Sevag Method Research Articles

ISOLATION, PURIFICATION AND PHYTOCHEMICAL SCREENING OF POLYSACCHARIDE FRACTIONS FROM APIUM GRAVEOLENS’ L. BOTANICAL FORMS

The study of natural polysaccharides is an important area of research due to their pharmacological effects, including antiviral activity and the ability to regulate metabolic disorders. In addition, these polymeric structures realize several properties (sorption, shaping, transport (and delivery systems), which increases the interest of scientists in their isolation and analysis. This study aims to comparatively investigate the polysaccharide fractions in different botanical forms of celery odouriferous. The objectives of the study include the isolation of polysaccharide fractions, their purification by the Sevag method, estimation of monomeric composition of the fractions after acid hydrolysis by the HPTLC method, and determination of structural characteristics of the molecules by IR spectroscopy. Alcohol-soluble polysaccharides (ASPS), water-soluble polysaccharides (WSPS), and pectin substances (PS) were isolated from leaf, petiole, and root botanical forms of celery. After purification, the obtained substances were amorphous powders of light brown or light beige colour and odourless. The root botanical form of celery showed the highest yield of the target compounds (total (13.54±1.07)% after purification), while the petiole form (total (5.51±0.04)% after purification) showed the lowest yield. The monomeric composition of pectins and WSPS showed a predominance of galactose and arabinose, whereas the alcoholic ones showed a predominance of fructose and glucose. The interpretation of IR spectra showed the presence of absorption bands characteristic of free and bound carboxyl groups, C-O-C valence vibrations, α-configuration of the glycosidic bond, and C1-α-conformation of galacturonic acid in different polysaccharide fractions, which allows us to draw some conclusions regarding the structure of the substances. A comparative study of polysaccharide fractions of different botanical forms of odorous celery was carried out for the first time. Based on the results obtained, it is possible to single out WSPS and PS of celery root crops as the most promising phytosubstances for further development of products of functional, specialized nutrition and potential medicines on their basis. The methods of analysis used in the study can be proposed as part of the regulatory documentation for the control of these products.

Characterization and anti-aging activities of polysaccharide from Rana dybowskii Guenther.

Introduction: Rana dybowskii Guenther (RDG), as a traditional Chinese medicine, has been shown to have antioxidant effects. However, studies on the anti-aging effect of RDG are still limited. Methods: In this study, we prepared polysaccharides from the skin of RDG (RDGP) by hot water extraction, alcohol precipitation, ion-exchange chromatography and gel chromatography. The proteins were removed using the Sevage method in combination with an enzymatic method. The structural features were analyzed using high-performance gel permeation chromatography, β-elimination reaction and Fourier transform infrared spectra. The anti-aging effect of RDGP was investigated by using D-Gal to establish an aging model in mice, and pathological changes in the hippocampus were observed under a microscope. Results: We obtained the crude polysaccharide DGP from the skin of RDG, with a yield of 61.8%. The free protein was then removed by the Sevage method to obtain DGPI and deproteinated by enzymatic hydrolysis combined with the Sevage method to further remove the bound protein to obtain the high-purity polysaccharide DGPII. Then, DGPIa (1.03 × 105Da) and DGPIIa (8.42 × 104Da) were obtained by gel chromatography, monosaccharide composition analysis showed that they were composed of Man, GlcA, GalNAc, Glc, Gal, Fuc with molar ratios of 1: 4.22 : 1.55: 0.18 : 8.05: 0.83 and 0.74 : 1.78: 1: 0.28: 5.37 : 0.36, respectively. The results of the β-elimination reaction indicated the presence of O-glycopeptide bonds in DGPIa. The Morris water maze test indicated that mice treated with DGPIIa exhibited a significantly shorter escape latency and increased time spent in the target quadrant as well as an increase in the number of times they traversed the platform. Pathologic damage to the hippocampus was alleviated in brain tissue stained with hematoxylin-eosin. In addition, DGPIIa enhanced the activities of SOD, CAT, and GSH-Px and inhibited the level of MDA in the serum and brain tissues of aging mice. Discussion: These results suggest that RDGP has potential as a natural antioxidant and provide useful scientific information for anti-aging research.

Preparation, Characterization, and Bioactivities of Polysaccharide-Nano-Selenium and Selenized Polysaccharides from Acanthopanax senticosus.

Acanthopanax senticosus polysaccharide-nano-selenium (ASPS-SENPS) and A. selenopanax selenized polysaccharides (Se-ASPS) were synthesized, and their characterization and biological properties were compared. The acid extraction method was used to extract the polysaccharides of A. selenopanax, followed by decolorization using the hydrogen peroxide method and deproteinization based on the Sevage method, and the purification of A. senticosus polysaccharides (ASPS) was carried out using the cellulose DEAE-52 ion column layer analysis method. An A. senticosus polysaccharide-nano-selenium complex was synthesized by a chemical reduction method using ASPS as dispersants. The selenization of polysaccharides from A. selenopanax was carried out using the HNO3-Na2SeO3 method. The chemical compositions, scanning electron microscopy images, infrared spectra, and antioxidant properties of ASPS-SENPS and Se-ASPS were studied, and they were also subjected to thermogravimetric analysis. The results indicated that the optimal conditions for the synthesis of ASPS-SENPS include the following: when ASPS accounts for 10%, the ratio of ascorbic acid and sodium selenium should be 4:1, the response time should be 4 h, and the reaction temperature should be 50 °C. The most favorable conditions for the synthesis of Se-ASPS were as follows: m (Na2SeO3):m (ASPS) = 4:5, response temperature = 50 °C, and response time = 11.0 h. In the in vitro antioxidant assay, when the mass concentration of Se-ASPS and ASPS-SENPS was 5 mg/mL, the removal rates for DPPH free radicals were 88.44 ± 2.83% and 98.89 ± 3.57%, respectively, and the removal rates for ABTS free radicals were 90.11 ± 3.43% and 98.99 ± 1.73%, respectively, stronger than those for ASPS. The current study compares the physiological and bioactivity effects of ASPS-SENPS and Se-ASPS, providing a basis for future studies on polysaccharides.

Chemical characteristics and anti-Escherichia coli mechanism of water-soluble extracts from yeast cell walls

BackgroundAntimicrobial resistance is a threat to global public health and requires the development of new antibiotic alternatives to treat bacterial infection. Infection with Escherichia coli (E. coli) is the most common cause of diarrhea in pigs, especially in weaning piglets. The objective of this study was to characterize chemical properties of water-soluble extract (WSE) obtained from yeast cell wall, and study its antibacterial activity and antimicrobial mechanisms against E. coli.ResultsWSE was extracted from yeast cell wall by hot water, followed by anhydrous ethanol precipitation and deproteinization by Sevag method to obtain water-soluble polysaccharides (WSP, accounting for 76.53 ± 5.91% by weight of WSE). WSP was purified by DEAE-52 cellulose column and Sephadex G-100 column to obtain three fractions of polysaccharides, WSNP, WSAP-1 and WSAP-2. Physicochemical properties of them were then characterized. The average molecular weights of WSNP, WSAP-1 and WSAP-2 were 105,130, 94,581 and 91,247 Da, respectively. WSNP was composed of glucosamine (GlcN), glucose (Glc), mannose (Man) and glucuronic acid (GlcA) in a molar ratio of 8.6:55.2:34.4:1.8, WSAP-1 was consisted of GlcN, Glc and Man in a molar ratio of 10.9:42.2:46.9, and WSAP-2 was consisted of GlcN, galactose (Gal), Glc and Man in a molar ratio of 9.0:1.0:55.1:34.9. Spectral analysis indicated that all three polysaccharides had pyran polysaccharides, triple-helix structure, α- and β-glycosidic bond. The minimum inhibitory concentrations of WSE and WSP on E.coli were 25 mg/mL and 100 mg/mL, respectively. WSE can not only disrupt the integrity of the cell wall and membrane of E.coli, but also increase the permeability of the outer membrane.ConclusionsOur research elucidated chemical composition and structure of WSNP, WSAP-1 and WSAP-2. WSE exerted its bactericidal activity by damaging the bacterial cell wall and membrane and increasing outer membrane permeability. Yeast cell wall products have been recommended as a potential alternative to antibiotics. This work provides more information, regarding chemical properties and anti-E. coli mechanism of WSE, and facilitates the application of yeast cell wall products as a promising antibiotic alternative in animal feed.

Preparation and analysis of polysaccharide from Solanum tuberdsm

The crude Solanum tuberdsm polysaccharides (STP) were extracted with hot water. In the process of extraction, proteins, pigments, small molecules and salts in the mixture were removed by Sevage reagent, diatomite and distilled water dialysis, respectively. In addition, the process conditions of protein removal by response surface methodology (RSM) were optimized, and the optimum process conditions of Sevage method were established as follows: ultrasound power 350 W, ultrasound time 20 min, deproteinization twice, volume ratio of polysaccharide solution to Sevage reagent 1:1 (mL/mL). Under these conditions, the protein removal rate was 93.14%.

Structural Characterization and Immunobiological Activity of Polysaccharides from Astragalus Oyster Mushroom.

When added to mushroom growing substrates, edible and medicinal herbs affect the mushrooms' nutritional and medicinal value. In this study, polysaccharides (P0OP-I and P15OP-I) were extracted and purified from oyster mushrooms grown on substrates supplemented with 0% and 15% Astragalus roots (P0 and P15), respectively, and their chemical structure and immunobiological activities were compared. P15OP-I and P0OP-I were extracted using ultrasound-assisted hot water and deproteinized with the Sevage method, depigmented with 30% H2O2, desalted with dialysis, and purified using DEAE-52 cellulose and Sephadex G-100 dextran column chromatography. The molecular weight of P0OP-I and P15OP-I was 21,706.96 and 20,172.65 Da, respectively. Both were composed of monosaccharides D-mannose, galacturonic acid, D-glucose, D-galactose, and L-arabinose but in different molar ratios, and both were connected by a pyranoside linkage. P15OP-I consisted of higher contents of mannose, glucose, galactose and arabinose and lower content of galacturonic acid as compared to P0OP-I. Both P0OP-I and P15OP-I induced NO and TNF-α production but did not show cytotoxic effect or induce ROS generation in RAW264.7 cells. P15OP-I showed a stronger ability to promote NO and TNF-α production relative to P0OP-I. In vitro experiments showed that the immunomodulatory activity of P0OP-I and P15OP-I in RAW264.7 macrophages were mediated by the JNK/MAPK, Erk/MAPK, and NF-κB signaling pathways. The results would be helpful for elucidation of the health promoting mechanism of Astragalus oyster mushrooms as a source of neutraceuticals.

Mass spectrometry-based metabolomics reveal Dendrobium huoshanense polysaccharide effects and potential mechanism of N-methyl-N'-nitro-N-nitrosoguanidine -induced damage in GES-1cells.

Dendrobium huoshanense C. Z. Tang et S. J. Cheng is an important edible medicinal plant that thickens the stomach and intestines, and its active ingredient, polysaccharide, can have anti-inflammatory, immunoregulatory, and antitumor effects. However, the gastroprotective effects and potential mechanisms of Dendrobium huoshanense polysaccharides (DHP) remain unclear. An N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced human gastric mucosal epithelial cells (GES-1) damage model was used in this research, aiming to investigate whether DHP has a protective effect on MNNG-induced GES-1cell injury and its underlying mechanism based on the combination of multiple methods. DHP was extracted using water extraction and alcohol precipitation methods, and the proteins were removed using the Sevag method. The morphology was observed using scanning electron microscopy. A MNNG-induced GES-1cell damage model was developed. Cell viability and proliferation of the experimental cells were investigated using a cell counting kit-8 (CCK-8). Cell nuclear morphology was detected using the fluorescent dye Hoechst 33342. Cell scratch wounds and migration were detected using a Transwell chamber. The expression levels of apoptosis proteins (Bcl-2, Bax, Caspase-3) in the experimental cells were detected by Western blotting. Ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was performed to investigate the potential mechanism of action of DHP. The CCK-8 kit analysis showed that DHP increased GES-1cell viability and ameliorated GES-1cell injury by MNNG. In addition, scratch assay and Transwell chambers results suggested that DHP improved the MNNG-induced motility and migration ability of GES-1cells. Likewise, the results of the apoptotic protein assay indicated that DHP had a protective effect against gastric mucosal epithelial cell injury. To further investigate the potential mechanism of action of DHP, we analyzed the metabolite differences between GES-1cells, GES-1cells with MNNG-induced injury, and DHP+MMNG-treated cells using UHPLC-HRMS. The results indicated that DHP upregulated 1-methylnicotinamide, famotidine, N4-acetylsulfamethoxazole, acetyl-L-carnitine, choline and cer (d18:1/19:0) metabolites and significantly down-regulated 6-O-desmethyldonepezil, valet hamate, L-cystine, propoxur, and oleic acid. DHP may protect against gastric mucosal cell injury through nicotinamide and energy metabolism-related pathways. This research may provide a useful reference for further in-depth studies on the treatment of gastric cancer, precancerous lesions, and other gastric diseases.

Preparation and Analysis of Pueraria lobata Polysaccharides.

Pueraria lobata polysaccharides (PLPs) were obtained by a hot water extraction method. Starting from the single factor experiment, the extraction was optimized by response surface methodology, and the following optimal extraction parameters were obtained: the extraction temperature was 84 °C, the liquid-solid ratio was 11 mL/g, the extraction time was 73 min, and the extraction rate of polysaccharides was 8.59%. The Sevag method was used to remove the protein soluble in water and H2O2 was used to remove the pigment; then PLPs were precipitated with three times of anhydrous ethanol, soluble salts and other small molecules were removed by dialysis, and finally refined PLPs were obtained by freeze-drying.

Extraction, moisturizing activity and potential application in skin cream of Akebia trifoliata (Thunb.) Koidz polysaccharide

Akebia trifoliata (Thunb.) Koidz is gaining popularity, because of its high nutritional, economic and medicinal values. The purpose of this study was to optimize the extraction conditions of A. trifoliata (Thunb.) Koidz polysaccharide (ATKP) and investigate moisturizing activity to prepare a novel moisturizing cosmetic material. It has been observed that using polysaccharide yield as the evaluation criterion showed optimal extraction at a material-liquid ratio of 1:20, an ultrasonic temperature of 80 °C, an ultrasonic time 50 min and ultrasonic power of 175 W. Furthermore, three deproteinization methods of ATKP process, including Sevag, enzymatic hydrolysis and their combination method, were evaluated. The deproteinization efficiency of the Sevag and enzymatic hydrolysis combination method was the highest, with a deproteinization efficiency of 87.52 %. The moisturizing capacity of ATKP was significantly higher than that of chitosan and sodium alginate. ATKP-containing skin cream, particularly the combination of 4.2 % glycerol and 0.8 % ATKP, had an excellent moisturizing effect, and was added to cosmetic formulations to form skin creams. The findings revealed that ATKP has the potential synergistic effect with glycerol in the development of cosmetic formulations, which could be used as a new material for natural moisturizers.

Optimization of ultrasonic-assisted polysaccharide extraction from Hyperici Perforati Herba using response surface methodology and assessment of its antioxidant activity

This study performed a comprehensive investigation of Hyperici Perforati Herba polysaccharide (HPHP) regarding the development and optimization of extraction methods, elucidation of structure and characteristics, and determination of antioxidant activities. An ultrasonic-assisted extraction method, which offered advantages in terms of the extraction yield and energy efficiency, was developed by response surface analysis. The following optimum conditions were determined: a crushing degree at 65 mesh, ultrasonic time at 50 min and temperature of 43 °C. Through enzyme-mediated deproteination via the Sevag method, activated carbon depigmentation, and DEAE-52 and Sephadex G-100 column elution, three HPHPs were obtained, and their monosaccharides mainly included mannose, galactose, glucose and arabinose. The molar weights were 8.347, 1.199 and 22.426 kDa, respectively. The HPHP structures were an amorphous aggregate of spherical-like shapes with a rough surface of pores and crevices, which presented characteristic Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectra of polysaccharides. Their main glucosidic linkage is the α-type configuration. Moreover, HPHPs exhibited strong scavenging activity for DPPH·, ABTS·+, OH· and O2·− radicals; good ferric reducing power; and effective protection against oxidative damage in human cells. Overall, the results of this work underpinned a fundamental understanding of HPHPs, thus providing a potential antioxidant for further research and development.

Effects of exopolysaccharides from Antrodia cinnamomea on inflammation and intestinal microbiota disturbance induced by antibiotics in mice

Antrodia cinnamomea is an important edible and medicinal mushroom, and it exhibits multiple biological activities, such as hepatoprotection, antitumor, antivirus, and immunoregulation. Polysaccharides are the main products of A. cinnamomea in submerged fermentation. In this work, exopolysaccharides from A. cinnamomea (AEPS) were extracted and purified by alcohol precipitation and Sevag method. Composition analysis revealed that AEPS were primarily composed of three distinct polysaccharides with mean molecular weights of 1,013, 233, and 28,743 kDa, accounting for 78%, 18%, and 4% of AEPS, respectively. The AEPS have β-type glycosidic bonds and relatively strong resistance to digestion. In vivo experiments showed that the intragastric administration of AEPS in mice with medium dose (0.25 g/kg body weight of mice) has the following effects: remarkably alleviate lincomycin hydrochloride (LIH)-induced injuries to immune organs; enhance the relative abundance of beneficial microorganisms such as Lactobacillus, Roseburia, Ligilactobacillus, and Lachnospiraceae_NK4A136_group in the intestinal tract; greatly reduce the levels of inflammatory cytokines IL-6 and TNF-α in serum and the relative abundances of harmful microbes such as Enterococcus and Shigella; regulate the balance of the gut microflora; and relieve LIH-induced symptoms such as diarrhea, inflammation, and weight loss. These findings might represent a new alternative to develop novel multifunctional carbohydrate prebiotics.

Asymmetrical flow field-flow fractionation combined with ultrafiltration: A novel and high-efficiency approach for separation, purification, and characterization of Ganoderma lucidum polysaccharides

Ganoderma lucidum polysaccharide (GLP) has attracted increasing attention owing to its multiple pharmacological effects. However, the establishment of a structure-bioactivity relationship is possible only if highly purified and accurately characterized GLP is used. In this study, a combined technique based on asymmetrical flow field-flow fractionation (AF4), ultrafiltration, and the Sevag method was developed to improve the purification process of GLP. Deionized water was used throughout this study which avoids the dialysis desalting process. Furthermore, the molecular weight (Mw) and radius of gyration (Rg) of the purified GLP were characterized by AF4 coupled online ultraviolet–visible (UV), multiangle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI). The results demonstrated that after removal of the larger size proteins, one highly purified GLP fraction (polysaccharide content of 108.37 ± 0.45%) and its structural information (i.e., Mw, Rg, and apparent density) were obtained in a single run owing to a combination of mechanisms of ultrafiltration and AF4 separation. The results suggested that the method developed in this study for separation, purification, and characterization of GLP is highly efficient, which could help to better understand its structure-function relationships.

The prebiotic properties of polysaccharides obtained by differentiated deproteinization methods from Flos Sophorae Immaturus on Lactobacillus fermentum.

The polysaccharides derived from various deproteinization methods were prepared from Flos Sophorae Immaturus (FSI) to investigate the prebiotic efficacy of Lactobacillus fermentum (L.f ). The implications of polysaccharides from FSI (PFSI) gained after purification performed by non-deproteinization and different deproteinization processes (Savage method, papain method, and TCA method) via one-factor optimization were firstly investigated for the influences on the growth of L.f. The utilization of carbohydrate sources and the synthesis of protein and lactate during its growth were analyzed, as well as the variations of LDH, SOD, and GSH- Px enzyme dynamics. The results showed that the one-factor optimization of the deproteinization process with the protein removal rate and polysaccharide retention rate as the indexes led to the optimal methods of the Sevage method with 5 elution times, papain method with 80 U/mL concentration, and TCA method with 2.5 ratio, respectively. In addition, the PFSI obtained with or without deproteinization purification had a certain effect on promoting L.f proliferation. Moreover, the PFSI gained by the third deproteinization purification, at a concentration of 10 g/L, significantly elevated L.f biomass and growth rate compared with the blank control, and the utilization of reducing sugars and the synthesis of protein and lactic acid were higher than the control (P < 0.05); improved LDH, SOD, and GSH-Px activity in L.f (P < 0.05), and the TCA method could be effectively applied to eliminate the proteins affecting FSI in probiotics, and PFSI may be a potentially beneficial prebiotic and intestinal reinforcer.

Effects of high-pressure homogenization extraction on the physicochemical properties and antioxidant activity of large-leaf yellow tea polysaccharide conjugates

This paper, discussed the high-pressure homogenization (HPH) method for the preparation of large-leaf yellow tea polysaccharide conjugates (LYTP), and the effects of LYTP on physicochemical properties as well as antioxidant activity were investigated. The results show that HPH extraction time was less than 0.5 h, and extraction rate reached 15.4%, which was nearly 1.8 times faster than that of hot water extraction (HWE). Component analysis showed that the total polysaccharide content of LYTP was higher, while the contents of polyphenols and free protein were lower than those of HWE. It is noteworthy that the 9.1% free protein content at 40 MPa was lower than that of the Sevag method (11.2%), indicating that HPH can eliminate the cumbersome protein removal process and is more environmentally. The small-molecule polysaccharide content extracted by HPH increased, but the monosaccharide composition did not change. Meanwhile, HPH decreased the particle size of the polysaccharides, and the changes in the surface topography of LYTP upon homogenization were confirmed with scanning electron microscopy. As a consequence, HPH extraction can improve the antioxidant capacity of LYTP. HPH extraction is an efficient, rapid and environmentally method for the preparation of polysaccharides, which could be widely used in the food industry.

Structure Characterization and Antioxidant Activity of Polysaccharides from Amomum villosum extracted with Alkaline Solution

In this study, A. villosum was used as the raw material, and the crude polysaccharides of A. villosum were extracted by 0.1 mol/L NaOH solution. After the protein was removed by the Sevag method, the polysaccharides of A. villosum were obtained. The extraction rate of polysaccharides was 3.97%, the total sugar content was 37.10%, the uronic acid content was 65.66%, the protein content was 4.36%, and the molecular weight of polysaccharide was 95.46 kDa. The results of high-performance liquid chromatography showed that A. villosum polysaccharides were mainly composed of arabinose, xylose, galactose and galacturonic acid. Fourier transform infrared spectroscopy(FTIR), scanning electron microscopy(SEM), X-ray diffraction(XRD), thermogravimetric analysis(TGA) results showed that it was an amorphous structure, had a pyran ring, and thermally stable sheet-like acidity heteropolysaccharides. The results of in vitro antioxidant models(DPPH, ABTS, hydroxyl free radicals) showed that the polysaccharides of A. villosum had good in vitro antioxidant capacity. This research aims to provide a theoretical basis for the application andproduct development of polysaccharides from A. villosum.

Extraction, isolation and identification of an enzymatic browning product from fresh white salted noodles

An enzymatic browning product (EBP) of fresh white salted noodles (FWSNs) was extracted, purified, isolated and identified. The optimum parameters of extraction were as follows: volume fraction of ethanol 70%, solid-liquid ratio 1 : 10 (g: mL, samples: ethanol solution), extraction time 10 days (20 °C). In the purified process, protein (99.86%) and carbohydrates (69.50%) impurities were removed from the crude extract using Sevag method, dialysis method and AB-8 macroporous resin column. The purified EBP was separated using 20% (v: v) methanol solution in a C18 chromatographic column. A monomer of EBP was isolated by high-performance liquid chromatography. Nuclear magnetic resonance and infrared spectroscopy analysis showed that the EBP monomer was 2-hydroxyindole-3-propylamine. This was the first time in the literature that 2-hydroxyindole-3-propylamine was found in the EBP of FWSNs. It would lay a material foundation for investigating the origin of EBPs and the revealing enzymatic browning mechanism of FWSNs.

Ganoderma lucidum polysaccharides promote T lymphocyte infiltration into tumor by regulating expression of ICAM-1 in endothelial cells

Polysaccharide is among the main active components of Ganoderma lucidum for tumor prevention and treatment. Howe-ver, it remains unclear whether it has synergy with tumor immunotherapy. This study evaluated the effect of G. lucidum polysaccharides(GLP) on the infiltration of T lymphocytes into tumor and the underlying mechanism, in order to provide a reference for its application in tumor immunotherapy. GLP were prepared by water extraction and alcohol precipitation combined with Sevag method and then given(intraperitoneal injection) to the mice bearing B16-F10 cells at 25, 50 and 100 mg kg~(-1), respectively, to evaluate the effect on tumor growth. The infiltration of CD3~+ and CD8~+ T cells and the expression of intercellular cell adhesion molecule-1(ICAM-1) in tumor were detected by immunohistochemistry. EA.hy926 cells were treated with 50, 100 and 200 μg·mL~(-1) GLP, and the expression of ICAM-1 was determined by Western blot. The adhesion of EA.hy926 cells treated with GLP was measured with fluorescence-labeled Jurkat cells. To analyze the mechanism based on NF-κB pathway, this study determined the protein levels of nuclear factor kappa-B(NF-κB) p65, alpha inhibitor of NF-κB(IκBα), p-NF-κB p65 and p-IκBα by Western blot. The results showed that GLP can significantly inhibit the tumor growth in mice bearing B16-F10 cells, promote the infiltration of CD3~+ and CD8~+ T cells in tumor, and increase the expression of ICAM-1 in tumor. Meanwhile, GLP could also enhance the expression of ICAM-1 in EA.hy926 cells, thus strengthen the adhesion to Jurkat cells, induce phosphorylation and protein degradation of IκBα, and raise the expression and phosphorylation level of NF-κB p65. These results suggested that GLP could promote the expression of ICAM-1 through NF-κB pathway and further enhance the infiltration of T lymphocytes into tumor, thereby inhibiting tumor growth. This study lays a foundation for the further application of GLP in tumor immunotherapy.

Anti-inflammatory and antioxidant activity of the hot water-soluble polysaccharides from Anacyclus pyrethrum (L.) Lag. roots

Ethnophamacological relevancethe roots of Anacyclus pyrethrum (L.) Lag. (Family: Asteraceae) are used in Algeria to treat respiratory infections, to cure chronic head and nostrils catarrh, and to clear the brain by stimulating the free flow of nasal mucous. They contain a high quantity of hot water-soluble polysaccharides. Aims of the studyThe study aims to evaluate the antioxidant and anti-inflammatory activity of polysaccharides extracted from Anacyclus pyrethrum roots (APPS). Materials and methodsThe APPS were extracted using boiling water, separated from proteins by the Sevag method then precipitated with 90% ethanol. The antioxidant effect of crude APPS was evaluated using FRAP assay. To investigate the anti-inflammatory potential, mice were treated with crude polysaccharides (25, 50, and 100 mg/kg, i.p.) for 3 days (14th, 15th, and 16th day of the experimentation). Respiratory inflammation was induced by HDM (House Dust Mite), mice were sensitized intranasally with 25 μg HDM suspended in 10 μl NaCl (5 μl/nostril) on days 0 and 7 then challenged with 5 μg HDM on days 14, 15, and 16. Mice were sacrificed 24 h after the last challenge. The number of immune cells in the blood in NL (Nasal Liquid) and in BAL (Broncho Alveolar Liquid) was enumerated, the spleen was removed to calculate the relative spleen weight and to count splénocytes, lungs histopathological examination was carried out to confirm the protective effect of APPS. Structural characterization of APPS was identified using FTIR (Fourier-Transform Infrared Spectroscopy) and SEM (Scanning Electron Microscopy). ResultsThe crude APPS possessed reducing power. In vivo assay, treatment with APPS causes a decrease in the number of blood leucocytes at all doses on the one hand, and in the relative spleen weight and splénocytes number on the other hand except at the dose of 50 mg/kg in which an enhancement of the number of splénocytes and immune cells in NL and BAL was significant. The histopathological examination showed clear protection of lung tissue damaged by HDM, after treatment with APPS mainly, at the dose of 50 mg/kg. ConclusionOur data clearly showed antioxidant and anti-inflammatory activity of APPS on HDM-challenged mice induced lungs inflammation by equilibrating the inflammatory reaction mostly, with an optimal dose of 50 mg/kg.

Characterization of polysaccharides from &lt;i&gt;Eremurus hissaricus&lt;/i&gt; roots by FTIR spectroscopy

The structure of water'soluble gluco' and galactomannans (GlcMan and GalMan) isolated from the roots of several Eremurus'related plant species has been studied previously. This article characterizes polysaccharides and other extraction products from the root tubers of Eremurus hissaricus by Fourier' transform infrared (FTIR) spectroscopy using an attenuated total reflectance (ATR) accessory. Polysaccharide samples were purified from protein substances with the Sevage method and discoloured through a polyamide column. In the FTIR spectra, the main peaks attributed to asymmetric and symmetric stretching vibrations of CH2 of the pyranose ring (for GluMan - 2886, 1373, 1244 cm-1; for GalGluMan - 2923, 1370, 1238 cm-1) were enhanced by purification after removing bound protein impurities. The KnownitAll and IR-Pal 2 software applications were used to examine the spectra of a sample of purified GalGluMan in the studied polysaccharides. The results show that the intensity of the absorption maximum decreased at 1732 cm-1. At the same time, at 1552.92 cm-1, a new band appeared that refers to the valence vibrations of carboxyl (CO) or nitroso (NO) groups in the purified polysaccharide. This band appeared as a result of the Maillard reaction between the protein and the reducing end of the polysaccharide unit. The intensity of the bands in the 1238-1244 cm-1 region also decreased in the purified samples, which may be due to the elimination of a small fraction of protein. The use of FTIR spectroscopy allows the process of purifying watersoluble GluMan and acid-soluble polysaccharide of GalGluMan from acrylamide impurities to be characterized timely and accurately. Additionally, this method allows a comparative estimation of functional groups in polysaccharides of the root tubers of E. hissaricus.

Antioxidant and Immunomodulatory Activities In Vitro of a Neutral Polysaccharide from Ginger (Zingiber Officinale)

AbstractIn this study, crude polysaccharides from ginger are obtained by ultrasonic‐assisted enzymatic extraction. After deproteinization using Sevag method, dialysis, and isolation on a DEAE52‐cellulose column and a Sephadex G‐100 column, a neutral heteropolysaccharide purified ginger polysaccharide (pGP) with the molecular weight of 62 500 Da is obtained. Four sugar residues are confirmed in pGP, including →3)‐α‐L‐Araf‐(1→, →2)‐α‐L‐Rhap, →3)‐β‐D‐Galp‐(1→, →6)‐α‐D‐Glcp‐(1→. Antioxidant activities analysis confirms that pGP possess good scavenging effects on DPPH, 2,2‐azino‐bis‐3‐ethylbenzthiazoline‐6‐sulphonic acid, and hydroxyl radicals. In addition, pGP significantly stimulates the phagocytic activity of RAW264.7 cells and has strong protection effect against H2O2‐induced damage. These results show that pGP can be used as a potential antioxidant agent in medicine or functional food.