Chromatographic Purification And Composition Analysis Research On Deproteinized Calf Serum Extract

Deproteinized calf serum extract is the calf serum which is extracted from Age-appropriate health and quarantine inspection qualified fresh calf blood,and is the protein removal small molecule multicomponent drug which is made after alcohol precipitation,centrifugalization,concentration,ultrafiltration,viral inactivation,etc.,technological processes.Because it has the function of strengthening the cells to glucose and oxygen ingestion and application,and could improve the metabolic balance,promote energy metabolism,and has broad clinical application.The pharmacology and drug efficacy of deproteinized calf serum extract is closely related to its bioactive components,this thesis has made researches on chromatography purification process and active ingredient analysis methods on the basis of current existed technology of the calf serum deproteinized extract ultra filtrate,with the purposes of isolating the active ingredient and establish detection methods,thus to guide the follow-up process improvements and the quality control standards promotion of the deproteinized calf serum extract.This thesis adopts Capto Q ImpRes and Capto SP Impres which is provided by GE Company to carry out the purification to the calf serum deproteinized extract ultra filtrate,and compared the impact effects of the two kinds of ion exchange medium on the content of peptides and respiratory activity,and finally adopts granularity to fill the Capto Q ImpRes of 40um.Capto Q ImpRes has extremely high resolution ratio,and has quick transfer rate and low back pressure under high flow-rate,and is the best anion-exchange filler of moderate purification and polishing purification.We optimize the chromatographic conditions and to conduct the sample elution under different gradient conditions,and finally choose to deliquate the sample 10 times by pure water,and the conductivity is reduced under 5,and use 20mM PB pH 7.5 to wash,in the end use 20mM PB pH 7.5+1M sodium chloride to elute;and we collect buffer rinse peak and sodium chloride elution peak,and to carry out vitality test after membrane concentration,and the acquired active peak all belong to penetrate peak,and the vigor index determination of stimulation reaches above 2.8.Then continue to use Sephadex G-10 to carry out the gel filtration chromatography to the calf serum deproteinized extract ultra filtrate,and the results show that the active ingredients appear before conductance peak,and some substances have hydrophobic effects,and the components appearance time is postponed,which has been certified that we could get the purified active componant of better separation effect by way of molecular sieve.The above researching results show that we take the content of peptides and respiratory activity as the index,and has initially identified the chromatography purification processes ways that have better separating effects to the active components of the calf serum deproteinized extract ultra filtrate are:negative ion Capto Q ImpRes under flowthrough method,membrane filtration enrichment,gel filtration.This thesis also carries out the LC-MS analysis to the calf serum deproteinized extract ultra filtrate.The sample is vacuum dried,and we directly conduct the LC-MS analysis after being dissolved by methyl alcohol;and the filtrate sample shall be analyzed by thin-layer chromatography,and to elute the sample thin layer flecks by methyl alcohol and ultrapure water,and to make LC-MS analysis to the spent regenerant;and the sample is analyzed by column chromatography,and collects different time spent regenerant to make LC-MS analysis.Through analysis method research,we could preliminary infer that the deproteinized calf serum extract contains glucose,fructose,choline and inositol,and the glucose content is higher than the fructose,and we also find the components of saccharides,choline,Adenosine monophosphate and small peptides.This also provides the basis for future chromatography purification and qualitative analysis,quantitative determination and quality specification formulation of active ingredients of the deproteinized calf serum extract.