Free radical scavenging and antiproliferative activities of peptide from hygroscopic earthstar astraeus hygrometricus

Abstract

Mushrooms have been used as food and for therapeutic purpose for decades. Indeed, various compounds derived from them have potential biological activities. The present study aims to explore the free radical scavenging properties of peptides derived from the edible mushroom Astraeus hygrometricus. In this work, peptides from A. hygrometricus protein hydrolysates attainable with microbial proteases (Alcalase®, Flavourzyme® and Neutrase®) were prepared and their antioxidant activities were determined. Peptide fractions derived from A. hygrometricus hydrolyzed by 1% Alcalase®, showed the highest DPPH and ABTS radical scavenging activities with IC50 values of 13.40±0.48 and 5.53±0.59 µg/mL, respectively. Peptide fractions were obtained using molecular weight cut-offs of 10, 5, 3 and 0.65 kDa membranes and their antioxidant properties were further analyzed. Among the fractions, MW < 0.65 kDa (F5 fraction) exhibited high levels of free radical scavenging activities towards DPPH and ABTS with IC50 values of 4.84±0.26 and 1.49±0.39 µg/mL, respectively. The F51 fraction from the Superdex® 75 column was identified as having the highest scavenging activities on DPPH and ABTS radicals with IC50 values of 32.47±1.16 and 15.58±0.73 µg/mL, respectively. Furthermore, the F51 fraction could protect hydroxyl radical-induced DNA damage as shown in pKS, pUC19, and pBR322. In addition, fraction F51 was used to determine antiproliferative activity. The results showed that purified peptide fraction F51 was not cytotoxic on normal and cancer cell lines, and the lung cancer cell line (Chago-k1 cell) had the highest antiproliferative activity. Moreover, fraction F51 was used for determining the induction of apoptosis that used flow cytometry and the caspase 3, 8 and 9 activities analysis method. The results indicate that purified peptide fraction F51 could induce the highest apoptotic cells and caspase 3, 8 and 9 activities on Chago-k1 cell at 72 hours. Furthermore, the peptide fraction F51 was purified using RP-HPLC and sub-fractions F51-1 to F51-5 collected. The result was found to possess the highest antioxidant activity on fraction F51-5. Finally, the amino acid sequences of sub-fractions F51-1 to F51-5 were identified using MS/MS (ESI-Q-TOF). The amino acid sequences of the purified peptide fractions F51-1 to F51-5 in respective order were as follows: Val-Thr-Thr-His-Lys-Thr-Val-Thr-Lys-His (VTTHKTVTKH, 1152 Da), Met-Pro-His-His-Tyr-Thr-Ile-Phe-Ala-Leu-Gly-Thr-Gln-Ser-Arg-Pro-Ser (MPHHYTIFALGTQSRPS, 1942 Da), Pro-Ser-Arg-Gly-Ser-Glu-Arg-Ala-Arg-Ala (PSRGSERARA, 1086 Da), Thr-Leu-Ser-Asn-Leu-Gly-Leu-Val-Leu-Val-His (TLSNLGLVLVH, 1165 Da) and Leu-Thr-Asn-Ser-Pro-Trp-Ala-His-Ala (LTNSPWAHA, 996 Da). Hence, the purified peptide fraction F51 could be used as a natural antioxidant for drug development.