ELICITATION, DETECTION AND BIOPROFILING OF SECONDARY METABOLITES IN MUSA SPECIES USING PLANT TISSUE CULTURE TECHNIQUES AND HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

Abstract

The search for new bioactive compounds from the wild often leads to conservation issues. Plant tissue culture, a less destructive source of plant materials is a priority technique. There is paucity of studies on potentials of plant tissue culture for the accumulation of secondary metabolites in wild and cultivated Musa species, with wide medicinal applications. This study was, therefore, designed to improve the phytoconstituents and profile the biological activities of selected Musa species. Fifteen taxonomic reference Musa species accessions (A-O) were micropropagated. Genomic DNA obtained from each accession was used to generate Single Nucleotide Polymorphic (SNP) markers using Diversity Array Sequencing (DArTseq) approach to analyse genetic fidelity. Antioxidant and anticholinesterase activities of field and micropropagated accessions were evaluated using spectrophotometric methods and High Performance Thin Layer Chromatography (HPTLC) hyphenated with High Resolution Mass Spectrometry (HRMS) for compounds identification. The Effect-Directed Analysis (EDA) of the Musa species was done on HPTLC plates with antioxidant, anticholinesterase, antidiabetic, and genotoxicity assays for bioprofiling. Gallic acid, eserine, acarbose, 4-nitroquinoline-1-oxide, were used as standards. Compounds were characterised with HRMS. Six of the accessions were subjected to elicitation experiments using different doses of sugar (30-50 g/L), temperature (15, 20 and 25 °C) and Jasmonic Acid (JA, 0–200 µM) as elicitors. The Total Phenolic Content (TPC), antioxidant profile and biological activities of the resulting elicited samples (71) were analysed with HPTLC. Data were analysed using one-way ANOVA followed by Dunnett’s multiple comparison at α0.05. The micropropagated accessions were shown to be true-to-type with the field samples. Micropropagated plants gave significantly higher TPC (2.43±0.30 to 124.52±12.72 mgGAE/g), DPPH antioxidant (10.68±0.27 to 154.42±6.44 µg/mL), ferric reducing antioxidant power (10.58±0.52 to 152.16±2.80 mgTE/g) and acetylcholinesterase (11.53±0.12 to 1181.00±91.25 µg/mL) activities than the field plants. iii Acetylcholinesterase (AChE) activity of micropropagated accession F (11.53±6.52 µg/mL) was significantly higher than that of eserine (31.23±7.33 µg/mL). Results of the HPTLC-EDA were similar to those of the spectrophotometric assays; accession F also had the highest AChE inhibition. Three multipotent compounds characterised by their base peaks (m/z) and molecular formula were identified as asparagine (1), aniracetam (2) and linolenic acid (3). All the compounds had AChE and butylrylcholinesterase (BuChE) inhibitory activities. Compounds 1 and 2 had antioxidant activity, while compound 1 exhibited α and β-glucosidase inhibition. None of the samples were genotoxic at the concentrations tested. The elicitation experiments increased the TPC and antioxidant activity of Musa species. In accession B, the TPC increased from 39.43 to 138.64 ngGAE/g (4.89 folds) when 30 and 50 g/L of sugar were used, respectively. Plants grown at 20 °C gave a higher TPC than other treatments. Addition of JA (200 µM) increased the TPC of accessions L and N by 2.24 and 1.68 folds, respectively. The plant tissue culture technique was validated as a suitable method for the production of genetically uniform Musa species and for sustainable supply of more bioactive metabolites. The identified compounds had antioxidant, anticholinesterase and antidiabetic activities. The diversity and titre of the phytoconstituents were also improved in the elicited accessions