Preparation Of IgY Capable Of Inhibiting α-Amylase And The Study Of Its Oral Drug Targeting

Egg yolk antibody（IgY）with the advantages of low cost,high specificity,good stability and oral safety has great application potential in developing a novelα-amylase inhibitor（α-AI）.At present,there are many researches about the target treatment of tumors with nano-Fe₃O₄,but little research has been done in the targeted therapy of diabetes.IgY was coupled to magnetic Fe₃O₄ nanoparticles to construct IgY nanoprobes with targeted function,which could improve the effective utilization of IgY.As a protein,IgY is easily degraded by gastric juice when administered orally.Calcium alginate/chitosan gel with low toxicity,stability and biocompatibility is able to ensure IgY nanoprobes to reach the intestines successfully and exert its hypoglycemic effect.First,A-IgY was prepared by immunizing the hens with porcine pancreaticα-amylase and extracted by water dilution and ammonium sulfate two-step precipitation method.The electrophoresis results showed that the molecular weights of heavy and light chains of A-IgY was 65 kDa and 25 kDa,respectively.The glycosylation modification mainly occurred in the heavy chain.The purity of A-IgY was 92%.The IgY concentration of A-IgY was 8.51 mg/mL and the extraction rate was 18.5 mg/mL egg yolk.Next,the inhibitory effect of A-IgY onα-amylase was analyzed by Bernfeld method.The results showed that the inhibition rate of A-IgY onα-amylase gradually increased after the first immunization and reached the maximum（98.90%）in the 10th week and then stabilized to the 17th week while N-IgY showed very low inhibitory activity（about 7%）.A-IgY inhibited porcine pancreaticα-amylase by a non-competitive inhibition with the half maximal inhibitory concentration(IC₅₀)of 1.189 mg/mL.The inhibition rate of A-IgY on human pancreaticα-amylase with the same enzyme activity was 82.76%.A-IgY can effectively inhibit the decomposition of starch in daily diet（α-amylase inhibition rates were all above85%）.The inhibition rate of A-IgY onα-amylase increased with the prolongation of the mixing time withα-amylase and was closed to 100%at 90 min.The inhibitory activity of A-IgY was highest at 30⁴0^oC and pH 6⁹ and significantly reduced in simulated gastric fluid but stabled in simulated intestinal fluid.Then,A-IgY nanoprobes were constructed by coupling A-IgY to Fe₃O₄@DMSA and Fe₃O₄@PEG magnetic nanoparticles,respectively.The characterization results showed that the four nanoparticles had good morphology,uniform size and good monodispersity.The hydrodynamic size of Fe₃O₄@DMSA and Fe₃O₄@PEG nanoparticles increased after coupling reaction from 18.21 nm to 51.39 nm and 24.36 nm to 54.50 nm,respectively.The Zeta potential of Fe₃O₄@DMSA and Fe₃O₄@PEG nanoparticles was-36.4 mV and-25.2 mV and decreased to-23.4 mV and-16.1 mV after coupling with IgY,respectively.The characteristic absorption peaks of the amide band and IgY in FT-IR images indicated that IgY was successfully coupled to the nanoparticles.The IgY loading rates of Fe₃O₄@DMSA@IgY and Fe₃O₄@PEG@IgY was 68.10%and 80.65%,respectively.The VSM results showed that Fe₃O₄@DMSA@IgY and Fe₃O₄@PEG@IgY nanoparticles were superparamagnetic and their saturation magnetizations was 54.08 emu/g and 64.05 emu/g,respectively.The inhibition rates of Fe₃O₄@DMSA@IgY and Fe₃O₄@PEG@IgY onα-amylase was 83.73%and 87.35%.The results abovementioned indicated that the coupling reaction did not affect the inhibitory activity of A-IgY.Finally,Fe₃O₄@PEG@IgY nanoparticles were embedded in calcium alginate/chitosan gel.The results showed that the Fe₃O₄@PEG@IgY gel beads were smooth spheres with great dispersibility and magnetic responsiveness had double-layer membrane structure and uniform size.The IgY gel beads became more compact after 2-hour simulated gastric fluid reaction and its IgY release rate was only 6.60%and then quickly increased to 90.86%after transfering to intestinal fluid for 2 hours.CT images were performed after the gel beads were targeted oral gavage for administration to healthy rats.The results showed that the gel beads entered the intestine after 2 hours of oral gavage and were basically eliminated after 48 hours.The gel beads were intragastrically administered to db/db rats.The postprandial blood glucose test showed that the hypoglycemic effect of Fe₃O₄@PEG@IgY gel bead was significantly better than that of Acarbose and was more durable.It is expected to be used to develop new and safe drugs of diabetes prevention and treatment.