Antioxidant And Anti-colon Cancer Activity Of Enzymatic Peptide From SweetPotato Protein

In this study, sweet potato proteins were isolated,and then hydrolyzed by different proteases to get sweetpotato protein hydrolyzates, of which the antioxidant activities were investegted and the enzymatic conditions were optimized; Under the optimum conditions,the antioxidative peptides from sweetpotato protein were separation, purification and identification;Meanwhile, the inhibition effect on the proliferation and metastasis of colon cancer cells HT-29for sweetpotato protein hydrolyzates were studied, as well as its mechanism, so to provide a theoretical basis for the development of antioxidant and antitumor health products.The aim of this research is to increase the additional value of sweet potato production,and provide theoretic support for the development of functional peptides foods with antioxidant and antitumor activities.Sweetpotato protein hydrolysates were prepared by six enzymes (alcalase, proleather FG-F, AS1.398,neutrase, papain and pepsin). The antioxidant activities and protective effect against oxidative DNA damage of sweetpotato protein hydrolysates were investigated. Alcalase hydrolysates exhibited the highest hydroxyl radical scavenging activity (1C501.74mg/mL) and Fe2+-chelating ability(IC501.54mg/mL)(P<0.05). Compared to other five hydrolysates, the hydrolysates obtained by alcalase had the most abundant<3kDa fractions. In addition,<3kDa fractions of alcalase hydrolysates showed the highest antioxidant activities and protective effects against DNA damage through both scavenging hydroxyl radicals and chelating Fe2+ion.The effect of pre-heating temperature and time on enzymolysis characteristics of sweetpotato protein by alcalase was investigated.On the basis of pre-heating treatment of sweetpotato protein, the response surface methodology was employed to study the preparation of antioxidative peptides from sweetpotato protein.The effects of enzyme to substrate concentration ratio, pH value and temperature on hydroxyl radicals scavenging activity and Fe2+-chelating ability were investigated. Experimental data were fitted to two quadratic polynomial models using multiple regression analysis.The validity of the model and the interactions of impact factors were analyzed.The results showed that the optimum conditions for preparing antioxidative peptides from sweetpotato protein were as follows:substrate concentration5%,enzyme to substrate concentration ratio4%, pH8.0, temperature57℃and time2h. Under the optimum conditions, the hydroxyl radicals scavenging activity and Fe2+-chelating ability were40.03±0.63%and74.08±1.53%.Sweetpotato protein hydrolysates obtained under the optimum conditions were separated into four fractions by ultrafiltration, and low-molecular-weight fraction F-IV (<3kDa) exhibited the strongest hydroxyl radical scavenging activity and Fe2+-chelating ability, which was59.74%and82.27%at concentration of3.0mg/mL, respectively. Fraction F-IV was then purified with size exclusion chromatography followed by reverse-phase high-performance liquid chromatography, by which two fractions IV-5c and IV-5i were obtained showing the highest hydroxyl radical scavenging activity and Fe2+-chelating ability,33.22%and45.01%at0.3mg/mL, separately. The amino acid sequence determination by LC-MS/MS identified five peptides in antioxidative fractions IV-5c and IV-5i,of which Thr-Tyr-Gln-Thr-Phe, Ser-Gly-Gln-Tyr-Phe-Leu and Tyr-Met-Val-Ser-Ala-Ile-Trp-Gly matched the sequence of sporamin A, while Tyr-Tyr-Ile-Val-Ser and Tyr-Tyr-Asp-Pro-Leu matched the sequence of sporamin B.Enzymatic peptides from sweetpotato protein exhibited certain inhibition effect on the proliferation and metastasis of colon cancer cells HT-29.<3kDa fractions of sweetpotato protein peptides could induced the expression of cyclin-dependent kinase inhibitory factor p21in HT-29cells, thereby blocking cell cycle progression, and promoted apoptosis in HT-29cells through activation of mitochondrial pathways as evidenced by increased expression of Bax and decreased expression of Bcl-2.At the same time,<3kDa fractions could significantly reduce the activity and decreased expression of uPA in HT-29cells, thereby inhibiting cell migration.