Anticancer and antioxidant potential of Amaranthus cruentus protein and its hydrolysates

Abstract

Traditionally, amaranth has been acknowledged to possess vital pharmacological properties with anticancer peptides having been found in Amaranthus cultivars. However, limited knowledge is available over the use of pepsin and alcalase enzymes to form hydrolysates. Thus, this study was aimed at comparing the in vitro anticancer effect of Amaranthus cruentus (grain) protein isolate and hydrolysates using alcalase, trypsin and pepsin. The safety of hydrolysates was investigated using the Ames mutagenicity and Brine Shrimp Lethality assay. Protein hydrolysates were thereafter investigated for their antioxidant potential using the FRAP, ABTS and DPPH assays. Subsequently, the protein hydrolysates were tested for their anticancer and apoptotic potential. The MTT assay was conducted to evaluate the cytotoxic potential of the protein hydrolysates using the HEK 293 (non-cancerous), A549 (cancerous) and MCF-7 (cancerous) cell lines. After that, morphological alterations were examined using the acridine orange and ethidium bromide double stain. Following this, the Annexin V apoptotic detection kit was used to quantify apoptosis together with the Glomax Caspase 3/7 kit to detect changes in the cell cycle Results show A. cruentus isolate and hydrolysates had no mutagenic response against Salmonella typhimurium TA 98 and TA 100 strains. The tested samples did not induce any significant increase in the death percentage of Artemia spp. in comparison to potassium dichromate (control). DPPH assay revealed that the hydrolysed samples had an enhanced scavenging activity compared to the unhydrolyzed sample, with pepsin having the greatest IC50 of 23.06 µg/ml. Amaranthus cruentus isolate (IC50 17.57 µg/ml) was a greater scavenger of the Fe+ ions compared to the control glutathione (IC50 79.81 µg/ml). For ABTS, all hydrolysates had a greater antioxidant scavenging potential compared to the isolate. The MTT cytotoxicity assay revealed that the isolate produced a greater cytotoxic effect on the MCF-7 and A549 cell line when compared to the control (camptothecin). For the non-cancerous cell line (HEK 293), trypsin hydrolysate had the highest toxicity. Apoptotic results revealed that trypsin hydrolysate was the most effective compared to the isolate, which was confirmed from morphological and Caspase 3/7 results. It may be concluded from the findings of this research that hydrolysates from food protein isolates have the potential for use as possible anticancer therapeutics. However, more research needs to be conducted to determine the peptides responsible for anticancer activity as well as the possible mechanism of action.