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Title: Antioxidant and anticancer properties of bioactive peptides from Lablab purpureus
Authors: Sipahli, Shivon 
Issue Date: May-2023
Cancer 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
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.
Submitted in complete fulfilment for the Degree of Doctor of Food Science and Technology Durban University of Technology, Durban, South Africa, 2023.
Appears in Collections:Theses and dissertations (Applied Sciences)

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