Neurodegenerative disorders like Parkinson’s and Alzheimer’s disease affect millions of people around the world. Oxidative stress has been implicated in the pathogenesis of a number of neurodegenerative disorders, cancer and ischemia. The brain is particularly vulnerable to oxidative damage because of its high utilisation of oxygen, high levels of polyunsaturated fatty acids, relatively high levels of redox transition metal ions and low levels of antioxidants. Oxidative stress occurs due to an imbalance in the pro–oxidant and antioxidant levels. Reactive oxygen/nitrogen species (ROS/RNS) is a collective term used for free radicals and related molecules, promoting oxidative stress within cells and ultimately leading to neurodegeneration. Antioxidants counteract the excess in ROS/RNS, and is therefore of interest in the treatment and prevention of neurodegenerative disorders. Monoamine oxidases, especially monoamine oxidase B (MAO–B), also play an important role in neurodegenerative disorders. MAO–B is the main enzyme responsible for the oxidative deamination of dopamine in the substantia nigra of the brain. By inhibiting MAO–B, dopamine is increased in the brain providing symptomatic relief in Parkinson’s disease. The focus of the current study was to synthesise multifunctional compounds that could be used in the treatment and/or prevention of neurodegenerative diseases. In this study flavonoids were selected because of their wide spectrum of biological activities, including antioxidant activity and its monoamine oxidase inhibition. Flavones and chalcones are both classified under flavonoids and both structures were included. The amantadine moiety was included because of its known ability to inhibit calcium flux through the N–methyl–D–aspartate (NMDA) receptor channel. Six amantadine–flavonoid derivatives were synthesised using standard laboratory procedures and structures were determined with standard methods such as NMR, IR and mass spectrometry. The synthesised compounds were tested in a selection of biological assays, to establish the relative antioxidant properties and MAO inhibitory activity. The biological assays employed to test antioxidant properties were the thiobarbituric acid (TBA) and nitro–blue tetrazolium (NBT) assays. The TBA assay relies on the assessment of lipid peroxidation, induced via hydroxyl anions (OH), generating a pink colour with the complex formation between malondialdehyde (MDA) and TBA, which is measured spectrophotometrically at 532 nm. The principal of the NBT assay is the reduction of NBT to nitro–blue diformazan (NBD), producing a purple colour in the presence of superoxide anions (O2 –). The synthesised compounds were also evaluated for their MAO inhibitory activity toward recombinant human MAO–A and -B and inhibition values were expressed as IC50 values. The experimental data obtained in the NBT and TBA assay indicated a weak but a significant ability to scavenge O2 – and OH. In the NBT assay N–(adamantan–1–yl)–2–{3–hydroxy–4–[(2E)– 3–(3–methoxyphenyl)pro–2–enoyl]phenoxy}acetamide (6) had the best results with a 50.47 ± 1.31 uM/mg protein reduction in NBD formation, indicating that the hydroxyl group contributed to activity. The synthesised compounds were compared to the toxin (KCN) with a reduction in NDB formation of 69.88 ± 1.59 uM/mg protein. Results obtained from the TBA assay indicated that the flavone moiety had better OH scavenging ability than that of the chalcone moiety with N–(adamantan–1–yl)–2–[(5–hydroxy–4–oxo–2–phenyl–4H–chromen–7– yl)oxy]acetamide (3) showing the best activity at 0.967 ± 0.063 nmol MDA/mg tissue. The synthesised compounds were compared to the toxin (H2O2) 1.316 ± 0.028 nmol MDA/mg tissue. None of the test compounds could be compared to the results obtained with Trolox®. The IC50 values obtained for inhibition of recombinant human MAO indicated that the chalcone moiety (N–(adamantan–1–yl)–4–[(1E)–3–oxo–3–phenylpro–1–en–1–yl]benzamide (5)) showed the best inhibition of MAO–B with an IC50 of 0.717 ± 0.009 M and of MAO–A with an IC50 of 24.987 ± 5.988 M. It was further confirmed that N–(adamantan–1–yl)–4–[(1E)–3–oxo–3– phenylpro–1–en–1–yl]benzamide (5) binds reversible to MAO–B and that the mode of inhibition is competitive. Docking studies revealed that N–(adamantan–1–yl)–4–[(1E)–3–oxo–3–phenylpro– 1–en–1–yl]benzamide (5) traverses both cavities of MAO–B with the chalcone moiety orientated towards the FAD co–factor while the amantadine moiety protrudes into the entrance cavity. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

South Africa is a country of great diversity. Different climate zones and a host of different habitats make South Africa the perfect platform for rich floral diversity. This floral diversity lends itself to the study of natural products by discovering new natural drugs that can be used in the treatment of many illnesses. Studies into the antioxidant properties of plants that are used in traditional medicine are an important aspect of research to determine the rationale of the use of plants by traditional healers. Many neurodegenerative diseases, like epilepsy, Parkinson s and Alzheimer s diseases, are linked to oxidative stress. Antioxidants could play a major role as neuroprotective agents and could alter the progression of these diseases. Epilepsy is one of the world s most prevalent central nervous system disorders and affects more than seventy per one thousand children in South Africa. Most of these cases are people in rural areas of South Africa where communities rely on the use of traditional medicine. Cotyledon orbiculata L. var orbiculata (Haw.) DC. is widely used in traditional medicine to treat epilepsy and other central nervous system disorders. The need to screen these plants for activity and toxicity is very important to understand the complex mechanism of action in the treatment of patients. In this study the methanol extract and three different fractions of the methanol extract of Cotyledon orbiculata were used to test for antioxidant activity and toxicity towards neuroblastoma cells. The freeze dried leaves of Cotyledon orbiculata were extracted with methanol using a Soxhlet apparatus. The concentrated extracts were analysed using HPLC (high pressure liquid chromatography) and three major peaks were selected for isolation. Three assays were performed to assess the antioxidant activity and toxicity of the isolated compounds. The thiobarbituric acid assay (TBA) quantifies the extent of the inhibition of lipid peroxidation in rat brain homogenates by the isolated fractions. All of the samples were able to attenuate lipid peroxidation as seen from the results obtained from the TBA assay. The methanol extract showed the best attenuation of lipid peroxidation in the rat brain homogenate with fraction 1 and 2 showing greater attenuation of lipid peroxidation than fraction 3. The nitroblue tetrazolium assay (NBT) quantifies the ability of the fractions to scavenge superoxide radicals in a rat brain homogenate. All samples were able to scavenge superoxide radicals as indicated by the NBT assay. The methanol extract showed the best superoxide scavenging abilities in the assay whereas fraction 1 showed better scavenging abilities than fraction 2 and 3. The 3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide assay (MTT) indicates the toxicity of the fractions towards neuroblastoma cells. The methanol extract and fraction 2 in the highest concentration of 10 mg/ml were the only samples that showed toxicity towards neuroblastoma cells. The molecular structure of a compound from fraction 2 was determined by using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), and mass spectroscopy (MS). This compound was identified as diethyl malate. Diethyl malate is an artefact that is generated in HPLC procedures in the presence of malic acid (which naturally occurs in the leaves of Cotyledon orbiculata) and ethanol. The methanol extract of Cotyledon orbiculata has high antioxidant activity and could be due to the presence of malic acid in the leaves of the plant. The rationale in the use of Cotyledon orbiculata in the treatment of epilepsy could not be determined due to the isolation of an artefact, diethyl malate, obtained from the fraction. Further research should include methods to prevent artefact formation and purification of the samples that are obtained. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

Neurodegenerative disorders like Parkinson’s and Alzheimer’s disease affect millions of people around the world. Oxidative stress has been implicated in the pathogenesis of a number of neurodegenerative disorders, cancer and ischemia. The brain is particularly vulnerable to oxidative damage because of its high utilisation of oxygen, high levels of polyunsaturated fatty acids, relatively high levels of redox transition metal ions and low levels of antioxidants. Oxidative stress occurs due to an imbalance in the pro–oxidant and antioxidant levels. Reactive oxygen/nitrogen species (ROS/RNS) is a collective term used for free radicals and related molecules, promoting oxidative stress within cells and ultimately leading to neurodegeneration. Antioxidants counteract the excess in ROS/RNS, and is therefore of interest in the treatment and prevention of neurodegenerative disorders. Monoamine oxidases, especially monoamine oxidase B (MAO–B), also play an important role in neurodegenerative disorders. MAO–B is the main enzyme responsible for the oxidative deamination of dopamine in the substantia nigra of the brain. By inhibiting MAO–B, dopamine is increased in the brain providing symptomatic relief in Parkinson’s disease. The focus of the current study was to synthesise multifunctional compounds that could be used in the treatment and/or prevention of neurodegenerative diseases. In this study flavonoids were selected because of their wide spectrum of biological activities, including antioxidant activity and its monoamine oxidase inhibition. Flavones and chalcones are both classified under flavonoids and both structures were included. The amantadine moiety was included because of its known ability to inhibit calcium flux through the N–methyl–D–aspartate (NMDA) receptor channel. Six amantadine–flavonoid derivatives were synthesised using standard laboratory procedures and structures were determined with standard methods such as NMR, IR and mass spectrometry. The synthesised compounds were tested in a selection of biological assays, to establish the relative antioxidant properties and MAO inhibitory activity. The biological assays employed to test antioxidant properties were the thiobarbituric acid (TBA) and nitro–blue tetrazolium (NBT) assays. The TBA assay relies on the assessment of lipid peroxidation, induced via hydroxyl anions (OH), generating a pink colour with the complex formation between malondialdehyde (MDA) and TBA, which is measured spectrophotometrically at 532 nm. The principal of the NBT assay is the reduction of NBT to nitro–blue diformazan (NBD), producing a purple colour in the presence of superoxide anions (O2 –). The synthesised compounds were also evaluated for their MAO inhibitory activity toward recombinant human MAO–A and -B and inhibition values were expressed as IC50 values. The experimental data obtained in the NBT and TBA assay indicated a weak but a significant ability to scavenge O2 – and OH. In the NBT assay N–(adamantan–1–yl)–2–{3–hydroxy–4–[(2E)– 3–(3–methoxyphenyl)pro–2–enoyl]phenoxy}acetamide (6) had the best results with a 50.47 ± 1.31 uM/mg protein reduction in NBD formation, indicating that the hydroxyl group contributed to activity. The synthesised compounds were compared to the toxin (KCN) with a reduction in NDB formation of 69.88 ± 1.59 uM/mg protein. Results obtained from the TBA assay indicated that the flavone moiety had better OH scavenging ability than that of the chalcone moiety with N–(adamantan–1–yl)–2–[(5–hydroxy–4–oxo–2–phenyl–4H–chromen–7– yl)oxy]acetamide (3) showing the best activity at 0.967 ± 0.063 nmol MDA/mg tissue. The synthesised compounds were compared to the toxin (H2O2) 1.316 ± 0.028 nmol MDA/mg tissue. None of the test compounds could be compared to the results obtained with Trolox®. The IC50 values obtained for inhibition of recombinant human MAO indicated that the chalcone moiety (N–(adamantan–1–yl)–4–[(1E)–3–oxo–3–phenylpro–1–en–1–yl]benzamide (5)) showed the best inhibition of MAO–B with an IC50 of 0.717 ± 0.009 M and of MAO–A with an IC50 of 24.987 ± 5.988 M. It was further confirmed that N–(adamantan–1–yl)–4–[(1E)–3–oxo–3– phenylpro–1–en–1–yl]benzamide (5) binds reversible to MAO–B and that the mode of inhibition is competitive. Docking studies revealed that N–(adamantan–1–yl)–4–[(1E)–3–oxo–3–phenylpro– 1–en–1–yl]benzamide (5) traverses both cavities of MAO–B with the chalcone moiety orientated towards the FAD co–factor while the amantadine moiety protrudes into the entrance cavity. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

South Africa is a country of great diversity. Different climate zones and a host of different habitats make South Africa the perfect platform for rich floral diversity. This floral diversity lends itself to the study of natural products by discovering new natural drugs that can be used in the treatment of many illnesses. Studies into the antioxidant properties of plants that are used in traditional medicine are an important aspect of research to determine the rationale of the use of plants by traditional healers. Many neurodegenerative diseases, like epilepsy, Parkinson s and Alzheimer s diseases, are linked to oxidative stress. Antioxidants could play a major role as neuroprotective agents and could alter the progression of these diseases. Epilepsy is one of the world s most prevalent central nervous system disorders and affects more than seventy per one thousand children in South Africa. Most of these cases are people in rural areas of South Africa where communities rely on the use of traditional medicine. Cotyledon orbiculata L. var orbiculata (Haw.) DC. is widely used in traditional medicine to treat epilepsy and other central nervous system disorders. The need to screen these plants for activity and toxicity is very important to understand the complex mechanism of action in the treatment of patients. In this study the methanol extract and three different fractions of the methanol extract of Cotyledon orbiculata were used to test for antioxidant activity and toxicity towards neuroblastoma cells. The freeze dried leaves of Cotyledon orbiculata were extracted with methanol using a Soxhlet apparatus. The concentrated extracts were analysed using HPLC (high pressure liquid chromatography) and three major peaks were selected for isolation. Three assays were performed to assess the antioxidant activity and toxicity of the isolated compounds. The thiobarbituric acid assay (TBA) quantifies the extent of the inhibition of lipid peroxidation in rat brain homogenates by the isolated fractions. All of the samples were able to attenuate lipid peroxidation as seen from the results obtained from the TBA assay. The methanol extract showed the best attenuation of lipid peroxidation in the rat brain homogenate with fraction 1 and 2 showing greater attenuation of lipid peroxidation than fraction 3. The nitroblue tetrazolium assay (NBT) quantifies the ability of the fractions to scavenge superoxide radicals in a rat brain homogenate. All samples were able to scavenge superoxide radicals as indicated by the NBT assay. The methanol extract showed the best superoxide scavenging abilities in the assay whereas fraction 1 showed better scavenging abilities than fraction 2 and 3. The 3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide assay (MTT) indicates the toxicity of the fractions towards neuroblastoma cells. The methanol extract and fraction 2 in the highest concentration of 10 mg/ml were the only samples that showed toxicity towards neuroblastoma cells. The molecular structure of a compound from fraction 2 was determined by using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), and mass spectroscopy (MS). This compound was identified as diethyl malate. Diethyl malate is an artefact that is generated in HPLC procedures in the presence of malic acid (which naturally occurs in the leaves of Cotyledon orbiculata) and ethanol. The methanol extract of Cotyledon orbiculata has high antioxidant activity and could be due to the presence of malic acid in the leaves of the plant. The rationale in the use of Cotyledon orbiculata in the treatment of epilepsy could not be determined due to the isolation of an artefact, diethyl malate, obtained from the fraction. Further research should include methods to prevent artefact formation and purification of the samples that are obtained. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

The progression of skin ageing in individuals is multifaceted and provoked by various aspects, including hereditary and a variety of environmental causes, for instance UV (ultra violet) radiation, resulting in the morphological modifications in the dermal layer of the skin (Makrantonaki & Zouboulis, 2007:40) Transformations caused by ageing skin, in which degenerative alterations exceed regenerative alterations are recognised by the thinning and wrinkling of the epidermis in conjunction with the appearance of lines, creases, crevices and furrows, particularly emphasised in lines of facial expressions (Aburjai & Natsheh, 2003:990). For human beings to continue to exist in a terrestrial atmosphere, the loss of water from the skin must be cautiously synchronised by the epidermis, a task dependent on the multifaceted character of the stratum corneum (Rawlings & Harding, 2004:43). The stratum corneum (SC) is responsible for the main resistance to the penetration of most compounds; nevertheless the skin represents as an appropriate target for delivery. The target site for anti-ageing treatment includes the epidermal and dermal layers of the skin. Therefore, the need to apply fatty materials to the skin is practically intuitive and may perhaps be as old as man’s existence itself (Lodén, 2005:672). Natural therapies have been used for several decades for taking care of skin illnesses and a wide variety of dermatological disorders, such as inflammation, phototoxicity, atopic dermatitis and alopecia areata (Aburjai & Natsheh, 2003:988). Using the skin as an alternative route for the administration of honeybush extracts for the treatment of ageing skin may be beneficial. Tea contains more than 500 chemical compounds, including, tannins, flavonoids, amino acids, vitamins, caffeine and polysaccharides. Tea polyphenols (flavonoids) have proven anti-inflammatory, antioxidant, antiallergic, antibacterial and antiviral effects (Aburjai & Natsheh, 2003:990). Unfortunately using the skin as an alternative route for administering drugs (transdermal drug delivery) has numerous limitations. One of these limitations is the barrier function of the skin (Naik et al., 2000:319). Because of the skin’s outstanding ability to protect the body against unwanted substances from its surroundings, it is necessary to use methods to enhance drug penetration through the skin. The aim of this study was to formulate two 2% semisolid formulations containing two different honeybush extracts as the active ingredient, and to determine which of the formulations deliver mangiferin and hesperidin best to the target site (dermis). Cosmetic formulations of a natural origin, is designed to protect the skin against exogenous or endogenous harmful agents, as well as to balance the dermal homeostatis lipids altered by dermatosis and ageing (Aburjai & Natsheh, 2003:988). Stability tests over a three month period were also performed on the different formulations. To determine the stability of the different semi-solid formulations, the formulated products were stored at 25 °C/60% RH (relative humidity), 30 °C/60% RH and 40 °C/75% RH. HPLC analysis was used to determine the concentrations of the ingredients in all the formulated products. Other stability tests included appearance, pH, viscosity, mass loss, zeta potential and particle size determination. Unfortunately a change in colour, viscosity, zeta potential, mass loss, particle size and concentration of the ingredients in both the formulations, indicated that the products were unstable from the first month of stability testing. A 2% Cyclopia maculata cream as well as a 2% Cyclopia genistoides cream was formulated. Franz cell diffusion studies as well as membrane release studies were performed over a 12 h period, followed by tape stripping experiments to determine which semi-solid formulation delivered mangiferin and hesperidin the best to the dermal layer of the skin. The results of the different formulations were compared. Unfortunately there was no significant penetration by any of the honeybush extracts. Results were inconclusive and unquantifiable due to unconvincing penetration results. The antioxidant properties of both the extracts and the active ingredients were calculated. Antioxidant studies by the use of the TBA-assay method were done to determine whether the honeybush extracts, mangiferin and hesperidin as well as their semisolid formulations had any antioxidant activities. Both the honeybush extracts and the semisolid formulations showed promising results. Mangiferin and hesperidin did not show any antioxidant activity on their own, therefore the assumption can be confirmed that plants do function synergistically. A clinical study was also conducted to see whether honeybush extracts have the potential to hydrate the skin, counteracting the symptoms and signs of skin ageing. Clinical efficacy studies were done to determine whether the honeybush formulations had any skin hydrating effects in the treatment against skin ageing. The results were statistically inconclusive and variations between the subjects were very high due to skin variations at different skin sites. There was however a trend that Cyclopia genistoides performed the best. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

Natural oils are regularly used in cosmetics and as treatment for numeral skin conditions (Nielsen, 2006:575). The natural products industry is a multibillion dollar industry and has grown tremendously over the past few years. Natural oils used in cosmetics contain a range of fatty acids which contribute to several valuable properties in cosmetic- and personal care products. Fatty acids are divided into saturated acids and unsaturated acids (Vermaak et al., 2011:920,922). Because of the popularity and wide diversity of skin care products, it is necessary to create products that will distinguish themselves from the rest of the commercial products. To include natural oils in skin care products is a new way to prevent skin ageing, as well as other dermatological conditions. In this study, a natural oil, namely crocodile oil was used. Crocodile oil is obtained from the fat of the Nile crocodile (Crocodylus niloticus). Crocodile oil has the same composition as human skin oil. It only differs with regard to the percentages of the ingredients present. Crocodile oil contains saturated and unsaturated fatty acids. Because of the similar composition as human skin oil, crocodile oil will rarely be allergenic when applied to human skin and therefore will be a very accepted and harmless product to use (Croc city, 2012). There are many claims of positive results when crocodile oil containing products have been used. It includes fading of freckles, treatment of acne and pimple marks, dark lines, wrinkles and laugh lines. It also includes vanishing of dark shadows, sun spots and other discolorations. It helps prevent discolorations from forming and makes the skin softer, brighter and more attractive. It also controls rashness and dryness (Croc city, 2012). Because of crocodile oil’s anti-ageing, anti-fungal and anti-bacterial effects claimed by crocodile oil suppliers, and due to the fact that little scientific data is available on crocodile oil, it was decided to investigate the claims. In this study, the aims and objectives were to use natural oil, namely crocodile oil, and investigate the fatty acid profile, anti-microbial and anti-fungal activity, anti-oxidant activity, toxicity studies, stability determination of crocodile oil lotion and clinical efficacy testing of the anti-ageing effects. To determine the fatty acid profile of crocodile oil, fatty acid methyl ester (FAME) analysis with gas chromatography were used. Identification of FAME peaks in the samples was made by comparing the relative retention times of FAME peaks from samples to those of reference standards. The composition of fatty acids in crocodile oil compared well to fatty acids found in human skin oil. Anti-microbial and anti-fungal tests were done by Envirocare Laboratories, North-West University, Potchefstroom. Staphylococcus aureus, Esterichia coli, Pseudomanas aeruginosa, Candida albicans, Brasiliensis, Propionibacterium acnes and Trichophyton rubrum cultures were used to determine the anti-microbial and anti-fungal activity of crocodile oil. Unfortunately no activity was observed. The anti-oxidant properties of crocodile oil and crocodile oil lotion were determined by using the most commonly used method for measuring Malondialdehyde (MDA) in biological samples, namely the thiobarbituric acid (TBA) test. This method is based on spectrophotometric quantification of the pink complex formed after reaction of MDA with two molecules of TBA. No anti-oxidant activity was observed in the oil or the lotion. Toxicity studies were performed by Dr. D. Goosen (BVSc Hons. Pret.) from Tswane University of Technology (Pretoria, South Africa). The studies showed that the lotion had no toxicity in the skin sensitisation, acute dermal toxicity and acute dermal irritation studies. To determine the stability of the crocodile oil lotion, the formulated products were store at 25 °C / 60% RH (relative humidity), 30 °C / 60% RH and 40 °C / 75% RH for 6 months in the original packaging as well as a glass container. The stability tests included pH, viscosity, visual appearance assessment, zeta-potential, droplet size and mass loss. The crocodile cream lotion was stable over the 6 months period in both containers. Clinical efficacy testing was performed at the CEL (Clinical Efficacy Laboratory) of the North-West University, Potchefstroom, South Africa. A short-term study over a period of 3 h was performed to investigate the hydrating effects of crocodile oil lotion. A long-term study over a period of 12 weeks was performed to examine the anti-ageing effects of crocodile oil lotion. An erythema study was also conducted to test the anti-erythema properties of crocodile oil lotion. Although the crocodile oil lotion as well as the placebo lotion showed an increase in skin hydration, there was no significant difference between the two treatments. Crocodile oil lotion also showed no anti-erythema properties. / Thesis (PhD (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

The progression of skin ageing in individuals is multifaceted and provoked by various aspects, including hereditary and a variety of environmental causes, for instance UV (ultra violet) radiation, resulting in the morphological modifications in the dermal layer of the skin (Makrantonaki & Zouboulis, 2007:40) Transformations caused by ageing skin, in which degenerative alterations exceed regenerative alterations are recognised by the thinning and wrinkling of the epidermis in conjunction with the appearance of lines, creases, crevices and furrows, particularly emphasised in lines of facial expressions (Aburjai & Natsheh, 2003:990). For human beings to continue to exist in a terrestrial atmosphere, the loss of water from the skin must be cautiously synchronised by the epidermis, a task dependent on the multifaceted character of the stratum corneum (Rawlings & Harding, 2004:43). The stratum corneum (SC) is responsible for the main resistance to the penetration of most compounds; nevertheless the skin represents as an appropriate target for delivery. The target site for anti-ageing treatment includes the epidermal and dermal layers of the skin. Therefore, the need to apply fatty materials to the skin is practically intuitive and may perhaps be as old as man’s existence itself (Lodén, 2005:672). Natural therapies have been used for several decades for taking care of skin illnesses and a wide variety of dermatological disorders, such as inflammation, phototoxicity, atopic dermatitis and alopecia areata (Aburjai & Natsheh, 2003:988). Using the skin as an alternative route for the administration of honeybush extracts for the treatment of ageing skin may be beneficial. Tea contains more than 500 chemical compounds, including, tannins, flavonoids, amino acids, vitamins, caffeine and polysaccharides. Tea polyphenols (flavonoids) have proven anti-inflammatory, antioxidant, antiallergic, antibacterial and antiviral effects (Aburjai & Natsheh, 2003:990). Unfortunately using the skin as an alternative route for administering drugs (transdermal drug delivery) has numerous limitations. One of these limitations is the barrier function of the skin (Naik et al., 2000:319). Because of the skin’s outstanding ability to protect the body against unwanted substances from its surroundings, it is necessary to use methods to enhance drug penetration through the skin. The aim of this study was to formulate two 2% semisolid formulations containing two different honeybush extracts as the active ingredient, and to determine which of the formulations deliver mangiferin and hesperidin best to the target site (dermis). Cosmetic formulations of a natural origin, is designed to protect the skin against exogenous or endogenous harmful agents, as well as to balance the dermal homeostatis lipids altered by dermatosis and ageing (Aburjai & Natsheh, 2003:988). Stability tests over a three month period were also performed on the different formulations. To determine the stability of the different semi-solid formulations, the formulated products were stored at 25 °C/60% RH (relative humidity), 30 °C/60% RH and 40 °C/75% RH. HPLC analysis was used to determine the concentrations of the ingredients in all the formulated products. Other stability tests included appearance, pH, viscosity, mass loss, zeta potential and particle size determination. Unfortunately a change in colour, viscosity, zeta potential, mass loss, particle size and concentration of the ingredients in both the formulations, indicated that the products were unstable from the first month of stability testing. A 2% Cyclopia maculata cream as well as a 2% Cyclopia genistoides cream was formulated. Franz cell diffusion studies as well as membrane release studies were performed over a 12 h period, followed by tape stripping experiments to determine which semi-solid formulation delivered mangiferin and hesperidin the best to the dermal layer of the skin. The results of the different formulations were compared. Unfortunately there was no significant penetration by any of the honeybush extracts. Results were inconclusive and unquantifiable due to unconvincing penetration results. The antioxidant properties of both the extracts and the active ingredients were calculated. Antioxidant studies by the use of the TBA-assay method were done to determine whether the honeybush extracts, mangiferin and hesperidin as well as their semisolid formulations had any antioxidant activities. Both the honeybush extracts and the semisolid formulations showed promising results. Mangiferin and hesperidin did not show any antioxidant activity on their own, therefore the assumption can be confirmed that plants do function synergistically. A clinical study was also conducted to see whether honeybush extracts have the potential to hydrate the skin, counteracting the symptoms and signs of skin ageing. Clinical efficacy studies were done to determine whether the honeybush formulations had any skin hydrating effects in the treatment against skin ageing. The results were statistically inconclusive and variations between the subjects were very high due to skin variations at different skin sites. There was however a trend that Cyclopia genistoides performed the best. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

Natural oils are regularly used in cosmetics and as treatment for numeral skin conditions (Nielsen, 2006:575). The natural products industry is a multibillion dollar industry and has grown tremendously over the past few years. Natural oils used in cosmetics contain a range of fatty acids which contribute to several valuable properties in cosmetic- and personal care products. Fatty acids are divided into saturated acids and unsaturated acids (Vermaak et al., 2011:920,922). Because of the popularity and wide diversity of skin care products, it is necessary to create products that will distinguish themselves from the rest of the commercial products. To include natural oils in skin care products is a new way to prevent skin ageing, as well as other dermatological conditions. In this study, a natural oil, namely crocodile oil was used. Crocodile oil is obtained from the fat of the Nile crocodile (Crocodylus niloticus). Crocodile oil has the same composition as human skin oil. It only differs with regard to the percentages of the ingredients present. Crocodile oil contains saturated and unsaturated fatty acids. Because of the similar composition as human skin oil, crocodile oil will rarely be allergenic when applied to human skin and therefore will be a very accepted and harmless product to use (Croc city, 2012). There are many claims of positive results when crocodile oil containing products have been used. It includes fading of freckles, treatment of acne and pimple marks, dark lines, wrinkles and laugh lines. It also includes vanishing of dark shadows, sun spots and other discolorations. It helps prevent discolorations from forming and makes the skin softer, brighter and more attractive. It also controls rashness and dryness (Croc city, 2012). Because of crocodile oil’s anti-ageing, anti-fungal and anti-bacterial effects claimed by crocodile oil suppliers, and due to the fact that little scientific data is available on crocodile oil, it was decided to investigate the claims. In this study, the aims and objectives were to use natural oil, namely crocodile oil, and investigate the fatty acid profile, anti-microbial and anti-fungal activity, anti-oxidant activity, toxicity studies, stability determination of crocodile oil lotion and clinical efficacy testing of the anti-ageing effects. To determine the fatty acid profile of crocodile oil, fatty acid methyl ester (FAME) analysis with gas chromatography were used. Identification of FAME peaks in the samples was made by comparing the relative retention times of FAME peaks from samples to those of reference standards. The composition of fatty acids in crocodile oil compared well to fatty acids found in human skin oil. Anti-microbial and anti-fungal tests were done by Envirocare Laboratories, North-West University, Potchefstroom. Staphylococcus aureus, Esterichia coli, Pseudomanas aeruginosa, Candida albicans, Brasiliensis, Propionibacterium acnes and Trichophyton rubrum cultures were used to determine the anti-microbial and anti-fungal activity of crocodile oil. Unfortunately no activity was observed. The anti-oxidant properties of crocodile oil and crocodile oil lotion were determined by using the most commonly used method for measuring Malondialdehyde (MDA) in biological samples, namely the thiobarbituric acid (TBA) test. This method is based on spectrophotometric quantification of the pink complex formed after reaction of MDA with two molecules of TBA. No anti-oxidant activity was observed in the oil or the lotion. Toxicity studies were performed by Dr. D. Goosen (BVSc Hons. Pret.) from Tswane University of Technology (Pretoria, South Africa). The studies showed that the lotion had no toxicity in the skin sensitisation, acute dermal toxicity and acute dermal irritation studies. To determine the stability of the crocodile oil lotion, the formulated products were store at 25 °C / 60% RH (relative humidity), 30 °C / 60% RH and 40 °C / 75% RH for 6 months in the original packaging as well as a glass container. The stability tests included pH, viscosity, visual appearance assessment, zeta-potential, droplet size and mass loss. The crocodile cream lotion was stable over the 6 months period in both containers. Clinical efficacy testing was performed at the CEL (Clinical Efficacy Laboratory) of the North-West University, Potchefstroom, South Africa. A short-term study over a period of 3 h was performed to investigate the hydrating effects of crocodile oil lotion. A long-term study over a period of 12 weeks was performed to examine the anti-ageing effects of crocodile oil lotion. An erythema study was also conducted to test the anti-erythema properties of crocodile oil lotion. Although the crocodile oil lotion as well as the placebo lotion showed an increase in skin hydration, there was no significant difference between the two treatments. Crocodile oil lotion also showed no anti-erythema properties. / Thesis (PhD (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

The evolution of aerobic metabolic processes unavoidably led to the production of reactive oxygen species (ROS). ROS have the ability to cause harmful oxidative damage to biomolecules. Increased ROS generation and subsequent oxidative stress have been associated with aging and neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases as a result of the extreme sensitivity of the central nervous system to damage from ROS. Antioxidant defence systems have co–evolved with aerobic metabolic processes to counteract oxidative damage inflicted by ROS. The impact of neurodegenerative disorders on society is increasing rapidly as the life expectancy of the global population increases. In this day and age, a much younger group of the population is also experiencing neurodegenerative symptoms as a result of the harmful effect of the human immunodeficiency virus (HIV) on the central nervous system. Plants are an invaluable source of medicinal compounds. The use of plants for their healing properties is rooted in ancient times. The aim of this study was to select from twenty one plants, the plant with the most promising antioxidant activity and to determine whether extracts of this plant could act as free radical scavengers, comparing the results to Trolox, a known free radical scavenger. The next step was to isolate and characterize a compound from an extract exhibiting promising antioxidant activity. Bioassay–guided fractionation was followed to achieve this. During screening trials, twenty one plants, namely Berula erecta, Heteromorpha arborescens, Tarchonanthus camphoratus, Vernonia oligocephala, Gymnosporia buxifolia, Acacia karroo, Elephantorrhiza elephantina, Erythrina zeyheri, Leonotis leonurus, Plectranthus ecklonii, P. rehmanii, P. venteri, Salvia auretia, S. runcinata, Solenostemon latifolius, S. rotundifolius, Plumbago auriculata, Clematis brachiata, Vangueria infausta, Physalis peruviana and Lippia javanica were selected from literature, based on reported antioxidant activity within the plant families, for screening of their antioxidant activity. One hundred and ten extracts were prepared from the leaves, using Soxhlet extraction and the solvents petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EtOAc) and ethanol (EtOH), consecutively. The focus during initial screening trials was on chemistry–based assays. The oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays were employed for the primary screening of the one hundred and ten leaf extracts. The ORAC assay was used to determine whether the plant extracts were able to scavenge peroxyl radicals and the FRAP assay was used to determine the reducing abilities of the extracts. Quantification of the peroxyl radical scavenging activity by the ORAC assay revealed that activity was observed for most of the extracts, with the ethyl acetate and ethanol extracts of L. javanica exhibiting the most promising activity. This pattern of activity was also found with the reducing capacity evaluated by the FRAP assay in which the EtOAc and EtOH extracts of L. javanica also exhibited the most promising activity. L. javanica was selected for further study by screening for biological activity, employing the nitro–blue tetrazolium (NBT) assay and thiobarbituric acid reactive substances (TBARS) assay. Using a cyanide model to induce neurotoxic effects in rat brain homogenate, the neuroprotective properties of the extracts of L. javanica leaves were examined using the NBT assay and compared to that of Trolox. The NBT assay determines the level of superoxide anions. All the extracts of L. javanica significantly reduced superoxide anion generation at all concentrations used. The petroleum ether and ethyl acetate extracts, at all concentrations, reduced superoxide anion generation to values lower than that of the control, suggesting that these extracts may be able to attenuate normal free radical processes in the brain. The petroleum ether extract exhibited the most promising activity at a concentration of 1.25 and 2.5 mg/ml and also exhibited similar results as the ethyl acetate extract at a lower concentration than the ethyl acetate extract (2.5 mg/ml compared to 5 mg/ml). A toxin–solution consisting of hydrogen peroxide (H2O2), iron(III)chloride (FeCl3) and ascorbic acid was used to induce lipid peroxidation and the ability of the extracts of the leaves of L. javanica to attenuate lipid peroxidation was investigated in rat brain homogenate and compared to that of Trolox. All of the extracts of L. javanica significantly attenuated toxininduced lipid peroxidation at all concentrations used. All of the extracts were also able to significantly attenuate toxin–induced lipid peroxidation to values lower than that of the control. These results suggest that all of the extracts of L. javanica possess the ability to attenuate not only toxin–induced lipid peroxidation, but also lipid peroxidation that occurs during normal processes in the brain. The petroleum ether extract was subjected to bioassay–guided fractionation using column and thin–layer chromatography and the NBT and TBARS assays. Fraction DD1 was investigated by means of nuclear magnetic resonance, infrared and mass spectrometry. The exact structure of fraction DD1 was not elucidated. Considering all the results, it is clear that L. javanica shows great potential as a medicinal plant with antioxidant activity and may therefore be beneficial in diminishing the destructive oxidative effects inflicted by free radicals. There are however still many compounds to be isolated from L. javanica. Key words: Verbenaceae, Lippia javanica, antioxidant, neurodegeneration, oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), nitro–blue tetrazolium assay (NBT), thiobarbituric acid reactive substances assay (TBARS). / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.

The evolution of aerobic metabolic processes unavoidably led to the production of reactive oxygen species (ROS). ROS have the ability to cause harmful oxidative damage to biomolecules. Increased ROS generation and subsequent oxidative stress have been associated with aging and neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases as a result of the extreme sensitivity of the central nervous system to damage from ROS. Antioxidant defence systems have co–evolved with aerobic metabolic processes to counteract oxidative damage inflicted by ROS. The impact of neurodegenerative disorders on society is increasing rapidly as the life expectancy of the global population increases. In this day and age, a much younger group of the population is also experiencing neurodegenerative symptoms as a result of the harmful effect of the human immunodeficiency virus (HIV) on the central nervous system. Plants are an invaluable source of medicinal compounds. The use of plants for their healing properties is rooted in ancient times. The aim of this study was to select from twenty one plants, the plant with the most promising antioxidant activity and to determine whether extracts of this plant could act as free radical scavengers, comparing the results to Trolox, a known free radical scavenger. The next step was to isolate and characterize a compound from an extract exhibiting promising antioxidant activity. Bioassay–guided fractionation was followed to achieve this. During screening trials, twenty one plants, namely Berula erecta, Heteromorpha arborescens, Tarchonanthus camphoratus, Vernonia oligocephala, Gymnosporia buxifolia, Acacia karroo, Elephantorrhiza elephantina, Erythrina zeyheri, Leonotis leonurus, Plectranthus ecklonii, P. rehmanii, P. venteri, Salvia auretia, S. runcinata, Solenostemon latifolius, S. rotundifolius, Plumbago auriculata, Clematis brachiata, Vangueria infausta, Physalis peruviana and Lippia javanica were selected from literature, based on reported antioxidant activity within the plant families, for screening of their antioxidant activity. One hundred and ten extracts were prepared from the leaves, using Soxhlet extraction and the solvents petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EtOAc) and ethanol (EtOH), consecutively. The focus during initial screening trials was on chemistry–based assays. The oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays were employed for the primary screening of the one hundred and ten leaf extracts. The ORAC assay was used to determine whether the plant extracts were able to scavenge peroxyl radicals and the FRAP assay was used to determine the reducing abilities of the extracts. Quantification of the peroxyl radical scavenging activity by the ORAC assay revealed that activity was observed for most of the extracts, with the ethyl acetate and ethanol extracts of L. javanica exhibiting the most promising activity. This pattern of activity was also found with the reducing capacity evaluated by the FRAP assay in which the EtOAc and EtOH extracts of L. javanica also exhibited the most promising activity. L. javanica was selected for further study by screening for biological activity, employing the nitro–blue tetrazolium (NBT) assay and thiobarbituric acid reactive substances (TBARS) assay. Using a cyanide model to induce neurotoxic effects in rat brain homogenate, the neuroprotective properties of the extracts of L. javanica leaves were examined using the NBT assay and compared to that of Trolox. The NBT assay determines the level of superoxide anions. All the extracts of L. javanica significantly reduced superoxide anion generation at all concentrations used. The petroleum ether and ethyl acetate extracts, at all concentrations, reduced superoxide anion generation to values lower than that of the control, suggesting that these extracts may be able to attenuate normal free radical processes in the brain. The petroleum ether extract exhibited the most promising activity at a concentration of 1.25 and 2.5 mg/ml and also exhibited similar results as the ethyl acetate extract at a lower concentration than the ethyl acetate extract (2.5 mg/ml compared to 5 mg/ml). A toxin–solution consisting of hydrogen peroxide (H2O2), iron(III)chloride (FeCl3) and ascorbic acid was used to induce lipid peroxidation and the ability of the extracts of the leaves of L. javanica to attenuate lipid peroxidation was investigated in rat brain homogenate and compared to that of Trolox. All of the extracts of L. javanica significantly attenuated toxininduced lipid peroxidation at all concentrations used. All of the extracts were also able to significantly attenuate toxin–induced lipid peroxidation to values lower than that of the control. These results suggest that all of the extracts of L. javanica possess the ability to attenuate not only toxin–induced lipid peroxidation, but also lipid peroxidation that occurs during normal processes in the brain. The petroleum ether extract was subjected to bioassay–guided fractionation using column and thin–layer chromatography and the NBT and TBARS assays. Fraction DD1 was investigated by means of nuclear magnetic resonance, infrared and mass spectrometry. The exact structure of fraction DD1 was not elucidated. Considering all the results, it is clear that L. javanica shows great potential as a medicinal plant with antioxidant activity and may therefore be beneficial in diminishing the destructive oxidative effects inflicted by free radicals. There are however still many compounds to be isolated from L. javanica. Key words: Verbenaceae, Lippia javanica, antioxidant, neurodegeneration, oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), nitro–blue tetrazolium assay (NBT), thiobarbituric acid reactive substances assay (TBARS). / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.