Study On Structure And Bioactivities Of Polysaccarides From Lobaria Kurokauae Yoshim

Lobaria Kurokauae Yoshim(LKY)is a typical lichen fungi,which belongs to Ascolichenes,Lecanorales,Stictaceae,Lobaria Hoffm.Research results show that the polysaccharide has strong antioxidant and anti-tumor activity,as well as significantly enhance the immune ability.The LKY as lichen resources,widely distributed in Yunnan Province,as well as it was used in the folk medicine and food.However,there are few studies on the structure identification and biological activities of polysaccharides from LKY.In this study,we systematically studied the extraction,isolation,purification,structure identification and biological activities of polysaccharides from the LKY.The main results are as follows:Ultrasonic assisted extraction technology of polysaccharides was investigated.Single factor screening and response surface analysis were used to analyze the best extraction technology conditions.The optimum conditions modified were ultrasonic power 86.8W,ultrasonic time 15.9min and liquid-solid ratio 26.6:1(mL/g),and the yield could come to4.48%.Taking into account the limitations of the actual operation,the parameters will be modified.The ultrasonic power is 80 W,ultrasonic time is 15 min and the ratio of liquid to material is 25:1.After 5 experiments,the results showed that the yield of the crude polysaccharides from the LKY was 4.37%.Lobaria Kurokauae Yoshim polysaccharides extracted liquid obtained in the best extraction condition conducted the following steps: decoloring by resins,deproteinizing with Sevag method,precipitating by ethanol alcohol,concentrating and drying.The crude polysaccharide was isolated by DEAE-52 ion-exchange column chromatography,which was used to get the LKY-I,LKY-II,LKY-III.LKY-I and LKY-?were homogeneous polysaccharides,the molecular weight Mw were 63127 Da and 122495 Da,respectively.LKY-? was miscellaneous polysaccharides,and the Mw of LKY-? was 697243 Da.It was found that LKY-I,LKY-?,LKY-? were like white cotton,canary yellow cotton,yellowish cotton,respectively.The protein contents were 0.13%,0.46%,0.91%;total sugar contents followed by 88.91%,74.05% and 59.18%;uronic acid contents followed by 13.21%,15.12%,16.02%,respectively.Structure analysis: LKY-I consists of rhamnose,xylose,mannose,glucose,galactose and molar ratio was 1.1:2.0:46.5:28.5:20.5.In addition,LKY-? was made of rhamnose,arabia sugar,xylose,mannose,glucose,galactose,and molar ratio of corresponding monosaccharide was 1.2:1:1.5:60.9:12.8:22.5.The infrared spectra show that LKY-I and LKY-? werepolysaccharides containing ?-D-mannose glucose.By periodate oxidation and Smith degradation methylation and NMR analysis,LKY-I contains 1?2,6 1?3 1?4 and 1?6glycoside bonds,and 1?6 glycosidic bond is the main chain.LKY-? contains 1?2 1?2,61?3,6 1?4 and 1?6 glycoside bonds,and 1?6 glycosidic bond is the main chain.The results of Congo red test and SEM showed that the conformation of LKY-I and LKY-II did not exist multiple spiral.In addition,they were irregular flakes,rod like clusters of different shapes,etc.The surface was relatively compact,but the bulk of the aggregate was not strong,indicating that LKY-I and LKY-II were amorphous structure.The antioxidant activity of the polysaccharides from LKY was measured by free radical scavenging ability and reducing power.The results showed that the DPPH free radical scavenging capacity and the ability of reducing Fe3+ of LKY-? was better than that of other samples.Moreover,the ABTS+ free radical scavenging capacity was the strongest of LKY-I.The purified polysaccharides from LKY had strong inhibitory effects on HepG-2,MCF-7 and HCC827,and they were positively correlated with the concentration.The antitumor activity of LKY-I had the best effect,comparable to the positive control 5-Fu uracil.The polysaccharides of LKY alone in mouse spleen lymphocytes could promote cell proliferation,but the degree of the improvement was lower.However,when LPS and ConA synergistic effect on cells,they could evidently promote proliferation,but between them has no significant difference.In 125~500?g/m L,phagocytosis of polysaccharides was stronger than the 1?g/mL LPS,and the role in setting the range of concentration significantly(p<0.05).Lobaria Kurokauae Yoshim polysaccharides could promote the secretion of NO,IL-6 and TNF-?,with the dose dependent effect.The expression of i NOS,IL-6 and TNF-?,mRNA gene in macrophages were detecte by RT-PCR.LKY-I could increase the expression of iNOS,IL-6 and TNF-? gene,which indicated that the LKY-I could promote the production of immune factors and improve the cellular immune activity.