Theses and dissertations (Applied Sciences)
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Item Anticancer activity of silver nanoparticles embedded in porous starch as a potential delivery system(2024-05) Mohan, Naaznee; Mellem, John JasonSilver nanoparticles have been proven to have anticancer abilities but they have been known to agglomerate and become toxic. Therefore, various studies have been conducted to explore ways of preventing aggregation using biopolymers such as starch. This study makes use of Lablab purpureus (hyacinth bean) porous starch to biosynthesize and encapsulate silver nanoparticles and then test its anticancer potential. Porous starches were produced from hyacinth bean using three different techniques. These were compared against the native starch with silver nanoparticles, then synthesized and encapsulated using the porous starch. In comparison to the native starch, the porous starches made through solvent exchange and enzyme hydrolysis had similar outcomes with granules exhibiting pores, as shown by the structural and chemical characteristics. The lack of pasting properties and extremely distinct chemical and structural graphs of the porous starch, produced by freeze-thaw procedures, may be related to the presence of mercaptosuccinic acid. It was decided to employ porous starch made by solvent-exchange (SE) for the manufacture of silver nanoparticles as it contained resistant starch. Nanoparticles were produced using the porous starch from solvent-exchange, characterised and tested for their anticancer potential. Silver nanoparticles were indicative of a colour change from clear to brown, as well as, the characteristic peak at 425 nm for silver nanoparticle formation. Silver nanoparticles were implanted into porous starch at a size of around 50 nm, as further evidenced by the particle size distribution and TEM images of spherical granules with dark spots within. The zeta potential for the silver nanoparticles was -34 mV, thereby indicating that aggregation was minimized and particles were stable. The nanoparticles demonstrated less cytotoxicity in the human colon (CACO) and cervical (HELA) cancer cell lines, but more inhibition in the human breast (MCF-7) cancer cell line than the positive control camptothecin. The human muscle (C2C12), normal cell line's capacity to sustain cell viability for silver nanoparticles demonstrated that AgNP were not toxic. However, to maximize the potential of the silver nanoparticles implanted in porous starch, more research is necessary.Item 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.