Characterisation of Opuntia phenolic extracts and enzymatic modification of selected compunds

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.