Extraction, Purification And Anticoagulant Activity Of The Polysaccharide From The Leaves Of Mulberry

With the improvement of people’s living standards and the change of diet, thrombotic disease have become a serious threat to people’s health. It should arouse our attention. Heparin has played a very important role in the treatment of thrombotic diseases. However, it also accompanied with the side effects of bleeding and thrombocytopenia. Exploring the main structure of anticoagulant active ingredients, which are traced from natural products, as lead compounds to develop new drugs or functional foods has extremely important significance.Glycobiology research shows that plant polysaccharides have a variety of pharmacological effects, such as hypoglycemic, anticancer and antioxidation, and so on. The polysaccharide is one of the main chemical components of mulberry leaves, therefore, the development and utilization of polysaccharide have a theoretical and practical significance. The main contents and results were as follows:1. Research the optimal extraction conditions of the crude polysaccharides from the leaves of mulberryThe crude mulberry polysaccharides were obtained using water extraction and alcohol precipitation. Single factor test was determined the range of raw material to water ratio, extraction time and extraction temperature. Respond surface methodology was employed to obtain the optimal extraction conditions of the crude polysaccharides from the leaves of mulberry. The optimum extraction conditions were as follows:raw material to water ratio of 1:30, extraction time of 113 min and extraction temperature of 80℃. The experimental yield was 3.49% under these conditions.2. Isolation and purification of the crude polysaccharides from the leaves of mulberryMulberry polysaccharides deproteinized by dynamic polyamide method. The polysaccharides retention rate was 88.3% and 87.7% of protein was removed. The mulberry polysaccharides was obtained by H2O2 oxidation bleaching and removing small molecular impurities. The yield of vacuum freeze dried mulberry polysaccharides was about 0.44%. It was isolated with DEAE sepharose CL-6B chromatograph column. MPS-1 (30.9%)、MPS-2 (29.1%) and MPS-3 (40.0%) were obtained. MPS-2 was further isolated with sepharose CL-6B chromatograph column. MPS-2a (13.4%)、MPS-2b (59.5%) and MPS-2c (27.1%) was separated.3. Preliminary structural characterization of five polysaccharide fractionsIn the UV-vis spectrum, absorption peaks were not found for five polysaccharide fractions at the specified wavelength, indicating absence of protein and nucleic acid. Five polysaccharide fractions showed the typical polysaccharides characterization by Fourier transform infrared analysis. The weight-average molecular of five polysaccharide fractions was 2.45×106,2.45×105,7.99×104,1.97×104 and 3.22×106 Da, respectively. The monosaccharide compositions of MPS-1 and MPS-2a were both contained galactose, glucose, rhamnose, arabinose, xylose and mannose. MPS-2b contained four monosaccharides and the ratio of Gal:Glc:Ara:Rha was approximately 5:3:2:1. MPS-2c was rich in galactose, arabinose, rhamnose, xylose and glucose. By contrast, MPS-3 was also contained mannose.4. Anticoagulant activity of five polysaccharide fractions outside the bodyThe anticoagulant activity of five polysaccharide fractions was assayed using APTT, PT and TT. The test results show five polysaccharide fractions have anticoagulant activity by the intrinsic pathway to influence the coagulation process. The fibrinogen was converted to fibrin to affect the clotting process in MPS-2a, MPS-2b and MPS-3. In five polysaccharide fractions, MPS-2b has the best anticoagulant activity in the APTT test. In the test concentration range, there was a increasingly enhanced anticoagulant trend with the polysaccharide concentration increase. Therefore, With current findings MPS-2b should be explored as a kind of natural potential anticoagulant.