• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.13-13
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• 2018

The anti-inflammatory (INF) compounds (1-15) were isolated from Agrimonia pilosa Ledeb. (APL) by activity-guided isolation technique. The isolated compounds (1-15) were identified as quercetin-7-O-rhanmoside (1), apigenin-7-O-glycoside (2), kaempferol-7-O-glycoside (3), apigenin-7-O-[6"-(butyl)-glycoside] (4), querceitn (5), kaempferol (6), apigenin (7), apigenin-7-O-[6"-(pentyl)-glycoside] (8), agrimonolide (9), agrimonolide-6-O-glucoside (10), desmethylagrimonolide (11), desmethylagrimonolide-6-O-glucoside (12), luteolin (13), vitexin (14) and isovitexin (15). Flavonoids, compound 2, 3, 11, and 14-15 have been found in APL for the first time. Furthermore, two novel flavone derivatives, compound 4 and 8, have been isolated inceptively in plant. In the no cytotoxicity concentration ranges of $0-20{\mu}M$, nitric oxide (NO) production level of 1-15 was estimated in LPS-treated Raw 264.7 macrophage cells. The flavone aglycones, 7 (apigenin, $IC_{50}=3.69{\pm}0.34{\mu}M$), 13 (luteolin, $IC_{50}=4.62{\pm}0.43{\mu}M$), 6 (kaempferol, $IC_{50}=14.43{\pm}0.23{\mu}M$) and 5 (quercetin, $IC_{50}=19.50{\pm}1.71{\mu}M$), exhibited excellent NO inhibitory (NOI) activity in dose-dependent manner. In the structure activity relationship (SAR) study of apigenin-derivatives (APD), apigenin; Api, apigenin-7-O-glucoside; Api-G, apignenin-7-O-[6"-(butyl)-glycoside]; Api-BG and apignenin-7-O-[6"-(pentyl)-glycoside]; Api-P, from APL on INF activity was investigated. The INF mediators level such as NO, INF-cytokines, NF-KB proteins, iNOS and COX-2 were sharply increased in Raw 264.7 cells by LPS. When pretreatment with APD in INF induced macrophages, NOI activity of Api was most effective than other APD with $IC_{50}$ values of $3.69{\pm}0.77{\mu}M$. And the NOI activity was declined in the following order: Api-BG ($IC_{50}=8.91{\pm}1.18{\mu}M$), Api-PG ($IC_{50}=13.52{\pm}0.85{\mu}M$) and API-G ($IC_{50}=17.30{\pm}0.66{\mu}M$). The NOI activity of two novel compounds, Api-PG and Api-BG were lower than their aglycone; Api, but more effective than Api-G (NOI: Api-PG and Api-BG). And their suppression ability on INF cytokines such as $TNF-{\alpha}$, $IL-1{\beta}$ and IL-6 mRNA showed the similar tendency. Therefore, the anti-INF mechanism study of Api-PG and Api-BG on nuclear factor-kappa B ($NF-{\kappa}B$) pathway, representative INF mechanism, was investigated and Api was used as positive control. Api-BF was more effectively prevent the than phosphorylation of $pI{\kappa}B$ kinase (p-IKK) and p65 than Api-PG in Raw 264.7 cells. In contrast, Api-PG and Api-BG were not reduced the phosphorylation of inhibitor of kappa B alpha ($I{\kappa}B{\alpha}$). Moreover, pretreatment with Api-PG and Api-BG, dose-dependently inhibited LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNAs and proteins in macrophage cells, and their expression were correlated with their NOI activity. Therefore, APL can be utilized to health promote agent associated with their AIN metabolites.

• Journal of Life Science
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• v.33 no.6
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• pp.463-470
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• 2023

Desmarestia tabacoides Okamura is a brown macroalgae that is found worldwide. Although several genera of Desmarestia have been reported as having anti-tumorigenic, anti-melanogenic, and photoprotective properties, the anti-inflammatory activity of D. tabacoides Okamura has not yet been evaluated. In this study, we analyzed the anti-inflammatory mechanisms of D. tabacoides Okamura ethanol extract (DTEE) via the inhibition of nitric oxide (NO) and prostaglandin (PG) E₂ production and the expression of their corresponding enzymes, inducible NO synthase (iNOS), and cyclooxygenase (COX)-2. In addition, their upstream signaling molecules were evaluated by Western blot analysis, such as nuclear factor (NF)-κB, mitogen-activated protein kinase (MAPK), and phosphoinositide-3-kinase (PI3K)/Akt, in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The DTEE treatment significantly inhibited LPS-induced NO and PGE₂ production as well as the expression of their corresponding enzymes, iNOS, and COX-2 without cytotoxicity. The stimulated transcription factor NF-κB and upstream signaling molecules extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK), and p38 were attenuated by the DTEE treatment, which was statistically significant, while Akt did not provide any inhibitory effect. Moreover, the DTEE treatment significantly mitigated the LPS-activated adaptor molecules, toll-like receptor 4 (TLR4), and myeloid differentiation primary response 88 (MyD88) in the RAW 264.7 cells. These results suggest that DTEE attenuates TLR4-mediated inflammatory responses by inhibiting NF-κB activation and suppressing MAPK phosphorylation in LPS-stimulated RAW 264.7 cells.

• Journal of Sasang Constitution and Immune Medicine
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• v.22 no.4
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• pp.56-64
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• 2010

1. Objectives 4 herbal formulas (Yanggyeoksanhwa-tang, Yeoldahanso-tang, Cheongsimyeonja-tang and Taeeumjowi-tang) were applied to investigate the anti-inflammatory activities. In many studies, plant-derived anti-inflammatory efficacies have been investigate for their potential inhibitory effects on lipopolysaccharide (LPS)-stimulated macrophages. This study was performed to examine the anti-inflammatory activities of 4 herbal formulas on LPS-stimulated RAW 264.7 cells. 2. Methods The productions of nitric oxide (NO), prostaglandin (PG)$E_2$, interleukin(IL)-6 and tumor necrosis factor (TNF)-${\alpha}$ were examined in the presence of the 4 herbal formulas in RAW 264.7 cells. The cells were incubated with LPS 1 ${\mu}g/mL$ and 4 herbal formulas for 18 hrs. The anti-inflammatory activity of 4 herbal formulas were investigate by carrageenin-induced paw edema in rats. The paw volume was measured at 2 and 4 hrs following carrageenininduced paw edema in rats. 3. Results Yanggyeoksanhwa-tang and Cheongsimyeonja-tang showed inhibitory effect on $PGE_2$ production in LPS-stimulated RAW 264.7 cells and a reduction in carrageenin-induced paw edema on rats. Yanggyeoksanhwa-tang showed inhibitory effect on IL-6 in LPS-stimulated RAW 264.7 cells. 4 herbal formulas not affect on NO and TNF-${\alpha}$ inhibition in LPS-stimulated RAW 264.7 cells. 4. Conclusions These results suggested that Yanggyeoksanhwa-tang and Cheongsimyeonja-tang have anti-inflammatory activity.

• The Journal of Korean Medicine
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• v.31 no.3
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• pp.47-54
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• 2010

Objective: To derive information on the efficacy of Sam-chul-kun-bi-tang (SKT), by evaluating its anti-inflammatory effect. SKT is a widely-used herbal formula in traditional Korean medicine. In man y studies, plant-derived anti-inflammatory efficacies have been investigated for their potential inhibitory effects on lipopolysaccharide (LPS)-stimulated macrophages. This study was performed to examine the anti-inflammatory effects of SKT extract on LPS-stimulated RAW 264.7 cells. Methods: The production of nitric oxide (NO), prostaglandin $(PG)E_2$, tumor necrosis factor (TNF)-$\alpha$ and interleukin (IL)-6 were examined in a macrophage cell line, RAW 264.7 cells, in the presence of SKT. RAW 264.7 cells were incubated with LPS 1 ${\mu}g/mL$ and SKT for 18 hrs. The anti-inflammatory activity of SKT was investigated by carrageenan-induced paw edema in rats. The paw volume was measured at 2 and 4 hrs following carrageenan-induced paw edema in rats. Results: SKT showed inhibitory effect on $PGE_2$, TNF-$\alpha$ and IL-6 in LPS-stimulated RAW 264.7 cells. But SKT was not inhibitory effect on NO by LPS-stimulated RAW 264.7 cells. Administration of SKT (1 g/kg) also showed a reduction in carrageenan-induced paw edema on rats. Conclusion: These results suggest that SKT has anti-inflammatory activities in both in vitro and in vivo models.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.3
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• pp.217-222
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• 2013

We reported that ailanthoidol, a neolignan from Zanthoxylum ailanthoides and Salvia miltiorrhiza Bunge, inhibited inflammatory reactions by macrophages and protected mice from endotoxin shock. We examined the anti-inflammatory activity of six synthetic ailanthoidol derivatives (compounds 1-6). Among them, compound 4, 2-(4-hydroxyphenyl)-5-(3-hydroxypropenyl)-7-methoxybenzofuran, had the lowest $IC_{50}$ value concerning nitric oxide (NO) release from lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Compound 4 suppressed the generation of prostaglandin (PG) $E_2$ and the expression of inducible NO synthase and cyclooxygenase (COX)-2 induced by LPS, and inhibited the release of LPS-induced pro-inflammatory cytokines from RAW264.7 cells. The underlying mechanism of compound 4 on anti-inflammatory action was correlated with the down-regulation of mitogen-activated protein kinase and activator protein-1 activation. Compound 4 is potentially an effective functional chemical candidate for the prevention of inflammatory diseases.

• Tuberculosis and Respiratory Diseases
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• v.45 no.4
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• pp.846-860
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• 1998

Background: Endotoxin (LPS : lipopolysaccharide), a potent activator of immune system, can induce acute and chronic inflammation through the production of cytokines by a variety of cells, such as monocytes, endothelial cells, lymphocytes, eosinophils, neutrophils and fibroblasts. LPS stimulate the mononucelar cells by two different pathway, the CD14 dependent and independent way, of which the former has been well documented, but not the latter. LPS binds to the LPS-binding protein (LBP), in serum, to make the LPS-LBP complex which interacts with CD14 molecules on the mononuclear cell surface in peripheral blood or is transported to the tissues. In case of high concentration of LPS, LPS can stimulate directly the macrophages without LBP. We investigated to detect the generation of proinflammatory cytokines such as interleukin 1 (IL-1), IL-6 and TNF-$\alpha$ and fibrogenic cytokine, TGF-$\beta$, by peripheral blood mononuclear cells (PBMC) after LPS stimulation under serum-free conditions, which lacks LBPs. Methods : PBMC were obtained by centrifugation on Ficoll Hypaque solution of peripheral venous bloods from healthy normal subjects, then stimulated in the presence of LPS (0.1 ${\mu}g/mL$ to 100 ${\mu}g/mL$ ). The activities of IL-1, IL-6, TNF, and TGF-$\beta$ were measured by bioassaies using cytokines - dependent proliferating or inhibiting cell lines. The cellular sources producing the cytokines was investigated by immunohistochemical stains and in situ hybridization. Results : PBMC started to produce IL-6, TNF-$\alpha$ and TGF-$\beta$ in 1 hr, 4 hrs and 8hrs, respectively, after LPS stimulation. The production of IL-6, TNF-$\alpha$ and TGF-$\beta$ continuously increased 96 hrs after stimulation of LPS. The amount of production was 19.8 ng/ml of IL-6 by $10^5$ PBMC, 4.1 ng/mL of TNF by $10^6$ PBMC and 34.4 pg/mL of TGF-$\beta$ by $2{\times}10^6$ PBMC. The immunoreactivity to IL-6, TNF-$\alpha$ and TGF-$\beta$ were detected on monocytes in LPS-stimulated PBMC. Some of lymphocytes showed positive immunoreactivity to TGF-$\beta$. Double immunohistochemical stain showed that IL-1$\beta$, IL-6, TNF-$\alpha$ expression was not associated with CD14 postivity on monocytes. IL-1$\beta$, IL-6, TNF-$\alpha$ and TGF-$\beta$mRNA expression were same as observed in immunoreactivity for each cytokines. Conclusion: When monocytes are stimulated with LPS under serum-free conditions, IL-6 and TNF-$\alpha$ are secreted in early stage of inflammation. In contrast, the secretion of TGF-$\beta$ arise in the late stages and that is maintained after 96 hrs. The main cells releasing IL-1$\beta$, IL-6, TNF-$\alpha$ and TGF-$\beta$ are monocytes, but also lymphocytes can secret TGF-$\beta$.

• Journal of the Korean Applied Science and Technology
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• v.30 no.1
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• pp.173-182
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• 2013

Atopic dermatitis is a chronic, relapsing inflammatory skin disease associated with dysfunction of skin barrier and cutaneous hyper-reactivity to environmental triggers. In the previous study, cytotoxicity, antioxidant, anti-inflammatory and anti-allergic activities were investigated for various herbal extracts such as Aloe vera L. (AV), Viola mandshurica W. Becker (VM), Punica granatum L. (PG), and Dendrobium nobile L. (DN) in order to develop effective therapeutic herbal extracts for atopic dermatitis, In this study, anti-inflammatory activities of these herb extracts in lipopolysaccharide (LPS)-induced macrophage RAW264.7 cells were further examined to find the underlying molecular mechanisms. The RT-PCR (reverse transcription polymerase chain reaction) analysis showed that PG, DN and AV inhibited effectively the gene expression of pro-inflammatory cytokines IL-6 and IL-$1{\beta}$ in LPS-stimulated macrophages, while VM did not. The transfection and luciferase analysis exhibited that all herbal extracts hindered the activation of transcription nuclear factor kappa B (NF-${\kappa}B$). The western blot analysis indicated that AV blocked the activation of only JNK MAP (c-Jun N-terminal kinase mitogen-activated protein) kinase not p38 MAP kinase, while VM, PG and DN did not show the activation of both JNK and p38 MAP kinases. These results suggest that AV, VM, PG, and DN have anti-inflammatory activities and thus have the potential to reduce and alleviate the symptoms of atopic dermatitis.

• Journal of The Korean Society of Integrative Medicine
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• v.11 no.3
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• pp.159-169
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• 2023

Purpose : Though other Citrus spp. have reported their anti-inflammatory and antioxidative activities in previous studies, the biological activity of Kiyomi (Citrus unshiu × C. sinensis) has not been reported yet. Therefore, this study attempted to analyze the anti-inflammatory mechanisms of Kiyomi leaf ethanol extract (KLEE) in lipopolysaccharide (LPS) stimulated RAW 264.7 cells. Methods : The cytotoxic effect of KLEE in RAW 264.7 cells was determined by WST-1 assay. Bacterial endotoxin, the concentration of nitric oxide (NO) was analyzed by the Griess reaction. In addition, Western blot analysis was applied to measure the protein expression level of inducible NO synthase (iNOS). The phosphorylated status of the critical inflammatory transcription factor, nuclear factor (NF)-𝜅B, and its upstream signaling molecules, phosphoinositide 3-kinase (PI3K)/Akt as well as mitogen-activated protein kinases (MAPKs), were also measured by Western blot analysis. Results : KLEE was not cytotoxic up to a concentration of 200 ㎍/㎖, and protein expression levels of iNOS and cyclooxygenase (COX)-2, enzymes that counteract NO and prostaglandin (PG) E2 production, were inhibited by KLEE treatment. The phosphorylated status of PI3K/Akt as well as MAPKs including extracellular regulated kinase (ERK), c-jun NH2kinase (JNK), and p38, were significantly attenuated by KLEE treatment in LPS stimulated RAW 264.7 cells. Moreover, one of phase II enzymes, heme oxygenase (HO)-1 which has known for its anti-inflammatory capacity, was strongly induced by KLEE treatment. Conclusion : Consequently, KLEE treatment significantly attenuated the production of NO as well as the expression levels of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. The inflammatory transcription factor, NF-𝜅B, as well as its upstream signaling molecules, PI3K/Akt and MAPKs, were also diminished by KLEE treatment with statistical significance in LPS-stimulated RAW 264.7 cells. These results suggest that KLEE might be a promising candidate for the attenuation of inflammatory disorders.

• Journal of The Korean Society of Integrative Medicine
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• v.11 no.2
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• pp.119-128
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• 2023

Purpose : The fruit of Actinidia polygama has been used in oriental medicine for the treatment of gout, rheumatoid arthritis, and inflammation. Though A. polygama exhibited anti-inflammatory activity in RAW 264.7 cells and carrageenan-induced rat paw edema, the exact mechanism for anti-inflammation was not evaluated yet. In this study, the anti-inflammatory mechanisms of A. polygama ethanol extract (APEE) in lipopolysaccharide (LPS) stimulated RAW 264.7 cells. Methods : WST-1 assay was applied to analyze the cytotoxic effect of APEE in RAW 264.7 cells. The productions of nitric oxide (NO) and prostaglandin (PG) E₂ were analyzed by the Griess reaction and enzyme immunoassay (EIA) assay, respectively. In addition, protein expressions for inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were measured by Western blot analysis. The activated status of an inflammatory transcription factor, NF-κ B, and its upstream signaling molecules, mitogen-activated protein kinases (MAPKs), was also evaluated by Western blot analysis. Results : As a result, APEE treatment did not exhibit any cytotoxicity until the concentration of 200 ㎍/㎖. APEE treatment significantly inhibited NO and PGE₂ productions as well as their enzymes, iNOS and COX-2 in a dose-dependent manner. The inflammatory transcription factor, NF-κ B, was also attenuated by APEE treatment. In addition, the phosphorylated status of MAPKs such as extracellular regulated kinase (ERK), c-jun NH2 kinase (JNK), and p38, were significantly diminished by APEE treatment in LPS stimulated RAW 264.7 cells. Conclusion : Consequently, APEE treatment significantly attenuated the production of inflammatory mediators and their enzyme expressions in LPS-stimulated RAW 264.7 cells. The inflammatory transcription factor, NF-κ B, and upstream signaling molecules, MAPKs, were also significantly attenuated by APEE treatment in LPS-activated RAW 264.7 cells. These results indicate that APEE might be a candidate to be utilized as a promising candidate for the treatment of inflammatory disorders.

• Journal of The Korean Society of Grassland and Forage Science
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• v.33 no.4
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• pp.313-318
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• 2013

A study was conducted to determine if subacute ruminal acidosis (SARA) induced by feeding high level of non-structural carbohydrates results in increases in lipopolysaccharides (LPS) of peripheral blood in dairy cattle. In this experiment four Holstein steers, two of which having ruminal canulae with indwelling probes placed for measuring long term pH changes, were alloted into two dietary treatments in a cross-over design, where an acidogenic diet (AD) was formulated by including high amount of non-structural carbohydrates (NSC) based on corn silage and corn flake as TMR ingredients. Data for ruminal pH change and plasma LPS concentration were compared against normal diet (ND) which contained grass hay as forage and low NSC ingredients. Feeding AD for more than 14 days to animals brought about a pH change as low as less than 5.8 for more than 4 hours, which made good contrast to ND fed animals. Decreased ruminal pH also had an effect on LPS concentrations which showed significantly higher level for AD compared with ND. Therefore, plasma LPS concentration may be used as an effective indicator to verify acidosis whether it is acute or chronic. Na-bentonite and Propylene glycol, which are frequently included in dairy TMR as additives, helped decrease ruminal pH by buffering and then ultimately alleviates SARA. Therefore, it could also be helpful to lower the occurrence of laminitis which is often caused by increased blood endotoxin (LPS) concentration.