Resistance of human pathogenic bacterial strains results in selective pressure against known antibiotic. However, plant derived compounds that possess antibacterial potential are currently being investigated for treatment of wound infections in diabetic patients as they are inexpensive and non-toxic. Hence, this dissertation was designed to evaluate two medicinal plants (Brachylaena elliptica and Brachylaena ilicifolia) traditionally used in the treatment of various diseases such as diabetes, and its secondary complications in diabetic patients. The in vitro antioxidant activity of both plants were evaluated using DPPH (1, 1-diphenylhydrazl), ferric reducing power, ABTS (2, 2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid), NO (nitric oxide) and H2O2 (hydrogen peroxide) techniques. The antibacterial test and Minimum inhibitory concentration (MIC) was determined by agar dilution method against 5 bacteria strains (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogene, Proteus vulgaris and Proteus mirabilis) infecting wounds in diabetic patients using amoxicillin and ciprofloxacin as positive control. The phytochemical analyses were assessed using standard published methods. Identification of bioactive components in essential oils of both plants were assessed using GCMS. The aqueous and ethanol extracts of both plants were also evaluated to identify bioactive components using LC-MS. The results of the phytochemical analysis revealed the presence of phenols, tannins, flavanoids, flavanols, proanthocyanidins, saponins and alkaloids in both plants. Both plants indicated strong antioxidant activities which might be due to the presence of bioactive compounds. The aqueous and ethanol leaf extracts of both plants demonstrated appreciable broad spectrum activities against these wound pathogens with MIC ranging between 5 and 0.3 mg/ml. The GC-MS analysis of the essential oils of both plants revealed the presence of monoterpenes, oxygenated sesquiterpenes, phenolics and esters. The LC-MS analysis of the aqueous and ethanol leaf extracts of both plants showed that both plants are rich in alkaloids, terpenes, terpenoids, monoterpernoids, and flavanoids. Conclusively, this study has partially justified the ethnomedicinal use of B. elliptica and B.licifolia leaves for the treatment of various diseases, including diabetes and wound infections caused by bacteria in diabetic patients. These may be attributed to the presence of antioxidant compound such as phenols, flavanoids, saponins, tannins, alkaloids and other phytochemical compounds.

M.Tech. / It is estimated that 59% of South African adult women and 29% of South African adult men are overweight (Department of Health, 2004). Significant risks arise from being overweight including: elevated cholesterol and the development of cardiovascular disease which increases with a greater gain in weight (Duyff, 2006). There is a lack of sufficient evidence supporting the safety and efficacy of many of the herbal weight-loss products currently available thus indicating that more research on herbal products and their efficacy in weight-loss is required (Lenz and Hamilton, 2004). The aim of this study is to determine the efficacy of a herbal complex (Aloe ferox, Fucus vesiculosis, Taraxacum officinale and Trigonella foenum– graecum) as an aid in weight loss in females utilising comparative measurements of the participants’ weight, Body Mass Index (BMI), body fat percentage and circumferential measurements of their hips, waist, thighs, upper arms and abdomen. The study was a quantitative, double blind placebo controlled study. The study involved thirty overweight female participants (BMI 25.5 - 30 kg/m²) between the ages of twenty and thirty five. The participants were recruited by means of advertisement posters placed at the University of Johannesburg, Homoeopathy Health Centre. The participants were randomly divided into two groups of fifteen. One group received the herbal complex (Aloe ferox, Fucus vesiculosis, Taraxacum officinale and Trigonella foenum– graecum) and the other group received the placebo. Participants from both groups attended an initial interview where they were screened by means of a questionnaire and physical examination, including the measurement of their height and weight, calculation of their Body Mass Index (BMI) and body fat percentage, as well as the circumferential measurement of their hips, waist, thighs, upper arms and abdomen. Each participant was given a weekly diary and instructed to take fifteen drops of the issued medication three times daily, after meals for the duration of the full eight week study. Participants were examined, weighed, and the measurement of their body circumference and fat percentage were recorded every second week for the duration of the eight week study. Data from each participant was collected and analysed using repeated measures analysis of variance (ANOVA). From statistical evaluation, it was determined that the herbal complex (Aloe ferox, Fucus vesiculosis, Taraxacum officinale and Trigonella foenum– graecum) was ineffective as an aid in weight loss in females.

何首烏為寥科多年生藤本植物，而中藥何首烏則為該植物的乾燥塊根。常用於治療白髮及與老年化相關的疾病。然而，何首烏的治療機理卻少有報導。本文對何首烏的粗提物及其主要生物活性成份2,3,5,4' -四羥基二萃乙烯-2-0-ß-D-葡萄糖苷(THSG) 在老鼠及人類的黑色素細胞中影響黑色素生成機理及細胞毒性進行了深入研究。 / 利用高效液相層析連接質譜儀的測定， THSG 在水及乙醇粗提物中含量分別為0.064 及0.75 的百分比。由於THSG 在水及乙醇粗提物中佔有一定份量，所以它對粗提物所產生的生物及生化反應有著重要的影響。在低於細胞毒範圍的劑量內，何首烏粗提物及THSG 能降低老鼠黑色素細胞株melan-a 的左旋多巴(L-DOPA)的轉化反應。在細胞毒性檢測中，水粗提物及THSG 在<100 μg/ml的劑量下均未有對至少5 種黑色素細胞株及黑色素瘤細胞造成傷害。然而，乙醇粗提物的細胞毒卻是水粗提物或THSG 的3-4 倍。 / 在無細胞系統中， THSG 在可逆轉的情況下抑制酪氨酸酶將左旋多巴轉化成黑色素。在細胞系統中，它也能阻止由蛋白激醋A (PKA)引發的黑色素生成反應。THSG 在老鼠及人類黑色素細胞中的中位值抑制率(lC₅₀)分別為123.0 μM及61.5 μM。 THSG 抑制酪氮酸醋的能力展現在黑色素細胞/角質細胞的共培養比在單黑色素細胞培養中更明顯。 / 調控酪氯酸臨可以在脫氧核糖核酸(DNA)轉錄及翻譯後修飾兩方面達成。在DNA 轉錄中，小眼球相關轉錄因數(MITF)的減少導致酪氨酸酶的表達隨著THSG 濃度而減少。翻譯後酪氮酸臨主要依靠蛋白激臨C-ß (PKC-ß)使其磷酸化，從而增加酪氯酸酶/酪氯酸酶相關蛋白-1(TRP-1 )組成複合蛋白。然而THSG 均減少蛋白激酶 C-ß的表達及酪氮酸酪/酪氮酸醋相關蛋白-1 所組成的複合蛋白。 另一方面， THSG 卻沒有影響酪氯酸酶蛋白在內質網/高爾基氏複合體內的糖基化及內涵體與溶酶體間的運輸。 / 總而言之，本文首次展示何首烏粗提物及THSG 在單細胞培養及共培養細胞的系統下抑制黑色素生成。THSG 能在可逆轉的抑制機制下阻止酪氯酸酶作出反應。而在PKA 引發的黑色素生成反應中， THSG 也能在DNA 轉錄及翻譯後修飾等過程中減低酪氯酸酶的活性。 / Radix Polygoni Multiflori, the dried root of Polygonum multiflorum (PM), is well documented for its clinical effects in treating various diseases associated with aging and hair graying, but the evidence based-mechanisms remain largely unknown. In this study, PM was extracted with water and 70% ethanol and a major constituent, 2,3,5,4'-tetrahydroxystilbene-2-0-I3-D-glucoside (THSG) of about 0.064% and 0.75%, respectively, were found in these extracts as analyzed by high-performance liquid chromatography (HPLC) coupled to mass spectrometry. The melanogenic properties and cytotoxicity of the two extracts and THSG were evaluated using murine and human melanocytes. / Both water and ethanol extracts of PM and THSG showed a dose-dependent anti-melanogenic activity in an in vitro murine melan-a melanocyte assay for reduction of L-DOPA conversion by tyrosinase. Of at least 5 melanoma and melanocyte cell lines tested, both water PM extract and THSG were relatively safe, which at doses <100 μg/ml did not demonstrate any significant cytotoxic effects. On the other hand, ethanol PM extract was about 3-4 folds more cytotoxic. / Tyrosinase is the rate-limiting enzyme for melanogenesis. In a cell-free kinetic analysis, THSG inhibited tyrosinase activity in a reversible and non-competitive manner. At the cellular level, this inhibition is mediated through a PKA-dependent melanogenic pathway, as well as in a dose-dependent manner, with IC₅₀ = 123.0 μM and 61.5 μM for murine and human melanocytes, respectively. Tyrosinase was much more sensitive to the inhibitory effect of THSG in the melanocytelkeratinocyte co-culture system than in the melanocyte mono-culture system. / Functional tyrosinase is regulated at both transcriptional and post-translational modification levels. At the transcription level, THSG reduced expression of microphthalmia-associated transcription factor (MITF) esulted in a down-regulation of tyrosinase expression. At the post-translational modification level, THSG inhibited expression of PKC-β which is responsible for tyrosinase phosphorylation, and enhanced tyrosinase/TRP-1 complex formation. On the hand, THSG did not affect glycosylation of tyrosinase nor its trafficking from ER/Golgi to endosomal/ lysosomal compartments. / Taken our results together, the anti-melanogenic property of PM extracts and THSG were firstly demonstrated in both mono- and co-culture system using murine or human melanocytes and keratinocytes. THSG is a reversible and competitive inhibitor, which lowered the tyrosinase activity at both transcription and post-translational modification levels via PKA-mediated melanogenesis. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheung, Wing Ki. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 160-174). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter 1 / General introduction / Chapter 1.1 --- Anatomy and Physiology of the Skin --- p.5 / Chapter 1.1.1 --- Epidermis --- p.5 / Chapter 1.1.1.1 --- Stratum basale --- p.6 / Chapter 1.1.1.2 --- Stratum spinosum --- p.6 / Chapter 1.1.1.3 --- stratum granulosum --- p.7 / Chapter 1.1.1.4 --- Stratum corneum --- p.7 / Chapter 1.1.2 --- Dermis --- p.8 / Chapter 1.2 --- Melanogenesis of the Skin --- p.9 / Chapter 1.2.1 --- History of melanogenesis study --- p.9 / Chapter 1.2.2 --- Today's melanogenesis study --- p.10 / Chapter 1.3 --- Hyperpigmentary Disorders --- p.14 / Chapter 1.3.1 --- Malasma --- p.14 / Chapter 1.3.2 --- Lentigines --- p.15 / Chapter 1.3.2.1 --- Lentigo simplex --- p.16 / Chapter 1.3.2.2 --- Lentigo senilis etActinicus --- p.16 / Chapter 1.3.3 --- Post-inflammatory hyperpigmentation --- p.17 / Chapter 1.4 --- Current Available Treatment for Hyperpigmentation --- p.18 / Chapter 1.4.1 --- Topical treatment and their strategies --- p.18 / Chapter 1.4.1.1 --- Inhibition of tyrosinase activity --- p.18 / Chapter 1.4.1.2 --- Antioxidation --- p.21 / Chapter 1.4.1.3 --- Melanosome transfer inhibition --- p.22 / Chapter 1.4.1.4 --- Stimulation of desquamation --- p.22 / Chapter 1.4.2 --- Laser treatment and their strategies --- p.23 / Chapter 1.4.3 --- Sunscreen --- p.24 / Chapter 1.5 --- Theories and the Treatment of Hyperpigmentation with Chinese Herbal Medicine --- p.25 / Chapter 1.6 --- Testing Systems --- p.26 / Chapter 1.7 --- Aims and Objectives of Study --- p.27 / Chapter 2 / Chemical Properties of THSG / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.1.1 --- Radix Polygoni multiflori --- p.30 / Chapter 2.1.2 --- 2,3,5,4'-tetrahydroxystilbene glucoside (THSG) --- p.31 / Chapter 2.1.2.1 --- Stilbene --- p.32 / Chapter 2.1.2.2 --- Chemical properties of THSG --- p.34 / Chapter 2.1.3 --- Objectives --- p.35 / Chapter 2.2 --- Materials and Methods --- p.36 / Chapter 2.2.1 --- Plant materials --- p.36 / Chapter 2.2.2 --- Extraction --- p.36 / Chapter 2.2.3 --- High performance liquid chromatography (HPLC) analysis --- p.36 / Chapter 2.2.4 --- Enzymatic hydrolysis of THSG and salicin --- p.37 / Chapter 2.2.5 --- HPLC/MS analysis --- p.38 / Chapter 2.2.6 --- Benedict's test --- p.38 / Chapter 2.2.7 --- Enzymatic oxidation --- p.38 / Chapter 2.2.8 --- Thin layer chromatography (TLC) analysis --- p.39 / Chapter 2.3 --- Results / Chapter 2.3.1 --- The THSG content in water and alcohol extracts of PM --- p.40 / Chapter 2.3.2 --- The stability of THSG against oxidation --- p.41 / Chapter 2.3.3 --- Enzymatic hydrolysis ofTHSG --- p.42 / Chapter 2.4 --- Discussion --- p.46 / Chapter 3 / The Melanogenic inhibitory mechanisms of Radix Polygonum multiflorum (PM) extracts and THSG in murine melanocyte / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.1.1 --- Murine melanocyte --- p.47 / Chapter 3.1.2 --- Melanogenesis --- p.48 / Chapter 3.1.2.1 --- Factors affecting Melanogenesis --- Oxidation --- p.49 / Chapter 3.1.2.2 --- Factors affecting Melanogenesis --- UV radiation --- p.50 / Chapter 3.1.2.3 --- Factors affecting melanogenesis---- Cellular regulation --- p.51 / Chapter 3.1.2.3.1 --- The regulation of transcription, translation, and post-translational modification of tyrosianse --- p.51 / Chapter 3.1.2.3.2 --- PKA-, PKC-, and PKG- melangenic pathways --- p.56 / Chapter 3.1.3 --- Kinetic analysis of tyrosinase --- p.57 / Chapter 3.1.4 --- Objectives --- p.58 / Chapter 3.2 --- Materials and Methods --- p.59 / Chapter 3.2.1 --- Cell culture --- p.59 / Chapter 3.2.2 --- Kinetic analysis of tyrosinase activity inhibition --- p.60 / Chapter 3.2.3 --- SRB assay --- p.61 / Chapter 3.2.4 --- L -DOPA conversion assay --- p.62 / Chapter 3.2.5 --- Melanin production measurement --- p.62 / Chapter 3.2.6 --- ROS detection by flow cytometer --- p.63 / Chapter 3.2.7 --- In-situ tyrosinase activity assay --- p.63 / Chapter 3.2.8 --- Western blotting (WB) analysis --- p.64 / Chapter 3.2.9 --- Immunofluorescence microscopy --- p.66 / Chapter 3.2.10 --- Glycosylation analysis --- p.67 / Chapter 3.2.11 --- Co-Immunoprecipitation --- p.68 / Chapter 3.2.12 --- Radix Polygonum Multiflorum and THSG metabolite collection from rat serum --- p.69 / Chapter 3.3 --- Results --- p.71 / Chapter 3.3.1 --- Enzyme kinetic study of the catalysis of L-DOPA by murine melanocyte lysate --- p.71 / Chapter 3.3.2 --- Inhibitory effect of crude PM preparations and THSG on tyrosinase activity and melanin synthesis in murine melan-a melanocytes --- p.74 / Chapter 3.3.3 --- Effect of THSG on H₂0₂- induced oxidation --- p.77 / Chapter 3.3.4 --- THSG inhibits PKA-induced melanogenesis --- p.78 / Chapter 3.3.5 --- Reduction of in situ tyrosinase activity in PKA-induced melanogenesis --- p.82 / Chapter 3.3.6 --- Alternation of melanogenic proteins --- p.85 / Chapter 3.3.7 --- THSG does not alter the tyrosinase trafficking in ER and Golgi --- p.89 / Chapter 3.3.8 --- THSG does not alter the tyrosinase trafficking in endosomal lysosomal compartments --- p.93 / Chapter 3.3.9 --- Glycosylation analysis --- p.95 / Chapter 3.3.10 --- Reduction of interaction between tyrosinase and TRP-1 to form heterodimeric complexes --- p.97 / Chapter 3.3.11 --- The metabolite of PM water extract and THSG maintained the in vitro tyrosinase activity --- p.101 / Chapter 3.4 --- Discussion --- p.103 / Chapter 4 / The Inhibitory Effect of THSG on Melanogenesis in Monolayer Culture of Human Melanocytes and in Co-culture of Melanocyte-Keratinocyte / Chapter 4.1 --- Introduction --- p.110 / Chapter 4.1.1 --- Human melanocyte --- p.110 / Chapter 4.1.1.1 --- The origin and the development of melanocyte --- p.110 / Chapter 4.1.1.2 --- Morphology, body site distribution and histological location --- p.111 / Chapter 4.1.1.3 --- In vitro growth of human melanocyte --- p.112 / Chapter 4.1.1.3.1 --- Lifespan vs. culture conditions --- p.113 / Chapter 4.1.1.3.2 --- Lifespan vs. donor age and skin type --- p.114 / Chapter 4.1.1.4 --- Modulation of pigmentation in response to stress --- p.114 / Chapter 4.1.1.5 --- Difference between human and murine TRPs --- p.115 / Chapter 4.1.2 --- Keratinocyte-Melanocyte interaction --- p.117 / Chapter 4.1.2.1 --- Release of melanogenic factors --- p.117 / Chapter 4.1.2.2 --- Release of survival and proliferating factors --- p.118 / Chapter 4.1.2.3 --- Melanosome transfer determines the cutaneous pigmentation --- p.118 / Chapter 4.1.2.3.1 --- Molecular events during melanosome transfer --- p.119 / Chapter 4.1.2.4 --- Others --- p.121 / Chapter 4.1.3 --- Objectives --- p.121 / Chapter 4.2 --- Materials and Methods --- p.122 / Chapter 4.2.1 --- Cell Culture --- p.122 / Chapter 4.2.1.1 --- Human melanocytes isolation and cultivation --- p.122 / Chapter 4.2.1.2 --- Immortalized keratinocytes - HaCaT cells --- p.123 / Chapter 4.2.1.3 --- Co-culture of melanocytes and HaCaT cells --- p.124 / Chapter 4.2.1.3.1 --- Monolayer co-culture --- p.124 / Chapter 4.2.1.3.2 --- Two-layer co-culture --- p.124 / Chapter 4.2.2 --- SRB assay --- p.125 / Chapter 4.2.3 --- L-DOPA conversion assay --- p.125 / Chapter 4.2.4 --- Western blotting (WB) analysis --- p.125 / Chapter 4.2.5 --- Light microscopy and immunofluorescent microscopy --- p.126 / Chapter 4.2.6 --- cAMP immunoassay --- p.126 / Chapter 4.3 --- Results --- p.128 / Chapter 4.3.1 --- The isolation and purification of human melanocytes --- p.128 / Chapter 4.3.2 --- Aging vs. Tyrosinase activity and melanin content --- p.131 / Chapter 4.3.3 --- Inhibitory effect of THSG in tyrosinase activity in human melanocyte --- p.133 / Chapter 4.3.4 --- Alternation of melanogenic proteins --- p.135 / Chapter 4.3.5 --- Sensitization of melanocytes to THSG treatment in co-culture system --- p.138 / Chapter 4.3.6 --- Induction of melanocyte dendricity in co-culture system --- p.140 / Chapter 4.3.7 --- THSG inhibited cAMP induction by forskolin and paracrine factors from keratinocytes --- p.141 / Chapter 4.4 --- Discussion --- p.143 / Discussion / Chapter 5.1 --- Discussion --- p.149 / References --- p.160

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Dentistry / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Chinese Medicine / Master / Master of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Ph.D. (Botany) / The most well-known tonic plants in South Africa have been used traditionally for the treatment of a great variety of ailments but aspects of their ethnobotany and chemistry remain poorly studied. Possible relationships between their ethnobotany and pharmacology are mostly speculative. In this study, literature reviews of the ethnobotany of these plants were combined with phytochemical screening studies and bitterness taste testing results in order to establish constituent patterns which may contribute to a scientific rationale for the claimed tonic (stimulating) properties of these plants. The tonic concept and definitions of terms associated with it are often used incorrectly and ambiguously. An analysis of literature on the traditional healing systems across the globe was used to establish the historical and cultural aspects relevant to tonics. This analysis revealed that sickness/illness is usually considered to be a result of imbalance in many cultures, whether this imbalance is between the patient and the environment or due to a lack of homeostasis in the body. In several healing cultures substances or mixtures of substances are used to rectify these imbalances through proposed effects on several bodily systems concurrently. According to some cultures, as in Eastern and Indian traditional medicine, tonic plants are considered superior to other medicinal plants in that they impart health, strength and a general sense of well-being, as well as being prophylactic. This definition of a tonic plant is consequently broad, but excludes plants merely used as multipurpose medicines. Where these tonics exhibit a specific mode of action, further classification is required, i.e. as bitter, adaptogenic, alterative, adjuvant or stimulant tonics. The South African traditional tonic plants studied were Agathosma species (Rutaceae), Aloe species (Asphodelaceae), Arctopus species (Apiaceae), Artemisia afra (Asteraceae), Balanites maughamii (Balanitacae), Dicoma species (Asteraceae), Harpagophytum procumbens (Pedaliaceae), Hypoxis hemerocallidea (Hypoxidaceae), Muraltia heisteria (Polygalaceae), Sutherlandia species (Fabaceae), Vernonia oligocephala (Asteraceae), Warburgia salutaris (Canellaceae), Withania somnifera (Solanaceae) and Ziziphus mucronata (Rhamnaceae). A detailed compendium of medicinal applications was compiled following a thorough, in-depth scrutiny of the historical and medicinal ethnobotany of each of these species. Such ethnobotanical data is important in understanding the cultural aspects of healing in southern Africa, and provides valuable direction and focus with regards to the phytochemical and pharmacological research of these plants.

by Frankie Tat-kwong Lau. / Bibliography: leaves 63-70 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1985