Department of Botany and Zoology

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Browsing Department of Botany and Zoology by Author "Adams, Tristan Kelsy"

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    The micropropagation and phytochemistry of commercially important medicinal plants
    (Stellenbosch : Stellenbosch University, 2022-03) Adams, Tristan Kelsy; Makunga, Nokwanda Pearl; Masondo, Nqobile; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.
    ENGLISH ABSTRACT: Cannabis sativa L. is a highly polymorphic, herbaceous plant species that originated from central Asia and has since dispersed all around the globe. This plant species has been domesticated for thousands of years due to its versatility, for use in medicines, oils, textiles and recreation. Tetrahydrocannabinol (THC) and Tetrahydrocannabinolic acid (THCA) are amongst the most common cannabinoids, and over one hundred different cannabinoids have been identified within different Cannabis varieties. Not only is Cannabis rich in cannabinoids but also contain over 150 terpenes. Cannabis owes its pharmacological activities to its large complement of secondary metabolites, and this has thus sparked significant industry interest in terms of the creation of various consumer products and the development of novel Cannabis- related drugs. The first aim of this study was therefore to do a metabolomic analyses on three industrially important Cannabis sativa strains (strain A, strain B and strain C), whose chemical profiles were previously unknown, to determine their metabolomic profiles and cannabinoid concentrations (THC and THCA). The phytochemistry of these three strains and their plant parts (leaf and bud) were compared using liquid chromatography- mass spectrometry (LC-MS) and headspace- solid phase microextraction- gas chromatography mass- spectrometry (HS- SPME-GC-MS). Twenty-seven different compounds were collectively tentatively identified using LC-MS tools in the negative and positive ionisation modes. The three Cannabis strains were clearly distinct from one another, and it was also apparent that tissue type plays a role in the separation of clusters. The buds of all three strains clustered together while leaf samples also formed a grouping. Strain A and C leaves and buds shared similar phytochemistry to one another while Strain B was qualitatively distinct. Isowertin 2”-rhamnoside and ∆⁹- tetrahydrocannabinol were amongst the top compounds responsible for the separation of groups. THC and THCA were found in higher concentrations in buds than in the leaves, and no cannabidiol (CBD) was observed in any of the strains. Forty-three different essential oil compounds were found in the samples using HS-SPME-GCMS, and when doing chemometric analyses using this data, strains were also visibly separate from one another. The leaves and buds of strain B were, however, more similar in their phytochemistry when compared to the other strains. This study verifies the use of metabolomics as a tool for the accurate discrimination and chemical classification of Cannabis strains. Due to the novel interest industry has placed on Cannabis and its pharmacological activity, and the known anti-cancer properties of Dodonaea viscosa, (family: Sapindaceae, order: Sapindales) the metabolomic analyses was followed by testing the anti-proliferative effects of these two plants in combination and individually to determine if combining the two extracts increases its potency against breast cancer cells. This combination extract would then serve as a potential adjuvant to breast cancer treatment. Liquid chromatography- mass spectrometry analysis of the extracts showed that cannabinoids such as cannabigerol (CBG) and THC, and dodonic acid (only found in Dodonaea) were the key drivers separating the extract groups. A CellTiter-Glo assay was used to determine the cell viability of two cell lines: 1) MDA-MB-231 and 2) MCF-10A. All extracts had anti-cancer effects, however, the Dodonaea extract demonstrated the highest anti-proliferative activity when administered on its own with an EC50 value of 14.32 µg/mL. When Cannabis extract was combined with the Dodonaea, anti-proliferative activity of the extract decreased. It is largely unknown which compounds drive the anti-cancer effects of these two plants, it is however suspected that it is as result of synergy between various compounds instead of the actions of a single compound. Another novel topic of research has been the development of a technique for the large- scale production of Cannabis and its variants, free from disease and somaclonal variation for commercial and pharmaceutical application. The final aim of this study was thus to develop an efficient tissue culture protocol for Cannabis, that can be applied in industry. A successful decontamination protocol for nodal explants was developed which included: 1) 70% ethanol (EtOH) + Tween®20 soak for 1 min 2) 3.5% sodium hypochlorite (NaOCl) diluted with distilled water (1:1 v/v) soak for 20min 3) 2% Benlate® soak for 15 min 4) distilled water rinse 5) 4% plant preservative mixture (PPM™) soak for 60 min. All bacterial and fungal contamination was eliminated by the addition of 500 mg/L cefotaxime and 0.1% PPM™ to the medium. The basal growth medium (MS medium supplemented with 0.1 g/L myo-inositol and 30 mg/L sucrose) was amended to include 10 mg/L agar. Plant growth regulators (6- Benzylaminopurine (BA), 1-Naphthaleneacetic acid (NAA), Kinetin (KN), Indole-3-acetic acid (IAA) and meta-Topolin (mT)) were added to the medium in different combinations at concentrations of (0.1, 0.5 and 1 mg/L). The explants that displayed the highest shoot proliferation with an average of 1.8 shoots and an average length of 1.8 cm were observed on medium supplemented with 0.1 mg/L mT. To further encourange shoot proliferation, explants were sub-cultured onto medium supplemented with 0.1 mg/L mT and double the original amount of nitrogen. Explants, however, were recalcitrant to tissue culture and did not survive in the medium containing twice the amount of nitrogen than the basal medium. This could owe to the fact that mineral nutrition in Cannabis may be strain specific and the current composition of the different media tested in this study thus need to be optimised.