Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Sutherlandia frutescens (L.) R. Br., also regarded as Lessertia frutescens, is a leguminous, perennial shrub indigenous to South Africa. Extracts prepared from the leaves have traditionally been used for the treatment of various diseases. Reports have also indicated that S. frutescens provides certain health benefits to cancer and HIV/AIDS patients. Analysis of extracts indicated the presence of several compounds (bitter triterpenoid glycosides, several flavonoids, amino acids, small amounts of saponins (no alkaloids though), asparagine, Larginine, canavanine, gamma-aminobutyric acid (GABA) and pinitol) which contribute to the medicinal properties of this plant. The first part of this study involved testing the effect of six treatments (light, dark, soaking of seeds, physical scarification, chemical scarification and flaming of seeds) on the in vitro germination of Sutherlandia seeds to elucidate the factors which control seed germination. Those treatments which removed the seed coat were most successful for germination with physical scarification being the most efficient method, resulting in 98.6% of the seeds germinating after 21 days. Although the organogenesis of Sutherlandia explants (cotyledons and hypocotyls) in vitro were investigated (results not included in this thesis), omitting plant growth regulators (PGR) in the cultivation medium was best for shoot multiplication. However, this PGR-free system successfully provided a continuous supply of plant material for further studies. It would be possible to successfully adopt it for commercial production of plants to assist with cultivation of Sutherlandia as a field crop. Another advantage of this system is spontaneous rooting with 85% of the in vitro microshoots rooting in PGR-free medium. These rooted plants were acclimated in the glasshouse using vented lids to harden off the shoots and this method resulted in 100% survival of plants. The second part of this study investigated the induction of hairy root cultures of S. frutescens using Agrobacterium-mediated transformation. The efficiency of three Agrobacterium strains (A4T, LBA9402 and C58C1) to transform different S. frutescens explants (cotyledons and hypocotyls) was analyzed. All three strains were equally efficient at inducing hairy roots in both hypocotyls and cotyledons. However, transformation of S. frutescens was dependent on the type of explant used with the hypocotyls being more efficiently transformed than the cotyledons. Overall the transformation of both the hypocotyl (93%) and cotyledon (47%) was highest when the strain A4T was used. Four hairy root clones were selected and their cultivation in a liquid system was optimized by investigating their growth in four different types of media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) and half strength MS medium). All the growth of hairy root clones was best in the B5 and MS medium, with White’s medium being the least effective cultivation medium. Molecular analysis of hairy roots was used to prove the transgenic status of these four putative transgenic clones. This was achieved using polymerase chain reaction (PCR) amplification of rol A (320 bp), B (780 bp) and C (600 bp) genes to determine the presence of the TL-DNA in the plant genome. During Southern hybridization a radioactively labeled rol A probe was used to determine the copy number of the rol A gene. The three rol genes were present in all four hairy root clones. The third part of this study focused on the effect of three abiotic stress factors (nitrogen availability, salinity and drought) on the synthesis of four metabolites (gamma-aminobutyric acid (GABA), asparagine, arginine and canavanine). The effect of nitrogen availability on metabolite synthesis and the morphology was determined using in vitro shoot cultures as well as the hairy root clone C58C1-g. Nitrogen availability studies were conducted by cultivating the microshoots or root tips on modified MS medium. The MS medium contained either the normal amount of nitrogen (1.9 g L-1 KNO3 and 1.65 g L-1 NH4NO3) in the MS medium (1x nitrogen), half the normal nitrogen concentration in MS medium (0.5x nitrogen) or twice the normal nitrogen concentration in MS medium (2x nitrogen). The arginine and asparagine levels in the roots and shoots and the canavanine level in the shoots were directly correlated with the amount of nitrogen in the medium (as the nitrogen level increased, the metabolite levels increased). The GABA level in the shoots was inversely correlated with the amount of nitrogen in the medium. Several reasons may explain these metabolic changes including the assimilation of extra nitrogen into asparagine, canavanine and arginine in the shoots. The reduced GABA levels may indicate the preferential flux of the free GABA into other nitrogen assimilatory pathways such as protein synthesis as well as its rapid utilization to replenish the tricarboxilic acid cycle intermediates. The effect of water (induced by including 3% (w/v) PEG in the medium) and salt stress (induced by including either 50 or 100 mM NaCl in the medium) was only investigated in the shoot cultures as the root cultures lacked the synthesis of canavanine. Water stress did not significantly alter the metabolite levels, but resulted in a significant decrease in the growth (fresh weight and total shoot length) and the rooting response of these microshoots. Salt stress only resulted in a significant increase in arginine levels with increasing salinity and also caused a reduction in the rooting and growth response. Lowered plant vigour may be the first visual sign of water stress. Addition of NaCl may lead to ion toxicity and requires osmotic adjustment resulting in changes at the metabolic level concomitant to physiological growth changes. Finally, the anti-bacterial activity and the phytochemistry of transgenic root cultures and untransformed in vitro and ex vitro plant material was examined. Only the extracts prepared from the wild harvested leaf material exhibited moderate anti-bacterial activity (1.25 mg ml-1) against all the bacteria (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus) tested. Changes to the secondary metabolism of hairy roots were investigated using TLC and LC-MS analysis. Several of the compounds in the hairy root extracts were present in higher levels than in the control root extracts. Transformation also increased the complexity of the phytochemical pattern of the hairy roots, either due the synthesis of novel compounds or upregulated synthesis of existing metabolic pathways. The production of hairy roots and the establishment in a liquid system during this study was an important step towards upscaling these cultures to a bioreactor. In future these roots can assist in developing cultures which produce a high yield of the desired metabolites. / AFRIKAANSE OPSOMMING: Sutherlandia frutescens (L.) R. Br., ook bekend as Lessertia frutescens is ‘n peulagtige meerjarige struik, inheems tot Suid Afrika. Ekstrakte wat van die blare voorberei word, is tradisioneel gebruik vir die behandeling van verskeie siektes. Berigte het ook daarop gedui, dat S. frutescens sekere gesondheidsvoordele vir kanker en HIV/VIGS pasiënte inhou. ‘n Ontleding van die ekstrakte, dui op die teenwoordigheid van verskeie verbindings (bitter triterpenoïed glikosiede, verskeie flavonoïede, aminosure, klein hoeveelhede saponiene (alhoewel geen alkaloïede), asparagien, L-arginien, canavanien, gamma-aminobottersuur (GABS) en pinitol) wat tot die medisinale eienskappe van hierdie plant bydrae. Die eerste deel van die studie het die effek van ses behandelings (lig, donker, week van sade, fisiese skarifikasie, chemiese skarifikasie en die vlam van sade) op die in vitro ontkieming van Sutherlandia sade getoets met die doel om die faktore wat saadontkieming beheer, te identifiseer. Die beste behandeling vir saadontkieming was dié behandelings wat die saadhuid verwyder het. Die mees effektiewe metode van saadhuidverwydering was die fisiese skarifikasie van sade, wat gelei het tot ‘n 98.6% ontkieming van sade na 21 dae. Alhoewel in vitro organogenese van Sutherlandia eksplante (kotiel en hipokotiel) ondersoek was (resultate nie ingesluit in die tesis nie), was plant groei reguleerders (PGR) uitgesluit in die groeimedium om stingelvermeerdering te bevorder. Nie te min was die PGR-vrye sisteem suksesvol om ‘n voortdurende bron van plant material vir verder studies te verskaf. Dit sou egter moontlik wees om die PGR-vrye sisteem suksesvol te kon aanpas vir die kommersiële produksie van plante met die doel om Sutherlandia as ‘n landbougewas te bevorder. ‘n Verdere voordeel van dié sisteem, is die spontane wortelvorming, met 85% van die in vitro mikrostingels wat wortels in die PGR-vrye medium produseer het. Hierdie bewortelde plante was in die glashuis geakklimatiseer met behulp van geventileerde deksels (vir stingel afharding) en het tot ‘n 100% oorlewing gelei. Die tweede deel van die studie het die induksie van S. frutescens harige wortelkulture met behulp van Agrobacterium-bemiddelde transformasie ondersoek. Die effektiwiteit van drie Agrobacterium stamme (A4T, C58C1 en LBA9402) om verskillende S. frutescens eksplante (kotiel en hipokotiele) te transformeer, was geanaliseer. Al drie stamme was ewe effektief om harige wortels op beide hipokotiel en kotiele te induseer. S. frutescens transformasie blyk egter tog van die tipe eksplant afhanklik te wees, aangesien die hipokotiele meer effektief as die kotiele getransformeer kon word. Met inagneming van beide die hipokotiel (93%) en kotiel vii (47%), was transformasie optimaal met die gebruik van die A4T stam. Vier harige wortelklone was geselekteer en hulle produksie in ‘n vloeibare sisteem was geoptimiseer deur hulle groei in vier verskillende tipe media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) en half-sterkte MS medium) te ondersoek. B5 en MS medium was beskou as die beste vir alle die harige wortelklone se groei, terwyl White’s medium die minste doeltreffende groeimedium was. Molekulêre analise van die harige wortels was gebruik ten einde die transgeniese status van die vier vermoedelike transgeniese klone te bewys. Dit was behaal deur polimerase kettingreaksie amplifisering (PKR) van die rol A, B en C gene ten einde die teenwoordigheid van die TL-DNS in die plant genoom aan te toon. Tydens Southern hibridisasie was ‘n radioaktief gemerkte peiler gebruik om die aantal rol A geen kopieë te bepaal. Die drie rol gene was teenwoordig in al vier harige wortelklone. Die derde deel van die studie het gefokus op die effek van drie abiotiese stress faktore (stikstof beskikbaarheid, sout- en droogte stres) op die produksie van vier metaboliete (GABS, asparagien, canavanien en arginien). Die effek van stikstof beskikbaarheid op die metaboliet produksie asook die morfologie was bestudeer deur gebruik te maak van in vitro mikrostingels asook die harige wortel kloon C58C1-g. Stikstof beskikbaarheidstudies was uitgevoer deur die mikrostingels of wortelpunte in ‘n gewysigde MS medium te groei. Die MS medium was aangepas om die normale hoeveelheid stikstof (1.9 g L-1 KNO3 en 1.65 g L-1 NH4NO3) in MS medium (1x stikstof), of die helfte van die normale stikstof konsentrasie (0.5x stikstof) of twee keer die normale stikstof konsentrasie in MS medium (2x stikstof) te bevat. Die arginien en asparagien vlakke in die wortels en stingels, asook die canavanien vlak in die stingels was positief gekorreleerd aan die stikstof konsentrasie in die medium. Die GABS vlak in die stingels was egter omgekeerd eweredig aan die stikstof konsentrasie in die medium. Verskeie redes kan aangevoer word om die metaboliet veranderinge te verduidelik, insluitende die assimilasie van addisionele stikstof in asparagien, canavanien en arginien in die stingels. Die verlaagde GABS vlakke kan dui op die voorkeur van vrye GABS vloei na ander stikstofassimilerende metaboliese paaie soos proteïen sintese, asook die snelle benutting van GABS ten einde die Trikarboksielsuursiklus intermediêre produkte aan te vul. Die effek van droogte (geïnduseer deur die byvoeging van 3% (m/v) PEG tot die medium) en sout stres (geïnduseer deur 50 of 100 mM NaCl byvoeging tot die medium) was slegs in die stingel kulture ondersoek weens die afwesigheid van canavanien produksie in die wortel kulture. Water stres het nie ‘n betekenisvolle verandering in die metaboliet vlakke meegebring nie, maar dit het wel tot ‘n beduidende afname in groei (vars massa en totale stingel lengte) en bewortelingsreaksie in die mikrostingels gelei. Sout stres het slegs tot ‘n betekenisvolle viii toename in arginien vlakke asook ‘n afname in die wortelvorming en groeireaksie tydens die toenemende sout vlakke gelei. ‘n Verlaging in plant groeikragtigheid mag ‘n eerste visuele teken van water stres wees. Die toevoeging van NaCl tot die medium kan tot ioontoksisiteit lei en plante reageer deur middel van osmotiese aanpassing wat tot veranderinge in die metaboliet vlakke asook veranderinge in fisiologiese groei, lei. Die finale deel van die studie het die anti-bakteriële aktiwiteit en die fitochemie van die transgeniese wortel kulture asook die ongetransformeerde in vitro en ex vitro plant materiaal ondersoek. Slegs die ekstrakte verkry vanaf blaar materiaal geoes uit die natuur, het matige anti-bakteriële aktiwiteit (1.25 mg ml-1) teen al die bakterië (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis en Staphylococcus aureus) wat ondersoek is, getoon. Aanpassings in die sekondêre metabolisme van die harige wortels is deur middel van dunlaag chromatografie (DLC) en vloeibare chromatografie-massa spektroskopiese (VC-MS) analise ondersoek. Verskeie verbindings was in hoër vlakke in die harige wortels teenwoordig, as in die kontrole wortel ekstrakte. Transformasie het ook die kompleksiteit van die harige wortels se fitochemiese patroon verhoog, moontlik weens die produksie van nuwe verbindings of weens die opregulasie van bestaande metaboliese paaie. Die produksie van harige wortels en die vestiging daarvan in ‘n vloeibare sisteem tydens hierdie studie word beskou as ‘n belangrike stap na die opskalering van die kulture na bioreaktore. Hierdie wortels kan toekomstig tot die ontwikkeling van kulture met ‘n hoë produksie van gewenste metaboliete lei.

For centuries Traditional Health Practitioners (THPs) used their indigenous knowledge (IK) in conserving medicinal plants and environments to maintain sustainability. With the rapid environmental, social, economic and political changes occurring in many areas inhabited by rural people exist the danger that the loss of biodiversity from habitat destruction and unsustainable harvesting practices will result in some species becoming extinct. The main aim of the study was to determine the natural habitat of extinction-prone traditional medicinal plants combining the insight of THPs with an ultimate goal of guiding research for the conservation, propagation and cultivation of traditional medicinal plants. Despite problems, opportunities and challenges expressed and identified by THPs, the analysis of data from interview schedule and personal observations, show that the THPs' practices are shaped by historical processes and local cultural values, social norms and their management strategies that are influenced by a broad range of factors. / Agriculture, Animal Health and Human Ecology / M.A. (Human Ecology)

Spices are of great importance in the indigenous culinary and traditional medicine systems of the people of Nkonkobe Municipality of the Eastern Cape, South Africa. The present investigation evaluated the ethnopharmacological potentials of two indigenous South African spices- Foeniculum vulgare and Lippia javanica. The pharmacological investigations on these two plant species include ultra-morphology, nutrient and mineral analysis, evaluation of the essential oil, phytochemical and antioxidant assays, antimicrobial, anti-inflammatory as well as anti-urolithiatic assay of the acetone and aqueous extracts of the two plants. The species were selected for study as the most cited plants after an ethnobotanical survey conducted on the indigenous knowledge of plants used as spices and medicine in Nkonkobe Municipality, Eastern Cape of South Africa. Among the plants cited Foeniculum vulgare(Apiaceae) and Lippia javanica (Verbenaceae) stood out as the most commonly used spices. Others were members of the families; Solanaceae, Apiaceae, Amaryllidaceae, Amaranthaceae and Lamiaceae. Ultra-morphological studies conducted on the leaves of the two selected plants using scanning electron microscope revealed the presence of non-glandular and glandular trichomes, stomata and crystals. The leaf surfaces of these herbs may serve as secretory sites where aromatic secondary metabolites are produced. Analyses of the proximate, mineral, vitamin and anti-nutrients contents of these two spices showed that both species are good sources of these phytochemicals and may be used to enrich the human diet. Lippia javanica possesses lower lipid (0.50 percent), fibre (5 percent) and carbohydrate (64.96 percent) contents than Foeniculum vulgare. On the other hand, protein (20.54 percent), ash (11.60 percent) and moisture content (11.69 percent) were higher in Foeniculum vulgare than in Lippia javanica. Foeniculum vulgare showed higher N (3286 mg/100g), Mg (386.7 mg/100g), K (3187 mg/100g) and Na (1383 mg/100g) content while Lippia javanica was higher in Ca (1833 mg/100g), Zn (4.7 mg/100g), Cu (0.9 mg/100g) and Fe (78.4 mg/100g). Vitamins Aand E were also higher in Lippia javanica (1.31 mg/100g; 2.52 mg/100g) while Foeniculum vulgare (0.45 mg/100g) had higher vitamin C content. No significant differences were observed in the phytate, oxalate and tannin contents of the two spices, but saponin and cyanide were significantly lower in Lippia javanica (268.5 mg/100g; 8.45 mg/100g) than in Foeniculum vulgare (1855 mg/100g; 10.5 mg/100g) Evaluation of the essential oil component, cytotoxicity and antimicrobial activities of both fresh and dried leaves of Lippia javanica and Foeniculum vulgare revealed that the dried samples yielded more oil and also contain more chemical than the fresh samples of both plants. The overall antimicrobial activity evaluated using susceptibility and microdilution assays revealed that the oils of F. vulgare and L. javanica exhibited high antifungal and antibacterial activity, compared to the reference drugs. In addition, essential oil from fresh leaves of both spices was less toxic compared to the oil from the dried leaves. Polyphenolic evaluation of the aqueous and acetone extracts of the plants revealed that the acetone extract had higher phenol, flavanol, flavonoid and proanthocyanidin contents than the aqueous extracts. The total phenolic content of acetone and aqueous extracts were 4.49 ± 0.411 mg/g and 3.73 ± 0.498 mg/g tannic acid equivalent (TAE) respectively for Lippia javanica. The same trend was also observed in Foeniculum vulgare with total phenolic content of acetone and aqueous extracts were 4.22 ± 0.325 mg/g and 4.17 ± 0.651 mg/g tannic acid equivalent (TAE) respectively. Further assessment of the antioxidant activity of the solvent extracts revealed that both plants exhibited promising free radical scavenging potentials against 1,1 diphenyl-2-picrylhydrazyl (DPPH), 2, 2’-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid (ABTS), reducing power, lipid peroxidation, nitric oxide, phosphomolybdate and hydrogen peroxide. Antimicrobial activities of the acetone and aqueous extracts of the two plants revealed moderate antibacterial and antifungal activities. They inhibited the growth of Microsporium canis and Trichophyton rubrum that were not susceptible to the standard antifungal drug used as control.

Helichrysum longifolium and H. pedunculatum belong to the Astereceae family and are used extensively in folkloric medicine in South Africa to manage stress-related ailments and as dressings for wounds normally encountered in circumcision rites, bruises, cuts and sores. The in vitro antibacterial time-kill studies, the synergistic potentials, the phytochemical screenings and antioxidant potentials as well as the isolation of the bioactive compounds from the extracts of these two plants were carried out in this study. The in vitro antibacterial activities and time kill regimes of crude extracts of H. pedunculatum was assessed. The extracts was active against both Gram positive and Gram negative bacteria tested at a concentration of 10 mg/ml. Minimum Inhibitory Concentration (MIC) values for all the susceptible bacteria ranged between 0.1 – 35 mg/ml. The average log reduction in viable cell count in time kill assay ranged between 0.17 Log10 to 6.37 Log10 cfu/ml after 6 h of interaction, and between 0.14 Log10 and 6.99 Log10 cfu/ml after 12 h interaction in 1 × MIC and 2 × MIC of the extract. The effect of the aqueous extract was only bacteriostatic on both reference and environmental strains and the clinical isolates were outrightly resistant to aqueous extract. This is worrisome and this could be one reason why, there is an incidence of high death rate resulting from circumcision wounds infection even after treating such wounds with H. pedunculatum leaf. In vitro antibacterial time kill studies of extracts of H. longifolium was assessed. All test bacteria were susceptible to the methanol extract, while none was susceptible to the aqueous extract. Two of the test bacteria were susceptible to the ethyl acetate extract, while ten and seven were susceptible to the acetone and chloroform extracts respectively at the test concentration of 5 mg/ml. The minimum inhibitory concentrations (MICs) ranged between 0.1 and 5.0 mg/ml, while minimum bactericidal concentrations (MBCs) ranged between 1.0 and >5 mg/ml for all the extracts. Average log reductions in viable cell counts for all the extracts ranged between 0.1 Log10 and 7.5 Log10 cfu/ml after 12 h interaction at 1 × MIC and 2 × MIC. Most of the extracts were rapidly bactericidal at 2 × MIC achieving a complete elimination of most of the test organisms within 12 h exposure time. The effect of combinations of the crude extracts of H. pedunculatum leaves and eight antibiotics was investigated by means of checkerboard and time-kill methods. In the checkerboard method, synergies of between 45.83-56.81 percent were observed and this is independent of Gram reaction, with combinations in the aqueous extract yielding largely antagonistic interactions (18.75 percent). The time kill assay also detected synergy that is independent of Gram reaction with a ≥ 3Log10 potentiation of the bactericidal activity of the test antibiotics. We conclude that the crude leaf extracts of H. pedunculatum could be potential source of broad spectrum antibiotics resistance modulating compounds. The interactions between crude extracts of H. longifolium in combination with six first-line antibiotics using both the time-kill and the checkerboard methods were carried out. The time-kill method revealed the highest bactericidal activity exemplified by a 6.7 Log10 reduction in cell density against Salmonella sp. when the extract and Penicillin G are combined at ½ × MIC. Synergistic response constituted about 65 percent, while indifference and antagonism constituted about 28.33 percent and 6.67 percent in the time kill assay, respectively. The checkerboard method also revealed that the extracts improved bactericidal effects of the antibiotics. About 61.67 percent of all the interactions were synergistic, while indifference interactions constituted about 26.67 percent and antagonistic interactions was observed in approximately 11.66 percent. The in vitro antioxidant property and phytochemical constituents of the aqueous crude leaf extracts of H. longifolium and H. pedunculatum was investigated. The scavenging activity on superoxide anions, DPPH, H2O2, NO and ABTS; and the reducing power were determined, as well as the flavonoid, proanthocyanidin and phenolic contents of the extracts. The extracts exhibited scavenging activity in all radicals tested due to the presence of relatively high total phenol and flavonoids contents in the extracts. Our findings suggest that H. longifolium and H. pedunculatum are endowed with antioxidant phytochemicals and could serve as a base for future drugs. Bioactivity-guided fractionation of the leaves of H. longifolium and H. pedunculatum yielded two known compounds. From the n-hexane fraction of H. longifolium a compound was isolated (Stigmasterol) and from the ethyl acetate fraction of H. pedunculatum another compound (β-sitosterol) was isolated. The compounds were isolated and identified using various techniques. The antimicrobial, anti-inflammatory, antioxidant, analgesic and anti-pyretic activities of these compounds have been reported in literatures. In general, the experiments and tests conducted in this study appear to have justified the folkloric medicinal uses of H. longifolium and H. pedunculatum for the treatment of stress related ailments and wound infections and make a substantial contribution to the knowledge base of the use of herbal medicine for the treatment of the microbial infections.

Pelargonium sidoides DC. (Geraniaceae) root extracts are used in the Eastern Cape Province of South Africa as a traditional medicine for the treatment of respiratory tract and gastro-intestinal infections. Ethanolic extracts are used globally as herbal treatments for bronchitis, asthma and as an immune system booster. Despite documented exploitation of wild populations by illegal harvesters, this species has not been awarded a protected status. The high level of harvest in the years preceding this study prompted this investigation of the prospects for sustainable root harvest through wild harvest and greenhouse cultivation. A novel method was developed for the purification of umckalin, a bioactive constituent in root extracts, such that the root umckalin concentrations of wild and cultivated plants could be quantified by HPLC. As part of the cultivation experiments, the concentration of umckalin in roots was measured for plants across part of the species’ distribution range in the Eastern Cape Province. This survey revealed that root umckalin concentrations were inversely related to the average annual rainfall of the collection site (r² = 0.94, p = 0.007) and directly related to soil pH (r² = 0.97, p = 0.002). Thus, the possibility of inducing high umckalin concentrations in greenhouse-cultivated plants was investigated by subjecting plants to rapid and prolonged water stress treatments. Two leaf applied hormone treatments (cytokinin and gibberellin) and a root competition treatment with a fast growing annual (Conyza albida) were also investigated based on the potential function of umckalin in P. sidoides plants. These five treatments did not significantly affect root umckalin concentrations compared to well-watered controls. The results of further experiments suggested that umckalin production may have been influenced by the geographical origin and genetics of plants rather than environmental variation. Following wild harvest experiments, the regrowth of replanted shoots from which a standard proportion of the root was harvested showed that water availability affected shoot survival but not root regrowth rate. Regrowth rates were low, questioning the viability of wild harvest. In contrast, greenhouse cultivated plants showed ca. six times greater growth rates, supporting the cultivation of roots to supply future market demand.

Secondary metabolites derived from plants, especially those used by traditional healers, are at the forefront of new drug development in combating diseases such as cancer and diabetes. Garlic is employed in indigenous medicine all over the world for the treatment of a variety of diseases. Dietary garlic has been recognized for its beneficial health effects. In particular, garlic consumption has been correlated with (i) reduction of risk factors for cardiovascular diseases and cancer, (ii) stimulation of immune function, (iii) enhanced detoxification of foreign compounds, (iv) hepatoprotection, (v) antimicrobial effects, (vi) antioxidant effects, and most importantly (vii) its hypoglycemic and anticoagulant properties. Due to these beneficial properties, garlic and its closely related genera which includes Tulbaghia violacea, may be useful as coadjuvant therapy in the treatment of type 2 diabetes and some of its physiological complications. The aim of this study was to determine if T. violacea has antidiabetic and anticoagulant properties. This was performed in vitro using both aqueous and organic extracts of the roots, leaves and bulbs. An organic extract was able to improve glucose-stimulated insulin secretion (GSIS) in INS-1 pancreatic β-cells and glucose uptake in Chang liver cells. The BO extract had no effect on the glucose uptake in 3T3-L1 an adipose cell line and reduced glucose utilisation in C2C12, a skeletal muscle cell line. Some of the properties displayed by T. violacea in this study are consistent with those found in similar studies with garlic extracts. It was observed that the BO extract increased the membrane potential and Glut-2 expression in INS-1 cells cultured at hyperglycemic levels, however, at normoglycemic levels a reduction was observed. The oxygen consumption increased at both glycemic levels due to treatment with the BO extract. Platelets were exposed to the extracts to determine their effects upon platelet aggregation, adhesion and protein secretion. Since the BO extract displayed the highest potential at inhibiting platelet aggregation and adhesion. A rat model was used in ex vivo studies to determine if the extract exhibited the same effect in a physiological model. It was noted that the BO extract exhibited a higher degree of inhibition on platelet aggregation and adhesion than the positive control, aspirin. The BO extract reduced clotting times in the prothrombin time (PT) test, but prolonged the clotting time in the actived partial thromboplastin time (APTT) assay in the ex vivo model; however, it had no affect on these clotting assays in the in vitro model using human blood. The BO extract increased the D-dimer and Fibrinogen-C levels in the in vitro model, but had no effect on the D-dimer concentrations and lowered the Fibrinogen-C levels in the ex vivo model. The active compounds in the extract remain to be elucidated.

The commercialisation of natural resources such as medicinal plants is a strategy that is increasingly being employed by poor people in developing countries such as South Africa, as a means of generating an income. Increasing economic hardship and a lack of employment opportunities in the formal sector of the economy has led to many urban dwellers seeking alternative means of meeting their everyday livelihood needs. Although the contribution of natural resources to subsistence economies is widely recognised, there are few studies that have examined the significance of the trade in medicinal plants to the livelihoods of poor and marginalised people. Thirty informal street traders were interviewed to investigate the role of the medicinal plant trade in sustaining the livelihoods of the traders who operate in the informal sector of the Nelson Mandela Bay Municipality, South Africa. The findings of the research indicate that the sale and collection of medicinal plants is an activity which is exploited by people with limited resources, few skills, little or no education and often without any other source of household income. Middle-aged women from impoverished, single income households dominated the trade. Although income from the trade is modest, it contributed substantially to the total household cash income of the traders. Other benefits of the trade included independence and having a sense of purpose in life, as the traders felt that they were not only helping their families but also the wider community. However, without the income from the trade, many of the traders and their families would be destitute and thus the medicinal plant trade is vital to the wellbeing of these people.

The ability of insulin to stimulate glucose uptake into muscle and adipose tissue is central to the maintenance of whole-body glucose homeostasis. Deregulation of insulin action manifests itself as insulin resistance, a key component of type 2 diabetes. Insulin resistance is also observed in HIV patients receiving protease inhibitors. An agent that can reversibly induce an insulin-resistant state would be a very useful tool in developing model systems that mimic the pathogenesis of type 2 diabetes. Insulin resistance can arise from defects in insulin signal transduction, changes in the expression of proteins or genes that are targets of insulin action, cross talk from other hormonal systems or metabolic abnormalities. Deterioration of the insulin-receptor-signalling pathway at different levels leading to decreased levels of signalling pathway intermediates and/or decreased activation through phosphorylation accounts for the evolution from an insulin-resistant state to type 2 diabetes. In addition, defects in GLUT4 glucose transporter translocation are observed, further fuelling impairments in skeletal muscle glucose uptake. Levels of insulin-induced GLUT4 translocation in the skeletal muscle of type 2 diabetic patients are typically reduced by 90%. Many cellular pathways & their intermediates are in some way or another linked to insulin signalling. This study focused on three of these namely the PI3-kinase/Akt pathway, the Mitogen Activated Protein Kinase (MAPK) cascade and the AMP Kinase pathway, with successful monitoring of the PI3-K pathway. Investigations involved observing and evaluating the effects of various compounds as well as an indigenous medicinal plant, Sutherlandia frutescens on the activities of key insulin signalling pathway intermediates within the three fore mentioned pathways including Akt, AMPK and MEK1/2 as well as membrane surface GLUT4 levels. Scientific research has in the past leant heavily on Western blotting as the method of choice for gaining vital information relating to signal transduction pathways, however for research into cellular mechanisms the negatives of this method outweigh the positives. The drawbacks include a need for large amount of cells, multiple washing steps which may be disadvantageous to any weak and transient interactions as well as lysing of cells which may interfere with the maintenance of the subcellular localisation of a specific signalling event. Based on these, the need for a better method in terms of speed & reliability to monitor phosphorylation states of signal transduction pathway intermediates & GLUT4 translocation was evident and was one VII of the main aims & successes of this study. The method created used the mouse muscle cell line C2C12 in conjunction with the quick, sensitive method of flow cytometry which allowed us to monitor these processes in these cells through immune-labelling. Adherent cell cultures such as the C2C12 cell line pose the problem of possible damage to plasma membrane receptors (including insulin receptors) during harvesting to obtain a cell suspension for flow cytometry. We however used C2C12 mouse myocytes to optimize a method yielding insulin responsive cells in suspension that were successfully used for flow cytometry after immunelabelling of insulin signalling intermediates. Insulin (0.1μM) significantly raised the levels of both P-Akt and GLUT4 above basal levels. This effect was shown to be dose dependent. At a concentration of 50μg/ml, Sutherlandia frutescens was able to act as an insulin-mimetic in terms of its ability to increase P-Akt levels, GLUT4 translocation and glucose utilisation in an acute manner. These increases could be reduced with the addition of wortmannin, a PI3-K inhibitor. Therefore, these results suggest the mechanism of the plant extract’s insulin-like activity may be in part due to the activation of the insulin signalling pathway leading to GLUT4 translocation, which involves the phosphorylation of insulin receptor- and subsequent PI3-K activity, leading to P-Akt activity. These results provide further evidence of this plant extract’s anti-diabetic potential. The effect of Sutherlandia frutescens on insulin secretion, calcium signalling and proliferation in INS-1 rat pancreatic cells was also investigated and it was found to increase the activities of all of these processes. However no change in the levels of GLUT2 glucose transporter was seen. Ritonavir is prescribed by the South African Department of Health in co-formulation with other protease inhibitors within its second regime in the treatment of HIV and AIDS. Using C2C12 cells, ritonavir decreased glucose uptake acutely and had no effect on GLUT4 translocation however surprisingly increased P-Akt levels. In conclusion, it was found that Sutherlandia frutescens has antidiabetic benefits, diverse in nature depending on tissue type as well as length of time administered. The establishment of novel flow cytometry techniques to assess antidiabetic properties using in vitro cell culture was achieved. These methods will be useful in the future for the assessment of insulin sensitivity and in the identification of novel compounds that stimulate the insulin signalling pathways.

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The chemical profiling, characterization of Aloe products and phytochemical properties of Aloe vera were studied. The adulteration of commercial products derived from medicinal plants has been a major muddle for both the society and the pharmaceuticalindustry. Economically motivated adulteration includes the potential for contaminated, sub‐potent or counterfeit medication to enter the supply chain at several levels, from the production of raw ingredients through to the point of retail sale. Darwin’s theory of evolution states that, species undergo genetic variation with time to adapt to environmental changes. Therefore, the same species growing in widely different habitats may drift from the original genetic makeup as a mechanism of adaptation and that may result in them having different chemical profiles. Therefore this study aimed at investigating the phytochemical properties of Aloe vera growing in South Africa. Also, this study aims to utilize Thin Layer Chromatography to profile this plant, as well as use Infra Red spectroscopy to characterize commercial Aloe vera products. A large quantity of Aloe vera plant was collected from AloeWay, Iphofolo Game Farm, Polokwane in the Limpopo province of South Africa. The identity of the plant was confirmedrom literature and authenticated by Professor DS Grierson of Botany Department, University of Fort Hare, Alice. The plant leaves were divided into two portions. One portion was extracted fresh while the other was cut into pieces and oven dried at 400C then and milled to a homogenous powder once dried completely. The phytochemical composition of the gel and leaf extracts revealed the presence of alkaloids, flavonoids, saponins, tannins and phenols at different concentrations. Results showed that the dry plant material yielded more phytochemicals than the fresh plant material. In particular, it was found that the acetone extract showed much more amounts ofphychemicals than the dichloromethane and aqueous extracts. The percentage compositions of phenols (71.86), flavonols (36.61), proanthocyanidins (82.71), saponins (37.73) and alkaloids (13.29) were significantly high in the acetone extract, followed by the dichloromthane extract with values of 46.85, 37.73, 49.51, 89.0 and 11.11 respectively, while the least composition was found in the aqueous extract. Furthermore, flavonoids were somewhat high in composition in both the aqueous extract of the dried and of the fresh plant material while others were very low. Tannins levels were significantly very low in all the solvent extracts. It was found that the acetone extract showed great amounts of phytochemicals than dichloromethane and aqueous extracts. Since A. vera is used in the treatment of different ailments such as skin wounds and abrasions, eczema, constipation, rheumatoid arthritis etc, the medicinal uses of this plant could be associated to such analysed bioactive compounds. Acetone, hexane, ethanol, water and dichloromethane were used to extract the Aloe vera leaf and the best solvent extract was determined. Thin layer chromatography was used to profile the leaf extracts with the aim of documenting the main phytochemicals present in the Aloe vera growing in South Africa. The best spraying reagent was determined. Fourier transform infrared spectrophotometer was used to validate the presence of Aloe vera ingredients in commercial products. The yield extraction ability of the solvent was the order: water>ethanol> hexane >dichloromethane and acetone for the dry portion. However, for the plant extracted fresh, the order of yield produced was ethanol-acetone-dichloromethane > and water. The different solvent systems separated the compounds differently. Hexane: acetone: ethanol (20 : 5: 2) and Benzene: ethanol: ammonium (80): ethanol (10): ammonium solvent systems were noted to be the best mobile phase as they gave the best separation compared to other systems.EMW [ethyl acetate (81): methanol (11): water (8)] showed better separation than the other two separating solvent systems. Vanillin- sulphuric acid spray was seen to be the best spraying reagent as compared to vanillin- phosphoric acid. Fourier transform infrared spectrophotometer validated the presence aloe ingredients in aloe vera commercial products.