Griess Reagent Assay Research Articles

Simultaneous determination of liquiritin, hesperidin, and glycyrrhizin by HPLC-photodiode array detection and the anti-inflammatory effect of Pyungwi-san

A high-performance liquid chromatographic method was developed and validated to determine liquiritin, hesperidin, and glycyrrhizin levels in a traditional Korean medicine, Pyungwi-san (PWS). Reverse-phase chromatography using a C18 column operating at 40oC, and photodiode array detection at 254 nm and 280 nm, were used for quantification of the three marker components of PWS. The mobile phase using gradient flow consisted of two solvent systems. Solvent A was 1.0% (v/v) aqueous acetic acid and solvent B was acetonitrile with 1.0% (v/v) acetic acid. Calibration curves were acquired with r (2) > 0.9999, and the relative standard deviation values (%) for intra- and inter-day precision were both less than 4.0%. The recovery of each compound was in the range 97.33-110.72%, with an relative standard deviation less than 6.0%. To provide information on the biological activity of PWS, anti-inflammatory action was evaluated. Production of nitric oxide and prostaglandin E(2) were measured using the Griess reagent and enzyme-linked immunosorbent assay, respectively. PWS showed inhibitory effect on prostaglandin E(2) production in LPS-treated RAW 264.7 cells.

Antioxidative and Anti-inflammatory Effects of Extracts from Different Organs of Cirsium japonicum var. ussuriense

Objective: The roots, leaves, flowers, stems and seeds of Cirsium japonicum var. ussuriense are often used in treatment of human diseases such as hemorrhage, blood congestion and inflammation. Focusing our attention on natural and bioavailable sources of antioxidants and anti-inflammation, we undertook to investigate the antioxidant and anti-inflammatory properties of Cirsium japonicum var. ussuriense used as a folk medicine in Korea. Methods: The extracts of the leaves, stems, flowers, seeds and roots from C. japonicum var. ussuriense were prepared by extracting with water or 80% ethanol. Total flavonoids and polyphenols were measured by a colorimetric assay. The free radical scavenging activity of the extract was analyzed by the DPPH (1,1-diphenyl-2-picryl hydrazyl), ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and Griess reagent assay. An oxidative product of nitric oxide (NO), was measured in the culture medium by the Griess reaction. The level of prostaglandin () was measured by enzyme-linked immunosorbent assay. The expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were measured by Western blot analysis. Results: Total flavonoid and polyphenol amounts of the leaves (CLE) and flowers (CFE) showed higher than those of the seed extract (CSE), stem extract (CSTE) and roots (CRE). CLE and CFE also showed the high antioxidant activities such as DPPH, NO-like and ABTS radical scavenging activity. An antioxidant activities of these water extracts showed higher than those of 80% ethanol extracts. We investigated the anti-inflammatory effects of CLE on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. CLE significantly suppressed the levels of the inflammatory mediators such as NO and prostaglandin () in dose dependant. Furthermore, the levels of iNOS and COX-2 protein expressions were markedly suppressed by the treatment with CLE extract in a dose dependent manner. Conclusions: These results suggest that CLE water extract has a higher anoxidant and anti-inflammatory activity, these properties may contribute to the oxidative and inflammatory related disease care.

Studies on Immuno modulating Acitivity of Fermented Sophorae Radix Extract

ABSTRACT Objectives：This study aims at examining the effect of the fermentative extract of root of Sophorae Radix on the immuno-modulating activity.Methods：Cell viabilities were measured by MTT assay. Effect of SFS on nitric oxide(NO), hydrogen peroxide production from RAW 264.7 cells was accessed by Griess reagent assay. Effect of SFS on productions of inflammatory cytokines such as TNF-α, IL-6 in LPS-induced RAW 264.7 cells was accessed by a multiplex bead array assay based on xMAP technology.Results：The results of the experiment are as follows.1. As a result of carrying out MTT assay to check the cellular toxicity of the fermentative extract of Sophorae Radix. There was not any excessive toxicity to the macrophage when the fermentative extract of root of Sophorae Radix was treated in different concentrations. 2. The fermentative extract of Sophorae Radix increased the generation of hydrogen peroxide in the macrophage and significantly restored the suppression of the generation of the hydrogen peroxide in the macrophage induced by LPS. 3. The fermentative extract of Sophorae Radix reduced the gener ation of NO in the macrophage and significantly suppressed the increase of the generation of NO in the macrophage induced by LPS. 4. The fermentative extract of Sophorae Radix significantly dec reased the amount of TNF-α generated in the macrophage induced by LPS when it was 25 ㎍/mL or higher.Conclusion：These results suggest that SFS has anti-inflammatory moiety rel ated with its inhibition of NO, hydrogen peroxide, TNF-α, IL-6, in macrophage led by LPS.Key words：Sophorae Radix, fermentation, immuno-modulating activity, react ive oxygen species(ROS)

Ginkgo biloba extract suppresses endotoxin-mediated monocyte activation by inhibiting nitric oxide- and tristetraprolin-mediated toll-like receptor 4 expression

Monocytes expressing toll-like receptor 4 (TLR4) play a major role in regulating the innate immune response and are involved in systemic inflammation. Previous studies have shown that Ginkgo biloba extract (GBE) may act as a therapeutic agent for some cardiovascular and neurological disorders. The objective of this study was to determine whether GBE could modulate immunity in human cells. The monocytic cell line THP-1 was used. Enzyme-linked immunosorbent assay results showed that lipopolysaccharide (LPS) induces the expression of monocyte chemotactic protein-1 (MIP-1), tumor necrosis factor-α, stromal cell-derived factor-1, and MIP-1α, and this induction may be repressed by GBE treatment due to TLR4 blockade. The Griess reagent assay and western blot analysis showed that GBE-mediated inhibition of TLR4 expression was associated with the activation of mitogen-activated protein kinase and production of nitric oxide (NO). Actinomycin D chase experiments demonstrated that GBE decreased the TLR4 mRNA stability in cells. Confocal microscopy and real-time polymerase chain reaction showed that GBE induced the expression of intracellular tristetraprolin (TTP). Transfection with TTP siRNA reversed the effects of GBE in naïve or TLR4-overexpressing cells. Treatment with SNAP (an NO donor) may increase intracellular TTP expression in cells. Immunoprecipitation analysis showed that GBE mediates TTP activation and increases the interaction of TTP with the 3′ untranslated region (UTR) of TLR4 mRNA by regulating NO production. Our findings indicate that GBE could decrease the sensitivity of monocytes to LPS. Utilizing TTP to control TLR4 expression may be a promising approach for controlling systemic inflammation, and GBE may have potential applications in the clinical treatment of immune diseases.

Anti-inflammatory Effects of Fermented Houttuyniae Herba Water Extract on LPS-induced Mouse Macrophage

Objectives : The purpose of this study is to investigate the effect of Fermented Houttuyniae Herba Water Extract (HL) on production of proinflammatory mediators in mous e macrophage RAW 264.7 cells.Methods : Cell viabilities were measured by MTT assay. Effect of HL on nitric oxide (NO) production from RAW 264.7 cells was accessed by Griess reagent assay. Effect of HL on productions of inflammatory cytokines such as interleukine (IL)-17, Interferon γ-inducible protein (IP)-10 , Eotaxin, IL-5, Monocyte Chemotactic Protein-3 (MCP-3), and IL-13 in LPS-induced RAW 264.7 cells was accessed by a multiplex bead array assay based on xMAP technology.Results : The results of the experiment are as follows.1.Incubation with HL for 24 hours showed significant increase i n cell viability of RAW 264.7 mouse macrophages (P < 0.05).2.HL showed to inhibit NO production from RAW 264.7 cells at the concentrations of 25 and 50 ug/mL significantly (P < 0.05). 3.HL inhibited significantly NO production in LPS-induced RAW 264.7 cells at the concentrations of 25, 50, 100 and 200 ug/mL (P < 0.05).4.HL inhibited significantly IL-17, IP-10 and Eotaxin in LPS-in duced RAW 264.7 cells at the concentrations of 25, 50, 100 and 200 ug/mL (P < 0.05).Conclusions : These results suggest that HL has anti-inflammatory moiety related with its inhibition of NO, IL-17, IP-10, and Eotaxin in macrophages.Key words : Houttuyniae Herba, Macrophage, Nitric oxide, IL-17, Eotaxin, IP-10.

유산균발효애엽이 독성물질들로 유발된 대식세포의 일산화질소생성 감소에 미치는 영향

Objectives : The purpose of this study is to investigate the effect of Water Extract from Lactobacillus pentosus-fermented ARTEMISIAE ARGI FOLIUM (AFL) on nitric oxide production of mouse macrophage Raw 264.7 cells impaired by various toxicants such as gallic acid, EtOH, nicotine, acetaminophen, and acetaldehyde. Methods : ARTEMISIAE ARGI FOLIUM was fermented with Lactobacillus pentosus and extracted by water. Nitric oxide production of mouse macrophage Raw 264.7 cells was measured by Griess reagent assay. Examined concentrations of AFL were 10, 50, 100, 200, 400 ug/mL. Results : The results of the experiment are as below. 1. AFL at the concentration of 400 ug/mL significantly recovered nitric oxide production which was reduced by gallic acid (100 uM) in Raw 264.7 cells. 2. AFL at the concentration of 200, 400 ug/mL significantly recovered nitric oxide production which was reduced by EtOH (100 uM) in Raw 264.7 cells. 3. AFL at the concentration of 400 ug/mL significantly recovered nitric oxide production which was reduced by nicotine (1mM) in Raw 264.7 cells. 4. AFL at the concentration of 200, 400 ug/mL significantly recovered nitric oxide production which was reduced by acetaminophen(2 mM) in Raw 264.7 cells. 5. AFL at the concentration of 200, 400 ug/mL significantly recovered nitric oxide production which was reduced by acetaldehyde (200 uM) in Raw 264.7 cells. Conclusions : AFL could be supposed to have the immune-enhancing activity related with nitric oxide production of macrophage impaired by various toxicants.

In vitro and in vivo immunomodulatory activity evaluation of Bacopa monniera, Scrophulariaceae

Bacopa monniera, also referred to as Bacopa monnieri, Herpestis monniera, water hyssop, and „Brahmi,“ (family Scrophulariaceae) has been used in the Ayurvedic system of medicine for centuries [1]. Aerial parts of Bacopa monniera were extracted with distilled water and then successively with methanol. Methanol extract was evaluated for immunomodulatory activity using in vitro and in vivo methodologies. Effect of Bacopa extract (832–6.5µg/ml) on secretion of mediators like nitric oxide (NO) and superoxide on isolated murine peritoneal macrophages were evaluated by using Griess reagent and nitro blue tetrazolium (NBT) dye reduction assay respectively. The extract showed significant (P&lt;0.05) stimulation of release of NO at 416µg/ml (SI, stimulation index=1.67) and 208µg/ml (SI=2.14) (P&lt;0.05). NBT reduction were significant at 52µg/ml (SI=1.34) as compared to control (SI=1). The extract was also evaluated for in vivo phagocytic activity by carbon clearance assay in mice and it showed significant increase in the phagocytic index (K) at 100 (K=0.104) and 200mg/kg (K=0.116) as compared to control (K=0.062). The extract significantly (P&lt;0.05) enhanced total leucocytes count at 50mg/kg (10.82×103cells/cmm) and for 100mg/kg (11.93×103cells/cmm) of cyclophosphamide induced myelosuppression (6.02×103cells/cmm) in mice (n=6). Data was analyzed by one way ANOVA followed by Dunnet's multiple comparisons test. The results suggest that Bacopa extract influences secretion of mediators in vitro and phagocytosis in vivo.

Valsartan regulates the interaction of angiotensin II type 1 receptor and endothelial nitric oxide synthase via Src/PI3K/Akt signalling

Valsartan, a selective angiotensin II type 1 receptor (AT1R) blocker, has beneficial effects in the cardiovascular system in part by its increase of nitric oxide (NO) bioavailability, yet the mechanisms are unclear. We investigated the molecular mechanisms underlying this effect in endothelial cells (ECs). NO production was examined by Griess reagent assay, DAF-2 DA fluorescence staining and cGMP ELISA kits. Protein interaction was determined by western blotting and immunoprecipitation. Treating bovine or human aortic ECs with valsartan increased NO production, as evidenced by elevated level of stable NO metabolites and intracellular cGMP. Valsartan increased the phosphorylation but not the protein level of endothelial NO synthase (eNOS). Inhibition of phosphoinositide-3 kinase (PI3K)/Akt and Src pathways by specific inhibitors suppressed valsartan-induced NO release. In addition, valsartan increased the tyrosine residue phosphorylation of AT1R, which was attenuated by inhibition of Src but not PI3K activities. Valsartan also suppressed the interaction of eNOS and AT1R, which was blocked by Src or PI3K inhibition. Valsartan-induced NO production in ECs is mediated through Src/PI3K/Akt-dependent phosphorylation of eNOS. Valsartan-induced AT1R phosphorylation depends on Src but not PI3K, whereas valsartan-induced suppression of AT1R-eNOS interaction depends on Src/PI3K/Akt signalling. These results indicate a novel vasoprotective mechanism of valsartan in upregulating NO production in ECs.

Role of Protease-Activated Receptor-1 in Endothelial Nitric Oxide Synthase-Thr495 Phosphorylation

Protease activated receptors (PARs) are G protein-coupled receptors that are known to regulate endothelial nitric oxide synthase (eNOS) activity in part by phosphorylating the enzyme at various sites. Ser1177 is a positive regulatory site, which leads to the enhanced production of nitric oxide (NO), a vasodilator of arteries. Thr495 is a negative regulatory site, which inhibits NO production. We have shown that thrombin, a PAR agonist, mediates eNOS-Ser1177 phosphorylation through Gq and a calcium and protein kinase C (PKC)-delta sensitive, but phosphatidylinositol 3-kinase (PI3K)/Akt-independent pathway. However, the mechanism for eNOS-Thr495 phosphorylation by PAR agonists is unknown. We used a specific synthetic PAR-1 activating peptide, TFLLR, and thrombin to assess the role of PAR-1 involvement in the phosphorylation of eNOS-Thr495 in human umbilical vein endothelial cells (HUVECs). Using Western blot analysis and the Griess Reagent assay, we found that both agonists phosphorylated Thr495 in a time- and dose-dependent manner and significantly decreased nitrite production, respectively. Pretreatment of cells with the PAR-1 inhibitor, SCH-79797, resulted in a significant decrease in thrombin- and TFLLR-induced phosphorylation of eNOS-Thr495 and an increase in nitrite production. We further demonstrated that inhibition of Rho with C3 exoenzyme or dominant negative (dn) RhoA, and inhibition of Rho-Kinase (ROCK) with Y-27632 caused a significant decrease in thrombin and TFLLR-induced Thr495 phosphorylation. Blockade of the Rho/ROCK pathway also caused an increase in nitrite production. This suggests that PAR-1 regulates eNOS activity via phosphorylation of eNOS-Thr495, which is dependent upon activation of the Rho/ROCK pathway. These findings will be beneficial in further understanding the signaling pathways that regulate eNOS-induced NO production, which plays an important role in endothelial dysfunction associated with cardiovascular disease.

Immunomodulatory effects of beta‐glucan from Coriolus versicolor on the phagocytosis of macrophages: involvement of SR‐B1 expression

Coriolus versicolor has been known to have effective immunomodulatory effects in Asia. Most of these studies have demonstrated that it appears to have significant antimicrobial, antiviral and antitumor properties when used as a supplement to chemotherapy and/or radiotherapy. To further understand the immunomodulatory effects of beta‐glucan, we examined tumoricidal activity, phagocytosis and nitric oxide (NO) production in beta‐glucan‐treated Raw264.7 cells. Our data showed that the treatment of beta‐glucan from C. versicolor induced a remarkable increase of phagocytosis and tumoricidal activity by macrophages. In addition, reverse transcription‐polymerase chain reaction (RT‐PCR) and Griess Reagent assay showed the increase of iNOS mRNA expression and slight induction of NO production at 1ug/ml concentration. We also examined whether SR‐A1 and SR‐B1 are involved in phagocytosis. The present data demonstrated that mRNA expression of SR‐A1 and SR‐B1 was increased respectively. In addition, the increase of SR‐B1 protein expression was observed. Moreover, beta‐glucan treatment resulted in increase of the expression of CK2 and Ikaros in Raw264.7 cells. Taken together, these results suggest that beta‐glucan from C. versicolor enhances phagocytosis via SR‐B1 expression and immunomoulatory effects of beta‐glucan might be related to CK2 and ikaros activation.

Platycodin D and D3 isolated from the root of Platycodon grandiflorum modulate the production of nitric oxide and secretion of TNF-α in activated RAW 264.7 cells

Platycodon D (PD) and D3 (PD3) isolated from Platycodon grandiflorum has been previously reported to show anti-inflammatory activities in rats. In this study, the production of proinflammatory cytokines, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) was examined in a macrophage like cell line, RAW 264.7 cells, in the presence of PD and PD3, oligosaccharide derivatives of oleanolic acid. RAW 264.7 cells activated with lipopolysaccharide (LPS; 1 μg/ml) and recombinant interferon-γ (rIFN-γ; 50 U/ml) were treated with various doses of PD and PD3 for 24 h. Supernatants were analyzed for the production of NO and TNF-α using Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. NO was inhibited in a dose-dependent manner by PD and PD3 (IC 50 of platycodin D≈15 uM, IC 50 PD3≈55 uM). The expression of inducible NOS (iNOS) was inhibited by these compounds, as measured by Western blot analysis, as well as the expression of iNOS mRNA, as measured by Northern blot analysis. RAW 264.7 cells were treated at various times after LPS and activation with PD. Treatment with PD up to 8 h after activation showed significant inhibition of NO, indicating that early signal transduction of NOS synthesis may be inhibited by PD. In contrast to NO, secretion of TNF-α as well as expression of TNF-α mRNA was increased by PD and PD3. TNF-α secretion from RAW 264.7 cells was measured at various times after LPS and rIFN-γ activation. Secretion of TNF-α was also increased up to 8 h postactivation, suggesting that PD may stimulate TNF-α synthesis or inhibit degradation of TNF-α mRNA. Oleanolic acid was without effect on both the production of NO and secretion of TNF-α. These data suggest a dichotomous regulation of these important proinflammatory mediators by PD and PD3.

Enhancement of Nitric Oxide Production by Corticotropin-releasing Hormone (CRH) in Murine Microglial Cells, BV2

Background: Microglial cells, major immune effector cells in the central nervous system, become activated in neurodegenerative disorders. Activated microglial cells produce proinflammatory mediators such as nitric oxide (NO), tumor necrosis factor- and interleukin-(IL-). These proinflammatory mediators have been shown to be significantly increased in the neurodegenerative disorders such as Alzhimer's disease and Pakinson's disease. It was known that one of the neurodegeneration source is stress and it is important to elucidate mechanisms of the stress response for understanding the stress-related disorders and developing improved treatments. Because one of the neuropeptide which plays a main role in regulating the stress response is corticotropin-releasing hormone (CRH), we analyzed the regulation of NO release by CRH in BV2 murine microglial cell as macrophage in the brain. Methods: First, we tested the CRH receptor expression in the mRNA levels by RT-PCR. To test the regulation of NO release by CRH, cells were treated with CRH and then NO release was measured by Griess reagent assay. Results: Our study demonstrated that CRH receptor 1 was expressed in BV2 murine microglial cells and CRH treatment enhanced NO production. Furthermore, additive effects of lipopolysaccaride (LPS) and CRH were confirmed in NO production time dependantly. Conclusion: Taken together, these data indicated that CRH is an important mediator to regulate NO release on microglial cells in the brain during stress.