<p>The aim of this study was to investigate the effects of Sutherlandia frutescens on the inflammatory response and T cell differentiation in vitro using cytokines as biomarkers. Whole blood cells containing various concentrations of Sutherlandia frutescens were stimulated in vitro with either Lipopolysaccharide (LPS) or Phytohaemagglutinin (PHA). Results show that Sutherlandia frutescens is not toxic at any of the concentrations tested. The addition of Sutherlandia frutescens at high concentrations to the stimulated whole blood cell cultures reflects a significant down regulation of Interleukin(IL) 6 and IL-10 compared to the control (P&lt / 0.05) hence suppressed the inflammatory and humoral immune response. Results obtained for Inteferon-gamma (IFN ) shows that Sutherlandia frutescens is donor specific as it reflects both up and down regulation in the release of IFN at the concentrations tested. The in vitro data generated by this study supports the use of Sutherlandia frutescens in the management of inflammatory conditions and allergies such as asthma. However the effects of Sutherlandia frutescens on cell mediated immunity was found to be donor specific. Further investigation of Sutherlandia frutescens on cellular immunity is advised.</p>

<p>The aim of this study was to investigate the effects of Sutherlandia frutescens on the inflammatory response and T cell differentiation in vitro using cytokines as biomarkers. Whole blood cells containing various concentrations of Sutherlandia frutescens were stimulated in vitro with either Lipopolysaccharide (LPS) or Phytohaemagglutinin (PHA). Results show that Sutherlandia frutescens is not toxic at any of the concentrations tested. The addition of Sutherlandia frutescens at high concentrations to the stimulated whole blood cell cultures reflects a significant down regulation of Interleukin(IL) 6 and IL-10 compared to the control (P&lt / 0.05) hence suppressed the inflammatory and humoral immune response. Results obtained for Inteferon-gamma (IFN ) shows that Sutherlandia frutescens is donor specific as it reflects both up and down regulation in the release of IFN at the concentrations tested. The in vitro data generated by this study supports the use of Sutherlandia frutescens in the management of inflammatory conditions and allergies such as asthma. However the effects of Sutherlandia frutescens on cell mediated immunity was found to be donor specific. Further investigation of Sutherlandia frutescens on cellular immunity is advised.</p>

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.

Cervical cancer is the most common malignancy after breast cancer in women worldwide. It accounts for 83% of all new cases and 85% cancer death in developing countries. In South Africa, cervical cancer is the common cancer in women with an annual crude incidence rate of 30.2 per 100,000 women and the highest rate were found to be in women between the ages of 66-69 years. Approximately 6800 women in S.A face new case of cervical cancer while accounting for 3700 cancer death annually. Because of unequal access to the health facilities, socio-economic differences, HPV and HIV infection, the rate of cervical cancer in black women is higher (42.1%) compared to the low rate in white women. Because of the name “cancer bush’ given to Sutherlandia Frutescence(S.F) plant by the traditional healers as well as Xhosas, Zulu, Sotho and cape Dutch for its anti-cancer activity, the plant was in this study to confirm its cytotoxic effect on the cervical cancer cell lines. Aim of the study: to evaluate the methanolic extracts of Sutherlandia Frutescens on cervical cancer cell lines. Materials and Methods: The MTT assay was performed to evaluate SiHa cell lines treated with methanolic extract of S.F (50μg/ml, 100μg/ml, 150μg/ml and 200μg/ml). The three compounds (Canavanine, GABA and Pinitol) were also evaluated for its anti-tumour activity. The cell growth was then showed in real time using Xcilligence. Flow cytometry was employed to determine the mode of action. Caspase 3/7 assay was performed to confirm if cell death was via caspase-dependent or independent and ATP was done to assess the ATP level in S.F treated cells. Results: MTT shows a significant decline in cells treated with 50μg/ml, 100μg/ml and 200μg/ml of the extract and 50μg/ml was concluded to be the concentration at which 50% of the cells die. The ATP results are inconsistent with MTT result; the ATP level increased in S.F treated cells. Cell index which represents the quantitative measure of cell growth in real time decline upon treatment with 50μg/ml. Flow cytometry showed cells are dying by apoptosis and the cell cycle arrest is mostly in the S phase. The cell death was caspase-dependent as it shows an increased luminescence which is proportional to the number of caspase. The concentrations of the compounds used, Canavanine (1000μM, 1500μM and 2500μM), GABA (100μM, 300μM and 500μM) and Pinitol (30μM, 90μM and 120μM) induce cell death and cell death shows to decrease after the maximum concentration. Conclusion: Sutherlandia Frutescence has proven with number of research that it induces cell death via apoptosis. After evaluating its cytotoxicity, the plant shows to be a promising anti-tumor agent that needs to be clinically proven. / Life Sciences / M. Sc. (Life Science)

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Endocrine disrupting compounds (EDCs) are exogenous compounds/chemicals which interfere with, or have adverse effects on the production, distribution and function of natural hormones, thereby affecting normal endocrine activity, health and quality of life of both humans and wildlife. The reproductive system is highly susceptible to EDCs due to it being controlled by an array of hormonal signals. The effects of EDCs on the male reproductive system include infertility, decreased sperm count, function and morphology, abnormal development of secondary sex characteristics, reproductive function and sexual behaviour as well as decreased libido. There are various sources by which EDCs enter the environment which include effluents from several industries (mining, agriculture, smelting, hazardous waste sites, manufacturing industries, etc.), sewage treatment effluents, urban and agricultural runoff and effluents which include natural and pharmaceutical chemicals excreted in the urine of humans and domestic livestock, pesticides, polychlorinated biphenyls, dioxins, plasticizers, surfactants, etc. Humans and animals can also be affected by EDCs by consuming food containing endocrine active substances. The growing concern regarding adverse effects due to EDC exposure of humans and wildlife, as well as the increased incidence of EDC contamination has prompted extensive research into the development and validation of screening tests to detect and monitor known EDCs and new substances with endocrine-disrupting capability. These screening tests involve assessing the effect of known and potential EDCs on reproductive function and development as well as hormone production. To assess the effect of EDCs on the reproductive system different methods are employed which include in vitro, in vivo and ex vivo methods. In vitro methods have been suggested as a suitable screening tool for EDC monitoring due to low costs, reduced animal usage, the use of standard and basic equipment as well as the ability to screen a large number of samples with multiple endpoints. Of the available in vitro methods, the minced testes method has been suggested as the most suitable method for screening EDCs and for this reason has been employed in this study. The aim of this study was thus to employ a minced testes method to screen samples for male reproductive toxicity using cell viability and hormone production (testosterone and estradiol) as endpoints.The first objective of this study was to optimize an in vitro testicular cell culture assay by determining both optimal luteinizing hormone (LH)&nbsp; concentration and incubation time needed for testosterone production. Testicular cell cultures were prepared and cells were treated with varying concentrations of LH (10, 1, 0.1, 0.01 and 0 mu/ml) and incubated for 4 hours and 20 hours. Testosterone production was evaluated for each incubation period. Testosterone production was significantly increased for both incubation periods at all LH concentrations tested as compared to the control. For both incubation periods, there was no significant difference in testosterone production between the different LH concentrations tested. From the data obtained, the 4 hour incubation period as well as the LH concentration of 10 mu/ml were selected as optimal for the testicular cell culture assay. The second objective of this study was to determine the effect of Tulbaghia violacea Harv. on the male reproductive system. T. violacea is a plant species indigenous to southern Africa and is used locally as a herbal remedy/medicine to treat several ailments. Cells were treated with varying concentrations of the T. violacea ethanol extract (with/without LH-treatment) and incubated for 4 hours. Hormone production and cell viability were evaluated. The results obtained from this pilot in vitro study demonstrated that the ethanol extract of T.violacea has androgenic properties by significantly increasing LH-induced testosterone production in mouse testes with no significant change in cell viability. The third objective of this study was to assess the effect of Sutherlandia frutescens(L.) R.Br and Artemisia afra Jacq. Ex Willd. on the male reproductive system. S. frutescens and A. afra are also plant species indigenous to southern Africa and used locally as a herbal remedy/medicine to treat several ailments. Ethanol extracts of each plant was prepared and cells were treated with varying concentrations of each extract (0, 156.25, 312.5, 625, 1250,2500 and 5000 &mu;g/ml) with or without LH-treatment and incubated for 4 hours. Cytotoxicity by LDH measurement and hormone production (testosterone and estradiol) were endpoints that were evaluated. The results obtained showed that the ethanol extracts of both plants are not cytotoxic to testicular cells and that A. afra decreases testosterone production at high concentrations. The fourth and final objective of this study was to assess the acute effect of four heavy metals, namely manganese, copper, cadmium and magnesium on the male reproductive system. These heavy metals are used extensively in manufacturing and mining industries. Cells were treated with varying concentrations of each metal salt (200, 100, 50, 25, 12.5, and 6.25 & mu;M) with or without LH-treatment and incubated for 4 hours. Endpoints evaluated included cell viability, testosterone and estradiol production. The results obtained showed that manganese, cadmium and copper are highly toxic to testicular cells in vitro and therefore may potentially cause reproductive toxicity. / South Africa

Magister Scientiae - MSc / The aim of this study was to investigate the effects of Sutherlandia frutescens on the inflammatory response and T cell differentiation in vitro using cytokines as biomarkers. Whole blood cells containing various concentrations of Sutherlandia frutescens were stimulated in vitro with either Lipopolysaccharide (LPS) or Phytohaemagglutinin (PHA). Results show that Sutherlandia frutescens is not toxic at any of the concentrations tested. The addition of Sutherlandia frutescens at high concentrations to the stimulated whole blood cell cultures reflects a significant down regulation of Interleukin(IL) 6 and IL-10 compared to the control (P&lt;0.05) hence suppressed the inflammatory and humoral immune response. Results obtained for Inteferon-gamma (IFN ) shows that Sutherlandia frutescens is donor specific as it reflects both up and down regulation in the release of IFN at the concentrations tested. The in vitro data generated by this study supports the use of Sutherlandia frutescens in the management of inflammatory conditions and allergies such as asthma. However the effects of Sutherlandia frutescens on cell mediated immunity was found to be donor specific. Further investigation of Sutherlandia frutescens on cellular immunity is advised. / South Africa

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2006. / This study aimed to assess the effect of mild psychological stress in male Wistar rats using incremental, intermittent stress on parameters of atrophy, including body mass, soleus and extensor digitorum longus (EDL) muscle mass, and mechanisms possibly contributing to atrophy. Serum corticosterone concentrations, 20s proteasome activity, glutamine synthetase (GS) and tyrosine amino-transferase (TAT) activities were determined. I also assessed whether Sutherlandia (Su) or resistance exercise was able to reverse the effects of stress on any of these parameters.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: The hypothalamic-pituitary-adrenal-(HPA) axis has long been closely associated with psychological stress-induced activation of the adrenal cortex and subsequent glucocorticoid production. Another, less known peripheral limb of the psychological stress response, is the sympatho adrenal medullary pathway. We hypothesized that the sympatho-adrenal medullary system constitutes the primary response to acute psychological stress, with the HPA-axis functioning as a secondary response. We tested our hypothesis by manipulating a model of acute mild psychological stress (restraint) by blocking IL-6, a valuable constituent of the sympatho-adrenal medullary system. Serum corticosterone concentration increased in response to stress (7 ± 3 vs. 57 ± 4 ng/ml; P<0.0001), a response attenuated when IL-6 was blocked (17 ± 7 ng/ml). Stress increased pituitary mass only when IL-6 was blocked (38 ± 3 vs. 65 ± 6 mg; P <0.001). Stress increased left adrenal mass only in the presence of IL-6 (34 ± 1 vs. 73 ± 8 mg; P <0.00001). Stress did not influence the circulating levels of TNF-α, IL-1β or IL-6 significantly. IL-1β and TNF-α concentrations in the unstressed rats were lower when IL-6 was blocked. We then manipulated the stress model by administering S. frutescens extract to elucidate both the central and peripheral effects of acute S. frutescens administration on the psychological stress response. Restraint caused decreases in hippocampal GR levels when compared to respective controls. S. frutescens administration and exposure to restraint synergistically decreased hippocampal GABAAR levels. In addition, exposure to both stress and S. frutescens led to a noteworthy increase in pituitary mass (P = 0.078), as well as pituitary ACTH levels (P < 0.01). Similarly, differences in circulating ACTH levels showed an effect of stress on ACTH secretion only in the presence S. frutescens (P < 0.05). Adrenal mass was significantly increased in S. frutescenstreated animals that were also exposed to restraint (P < 0.05). Adrenal levels of ACTH showed a reciprocal trend to pituitary and circulating ACTH levels. No statistically significant differences were seen in adrenal IL-6 content. However, marked increases in IL-6 levels were seen at this level with administration of S. frutescens stress exposure and a cumulative increase seen with both S. frutescens-treatment and stress exposure. Hippocampal GABAAR, pituitary mass, pituitary ACTH and circulating ACTH levels showed a similar trend towards a synergistic effect of S. frutescens and restraint in activation of the psychological stress response, while adrenal ACTH levels showed an inverse trend. Hippocampal GR did not show any effect of stress or S. frutescens-treatment. The results from these two experiments indicate that the sympatho-adrenal medullary system constitutes the primary response to acute mild psychological stress and that the HPA-axis is only activated during an exacerbated stress response or when the sympatho-adrenal medullary contribution is inadequate. Furthermore, the acute administration of S. frutescens possibly led to a functional shift in GABAergic function, resulting in activation of the stress response. The anecdotal reports of a “docile” effect of S. frutescens most likely results from activation of the mesolimbic dopaminergic system by the hippocampus and amygdala. These results have dramatic consequence in GABA-based anxiety-treatments. / AFRIKAANSE OPSOMMING: Die hipotalamo-pituïtêre-adrenale (HPA)-as is lank bekend as ‘n primêre rolspeler in die respons op emosionele stres en daaropvolgende glukokortikoïed produksie. ‘n Ander, minder bekende arm van die sielkundige stres respons is die simpatiese bynier-medulla-sisteem. Ons hipotese was dat die laasgenoemde simpatiese bynier-medulla-sisteem die primêre respons tot sielkundige stres behartig terwyl die HPA-as ‘n sekondêre respons bied. Ons het ons hipotese getoets deur die manupilering van ‘n beproefde stres model waar ons IL-6, ‘n waardevolle rolspeler in die simpatiese bynier-medulla-sisteem, onderdruk het. In respons op stress, het serum kortikosteroon konsentrasies toegeneem slegs in die teenwoordigheid van IL-6 (7 ± 3 vs. 57 ± 4 ng/ml; P<0.0001), maar nie wanneer IL-6 onderdruk is nie (17 ± 7 ng/ml). Stres het ‘n verhoging in hipofise massa teweeggebring slegs tydens die onderdrukking van IL-6 (38 ± 3 vs. 65 ± 6 mg; P <0.001). Stres het ook linker-byniermassa verhoog slegs wanneer voldoende IL-6 beskikbaar was (34 ± 1 vs. 73 ± 8 mg; P <0.00001). Stres alleen het geen invloed gehad op serum IL-1β, IL-6 of TNF-α nie, maar die onderdrukking van IL-6 het wel ‘n inhiberende effek op basale IL-1β en TNF-α gehad. Daarna het ons weer eens die stresmodel manipuleer deur die rotte ‘n S. frutescens ekstrak te gee in ‘n poging om beide die sentrale en perifere effekte daarvan op die sielkundige stres respons te evalueer. Stres alleen het gelei tot ‘n afname in GR terwyl ‘n kombinasie van stres en S. frutescens administrasie tot ‘n afname in GABAARα1 in die hippokampus gelei het. Hierdie kombinasie het ook tot ‘n merkwaardige toename in hipofise massa (P = 0.078) sowel as ACTH-inhoud van die hipofise (P < 0.01) gelei. ‘n Soortgelyke patroon is waargeneem betreffende sirkulerende ACTH en byniermassa met P < 0.05 vir elk. Bynier ACTH inhoud, aan die ander kant, het ‘n omgekeerd eweredige verhouding met ACTH in die hipofise en in sirkulasie getoon. Bynier IL- 6 inhoud het geen statisties beduidende verskille getoon nie, maar ‘n merkwaardige verhoging is weereens gesien met ‘n kombinasie van stres en S. frutescens administrasie. Die soortgelyke tendens wat waargeneem word in GABAAR in die hippokampus, asook hipofise- en sirkulerende ACTH vlakke, en dui op ‘n samewerkende rol van stres en S. frutescens in die aktivering van die sielkundige stres respons. GR in die hippokampus toon geen veranderinge nie. Die resultate van die twee eksperimente dui op ‘n primêre rol van die simpatiese bynier-medulla-sisteem in die respons op ‘n akute stressor en dat die HPA-as net geaktiveer word tydens ‘n ooreiste stres reaksie of indien die simpatiese bynier-medulla-sisteem onderdruk word. Die waargenome “verdowings”-effek van S. frutescens word moontlik deur aktivering van die mesolimbiese dopamien pad deur die hippokampus en amigdala bewerkstellig. Die resultate mag ook lei tot die heroorweging van GABA-gebaseerde angs medikasies.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / Sutherlandia frutescens (S. frutescens), sub-species microphylla, is a member of the Fabacea family and is used as a herbal remedy for the treatment of several ailments which include influenza, diabetes, cancer, tuberculosis, chronic fatigue syndrome, rheumatoid arthritis, anxiety, clinical depression, and more recently, those living with human immunodeficiency virus/ acquired immune deficiency syndrome (HIV/AIDS) (1-4). Many of the symptoms of these ailments are associated with a perturbation of the stress response which may be associated with disorders of the endocrine system. Of all the traditional plants in South Africa, S. frutescens is regarded the most profound in that it is a multipurpose traditional remedy. The plant has enjoyed a long history of use and reports indicating its efficacy as a safe treatment for various health conditions have added to the popularity of this medicinal plant. The extracts of S. frutescens have been shown to exhibit anti-proliferative effects on cancer cells, antioxidant activity, and to possess anti-diabetic and anti-inflammatory potential (5, 6), providing scientific evidence for its therapeutic use in the treatment of cancer and diabetes. However, this study focuses on the potential use of this medicinal plant in the treatment of stress and stress related diseases. Chronic stress is characterized by elevated plasma levels of glucocorticoids. These steroid hormones are synthesized in the adrenal cortex in a series of reactions involving the steroidogenic enzymes. The major aim of this thesis was the determination of the influence of S. frutescens extracts on the adrenal cytochrome P450 enzymes. Aqueous, methanol and chloroform S. frutescens extracts were prepared and the interaction with the cytochrome P450 enzymes was investigated. The effect of these extracts towards progesterone (PROG), deoxycortisol and deoxycorticosterone (DOC) binding to the cytochrome P450 enzymes as well as their influence on the metabolism of these steroid substrates was investigated. A similar study (7) showed that compounds from the S. frutescens extracts could interact with these enzymes and possibly affect adrenal steroidogenesis. This study further investigates the bioactive properties of the plant material in terms of the influence of S. frutescens on the cytochrome P450 enzymes and the effect of the manufacturing process on the bioactivity of the plant.

<p>Endocrine&ndash / disrupting compounds (EDCs) are exogenous compounds/chemicals which interfere with, or have adverse effects on the production, distribution and function of natural hormones, thereby affecting normal endocrine activity, health and quality of life of both humans and wildlife. The reproductive system is highly susceptible to EDCs due to it being controlled by an array of hormonal signals. The effects of EDCs on the male reproductive system include infertility, decreased sperm count, function and morphology, abnormal development of secondary sex characteristics, reproductive function and sexual behaviour as well as decreased libido. There are various sources by which EDCs enter the environment which include effluents from several industries (mining, agriculture, smelting, hazardous waste sites, manufacturing industries, etc.), sewage treatment effluents, urban and agricultural runoff and effluents which include natural and pharmaceutical chemicals excreted in the urine of humans and domestic livestock, pesticides, polychlorinated biphenyls, dioxins, plasticizers, surfactants, etc. Humans and animals can also be affected by EDCs by consuming food containing endocrine active substances. The growing concern regarding adverse effects due to EDC exposure of humans and wildlife, as well as the increased incidence of EDC contamination has prompted extensive research into the development and validation of screening tests to detect and monitor known EDCs and new substances with endocrine-disrupting capability. These screening tests involve assessing the effect of known and potential EDCs on reproductive function and development as well as&nbsp / hormone production. To assess the effect of EDCs on the reproductive system different methods are employed which include in vitro, in vivo and ex vivo methods. In vitro methods have been suggested as a suitable screening tool for EDC monitoring due to low costs, reduced animal usage, the use of standard and basic equipment as well as the ability to screen a large number of samples with multiple endpoints. Of the available in vitro methods, the minced testes method has been suggested as the most suitable method for screening EDCs and for this reason has been employed in this study. The aim of this study was thus to employ a minced testes method to screen samples for male reproductive toxicity using cell viability and hormone production (testosterone and estradiol) as endpoints.The first objective of this study was to optimize an in vitro testicular cell culture assay by determining both optimal luteinizing hormone (LH)&nbsp / concentration and incubation time needed for testosterone production. Testicular cell cultures were prepared and cells were treated with varying concentrations of LH (10, 1, 0.1, 0.01 and 0 mu/ml) and incubated for 4 hours and 20 hours. Testosterone production was evaluated for each incubation period. Testosterone production was significantly increased for both incubation periods at all LH concentrations tested as compared to the control. For both incubation periods, there was no significant difference in testosterone production between the different LH concentrations tested. From the data obtained, the 4 hour incubation period as well as the LH concentration of 10 mu/ml were selected as optimal for the testicular cell culture assay. The second objective of this study was to determine the effect of Tulbaghia violacea Harv. on the male reproductive system. T. violacea is a plant species indigenous to southern Africa and is used locally as a herbal remedy/medicine to treat several ailments. Cells were treated with varying concentrations of the T. violacea ethanol extract (with/without LH-treatment) and incubated for 4 hours. Hormone production and cell viability were evaluated. The results obtained from this pilot in vitro study demonstrated that the ethanol extract of T.violacea has androgenic properties by significantly increasing LH-induced testosterone production in mouse testes with no significant change in cell viability. The third objective of this study was to assess the effect of Sutherlandia frutescens(L.) R.Br and Artemisia afra Jacq. Ex Willd. on the male reproductive system. S. frutescens and A. afra are also plant species indigenous to southern Africa and used locally as a herbal remedy/medicine to treat several ailments. Ethanol extracts of each plant was prepared and cells were treated with varying concentrations of each extract (0, 156.25, 312.5, 625, 1250,2500 and 5000 &mu / g/ml) with or without LH-treatment and incubated for 4 hours. Cytotoxicity by LDH measurement and hormone production (testosterone and estradiol) were endpoints that were evaluated. The results obtained showed that the ethanol extracts of both plants are not cytotoxic to testicular cells and that A. afra decreases testosterone production at high concentrations. The fourth and final objective of this study was to assess the acute effect of four heavy metals, namely manganese, copper, cadmium and magnesium on the male reproductive system. These heavy metals are used extensively in manufacturing and mining industries. Cells were treated with varying concentrations of each metal salt (200, 100, 50, 25, 12.5, and 6.25&nbsp / &mu / M) with or without LH-treatment and incubated for 4 hours. Endpoints evaluated included cell viability, testosterone and estradiol production. The results obtained showed that manganese, cadmium and copper are highly toxic to testicular cells in vitro and therefore may potentially cause reproductive toxicity.</p>