Faculty of Applied Sciences
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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.Item Laccase-mediated biotransformation of phenolic compounds for the synthesis of new antioxidants(2020) Mazibuko, Bodine; Kudanga, TukayiThe increased incidences, mortality rate and economic impact of noncommunicable diseases (e.g. high blood pressure and diabetes) associated with oxidative stress, have led to the higher demand for antioxidant supplements for their prevention. The use of naturally occurring antioxidants is becoming a more attractive option due to the health risks associated with synthetic antioxidants. Phenolic compounds from plants have been shown to have antioxidant properties with the potential to be used as substitutes to synthetic antioxidants. However, monomeric phenolic compounds have several short comings such as low bioavailability, poor solubility, and low antioxidant capacity while some have pro-oxidant properties at high concentrations. Hence there has been increasing research focused on the biotransformation of these phenolic antioxidants through enzymatic oligomerisation to higher molecular weight compounds with improved antioxidant capacity and stability. Of the investigated enzymes, laccases have shown the most promise owing to their green catalytic properties. Their reaction mechanism involves the use of molecular oxygen as a co- substrate in oxidising phenolic compounds to corresponding radicals, with water as the only by- product. This study focused on the synthesis of antioxidants with enhanced antioxidant capacity using a laccase from Trametes pubescens as biocatalyst. To establish the potential of the phenolic compounds for use as substrates for the coupling reactions, a preliminary screening process was done. Guaiacol, caffeic acid, vanillic acid, eugenol, catechol, gallic acid, ferulic acid and quercetin hydrate were identified as suitable substrates for the laccase enzyme. However, only products from eugenol, coumaric acid and quercetin could be isolated, hence coupling reactions were carried out using these substrates in monophasic systems. Reaction products were monitored using thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Purification was carried out using preparative TLC and characterisation using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). The antioxidant capacities of reaction products were determined using ABTS (2,2’-Azinobis 3- ethylbenzthiazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric- reducing antioxidant power) assays. Quercetin hydrate oxidation produced one product which was purified and characterised. The product had an Rf of 0.68, tR 13.567 and m/z 601 in negative mode, indicating that it was a dimeric form of quercetin. Oxidation of ρ-coumaric acid resulted in the production of two products designated P1 (Rf 0.47) and P2 (Rf 0.42). Further characterisation was done on product P2 since product P1 could not be successfully purified. P2 had a retention time of 11.295 and m/z 325, indicating that it was a dimer of ρ-coumaric. The ρ-coumaric dimer had an enhanced antioxidant capacity, approximately 2-fold, 3-fold and 6-fold higher compared to the substrate, as demonstrated by the ABTS, DPPH and FRAP assays, respectively. A symmetrical 5-5 eugenol dimer (m/z 325, [M] =326), bis-eugenol, was produced from eugenol oxidation. Maximum product formation (50% yield) was obtained in a monophasic system with 40% v/v dioxane as co-solvent after incubation for 18 h. The bis- eugenol dimer had an improved antioxidant capacity of up to three and four times that of eugenol as demonstrated by the ABTS and DPPH assays, respectively. In conclusion, two dimers with high antioxidant capacity were successfully produced, purified and characterised. The study has demonstrated the potential of the T. pubescens laccase as a catalyst for the synthesis of phenolic compounds with enhanced antioxidant capacity.Item An investigation of the voltammetric behaviour of antioxidants in flavonoids(2020-04) Ramsarup, Lee-Ann; Bisetty, Krishna; Kanchi, SuvardhanThe two case studies in this work involve the development and fabrication of an electrochemical biosensor using various enzymes for the evaluation of the electrochemical responses, relating to the total phenolic (TP) content and the antioxidant activities in wine and tea samples respectively. The modification of the glassy carbon electrode (GCE) was carried out using green apple as an enzymatic source of polyphenol oxidase and laccase enzyme. The experimental variables were optimized using the Box-Behnken experimental design as a predictive model, for a better understanding of the parameters and their interaction responses with each other during an electrochemical analysis. This multivariate optimization method is based on a factorial design, where the three most influential factors include the electrolyte pH, the deposition time (td) and the scan rate (sr). The design was run in a single block fashion while random order of experiment was selected to provide greater protection against the effects of outlying variables. The optimized results obtained yielded the most suitable conditions for the determination of the TP content in wine samples. They were selected as follows: phosphate buffer of pH 7.65 as supporting electrolyte, td 29.8 s and sr 25.0 mV/s respectively. The method was optimized for the current signal at a deposition potential of 0.2 V and within an oxidation potential of -0.2 V to 0.6 V. Good analytical responses were obtained with apple sensors for the detection of TP content in wine samples, with a higher concentration in red wines than in white wines. Differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were used to establish and interpret the redox mechanisms of flavonoids present in alcoholic and non-alcoholic beverages. The sensor responses were evaluated by first, investigating the changes in the total phenolic (TP) content in wine samples using catechin as a standard. Thereafter, the electrochemical behaviour of rutin and ascorbic acid, antioxidant capacities (trolox reagents) were established in tea samples, yielding a positive linear correlation between the trolox equivalent antioxidant capacities (TEAC) and TP content (R2 = 0.9812 ± 0.012). DPV was applied to the laccase modified GCE, and the experimental results indicate that this sensor shows good reducing properties. The scavenging ability of 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) a diammonium salt, was assessed in the sample extracts, which yielded half-maximal effective concentration (EC50) values of 10.80 μg/ml and 11.62 μg/ml for ascorbic acid and rutin respectively. These findings indicated that the experimental design was a convenient method to evaluate the statistical significance of the optimised parameters, and the positive linearity for the TEAC and the TP content confirms the robustness of this methodology.Item Characterisation of Opuntia phenolic extracts and enzymatic modification of selected compunds(2019) Aruwa, Christiana Eleojo; Kudanga, Tukayi; Amoo, Stephen O.Opuntia species are utilised as local medicinal interventions for chronic diseases and as food sources. The phytochemical profile varies within and across Opuntia species and has been related to differences in cultivar and geographical location. Macromolecular antioxidant (MA) fractions are also largely ignored from most conventional extractive processes compared to the well-known extractable polyphenol fractions. This study characterised subtropical spineless cladode, fruit pulp and peel extracts and selected phenolic compounds for enzymatic modification using a laccase from Trametes pubescens. MA extracts were also characterised in comparison with extractable fractions. The effects of drying methods and extraction solvent on extract yields and bioactivities were also studied. Extracts were assayed for phenolic content and antioxidant activities were determined using standard 2,2’-diphenyl-1-picrylhydrazyl (DPPH), 2,2,-azinobis3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) assays. Antimicrobial activities and mode of antibacterial action were assessed against type-bacterial cultures. Minimum inhibitory concentration (MIC) values were recorded for the extracts and compounds. Compound profiling was achieved using liquid chromatography-time of flight-mass spectrometry (LC-TOF/MS) in negative ionisation mode. Antibacterial and antioxidant activities were higher in MA, hydrolysed and hydroalcoholic cladode and fruit extracts than in aqueous fractions. Ethanolic, methanolic and hexane extracts of freeze-dried Opuntia cladode, MA and peel samples showed higher total phenolic content, and in vitro antioxidant and antimicrobial activities than the oven-dried extracts. Cladode extracts inhibited growth of both Gram-positive and Gram-negative microorganisms (MIC range of 25 to 250 mg/mL). Likewise, fruit extracts inhibited both Gram-positive and Gram- negative microorganisms (MIC range of 2.5 to 18.75 mg/mL). Cladode and fruit extract profiles showed the presence of mainly phenolic acids and flavonoid derivatives. Isovitexin 7-O- xyloside-2"-O-glucoside, polyhydroxypregnane glycoside and neohancoside C in Opuntia cladode, and pinellic acid in Opuntia fruit were identified for the first time in this study. Some compounds, however, remained unidentified. Thereafter, selected Opuntia cladode and fruit phenolic compounds (isorhamnetin and luteolin) were used for enzymatic (laccase) transformation after preliminary screening reactions. Laccase-catalysed oxidation of luteolin in a monophasic system containing sodium acetate buffer (pH 5.0) and ethanol (50%, v/v) as co- solvent, resulted in the production of a dimer (m/z 569, M=570). Using a similar approach, oxidative coupling of isorhamnetin produced two main products, IP1 which was a dimer (m/z 629, M=630) and IP2 (m/z 457, M=458) which was most likely a result of coupling of an oxidative cleavage product and the isorhamnetin monomer. Dimers showed up to two-fold improvement in antioxidant and antimicrobial activities, compared to their respective substrates. The synthesised products showed a bactericidal mode of action as demonstrated by time-kill and bacterial cell integrity assays. The bactericidal action was further confirmed by scanning electron microscopy (SEM) which showed that treatment of bacterial cells with the synthesised compounds resulted in deformed, pitted, broken or fragmented cells, indicating strong bactericidal action. In conclusion, this study showed that Opuntia fruit pulp, peel and cladode extractable and MA extracts have potential as sources of phenolic compounds with antioxidant and antimicrobial activities. Laccase catalysis has potential to transform the phenolic compounds into coupling products with higher biological activities. The synthesised products have potential for application in the food, nutraceutical and other relevant industries.Item Anti-inflammatory, anti-oxidant and wound-healing properties of selected South Africa medicinal plants(2017) Mzindle, Nonkululeko Betty; Odhav, BhartiSouth Africa has a wide range of medicinal plants that are used traditionally by black Zulu South Africans for the treatment of a range of illnesses, including inflammatory ailments; disease conditions caused by oxidative stress and wound healing. It has been indicated that bioactive compounds isolated from plants contribute to their anti-inflammatory, antioxidant and wound healing properties; hence, herbal remedies have been widely used traditionally in many countries in the management and treatment of wounds. Inflammation is the main condition that relates to a variety of diseases affecting most of the world’s population. It is the body’s immune response to infection and injury and is induced by the release of pro-inflammatory mediator’s —prostaglandins and leukotrienes—following wound occurrence. Wounds result in disruption of living tissue caused by oxidative stress. Anti-inflammatory agents, antioxidants, and antimicrobials play an important role in the wound healing process and they prevent aggravated wound conditions.Controlling inflammation during wound repair is important to minimize any additional complications that may result; hence, chemical agents such as non-steroidal anti-inflammatory drugs (NSAIDS), synthetic antioxidantsand steroids are frequently used. These drugs block the enzymes that are responsible for prostaglandin synthesis in inflammation, react with free radicals thereby interfering with oxidation process as a result affect one or more phases of wound healing. The use of these drugs, however, has been limited as they can cause detrimental side effects when used over long periods of time.There is, consequently, a need to find alternative natural therapeutic drugs. Studies on medicinal plants confirmed that herbal drugs exhibit fewer side effects in comparison with chemical agents and are more cost-effective.Thus the aim of this study was to investigate South African medicinal plants, for anti-inflammatory, antioxidant and wound healing properties. Dissolved extracts of thirty-eight medicinal plants were evaluated for theiranti-inflammatory activity using the 5-lipoxygenase assay as well as free radical scavenging activity using the 1; 1-diphenyl-2-picrylhydrazyl (DPPH) assay.Their safety was evaluated using brine shrimp lethality assay. Proliferation and viability of fibroblast cells was determined by the3-(4, 5-dimethylthiazolyl)-2, 5-diphenyltetrazolium bromide(MTT) assay furthermore a scratch wound assay was used to study the properties of wound healing in vitro and to confirm the anti-inflammatory activities of the dissolved extracts. Migration rate was evaluated quantitatively by an image analyzer. Methanol was chosen for extraction because it completely dissolves extracts. Anova was used for statistical analysis. Almost all aqueous extracts were found to be effective in inhibiting lipoxygenase enzyme when compared to nordihydroguaiaretic acid (NDGA). Aqueous extracts exhibited remarkably high percentage inhibition of lipoxygenase with most above 100% when compared to methanolic extracts. Amaranthus dubius and Portulaca oleracea were found to have good biological activities in the inhibition of 5-lipoxygenase enzymes when compared to the other plants. However, Galinsoga parviflora and Syzygium cordatumwere least effective in inhibiting enzyme activity with percentages as low as -2% and 34% respectively. Percentage inhibitions for methanolic extracts were lower than that of aqueous extracts. Amaranthus spinosus had the highest percentage inhibition among all the methanolic extracts andGalinsoga parviflorahad the lowest. The methanolic plant extracts were found to be more effective in scavenging DPPH free radicals than the corresponding aqueous extracts. All the methanolic extracts exhibited free radical scavenging ability in the range of 60%–104%. Asystasia gangetica, Ficus sur, Heteropyxis natalensis, Hibiscus sabdariffa, Pelargonium sp. showed notably higher scavenging abilities, ranging from 101%–104% compared to Rutin. Methanolic extracts of Heteropyxis natalensis and Hibiscus sabdariffa exhibited scavenging ability even at the lowest concentration of 10μg/ml. Furthermore, aqueous extracts displayed remarkably lower activities than methanolic extracts with thirty-one extracts having a scavenging capacity ranging from 22%—59%. None of the extracts were found to be detrimental to brine shrimp. Almost all the extracts were shown to stimulate the growth of fibroblast cells except the methanolic extract of Solanum nodiflorum, which was shown to be killing the cells at high concentrations with a percentage viability of 46%.As the concentration decreased, however, the viability of cells with this extract increased to 143%. An increase in the number of fibroblast cells was observed in the scratched area of the treated cells and a significant migration rate was also noted with some of the extracts. Aqueous extracts of Sonchus oleraceus (86%), Justicia flava (85%) and Dichrostachys cinerea (85%) and methanolic extracts of Senna occidentalis and Hibiscus sabdariffa were found to have the highest migration rate compared to untreated cells that served as a control. No cell migration was observed with methanolic extract of Solanum nodiflorum.Instead, the extract was found to be toxic to the cells. Some of the plants evaluated in this study have been studied for either anti-inflammatory, antioxidantand wound healing properties in vivo, however, no work has been conducted to demonstrate a correlation between anti-inflammatory, antioxidant and wound healing properties of plant species in vitro. The current study was, therefore, conducted to review medicinal herbs considered as anti-inflammatory, antioxidants and wound healing agents as well as collecting evidence for their effectiveness and pharmacological mechanisms in modern science. In the plant species investigated Amaranthus dubius, Asystasia gangetica, Bidens pilosa, Buddleja saligna, Carpobrotus dimidiatus, Chenopodium album, Dichrostachys cinerea, Emex australis, Ficus sur, Guilleminea densa, Hibiscus sabdariffa, Physalis viscose, Syzygium cordatum, Taraxacum officinale and Tulbaghia violacea demonstrated good anti-inflammatory and wound healing properties.In conclusion the results from this study demonstrated promising anti-inflammatory and antioxidantactivities as well as wound healing properties,furthermoreit was aslo shown that the plant extracts were not toxic to the cells hencethis suggested that the plants investigated, can be used as substitutes or to formulate wound healing agents that are safe to use in primary healthcare.