Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: This study: 1. describes the preparation of a methanol extract of Sutherlandia frutescens and the HPLC fractionation of the methanol extract. 2. investigates the influence of S. frutescens on the binding properties of mitochondrial cytochrome 11 -hydroxylase (CYP11B1) to deoxycorticosterone (DOC) and deoxycortisol, demonstrating that methanol extracts of S. frutescens inhibit the Type I substrate-induced difference spectra. 3. investigates the influence of S. frutescens on the catalytic activity of CYP11B1 expressed in COS1 cells, demonstrating that the methanol extract of S. frutescens inhibits the conversion of DOC and deoxycortisol. 4. describes the sequential extraction of the methanol extract of S. frutescens using organic solvents and the inhibition of the conversion of DOC by CYP11B1 expressed in COS1 cells in the presence of these extracts. 5. describes the inhibition of the binding of DOC to CYP11B1 in ovine adrenal mitochondria, and the conversion of DOC by CYP11B1 expressed in COS1 cells by these fractions. 6. identifies the presence of the flavonoid compounds, orientin vitexin and rutin, in S. frutescens. 7. investigates the influence of the flavonoid compounds on the binding of DOC to CYP11B1 and on the catalytic activity of DOC by CYP11B1 expressed in COS1 cells. 8. identifies the presence of the triterpenoid, sutherlandioside A (SU1), in S. frutescens extracts and investigates its effect on the binding of DOC to CYP11B1. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: 1. die voorbereiding van ‘n metanol ekstraksie van Sutherlandia frutescens en die HPLC fraksionering van die metanol ekstrakte. 2. ‘n ondersoek na die invloed van S. frutescens op die bindingseienskappe van sitochroom P450 11 -hidroksilase (CYP11B1) in skaap bynier mitochondria en demonstreer dat S. frutescens metanol ekstrakte die vorming van steroïed-geinduseerde tipe I verskil spektra van deoksiekortisol en deoksikortikosteroon (DOC) inhibeer. 3. ‘n ondersoek na die invloed van S. frutescens op die katalitiese aktiwiteit van CYP11B1 in COS1 selle en demonstreer die inhibisie van DOC en deoksikortisol omsetting na hul produkte deur die methanol ekstrakte. 4. die opeenvolgende ekstraksie van methanol extrakte van S. frutescens met organiese oplosmiddels en beskryf die inhibisie van die CYP11B1 gekataliseerde omsetting van DOC in COS1selle in die teenwoordigheid van die ekstrakte. 5. die inhibeerende effek op die binding van DOC aan CYP11B1 in skaap bynier mitochondria en die inhibisie van die CYP11B1 gekataliseerde omsetting van DOC in COS1selle. 6. die identifisering van flavonoïed verbindings, orientin vitexin en rutin in S. frutescens. 7. ‘n ondersoek na die invloed van die flavonoïed verbindings op die binding van DOC aan CYP11B1 en op die katalitiese aktiwiteit van CYP11B1 in COS1 selle. 8. die indentifisering van die triterpenoïed, sutherlandiosied A (SU1), in S. frutescens en ondersoek die invloed van SU1 op die binding van DOC aan CYP11B1.

The overall aims of this project were to assess the pharmaceutical quality and consistency of commercially available solid dose Sutherlandia frutescens containing products (viz. tablets & capsules) by exploring the use of monitoring the pharmaceutical presentation, flavonoid profile and antioxidant activity levels and to develop/or adapt methods and specifications that may be used for the quality control of such products.Stability tests were conducted on all of the selected SCP. The products were stored under elevated temperatures and environmental humidity conditions and total phenol, antioxidant and chromatographic analysis was conducted on these samples. Samples of each of the SCP were hydrolyzed using HCL and then analyzed using HPLC to test the stability of the flavonoids present in each product. The SCP investigated in this study physically appeared to be of quite good “pharmaceutical” quality, but generally lacked information on the date of manufacture and lacked package inserts, or when these were present they contained insufficient information.Based on the results obtained, it is recommended that, the manufacturers of SCP pay more attention to the information provided on the package inserts and the storage conditions for their products. Further the levels of antioxidant activity, total phenols and flavonoid (sutherlandins A to D) be used as specifications to control the quality of commercially available solid dose Sutherlandia frutescens containing preparations on an individual basis.

The overall aims of this project were to assess the pharmaceutical quality and consistency of commercially available solid dose Sutherlandia frutescens containing products (viz. tablets & capsules) by exploring the use of monitoring the pharmaceutical presentation, flavonoid profile and antioxidant activity levels and to develop/or adapt methods and specifications that may be used for the quality control of such products.Stability tests were conducted on all of the selected SCP. The products were stored under elevated temperatures and environmental humidity conditions and total phenol, antioxidant and chromatographic analysis was conducted on these samples. Samples of each of the SCP were hydrolyzed using HCL and then analyzed using HPLC to test the stability of the flavonoids present in each product. The SCP investigated in this study physically appeared to be of quite good “pharmaceutical” quality, but generally lacked information on the date of manufacture and lacked package inserts, or when these were present they contained insufficient information.Based on the results obtained, it is recommended that, the manufacturers of SCP pay more attention to the information provided on the package inserts and the storage conditions for their products. Further the levels of antioxidant activity, total phenols and flavonoid (sutherlandins A to D) be used as specifications to control the quality of commercially available solid dose Sutherlandia frutescens containing preparations on an individual basis.

Indigenous plants have long been used by African populations in their cultural lives and health care. Sutherlandia frutescens (SF) is a popular traditional medicinal plant found in various parts of southern Africa and used for treatment or management of different diseases, including cancer and HIV/AIDS. In this study, the biochemical effects of various dilutions (1/50, 1/150, 1/200, and 1/300) of SF 70% ethanol (SFE) and deionised water (SFW) extracts in cancerous H9 and normal T cells were examined. Untreated, 70% ethanol-treated and camptothecin (CPT, 20jiiM) treated cells were used as reference samples for comparison. Cytotoxicity, apoptotic enzymes activity, oxidant scavenging and antioxidant promoting abilities, cellular morphology and cytokine signalling effects were assessed using the methylthiazol tetrazolium (MTT) assay, adenosine triphosphate (ATP) assay, caspase-3/-7 activity assay, thiobarbituric acid reactant substance (TBARS) and glutathione (GSH) assays, fluorescence microscopy and an ELISAbased cytokine analyses assay respectively. Sutherlandia frutescens ethanol and water extract dilutions (1/50 and 1/200) were shown to be cytotoxic to H9 T cells in a dose- and time-dependent manner with the SFE extract having an average IC50 of 1/40 after 24 hours while SFW extract reached a similar IC50 only after 48 hours. In normal T cells, the SFE extract induced proliferation after 24 hours but this was reverse after 48 hours. The SFW extract dilutions did not significantly change cell viability after 24 hours but significantly increased cell viability after 48 hours. Both SFE and SFW extracts dilutions induced a dose- and time-dependent inhibition of caspase-3/-7 activity in both H9 and normal T cells. Both types of extracts were also shown to efficiently remove lipid peroxides from supernatants of treated cell lines, with SFW extract having a more lasting effect. In the GSH assay, the SFE and SFW extract dilutions reduced GSH levels in H9 T cells, with the SFW extract dilutions being more effective. In normal T cells, the higher dilutions (1/150 and 1/300) of SFW extract increased GSH levels significantly while lower dilutions (1/50) of both SFE and SFW extracts significantly inhibited GSH levels. Lower dilutions (1/50) of SFE and SFW extracts induced chromatin condensation in both H9 and normal T cells after 48 hours incubation. Using treated peripheral blood mononuclear cells (PBMCs) supernatants, SFE and SFW extract dilutions were shown to reduce the levels of pro-inflammatory cytokines IL 1 p and TNF-a in a dose-dependent manner. These results further confirmed the anticancer abilities of SF and showed that higher concentrations of this medicinal plant can be toxic to normal T cells in vitro while lower concentrations can stimulate the immune cells. Therefore further studies should be conducted with regards to the effects of SF on the immune system in both in vitro and in vivo systems. / Thesis (M.Med.Sci.)-University of KwaZulu-Natal, 2008.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Sutherlandia frutescens (L.) R. Br., also known as Lessertia frutescens, is a leguminous shrub indigenous to southern Africa. Traditionally this plant has been used for the treatment of various ailments; current interest in this plant has escalated after it was announced that extracts could aid in the relief and treatment of HIV/AIDS. These extracts contain an array of metabolites, including sutherlandins, sutherlandiosides L-arginine, L-canavanine, asparagine, gamma-aminobutyric acid (GABA), and various other amino acids, which have been linked to medicinal uses. This study focused on the use of hormones to promote the growth and metabolite production of S. frutescens in vitro cultures. The growth promoting substances used in this study were synthetic analogues of strigolactones, GR24 and Nijmegen-1, and auxins, indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA). The first part of this study focused on the effects strigolactones and auxins, alone and combined, had on the growth of S. frutescens in vitro nodal explants. The S. frutescens nodal explants had the most significant improvement in growth with treatments that contained 1 mg/L NAA. These treatments increased growth via fresh and dry mass and plant length. The metabolite content of these nodal explant cultures was evaluated using liquid chromatography/mass spectrometry (LC/MS) metabolite analysis. The treatments that contained 1 mg/L NAA differed in metabolite composition and showed an increase in metabolite quantity. The SU1 content of the treated plants was also quantified using LC/MS techniques and a combination of 1 mg/L NAA and Nijmegen-1 doubled the amount of SU1. The effect of strigolactones was also studied using hairy root cultures of S. frutescens. Strigolactones alone slightly inhibited the formation of lateral transgenic roots, but when these chemicals were used in combination with auxins, significant reduction in dry mass and lateral root outgrowth resulted. Of the treatments tested in this study, 0.1 mg/L IBA caused noticeable alterations to the metabolite pool, with amino acids such as GABA and arginine accumulating at higher levels than the control explants. The exploitation of hormones to up-regulate the growth and metabolism of the medicinally important plant, Sutherlandia frutescens, proved successful in this study. The use of in vitro nodal explants along with hairy root cultures has assisted in the establishment of a stable system for the up-regulation of metabolites. / AFRIKAANSE OPSOMMING: Sutherlandia frutescens (L.) R. Br., ook bekend as Lessertia frutescens, is 'n peulagtige struik inheems tot suider Afrika. Tradisioneel is die plant vir 'n groot verskeidenheid van kwale gebruik; huidige belangstelling in die plant het toegeneem nadat dit bekend gemaak was dat ekstraksies vanaf hierdie plant verligting kan bied vir MIV/VIGS. Hierdie ekstrakte bevat 'n verskeidenheid van metaboliete, insluitend sutherlandins, sutherlandiosiede L-arginien, L-kanavanien, asparagien, gamma-aminobottersuur (GABS), asook verskeie ander aminosure wat medisinale gebruike het. Die studie het gefokus op die gebruik van hormone om die groei en metaboliete van S. frutescens in vitro kulture te vermeerder. Die groei reguleerders wat in hierdie studie gebruik was, was die sintetiese analoë van strigolaktoon, GR24 en Nijmegen-1, asook die ouksiene, indool-3-bottersuur (IBS) en naftaleen asynsuur (NAS). Die eerste deel van die studie het gefokus op die effek van strigolaktoon en ouksien, alleen en in kombinasie, op die groei van S. frutescens in vitro nodale mikrostingels. Die S. frutescens nodale mikrostingels wat behandel was met 1 mg/L NAS het die aansienlikste toename in groei getoon. Hierdie behandeling het groei bevorder deur middel van vars en droë massa en plant lengte. Die metaboliet inhoud van die behandelde mikrostingels was met behulp van vloeistofchromatografie/massa spektrometrie (VC/MS) ondersoek. Al die behandelinge wat 1 mg/L NAS bevat het, het in metaboliet samestelling verskil en het ook 'n toename in metaboliet hoeveelheid getoon. Die SU1 inhoud van die behandelde plante was ook met behulp van VC/MS tegnieke gekwantifiseer en dit was gevind dat 'n kombinasie van 1 mg/L NAS en Nijmegen-1 die hoeveelheid SU1 verdubbel het. Die effek van strigolaktoon op harige wortel kulture van S. frutescens was ook ondersoek. Strigolaktoon alleen het die formasie van laterale transgeniese wortels effens inhibeer, maar wanneer hierdie chemikalieë saam met ouksiene gebruik was, was die aansienlike afname van die massa en inhibisie van die laterale wortel uitgroeisels meer prominent. Van al die behandelinge wat in hierdie studie getoets is, het 0.1 mg/L IBS die mees merkbare veranderinge in metaboliete meegebring en aminosure soos GABS en arginien het teen hoër vlakke versamel. Die uitbuiting van hormone om groei en metaboliet produksie te bevorder in die belangrike medisinale plant, Sutherlandia frutescens, was suksesvol in hierdie studie. Die gebruik van nodale mikrostingels asook harige wortel kulture het bygedra om 'n stabiele sisteem te vestig vir die vermeerdering van metaboliete.

Magister Scientiae - MSc / The overall aims of this project were to assess the pharmaceutical quality and consistency of commercially available solid dose Sutherlandia frutescens containing products (viz. tablets & capsules) by exploring the use of monitoring the pharmaceutical presentation, flavonoid profile and antioxidant activity levels and to develop/or adapt methods and specifications that may be used for the quality control of such products.Stability tests were conducted on all of the selected SCP. The products were stored under elevated temperatures and environmental humidity conditions and total phenol, antioxidant and chromatographic analysis was conducted on these samples. Samples of each of the SCP were hydrolyzed using HCL and then analyzed using HPLC to test the stability of the flavonoids present in each product. The SCP investigated in this study physically appeared to be of quite good “pharmaceutical” quality, but generally lacked information on the date of manufacture and lacked package inserts, or when these were present they contained insufficient information. Based on the results obtained, it is recommended that, the manufacturers of SCP pay more attention to the information provided on the package inserts and the storage conditions for their products. Further the levels of antioxidant activity, total phenols and flavonoid (sutherlandins A to D) be used as specifications to control the quality of commercially available solid dose Sutherlandia frutescens containing preparations on an individual basis. / South Africa

Philosophiae Doctor - PhD / Various formulations of the popular South African medicinal plant, Sutherlandia frutescens,are commercially available, with no documented specifications for quality assessment. With plans already underway for a clinical trial to assess its efficacy in HIV patients, there is a need for scientifically validated tests for the quality control of products of this plant. Chemical constituents of the plant are many and varied but it is still unclear which might be the most appropriate ones to monitor for activity or to describe the quality of the plant’s products. For quality control and regulatory purposes, the content and dissolution of flavonoids in the plant products can be assessed. However, these compounds are not monitored for regulation and there are as yet no HPLC or dissolution methods that can be employed for quality control of herbals like S. frutescens. Therefore, the objectives of this study were to assess the suitability of its flavonoid constituents as quality control (QC) marker compounds, and the suitability of content levels and dissolution tests of flavonoids as QC tools for S. frutescens products. To realise the afore-mentioned objectives, non-commercially available flavonoid compounds (sutherlandins) that could be used as marker compounds were isolated from S. frutescens. An HPLC assay was developed and validated for determination of flavonoid content in solution. Five S. frutescens materials viz leaf powder (LP), spray-dried aqueous extract (SDAE) and freeze-dried aqueous extracts (FDAE) were analysed for flavonoid content and dissolution. Dissolution tests were conducted for different S. frutescens materials and dissolution profiles of flavonoids in capsules containing these materials were compared using Q-release values, the similarity factor (f2) and mathematical models. To predict in vivo bioavailability of the flavonoids, in silico assessment of in vivo bioavailability of flavonoids (glycosides and aglycones) that may be contained in different S. frutescens materials was conducted. Sutherlandins A, B, C and D were successfully isolated (percentage purity approximately99 % for sutherlandins A, C and D, and 90 % for sutherlandin B) and identified, and used, along with other flavonoid compounds, for the development of a simple and robust HPLC method. Content of sutherlandins A, B, C and D, quercetin and kaempferol in different plant materials were 0.4 ± 0.3, 0.8 ± 0.2, 1.3 ± 0.2, 0.6 ± 0.1, 0.01 ± 0.02 and 0.08 ±0.1 %,respectively, and differed significantly (p < 0.001). In vitro dissolution showed faster dissolution of flavoniod glycosides compared to aglycones. The flavonoids from the LP and SDAE materials showed characteristics of immediate release with Q75 in ≤ 45 minutes, and delayed release from the FDAE material, i.e. Q75 > 45 minutes. The dissolution profiles of each flavonoid compared from different S. frutescens materials were different as signified by their f2 values which were all below 50. The mathematical models describing release were also different for each flavonoid from the different S. frutescens materials. For in vivo bioavailability modelling and prediction studies, the flavonoid aglycones met the conditions for oral bioavailability while the flavonoid glycosides did not. In conclusion, the sutherlandins isolated from S. frutescens proved to be good markers for HPLC assay and dissolution tests of S. frutescens materials. The HPLC method was suitable for assessing flavonoid levels in S. frutescens materials, and also showed differences in flavonoid content in these materials. The dissolution method was simple and reproducible, and Q-release values, the f2 and mathematical models proved to be good tools for differentiating between S. frutescens materials. In silico modelling showed that the flavonoid glycosides and aglycones differed in oral bioavailability. Although not presently required by the Medicines Control Council (MCC), quantification, release and dissolution studies and specifications may be employed as tools for routine analysis and for quality control of herbal drug formulations containing S. frutescens.

Magister Pharmaceuticae - MPharm / Sutherlandia frutescens is a traditional plant medicine widely used in South Africa. Traditionally, the leaves of S. frutescens are mainly used as a tea, but these traditional dosage forms have several disadvantages, including that they are not particularly convenient to prepare and store, encourage dosage inaccuracy and are highly susceptible to microbial contamination. To solve these problems, dried aqueous extract forms, e.g. freeze dried aqueous extract (FDAE) of S. frutescens were prepared, but they, in turn, may still suffer from instability and contain mainly hydrophilic phytoconstituents that are poorly absorbed and delivered for in vivo activity. Modified forms of the FDAE, i.e. the active phytopharmaceutical ingredient (API), may be a better solution. Therefore this study sought to prepare liposomes and phytosomes of the freeze dried aqueous extract of Sutherlandia frutescens, as a means of increasing the total the surface area of the API, thus improving its release and dissolution in gastrointestinal fluids. Liposomes and phytosomes of the FDAE of Sutherlandia frutescens obtained were prepared using a thin film hydration method at ratios of lecithin: S. frutescens (3:1) and phosphatidylcholine: S. frutescens (2:1) respectively. The physical characteristics (i.e. particle size, size distribution, zeta potential, and morphology), of flavonoid glycosides (i.e. sutherlandins A to D; API) as well as content and release profiles of each dosage form (i.e. FDAE liposome or phytosomes) at pH 1.2 and pH 6.8 was determined. A validated HPLC assay was used to determine and compare the flavonoid glycoside content and release profiles of the liposomes and phytosomes. Both liposomes and phytosomes were successfully prepared, in moderate yields (± 30 %, and ± 50 %, respectively), using the thin film hydration method. The liposomes had a significantly smaller size, lower size distribution, higher zeta potential and better stability than the phytosomes (p < 0.05). The phytosomes, however, had significantly higher flavonoid glycoside encapsulation efficiency than the liposomes (±50 % vs ±26 %; p < 0.01). In addition, the release at 120 minutes, of flavonoid glycosides from the liposomes (63%, 58%, 76% and 46% % at pH 1.2, and 78%, 76%, 87% and 89 % at pH 6.8 for sutherlandins A, B, C and D, respectively) was significantly higher and faster than that of the phytosomes (52%, 41%, 51% and 39 % at pH 1.2, and 31% 31%, 33%and 45% % at pH 6.8, for sutherlandins A, B, C and D, respectively). The differences in release were likely due to differences in particle size and size distribution of the two modified API forms. Overall, liposomes and phytosomes can be considered promising vehicles for delayed delivery of herbal crude extracts. Based on its characteristics (i.e. narrower size distribution, and better stability), the liposomes were preferred compared to the phytosomes offering a better kinetic release profile. The phytosomes had higher encapsulation than the liposomes that may be due to complex formation between the API and the lipid.

>Magister Scientiae - MSc / Hyperpigmentation disorders such as melasma, freckles and black-pigmented spots on the surface of the skin are often a result of increased over production and accumulation of melanin pigments in the skin. In melanin biogenesis, tyrosinase is the key enzyme that catalysis the synthesis of melanin, thus the most effective and easiest way to reduce melanin synthesis is by inhibiting tyrosinase. There are a large number of reported tyrosinase inhibitors, their identification and isolation from natural sources is highly important because when natural tyrosinase inhibitors are identified in natural sources, their production is relatively low in cost. Tyrosinase inhibitors are highly sought in the cosmetic industry because of their skin – whitening effects. Most common used tyrosinase inhibitors are kojic acid (KA), arbutin, hydroquinone and ascorbic acid. However, these inhibitors have side effects and lack clinical efficiency. These facts led us to focus our research work on the exploration of natural tyrosinase inhibitors. Due to the therapeutic potential of medical plants researchers are not only concerned with validating ethnopharmacological usage of plants, but also with identification, isolation and characterization of bioactive components. Sutherlandia frutescens and Psoralea aphylla are both examples of indigenous fynbos species, which have been applied by indigenous people for the benefit of their medicinal properties.

The medicinal plant Sutherlandia frutescens (SF) is commonly consumed in South Africa, and is traditionally applied to a range of ailments. Yet its popularity stems from the use of SF as a cancer treatment. This plant contains a range of active compounds including L-canavanine (L-CAV), D-pinitol and gamma (γ)-aminobutyric acid, all of which contribute to the therapeutic properties of SF. It is also endorsed by the South African Ministry of Health as a supplementary treatment for HIV/AIDS. Maize is the staple crop of South Africa, and can be frequently contaminated by the mycotoxin fumonisin B1 (FB1). The mycotoxin is linked to an extensive list of livestock diseases. Although little is known about its role in human disease, FB1 has been epidemiologically linked to oesophageal cancer in South Africa. Both SF and FB1 have been shown to promote apoptosis, and the effect(s) of consuming both in combination is currently unknown. The principle aim of this study was to determine whether SF and FB1 had either synergistic or antagonising effects in combination, by investigating immune cell toxicity Jurkat cells. Apoptotic parameters such as caspase activation, mitochondrial depolarisation, phosphatidylserine (PS) externalisation and ATP quantification were analysed. Levels of caspase activation were highest in cells treated with SF only (caspase-3: 86.79 RLU, no significance compared to other treatments; caspase-8: 40.1 RLU, significance compared to other treatments [p<0.05]; caspase-9: 11.07 RLU, significance compared to FB1 and control treatments [p<0.05]). ATP levels were significantly highest in SF-treated cells compared to other treatments (8.17 RLU, [p<0.05]). Mitochondrial depolarisation was also highest in SF-treated Jurkat cells at 18.5% depolarisation with no significance compared to other treatments, however PS externalisation were significantly lower in SF-treated cells compared with other treatments (3.69% [p<0.05]). Oxidative stress parameters were also investigated, including thiobutyric acid reactive species (TBARS), Glutathione (GSH) and Reactive Nitrogen Species (RNS) assays. TBARS levels were significantly higher in FB1 treated cells (OD 1.95, [p<0.05]) compared to SF and control. Glutathione and RNS levels were also lowest in FB1-treated cells. The data suggests that SF induces apoptosis, characteristic of its nature as an anti-cancer treatment, and FB1 induces oxidative stress, which is characteristic of its carcinogenic properties. Based on this preliminary study, it appears that FB1 and SF both synergises and antagonises the other in combination, yet further investigation is needed into its effects in vivo. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, 2011.