Isolation and structural characterization of exopolysaccharide from the Cordyceps cicadae and the immunomodulatory activity on RAW264.7 cells.

A new exopolysaccharide component named as PC-EPS was isolated from Cordyceps cicadae, and its structure was determined. PC-EPS was identified to be constituted of mannose, glucose, and galactose (28.84:1:19.42), with an average molecular weight of 3.72 × 106  Da, according to the results of monosaccharide composition, Fourier transform infrared, nuclear magnetic resonance, periodate oxidation and Smith degradation, and methylation studies. According to structural characterization, PC-EPS's connection type was made up of →6) -α-d-Manp (1→, →2) -ß-d-Manp (1→, →4) -α-d-Manp (1→, →2) -α-d-Galf (1→, and →4) -α-d-Galp (1→. PC-EPS may significantly increase phagocytosis and RAW264.7 cell proliferation. Additionally, by boosting intracellular lysozyme, cellular acid phosphatase, and cellular superoxide dismutase enzyme concentrations, as well as by promoting the generation of cellular NO, it is the potential to regulate the immunological activity of RAW264.7 cells. Additionally, the effects of PC-EPS on RAW264.7 cells increased their capacities to create tumor necrosis factor-α and interleukin 6 cytokines, all of which suggested that PC-EPS had the potential to improve immunomodulatory activity.

Preparation and antibacterial effect of chitooligosaccharides monomers with different polymerization degrees from crab shell chitosan by enzymatic hydrolysis.

This study aimed to explore the structure and antibacterial properties of chitooligosaccharide monomers with different polymerization degrees and to provide a theoretical basis for inhibiting Salmonella infection. Chitosan was used as a raw material to prepare and separate low-molecular-weight chitooligosaccharides. Chitobiose, chitotriose, and chitotetraose were obtained by gradient elution with cation exchange resin. The molecular weights and acetyl groups of the three monomers were determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and nuclear magnetic resonance (NMR), respectively. Three chitooligosaccharide monomers were used to explore the antibacterial effect on Salmonella. The results showed that the degree of deacetylation of chitosan was 92.6%, and the enzyme activity of chitosanase was 102.53 U/g. Within 18 h, chitosan was enzymatically hydrolyzed to chitooligosaccharides containing chitobiose, chitotriose, and chitotetraose, which were analyzed by thin-layer chromatography (TLC) and MALDI-TOF. MALD-TOF and TLC showed that the separation of monomers with ion exchange resins was effective, and NMR showed that there was no acetyl group. Chitobiose had a poor inhibitory effect on Salmonella, and chitotriose and chitotetraose had equivalent antibacterial effects.

Structural characterization of a polysaccharide from Abelmoschus esculentus L. Moench (okra) and its hypoglycemic effect and mechanism on type 2 diabetes mellitus.

A novel acidic polysaccharide named AeP-P-1 was prepared from Abelmoschus esculentus L. Moench (okra). AeP-P-1 is a heteropolysaccharide with a molecular weight of 3.02 × 103 kDa and is composed of L-rhamnose, D-galactose, and D-galacturonic acid in the ratio 1.87 : 3.58 : 1.00. Structural characterization based on methylation and 1D/2D NMR analyses indicated that AeP-P-1 is composed of T-linked-Rhap, T-linked-Galp, 1,2,4-linked-Rhap, 1,4-linked-Galp, 1,6-linked-Galp, and 1,3,4-linked-Galp in a molar ratio of 2.42 : 3.36 : 6.46 : 13.31 : 3.12 : 1, respectively. The hypoglycemic effect and mechanism of AeP-P-1 on type 2 diabetes mellitus were also explored. Firstly, AeP-P-1 can reduce blood lipids and liver and kidney damage caused by T2DM. Finally, AeP-P-1 induces the phosphorylation of GSK3ß, maintains the activity of glycogen synthase (GCS), and promotes glycogen synthesis by regulating the expression of insulin/PI3K/Akt pathway proteins. These results indicated that AeP-P-1 could be developed as a potential ingredient in immunostimulatory agents.

Rheological properties of polysaccharides from Pholiota nameko with different temperature extraction: Concentration, pH, temperature, and saltion.

Cold and hot water extracted polysaccharides (CW-PNPs and HW-PNPs) were isolated from Pholiota nameko. The rheological properties of PNPs were investigated by steady shear and oscillatory rheological measurements. The PNPs exhibited typical non-Newtonian and shear-thinning behavior, which are affected by PNP concentration, temperature, pH value, salt ion, and concentration. Specifically, the apparent viscosity of the two PNPs solutions at concentration of 1% (w/w) was shown as HW-PNPs > CW-PNPs. The apparent viscosity of PNPs decreases under acidic and alkaline conditions and when the temperature rises; K+ and Na+ cause the apparent viscosity of CW-PNPs to decrease, while Ca2+ and Al3+ are opposite. The addition of four different salt ions all caused the apparent viscosity of the HW-PNPs to decrease. The results of dynamic rheological experiments show that G' and G″ showed slightly frequency dependency with G' exceeding G″ throughout the accessible range of frequency for CW-PNPs and HW-PNPs.

Structural characterization and prebiotic potential of an acidic polysaccharide from Imperial Chrysanthemum.

A novel water-soluble polysaccharide, named ICP-1, was isolated and purified by Sephadex G-200 after extracting the crude polysaccharide (ICP) from Imperial Chrysanthemum. The structural characterization of ICP-1 was determined by physical and chemical methods, FT-IR, NMR, SEM, HPGPC, periodate oxidation, Smith degradation, methylation and Congo red test. Then, acid production and proliferation of lactic acid bacteria and the tolerance tests of simulated gastrointestinal fluid were measured to investigate the activity of prebiotic potential. The results showed that ICP-1 was an acidic hetero-polysaccharide with an average molecular weight of 2.98 × 103 kDa and a specific optical rotation of +155°. The glycosyl residues of ICP-1 were composed of (1→), (1→4) and (1→6) glucose, (1→5) arabinose, (1→4) galacturonic acid and (1→3,6) mannose. Besides, ICP-1 can speed up the acid production of lactic acid bacteria and promote the growth and proliferation of lactic acid bacteria effectively.

Structure, antioxidant property and protection on PC12 of a polysaccharide isolated and screened from Abelmoschus esculentus L.Moench (okra).

AeP-P-2, a pectic polysaccharide, was extracted from the fruit pod of okra. It composed of rhamnose (Rha), arabinose (Ara), glucose (Glc), galactose (Gal) and galacturonic acid (GalA) with the ratio of 4.75:2.01:1.00:4.91:7.24. The main structural feature of AeP-P-2 are 1,4-linked galacturonan units (homogalacturonan backbone) and (1 → 2) and (1 → 2,4) linked Rha (rhamnogalacturonan I region). And the other side chains contained →1)-linked Ara, (1 → 5)-linked Ara, (1 → 4)-linked Glc, (1 → 6)-linked Gal, (1 → 4)-linked Rha, (1 → 2,4)-linked Rha, →1)-linked Ara and →1)-linked Gal. When the concentration of AeP-P-2 was 3.2 mg/mL, the scavenging rates on DPPH·, ABTS, O2-· and ·OH reached to 61.88%, 87.10%, 52.17% and 60.32%, respectively. AeP-P-2 also could protect PC12 cells from the damage of H2O2 and reduce apoptosis caused by oxidative damage by decreasing the level of ROS. The findings indicated that okra was a functional vegetable and AeP-P-2 was worth studying and developing into antioxidant component.

Effect of steam explosion pretreatment on the structure and bioactivity of Ampelopsis grossedentata polysaccharides.

Steam explosion (SE) was a friendly environmentally pretreatment method. In this study, the effect of steam explosion (SE) pretreatment on structure and α-glucosidase inhibitory activity of Ampelopsis grossedentata polysaccharides was evaluated. Two novel polysaccharides (AGP and AGP-SE) were extracted, isolated, purified and analyzed by NMR, FT-IR and methylation. The results indicated that AGP mainly consisted of Rha, Xyl, Glc, and Ara with a molecular weight of 2.74 × 103 kDa and AGP-SE mainly consisted of Man, Ara, and Gal with a molecular weight of 2.14 × 103 kDa. Furthermore, the backbone of AGP and AGP-SE were mainly composed of 5)-Araf-(1→, -Glcp-(1→, 6)-Glcp-(1→, 6)-Galp-(1→, 3,6)-Manp-(1→, and 2,3,6)-Glcp-(1→. Finally, we demonstrated that all polysaccharides exhibited obviously α-glucosidase inhibition activity and mixed type inhibition. AGP-SE had better α-glucosidase inhibition activity and the binding affinity KD on α-glucosidase by using Surface Plasmon Resonance (SPR) than AGP. Overall, SE pretreatment is an effective method for extracting polysaccharide and provides a new idea into the improvement of biological activity.

Synthesis and inhibition of α-glucosidase of methyl glycyrrhetinate glycosides.

The synthesis of the methyl glycyrrhetinate glycosides and inhibition of α-glucosidase were studied. The carboxyl group of glycyrrhetinic acid was methylated, and glucose and galactose were introduced into the hydroxyl group to obtain compounds 7 and 12. Compound 1, 2, 7, 12 and glycyrrhizic acid (GL) were evaluated for their inhibitory activities against α-glucosidase. As a result, Compound 1, 2, 7, 12 and GL all showed significant α-glucosidase inhibitory activity and IC50 values were 0.465, 1.352, 0.759, 0.687 and 2.085 mM, respectively, and acted as non-competitive inhibitors. The activity of the compound 2, 7, 12 was lower than compound 1, but significantly higher than GL. Therefore, it was concluded that the change of structure in glycyrrhetinic acid by chemical modification had certain effect on bioactivity, and the change of carboxyl group, hydroxyl group and the type of monosaccharide introduced were the influencing factors.

Enzymatic characterization and validation of gene expression of phosphoglucomutase from Cordyceps militaris.

The purification and characterization of PGM (Phosphoglucomutase) from Cordyceps militaris (C. militaris) was investigated. PGM was purified using a combination of ultrafiltration, salting-out and ion exchange chromatography resulting in 4.23-fold enhancement of activity with a recovery of 20.01%. Molecular mass was 50.01 kDa by SDS-PAGE. The optimal activity was achieved at pH 7.5 and 30 °C with NADPH as substrate. The results showed that SDS, DTT Li+, Cu2+, Na+, Mn2+ and Al3+ were effective PGM inhibitors; whereas glycerol, Zn2+, Mg2+, Ca2+, Fe2+ and Fe3+ could enhance the activity of PGM, and the Km and Vmax values were 11.62 mmol/L and 416.67 U/mL, respectively. At the same time, qRT-PCR was used to test the changes of mRNA transcription level of PGM gene encoding under two fermentation conditions: basic medium and optimized medium. The relative quantitative results of PGM target genes resulting in 2.60-fold enhancement than the control group.

Chemical structure and antioxidant activity of a polysaccharide from Siraitia grosvenorii.

A novel polysaccharide from Siraitia grosvenorii residues (SGP, molecular weight 1.93 × 103 KDa) was isolated and purified. SGP was composed of α-L-Arabinose, α-D-Mannose, α-d-Glucose, α-D-Galactose, Glucuronic acid, and Galacturonic acid with the ratio of 1: 1.92: 3.98: 7.63: 1.85: 7.34. The backbone of SGP was consist of galactoses and linked by α-(1,4)-glycosidic bond. The branch chains including α-1,6 linked glucose branch, α-1,6 linked mannose branch, α-1,3 linked galactose branch and arabinose branched (α-L-Ara(1→). The results of bioactivity experiments suggested that SGP had antioxidant in vitro, especially on scavenging DPPH radicals. Besides, SGP resulted in the decrease of ROS and the percentage of apoptotic and necrotic cells in a dose-dependent manner in H2O2 oxide injury PC12 cells. This research could help to develop the potential value and utilization of Siraitia grosvenorii.

Hypoglycemic effect of glycyrrhizic acid, a natural non-carbohydrate sweetener, on streptozotocin-induced diabetic mice.

In this paper, glycyrrhizic acid (GZA) was extracted from the stem of licorice by enzymatic hydrolysis, separated and purified by silica gel column chromatography, its purity was determined by high performance liquid chromatography (HPLC), and the structure was identified by Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The hypoglycemic activity of GZA was measured by α-glucosidase inhibition in vitro and the establishment of a T2DM diabetic mouse model in vivo. The experimental results showed that the extraction rate and purity of GZA were 92.4% and 93.53%, respectively, and the IR spectrum and NMR spectrum were consistent with the standard and literature, respectively. The IC50 value of GZA for α-glucosidase inhibition was 1.88 mmol L-1, which was close to the positive control (acarbose). In addition, GZA could reduce the fasting blood glucose of T2DM mice by increasing insulin sensitivity, increasing glucose tolerance, significantly decreasing serum triglyceride (TG), total cholesterol (TC), and low density lipoprotein cholesterol (LDL-C) contents, and increasing high density lipoprotein cholesterol (HDL-C) content. At the same time, it could also repair damaged organs to some extent. Therefore, GZA has broad application prospects in the treatment of T2DM.

Chemical structure and effects of antioxidation and against α-glucosidase of natural polysaccharide from Glycyrrhiza inflata Batalin.

GIBP, a homogeneous polysaccharide extracted from Glycyrrhiza inflata Batalin with a molecular weight 1.96 × 103 kDa, had a triple helix structure, smooth and sheet-like structure. Comprehensive analysis showed that the main chain of GIBP was composed of α-D-1,4 linked glucose, branch points were composed of α-D-1,3,6 and α-D-1,2,3,6 linked glucoses, and side chains were composed of α-D-1,3 and ß-D-1,6 linked galactose, ß-L-1,2 linked arabinose, α-D-1,3 and ß-D-1,3 mannose. The scavenging abilities of GIBP (3 mg/mL) against DPPH radical, OH radical, O2- radical and ABTS were 50.75 ± 0.13% and 52.32 ± 0.13, 25.84 ± 0.35% and 44.57 ± 0.15% and it also demonstrated an obvious dose-effect relationship. The inhibitory activity of α-glucosidase showed that the inhibitory effect of GIBP was enhanced with the increase of concentration. When the concentration reached 6 mg/mL, the inhibition rate of α-glucosidase activity reached 64.77%. And the ka, kd and KD were 6.472 × 104 1/Ms., 2.934 × 10-3 1/s and 4.534 × 10-8 M.

Carboxymethylation and acetylation of the polysaccharide from Cordyceps militaris and their α-glucosidase inhibitory activities.

The crude polysaccharide extracted from Cordyceps militaris was chemically modified to obtain carboxymethylated derivatives (CM-CPS) and acetylated derivatives (AC-CPS). The physicochemical characterizations were comparatively investigated by chemical methods, high-performance gel permeation chromatography, FT-IR spectra, NMR analysis, Congo red test, scanning electron microscopy, atomic force microscopy and differential scanning calorimetry. Then α-glucosidase inhibitory activities were conducted to determine the structure-bioactivity relationship. Results indicated that carboxymethylation and acetylation modification of polysaccharide were successful with the carboxymethyl substitutions might being C-6, C-2 and acetyl substitutions at C-3, C-6 inferred from NMR analysis. In addition, the tertiary structure, ultrastructure, melting properties were also different from native polysaccharide. Besides, α-glucosidase inhibitory activities of derivatives exhibited differently with CM-CPS to be the lowest. Therefore, it was concluded that change of structure in polysaccharide had certain effect on bioactivity with degree of substitution and substituents position being the influence factors.

1H NMR-based metabonomics of the hypoglycemic effect of polysaccharides from Cordyceps militaris on streptozotocin-induced diabetes in mice.

The crude polysaccharide was extracted from Cordyceps militaris. Material ratio of powder and water was 1:10. The polysaccharide was successively purified by Sevag and chromatography on Sephadex G-100 column to produce a polysaccharide fraction termed CBPS-II. The average molecular weight of CBPS-II was 1.273 × 103 kDa. The study was conducted to investigate the hypoglycemic effect of Cordyceps militaris polysaccharide on diabetic mice. Analysis of the clinical chemistry of the serum samples included serum creatinine (CRE), urea nitrogen (BUN), triglyceride (TG) and total cholesterol (TC). Results revealed that a certain dose of polysaccharide can alleviate the symptoms of metabolic disorders of diabetes, contributing to the body to restore the normal levels. The metabolic profiling method was adopted to find the related biomarkers and the metabolic pathway of diabetes. Moreover, results showed that 100 mg·kg-1 of Cordyceps polysaccharides can effectively reduce the blood glucose level of diabetic mice, thus regulating the metabolism of their energy, amino acids and intestinal microbes. The biomarkers noted in their metabolism were glucose, lactic acid, 3-hydroxy butyric acid, creatine, glutamate, valine, leucine, isoleucine and very low density lipoprotein (VLDL).

Comparisons of the anti-tumor activity of polysaccharides from fermented mycelia and cultivated fruiting bodies of Cordyceps militaris in vitro.

A comparison of the anti-tumor activity of CMPS-II and CBPS-II polysaccharides, respectively is obtained from the fermented mycelium and cultivated fruiting bodies of Cordyceps militaris. This in vitro anti-tumor activity is investigated using an MTT assay, immunofluorescence staining, a Western Blot assay, a qRT-PCR assay, and Annexin V-FITC/PI double staining. The experimental results indicate that the inhibition rate of CMPS-II on H1299 tumor cells is higher than that of CBPS-II. With a concentration of 500 µâ€¯g/mL, the inhibition rate of CMPS-II and CBPS-II were 54.55% and 34.80%, respectively. Both CMPS-II and CBPS-II can increase the protein and mRNA expression level of cell apoptosis factors Caspase-3, Caspase-9, and p53, while reducing the protein and mRNA expression levels of proliferating cell nuclear antigen (PCNA), to induce tumor cells apoptosis. The induction effect of CMPS-II was stronger than CBPS-II. These results suggest that CMPS-II is superior to CBPS-II regarding the inhibition of H1299 lung cancer cells. Furthermore, CMPS-II is a potentially useful substitution for CBPS-II in the treatment of lung cancer and provides new insights into the mechanism of its anti-tumor activity.

The effect of fermentation conditions on the structure and anti-tumor activity of polysaccharides from Cordyceps gunnii.

This study investigates the effect of fermentation conditions on the structure and anti-tumor activity of intracellular polysaccharides (IPS) of Cordyceps gunnii (C. gunnii) in submerged fermentation. The environmental and nutritional conditions are determined in a shaker flask by a single factor test. The inhibition of IPS on S180 cells was as an optimization index. The results show that the optimal fermentation conditions of C. gunnii are an initial pH value of 6, a temperature of 25 °C, a rotation speed of 150 rpm, 4% glucose, and 1.0% peptone. Under these conditions, the macro molecular weight (M w) polysaccharide content and anti-tumor activity of IPS are significantly higher than that in the basal culture medium. GC, HPGPC, periodate oxidation-Smith degradation, NMR, and FT-IR determine the structural characteristics of CPS-JC and CPS-YH (pure IPS cultured in basal culture medium and optimal culture medium, respectively). The results indicate that CPS-JC is mainly composed of α-d-glucans, whereas CPS-YH primarily contain α-d-glucans with a trace amount of ß-d-glucans.

Structural properties and antioxidant activities of polysaccharide from fruit bodies of Pholiota nameko.

A polysaccharide named PNP was extracted and purified from Pholiota nameko. The total sugar content of PNP was 95.29% and the molecular weight was 1.89 × 103 kDa. The structural features of PNP were investigated by the combination of chemical and instrumental analysis such as UV spectrophotometer, specific rotation determination, FT-IR, methylisation analysis and Congo red. The results showed that the optical rotation of PNP was +120° and that it had a triple-helical structure. Besides, PNP was mainly composed of glucose and mannose at the molar ratio of 4.24:1.00. The backbone of PNP was composed of (1→3)-linked-Glc and (1→3)-linked-Man whereas the branches of (1→3,6)-linked- Glc, (1→3,6)-linked-Man and T- Glc. Consistenting with the results of UV-Vis spectra, FT-IR spectroscopy and 1H NMR, indicated that PNP was a complex of polysaccharides and polyphenols. In vitro antioxidant results suggested that PNP was processed with certain scavenging capacity.

Structural characterisation and ACE-inhibitory activities of polysaccharide from Gastrodia elata Blume.

The structural properties and Angiotensin-I converting enzyme (ACE) inhibition activities of a polysaccharide (PGE) extracted from Gastrodia elata Blume were investigated. PGE was extracted using hot water and purified by Sephadex G-200 followed by ultra-filtration. The structural characterisation of PGE was analysed by FT-IR, NMR spectroscopy, specific rotation determination, periodate oxidation-smith degradation, methylation analysis, GC-MS and Congo red test. The results revealed that PGE was composed by glucose, with an average molecular weight of 1.54 × 103 kDa. The structure of PGE was 1→3 and 1→4,6-branched-glucopyranose that had a linear backbone of (1 → 4)-linked-d-glucopyranose (Glcp). ACE-inhibitory activity results showed that PGE was efficient to inhibit ACE and the IC50 value was 0.66 mg/mL.

Structure analysis and anti-fatigue activity of a polysaccharide from Lepidium meyenii Walp.

A polysaccharide was obtained from Lepidium meyenii Walp by hot water extraction and purification by Millipore (100 kD) and Sephadex G-200. The content of polysaccharide was examined to be 89.9% with phenol-sulfuric acid method. Its average molecular weight was estimated to be 2.213 × 106 Da by High Performance Gel Permeation Chromatography (HPGPC). Monosaccharide analysis showed that the polysaccharide was composed of arabinose, mannose, glucose and galactose with the molar ratio of 2.134: 1: 2.78: 2.82. After Smith degradation, methylation, infrared spectroscopy and NMR, the primary structure of the polysaccharide was identified. The backbone of the polysaccharide was composed of →4)-ß-D-Galp-(1→ and →4)-α-D-Galp-(1→, while the branches were comprised of →6)-ß-D-Glup-(1→, →5)- ß-D-Araf-(1→, →3,6)-α-D-Manp-(1→, →3)-α-D-Galp-(1→, and α-D-Glup-(1→. The anti-fatigue effect of the polysaccharide was evaluated using exhaustive swimming test and biochemical indexes. The results indicated the polysaccharide has anti-fatigue effect.

Structural analysis and antioxidant activity of the glycoside from Imperial Chrysanthemum.

A new glycoside, named as CG-1, was separated from Imperial Chrysanthemum with silica gel column chromatography. The purity was detected by TLC and HPLC. The crystal shape of CG-1 was consisting of a quadrangular and two rectangular pyramids. The analysis of DSC and TGA showed that the melting point of CG-1 crystal was 150.22 °C and had good thermal stability. The monosaccharide conformation analysis showed that it was d-glucose. The structure characteristics were compared by FT-IR, NMR spectroscopy and LC-MS and a molecular structure has been deduced which consistent with spectroscopic results. In vitro antioxidant results suggested that the glycoside extracted from Imperial Chrysanthemum could be effectively employed as natural antioxidant in health or functional food. This work is of significance to keep the antioxidant activity in the processing and application of Imperial Chrysanthemum.