본 연구는 국내산 가시오가피의 건강기능식품 소재로서 의 가능성을 확인하기 위해 산지별 채취한 가시오가피의 유효물질 함량 및 면역 증강 효과을 평가하였다. 태백, 철 원, 삼척, 강원도 농업기술원에서 수확한 가시오가피의 지 표성분인 eleutheroside B 및 eleutheroside E의 분석을 수 행하였으며, 면역 증강에 대한 효과를 관찰하기 위하여 MTT 세포독성 평가, NO 생성량과 cytokine 생성량을 측 정하였다. 지표성분 eleutheroside B의 함량은 채취 지역별 로 70% 에탄올 추출물에서는 2.96±0.11-6.24±0.05 mg/g로 태백에서 가장 높은 함량을 나타냈으며, 열수 추출물에서 는 1.11±0.05-2.11±0.03 mg/g로 태백에서 가장 높은 함량 을 나타냈다. Eleutheroside E 함량은 채취 지역별로 70% 에탄올 추출물에서는 4.93±0.20-10.79±0.03 mg/g을 나타냈으 며 철원에서 가장 높은 함량을 나타냈고, 열수 추출물에서 는 1.75±0.14-3.64±0.05 mg/g로 철원과 농업기술원에서 가장 높은 함량을 나타냈다. 또한, eleutheroside B 및 E 함량은 열수 추출물보다 70% 에탄올 추출물에서 더 높은 함량을 나타냈다. 채취 지역별 가시오가피의 70% 에탄올 추출물은 50-200 μg/mL 농도에서, 열수 추출물은 100-500 μg/mL 농도 에서 RAW 264.7 대식세포에 대한 세포독성을 나타내지 않 았으며, 대식세포의 활성화로 방출되는 NO 성성량을 측정 한 결과, 가시오가피 줄기 추출물에서 NO 생성량이 증가하 는 것을 확인하였으며, TNF-α, IL-6, IL-1β을 포함하는 cytokine의 방출을 측정한 결과 유의적인 증가를 나타냈다. 따라서 가시오가피 줄기는 면역 관련 질환의 개선을 위한 건강기능식품 소재로 활용 가능할 것으로 사료된다.

Macrophages secrete various cytokines and inflammatory mediators, resulting in playing critical roles in inflammation and immunity. In this study, we investigated anti-inflammatory and immune enhancing properties of PB203, which is a water-soluble extract powder from the fruit of Actinidia polygama, in macrophages. A. polygama is a medicinal plant traditionally known to treat abdominal pain, stroke and rheumatoid arthritis. However, the molecular mechanism for the immune modulation of PB203 is still unclear. Therefore, we assessed the effects of PB203 on the lipopolysaccharide (LPS)-induced inflammation and immune activation, and elucidated its action mechanism in mouse macrophage, RAW264.7 cells. PB203 significantly suppressed not only the levels of nitric oxide (NO), prostaglandin E2, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), but also the mRNA expression of inducible NO synthase, cyclooxygenase-2, TNF-α and IL-1β in LPS-stimulated RAW264.7 cells. We also found that these anti-inflammatory activities of PB203 were mediated through the inhibition of toll-like receptor 4 and nuclear factor kappa B (NF-κB) induced by LPS. On the other hand, in normal macrophages, PB203 dose-dependently elevated the gene expression of immunomodulators including granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1 and TNF-α in a statistically significant manner. The expression of IL-10, IL-1β, IL-6, and interferon-γ were also remarkably upregulated by the treatment of 500 μg/mL PB203. In addition, PB203-mediated production of NO and TNF-α was attenuated by NF-κB inhibition in RAW264.7 cells. Interestingly, PB203 promoted the production of nuclear factor erythroid-2-related factor 2, resulting in the increased level of heme oxygenase-1, which is a representative antioxidant enzyme, in both LPS-stimulated and normal RAW264.7 cells. Taken all together, these results suggest that PB203 may have great potential as the candidate of anti-inflammatory agent for improving inflammatory diseases or immune enhancing agent for preventing infectious diseases. Keywords: Actinidia polygama extract (PB203); macrophages; immunomodulator; nuclear factor kappa B (NF-κB); heme oxygenase-1 (HO-1)

Coffee (Coffea spp.) is one of the most important agricultural commodities, being widely consumed in the world. Various beneficial health effects of coffee have been extensively investigated, but data on habitual coffee consumption and its bio-physiological effect have not been clearly explained as well as it is not proved the cause and effect between drinking coffee and its bio-physiological reactions. We made the dialyzed coffee extract (DCE), which is absorbable through gastrointestinal tract, in order to elucidate the cellular effect of whole small coffee molecules. RAW 264.7 cells, a murine macrophage lineage, were directly treated with DCE, i.e., DCE-2.5 (equivalent to 2.5 cups of coffee a day), DCE-5, and DCE-10, for 12 hours, and their protein extracts were examined by immunoprecipitation high performance liquid chromatography (IP-HPLC). RAW 264.7 cells differently expressed the inflammation-related proteins depending on the doses of DCE. RAW 264.7 cells treated with DCE showed marked increase of cathepsin C, cathepsin G, CD20, CD28, CD31, CD68, indicating the activation of innate immunity. Particularly, the macrophage biomarkers, cathepsin G, cathepsin C, CD31, and CD68 were markedly increased after DCE-5 and DCE-10 treatments, and the lymphocyte biomarkers, CD20 and CD28 were consistently increased and became marked after DCE-10 treatment. On the other hand, RAW 264.7 cells treated with DCE showed consistent increase of IL-10, an anti-inflammatory factor, but gradual decreases of different pro-inflammatory proteins including TNFα, COX-2, lysozyme, MMP-2, and MMP-3. In particular, the cellular signaling of inflammation was gradually mitigated by the reduction of TNFα, COX-2, IL-12, and M-CSF, and also the matrix inflammatory reaction was reduced by marked deceases of MMP-2, MMP-3, and lysozyme. These anti-inflammatory expressions were consistently found until DCE-10 treatment. Therefore, it is presumed that DCE may have dynamic effects of innate immunity activation and pro-inflammation suppression on RAW264.7 cells simultaneously. These effects were consistently found in the highest dose of coffee, DCE-10 (equivalent to 10 cups of coffee a day in man), that might imply the small coffee molecules were accumulated in RAW 264.7 cells after DCE-10 treatment and produce synergistic cytokine effects for innate immunity activation and anti-inflammatory reaction concurrently.

Macrophages can recognize antigens and microorganisms, and then initiate an appropriate defense. However, there has been a lack of comprehensive information regarding the genes that are modulated by commensal yeasts, including Saccharomyces cerevisiae or Saccharomyces exiguus. In addition, it is not clear to what extent the beneficial yeasts modulate the immune response against microbes and/or microbial toxins. Using DNA microarray, which contains approximately 25,000 genes, we studied interactions between host cells and yeast/bacterial toxin (LPS) by analyzing the transcriptional response of macrophages stimulated by Saccharomyces exiguus and/or Lipopolysaccharides. Thirty three genes were identified to be modulated by more than two folds between groups of macrophage cells. Pathway analysis provided insight into the mutual interactions. Of particular interest was the responses elicited by fungus in murine macrophage cells, including modulation of immunity/defense, cellular signal transduction, cell proliferation/differentiation, and transport. This finding indicates that the yeast induces immune response pathways as well as those associated with cell proliferation and transport. Among the 33 genes identified from the DNA microarray screening, eight genes were further checked by RT-PCR analysis using gene specific primers. Compared to those of negative control, sequential treatment with the yeast strain followed by LPS apparently induced expression of Tnfaip3, IL7R, and CD86, while it inhibited expression of Cxcl10 and CD83. In conclusion, this study identified the genes that are up-regulated by Saccharomyces exiguus. A further study is needed in order to determine whether these genes are modulated at the protein level, and also for their roles in control of immune responses.

Salmonellosis is the commonest zoonosis worldwide that generally causes enterocolitis and foodborne poisoning which represents a considerable public health burden. Salmonella spp. are potential enteric pathogens and intracellularly replicates in host cells resulting in chronic infections. The medical treatments for salmonellosis have been difficult yet and had a serious problem including the increasing emergence of antibiotic resistance. The present report was designated to investigate the antibacterial effects of Saururus chinensis Baill ethanol extract (SCEE) on pure culture and infection with Salmonella enterica serovar Typhimurium (S. typhimurium) in murine derived macrophage RAW 264.7 cells. In determination of antibacterial activity of SCEE against S. typhimurium, bacterial viability was markedly decreased compared to the control. Also, SCEE significantly induced morphological change (p<0.05) of RAW 264.7 cells. In infection assay of S. typhimurium in RAW 264.7 cells pretreated with 100㎍/㎖ of SCEE, which is a non-cytotoxic concentration, bacterial uptake ability of macrophage was increased corresponding with morphological change, whereas bacterial survival rates within macrophage were markedly reduced compared with untreated control. Furthermore, nitric oxide (NO) production in SCEE-treated cells was slightly increased until 2 h but showed a tendency of decrease after 4 h until 24 h post infection compared with untreated control with S. typhimurium infection. Taken together, these findings demonstrated that SCEE has the antibacterial activity for S. typhimurium and the protective effects against S. typhimurium infection through activating murine macrophage independent on NO, suggesting that SCEE may be beneficial on the disease caused by intracellularly replicating pathogens as a safe alternatives of conventional chemotherapies.

Recently, as a natural substance has been emphasized interest in research to enhance the immune function. Green lettuce (Lactuca sativa L.) is a popular vegetable used fresh and it contains various phytochemicals and antioxidant compounds, and has been reported to have various physiological activities such as antibacterial, antioxidant, antitumor and anti-mutagenic. However, only a few studies have investigated on the mechanism of action of immune-enhancing activity of lettuce. Therefore, in this study, the immunomodulatory activities and potential mechanism of action of Green lettuce extracts (GLE) were evaluated in the murine macrophage cell line RAW264.7. GLE significantly increased NO levels by RAW264.7 cells, as well as expressions of immunomodulators such as iNOS, COX-2, IL-1β, IL-6, IL-12, TNF-α and MCP-1. Although GLE activated ERK1/2, p38, JNK and NF-κB, GLE-mediated expressions of immunomodulators was dependent on p38, JNK and NF-κB. In addition, TLR4 inhibition blocked GLE-mediated expressions of immunomodulators and activation of p38, JNK and NF-κB. Taken together, these results demonstrated that TLR4-MAPK/NF-κB signalling pathways participated in GLE-induced macrophage activation and GLE could be developed as a potential immunomodulating functional food.

In this study, a preliminary evaluation of the antioxidant and anti-inflammatory activity of the Ficus erecta var. sieboldii (Miq.) King (FES) leaf extract has been performed to assess its potential as a natural resource for food and medicinal materials. FES was extracted using 70% EtOH and then fractionated sequentially using n-hexane, CH2Cl2, EtOAc, and n-BuOH. To screen for antioxidant and anti-inflammatory agents effectively, the inhibitory effect of the FES extracts on the production of oxidant stresses (DPPH, xanthine oxidase, and superoxide) and pro-inflammatory factors (NO, iNOS, COX-2, PGE2, IL-6, and IL-β) in the murine macrophage cell line RAW 264.7 activated with lipopolysaccharide (LPS) was examined. Among the sequential solvent fractions of FES, the CH2Cl2 and EtOAc fractions showed decreased production of oxidant stresses (DPPH, xanthine oxidase and superoxide), and the hexane and CH2Cl2 fractions of FES inhibited the production of pro-inflammatory factors (NO, iNOS, COX-2, and PGE2). The CH2Cl2 fraction also inhibited the production of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β). These results suggest that FES has a significant effects on the production of oxidant stresses and pro-inflammatory factors and may be used a natural resource for antioxidant and anti-inflammatory agents.

스테비아(Stevia rebaudiana)는 남아메리카 지역이 원산지인 국화과 스테비아 속의 다년생 식물로 스테 비올(steviol)을 기본 구조로 하는 다양한 배당체가 존재하며 스테비오사이드(stevioside)와 리바우디오사이드 (rebaudioside) A 등이 주성분이다. 스테비올 배당체들은 설탕보다 단맛이 월등히 뛰어나 감미료로 널리 사용 되어지고 있다. 최근 여러 논문들에서 스테비올 배당체들이 미백 및 항염 효과 뿐 아니라 피부장벽 타이트정션 단백질 조절에 연관되어 있다는 보고가 있었다. 따라서 본 연구에서는 스테비올 배당체인 리바우디오사이드 A 의 항염 효과 연구를 통해 향후 아토피 피부염 개선 화장품 원료 개발 가능성을 확인하고자 하였다. 항염 연구를 위해 마우스 대식세포인 RAW264.7 세포를 이용하여 cell viability 및 염증 유발 사이토카인 mRNA 발현량을 분석하였다. 우선 cell viability 측정을 위해 cell counting kit-8 (CCK-8) assay를 수행하였고 세포독성이 없는 최대 농도를 250 μ M로 설정하여 이후 모든 실험을 진행하였다. 리바우디오사이드 A의 염증 조절 기능 연구는 주로 정량적 real-time RT-PCR 방법을 이용하였다. LPS에 의해 활성화된 RAW264.7 대식세포에서 리바우디오사이드 A 처리 결과 LPS 처리군 대비 iNOS 발현량은 약 47% 감소하였고, COX-2 또한 41% 감소 하였다. 생성된 NO의 양 또한 농도 의존적으로 감소하였다. 대식세포를 LPS로 활성화시킨 조건에서 염증 관련 사이토카인 유전자인 interleukin (IL)-1α, IL-1β, IL-6의 발현량 조절을 확인한 결과 사이토카인(IL-1α, 1β, 6) 발현이 LPS 처리군 대비 40%, 45%, 59%로 농도 의존적 유의성 있게 감소하였다. 결론적으로 스테비 올 배당체인 리바우디오사이드 A는 NO 생성 및 사이토카인 분비 억제를 통해 염증 반응을 저해하였다. 이러한 리바우디오사이드 A의 신규 항염증 조절 기능을 통해 아토피성피부염 개선 소재로의 개발이 기대된다.

The purpose of this paper is to investigate potential anti-inflammatory and anti-oxidant effects of Tenebrio molitor. Macrophage cell response by outside stimulation leads expression of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and trigger expression of genes which are affected by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), resulting in formation of inflammatory factors like nitric oxide (NO) and Prostaglandin E2 (PGE2). Cell viability was determined by MTT assay. In order to investigate anti-inflammatory agents, the inhibitory effects on the production of lipopolysaccharide (LPS)-induced NO in RAW 264.7 cells were examined. T. Molitor significantly decreased the production of NO in a dose-dependent manner, and also reduced the expression of iNOS, a COX-2 protein. As a result, the levels of protein such as PGE2, iNOS, COX-2 and MARKs were significantly reduced compared to non-treated group in T. Molitor water extract (TDW) treated group. Also, antioxidant effect of T. Molitor were investigated using DPPH, ABTS+ and superoxide anion radical scavenging activity tests in cell-free system. Antioxidant activity of T. molitor was found low in the DPPH radical scavenging test while high in the ABTS+ and superoxide anion radical scavenging activity tests. These results show that TDW could be an effective anti-pro-inflammatory and anti-oxidant agent.

The antioxidant and anti-inflammatory effects of Corni fructus extracts (CEF, EtOAc extraction; CBF, buthanol extraction; CWF, water extraction) were investigated. The total phenolics of CEF (173.3 mg TAE/g) were significantly higher than those of CWF (26.7 mg TAE/g) and CBF (94.8 mg TAE/g). DPPH and ABTS free radical scavenging activity of CEF (DPPH: RH50; 25.1 μg/mL, ABTS: RC50; 36.1 μg/mL) showed even higher than that of BHA and α-tocopherol used as positive control. All three Corni fructus extracts in the concentration of 1~100 μg/mL were effective inhibitors of NO and prostaglandin E2 (PGE2). NO production was inhibited 71.3~92.2% by CEF, 76.8~85.5% by CBF and 74.4~96.9% by CWF, respectively. CEF, CBF and CWF (1~100 μg/mL) inhibited also pro-inflammatory cytokines like TNF-α, IL-1β and IL-6 very effectively. TNF-α was inhibited up to 51.2% by CWF and IL-1β was inhibited up to 67.1% by CEF. IL-6 was best inhibited by CEF up to 58.9%. This study suggested the potential of Corni fructus for use as an excellent antioxidant substance and inflammatory inhibiting mediators. Therefore CEF, CBF and CWF Corni fructus extracts may be used for therapeutic approach to various inflammatory diseases.

Background : Ganoderma lucidum is a non-toxic, medicinal mushroom, which is known to possess anti-inflammatory and immunomodulating activities. However, the effects and mechanism of action of Ganoderma lucidum on lipopolysaccharide (LPS) and interferon-gamma (IFN-γ)-induced nitric oxide (NO) production and its-related cytokine expression are not yet fully understood. This study aimed to evaluate the effect of Ganoderma lucidum on NO production and NO-mediated pro-inflammatory cytokine expression in LPS/IFN-γ-induced RAW 264.7 macrophage-like cells. Methods and Results : The results showed that Ganoderma lucidum inhibited inducible NO synthase (iNOS) expression of RAW 264.7 macrophage-like cells at non-cytotoxic concentrations probably through the reduction of reactive oxygen species (ROS) production. After pre-treatment of cells with non-toxic doses of Ganoderma lucidum; NO production was significantly decreased. Moreover, Ganoderma lucidum treatment suppressed LPS/IFN-γ -stimulated pro-inflammatory cytokine secretion, including interleukin-1β and interleukin-6, in a dose-dependent manner. Conclusion : Taken together, these results indicate that the anti-inflammatory activation of Ganoderma lucidum in LPS/IFN-γ-stimulated macrophages might be due to abrogation of NO-dependent cytokine release by impairment of iNOS expression via ROS generation.

This study describes a preliminary evaluation of the anti-inflammatory activity of Acrosorium yendoi Yamada extracts. A. yendoi Yamada was extracted using 80% ethanol and then fractionated sequentially with n-hexane, ethyl acetate and butanol. To screen for anti-inflammatory agents effectively, we first examined the inhibitory effect of 80% EtOH extract and solvent fractions of A. yendoi Yamada on the production of pro-inflammatory factors and cytokines stimulated with lipopolysaccharide. In addition, we examined the inhibitory effect of 80% EtOH extract and solvent fractions of A. yendoi Yamada on pro-inflammatory mediators (NO, iNOS, PGE2, and COX-2) in RAW 264.7 cells. In the sequential fractions of n-hexane and EtOAc inhibited the NO and PGE2 production and the protein level of iNOS and COX-2, and protein expression of pro-inflammatory cytokines (TNF-α, and IL-6). These results suggest that A. yendoi Yamada may have significant effects on inflammatory factors and may be provided as possible anti-inflammatory therapeutic seaweed.

다양한 제품들에 사용되고 있는 천연물 소재들의 효능에 대한 체계적이고 과학적인 증거자료와 임상자료는 매우 부족한 실정이다. 이러한 천연물 소재의 과학적 연구는 국 민보건과 건강증진을 위한 다양한 제품개발의 기초자료로 활용할 수 있는 중요한 자료이다. 따라서 본 연구에서는 보리순 추출물을 이용하여 기능성 천연물 소재로서의 가능 성을 검토하였다. 항염증 활성을 조사하기 위해서 대식세 포 RAW264.7에 LPS로 자극시켜 유도된 염증반응에서 보 리순 에탄올 추출물의 매개체 억제 효과를 수행하였고 oxazolone을 이용하여 hairless 마우스에 접촉성 피부염을 유도하여 보리순 추출물의 항염 효과를 확인하였다. 연구 의 결과에서, LPS로 자극한 RAW264.7 세포에서 COX-II, iNOS와 같은 염증성 매개체뿐만 아니라 염증성 사이토카 인의 생성 및 발현이 보리순 에탄올 추출물에 의하여 현저 히 억제됨을 확인하였다. 이러한 보리순 에탄올 추출물의 억제효과는 IκB의 분해반응을 억제함으로써 NF-κB의 핵 으로 이동을 억제하여 신호전달체계를 불활성화시키는 것 과 관련이 있는 것으로 나타났다. 또한 보리순 에탄올 추출 물은 NF-κB의 상위 신호전달경로인 MAPKs에도 영향을 미치는 것으로 증명되었다. 대식세포에서의 실험결과를 토 대로 hairless 마우스에 oxazolone으로 접촉성 피부염을 유 발시킨 모델에서 보리순 에탄올 추출물을 처리하여 2주간 피부 병변을 관찰한 결과 염증반응이 현저히 감소됨을 확인 하였다. 이상의 결과에서 보리순 추출물이 항염증 효능을 가진 천연물 소재로서의 가능성을 제시하고 있다. 기존 연 구에 의하면, 보리 추출물에는 항산화 효과가 뛰어난 폴리 페놀류인 루테오린(luteolin), 사포나린(saponarin) 등이 풍 부하며, 항염증 효과가 우수한 페루릭산(ferulic acid), 루토 나린(lutonarin), 그리고 각종 비타민, 미네랄 등이 풍부하다 고 알려져 있다. 또한 superoxide와 hydroxyl 라디칼 생성을 억제할 수 있는 2"-O-glycosylisovitexin이 함유되어 있는 것으로 밝혀졌다. 이러한 성분들에 의하여 보리순 추출물 의 항염 효과가 나타날 것으로 생각된다. 다양한 염증성 질환에서 여러 매개체들의 과도한 발현이 그 질환의 원인임 을 생각해 볼 때 보리순 추출물은 항염증에 관련된 여러 제품들에서 다양하게 활용할 수 있는 천연물 소재가 될 것으로 판단되며 앞으로 보리순 추출물에서 항염 효과를 나타내는 유효화합물의 분리동정 및 생리활성평가에 대한 연구가 진행되어야 할 것으로 사료된다.

A variety of herbs and plants have been traditionally used in oriental folk medicine for the treatment of inflammatory diseases. In our attempt to search for anti-inflammatory agents from natural products, we investigated 64 methanol extracts from 42 medicinal plants belonging to 10 families which were evaluated for inhibitory activities of NO production in lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells. Among them, 16 extracts exhibited inhibitory activities of NO production (IC50 values ranging from 59.6 to 94.7 μg/ml). Only the extract from aerial parts of Hosta lancifolia (H. lancifolia) did not exert cytotoxic effects at the concentrations tested. The extract from H. lancifolia decreased the mRNA and protein levels of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines in activated macrophage RAW 264.7 cells in dose-dependent manner. The results suggest that the extract may contain bioactive compounds that suppress expression of pro-inflammatory cytokines, which may prove beneficial with regard to the development of natural agents for prevention and treatment of inflammatory diseases.

The present study describes the preliminary evaluation of the anti-inflammatory activities of Phellinus linteus (PL) and Phellinus linteus Grow in Germinated Brown Rice (BRPL). In order to effectively screen for anti-inflammatory agents, we first examined the extracts' inhibitory effects on the expression of pro-inflammatory cytokines activated with lipopolysaccharide. Moreover, we examined the inhibitory effects of the PL and BRPL extracts on pro-inflammatory factors such as NO, iNOS, TNF-α and IFN-γ in murine macrophage RAW 264.7 cells. NO production and iNOS expression was significantly augmented in LPS treated cell, the production of NO and iNOS was greater in the BRPL than in the PL group. In addition, protein and mRNA levels of TNF-α and IFN-γ in BRPL showed relatively more potent pro-inflammatory-activity inhibition compared to that of PL. These results suggest that BRPL may have significant effects on inflammatory factors, and may be a potential anti-inflammatory therapeutic materials.