Reversible pressure denaturation occurs at pressures below 300 MPa, and higher pressures are needed to cause irreversible

denaturation of the protein. High pressure also causes deprotonation of charged groups and the disruption of salt bridges and hydrophobic bonds, resulting in conformational changes and protein denaturation under high pressure >300 MPa [32]. Most enzymes also lose their catalytic activities with pressure exceeds 300 MPa, resulting in changes in the substrate property or producing rate-limiting conformational changes. In this study, therefore, we have examined the optimal conditions of HHP treatment (<100 MPa) combined with enzymatic hydrolysis to extract CS from fresh antler cartilage. A high pressure (100 MPa) used in this study noticeably accelerates papain catalytic activity. Because HHP technology has been commercially available for many years for industrial-scale applications, Staurosporine it is worthwhile to investigate other enzymes for digesting

various sources of cartilage components. Antler CS fractions treated by HHP-EH process were examined for their capabilities to interact with hyaluronic acid to form high molecular weight aggregates (Fig. 6). The chromatography of the antler CS fraction following incubation with exogenous hyaluronic acid showed an absence of the peak from the column (Fig. 6a). However, the bovine articular cartilage aggrecan interacted with hyaluronic acid, which is evidenced by the appearance of a peak excluded from Sepharose CL-2B (Fig. 6b), indicating an interaction of the CS fraction with hyaluronic acid. The binding ability shows that aggrecan possesses the G1 domain containing the hyaluronic acid binding Doxorubicin molecular weight region, which is located at the N-terminus [22], and constitutes about one-quarter to Dynein one-third of the total core protein [15]. The antler CS fraction shows a lack of the G1 domain specific to hyaluronic acid with the formation of macromolecular aggregates [22]. Although antlers have been used as a Chinese medicine for many years, only limited

information is available on the chemical compositions, bioactive ingredients, extraction methods and pharmacological effects [28] and [30]. We have also showed the chemical analyses of high hydrostatic pressure and papain digests from antler cartilage (Table 2). Increasing evidence indicates that acidic polysaccharides, which are widely distributed in animals, possess potential antioxidant activity by scavenging free radicals [2]. Although the antler CS fraction was not superior to ascorbic acid and BHT for DPPH scavenging activity, its antioxidative activity was much higher than that of bovine and shark CS, indicating greater potential as antioxidant components, because much attention has been given to antioxidants in preventing free radical-induced damage. The difference in the DPPH radical scavenging activity of the HHP-EH-treated antler CS from bovine and shark CS requires further investigation.