Sephadex G-100 Chromatography Research Articles

Structural analysis, acetylcholinesterase inhibitory activity and immunoregulatory activity of two acidic polysaccharides from Chrysanthemum morifolium cv. Gongju

The structural characteristics, acetylcholinesterase inhibitory activity, and immune effects of two purified acid polysaccharides from Chrysanthemum morifolium cv. Gongju were investigated. GJP-1 and GJP-2 were isolated and purified using DEAE-sepharose and Sephadex G-100 chromatography. GJP-1 consisted of Man, Rha, Ara, Glc, Gal, and GalA, with a ratio of 1.99:3.98:81.2:2.65:7.03:3.15, while GJP-2 comprised of Man, Rha, Ara, Glc, Gal, and GalA in a ratio of 0.27:2.36:8.03:38.44:2.38:48.52. GJP-2 is a kind of pectin-type polysaccharide. GJP-1 and GJP-2 differed in average molecular weight of 5.86 kDa and 4.92 kDa. The results indicated that GJP-1 and GJP-2 significantly inhibited the activity of acetylcholinesterase and had good immunomodulatory activities. GJP-1 and GJP-2 are bioactive polysaccharides with the potential to improve cognitive function. GJP-2 promoted more proliferation, as well as the release of tumor necrosis factor-α and interferon γ. Clarifying the structures and bioactivities of polysaccharides could lay a foundation for the application of Gongju in functional foods and medicines production.

Purification and characterization of a thermostable Galium aparine β-galactosidase: A competent agent with enhanced cytotoxic activity against MCF-7 cell line

β-galactosidases (EC 3.2.1.23) are capable of catalysing two distinct types of reactions: transgalactosylation and hydrolysis. It is very important for the senescence of cancer cells. It is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates by sequential release of β-linked terminal galactosyl residues. It has profound significance in cancer cell senescence. It can be derived from various sources including plants, animals, bacteria, yeasts, and filamentous fungi. Purification and thermodynamic characterization of β-galactosidase from Galium aparine seeds, and in vitro efficacy assessment of a breast cancer cell line. The enzyme was purified by using ammonium sulphate precipitation, dialysis, DEAE cellulose, and Sephadex G-100 chromatography. The enzyme was purified to 6.4 fold with a yield of 14 % and specific activity of 11.6 U/mg of protein. SDS-PAGE and MALDI-TOF analysis revealed that β-galactosidase is a monomer with a molecular weight of 35 kDa. The optimum pH and temperature for the purified enzyme were 6.0 and 50 ºC. The enzyme had Km and Vmax values of 0.25 mM and 32 µmol/min, respectively. The thermodynamic parameters (ΔHº, ΔGº, ΔSº) for the irreversible denaturation of the enzyme were also investigated at different temperatures (50–75 ºC). Lactose in milk was successfully hydrolyzed using a purified enzyme at a rate of 0.0121 min−1 and 12.12 min. This is better than other studied β-galactosidases. These results suggest the use of β-galactosidase from G. aparine for producing delactosed milk for the lactose-intolerant population and prebiotic synthesis. pH and optimum temperature and its activation by Ca2+ shows that it is suitable for milk processing. As a result, the enzyme was stable even at higher temperatures and had the potential to undergo catalysis, making it economically viable, primarily in the food and pharmaceutical industries. The present study is aimed to purification and thermodynamic characterization of enzyme to assess in vitro efficacy of β-galactosidase on MCF-7 cell line to delineate its therapeutic efficacy.

Divalent metal ion in the active site of purple acid phosphatase modulates substrate binding: Kinetic and thermodynamic properties

The purple acid phosphatase was purified from 5.9-fold to apparent homogeneity from Anagelis arvensis seeds using SP-Sephadex C-50 and Sephadex G-100 chromatography. The results of residual activity tests conducted using different temperature ranges (50–70 °C) were calculated as the activation energy (Ed = 72 kJ/mol), enthalpy (69.31 ≤ (ΔH° ≤ 69.10 kJ/mol), entropy (−122.48 ≤ ΔS° ≤ −121.13 J/mol·K), and Gibbs free energy (108.87 ≤ ΔG° ≤ 111.25 kJ/mol) of the enzyme irreversible denaturation. These thermodynamic parameters indicate that this novel PAP is highly thermostable and may be significant for use in industrial applications. However, it may be confirmed by stopped-flow measurements that this substitution produces a chromophoric Fe3+ site and a Pi-substrate interaction that is about ten times faster. Additionally, these data show that phenyl phosphate hydrolysis proceeds more rapidly in metal form of A. arvensis PAP than the creation of a μ-1,3 phosphate complex. The Fe3+ site in the native Fe3+-Mn2+ derivative interacts with it at a faster rate than in the Fe3+-Fe2+ form. This is most likely caused by a network of hydrogen bonds between the first and second coordination spheres. This suggests that the choice of metal ions plays a significant role in regulating the activity of this enzyme.

Immunomodulatory Effect of Cordyceps militaris Polysaccharide on RAW 264.7 Macrophages by Regulating MAPK Signaling Pathways.

Polysaccharide is one of the principal bioactive components found in medicinal mushrooms and has been proven to enhance host immunity. However, the possible mechanism of immunomodulatory activity of Cordyceps militaris polysaccharide is not fully understood. Hot water extraction and alcohol precipitation, DEAE-Sephadex A-25 chromatography, and Sephadex G-100 chromatography were used to isolate polysaccharide from C. militaris. A high-molecular-weight polysaccharide isolated from C. militaris was designated as HCMP, which had an Mw of 6.18 × 105 Da and was composed of arabinose, galactose, glucose, mannose, and xylose in a mole ratio of 2.00:8.01:72.54:15.98:1.02. The polysaccharide content of HCMP was 91.2% ± 0.16. The test in vitro showed that HCMP activated mouse macrophage RAW 264.7 cells by enhancing phagocytosis and NO production, and by regulating mRNA expressions of inflammation-related molecules in RAW 264.7 cells. Western blotting revealed that HCMP induced the phosphorylation of mitogen-activated protein kinases (MAPKs). Moreover, using inhibitors of MAPKs decreased the mRNA levels of inflammation-related molecules induced by HCMP. These data evidenced that the immunomodulatory effect of HCMP on RAW 264.7 macrophages was mediated via the MAPK signaling pathway. These findings suggested that HCMP could be developed as a potent immunomodulatory agent for use in functional foods and dietary supplements.

Structural Characterization of Polygonatum Cyrtonema Polysaccharide and Its Immunomodulatory Effects on Macrophages.

A neutral Polygonatum cyrtonema polysaccharide (NPCP) was isolated and purified from Polygonatum cyrtonema by various chromatographic techniques, including DEAE-52 and Sephadex-G100 chromatography. The structure of NPCP was characterized by HPLC, HPGPC, GC-MS, FT-IR, NMR, and SEM. Results showed that NPCP is composed of glucose (55.4%) and galactose (44.6%) with a molecular weight of 3.2 kDa, and the sugar chain of NPCP was →1)-α-D-Glc-(4→1)-β-D-Gal-(3→. In vitro bioactivity experiments demonstrated that NPCP significantly enhanced macrophages proliferation and phagocytosis while inhibiting the M1 polarization induced by LPS as well as the M2 polarization induced by IL-4 and IL-13 in macrophages. Additionally, NPCP suppressed the secretion of IL-6 and TNF-α in both M1 and M2 cells but promoted the secretion of IL-10. These results suggest that NPCP could serve as an immunomodulatory agent with potential applications in anti-inflammatory therapy.

Ultrasound assisted hot water extraction of polysaccharides from Taraxacum mongolicum: Optimization, purification, structure characterization, and antioxidant activity.

Ultrasound assisted hot water extraction (UAHWE) was applied to extraction of polysaccharides from Taraxacum mongolicum with hot water as extract solvent. Experimental factors in UAHWE process were optimized by response surface methodology. The optimal extraction parameters to achieve the highest Taraxacum mongolicum polysaccharides (TMPs) yield (12.08±0.14)% by UAHWE were obtained under the ultrasound power of 200W, extraction temperature of 62°C, solid-to-liquid ratio of 1:20g/mL, and extraction time of 40min, and then the crude TMPs were further purified by DEAE-52 and Sephadex G-100 chromatography to obtain a homogenous polysaccharide fraction (TMPs-1-SG). Subsequently, the structure of TMPs-1-SG was characterized by UV-vis, Fourier transform infrared spectroscopy (FT-IR), high performance gel permeation chromatography (HPGPC), high performance liquid chromatography (HPLC), scanning electron microscope (SEM), transmission electron microscopy (TEM), and Congo red test. The results display that TMPs-1-SG with an average molecular weight of 5.49 × 104Da was comprised of mannose (Man), galactose (Gal), xylose (Xyl), and arabinose (Ara) with a molar ratio of 39.85:52.61:27.14:6.30. Moreover, TMPs-1-SG did not contain a triple helix structure. Furthermore, TMPs-1-SG and TEM presented a sheet-like, rod-shaped, and irregular structure. Finally, the antioxidant activity of TMPs-1-SG was evaluated by in vitro experiment. The IC50 values of scavenging DPPH and OH radicals for TMPs-1-SG achieved 0.71mg/mL and 0.75mg/mL, respectively. The findings can provide an effective method for extracting polysaccharides from natural resources.

Purification, kinetic characterization of thermostable multicopper oxidase from the oyster mushroom and its versatility for greener agro-pulp bio bleaching in the paper industry.

Production of a thermostable laccase from Pleurotus florida was reported for the first time, both in submerged and solid-state fermentation using agro-industrial residues. This enzyme was purified using ammonium sulphate precipitation (60-90%), Sephadex G-100 and DEAE column ion exchange chromatography, respectively. The laccase was purified to 21.49 fold with an apparent molecular weight of 66 kDa and had an optimal pH of 5 with temperature stability at 60°C. Metal ions such as Cu2+ (91.26 µmole/mL/min), Mg2+ (68.15 µmole/mL/min), and Fe2+ (1.73 µmole/mL/min) enhanced the laccase activity, but Fe2+ (1.73µmole/mL/min) inhibited the enzyme activity. The purified laccase had Km and Vmax of 16.68 mM and 26.73 µmole/mL/min for guaiacol as a substrate. The isolated enzyme was characterized by FT-IR which revealed bands at 3655.0 cm-1, 2894.7 cm-1, and 1151.7 cm-1 corresponding to primary amines, C-H stretch, and amide -III, respectively. The enzymatic bio bleaching of paddy straw pulp was found to be most effective which resulted in a lowering of kappa number and yellowness by 19.47% & 17.84% whereas an increase in brightness and whiteness by 41.92%. & -19.61%. Thus, this might be stated that the crude laccase from P. florida can be exploited to reduce the toxic waste load for managing environmental pollution and helps in enhancing the yield and quality of the paper.

Isolation, Purification, Fractionation, and Hepatoprotective Activity of Polygonatum Polysaccharides.

In this study, three homogeneous fractions, PSP-N-b-1, PSP-N-b-2, and PSP-N-c-1, were obtained from an aqueous extract of Polygonatum using DEAE cellulose column chromatography, CL-6B agarose gel chromatography, and Sephadex G100 chromatography. Their monosaccharide compositions and molecular weights were analyzed. The results revealed that PSP-N-b-1, PSP-N-b-2, and PSP-N-c-1 are primarily composed of six monosaccharides: Man (mannose), GlcA (glucuronic acid), Rha (rhamnose), GalA (galacturonic acid), Glc (glucose), and Ara (arabinose), with molecular weights of 6.3 KDa, 5.78 KDa, and 3.45 KDa, respectively. Furthermore, we observed that Polygonatum polysaccharides exhibited protective effects against CCL4-induced liver damage in HepG2 cells in vitro, operating through both anti-oxidant and anti-inflammatory mechanisms. Our research findings suggest that Polygonatum polysaccharides may emerge as a promising option in the development of hepatoprotective drugs or functional foods with anti-inflammatory and antioxidant properties.

Extraction, purification, and investigation of the antibacterial potential of lysozyme isolated from the latex of Calotropis procera

Calotropis procera produces a latex used in traditional medicine because of its components which are found to be biologically active. The latex of C. procera has many benefits such as pain reduction, antimicrobial attitude and other precious attributes. Latex lysozyme (LL) is a particular protein contained in C. procera plant having a promising antibacterial property. The process applied for extraction implicated ammonium sulfate precipitation, water extraction, ion exchange and gel filtration column chromatography evidencing 14.4 kDa for LL as a molecular mass. LL after purification showed 397.18 U mg−1 as specific activity (10.30 purification fold), and with 37.9% as a yield. In addition, the chemical and physical analyses showed that LL had its best realization when the pH was 5.5 at 50 °C. Moreover when metal ions such as Ca2+ and Mg2+ were incorporated the relative activity of the enzyme resulted ampliflied. When a purification was conducted with CM-Cellulose and Sephadex G-100 chromatography, LL revealed an antibacterial property versus both Escherichia coli (MICs 14 µg ml−1) and Bacillus cereus (MICs 13 µg ml−1). HR-TEM analysis displayed an antimicrobial potential of LL after its purification; that analysis showed the deterioration of the cell wall (external membrane of the cell) of both E. coli and B. cereus. Therefore, the LL obtained from C. procera maintains ability as an antibacterial enzyme applicable in various domains such as biotechnological and pharmaceutical industries.Graphical abstract

Purification and partial characterization of a thermostable peroxidase isoenzyme from Bambara groundnut (Vigna subterranea L. Verdc) seedlings

Thermostable isoperoxidases, especially peroxidase A6, have already been found in seedlings of Vigna sp, indicating their potential for biotechnological applications. In this study, peroxidase A6 was purified from the roots of Bambara groundnut (Vigna subterranea L. Verdc) seedlings by a combination of gel filtration on Sephadex G-100, heat treatment, and ion exchange chromatography on CM cellulose and DEAE cellulose. The 41-kDa enzyme shows high catalytic efficiency in the oxidation of O-dianisidine, ABTS, TMB, DAB and OPD at acidic pH optimum and the reduction of H2O2. It has an optimum temperature of activity of 60 °C and a calculated activation energy of 221.5 KJ/mol for its thermal inactivation at pH 8. Mg2+ inhibits the enzyme. Ca2+ strongly increases its thermal stability, while Mn2+ and Zn2+ decrease it. The enzyme is inhibited by sodium azide at concentrations above 1 µM, with an IC50 value of about 10 µM. This inhibition, in addition to the RZ value of 2.4 (A403nm/A280nm), confirms the presence of a haem group in the active site, which is common for class III plant peroxidases. Due to its unique catalytic and thermal properties, peroxidase A6 has the potential to be a valuable tool for various biotechnological applications, especially for enzyme immunoassays and clinical diagnosis.

Antibacterial and anticancer activities of (free and immobilized) elastase produced by Klebsiella pneumoniae

Elastase is a type of protease that degrades explicitly elastin. The elastase produced by Klebsiella pneumoniae isolates was purified by three steps: ammonium sulfate precipitation, ion-exchange chromatography, and Sephadex G-150 chromatography. The optimal condition for elastase production showed high specific activity with starch (3.8 U/mg protein) and casein as a nitrogen source with a specific activity reaching (3.3 U/mg protein). The maximum elastase production was obtained when the pH value was (7.5) with specific activity (4.4 U/mg protein). Elastase (free and immobilized on TiO2- NPs) was used in application as antibacterial and anticancer, and results showed high antibacterial activity against pathogenic isolates, especially Lactobacillus acidophilus and Pseudomonas aeruginosa were affected by immobilized elastase. Free and immobilized elastase have anticancer activity against lung cancer using the A549 cell line, and immobilized elastase had the potent cytotoxic effect on A549 cells with IC50 142.8 µg/ ml compared with IC50 of normal cell line HdFn on 655.0 µg /ml. Key Words: Klebsiella pneumoniae, Elastase, Immobilization, TiO2-Nps- Antibacterial, Anticancer.

A novel heteropolysaccharide isolated from custard apple pulp and its immunomodulatory activity in mouse macrophages and dendritic cells

In this study, a novel heteropolysaccharide (ASPA80-1) with an average molecular weight of 5.48 × 104 Da was isolated and structurally elucidated from custard apple pulp (Annona squamosa) through DEAE-cellulose, Sephadex G-100 and Sephacryl S-300 HR chromatography and spectral analysis. ASPA80-1 is a water-soluble polysaccharide and it is a polymer consisting of predominant amounts of (1 → 3)-linked-L-arabinose (Ara) residues, small amounts of (1 → 6)-linked-D-galactose (Gal), (1 → 3,5)-linked-L-arabinose (Ara) residues and terminal linked-L-arabinose (Ara) residues, trace amount of (1 → 4)-linked-D-glucose (Glc) residues and (1 → 2)-linked-L-rhamnose (Rham) residues. ASPA80-1 showed significant effect on antigen-presenting cells (APCs) activation. On the one hand, ASPA80-1 activated RAW264.7 macrophage cells by inducing morphology change, enhancing phagocytic ability, increasing nitric oxide (NO) secretion and promoting expression of major histocompatibility complex class II (MHC II) and cluster of differentiation 86 (CD 86). On the other hand, ASPA80-1 promoted the maturation of dendritic cells (DCs) by inducing longer dendrites, decreasing phagocytic ability and increasing MHC II and CD86 expression. Furthermore, mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) signaling pathways were activated after the intervention of ASPA80-1 on RAW264.7 cells or DCs. Thus, the novel heteropolysaccharide ASPA80-1 has the potential to be used as an immunoenhancing component in functional foods.

Production, optimization, and purification of alkaline thermotolerant protease from newly isolated Phalaris minor seeds

The present work aims to purify and perform a preliminary analysis on a thermostable serine alkaline protease from a recently identified P. minor. The enzyme was purified 2.7-fold with a 12.4 % recovery using Sephadex G-100 chromatography, DEAE-cellulose, and ammonium sulphate precipitation. The isolated enzyme has a specific activity of 473 U/mg. The purified protease had a molecular mass of 29 kDa, and just one band was seen, which matched the band obtained using SDS-PAGE. High thermostability was demonstrated by the enzymes, which had half-lives of 31.79 and 6.0 min (a 5.3-fold improvement), enthalpies of denaturation (ΔH°) of 119.53 and 119.35 KJ mol−1, entropies of denaturation (ΔS°) of 32.96 and 41.11 J/mol·K, and free energies of denaturation (ΔG°) of 108.87 and 105.58 KJ mol−1 for the protease enzyme. Studies on the folding and stability of alkaline proteases are important since their use in biotechnology requires that they operate in settings of extreme pH and temperature. According to the kinetic and thermodynamic properties, the protease produced by P. minor is superior to that produced by other sources and previously described plants, and it might find utility in a variety of industrial fields.

Preparation of nano-selenium from chestnut polysaccharide and characterization of its antioxidant activity.

Chestnut is widely cultivated and has high nutritional value due to its richness in polysaccharides. In order to improve the antioxidant activity of chestnut polysaccharide, chestnut polysaccharide (CP) was extracted by ultrasonic-assisted water extraction and alcohol precipitation and purified by cellulose DEAE-52 exchange and Sephadex G-100 chromatography in this study. CP isolates were characterized by I2-KI reaction, three-strand helical structure analysis, infrared spectrum analysis, and nuclear magnetic resonance detection. The results showed that CP is a pyrylan sugar with triple helical structure and connected by α-glycosidic bonds, with sugar residues 1,4-α-D-Glcp, 1,6-α-D-Galp, 1,5-α-L-Araf, 1,4-α-L-Rhap, and 1,4-β-D-Glcp in the CP backbone. After purification, the branching structure, rod, and spherical structure were significantly increased, with reduced lamellar structure. The in vitro scavenging rates of CP at 10 mg·mL-1 against DPPH, hydroxyl radicals, and ABTS were 88.95, 41.38, and 48.16%, respectively. The DPPH free radical scavenging rate of purified polysaccharide fraction CP-1a was slightly enhanced, and the other rates showed a small decrease. Selenized chestnut polysaccharide (CP-Se) was prepared using nano-selenium method. The selenization method was optimized and stable Se-CP was obtained. When the concentration was 5 mg·mL-1, Se-CP had significantly higher scavenging abilities 89.81 ± 2.33, 58.50 ± 1.60, and 40.66 ± 1.91% for DPPH, hydroxyl radical, and ABTS radicals, respectively, than those of CP. The results of this study provide insight into the effects purification and selenization of chestnut polysaccharide on antioxidant activity, and also provide a theoretical basis for the development of chestnut polysaccharide for use in functional foods or health products.

Purification and biochemical characterization of two laccase isoenzymes isolated from Trichoderma harzianum S7113 and its application for bisphenol A degradation

Two laccase isoenzymes (LacA and LacB) were isolated from a novel Trichoderma harzianum S7113 isolate employing ammonium sulfate precipitation, Sephadex G100, and DEAE Sepharose ion exchange chromatography. The molecular weights of the purified LacA and LacB laccases were estimated to be 63 and 48 kDa, respectively. The two isoenzymes had their optimum activities at the same temperature (50 °C), but at slightly different pH values (pH 3.0 for LacA and pH 2.5 for LacB). LacA and LacB had the same thermal stability at 40 °C and pH stability at pH 9.0. The two isoenzymes also showed a high level of specific activity toward ABTS, where the Km values of LacA and LacB were 0.100 and 0.065 mM, whereas their Vmax values were 0.603 and 0.182 µmol min−1, respectively. LacA and LacB catalytic activity was stimulated by Mg2+, Zn2+, K+, and Ni2+, whereas it was inhibited by Hg2+ and Pb2+, β-mercaptoethanol, EDTA, and SDS, and completely inhibited by sodium azide. Our findings indicate that purified laccase has a promising capacity for bisphenol A (BPA) bioremediation across a broad pH range. This finding opens up new opportunities for the commercialization of this technique in a variety of biotechnology-based applications, particularly for removing endocrine chemicals from the environment.

A novel cyclic dipeptide from Lactiplantibacillus plantarum MC39 inhibits proliferation of multidrug-resistant Klebsiella pneumoniae W8 and Enterobacter hormaechei U25

The present study pays special attention to lactic acid bacteria products, cyclic dipeptides (CDPs), as an effective prevention strategy that can be used to eradicate the threats posed by multidrug-resistant (MDR) Enterobacteriaceae. Herein, Lactiplantibacillus plantarum MC39, a strain isolated from an artisanal Egyptian cheese, was a producer of a novel CDP that could strongly inhibit, in vitro, MDR Klebsiella pneumoniae W8 and Enterobacter hormaechei U25, for the first time. Following partial purification with 80% ammonium sulphate precipitation and Sephadex G-100 chromatography, this biomolecule was identified as the cyclic dipeptide cyclo(Ala–Glu) using IR, HNMR, and LC–MS. Cyclo(Ala–Glu) was thermostable up to 121 °C, insensitive to pepsin and α-chymotrypsin, tolerant to organic solvents, active at acidic pH, and stable for 45 days of storage. Thus, it shows clear evidence to be usable as a bio-preservative in dairy products and as a potential therapeutic agent, controlling MDR K. pneumoniae and E. hormaechei.

Purification, structural characterization and immunological activity of Sibiraea laexigata (L.) Maxim polysaccharide.

Sibiraea laexigata (L.) Maxim (SLM) has been used as an herbal tea for treating stomach discomfort and indigestion for a long time in china. Polysaccharides have been identified as one of the major bioactive compounds in the SLM. In the present paper, ultrasonic-assisted enzymatic extraction (UAEE) method was employed in polysaccharides extraction derived from SLM using polyethylene glycol (PEG) as extraction solvent, two SLM polysaccharides (SLMPs) fractions (SLMPs-1-1 and SLMPs-2-1) were purified by DEAE Cellulose-52 and Sephadex G-100 chromatography in sequence. Then, the preliminarily structure of the two factions were characterized by chemical composition analysis, molecular weight measurement, UVS, HPLC-PMP, FT-IR, nuclear magnetic resonance (NMR) spectra analysis and SEM. The results showed that SLMPs-1-1 and SLMPs-2-1 with different molecular weights of 1.03 and 1.02 kDa, mainly composed of glucose (46.76 and 46.79%), respectively. The results of structural characterization from FT-IR, 1H NMR, and SEM revealed that SLMPs-1-1 and SLMPs-2-1 contained the typical pyranoid polysaccharide with α-glycosidic bond and β-glycosidic bond. Furthermore, it was found that SLMPs-1-1 could increase the levels of tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2), and alleviated the immune organs tissue damage of cyclophosphamide (Cy)-treated mice. RT-qPCR and Western-Blot analysis showed that SLMPs-1-1 could significantly up-regulated the levels of NF-κB, TLR4, which revealed that SLMPs-1-1 could participate in immunosuppressive protection of Cy-treated mice. These findings suggested that the potential of SLMPs-1-1 as an alternative immunostimulator could be used in food and pharmaceutical industries.

Microwave assisted extraction, characterization of a polysaccharide from Salvia miltiorrhiza Bunge and its antioxidant effects via ferroptosis-mediated activation of the Nrf2/HO-1 pathway

A microwave-assisted extraction procedure for the crude Salvia miltiorrhiza polysaccharides (SMPs) obtained from Salvia miltiorrhiza Bunge was optimized. Four independent variables were studied: microwave power, extraction time, solvent-to-solid ratio, and concentration of ethanol, with optimal settings of 1200 W, 12 min, 38, and 86 %, respectively. The SMPs were successively purified by DEAE Sepharose Fast Flow and Sephadex G-100 chromatography to produce a novel polysaccharide termed SMP1. The SMP1 was composed of glucose, galactose, and fructose in a molar ratio of 1:1.67:1.12 with an average molecular weight of 6087 Da. Pharmacological studies showed that SMP1 protected from OGD/R-induced ferroptosis and lipid peroxidation by activating Nrf2/HO-1 pathway in PC12 cells. Our research systematically indicated that polysaccharide could inhibit ferroptosis to alleviate oxidative stress injury, which laid the foundation for the future clinical application of Salvia miltiorrhiza polysaccharide.

Purification of wild type and recombinant xylanases and evaluation of their arabinoxylan hydrolysis ability

Arabinoxylan hydrolyzing enzymes are very rich and diversified groups, among these, endo-1,4-β-xylanase is the most important group which catalyzes the cleavage of xylan main skeleton to release arabinoxylan oligosaccharides such as D-xylose, xylobiose, L-arabinose, xylotetraose, xylopentose. This group of enzyme was efficiently produced from natural microorganism including bacteria, yeast and fungi and recombinant strains. In this study, we have purified wild-type and recombinant xylanase to compare the difference in hydrolysis products of these two enzymes. Using sephadex G-100 and DEAE-sephadex column chromatography, we have purified xylanase from wild-type Aspergillus niger DSM1957 with molecular weight of 25 kDa, specific activity of 3240 IU/mL, the purity increases 1.68 folds compare to crude extract, and a recovery rate of 36%. The recombinant enzyme from Pichia pastoris GS115/pPXlnA was purified by ProbondTM affinity chromatography column, with a molecular weight of 36 kDa; the purity increased 3.2 folds than the crude extract, and with the recovery efficiency of 21.1%. The activity of commercial wild-type xylanase was better than that of our recombinant and wild-type enzymes. Our wild-type xylanase could hydrolyze arabinoxylan into L-arabinose in 50 mM CH3COONa pH 5.0. Our results showed that xylanases purified from different sources were stable and highly specific to xylan. Xylanase has potential application in the production of high quality biotechnological products.

Modeling, optimization, purification, and characterization of polysaccharides from Lilium lancifolium Thunb

Ultrasound assisted aqueous two-phase extraction of polysaccharides from Lilium lancifolium Thunb was modeled by response surface methodology (RSM) and artificial neural network (ANN), and optimized using genetic algorithm coupled with ANN (GA-ANN). Statistical analysis showed that the models obtained by RSM and ANN could accurately predict the Lilium lancifolium Thunb polysaccharides (LLPs) yield. However, ANN prediction was more accurate than RSM. The optimum extraction parameters to achieve the highest LLPs yield (15.17 ± 0.21)% was obtained at the ultrasound power of 250 W, extraction temperature of 63 °C, liquid-to-solid ratio of 21 mL/g, and extraction time of 32 min. Subsequently, the crude LLPs were further purified via DEAE-52 and Sephadex G-100 chromatography to obtain a homogenous fraction (LLPs-2-SG, 421.41 kDa) that contained mannose, glucose, and galactose in a molar ratio of 10.52:23.06:7.19. The structure of LLPs-2-SG was characterized with UV–vis, FT–IR, AFM, and SEM. The findings provide a critical method for the extraction, separation and purification of polysaccharides from natural resources.