Skip to main content


Log in

Structural Modifications and Solution Behavior of Hyaluronic Acid Degraded with High pH and Temperature

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript


Hyaluronic acid (HA) is a macromolecule with valuable benefits over its range of molar masses (MM). Degradation studies are relevant to maintain the same purity level in biomedical studies when using HA of different MM. We degraded HA via high pH and temperature and evaluated its MM, solution behavior, and structure over time. After 24 h, low MM HA was predominant, and the MM decreased from 753 to 36.2 kDa. Dynamic light scattering (DLS) showed a decrease in the number of HA populations, and the solution tended to be less polydispersed. The zeta potential varied from − 10 to − 30 mV, close to the stable range. FTIR showed that the primary structure of HA was affected after only 48 h of reaction. These results are relevant for the production of low MM HA to be used or mixed with high MM HA, generating structured biomaterials for biomedical applications.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others


  1. Garg, H. G., & Hales, C. A. (2004). Chemistry and biology of hyaluronan. Elsevier.

  2. Lee, H. G., & Cowman, M. K. (1994). An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution. Anal.Biochem., 219(2), 278–287.

    Article  CAS  PubMed  Google Scholar 

  3. Slevin, M., Kumar, S., & Gaffney, J. (2002). Angiogenic oligosaccharides of hyaluronan induce multiple signaling pathways affecting vascular endothelial cell mitogenic and wound healing responses. The Journal of Biological Chemistry, 277(43), 41046–41059.

    Article  CAS  PubMed  Google Scholar 

  4. West, D. C., Hampson, I. N., Arnold, F., & Kumar, S. (1985). Angiogenesis induced by degradation products of hyaluronic acid. Science., 228(4705), 1324–1326.

    Article  CAS  PubMed  Google Scholar 

  5. R. Stern, A. a. Asari, K.N. Sugahara, Hyaluronan fragments: an information-rich system, European Journal of Cell Biology 85 (2006) 699–715., 8

  6. Sattar, A., Kumar, S., & West, D. C. (1992). Does hyaluronan have a role in endothelial cell proliferation of the synovium? Seminars in Arthritis and Rheumatism, 22(1), 37–43.

    Article  CAS  PubMed  Google Scholar 

  7. P.W. Noble, Hyaluronan and its catabolic products in tissue injury and repair, Matrix Biol. 21 (2002) 25–29.

  8. Rooney, P., Wang, M., Kumar, P., & Kumar, S. (1993). Angiogenic oligosaccharides of hyaluronan enhance the production of collagens by endothelial cells. Journal of Cell Science, 105(Pt 1), 213–218

    CAS  PubMed  Google Scholar 

  9. Gao, F., Liu, Y., He, Y., Yang, C., Wang, Y., Shi, X., & Wei, G. (2010). Hyaluronan oligosaccharides promote excisional wound healing through enhanced angiogenesis. Matrix Biology, 29(2), 107–116.

    Article  CAS  PubMed  Google Scholar 

  10. Schlesinger, T., & Powell, C. R. (2012). Efficacy and safety of a low-molecular weight hyaluronic acid topical gel in the treatment of facial seborrheic dermatitis final report. J. Clin. Aesthetic Dermatology., 7, 15–18.

    Google Scholar 

  11. Shewale, A. R., Barnes, C. L., Fischbach, L. A., Ounpraseuth, S., Painter, J. T., & Martin, B. C. (2017). Comparative effectiveness of low, moderate and high molecular weight hyaluronic acid injections in delaying time to knee surgery. The Journal of Arthroplasty, 32(17), 2952–2957.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ghosh, P., & Guidolin, D. (2002). Potential mechanism of action of intra-articular hyaluronan therapy in osteoarthritis: are the effects molecular weight dependent? Seminars in Arthritis and Rheumatism, 32(6), 10–37.

    Article  CAS  PubMed  Google Scholar 

  13. Euppayo, T., Siengdee, P., Buddhachat, K., Pradit, W., Viriyakhasem, N., Chomdej, S., Ongchai, S., Harada, Y., & Nganvogpanit, K. (2015). Effects of low molecular weight hyaluronan combined with carprofen on canine osteoarthritis articular chondrocytes and cartilage explants in vitro. In Vitro Cellular & Developmental Biology. Animal, 51(8), 857–865.

    Article  CAS  Google Scholar 

  14. Petrella, R. J., Decaria, J., & Petrella, M. J. (2011). Long term efficacy and safety of a combined low and high of osteoarthritis of the knee. Rheumatol. Reports., 3(1), 4.

    Article  CAS  Google Scholar 

  15. Stern, R., Kogan, G., Jedrzejas, M. J., & Šoltés, L. (2007). The many ways to cleave hyaluronan. Biotechnology Advances, 25(6), 537–557.

    Article  CAS  PubMed  Google Scholar 

  16. Cardoso, M. J., Caridade, S. G., Costa, R. R., & Mano, J. F. (2016). Enzymatic degradation of polysaccharide-based layer-by-layer structures. Biomacromolecules., 17(4), 1347–1357.

    Article  CAS  PubMed  Google Scholar 

  17. Tokita, Y., & Okamoto, A. (1995). Hydrolytic degradation of hyaluronic. Polymer Degradation and Stability, 48(2), 269–273.

    Article  CAS  Google Scholar 

  18. Gatej, I., Popa, M., & Rinaudo, M. (2004). Role of the pH on hyaluronan behavior in aqueous solution. Biomacromolecules., 6(1), 61–67.

    Article  CAS  Google Scholar 

  19. Maleki, A., Kjøniksen, A. L., & Nyström, B. (2008). Effect of pH on the behavior of hyaluronic acid in dilute and semidilute aqueous solutions. Macromolecular Symposia, 274(1), 131–140.

    Article  CAS  Google Scholar 

  20. Tømmeraas, K., & Melander, C. (2008). Kinetics of hyaluronan hydrolysis in acidic solution at various pH values. Biomacromolecules., 9(6), 1535–1540.

    Article  CAS  PubMed  Google Scholar 

  21. Šoltés, L., Mendichi, R., Kogan, G., Schiller, J., Stankovská, M., & Arnhold, J. (2006). Degradative action of reactive oxygen species on hyaluronan. Biomacromolecules., 7(3), 659–668.

    Article  CAS  PubMed  Google Scholar 

  22. Caspersen, M. B., Roubroeks, J. P., Liu, Q., Huang, S., Fogh, J., Zhao, R., & Tømmeraas, K. (2014). Thermal degradation and stability of sodium hyaluronate in solid state. Carbohydrate Polymers, 107, 25–30.

    Article  CAS  PubMed  Google Scholar 

  23. Mondek, J., Kalina, M., Simulescu, V., & Pekař, M. (2015). Thermal degradation of high molar mass hyaluronan in solution and in powder; comparison with BSA. Polymer Degradation and Stability, 120, 107–113.

    Article  CAS  Google Scholar 

  24. Dřímalová, E., Velebný, V., Sasinková, V., Hromádková, Z., & Ebringerová, A. (2005). Degradation of hyaluronan by ultrasonication in comparison to microwave and conventional heating. Carbohydrate Polymers, 61(4), 420–426.

    Article  CAS  Google Scholar 

  25. Gura, E., Hückel, M., & Müller, P.-J. (1998). Specific degradation of hyaluronic acid and its rheological properties. Polymer Degradation and Stability, 59(1-3), 297–302.

    Article  CAS  Google Scholar 

  26. Wu, Y. (2012). Preparation of low-molecular-weight hyaluronic acid by ozone treatment. Carbohydrate Polymers, 89(2), 709–712.

    Article  CAS  PubMed  Google Scholar 

  27. il Choi, J., Kim, J. K., Kim, J. H., Kweon, D. K., & Lee, J. W. (2010). Degradation of hyaluronic acid powder by electron beam irradiation, gamma ray irradiation, microwave irradiation and thermal treatment: a comparative study. Carbohydrate Polymers, 79(4), 1080–1085.

    Article  CAS  Google Scholar 

  28. Chen, S., Chen, H., Gao, R., Li, L., Yang, X., Wu, Y., & Hu, X. (2015). Degradation of hyaluronic acid derived from tilapia eyeballs by a combinatorial method of microwave, hydrogen peroxide, and ascorbic acid. Polymer Degradation and Stability, 112, 117–121.

    Article  CAS  Google Scholar 

  29. Simulescu, V., Kalina, M., Mondek, J., & Pekař, M. (2016). Long-term degradation study of hyaluronic acid in aqueous solutions without protection against microorganisms. Carbohydrate Polymers, 137, 664–668.

    Article  CAS  PubMed  Google Scholar 

  30. Laurent, T. C., Ryan, M., & Pietruszkiewicz, A. (1960). Fractionation of hyaluronic acid. The polydispersity of hyaluronic acid from the bovine vitreous body. Biochim. Biophys. Acta - Mol. Cell Res., 42, 476–485.

    Article  CAS  Google Scholar 

  31. Sun, J., Wang, M., Chen, Y., Shang, F., Ye, H., & Tan, T. (2012). Understanding the influence of phosphatidylcholine on the molecular weight of hyaluronic acid synthesized by Streptococcus zooepidemicus. Applied Biochemistry and Biotechnology, 168(1), 47–57.

    Article  CAS  PubMed  Google Scholar 

  32. Schiraldi, C., Andreozzi, L., Marzaioli, I., Vinciguerra, S., D’Avino, A., Volpe, F., Panariello, A., & De Rosa, M. (2010). Hyaluronic acid degradation during initial steps of downstream processing. Biocatalysis and Biotransformation, 28(1), 83–89.

    Article  CAS  Google Scholar 

  33. Cavalcanti, A. D. D., Melo, B. A. G., & Oliveira, R. C. (2018). Recovery and purity of high molar mass bio-hyaluronic acid via precipitation strategies modulated by pH and sodium chloride. Applied Biochemistry and Biotechnology.

  34. Chen, Y. H., & Wang, Q. (2009). Establishment of CTAB turbidimetric method to determine hyaluronic acid content in fermentation broth. Carbohydrate Polymers, 78(1), 178–181.

    Article  CAS  Google Scholar 

  35. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., & Klenk, D. (1985). Measurement of protein using bicinchoninic acid. pdf, Anal. Chem., 150, 76–85.

    CAS  Google Scholar 

  36. S.T. Balke, A.E. Hamielec, B.P. Leclair, S.L. Pearce, Gel permeation chromatography: calibration curve from polydisperse standards, I EC Prod. Res. Dev. 8 (1969) 54–57., 1.

  37. Pires, A. M. B., & Eguchi, S. Y. (2010). The influence of mineral ions on the microbial production and molecular weight of hyaluronic acid. Applied Biochemistry and Biotechnology, 162(8), 2125–2135.

    Article  CAS  PubMed  Google Scholar 

  38. Pan, N. C., Cristina, H., Pereira, B., De Lourdes, M., Flora, A., Vasconcelos, D., Antonia, M., & Colabone, P. (2017). Improvement production of hyaluronic acid by Streptococcus zooepidemicus in sugarcane molasses. Applied Biochemistry and Biotechnology, 182(1), 276–293.

    Article  CAS  PubMed  Google Scholar 

  39. Pires, A. M. B., & Santana, M. H. A. (2011). Rheological aspects of microbial hyaluronic acid. Journal of Applied Polymer Science, 122(1), 126–133.

    Article  CAS  Google Scholar 

  40. Reháková, M., Bakoš, D., Soldán, M., & Vizárová, K. (1994). Depolymerization reactions of hyaluronic acid in solution. International Journal of Biological Macromolecules, 16(3), 121–124.

    Article  PubMed  Google Scholar 

  41. Morris, E. R., Rees, D. A., & Welsh, E. J. (1980). Conformation and dynamic interactions in hyaluronate solutions. Journal of Molecular Biology, 138(2), 383–400.

    Article  CAS  PubMed  Google Scholar 

  42. Ghosh, S., Kobal, I., Zanette, D., & Reed, W. F. (1993). Conformational contraction and hydrolysis of hyaluronate in sodium hydroxide solutions. Macromolecules., 26(17), 4685–4693.

    Article  CAS  Google Scholar 

  43. Bothner, H., & Waaler, T. (1988). Limiting viscosity number and weight average molecular weight of hyaluronate samples produced by heat degradation. International Journal of Biological Macromolecules, 10(5), 287–291.

    Article  CAS  Google Scholar 

  44. Laurent, T. C., Laurent, U. B., & Fraser, J. R. E. (1996). The structure and function of hyaluronan: an overview. Immunology and Cell Biology, 74(2), A1–A7.

    Article  CAS  PubMed  Google Scholar 

  45. Lapčík, L., Lapčík, L., De Smedt, S., Demeester, J., & Chabreček, P. (1998). Hyaluronan: preparation, structure, properties, and applications. Chemical Reviews, 98(8), 2663–2684.

    Article  PubMed  Google Scholar 

  46. Scott, J. E., & Heatley, F. (1999). Hyaluronan forms specific stable tertiary structures in aqueous solution: a 13 C NMR study. Biochemistry., 96(9), 4850–4855.

    Article  CAS  Google Scholar 

  47. Hunter, R. J., Ottewill, R. H., & Rowell, R. L. (1981). Preface. In Zeta potential in colloid science (3rd ed.). Academic Press.

  48. H. Günzler, H.-U. Gremlich, IR spectroscopy: an introduction, Wiley-VCH, 2002. ISBN: 978-3-527-28896-0.

  49. Gilli, R., Kacuráková, M., Mathlouthi, M., Navarini, L., & Paoletti, S. (1994). FTIR studies of sodium hyaluronate and its oligomers in the amorphous solid phase and in aqueous solution. Carbohydrate Research, 263(2), 315–326.

    Article  CAS  PubMed  Google Scholar 

  50. Madejová, J. (2003). FTIR technique in clay mineral studies. Vibrational Spectroscopy, 31(1), 1–10.

    Article  Google Scholar 

Download references


This study was financially supported by Fapesp (São Paulo Research Foundation), grant numbers 2015/23134-8 and 2016/10132-0.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Maria Helena Andrade Santana.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflicts of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

de Melo, B.A.G., Santana, M.H.A. Structural Modifications and Solution Behavior of Hyaluronic Acid Degraded with High pH and Temperature. Appl Biochem Biotechnol 189, 424–436 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: