Study On Extraction,purification And Anticoagulant Activity Of Polysaccharides From Seeds Of Toona Sinensis

The seeds of Toona sinensis,the fruit of Toona sinensis(A.Juss.)M.Roem,are generally picked in autumn and dried for medicinal materials.In a variety of active components of toona sinensis seeds,the polysaccharide is an important active substance.In the paper,the seeds of Toona sinensis were as the raw material to research the extraction technology,separation and purification,physicochemical properties of polysaccharides from seeds of Toona sinensis(STSP)and the anticoagulant activity in vitro of the purified polysaccharide fractions.The main research contents in the study were as follows:1.Optimization of extraction process of crude polysaccharides fromToona sinensis seeds In the study,the crude STSP were obtained by the hot water extractionmethod.And the extraction conditions of polysaccharides were optimized by using response surface software(Design expert 8.60),the optimal extraction conditions were obtained as follows:extraction temperature 82.5?,extraction time 92 min,material-liquid ratio1:25 g/mL.Under the process condition,the yield of CSTSP was 5.08%.2.Separation and purification of polysaccharides from Toona sinensis seedsThe deproteinization effects of Sevag method,TCA method and polyamide method on crude STSP were compared.Comprehensive the protein removal rate and the polysaccharide retention rate two aspects,the results showed that polyamide method was the best deproteinization method with the protein removal rate of 68.15%and the polysaccharide retention rate of 88.09%.Then,using activated carbon method to remove the pigment from polysaccharides which have deproteinized,the effects of activated carbon dosage,decolorization temperature,decolorization time and pH on the decolorization of polysaccharides were studied by orthogonal experiment design L9(34),the results showed that the optimal decolorization conditions of activated carbon method were added 2.0%activated carbon at sample pH 4,with temperature at 60? for 1 h,respectively.Under the conditions,the decolorization rate was reached to 64.58%,and the retention rate of polysaccharide was 80.23%.After deproteinization and decolorization,STSP were further purified by DEAE Sepharose CL-6 column.The results showed that four polysaccharide components were obtained after purification which named STSP-1,STSP-2,STSP-3 and STSP-4 with yields of 13.3%,10.38%,25.12%and 26.39%respectively.The total recovery was 75.19%.3.The physicochemical properties of polysaccharides from Toona sinensis seedsThe structure of purified polysaccharides was preliminarily analyzed.The UV spectrum analysis showed that there were no proteins and nucleic acids in STSP-1,STSP-2,STSP-3 and STSP-4.The FT-IR spectrum analysis showed that four purified fractions of STSP were similar all of which were carbohydrate compounds.The high efficiency gel filtration chromatography(HPGFC)showed that the STSP-2 and STSP-3 were single peaks and uniform polysaccharides,their weight-average molecular weight were 21.4 kDa and 43.7 kDa,separately.By ion chromatography,the monosaccharide compositions of STSP-1 were mainly rhamnose,glucosamine,galactose and xylose.STSP-2 was mainly composed of galactose and glucose.STSP-3 was mainly composed of rhamnose and galactose.Arabinose,rhamnose and galactose were mainly monosaccharides of STSP-4.The results of scanning electron microscope(SEM)showed that STSP-1 exhibited the mainly a square block crystal structure with smooth surface,STSP-2 had lamellar structure with rough surface,STSP-3 had an uneven spherical particle with compact surface structure,and STSP-4 exhibited the mainly long rodlike structure.The Congo red analysis showed that STSP-2 and STSP-3 had triple-helical structure,while STSP-1 and STSP-4 had no triple-helical structure.4.The anticoagulant activity in vitro of polysaccharides from seeds of Toona sinensisTaking activated partial thromboplastin time(APTT),prothrombin time(PT)and thrombin time(TT)as the detection index to research the anticoagulant activity in vitro of STSP and its components,and heparin sodium(positive)and normal saline(negative)were taken as the reference,separately.The results showed that the STSP and its components had no effect on APPT compared with saline,indicating that it did not affect the coagulation system through the endogenous pathway.However,STSP and polysaccharide fractions could prolong the time of TT.And STSP-3 could significantly extend PT and TT,when STSP-3 was 4 mg/mL,the time of PT and TT were prolonged by 27.36%and 60%,respectively,indicating that it can affect the coagulation process through exogenous and common pathways.