Doctoral Degrees (Botany and Zoology)
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Browsing Doctoral Degrees (Botany and Zoology) by Subject "Alkaloid biosynthesis"
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- ItemSceletium tortuosum (L.) N.E. Br.: mesembrine alkaloid chemotype signatures in wild type, cultivated and in vitro tissues(Stellenbosch : Stellenbosch University, 2021-04) Glyn-Woods, Christina; Makunga, Nokwanda P.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.ENGLISH ABSTRACT: The South African indigenous, medicinal succulent Sceletium tortuosum (L.) N.E. Br., has entered the world stage, as increasing evidence of the anxiolytic and human health applications of its mesembrine alkaloid suite emerges. Clinical trials, confirming ethnopharmacogical accounts linked to their bioactivity, has ensured the demand for these metabolites will increase. Concommittal with this global market opportunity, is the need to optimize and tailor existing horticultural production systems, and the parallel development of alternative, high fidelity, phytopharmaceutical production platforms to meet this demand. To accomplish this, greater insight into the fundamental triggers, endogenous and environmental, determining mesembrine alkaloid signatures in planta is needed. The driving rationale behind this research, therefore, was to populate this current gap in knowledge of the in planta aspects of mesembrine alkaloid biosynthesis. Using the resolving power of ultra-high-performance liquid chromatography mass spectroscopy (UHPLC-MS), to quantify; and multivariate principal component analysis (PCA), to visualise, a quantitative and qualitative examination of the factors shaping chemotype signatures in living tissue was undertaken. Firstly, a global view of the seasonal accumulation patterns of the different mesembrine alkaloids was determined. Chemotype profiles of a single, limited range, wild type population were sampled across the 4 seasons of one year. Correlation with physiological plant growth parameters revealed the accompanying growth phase mesembrine alkaloid expression patterns. During summer, the major alkaloids mesembrine (4.01 ±5.62) mg/kg DW, mesembrenol (2.49 ±2.85) mg/kg and mesembranol (52.79 ±51.29) mg/kg DW and the isomers 6-epimesembrenol (2.10 ±2.19) mg/kg DW and 6-epimesembranol (1.45 ±1.41) mg/kg DW exhibited maximal tissue accumulation. This season, of extreme high temperatures and water scarcity, coincided with greatly curtailed plant growth. Conversely during autumn and winter, during which plant growth is initiated and active, respectively, accumulation minima were recorded. This first report of the seasonal accumulation patterns of the mesembrine alkaloids of S. tortuosum, corroborates and explains for the first time the rational of the traditional practice of the summer harvest of these plants. Significant seasonal alkaloid specific accumulation differences were found, however, large within season variation in alkaloid content was also observed. It is proposed that this variation is underpinned by a large, inherent, intra-population chemical heterogeneity occurring amongst individuals. It is conjectured, based on these observations, that additional significant factors, besides seasonality, are determinate in shaping chemotypic variability of the wild growing material and the suggested possibility of inducibility of these alkaloids, awaits further investigations. A potential photoprotective role of the mesembrine alkaloids is proposed. This conjecture is grounded in the high relative amounts of key mesembrines recorded in summer, the spatial accumulation of specific mesembrines in the outer leaves of the bud growth structures and the inherent photochemical UV quenching properties of the mesembrine alkaloids themselves. The high irradiance and incident UV light, inherent to the natural habitats of these plants, would render such a functionality of these compounds an evolutionary advantage. Additionally, in wild type plants an upregulation of betalain pigments and key mesembrine alkaloids over the year, reaching a maximum in summer, was observed. These betalain pigments, made up of the red to maroon betacyanins and yellow betaxanthins groups, occur exclusively in Caryophyllaceae order plants, to which S. tortuosum belongs. As aromatic ring chromophores, these pigments, besides antioxidant and osmotic protectant properties, also possess inherent absorption maxima in both the UV, and visible region, and are functional analogues of the more ubiquitous anthocyanins, with which they share similar photochemical properties. It was interesting to realise, that the nitrogenous mesembrines and betalains share a common primary metabolism pathway precursor, the amino acid tyrosine. While speculative, an interaction of these two metabolite pathways is hypothesised, and may hold clues as to their respective co-evolution, and in planta role, in plants such as S. tortuosum, and the Caryophyllaceae order at large. The second branch of the study examined mesembrine alkaloid accumulation patterns from a tissue developmental perspective. A spatiotemporal study of the chemotypic variation in developing leaves of tunnel grown plants, charted the accumulation of the mesembrine alkaloids to specific leaf age, developmental stages. Mesembrine (5156.7 ±1276.5) mg/kg DW was associated with actively growing leaves, whereas �⁴-mesembrenone (255.9 ±301.15) mg/kg DW, mesembranol (551.52 ±56.4) mg/kg DW and 6-epimesembranol (612.4 ±64.7) mg/kg DW were present at highest levels with the onset and progression of senescent aging of leaves. This new information linked to improved understanding of the developmental and spatial accumulation of these alkaloids will inform biosynthetic pathway investigations and pave the way to defining the possible ecological and/or biological significance of these accumulation patterns. In conclusion, the final branch of the study sought to rapidly and sustainably meet the immediate and future demand for the high value phytochemicals of S. tortuosum, through the development of a suitable in vitro, high throughput, metabolite production technology. Increased scientific and mainstream reports and growing public awareness of the psychoactive properties of S. tortuosum, has exposed wild populations to an increased threat of unscrupulous harvesting. By establishing a biotechnological platform for future production of these compounds, it was envisaged that the current pressure on wild populations could be assuaged. Using in vitro microshoot cultures, a two-phase regimen employing a tissue dehydration pre-treatment step, prior to metabolite extraction, produced commercially exploitable levels of mesembrine (3270.9 ±981.5) mg/kg DW, mesembrenol (333.1 ±36.1) mg/kg DW, mesembranol (4738.8 ±172.9) mg/kg DW and �⁴-mesembrenone (29.9 ±0.5) mg/kg DW in tissue extracts. Additionally, select liquid callus culture lines, once established, accumulated mesembranol (64.6 ±8.5) mg/kg DW as the maJor alkaloid component, and exhibited a retained significant biosynthetic capacity for mesembrine, mesembranol, mesembrenol and �⁴-mesembrenone. These in vitro platforms present a potentially scalable, high throughput alternative, to access mesembrine alkaloids for the various phytopharmaceutical industries. Additionally, the clonal tissue uniformity and exogenous growth parameter control inherent to such technologies have not been previously accessible for this plant and will greatly benefit advances in metabolic pathway and molecular biology investigations. As various key mesembrines are poised to become more prominent in the phytopharmaceutical arena, South Africa, as the home of the Sceletium plant, holds a unique position of responsibility in the global marketplace. The research presented here, on both the seasonal and spatiotemporal chemotype development in this plant, begins to answer for the first-time fundamental question of inherent biological timing and context of mesembrine alkaloid signatures necessary to optimise their harvesting. Sceletium has shared a long history of use within the San culture, and as the industry surrounding this plant grows in this country, and globally, we should remain cognisant of fairness, and of the original knowledge keepers' stake. The production of mesembrine alkaloids using biotechnology as a system, provides a novel manufacturing platform for this species that has the potential to become highly desired in the industry. As the lucrative healing potential of Sceletium unfolds, communities with San heritage are in an opportune position to benefit from their intellectual property and its availability to the world at large, through the commercialisation linked to this work.