The lyophilized skin was soaked into 0.5 M acetic acid (1:3 weights per volume) for 3 days and the extracts were centrifuged at 20,000 × g for 1 h at 4 ◦C. The supernatant was collected and the residue was re-extracted by same procedure. The supernatant was mixed and desalted out by adding NaCl to a final concentration of 0.8 M and followed by precipitation of collagen by the addition of NaCl (final concentration of 2.3 M) at a neutral pH (0.05 M Tris–HCl, pH 7.5). The precipitates were collected and re-dissolved in 0.5 M acetic acid and dialyzed against 0.1 M acetic acid and distilled water for 2 days until the neutral pH was obtained. The dialyzed sample was lyophilized and referred to ASC.