Preparation And Quality Of Succinic Anhydride Acylated Collagen Freeze-Dried Solid

Objective As a unique biomaterial,collagen has low immunogenicity,high biocompatibility and repair function,so it is widely used in medical,health care,beauty and other fields.The traditional extraction of collagen is mainly based on animal skin,but less on animal achilles tendon.China does not belong to the epidemic area of BSE,and the cattle resources are safe and abundant.Therefore,in this paper,bovine achilles tendon was used as raw material,and the best extraction process was obtained through the screening of extraction conditions.Through the acylation reaction between succinic anhydride and collagen,the problem of poor solubility of natural collagen in physiological environment was solved,and a material with good solubility and tissue repair function is prepared.Methods ①In this paper,the best extraction conditions of bovine achilles tendon collagen were screened by single factor experiment.The triple helix structure and characteristics of collagen were analyzed by amino acid analysis,SDS-PAGE gel electrophoresis,thermal denaturation temperature,circular dichroism and infrared spectrum.②The regeneration of zebrafish caudal fin and the relative expression of zebrafish coll alb gene were used to demonstrate the effect of collagen in promoting tissue regeneration.The anti-inflammatory effect of collagen was demonstrated by the model of zebrafish inflammation induced by sodium dodecyl sulfate.③The optimum reaction conditions of succinic anhydride acylated collagen were screened by single factor experiment,and the freeze-dried solid of succinic anhydride acylated collagen was prepared.Through the isoelectric point,dissolution time,SDS-PAGE gel electrophoresis,thermal denaturation temperature,circular dichroism and infrared spectrum of succinic anhydride acylated collagen,the triple helix structure and characteristics of acylated collagen were analyzed.④ The safety of succinic anhydride acylated collagen freeze-dried solid was demonstrated by skin irritation test in New Zealand rabbits,skin sensitization test in albino guinea pigs and cytotoxicity test in vitro.Results ①The optimal extraction time of collagen from bovine achilles tendon is 72h,the optimal temperature is 25 ℃,and the optimal enzyme dosage is 3%.Through amino acid analysis,the characteristic strong proline content is 13.5%;The molecular weight of bovine achilles tendon collagen obtained by SDS-PAGE gel electrophoresis is close to 300kd,and the thermal denaturation temperature is 69.9℃.Circular dichroism and infrared spectrum all have characteristic peaks of collagen;②The regeneration efficiency of zebrafish caudal fin was 23%,P<0.01;The relative expression of col1a1b gene in zebrafish was 1.27,P<0.01;The anti-inflammatory effect of zebrafish was 32%,P<0.41.③ The optimum reaction conditions for acylation of collagen with succinic anhydride are pH9,reaction temperature 25℃ and the dosage of succinic anhydride 20%.The isoelectric point of the prepared succinic anhydride acylated collagen freeze-dried solid was 3.82,the dissolution time was 60s-90s,the molecular weight was high by SDS-PAGE gel electrophoresis,and the thermal denaturation temperature was 67.4℃.Circular dichroism and infrared spectrum all have characteristic peaks of collagen.④ The results of animal experiments show that the succinic anhydride acylated collagen freeze-dried solid has little irritation,no allergic reaction and no cytotoxicity test.Conclusion In this paper,the extraction scheme of bovine achilles tendon collagen was designed and optimized,and type I collagen was obtained.The triple helix structure of bovine achilles tendon collagen was verified by thermal denaturation temperature,circular dichroism,and infrared spectrum.In addition,The zebrafish animal experiment proved that bovine achilles tendon collagen has the effects of promoting tissue repair and anti-inflammation.Modified collagen was obtained by acylation of collagen with succinic anhydride.Collagen acylated by succinic anhydride has low isoelectric point and high solubility.The thermal denaturation temperature,circular dichroism and infrared spectrum were also used to verify that the prepared succinic anhydride acylated collagen freeze-dried solid still has a triple helix structure,indicating that its main biological activity remains.In addition,skin irritation test,skin sensitization test and cytotoxicity test proved that succinic anhydride acylated collagen freeze-dried solid has high safety.