The micropropagation and phytochemistry of commercially important medicinal plants

Thesis (MSc)--Stellenbosch University, 2022.

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.

AFRIKAANSE OPSOMMING: Cannabis sativa L., 'n hoogspolimorfiese, kruidagtige plantspesie met sy oorsprong in Sentraal- Asië, het sedertdien oor die hele wêreld versprei. Hierdie plantspesie is al vir duisende jare mak gemaak as gevolg van sy veelsydigheid in die gebruik van medisyne, olies, tekstiele en ontspanning. Tetrahidrokannabinol (THC) en THC-suur (THCA) is van die mees algemene cannabinoïde. Meer as honderd verskillende cannabinoïde is in verskillende Cannabis- variëteite geïdentifiseer. Cannabis is nie net ryk aan cannabinoïde nie, maar bevat ook meer as 150 terpene. Cannabis het sy farmakologiese aktiwiteite te danke aan sy groot komplement van sekondêre metaboliete. Dít het dus aansienlike belangstelling in die industrie ontlok in terme van die skepping van verskeie verbruikersprodukte en die ontwikkeling van nuwe Cannabis-verwante middels en medisyne. Die eerste doel van hierdie studie was dus om 'n metabolomiese ontleding op drie industrieel-belangrike Cannabis sativa-variante (variant A, variant B en variant C), wie se chemiese profiele voorheen onbekend was, te doen. Daardeur sou hul metabolomiese profiele en kannabinoïedkonsentrasies (THC en THCA) bepaal word. Die fitochemie van hierdie drie stamme en hul plantdele (blaar en knop) is vergelyk met behulp van vloeistofchromatografie- massaspektrometrie (LC-MS) en kopruimte- vastefase-mikro-ekstraksie-gaschromatografie- massaspektrometrie (HS-SPME-GC-MS). Sewe-en-twintig verskillende verbindings is gesamentlik tentatief geïdentifiseer deur gebruik te maak van LC-MS sagteware in die negatiewe en positiewe ionisasie modusse. Die drie Cannabis- variante was duidelik van mekaar onderskei, en dit was ook duidelik dat weefseltipe 'n rol speel in die skeiding van trosse. Die knoppe van al drie stamme het saam gegroepeer terwyl blaarmonsters ook 'n groepering gevorm het. Variant A en C se blare en knoppe het soortgelyke fitochemie aan mekaar gedeel terwyl variant B kwalitatief onderskeibaar was. Isowertin 2”-rhamnoside en ∆9- tetrahidrokannabinol was van die topverbindings verantwoordelik vir die skeiding van groepe. THC en THCA is in hoër konsentrasies in knoppe as in die blare gevind, en geen kannibidiol (CBD) is in enige van die variante waargeneem nie. Drie-en-veertig verskillende essensiële olieverbindings is in die monsters gevind met behulp van HS-SPME-GCMS. Wanneer chemometriese ontledings met behulp van hierdie data gedoen is, was stamme ook duidelik van mekaar geskei. Die blare en knoppe van variant B was egter meer soortgelyk in hul fitochemie in vergelyking met die ander stamme. Hierdie studie bevestig die gebruik van metabolomika as 'n instrument vir die akkurate diskriminasie en chemiese klassifikasie van Cannabis- variante. As gevolg van die nuwe belangstelling wat die industrie op Cannabis en sy farmakologiese aktiwiteit geplaas het, en die bekende anti-kanker eienskappe van Dodonaea viscosa (familie: Sapindaceae, orde: Sapindales), is die anti-proliferatiewe effekte van hierdie twee plante saam en individueel, na die metabolomiese ontledings. getoets. Dit was om te bepaal of die kombinasie van die twee uittreksels sy sterkte teen borskankerselle verhoog. Hierdie kombinasie uittreksel sal dan dien as 'n potensiële hulpmiddel vir borskanker behandeling. Vloeistofchromatografie-massaspektrometrie-analise van die uittreksels het getoon dat cannabinoïde soos cannabigerol (CBG) en THC, en dodonsuur (slegs in Dodonaea gevind) die sleuteldrywers was wat die uittrekselgroepe geskei het. 'n CellTiter-Glo-toets is gebruik om die sellewensvatbaarheid van twee sellyne te bepaal: 1) MDA-MB-231 en 2) MCF- 10A. Alle uittreksels het anti-kanker effekte gehad, maar die Dodonaea-uittreksel het die hoogste anti-proliferatiewe aktiwiteit getoon wanneer dit op sy eie toegedien is met 'n EC50- waarde van 14.32 µg/mL. Wanneer Cannabis-uittreksel met die Dodonaea gekombineer is, het die anti-proliferatiewe aktiwiteit van die uittreksel afgeneem. Dit is grootliks onbekend watter verbindings die teenkanker-effekte van hierdie twee plante aandryf, maar daar word vermoed dat dit 'n gevolg is van sinergie tussen verskeie verbindings in plaas van die werking van 'n enkele verbinding. Nog 'n nuwe onderwerp van navorsing was die ontwikkeling van 'n tegniek vir die grootskaalse produksie van Cannabis en sy variante, vry van siektes en somaklonale variasie vir kommersiële en farmaseutiese toepassing. Die uiteindelike doel van hierdie studie was dus om 'n doeltreffende weefselkultuurprotokol vir Cannabis te ontwikkel, wat in die industrie toegepas kan word. 'n Suksesvolle dekontaminasieprotokol vir nodale uitplantings is ontwikkel wat die volgende insluit: 1) 70% etanol (EtOH) + Tween®20 week vir 1 min 2) 3.5% natriumhipochloriet (NaOCl) verdun met gedistilleerde water (1:1 v/v) week vir 20 min 3) 2% Benlate® week vir 15 min 4) gedistilleerde water spoel 5) 4% plant-preserveermiddel mengsel (PPM™) week vir 60 min. Alle bakteriese en swamkontaminasie is uitgeskakel deur 500 mg/L kefotaksim en 0.1% PPM™ by die medium by te voeg. Die basalegroeimedium (MS-medium aangevul met 0.1 g/L mio-inositol en 30 mg/L sukrose) is gewysig om 10 mg/L agar in te sluit. Plantgroeireguleerders (6-Benzielaminopurien (BA), 1-Naftaleenasynsuur (NAA), Kinetin (KN), Indool-3-asynsuur (IAA) en meta-Topolien (mT)) is in verskillende kombinasies by die medium gevoeg by konsentrasies van (0,1, 0,5 en 1 mg/L). Die uitplantings wat die hoogste lootproliferasie getoon het met 'n gemiddeld van 1.8 lote en 'n gemiddelde lengte van 1.8 cm is waargeneem op medium aangevul met 0.1 mg/L mT. Om lootproliferasie verder aan te moedig, is uitplantings sub-gekweek op medium aangevul met 0.1 mg/L mT en dubbel die oorspronklike hoeveelheid stikstof. Uitplantings was egter weerbarstig teenoor weefselkultuur en het nie oorleef in die medium wat twee keer die hoeveelheid stikstof bevat het as die basale medium nie. Dit kan te danke wees aan die feit dat mineraalvoeding in Cannabis moontlik stamspesifiek is en die huidige samestelling van die verskillende media wat in hierdie studie getoets is, moet dus geoptimaliseer word.

Masters