| Bletilla striata(Thunb.)Reichb..f,also known as "baiji",is a perennial herb of Bletilla in Orchidaceae.It has the effects of astringent hemostasis,anti-inflammatory and muscle growth.Bletilla striata contains active substances such as polysaccharides,mucus,steroids,terpenes,esters,ethers and diphenyl,and Polysaccharide is the main active substance with the largest content.Because the water extract of polysaccharide is sticky,it is often called "Bletilla striata glue".The research on Bletilla striata polysaccharide focuses on the polysaccharide of Bletilla striata,but there are few reports on alcohol precipitation and classification of Bletilla striata polysaccharide and its biological activity.In this paper,the process optimization,alcohol precipitation classification,physicochemical properties and structural characterization analysis,antioxidant,moisture absorption and moisturizing,in vitro coagulation test of Bletilla striata polysaccharide were carried out,the process parameters to improve the extraction rate of Bletilla striata polysaccharide were determined,and the basic differences of physicochemical properties and biological activities of the three components of Bletilla striata polysaccharide after alcohol precipitation classification were preliminarily determined.The following results were obtained:(1)The results showed that the extraction temperature have a highly significant effect on the extraction rate of Bletilla striata polysaccharide,and the extraction time have a significant effect on the extraction rate of Bletilla striata polysaccharide.The optimum extraction process is that the ratio of liquid to material is 59.77:1(m L/g),,the extraction time is 1.42 h,the extraction temperature is 67.79 ℃,and the extraction rate of Bletilla striata polysaccharide is 40.99%.Three components of Bletilla striata polysaccharide were obtained by graded gradient alcohol precipitation of BSPs,and the yield was BSP-50 > BSP-70 > BSP-30.(2)The study on the physical and chemical properties of Bletilla striata polysaccharide by alcohol precipitation showed that the total sugar contents of BSP-30,BSP-50,BSP-70 and BSPs were 61.57 ± 3.12%,62.47 ± 3.10%,69.33 ±3.32% and 61.89± 1.18% respectively,and the protein contents were 2.27±0.03%、1.97±0.03%、0.35±0.04% and 4.04±0.08% respectively;The contents of reducing sugar were 8.70±0.89%,7.86 ± 0.12%,14.18 ± 1.46% and 3.50 ± 0.78%,respectively.The results of p H measurement showed that Bletilla striata polysaccharide was neutral polysaccharide;The viscosity of BSP-70(4.03 ± 0.45 m Pa.S)decreased by more than 30 times compared with BSPs(144.73 ± 1.53 mpa.S),while the viscosity of BSP-30 and BSP-50 was 177.67 ± 1.04 m Pa s、151.43±0.36 mpa.s。 Solubility test results: between 25 ℃ and 100 ℃,the dissolution time of polysaccharide gradually shortened with the increase of temperature.After graded alcohol precipitation,the complete dissolution time of Bletilla striata polysaccharide was shortened by about9 min,17min and 28 min respectively.Among them,the dissolution rate of BSP-70 component was more than 4 times that of BSPs,indicating that the graded alcohol precipitation method can improve the solubility of Bletilla striata polysaccharide.The water absorption properties of Bletilla striata polysaccharide(BSP-30,BSP-50 and BSP-70)and non graded Bletilla striata polysaccharide(BSPs)were 315.00 ± 2.33%,200.22 ± 1.68%,181.11 ± 1.92% and 250.33 ± 1.53% respectively.It can be seen that each component of Bletilla striata polysaccharide has good water absorption,and BSP-30 has the highest water absorption.(3)The infrared detection results showed that the four polysaccharides have polysaccharide basic structure absorption peaks and β-Characteristic absorption peak of dextran;The graded alcohol precipitation method have little effect on the thermal stability of Bletilla striata polysaccharide and slightly enhanced the thermal stability;The four polysaccharides showed low diffraction maxima between 10°and 30°,indicating that their structures were amorphous;Congo red test shows that the trichelix structure of Bletilla striata polysaccharide is three helix structure,while the trichelix structure of BSP-70 changes to a free bending structure in 0.1mol/m L Na OH.The microscopic morphology of each component of Bletilla striata polysaccharide after graded alcohol precipitation is different.After magnification of 20000 and 50000 times,the surface of BSP-30 is compact and colloidal,and the surfaces of BSP-50,BSP-70 and BSPs have different sizes and tight pores.High performance gel permeation chromatography(HPLC)and high performance liquid chromatography(HPLC)showed that the composition of Bletilla striata polysaccharides was similar to that of mannitol and glucose,which accounted for more than 98%.The ratio of mannose and glucose was about 4:1,and the structure of monosaccharide composition changed.After grading,the minimum molecular weight of polysaccharide BSP-70 component is 3.6831 x104 Da,and the maximum molecular weight is BSP-30,which is 8.5643 x104 Da.(4)The results of antioxidant,hygroscopic and moisturizing activities and in vitro coagulation tests of Bletilla striata polysaccharide showed that the antioxidant activity of Bletilla striata polysaccharide is dependent on the concentration of polysaccharide in a certain concentration range.Among them,the antioxidant activity of BSP-70 is the strongest,while the antioxidant activity of BSP-30 and BSP-50 Bletilla striata polysaccharide is weakened,showing that the polysaccharide with low molecular weight may have better free radical scavenging activity.The moisture absorption and moisturizing experiment showed that the four Bletilla striata polysaccharide had certain moisture absorption capacity in the environment of RH = 43% and RH = 81%,which were higher than that of hyaluronic acid(8.09±0.18%,31.90±1.67%)and sodium alginate(7.00±0.38%,15.00±1.01%).Among the four different components of Bletilla striata polysaccharide,BSP-70 has the best moisture absorption performance,and the maximum moisture absorption rates are 12.00 ± 0.46% and31.90 ± 1.50% respectively.The moisturizing test results show that the optimal moisturizing rate of BSP-30 is 85.77±0.87%,which is equivalent to 85.6±0.75% of hyaluronic acid.It has large molecular weight and shows good moisturizing performance.In vitro coagulation test showed that the four polysaccharide could promote coagulation through TT,PT and APPT,and the coagulation promoting effect was concentration dependent.Among them,BSP-70 mainly promoted coagulation through PT,and the exogenous coagulation time was shortened by about 27.14%.After graded alcohol precipitation,each component of Bletilla striata polysaccharide still has the effect of promoting coagulation and hemostasis,but there is no significant change in the effect of promoting coagulation compared with non graded BSPs.In this experiment,the traditional water extraction and alcohol precipitation method was used to extract polysaccharides from Bletilla striata.The effects of liquid-solid ratio,extraction time and extraction temperature on the extraction rate of polysaccharides were investigated by single factor test.Combined with the response surface optimization and the feasibility of the experiment,The optimum extraction process is that the ratio of liquid to material is 60:1(m L/g),The extraction time is 1.5h and the extraction temperature was 70℃.Under these conditions,the extraction yield of polysaccharides from Bletilla striata is 40.82 ± 0.28%.Under the optimal conditions,the polysaccharides of Bletilla striata were classified into BSP-30,BSP-50 and BSP-70 by ethyl alcohol precipitation classification method.The yield of polysaccharides was BSP-50 >BSP-70>BSP-30.The three graded polysaccharides is neutral,and the graded alcohol precipitation method could change the chemical composition,water absorption,viscosity and solubility of polysaccharides.The viscosity of BSP-70(4.03±0.45 Mpa.s)was more than 30 times lower than that of BSPs(144.73 ± 1.53 Mpa.s).BSP-30 polysaccharide component have the highest water absorption.The dissolution rate of BSP-70 components is more than 4 times that of BSPs.Graded alcohol precipitation had little effect on the thermal stability of absorption peaks of polysaccharides in Bletilla striata.All the structures are amorphous.The four polysaccharide samples have triple helix structure,while the triple helix structure of BSP-70 changed when the concentration of Na OH solution was 0.1mol/m LNa OH and became free bending structure.The micromorphology and monosaccharide composition of Bletilla striata polysaccharides changed after fractional alcohol precipitation.The ratio of mannose and glucose is about 4:1,accounting for more than 98%,Among them,BSP-70 have the strongest oxidation resistance and the best hygroscopicity,with the maximum hygroscopicity of 12.00±0.46% and 31.90 ± 1.67% at RH=43% and RH=81%;The results of moisturizing experiment show that the optimal moisturizing rate of BSP-30 is 85.77 ± 0.87%,which is equivalent to that of hyaluronic acid(85.60±0.75%).Each component of Bletilla striata polysaccharide still have the effect of promoting coagulation and hemostasis,but compared with ungraded BSPs,the effect of promoting coagulation have no obvious change.Therefore,there is a certain relationship between polysaccharide activity and its physical and chemical properties and basic structure,and the structure-activity relationship needs further analysis and study. |
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