title: Extraction and identification of collagen-derived peptides with hematopoietic activity from Colla Corii Asini.
abstract: BACKGROUND
  Colla Corii Asini is a widely used traditional Chinese medicine to treat anemia with a long history due to its stimulating effect in hematopoiesis, but the components contributing to this effect are still unknown. In this study, we aimed to establish a methodology to isolate the bioactive components and provide pharmacological basis for its usage in treating anemia.
  
  
  METHODS
  5-FU and γ-ray radiation induced anemic mice models were generated by treating with 5-FU at 150mg/kg body weight and γ-rays by a 4MV linear accelerator by total body irradiation using female ICR mice respectively. Oral administration of fraction A was performed by gastric lavage at 1g/kg and 2g/kg body weight for 12 days and 25 days and peripheral blood sample was collected from ocular sinus red blood cell (RBC) and white blood cell (WBC) counts every 3 days and 5 days for 5-FU and radiation induced models, respectively. Next, fraction A was separated to A1 and A2 using cation exchange chromatography (IEC) based on ionic strength. Fraction A1 was further separated using reverse phase chromatography (RPC) based on the hydrophobicity first with 0-10% linear gradient, then 20%, 30%, 50% constant gradient of 60% acetonitrile in neutral Na2HPO4 buffer. Peak fractions were pooled, evaporatively dried, and dissolved in ultrapure water. Finally, fraction A11 was analyzed combining tandem mass spectrometry and proteomic tools and two peptides (peptide 11 and 16) were identified. The hematopoietic effects of multiple fractions and the two peptides were measured using colony-forming units-erythroid (CFU-E), an indication of late erythroid progenitor cells and colony-forming units granulocyte-monocyte (CFU-GM), an indication of granulocyte and monocyte progenitor cells respectively on hematopoietic progenitor cells prepared from bone marrow (Till and Mcculloch 1961).
  
  
  RESULTS
  Fraction A at 1g/kg and 2g/kg could increase RBC and WBC counts in 5-FU and radiation induced anemic mice models. Fraction A1 at 0.1mg/ml and 0.5mg/ml, exhibited stronger hematopoietic activity than fraction A2, both of which were subfractions from fraction A using IEX, by elevated CFU-E and CFU-GM of mouse bone marrow cells. Furthermore, fraction A11 at 0.1mg/ml showed stronger CFU-E and CFU-GM than fractions A12 to A14 from RPC separation. Finally, peptide 11 and peptide 16 were identified from tandem mass spectrometry and peptide 11 increased CFU-E and CFU-GM in a dose dependent manner.
  
  
  CONCLUSIONS
  We combined multiple approaches including chromatography, mass spectrometry, cell-based assays, as well as animal studies to identify and demonstrate that the hematopoietic effect of Colla Corii Asini is at least in part from the peptidic components identified using our methodology. This is the first time to isolate peptidic components from Colla Corii Asini, and to provide molecular basis for its usage in treating anemia, which may particularly have the potential to benefit cancer patients suffering from myelosuppression due to radiotherapy or chemotherapy.
authors.affiliation: The Pharmacology School of East China University of Science and Technology, Shanghai 200237, China; HUYA Bioscience International, Shanghai 201203, China. Electronic address: Thomaswhz@hotmail.com.
authors.forename: Hongzhong
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address: chunyan.ren@mssm.edu.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Ren
authors.affiliation: The Pharmacology School of East China University of Science and Technology, Shanghai 200237, China; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Yang
authors.affiliation: Shandong Dongeejiao Group, Shandong 252201, China.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Yuanxing
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: The Pharmacology School of East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Jianwen
authors.initials: J
authors.lastname: Liu
mesh_terms: D000740:Anemia / Q000097:blood / Q000139:chemically induced / Q000188:drug therapy*; D000818:Animals; D001772:Blood Cell Count; D003094:Collagen / Q000737:chemistry*; D004365:Drugs, Chinese Herbal / Q000494:pharmacology / Q000627:therapeutic use*; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects; D005260:Female; D005472:Fluorouracil; D005720:Gamma Rays; D005780:Gelatin / Q000494:pharmacology / Q000627:therapeutic use*; D055014:Granulocyte-Macrophage Progenitor Cells / Q000166:cytology / Q000187:drug effects; D006397:Hematinics / Q000032:analysis / Q000494:pharmacology / Q000627:therapeutic use*; D006410:Hematopoiesis / Q000187:drug effects; D008516:Medicine, Chinese Traditional; D008813:Mice, Inbred ICR; D010455:Peptides / Q000032:analysis / Q000494:pharmacology / Q000627:therapeutic use*
chemical_list: D004365:Drugs, Chinese Herbal; D006397:Hematinics; D010455:Peptides; C000612945:colla corii asini; D005780:Gelatin; D003094:Collagen; D005472:Fluorouracil
keywords: Anemia; Cancer; Colla Corii Asini; E-jiao; Hematopoiesis; Traditional Chinese medicine