Nitric Oxide Synthesis In RAW Research Articles

Chemical and Biological Changes Under Force Degradation and Acceleration Condition of the Combination of Ha-Rak Remedy, Piper betle, and Garcinia mangostana Extracts for Atopic Dermatitis.

Herbal medicine could be an option for atopic dermatitis (AD) treatment for those suffering from global public health. HMB is a new combination of three herb extracts, consisting of the Ha-Rak (HR) remedy extract, Piper betle (PB) extract, and Garcinia mangostana (GM) extract in equal proportions, using Thai traditional medicine theory, that uses a combination of medications that can improve therapeutic efficacy and reduce side effects and toxicity. HMB extract has anti-inflammatory and antiallergic properties, is a component for AD treatment, and tends to develop topical products. Drug registration requires stability data. Results from drug stability testing affect not only the efficacy of the drug but also its safety. The aim of this study was to investigate stability through forced degradation and an accelerated study of extracts. Chemical content analysis and in vitro biological activities such as anti-inflammatory and antiallergic activities determined the effects of all examined samples. Anti-inflammatory and antiallergic effects were assessed by inhibiting nitric oxide synthesis in RAW 264.7 cells and β-hexosaminidase release in RBL-2H3 cells, respectively. High-performance liquid chromatography (HPLC) assessed content indicators. Moisture and temperature hydrolysis had no significant differences in the chemical or biological properties of the HMB. However, the HMB demonstrated sensitivity to alkaline hydrolysis, showed low anti-inflammatory activity, and decreased hydroxychavicol, eugenol, and α-mangostin contents. The contents of the three compounds also decrease with acid hydrolysis. For the accelerated study, anti-inflammatory and antiallergic effects and hydroxychavicol amount were not significantly different after 180 days at 40°C and 75% RH. Therefore, the contents of eugenol and α-mangostin were changed. Eugenol in HMB decreased significantly from the 15th day until the 180th day of storage. In addition, α-mangostin amounts in HMB decreased slightly on 180th day. Fortunately, reducing the two chemicals did not affect anti-inflammatory or antiallergic effects. For stability, combination extract should be stored in a closed container in the refrigerator at a low temperature and protected from light, high temperature, oxygen, and pH. Further HMB development should avoid pH or oxidation processes or components.

First isolation of glutinol and a bioactive fraction with good anti-inflammatory activity from n-hexane fraction of Peltophorum africanum leaf.

To investigate the anti-inflammatory activity of different fractions and glutinol (isolated compound), using nitric oxide synthase and cyclooxygenase (COX) inhibition as an indication of anti-inflammatory activity. Anti-inflammatory activity was evaluated using an invitro assay determining the inhibition of the activity of pro-inflammatory enzyme model. Cyclooxygenases and inducible nitric oxide synthase are crucial enzymes involved in the pathogenesis of many chronic inflammatory conditions. Sub-fraction F3.3 that was derived from n-hexane fraction of PA leaves significantly inhibited (P=0.01) the catalytic activity of COX-2 (IC50=0.67μg/mL) better than isolated compound, glutinol (IC50=1.22μg/mL), compound 2 (CP2) (IC50=1.71μg/mL) and sub-fraction F3.3.0 (IC50=1.30μg/mL). A similar trend was observed in investigation of the inhibition of nitric oxide synthesis in RAW 264.7 cells by F3.3, glutinol, CP2 and F3.3.0. Inducible COX-2 and inducible nitric oxide synthase are among potent signalling enzymes that exacerbate inflammation. Bioactive sub-fractions (F3.3 and F3.3.0) derived from the n-hexane fraction of PA had good anti-inflammatory activity, and the isolated compound, and glutinol may be useful as a template for the development of new anti-inflammatory drugs.

Comparative analysis on immune response of combination with Astragali Radix and Cinnamomi Cortex

ABSTRACT Objective：Astragali Radix (AR) and Cinnamomi Cortex (CC) are used to enhance immune response in Asian traditional medicine. Immuno-potentiation of the combination of AR and CC were evaluated on the cellular and humoral immune response using murine macrophage cell line (RAW 264.7) and OVA-immunized mice.Methods：This study was designed to investigate the immuno-potentiative effects of AR, CC, and AR with CC on nitric oxide synthesis in RAW 264.7 cells and proliferation and production levels of Intereukin-2 (IL-2) in mouse splenocytes. In addition, we evaluated the plasma-specific antibody responses and splenocyte proliferation on ovalbumin (OVA)-immunized mice treated with he rbal extracts. Results：Combination treatment with AR and CC increased nitric oxide syn thesis in RAW 264.7 cells and IL-2 level in splenocytes (p<0.001). Combination of AR and CC signif icantly enhanced the Concanavalin A- (Con A； T cell mitogen) and lipopolysaccharide-(LPS；B cell mitogen) induced splenocyte proliferation on the OVA-immunized mice. Combination of AR and CC also significantly enhanced plasma levels of OVA-specific IgG (p<0.01), IgG1 (p<0.05) and total IgM (p<0.01) compared wit h the OVA-immunized control group.Conclusion：These results suggest that combination of AR and CC could be us ed as therapeutic profile on activation of immune response.Key words：Astragali Radix, Cinnamomi Cortex, immune response, synergistic effect

Inhibitory effect of DCDC on lipopolysaccharide-induced nitric oxide synthesis in RAW 264.7 cells

In the present study we have examined the effect of DCDC (2′, 5′, -dihydroxy-4-chloro-dihydrochalcone) on lipopolysaccharide (LPS)-induced responses in murine macrophage cell line RAW 264.7. Exposure of LPS-stimulated cells to DCDC inhibited the nitrite accumulation in culture medium. DCDC also concentration-dependently inhibited LPS-stimulated increase of iNOS expression; however, it had little effect on iNOS enzyme activity, suggesting that the inhibitory action to DCDC is mainly due to the inhibition on iNOS expression rather than iNOS enzyme activity. DCDC significantly inhibited LPS-evoked degradation of IκB-α and the nuclear translocation of NF-κB; it also exhibited the activity of scavenging the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). DCDC also inhibited cyclooxygenase-2 activity in RAW 264.7 cells with an IC 50 of 3.0 μM; furthermore, it also significantly decreased LPS-induced mortality rate in mice. Taken together, we demonstrate that DCDC exhibits inhibitory effects on nitric oxide production through the inhibition of IκB-α degradation and NF-κB activation, and therefore the suppression of iNOS expression. DCDC also shows the antioxidant activity and COX-2 inhibitory action. Moreover, it improves survival in a murine model of endotoxaemia suggesting that DCDC may be potential in the therapy of septic shock.

Structural requirements of sesquiterpene lactones to inhibit LPS-induced nitric oxide synthesis in RAW 264.7 macrophages

Some sesquiterpene lactones were recently demonstrated to inhibit inducible nitric oxide synthase (iNOS)-dependent nitric oxide (NO) synthesis. The primary objective of the present study was, therefore, to find evidence for structural requirements of sesquiterpene lactones regarding their capability to inhibit iNOS-dependent NO synthesis. Sesquiterpene lactones 1– 11 were examined for their influence on nitrite accumulation in cell culture supernatants of LPS-induced RAW 264.7 macrophages. Except the taraxinic acid β- d-glucopyranosylester 8 all compounds showed a dose-dependent inhibition of nitrite accumulation in cell culture supernatants with IC 50 values ranging from 0.5 to 36.8 μM. High activity seemed to be dependent on an α-methylene-γ-lactone functionality. Cytotoxicity and the ability to inhibit activation of transcription factor NF-κB are further biological activities of sesquiterpene lactones. The second point of interest was, therefore, whether the structural requirements of sesquiterpene lactones for these activities may differ or be the same for those needed to inhibit iNOS-dependent NO synthesis. Using concentrations of 1– 11 required to inhibit NO synthesis cell viability was determined and NF-κB binding activity was measured by gel-shift experiments. Interestingly, compounds almost equally effective in inhibiting nitrite accumulation did not show the same cytotoxic potential, and most sesquiterpene lactones inhibited nitrite accumulation at concentrations where inhibition of NF-κB activation was not significant. These results suggest that different biological activities of sesquiterpene lactones have different structural requirements.

Mechanisms of suppression of inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells by andrographolide.

Andrographolide, an active component found in leaves of Andrographis paniculata, has been reported to exhibit nitric oxide (NO) inhibitory property in endotoxin-stimulated macrophages, however, the detailed mechanisms remain unclear. In the present study we investigated the effect of andrographolide on the expression of inducible NO synthase (iNOS) mRNA, protein, and enzyme activity in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). RAW 264.7 cells stimulated with LPS/IFN-gamma activated NO production; in this condition andrographolide (1-100 microM) inhibited NO production in a dose-dependent manner with an IC(50) value of 17.4+/-1.1 microM. Andrographolide also reduces the expression of iNOS protein level but without a significant effect on iNOS mRNA. The reduction of iNOS activity is thought to be caused by decreased expression of iNOS protein. In a protein stability assay, andrographolide moderately but significantly reduced the amount of iNOS protein as suggested by accelerating degradation. Furthermore, andrographolide also inhibited total protein de novo synthesis as demonstrated by [(35)S]-methionine incorporation. As a whole, these data suggest that andrographolide inhibits NO synthesis in RAW 264.7 cells by reducing the expression of iNOS protein and the reduction could occur through two additional mechanisms: prevention of the de novo protein synthesis and decreasing the protein stability via a post-transcriptional mechanism. It is also possible that inhibition of iNOS protein expression and NO production under immune stimulation and/or bacteria infection may explain, in part, the beneficial effects of andrographolide as an anti-inflammatory agent.