The dynastid beetle Allomyrina dichotoma has been used as a herbal medicine. Recently, we performed de novo RNAsequencing of Allomyrina dichotoma and identified several antimicrobial peptide candidates based on in silico analysis.Among them, cationic antimicrobial peptide, Allomyrinasin, was selected and we assessed the anti-inflammatory activitiesof Allomyrinasin against mouse macrophage Raw264.7 cells. The results showed that Allomyrinasin decreased the nitricoxide production of the lipopolysaccharide-induced Raw264.7 cells. In addition, quantitative RT-PCR, ELISA and Westernblot analysis revealed that Allomyrinasin reduced cytokine expression levels in the Raw264.7 cells. Taken together, thesedata indicated that Allomyrinasin had anti-inflammatory activity in the lipopolysaccharide-induced Raw264.7 cells.

King oyster mushroom (Pleurotus eryngii), an improved species of oyster mushroom, is a popular ingredient in Asian cuisine. Spleen cells were treated with various concentrations (0, 5, 10, 50, 100, 250, 500, and 1,000 μg/mL) of king oyster water extracts (KOWE); then, the proliferation of the cells was measured 24, 48, and 72 h after each treatment. Also, type 1 T helper cytokine productions (TNF-α, IFN-γ, and IL-2) were measured in activated macrophage by KOWE in seven concentrations. Under the condition of its 50, 100, 250, and 1,000 μg/mL for 48 h, the proliferation of cells was increased. However, there was no significant fluctuation in the spleen cells proliferation for 24 and 72 h-long KOWE exposure. To determine cytokine (TNF-α, IFN-γ, IL-2) productions of type 1 T helper cells, macrophage was stimulated by KOWE for 48 h. Treatment of KOWE gave a rise to the levels of TNF-α and IFN-γ, but not in that of IL-2 productions. These results suggest that king oyster mushroom water extracts may be beneficial for enhancing immune functions in its high concentration.

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.

In vitro 실험을 통한 가시오가피 물 추출물 첨가가 마우스 의 면역세포 증식에 미치는 영향에 대한 연구 결과, 음의 대 조군에 비해 가시오가피 물 추출물을 첨가한 모든 농도에서 비장세포 증식능이 증가하였으며, 특히 고농도인 500~1,000 μg/ mL 농도에서 유의적으로 증가하였다. 반면, 복강 대식세 포로부터 유도된 사이토카인 생성의 경우, IL-2, IFN-γ, TNF- α 사이토카인 생성량을 측정한 결과, TNF-α 사이토카인은 가시오가피 물 추출물 50 μg/mL 농도와 250~1,000 μg/mL 농 도에서 대조군보다 유의적으로 높은 분비량을 보인 반면, IFN-γ에서는 변화를 보이지 않았다. IL-2의 경우, 100~500 μg/ mL에서 유의적으로 증가하는 경향을 보여주었다. 이상의 결 과에 의하면 가시오가피 물 추출물은 마우스 비장 세포를 증 식시키고, 사이토카인 분비량에도 영향을 줄 것으로 보이며, 면역 기관의 주요 기능을 증진시킬 가능성이 있을 것으로 사료된다. 따라서 가시오가피 물 추출물이 면역 증진 기능성식 품 개발의 소재로 활용될 가능성이 있을 것으로 기대된다

본 연구에서는 일본식으로 제조된 상업용 간장과 우리나 라 전통방식으로 제조된 재래식 간장으로부터 다당을 분리 하여 RAW 264.7 대식세포주를 이용한 면역 증진활성을 비교 하였다. 간장 유래 다당, CSP-0 및 KTSP-0는 RAW 264.7 세 포주에 대해 모든 농도에서 세포 독성을 나타내지 않았다. 또 한 재래식 간장 유래 다당인 KTSP-0는 상업용 간장 유래 다 당 CSP-0보다 대식세포에 의한 NO 및 ROS의 생산을 농도의 존적으로 증가시켰으며, KTSP-0를 1,000 ㎍/㎖ 처리하였을 시 가장 높은 생산능을 나타내었다. 간장 유래 다당의 RAW 264.7 세포로부터 면역반응에 중요한 cytokine인 IL-6와 TNF-α의 cytokine mRNA의 발현량과 해당 단백질의 생산을 각각 real-time PCR과 ELISA로 확인한 결과, CSP-0는 IL-6와 TNF- α mRNA의 발현 및 생산에 유의적인 영향을 미치지 않았지 만, KTSP-0는 농도의존적으로 IL-6와 TNF-α mRNA 발현 및 생산을 증가시키는 것을 확인하였으며, 1,000 ㎍/㎖ 처리 시 최대 활성을 보였다. 한편, 대식세포의 탐식작용에서 중요한 역할을 하는 Fc 수용체의 발현 증가를 RT-PCR로 확인한 결 과, CSP-0는 FcR I, II의 발현에 모두 영향을 미치지 않았지만, KTSP-0는 FcR I의 발현을 선택적으로 증가시킴이 확인되어 항원에 결합한 IgG와 강하게 결합하여 탐식작용을 촉진시킬 것으로 예상되었다. 본 연구를 통해 상업용 간장에서 분리한 다당, CSP-0보다 우리나라 전통 재래식 간장에서 분리한 다 당, KTSP-0가 대식세포를 활성화하여 높은 면역 증진 효과를 나타내는 것으로 결론 지을 수 있었다.

This study was designed to investigate the effect of fucoidan on the activation of macrophage and on induction of apoptosis in AGS cell. To measure the activity of macrophages, NO and TNF-α assays were performed in Raw 264.7 cell. Treatment with fucoidan significantly increased production of NO and TNF-α, indicating activation of macrophages. The result of MTT assay shows that cell viability was significantly decreased in a dose and time-dependent manner. Fucoidan increased to enhance mitochondrial membrane permeability, as well as the cytochrome c release from the mitochondria. Fucoidan decreased Bcl-2 and XIAP expression, whereas the expression of Bax was increased in a time-dependent manner compared to the control. In addition, the active forms of caspase-9 were increased, and the inactivation of Akt was decreased in a time-dependent manner. Caspase inhibitor, z-VADFMK, canceled the apoptosis of fucoidan, expression of Bax and caspase-9 were decrease. These results indicate that fucoidan induces activation of macrophage and apoptosis through activation of caspase on AGS cell.

To suggest the development possibility of immunostimulating materials from lactic acid bacteria (LAB) postbiotics, Lactobacillus sakei/Leuconostoc mesenteroides were isolated from Kimchi. Next, two isolated LAB were cultured to prepare postbiotics (LABP) to measure macrophage activity. LABP significantly increased the production of macrophage stimulating factors such as nitric oxide, tumor necrosis factor-α, and interleukin-6. LABP-crude polysaccharides (CP) fractionated from LABP by EtOH precipitation not only showed more potent macrophage stimulating activity but also induced phagocytic activity in a dose-dependent manner. In addition, LABP-CP was identified as polysaccharides with a major monosaccharide composition of mannose (59.1%), glucose (23.0%), and galactose (14.0%) with high-molecular-weight of 103.5~126.7 kDa. In conclusion, postbiotics prepared from LAB isolated from Kimchi were confirmed to have industrial applications as a functional material with immunostimulating activity.