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.

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.