| Raspberry(Rubus idaeus L.) is Rosaceae Rubus plants, bush fruit. Raspberry tender and juicy, inviting color, unique flavor. It is not only used in fresh-eating and processed products, but also a kind of important health-beneficial fruit, which is confirmed that it has many improtant health benefits such as antiaging, anticancer, analgesic and antipyretic, protect the liver, reducing cardiovascular disease. It is rich in vitamins and mineral elements, especially rich in active substance such as polysaccharides, flavonoids, SOD, ellagic acid and other active substances. With the raspberry(Qiu Fu) fruits as raw materials, degradation, extractions, purification, structure as well as antioxidation, antidiabetic activities have been studied in the experiment in vitro. Degraded polysaccharides from raspberry fruit have been preparated by the Fenton Reagent Method at the first time, which is reduced cost, simplified manipulation, material readily available and has narrow Product molecular weight distribution. It provide the reference basis for in-depth study of raspberry polysaccharide health benefits and polysaccharide product.The polysaccharides were extracted by method of hot water extraction. The extraction conditions were temprature 80 ℃, time 1h and liquid to solid ratios 10:1(mL/g). The yield of polysaccharides was 4.42±0.56%. The polysaccharides were precipitated with EtOH to a final concentration of 40%(v/v) and lyophilized(crude polysaccharide). The purity was 29.23±0.72% detected by the phenol-sulfuric acid. Macroporous resins D4020 was used to purify the crude polysaccharide which were lyophilized(RCPI). The purity was 42.39±1.14% which was 1.45 times of crude polysaccharide. The recovery of RCPI was 72.16±0.39% indicating the low loss rate of polysaccharides. RCPI was degraded with two different Fenton reagent methods, getting two pale green degraded polysaccharides(DRCPI, DRCPII). The purity was 43.53±1.03%, 46.74±0.87% detected by the phenol-sulfuric acid, which was 1.03,1.10 times of RCPI. After the determination of protein content, polyphenol content and anthocyanin content, RCPI, DRCPI and DRCPII were found to contain almost no protein, polyphenols and anthocyanins. Determination of uronic acid content of RCPI, DRCPI, DRCPII showed that the uronic acid content is 29.28 ± 0.42%, 35.07 ± 0.81% and 33.60 ± 0.54%, indicating that the content of uronic acid increased after degradation.In the determination of physical properties, RCPI, DRCPI and DRCPII are soluble in water, insoluble in alcohol, ether, ethyl acetate, acetone, chloroform and other organic solvents. But the water soluble was different between polysaccharide and degraded polysaccharides. The degradation process increases the solubility of polysaccharide. The determination of molecular weight of the polysaccharide by HPLC showed that the polysaccharide RCPI is not homogeneous, and weight average molecular weight of the main component of RCPI is 745983 Da. Two degraded polysaccharide DRCPI and DRCPII are homogeneous components, the weight average molecular weight is 16421 Da and 11832 Da, respectively. The antioxidant activities of RCPI, DRCPI and DRCPII were studied. The results showed that except for the weak metal ion chelating ability is weak, whether it is before degradation more sugar RCPI or degradation of polysaccharides DRCPI, DRCPII are showed out the certain scavenging free radical activity and total reducing capacity, and the effect is obvious. Degradation of raspberry polysaccharide were clearly demonstrated superior to polysaccharides of antioxidant capacity, and antioxidant activity of DRCPII were higher than DRCPI. Further purification, structural characterization and activity of DRCPII were determined in terms of purity, solubility, molecular weight and so on.The polysaccharide RCPI and DRCPII was separated and purified by agarose gel Sepharose-6B. The concentration of sample was 15 mg/mL and 20 mg/mL, the sample volume was 1 mL, the elution flow rate was 0.80 mL/min, two components RCPI-2, DRCPII-2 and were collected, concentrated and freeze-dried because that it is more abundant and the peak shape was sharp and symmetrical. The purity was 59.82±0.54% and 61.83±0.72% detected by the phenol-sulfuric acid. Using Sepharose-6B gel column chromatography, freeze thawing method and UV spectral analysis of RCPI-2 and DRCPII-2 identification showed that RCPI-2 and DRCPII-2 were homogeneous components.Polysaccharides RCPI-2 and DRCPII-2 were white, pale green floc, respectively. RCPI-2 and DRCPII-2 are soluble in water, insoluble in organic solvents. The pH of RCPI-2 and DRCPII-2 solution was 4.96±0.38, 4.83±0.62, indicating that RCPI-2 and DRCPII-2 were acid polysaccharides. The relative molecular weight of RCPI-2 and DRCPII-2 were 410912 Da and 8019 Da determined by HPLC. The polysaccharide molecular weight decreased compared with the polysaccharide before purified.The meteorological chromatogram showed that the monosaccharide composition of RCPI-2 in a molar ratio of galacturonic acid: rhamnose: arabinose: xylose: mannose: glucose: galactose =1: 0.148: 0.647: 0.258: 0.110: 0.101: 0.445 and the monosaccharide composition of DRCPII-2 in a molar ratio of galacturonic acid: rhamnose: arabinose: xylose, mannose: glucose: galactose =1 : 0.211 : 0.387 : 0.188 : 0.358 : 0.419 : 0.427. The infrared spectrum analysis showed that RCPI-2 and DRCPII-2 were mainly composed of furan ring and α-configurations of sugar units, which were composed of uronic acid. In the structure of determination, scanning electron micrograph shows polysaccharide DRCPII-2 have a more flat surface and smaller plate structure. The results of Congo red test showed that the polysaccharide had no three helical structure before and after the degradation, and the structure was consistent with the observed structure.Study on hypoglycemic activity of raspberry fruit polysaccharide DRCPII include test of glycosylation resistance(Amadori product inhibition test, dicarbonyl compounds inhibition test and ages inhibition test) and alpha amylase activity inhibition test. The results of test of glycosylation resistance showed that DRCPII could inhibit certain anti-glycation ability from three periods, but lower than that of the control aminoguanidine. In addition, polysaccharide DRCPII showed obviously inhibitory effects on the formation of Amadori compounds which were stronger than that of dicarbonyl compounds and AGEs, indicating that DRCPII inhibit the glycation mainly occurred in the first period. The results were consistent with the aminoguanidine control group. There was a significant difference between different concentrations of DRCPII in the maximum inhibition rate, which indicated that the concentration had significant effect on the formation of the products in the three stages of inhibition of glycosylation. The α-amylase inhibition activities of DRCPII was studied. The result showed that DRCPII exhibited some inhibitory activity, but lower than the control of aminoguanidine. Besides, the inhibitory effect of DRCPII was dose-dependent in a certain concentration range. |
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