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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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  9. Mak P, Wojcik K, Wicherek L, Suder P, Dubin A. 2004. Antimicrobial hemoglobin peptides in human menstrual blood. Peptides 25: 1893-1947.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    CrossRef
  13. Chang CY, Wu KC, Chiang SH. 2007. Antioxidant properties and protein compositions of porcine haemoglobin hydrolysates. Food Chem. 100: 1537-1543.
    CrossRef
  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.
    CrossRef
  15. Hordur GK, Barbara AR. 2000. Fish protein hydrolysates:production, biochemical, and functional properties. Crit. Rev. Food Sci. Nutr. 40: 43-81.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  17. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
    Pubmed CrossRef
  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.
    CrossRef
  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.
    CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    CrossRef
  24. Moosman B, Behl C. 2002. Secretory peptide hormones are biochemical antioxidants: structure-activity relationship. Mol. Pharm. 61: 260-268.
    CrossRef
  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.
    Pubmed CrossRef
  26. Freer E, Pizarro-Cerdá J, Weintraub A, Bengoechea J, Moriyón 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.
    Pubmed PMC
  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.
    Pubmed
  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.
    CrossRef
  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.
    Pubmed PMC CrossRef
  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.
    Pubmed CrossRef
  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.
    CrossRef
  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.
    Pubmed PMC CrossRef
  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.
    CrossRef
  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.
    CrossRef
  35. Kurahashi T, Fujii J. 2015. Roles of anti-oxidative enzymes in wound healing. J. Dev. Biol. 3: 57-70.
    CrossRef
  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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Article

Research article

J. Microbiol. Biotechnol. 2017; 27(1): 26-35

Published online January 28, 2017 https://doi.org/10.4014/jmb.1603.03046

Copyright © The Korean Society for Microbiology and Biotechnology.

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

Anawat Pakdeesuwan 1, 2, Tomohiro Araki 3, Sakda Daduang 2, 4, Wisarut Payoungkiattikun 2, Nisachon Jangpromma 2, 5 and Sompong Klaynongsruang 1, 2*

1Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand, 2The Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand, 3Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto 869-140, Japan, 4Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand, 5Office of the Dean, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand

Received: March 21, 2016; Accepted: September 26, 2016

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 μg. 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: Crocodile hemoglobin, chemical digestion, antibacterial peptide, fragmented hemoglobin

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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  9. Mak P, Wojcik K, Wicherek L, Suder P, Dubin A. 2004. Antimicrobial hemoglobin peptides in human menstrual blood. Peptides 25: 1893-1947.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    CrossRef
  13. Chang CY, Wu KC, Chiang SH. 2007. Antioxidant properties and protein compositions of porcine haemoglobin hydrolysates. Food Chem. 100: 1537-1543.
    CrossRef
  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.
    CrossRef
  15. Hordur GK, Barbara AR. 2000. Fish protein hydrolysates:production, biochemical, and functional properties. Crit. Rev. Food Sci. Nutr. 40: 43-81.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  17. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
    Pubmed CrossRef
  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.
    CrossRef
  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.
    CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    Pubmed CrossRef
  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.
    CrossRef
  24. Moosman B, Behl C. 2002. Secretory peptide hormones are biochemical antioxidants: structure-activity relationship. Mol. Pharm. 61: 260-268.
    CrossRef
  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.
    Pubmed CrossRef
  26. Freer E, Pizarro-Cerdá J, Weintraub A, Bengoechea J, Moriyón 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.
    Pubmed KoreaMed
  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.
    Pubmed
  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.
    CrossRef
  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.
    Pubmed KoreaMed CrossRef
  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.
    Pubmed CrossRef
  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.
    CrossRef
  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.
    Pubmed KoreaMed CrossRef
  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.
    CrossRef
  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.
    CrossRef
  35. Kurahashi T, Fujii J. 2015. Roles of anti-oxidative enzymes in wound healing. J. Dev. Biol. 3: 57-70.
    CrossRef
  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.