Separation And Purification Of Polysaccharides In Mulberry Leaves And Composition Identification

Mulberry leaves polysaccharides (MLPs) are considered to be an excellent competitive inhibitor ofα-glycosidase for glucose releasing. Consequently, it has good application prospect in the treatment of diabetes with mulberry leaves polysaccharides, and it is realistic and meaningful to develop the MLPs into a medicine for diabetes.Based on the previous work done by the other members of our lab, the method for the determination of polysaccharides was improved, and the mulberry leaves polysaccharides were further purified by a sequence of purification processes. Preliminary study on the monosaccharide composition of MLPs and analyse of the Infrared Spectrum Characteristics analysis of MLPs was also carried out.Firstly, a method for the quantitative determination of uronic acid with m-hydroxydiphenyl was established, and the optimum conditions of determination were studied. The optimum Na2BB4O7–H2SO4 dosage of 6.0ml, heating time of 10min, m-hydroxydiphenyl dosage of 100ul, and coloration time of 40min were acquired. Besides, the interaction between the determination of neutral sugars and uronic acid was studied. The experimental results indicated that: m-Hydroxydiphenyl method was not affected by neutral sugars, but Anthrone-sulfuric method had some influences on the content of uronic acid. A correlation method was presented which make the methods of determination of the neutral sugars and uronic acid more effective.Secondly, the content of neutral sugars and uronic acid of the mulberry leaves coming from four different producing areas were determined. It was found that the mulberry leaves coming from He Nan Province had the highest content. The polysaccharides were separated and purified from mulberry leaves by a sequence of processes. The experimental results indicated that, both the procedures of flocculation with natural clarifying agents of ZTC1+1 and multiple alcohol deposit were unsuitable for the purification of MLPs, since the content of polysaccharides didn't increase evidently, but a serious polysaccharides loss was taken place. However, the staged alcohol deposit procedures indicated a good separating effect. With 20% ethanol deposit the content of neutral sugars and uronic acid content in the precipitation was 14.09% and 45.53% respectively, which are the main components of MLPs. Processing conditions of particle active charcoal for discoloration of polysaccharides extracts were studied. The results showed that optimum discoloration effect was obtained from the following conditions: temperature of 70℃, solution pH value of 6.0, quantity of adsorption time of 60min, and activated carbon of 4%. After discoloration, the content the neutral sugars and uronic acid in the precipitation with 20% ethanol deposit was 21.87% and 57.43% respectively, the loss rate of polysaccharide was only 8.35%. The separated polysaccharides were further purified with DEAE-cellulose ion-exchange chromatography, and a higher purity of product was obtained, in which the content of neutral sugars was 24.21% and uronic acid 64.23%, respectively.Finally, the composition of MLPs was studied by thin layer chromatography (TLC) and gas chromatography (GC). It found that the precipitation with 20% ethanol deposit was mainly composed of rhamnose and galacturonic acid. Infrared spectrum analysis further showed that it should contain uronic acid, in which the monosaccharide should be pyranose.