*3.5. RP-HPLC*

The purity of extracted collagen was confirmed by RP-HPLC as per our earlier method [34]. In brief, a collagen sample dissolved in acetic acid was loaded on a ZORMAX 300 SM-C18 (Agilent, Shanghai, China) column of LaChrom Elite HPLC system (Hitachi, Tokyo, Japan) equipped with an

L-2130 pump, L-2300 column oven, L-2400 UV detector and L-2200 auto-sampler; and eluted with a two gradient solvent system using acetonitrile and trifluoroacetic acid with the peak absorbance at 230 nm using the HITACHI D-2000 Elite workstation software (Hitachi, Tokyo, Japan).
