Flavonoids from Machilus japonica stems and their inhibitory effects on LDL oxidation.

Stems of Machilus japonica were extracted with 80% aqueous methanol (MeOH) and the concentrated extract was successively extracted with ethyl acetate (EtOAc), normal butanol (n-BuOH), and water. Six flavonoids were isolated from the EtOAc fraction: (+)-taxifolin, afzelin, (-)-epicatechin, 5,3'-di-O-methyl-(-)-epicatechin, 5,7,3'-tri-O-methyl-(-)-epicatechin, and 5,7-di-O-methyl-3',4'-methylenedioxyflavan-3-ol. The chemical structures were identified using spectroscopic data including NMR, mass spectrometry and infrared spectroscopy. This is the first report of isolation of these six compounds from M. japonica. The compounds were evaluated for their diphenyl picryl hydrazinyl scavenging activity and inhibitory effects on low-density lipoprotein oxidation. Compounds 1 and 3-6 exhibited DPPH antioxidant activity equivalent with that of ascorbic acid, with half maximal inhibitory concentration (IC50) values of 0.16, 0.21, 0.17, 0.15 and 0.07 mM, respectively. The activity of compound 1 was similar to the positive control butylated hydroxytoluene, which had an IC50 value of 1.9 µM, while compounds 3 and 5 showed little activity. Compounds 1, 3, and 5 exhibited LDL antioxidant activity with IC50 values of 2.8, 7.1, and 4.6 µM, respectively.

Effect of novel cyclohexane diester and benzene diester derivatives on melanogenesis.

In order to investigate potent whitening agents, we synthesized 15 cyclohexane diester derivatives and 15 benzene diester derivatives. To evaluate their structure-cytotoxicity relationships, we performed cell cytotoxicity tests on B16F10 mouse melanoma cells. To understand their whitening effects, melanin synthesis inhibitory activities in B16F10 cells and mushroom tyrosinase inhibitory activities were performed. In most cases, cell cytotoxicity was observed to be lower in 1,3-diester than in 1,2- and 1,4-diesters; when it came to the structural isomer of the side chain, all derivatives except the 1,2-cyclohexane diester derivatives showed lower cell cytotoxicity in the branch type of the side chain than in the linear type. Among the compounds evaluated, the compounds cyclohexane-1,3-diyl bis(decanoate), cyclohexane-1,4-diyl dioctanoate, and 1,3-phenylene bis (2-ethylhexanoate) emerged as potent melanin synthesis inhibitors. Our goal was to determine the expression levels of proteins involved in melanogenesis, Western blotting and RT-PCR showing that these compounds decreased tyrosinase, TRP-1, and TRP-2 while demonstrating significantly low cytotoxicity.

Matrine inhibits PMA-induced MMP-1 expression in human dermal fibroblasts.

Matrix metalloproteinase-1 (MMP-1) plays an important role in the maintenance and turnover of extracellular matrix (ECM) macromolecules. Remodelling of extracellular matrix by MMPs is a hallmark feature of physiological and pathological processes. In this study, in order to establish the therapeutic potential of matrine, we investigated its effect on MMP-1 expression in human dermal fibroblast cells. We found that matrine inhibited both MMP-1 mRNA and protein expression induced by PMA (phorbol myristate acetate). Therefore, we characterized the inhibitory mechanism of matrine on PMA-induced MMP-1 expression. Matrine inhibited PMA-induced activation of the AP-1 promoter, an important nuclear transcription factor in MMP-1 expression. Additionally, we detected that matrine suppressed the PMA-induced phosphorylation of two mitogen-activated protein kinases, extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but did not suppress the PMA-induced phosphorylation of p38 kinase. These results suggest that matrine suppresses PMA-induced MMP-1 expression through inhibition of the AP-1 signaling pathway and also may be beneficial for treatment of some inflammatory skin disorders.

Red pigment from Lithospermum erythrorhizon by supercritical CO2 extraction.

In this study, a stable red pigment was prepared from Lithospermum erythrorhizon via supercritical carbon dioxide extraction. The optimal extraction conditions were 400 bar and 60 degrees C. The patch tests indicated that up to 10% of the red pigment was acceptable from a skin irritation standpoint. According to the results of the CIE LAB chromaticity test, the color difference was acceptable when compared to commercial synthetic red pigments. The light-illuminated color stability test indicated that the pigment was more stable than the red pigment extracted with ethanol. The higher stability was also demonstrated in the DPPH antioxidant activity test. The supercritical red pigment harbored elevated amounts of shikonin and derivatives, and appears to be usable as a stable red pigment for cosmetic color products.

The ubiquitin ligase COP1 regulates cell cycle and apoptosis by affecting p53 function in human breast cancer cell lines.

BACKGROUND: The E3 ubiquitin ligase constitutive photomorphogenic 1 (COP1) mediates cell survival, growth, and development, and interacts with the tumor suppressor protein p53 to induce its ubiquitination and degradation. Recent studies reported that COP1 overexpression is associated with increased cell proliferation, transformation, and disease progression in a variety of cancer types. In this study, we investigated whether COP1 regulates p53-mediated cell cycle arrest and apoptosis in human breast cancer cell lines. METHODS: We downregulated COP1 expression using lentiviral particles expressing short hairpin RNA (shRNA) targeting COP1 and measured the effects of the knockdown in three different breast cancer cell lines. RESULTS: COP1 silencing resulted in p53 activation, which induced the expression of p21 and p53-upregulated modulator of apoptosis (PUMA) expression, and reduced the levels of cyclin-dependent kinase 2 (CDK2). Notably, knockdown of COP1 was associated with cell cycle arrest during the G0/G1 phase. CONCLUSIONS: The COP1-mediated degradation of p53 regulates cancer cell growth and apoptosis. Our results indicate that COP1 regulates human breast cancer cell proliferation and apoptosis in a p53-dependent manner. These findings suggest that COP1 might be a promising potential target for breast cancer-related gene therapy.

Ferritin immunosensing on microfabricated electrodes based on the integration of immunoprecipitation and electrochemical signaling reactions.

A signal registration strategy from micropatterned immunosensors that converts antigen-antibody binding reactions into electrochemical signals was demonstrated. An array-type micropatterned gold electrode on a silicon wafer was fabricated, containing two electrode geometries of rectangular (100 microm x 500 microm) and circular (r. 50 microm) types, exhibiting electrochemical characteristics of bulk and micro-electrodes, respectively. Ferritin was employed as a model analyte for immunosensing because it has an advantageous molecular structure for functionalization to the sensing interface, and is regarded as a general marker protein for tumors and cancer recurrence. With the fabricated and ferritin-functionalized immunosensors, biospecific interactions were performed with antiferritin antiserum and secondary antibody samples, followed by electrochemical signaling via an immunoprecipitation reaction by the label enzyme. Under the optimized affinity-surface construction steps and reaction conditions, both types of microfabricated electrodes exhibited well-defined calibration results as a function of the protein concentration in antiserum samples. Furthermore, circular-type micropatterned immunoelectrodes exhibited voltammetric characteristics of microelectrodes, which is advantageous in terms of sensor operation under a fixed potential and low signal drift during the signaling reaction compared with the bulk-type electrodes. The results support that the employed signaling method with the proposed immunosensor configuration is fit for sensor miniaturization and integration to future biomicrosystems.

Oral and topical pharmacokinetic studies of a novel TRPV1 antagonist, PAC-14028 in rats and minipigs using liquid chromatography/tandem mass spectrometric method.

PAC-14028 ((E)-N-((R)-1-(3,5-difluoro-4-methanesulfonylamino-phenyl)-ethyl)-3-(2-propyl-6-trifluoromethyl-pyridine-3-yl)-acrylamide) is a novel and potent transient receptor potential vanilloid type I (TRPV1) antagonist. We developed and validated a rapid, sensitive and selective liquid chromatography/tandem mass spectrometric method for determination of PAC-14028 in rat and minipig plasma. After protein precipitation PAC-14028 and internal standard (methylated analog, PAC-14026) were separated on a Symmetry C(18) column (4.6 mm × 75 mm, 3.5 µm) with an isocratic mobile phase, acetonitrile: water (8:2, v/v) containing 0.2% formic acid and monitored by electrospray positive ionization with multiple reaction monitoring mode (PAC-14028, 492→156; IS, 506→156, m/z). The calibration curve was linear over the range of 1.0-500 ng/ml (r(2)>0.999) and lower limit of quantitation (LLOQ) was 1 ng/ml. The precision and accuracy were within ± 15% and the stability was acceptable during bench-top, auto-sampler, 3 freeze-thaw cycles and 4-week storage in a freezer at -80°C. This method was successfully applied to the intravenous, oral and topical pharmacokinetic studies of PAC-14028 in rats and minipigs, which showed comparable pharmacokinetic parameters (T1/2, 2.1h and 3.8h; F%, 52.7% and 64.2% for rats and minipigs, respectively). Percutaneous absorption of PAC-14028 was negligible after topical application (F% 0.2-1.7%).

Isorhamnetin represses adipogenesis in 3T3-L1 cells.

Adipocyte dysfunction is strongly associated with the development of obesity, which is a major risk factor for many disorders including diabetes, hypertension, and heart disease. It is generally accepted that the regulation of adipogenesis or adipokines expression prevents obesity. In this study, we show that isorhamnetin inhibits adipocyte differentiation, as evidenced by reduced triglyceride (TG) accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity. At the molecular level, the mRNA expression levels of peroxidase proliferator-activated receptor-gamma (PPAR-gamma) and CCAAT/enhancer-binding protein-alpha (C/EBP-alpha), which are the major adipogenic transcription factors, were markedly reduced by isorhamnetin. However, the mRNA levels of C/EBP-beta and -delta, the upstream regulators of PPAR-gamma and C/EBP-alpha, were not reduced by isorhamnetin. Moreover, the mRNA levels of PPAR-gamma target genes such as lipoprotein lipase (LPL), CD36, aP2, and liver X receptor-alpha (LXR-alpha) were downregulated by isorhamnetin. We also showed that isorhamnetin inhibits the expression and secretion of adiponectin, and the results of adiponectin promoter assays suggest the inhibition of PPAR-gamma expression as a possible mechanism underlying the isorhamnetin-mediated effects. Taken together, these results indicate that isorhamnetin inhibits adipogenesis through downregulation of PPAR-gamma and C/EBP-alpha.

Bioelectrocatalytic signaling from immunosensors with back-filling immobilization of glucose oxidase on biorecognition surfaces.

We report a novel method of electrochemical signaling from antigen-antibody interactions at immunoelectrodes with bioelectrocatalyzed enzymatic signal amplification. For the immunosensing surface construction, a poly(amidoamine) G4-dendrimer was employed not only as a building block for the electrode surface modification but also as a matrix for ligand functionalization. As a model biorecognition reaction, the dinitrophenyl (DNP) antigen-functionalized electrode was fabricated and an anti-DNP antibody was used. Glucose oxidase (GOX) was chosen to amplify electrochemical signal by enzymatic catalysis. The signal amplification strategy introduced in this study is based on the back-filling immobilization of biocatalytic enzyme to the immunosensor surface, circumventing the use of an enzyme-labeled antibody. The non-labeled native antibody was biospecifically bound to the immobilized ligand, and the activated enzyme (periodate-treated GOX) reacted and "back-filled" the remaining surface amine groups on the dendrimer layer by an imine formation reaction. From the bioelectrocatalyzed signal registration with the immobilized GOX, the surface density of biospecifically bound antibody could be estimated. The DNP functionalization reaction was optimized to facilitate the antibody recognition and signaling reactions, and approximately 6% displacement of surface amine to DNP was found to be an optimum. From quartz crystal microbalance measurement, immunosensing reaction timing and the surface inertness to the nonspecific biomolecular binding were tested. By changing the surface functionalization level of DNP in the calibration experiments, immunosensors exhibited different dynamic detection ranges and limits of detection, supporting the capability of parameters modulation for the immunosensors. For the anti-DNP antibody assay, the fabricated immunosensor having 65% functionalization ratio exhibited the linear detection range of 10(-4) to 0.1 g/L protein and a limit of detection around 2 x 10(-5) g/L.