A novel peptide derived from Zingiber cassumunar rhizomes exhibits anticancer activity against the colon adenocarcinoma cells (Caco-2) via the induction of intrinsic apoptosis signaling

Abstract

This paper presents the initial exploration of the free radical scavenging capabilities of peptides derived from protein hydrolysates (PPH) obtained from Zingiber cassumunar rhizomes (Phlai). To replicate the conditions of gastrointestinal digestion, a combination of pepsin and pancreatin proteolysis was employed to generate these hydrolysates. Subsequently, the hydrolysate underwent fractionation using molecular weight cut-off membranes at 10, 5, 3, and 0.65 kDa. The fraction with a molecular weight less than 0.65 kDa exhibited the highest levels ABTS, DPPH, FRAP, and NO radical scavenging activity. Following this, RP-HPLC was used to further separate the fraction with a molecular weight less than 0.65 kDa into three sub-fractions. Among these, the F5 sub-fraction displayed the most prominent radical-scavenging properties. De novo peptide sequencing via quadrupole-time-of-flight-electron spin induction-mass spectrometry identified a pair of novel peptides: Asp-Gly-Ile-Phe-Val-Leu-Asn-Tyr (DGIFVLNY or DY-8) and Ile-Pro-Thr-Asp-Glu-Lys (IPTDEK or IK-6). Database analysis confirmed various properties, including biological activity, toxicity, hydrophilicity, solubility, and potential allergy concerns. Furthermore, when tested on the human adenocarcinoma colon (Caco-2) cell line, two synthetic peptides demonstrated cellular antioxidant activity in a concentration-dependent manner. These peptides were also assessed using the FITC Annexin V apoptosis detection kit with PI, confirming the induction of apoptosis. Notably, the DY-8 peptide induced apoptosis, upregulated mRNA levels of caspase-3, -8, and -9, and downregulated Bcl-2, as confirmed by real-time quantitative polymerase chain reaction (RT-qPCR). Western blot analysis indicated increased pro-apoptotic Bax expression and decreased anti-apoptotic Bcl-2 expression in Caco-2 cells exposed to the DY-8 peptide. Molecular docking analysis revealed that the DY-8 peptide exhibited binding affinity with Bcl-2, Bcl-xL, and Mcl-1, suggesting potential utility in combating colon cancer as functional food ingredients.