Extraction,Structure Analysis And Biological Activity Of Polysaccharides From Ramulus Mori

Mulberry is a perennial woody plant,which has vrious applications in vegetation restoration,rocky desertification control,rational use of land,and ecological landscape reconstruction in the Three Gorges reservoir area.As a traditional Chinese medicine,mulberry has high medicinal values.Modern phytochemical studies have shown that mulberry contains flavonoids,alkaloids,polysaccharides,and other active ingredients.Ramulus mori polysaccharides?RMP?has outstanding curative effects such as diabetes al eviating and liver protecting.The present study has investigated the content and monosaccharide composition of RMP from different mulberry resources.The structural characteristics and biological activities of RMP were then analyzed to screen out the mulberry resources available for medicinal purposes.The data obtained in this study provided the useful information for the breeding of mulberry varieties.1.Optimization and content determination of RMP.The contents of polysaccharides in mulberry leaves,fruit and branches were determined by sulfuric acid phenol method in four mulberry resources?Mengsang,Jinqiang 63,Black mulberry and Baiyuwang?.The results showed that the contents of polysaccharides from ramulus mori were the highest in all mulberry resources.There was no significant difference in the content of polysaccharide from ramulus mori before and after treatment with cel ulose,suggesting that RMP was not cel ulose.The content of RMP in dry mulberry was significantly higher than that in fresh mulberry.Ultrasound-assisted extraction of polysaccharides from ramulus mori was established as the conditions as follows:ultrasonic processing temperature 80?,ultrasonic processing time50 min,solvent-to-sample ratio 1:40?g:mL?.Contents of polysaccharide from 99 different mulberry resources obtained from mulberry breeding center of Southwest University were extracted by the method described above.The content of polysaccharide from ramulus mori was mainly distributed in 3%⁴%,and the content of polysaccharide from Kuisang was the highest?6.568%?and that in Tuo Sang?1.052%?was the lowest in 99 different mulberry resources.2.Determination of monosaccharide composition and structural analysis of RMP.The result of UPLC showed that mulberry polysaccharides mainly consist of mannose,rhamnose,glucuronic acid,glucose,xylose,galactose,and arabinose with molar ratios:1.21:1.55:0.17:73.72:10.20:2.45,respectively.Seven species of mulberry germplasm with different composition of monosaccharide were selected?Kuisang,Huosang,6071,Shanxitiansang,Dazuchangguosang,Jinqiang 82,Baokan 61?according to the results of UPLC.The thermal properties of RMP were analyzed by differential scanning calorimetry?DSC?.The main functional groups of RMP were analyzed by FT-IR and SEM.The results showed that the glass transition temperature of RMP was more than 80?,which indicated that the structure of RMP did not change during extraction process.FT-IR results showed that RMP was?-type polysaccharides and was the main functional groups of the carbohydrate.Under the observation of SEM,the surface of RMP appeared porous and sponge-like after freeze-drying.Different RMP from different mulberry resources had different pore sizes,which may lead to differences in their adhesive ability.3.Exploring the bioactivity of RMP.Antioxidant test showed that RMP has antioxidant activity,but lower than ascorbic acid?VC?.RMP from Kuisang,Dazuchangguosang,6071 have relatively higher reducing power(A₇₀₀=0.46),rate of hydroxyl radical scavenging?69.75%?and superoxide anion scavenging?46.23%?,respectively.The antimicrobial test results indicated that the inhibitory effects of RMP on Staphylococcus aureus,Pseudomonas aeruginosa,Shigella flexneri,and Escherichia coli were significant.RMP from Huosang and Baokan 61 had inhibitory effects on the proliferation of Candida albicans.The results revealed that the antibacterial effect of RMP from Jinjiang 82was the best.The results showed that the content of NO in the supernatant of inflammatory cel s was significantly decreased after incubated with RMP.The inhibitory rate of NO was the highest?98%?,and the survival rate of cel s was more than 80%after treatment with RMP?except for Baokan 61?,which indicated that the RMP had few toxicity on cells.RMP from Huosang was resistant to the proliferation of HeLa cel s(IC₅₀=31.87 mg/mL)and Caski cel s(IC₅₀=28.50 mg/mL)in a dose-dependent manner.Compared with the normal control group,with the increase of the concentration of RMP,the cell morphology changed from normal polygons to round shape with the cell spacing increased,and the cell debris in supernatant was increased observed by the inverted microscope.High concentration insulin added into HepG2 cel s induced the abnormal glucose metabolism.Insulin resistant HepG2 cel s?HepG2/IR?model was then established by treating with insulin?10?g/mL?for 48h.Compared with HepG2/IR group,treatment with RMP could not only significantly al eviated insulin resistant HepG2 cel s,but increased cell activity?except for Jinqiang 82?.The type 2 diabetic mice were induced by STZ combined with high fat diet.RMP can significantly reduce the blood glucose concentration of the diabetic model mice.Compared with the model group,RMP treatment increased SOD activity?P<0.01?and insulin sensitivity?P<0.01?in serum and reduce the content of TG,TCH and MDA in serum?P<0.01,P<0.01,P<0.01?.