Study On The Protective Effect Of Deer Whiptail Polypeptide Microcapsules On Osteoblasts

Objective:Osteoporosis（OP）is a disease with high morbidity and prevalence.As the main functional cell of bone formation,osteoblast（MC3T3-E1）is the main cause of osteoporosis.Traditional Chinese medicine has certain advantages in protecting osteoblasts.Therefore,in this paper,the raw materials of deer whip tail were extracted and hydrolyzed by peptides,and the process was optimized to determine the best technological conditions of extraction and enzymatic hydrolysis of deer whip tail peptides.The preparation of deer whip tail polypeptide microcapsule was carried out,and the best preparation process was determined and optimized.the morphology,the release in stomach and intestine and the effect of deer whip tail peptide microcapsule on MC3T3-E1 cells were observed,which provided a theoretical basis for the research and development of deer whip tail.Methods:The best extraction technology of whip tail polypeptide protein was determined by single factor method and orthogonal experiment,taking protein yield and protein content as indexes,three peptides and one protein segment were separated by ultrafiltration fractionation,and the peptide with the highest protein content was determined as the substrate of enzymatic hydrolysis products,and the components of deer whip tail polypeptides were determined by amino acid analysis.With the degree of hydrolysis as the index,the best enzymatic hydrolysis process of deer whip tail polypeptide protein was determined by single factor method and orthogonal experiment,and the entrapment efficiency was taken as the index by single factor method and orthogonal experiment.the best dripping process of deer whip tail peptide microcapsules was determined,and the surface structure and morphology of deer whip tail peptide microcapsules were observed by microscope.The release in vitro was detected by simulating artificial gastric juice（USP）and artificial intestinal fluid environment.The LPS-induced injury model of MC3T3-E1 cells induced by deer whip tail polypeptide protein,enzymatic hydrolysate and microcapsule product in vitro was established by MTT method,and its protective effect on MC3T3-E1 cells was observed,and its pharmacodynamics was investigated in vitro.Results:the best extraction technology of deer whip tail polypeptide protein was that water was used as extraction solvent,and Flash extraction method was used for extraction.When the ratio of material to liquid was 1:10,the extraction time was 1minute,and the extraction times was 5 times,the yield of protein was 38.27%.In the ultrafiltration fractionation,three peptides（<1 k Da,1-3 k Da,3-10 k Da,>10 k Da）and one protein segment were separated,and the protein content of 3-10 k Da was the highest,and it was selected as the substrate of the enzymatic hydrolysis product.Deer whip tail peptide contains 18 kinds of amino acids.Alkaline protease was selected as the best enzyme for enzymatic hydrolysis of deer whip tail polypeptide protein,and the optimum enzymatic hydrolysis conditions were determined as follows:p H 9.0,temperature 60℃,enzymatic hydrolysis 5 h,enzyme bottom ratio 6%.The degree of hydrolysis of the hydrolyzed product is the highest,which is 44.5%.When the concentration of chitosan was 0.3%,the ratio of whiplash polypeptide to sodium alginate was 1:6,the concentration of sodium alginate was 2%and the concentration of Ca Cl₂was 2%.The entrapment efficiency was 90.56%.Under the best preparation conditions,its morphology was investigated,which was spherical,no special aggregation and independent of each other.The color is yellowish and there is no pungent smell.The release of deer whip tail polypeptide microcapsules is small in artificial gastric juice and increased in artificial intestinal juice,that is,the effect is obvious in artificial intestinal juice,indicating that microcapsules can protect the core under the best preparation conditions.and the sustained release performance is good.Establishment of lipopolysaccharide（LPS）cell model:mouse osteoblast MC3T3-E1was treated with different concentrations of LPS（25μg/L,50μg/L,100μg/L,200μg/L,400μg/L,800μg/L）for 4 h.The cell survival rate induced by 100μg/L LPS was about 70%of that of the normal control.Therefore,an ideal cell injury model was established by inducing MC3T3-E1 cells with 100μg/L LPS for 4 hours.The three samples of deer whip tail enzymatic hydrolyzed peptide,deer whip tail peptide microcapsule and deer whip tail peptide protein 3-10 k Da could protect and improve the damage state of MC3T3-E1 cells induced by LPS,and the survival rate was increased but not higher than that of the control group（P<0.05）.Conclusion:it is feasible to select deer whip tail peptide microcapsules as the research direction of anti-osteoporosis therapy,and deer whip tail peptide microcapsules have obvious protective effect on MC3T3-E1 cells.Through the preparation of deer whip tail polypeptide microcapsules,the best preparation conditions were determined.As a new dosage form,microcapsules can protect the core drug,have good sustained release performance,and are suitable for release in the intestinal tract.Therefore,the preparation of its raw materials into new dosage forms of microcapsules has a certain development and clinical value.