| Wet ultra-fine grinding process is a kind of rapid extracting high-technology from Chinese herbs,as an inprovement and innovation from grinding technology.Wet ultra-fine grinding process as follow:adding herbs and solvent into ultra-fine grinding equipment for mixing firstly,attributed to herbs particle size decreasing through strong mechanical vibration grinding tissue structure as ultra-fine ginding time extention,dissolved path of components to be shorten and dissolved area increasing.finally,extracting solvent reached internal tissues immediately.Therefore,ultra-fine grinding and extracting could be completed synchronously,saving extracted time and improving extracted efficiency perfectly.The whole extracted process could be operated easily and without heating,it could protect heat-sentive drug from damage fully.Therefore,Wet ultra-fine grinding technology applied to animal drugs in Tradional Chinese Medine has a specific advantage.The active ingredients of animal drugs ofen existed in complex mixed system.However,it was poor stability,while quite sensitive to temperature,pH,organic reagents and so easy to degeneration.In addition,their separation and purification were carried out through precipitation,centrifugation,extraction,chromatogram,electrophoresis and other methods.What's more,the process is complex and expensive.While,membrane separation technology has a specific advantage,as for simple equipmet,operating at room temperature,no phase or chemical change,high selectivity and low energy consumption.As a novel separation technology,it has aroused more and more atteition.Protein,peptide even some small molecular substance could be perfectly separated and concentrated through selecting appropriate membrane module and establishing accurate operating parameters.Therefore,this project intended to choose earthworm as a representation of animal drug,aiming to extacte and refine its active ingredient using a coupling system,Wet ultra-fine grinding technology-Membrane separation techonogy.Firstly,earthworm chemical compositon was extracted with wet ultra-fine grinding technology and heating reflux respectively and anti-thrombotic efficacy in vivo or vitro was analyzed independently.The results showed that earthworm extract using Wet ultra-fine grinding technology contain dual role of anticoagulant and thrombolytic,while decoction has a sigle anticoagulant effect.Finally,compositions and efficacy were linked to make a comprehensive analysis.We speculated that playing the role of anticoagulation may be some peptid substances,while earthworm protein contained thrombolytic activity.Howervr,owing to taking oral administration mostly in trational medicine,therefore,playing antithrombotic effect may be some free peptide or protein degradation products,while,thrombolysis effect of prototype protein has not been developed so far.The second part:A research on the extract degradation in gastrointestinal environment.The results indicated that small molecular substances degradation did not occur in artificial gastric juice,while degraded obviously in artificial intestinal fluid,even appeared new small molecule substance.Whther it has relationship to antithrombotic efficacy,pending for further research.Earthworm protein degraded completely in artificial gastric juice,and the degradation has no thrombolytic activity by fibrin plates assay.Finally we concluded that the substance playing the role on therapeutic effects in vivo may be some degraded peptide,amino acid and small molecules stable existence in artificial intestinal fluid in tradional oral administration.What's more,prototype protein did not play the role in aitithrombotic efficacy.The third part:A research on comparison of earthworm "smash-dissolution"dynamics using conventional homogenized and wet ultrafine grinding process respectively.The results shows that the peak shapes of the two process' extracts were basically the same,while composition peak area extracted with Wet ultra-fine grinding process were significantly higher than conventional homogenized process.It indicates that earthworm extracted efficiency using wet ultrafine grinding process were obviously higher than conventional homogenized process,wet ultrafine grinding can be used to extract earthworm compositions,and the whole process is quick,simple and suitable for large-scale promotion.The fourth part:A study of optimizing wet ultrafine grinding technology extraction process of earthworm protein by response surface methodology(RSM).The results as follows:soaked 4.7h,solid-liquid(1:11,g/mL),extracted temperature 17?,grinding for 10 min.Protein obtained rate can be achieved 3.46%.The optimum extraction technology is stable,feasible and suitable for large-scale promotion.The fifth part:A rapid separation of fibrinolysis protein from Lumbricus rubellus using a coupling purification system of hollow fiber membrane and size exclusion chromatography The result showed that the large molecular weigh protein(>50 kDa)in extract using Wet ultra-fine technology could basically be removed through MWCO 50kDa PES,and peptide and some nucleic acid could basically be removed through MWCO 6kDa PES equally.The crude protein was purified using Sephadex G-75 as follow.Finally,we obtained three groups of high-purity protein,among of which named LK1 containing fibrinolytic activity.The peptide mass fingerprinting(PMF)of LK1 digested with trypsin was analyzed using matrix-assisted laser desorption inoization-time of flight mass spectrometry(MALDI-TOF),the sequencing of LK1 was identified using mach known PMF from NCBI.And inferred the homologous protein was gil 157103994,a Nicotinate phosphoribosyltransferase from Aedes aegypti.As compared with traditional protein purified method,purified step was simplied greatly,and efficiency has been greatly improved as well.The results indicated that this new concept was feasible,and it may be promoted to purify other animal tissues extract in futher. |
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