Extraction,Purification,Structural Characterization And Bioactivity Of Polysaccharides From Rehmannia Glutinosa Libosch

Rehmannia glutinosa Libosch is one of the traditional bulk medicinal materials,and its root is used as one of the traditional Chinese medicine.Polysaccharide,as the main active component in Rehmannia,has been widely concerned because of its antioxidant,antitumor and immunomodulatory activities.At present,the researches on polysaccharides from Rehmannia glutinosa Libosch mainly focus on single extraction method and biological activity,but there is no report on the researches on polysaccharides from Rehmannia glutinosa Libosch with different selective extraction methods.In this paper,the extraction and purification,structural characterization,in vitro biological activities(antioxidant,hypoglycemic,anti-allergic,immune regulation)of Rehmannia glutinosa Libosch polysaccharides were systematically studied.The aim is to provide theoretical basis for the development of the bioactivity and mechanism of Rehmannia glutinosa Libosch polysaccharides in vivo and scientific basis for the development of functional food related to Rehmannia glutinosa Libosch polysaccharides.The main contents and results are as follows:1.Preparation and chemical composition analysis of polysaccharides from Rehmannia glutinosa Libosch based on different extraction methods.In this paper,six selective extraction methods were developed to extract Rehmannia glutinosa Libosch polysaccharides: Cold water extraction at room temperature(RGP-1),boiling water extraction at room temperature(RGP-2),50% ultrasonic alcohol extraction at room temperature(RGP-3),25% ultrasonic alcohol extraction at room temperature 50% ultrasonic alcohol extraction(RGP-4),25% ultrasonic water extraction at room temperature(RGP-5),80℃ hot water extraction(RGP-6).The crude polysaccharides of Rehmannia glutinosa Libosch were isolated and purified by trichloroacetic acid deproteinization,alcohol precipitation,membrane dialysis,hydrogen peroxide decolorization,alcohol precipitation,and six different extraction methods.The yield of crude polysaccharide from Rehmannia glutinosa Libosch by six different extraction methods was RGP-3>RGP-6>RGP-1>RGP-2>RGP-4>RGP-5.The chemical constituents of polysaccharide components 1-6 of Rehmannia glutinosa Libosch were determined: the content of polysaccharide was 90.19±1.86%,80.22±1.90%,89.33±0.72%,80.97±0.72%,75.43±2.70%,89.89±0.93%.The protein content of six components of Rehmannia glutinosa Libosch polysaccharide was within0.1%.The content of RGP-5 uronic acid in six polysaccharide components of Rehmannia glutinosa Libosch was 14.45±0.45%.Congo red experiment showed that Rehmannia glutinosa Libosch polysaccharide had no triple helix structure.2.Structure analysis of different polysaccharides from Rehmannia glutinosa LiboschThe monosaccharides of RGP-1,RGP-3 and RGP-6 were glucose,galactose and glucuronic acid,and the molar ratio of monosaccharides were 32.56:67.19:0.24,37.74:65.15:0.89,32.71:66.56:0.73,respectively.The monosaccharides of RGP-2 and RGP-4 were glucose,galactose,galacuronic acid and rhamnose-the molar ratios of monosaccharides were34.39:64.16:11.29:0.314 and 32.23:63.28:1.08:0.34,respectively.RGP-5is mainly composed of glucose and galactose,arabinose,galacturonic acid,rhamnose,the mole ratio is: 31.77:58.99:3.96:4.70:0.34.Infrared and nuclear magnetic resonance spectra showed that Rehmannia glutinosa Libosch polysaccharides obtained by six different selective extraction methods were pyranose containing α-type and β-type glycosidic bonds.The results of scanning electron microscopy showed that the six groups were divided into lamellar structures.Thermogravimetric analysis showed that different extraction methods of Rehmannia glutinosa Libosch polysaccharide had little effect on thermogravimetric loss.3.Bioactivity analysis of different polysaccharides from Rehmannia glutinosa Libosch(1)RGP-4 and RGP-6 were the dominant components of antioxidant activity among the six Rehmannia glutinosa Libosch components.For different extraction methods,the hydroxyl radical results showed that,RGP-6> RGP-1> RGP-5> RGP-4> RGP-2> RGP-3;The highest clearance rate of RGP-6 was 96.76±0.29%,and the highest clearance rate of RGP-3was 65.29±0.83%.Superoxide radical activity sequence: RGP-4> RGP-2>RGP-6> RGP-1> RGP-5> RGP-3;The highest clearance rate of RGP-4was 94.52±0.57%,and that of RGP-3 was 81.31±1.17%.DPPH radical analysis showed that the scavenging rate of RGP-5 as the highest active component was 46.46±0.39%.The results of ABTS free radical analysis showed that the scavenging rate of RGP-4 as the highest active component was 19.32±0.91%.Analysis of antioxidant activity of different polysaccharides from RGP-4 and RGP-6 were the dominant components of antioxidant activity among the six Rehmannia glutinosa Libosch components.(2)Analysis of immunomodulatory activity of different polysaccharides from RGP-4 and RGP-6 were the dominant components of antioxidant activity among the six Rehmannia glutinosa Libosch components.The results showed that the safe concentration ranges of RAW264.7 cell proliferation were RGP-1,RGP-3 and RGP-5 of 1-400μg/m L,and RGP-2,RGP-4 and RGP-6 of 1-200 μg/m L,respectively.Different RGP significantly promoted the phagocytic ability of RAW264.7and the ability to secrete NO in the safe concentration range.RGP-1 had the strongest phagocytic ability among different polysaccharide components of Rehmannia glutinosa Libosch,and RGP-5 had the best ability to enhance immune ability by releasing NO.(3)Antiallergic activity of different polysaccharide components of Rehmannia glutinosa Libosch.The inhibition rate of hyaluronidase was RGP-4> RGP-3> RGP-2> RGP-5> RGP-6> RGP-1;The highest clearance rate of RGP-4 was 88.90±0.18%,and that of RGP-1 was 79.97±0.61%.The inhibition rate of six components of Rehmannia glutinosa Libosch polysaccharide on hyaluronase was more than 70%,and Rehmannia glutinosa Libosch polysaccharide had good antiallergic activity.(4)Hypoglycemic activity of different polysaccharide components of Rehmannia glutinosa Libosch.Two blood glucose indexes of α-glucosidase and α-amylase were determined.The best α-amylase inhibition rate was RGP-3 and the highest clearance rate was 30.01±0.88%.The inhibition rate of α-glucosidase was the best,and the highest clearance rate of RGP-6 was 16.33±0.24%.The hypoglycemic activity of Rehmannia glutinosa Libosch polysaccharide was poor.