Sepharose Fast Flow Column Research Articles

Isolation and purification of bovine N‐glycans from whey protein concentrate (WPC 70)

N‐glycans present in milk glycoproteins (e.g. lactoferrin and immunoglobulin G) can provide infants with nutritional and protective effects similar to breast milk. In this study, we enriched N‐glycoproteins from WPC 70 using ultrafiltration and anion exchange chromatography on DEAE Sepharose Fast Flow column, with N‐glycan content and N‐glycoprotein purity as evaluation indicators. The results showed that changing feed flow rate and molecular weight cut‐off was more effective to improve ultrafiltration separation than changing feed concentration. When using a 100 kDa ultrafiltration membrane with a feed flow rate of 10 rpm and a feed concentration of 10 mg/mL, N‐glycan content could be increased by 200%. However, anion exchange chromatography was more efficient for purification, increasing N‐glycan content by 700%. This study provided a reliable method for N‐glycan enrichment and a scientific basis for further research and potential application of N‐glycans in infant formula and whey products.

Separation and purification of bovine nasal cartilage-derived chondroitin sulfate and evaluation of its binding to bovine serum albumin

This study employs an optimized and environmentally friendly method to extract and purify chondroitin sulfate (CS) from bovine nasal cartilage using enzymatic hydrolysis, ethanol precipitation, and DEAE Sepharose Fast Flow column chromatography. The extracted CS, representing 44.67 % ± 0.0016 of the cartilage, has a molecular weight of 7.62 kDa. Characterization through UV, FT-IR, NMR spectroscopy, and 2-aminoacridone derivatization HPLC revealed a high content of sulfated disaccharides, particularly ΔDi4S (73.59 %) and ΔDi6S (20.61 %). Interaction studies with bovine serum albumin (BSA) using fluorescence spectroscopy and molecular docking confirmed a high-affinity, static quenching interaction with a single binding site, primarily mediated by van der Waals forces and hydrogen bonding. The interaction did not significantly alter the polarity or hydrophobicity of BSA aromatic amino acids. These findings provide a strong foundation for exploring the application of CS in tissue engineering and drug delivery systems, leveraging its unique interaction with BSA for targeted delivery and enhanced efficacy.

Enzyme-Assisted Ultrasonic Extraction and Antioxidant Activities of Polysaccharides from Schizochytrium limacinum Meal.

Enzyme-assisted ultrasonic extraction (EAUE) was utilized and optimized for extracting polysaccharides from Schizochytrium limacinum meal (SLMPs) via the response surface methodology. The optimal EAUE conditions were determined as follows: enzyme concentration at 5.18%, ultrasonic temperature at 53 °C, ultrasonic duration of 40 min, ultrasonic power at 60 W, and a liquid-to-material ratio of 34 mL/g, achieving a polysaccharide extraction yield of 11.86 ± 0.61%. The purified polysaccharide component, SLMP1-1, isolated using DEAE Sepharose Fast Flow and Sephadex G-100 columns, exhibited potent antioxidant activity. SLMP1-1, with a molecular weight of 25.5 kDa, comprises glucose, mannose, arabinose, and galactose in a molar ratio of 16.39:14.75:1:693.03. 1H NMR analysis revealed the α configuration of SLMP1-1. Antioxidant assessments, including DPPH, ABTS, and ferric ion reduction assays, were detected with inhibitory values at 21.82-82.98%, 38.21-98.46%, and 3.30-20.30% at 0.2-1.0 mg/mL. This confirmed the effective antioxidant capacity of SLMP1-1, which is notably enhanced post oral and gastric digestion. The findings suggest that polysaccharides extracted from Schizochytrium limacinum meal hold significant promise as natural antioxidants.

Structural characterization of chia seed polysaccharides and evaluation of its immunomodulatory and antioxidant activities

This study aims to extract an active heteropolysaccharide Chia seed polysaccharide (CSP-A) and further purified by DEAE Sepharose Fast Flow and Sepharose CL-6B chromatographic column, characterize its structure, and evaluate its antioxidant and immunomodulatory activities. Structural analysis revealed that CSP-A was composed of d-mannose, d-glucuronic acid and d-xylose in a molar ratio of 1:3:4 with molecular weight of 1.688 × 105 Da, owning 4 sugar residues of β-d-Manp-(1→, →4)-α-d-GlcpA-(1→, →2,4)-β-d-Xylp-(1→, and → 4)-β-d-Manp-(1 →. Congo red assay and microscopic characteristics showed that CSP-A in its solution may possess a helical conformation. In vitro experiments showed that CSP-A had moderate DPPH· and OH· scavenging activities. CSP-A also enhanced the phagocytosis ability of RAW 264.7 cells and prompted the release of NO, TNF-α, IL-6 and IL-1β from RAW 264.7 cells, which indicated CSP-A had immune regulation effect. This experiment provides scientific basis for further utilization and development of chia seeds, a kind of functional food.

Structural properties and immune-enhancing activities of galactan isolated from red seaweed Grateloupia filicina.

A water-soluble polysaccharide (GFP) was isolated from Grateloupia filicina and fractionated using a DEAE Sepharose Fast Flow column to evaluate immunostimulatory activity. Carbohydrates (62.0%-68.4%) and sulfates (29.3%-34.3%) were the major components of GFP and its fractions (GFP-1 and GFP-2), with relatively lower levels of proteins (4.5%-15.4%) and uronic acid (1.4%-3.9%). The average molecular weight (Mw ) for GFP and its fractions was calculated between 98.2%-243.7 kDa. The polysaccharides were composed of galactose (62.1%-87.2%), glucose (4.5%-33.2%), xylose (3.1%-5.3%), mannose (1.4%-2.2%), rhamnose (1.2%-2.0%), and arabinose (0.9%-1.7%) units connected through →3)-Galp-(1→, →4)-Galp-(1→, →2)-Galp-(1→, →6)-Galp-(1→, →3,4)-Galp -(1→, →3,6)-Galp-(1→, →4,6)-Galp-(1→, →3,4,6)-Galp-(1→, →2,3)-Galp-(1→, →2,4)-Galp-(1→, →4)-Glcp-(1→, →6)-Glcp-(1→ and →4,6)-Glcp-(1→residues. The isolated polysaccharides effectively induced RAW264.7 murine macrophages by releasing nitric oxide (NO) and various cytokines via nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Further, the expression of toll-like receptor-2 (TLR-2) and TLR-4 in RAW264.7 cells indicated their activation through TLR-2 and TLR-4 binding receptors. Among the polysaccharides, GFP-1 highly stimulated the activation of RAW264.7 cells, which was mainly constituted of (→1) terminal-D-galactopyranosyl, (1→3)-linked-ᴅ-galactopyranosyl, (1→4)-linked-ᴅ-galactopyranosyl and (1→3,4) -linked-ᴅ-galactopyranosyl residues. These findings demonstrate that GFP-1 from G. filicina are effective at stimulating the immune system and this warrants further investigation to determine potential biomedical applications.

Extraction, characterization, and biological activities of exopolysaccharides from plant root soil fungus Fusarium merismoides A6.

The exploration of polysaccharides from microorganisms is of great importance. In this study, a new type of exopolysaccharide excreted by Fusarium merismoides A6 (FM-EPS) was isolated, and the extraction conditions were optimized using a response surface methodology (RSM). The extraction temperature at 0°C, a precipitation time of 7.83h, and an ethanol precipitation concentration of 77.64% were predicted and proved to be the best extraction conditions with the maximum extraction yield of 0.74g/mL. Then, two fractions of F. merismoides A6 exopolysaccharides (FM-EPS1 and FM-EPS2) were obtained through DEAE Sepharose fast flow column chromatography. As indicated by monosaccharide composition analysis, both fractions mainly consisted of mannose, glucose, galactose, and ribose, with an average molecular weight of 5.14 × 104 and 6.50 × 104g/mol, respectively. FT-IR and NMR spectroscopy indicated the FM-EPSs had both α- and β-glycosidic bonds. Moreover, the determination of antioxidant and antiproliferative activities in vitro proved that FM-EPSs had good antioxidant activities and antiproliferation activities. FM-EPS1 showed stronger antioxidant activities than FM-EPS2. FM-EPS2 showed antiproliferation activities on HeLa and HepG2 cells, while FM-EPS1 had no obvious antiproliferative activity. Therefore, FM-EPSs could be explored as potential antioxidant and anticancer agent applied in food, feed, nutraceutical, pharmaceutical, cosmetics, and chemical industries.

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.

The antifungal activity of a serine protease and the enzyme production of characteristics of Bacillus licheniformis TG116

The growth of Phytophthora capsica, Rhizoctonia solani, Fusarium graminearum, Fusarium oxysporum and Botrytis cinerea were all inhibited by the fermentation supernatant of Bacillus licheniformis TG116, a biocontrol strain isolated from Typhonium giganteum Engl. previously with broad-spectrum resistance to plant pathogens. The fermentation supernatant of the TG116 has a great stability on temperature and UV, and shows the biological activity of protease and cellulase. The antifungal protease produced by B. licheniformis TG116 was purified to homogeneity by ammonium sulfate precipitation, DEAE Sepharose Fast Flow column chromatography and Sephadex G-50 column chromatography. The inhibition of protease by the three surfactants increased with increasing concentration inhibition. Among these surfactants, EDTA showed the strongest inhibition, with only 25% protein activity at a concentration of 1.1mmol·L-1. Gene amplification verified the presence of a gene fragment of serine protease in the strain TG116. The antimicrobial substance isolated from the fermentation broth of TG116 is a serine protease component.

The structural characteristic of bamboo shoot shell polysaccharides extracted using ultrasound‐assisted phosphotungstic acid hydrolysis and its protection against cell oxidative injury

SummaryIn this study, crude polysaccharide (BSSP) was extracted from bamboo (Phyllostachys edulis) shoot shell using the ultrasonic‐assisted phosphotungstic acid hydrolysis method. Then, BSSP was further fractionated by DEAE Sepharose fast flow column and serial NaCl solution elutions to obtain two fractions, BSSP‐A and BSSP‐B. The results showed that the maximum yield of BSSP was 195.2 mg g−1 (19.52%) under optimised conditions. Structural analysis indicated that both BSSP‐A and BSSP‐B were heteropolysaccharides with α‐pyranoside bond structure and mainly consisted of xylose by scanning electronic microscopy, Fourier transform infrared spectroscopy, ultraviolet–visible spectroscopy, thermal characterisation and nuclear magnetic resonance spectroscopy. BSSP‐B showed a better protective effect than BSSP‐A against H2O2‐induced oxidative injury in WRL‐68 cells by protecting the cell membrane structure, maintaining mitochondrial membrane potential and increasing intracellular antioxidant enzymes activities. Immunoblotting indicated that polysaccharides might up‐regulate the expression of antioxidant enzymes through the Keap1/Nrf2 signalling pathway and thus alleviate H2O2‐induced cell injury. The findings of this study suggested that polysaccharides from bamboo shoot shell may act as potent antioxidants in functional food industries.

Structural Characterization and Anti-inflammatory Activity of a Galactorhamnan Polysaccharide From Citrus medica L. var. sarcodactylis.

This study aimed to extract polysaccharides from Citrus medica L. var. sarcodactylis (finger citron fruits) and analyze their structures and potential bioactivities. A new polysaccharide named K-CMLP was isolated and purified by Diethylaminoethylcellulose (DEAE)-Sepharose Fast Flow and DEAE-52 cellulose column chromatography with an average molecular weight of 3.76 × 103 kDa. Monosaccharide composition analysis revealed that K-CLMP consisted of rhamnose, galactose, and glucose, with a molar ratio of 6.75:5.87:1.00. Co-resolved by methylation and two-dimensional nuclear magnetic resonance (NMR), K-CLMP was alternately connected with 1, 2-Rha and 1, 4-Gal to form the backbone, and a small number of glucose residues was connected to O-4 of rhamnose. The results of DPPH⋅ and ABTS+⋅ radical scavenging assays indicated that both crude polysaccharide Citrus medica L. var. polysaccharide (CMLP) and K-CLMP exhibited strong free-radical-scavenging properties in a dose-dependent manner. In addition, K-CMLP significantly inhibited the production of pro-inflammatory cytokines (IL-6 and TNF-α) and reactive oxygen species (ROS) in RAW 264.7 cells treated with LPS. These results provide a basis for further use as one of the potential functions of food or natural medicine.

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.

Isolation, purification, structural characterization and antitumor activities of a polysaccharide from Lilium davidii var. unicolor Cotton

The water-soluble polysaccharide from Lilium davidii var. unicolor Cotton (LP60-1) was extracted by hot water, precipitated by fractional alcohol, separated by DEAE Sepharose Fast Flow column and purified though Sephacryl S-400 HR. The HPGPC result showed LP60-1 was a homogeneous polysaccharide with the MW of 6.5 × 103 Da. LP60-1 was composed of mannose and glucose with the ratio of 1.26: 1. Methylation and NMR analysis showed that the backbone of LP60-1 was consisted of β-(1→4)-linked mannose residues and β-(1→4)-linked glucose residues. And the substitution site of acetyl groups was O-2 or O-3 of mannose and the degree of acetylation was 0.2. In addition, the higher structure of LP60-1 has been preliminarily explored through CD, SEM, and AFM. LP60-1 had obvious anti-tumor activity, and the inhibition rate of HepG2 cell was 81.45% (IC50 = 123.8 µg/mL). The results proposed that LP60-1 can be researched and developed as a new antitumor drug.

English

The stomach lysozymes of ruminants play a paramount role in digestion and offer the chance to probe evolutionary changes in complex organisms on a biochemical basis. In this paper, we focus on the characterization of the catalytic properties of Tibetan ruminant (TR) stomach lysozymes and provide a comparative study of TR stomach lysozymes and nonplateau ruminant stomach lysozymes. The stomach lysozymes were purified with a Carboxymethyl (CM) Sepharose Fast Flow (FF) column and a Bio-Gel P-100 column. The purified stomach lysozymes were characterized by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) and matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) analyses. The antimicrobial activity was evaluated using an agar diffusion method, and the pH, ion strength, temperature, pepsin and trypsin effects on enzymatic activities were evaluated by normal biochemistry methods. The stomach lysozymes of TRs (cattle:TC, yak:TY, and sheep:TS) exhibit maximum activity at pH 5 and ionic strengths below 0.02. TC and TS stomach lysozymes are more resistant to higher temperatures than nonplateau lysozymes. Stomach lysozymes of TRs exhibit greater enzymatic activity than nonplateau lysozymes, in the presence of pepsin and trypsin. Stomach lysozymes from TS retain 86% of their initial enzymatic activity against pepsin inactivation. After trypsin treatment, the stomach lysozymal activities of TRs were significantly affected by trypsin inactivation, with the exception of TS, whose enzymatic activities in the presence of trypsin were 5- and 10-fold higher than those of nonplateau lysozymes. The stomach lysozymes of TRs are more resistant to environmental factors such as pH, temperature, pepsin, and trypsin than nonplateau lysozyme C.

Sulphation and Hydrolysis Improvements of Bioactivities, and Immuno-Modulatory Properties of Edible Amanita hemibapha Subspecies javanica (Corner and Bas) Mucilage Polysaccharide as a Potential in Personalized Functional Foods.

In this study, the mucilage polysaccharide (MP) from Amanita hemibapha subspecies javanica was prepared by hot water extraction and ethanol precipitation and then fractionated using anion-exchange chromatography equipped with a DEAE Sepharose fast flow column. The most immune-enhancing polysaccharide fraction 2 (MPF2) was subjected to a structural modification such as hydrolysis or over-sulphation. The sulphate and molecular weight (Mw) of over-sulphated (OS1-3) and hydrolysed (HS1-3) derivatives of MPF2 differed between 9.85% and 14.2% and 32.8 and 88.1 × 103 g/mol, respectively. Further, the immune-enhancing properties of MPF2 and its derivatives were tested on RAW264.7 and NK cells through various in vitro assays. Interestingly, a low molecular weight of HS1-3 significantly increased the nitric oxide (NO) production (p < 0.05) more than MPF2, indicating that Mw is a major factor in RAW264.7 cell stimulation. In addition, RAW264.7 cells produced various cytokines by up-regulating mRNA expression levels and the activation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. On the other hand, OS1-3-treated natural killer (NK) cells induced cytotoxicity in HepG2 cells through the expression of IFN-γ, Grandzyme-B, perforin, NKp30, and FasL. These results demonstrated that sulphate derivatives play an important role in NK cell activation. Further, this study also explores how polysaccharide binds to RAW264.7 and NK cells. MPF2 and HS3 may activate RAW264.7 cells via binding to TLR4 receptors, and OS2 could be activated through the CR3 signalling pathways.

A Novel Biochemical Study of Anti-Dermal Fibroblast Replicative Senescence Potential of Panax Notoginseng Oligosaccharides.

Dermal fibroblast replicative senescence that often occurs in aging skin is characterized by loss of cell proliferative capacity, cell cycle arrest, decreased cell elongation, and decreased synthesis of dermal extracellular matrix (ECM) components. Although Panax notoginseng is known for its effectiveness in alleviating many age-related degenerative diseases, few studies have evaluated P. notoginseng components for efficacy or mechanisms of action in delaying cell replicative senescence. In this study, P. notoginseng oligosaccharides (PNO) were isolated using a stepwise purification procedure involving water extraction and alcohol precipitation followed by DEAE Sepharose Fast Flow column chromatography, preparative high performance liquid chromatography, and size-exclusion chromatography. Monosaccharides detected in PNO constituents included mannose, galactose, and sorbitose in relative molar proportions of 14.2:12.3:1, respectively, aligning with PNO absorption spectrum results resembling typical known spectra for sugars. In vitro, PNO treatment of replicative senescent NIH-3T3 fibroblasts significantly promoted cell vitality, inhibited SA-β-galactosidase (SA-β-Gal) activity, and reduced p16 and p21 protein-level expression. Moreover, PNO treatment of senescent fibroblasts led to a lower proportion of G1 phase cells and higher proportion of S phase cells, while also inducing aging NIH-3T3 cells to migrate and synthesize collagen-I (CoL-I). Mechanistically, PNO treatment up-regulated expression of proliferating cell nuclear antigen (PCNA), cyclin E, cyclin D1, and cyclin-dependent kinase 4 (CDK4) proteins and promoted phosphorylation of MEK, p38, and ERK1/2 to trigger cell cycle progression. Additionally, PNO treatment also up-regulated protein-level expression of TGF-β1 and levels of p-Smad2/3, p-FAK, and p-Pax to trigger CoL-I synthesis and cell migration. Taken together, these findings demonstrate that oligosaccharides purified from P. notoginseng could reverse fibroblast replicative senescence by promoting fibroblast cell proliferation, migration, and CoL-I production.

Optimization of ultrasonic-assisted extraction of polysaccharides from Hemerocallis citrina and the antioxidant activity study.

The present study discussed the optimization of the ultrasonic-assisted extraction of polysaccharides from daylily polysaccharides (DPs). The extracted crude polysaccharides were further separated and purified, and the antioxidant activities including 1,1-diphenyl-2-111 picrylhydrazyl (DPPH) radical scavenging, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) radical scavenging, hydroxyl radical scavenging, and ferric-reducing antioxidant power (FRAP) activities of the obtained fractions were also evaluated. The results showed that the optimal ultrasonic-assisted extraction parameters with DPs yield of 15.25 ± 1.13% were water to powder ratio of 25 ml/g, extraction power of 694 W, extraction temperature of 71°C, extraction time of 38 min, and three times extraction. By DEAE Sepharose Fast Flow column, four water-soluble polysaccharide fractions (DP-1, DP-2, DP-3, and DP-4) were successfully obtained. Monosaccharide component analysis showed that the four obtained fractions were all hetero-polysaccharides that mainly contained rhamnose, arabinose, fructose, galactose, glucose, galacturonic acid, and glucuronic acid in different molar ratios. All the four DP fractions did show obvious antioxidant activities in vitro, and the DP-3 component had relatively high ABTS free radical scavenging activity. Overall, our research showed that DPs could provide cheap raw materials for the development of natural antioxidants in medicines, functional foods, and even cosmetics. PRACTICAL APPLICATION: This article deals with the optimization of the ultrasonic-assisted extraction of polysaccharides from daylily and its antioxidant activities. The results showed that the optimal ultrasonic-assisted extraction yield of DPs was 15.25 ± 1.13%. By DEAE Sepharose Fast Flow column, four water-soluble polysaccharide fractions were successfully obtained, and all the four DP fractions did show obvious antioxidant activities in vitro. Daylily polysaccharides could provide cheap raw materials for the development of natural antioxidants in medicines, functional foods, and even cosmetics.

Purification and characterization of the receptor-binding domain of SARS-CoV-2 spike protein from Escherichia coli.

SARS‐CoV‐2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID‐19. Spike protein of SARS‐CoV‐2 mediates viral entry into host cells by binding ACE2 through the receptor‐binding domain (RBD). RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines. RBD expressed in mammalian cells suffers from low expression yield and high cost. E. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. However, RBD expressed by E. coli (RBD‐1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. In the present study, RBD‐1 was expressed by E. coli and purified by a Ni Sepharose Fast Flow column. RBD‐1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD‐2). The secondary structure and tertiary structure of RBD‐1 were largely maintained without glycosylation. In particular, the major β‐sheet content of RBD‐1 was almost unaltered. RBD‐1 could strongly bind ACE2 with a dissociation constant (KD) of 2.98 × 10–8 M. Thus, RBD‐1 was expected to apply in the vaccine development, screening drugs and virus test kit.

Structural characterization and hypoglycemic activity of an intracellular polysaccharide from Sanghuangporus sanghuang mycelia

A neutral polysaccharide (SSIPS1) was isolated and purified from cultured mycelia of Sanghuangporus sanghuang by DEAE Sepharose Fast Flow and Sephacryl S-100 columns. Basic monosaccharide composition indicated that SSIPS1 was mainly composed of d-glucose. The results of methylation and 2D-NMR analysis suggested that the glycosidic linkages of SSIPS1 were elucidated to consisted of 1,4-linked α-d-glucopyranose (Glcp) residues with two branched points at O-6. The two branches were composed of 1,4-linked α-D-Glcp terminated with α-D-Glcp, 1,4-linked α-D-Glcp and 1,4-linked β-Galp terminated by α-D-Glcp. Moreover, its chain conformation was revealed to present a flexible chain conformation in 0.1 NaNO3 with a hydrodynamic radius and radius of gyration of 3.26 and 6.45 nm by multi-angle laser light scattering, with a single chain of 0.559 nm observed by atomic force microscopy. Further, SSIPS1 exhibited a potential inhibitory activity against α-amylase and α-glucosidase, and it had hypoglycemic effects on in vitro insulin resistance of HepG2 cells as well.

Extraction, purification and characterization of sulphated polysaccharide from Bellamya quadrata and its stabilization roles on atherosclerotic plaque

The aim of this paper was to investigate the extraction, purification and characterization of sulphated polysaccharide (BQPS) from Bellamya quadrata and its stabilization roles on atherosclerotic plaque. Firstly, crude polysaccharide (CBQP) from Bellamya quadrata was extracted by protease enzyme assisted extraction. Moreover, its optimal parameters were obtained by the response surface method as follows: the ratio of water to raw material of 24:1, enzyme dosage of 285 U/g, enzymolysis pH value of 4.7 and temperature of 67 °C. Secondly, CBQP was further purified to obtain the target polysaccharide BQPS by Q Sepharose Fast Flow and Sephacryl S-400 gel column chromatography. Then, the characterization of BQPS revealed that it possessed a total polysaccharide content of 91.88 ± 1.23%, sulfuric acid group content of 9.12 ± 1.59% and molecular weight of 91.1 kDa. BQPS was only consisted of glucose without any proteins. Finally, BQPS was confirmed to have a significant stabilizing effect on atherosclerotic plaque and its mechanism was related to the selective promotion of autophagy with the precisely right strength.

Mycobacterium tuberculosis Ag85b:hfcγ1 recombinant fusion protein as a selective receptor-dependent delivery system for antigen presentation

Introducing an effective vaccine for tuberculosis (TB) is an urgent need. Mycobacterium tuberculosis (Mtb) Ag85 complex is suggested for making protective immunodominant antigens for design and development of novel TB vaccine. In the present study, a pDR2EF1-Fcγ1 vector has been used to make Ag85B:hFcγ1 recombinant fusion protein. Briefly, specific XbaI and NotI site incorporated primers were used to amplify Mtb-fbpB gene by PCR, TA-cloned and amplified in E.coli DH5α. The resulting vector then subcloned into the pDR2EF1.Fcγ1 vector and transferred to Chinese hamster ovary (CHO) cell line. DNA sequencing was performed to confirm that Ag85B:hFcγ1 construct is precise and in-frame. Then, Ag85B:hFcγ1 protein was produced by CHO expression system and recombinant protein was purified using HiTrap rProtein A Sepharose Fast Flow column. The presence of recombinant fusion protein confirmed by immunofluorescence (IFA) and Western blotting (WB). This fusion protein containing Fc fragment of human IgG1, apart from stability and adjuvanticity potential, could bind to FcRγI (CD64) on the surface of antigen-presenting cells (APCs) and induce cross-presentation in favour of host immune response and can be used as a potential candidate along with other subunit vaccines against Mtb.