Faculty of Applied Sciences

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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.
Nutritional value and bioactive properties of Opuntia ficus-indica cladodes
(2020) Mabotja, Mologadi Bernard; Kudanga, Tukayi; Amoo, Stephen O.
Medicinal properties and pharmacological activities of plants have been attributed to their bioactive compounds. However, research has shown that bioactive compound concentrations, biological activities and nutritional profile are influenced by cultivar type. The aim of the study was to characterize the cladodes of 42 spineless cultivars of Opuntia ficus-indica at the Agricultural Research Council in terms of their chemical, nutritional and medicinal properties. The antidiabetic potential of selected extracts was investigated in vitro against alpha-glucosidase enzyme. Aqueous methanol extracts were assayed for total phenolic and flavonoid content, and antioxidant activities using 1,1-Diphenyl-2- picrylhydrazyl (DPPH) and β-carotene linoleic acid system assays. Antibacterial activities of different extracts were assessed against two Gram (-) and two Gram (+) bacteria and their minimum inhibitory concentration (MIC) values recorded. Vitamin C, β-carotene and individual phenolic acid contents were analysed using a Shimadzu HPLC (LC-2030C 3D) equipped with a C18 Luna® column. Quantification was achieved by calibration curve plotted using different standards. Mineral elements were quantified using inductively coupled plasma - optical emission spectrometry. There were variations in the yield depending on the cultivars, however, 50% methanol extracts generally had higher yield as compared to petroleum ether extracts. Significant variation in phytochemical composition, pharmacological activities and nutritional value was observed in the different cultivars studied. The total phenolic content of Berg x Mexican was about 5 times higher than that of Robusta and Montery. Similarly, the flavonoid content of Turpin and Berg x Mexican was about six folds higher than that of Corfu, Montery and Amersfoort. Different cultivars showed different percentage antioxidant activities. Many of the cultivars exhibited antioxidant activity comparable to that of butylated hydroxytoluene (BHT), a synthetic antioxidant usually used as a food additive to prevent the damage caused by free radicals during oxidation processes. Alpha glucosidase inhibitory assay revealed a dose dependent activity with IC50 values ranging from 0.06 - 1.85 mg/ml and 27 of the cultivars showing IC50 values lower than that of acarbose. Noteworthy antibacterial activity was observed against Bacillus subtilis and Escherichia coli with MIC values below 1 mg/ml. The poorest activity was observed against Klebsiella pneumoniae. Petroleum ether extracts generally had the best antibacterial activity when compared to 50% methanol extracts. Compounds profiling indicated that catechin and gallic acid were found to be present in significant quantities in all the cultivars investigated, whilst the least occurring compound was quercetin. Vitamin C content ranged from a lowest of 8.95 mg/100 g to a highest of 124.10 mg/100 g. β-carotene content ranged from a lowest of 3.9 mg/100 g and highest of 31.4 mg/100 g. Potassium and calcium were the most abundant elements present in the Opuntia ficus-indica with a highest of 4980.00 mg/100 g for potassium, whilst iron was found to be the least present with a range of 0.20 to 54.67 mg/100 g. In conclusion, the observed findings indicate that the spineless cladodes of Opuntia ficusindica cultivars are important sources of nutrients and bioactive properties and can be considered as functional foods. Although no cultivar could be singled out as the best as each pharmacological, phytochemical and nutritional trait was different in each cultivar, the observed findings indicate the need for careful cultivar selection when using spineless cactus pear cultivars for product development to ensure product integrity.