Extraction, Structural Characterization And Biological Activities Of Polysaccharides From Epimedium Acuminatum

Epimedium (Berberidaceae) is a perennial flowering plants. The clinical therapy effect of this kind of traditional Chinese medicine herb was firstly recorded at "Shen Nong Ben Cao Jing" which reported that Epimedium can be used to treat various diseases and health disorders, including enhance kidney function, strengthen muscles, bones and heart.Nowadays, Epimedium was reported to perform well in cardiovascular and cancer therapy by modern pharmacology study. The Epimedium study is focus on total flavonoids and the quality of the herb is only related to the content of total flavonoids and icariin, which leads the shortage of Epimedium polysaccharide study. Epimedium acuminatum Franch. is not recorded at "Chinese pharmacopoeia" due to the low content of total flavonoids and icariin, but it has an abundant resource and a wide distribution in a wide range place in China, such as sichuan province, etc. Therefore, the development of Epimedium acuminatum polysaccharide is quite promising. This research is to extract polysaccharide from Epimedium acuminatum by hot water extraction, ultrasonic-assisted extraction, enzyme extraction, and microwave-assisted extraction. Aim to investigate the influence of extraction method on the physicochemical properties and biological properties, optimize the extraction process to improve the yield rate, and analyze the structure-activity relationship. The main results were as follows:1. The polysaccharides were extracted from Epimedium acuminatum by hot water extraction, ultrasonic-assisted extraction, enzyme extraction, and microwave-assisted extraction. The small molecular impurity and protein were removed by aether, acetone and Savage assay, and the crude polysaccharide was obtained after concentrating and drying.2. Chemical analysis, FT-IR and Scanning electron microscope were employed to study the properties of crude Epimedium polysaccharides. Results indicated that the chemical composition and functional groups of four Epimedium polysaccharides is similar, but the terminal glycosidic bond is different. The polysaccharide extracted by hot water extraction, enzyme extraction, and microwave-assisted extraction has β-glycosides linkage, but the polysaccharide extracted by ultrasonic-assisted extraction has a-glycosides linkage. The SEM results indicate that surface of polysaccharide extracted by hot water extraction and enzyme extraction is smooth and neat, but the one by ultrasonic-assisted extraction and microwave-assisted extraction is rough and rugged with many caves. The in vitro antioxidant assay indication that the antioxidant ability of polysaccharide extracted by hot water extraction are significant higher than other three.3. The 3 level and 3 factors CCD was employed to optimize the hot water extraction of polysaccharide. Total of 20 independent experiment was conducted to calculate the quadratic polynomial of polysaccharide yield. The optimal conditions of the extraction were determined to be the ratio of water to raw material of 29.61, extraction temperature of 85.67℃ and extraction time of 3.57 h. The yield, under selected optimal condition, is 8.23%.4. Purified by gradient ethanol, anion exchange chromatography and gel permeation chromatography, the crude Epimedium polysaccharide was fractionated as three polysaccharide fraction EAP-40-1、EAP-60-1 和 EAP-80-2, according to their different polarity, different amount of charge and different relative molecular weight. The chemical composition and functional groups of three polysaccharide fraction were similar, but the monosaccharide compositions and relative molecular weights were different. The relative molecular weight of EAP-40-1 was 138kD, which was mainly consist of glucose and galactose; The relative molecular weight of EAP-60-1 was 114kD, which was mainly consist of glucose and galactose glucose and mannose; The relative molecular weight of EAP-80-2 was 65kD, which was mainly consist of galacotse.5. DPPH assay, ABTS assay, FRAP assay, hydroxyl radical scavenging ability, erythrocyte hemolysis inhibition ability, and lipid peroxidation inhibition ability were employed to study the antioxidant ability of three Epimedium polysaccharide fractions. Results indicated that all of the three fractions exhibited significant antioxidant ability in a dose-dependent manner. EAP-40-1 exhibited the highest DPPH radical scavenging ability, hydroxyl radical scavenging ability and ferric reducing ability. EAP-60-1 showed the highest ABTS radical scavenging ability, erythrocyte hemolysis inhibition ability and membrane lipid peroxidation inhibition ability. EAP-80-2 showed relatively weaker antioxidant abilities, and the membrane lipid peroxidation inhibition ability of this fraction is not significant.6. All of the three Epimedium polysaccharide fraction exhibit significant antimicrobial ability against Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae. But the antimicrobial ability against Aspergillus niger of the three Epimedium polysaccharide fraction is not significant. EAP-60-1 showed the highest antimicrobial ability among the three polysaccharide fractions.