To utilize pepper (Piper nigrum) as an immunostimulatory agent, we isolated macrophage stimulating polysaccharides from pepper and investigated their macrophage activating activities. Hot-water extracts (HW) of black pepper (BP) and white pepper (WP) were prepared, and their macrophage stimulating activities were evaluated using RAW 264.7 cells. BP-HW significantly promoted the secretion of macrophage stimulating factors such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-12 compared to WP-HW. When BP and WP-HW were fractionated into crude polysaccharides (CP) and low molecules (LM) by ethanol precipitation, BP-CP demonstrated significantly more potent activity. Furthermore, BP-CP not only induced mRNA gene expression of macrophage activation factors, but also promoted nuclear localization of p65 and c-Jun. In addition, component sugar analysis revealed that glucan-type polysaccharides in BP-CP played a crucial role in macrophage activation. Taken together, these findings suggest that black pepper has industrial applicability not only as a spice, but also as an immunostimulatory functional material.

This study investigated major constituents and anti-inflammatory effects of an ethanol extract of Platycodon grandiflorum leaves. Through HPLC analysis, chlorogenic acid and luteolin-7-O-glucoside were identified as predominant constituents in the ethanol extract. Their anti-inflammatory effects were evaluated using murine macrophage (RAW 264.7 cells) and human lung carcinoma cells (NCI-H292 & A549). The ethanol extract significantly (p<0.01) inhibited the production of nitrite, interleukin-6 (IL-6), and prostaglandin E2 (PGE2) induced by lipopolysaccharide (LPS) in RAW 264.7 cells. Furthermore, the ethanol extract suppressed the expression of cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) proteins in RAW 264.7 cells stimulated with LPS. In NCI-H292 and A549 cells, treatment with the ethanol extract significantly (p<0.05) decreased levels of pro-inflammatory cytokines IL-6 and IL-8 induced by IL-1β. The phosphorylation of ERK rather than JNK in the mitogen-activated protein kinase signaling pathway was observed to be a more important mediator in the down-regulation of pro-inflammatory cytokines in NCI-H292 cells. These findings suggest that the ethanol extract of Platycodon grandiflorum leaves containing luteolin-7-O-glucoside exhibits promising anti-inflammatory properties.

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.

To increase industrial applicability of Astragalus membranaceus (AM) as immunostimulating materials, hot-water extract (AME) was prepared from AM and fermented with Kimchi-lactic acid bacteria (Lactobacillus sakei & Leuconostoc mesenteroides) to prepare fermented AM-postbiotics (FAME). Although FAME prepared from AM-postbiotics did not show a significant enhancement in macrophage stimulating activity compared to non-fermented AME, crude polysaccharide (FAME-CP) fractionated by EtOH precipitation from FAME showed significantly higher macrophage stimulating activity than AME-CP. Compared to AME-CP, FAME-CP showed dramatic changes in component sugar and molecular weight distribution. FAME-CP was a polysaccharide with a major molecular weight distribution of 113.4 kDa containing Man (44.2%), Glc (19.3%), Gal (10.2%), GalA (10.2%), and Ara (7.4%) as sugar components. FAME-CP with enhanced macrophage stimulatory activity not only increased expression levels of mRNA genes encoding macrophage-activated factors (iNOS, TNF-α, MCP-1, IL-6, and COX-2), but also led the nuclear translocation of activated p65 and c-Jun. In conclusion, crude polysaccharide from AM-postbiotics fermented with lactic acid bacteria could increase industrial applicability as a functional material with enhanced immunostimulating activity than AME-CP.

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)

To enhance the bioavailability and bioactivities of mixed herbal medicines (RW), they were fermented with lactic-acid bacteria isolated from kimchi into postbiotics (FRW). Then, from the results of the 16s rRNA sequencing analysis, lactic acid bacteria isolated from kimchi were identified to be of two species, namely Lactobacillus sakei and Leuconostoc mesenteroides. The FRW prepared from the RW were extracted using hot water (HW) and 70% EtOH (EtOH) for comparison of their macrophage-stimulating activities. Based on a comparison of the activities of the FRW extracts, nitric oxide (NO) production of HW was significantly higher than that in EtOH. An analysis of the chemical properties of the extracts showed that HW had higher contents of neutral sugar and uronic acid than EtOH as well as contained a large amount of glucose. In addition, crude polysaccharide (CP) was prepared to enhance the macrophage-stimulating activity. The FRW-CP not only secreted immunostimulatory mediators but also increased the expression of immunostimulatory genes (iNOS, TNF-α, MCP-1, and IL-6). The fractionated FRW-CP contained about 90% neutral sugars, and these sugars were mainly composed of glucose, galacturonic acid, and arabinose. Thus, FRW prepared by fermentation of RW with kimchi lactic acid bacteria were found to be immunostimulatory modulators.

Asthma is a chronic inflammatory disease characterized by recurring symptoms, airflow obstruction, and bronchial hyper-responsiveness. The onset of asthma for most patients begins early in life, and current asthma treatment with anti-inflammatory agents can have adverse effects, eventually leading to impaired quality of life. In the pathogenesis of asthma, macrophages and basophils play a vital role during progression. Macrophages not only induce inflammation by secreting inflammatory cytokines but also promote DNA damage and mucus production through nitric oxide (NO) production. Basophils enhance eosinophil recruitment and aggravate asthma through the FcεRIα receptor with high affinity for histamine and IgE. Therefore, in this study, we investigated whether the activation of macrophages and basophils is suppressed by the individual extracts of 28 natural products. RAW 264.7 cells (mouse macrophages) were treated with the natural products in LPS, and 4 natural product extracts resulted in decreased NO production. In β-hexosaminidase assay using RBL-2H3 cells (rat basophils), 19 natural product extracts decreased β-hexosaminidase production. In NO production and β-hexosaminidase assay using macrophages and basophils, 3 natural product extracts (Plantago asiatica, Centella asiatica, and Perilla frutescens var. japonica) significantly inhibited NO production and β-hexosaminidase release. Overall, we examined the inhibitory effects of 28 natural product extracts on macrophage and basophil activity, and the findings demonstrated the potential of natural product extracts for treating asthma and macrophage- and basophil-related diseases.

Maca roots (Lepidium meyenii) are an important medicinal herb that have long been used by the Andes-indigenous peoples and South Americans. In Korea, recently, it has attracted attention as a health food material because of nutritional values and physiological activities. The purpose of this study was to investigate the industrial applicability of maca (red and golden varieties; R&G) as immunostimulating materials. In the macrophage stimulating assay using RAW 264.7 cells at 125~500 μg/mL of non-cytotoxicity doses, G-HW showed the most potent production of TNF-α, IL-6 and nitric oxide compared to red maca or the other extracts. The general component analysis results showed that all extracts comprised more than 90% neutral sugars with small amounts of uronic acid and protein. Meanwhile, component sugar analysis showed the difference in the content of uronic acids of cold- and hot-water extract. Additionally, the further fractionation of G-HW into crude polysaccharide (G-CP) greatly enhanced the macrophage stimulating activity, and G-CP contained macromolecules over 144 kDa, comprised mainly of glucose and galacturonic acid (51.0 and 34.9%). In conclusion, crude polysaccharide from maca showed industrial applicability as immunostimulating material, and especially golden maca showed higher macrophage stimulating activity than red maca.

Turmeric is known for its ability to enhance immunity via anti-inflammatory and anti-oxidant effects. Salmonella enterica species contain a large number of pathogenic serotypes that are adapted to a broad range of vertebrate hosts. Our previous study revealed that bioprocessed polysaccharides from the liquid culture Lentinus edodes fungal mycelia containing turmeric (BPP-turmeric) is able to alter chicken macrophage responses and increases chick survival against Salmonella enterica infection. In this study, we examined the immunomodulatory effects of BPP-turmeric on the porcine macrophage 3D4/31 cell line infected with Salmonella enterica subsp. enterica serovar Choleraesuis (S. Choleraesuis) or S. Enteritidis. Our experimental analyses demonstrated that BPP-turmeric (i) does not alter phagocytic and killing activity of 3D4/31 against either Salmonella serotypes, but that it (ii) represses mRNA transcription of interleukin (IL)-6, IL-8, and tumor necrosis factor α in response to Salmonella infection. Collectively, these results imply that BPP-turmeric has an immunomodulatory effect that represses pro-inflammatory cytokine expression in porcine macrophages, suggesting that it may protect swine from salmonellosis via controlling Salmonella-induced hyperinflammation.

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.

Agricultural by-productsis a rich source of dietary fibers including arabinoxylans, which is found mainly in water-insoluble form and reported to have immunomodulatory activities. We investiglatedwhether solublearabinoxylan (AX)-enriched byproducts alters macrophage activity.Agricultural by-products wereorally given to mice for 4 weeks. Peritoneal macrophages from mice were assayed for scavenger receptor expression, phagocytosis, and lipopolysaccharide (LPS)-stimulated responses. Serum levels of inflammatory cytokines were determined 1 hour after intraperitoneal injection of LPS. Agricultural by-productstreated mice showed a higher expression of SRA and CD36, accompanied by enhanced phagocytosis of latex beads, relative to control mice. Upon stimulation with LPS ex vivo, macrophages from the byproducts group showed lower levels of CD86 and CD40 at the surface protein level and lower production of NO, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, compared with the control group. Theagricultural by-products group showed a decrease in serum levels of LPS-induced TNF-αand IL-6, while mice given crude byproducts, which was prepared without enzymatic hydrolysis, showed a reduction only in serum TNF-α,indicating that soluble arabinoxylans contributes to the immunomodulatory effect ofagricultural by-products. Our data present evidence that soluble arabinoxylan-enriched agricultural by-products can be an immune enhancing functional food.

Macrophages play a pivotal role in the innate and adaptive immune systems. This study investigated the immuno-modulatory activities of polysaccharides separated from Chrysanthemum zawadskii var. latilobum (CZPS) in macrophages. Polysaccharides from Chrysanthemum zawadskii var. latilobum were extracted by the ethanol precipitation method. RAW 264.7 mouse macrophage cell line was treated with CZPS (4 to 128 μg/mL), and there was no cytotoxicity at a dose below 32 μg/mL. The levels of nitric oxide (NO) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-6, IL-1β) production in the CZPS treated group (32 μg/mL) were 6.5±0.12 μM (NO), 1252.8±79.85 (TNF-α), 305.4±29.41 (IL-6), and 683.3±59.71 (IL-1β), respectively, and they were significantly increased when compared to the control group; 2.2±0.03 μM (NO), 452.3±38.34 (TNF-α), 31.7±5.75 (IL-6), and 184.1±11.52 (IL-1β). Additionally, protein expression of inducible nitric oxide synthase (iNOS) and phosphorylation of MAPKs and NF-κB expression were significantly increased upon CZPS treatment. Therefore, these results indicated that polysaccharides separated from Chrysanthemum zawadskii var. latilobum (CZPS) may have a potential immunomodulatory activity in macrophages through MAPKs and NF-κB signaling, and this information is useful for the development of immune enhancing adjuvant materials using a natural ingredient.