Changes in the component sugar and immunostimulating activity of polysaccharides isolated from Dendrobium officinale in the pretreatments.

BACKGROUND: To isolate polysaccharides with enhanced immunostimulatory activity from Dendrobium officinale, which is used as a herbal medicine in China and Southeast Asia, D. officinale (DO) was pretreated with organic solvents (DOOS) or puffing at 7.5 and 9.0 kgf (7.5DO and 9DO). Hot-water extracts (DOOS-HW, 7.5DO-HW and 9DO-HW) were prepared from each pretreated DO, along with non-pretreated DO, and crude polysaccharides (DO-CP, DOOS-CP, 7.5DO-CP and 9DO-CP) were fractionated from each hot-water extract using ethanol (five volumes). RESULTS: When their immunostimulatory activities were compared by macrophage stimulation and intestinal immune system modulation via Peyer's patches, DOOS-CP showed more potent activity than DO-CP. However, crude polysaccharides fractionated from puffed DO showed significantly lower activity than non-puffed DO and DOOS. The most active polysaccharide contained 95% or more neutral sugar, and the composition ratio of mannose and glucose was 3.0, whereas the lowest polysaccharide content was 2.0 or less. In addition, DOOS-CP was a somewhat refined fraction containing a major peak, representing a molecular weight of 250 kDa, despite being a crude polysaccharide. CONCLUSION: These results suggest that pretreatment of D. officinale with organic solvents may enhance the immunostimulatory activity of polysaccharides and affect the mannose/glucose ratio of polysaccharides, which plays an important role in immunostimulation. © 2021 Society of Chemical Industry.

Biotransformation of catechin and extraction of active polysaccharide from green tea leaves via simultaneous treatment with tannase and pectinase.

BACKGROUND: Green tea is a dietary source of bioactive compounds for human health. Enzymatic treatments induce the bioconversion of bioactive components, which can improve biological activities. In this study, we investigated the effect of simultaneous treatment with tannase and Rapidase on biotransformation of catechins and extraction of polysaccharide from green tea extract (GTE). RESULTS: Tannase and pectinase treatments induced the biotransformation of catechins and altered tea polysaccharide () content. The addition of GTE to the enzyme reaction resulted in a significant increase in degallated catechins, including gallic acid, a product of the tannase reaction (314.5-4076.0 µg mL(-1)) and a reduction in epigallocatechin gallate (EGCG). Biotransformation of catechins improved the radical scavenging activity of GTE. Pectinase treatment led to change of TPS composition in GTE by hydrolyzing polysaccharides. In addition, pectinase-driven hydrolysis in polysaccharides significantly increased TPS-induced Interleukin 6 (IL-6) production in macrophages. In particular, treatment of Rapidase (TPS-Ra) led to the highest IL-6 production among TPS samples, similar to treatment of highly purified pectinase (TPS-GTE), a positive control. CONCLUSION: Simultaneous processing with tannase and Rapidase can be an efficient method for the extraction of bioactive polysaccharides and biotransformation of catechins with enhanced radical scavenging activity from green tea.

Enzyme-assisted extraction of cactus bioactive molecules under high hydrostatic pressure.

BACKGROUND: To improve the extraction and recovery of bioactive materials from cactus, the present study investigated the effect of polysaccharide-degrading enzymes [Rapidase-Viscozyme mixture, 1/3 (v/v)] treatment under high hydrostatic pressure (HHP). RESULTS: The dry weight of the extract increased with the use of increasing pressure regardless of enzyme treatment. However, the polyphenol content showed a tendency to decrease with the increase in pressure in the cactus extract with or without enzyme treatment. The enzyme-assisted extraction resulted in an increase of dry weight and polyphenol content in the cactus extract. The total sugar and reducing sugar contents of the cactus extract increased with increasing pressure in enzyme-assisted extraction. The uronic acid content of the cactus extract showed a pattern similar to that of the reducing sugars. The enzyme-assisted extraction also increased the contents of taxifolin, quercetin and isorhametin. The cactus extract obtained through enzyme-assisted extraction showed intense scavenging activity of both DPPH and ABTS radicals. The crude polysaccharides isolated from the extract (51.2% at 1000 µg mL⁻¹ for HHP extraction at 300 MPa) had higher anti-complementary activity than the others except for lipopolysaccharide (60.00% at 1000 µg mL⁻¹). HHP extraction and enzyme-assisted extraction using HHP showed an increase of anti-complementary activity compared with the heat and enzyme controls, respectively. CONCLUSION: Overall, the use of HHP in enzyme-assisted extraction resulted in more efficient extraction than the use of enzyme treatment alone.

Enzymatic improvement in the polyphenol extractability and antioxidant activity of green tea extracts.

This study describes increases in extraction efficiency and the bioconversion of catechins after treatment with several commercial enzymes. Tannase was also used to improve the anti-radical activities of green tea extracts. Enzymatic treatment with various commercial enzymes was introduced to improve the extraction efficiency of polyphenols. The total polyphenol, flavonoid, and catechin contents and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of the green tea extract treated with Viscozyme (VG) were significantly higher than those treated with other commercial enzymatic extractions (p<0.05). More than 95% of the epigallocatechingallate (EGCG) and of the epicatechingallate (ECG) was hydrolyzed to epigallocatechin (EGC) and to epicatechin (EC) in successive 20 min treatments with Viscozyme and tannase (TG). Due to its hydrolytic activity, treatment involving tannase resulted in a significant release of gallic acid (GA), EGC, and EC, leading to greater radical scavenging activities. Regarding the IC(50) values of the DPPH and 2,2-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, the green tea extract treated with TG showed values of 131.23 and 28.83 µg/mL, VG showed values of 224.70 and 32.54 µg/mL, and normal green tea extract (NG) showed values of 241.11 and 66.27 µg/mL, respectively. These results indicate that successive treatment with Viscozyme and tannase improves the extraction efficiency of polyphenols and increases radical scavenging activities.

Modulation of gut microbiota ecosystem by a glucan-rich snail mucin heteropolysaccharide attenuates loperamide-induced constipation.

The present study aimed to investigate the effect of oral administration of snail-derived mucin extract (SM) on ameliorating constipation symptoms of loperamide-induced constipated rats (n = 6). The analytical results indicated that SM mainly contains a glucan-rich snail mucin heteropolysaccharide with high molecular weights (108.5-267.9 kDa), comprising primarily of glucose (64.9 %) and galactose (22.4 %) with some deoxyhexoses (5.0 %) and hexosamines (4.9 %). Daily SM administration at doses of 10-40 mg/kg/day to the loperamide-induced constipated rats significantly (p < 0.05) ameliorated the deterioration in fecal parameters, such as numbers and weight of feces, fecal water contents, and gastrointestinal transit ratio. The histomorphometric results showed that the loperamide-induced decreases in the thickness of mucosal and muscularis mucosae layers as well as the distribution of mucin and c-KIT-positive areas were significantly (p < 0.05) improved via SM consumption at all doses tested. SM administration at all doses significantly increased the expression of genes encoding tryptophan hydroxylases (TPH1 and TPH2; p < 0.05), tight junction molecules (OCLN, CLDN1, and TJP1; p < 0.05), and mucin (MUC2 and MUC4; p < 0.05), but significantly decreased the aquaporin-encoding genes (AQP3 and AQP8; p < 0.05). Gut microbial community analysis indicated that SM administration could modulate loperamide-induced dysbiosis by increasing the phyla Actinobacteria (11.72-12.64 % at 10-40 mg/kg doses; p < 0.05) and Firmicutes (79.33 % and 74.24 % at 20 and 40 mg/kg doses; p < 0.05) and decreasing the phyla Bacteroidetes (5.98-12.47 % at 10-40 mg/kg doses; p < 0.05) and Verrucomicrobia (2.21 % and 2.78 % at 20 and 40 mg/kg doses; p < 0.05), suggesting that SM administration is effective in ameliorating constipation by controlling gut microbial communities. These findings can be utilized as fundamental data for developing novel functional materials using SM to prevent or treat constipation.

Identification of galacturonic acid-rich polysaccharide with intestinal immune system modulating activity via Peyer's patch from postbiotics of Phellinus linteus mycelial submerged culture.

Interests in the development and exploration of industrial applications of medicinal mushrooms as postbiotics have lately increased. We recently reported the potential use of Phellinus linteus mycelial-containing whole culture extract (PLME) prepared by submerged cultivation as a postbiotic that promotes immune system activation. Here, we aimed to isolate and structurally elucidate the active ingredients in PLME by activity-guided fractionation. The intestinal immunostimulatory activity was evaluated by bone marrow (BM) cell proliferation activity and related cytokine production in C3H-HeN mouse-derived Peyer's patch (PP) cells treated with polysaccharide fractions. The initially crude polysaccharide (PLME-CP) of PLME prepared using ethanol precipitation was further fractionated into four fractions (PLME-CP-0 to -III) by anion-exchange column chromatography. BM cell proliferation and cytokine production of PLME-CP-III were significantly improved compared to those of PLME-CP. PLME-CP-III was then fractionated into PLME-CP-III-1 and PLME-CP-III-2 by gel filtration chromatography. Based on the molecular weight distribution, monosaccharide, and glycosyl linkage analyses, PLME-CP-III-1 was revealed as a novel galacturonic acid-rich acidic polysaccharide and further shown to play an important role in facilitating PP-mediated intestinal immunostimulatory activity. This is the first study demonstrating the structural characteristics of a novel intestinal immune system modulating acidic polysaccharide from P. linteus mycelium-containing whole culture broth postbiotics.

Anti-complementary activity of enzyme-treated traditional Korean rice wine (Makgeolli) hydrolysates.

BACKGROUND: Makgeolli brewed from rice contains about 150 g kg(-1) alcohol and has a fragrance as well as an acidic and sweet taste. During the brewing process, by-products such as rice bran and brewery cake are produced. At the end of fermentation the matured mash is transferred to a filter cloth and the Makgeolli is squeezed out from the cake, leaving the lees of the mash. These by-products have continued to increase every year, resulting in an ecological problem. It is therefore important to develop new uses for them. The objective of this study was to use the by-products from the brewing of Makgeolli as a valuable functional food or nutraceutical. RESULTS: The anti-complementary activities of crude polysaccharides isolated from Cytolase hydrolysates of Makgeolli lees at concentrations of 1000 and 500 µg mL(-1) were 84.15 and 78.70% respectively. The activity of polysaccharide krestin (PSK) was 60.00% at 1000 µg mL(-1). The active polysaccharide obtained with Cytolase comprised mainly glucose and mannose (molar ratio 1.00:0.62). CONCLUSION: Glucose- and mannose-rich crude polysaccharides were isolated from the Cytolase hydrolysate of Makgeolli lees. The polysaccharides retain anti-complementary activity to enhance the immune system as a functional food or nutraceutical.

Ameliorating the efficacy of galacto-oligosaccharides on ovalumin-induced allergic dermatitis symptoms in Balb/c mice by regulating Th2 immune response and the ecosystem of gut microbiota.

In this study, we evaluated the effect of oral administration of galacto-oligosaccharide (GOS), famous biological molecules that are comprised of galactose and lactose, on ovalbumin (OVA)-induced allergic dermatitis. OVA-induced mice were divided into the OVA-administered group (OVA-C), promethazine hydrochloride-administered group (PC), and 100 and 200 mg kg-1 GOS-administered groups (GL and GH, respectively). GOS administration significantly improved epidermal thickness and decreased CD4+ cell numbers. The concentrations of IgE, OVA-specific IgE, and inflammatory cytokines (IL-4, IL-5, and INF-γ) in GH group mice were significantly lower than those in OVA-C group mice. Firmicutes and Bacteroidetes were identified as the major phyla in the intestinal microbiota in mice, and the relative abundance of Deferribacteres was significantly lower in the GH group than in the OVA-C group. Deferribacteraceae and Mucispirillum species were significantly lower in the GH group than in the OVA-C group. The relative abundance of Muribaculum species was significantly lower, but those of Lachnospira and Lactococcus species were significantly higher in the GH group than in the OVA-C group. Our results suggest that the alleviation effect of GOS on allergic dermatitis induced by OVA sensitization was achieved by regulating hypersensitive immune responses by improving the intestinal microbial ecosystem.

Antioxidant and Anti-Inflammatory Activities of Sargassum macrocarpum Extracts.

Oxidative stress and the inflammatory response are known to be the most important pathological factors for aging skin cells. Therefore, substances that protect skin cells from oxidative stress and inflammatory reactions of the skin have potential as functional ingredients for skin care. In the present study, we investigated the potential of Sargassum macrocarpum as an anti-inflammatory candidate for inflammatory skin disease. Antioxidant and anti-inflammatory activities are desirable properties in such functional materials. The total polyphenol content as well as antioxidant and anti-inflammatory activities were evaluated in hot-water (HES) and ethanol (EES) extracts of S. macrocarpum. The polyphenol content was higher in the HES (HES: 115.9 ± 15.3 mg GA/g, EES: 3.9 ± 0.5 mg GA/g), and the HES also had ABTS (HES: IC50 1.0 ± 0.0 mg/mL, EES: IC50 16.09 ± 0.7 mg/mL) and DPPH (HES: IC50 6.50 ± 0.3 mg/mL, EES: IC50 35.3 ± 3.1 mg/mL) radical scavenging capacities as well as FRAP activity (HES: IC50 18.8 ± 0.4 mg/mL, EES: IC50 n.d.). Compared with the EES at the equivalent concentration range (1.25-20 µg/mL), the HES exerted a more potent inhibitory activity on LPS-stimulated nitric oxide (10.3-43.1%), IL-6 (15.7-45.0%), and TNF-α (14.1-20.8%) in RAW 264.7 macrophage cells in addition to TNF-α and IFN-γ-facilitated IL-6 (10.9-84.1%) and IL-8 (7.7-73.2%) in HaCaT keratinocytes. These results suggested that water-soluble materials might be deeply involved in the antioxidant and anti-inflammatory activity in S. macrocarpum. General composition analysis indicated that the HES contains more carbohydrates and polyphenols than the EES, and the monosaccharide composition analysis suggested that fucose-containing sulfated polysaccharide and ß-glucan might be potent anti-inflammatory candidates in the HES. The present study presents important preliminary results and a valuable strategy for developing novel anti-skin dermatitis candidates using a hot-water extract of S. macrocarpum.

Immunostimulatory Effect of Postbiotics Prepared from Phellinus linteus Mycelial Submerged Culture via Activation of Spleen and Peyer's Patch in C3H/HeN Mice.

Medicinal mushrooms are an important natural resource promoting health benefits. Herein, Phellinus linteus mycelia were prepared under submerged cultivation, the mycelium-containing culture broth was extracted as a whole to obtain the postbiotic materials (PLME), and its effect on the immune system was evaluated in normal C3H/HeN mice. Oral administration of PLME for 4 weeks was well tolerated and safe. In the PLME-administered groups, in addition to the production of immunostimulatory cytokines, such as interferon gamma (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), the mitogenic activity was significantly increased. PLME administration also significantly increased the levels of serum immunoglobulin G (IgG) and IgA in the small intestinal fluid and Peyer's patches and enhanced Peyer's patch-mediated bone marrow cell proliferation activity and cytokine production (IL-2, IL-6, and IFN-γ). Histomorphometric analyses showed an increase in immune cells in the spleen and small intestinal tissues of mice administered PLME, supporting the rationale for its immune system activation. PLME mainly contained neutral sugar (969.1 mg/g), comprising primarily of glucose as a monosaccharide unit. The ß-glucan content was 88.5 mg/g. Data suggest that PLME effectively promote immune function by stimulating the systemic immune system through the spleen and intestinal immune tissues. PLME can thus be developed as a functional ingredient to enhance immune functions.

Association of the Healthy Eating Index with Metabolic Abnormalities among Middle-Aged Adults Living in Single-Person Households in Korea.

This study aimed to analyze the association between the dietary lifestyles and health outcomes among middle-aged (40-64 years old) and elderly (65 years old and older) individuals living alone using the Korean Healthy Eating Index (KHEI). The study was conducted with 1442 participants (475 men and 967 women) aged 40 years and older living in single-person households using the Korea National Health and Nutrition Examination Survey from 2016 to 2018. The KHEI scores were calculated based on the 24-h recall data of dietary intake. Among women living alone, the total KHEI score of the participants aged 40-64 years was 65.92, which was significantly lower than the 70.66 of those aged 65 years and older (p = 0.0152). In addition, the total score in the adequacy domain was significantly lower among the 40~64-year-old group than those aged 65 years and older (p = 0.0011). Among the elderly in single-person households, the odds of diabetes in the T1 group were 2.08 times higher than those in the T3 group according to the KHEI (95% confidence interval: 1.36-3.17). The results of this study are expected to be used as baseline data to establish nutrition, home meal replacement utilization, and health policies for the elderly living alone.

Polysaccharides fractionated from enzyme digests of Korean red ginseng water extracts enhance the immunostimulatory activity.

Polysaccharide with the enhanced immunostimulatory activities including intestinal immune system modulation was fractionated from Korean red ginseng (KRG) and its characteristics were investigated in the present experiment. When the water extracts were digested with α-amylase and amyloglucosidase and precipitated by ethanol to enhance immunostimulatory activity, enzyme digested-crude polysaccharides enhanced the macrophage and intestinal immune system via Peyer's patches compared to non-enzymatic crude polysaccharides. Starch-like polysaccharide also potently decreased in enzyme digested-crude polysaccharides. Especially, crude polysaccharide (RG-CW-EZ-CP) from the digest of cold water extracts showed significantly the most active immunostimulatory activities. By precipitation using ethanol concentrations (distilled water:ethanol = 1:4 and 1:8), two immunostimulatory polysaccharides (RG-CW-EZ-CP-4 and RG-CW-EZ-CP-8) were further fractionated from RG-CW-EZ-CP. In chemical analysis, RG-CW-EZ-CP-4 and RG-CW-EZ-CP-8 seems to be a pectic-like acidic polysaccharide and arabinose-rich polysaccharide, and heat treatment of polysaccharides (RG-CW-EZ-CP-4 and RG-CW-EZ-CP-8) did not significantly affect the intestinal immune system-modulating activity. RG-CW-EZ-CP-8 also significantly upregulated the phosphorylation of three major mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38. Thus, enzymatic digestion of KRG cold water extracts played a very important role in the isolation of the enhanced immunostimulatory polysaccharides from KRG.

Innate immune stimulation of exo-polymers prepared from Cordyceps sinensis by submerged culture.

After we prepared exo-polymers (EPS) from Cordyceps sinensis by submerged culture, prophylactic intravenous administration (i.v.) of EPS significantly inhibited metastasis in experimental lung metastasis of colon 26-M3.1 carcinoma. Cytotoxicity against Yac-1 tumor cells of natural killer (NK) cell, which was prepared by i.v. of EPS (100 mug/mouse), significantly augmented 2 days after EPS treatment. When NK cells were depleted by rabbit anti-asialo GM1 serum, even the EPS group totally abolished the inhibitory effect on lung metastasis of colon 26-M3.1 cells. EPS can stimulate innate immune system to inhibit tumor metastasis, and its anti-tumor metastasis is associated with macrophage and NK cell activation.

Nonpungent Capsicum fermentation by Bacillus subtilis and the addition of Rapidase.

To reduce the pungency of Capsicum without the loss of its biological activity, a Capsicum sp. was fermented by Bacillus subtilis with the addition of Rapidase enzyme. At 1 day of fermentation, the capsaicin content of the Capsicum ferment with Rapidase had sharply decreased from an initial content of 11.8 to 2.8 microg/ml. The Rapidase-fermented Capsicum had higher total flavonoid and phenolic contents than the Capsicum ferment without Rapidase. In addition, ABTS radical scavenging activity was enhanced in the Rapidase-fermented Capsicum as compared to that without Rapidase. Overall, fermentation using B. subtilis and Rapidase was an efficient method to produce a non-pungent Capsicum with antioxidant properties.

Antitumor and antimetastatic activities of rhamnogalacturonan-II-type polysaccharide isolated from mature leaves of green tea via activation of macrophages and natural killer cells.

To investigate the antitumor and antimetastatic polysaccharide from the mature leaves of green tea, GTE-II was purified using size exclusion chromatography. GTE-II consisted of 15 different sugars including rarely observed sugars such as 2-O-methyl-fucose, 2-O-methyl-xylose, apiose, aceric acid, 3-deoxy-d-manno-2-octulosonic acid, and 3-deoxy-d-lyxo-2-heptulosaric acid, which were characteristics of pectic polysaccharide rhamnogalacturonan-II. Treatment of peritoneal macrophages with GTE-II not only increased interleukin (IL)-6 and IL-12 production, but also had significantly increased tumoricidal activity against Yac-1 tumor cells than those obtained from untreated mice. In an assay of natural killer (NK) cell activity, intravenous administration of GTE-II significantly stimulated NK cytotoxicity against Yac-1 tumor cells. Furthermore, the depletion of NK cells by injection of rabbit anti-asialo GM1 serum eliminated the inhibitory effect of GTE-II on B16BL6 melanoma cells. These data suggest that GTE-II inhibits tumor metastasis, and its antitumor effect is associated with activation of macrophages and NK cells.

Anti-cancer effects of traditional Korean wild vegetables in complementary and alternative medicine.

This research study explored the anti-cancer effects of natural materials in South Korea. Although South Korea has a long history of traditional medicine, many natural materials of South Korea have not yet been introduced to the rest of the world because of language barriers and inconsistent study conditions. In the past 3 years, 56 papers introducing 56 natural materials, which have anti-cancer effects, have been published by scientists in South Korea. Further, these studies have introduced five kinds of natural materials presented in research papers that were written in Korean and are therefore virtually unknown overseas. The anti-cancer effects were confirmed by 2-3 cancer markers in the majority of the studies, with the most common targets being breast cancer cells and gastric cancer cells. These cancers have the greatest incidence in South Korea. The natural materials studied not only exhibit anti-cancer activity but also display anti-inflammatory, anti-oxidative stress, and anti-diabetic activities. They have not yet been used for the direct treatment of disease but have potential as medicinal materials for alternative and complementary medicine for the treatment of many modern diseases. Many natural materials of South Korea are already known all over the world, and with this study, we hope to further future research to learn more about these natural medicines.

Immunostimulation activity of a polysaccharide from fermented ginseng with Hericium erinaceum mycelia in solid-state culture.

Korean fresh ginseng was cultured with Hericium erinaceum mycelia (HE) in solid-state culture (SSC) to enhance the immunomodulation activity. Hot-water extracts (FG-HE-HW) of H. erinaceum-fermented ginseng (FG-HE) fractionated into a crude polysaccharide (FG-HE-CP) showed higher mitogenic, macrophage stimulation, and intestinal immune system modulation activities (1.52, 1.63, and 1.35x relative to a saline control at 100 µg/mL, respectively) than the crude polysaccharide from non-fermented ginseng (NG-CP; 1.35, 1.50, and 1.18x) and H. erinaceum mycelia alone (HE-CP; 1.26, 1.46, and 1.27x). After FG-HE-CP was further fractionated on a DEAE-Sepharose CL-6B column, the polysaccharide fraction (FG-HE-CP-III) exhibited larger activity increases than any subfraction from NG-CP or HE-CP. FG-HE-CP-III mainly consisted of Ara, Gal, Glc, and GalA (molar ratios of 0.45:1.00: 0.51:0.73). NaIO4 oxidation decreased the activities of FG-HE-CP-III (51.0-76.9%). The polysaccharide from H. erinaceum-fermented ginseng using SSC plays an important role for enhancement of immunomodulation activities of fresh ginseng.

Anti-metastatic activity of glycoprotein fractionated from Acanthopanax senticosus, involvement of NK-cell and macrophage activation.

Previously, we reported that water-extracted Acanthopanax senticosus exhibited anti-metastatic activity by stimulating the immune system. In this study, we fractionated glycoproteins (EN-SP) from the soluble protein layer (GF-AS) of A. senticosus and determined their basic chemical properties. We also investigated the anti-tumor and immunostimulating activities of the fractionated glycoprotein, EN-SP. We found that intravenous (i.v.) administration of GF-AS dramatically inhibited metastasis of colon26-M3.1 carcinoma cells to the lung in a dose-dependent manner. In vitro analysis showed GF-AS to enhance the proliferation of splenocytes. GF-AS also stimulated peritoneal macrophage, which was followed by the production of various cytokines such as IL-1beta, TNF-alpha, IL-12 and IFN-gamma. Furthermore, the production of these cytokines was partially blocked when peritoneal macrophage was cultured with the polyclonal antibodies against GF-AS. The depletion of NK cells by rabbit anti-asialo GM1 serum partly abolished the inhibitory effect of GF-AS on lung metastasis of colon26-M3.1 cells. Using gel filtration, EN-SP, an active glycoprotein fraction, is isolated from GF-AS. While both GF-AS and EN-SP stimulated the proliferatation of splenocytes of normal mice, EN-SP showed higher anti-metastatic activity and more potently stimulated the proliferation of splenocytes compared to GF-AS. These results suggest the use of EN-SP, the fractionated glycoprotein from A. senticosus, can be used as a therapeutical reagent to prevent or inhibit tumor metastasis.

Immunological activity of ginseng is enhanced by solid-state culture with Ganoderma lucidum mycelium.

Fresh ginseng was fermented with Ganoderma lucidum mycelium (GL) in solid-state culture to enhance its immunomodulatory activity. When crude polysaccharide (FG-GL-CP) was fractionated from lyophilized fermented ginseng (FG-GL), FG-GL-CP showed significantly higher mitogenic, macrophage stimulating, and intestinal immune system modulating activity (1.52-, 1.52- and 1.46-fold, respectively) than those of crude polysaccharide from nonfermented ginseng (NG-CP, 1.36-, 1.37- and 1.32-fold) and G. lucidum mycelium alone (GL-CP, 1.48-, 1.40- and 1.31-fold). After FG-GL-CP was further fractionated into two active carbohydrate-rich fractions (FG-GL-CP-II and III) on DEAE-Sepharose CL-6B (Cl⁻ form), the most active polysaccharide (FG-GL-CP-III-1) was purified as a single peak with a molecular weight of 71 kDa. FG-GL-CP-III-1 mainly consists of Ara, Man, Gal, and Glc (molar ratio: 0.85:1.00:0.81:0.76) in addition to a small amount of uronic acid. Methylation analysis indicates that FG-GL-CP-III-1 is composed mainly of terminal/4- or 5-linked Ara(f), terminal/4-linked Man, terminal/3,6-branched Gal, and terminal/4-linked Glc. NaIO4 oxidation of FG-GL-CP-III significantly decreased its activity (64.0-79.2%). These results suggest that solid-state culture of ginseng with G. lucidum may enhance the immunomodulatory activity and that neutral polysaccharides containing Ara, Man, Gal, and Glc may be important for the activity of fermented ginseng.

Peyer's patch-mediated intestinal immune system modulating activity of pectic-type polysaccharide from peel of Citrus unshiu.

An intestinal immune system modulating polysaccharide (CUI-3IIb-3-2, 18kDa) was purified from Citrus unshiu peel. CUI-3IIb-3-2 mainly comprised GalA, GlcA, Ara, Gal and Rha, and it consisted of 4-linked GalA, terminal Araf, 4- or 5-linked/3,4- or 3,5-branched Ara, terminal Gal, and 2-linked/2,4-branched Rha. After CUI-3IIb-3-2 digestion by endo-α-d-(1→4)-polygalacturonase, its hydrolysate was fractionated into PG-1 and PG-2. Methylation analyses of PG-1 and PG-2 using base-catalysed ß-elimination suggested that CUI-3IIb-3-2 be assumed as pectic-type polysaccharide. Since the activities of PG-1 and PG-2 were potently decreased, the whole polysaccharide structure of CUI-3IIb-3-2 would be essential to maintain the activity. Meanwhile, when CUI-3IIb was orally administered in mice, bone marrow cell proliferation and GM-CSF/IL-6 production from Peyer's patch cell were significantly higher (1.76- and 2.03/2.51-fold, respectively) than a saline. Therefore, a pectic-type polysaccharide from citrus peel could stimulate Peyer's patches and produce hematopoietic growth factors resulted in bone marrow cell proliferation.