• Title/Summary/Keyword: iNOS assay

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Effects of Corydalis Tuber on Synthesis of NO and $PGE_2$ in Murine Macrophage RAW 264.7 Cells Stimulated by LPS

  • Lee, Ki-Young;Park, Se-Keun;Kim, Jeong-Seon;Jang, Mi-Hyeon;Kim, Chang-Ju;Choi, Sun-Mi;Lee, Hye-Jung;Kim, Ee-Hwa
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.19 no.3
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    • pp.785-791
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    • 2005
  • Corydalis Tuber has traditionally been used for the treatment of water retention in the body. Administration of the aqueous extract of Corydalis Tuber has been known to be effective for the control of pain and treatment of arthritis. It was reported that Corydalis Tuber possesses anti-inflammatory activity and modulates the intestinal immune system. The effect of Corydalis Tuber against LPS-stimulated expressions of COX-2, iNOS, and $IL-1{\beta}$ in cells of the murine RAW 264.7 macrophages was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reverse transcription-polymerase chain reaction (RT-PCR), $PGE_2$ immunoassay, and NO detection. The aqueous extract of Corydalis Tuber was shown to suppress $PGE_2$ production by inhibition on the LPS-stimulated enhancement of COX-2 enzyme activity, $IL-1{\beta}$, and iNOS expression in the RAW 264.7 macrophages. Present results suggest that Corydalis Tuber exerts anti-inflammatory and analgesic effects probably by suppressing of COX-2, iNOS, and $IL-1{\beta}$ expressions, resulting in inhibition of $PGE_2$ synthesis. Corydalis Tuber has anti-inflammatory and analgesic effects probably by suppressing of COX-2, iNOS, and $IL-1{\beta}$ mRNA expressions, resulting in inhibition of $PGE_2$ and NO synthesis.

Anti-inflammatory and Anti-oxidant Studies of Osung-tang Extracts in LPS-Induced RAW 264.7 Cells (LPS로 유도된 RAW 264.7 세포에서 오성탕(五聖湯) 추출물의 항염증 및 항산화 연구)

  • Kim, So-Jeong;kim, Tae-Jun;Kim, Ee-Hwa;Kim, Yong-Min
    • The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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    • v.33 no.1
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    • pp.1-11
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    • 2020
  • Objectives : The purpose of this study was to investigate the anti-inflammatory and anti-oxidative effects of Osung-tang(OST) extract. Methods : MTT assay was performed to confirm the survival rate of RAW 264.7 cells treated with OST extract(50-400㎍/㎖) and the production of NO from LPS-induced RAW 264.7 cells was confirmed. The mRNA expression of iNOS, COX-2, and NF-κB were measured by real-time PCR. The protein expression of iNOS, COX-2 and p-IκB were measured by western blot and the anti-oxidant activity of OST extract(50-400㎍/㎖) was investigated by measuring DPPH scavenging activity. Results : OST extract showed a cell survival rate of 90% or more at 50-400㎍/㎖. The NO production was inhibited in a dose-dependent manner in RAW 264.7 cells treated with LPS on OST treated group. mRNA expression levels of iNOS, COX-2 and NF-κB decreased in a concentration-dependent manner after treatment with OST(50-400㎍/㎖). Protein expression levels of iNOS, COX-2 and p-IκB decreased in a concentration-dependent manner after treatment with OST(50-400㎍/㎖). It was found that OST has a high free-radical scavenging ability. Conclusions : These results suggest that OST extract can be used as a treatment for various skin diseases by demonstrating its anti-inflammatory and anti-oxidant effects.

Anti-inflammatory Effects of Tanghwajitong-san through Inhibition of NF-κB and MAPK (탕화지통산(湯火止痛散)의 NF-κB 및 MAPK 억제를 통한 항염증 효과)

  • Min Jung Ko;Seon Young Jee;Min Hwangbo
    • The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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    • v.36 no.1
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    • pp.1-20
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    • 2023
  • Objectives : The purpose of this study was to evaluate the anti-inflammatory effect of Tanghwajitongsan(THJTS) through inhibition of NF-κB and MAPK. Methods : We evaluated cell survival rate by MTT assay, NO production by nitrite content in the culture medium. We quantified TNF-α, IL-1β, IL-6 and PGE2 of the cultured supernatant by ELISA. And we evaluated the effect of THJTS on protein expression of NF-κB, MAPK, iNOS and COX-2 by Western blot analysis. THJTS ameliorates LPS-activated alterations in protein expression of NF-κB, p-38, iNOS and COX-2 and production of NO, pro-inflammatory cytokines and PGE2. Also, THJTS ameliorates LOX, PGN and FLA-activated alterations in protein expression of NO, iNOS. THJTS ameliorates only PGN-activated alterations in protein expression of COX-2. Results : THJTS ameliorates LPS-activated alterations in protein expression of NF-κB, p-38, iNOS and COX-2 and production of NO, pro-inflammatory cytokines and PGE2. Also, THJTS ameliorates LOX, PGN and FLA-activated alterations in protein expression of NO, iNOS. THJTS ameliorates only PGN-activated alterations in protein expression of COX-2. Conclusions : This study can provide scientific evidence for the anti-inflammatory effects and underlying mechanisms of THJTS.

Comparative Study on Biological Activities of Colored Potatoes, Hongyoung and Jayoung Cultivar (유색감자 홍영 및 자영 추출물의 생물학적 활성 비교)

  • Kang, Se-Chan; Choung, Myoung-Gun
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.53 no.2
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    • pp.233-238
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    • 2008
  • This experiment was conducted to enhance the colored potatoes utilization and to determine the biological activity of colored potato extracts. In order to understand the factors responsible for the potent anti-oxidant ability of colored potatoes, it has been evaluated for anti-oxidative activity using oxygen radical absorbance capacity (ORAC) assay. 'Hongyoung' extract was significant anti-oxidant activities in ORAC assay. About two-fold higher radical absorbance capacity was found in 'Hongyoung' compared to that in 'Jayoung'. The ability of 80% ethanol extracts from colored potatoes to influence the inhibitory activity of nitric oxide (NO) and nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) has also been investigated. The various therapeutic benefit claims in the new functional medicinal usage of colored potatoes ascribed to the phenolic compounds and anthocyanin. This result revealed that the extracts of colored potatoes are expected to be good candidate for development into sources of free radical scavenger or COX-2 inhibiting agents.

Effect of aging on expression of nitric oxide and inducible nitric oxide synthase in human gingival fibroblasts (노화가 사람 치은섬유아세포의 nitric oxide와 inducible nitric oxide synthase 발현에 끼치는 영향)

  • Ji, Suk;Kook, Jung-Ki;Park, Joo-Cheol;Kim, Heung-Joong;Jang, Hyun-Seon;Kim, Chong-Kwan;Kim, Byung-Ock
    • Journal of Periodontal and Implant Science
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    • v.36 no.2
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    • pp.361-373
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    • 2006
  • 치주질환의 진행이 나이에 의해 영향을 받는다는 사실은 알려져 있으나 노화에 따른 치주조직 세포의 기능적인 변화에 관한 사실은 많이 알려져 있지 않다. 노화에 따른 세포의 노화가 치주질환의 진행에 어떠한 여향을 끼치는가를 아는 것은 중요하다. 염증 상태에서 nitric oxide (NO)는 조직 파괴에 관여하는 인자로 작용하여 치주질환의 진행에 관여하는 것으로 알려져 있다. 따라서 이 연구는 사람의 치은에서 배양된 치은섬유아세포를 이용하여 세포의 노화에 따른 NO와 이의 합성효소인 inducible nitric oxide synthase (iNOS)의 발현을 알아봄으로써 세포의 노화가 치주질환의 진행에 끼치는 영향에 대해 알아보고자 하였다. 10세의 환자와 55세의 환자에서 각각 채취한 치은에서 배양된 세포와 10세의 환자에서 채취한 세포를 계속적인 계대배양을 통해 얻은 실험실 상 노화된 세포를 포함하여 총 3 종류의 치은섬유세포를 실험에 이용하였다. Hot phenol-water extraction을 통해 추출된 Porphyromonas, gingivalis ATCC 33277 lipopolysaccharide (LPS)와 재조합 $IFN-{\gamma}$ 를 세포에 적용시켜 Griess assay를 통해 조건화된 배지에서 NO를 측정하였다. 20세와 55세의 환자에서 채취된 치은 조직과 총 3 종류의 배양된 세포에 NOS-II 항체를 적용시켜 iNOS 단백질 발현을 관찰하였다. Total RNA를 추출하여 RT-PCR를 통해 iNOS mRNA의 발현을 분석하였다. 치은섬유아세포에서 NO는 자발적으로 발생되었고, 이러한 발현은 젊은 세포보다 노화된 세포에서 강하였다. P, gingivalis LPS와 제조합 $IFN-{\gamma}$는 치은섬유아세포에서 NO의 발현을 증가시켰고, 이러한 발현은 젊은 세포보다 노화된 세포에서 강하였다. 면역조직화학 염색에서 iNOS 단백질은 젊은 사람과 노화된 사람의 치은 조직 모두에서 치은섬유아세포와 상피의 기저층 세포와 염증세포에서 발현되었으나 노화에 따른 발현의 차이를 구별할 수는 없었다. 세포의 면역염색에서 iNOS 단백질은 노화된 세포에서 강하게 발현되었고 이러한 발현은 LPS와 $IFN-{\gamma}$ 에 의해 강화되었다. LPS와 $INF-{\gamma}$ 의 조건이 주어지지 않은 상태에서 iNOS mRNA는 젊은 세포에서보다 노화된 세포에서 강하게 발현되었다. 이러한 결과를 통해 세포의 노화가 NO와 iNOS 발현을 증가시킴으로서 치주질환의 진행에 영향을 끼칠 수 있음을 시사하였다.

Effects of solvents on the anti-aging activity of Salvia miltiorrhiza extract (추출용매에 따른 단삼 추출물의 항노화 활성)

  • Guo, nan;Lee, Ji-An
    • Journal of Convergence for Information Technology
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    • v.12 no.5
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    • pp.290-297
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    • 2022
  • This study was conducted to determine the cell cytotoxicity, astringency, nitrite oxide scavenging, iNOS protein expression level, pro-inflammatory cytokine, elastase inhibition, and type I pro-collagen synthesis as a functional cosmetics material of Salvia miltiorrhiza root. We prepared the 80% ethanol(SE) and hot-distilled water(SW), respectively. Both SE and SW showed no toxicity from 0.05 to 0.5 mg/mL concentration as a result of MTT assay in NHDF or RAW264.7 cells. In the measurement of astringent effect, SE reveled 74.6% of astringent activity in 10 mg/mL. SE showed that LPS-induced nitric oxide production, iNOS protein expression, and cytokines were inhibited in a dose-dependent manner. Furthermore, two extracts significantly inhibited elastase activity and increased the type I pro-collagen production. Therefore, it is expected that Salvia miltiorrhiza extract is used as a natural material for functional cosmetics that can effectively prevent skin-related inflammation and wrinkles, and aging.

Anti-inflammatory and Antioxidant Effects of Hot Water Extracts from Kaempferia Galanga L (삼내자 열수추출물의 항산화 및 항염 효과)

  • Chan, Ching Yuen Venus;Lee, Ji-An
    • Journal of Convergence for Information Technology
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    • v.9 no.6
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    • pp.218-226
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    • 2019
  • In this study, we investigated the possibility of Kaempferia Galanga(KG) hot water extract on the antioxidant, cytotoxic and anti-inflammatory efficacy as a cosmetic ingredient. Antioxidant effects were evaluated based on DPPH and ABTS radical scavenging activity, FRAP assay, and total polyphenol contents. The MTT assay was used to confirm the cell toxicity in mouse macrophage RAW264.7 cells. Anti-inflammatory effects were also investigated in LPS-induced RAW264.7 cells by measuring secretion of NO, $TNF-{\alpha}$ and iNOS, $TNF-{\alpha}$ mRNA expression level. As a result, DPPH and ABTS radical scavenging activities were increased in a concentration-dependent manner. The ferric reducing antioxidant power(FRAP) was the highest at 5 mg/mL as 24.5 uM. The measurements of total polyphenol content was $1.28{\pm}0.064mg\;GAE/g$. The cytotoxicity of the KG extract results showed no cytotoxicity at concentration of 0.625 to 2.5 mg/mL. In addition, the extract of KG significantly suppressed the LPS-induced nitrite, $TNF-{\alpha}$ secretion and the mRNA expression of iNOS, $TNF-{\alpha}$ in RAW264.7 cells. Taken together, these data suggest that the KG hot water extracts can be used as a safe and functional cosmetic raw material.

Anti-inflammatory and Antioxidant Effects of Water Extracts of Sasangja-tang(SSJ) and Gami-sasangja-tang(GSJ) (사상자탕과 가미사상자탕의 항염증 및 항산화 효과 비교 연구)

  • Choi, Jung-Eun;Park, Bo-Kyung;Jin, Mirim
    • Journal of Haehwa Medicine
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    • v.23 no.2
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    • pp.5-13
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    • 2015
  • Objectives : The anti-inflammatory and antioxidant effects of water extracts of Sasangja-tang(SSJ) and Gami-sasangja-tang(GSJ) were investigated. The effects of SSJ and GSJ were compared. Methods : We performed cell viability assay in HaCaT cells and RAW 264.7 cells using 3-(4,5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide(MTT) assay. We measured chemokines(regulated on activation normal T-cell expression and secreted ; RANTES/CCL5, interferon-inducible protein; IP-10/CXCL10, macrophage-derived chemokine; MDC/CCL22) in HaCat cells, also we measured cytokines (tumor necrosis factor-${\alpha}$; TNF-${\alpha}$, interleukin-6; IL-6) and nitric oxide(NO) production in RAW 264.7 cells using enzyme-linked immunosorbent assay(ELISA) and NO assay. Western blot assay was used to evaluate the expression for inducible nitric oxide synthase(iNOS) in RAW 264.7 cells. Results : SSJ and GSJ did not affect the cell viability at the concentrations treated ($0-800{\mu}g/ml$). As a result of SSJ and GSJ treatment in HaCat cells stimulated by TNF-${\alpha}$(10 ng/ml) and interferon(IFN)-${\gamma}$(10 ng/ml), the production of RANTES and IP-10 was inhibited significantly. However there was no significant difference in the secretion of MDC. And in RAW 264. 7 cells stimulated by lipopolysaccharide(LPS, $1{\mu}g/ml$), SSJ and GSJ treatment significantly inhibited the secretion of TNF-${\alpha}$ and IL-6 and the production of NO. The expression of iNOS was also decresed by SSJ and GSJ treatment in RAW 264. 7 cells. Compared with SSJ, GSJ was superior to SSJ in inhibition of RANTES, IP-10, TNF-${\alpha}$, IL-6 and NO production at the concentration of $200{\mu}g/ml$. Conclusion : Both SSJ and GSJ have anti-inflammatory and antioxidant effects. And GSJ has better effects than SSJ.

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Antioxidant, Antimicrobial and Anti-inflammatory of Mixed Medicinal Herb Extract (한약재 복합 추출물의 항산화, 항균 및 항염 효과)

  • Lee, In-Chul;Kim, Mee-Kyung
    • The Korea Journal of Herbology
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    • v.30 no.5
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    • pp.51-58
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    • 2015
  • Objectives : This paper aimed to verify the applicability of mixed extract ofAngelica gigasNakai,Cnidium officinaleMakino,Paeoniala ctifloraPall,Rechmannia glutinosaLibosch,Scutellaria baicalensisGeorgi, which were prescribed for improving inflammation in Donguibogam, as the materials for beauty food and functional medicinal herb cosmetics by manufacturing such mixed extract and evaluating the biological activity of the extract.Methods : The mixed medicinal herb water extract(MMW) and ethanol extract(MME) were freeze-dried to be used as the specimen. We performed electron donating ability, lipid acidification inhibitory activity, anti-inflammatory activity against skin flora, MTT assay, NO inhibitory activity and the protein expression inhibitory activity of iNOS and COX-2.Results : For anti-oxidation experimentation, the electron donating abilities of MMW and MME were above 60.0% and 90.0% at 500 μg/ml, respectively. In the inhibition rate of lipid peroxidation, MMW and MME showed 43.1% and 52.1% at 1,000 μg/ml, respectively. As a result of antimicrobial activity, both the MMW and MME showed significant clear zones forPropionibacterium acnesat 4 mg/disc, but did not indicated the clearzones forStaphylococcus aureus, Escherichia coliandStaphylococcus epidermidis. Anti-inflammatory activity by NO assay showed LPS-induced NO was significantly inhibited in a concentration-dependent manner. Also, the expression of iNOS and COX-2 proteins were significantly inhibited following treatment with MMW and MME of 50 μg/ml.Conclusions : Accordingly, it can be concluded that mixed medicinal herb extract has the potential to beused as a functional food and cosmetic material.

Effects of Berberine on the production of inflammatory mediators from LPS-stimulated BV2 microglial cells (황련(黃連)의 주성분인 Berberine의 뇌신경소교세포로부터 LPS에 의해 유도되는 염증매개물질 생성억제효과)

  • Park, Yong-Ki;Jung, Hyo-Won;Kim, Chang-Min;Choi, Jae-Sue;Kim, Yeong-Shik
    • The Korea Journal of Herbology
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    • v.22 no.4
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    • pp.117-125
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    • 2007
  • Objectives : Berberine, a main alkaloid component of Coptidis rhizoma, has an antimicrobial and anti-tumor activities and antiinflammatory effect. In the present study, we investigated effect of berberine on the production of inflammatory mediators such as nitric oxide(NO), prostaglandin E2(PGE2), TNF-${\alpha}$ and IL-1${\beta}$ in LPS-stimulated BV2 microglial cells, Methods : BV2 cells were pre-treated with berberine and then stimulated with LPS. The cytotoxicity of berberine was determined by MTT assay. The NO production was measured by Griess assay. The mRNA expression and protein levels of inducible nirtic oxide synthase(iNOS) were determined by RT-PCR and Western blot. The production of PGE2 and cytokines was measured by ELISA. Results : Berberine inhibited the production of NO, PGE2 and pro inflammatory cytokines, TNF-${\alpha}$ and IL-1${\beta}$ in a dose dependent manner in LPS-stimulated BV2 cells. In addition, berebrine greatly suppressed the mRNA expression and protein levels of iNOS and inflammatory cytokines induced by LPS stimulation. These results indicate that the post-transcriptional regulatory mechanism of iNOS and/or inflammatory cytokine gene expression by berberine is involved in its anti-inflammatory effects, respectively. Conclusion : The present study suggests that berberine can be useful as a potential anti-inflammatory agent for treatment of various neurodegenerative diseases such as Alsheimer's disease, Parkinson's disease and stroke.

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