In vitro antioksidacijska, citotoksična i antidijabetička aktivnost hidrolizata proteina iz Reevesove barske kornjače (Chinemys reevesii)

Research background. Cardiovascular diseases and diabetes are the biggest causes of death globally. Bioactive peptides derived from many food proteins using enzymatic proteolysis and food processing have a positive impact on the prevention of these diseases. The bioactivity of Chinese pond turtle muscle proteins and their enzymatic hydrolysates has not received much attention, thus this study aims to investigate their antioxidant, antidiabetic and cytotoxic activities. Experimental approach. Chinese pond turtle muscles were hydrolysed using four proteolytic enzymes (Alcalase, Flavourzyme, trypsin and bromelain) and the degrees of hydrolysis were measured. High-performance liquid chromatography (HPLC) was conducted to explore the amino acid profiles and molecular mass distribution of the hydrolysates. The antioxidant activities were evaluated using various in vitro tests, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydroxyl radical scavenging activity, reducing capacity, chelating Fe2+ and lipid peroxide inhibition activity. Antidiabetic activity was evaluated using α-amylase inhibition and α-glucosidase inhibition assays. Besides, cytotoxic effect of hydrolysates on human colon cancer (HT-29) cells was assessed. Results and conclusions. The amino acid composition of the hydrolysates revealed higher mass fractions of glutamic, aspartic, lysine, hydroxyproline and hydrophobic amino acids. Significantly highest inhibition of lipid peroxidation was achieved when hydrolysate obtained with Alcalase was used. Protein hydrolysate produced with Flavourzyme had the highest radical scavenging activity measured by DPPH (68.32%), ABTS (74.12%) and FRAP (A700 nm=0.300) assays, α-glucosidase (61.80%) inhibition and cytotoxic effect (82.26%) on HT-29 cell line at 550 µg/mL. Hydrolysates obtained with trypsin and bromelain had significantly highest (p<0.05) hydroxyl radical scavenging (92.70%) and Fe2+ metal chelating (63.29%) activities, respectively. The highest α-amylase (76.89%) inhibition was recorded when using hydrolysates obtained with bromelain and Flavourzyme. Novelty and scientific contribution. Enzymatic hydrolysates of Chinese pond turtle muscle protein had high antioxidant, cytotoxic and antidiabetic activities. The findings of this study indicated that the bioactive hydrolysates or peptides from Chinese pond turtle muscle protein can be potential ingredients in pharmaceuticals and functional food formulations.Pozadina istraživanja. Kardiovaskularne bolesti i dijabetes najčešći su uzroci smrti na svijetu. Bioaktivni peptidi dobiveni proteolizom i preradom hrane imaju pozitivan učinak na prevenciju tih bolesti. Biološka aktivnost proteina iz mišića Reevesove barske kornjače i njihovih hidrolizata nije dovoljno istražena, stoga je svrha ovoga rada bila ispitati njihovu antioksidacijsku, antidijabetičku i citotoksičnu aktivnost. Eksperimentalni pristup. Mišići Reevesove barske kornjače hidrolizirani su pomoću proteolitičkih enzima (Alcalase, Flavourzyme, tripsin i bromelain), te su mjereni stupnjevi hidrolize proteina. Aminokiselinski sastav i distribucija molekularne mase hidrolizata ispitani su pomoću visokodjelotvorne tekućinske kromatografije. Antioksidacijska aktivnost određena je različitim testovima in vitro, uključujući sposobnost uklanjanja 1,1-difenil-2-pikrilhidrazila (DPPH), 2,2’-azino-bis(3-etilbenzotiazolin-6-sumporne kiseline) (ABTS) i hidroksil radikala, keliranja Fe2+ i inhibicije lipidne peroksidacije. Antidijabetička aktivnost ispitana je testovima inhibicije α-amilaze i α-glukozidaze. Osim toga, analiziran je citotoksični učinak hidrolizata na stanice tumora debelog crijeva (HT-29). Rezultati i zaključci. Analizom aminokiselinskog sastava hidrolizata pronađeni su veći maseni udjeli glutaminske i asparaginske kiseline, lizina, hidroksiprolina te hidrofobnih aminokiselina od onih u nehidroliziranim proteinima. Hidrolizat proteina dobiven pomoću proteolitičkog enzima Alcalase bitno je inhibirao peroksidaciju lipida. Pri koncentraciji od 550 µg/mL, hidrolizat proteina dobiven pomoću enzima Flavourzyme imao je najveću sposobnost uklanjanja slobodnih radikala mjerenu pomoću DPPH (68,32 %), ABTS (74,12 %) i FRAP (A700 nm=0,300) metoda, inhibicije α-glukozidaze (61,80 %) te najveći citotoksični učinak na stanične linije HT-29 (82.26 %). Hidrolizat proteina dobiven pomoću tripsina imao je znatnu (p