Characterization of enzymatically digested hyaluronic acid using NMR, Raman, IR, and UV-Vis spectroscopies

Hyaluronic acid (HA) is a linear polysaccharide formed from disaccharide units containing N-acetylglucosamine and glucuronic acid. When HA was digested with the enzyme hyaluronidase, a double bond is formed. It is known that this double bond forms a complex (radical scavenger) with the radicals (ROO, HO) during UV irradiation, and reduced the toxicity of the radicals before they are absorbed in the human skin. Therefore, the characterization of the double bond formed after the enzymatic digestion of HA is very important. In this study, 1H NMR, 13C NMR, Raman, infrared (IR), and UV-Vis spectroscopies were used for characterization of the double bond of HA after enzymatic digestion. HA derivatives in shape of films were tested using Raman and infrared (IR) spectroscopies and the wavenumber of the double bond and some other assignment were determined. The 1H and 13C NMR spectra were measured for HA derivatives in D(2)O solutions. The chemical shifts and coupling constant of 1H and 13C were assigned to the CH=C fragment. The relative amount of olefinic proportion in the mixture was obtained from 1H and 13C NMR spectra. The spectroscopy measurement showed an increase in the double bond amount with increasing enzymatic digestion time.