In a previous study, we fractionated crude polysaccharide (AME-CP) with macrophage-stimulating activity from a hot-water extract (AME) of Astragalus membranaceus. AME-CP contained glucose (Glc) as a main component sugar, suggesting that it might be rich in starch-like compounds (SLC). To enhance the immunostimulating activity of AME-CP by pruning SLC rarely known to contribute to activity, hydrolysate (AME-SH) was prepared by digesting with starch-related enzymes, including α-amylase and amyloglucosidase. AME-SH was found to contain substances with molecular weights ranging from 3.9 to 84.4 kDa. These substances were primarily composed of galactose, galacturonic acid, Glc, arabinose, rhamnose, and mannose. AME-SH significantly enhanced the production of macrophage-stimulating factors, including nitric oxide (NO), interleukin (IL)-6, and IL-12, in RAW 264.7 cells compared to AME-CP. Treatment of splenocytes isolated from C3H/HeN mice with AME-SH not only promoted IL-6 secretion, but also induced mitogenic activity. In addition, AME-SH promoted the secretion of hematopoietic growth factors including IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in Peyer's patch (PP) cells and stimulated bone marrow cell proliferation through these PP cells. In conclusion, hydrolysate (AME-SH) digested from AME-CP with starch-related enzymes could be used as a potential immunostimulant.

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.

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.

To produce an intestinal immunomodulatory beverage containing Centella asiatica extract (CAE), three types of CAE-added beverage prototypes were prepared, and their immunomodulatory activities and marker compounds were analyzed. As a result of the cytotoxicity assessment, all the beverages did not show significant toxicity compared to the control group. Next, the immunomodulatory activities of the beverage prototype were evaluated using the inflammatory model of IL-1β-induced intestinal epithelial cell line. All the samples significantly reduced the production of IL-6, IL-8, and MCP-1 in a CAE concentration-dependent manner. In addition, CAE-added beverages inhibited NO, IL-6, and IL-12 production in LPS-induced RAW 264.7 cells. When the major triterpenoids, as marker compounds for the production of CAE-added beverages, were analyzed by HPLC-DAD, only asiaticoside was detected beyond the limit of quantification, while madecassoside, madecassic acid, and asiatic acid were not detected. The amounts of asiaticoside in CAE-added beverage prototypes were confirmed in No. 1 (19.39 μg/mL), 2 (19.25 μg/mL), and 3 (19.98 μg/mL). In conclusion, the results of this study suggested that CAE-added beverage prototypes induced immunomodulatory effects in the intestinal inflammatory cell line models and asiaticoside could be used as a marker compound for CAE-added beverage production.

Centella asiatica (C. asiatica) has been widely used in food, cosmetics, and pharmaceutical industry as a functional material. In a previous study, we have investigated not only pharmacological effects such as antioxidative and anti-inflammatory effects, but also analyzed various functional ingredients. In this study, triterpenoids were analyzed using HPLC-DAD to determine marker compounds among functional ingredients. When triterpenoids were analyzed, asiaticoside from C. asiatica was determined as an optimal marker compound. Next, specificity, linearity, limited of detection (LOD), limited of quantification (LOQ), precision, accuracy, and range were evaluated using HPLC-DAD to determine asiaticoside contents in C. asiatica juice and extracts. The specificity was elucidated by chromatogram and retention time using an established analytical method. The coefficient of correlation obtained was 0.9996. LOD was 4.99 μg/mL and LOQ was 15.12 μg/mL. Intra- and inter-day precision of asiaticoside were determined to be 0.48~1.68% and 0.08~1.09%, respectively. Furthermore, the recovery rate of asiaticoside was 98.88% and the analytical range of Field-70E was determined to be 0.625~10 mg/mL. As a results of evaluating ABTS, DPPH, and FRAP antioxidative effect, Field-70E showed potent antioxidant activities. Results of this study could be used as basic data for quality standardization of C. astiatica juice and extracts.

To investigate the anti-inflammatory activity of submerged culture using Cordyceps militaris mycelium, culture-including mycelia was extracted and lyophilized into postbiotics (hot-water extract; CM-HW). HW was fractionated into crude polysaccharide (CM-CP) by ethanol precipitation, and CM-CP was further dialyzed into CM-DCP by dialysis with running water using 12~14 kDa dialysis tube. When the cytotoxicity of subfractions against cells was assessed, no subfraction had a cytotoxic impact that was substantially different from the control groups. In an inflammatory model using LPS-stimulated RAW 264.7 cells, CM-DCP significantly decreased IL-6 and MCP-1 production levels compared to the LPS-control group. CM-DCP also inhibited IL-6 and IL-8 secretion in HaCaT keratinocytes stimulated with TNF-α and IFN-γ. In the meanwhile, the neutral sugar content and mannose ratio of anti-inflammatory CM-DCP were higher than the other fractions, and CM-DCP contained β-1,3/1,6-glucan of 216.1 mg/g. High pressure size exclusion chromatography revealed that CM-DCP contained molecules with a molecular weight range of 5.6 to 144.0 kDa. In conclusion, postbiotics of C. militaris mycelium significantly promoted anti-inflammatory activity, suggesting that neutral polysaccharides including Glc and Man contribute to the anti-inflammation in RAW 264.7 or HaCaT cells.

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.

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.

Compound K (20-O-β-(D-glucopyranosyl)-20(S)-protopanaxadiol)는 진세노사이드의 활 성성분이다. Compound K는 경구 투여 후 Rb1, Rb2 및 Rc가 사람의 장내 미생물의 β-glucosidase에 의 해 생물전환 과정을 거쳐 생성된다고 알려져 있다. 본 연구는 생물전환된 인삼농축액에서 얻은 compound K를 이용해 항염증 및 독성을 조사하였다. 세포독성평가 결과, compound K는 0.001∼1 μg/mL의 농도 범위에서 유의적인 세포독성은 나타나지 않았으며, LPS로 염증이 유발된 RAW 264.7 세포에서 TNF-α, MCP-1, IL-6 및 NO의 생성을 억제하는 것으로 확인되었다. 동일 농도범위에서 TNF-α 및 IFN-γ로 염증이 유발된 HaCaT 세포는 compound K의 처리로 인해 IL-8의 생성을 감소시키는 것으로 나타났지만, IL-6의 경우 일부 농도에서 생성을 감소시켰으나, 통계적인 유의성은 나타나지 않았다. Brine shrimp를 이 용한 치사율 검정법에서 compound K의 LC50는 0.37mg/mL로 다소의 독성을 함유하고 있는 것으로 나타 났으나 compound K가 35% 고함유된 생물전환물은 LC50가 0.87mg/mL로 나타나 상대적으로 낮은 독성 을 보였다. 따라서 이 생성물은 향후 여드름 완화용 화장품 개발에 사용할 수 있는 매우 우수한 기능성 소 재가 될 수 있을 것으로 기대된다.

To investigate the industrial availability of liquid fermentation (PL-ferment) by Phellinus linteus mycelium as a postbiotics for the inhibition of inflammation, PL-ferment was fractionated into culture supernatant (CS), hot-water extract (HW) from PL-ferment, EtOH-precipitate (CP) fractionated from HW, and the dialysate (DCP) of CP. Compared to the other fractions, DCP which is expected to contain exopolysaccharide (EPS) as the major component, significantly decreased the production of NO, IL-6, and MCP-1 in LPS-induced RAW 264.7 cells, and IL-6 and IL-8 in TNF-α and IFN-γ-induced HaCaT cells. The general component analysis results showed that no significant difference in components was observed between the fractions, whereas sugar composition analysis revealed that DCP had decreased glucose and increased mannose contents compared to the other fractions. This suggests that mannose played an important role in the anti-inflammatory activity of the active fraction, DCP. Molecular weight distribution analysis revealed that DCP was mainly composed of low-molecular-weight material-removed high-molecular-weight polysaccharides of 18–638 kDa, suggesting that EPS originated from P. linteus EPS. In conclusion, our results suggest that the DCP of P. linteus mycelium fermentation using the anti-inflammatory activity could be used industrially as postbiotic material.

To utilize Malus pruniforia Borkh. as a functional material, cold-water (CW), hot-water (HW), and 70% ethanol (EtOH) extracts were prepared, and their antioxidant and anti-inflammatory activities were compared. The antioxidant activity of the HW extract evaluated by ABTS and DPPH radical scavenging and FRAP activity was significantly effective. The total polyphenol content of the HW extract was also higher by 15.5±0.7 mg GAE/g extract compared to other extracts. The EtOH extract showed significantly decreased TNF-α (39.8%), IL-6 (65.5%), and NO (34.9%) levels in RAW 264.7 cells compared to the LPS-induced control group. The levels of IL-6 (21.1%) and IL-8 (19.3%) were significantly decreased by treatment of EtOH extract in HaCaT keratinocytes induced with TNF-α and IFN-γ. The UHPLC-MS results indicated that the EtOH extract might have chlorogenic acid and phlorizin as the major compounds. This was validated using HPLC-DAD, which showed that the EtOH extract had higher levels of chlorogenic acid and phlorizin (1,185±58 and 470±10 μg/g extract, respectively). In conclusion, the present study suggested that the anti-inflammatory activity of the EtOH extract was more effective than the CW and HW extracts, and chlorogenic acid and phlorizin could be used as indicator compounds and functional substances.

Centella asiatica (CA) has been used as a nutritional plant as well as a traditional herbal medicine around the world. This study, quality component (proximate composition, total polyphenol, and triterpenoid compound), and antioxidant and anti-inflammatory activities of CA dried using various methods were investigated. Proximate compositions of CA with different drying methods included a large amount of carbohydrates, crude protein, crude ash, and crude fiber. Among the drying methods, cold drying provided the highest total polyphenol content and antioxidant activity, while hot-air drying at 75℃ provided the lowest total polyphenol content and antioxidant activity. In addition, when the major triterpenoid compounds of CA were analyzed, the highest content of asiaticoside of triterpenoid glycoside was obtained with all drying methods. With respect to the total triterpenoid, the highest content was obtained with cold drying (68.8 mg/g) whereas natural drying (31.4 mg/g) provided the lowest content. In anti-inflammatory activity of LPS-stimulated RAW 264.7 cells, EtOH extract of cold drying showed a significantly higher inhibitory activity in comparison to the other drying methods. In conclusion, it is considered that the cold drying method is suitable for industrial preparation of functional materials with high physiological ingredients, and antioxidant and anti-inflammatory activities from CA.

After ethanol (BM-E and RW-E) and hot-water (BM-HW and RW-HW) extracts were fractionated from two herbal mixtures (BM and RW), their physiological activities were investigated. All extracts consisted of more than 50% of neutral sugar, with their total polyphenol levels higher than flavonoid levels. Radical scavenging activities of EtOH extracts remained significantly higher compared to that of hot-water extracts, and in particular, RW-E showed consistently higher antioxidant activity than BM-E. When anti-inflammatory activities of the extracts were evaluated by LPS-stimulated RAW 264.7 cells at 10~500 μg/mL non-cytotoxicity doses, BM-E showed significantly higher levels of TNF-α, IL-1β, IL-6, and nitric oxide inhibitory activity than those of hot-water extracts and RW-E. Murine peritoneal macrophage cells were shown to be enhanced in crude polysaccharides (BM-CP and RW-CP fractionated from BM-HW and RW-HW) compared to hot-water extracts and polysaccharide K (PSK, positive control). Especially, RW-CP exhibited higher activity than BM-CP, and component sugar analysis showed that BM-CP mainly contained galacturonic acid, glucose, arabinose, galactose, and xylose (34.5%, 33.9%, 16.1%, 7.1%, and 6.3%, respectively), whereas RW-CP showed different measurements (29.5%, 59.2%, 5.0%, 4.5%, and 0.2%). In conclusion, two herbal mixtures could contain varying sets of physiological activities dependent on different extraction and fractionation methods.

Centella asiatica (CA) has been widely used as herbal plants. It is a valuable resources. The aim of the present study was to evaluate physiological activities of solvent extracts from CA cultivated in Chungju, Korea (Good tiger care). After preparing water (cold-water, CA-WE; hot-water, CA-HWE) and EtOH extracts (50% EtOH, CA-50E; 70% EtOH, CA-70E), their total polyphenol, flavonoid, and triterpenoid contents, and anti-oxidant activities, and anti-inflammatory activities were examined and compared. CA-70E showed the most potent anti-oxidant activity based on ABTS radical scavenging activity and reducing ability assays, while CA-50E and CA-70E showed the highest DPPH radical scavenging activity. Among major triterpenoid glycosides present in CA, madecassoside and asiaticoside contents were found to be the highest in CA-70E, and madecassic acid and asiatic acid were the highest in CA-50E. In LPS-stimulated RAW 264.7 cells, CA-70E showed the highest TNF-α inhibitory activity, although CA-50E and CA-70E similarly inhibited nitric oxide production. Ethanol extracts significantly inhibited IL-6 and IL-8 production more than water extracts using TNF-α/IFN-γ-stimulated HaCaT cells, indicating its better inhibitory against skin inflammation. Therefore, Chungju-CA EtOH extract, especially 70% EtOH extract, has high physiologically active ingredients and potent anti-oxidant and anti-inflammatory activities, suggesting its industrial application as a functional material.

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.

After liquid culture of Phellinus baumii (P. baumii) mycelium (LPBM) was prepared, LPBM was fractionated into A∼E fraction (A; hot-water extract of liquid culture including mycelia, B; crude polysaccharide of A, C; hot-water extract of mycelia, D; crude polysaccharide of C, and E; crude polysaccharide of culture broth) to evaluate for possibility as functional materials with immunostimulatory activity. In macrophage stimulatory activity, E fraction as postbiotics significantly increased secretion of NO and IL-12 from RAW 264.7 cells. Next, when the splenocytes of C3H/HeN mice were primary cultured, E fraction showed significantly mitogenic activity with enhancing mitogen-related cytokines (IFN-γ and TNF-α) production from splenocyte. E fraction also potently stimulated GM-CSF production from Peyer’s patch cells as well as Peyer’s patch-mediated bone marrow cell proliferation. In addition, the immunostimularoy E fraction contained neutral sugar (73.8%), uronic acid (10.6%), protein (7.8%), and polyphenol (7.5%), and mainly consisted of glucose (39.1%), galactose (21.7%), mannose (11.1%), galacturonic acid (9.9%), and arabinose (8.9%) as component sugars. In conclusion, it was demonstrated that postbiotics including exopolysaccharide fractionated from liquid culture of the P. baumii mycelium could enhanced immunostimulatory activity.

교대뒷채움 및 벽체 배면에 되메우기 시공시 협소한 장소로 인한 다짐장비 운용이 원활치 않아 시방서 기준의 다짐도를 만족하지 못하는 경우가 빈번이 발생하고 있으며, 이러한 경우 시공관리를 위하여 고형화제를 첨가하여 시공하게 된다. 하지만 일반적인 고형화제의 적용시, 대상 지반의 적용에 있어 균질한 지반을 형성하기 어려운 단점이 있다. 이러한 단점을 보완하기 위하여 유동성이 확보된 고형화제를 첨가하여 사용하게 된다. 하지만 이러한 유동성이 확보된 고형화제가 첨가된 지반의 강도 및 저변형률에서의 변형 특성에 대한 연구는 아직 미진한 실정으로, 특히 진동을 유발하는 기계의 기초 지반에 적용하는 경우, 유동성이 확보된 고형화제 첨가 지반의 동적인 거동 특성을 규명하는 것이 매우 중요하다. 따라서 본 연구에서는 산업부산물인 매립석탄회와 점토, 황토를 이용한 유동화처리토의 강도 및 동적 특성 변화를 알아보기 위하여 일축압축시험과 공진주시험을 실시하였다.