Extraction and characterization of polysaccharides from blackcurrant fruits and its inhibitory effects on acetylcholinesterase.

Microwave assisted aqueous two-phase system (MA-ATPS) was used to simultaneously extract two polysaccharides from blackcurrant. Under the suitable ATPS (ethanol/(NH4)2SO4, 26.75 %/18.98 %) combining with the optimal MA conditions (liquid-to-material ratio 58.5 mL/g, time 9.5 min, temperature 60.5 °C, power 587 W) predicted by response surface methodology, the yields of the top/bottom phase polysaccharides were 13.08 ± 0.37 % and 42.65 ± 0.89 %, respectively. After purification through column chromatography, the top phase polysaccharide (PRTP) and bottom phase polysaccharide (PRBP) were obtained. FT-IR, methylation and NMR analyses confirmed that the repeating unit in the backbone of PRTP was →2, 5)-α-L-Araf-(1 â†’ 3)-α-D-Manp-(1 â†’ 6)-ß-D-Galp-(1 â†’ 6)-α-D-Glcp-(1 â†’ 4)-α-L-Rhap-(1 â†’ 4)-α-D-GalAp-(1→, while the possible unit in PRBP was →4)-α-L-Rhap-(1 â†’ 3)-α-D-Manp-(1 â†’ 6)-ß-D-Galp-(1 â†’ 6)-α-D-Glcp-(1 â†’ 2, 5)-α-L-Araf-(1 â†’ 4)-α-D-GalAp-(1→. PRBP with relatively low molecular weight exhibited better stability, rheological property, free radical scavenging and acetylcholinesterase (AChE) inhibitory activities than PRTP. PRTP and PRBP were reversible mixed-type inhibitors for AChE, and the conformation of AChE was changed after binding with the polysaccharides. Molecular docking, fluorescence and isothermal titration calorimetry assays revealed that PRTP and PRBP quenched the fluorescence through static quenching mechanism, and the van der Waals interactions and hydrogen bonding played key roles in the stability of polysaccharide-enzyme complexes. This study provided a theoretical basis for blackcurrant polysaccharides as AChE inhibitors to treat Alzheimer's disease.

Preparation, characteristics, and antioxidant activities of carboxymethylated polysaccharides from blackcurrant fruits.

In the present study, the native polysaccharide (RNP) extracted from blackcurrant fruits was carboxymethylated. Physicochemical characteristics and antioxidant activities in vitro of RNP and three carboxymethylated polysaccharides (CRNPs) were determined. GC analysis proved that RNP and CRNPs were composed of the same six monosaccharides (galacturonic acid, rhamnose, arabinose, mannose, glucose and galactose), but the molar ratios of monosaccharides were different. HPLC demonstrated that the molecular weights of CRNPs were improved. The assays of the antioxidant properties indicated that CRNPs possessed stronger scavenging activities on radicals (hydroxyl and superoxide radicals) and better anti-lipid peroxidation activities, as well as better protection effects on erythrocyte hemolyses in vitro compared with RNP. The activities of CRNPs were significantly enhanced with the increase of the degree of substitution (DS). These results proved that the carboxymethylation could effectively increase the antioxidant activities of the polysaccharide.

Effects of ultrasound irradiation on the characterization and bioactivities of the polysaccharide from blackcurrant fruits.

In this study, the influence of ultrasound irradiation on the characterization and bioactivities of the polysaccharide from blackcurrant fruits (BCP, molecular weight: Mw = 3.26 × 104 kDa) was investigated. Two degraded polysaccharides (U-400, Mw = 1.89 × 104 kDa, and U-600, Mw = 1.32 × 104 kDa) were obtained by different ultrasound powers of 400 W and 600 W, respectively. Compared with BCP, U-400 and U-600 showed 63.52% and 68.85% reductions in the particle size (Zavg), respectively; moreover, the dynamic viscosity of BCP was reduced by 27.88%, and 33.63%, separately. The reducing sugar content and thermal stability increased with the increase of ultrasound intensity. The degraded polysaccharides contained the same monosaccharide species as those of BCP but at different molar ratios. Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopic analysis confirmed that the degraded polysaccharides and BCP exhibited the similar structural features, which were mainly composed of six glycosidic bonds. A reduction in surface area of the flake-like structure was observed in the degraded polysaccharides compared to that of BCP, and they had no triple helix structure. Furthermore, the precise structural characteristics of U-600 were identified by 2D NMR analysis. The results of the bioactivity assays indicated that the ultrasound irradiation could evidently enhance the antioxidant (hydroxyl and superoxide radicals scavenging, lipid peroxidation inhibition, and DNA damage protection activities), α-amylase and α-glucosidase inhibition activities of BCP. These activities increased in the order of U-600 > U-400 > BCP. In particular, the DNA protection and α-amylase inhibition activities for U-600 were 52.19 ±â€¯1.34% and 75.98 ±â€¯0.77%, respectively, which were 2 times higher than those of BCP. U-600 prepared with the higher-intensity ultrasound exhibited the best physicochemical properties and bioactivities among the three polysaccharides. These results suggested that ultrasound irradiation was an efficient, green method to produce value-added polysaccharide for use in functional food or medicine.

Sulfated modification of the polysaccharides from blackcurrant and their antioxidant and α-amylase inhibitory activities.

Sulfated modification was conducted to modify a homogenous polysaccharide from blackcurrant (BCP). The sulfated polysaccharides (SBCPs) with different degree of substitution (DS) were synthesized using the aminosulfonic acid (ASA)/4-dimethylaminopyridine method by varying reaction conditions such as the mass ratio of ASA to BCP, temperature, and time. Three sulfated derivatives were chosen for high-performance gel-permeation chromatography, gas chromatography, fourier-transform infrared (FT-IR) spectroscopy, and nuclear magnetic resonance (NMR), and activity studies, designated as SBCP-1, SBCP-2, and SBCP-3 with DS of 1.28, 0.95, and 0.53, respectively. Results showed that the sulfated modification was successful, and SBCPs had an increase in molecular weight compared to BCP. Both SBCPs and BCP were composed of rhamnose, arabinose, xylose, mannose, galactose, and glucose, with different molar ratios. Sulfate substitution was further confirmed by FT-IR and 13C NMR analysis. SBCPs exhibited excellent antioxidant capacities (DPPH, hydroxyl, and superoxide radical scavenging, reducing power, and ferrous metal-chelating capacities) and α-amylase inhibitory activity in vitro, and the activities of SBCPs were significantly improved in positive correlation with the DS value. This study suggested that SBCPs could serve as potential antioxidant agents to be used as alternative supplements or functional foods.

Optimization of ultrasound-assisted compound enzymatic extraction and characterization of polysaccharides from blackcurrant.

In the present study, an efficient procedure for ultrasound-assisted compound enzymatic extraction of polysaccharides from blackcurrant fruits was investigated using response surface methodology (RSM). The Box-Behnken design was applied to optimize the effects of enzyme concentration (X1), pH (X2) and ultrasonic time (X3). The statistical analysis indicated that the independent variables (X1) and the quadratic terms (X1(2) and X3(2)) had significant effects on the yield of blackcurrant polysaccharides (BCP). The optimal conditions were: enzyme concentration 1.575%, pH 5.3, and ultrasonic time 25.6 min. The experimental yield of BCP was 14.28±0.06%, which was closely matched with the predicted yield of 14.31%. After preliminary purification, BCP I was obtained and characterized by GC, HPLC, and IR. BCP I comprised rhamnose, arabinose, xylose, mannose, glucose, and galactose in a molar ratio of 1.818:1.362:0.377:0.501:1.581:1.722 and its molecular weight was 8146 kDa. BCP I showed notable α-amylase inhibitory activity.