Ligament/drug effects , Transforming Growth Factor beta/pharmacology , Cells, Cultured , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Humans , Periodontal Ligament/cytology , Periodontal Ligament/metabolism , RNA, Messenger/biosynthesis , Transforming Growth Factor beta1ABSTRACT
BACKGROUND: Osteonectin/secreted protein, acidic and rich in cysteine (SPARC) is expressed in periodontal ligaments. Therefore, a better understanding of the action of SPARC on periodontal ligament cells could help to elucidate remodelling and repair mechanisms in periodontal tissue. In the present study, we examined the effects of human platelet-derived SPARC (hp-SPARC) on the expressions of SPARC and osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF), alkaline phosphatase (ALPase) activity, matrix metalloproteinase-2 (MMP-2) production and DNA synthesis in cultures of human periodontal ligament (HPL) cells. METHODS: HPL cells at the sixth passage were exposed to hp-SPARC. The expression of OPG/OCIF and SPARC mRNAs was examined by Northern blot analysis. The protein levels for OPG/OCIF and MMP-2 were determined by Western blot analysis. ALPase activity was measured by the method of Bessey et al. DNA synthesis was estimated by incorporation of [3H] thymidine. RESULTS: Hp-SPARC enhanced OPG/OCIF synthesis at the protein and mRNA levels. Hp-SPARC also enhanced DNA and MMP-2 synthesis dose-dependently, but had little effect on ALPase activity and SPARC mRNA expression. CONCLUSION: SPARC may play a role in remodelling and repair of periodontal tissue by promoting proliferation and MMP-2 production. It may also regulate osteoclast formation through OPG/OCIF in periodontal ligament cells.
Subject(s)
Osteonectin/metabolism , Periodontal Ligament/metabolism , Alkaline Phosphatase/metabolism , Analysis of Variance , Blotting, Northern , Blotting, Western , Bone Remodeling , Cell Count , Cells, Cultured , DNA/biosynthesis , Glycoproteins/biosynthesis , Humans , Matrix Metalloproteinase 2/biosynthesis , Osteoprotegerin , Periodontal Ligament/cytology , RNA, Messenger/analysis , Receptors, Cytoplasmic and Nuclear/biosynthesis , Receptors, Tumor Necrosis Factor , RegenerationABSTRACT
Osteoprotegerin (OPG) is a novel tumor necrosis factor receptor superfamily that inhibits osteoclast differentiation, activity, and survival. Interleukin-1beta (IL-1beta) increases OPG expression. IL-1beta also increases prostaglandin E(2) (PGE(2)) production and stimulates bone resorption. In the present study, we examined the involvement of PGE(2) in IL-1beta-induced increases in OPG levels in human periodontal ligament cells (HPL cells) in an effort to clarify apparently conflicting IL-1beta actions on bone resorption and understand IL-1beta-induced increases in secretion of OPG and PGE(2) in HPL cells. 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, a mRNA synthesis inhibitor, partly inhibited the increase in OPG mRNA levels induced by IL-1beta. Cycloheximide, a protein synthesis inhibitor, enhanced the stimulatory effect of IL-1beta. Etodolac, a selective cyclooxygenase-2 inhibitor, suppressed the increase in PGE(2) levels. Furthermore, etodolac reinforced the promotion of OPG expression by IL-1beta at the mRNA and protein levels. PGE(2) added to cultures of HPL cells decreased OPG mRNA levels in a dose- and time- dependent manner. These findings suggest that the increase in OPG levels induced by IL-1beta in HPL cells is suppressed through PGE(2) synthesized de novo.
