Polysaccharide-containing Fractions Research Articles

Polysaccharide-containing fraction from Artemisia argyi inhibits tumor cell-induced platelet aggregation by blocking interaction of podoplanin with C-type lectin-like receptor 2

Hepatocellular carcinoma (HCC) is one of the most common malignancies in Taiwan. Many risks factors induce liver chronic inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Mulberry fruits containing polyphenols to remove free radicals and mitigate inflammation has been reported to not only against gastric cancer, melanoma and leukemia but also prevent liver injury induced by alcohol or CCl4 in previous researches. The aim of this study is to examine whether Mulberry could inhibit hepatocarcinogenesis. In animal experiment, diethylnitrosamine (DEN) was used to induce hepatic tumorgenesis. After injecting DEN, the rats treated with mulberry water extracts (MWE) had less and smaller tumor than others without MWE. Moreover, MWE reduced the serum ALT and AST, HCC marker, cleavage caspases, Ser-15-p53 and Ser46-p53 induced by DEN. Further, we observed that mulberry polyphenol extracts (MPE) inhibited the cell growth of HepG2 cell and Hep3B cell. By using flow cytometry and western blotting methods, MPE induced HepG2 cell apoptosis by increase subG1 cells and the elevated expression of caspase-3/8/9. Instead of apoptosis, MPE caused Hep3B cells autophagy by inhibiting Akt and mTOR phosphorylation. Comprehensively, mulberry extracts has a potential to be a health supplement to prevent hepatocarcinogenesis in the future. © 2019

A mechanistic and empirical review of antcins, a new class of phytosterols of formosan fungi origin

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism that involves the protection of tumor cells in the circulation and the promotion of tumor cell invasion and metastases. The C-type lectin-like receptor 2 (CLEC-2) that binds podoplanin (PDPN) is on the platelet surface and facilitates the TCIPA. Selective blockage of the PDPN-mediated platelet-tumor cell interaction is thereby a plausible strategy for inhibiting metastases. In a search for antagonists of PDPN- and tumor cell-induced platelet aggregation, traditional Chinese medicines were screened and it was found that the water extract of Artemisia argyi leaves selectively inhibited the PDPN-induced platelet aggregation. Bioactivity-guided fractionation analysis was performed for defining a polysaccharide-containing fraction (AAWAP) characterized by inhibition of PDPN activity and tumor cell-induced platelet aggregation. The pharmacological effects of AAWAP on PDPN-activated CLEC-2 signaling were determined by using Western blot and alpha screening analyses. AAWAP was non-toxic to the cells and platelets and it suppressed PDPN- and tumor cell-induced platelet aggregation by irreversibly blocking the interaction between PDPN and CLEC-2 in a dose-dependent manner. These findings indicate that AAWAP is an antagonist of the PDPN-CLEC-2 interaction. This action by AAWAP may result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients. © 2019

Cryoprotective Properties of the Polysaccharide Fraction of the Mushroom Hericium erinaceus BP 16

The natural isolate of xylotrophic basidiomycetes Hericium erinaceus BP 16 was cultivated artificially to produce fruiting bodies from which the polysaccharide-containing fraction (PF) was isolated by extraction with hot water (70°C), followed by precipitation with 96% ethanol. Residues of rhamnose, fructose, xylose, arabinose, mannose, glucose, and galactose in the ratio of 7 : 8 : 1 : 19 : 14 : 26 : 27, respectively, were identified in the composition of the PF carbohydrate chains. The effect of adding PF to aqueous solutions of cryoprotectants during water freezing in human venous blood during its cryopreservation has been demonstrated. It was found that the presence of PF (0.25%) in a glycerin-containing (3.5%) preservative ensures the safety of human nuclear blood cells at −20 and −80°C, which indicated the cryoprotective activity of PF H. erinaceus BP 16. Thus, this property of PF may be used in the development of new substances to freeze biological samples.

Polysaccharide-containing fraction from Artemisia argyi inhibits tumor cell-induced platelet aggregation by blocking interaction of podoplanin with C-type lectin-like receptor 2

Tumor cell-induced platelet aggregation (TCIPA) is a mechanism that involves the protection of tumor cells in the circulation and the promotion of tumor cell invasion and metastases. The C-type lectin-like receptor 2 (CLEC-2) that binds podoplanin (PDPN) is on the platelet surface and facilitates the TCIPA. Selective blockage of the PDPN-mediated platelet-tumor cell interaction is thereby a plausible strategy for inhibiting metastases. In a search for antagonists of PDPN- and tumor cell-induced platelet aggregation, traditional Chinese medicines were screened and it was found that the water extract of Artemisia argyi leaves selectively inhibited the PDPN-induced platelet aggregation. Bioactivity-guided fractionation analysis was performed for defining a polysaccharide-containing fraction (AAWAP) characterized by inhibition of PDPN activity and tumor cell-induced platelet aggregation. The pharmacological effects of AAWAP on PDPN-activated CLEC-2 signaling were determined by using Western blot and alpha screening analyses. AAWAP was non-toxic to the cells and platelets and it suppressed PDPN- and tumor cell-induced platelet aggregation by irreversibly blocking the interaction between PDPN and CLEC-2 in a dose-dependent manner. These findings indicate that AAWAP is an antagonist of the PDPN-CLEC-2 interaction. This action by AAWAP may result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients.

Structural Elements of an Antioxidative Pectic Arabinogalactan from Solanum virginianum

The water-soluble polysaccharide-containing fraction from Solanum virginianum leaves upon anion exchange chromatography yielded a highly branched pectic arabinogalactan-containing fraction (F2). Herein, direct evidences for the (i) presence of a chain having 1,6-linked Gal units substituted at O-3, (ii) coexistence of Ara and Gal residues in the same molecule, (iii) existence of a chain containing 1,3-linked Gal p residues substituted at C-6, and (iv) occurrence of 1,5-linked Ara f residues substituted at O-3 were presented. This polysaccharide that showed a dose-dependent antioxidative property formed a water-soluble complex with bovine serum albumin. Thus, F2 could be used as a natural ingredient in functional food and pharmaceutical products to mollify oxidative stress.

Structural Features and Complement-Fixing Activity of Pectin from Three Brassica oleracea Varieties: White Cabbage, Kale, and Red Kale

Leaves of different cabbage species are used both as food and as wound healing remedies in traditional medicine. This supposed wound healing activity might be connected to presence of immunomodulating water soluble polysaccharides. To study this, three different cabbage varieties, white cabbage (W), kale (K), and red kale (RK), were pretreated with 80% ethanol and then extracted with water at 50 degrees C and 100 degrees C for isolation of polysaccharide-containing fractions. The fractions were analyzed for monosaccharide composition, glycosidic linkages, Mw distribution, protein content, and phenolic compounds and then tested for complement-fixing activity. All fractions contained pectin type polysaccharides with linkages corresponding to homogalacturonan and hairy regions. Those extracted at 50 degrees C contained higher amounts of neutral side chains and were more active in the complement-fixation test than those extracted at 100 degrees C. The fractions can be ranged by decreasing activity: K-50 > RK-50 > W-50 approximately = K-100 > RK100 approximately = W-100. Studies on structure-activity relationships (SAR) employing multivariate statistical analysis strongly suggest that the magnitude of the measured activity is influenced by the content of certain side chains in the polymers. High activity correlates to large neutral side chains with high amounts of (1-->6)- and (1-->3,6)-linked Gal and low amounts of (1-->4)-linked GalA but not on molecular weight distribution of the polymers.

Se-Enriched Mycelia of Pleurotus ostreatus: Distribution of Selenium in Cell Walls and Cell Membranes/Cytosol

The incorporation of Se to fungi has been studied, focusing on element distribution among different cellular compartments and, in particular, polysaccharide structures contained in cell walls. Se-enriched mycelia of Pleurotus ostreatus were obtained in submerged cultures. The incorporation of selenium from the growth medium to mycelia was observed with the relative distribution between cytosol plus cell membranes fraction (CCM) and cell walls fraction (CW) of about 44 and 56%, respectively. CCM fractions were analyzed by size exclusion chromatography with on-line UV (280 nm) and ICP-MS detection (80Se). The results obtained showed selenium binding to components of different molecular masses (about 24% of total selenium coeluted with the compounds of molecular mass > 10 kDa). A polysaccharide-containing fraction of mycelia was treated alternatively with Tris-HCl at pH 7.5 or with chitinase. Better solubility and increased contribution of low molecular mass compounds were observed in chitinase extracts (UV detection), confirming the degradation of polysacharides by the enzyme. The total area under the ICP-MS chromatogram of chitinase extract was 2 times higher with respect to the area for Tris-HCl extract. Furthermore, the relative contribution of selenium in the low molecular mass fraction (molecular mass < 1 kDa) in chitinase extract was 72% as compared to 45% in Tris-HCl extract (based on peak area measurements with respect to total area under the chromatogram). The results obtained suggest selenium binding to chitin-containing polysaccharide structures in fungi cell walls.

Synergistic Antioxidative Effects of Alkamides, Caffeic Acid Derivatives, and Polysaccharide Fractions from Echinacea purpurea on in Vitro Oxidation of Human Low-Density Lipoproteins

Preparations of Echinacea are widely used as alternative remedies to prevent the common cold and infections in the upper respiratory tract. After extraction, fractionation, and isolation, the antioxidant activity of three extracts, one alkamide fraction, four polysaccharide-containing fractions, and three caffeic acid derivatives from Echinacea purpurea root was evaluated by measuring their inhibition of in vitro Cu(II)-catalyzed oxidation of human low-density lipoprotein (LDL). The antioxidant activities of the isolated caffeic acid derivatives were compared to those of echinacoside, caffeic acid, and rosmarinic acid for reference. The order of antioxidant activity of the tested substances was cichoric acid > echinacoside > or = derivative II > or = caffeic acid > or = rosmarinic acid > derivative I. Among the extracts the 80% aqueous ethanolic extract exhibited a 10 times longer lag phase prolongation (LPP) than the 50% ethanolic extract, which in turn exhibited a longer LPP than the water extract. Following ion-exchange chromatography of the water extract, the majority of its antioxidant activity was found in the latest eluted fraction (H2O-acidic 3). The antioxidant activity of the tested Echinacea extracts, fractions, and isolated compounds was dose dependent. Synergistic antioxidant effects of Echinacea constituents were found when cichoric acid (major caffeic acid derivative in E. purpurea) or echinacoside (major caffeic acid derivative in Echinacea pallida and Echinacea angustifolia) were combined with a natural mixture of alkamides and/or a water extract containing the high molecular weight compounds. This contributes to the hypothesis that the physiologically beneficial effects of Echinacea are exerted by the multitude of constituents present in the preparations.

Linear β-mannose-containing polysaccharide, β-xylan, and amylose from the cultured photobiont Trebouxia sp. of the ascolichen Ramalina celastri

The cultured photobiont Trebouxia sp. of Ramalina celastri was successively extracted at 100°C with hot water, 2% aqueous KOH, and 10% aqueous KOH to give polysaccharide-containing fractions A (2.9%), B (3.9%), and C (0.9% yield) respectively. The intact biont contained 3.8% amylose, which was present in each fraction, and was identified by a blue color formed with iodine solution. In fraction A, and following retrogradation from aqueous solution, it was characterized by 13C-NMR spectroscopy. Fraction B was treated with α-amylase to give a water-soluble fraction consisting mainly of β-mannose-containing polysaccharides (1.5% yield), whose main component had d n/d c 0.162 and M r 17 kDa. Fraction C was subjected to freeze–thawing and the precipitate was treated with α-amylase to give a resistant, linear, low molecular mass (1→4)-linked β-xylan. The β- D-mannopyranan preparation contained mainly of 3-O- (28%), 4-O- (11%), and 6-O-substituted Man p units (35%), with 3-O-substituted Rha p units (11%). A controlled Smith degradation provided a β-mannan with nonreducing end- (8%), 3-O- (85%) and 6-O-substituted units, showing (1→3)- and (1→6)-linked structures in the original polysaccharide. These could be present as block-type structures.

A natural inhibitor of lignin peroxidase activity from Phanerochaete chrysosporium, active at low pH and inactivated by divalent metal ions

The pH optimum of a crude preparation of lignin peroxidase was pH 3.1, whereas those of the three main isozymes of the enzyme purified from it were pH 2.2, pH 2.7 and pH 2.0. During the purification of the crude enzyme, an anionic polysaccharide containing fraction (PCF) was also separated. The latter was found to inhibit lignin peroxidase activity at pH values less than pH 3.2, thus resulting in a shift in the pH optimum of the purified isozymes back to a similar value as that obtained for the crude enzyme. Addition of divalent metal ions at 1.0 mM relieved the inhibition.

Serological characterization of Streptococcus mutans serotype polysaccharide g and its different molecular weight forms.

The serotype polysaccharide g from Streptococcus mutans 6715 was found to cross-react with serotype polysaccharide a from S. mutans HS6 and serotype polysaccharide d from S. mutans B13. Double immunodiffusion experiments indicated that the serotype polysaccharide g consisted of the following: (i) the type-specific g site; (ii) a cross-reactive site g-a that was in common with polysaccharide a; (iii) a cross-reactive site g-d that was in common with polysaccharide d; and (iv) a cross-reactive site g-(a-d) that was in common with both polysaccharides a and d. Moreover, by a procedure involving several column chromatography steps, six polysaccharide-containing fractions showing reactivity with anti-g serum were found. By gel filtration, the molecular weight estimates of fractions LI, LII, LIII, SI, SII, and SIII were 2 X 10(6), 5 X 10(5), 6 X 10(4), 3 X 10(4), 1.4 X 10(4), and 1 X 10(4), respectively. Double immunodiffusion analysis indicated that LI, LII, and LIII contained the four antigenic sites of the putative polysaccharide g. LII also contained another additional immunodominant region, designated site x. The analysis also suggested that fraction SI lacked the type-specific site g, fraction SII lacked sites g and g-a, and fraction SIII lacked sites g, g-a, and g-d.

Survey of taurine uptake and metabolism in Staphylococcus aureus.

Taurine has been reported to be a component of the capsular polysaccharide of the encapsulated M strain of Staphylococcus aureus. This led to a study of the uptake and metabolism of [1,2-14C]taurine in a variety of encapsulated and unencapsulated S. aureus strains. Taurine was taken up by all strains studied. A discrepancy between uptake measured as depletion of radioactivity from growth medium and as cell-associated radioactivity suggested that taurine may be catabolized to CO2 in some strains. In most strains, cell-associated radioactivity was located mainly in cold TCA-soluble (pool metabolites) fractions. About 90% of the cell-associated radioactivity was present in the pool metabolites fraction in the M strain, and about 10% in hot TCA-soluble (nucleic acid-teichoic acid-capsular polysaccharide) fraction. Radioactivity in spent medium and the capsular polysaccharide-containing fraction appeared to be present as taurine in this strain. Radioactivity in the pool metabolites fraction of three of the strains examined did not chromatograph as taurine, indicating that taurine was converted into other cell metabolites. One strain incorporated radioactivity from taurine into cellular macromolecules, thus revealing a heterogeneity of staphylococcal taurine metabolism.

The affinity of bacterial polysaccharide-containing fractions for mammalian cell membranes and its relationship to immunopotentiating activity

The natural affinity of various bacterial glycopeptides and lipopolysaccharides for mammalian cell membranes was estimated quantitatively by comparison with the adsorption of lipopolysaccharide from Escherichia coli NCTC 8623 to erythrocytes, thymocytes, bone marrow cells, spleen cells, peritoneal lymphocytes and macrophages. Immunopotentiating activity was estimated by measuring the ability of the bacterial fractions to stimulate a humoral response to ovalbumin in HAM/1CR mice. When the affinity for mammalian cell membranes was compared with the stimulation of the antibody response, it was found that a negative correlation for peritoneal macrophages ( r s =−0.94, P<0.0005 ) and a positive correlation for peritoneal lymphocytes ( r s =+0.97,P<0.0005 ) and spleen cells ( r s =+0.76,P<0.005 ) existed.

Lectin, a possible basis for symbiosis between bacteria and sponges.

From the marine sponge Halichondria panicea a lectin was isolated and characterized. The homogeneous lectin (composed of protein to 80.7% and of neutral carbohydrates to 14.1%) had a molecular weight of 78,000 (determined by gel filtration) and consisted of four subunits with a molecular weight of 21,000 each (determined by gel electrophoresis in the presence of sodium dodecyl sulfate). The hemagglutinating activity was only slightly dependent upon ionic strength and incubation temperature and did not require divalent cations, but it was inhibited by reagents for thiol groups. The Halichondria lectin was completely inhibited in hemagglutination competition experiments in the presence of fetuin, D-galacturonic acid, D-glucuronic acid, polygalacturonic acid, or L-fucose. The purified Halichondria lectin did not cause reaggregation of dissociated H. panicea cells. From the same sponge species bacteria were isolated and identified as Pseudomonas insolita. These bacteria were cultivated in marine broth 2216. Under these culture conditions the bacteria grew only in the presence of the homologous lectin; the lectin-caused effect was not abolished by D-glucuronic acid or D-galacturonic acid. However, after addition of a polysaccharide-containing fraction isolated from P. insolita, the lectin-caused, growth-promoting effect was abolished. Other lectins were found to exhibit no growth-promoting effect. On the basis of colony counts, P. insolita was the predominant bacterial species in the sponge extract; 1.9 X 10(6) Pseudomonas colonies were measured in extracts isolated from 1 g of sponge. The assumption of an interrelationship between the sponge and the bacterium is supported by the results indicating that the Halichondria lectin has no effect on the growth of such bacteria isolated from six other marine sponge species. Evidence is presented which indicates that the Halichondria lectin is not utilized during growth of the Pseudomonas species. Lectin activity was detected on the surface of mucoid cells from H. panicea. From the data obtained the possibility is discussed that the Halichondria lectin is a basis for a symbiotic relationship between the sponge and the bacterium.

A Carbohydrate-Containing Antigen from Trypanosoma cruzi and Its Detection in the Circulation of Infected Mice

Abstract A polysaccharide-containing fraction has been prepared from the water-soluble material of a phenol-water extract of epimastigote forms of Trypanosoma cruzi. Sera from rabbits immunized with this material have been used to detect trypanosome-derived polysaccharide in plasma of mice acutely infected with this parasite. The polysaccharide nature of the circulating antigen is suggested by its heat stability, resistance to proteinase and nucleases, and destruction by sodium metaperiodate, as well as by its periodic acid-Schiff staining and precipitation with concanavalin A. The antigen appears to be present in different strains of the parasite and in different stages in the life cycle of the parasite.

Heterogeneity of the lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from pseudomonas aeruginosa PAC1 and its phage-resistant mutant was degraded by mild acid hydrolysis into lipid A and three major polysaccharide-containing fractions which were separated on Sephadex G-75. The low-molecular-weight fraction contained glucose, rhamnose, heptose, galactosamine, alanine and phosphate. The higher-molecular-weight fractions consisted mainly of glucose, rhamnose and glucosamine together with amino compounds. Alkaline degradation of the lipopolysaccharide produced at least four different species each of which contained a low-molecular-weight polysaccharide similar if not identical to that produced by acid hydrolysis. Under certain growth conditions an abnormal lipopolysaccharide was produced which was defective in the low-molecular-weight polysaccharide and contained mainly high-molecular-weight material. Strains of different serotype yielded lipopolysaccharides which also exhibited heterogeneity but contained a low-molecular-weight polysaccharide similar to that obtained from strain PAC1 and PAC1R. It is suggested that each strain of P. aeruginosa may produce several lipopolysaccharides each containing a polysaccharide common to all. The relative proportions of the various lipopolysaccharides may be changed by growth conditions.

Incorporation of Glucose-14C, in vitro, into carbohydrates of intestinal mucosae of sarcoma (subcutaneous Yoshida-sarcoma)-bearing rats.

Scrapings of intestinal mucosae from sarcoma (subcutaneous Yoshida-sarcoma)-bearing rats were incubated, in vitro, with U-14C-glucose, comparing with those from normal rats. The resulting 14C-labelled products were fractionated into three carbohydrate fractions: polysaccharide-containing fraction (PS); carbohydrate fraction eluted with 50% aqueous ethanol from the charcoal-absorbed substances (Et); carbohydrate fraction eluted with aqueous ethanol-ammonia from the charcoal-absorbed substances (Et-Am). Monosaccharide components in these carbohydrate fractions were separated by paper chromatography after acidic hydrolysis and the radioactivities incorporated into these sugars were determined. The results showed that glucose-14C incorporated much more into monosac-charide components of the three carbohydrate fractions from the sarcoma-bearing animals than those from normal rats. No remarkable difference was found on the ratios of radioactivities incorporated into the fractions of glucosamine and galactosamine and also on the analyzed three monosaccharides contents between normal and the sarcoma-bearing conditions. The effects of sarcoma on the metabolism of hexosamine-containing carbohydrates were discussed.

Incorporation of glucose-14C, in vitro, into carbohydrates of colonic mucosa of liver tumor (3'-Me-DAB-induced liver tumor)-bearing rats.

Incorporation of Glucose-14C, in vitro, into the sugar components of the three carbohydrate fractions of colonic mucosa from liver tumor (3'-methyl-4-dimethyl-aminoazobenzene-induced liver tumor)-bearing rats was studied, comparing with that from normal younger rats. The results showed that the rate of incorporation of glucose-14C into the sugar components of polysaccharide-containing fraction (PS) was small, but those of carbohydrate fraction eluted with 50% aqueous ethanol from the charcoal-absorbed substances (Et) and of carbohydrate fraction eluted with aqueous ethanol-ammonia from the charcoal-absorbed substances (Et-Am) were fairly large. And glucose-14C incorporated much more into these carbohydrate fractions from the tumor-bearing rats than those from normal animals.

Polysaccharide of Coccidioides immitis.

Pappagianis, D. (University of California, Berkeley), E. W. Putman, and G. S. Kobayashi. Polysaccharide of Coccidioides immitis. J. Bacteriol. 82:714-723. 1961.-Soluble polysaccharide from mycelia or culture filtrates of Coccidioides immitis was found to consist mainly of mannose, but also included small quantities of galactose and another reducing sugar. Isolation of the polysaccharide by ethanol precipitation provided accompanying nitrogenous material. There was 3 to 4% nitrogen, present in amino acids, but in a nondialyzable (possibly protein or peptide) form. The average molecular weight of the complex was 31,700. Attempts to separate the nitrogenous and polysaccharide materials by chemical methods or by moving boundary electrophoresis were unsuccessful; ultracentrifugation showed a single peak. Despite these findings, there appeared to be several antigenic species present, as indicated by multiple lines of precipitation in double diffusion and quantitative precipitin tests. In the latter, polysaccharide-containing fractions gave precipitates without measurable carbohydrate, suggesting separability of nitrogenous and polysaccharide components.

Pneumococcal Antigenic Polysaccharide Substances from Animal Tissues

Summary Polysaccharide-containing fractions have been prepared from 12 organs and tissues of mice 2 to 3 weeks after injection of 0.5 mg of specific pneumococcus polysaccharide type I, II, or III, an amount known to produce immunological paralysis. The materials stimulated a high degree of immunity in mice against the homologous pneumococcus infection. Similar fractions prepared from tissues of normal mice were devoid of immunizing ability. Those from most tissues of immunized mice (after a 0.002-mg dose of specific polysaccharide) were found to stimulate a low degree of immunity. Tests of similar polysaccharide-containing fractions from tissues of albino and C3H mice 8 to 9 months after a “paralyzing” injection of specific polysaccharide showed that but little type I antigenicity remained, and no type III, but that high titer immunity against type II could still be induced by the homologous polysaccharides isolated from both albino and C3H strains of mice. In one experiment similar fractions prepared from organs and tissues of guinea pigs and rabbits injected with pneumococcal polysaccharides, especially type II, stimulated some immunity in mice against homologous pneumococcus infection.