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Metabolomic profiling, computational and experimental validation of sunflower seeds as therapeutics against type-2diabetes mellitus
No full textSubmitted in fulfilment for the Degree of Master of Applied Science in Biotechnology, Durban University of Technology, Durban, South Africa, 2025.Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by impaired
glucose metabolism due to insufficient insulin secretion or insulin resistance. This global health
crisis is projected to affect an estimated 7079 individuals per 100,000 by 2030. While
medications like metformin are effective, accessibility and affordability are issues consistent
with low-income populations alongside potential side effects like hypoglycaemia, nausea and
gastrointestinal issues that have limited their use in clinical practice. More importantly,
uncontrolled T2DM can lead to serious complications like retinopathy, nephropathy,
neuropathy, and delayed wound healing. Therefore, this prompts the search for alternative
management options that are safer, easily accessible, affordable and with minimal side effects.
Plants and their products are becoming increasingly important due to their relative ease of
accessibility, affordability and potential health benefits. Sunflower seed, a popular dietary
snack, has rich nutritional profile and has found significant health benefits as an antiinflammatory, antioxidant, anticancer, antimicrobial, and antidiabetic agent. While the
antidiabetic potential of sunflower seeds has been explored, there remains a lack of
understanding on its mechanism of action. This study addressed this knowledge gap by
establishing the comprehensive metabolite profiles and investigating the antidiabetic efficacy
of sunflower seed extracts through a two-pronged approach: targeted enzyme inhibition and
network pharmacology analyses complemented with experimental validation in vitro.
Metabolomic profiling of six cultivars of sunflower seeds commonly consumed in South
Africa, namely, AGSUN 8251, 5270, 5101 CLP, 5103 CLP, 5106 CLP and 5108 CLP was
performed using Liquid chromatography – mass spectrometry (LC-MS) and Gas
chromatography – mass spectrometry (GC-MS) techniques. A total of 94 metabolites were
identified, with LC-MS analysis revealing 44 phenolic compounds across the six cultivars with
a minor variance of 39.7%, while GC-MS analysis revealed the presence of volatile compounds
such as organic acids, alkanes, alcohols, terpenes, heterocyclic compounds and hydrocarbons
in all the cultivars in similar abundance. Noteworthily, 84 of the 94 metabolites profiled passed
Lipinski’s rule of five and were selected for further analysis.
For the enzyme inhibition study, molecular docking analysis was initially used to screen the
profiled metabolites against the key enzymes [α-amylase (AAMY), α-glucosidase (AGLU),
aldose reductase (AR), sorbitol dehydrogenase (SDH), dipeptidyl peptidase 4 (DPP-4) and protein tyrosine phosphatase 1B (PTP1B)] implicated in T2DM pathogenesis and its secondary
complications. The top-ranked metabolites against each enzyme were further subjected to
molecular dynamics (MD) simulation to identify putative leads with the strongest binding
affinity, and unperturbed structural integrity through evaluation of their stability, compactness
and intermolecular interactions. This aspect of the study identified sonchuside I (SON I) -
AAMY (–47.26 kcal/mol), sacranoside A (SAC A) - α-glucosidase (–40.10 kcal/mol),
pelatoside A (PLT) - AR (–58.84 kcal/mol), sacranoside A (SAC A) - SDH (–48.03 kcal/mol),
4α,6S,7α)-6α-[6-O-(4-Hydroxybenzoyl)-β-D-glucopyranosyloxy]-7βmethyloctahydrocyclopenta[c]pyran-1-one) (PYR) -DPP-4 (–37.93 kcal/mol) and chlorogenic
acid (CGA)-PTP1B (–24.32 kcal/mol) as potential lead inhibitors of the respective enzyme
relative to their respective reference standards. This was further supported by their improved
thermodynamic properties and favourable post-dynamic simulation parameters such as
improved stability and compactness of their resulting complexes. These observations are
suggestive of multiple mechanisms by which sunflower seed may exert its antidiabetic effects
such as anti-hyperglycaemia (α-amylase and α-glucosidase), prevention and management of
diabetic complications (AR and SDH), increasing insulin signalling (DPP-4) and sensitivity
(PTP1B) by the respective putative leads.
For network pharmacology analysis, the filtered sunflower seed metabolites were used to create
a gene-compound library that was subsequently used to identify genes commonly associated
with both the metabolites and T2DM. Thereafter, Kyoto Encyclopaedia of Genes and Genomes
(KEGG) pathway enrichment analysis was performed to identify the most significantly
enriched pathways with key target genes for molecular docking and MD simulations to identify
lead metabolites. Finally, the antidiabetic activity of sunflower seed extracts and the findings
from the network pharmacology analysis were validated using insulin-resistant HepG2 cells
where glucose consumption assay and gene expression analysis were performed. The network
pharmacology analysis revealed a total of 87 genes common to sunflower seeds metabolites
and T2DM, whereas KEGG enrichment analysis highlighted 35 signalling pathways
potentially influenced by the metabolites. Of these, the Peroxisome proliferator-activated
receptor (PPAR) signalling pathway and its hub receptors, Matrix metalloproteinase-1(MMPI)
and peroxisome proliferator-activated receptor alpha (PPAR) were selected as the most
significant. These receptors interacted mostly with the identified metabolites, with CGA (–
43.74 kcal/mol), GPA (–41.62 kcal/mol), and CFG (–45.36 kcal/mol) having lower binding
free energy than both reference standards, rosiglitazone (ROS) and metformin (MET) against MMP1 after 100 000 ps MD simulation. In contrast, ROS (–46.98 kcal/mol) had better affinity
against PPARA compared to the top-hits derived from sunflower seeds. However, against both
genes, the top-hits had significant thermodynamic stability, flexibility, and compactness, which
are attributable to their bond interactions and molecular orbital properties. These findings are
suggestive of the essential role of the top-hits in the antidiabetic potential of sunflower seeds
through activation of the PPAR signalling pathway and most especially MMP1. In this regard,
the modulation of MMP1 and PPARA genes by the identified metabolites of sunflower seeds
may enhance insulin sensitivity and glucose homeostasis in the management of T2DM. Finally,
the in vitro validation using insulin-resistant HepG2 cells revealed cultivar-specific effects on
cell viability, with each cultivar having a unique optimal concentration. Overall, all cultivars
demonstrated the ability to stimulate glucose consumption, suggesting their potential
antihyperglycemic activity. Among the cultivars, AGSUN 5103 CLP (14.4 mmol/L), 8251
(14.6 mmol/L), and 5101 CLP (13.7 mmol/L) exhibited the most pronounced glucose lowering
action compared to the untreated cells (23.3 mmol/L) after 24 h, highlighting their promising
antidiabetic effects. These three cultivars also modulate the PPAR signalling pathway, as
evidenced by the upregulation of MMP1 and PPARA expression. Specifically, AGSUN 5101
CLP emerged as a particularly promising candidate based on its superior glucose lowering
potential and higher fold increase expression of MMP1 (1.88) and PPARA (4.59) compared to
the effect observed with the untreated cells (1.00).
In conclusion, this study provides compelling evidence for the antidiabetic potential of
sunflower seeds. The observed effects on enzyme inhibition, activation of the PPAR signalling
pathway, and stimulation of glucose uptake in HepG2 cells suggest a multifaceted approach by
the seeds in regulating blood sugar levels. The identification of cultivar-specific effects and
promising lead compounds warrants further investigation to explore the therapeutic potential
of sunflower seeds in managing T2DM.
African medicinal flora in the limelight
Get PDFThe original publication is available at http://www.sajs.co.za/In the past few years, African medicinal plants have received considerable attention, and it has
been lamented that the documentation of the continent’s species that are used in traditional
medicine lags behind China and India in terms of ‘internationally recognised phytochemical
standards’. This book not only redresses this issue, but is the first to include plants from the south,
north, east and west of Africa. In South Africa alone, there are over 3000 species that are used for
medicinal purposes, with over 70% of the Black African population relying on medicinal flora as
part of their primary health care and 84% of clinic patients confirming their preference for wildcrafted
raw herbal medicines in spite of having access to western health care.1 Both traditional
and Western healing systems are used – many educated Black people retain traditional practices
as they are regarded as an important cultural link to their predecessors. Throughout Africa, plants
are viewed as contributors to health; they are also used in religious and cultural ceremonies. The
African continent has a rich biodiversity and this is matched by a commensurate proliferation of
medicinal plant use. So the trade of medicinal plants in Africa is substantial, but largely informal,
and consists of plant collectors as well as traders at herbal markets.Publishers' versio
Genetic transformation and micropropagation of Thapsia garganica L. - a medicinal plant.
Get PDFThesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.No abstract available
Genetic modification in Pinus patula using transgenic technology.
Get PDFThesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.Progress in tree biotechnology initially trailed behind agricultural crops due to their long life cycle, difficult tissue culture and regeneration protocols, and their abundance in natural forests. However, rapid global deforestation rates, together with an increased world demand for pulp, paper and timber products, have prompted scientific and commercial focus to improve genetic timber stocks. South Africa, a tree-poor country (where indigenous forests are protected), has relied almost solely on exotic plantations to meet its demand for timber. A pioneer study investigating the feasibility of using direct (biolistic) and indirect (Agrobacterium-mediated) methods for gene transfer was undertaken in Pinus patula Schiede et Deppe, a Mexican softwood and a forerunner for saw timber,
pulpwood and paper in the South African forest industries. The aim of the
transformation methods was to impart herbicide resistance to the trees. This was achieved via the introduction of a bar-GUS pAHC25 cassette under the control of the ubiquitin promoter. To provide target material for transformation, two in vitro micropropagation pathways were used: somatic embryogenesis and organogenesis. Both embryonal suspensor masses (ESM) and somatic embryos at various stages of development were initially used as target explants for the biolistic study using an
established in vitro protocol. A stepwise selection was implemented in order to allow transformed (particularly bombarded) cultures the opportunity to regenerate under selection pressure using MSG3 maintenance medium supplemented with BASTA® herbicide at 1 mg l ¯¹ followed by 3 mg l¯¹ active ingredient at the next subculture. Biolistic transgene delivery was more efficient when sorbitol was included in the pre-bombardment medium enabling use of higher vacuum and shooting pressures, without lowering the regeneration potential of ESM significantly. Bombarded material from two genotypes (Lines 2 and 3) was regenerated to produce mature somatic embryos using an optimized regeneration
regimen. The indirect study with Agrobacterium tumefaciens (LBA4404),
transformed with the pAHC25 vector via triparental mating or heat shock, used a variety of target tissues including: mature somatic embryos, ESM and mature zygotic embryos (MZE's) - a novel in vitro system for P. patula. The Agrobacterium-mediated method resulted in optimized decontamination conditions using a combination of liquid MSG3 (or sterile dH₂O for mature embryos) supplemented with 500 mg l ¯¹ cefotaxime, with rotation, and sterile 65 mm Whatman No. 3 filter paper stacks, which avoided excess filtering and stress to transformation material. Further efforts to aid regeneration during the indirect study included L-proline post-transformation, though no mature somatic embryos were regenerated at the conclusion of the Agrobacterium-mediated study. Recovery of transformed ESM in both studies was best during the active growth
phase 4-6 d after subculture. Regeneration with good somatic embryo potential was an exigent aspect in both transformation studies. Expression of positive histochemical GUS activity in all transformed material was
confirmed by polymerase chain reaction (PCR) analysis indicating that Pinus
patula tissue was amenable to transformation. A new bar PCR regime was
implemented in P. patula. In the biolistic study, a higher transformation efficiency of bar amplicons (53%) than GUS amplicons (45%) was observed, reflecting their non-linked status on the pAHC25 transformation vector. This is the first report of biolistic transformation of P. patula that will allow for the production of transgenic ESM. The production of transgenic P. patula holds great promise for commercial development in the South African forestry industry. The application of transgenic trees in the timber industry is numerous but the aims most relevant to P. patula include wood modification and disease resistance to pathogens like pitch canker fungus
Molecular basis of anthocyanin production in callus and cell cultures of Oxalis reclinata.
No full textThesis (M.Sc.)-Univesity of Natal, Pietermaritzburg, 1996.Oxalis reclinata Jacq., is a dicotyledonous plant. O. reclinata belongs to the family Oxalidaceae. This plant produced callus which accumulated red coloured
anthocyanin pigments when cultured in vitro. The levels of anthocyanin
accumulated by O. reclinata callus were higher than in the intact plant. The
major pigment was isolated and identified as cyanjdin-3-glucoside (CROUCH,
VAN STADEN, VAN STADEN, DREWES & MEYER, 1993). In nature,
anthocyanins are responsible for orange, red, purple and blue colouration of
certain tissues of higher plant s. Due to the toxicity of many synthetic red
colouring agents, anthocyanins are regarded as potential substitutes for
synthetic food colourants. This research was aimed at investigating
mechanisms which induce pigment production as well as to optimize
anthocyanin yield from callus cultures of O. reclinata, once anthocyanin
production was stimulated.
Pigmented and non-pigmented callus lines were generated from O. reclinata
(CROUCH & VAN STADEN, 1994) and maintained on MURASHIGE & SKOOG
(1962) agar medium (O.8% [w/v], pH 5.7) supplemented with 0.5 mgℓ ¯¹ BA,
5 mgℓ ¯¹ NAA, 30 gℓ ¯¹ sucrose and 0.1 gℓ ¯¹ myo-inositol. Plant tissue culture
studies were conducted on red and white lines of O. reclinata to optimize callus
yield and anthocyanin production in vitro. This involved manipulating
contributory factors of the culture environment (carbohydrates, nitrates,
phosphates, phytohormones, light and temperature).
In vitro studies showed that, light played an inductive role in anthocyanin
production in callus cultures of O. reclinata. The auxin, 2,4-
dichlorophenoxyacetic acid (2,4-D) reduced pigment production but increased
callus biomass. This hormone probably exerted its effect by reducing the pool
of anthocyanin precursors, such as phenylalanine, resulting in increased primary
metabolic activity. Suspension cultures were shown to be a viable means of
propagating pigmented callus cells of O. reclinata. The growth curves for red
and white callus cells were determined using the settled cell volume (SCV) method. Pigmented cell cultures grew for longer periods compared to nonpigmented
cells of O. reclinata. White callus cells reached the stationary phase
after 18 days. Red callus cells continued growing exponentially for an extra
three days compared to white callus cells. The vacuole was identified as the
organelle where anthocyanins accumulate using the light microscope.
The molecular techniques of two-dimensional electrophoresis and in vitro
translation were utilized to analyze differences in gene expression between
white and red callus cultures of O. reclinata. Thus far, two-dimensional
electrophoresis has shown that the red callus of O. reclinata had more
polypeptides compared to the white callus. The level of gene expression was
higher in the red callus compared to white callus, as revealed by nonradioactive
in vitro translation. With optimization of radioactive in vitro
translation, identification of specific structural anthocyanin genes which are
under regulatory control should be possible.
Future research should aim at acquiring a better understanding about the
genetic control of anthocyanin biosynthesis in order to manipulate this pathway
effectively
In vitro conservation of endangered Dierama species.
Get PDFThesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.No abstract available
Molecular analyses of Salvia Africana-Lutea L. transgenic hairy root clones for secondary bioactives
No full textThesis (MSc)--Stellenbosch University, 2009.ENGLISH ABSTRACT: Biotechnological applications are useful for adding value to the local medicinal plants
and may provide an alternative source of pharmacologically-active compounds thus
assisting with the conservation of wild populations. Salvia africana-lutea aromatic herb
has long been used in folk medicine by traditional healers in the Western Cape Province
(South Africa) for various ailments. As an in vitro conservation strategy, a continuous S.
africana-lutea shoot culture was successfully established in solid MS medium containing
BA (0.5 mg L-1) and NAA (0.2 mg L-1). The regeneration rate of the S. africana-lutea
plants was high which produced approximately 720 plantlets in 20 culture bottles over a
four week cycle. The microshoots were rooted in the MS medium without PGRs prior to
acclimatisation. A survival rate of 92% was recorded for the greenhouse-acclimatised
shoots.AFRIKAANS OPSOMMING: Biotegnologiese toepassings is nuttig vir waarde toevoeging tot inheemse medisinale
plante en kan ‘n alternatiewe bron van farmakologies aktiewe verbindings verskaf wat
bydrae tot die bewaring van populasies in die natuur. Die aromatiese krui Salvia.
Africana-lutea is reeds vir ‘n lang tydperk in volks medisyne deur tradisionele
geneesheers in die Wes Kaap provinsie (Suid Afrika) vir ‘n verskeidenheid kwale
gebruik. ‘n Kontinu S. africana-lutea lootkultuur in soliede Murashige en Skoog (1962)
(MS) media wat BA (0.5 mg L-1) en NAA (0.2 mg L-1) bevat, is suksesvol as ‘n in vitro
konservasie strategie ontwikkel. Die regenerasie tempo van die S. africana-lutea plante
was hoog en het ongeveer 720 plante in 20 kultuur bottels tydens ‘n vier week siklus
gelewer. Die mikrolote is op plant groei reguleerder vrye MS media gewortel voordat
plante geaklimatiseer is. ’n Oorlewingstempo van 92% is vir die glashuis
geaklimatiseerde lote waargeneem.Master
In vitro growth and development of the sweet medicinal plant Stevia rebaudiana Bertoni
Get PDFThesis (MSc)--Stellenbosch University, 2017.ENGLISH ABSTRACT: Stevia rebaudiana Bertoni is a plant native to the Amambay region located north-east of Paraguay in South America. S. rebaudiana is a natural, sweet perennial herb that contains ent-kaurene diterpene glycosides in its leaves. There are over 9 ent-kaurene diterpene glycosides and stevioside is the most abundant but rebaudioside A is the sweetest. S. rebaudiana also commonly known as Stevia is recognized to have great economic and scientific value around the world due to its sweetness and reported therapeutic properties. As a result, it is cultivated commercially in certain parts of the world. This, however, excludes southern Africa. South Africa has an opportunity to cultivate S. rebaudiana as a new crop for the agricultural sector. The aim of this study was to establish a protocol to determine the best treatment for optimal seed germination using acid scarification, smoke-water, a combination of acid scarification and smoke-water and gibberellic acid. To study the macronutritional requirements of S. rebaudiana plants utilizing nitrogen and phosphate manipulation in vitro. To determine if in vitro derived plant extracts differ in metabolite profiles regarding the main bio-actives (diterpene glycosides) using a metabolomic approach that involved the application of LC-MS and GC-MS technology. To determine the effects of drought and salinity stress on the growth of S. rebaudiana using different concentrations of (w/v) polyethylene glycol 6000 (PEG 6000) and sodium chloride (NaCl) as osmotica.
This plant exhibits a low seed germination rate which is a great challenge towards large scale propagation thus making its production expensive. Using a tissue culture system as a propagation study tool, germination of Stevia seeds was tested using 1% (w/v) 2, 3, 5-triphenyl tetrazolium chloride solution in this study. This showed a low viability of 19%. S. rebaudiana seeds were subjected to four variables namely: smoke water extract, chemical scarification using 70% (v/v) sulfuric acid for 30 seconds, a combination of smoke water extract and 70% (v/v) sulfuric acid and gibberellic acid were tested as a means of improving germination in vitro. The smoke treatment was highly efficacious in producing a significant germination percentage (P < 0.05) while seeds scarified using 70% (v/v) H2SO4 had the lowest germination rate.
To test the effect of macronutrients (nitrogen and phosphate), various levels of nitrogen and phosphate were added to the growth medium. Thereafter, liquid chromatography-mass spectrometry was used to analyze the effects on the metabolomic profile. All other 85 nutritional elements were kept similar to the control which contained similar concentrations as Murashige and Skoog (1962) medium (MS) with both nitrogen (NH4NO3 at 20.61 mM and KNO3 at 18.79 mM) and phosphate (KH2PO4 at 1.25 mM). Two distinct clusters were revealed after principal component analysis of the metabolite profiles. The orthogonal partial least squares discriminant analysis was also applied. This allowed the organization of the clusters into two distinct groups. Steviol hydrate, stevioside hydrate and rebaudioside A contributed significantly to the distinct separation of phosphate-treated plants from the nitrogen-treated plants. The clustering suggests different chemical influences at enzyme and gene level on secondary metabolism resulting in different chemical profiles. Reducing the nitrogen level to half (0.5 N) in the MS medium led to the tallest plants. Reduction in the roots was observed with increasing levels of nitrogen and phosphate. I further assessed the effects of drought and salinity stress by using polyethylene glycol 6000 (PEG) and sodium chloride (NaCl) at different concentrations, respectively. Higher concentrations of PEG 6000 (7.5 and 10%) and NaCl (75 and 100 mM) resulted in a decline in both ent-kaurene diterpene glycosides and terpenes present in the treated Stevia leaves. Headspace solid phase microextraction gas chromatography spectrometry revealed an abundance of α-pinene, β-pinene and sabinene in all treated plants except in the plants exposed to 10% PEG 6000 which showed no growth. The addition of PEG 6000 decreased the concentrations of rebaudioside A and stevioside significantly.
In conclusion, this study has revealed the importance of nitrogen and phosphate in the manipulation of ent-kaurene diterpene glycoside production in Stevia microplants, setting a platform to test these effects ex vitro.AFRIKAANSE OPSOMMING: Stevia rebaudiana Bertoni is ‘n inheemse plant van die Amambay streek, Noord-Oos van Paraguay in Suid-Amerika. S. rebaudiana is ‘n natuurlike soet, meerjarige krui wat ent-kaurene diterpeen glikosiedes bevat in die blare. Daar is meer as 9 ent-kaurene diterpeen glikosiedes waarvan steviosied die mees volop is, maar rebaudiosied A is die soetste. S. rebaudiana (Stevia) is bekend vir sy merkwaardige ekonomiese en wetenskaplike waarde wêreldwyd, as gevolg van sy soetheid en berigte terapeutiese eienskappe. As gevolg word dit kommersiëel vervaardig in sekere dele van die wêreld. Hierdie sluit alhoewel nie Suid-Afrika in nie, en dus is daar ‘n geleentheid vir vervaardiging van S. rebaudiana as ‘n nuwe kropgewas in die lanbousektor.
Die doel van hierdie studie was om 'n protokol op te stel vir die beste behandeling vir optimale ontkieming met behulp van suurbeskadiging, rookwater, 'n kombinasie van suurbeskadiging en rookwater en gibberelliensuur. Om die makronutriënt vereistes van S. rebaudiana plante te bestudeer deur gebruik te maak van in vitro stikstof en fosfaat manipulasie. Om te bepaal of plant ekstrakte wat in vitro versamel is, verskil in metaboliet profiele met betrekking tot die belangrikste bio-aktiewe molekules (diterpeen glikosiede) met behulp van 'n metabolomiese benadering wat die toepassing van LC-MS en GC-MS tegnologieë gebruik. Om die uitwerking wat droogte en soutgehalte stres het op die groei van S. rebaudiana te bepaal met behulp van verskillende konsentrasies van (w/v) poliëtileenglikol 6000 (PEG 6000) en natriumchloried (NaCl) as osmotika.
Hierdie plant het laë ontkiemings-sukses, wat ‘n groot uitdaging is vir groot-skaal boerdery, en as gevolg produksie duur maak. Na aanvang van ‘n tetrasolium toets met gebruik van 1% (w/v) 2, 3, 5-trifeniel tetrasolium chloried oplossing, was ‘n weefselkultuur sisteem gebruik as ‘n wetenskaplike werktuig om ontkieming-sukses van Stevia saad te toets. Lewensvatbaarheid na hierdie toets was slegs 19%. S. rebaudiana sade was behandel met vier veranderlikes naamlik: rookwaterekstrak; chemiese skade met gebruik van 70% (v/v) swaelsuur vir 30 sekondes; ‘n kombinasie van rookwaterekstrak en 70% (v/v) swaelsuur and gibberelliensuur. Hierdie veranderlikes was getoets as ‘n metode vir die verbetering van in vitro ontkieming. Die rook behandeling was hoogs effektief met ‘n beduidende ontkieming persentasie (P < 0.05), terwyl saaddoppe wat verswak was met 70% (v/v) swaelsuur, die laagste ontkiemings-sukses gehad het.
Om die effek van makronutriënte te toets, was verskeie vlakke van stikstof en fosfaat by die groeimedium gevoeg. Dit was gevolg deur vloeistofchromatografie-massaspektrometrie te gebruik om die effekte op die metaboliese profile te ontleed. Al die ander 85 voedingstowwe was behou soortgelyk aan die kontrole, wat dieselfde konsentrasie Murashige and Skoog (1962) medium (MS) met albei stikstof (NH4NO3 van 20.61 mM en KNO3 van 18.79 mM) en fosfaat (KH2PO4 van 1.25 mM). Twee onderskeie groepe was merkbaar na hoofkomponentanalise van die metaboliese profiele. Die ortogonale gedeeltelike kleinste kwadrate diskriminantontleding was ook gebruik. Dit het die groepering van twee onderskeie groepe moontlik gemaak. Steviol hidraat, steviosied hidraat en rebaudiosied A het beduidende bedrae tot die verdeling van fosfaat-behandelde plante van die stikstof-behandelde plante. Die groepering stel verskeie chemiese invloede op ensiem en geen vlak vir sekondêre metabolism, wat lei tot unieke chemiese profiele. Die halvering van die stikstof konsentrasie (0.5 N) in die MS medium, het gelei tot die hoogste lengte plante. Verminderde wortellengte was waargeneem deur verhoogde vlakke van stikstof en fosfaat. Die effekte van droogte en soutgehalte stres was geëvalueer deur gebruik van poliëtileenglikol 6000 (PEG) en natriumchloried (NaCl) teen verskeie konsentrasies, onderskeidelik. Hoër konsentrasies van PEG 6000 (7.5 en 10%) en NaCl (75 en 100 mM) het gelei tot ‘n vermindering in beide ent-kaurene diterpeen glikosiedes en terpene teenwoording in Stevia blare. Kopspasie vastestoffase mikroekstraksie gaschromatografie spektrometrie het ’n oorvloed van α-pineen, β-pineen en sabineen in alle behandelde plante waargeneem behalwe dié blootgestel aan 10% PEG 6000, wat geen groei aangetoon het nie. Die byvoeging van PEG 6000 het die konsentrasies van rebaudiosied A en steviosied drasties verminder.
Hierdie studie het die belang van stikstof en fosfaat in die manipulasie van ent-kaurene diterpeen glikosied vervaardiging in Stevia mikroplante bewys, wat ‘n platform skep om hierdie effekte ex vitro te toets
Metabolomic profiling and micropropagation of Salvia africanalutea L. potent against Fusarium verticillioides
Get PDFThesis (PhD)--Stellenbosch University, 2014.ENGLISH ABSTRACT: Salvia africana lutea is one of 26 Sage species indigenous to Southern Africa from a total of 900
worldwide. The genus Salvia belongs to the Lamiaceae family. Labeled a ‘broad spectrum remedy’ S.
africana lutea amongst other sage species is medicinally important. Reports are many highlighting its
benefits, which include from alleviating coughs and colds to gynaecological complaints. Studies have
revealed in vitro antimicrobial, anti-cancer and antioxidant activity.
Plant secondary metabolites fundamentally have a strong bearing on the phytochemical activities a
plant may possess. Consequently the environment indirectly affects the phytochemical properties as it
influences the variation in the plant metabolome via plant-environment interactions. Five S. africana
lutea plant populations, within the Western Cape province of South Africa were sampled and
chemotypes and bioactivity tested. Four populations were wild growing in protected areas namely;
Brackenfell, Koeberg, Silwerstroomstrand and Yzerfontein, while the fifth was a garden growing
population from Stellenbosch.
Using gas chromatography hyphenated with mass spectrometry (GC-MS), compounds such as
monosaccharides, carboxylic acids and fatty acids were detected. Variation of compounds identified
with 80% certainty was compared across all populations. Stellenbosch population showed some
compounds that were not present in the other four sites. These compounds were namely; propanoic
acid, rythronic acid, 2-keto-1-gluconic acid and 1,3-dibromobicyclon, while this population also did not
have xylitol that was detected in all the other four populations. To consolidate the GC-MS findings, analysis on the metabolite profiles (utilizing liquid
chromatography linked with mass spectrometry (LC-MS) and nuclear magnetic resonance (1H NMR))
was done. Principal component analysis (PCA) was applied to the NMR data. The partial least squaresdiscriminant
analysis (PLS-DA) was used to integrate LC-MS and NMR data sets. All statistics were
performed with the SIMCA-P+ 12.0 software. By integrating LC-MS and 1H NMR analyses, large
chemotype differences leading to samples grouping by site, suggested strong plant-environment
interactions as factors influencing metabolite composition. Signals distinguishing the Stellenbosch
profile were in the aromatic part of the 1H NMR spectra. Antimicrobial activity was tested against two Fusarium species. Fusarium is a plant pathogenic species
that causes large agricultural losses particularly in the maize crop, one of the staple foods in the African
continent. Some species also produce mycotoxins in infected crop and lead to a significant increase in
the risk factor of cancers when contaminated foods are consumed. Four high-mycotoxin producing
strains from two species F. verticillioides (MRC 826 and MRC 8267); F. proliferatum (MRC 7140 and
MRC 6809) were utilized in all in vitro antifungal assays in this study. A preliminary assay using
dichloromethane: methanol (1:1 v/v) crude plant extracts of the five populations; Stellenbosch,
Brackenfell, Koeberg, Silwerstoomstrand and Yzerfontein, from 2009 and 2011. All test samples
exhibited good activity as the minimum inhibitory concentrations (MIC) values ranged from 0.031 mg
ml-1 to 0.5 mg ml-1, values below the latter are regarded as strong inhibitors. The Stellenbosch extracts
were the most active for both 2009 and 2011 collections, with the best activity against F. verticillioides
MRC 8267 and MRC 826 at 0.031 mg ml-1. While the least activity, albeit still a strong inhibitor, was
observed from the Yzerfontein extracts with an MIC value of 0.5 mg ml-1. Generally comparison
between the two years revealed that samples collected in 2011 were more potent than those in 2009,
possibly due to prolonged storage that may have resulted in chemical decomposition. As the Stellenbosch population had shown the best activity as well as a relatively different chemical
profile, leaves from these plants were then introduced into tissue culture conditions. Leaf explants were
placed on solid plant growth regulator (PGR)-free Murashige and Skoog media and that supplemented
with hormones in various combinations. (two concentrations of benzyl adenine (BA) utilized
individually and in combination with naphthalene acetic acid (NAA) 4.4 and 8.8, while for NAA 0.27,
2.7 and 5.4.) Namely: 4.4 BA, 8.8 BA, 0.27 NAA: 4.4 BA, 2.7 NAA: 4.4 BA, 5.4 NAA: 4.4 BA, 2.7
NAA: 8.8 BA and 5.4 NAA: 8.8 BA. The PGR combinations did not induce shooting nor rooting, only
callus on PGR-free MS media. Antifungal activity of the callus extract was in the same range as the
whole plant extracts from which the leaf explants were harvested from, showing no ‘loss’ of activity
after introduction to tissue culture conditions. Metabolite profiles using LC-MS, however, did reveal
qualitative and quantitative differences, though they appear to not have any bearing on the activity.
A bioassay-guided fractionation was then conducted on samples collected from Stellenbosch. This led
to the identification of carnosol and carnosic acid being involved in the anti-Fusarium activity of S
africana-lutea. A combinational study revealed no synergistic activity of the two compounds against
four Fusarium test strains, with fractional inhibitory concentration (FIC) values of 1.5 and 3.0. Antifungal activity of carnosol and carnosic acid was observed to be in the same range (strong
inhibitor) as was the callus and whole plant extracts.
The study showed variation in population chemotypes and identified two compounds that are involved
in S. africana-lutea activity against Fusarium species. It also provided a tissue culture system onto
which mass production of the two bioactives may be achieved from, in the development of new
fungicides.AFRIKAANSE OPSOMMING: Salvia Africana lutea is een van die 26 Salie spesies wat inheems aan Suid-Afrika is uit ‘n totaal van
900 spesies wêreldwyd. Die genus Salvia hoort tot die Lamiaceae familie. S. africana lutea word
geklassifiseer as ‘n “breë spektrum geneesmiddel”, en medisinaal as belangrik geag tussen die ander
salie spesies, want volgens verslag word dit gebruik vir die verligting van hoes en verkoues tot selfs
ginekologiese ongesteldhede. Definitiewe biologiese aktiwiteit eksperimente het anti-mikrobiese, antikanker
en anti-oksidant aktiwiteite aan die lig gebring.
Plant sekondêre metaboliete het fundamenteel ‘n baie sterk verband met die fitochemiese aktiwiteite
van ‘n plant. Gevolglik affekteer die omgewing indirek die fitochemiese eienskappe, want dit
beïnvloed die variasie in die plant metaboloom deur die interaksies van die plant met die omgewing. In
vyf streke binne die Wes-Kaap van Suid-Afrika waar S. africana lutea bevolkings voorkom, is
steekproewe gedoen en chemotipes en bioaktiwiteit getoets. Vier bevolkings was wild-groeiende
bevolkings in beskermde areas, naamlik; Brackenfell, Koeberg, Silwerstroomstrand en Yzerfontein,
terwyl die vyfde uit ‘n tuingroeiende bevolking in Stellenbosch geneem is.
Deur gas chromatografie gekoppel met massa spektrometrie te gebruik, is primêre samestellings soos
monosakkariede, karboksielsure en vetsure gevind. Variasies van samestellings wat met 80% sekerheid
geïdentifiseer is, is oorkruis met al die bevolkings vergelyk. Die Stellenbosch bevolking het ‘n paar
samestellings geopenbaar wat nie aanwesig was in die ander vier terreine nie. Hierdie samestellings
was: propanoësuur, erythroniese suur, 2-keto-1-glukoniese suur en 1,3-dibromobicyclon. Verder het
hierdie bevolking geen xylitol gehad nie en dit is in al vier die ander bevolkings gevind. Verdere studies was gedoen met die gebruik van vloeibare chromatografie gekoppel met massa
spektrometrie (LC-MS) sowel as kern magnetiese resonansie (1H NMR). Chemiese profiele het hoë
variasies getoon, en dus deur te fokus op die aromatiese samestelling streke, het die Stellenbosch
terrein duidelik merkbare verskille en punte op die PLS-DA aangetoon. Met die koppeling van NMR
data met LC-MS data, is daar gevind dat onderskeidende punte van die NMR PLS-DA wat gegroepeer
is met retensie tye die skeiding van die Stellenbosch terrein van ander terreine gedryf het. Dit het
onweerlegbaar bewys dat daar variasie binne die vyf bevolkings voorkom en dat Stellenbosch die mees
noemenswaardige chemotipe variasie het. Dit blyk uit die anti-mikrobiese eksperimente dat aktiwiteit teen Fusarium heel nuwe belangstelling
wek. Fusarium is ‘n plant-patogeniese spesie wat groot landbou verliese veroorsaak veral in die mielie
gewasse, een van die stapelvoedsels van die Afrika kontinent. Dit produseer ook mikotoksiene in
aangetaste gewasse en hierdie kan lei tot die ontstaan van kankers wanneer besmette voedsel op groot
skaal verbruik word. Vier hoë-mikotoksien produserende swamlyne van twee spesies, naamlik F.
verticillioides (MRC 826 en MRC 8267) en F. proliferatum (MRC 7140 en MRC 6809) is gebruik in
alle in vitro anti-swam ondersoeke in hierdie studie. Die eerste analise het dichloromethan: methanol
(1:1 v/v) ongesuiwerde plant ekstrakte bevat van die vyf bevolkings: Stellenbosch, Brackenfell,
Koeberg, Silwerstroomstrand en Yzerfontein, geneem gedurende 2009 en 2011. Al hierdie toets
monsters het goeie aktiwiteit getoon waar die minimum beperkende konsentrasie (MIC) waardes van
0.031 mg ml-1 tot 0.5 mg ml-1 gevarieer het. Waardes laer as laasgenoemde word beskou as sterk
inhibeerders. Die Stellenbosch ekstrakte was die mees aktief vir albei jare hierbo genoem, met die
beste aktiwiteit teen F. verticillioides MRC 8267 en MCR 826 by 0.031 mg ml-1. Die minste aktiwiteit
(hoewel nog ‘n sterk inhibeerder) waargeneem was van die Yzerfontein ekstrakte, met ‘n MIC waarde
van 0.5 mg ml-1. Oor die algemeen het ‘n vergelyking tussen die twee jare aangetoon dat die monsters
wat in 2011 versamel is veel sterker was dan dié van 2009, moontlik te wyte aan ‘n verlengde
bewaringstyd wat moontlik ‘n chemiese dekomposisie ten gevolge gehad het. Omdat die Stellenbosch bevolking die beste aktiwiteit getoon het sowel as ‘n relatief afwykende
chemiese profiel, is blare van hierdie plante toe bekendgestel aan weefselkultuur kondisies. Blaar
eksplante is op soliede hormoonvrye Murashige en Skoog media geplaas en dit is aangevul met
sintetiese auksien Naftaleen asynsuur (NAA) en sitokien Bensiel adenien (BA) individueel en in
verskillende kombinasies. Geen wortels of uitloopsels is waargeneem in al die hormoon kombinasies
nie maar in die hormoonvrye media het daar egter Kallus in twintig persent van die eksplante
voorgekom. Kallus is toe as subkultuur van hormoonvrye MS media gekweek en saamgevoeg en
dichloromethan: methanol (1:1v/v) ekstrakte is getoets teen die volgende Fusarium swamlyne MRC
826; MRC 8267; MRC 7140 en MRC 6809. MIC waardes het sterk inhiberende eienskappe getoon met
die laagste waarde as 0.025 mg ml-1 teen drie swamlyne: MRC 1740, MRC 8267 en MRC 826, en die
hoogste was 0.25 mg ml-1 na 48 uur. Die minimum inhiberende konsentrasie waardes het gestyg na
0.5mg ml-1 na 60 uur, wat ‘n fungistatiese aksie getoon het. Maar van 60 tot 92 uu het waardes egter ‘n
swamdodende aksie aangetoon met geen verandering van 0.5mg ml-1 nie. In die identifisering van die bioaktiewe komponente, is die ekstraksie van Stellenbosch se bevolking in
dichloromethan: methanol (1:1 v/v) uitgevoer, en met gebruik van vyftig gram van die ekstrak is
bioanalise-geleide fraksionering gedoen deur gebruik van ‘n VersaFlash®. Die mees aktiewe fraksie is
verder gefraksioneer deur die gebruik van ‘n konvensionele silikajel kolom. Aktiewe fraksies is getoets
deur LC-MS te gebruik, en twee verbindings, carnosol en carnosic suur, is geïdentifiseer.
Voorbereidende TLC is gebruik om identiteit te bevestig, want fraksies was naas die kommersiele
standaarde van die twee verbindings getoets. Sinergistiese aktiwiteit van die twee samestellings is
ondersoek deur ‘n antiswam ontleding teen die vier swamlyne uit te voer.
Hierdie studie het dus die veronderstelde bestaan van verskillende chemotipes tussen die bevolkings
waarvan voorbeelde geneem is, bekend gestel. Veral die Stellenbosch se bevolking het die meeste
verskil, heel moontlik omrede die verlengde en hoër versteurings deur die nabyheid van mense. Plant–
omgewing interaksies speel ‘n belangrike rol in die metaboloom van plante, wat dan indirek hul
eienskappe verander, en in hierdie geval die antiswam aktiwiteit. Die tuingroeiende bevolking was die
mees aktief, heel moontlik omrede hierdie aspek. Nietemin was geen bioaktiwiteit verloor waar die
mees kragtige bevolking met weefsel kultuur kondisies in aanraking gebring is nie. Dus is dit ideaal vir
kommersialisering. Een nuwe belangrike bevinding was die carnosol en carnosic suur wat twee
welbekende samestellings is wat meesal geassosieer is met Rosmarinus officinalis en gedokumenteer is
vir antioksidant aktiwiteit. Hier dui laasgenoemde samestellings ‘n antiswam aktiwiteit aan teen die
getoetste Fusarium swamlyne. Met ‘n gevestigde weefsel kultuur sisteem alreeds in plek, voorsien dit
‘n beginpunt vir die bestudering van hoe hierdie bioaktiewe komponente in massa geproduseer kan
word in die ontwikkeling van nuwe swamdodende produkte.Doctora
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