A Study On Major Constituent Proteins Of Raw Material Of Ejiao And Their Bioactivities

Ejiao,or Colla Corri Asini, is one of the most important traditional Chinese medicine with a history of more than 2,000 years. Because of insufficient studies on the basic questions such as the constituting composition of its raw material, the function mechanism of the medicine remains ambiguous. It is therefore, the goal to the current study to elucidate the major constituent proteins of the donkey skin which is Ejiao's raw material, and further testing their bioactivities, in hope to eventually decipher the biochemical mechanism behind Ejiao's prominent medicinal efficacy.Extraction of total proteins in donkey skin was carried out by two methods: the first by using TriPure Isolation Reagent to extract the total proteins in donkey skin, and another by dissolving the skin sample in 1% SDS at 100℃ overnight. SDS-PAGE and capillary HPLC were used to analyze the component of the extracted protein samples. The results of SDS-PAGE of both samples prepared by different methods indicate that donkey serum albumin(DkSA) is also abundant in donkey skin in addition to the well expected collagen α₁ (I) and collagen α₂ (I). Moreover collagen subunits and serum albumin were found to be bound together in donkey skin through analysis with capillary HPLC(cLC). Thus Ejiao was made up of type I collagen and DkSA and their hydrolysates. It's reasonable to assume that DkSA may play a more important role in the medicine's efficacy considering its much more complex structure and versatile function than those of collagens.Based on the assumption that DkSA found in the skin must be derived from the plasma, donkey plasma abundant of albumin was used as the raw material for isolation and purification of DkSA. Donkey serum from donkey plasma prepared by means of freezing, centrifugation, dialysis and filtration by 0.22 μ m membrane was fractionated by two steps of anion-exchange chromatography of Source 15Q on HPLC and three peaks of albumin were obtained finally. The purity of the obtained albumin sample was determined by cLC , SDS-PAGE and PAGE and the results show that the purified albumins were all single constituent. Molecular weight determined to be 66.0 kDa by SDS-PAGE.The influence of DkSA on proliferation of osteoblast-like UMR-106 cells was investigated. A prominent osteogenic effect was observed with DkSA at different concentrations. Since type I collagen could suppress the proliferation and promote the differentiation of osteoblast cells, DkSA and type I collagen might work synergically on osteopososis when treated by Ejiao.The tryptic hydrolysate of DkSA was shown to inhibit ACE activity in vitro. Virtual tryptic digestion of DkSA using bioinformatic software renders a clue to a hepta-peptide(YLYEVAR) with potential ACE inhibitory activity.In Summary, the present work has provided a new angle of view on Ejiao's pharmaceutical efficacy. Further studies on the constituent proteins and bioactive peptides derived from these proteins can possibly result in elucidation of the machanism of medicine efficacy of Ejiao.