Sephacryl S-200 Column Research Articles

Purification, Characterization and Spectrofluorometric Study of Buffalo Lactoferrin Coupled to Amoxicillin

Antibiotic resistance raises concerns about their residues in food and the environment. To produce antibiotic-free dairy products, countries explore new strategies to reduce antibiotic doses in mastitis treatment. Lactoferrins (LF), iron-binding glycoproteins, exhibit species-specific glycosylation affecting their properties. While formulations of bovine lactoferrin have been tested with antibiotics showed synergistic properties increasing the antibacterial properties, other interactions of lactoferrin isoforms with antibiotics remain unclear. This study isolates, purifies, and characterizes buffalo lactoferrin (Bf-LF) and its interaction with amoxicillin. Bf-LF was purified using a Sephacryl S-100 column and monitored spectrofluorometrically. Spectroscopic analysis of Bf-LF interaction with amoxicillin reveals that increasing amoxicillin concentration reduces Bf-LF fluorescence, indicating conformational changes. The Stern-Volmer quenching constant (KSV) and binding constant (Ka) measured at 298 K shows values around (2.79 ± 0.29) × 104 L mol-1 and (0.300 ± 0.04) × 104 L mol−1, respectively. UV-Vis spectroscopy shows a red shift (258.08 to 271.83 nm) implying Bf-LF-amoxicillin binding. In silico studies suggest polar interactions between amoxicillin and specific Bf-LF amino acids.

PURIFICATION AND CHARACTERIZATION OF MUCOR JANSENII LIPASE ISOLATED FROM COCOA PROCESSING PLANT EFFLUENTS IN ILE-OLUJI, ONDO STATE, NIGERIA

This study purified and characterized lipases secreted by a fungus Mucor jansenii isolated from the effluent of a cocoa processing plant. This was done in an effort to investigate the fungus' ability to produce lipase for industrial and biotechnological uses. The fungus isolated was identified macroscopically and microscopically using Lactophenol cotton blue stain. The enzyme produced was purified partly by ion-exchange chromatography on QAE-Sephadex A-25. Specific activity of the partially purified Mucor jansenii B6 lipase was 680.33 units/mg protein, 350.11 units/mg protein, and 342.19 units/mg protein, respectively, for isoforms A, B, and C. Partly purified M. jansenii B6 lipase had a molecular weight of 127 kDa, as determined by gel fitment on the Sephacryl S-200 column. The optimum pH and temperature for enzyme activity were 11.0 and 50 oC, respectively. The enzyme activity was enhanced by Ba2+, Na+, Ca2+, and Mg2+ at 5 mM and 10 mM, while Al3+ reduced the catalytic activity. Activity of the enzyme increased in acetone but decreased in ethyl acetate. The Michaelis constant (Km) and maximum velocity (Vmax) of the enzyme were found to be 0.5 mM and 16.6 units/mg protein, respectively. The study concluded that Mucor jansenii B6 lipase is a potential alkaline and thermostable lipase suitable for industrial and biotechnological applications.

Structural characteristics and antioxidant activity of a low-molecular-weight jujube polysaccharide by ultrasound assisted metal-free Fenton reaction

This study used an ultrasonically accelerated metal-free Fenton (H2O2-Vc system) reaction to promote water-extracted degrading polysaccharides from Ziziphus Jujuba cv. Muzao (DZMP). A novel jujube polysaccharide (DPZMP3) was obtained by degradation using DEAE-Sepharose Fast Flow and Sephacryl S-100 column chromatography. Methylation analysis, HPGPC, ion chromatography, FT-IR, and NMR spectroscopies were used to clarify the chemical structures of DPZMP3. Monosaccharide compositional analysis of DPZMP3 revealed the presence of Rha, Ara, Gal, and GalA at a molar ratio of 1.00:1.49:1.60:7.68, and the HPGPC data demonstrated the average Mw of 34.3 kDa. Based on the structural and linkage research using NMR spectroscopy and GC–MS, it was determined that DPZMP3 was a homogalacturonan pectic polysaccharide with a (1 → 4)-Galp branch at C-6 and a small amount of Araf and Rhap residues. The ultrasonic-aided Fenton treatment did not significantly alter the structure of DPZMP3. It may also be useful for DZMP and enhancing their antioxidant activity in vitro. The current study's findings could pave the way for the food sector to use jujube polysaccharides obtained by degradation as a functional food component.

Isolation and characterization of polygalacturonase producing thermophilic Aspergillus niger isolated from decayed tomato fruits

This study aimed to isolate a fungal strain capable of producing acidophilic and thermostable polygalacturonase. In this study, the fungal isolate was isolated from decaying tomatoes. Based on the colony characteristics, microscopic and morphological observations, the isolated fungal pathogen has been identified as Aspergillus niger. The isolated fungus was used in solid-state fermentation to produce an acidic polygalacturonase enzyme. The enzyme was then purified using ammonium sulphate precipitation and column chromatography, and its activity was assayed by measuring the releasing sugar group from citrus pectin using a 3, 5-dinitrosalicylic acid (DNSA) reagent assay. The crude extract obtained from solid-state fermentation had an activity of 94.6 U/mL. Ammonium sulphate precipitation increased the enzyme's specific activity from 6.89 U/mg to 12.42 U/mg. Sephadex G-200 was used to purify the enzyme 3.58 times, and its specific activity was determined to be 24.66 U/mg. The Sephacryl S-100 column achieved a final fold purification of 9.93 times and a specific activity of 68.41 U/mg. The purified enzyme performed best when polygalacturonic acid was used as a substrate. The enzyme's optimum temperature and pH were 55°C and 5, respectively. CaCl2 was found to be the best chelating ion for the enzyme. This enzyme is recommended for use in a variety of industrial applications as the enzyme was found to be stable at acidic pH and high temperature.

Physicochemical properties and in vitro prebiotic activity of Ulva rigida polysaccharides

The physicochemical properties and prebiotic potential of polysaccharide extract from the green seaweed Ulva Rigida were explored. The UPS PII-1 fraction purified by DEAE-Sephacel and Sephacryl S-400 columns was identified as a high molecular weight heteropolysaccharide (1.63 × 103 kDa) composed of rhamnose, glucose, mannose, galactose, arabinose, and fructose. FTIR analysis showed typical absorption bands of the structure of ulvan polysaccharides. In vitro prebiotic studies revealed that U. rigida polysaccharides (UPS) were highly resistant to gastric and intestinal juice hydrolysis. UPS also enhanced the growth of probiotic bacteria including Bifidobacterium and Lactobacillus as a potential source of prebiotics in functional food.

Structural characterization of a glycoprotein from white jade snails (Achatina Fulica) and its wound healing activity

Snail mucus is rich in proteins and polysaccharides, which has been proved to promote wound healing in mice in our previous research. The aim of this study was to investigate the effective component in snail mucus that can exert the wound healing potential and its structural characterization. Here, the glycoprotein from the snail mucus (SM1S) was obtained by DEAE-Sepharose Fast Flow and Sephacryl S-300 columns. The structural characteristics of SM1S were investigated via chromatographic techniques, periodic acid oxidation, FT-IR spectroscopy and NMR spectroscopy. Results showed that SM1S was a glycoprotein with a molecular weight of 3.8 kDa (83.23 %), consists of mannose, glucuronic acid, glucose, galactose, xylose, arabinose, fucose at a ratio of 13.180:4.875:1043.173:7.552:1:3.501:2.058. In addition, the periodic acid oxidation and NMR analysis showed that SM1S contained 1,6-glycosidic bonds, and might also contain 1 → 4 and 1 → 2 glycosidic or 1 → 3 glycosidic bonds. Furthermore, the migration experiment of human skin fibroblasts in vitro suggested that SM1S had a good effect to accelerate the scratch healing of cells. This study suggested that SM1S may be a prospective candidate as a natural wound dressing for the development of snail mucus products.

Sulfated polysaccharides from Caulerpa lentillifera: Optimizing the process of extraction, structural characteristics, antioxidant capabilities, and anti-glycation properties

The polysaccharides found in Caulerpa lentillifera (sea grape algae) are potentially an important bioactive resource. This study makes use of RSM (response surface methodology) to determine the optimal conditions for the extraction of valuable SGP (sea grape polysaccharides). The findings indicated that a water/raw material ratio of 10:1 mL/g, temperature of 90 °C, and extraction time of 45 min would maximize the yield, with experimentation achieving a yield of 21.576 %. After undergoing purification through DEAE-52 cellulose and Sephacryl S-100 column chromatography, three distinct fractions were obtained, namely SGP11, SGP21, and SGP31, each possessing average molecular weights of 38.24 kDa, 30.13 kDa, and 30.65 kDa, respectively. Following characterization, the fractions were shown to comprise glucose, galacturonic acid, xylose, and mannose, while the sulfate content was in the range of 12.2–21.8 %. Using Fourier transform infrared spectroscopy (FT-IR) it was possible to confirm with absolute certainty the sulfate polysaccharide attributes of SGP11, SGP21, and SGP31. NMR (nuclear magnetic resonance) findings made it clear that SGP11 exhibited α-glycosidic configurations, while the configurations of SGP21 and SGP31 were instead β-glycosidic. The in vitro antioxidant assays which were conducted revealed that each of the fractions was able to demonstrate detectable scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cations. All fractions were also found to exhibit the capacity to scavenge NO radicals in a dose-dependent manner. SGP11, SGP21, and SGP31 were also able to display cellular antioxidant activity (CAA) against the human adenocarcinoma colon (Caco-2) cell line when oxidative damage was induced. The concentration levels were found to govern the extent of such activity. Moreover, purified SGP were found to exert strong inhibitory effects upon glycation, with the responses dependent upon dosage, thus confirming the potential for SGP to find a role as a natural resource for the production of polysaccharide-based antioxidant drugs, or products to promote improved health.

Purification, characterization of a novel α-glucosidase from Debaryomyces hansenii strain MCC 0202 and chromatographic separation for high purity isomalto-oligosaccharides production

This study characterized a novel intracellular α-glucosidase, produced by Debaryomyces hansenii. The enzyme was purified through ultra-filtration, ammonium sulfate precipitation, chromatography Q-Sepharose and Sephacryl S-200 column. SDS-PAGE analysis confirmed the molecular weight of the purified protein to be 125 kDa. The enzyme exhibited maximum activity at pH 4.5and 50 °C and stability at pH 4.0 – 6.0 at 30 – 60 °C. The optimum isomalto-oligosaccharides (IMOs) bio-synthesis temperature using 400 g/L maltose was evaluated to be 35 °C with an optimum enzyme unit determined as 4.34 U g−1. The enzyme exhibits maximum maltose utilization of 78%, with or without inorganic salts, and was inhibited by ZnCl2, CoCl2 and CuSO4. Using maltose, the Km and Vmax values were 7.55 µM and 909 µmol/ml/mg respectively. In 3 L bioreactor, 4.34 U of enzyme produced IMOs with 47% purity utilizing 400 g/L maltose at 35 °C for 14 h. Furthermore, low purity IMOs was passed through cationic ion exchange resins to obtain high purity IMOs by separating glucose and maltose. This pioneering study produced high purity IMOs with 89% purity and 60% yield with greater conversion efficiency using a purified enzyme with a higher concentration of maltose and chromatographic method, which were confirmed by HPLC.

Purification and characterization of acid phosphatase from larvae of the camel tick Hyalomma dromedarii: inhibition kinetics by sodium fluoride

ABSTRACTAcid phosphatases (ACPs) perform a fundamental role in the digestion of egg proteins (vitellins), ensuring the survival of ticks by providing nutrients necessary for their oogenesis. Here, acid phosphatase activity is detected during embryogenesis of the camel tick Hyalomma dromedarii and reached its highest activity level in the larval stage. The tick larvae acid phosphatase which was named TLACP is homogeneously purified using CM cellulose cation exchange chromatography followed by gel filtration on Sephacryl S-300 column. TLACP is purified to 11 times purification folds and 51.3% yield with a specific activity of 12.9 U mg−1 protein. The molecular weight of TLACP was derived from both the gel filtration column and SDS-PAGE as monomer protein of 40 kDa. The Km of TLACP for p-nitrophenyl phosphate (p-NPP) was 1.25 mM and Vmax was 6.4 Umg−1. TLACP exhibited its optimum enzymatic activity at pH 4.8. TLACP was found highly specific to p-NPP followed by ATP, ADP, glucose-6-phosphate, naphthyl phosphate, phospho-enol-pyruvate, creatine phosphate, AMP, and GMP. The ions of Ca2+, Co2+, Mg2+, Ni2+, and Zn2+ motivated TLACP activity while Mn2+, Cu2+, and Fe2+ suppressed it. TLACP is noncompetitively inhibited with sodium fluoride with Ki value 0.15 mM. Thus, the presence of TLACP in tick embryonic and larval cells classifies this enzyme as an important molecular target for evolving potential vaccines in further studies aiming to find new strategies to control ticks.

Purification, structural characteristics and anti-atherosclerosis activity of a novel green tea polysaccharide

A new homogeneous polysaccharide (TPS3A) was isolated and purified from Tianzhu Xianyue fried green tea by DEAE-52 cellulose and Sephacryl S-500 column chromatography. Structural characterization indicated that TPS3A mainly consisted of arabinose, galactose, galacturonic acid and rhamnose in a molar ratio of 5.84: 4.15: 2.06: 1, with an average molecular weight of 1.596 × 104 kDa. The structure of TPS3A was characterized as a repeating unit consisting of 1,3-Galp, 1,4-Galp, 1,3,6-Galp, 1,3-Araf, 1,5-Araf, 1,2,4-Rhap and 1-GalpA, with two branches on the C6 of 1,3,6-Galp and C2 of 1,2,4-Rhap, respectively. To investigate the preventive effects of TPS3A on atherosclerosis, TPS3A was administered orally to ApoE-deficient (ApoE−/−) mice. Results revealed that TPS3A intervention could effectively delay the atherosclerotic plaque progression, modulate dyslipidemia, and reduce the transformation of vascular smooth muscle cells (VSMCs) from contractile phenotype to synthetic phenotype by activating the expression of contractile marker alpha-smooth muscle actin (α-SMA) and inhibiting the expression of synthetic marker osteopontin (OPN) in high-fat diet-induced ApoE−/− mice. Our findings suggested that TPS3A markedly alleviated atherosclerosis by regulating dyslipidemia and phenotypic transition of VSMCs, and might be used as a novel functional ingredient to promote cardiovascular health.

Purification and characterization of amylases from three freshwater fish species providing new insight application as enzyme molecular markers for zymography.

Purification of amylases from digestive tracts of three freshwater fish species with Q-Sepharose Fast Flow and Sephacryl S-200 columns displayed two isoforms of amylases from Osteochilus hasselti (O1, O2) and three isoforms of those from both Hampala dispar (UB, H1, H2) and Puntioplites proctozystron (P1, P2, P3). The optimum pH values displayed at 7.0 and 8.0, while the optimum temperatures revealed at 40 and 50°C. Almost isoenzyme activities were activated by NaCl and CaCl2, whereas EDTA and SDS strongly inhibited all enzymatic activities. Verification with an atomic absorption spectrophotometry exhibited the presence of Ca2+ ions in the range of 0.02-13.53ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25kDa validated with LC-MS/MS were selected. Three certain enzymes revealed high stability in a sample buffer after five cycles of freeze-thawing process upon storage at - 20°C for 12weeks. No protein degradation was observed on polyacrylamide gel, and the enzymes still displayed sharp and clear bands on zymograms. The result suggested that the purified fish amylases, which expressed high activities and stabilities, were potentially used as enzyme molecular weight markers for zymography.

Optimization of ultrasonic-assisted extraction of Platycodon grandiflorum polysaccharides and evaluation of its structural, antioxidant and hypoglycemic activity

The study aimed to improve the extraction rate of Platycodon grandiflorum roots polysaccharides (PGPs) using ultrasound-assisted extraction (UAE). A comparative analysis was undertaken to evaluate polysaccharides content, molecular weight distribution, monosaccharide composition, preliminary structure, antioxidant, and hypoglycemic activity of UAE in comparison with heating water extraction (HWE). The optimum extraction conditions included a liquid-to-material ratio of 20 mL/g, ultrasonic power of 150 W, extraction temperature of 70 ℃, and extraction time of 20 min, resulting in a significantly greater polysaccharides (12.011 ± 0.91 %) compared to HWE (7.62 ± 0.18 %). Through Sephacryl S-100 column elution, two homogenous fraction (PGP-U extracted with UAE and PGP-H extracted with HAE) were obtained.The molecular weight of PGP-U and PGP-H was 3.14 kDa and 3.44 kDa, respectively, mainly composed of different proportions of fourteen monosaccharides. Fourier transform infrared spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR) spectra experiment results showed that the two polysaccharides were pyranose ring with α- and β-glycoside bond. PGP-U and PGP-H exhibited specific antioxidant activities, encompassing total reducing force, scavenging of DPPH radicals, ABTs radicals and hydroxyl radicals in vitro, along with mitigation of H2O2-induced damage in HepG2 cells. Moreover, PGP-U exerted significantly stronger inhibitory activities against α-amylase and α-glucosidase and could significantly enhances the glucose uptake capacity and intracellular glycogen content of insulin-resistant HepG2 (IR-HepG2) cells.

Production of laccase enzyme from Curvularia lunata MY3: purification and characterization

Laccase-producing fungus (MY3) was successfully isolated from soil samples collected from Mansoura Governorate, Egypt. This fungal isolate has shown a high laccase production level over other isolated fungi. The identity of this isolate was determined by the molecular technique 18SrRNA as Curvularia lunata MY3. The enzyme purification was performed using ammonium sulfate precipitation followed by Sephacryl S-200 and DEAE-Sepharose column chromatography. The denatured enzyme using SDS-PAGE had a molar mass of 65 kDa. The purified laccase had an optimum temperature at 40 °C for enzyme activity with 57.3 kJ/mol activation energy for 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidation. The enzyme had an optimum pH of 5.0, and it has shown a high stability at the acidic range (4.5 to 5.5). Mn2+ and Mg2+ ions enhanced the enzyme activity, while most of the enzyme activity was inhibited by Hg2+. Some compounds such as 2-mercaptoethanol, L-cysteine, and sodium azide at a concentration of 10 mmol/L had shown a high suppression effect on the enzyme activity. The enzyme strongly oxidized ABTS and syringaldazine and moderately oxidized DMP and guaiacol. The antimicrobial activity of the purified enzyme towards three pathogenic strains (Escherichia coli ATCC-25922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC-10231) was evaluated for the potential use as an antimicrobial therapeutic enzyme.

Identification and Characterization of Mosquitocidal Toxins From <i>Bacillus cereus</i> VCRC-641

Bacillus cereus VCRC 641 was found to be an alternate larvicidal bacterial agent which was newly isolated from fresh water fish, Clarias batrachus (walking cat fish). The protein responsible for mosquito toxic effect was separated and purified by Sephacryl S-200 column chromatography and NATIVE PAGE. It was observed that a unique protein with molecular weight of 70kDa was the responsible factor for mosquito killing effect. Further analysis of the protein by liquid chromatography-mass spectrometry (LC-MS) it was identified as “Uncharacterized Protein”. The length of the uncharacterized protein was around 158bp. The uncharacterized protein was expected to have a molecular mass of 70 kDa, 51 amino acids, confidence score of 78.45 and mass of 206772.57. Therefore, in the present study we report for the first time that uncharacterized protein (70kDa) was the responsible factor for the mosquito killing effect.

Bosentan attenuates sickle cell disease erythrocyte HbS polymerization and impaired deformability induced by endothelin-1.

The effects of endothelin-1 (ET-1) on erythrocytes from sickle cell disease (SCD) patients have been described, but mechanisms of ET-1 regarding primary erythrocyte functions remain unknown. ET-1 is a vasoconstrictor peptide produced by endothelial cells, and the expression of ET-1 is increased in SCD. The present study used ex vivo experiments with sickle cell erythrocytes, ET-1, and bosentan, a dual antagonist of ETA and ETB receptors. We performed a hemoglobin S (HbS) polymerization assay with three concentrations of ET-1 (1, 20, and 50 pg/mL) and bosentan (100nmol/L). ET-1 increased HbS polymerization at all concentrations, and this effect was suppressed by bosentan. For the deformability assay, red blood cells (RBCs) were incubated on a Sephacryl column with the same concentrations of ET-1 and bosentan. ET-1 decreased deformability, and this effect was reversed by bosentan. To observe erythrocyte adhesion, ET-1 and bosentan were incubated with RBCs in thrombospondin-coated 96-well plate, which demonstrated that ET-1 decreased adhesion but that bosentan enhanced adhesion. We also assessed erythrocyte apoptosis and observed decreased eryptosis induced by ET-1, and these effects were inhibited bosentan. Thus, these findings demonstrated that ET-1 modulates HbS polymerization, erythrocyte deformability, adhesion to thrombospondin, and eryptosis, and these effects were suppressed or enhanced by bosentan.

Systematic Comparison of Structural Characterization of Polysaccharides from Ziziphus Jujuba cv. Muzao.

To investigate the structural information differences of Ziziphus Jujuba cv. Muzao polysaccharides, ten samples were successfully extracted from aqueous and alkaline solutions, prepared via DEAE-Sepharose Fast Flow through different eluents and Sephacryl S-300 columns, and systematically analyzed. Their characteristics were studied and then compared using chemical testing, high-performance gel permeation chromatography (HPGPC), gas chromatography (GC), methylation analysis, and NMR spectroscopy. The data achieved demonstrated that different jujube polysaccharide fractions possessed different structural characteristics, and most of them belonged to pectic polysaccharides. Overall, the structural information difference of jujube polysaccharides was preliminarily illuminated, which could not only promote the potential application of Z. Jujuba cv. Muzao polysaccharides but also provide an effective way to analyze the structures of polysaccharides from other genera jujube fruit.

Structural characterization of a pectic polysaccharide from laoshan green tea and its inhibitory effects on the production of NO, TNF-α and IL-6

A novel pectic polysaccharide, named GTPS3-1, was isolated and purified from Laoshan green tea polysaccharide (GTPS) through DEAE Sepharose Fast Flow and Sephacryl S-300 columns, its structure was characterized and its anti-inflammatory activity was explored. GTPS3-1, with a molecular weight of 26.05 kDa, was mainly composed of galacturonic acid, galactose, rhamnose and arabinose in a molar ratio of 4.72:2.5:1.68:1 on the basis of monosaccharide composition. Structural analysis results revealed that GTPS3-1 was a highly branched pectin consisting of →3)-Galp-(1→, →2)-Rhap-(1→, →3,5)-Araf-(1→, →3)-Rhap-(1→, GalpA-(1→, →3,4)-Galp-(1→, →4)-GalpA-(1→, →5)-Araf-(1→, →2,4)-Rhap-(1→, Rhap-(1→ and Araf-(1→ according to FT-IR, methylation and NMR analyses. In addition, GTPS3-1 inhibited the production of NO, TNF-α and IL-6 in a dose-dependent manner, which resulted in the amelioration of inflammatory injury in LPS-induced RAW 264.7 cells. These results would provide a theoretical basis for practical application of the novel polysaccharide as an anti-inflammatory adjuvant.

Structural Characterization of a Low Molecular Weight HG-Type Pectin From Gougunao Green Tea.

Tea is a popular beverage with a long history of safe and healthy use. Tea polysaccharide is a bioactive component extracted from tea, which has attracted more and more attention in recent decades. In this article, an acidic polysaccharide Gougunao tea polysaccharide (GPS) was isolated from Gougunao green tea by hot water extraction and ethanol precipitation. After purification by a diethylaminoethyl (DEAE) Sepharose Fast Flow column and a Sephacryl S-400 column, several homogalacturonan (HG) and rhamnogalacturonan-I (RG-I) fractions were obtained. Fraction GPS2b with the highest yield was selected for structural characterization by methylation and nuclear magnetic resonance (NMR) analysis. GPS2b was found to be an HG-type pectic polysaccharide (degree of methyl esterification [DE], 51.6%) with low molecular weight (Mw, 36.8 kDa). It was mainly composed of →4)-α-GalpA- (1→ and →4)-α-GalpA-6-OMe-(1→. In addition, a minor highly branched RG-I domain was identified in this fraction. The investigation of structural features of tea polysaccharides can provide insights to understand their structure-bioactivity relationship.

Extraction, Structural Characterization, and Immunomodulatory Activity of a High Molecular Weight Polysaccharide From Ganoderma lucidum.

Ganoderma lucidum polysaccharides (GLP) exhibited excellent immunomodulatory activity. Unfortunately, the structure and immunomodulatory activity of GLP are still unclear. GLP was separated into two fractions [high Mw Restriction Fragment Length Polymorphism (RGLP) and low Mw EGLP] using 10 kDa cut-off ultrafiltration membrane. Although the RGLP content was low in GLP, the immunomodulatory activity in RGLP was significantly higher than that of EGLP. Moreover, RGLP was further separated via the Sephacryl column to obtain RGLP-1 showed the best immunomodulatory activity in the macrophage RAW264.7 model. Structural analysis revealed that RGLP-1 was 3,978 kDa and mainly consisted of glucose. Periodate oxidation, Smith degradation, and methylation results indicated that RGLP-1 is a β-pyran polysaccharide mainly with 1→3, 1→4, 1→6, and 1→3, 6 glycosyl bonds at a molar ratio of 40.08: 8.11: 5.62: 17.81. Scanning electron microscopy, atomic force microscopy, and Congo red experiments revealed that RGLP-1 intertwined with each other to form circular aggregates and might possess a globular structure with triple-helix conformation in water. Overall, these results provide RGLP-1 as a potential functional food ingredient or pharmaceutical for immunomodulatory.

Purification and characterization of Se-enriched Grifola frondosa glycoprotein, and evaluating its amelioration effect on As3+ -induced immune toxicity.

Selenium (Se)-enriched glycoproteins have been a research highlight for the role of both Se and glycoproteins in immunoregulation. Arsenic (As) is a toxicant that is potentially toxic to the immune function and consequently to human health. Several reports suggested that Se could reduce the toxicity of heavy metals. Moreover, more and more nutrients in food had been applied to relieve As-induced toxicity. Hence glycoproteins were isolated and purified from Se-enriched Grifola frondosa, and their preliminary characteristics as well as amelioration effect and mechanism on As3+ -induced immune toxicity were evaluated. Four factions, namely Se-GPr11 (electrophoresis analysis exhibited one band: 14.32 kDa), Se-GPr22 (two bands: 20.57 and 31.12 kDa), Se-GPr33 (three bands: 15.08, 20.57 and 32.78 kDa) and Se-GPr44 (three bands: 16.73, 32.78 and 42.46 kDa), were obtained from Se-enriched G. frondosa via DEAE-52 and Sephacryl S-400 column. In addition, Se-GPr11 and Se-GPr44 are ideal proteins that contain high amounts of almost all essential amino acids. Thereafter, the RAW264.7 macrophage model was adopted to estimate the effect of Se-GPr11 and Se-GPr44 on As3+ -induced immune toxicity. The results showed that the pre-intervention method was the best consequent and the potential mechanisms were, first, by improving the oxidative stress state (enhancing the activity of superoxide dismutase and glutathione peroxidase, decreasing the levels of reactive oxygen species and malondialdehyde); secondly, through nuclear factor-κB and mitogen-activated protein kinase-mediated upregulation cytokines (interleukin-2 and interferon-γ) secretion induced by As3+ . The results suggested Se-enriched G. frondosa may be a feasible supplement to improve health level of the As3+ pollution population. © 2021 Society of Chemical Industry.