• Journal of Life Science
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• v.27 no.6
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• pp.640-645
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• 2017

Clerodendrum trichotomum (CT) leaves and stems have been used in folk medicine for their anti-hypertension, arthritis, rheumatism, and anti-inflammatory properties. This study was performed to evaluate the potential of CT as an anti-oxidant and anti-inflammatory agent. CT leaves were extracted using 70% ethanol (EtOH). Then, using this extract, a hexane, chloroform ($CHCl_3$), ethyl acetate (EtOAc), and n-butanol (BuOH) fraction was prepared. The polyphenol contents were higher in the EtOAc fraction ($78.08{\mu}g/mg$) and BuOH fraction ($77.54{\mu}g/mg$) compared to the other fractions. Also, these two fractions exhibited strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activities. Xanthine oxidase inhibitory activities were higher in the $CHCl_3$ fraction ($IC_{50}=4.43{\mu}g/ml$) and EtOAc fraction ($IC_{50}=5.69{\mu}g/ml$). Moreover, the EtOAc fraction effectively inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells ($IC_{50}=18.87{\mu}g/ml$). Thus, we investigated the effects of the EtOAc fraction on the expression of pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 cells. The treatment of the EtOAc fraction ($100{\mu}g/ml$) effectively decreased the levels of the tumor necrosis factor ${\alpha}$ ($TNF-{\alpha}$) and interleukin-6 (IL-6), and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). These results suggest the potential for CT extract and fractions as promising anti-oxidant and anti-inflammatory agents.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.4
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• pp.343-351
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• 2001

This study was undertaken to investigate the mechanism of lipopolysaccharide (LPS) and nitric oxide (NO) as a regulator of vascular smooth muscle cell (VSMC) proliferation. VSMC was primarily cultured from rat aorta and confirmed by the immunocytochemistry with anti-smooth muscle myosin antibody. The number of viable VSMCs were counted, and lactate dehydrogenase (LDH) activity was measured to assess the degree of cell death. Concentrations of nitrite in the culture medium were measured as an indicator of NO production. LPS was introduced into the medium to induce the inducible nitric oxide synthase (iNOS) in VSMC, and Western blot for iNOS protein and RT-PCR for iNOS mRNA were performed to confirm the presence of iNOS. Inhibitors of iNOS and soluble guanylate cyclase (sGC), sodium nitroprusside (SNP) and L-arginine were employed to observe the action of LPS on the iNOS-NO-cGMP signalling pathway. LPS and SNP decreased number of VSMCs and increased the nitrite concentration in the culture medium, but there was no significant change in LDH activity. A cell permeable cGMP derivative, 8-Bromo-cGMP, decreased the number of VSMCs with no significant change in LDH activity. L-arginine, an NO substrate, alone tended to reduce cell count without affecting nitrite concentration or LDH level. Aminoguanidine, an iNOS specific inhibitor, inhibited LPS-induced reduction of cell numbers and reduced the nitrite concentration in the culture medium. LY 83583, a guanylate cyclase inhibitor, suppressed the inhibitory actions of LPS and SNP on VSMC proliferation. LPS increased amounts of iNOS protein and iNOS mRNA in a concentration-dependent manner. These results suggest that LPS inhibits the VSMC proliferation via production of NO by inducing iNOS gene expression. The cGMP which is produced by subsequent activation of guanylate cyclase would be a major mediator in the inhibitory action of iNOS-NO signalling on VSMC proliferation.

• Journal of the Korean Society of Food Science and Nutrition
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• v.40 no.11
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• pp.1507-1511
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• 2011

This study investigated the anti-inflammatory effect of Erigeron annuus L. flower (EAF) methanol extract. We examined the involvement of heme oxygenase-1 (HO-1) in the inhibitory activities of EAF methanol extract on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Cell viability and NO assays were performed. In addition, inducible nitric oxide synthase (iNOS) and HO-1 expressions were detected by Western blotting and blocking HO-1 activity on NO production. EAF methanol extract (25, 50, 100, 200 ${\mu}g$/mL) significantly inhibited LPS-stimulated NO production (p<0.05; 12.82, 9.61, 6.83, 2.52 ${\mu}m$) in a concentration-dependent manner. EAF methanol extract also reduced the expression of iNOS protein. The EAF methanol extract induced the expression of HO-1 in a dose-dependent manner. Blockage of HO-1 activity by zinc protoporphyrin suppressed EAF methanol extract-induced reductions in the production of NO. The present results suggest that EAF methanol extract has a potent anti-inflammatory effect in RAW264.7 macrophages through the induction of HO-1.

• Journal of Trauma and Injury
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• v.18 no.1
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• pp.53-63
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• 2005

Purpose: This study was designed to determine if methylene blue inhibited the lipid peroxidation, the production of NO, and the gene expression of iNOS in acute lung injury induced by paraquat and if the inhibitory effect was dose dependent. Methods: Female Sprague-Dawley rats were divided into four groups: the control group, the group treated with paraquat only, the group treated with paraquat and a low dose of methylene blue (2 mg/kg), and the group treated with paraquat and a high dose of methylene blue (20 mg/kg). Methylene blue was administered via the jugular vein 1 h after paraquat administration, and animals were sacrificed 6 and 24 h after paraquat administration. Malondialdehyde (MDA) as lipid peroxidation, reduced glutathione (GSH) as an antioxidant defense, the plasma NO concentration, and the expression of iNOS mRNA in the lung tissue were measured Results: Lung MDA contents decreased, with no significant difference between the methylene-blue groups and the paraquat-only group. Lung GSH contents were significantly elevated at 24 h in the methylene-blue groups compared with the paraquat-only group. Plasma NO concentrations were significantly reduced at 6 and 24 h in the methylene-blue groups compared with the paraquat-only group. There was also a significant decrease in the plasma NO concentration at 6 h in the high-dose methylene-blue group compared with the low-dose methylene-blue group. The expression of iNOS mRNA in the lung tissue was slightly decreased in the methylene-blue groups. It was also markedly increased at 24 h in the paraquat-only group compared with the methylene-blue groups. The gene expression was relatively decreased in the high-dose methylene-blue group compared with the low-dose methylene-blue group. Conclusion: This study suggests that methylene blue has an inhibitory effect on the plasma NO concentration and the expression of iNOS mRNA in lung injury induced by paraquat. No inhibitory effect of methylene blue on lipid peroxidation or dose-dependent inhibitory effects were clearly shown.

• Journal of Yeungnam Medical Science
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• v.13 no.2
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• pp.159-172
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• 1996

대기오염물질이면서 동시에 생체내 화학반응의 산물이기도 한 nitric oxide(NO)는 그 생체내 분포가 광범위하고 생리적 역할이 다양하여, 최근의 생명과학 분야에서 가장 크게 주목받는 몇가지 연구대상 중 하나이다. 세포에서의 NO 산생은 nitric oxide synthase (NOS)에 의해 촉매되는데, 이들은 brain form (bNOS, neuronal; nNOS, NOS I), inducible form (iNOS), 및 endothelial form(eNOS)로 구분되는데, 이중 bNOS(nNOS)와 eNOS는 inducible form에 대비되는 constitutive form(cNOS)에 해당하므로 각각 ncNOS 와 ecNOS로도 불리운다. NOS는 아미노산인 L-arginine을 산소와 결합시켜 L-citrulline으로 변환시키면서 NO를 유리하고, 이 NO는 세포내의 guanylate cyclase를 활성화하여 cyclic GMP를 생산하거나, superoxide(O2-) 및 수소이온과 차례로 결합하여 반응성이 매우 높은 수산화기(-OH)를 발생시켜 세포독작용을 유발하기도 한다. 정상상태에서 뇌혈관내피세포의 ecNOS로 부터 유리된 NO는 혈관을 확장시켜 신경세포에 대한 산소공급을 원활히 유지해 주지만, 순환장애를 일으켰을 때는 뇌조직내의 iNOS로부터 대량의 NO가 유출되어 신경세포의 손상을 가져온다. 호흡기에서는 NO가 기도평활근을 이완시키고 폐혈류를 개선하므로, 미숙아나 성인의 호흡장애시에 소량의 NO를 흡입시키면 oxygenation을 호전시킬 수 있다. 그러나 대기오염이나 흡연 등으로 대량의 NO를 흡입할 경우 치명적인 폐부종이나 methemoglobin혈종을 일으킬 수 있다. 순환계에서는 cNOS가 혈관을 확장시켜 조직의 혈류를 유지하는데 일익을 담당한다. 세균내 독소(lipopolysaccharide; LPS)나 각종 명역조절물질들이 혈관내피세포와 혈관평활근세포로 부터 과다한 NO를 유리시키면 혈압이 급격히 떨어져 순환허탈상태에 빠지게 된다. 심장에서는 관상혈관 내피세포의 eNOS가 심근의 혈류를 유지해 주지만 허혈이나 세균내독소 또는 면역조절물질 등에 의하여 심근세포나 침윤된 대식세포의 iNOS로 부터 과량의 NO가 유리되면 심근세포의 손상이 초래된다. 신장에서는 내피세포의 cNOS에 의하여 사구체여과가 조절되고 있는데, 세균내독소나 면역 조절물질 등에 의하여 사구체관막세포(mesangial cell)등의 iNOS로 부터 과량의 NO가 유리되면 신조직과 사구체의 손상을 초래한다. 위와 같이 대부분의 장기에서 ecNOS는 조직의 혈류를 유지하는 역할을 하며, iNOS는 애초 세균 등 침입자에 대한 세포독작용이 그 존재 목적이라고 풀이할 수 있겠으나 일종의 부작용으로 자체조직의 손상을 초래하게 되는 것으로 본다. 따라서 NO와 관련된 각종 병변의 치료를 위해서는 NOS의 비선택성 억제제인 arginine 유도체 보다는 iNOS에 대한 선택적 억제제인 S-methylisothiourea(SMT), aminoethylisothiourea(AETU), aminoguanidine (AMG), agmatine, L-canavanine, transforming growth factor b1(TGF-b1) 등의 사용을 검토해 보는 것이 타당할 것으로 사료된다.

• Journal of Ginseng Research
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• v.37 no.1
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• pp.54-63
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• 2013

Ginsenoside Rd is a primary constituent of the ginseng rhizome and has been shown to participate in the regulation of diabetes and in tumor formation. Reports also show that ginsenoside Rd exerts anti-oxidative effects by activating anti-oxidant enzymes. Treatment with ginsenoside Rd decreased nitric oxide and prostaglandin $E_2$ ($PGE_2$) in lipopolysaccharides (LPS)-challenged RAW264.7 cells and in ICR mouse livers (5 mg/kg LPS; LPS + ginsenoside Rd [2, 10, and 50 mg/kg]). Furthermore, these decreases were associated with the down-regulations of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 and of nuclear factor (NF)-${\kappa}B$ activity in vitro and in vivo. Our results indicate that ginsenoside Rd treatment decreases; 1) nitric oxide production (40% inhibition); 2) $PGE_2$ synthesis (69% to 93% inhibition); 3) NF-${\kappa}B$ activity; and 4) the NF-${\kappa}B$-regulated expressions of iNOS and COX-2. Taken together, our results suggest that the anti-inflammatory effects of ginsenoside Rd are due to the down-regulation of NF-${\kappa}B$ and the consequent expressional suppressions of iNOS and COX-2.

• Journal of Chest Surgery
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• v.32 no.2
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• pp.138-143
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• 1999

Background: The endothelium-dependent vasorelaxation has been largely accounted for by the release of nitric oxide (NO). Three distinct isoforms of NO synthases (NOS) have been characterized, i.e., brain(bNOS), inducible (iNOS), and endothelial constitutive (ecNOS). Although hypertension hasbeen associated with a vascular endothelial dysfunction, changes in the vascular expression of NOS isoforms have not been established. The present study was aimed at exploring the vascular expression of NOS isozymes in hypertension. Material and Method: Two-kidney, one clip (2K1C) and deoxycorticosterone acetate (DOCA)-salt hypertension were induced in rats. The expression of different NOS isozymes in the thoracic aorta was determined by Western blot analysis. The vascular tissue contents of nitrites were measured by colorimetric assay. Result: Arterial blood pressure was significantly higher in experimental groups of 2K1C and DOCA-salt rats compared with their corresponding control rats. The vascular expression of bNOS as well as that of ecNOS was decreased in both models of hypertension. iNOS was not changed in DOCA-salt hypertension, but was also decreased in 2K1C hypertension. The vascular contents of nitrites were significantly decreased in DOCA-salt as well as in 2K1C hypertension. Conclusion: These results suggest that 2K1C and DOCA-salt hypertension are associated with decreases in the vascular expression of NOS isozymes and nitrite contents.

• Biomolecules & Therapeutics
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• v.17 no.1
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• pp.70-78
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• 2009

Coptis japonica (C. japonica) is a perennial medicinal plant that has anti-inflammatory activity. C. japonica contains numerous biologically active alkaloids including berberine, palmatine, epi-berberine, and coptisine. The most well-known anti-inflammatory principal in C. japonica is berberine. For example, berberine has been implicated in the inhibition of iNOS induction by cytokines in microglial cells. However, the efficacies of other alkaloids components on microglial activation were not investigated yet. In this study, we investigated the effects of three alkaloids (palmatine, epi-berberine and coptisine) from C. japonica on lipopolysaccharide (LPS)-induced microglial activation. BV2 microglial cells were immunostimulated with LPS and then the production of several inflammatory mediators such as nitric oxide (NO), reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) were examined as well as the phosphorylation status of Erk1/2 mitogen activated protein kinase (MAPK). Palmatine and to a lesser extent epi-berberine and coptisine, significantly reduced the release of NO, which was mediated by the inhibition of LPS-stimulated mRNA and protein induction of inducible nitric oxide synthase (iNOS) from BV2 microglia. In addition to NO, palmatine inhibited MMP-9 enzymatic activity and mRNA induction by LPS. Palmatine also inhibited the increase in the LPS-induced MMP-9 promoter activity determined by MMP-9 promoter luciferase reporter assay. LPS stimulation increased Erk1/2 phosphorylation in BV2 cells and these alkaloids inhibited the LPS-induced phosphorylation of Erk1/2. The anti-inflammatory effect of palmatine in LPS-stimulated microglia may suggest the potential use of the alkaloids in the modulation of neuroinflammatory responses, which might be important in the pathophysiological events of several neurological diseases including Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD) and stroke.

• Journal of Life Science
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• v.25 no.12
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• pp.1377-1383
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• 2015

Scopoletin is a component of several plant such as Erycibe obtusifolia, Aster tataricus, Foeniculum vulgare and Brunfelsia grandiflora. It was reported to have anti-angiogenesis and anti-allergy effects. In this study, the anti-inflammatory effect of scopoletin was investigated in Raw264.7 cells, mouse macrophages. The effects of scopoletin on phagocytosis and nitric oxide (NO) production were investigated in lipopolysaccharide (LPS)-induced inflammatory responses. It was observed that scopoletin exerted inhibitory effects on both phagocytosis and NO production. In addition, scopoletin decreased the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) which were related to NO and prostaglandin E2 (PGE2) production. In particular, the expression of pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The expression levels of IL-1β, IL-6 were remarkably decreased by treatment with scopoletin. Furthermore, the content of TNFα produced by macrophage was decreased in the presence of scopoletin at 8 hr. These results indicate that the anti-inflammatory effect of scopoletin could exert by inhibiting the expression of pro-inflammatory cytokines in Raw264.7 cells stimulated with LPS. The above results suggest scopoletin could be a new remedial agent for anti-inflammation through inhibition of iNOS, COX-2, IL-1β, IL-6 and TNF-α expressions as well as supression of phagocytosis and NO production.

• Microbiology and Biotechnology Letters
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• v.45 no.1
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• pp.35-42
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• 2017

This study aimed to investigate the effects of probiotic fermentation by comparing the skin antioxidant and anti-inflammatory properties of non-fermented (ARE) and fermented (ARE-F) hot water extracts of Agastache rugosa leaves. ARE-F was obtained via ARE fermentation using Lactobacillus rhamnosus HK-9. In vitro, anti-inflammatory properties were evaluated by analyzing the levels of nitric oxide (NO), reactive oxygen species (ROS), and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated HaCaT keratinocytes. In vitro antiradical activity was measured using 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. Attenuation of LPS-stimulated NO (p < 0.01), ROS (p < 0.001) and iNOS (p < 0.05) levels by ARE-F was significantly stronger than that by ARE in HaCaT keratinocytes. However, no differences were observed between the DPPH radical scavenging activities of ARE and ARE-F. ARE-F possesses enhanced skin antioxidant and anti-inflammatory properties, suggesting that probiotic bacterial fermentation can be considered an effective tool for augmenting some pharmacological properties of A. rugosa leaves. In brief, the skin antioxidant and anti-inflammatory potentials of A. rugosa leaf extract are augmented by the fermentation with L. rhamnosus HK-9, a probiotic bacterium.