Effects of slit divergent ultrasound and enzymatic treatment on the structure and antioxidant activity of arrowhead protein

The arrowhead has attracted great research interest for their potential applications in pharmacy, food and biomedical areas. However, no information is reported about the nature and structure of the arrowhead protein (AP). Herein, effects of slit divergent ultrasound (28, 33, 40 KHz frequencies at 30-50 °C) and enzymatic (pepsin, trypsin, and alcalase) treatment on structure of AP were studied. In addition, changes in antioxidant activity of AP treated with ultrasound and enzymes were measured by chemical and cellular-based assays. The results showed that ultrasound treatment had considerable impact on the structure of AP and increased the susceptibility of AP to pepsin, trypsin and alcalase proteolysis. The changes in UV-Vis spectra, free sulfhydryl (SH) and disulfide bonds (SS) groups indicated that the structure of AP unfolded after ultrasound treatment. Besides, intrinsic fluorescence intensity of AP was increased by ultrasound treatment and then decreased after following enzymatic treatment. The circular dichroism (CD) analysis showed that ultrasound and enzymatic treatment decreased α-helix, β-turn of content of AP. However, the β-sheet and random coil content of AP increased. Interestingly, the AP after ultrasound and enzymatic treatment showed significant higher anti-oxidative activity in RAW 264.7 cells (p < 0.05) in comparison with control. In conclusion, the slit divergent ultrasonic provides a powerful endorsement for increasing the proteolysis of AP. Moreover, the improvement of the antioxidant activity of AP enzymatic hydrolysates provides a foundation of developing new type of plant-derived antioxidant peptides application.