Immunostimulating Polysaccharide Research Articles

Structural elucidation of immuno-stimulating polysaccharide, galactomannan isolated from Colocasia esculenta

Structural elucidation of immuno-stimulating polysaccharide, galactomannan isolated from Colocasia esculenta

Purification, structural analysis and mechanism of murine macrophage cell activation by sulfated polysaccharides from Cystoseira indica

Sulfated polysaccharides were isolated and purified from the water extract of Cystoseira indica using DEAE Sepharose Fast Flow column to evaluate their structure and macrophage stimulating capacity. Crude and fractionated polysaccharides, CIF1 and CIF2, were mostly composed of neutral sugars (73.1%–78.6%) with relatively lower amounts of acidic sugars (1.3%–9.0%) and sulfate esters (6.9%–9.7%). The polymer chains of polysaccharides were mainly built of different levels of glucose (2.1%–30.8%), fucose (17.2%–24.4%), mannose (17.8%–20.6%) and galactose (16.7%–17.3%). The weight average molecular weight (Mw) of polysaccharides varied between 573.1 × 103 g/mol to 1146.6 × 103 g/mol. The CIF2 polysaccharide, as the most immunostimulating polysaccharide, remarkably induced the release of nitric oxide and inflammatory cytokines including TNF-α, IL-1β, IL-6 and IL-10 from RAW264.7 murine macrophage cells through NF-κB and PAMKs transduction signaling pathways via cell surface TLR4. The interconnections of sugars in CIF2 polysaccharide were complex with (1→3)-fucopyranose, (1→2,3,4)-glucopyranose, (→1)-galactopyranose, (→1)-xylopyranose, (1→2)-rhamnopyranose and (1→2,3)-mannopyranose units being the most predominant residues.

Isolation and chemical characterization of a novel immunostimulating galactofucan from freshwater Azolla filiculoides

Water-soluble polysaccharides were isolated from Azolla filiculoides to determine their chemical and structural characteristics as well as anticancer and immunostimulatory activities. Crude and fractions (F1 and F2) were mainly composed of neutral sugars (70.0–80.1%), proteins (2.1–14.2%) and uronic acids (1.2–10.8%). The polysaccharides were mostly formed of different levels of fucose (23.8–61.2%), galactose (28.5–38.7%), mannose (7.5–16.7%), xylose (13.3–13.6%), glucose (12.7–13.3%), arabinose (5.5–11.6%) and rhamnose (8.0–9.5%) units. The polysaccharide molecules contained one or more sub-fractions with average molecular weight ranging from 992 to 2162 × 103 g/mol. Crude and fractionated polysaccharides induced RAW264.7 macrophages to release pro-inflammatory mediators and cytokines including nitric oxide, IL-1β, TNF-α, IL-6, IL-10 and IL-12 through NF-κB and MAPKs signaling pathways as confirmed by the presence of p-NK-κB, p-JNK, p-ERK and p-38 proteins in the cell cytoplasm. The most immunostimulating polysaccharide, F2, consisted of alternating →3)-Fuc-(1→ and →4)-Fuc-(1 → residues.

Water-soluble polysaccharides from Ulva intestinalis: Molecular properties, structural elucidation and immunomodulatory activities

Water-soluble sulfated polysaccharides extracted from Ulva intestinalis and fractionated using DEAE Sepharose fast flow column to identify their molecular properties and macrophage cells stimulating activities. Crude and fractions (F1 and F2) were formed of neutral sugars (58.7–74.7%), sulfates (6.2–24.5%), uronic acids (4.9–5.9%) and proteins (3.2–10.4%). Different levels of sugar constituents including rhamnose (30.1–39.1%), glucose (39.0–48.4%), galactose (0.0–15.8%), xylose (8.5–11.3) and arabinose (0.0–5.1%). The molecular weight (Mw) of crude and fractionated polysaccharides ranged from 87.1 × 103 to 194.1 × 103 (g/mol). Crude polysaccharides were not toxic to RAW264.7 cells and fractions induced cell proliferation. Fraction F1 stimulated RAW264.7 cells to release considerable amounts of nitric oxide, IL-1β, TNF-α, IL-6, IL-10 and IL-12 cytokines. The main backbone of the most immunostimulating polysaccharide (F1) was consisted of mixed linkages of (1 → 2)-linked rhamnose and (1 → 2)-linked glucose residues.

Structural characterization of an immunostimulating polysaccharide from the stems of a new medicinal Dendrobium species: Dendrobium Taiseed Tosnobile

Dendrobium Taiseed Tosnobile, a new Dendrobium species developed by crossbreeding Dendrobium tosaense and Dendrobium nobile, exhibits the characteristics of high mass production and high polysaccharide content. This study investigated the structural characterization and immunostimulating effects of a polysaccharide isolated from D. Taiseed Tosnobile (DTTPS). DTTPS was fractioned using a DEAE–650M column to obtain the major neutral polysaccharide (DTTPS-N). The structural characteristics of DTTPS-N were investigated through high-performance anion exchange chromatography, high-performance size exclusion chromatography, gas chromatography–mass spectrometry, and nuclear magnetic resonance spectroscopy. In the immunostimulating experiment, BALB/c mice were administered DTTPS (100 and 300mg/kg) daily for 3 weeks. The results revealed that DTTPS-N comprised arabinose, galactose, glucose, mannose, and xylose at a ratio of 1:1.5:3.0:29.9:1.3. DTTPS-N comprised (1→3; 1→4)-Man as the backbone, and its average molecular weight was 281kDa. Pharmacological experiments demonstrated that DTTPS substantially increased the population of splenic natural killer (NK) cells, NK cytotoxicity, macrophage phagocytosis, and cytokine induction. This is the first study to demonstrate the structural characteristics and immunopharmacological effects of an active polysaccharide derived from D. Taiseed Tosnobile.

Structure elucidation of an immunostimulatory arabinoxylan-type polysaccharide prepared from young barley leaves (Hordeum vulgare L.)

We recently isolated an immunostimulating polysaccharide (BLE-P-I), which possesses a large proportion of arabinoxylan, from young barley leaves. In the present study, to elucidate the structural details of BLE-P-I, it was fractionated into enzyme-resistant fraction (BLE-P-I-X1) and arabinoxylan-rich oligosaccharide fraction (BLE-P-I-X2) after endo-xylanase hydrolysis. Commercial wheat arabinoxylan was also fractionated into WAX-XI and WAX-X2 after the same treatment. BLE-P-I-X2 consisted of arabinose and xylose, including 11 types of neutral glycosidic linkages, whereas WAX-X2 had less, with 6 types of linkages. Mass spectrometric results indicated that WAX-X2 was composed of a xylan backbone bearing only arabinose. In contrast, BLE-P-I-X2 consisted of a xylan backbone with acetate, arabinose, galactose, glucuronic acid, and 4-O-methylglucuronic acid, resembling structural characteristics as glucuronoarabinoxylan. Macrophage-stimulatory activity showed that BLE-P-I-X2 has a role in the expression of the activity. These results indicate that the structural complexity of arabinoxylan seems to be responsible for the immunomodulatory activity in barley leaf.

Isolation and purification of a polysaccharide from the caterpillar medicinal mushroom Cordyceps militaris (Ascomycetes) fruit bodies and its immunomodulation of RAW 264.7 macrophages.

A novel polysaccharide (CP2-S) was purified from Cordyceps militaris fruit bodies by hot water extraction, ethanol precipitation, DEAE-Sepharose Fast Flow and Sephacryl S-400 high-resolution chromatography. The polysaccharide had a molecular weight of 5.938 × 10(6) g/mol and was mainly composed of glucose. CP2-S had carbohydrate content estimated to be 100% using the phenol-sulfuric acid method. Immunostimulating experiments in vitro indicated that CP2-S could stimulate nitric oxide production, phagocytosis, respiratory burst activity, and secretion of interleukin-1β and interleukin-2 of macrophages, suggesting that this water-soluble polysaccharide from the fruit body of C. militaris is a natural immunostimulating polysaccharide with potential for further application.

감잎에서 분리한 다당의 면역자극 활성

Abstract In order to develop new physiologically active polysaccharides from persimmon leaves, two different crude polysaccharides were prepared using hot water (PLW-0) and pectinase digestion (PLE-0) and their immuno-stimulating activities were estimated. PLW-0 and PLE-0 showed similar sugar compositions with 15 different sugars, including rarely observed sugars in general polysaccharides 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, but the uronic acid content of PLE-0 was lower than that of PLW-0 caused by pectinase treatment. Both PLW-0 and PLE-0 showed potent anti-complementary activity in a dose-dependent manner which was similar to a known immuno-stimulating polysaccharide, PSK, from Coriolus versicolor. The activity of PLE-0 at a low concentration (100 ㎍/㎖) was higher than that of PLW-0. In an in vitro cytotoxicity analysis, PLW-0 and PLE-0 (up to 1,000 ㎍/㎖) did not affect the growth of peritoneal macrophages and Colon 26-M3.1 carcinoma cells. In contrast, they enhanced lymphocyte proliferation activity. Peritoneal macrophages stimulated with PLW-0 and PLE-0 produced various cytokines, such as IL-6 and IL-12. However, PLE-0 was more effective on the cytokine production. Intravenous administration of PLW-0 and PLE-0 significantly augmented natural killer (NK) cell cytotoxicity against Yac-1 tumor cells 3 days after the treatment of polysaccharide fractions. But NK cells obtained from the PLE-treated group showed higher tumoricidal activity even at a low dose of 40 ㎍/mouse. In experimental lung metastasis of Colon 26-M3.1 carcinoma cells, prophylactic administration of PLW-0 and PLE-0 significantly inhibited lung metastasis in a dose-dependent manner and PLE-0 was more effective on the inhibition of cancer metasasis. The results lead us to conclude that the pectinase-treated process is indispensable to preparing polysaccharides with higher immune-stimulating activity from persimmon leaves.Key words: persimmon leaves, polysaccharide, immune-stimulating activity, anti-metastatic activity

추출조건에 따른 차가버섯 생리활성 및 면역활성 다당

To investigate the pharmacological activity of chaga mushroom (Inonotus obliquus) on extraction conditions, chaga was extracted using water (reflux at 50℃, decoction over 90℃, pressure at 121℃) or ethanol (reflux at 50, 70, or 90℃). When water extract was further fractionated into crude polysaccharide (IO-CP), yields of IO-CP (4.8～16.8%) were higher than those of ethanolic extracts (IO-E, 1.9～2.7%) at increased temperature. For antioxidant activity, crude polysaccharide (IO-CP-121) obtained by pressurized extraction showed the highest polyphenolic and flavonoid contents (35.10 mg TAE/g and 18.48 mg QE/g, respectively) as well as DPPH and ABTS free radical scavenging activities (26.08 and 27.99 mg AEAC/100 mg, respectively). Meanwhile, IO-CP-D (decoction) and IO-CP-50 (reflux) had more potent mitogenic effects (2.10- and 1.95-fold of saline control at 100 μg/mL) as well as intestinal immune system modulating activities (6.30- and 5.74-fold) compared to IO-CP-121, whereas ethanolic extracts showed no activity. Although no IO-CP showed cytotoxicity against RAW 264.7 cells at 0.1 mg/mL, IO-CP-121 significantly inhibited TNF-α and NO production as pro-inflammatory factors in LPS-stimulated RAW 264.7 cells (29.2 and 63.5%, respectively). Ethanolic extracts also showed no cytotoxicity at 0.1 mg/mL, whereas inhibition of TNF-α and NO production was significantly low compared to that of IO-CP-121. In addition, active IO-CP-D was further fractionated into an unadsorbed (IO-CP-I) and seven adsorbed fractions (IO-CP-II～VIII) by DEAE-Sepharose CL-6B column chromatography in order to isolate immunostimulating polysaccharide. IO-CP-II showed the most potent mitogenic effect and macrophage stimulating activity (4.51- and 1.64-fold, respectively). IO-CP-II mainly contained neutral sugars (61.86%) in addition to a small amount of uronic acid (2.96%), and component sugar analysis showed that IO-CP-II consisted mainly of Glc, Gal, and Man (molar ratio of 1.00:0.55:0.31). Therefore, extraction conditions affect the physiological activity of chaga, and immunostimulating polysaccharide fractionated from chaga by decoction is composed mainly of neutral sugars.

Isolation and characterization of immuno-stimulating polysaccharide in Korean black raspberry wine

To characterize polysaccharides existing as soluble form in Korean fruit wines, polysaccharides were isolated from Korean black raspberry wine, and their immuno-stimulating activities were examined. A complex pectic polysaccharide, RB-2 was purified from the crude polysaccharide (RB-0) in raspberry wine by subsequent size-exclusion chromatography. RB-2 had relatively potent macrophage activity and splenocyte mitogenic activity in vitro. The intravenous administration of RB-2 significantly augmented the cytotoxicity of natural killer cells against Yac-1 tumor cells. RB-2 consisted of 15 monosaccharides including rarely observed sugars in general polysaccharides, 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. Methylation analysis indicated that RB-2 comprised at least 20 different glycosyl linkages such as 3,4-linked fucose, 2,3,4-linked rhamnose, 3′-linked apiose, and 2,3,3′-linked apiose, characteristic of rhamnogalacturonan II (RGII). RB-2 was eluted as two peaks having a high (9,000 Da) and a low molecular mass (5,000 Da) on high performance size exclusion chromatography, and the peak of high molecular mass was down shifted to the low molecular mass under the acidic pH conditions less than 2.0. Results indicated higher molecular weight polysaccharide in RB-2 was mainly presented as an RG-II dimer, and Korean black raspberry wine contains selected polysaccharides, which provide immunostimulating activities beneficial to human health.

Structural characterization of immunostimulating polysaccharide from cultured mycelia of Cordyceps militaris

The water soluble crude polysaccharide obtained from cultured mycelia of Cordyceps militaris (CPM) by hot water extraction followed by ethanol precipitation was fractionated by DEAE cellulose and Sepharose CL-6B column chromatography. This fractionation process resulted in four polysaccharide fractions that were termed CPMN Fr I, CPMN Fr II, CPMN Fr III, and CPMN Fr IV. Of these fractions, CPMN Fr III was able to upregulate the functional events mediated by activated macrophages, such as production of nitric oxide (NO) and expression of cytokines (IL-1β and TNF-α). Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and gas chromatography–mass spectrometry (GC–MS). Results indicate that CPMN Fr III was a high molecular mass polysaccharide with a random coil conformation of the β-1,4-branched-β-1,6-galactoglucomannan.

Analysis of the Chemical Constituents of Agaricus brasiliensis

This study examined the chemical composition of A. blasiliensis and the chemical structural properties of an immuno-stimulating polysaccharide. The amino acids, free sugars, and organic acids by HPLC and fatty acids by GC were analyzed. The immuno-stimulating substance from A. blasiliensis was extracted with hot water and purified by ethanol precipitation. It underwent ion exchange chromatography on DEAE-cellulose and gel filtration on Toyopearl HW 65 F. Through GP-HPLC, the substance was found to be homogeneous. Its chemical structure was determined by 13 C-NMR. Fatty acids, organic acids, and sugar alcohol composition consisted exclusively of linoleic acid, fumaric acid and mannitol, respectively. The amino acids were mainly glutamic acid, glycine, and arginine. By 13 C-NMR analysis, the immuno-stimulating substance was identified as β-(1→3) (1→6)-glucan, composed of a backbone with (1→3)-linked D-glucopyranosyl residues branching a (1→6)-linked D-glucopyranosyl residue. The β-glucan from A. blasiliensis showed pronounced immuno-stimulating activity on the antibody-production ability of B-lymphocytes by the hemolytic suspension assay. In these results, A. blasiliensis was estimated to have potent pharmacological properties and potential nutritional values.

Isolation and structural characterization of an immunostimulating polysaccharide from fuzi, Aconitum carmichaeli

A water-soluble polysaccharide named as FPS-1 was isolated from fuzi, the root of Aconitum carmichaeli Debx. by hot-water extraction, anion-exchange and gel-permeation chromatography and tested for its pharmacological activities. Its structural characteristics were investigated by FTIR, HPLC, NMR spectroscopy, methylation analysis and GLC–MS. Based on the data obtained, FPS-1 was determined to be an α-(1→6)- d-glucan, with a weight-average molecular weight of about 14,000 Da. The glucan is highly branched with a single glucose at the C-3 position every four residues, on average, along the main chain. In immunopharmacological studies, FPS-1 showed potent stimulating effects on murine lymphocyte proliferation induced by concanavalin A or lipopolysaccharide both in vitro and in vivo as well as on splenocyte antibody production.

Immuno-stimulating effect of the endo-polysaccharide produced by submerged culture of Inonotus obliquus

Inonotus obliquus BELYU1102 was selected from 12 different strains of Inonotus as a producer of immuno-stimulating polysaccharide. After a batch fermentation of I. obliquus BELYU1102 was carried out in a 300 l pilot vessel, endo-polysaccharide and exo-polysaccharide were both obtained. The proliferation activity of endo-polysaccharide for splenic cells was much higher than the activity of exo-polysaccharide. The active endo-polysaccharide was produced primarily during the late stationary phase. Enhanced proliferation and polyclonal IgM antibody production were observed in B cells by purified water-soluble endo-polysaccharide. Nitrite production and expression of IL-1β, IL-6, TNF-α, and iNOS in macrophages were also enhanced. However, the endo-polysaccharide did not affect the proliferation of T cells, the IL-2 expression of Th1 cells, or the IL-4 expression of Th2 cells. The endo-polysaccharide showed activities similar to lipopolysaccharide (LPS) for B cells and macrophages, but there was a large difference between the two polysaccharides because cellular activations induced by endo-polysaccharide were not affected by polymyxin B, a specific inhibitor of LPS. The endo-polysaccharide appeared to have other cellular binding sites with TLR-4 and did not show a direct toxicity against tumor cells. However, indirect anti-cancer effects via immuno-stimulation were observed. The mycelial endo-polysaccharide of I. obliquus is a candidate for use as an immune response modifier. Submerged mycelial cultures are advantageous for industrial production of polysaccharides.

Natural vitamin E enrichment of Artemia salina fed freshwater and marine microalgae

Three species of microalga, the freshwater Euglena gracilis and the marine Dunaliella salina and Tetraselmis suecica, were compared in terms of vitamin E enrichment and survival and growth of the brine shrimp Artemia salina. The tocopherol content was investigated using HPLC for the post-larval and pre-adult stages of Artemia fed the microalgae and the results compared to the initial content of unfed newly hatched nauplii. There was a markedly higher content of tocopherols (about two-fold) in Artemia fed Euglena. Since this microalga contains other antioxidants such as β-carotene, vitamin C and glutathione, bioactive molecules such as PUFA, and the immunostimulant polysaccharide β-glucan, it represents a valuable alternative for enriching the diets of Artemia and increase its nutritional value as a food item.

Synthesis of the HSA-conjugate of the S-linked thiomimetic of the branched tetrasaccharide repeating unit of the immunostimulant polysaccharide, schizophyllan. Evaluation as potential immunomodulator

The conjugate with human serum albumin (HSA) of the S-linked thioanalogue of the branched tetrasaccharide repeating unit of the polysaccharide, schizophyllan, was synthesized from 1,2,4,6-tetra- O-acetyl-3- S-[2,4-di- O-acetyl-3,6-di- S-(2,3,4,6-tetra- O-acetyl-β- d-glucopyranosyl)-3,6-dithio-β- d-glucopyranosyl]-3-thio-β- d-glucopyranose [M.O. Contour-Galcera et al., Carbohydr. Res., 281 (1996) 119–128] in five steps, and its potential immunomodulatory activity was evaluated in human blood mononuclear cells. The protein glycoconjugate did not effect proliferation or production of IL-4, IL-5 and IFN-g in a significant way.

Immunostimulating polysaccharide from cell culture of Angelica gigas Nakai

An immunostimulating polysaccharide was produced extracellularly by suspension cell culture of Angelica gigas Nakai. The polysaccharide was larger in molecular weight than that obtained from the extract of A. gigas root. It was mainly composed of arabinose, galactose, galacturonic acid, protein, Ca2+ and Mg2+, and stimulated the mixed lymphocyte reaction activity as potently as the polysaccharide obtained from the plant root.

Stereocontrolled synthesis of sulfur-linked analogues of the branched tetrasaccharide repeating-unit of the immunostimulant polysaccharide schizophyllan and of its β-(1 → 3)-branched, β-(1 → 6)-linked isomer

The branched, sulfur-linked tetrasaccharide S-(β- d- glucopyranosyl)-(1 → 3)-S-[(6-S-β- d- glucopyranosyl)-3,6- dithio-β- d- glucopyranosyl]-(1 → 3)-S-3- thio- d- glucopyranose ( 9) has been conveniently prepared by S N2 displacement of the triflate group in 1,2:5,6- di-O- isopropylidene-3-O- trifluoromethylsulfonyl-α- d- allofuranose with the sodium salt of 2,4- di-O- acetyl-3,6- di-S-(2,3,4,6- tetra-O- acetyl-β- d- glucopyranosyl)-1,3,6- trithio-β- d- glucopyranose ( 5). Conversely, reaction of the sodium salt of 5 with 1,2,3,4- tetra-O- acetyl-6- deoxy-6- iodo-β- d- glucopyranose afforded the positional isomer S-(β- d- glucopyranosyl)-(1 → 6)-S-[(3-S-β- d- glucopyranosyl)-3,6- dithio-β- d- glucopyranosyl]-(1 → 6)-S-6- thin- d- glucopyranose ( 12).

Lymphocyte activation by a polysaccharide fraction separated from hot water extracts of Angelica acutiloba Kitagawa.

The action of a polysaccharide fraction obtained from hot water extracts of Angelica acutiloba Kitagawa, termed as Angelica immunostimulating polysaccharide (AIP) fraction, on murine lymphocytes participating in antibody responses was investigated. When AIP fraction was injected concomitantly into mice immunized with antigens, immunoglobulin M (IgM) and IgG antibody responses against sheep erythrocytes (SRBC) increased significantly, but IgM response against T-independent antigens such as trinitrophenylated lipopolysaccharide (TNP-LPS) and TNP-Ficoll did not augment. Murine B lymphocytes were polyclonally activated in vitro and in vivo by AIP fraction to differentiate into antibody-forming cells as functionally matured cells. The differentiation of B lymphocytes to an intermediate stage capable of responding to helper T lymphocytes was also stimulated by the administration of AIP fraction into CDF1 and C3H/HeJ mice. A concomitant injection of AIP fraction with SRBC for carrier priming resulted in the increment of anti-TNP IgM antibody response in cultured reconstituted with unprimed B and SRBC-primed T lymphocytes, indicating that AIP fraction can stimulate T lymphocytes.

Activation of the classical complement pathway by a polysaccharide from sugar cane

The effects of an immunostimulating polysaccharide, Bo, from sugar cane, on the complement system have been investigated. Bo, a glucan of about 10,000 mol wt, was found to activate complement in whole human and guinea pig serum in vitro by the classical pathway. Complement consumption was also demonstrated in guinea pigs upon intravenous injection. Specifically, C1 is activated, and C4 and C2, as well as C3, are consumed. The activation is prevented when Ca++ ions are chelated by ethyleneglycoltetraacetic acid, and when C1q is lacking. Hence, it does not rest on direct activation of C1s. Supplementation of C1q-deficient human serum with purified C1q restores the ability to be activated by Bo. The alternative pathway of complement is little if at all affected by the polysaccharide. The activation of C1 seems to be mediated by immune complex formation between Bo and naturally occurring immunoglobulins. Complement in sera from two severely hypogammaglobulinemic patients was not activated by Bo, but was made reactive by addition of purified human immunoglobulin G.