Separation And Purification Of Bletilla Striata Polysaccharide And Its Application In The Treatment Of Inflammation

Reactive oxygen species(ROS)is a general term for oxygen-containing free radicals,which are highly unstable and active and play an important role in cell signaling.However,excess ROS irreversibly damages cells and biomolecules,further leading to inflammatory responses.Intake of antioxidants in vitro to maintain REDOX homeostasis and basic ROS level in vivo is an effective method to avoid excessive accumulation of ROS.However,conventional antioxidants face many problems,such as poor safety in vivo,non-degradation in vivo,and the complexity of constructing antioxidant carriers.Based on small molecules luminescent material Luminol(Luminol)is a functional unit,with the good biocompatibility of Bletilla polysaccharide(Bletilla striata polysaccharide,BSP)bonding get Myeloperoxidase,Myeloperoxidase(MPO)response of nanoparticles(LBSP NP),by eliminating the high level of ROS,and inhibit the secretion of macrophage in MPO implements the effective treatment,also confirmed the experiments in vivo and in vitro LBSP NP has good biological safety.Thus,it overcomes the deficiency of traditional antioxidants in terms of safety and functional molecular targeting to pathological tissue and provides a possible new strategy for the treatment of inflammation.In the first chapter of this paper,the harms caused by excessive ROS in inflammatory diseases and the relevant strategies and latest progress of anti-inflammatory and antioxidant therapy are briefly described,with emphasis on the advantages and existing problems of cyclodextrin-based antioxidant therapy.The structure and intrinsic biological activity of polysaccharides in Bletilla striata and the application of nanomaterials based on saccharide in anti-inflammatory and anti-oxidation therapy were further expounded.In the second chapter,we designed a simple and rapid method to separate three polysaccharides from Bletilla tuber: BSP-1,BSP-2 and BSP-3,with yield of 0.4%,0.6% and0.7%,respectively.The molecular weight of the polysaccharides was analyzed by high performance gel chromatography and high performance liquid chromatography.The basic structural characteristics of the polysaccharides were identified by nuclear magnetic resonance spectroscopy and Fourier transform infrared absorption spectroscopy.The biological activities of three polysaccharides were also evaluated.The results showed that the polysaccharides of Bletilla striata had characteristic peaks of polysaccharide.The lower the concentration of alcohol,the higher the molecular weight of polysaccharide.When the alcohol concentration was 30%,the molecular weight of the extracted BSP-1 was 128.34 k Da.When the alcohol concentration reached 50%,the molecular weight of BSP-2 was 9.97 k Da.When the alcohol concentration reached 90%,the molecular weight of BSP-3 was 9.26 k Da.Different molecular weights endow BSP with different antioxidant activities.The ability of BSP to scavenge DPPH free radicals is directly proportional to the molecular weight.The antioxidant activity of BSP-1 with molecular weight of 128.34 k Da is the highest,which is1.5 times of that of BSP-3 with low molecular weight of 9.26 k Da.In vitro safety experiment and bacteriostatic experiment confirmed that Bletilla striata polysaccharide has good safety in vivo and certain bacteriostatic ability,which is expected to be used in anti-inflammatory and anti-oxidation therapy.It was found that BSP-1 with high molecular weight had poor water solubility and was insoluble in N,N-dimethylformamide(DMF),so it was difficult to modify it.The lower molecular weight BSP-3 had better water solubility and soluble in DMF.On this basis,aiming at the problem of inflammatory response caused by excessive ROS,Chapter 3 successfully synthesized a Luminol/Bletilla striata polysaccharide nanomaterial that can deliver functional molecules to inflammatory tissues for anti-inflammatory and anti-oxidative treatment.Firstly,functional material LBSP was prepared by combining Luminol with Bletilla striata polysaccharide by a simple synthesis method.Then,LBSP NP nanoparticles were prepared by nano precipitation method.The morphology of LBSP NP nanoparticles was determined to be nanoporbular structure within 200 nm size.Laser confocal microscopy showed time-dependent intracellular distribution.Cy5/LBSP NP was absorbed by macrophages in a dose-dependent and time-dependent manner,suggesting that LBSP NP could be effectively internalized by inflammatory cells.Preliminary studies on the mechanism in vitro confirmed that LBSP NP can clear high levels of MPO and significantly inhibit the secretion of MPO in macrophages.Similarly,the antioxidant activity of LBSP NP in vitro showed that LBSP NP had certain antioxidant activity,and the scavenging rate was 81.3%,which made up for the deficiency of BSP-3.At the same time,LBSP NP has good biosafety and has no obvious toxic and side effects on the body,which makes up for the poor safety of Luminol in vivo.Therefore,the LBSP NP nanoparticles constructed based on Luminol/Bletilla striata polysaccharide in this study can be used as a highly effective nanomaterial with high biological safety to inhibit macrophage mediated inflammatory response and achieve effective treatment,which is expected to be used in the imaging and treatment of other related diseases...