Structural characterization and biological properties of degradation byproducts from hyaluronan after acid hydrolysis

The generation of degradation byproducts from hyaluronan during its acid hydrolysis was studied. The degradation byproducts were removed from reaction mixture by ultrafiltration, followed by ionex separation and purification on a reversed-phase HPLC column. Isolated fractions were structurally analyzed by NMR and mass spectrometry. Results indicated that when acid hydrolysis is performed at 100 °C for 24 h, about 0.7% of high molar mass hyaluronan (1.2 × 106 g mol−1) was converted into furan-like derivatives. Other major degradation byproducts present in 0.2 wt% amounts were identified as derivatives of cyclopentanone. Cell viability tests indicated that most of the degradation byproducts have negative effect on cell growth. These tests were performed using mouse fibroblast cell line 3T3 and human keranocytes cell line HaCaT. For this reason, it is important to evaluate the amount of degradation byproducts in low molar mass hyaluronan produced by acid hydrolysis, mainly when these products are used in pharmaceutical and cosmetic applications. During this study it was found out that the unwanted degradation byproducts can be completely removed when an ultrafiltration step is introduced after acid hydrolysis prior hyaluronan isolation.