Study On The Active Components Of Colla Corii Asini Promoting Iron Absorption And Hematopoietic Effect Of Their Iron Chelates

Iron deficiency anemia(IDA)is one of the nutritional diseases with the highest incidence,which can cause dysfunction of cognitive ability,growth and development and immune system.Colla Corii Asini(Ejiao)as a kind of medicine and food resource with the hematopoietic effect.It is very important to study its active components and mechanism of action for scientific understanding of the hematopoietic effect of Ejiao.However,the research on the active ingredients of Ejiao have not been fully clarified.The study employed in vitro simulated digestion and Ussing chamber absorption model to evaluate the iron chelating ability and iron absorption facilitation of Ejiao.After targeted isolation of Ejiao hydrolysates on immobilized metal affinity chromatography(IMAC)to obtain iron chelating peptides.To prepare Ejiao peptide-iron chelates(EPI)and analyze the structural characteristics.Based on the IDA mouse model to evaluate the hematopoietic effect of EPI and utilize metabonomics methods to explore the metabolic regulation effect of EPI on IDA.The research results have guiding significance for elucidating the hematopoietic active components of Ejiao and promoting the development of donkey collagen products.Firstly,the form and content of iron in Ejiao were analyzed,and the effects of digestion process on iron chelating and absorption capacity of Ejiao was evaluated.The results showed that the iron ions in Ejiao mainly exists in free and chelated forms,and iron chelating rate is46.45%.With the addition of dietary iron,the amount of iron chelation increased.Compared with pre-digestion,the iron chelating ability of Ejiao was significantly increased by 16.22 times(P<0.05)after gastrointestinal two-step digestion.The Ussing chamber absorption experiment indicated that the iron transport and absorption rate of Ejiao gastrointestinal digestion products were higher than Fe SO₄(P<0.05),and the apparent permeability coefficient increased from5.15×10^-6cm/s to 6.18×10^-6cm/s,which promoted Fe²⁺absorption in intestinal mucosa,suggesting that iron chelates formed after digestion of Ejiao combined with dietary iron has a potential iron supplemental activity.Secondly,IMAC was used to separate iron chelating peptides(F2)from the Ejiao hydrolysates and characterized the structure of EPI.The results showed that iron chelating capacity of the separated F2 fraction was 23.58±0.70?g/mg,which was 27.15 times higher than before separation.The content with relative molecular mass less than 5000 in F2 with iron chelating capacity reached 97.01%,and mainly distributed in 180?2000(70.51%).The contents of acidic and basic amino acids in F2 are 20.08% and 14.51%,respectively.Moreover the contents of glycine(Gly),glutamic acid(Glu)and proline(Pro)are higher,accounting for25.14%,13.25%and 11.22%of the total amino acid contents,respectively.Six high intensity peptides were sequenced as AGPPGADGQPGAK,LQGMPGERG,GPAGPIGPV,VGPVGPA,IDGRPGPIGPA and LRGPRGDQGPVGRA,which are derived from collagen?-1(I)chain and collagen?-2(I)chain,respectively.The structure of EPI was characterized by infrared spectroscopy,circular dichroic spectroscopy,scanning electron microscope etc.The results revealed that iron ion is mainly chelated with carboxyl oxygen and amino nitrogen in the peptide chain,and could neutralize the negative charge.After chelating,peptides transformed into an orderly and stable structure,and the chelates formed fold and aggregate.Finally,the hematopoietic effect of EPI was evaluated based on IDA mouse model,and metabonomic analysis of LC-MS was used to explore the metabolic regulation effect of EPI on IDA.The results of indicators such as mice weight growth rate,organ index and blood basic value showed that EPI could effectively improve IDA,promote the growth of IDA mice,restore the organ index and basic blood indicators to normal levels.And there was no significant difference between EPI and Ejiao hydrolysates-iron chelates(EJI),indicating that EPI can improve iron deficiency anemia and has the effect of enriching blood.By calculating the relative bioavailability of iron and analyzing the contents of serum iron,ferritin,and tissue iron in mice,it was found that compared with Fe SO₄,the relative bioavailability of iron in EPI was increased to 123.45%,which could promote iron absorption and effectively improve the iron homeostasis level in body.Further studies found that EPI is mainly involved in the tricarboxylic acid cycle,pyruvate metabolism,glycolysis and gluconeogenesis,arachidonic acid metabolism,histidine metabolism,cysteine and methionine metabolism pathways to regulate IDA induced metabolic disorders in mice,and can improve the iron level of mice,restore iron homeostasis and improve the organs state.In conclusion,the Ejiao peptides with iron chelating ability produced in the process of digestion could promote the intestinal iron absorption as the active component of Ejiao,and chelated with dietary iron through carboxyl oxygen and amino nitrogen to form EPI.EPI could improve the bioavailability of iron.After being absorbed by the body,EPI could improve the metabolic disorder caused by IDA mainly by regulating energy metabolism,lipid metabolism and amino acid metabolism,and protect tissues and organs while replenishing blood.