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In Vivo Wound Healing Activity of Crocodile (Crocodylus siamensis) Hemoglobin and Evaluation of Antibacterial and Antioxidant Properties of Hemoglobin and Hemoglobin Hydrolysate

  • Pakdeesuwan, Anawat (Department of Biochemistry, Faculty of Science, Khon Kaen University) ;
  • Araki, Tomohiro (Department of Bioscience, School of Agriculture, Tokai University) ;
  • Daduang, Sakda (The Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Faculty of Science, Khon Kaen University) ;
  • Payoungkiattikun, Wisarut (The Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Faculty of Science, Khon Kaen University) ;
  • Jangpromma, Nisachon (The Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Faculty of Science, Khon Kaen University) ;
  • Klaynongsruang, Sompong (Department of Biochemistry, Faculty of Science, Khon Kaen University)
  • Received : 2016.03.21
  • Accepted : 2016.09.26
  • Published : 2017.01.28

Abstract

The hydrolysis of proteins constitutes an invaluable tool, granting access to a variety of peptide fragments with potentially interesting biological properties. Therefore, a hemoglobin (Hb) hydrolysate of Crocodylus siamensis was generated by digestion under acidic conditions. The antibacterial and antioxidant activities of the Hb hydrolysate were assessed in comparison with intact Hb. A disc diffusion assay revealed that the Hb hydrolysate exhibited antibacterial activity against eight strains of gram-positive bacteria and showed a higher efficacy than intact Hb. Moreover, the antioxidant activity of intact Hb and its hydrolysate was evaluated using ABTS and DPPH radical scavenging assays. The Hb hydrolysate exhibited free radical scavenging rates of 6-32%, whereas intact Hb showed a slightly higher activity. In addition, non-toxicity to human erythrocytes was observed after treatment with quantities of Hb hydrolysate up to $10{\mu}g$. Moreover, active fragmented Hb (P3) was obtained after purifying the Hb hydrolysate by reversed-phase HPLC. Scanning electron microscopy demonstrated the induction of bacterial cell membrane abnormalities after exposure to P3. Antibacterial and antioxidant activities play crucial roles for supporting the wound healing activity. Consequently, an in vivo mice excisional skin wound healing assay was carried out to investigate the effects of intact Hb treatment on wound healing in more detail. The results clearly demonstrate that intact Hb is capable of promoting 75% wound closure within 6 days. These findings imply that intact Hb of C. siamensis and its acid hydrolysate may serve as valuable precursors for food supplementary products benefitting human health.

Keywords

References

  1. Karuppusamy A, Thangaraj P. 2013. Anti-inflammatory, wound healing and in-vivo antioxidant properties of the leaves of Ficus amplissima Smith. J. Ethnopharmacol. 145: 139-145. https://doi.org/10.1016/j.jep.2012.10.041
  2. Phosri S, Mahakunakorn P, Lueangsakulthai J, Jangpromma N, Swatsitang P, Daduang S, et al. 2014. An investigation of antioxidant and anti-inflammatory activities from blood components of crocodile (Crocodylus siamensis). Protein J. 33: 484-492. https://doi.org/10.1007/s10930-014-9581-y
  3. Kim SY, Je JY, Kim SK. 2007. Purification and characterization of antioxidant peptide from hoki (Johnius belengerii) frame protein by gastrointestinal digestion. J. Nutr. Biochem. 18: 31-38. https://doi.org/10.1016/j.jnutbio.2006.02.006
  4. Jangpromma N, Preecharram S, Srilert T, Maijaroen S, Mahakunakorn P, Nualkaew N, et al. 2016. In vitro and in vivo wound healing properties of plasma and serum from Crocodylus siamensis blood. J. Microbiol. Biotechnol. 26: 1140-1147. https://doi.org/10.4014/jmb.1601.01054
  5. Parish CA, Jiang H, Tokiwa Y, Berova N, Nakanishi K, McCabe D, et al. 2001. Broad-spectrum antimicrobial activity of hemoglobin. Bioorg. Med. Chem. 9: 377-382. https://doi.org/10.1016/S0968-0896(00)00263-7
  6. Theansungnoen T, Yaraksa N, Daduang S, Dhiravisit A, Thammasirirak S. 2014. Purification and characterization of antioxidant peptides from leukocyte extract of Crocodylus siamensis. Protein J. 33: 24-31. https://doi.org/10.1007/s10930-013-9536-8
  7. Jandaruang J, Siritapetawee J, Songsiriritthigul C, Daduang S, Dhiravisit A, Thumanu K, et al. 2012. The effects of temperature and pH on secondary structure and antioxidant activity of Crocodylus siamensis hemoglobin. Protein J. 31: 43-50. https://doi.org/10.1007/s10930-011-9372-7
  8. Daoud R, Dubois V, Bors-Dodita L, Nedjar-Arroume N, Krier F, Chihib NE, et al. 2005. New antibacterial peptide derived from bovine hemoglobin. Peptides 26: 713-719. https://doi.org/10.1016/j.peptides.2004.12.008
  9. Mak P, Wojcik K, Wicherek L, Suder P, Dubin A. 2004. Antimicrobial hemoglobin peptides in human menstrual blood. Peptides 25: 1893-1947.
  10. Nedjar-Arroume N, Dubois-Delval V, Adje EY, Traisnel J, Krier F, Mary P, et al. 2008. Bovine hemoglobin: An attractive source of antibacterial peptide. Peptides 29: 969-977. https://doi.org/10.1016/j.peptides.2008.01.011
  11. Srihongthong S, Pakdeesuwan A, Daduang S, Araki T, Dhiravisit A, Thammasirirak S. 2012. Complete amino acid sequence of globin chains and biological activity of fragmented crocodile hemoglobin (Crocodylus siamensis). Protein J. 31: 466-476. https://doi.org/10.1007/s10930-012-9424-7
  12. Nyberg F, Sanderson K, Glämsta EL. 1997. The hemorphins: a new class of opioid peptides derived from the blood protein haemoglobin. Biopolymers 43: 147-156. https://doi.org/10.1002/(SICI)1097-0282(1997)43:2<147::AID-BIP8>3.0.CO;2-V
  13. Chang CY, Wu KC, Chiang SH. 2007. Antioxidant properties and protein compositions of porcine haemoglobin hydrolysates. Food Chem. 100: 1537-1543. https://doi.org/10.1016/j.foodchem.2005.12.019
  14. Liu Q, Kong B, Jiang L, Cui X, Liu J. 2009. Free radical scavenging activity of porcine plasma protein hydrolysates determined by electron spin resonance spectrometer. LWT Food Sci. Technol. 42: 956-962. https://doi.org/10.1016/j.lwt.2008.12.007
  15. Hordur GK, Barbara AR. 2000. Fish protein hydrolysates: production, biochemical, and functional properties. Crit. Rev. Food Sci. Nutr. 40: 43-81. https://doi.org/10.1080/10408690091189266
  16. Pata S, Yaraksa N, Daduang S, Temsiripong Y, Svasti J, Araki T, et al. 2011. Characterization of the novel antibacterial peptide leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts. Dev. Comp. Immunol. 35: 545-553. https://doi.org/10.1016/j.dci.2010.12.011
  17. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. https://doi.org/10.1038/227680a0
  18. Bradford MM. 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  19. Memarpoor-Yazdi M, Asoodeh A, Chamani J. 2012. A novel antioxidant and antimicrobial peptide from hen egg white lysozyme hydrolysates. J. Funct. Foods 4: 278-286. https://doi.org/10.1016/j.jff.2011.12.004
  20. Strub JM, Goumon Y, Lugardon K, Capon C, Lopez M, Moniatte M. 1996. Antibacterial activity of glycosylated and phosphorylated chromogranin A-derived peptide from bovine adrenal medullary chromaffin granules. J. Biol. Chem. 271: 28533-28540. https://doi.org/10.1074/jbc.271.45.28533
  21. Lau SKP, Woo PCY, Woo GKS, Fung AMY, Wong MKM, Chan KM, et al. 2004. Eggerthella hongkongensis sp. nov. and Eggerthella sinesis sp. nov., two novel Eggerthella species, account for half of the cases of Eggerthella bacteremia. Diagn. Microbiol. Infect. Dis. 49: 255-263. https://doi.org/10.1016/j.diagmicrobio.2004.04.012
  22. Lodhi S, Rajesh SP, Alok PJ, Singhai AK. 2006. Wound healing potential of Tephrosia purpurea (Linn.) Pers. in rat. J. Ethnopharmacol. 108: 204-210. https://doi.org/10.1016/j.jep.2006.05.011
  23. Li B, Chen F, Wang X, Ji BP, Wu YN. 2007. Isolation and identification of antioxidative peptides from porcine collagen hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry. Food Chem. 102: 1135-1143. https://doi.org/10.1016/j.foodchem.2006.07.002
  24. Moosman B, Behl C. 2002. Secretory peptide hormones are biochemical antioxidants: structure-activity relationship. Mol. Pharm. 61: 260-268. https://doi.org/10.1124/mol.61.2.260
  25. Arzese A, Skerlavaj B, Tomasinsing L, Gennaro, R, Zanetti M. 2003. Antimicrobial activity of SMAP-29 against the Bacteroides fragilis group and Clostridia. J. Antimicrob. Chemother. 52: 375-381. https://doi.org/10.1093/jac/dkg372
  26. Freer E, Pizarro-Cerda J, Weintraub A, Bengoechea J, Moriyon I, Hultenby K, et al. 1999. The outer membrane of Brucella ovis shows increased permeability to hydrophobic probes and is more susceptible to cationic peptides than are the outer membranes of mutant rough Brucella abortus strains. Infect. Immun. 67: 6181-6186.
  27. Henk WG, Todd WJ, Enright FM, Mitchell PS. 1995. The morphological effect of two antimicrobial peptides, hecate-1 and melittin, on Escherichia coli. Scanning Microsc 9: 501-507.
  28. Oren Z, Hong J, Shai Y. 1999. A comparative study on the structure and function of a cytolytic alpha-helical peptide and its antimicrobial beta-sheet diastereomer. Cent. Eur. J. Biol. 259: 360-369. https://doi.org/10.1046/j.1432-1327.1999.00047.x
  29. Oren Z, Lerman JC, Gudmundsson GH, Agerberth B, Shai Y. 1999. Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membrances: relevance to the molecular basis for its non-cell-selective activity. Biochem. J. 341: 501-513. https://doi.org/10.1042/bj3410501
  30. Tiozzo E, Rocco G, Tossi A, Romeo D. 1998. Wide-spectrum antibiotic activity of synthetic, amphipathic peptides. Biochem. Biophys. Res. Commun. 249: 202-206. https://doi.org/10.1006/bbrc.1998.9114
  31. Moure A, Dominguez H, Parajo JC. 2006. Antioxidant properties of ultrafiltration-recovered soy protein fractions from industrial effluents and their hydrolysates. Process Biochem. 41: 447-456. https://doi.org/10.1016/j.procbio.2005.07.014
  32. Sun Q, Shen H, Luo Y. 2011. Antioxidant activity of hydrolysates and peptide fractions derived from porcine hemoglobin. J. Food Sci. Technol. 48: 53-60. https://doi.org/10.1007/s13197-010-0115-0
  33. Jeon YJ, Byun HG, Kim SK. 2000. Improvement of functional properties of cod frame protein hydrolysates using ultrafiltration membranes. Process Biochem. 35: 471-478.
  34. Hsu A, Thomas A. 2010. The Principles of wound healing, pp. 3-7. In Weinzweig J (ed.). Plastic Surgery Secrets, Ch. 1. 2nd Ed. Mosby, Elsevier, Amsterdam.
  35. Kurahashi T, Fujii J. 2015. Roles of anti-oxidative enzymes in wound healing. J. Dev. Biol. 3: 57-70. https://doi.org/10.3390/jdb3020057
  36. Arenbergerova M, Engels P, Gkapakiotis S, Dubska Z, Arenberger P. 2013. Effect of topical haemoglobin on venous leg ulcer healing. EWMA J. 13: 25-30.

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