Theses and dissertations (Applied Sciences)
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Item Anticancer and anti-reactive oxygen species activity of bioactive peptides isolated from vigna unguiculata(2024-05) Ramsookmohan, Sonaal; Mellem, John Jason; Dwarka, DepikaCancer is a major cause of death globally and continues to escalate with current anticancer drugs associated with severe side effects and resistance driving the need for safer alternative therapeutics. Food proteins, from legumes, are a source of bioactive peptides and studies revealed that they are associated with various therapeutic properties. Cowpea (Vigna unguiculata) is an underutilized nutritious legume crop with promising potential due to its documented protein profile. Therefore, this study evaluated the in vitro anticancer effect of V. unguiculata peptides derived from alcalase and flavourzyme. Physicochemical properties such as water and oil absorption capacities, emulsifying properties, sub-unit composition, amino acid composition among others, were also assessed. Peptides were also evaluated for their antioxidant activity using superoxide radical scavenging, 1,1-diphenyl-2-picrylhydrazil (DPPH) and 2,2’-azino-bis (3 ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays as well as for their apoptotic potential using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), reactive oxygen species (ROS) and caspase 3/7 assays on cancerous (Caco-2 and MCF 7) and healthy (C2C12) cell lines. From results obtained it was observed that the foam capacity for the peptides derived from alcalase and flavourzyme were 78.34 and 82.39%, respectively, which was noted to be significantly different. The physicochemical properties determined their potential application in food industries. Glutamic acid was the most abundant amino acid in all samples while methionine was noted to be the least concentrated amino acid in the flour and alcalase derived sample while proline was the least concentrated in the flavourzyme sample. Results from this study suggest that cowpea samples have antioxidant capabilities with enzymatic hydrolysis contributing to a higher capacity compared to that of raw flour flour samples. From the cowpea flour, the peptide sample derived from alcalase demonstrated the highest DPPH free radical scavenging activity (70.88-80.47%), followed by flavourzyme (67.27-75.84%), while the raw flour sample showed the lowest activity (24.28-66.17%). The ABTS scavenging capacity of the alcalase peptide was in the range of 35.26-85.92%. The MTT cytotoxicity assay revealed that the cowpea peptides and camptothecin showed different sensitivities on the MCF-7 cell lines. The IC50 values of flavourzyme peptide, alcalase peptide and camptothecin were 0.07, 0.09 and 0.07 µg/mL respectively. Cell viability of the cowpea peptides and camptothecin (control) on the Caco-2 cells varied with the different concentrations. Alcalase and flavourzyme samples had IC50 values of 0.15 and 0.11 µg/mL respectively. The apoptotic potential of the peptides was further shown by the caspase 3/7 activity. From the results in this study, it can be ascertained that the cowpea peptides have potential as an anticancer therapeutic agent. Further research is necessary to determine mechanism of action and to conduct in vivo evaluations of these peptides using animal modelsItem Antioxidant and anticancer properties of bioactive peptides from Lablab purpureus(2023-05) Sipahli, Shivon; Mellem, John JasonCancer can be described as a non-communicable disease that develops from defective cells in the human body and grows uncontrollably. Globally in 2020, statistics revealed that the disease had affected approximately 19.3 million people. With about 51% of these cases resulting in death. Cancer treatments usually comprise surgery, chemotherapy, radiotherapy, or a combination of the three. Traditional therapies such as chemotherapy and radiotherapy drugs are effective at shrinking tumours. However, a key disadvantage is that these drugs are unable to distinguish between cancerous and healthy cells. Subsequently, the human body experiences many adverse side effects such as hair loss, vomiting, lowered immunity, and a general deterioration of health. Drug resistance and rejection are also major disadvantages of these traditional therapies. Alternative therapies are required to mitigate these drawbacks. The vital factor to consider for alternative treatments should be to selectively target cancer cells thereby alleviating the unwanted side effects. Compounds derived from non-toxic edible plants have shown to have bioactive potential. These plants are regarded as non-toxic to the human body therefore they would be able to target the tumour cells alone. Plant compounds also provide additional protection such as their antioxidant abilities and apoptotic potential. Evidence suggests that bioactive peptides derived from legumes can act as both anticancer agents and strong antioxidants. This study investigated the bioactive potential of peptides derived from Lablab purpureus. This investigation began by assessing the antioxidant capacity (2,2-diphenyl-1-picrylhydrazyl-hydrate (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic (ABTS), superoxide radical scavenging and Ferric Reducing Antioxidant Power (FRAP) assays) and antiproliferative abilities (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) of L. purpureus protein isolate and hydrolysates (alcalase, trypsin and pepsin). The hydrolysate and fractions of interest were selected based on the MTT assay with the pepsin hydrolysate selected for further apoptotic studies (caspase-3 and -7, and annexin V-PI). Thereafter, the pepsin hydrolysate was fractionated by ultrafiltration (molecular weight cut-off: <1, 3, 5, 10, >10 kDa). The 3 kDa fraction was further fractionated by RP-HPLC. Five peaks appeared on the chromatogram, however, fraction 2 was selected, for apoptotic investigations (caspase -3 and -9, p53 and annexin V-PI). Antioxidant studies are a good measure of the isolate or hydrolysate's ability to perform as a bioactive compound. The 50% inhibitory concentration (IC50) observed for the respective antioxidant studies showed the radical scavenging ability of the isolate and hydrolysates to be 1.81-4.47 mg/mL (DPPH), 1.73-2.42 mg/mL (ABTS), 1.36-4.4 mg/mL (superoxide radical scavenging) and 19.20-21.94 mg/mL (FRAP). Anticancer activity was substantiated by the peptides' ability to induce apoptosis. The pepsin hydrolysate was selected using the MTT assay (IC50 values of A549, 119.6; MCF7, 9.80 and HEK293, 13.86 µg/mL). Pepsin hydrolysate inhibited cancerous cells (A549 and MCF-7) while causing minimal damage to healthy cells (HEK293). Thereafter apoptotic markers, caspase 3/7 and annexin V-PI were quantified. Visualisation of cells in different stages of apoptosis was investigated by Annexin V-PI staining quantified by flow cytometry. During early apoptosis; A549, 42%; MCF-7, 17%; HEK293, 34%. Caspase 3/7 assay verified that the pepsin hydrolysate caused an increase in apoptotic activity. Caspase-3 and -9 activity of cells, determined by ELISA showed that Fraction 2 treated cancer cells (A549 - 0.067 ng/mL, 21.966 ng/mL, and MCF-7 - 0.137 ng/mL, 0.205 ng/mL respectively) had a greater caspase concentration over camptothecin (A549 - 0.029 ng/mL, 20.486 ng/mL and MCF-7 - 0.051 ng/mL, 0.112 ng/mL respectively). Tumour suppressor protein, p53, acts as a protective mechanism by initiating apoptosis in ‘suspicious’ cells. The A549 cell line showed the greatest p53 expression compared to MCF-7 and HEK293. Increased p53 can regulate signalling pathways leading to targeted apoptosis. Finally, annexin V-PI confirmed that Fraction 2 did induce apoptosis in the cells (cells in early apoptosis, A549, 85%; MCF-7, 90%; HEK293, 94%). Results from this study have shown that peptides derived from L. purpureus (specifically fraction 2) have potential anticancer abilities which may be attributed to their antioxidant and apoptotic abilities.