Thesis (Ph. D. (Botany)) -- University of Limpopo, 2014 / Refer to document

Lim Sze Nee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 165-176). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese Version) --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.xi / List of Figures --- p.xiii / List of Abbreviation --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1. --- General Introduction --- p.1 / Chapter 1.1 --- Classification of algae --- p.2 / Chapter 1.2 --- Chemical and mineral composition of marine macroalgae --- p.4 / Chapter 1.3 --- Uses of marine macroalgae --- p.7 / Chapter 1.3.1 --- Food --- p.7 / Chapter 1.3.2 --- Industrial uses --- p.8 / Chapter 1.3.3 --- Agricultural uses --- p.9 / Chapter 1.3.3.1 --- Fertilizer --- p.9 / Chapter 1.3.3.2 --- Fodder --- p.9 / Chapter 1.3.4 --- Medicinal properties --- p.10 / Chapter 1.4 --- Pharmacological effects of marine macroalgae --- p.11 / Chapter 1.4.1 --- Antioxidant activity --- p.11 / Chapter 1.4.2 --- Hypotensive activity --- p.11 / Chapter 1.4.3 --- Antiviral activity --- p.12 / Chapter 1.4.4 --- Antimicrobial activity --- p.12 / Chapter 1.4.5 --- Antitumor activity --- p.13 / Chapter 1.4.6 --- Hypocholesterolemic activity --- p.14 / Chapter 1.5 --- Objectives --- p.14 / Chapter CHAPTER 2 --- Free Radical Scavenging and Antioxidative Activities of Marine Macroalgae --- p.16 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.1.1 --- Free radicals: definition and sources --- p.16 / Chapter 2.1.2 --- Free radical-induced damage --- p.16 / Chapter 2.1.2.1 --- Biological lipid peroxidation --- p.16 / Chapter 2.1.2.2 --- Lipid oxidation of foods --- p.18 / Chapter 2.1.3 --- Antioxidants --- p.19 / Chapter 2.1.3.1 --- Antioxidants --- p.19 / Chapter 2.1.3.2 --- Antioxidant mechanisms --- p.20 / Chapter 2.1.4 --- Synthetic antioxidants --- p.21 / Chapter 2.1.5 --- Natural antioxidants --- p.24 / Chapter 2.1.6 --- Objectives --- p.27 / Chapter 2.2 --- Methods and Materials --- p.28 / Chapter 2.2.1 --- Preparation of algae extracts --- p.28 / Chapter 2.2.2 --- Determination of free radical scavenging activities --- p.32 / Chapter 2.2.2.1 --- Superoxide anions scavenging activity --- p.32 / Chapter 2.2.3 --- Antioxidative activity using hemolysis assay --- p.33 / Chapter 2.2.3.1 --- Preparation of red blood cell (RBC) --- p.33 / Chapter 2.2.3.2 --- Hemolysis assay --- p.33 / Chapter 2.2.4 --- Lipid peroxidation assay --- p.34 / Chapter 2.2.4.1 --- Preparation of rat brain homogenates --- p.34 / Chapter 2.2.4.2 --- Measurement of lipid peroxidation --- p.34 / Chapter 2.2.5 --- Statistics --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Superoxide radical scavenging activity of algal extracts --- p.36 / Chapter 2.3.2 --- Effects of algae extracts on hemolysis assay --- p.41 / Chapter 2.3.3 --- Effects of algae extracts on lipid peroxidation --- p.44 / Chapter 2.4 --- Discussion --- p.50 / Chapter CHAPTER 3 --- Isolation of Antioxidative Phenolic Compounds from Sargassum siliquastrum --- p.60 / Chapter 3.1 --- Introduction --- p.60 / Chapter 3.1.1 --- Phenolic compounds --- p.60 / Chapter 3.1.2 --- Major classes of phenolic compounds --- p.60 / Chapter 3.1.3 --- Functional aspects of phenolic compounds --- p.61 / Chapter 3.1.3.1 --- Functions of phenolic compounds in plants --- p.61 / Chapter 3.1.3.2 --- Biological and pharmacological activities --- p.64 / Chapter 3.1.3.3 --- Food industry --- p.65 / Chapter 3.1.4 --- Polyphenolic compounds in brown algae --- p.66 / Chapter 3.1.5 --- Objectives --- p.68 / Chapter 3.2 --- Methods and Materials --- p.69 / Chapter 3.2.1 --- Extraction and isolation of antioxidant components from S siliquastrum --- p.69 / Chapter 3.2.2 --- Thin-Layer chromatography --- p.70 / Chapter 3.2.3 --- Antioxidant activity --- p.71 / Chapter 3.2.4 --- Determination of total phenolics --- p.71 / Chapter 3.2.5 --- Infrared spectra --- p.72 / Chapter 3.2.6 --- Ultra-violet and visible (UV-vis) spectrophotometry --- p.72 / Chapter 3.2.7 --- Statistics --- p.73 / Chapter 3.3 --- Results --- p.73 / Chapter 3.3.1 --- Identification of phenolic compounds from various solvent extracts of S. siliquastrum --- p.73 / Chapter 3.3.2 --- Isolation of dichloromethane fraction by liquid chromatography --- p.81 / Chapter 3.3.3 --- Phenolic content of isolated compounds --- p.86 / Chapter 3.3.4 --- IR and UV-vis spectra --- p.86 / Chapter 3.4 --- Discussion --- p.92 / Chapter 3.4.1 --- Antioxidative activities --- p.92 / Chapter 3.4.2 --- Relationship between phenolic contents and antioxidant activity --- p.95 / Chapter 3.4.3 --- Identification of antioxidant compounds --- p.97 / Chapter CHAPTER 4 --- Hypotensive Activities of Marine Algae in the Rat --- p.102 / Chapter 4.1 --- Introduction --- p.102 / Chapter 4.1.1 --- Basic principles of cardiovascular system --- p.102 / Chapter 4.1.2 --- Regulation of arterial pressure --- p.105 / Chapter 4.1.2.1 --- Short-term regulation of arterial pressure --- p.105 / Chapter 4.1.2.2 --- Long-term regulation of arterial pressure --- p.107 / Chapter 4.1.3 --- Hypertension --- p.108 / Chapter 4.1.3.1 --- Causes of hypertension --- p.109 / Chapter 4.1.3.2 --- Where do antihypertensive or hypotensive agents act? --- p.114 / Chapter 4.1.3.2.1 --- Sympathetic nervous system inhibitors --- p.115 / Chapter 4.1.3.2.2 --- Diuretics --- p.120 / Chapter 4.1.3.2.3 --- Vasodilators --- p.121 / Chapter 4.1.3.2.4 --- Calcium antagonist (Calcium channel blockers) --- p.121 / Chapter 4.1.3.2.5 --- Angiotensin-converting enzyme (ACE) inhibitors --- p.122 / Chapter 4.1.3.2.6 --- Antihypertensive drug combination --- p.122 / Chapter 4.1.4 --- The relationship between hypertension and free radicals --- p.123 / Chapter 4.1.5 --- Development of new antihypertensive agenrs --- p.124 / Chapter 4.2 --- Materials and methods --- p.125 / Chapter 4.2.1 --- Animal care --- p.125 / Chapter 4.2.2 --- Preparation of the blood pressure measurement in rats --- p.125 / Chapter 4.2.2.1 --- Effects of seaweed extracts on arterial blood pressure of rat --- p.126 / Chapter 4.2.2.1.1 --- Single-dose response curve --- p.126 / Chapter 4.2.2.1.2 --- Cumulative-dose response curve --- p.126 / Chapter 4.2.2.2 --- Pharmacological blocker studies --- p.128 / Chapter 4.2.3 --- Statistics --- p.131 / Chapter 4.3 --- Results --- p.131 / Chapter 4.3.1 --- Hypotensive effects of marine algal extracts --- p.131 / Chapter 4.3.2 --- Effects of pharmacological blockers on MAP --- p.135 / Chapter 4.4 --- Discussion --- p.150 / Chapter 4.4.1 --- Hypotensive effects of the marine algal extracts --- p.150 / Chapter 4.4.2 --- Pharmacological action of marine algal extracts --- p.152 / Chapter CHAPTER 5 --- Conclusion --- p.160 / REFERENCES --- p.165 / RELATED PUBLICATIONS --- p.177

鉤藤是眾多用於治療神經性退行性疾病的傳統中藥複方的組成成份之一。文獻研究發現鉤藤提取物能夠顯著抑制β澱粉樣蛋白纖維的形成和拆卸預製β澱粉樣蛋白纖維。然而鉤藤作用於老年性癡呆模型的實驗研究還未見報道。本課題的研究目的是探討鉤藤提取物對認知功能的改善作用，從而篩選出鉤藤抗老年性癡呆的有效化學成份及探討鉤藤抗老年性癡呆有效化學成份的神經保護作用及其作用機理。 / 首先我們探討了70%乙醇鉤藤提取物對D-半乳糖引起小鼠認知功能障礙的改善作用。水迷宮試驗結果顯示鉤藤提取物(200 和400毫克/千克)能顯著改善D-半乳糖處理小鼠的空間學習和記憶能力。此外，鉤藤提取物(200 和400毫克/千克)還顯著提高D-半乳糖處理小鼠腦組織中乙醯膽鹼和還原型穀胱甘肽的含量，以及超氧化物歧化酶和過氧化氫酶的活性，同時也能降低D-半乳糖處理小鼠腦組織中乙醯膽鹼酯酶的活性和丙二醛的含量。以上研究結果表明鉤藤提取物能改善D-半乳糖處理小鼠認知功能障礙的作用可能是通過抑制腦組織中乙醯膽鹼酯酶的活性和提高腦組織的氧化能力而達成的。 / 其次，我們選用β澱粉樣蛋白引致PC12細胞神經毒性的體外細胞模型來跟蹤篩選出鉤藤提取物中抗老年性癡呆的有效活性成分。結果顯示從鉤藤提取物中分離出六個生物鹼，分別為柯諾辛堿，柯諾辛堿B，去氫鉤藤堿，異鉤藤堿，異去氫鉤藤堿和鉤藤堿。在這六個生物鹼中，只有鉤藤堿和異鉤藤堿具有顯著降低β澱粉樣蛋白導致PC12細胞的死亡，而異鉤藤堿是鉤藤提取物中對β澱粉樣蛋白所致的PC12細胞損傷有最強的保護作用。 / 在明確異鉤藤堿是鉤藤提取物中抗老年性癡呆的主要有效成分的研究基礎上，我們應用β澱粉樣蛋白所致PC12細胞的神經毒性的體外實驗模型來探討異鉤藤堿的神經保護作用及其作用機理。實驗結果顯示異鉤藤堿對β澱粉樣蛋白引起PC12細胞的神經毒性的保護作用呈良好的量效關係。異鉤藤堿對β澱粉樣蛋白引起PC12細胞的神經毒性的保護作用是通過抑制細胞內鈣離子的超載，氧化應激，tau蛋白的過度磷酸化和線粒體細胞凋亡。 此外，異鉤藤堿還顯著抑制3β糖原合成酶激酶的活性，同時啟動磷酸化磷脂醯肌醇3-激酶底物Akt，提示異鉤藤堿對β澱粉樣蛋白所致的PC12細胞的神經毒性的保護作用與PI3K/Akt/GSK3信號通路相關密切相關。 / 最後，我們進一步探討了異鉤藤堿對β澱粉樣蛋白致大鼠認知功能障礙的改善作用及其作用機理。研究結果表明異鉤藤堿(20和40毫克/千克/天)能顯著改善β澱粉樣蛋白所致的大鼠認知功能障礙（用水迷宮試驗來評價）及明顯增加海馬CA1區錐體細胞數目。同時，異鉤藤堿能顯著抑制β澱粉樣蛋白導致大鼠海馬的氧化應激，神經元凋亡以及tau蛋白過度磷酸化。此外，異鉤藤堿能顯著抑制3β糖原合成酶激酶的活性，啟動磷酸化磷脂醯肌醇3-激酶底物Akt，提示異鉤藤堿改善β澱粉樣蛋白導致大鼠認知功能障礙的作用機理與PI3K/Akt/GSK3信號通路相關。 / 綜上所述，鉤藤和異鉤藤堿具有顯著的抗老年癡呆的作用。異鉤藤堿的神經保護作用與其抑制β澱粉樣蛋白導致PC12細胞和大鼠海馬的氧化應激，神經元凋亡以及tau蛋白的過度磷酸化有關。異鉤藤堿神經保護的作用機理與PI3K/Akt/GSK3信號通路密切相關。以上研究結果提示異鉤藤堿具有很好的進一步開發成新的抗老年性癡呆製劑的應用前景。 / The stem with hooks of Uncaria rhynchophylla (Ramulus Uncariae cum Uncis) is a component herb of many traditional formulae for the treatment of neurodegenerative diseases. Previous studies have demonstrated that the extract of U. rhynchophylla inhibited beta-amyloid (Aβ) fibril formation and disassemble preformed Aβ fibrils. However, scientific evidence concerning the efficacy of U. rhynchophylla in Alzheimer’s disease (AD) experimental models is lacking. The present study aimed at investigating the cognition-improving effect of U. rhynchophylla, identifying the active anti-AD chemical constituents and elucidating the underlying mechanisms of neuroprotective action. / Firstly, we investigated whether 70% aqueous ethanol extract of U. rhynchophylla (EUR) could protect against D-galactose (D-gal)-induced cognitive deficits in mice. Mice were given a subcutaneous injection of D-gal (50 mg/kg) and orally administered EUR (100, 200, or 400 mg/kg) daily for 8 weeks. The results showed that EUR (200 or 400 mg/kg) significantly improved spatial learning and memory function in D-gal-treated mice as assessed by the Morris water maze test. In addition, EUR (200 or 400 mg/kg) significantly increased the levels of acetylcholine and glutathione, and the activities of superoxide dismutase and catalase, while it decreased the activity of acetylcholinesterase and the level of malondialdehyde in the brains of D-gal-treated mice. These results indicate that EUR was able to ameliorate cognitive deficits induced by D-gal in mice, and the observed pharmacological action may be mediated, at least in part, by the inhibition of acetylcholinesterase activity and the enhancement of the antioxidant status of the brain tissues. / Secondly, we tried to identify the active ingredients of U. rhynchophylla by a bioassay-guided fractionation approach using beta-amyloid (Aβ)-induced neurotoxicity in rat pheochromocytoma (PC12) cells, a well established cellular model of AD. As a result of this work, six alkaloids, namely corynoxine, corynoxine B, corynoxeine, isorhynchophylline, isocorynoxeine and rhynchophylline were isolated from the extract of U. rhynchophylla. Among them, only rhynchophylline and isorhynchophylline could significantly decrease Aβ-induced cell death in PC12 cells. Moreover, isorhynchophylline (IRN) was found to be the most active ingredient responsible for the protective action of U. rhynchophylla against Aβ₂₅₋₃₅-induced cell death. / Thirdly, the neuroprotective effects and its action mechanism of IRN against Aβ₂₅₋₃₅-induced neurotoxicity in PC12 cells, an in vitro experimental model of AD, were examined. The results showed that treatment with IRN dose-dependently protected PC12 cells against Aβ₂₅₋₃₅-induced neurotoxicity. The neuroprotective effect of IRN may be mediated, at least in part, by inhibiting the intracellular calcium overloading, oxidative stress, tau protein hyperphosphorylation and mitochondrial cellular apoptosis induced by Aβ₂₅₋₃₅. Moreover, IRN also inhibited the activity of glycogen synthase kinase (GSK)-3β, an important kinase responsible for tau protein hyperphosphorylation in the development of AD; and activated the phosphorylation of phosphatidylinositol 3-kinase (PI3K) substrate Akt, suggesting that the neuroprotective action of IRN is associated with inhibition of GSK-3β activity and activation of PI3K/Akt signaling pathway. / Finally, the ameliorating effect on cognitive deficits of IRN and its underlying mechanism of action in Aβ₂₅₋₃₅-treated rats were investigated. The results showed that oral administration of IRN with two different doses (20 or 40 mg/kg) for 21 days significantly ameliorated cognitive impairments and suppressed the oxidative stress, neuronal apoptosis, and tau protein hyperphosphorylation in the hippocampus of Aβ₂₅₋₃₅-treated rats. In addition, IRN also inhibited the activity of GSK-3β, and activated phosphorylation of phosphatidylinositol 3-kinase (PI3K) substrate Akt, suggesting that the amelioration of cognitive deficits by IRN is associated with inhibition of GSK-3β activity and activation of PI3K/Akt signaling pathway. / Taken together, these results confirmed the anti-AD effects of U. rhynchophylla and IRN. The neuroprotective action of IRN may be mediated via inhibition of oxidative stress, neuronal apoptosis and hyperphosphorylation tau protein induced by Aβ₂₅₋₃₅ in vitro and in vivo. The neuroprotective action of IRN is associated with the inhibition of GSK-3β activity and the activation of PI3K/Akt signaling pathway. These experimental findings render IRN a promising candidate worthy of further development into anti-AD pharmaceutical agents. / 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. / Detailed summary in vernacular field only. / Xian, Yanfang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 242-278). / Abstracts also in Chinese. / Abstract (English) --- p.I / 摘要 --- p.IV / Publications --- p.VII / Acknowledgements --- p.IX / Table of Contents --- p.X / List of Figures --- p.XXI / List of Tables --- p.XXVI / List of Abbreviation --- p.XXVII / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Alzheimer’s Disease --- p.2 / Chapter 1.1.1 --- Symptoms --- p.2 / Chapter 1.1.2 --- Epidemiology --- p.4 / Chapter 1.1.3 --- Pathology --- p.5 / Chapter 1.1.4 --- Risk factors --- p.6 / Chapter 1.2 --- Pathogenesis of AD --- p.10 / Chapter 1.2.1 --- Neurotransmitter dysfunction --- p.10 / Chapter 1.2.1.1 --- Cholinergic system dysfunction --- p.10 / Chapter 1.2.1.2 --- Glutamatergic system dysfunction --- p.11 / Chapter 1.2.2 --- Hippocampus atrophy --- p.15 / Chapter 1.2.3 --- “Amyloid Cascade hypothesis --- p.18 / Chapter 1.2.4 --- Increased oxidative stress --- p.21 / Chapter 1.2.5 --- Increased neuronal apoptosis --- p.23 / Chapter 1.2.6 --- Mitochondrial dysfunction --- p.27 / Chapter 1.2.7 --- Calcium dysregulation --- p.31 / Chapter 1.2.8 --- Increased tau protein hyperphosphorylation --- p.34 / Chapter 1.2.9 --- GSK3 hypothesis of AD --- p.37 / Chapter 1.3 --- Animal Models of AD --- p.41 / Chapter 1.3.1 --- Non-transgenic animal models of AD --- p.42 / Chapter 1.3.1.1 --- Spontaneous models --- p.42 / Chapter 1.3.1.2 --- Scopolamine-induced rodent models --- p.43 / Chapter 1.3.1.3 --- Aluminum-induced rodent models --- p.44 / Chapter 1.3.1.4 --- D-galactose-induced rodent models --- p.45 / Chapter 1.3.1.5 --- Aβ infusion rodent models --- p.46 / Chapter 1.3.2 --- Transgenic animal models of AD --- p.48 / Chapter 1.3.2.1 --- Transgenic rodent models for AD --- p.49 / Chapter 1.3.2.2 --- AD models in D. rerio --- p.53 / Chapter 1.3.2.3 --- AD models in D. melanogaster --- p.54 / Chapter 1.3.2.4 --- AD models in C. elegans --- p.54 / Chapter 1.4 --- Treatments for AD --- p.55 / Chapter 1.4.1 --- Current symptomatic treatments --- p.56 / Chapter 1.4.1.1 --- AChEIs --- p.56 / Chapter 1.4.1.2 --- NMDA antagonist --- p.57 / Chapter 1.4.2 --- Disease-modifying approaches --- p.61 / Chapter 1.4.2.1 --- Amyloid-directed therapies --- p.61 / Chapter 1.4.2.2 --- Tau-directed therapies --- p.61 / Chapter 1.4.2.3 --- Anti-oxidant agents --- p.62 / Chapter 1.4.2.4 --- NSAIDs --- p.63 / Chapter 1.4.2.5 --- Estrogen replacement therapy (ERT) --- p.64 / Chapter 1.4.3 --- Herbal medicines --- p.67 / Chapter 1.5 --- Uncaria rhynchophylla --- p.69 / Chapter 1.5.1 --- Chemical constituents --- p.69 / Chapter 1.5.2 --- Alkaloids --- p.72 / Chapter 1.6 --- Pharmacological Activities of Uncaria rhynchophylla and Its Alkaloids --- p.75 / Chapter 1.6.1 --- Effects on cardiovascular system --- p.75 / Chapter 1.6.2 --- Effects on central nervous system --- p.77 / Chapter 1.6.3 --- Antioxidant activities --- p.79 / Chapter 1.6.4 --- Anti-inflammatory and analgesic effects --- p.80 / Chapter 1.6.5 --- Effects on platelet aggregation and thrombosis --- p.81 / Chapter 1.6.6 --- Other pharmacological effects --- p.81 / Chapter 1.7 --- Hypothesis and Objectives of the Present Study --- p.83 / Chapter Chapter Two --- Uncaria rhynchophylla Ameliorates Cognitive Deficits Induced by D-galactose in Mice / Chapter 2.1 --- Introduction --- p.86 / Chapter 2.2 --- Materials and Methods --- p.88 / Chapter 2.2.1 --- Drugs and chemical reagents --- p.88 / Chapter 2.2.2 --- Plant materials and extraction --- p.89 / Chapter 2.2.3 --- Animals --- p.90 / Chapter 2.2.4 --- Experimental design and drugs treatment --- p.90 / Chapter 2.2.5 --- Morris water maze test --- p.91 / Chapter 2.2.6 --- Preparation of brain tissue samples --- p.92 / Chapter 2.2.7 --- Measurement of intracellular ROS level --- p.92 / Chapter 2.2.8 --- Assay of MDA level --- p.92 / Chapter 2.2.9 --- Assay of GSH level --- p.93 / Chapter 2.2.10 --- Measurement of SOD activity --- p.93 / Chapter 2.2.11 --- Measurement of CAT activity --- p.94 / Chapter 2.2.12 --- Assay of Ach level --- p.94 / Chapter 2.2.13 --- Measurement of AChE activity --- p.95 / Chapter 2.2.14 --- Statistical analysis --- p.95 / Chapter 2.3 --- Results --- p.95 / Chapter 2.3.1 --- Quality determination of EUR --- p.95 / Chapter 2.3.2 --- Effects of EUR on Morris water maze in D-gal-treated mice --- p.97 / Chapter 2.3.3 --- Effects of EUR on the level of intracellular ROS in the brains of D-gal-treated mice --- p.101 / Chapter 2.3.4 --- Effects of EUR on the levels of GSH and MDA in the brains of D-gal-treated mice --- p.103 / Chapter 2.3.5 --- Effects of EUR on the activities of SOD and CAT in the brains of D-gal-treated mice --- p.105 / Chapter 2.3.6 --- Effects of EUR on the level of ACh and the activity of AChE in the brains of D-gal-treated mice --- p.107 / Chapter 2.4 --- Discussion --- p.109 / Chapter Chapter Three --- Bioassay-Guided Isolation of Neuroprotective Compounds from Uncaria rhynchophylla Against Beta-Amyloid-Induced Neurotoxicity / Chapter 3.1 --- Introduction --- p.113 / Chapter 3.2 --- Materials and Methods --- p.114 / Chapter 3.2.2 --- Drugs and chemical reagents --- p.114 / Chapter 3.2.2 --- Preparation of aggregated Aβ₂₅₋₃₅ --- p.115 / Chapter 3.2.3 --- Extraction, fractionation, isolation and identification processes --- p.115 / Chapter 3.2.4 --- Cell culture and drug treatment --- p.119 / Chapter 3.2.5 --- Cell viability assay --- p.119 / Chapter 3.2.6 --- Statistical analysis --- p.120 / Chapter 3.3 --- Results --- p.120 / Chapter 3.3.1 --- Isolation and structural determination of the isolated compounds --- p.120 / Chapter 3.3.2 --- Effects of different fractions and isolated compounds on Aβ₂₅₋₃₅-induced cells death in PC12 cells --- p.122 / Chapter 3.4 --- Discussion --- p.126 / Chapter Chapter Four --- Neuroprotective Effects of Isorhynchophylline Against Beta-Amyloid-Induced Neurotoxicity in PC12 Cells and Its Possible Mechanisms / Chapter 4.1 --- Introduction --- p.130 / Chapter 4.2 --- Materials and Methods --- p.131 / Chapter 4.2.1 --- Drugs and chemical reagents --- p.131 / Chapter 4.2.2 --- Cell culture and drugs treatment --- p.134 / Chapter 4.2.3 --- Cell viability assay --- p.134 / Chapter 4.2.4 --- Lactate dehydrogenase (LDH) activity assay --- p.135 / Chapter 4.2.5 --- Measurement of intracellular ROS production --- p.135 / Chapter 4.2.6 --- Malondialdehyde (MDA) and glutathione (GSH) assay --- p.136 / Chapter 4.2.7 --- Measurement of SOD activity --- p.137 / Chapter 4.2.8 --- Measurement of CAT activity --- p.137 / Chapter 4.2.9 --- Measurement of intracellular calcium concentration --- p.138 / Chapter 4.2.10 --- Measurement of mitochondrial membrane potential --- p.139 / Chapter 4.2.11 --- Quantification of DNA fragmentation --- p.139 / Chapter 4.2.12 --- Cytochrome c assay --- p.140 / Chapter 4.2.13 --- Western blotting analysis --- p.140 / Chapter 4.2.14 --- Real time-polymerase chain reaction (RT-PCR) analysis --- p.141 / Chapter 4.2.15 --- Statistical analysis --- p.142 / Chapter 4.3 --- Results --- p.143 / Chapter 4.3.1 --- Effects of IRN on Aβ₂₅₋₃₅-induced cytotoxicity in PC12 cells --- p.143 / Chapter 4.3.2 --- Effects of IRN on the level of intracellular ROS in Aβ₂₅₋₃₅-treated PC12 cells --- p.145 / Chapter 4.3.3 --- Effects of IRN on the levels of GSH and MDA in Aβ₂₅₋₃₅-treated PC12 cells --- p.147 / Chapter 4.3.4 --- Effects of IRN on the activities of SOD and CAT in Aβ₂₅₋₃₅-treated PC12 cells --- p.149 / Chapter 4.3.5 --- Effects of IRN on intracellular calcium level in Aβ₂₅₋₃₅-treated PC12 Cells --- p.151 / Chapter 4.3.6 --- Effects of IRN on MMP in Aβ₂₅₋₃₅-treated PC12 cells --- p.153 / Chapter 4.3.7 --- Effects of IRN on DNA fragmentation in Aβ₂₅₋₃₅-treated PC12 cells --- p.155 / Chapter 4.3.8 --- Effects of IRN on the release of cytochrome c in Aβ₂₅₋₃₅-treated PC12 cells --- p.157 / Chapter 4.3.9 --- Effects of IRN on the protein and mRNA levels of the ratio of Bcl-2/Bax in Aβ₂₅₋₃₅-treated PC12 cells --- p.159 / Chapter 4.3.10 --- Effects of IRN on the protein and mRNA levels of cleaved caspase-3 and caspase-9 in Aβ₂₅₋₃₅-treated PC12 cells --- p.162 / Chapter 4.3.11 --- Effects of IRN on the protein of pro-caspase-8 and mRNA levels of the full length of caspase-8 in Aβ₂₅₋₃₅-treated PC12 cells --- p.165 / Chapter 4.3.12 --- Effects of IRN on tau protein hyperphosphorylation in Aβ₂₅₋₃₅-treated PC12 Cells --- p.168 / Chapter 4.3.13 --- Effects of IRN on Aβ₂₅₋₃₅-induced activation of GSK-3β in PC12 cells --- p.170 / Chapter 4.3.14 --- Effects of IRN on Aβ₂₅₋₃₅-induced inactivation of PI3K/Akt pathway --- p.173 / Chapter 4.4 --- Discussion --- p.177 / Chapter Chapter Five --- Isorhynchophylline Treatment Improves Cognitive Deficits Induced by Beta-Amyloid in Rats: Involvement of PI3K/Akt Signaling Pathway / Chapter 5.1 --- Introduction --- p.186 / Chapter 5.2 --- Materials and Methods --- p.187 / Chapter 5.2.1 --- Drugs and chemical reagents --- p.187 / Chapter 5.2.2 --- Animals --- p.188 / Chapter 5.2.3 --- Aβ₂₅₋₃₅ injections --- p.188 / Chapter 5.2.4 --- Experimental design and drugs treatment --- p.189 / Chapter 5.2.5 --- Morris water maze test --- p.190 / Chapter 5.2.6 --- Nissl’s staining for neurons --- p.193 / Chapter 5.2.7 --- Preparation of brain tissue samples --- p.193 / Chapter 5.2.8 --- Measurement of intracellular ROS level --- p.194 / Chapter 5.2.9 --- Assay of MDA level --- p.194 / Chapter 5.2.10 --- Assay of GSH level --- p.195 / Chapter 5.2.11 --- Measurement of SOD activity --- p.195 / Chapter 5.2.12 --- Measurement of CAT activity --- p.195 / Chapter 5.2.13 --- Cytochrome c assay --- p.196 / Chapter 5.2.14 --- Western blotting analysis --- p.196 / Chapter 5.2.15 --- RT-PCR analysis --- p.197 / Chapter 5.2.16 --- Statistical analysis --- p.198 / Chapter 5.3 --- Results --- p.199 / Chapter 5.3.1 --- IRN treatment rescued behavioral impairment in the Morris water maze test --- p.199 / Chapter 5.3.2 --- Effects of IRN on the number of pyramidal neuronal cells in the hippocampal CA1 region of Aβ₂₅₋₃₅-treated rats --- p.203 / Chapter 5.3.3 --- Effects of IRN on the intracellular ROS level in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.205 / Chapter 5.3.4 --- Effects of IRN on the levels of GSH and MDA in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.207 / Chapter 5.3.5 --- Effects of IRN on the activities of SOD and CAT in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.209 / Chapter 5.3.6 --- Effects of IRN on cytochrome c in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.211 / Chapter 5.3.7 --- Effects of IRN on the protein and mRNA level of the ratio of Bcl-2/Bax in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.213 / Chapter 5.3.8 --- Effects of IRN on the protein and mRNA levels of cleaved caspase-3 and caspase-9 in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.216 / Chapter 5.3.9 --- Effects of IRN on the protein and mRNA levels of caspase-8 in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.219 / Chapter 5.3.10 --- Effects of IRN on the tau protein hyperphosphorylation in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.222 / Chapter 5.3.11 --- Effects of IRN on the activation of GSK-3β in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.224 / Chapter 5.3.12 --- Effects of IRN on the PI3K/Akt pathway in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.226 / Chapter 5.4 --- Discussion --- p.228 / Chapter Chapter Six --- General Discussion and Future Perspectives / Chapter 6.1 --- General Discussion and Conclusions --- p.237 / Chapter 6.2 --- Future Perspectives --- p.243 / References by Alphabetical Order --- p.246

Finally, the neuroprotective effects of TGP against corticosterone-induced neurotoxicity in rat pheochromocytoma (PC12) cells, an in vitro experimental model of depression were studied. The results showed TGP treatment dose-dependently protected the cells against corticosterone-induced toxicity. The cytoprotection afforded by TGP treatment was shown to be associated with an enhanced antioxidant activity, and increased expressions of neurotrophins including brain-derived neurotrophic factor, nerve growth factor and neurotrophin-3. / Secondly, the antidepressant-like effect of TGP was evaluated by a rat model of depression induced by chronic unpredictable mild stress (CUMS). The results showed that a 5-week CUMS caused depression-like behavior in rats, as indicated by a significant decreases in sucrose consumption (assessed by sucrose preference test) and locomotor activity (assessed by open-field test), and an increase in immobility time (assessed by forced swim test). Intragastric administration of TGP during the five weeks of CUMS procedure significantly suppressed these behavioral changes induced by CUMS. / Taken together, the results confirmed the antidepressant-like effect of TGP. The antidepressive action of TGP may be mediated by the modulation of the hypothalamic-pituitary-adrenal axis function, the inhibition of oxidative stress, and the up-regulation of neurotrophins, thereby leading to the neuroprotective effects. / The antidepressant-like effect of TGP was firstly evaluated by the behavioral despair test, forced swim test and tail suspension test. The results showed that intragastric administration of TGP caused a significant reduction of immobility time in both forced swim and tail suspension tests in mice. TGP treatment also significantly reduced the duration of immobility time in the forced swim test in rats. / The root of Paeonia lactiflora Pall. (Family: Ranunculaceae), commonly known as peony, is a component herb of many traditional formulae for the treatment of depression-like disorders. Previous studies have demonstrated the antidepressive effect of peony extract in mouse models of depression. Total glycosides of peony (TGP) is regarded as the major active ingredients of peony. The present study aims to confirm the antidepressive potential of TGP and evaluate its action mechanisms. / Thirdly, the neuroprotective effects of TGP on CUMS-treated rats and its possible mechanisms were investigated. The results showed that treatment with TGP for 5 weeks produced neuroprotective effects on the hippocampus of CUMS-treated rats. This effect was associated with the attenuation of hypothalamic-pituitary-adrenal axis hyperactivation (characterized by a decreased serum corticosterone level and an increased hippocampal glucocorticoid receptor expression), an inhibition of oxidative stress, and up-regulation of neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3 in the hippocampus. / Mao, Qingqiu. / Adviser: Che Chun-Tao. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 158-186). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

EGb761 consists of two major groups of substances, flavonoids and terpenoids. Using human neuroblastoma SH-SY5Y cells, the present study demonstrated that, EGb761 could block Abeta-42 (a 42-amino acid cytoxic form of beta amyloid protein)-induced cell apoptosis, reactive oxygen species (ROS) accumulation, mitochondrial dysfunction and activation of c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt signaling pathways, possibly via its antioxidant and platelet activating factor (PAF) antagonizing activities. Two active constituents of EGb761, quercetin (a flavonoid) and ginkgolide B (a terpenoid) might contribute to the protective effects of EGb761. Quercetin but not ginkgolide B might be responsible for the antioxidant action of EGb761. Both compounds might be involved in the PAF antagonist activity of EGb761. / EGb761, a Ginkgo biloba extract, is a medicinal product for the treatment and prevention of cardiovascular and neuronal diseases, including Alzheimer's disease (AD). While considerable researches have documented its neuroprotective effects, its clinical effect is inconclusive and the precise neuroprotective mechanisms are not clearly known. / In conclusion, EGb761 may have beneficial effects in treatment and prevention of neurodegenerative diseases like AD. Its neuroprotective effects may be associated with constituent multiplicity, the dosage and BBB permeability. / The ability of EGb761 to cross the blood brain barrier (BBB) is unclear. In this study, the ability of EGb761 to cross the BBB was speculated through comparison of the effects of EGb761 on mitochondrial function between platelets and central nervous system in two animal models, the senescence accelerated prone 8 (SAMP8) mouse strain and ovariectomized rats. Mitochondrial function was evaluated as cytochrome c oxidase (COX) activity, mitochondrial ATP content and mitochondrial glutathione (GSH) content. SAMP8 mice have been widely used as a model of age-related cognitive decline with relevance to biochemical and genetic alterations in AD. Using two age groups (3-week-old and 40-week-old) of SAMP8 mice, this study found that, EGb761 protected against mitochondrial dysfunction in both platelets and hippocampi of old mice, but only showed protective effects on platelet mitochondria of young mice. Estrogen withdrawal was suggested to play a primary role in the onset of post-menopausal AD. Using ovariectomized middle-aged rats to mimic the post-menopausal pathophysiological changes, this study also demonstrated that, EGb761 protected against mitochondrial dysfunction in both platelets and hippocampi of ovariectomized rats. In contrast, in sham-operated rats, EGb761 increased mitochondrial GSH content in platelets but failed to show similar effect on hippocampi. These results suggested that the effects of EGb761 on the brain might be interfered by the BBB permeability. / The effective dosage of EGb761 in the brain remains undetermined. Using SH-SY5Y cells, this study demonstrated that low doses of EGb761 (50--100 mug/ml) inhibited hydrogen peroxide (H2O2)-induced cell apoptosis via inactivation of Alet, JNK and caspase 3 while high doses of EGb761 (250--500 flg/ml) enhanced H2O2 toxicities via inactivation of Akt and enhancement of activation of JNK and caspase 3. Additional experiments suggested that the dosage effect of EGb761 on apoptotic signaling proteins might be correlated with regulation of the cell redox state. / Shi, Chun. / Adviser: Lee Ka Ho Kenneth. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 81-99). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Fung Yin Lee, Annie. / "January 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 132-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

The following contains two projects focused on the fungal pathogen, Fusarium oxysporum. The first project was purely computational in the examination of transposable elements (TEs), which are mobile sequences with the ability to multiply and move in their host genome. In F. oxysporum, TEs such as miniature impala elements are associated with the secreted in xylem gene that are related to its virulence over its host. The F. oxysporum species complex can be utilized as a model system for the examination of TE content and TE expression during the infection cycle. To find whether TEs play a role in the infection process and if their expression changes when fungi are in planta, a comparison was made using RNA-seq data from a pathogenic (Fo5176) and a non-pathogenic strain (Fo47) of F. oxysporum interacting with the model plant Arabidopsis thaliana. Complementary to this, the copy numbers of the same TEs were calculated in the two aforementioned strains and in F. oxysporum f.sp. lycopersici 4287 (Fo4287) to find if there was a correlation between expression and copy number. Using these two different datasets together showed that TE expression and copy number are lower in the non-pathogenic strain and unlinked in the infection course. The second project examined the growth inhibition of Fusarium oxysporum isolates Fo32931 (the isolate pathogenic to immunocompromised humans) and Fo4287 with the use of extracts from chilies of Capsicum chinense. Pepper plants were grown from seed and the peppers were harvested for an ethanol (100%) extraction. After preparation, the optical density of growth of the F. oxysporum isolates was measured for a 48-hour period with 96-well plate containing varying concentrations of the extracts and controls. Growth curves were analyzed and normalized to a growth control. After doing High Performance Liquid Chromatography, an estimated concentration of capsaicin (the causal agent of the burning sensation from hot chilis) was established. A correlation between the amount of growth inhibition and the concentration of capsaicin was made. Taken together, the data suggests that an increase of capsaicin concentration in extracts is correlated with reduced growth for the two tested isolates of F. oxysporum.

Malaria, caused by the parasite Plasmodium falciparum, has a long history as a global health threat. The vector-borne disease causes millions of deaths yearly, especially in developing countries with tropical climates that facilitate transmission. Compounding the problem is the emergence of drug-resistant strains due to overuse of outdated treatments. New compounds with antiplasmodial activity are needed to be developed as effective drugs against malaria. The hypothesis for this project is that marine microorganisms have a high likelihood of yielding novel antiplasmodial chemotypes because of their high diversity, which has not yet been explored for antimalarial development. In this project, microbes harvested and fermented by the Harbor Branch Oceanographic Institute in Fort Pierce, Florida were explored as sources for antiplasmodial natural products. Using a SYBR Green I fluorescence-based assay, 1,000 microbial extracts were screened for inhibition of the multidrug-resistant Plasmodium falciparum strain Dd2. Dose-response analysis was performed on 46 fractions from isolates whose extracts demonstrated greater-than or equal to] 70% inhibition of Dd2 at 1 micro]g/mL. To evaluate cytotoxicity, the MTS cell viability assay was used to calculate IC50 of extracts from active isolates in NIH/3T3 embryonic mouse fibroblasts. Several extracts demonstrated low IC50 in Dd2 and high IC50 in 3T3, suggesting that they contain potential lead antimalarial compounds. Extracts with high selectivity indices (potent plasmodial inhibition with low mammalian toxicity) have been prioritized for dereplication, with the goal of identifying novel active components that can be developed as antimalarial drugs.

Infectious diarrhoea is the second largest single cause of mortality in children under the age of five globally. Bacteria are responsible for most diarrhoeal episodes especially in developing countries, and progressive increase in antimicrobial resistance has given rise to the need to investigate other sources of therapy such as medicinal plants. Ten plant extracts were analysed for their antimicrobial activities using the agar well diffusion and broth microdilution method. Their phytochemical contents were screened, and their effect on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was used to assess their antioxidant activities. Their toxicity profiles were evaluated using the XTT Cytotoxicity Assay. Water and methanol extracts of Adansonia digitata v ABSTRACT Infectious diarrhoea is the second largest single cause of mortality in children under the age of five globally. Bacteria are responsible for most diarrhoeal episodes especially in developing countries, and progressive increase in antimicrobial resistance has given rise to the need to investigate other sources of therapy such as medicinal plants. Ten plant extracts were analysed for their antimicrobial activities using the agar well diffusion and broth microdilution method. Their phytochemical contents were screened, and their effect on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was used to assess their antioxidant activities. Their toxicity profiles were evaluated using the XTT Cytotoxicity Assay. Water and methanol extracts of Adansonia digitata seeds and pulp showed no inhibition against all the test organisms, while water and methanol extracts of A. digitata leaves showed inhibition, with minimum inhibitory concentration (MIC) ranging from 0.39 to 6.25mg/ml. Water and methanol extracts of Garcinia livingstonei and Sclerocarya birrea barks showed good activity against all the test organisms, with MICs between 0.39 and 1.56 mg/ml. Alkaloids, phenols, flavonoids, saponins, tannins, and terpenoids were found in one or more of the plant extracts, and all the plant extracts demonstrated scavenging power against DPPH.The cytotoxicity of extracts of Garcinia livingstonei, and Sclerocarya birrea barks ranged between 105.9 μg/ml and 769.9 μg/ml. The results obtained in this study validate the traditional use of A. digitata leaves, G. livingstonei and S. birrea bark in treating bacteria causing diarrhoea. / Life Sciences / M. Sc. (Life Sciences)

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Apples are an important export commodity for the South African market, and postharvest losses that occur as a result of decay due to infection with pathogenic fungi such as Botrytis cinerea Pers., Penicillium expansum (Link) Thom. and Neofabraea alba (E.J. Guthrie) are of major concern for all parties concerned with fruit production and distribution. Decay control of these fungi is primarily managed through the use of synthetic fungicides; however, pathogen development of resistance to these fungicides and recent worldwide concern over healthier living and a greener environment has called for the discriminate use of synthetic chemicals. This has opened up an avenue for the development of safer and more environmentally friendly alternatives to control postharvest decays. The use of plant extracts and essential oils are favoured as natural sources of antimicrobials whilst still being safe for human consumption and having no negative impact on the environment. Allium sativum (garlic) is one such plant species that is well documented for its value in improving human health and is readily available for consumption not just as a flavour component of food but also to be taken as a daily herbal diet supplement. Given the antimicrobial effectiveness of garlic against human pathogens and ailments, its value as an antifungal agent against postharvest pathogens causing grey mould, blue mould and bull’s eye rot of apples was investigated in vitro and in vivo within this study. Furthermore, an attempt was made to elucidate the chemical components of garlic extracts by gas chromatography-mass spectrometry (GC-MS). All experiments in this study were carried out with garlic extracts prepared from fresh garlic bulbs. For the in vitro experiments, two extract preparations of garlic, one containing ethanol (Extract 1) and one where ethanol had been removed by evaporation (Extract 2), was tested for antifungal action within an amended media experimental design. Both extract preparations were each subjected to two dilution series (0-80% garlic extract) with water and ethanol as diluents. Both extract preparations were successful at retarding pathogen mycelial growth and spore germination; however, concentrations of Extract 2 (ethanol evaporated) and diluted with distilled water provided markedly better inhibition of B. cinerea and P. expansum than the ethanolic dilutions of extract 2. Both extract preparations yielded similar inhibitory results when tested against N. alba. Due to the results achieved in the amended media experiments, the use of a crude garlic extract without ethanol and diluted in water was considered to be the best option for further tests throughout the remainder of the study. In vitro volatile effects of crude garlic extracts at concentrations between 0 and 40% garlic extract were subsequently tested. Garlic volatiles were effective in inhibiting pathogen mycelial growth and spore germination of all three pathogens, at lower concentrations compared to the amended media experiments. In vitro volatile exposure with garlic extracts was more effective at inhibiting N. alba than direct application of the extracts. Curative and protective application of garlic extracts and clove oil for increased fungal inhibition through synergism was tested by direct and volatile exposure to the pathogens in vivo on three economically important apple cultivars; ‘Granny Smith’, ‘Golden Delicious’, and ‘Pink Lady’. Direct exposure of artificially wounded and inoculated fruit to the garlic extract and clove oil revealed that garlic extracts applied curatively but not protectively effectively controlled decay caused by B. cinerea and P. expansum on all apple cultivars. Both curative and protective applications were ineffective in controlling N. alba. In vivo volatile exposure to the garlic extracts and clove oil did not inhibit decay on any of the cultivars and was not effective against any of the three pathogens investigated. A full chemical profile analysis was done by GC-MS analysis of garlic extract samples. The compounds diallyl disulphide, allyl methyl trisulphide, allyl methyl disulphide and dimethyl trisulphide were detected in relatively high amounts. This result suggests that the abundance of sulphur and sulphur related compounds detected may be responsible for the antifungal action noted in the experimental studies. In conclusion, garlic was shown to have antifungal activity against B. cinerea, P. expansum and N. alba. The pathogens used in this study were not compared with each other, but undoubtedly each pathogens reacts differently to exposure to the garlic extracts. It would therefore be advisable to investigate the effects of the extracts on each of the pathogens in a more in-depth study. More investigations into the application of the garlic extracts is required before it may be recommended for use; however, results for the use of garlic extracts against these postharvest pathogens and the postharvest decay they cause are promising. / AFRIKAANSE OPSOMMING: Appels is ‘n belangrike uitvoerproduk vir die Suid-Afrikaanse vrugtebedryf, maar noemenswaardige na-oes verliese word weens bederf deur patogeniese swamme soos Botrytis cinerea Pers., Penicillium expansum (Link) Thom. en Neofabraea alba (E.J. Guthrie) ervaar. Dit raak alle partye betrokke met die produksie en verspreiding van hierdie vrugsoort. Hierdie swamme word hoofsaaklik met behulp van kunsmatige swamdoders beheer, alhoewel weerstand-ontwikkeling en wêreldwye bewusmaking van ‘n gesonder leefstyl en omgewing die gebruik van kunsmatige middels streng aanspreek en die ontwikkeling van veiliger en meer omgewingsvriendelike alternatiewe middels verlang. Plant-ekstrakte en essensiële olies kan dien as sulke middels en is natuurlike bronne van anti-mikrobiese aktiwiteit, is veilig vir menslike verbruik en het ook geen negatiewe invloed op die omgewing nie. Allium sativum (knoffel) is so ‘n plantspesie wat as alternatiewe middel gebruik kan word. Dit is bekend vir sy waarde in die verbetering van menslike gesondheid, is maklik bekombaar en word nie net as ‘n geurmiddel vir voedsel gebruik nie, maar ook as ‘n daaglikse krui-aanvulling. Gegewe die anti-mikrobiese doeltreffendheid van knoffel teenoor menslike patogene en kwale, is die werking (in vitro en in vivo) teen na-oes patogene wat grys skimmel, blou skimmel en teikenvrot in appels veroorsaak, in hierdie studie ondersoek. Bepaling van die chemiese samestelling van die knoffel-ekstrak is ook met behulp van gaschromatografie massa spektrometrie (GK-MS) onderneem.Vars knoffelbolle is vir elke eksperiment in hierdie studie gebruik met die voorbereiding van die knoffel-ekstrak. Vir die in vitro eksperiment is twee knoffel-ekstrakte voorberei, naamlik: ‘n ekstrak wat etanol bevat (Ekstrak 1) en een waarvan die etanol verwyder is met verdamping (Ekstrak 2). Die ekstrakte is getoets vir werking teen fungi in kultuur-medium.. Albei ekstrakte is verdun tot twee konsentrasie reekse (0-80%) met water en etanol as verdunningsmiddels. Albei ekstrakte het suksesvolle werking getoon teenoor die patogene ten opsigte van vertraging van miseliumgroei en spoor-ontkieming, alhoewel konsentrasies van Ekstrak 2, verdun met gesuiwerede water, patogene B. cinerea en P. expansum beter onderdruk het as Ekstrak 2 verdunnings met etanol.. Beide ekstrakte en hul afsonderlike verdunnings met etanol en water het soortgelyke resultate gelewer met onderdrukking van N. alba. Volgens resultate wat verkry is van die kultuur-medium eksperimente, is Ekstrak 2 verdun met gesuiwerde water beskou as die geskikste vir verdere toetse in hierdie studie. Die vlugtige effek van Ekstrak 2 is in vitro getoets by konsentrasies tussen 0 tot 40%. Die vlugtige stowwe van knoffel het al drie patogene se groei en spoor-ontkieming effektief onderdrukby laer konsentrasies as wat gebruik is in die kultuur-medium eksperiment. Dus is in vitro blootstelling van N. alba aan die vlugtige stowwe meer effektief as direkte toediening van die ekstrakte. Die voorkomende en beskermende effek van die knoffel-ekstrak, asook naeltjie-olie, is in vivo ondersoek om te bepaal of die stowwe saam sterker onderdrukking van die patogene kon bewerkstellig. Direkte en vlugtige blootstelling is op drie ekonomies-belangrike appel-kultivars getoets, naamlik: ‘Granny Smith’, ‘Golden Delicious’ en ‘Pink Lady’. Direkte blootstelling met die knoffel-ekstrak en naeltjie-olie aan gewonde en ge-inokuleerde vrugte het aangedui dat B. cinerea- en P. Expansum-bederf net beheer kon word indien knoffel voorkomend toegedien is vir al die ondersoekte appel-variëteite. Voorkomende en beskermende toediening was onsuksesvolle om N. alba te beheer. In vivo blootstelling van die drie patogene aan die knoffel-ekstrak en naeltjie-olie se vlugtige stowwe kon nie enige van die patogene effektief onderdruk nie en was onsuksesvol in bederf-beheer. ‘n Volledige chemiese profiel is saamgestel deur GK-MS ontleding van die knoffelekstrakte. Hoë vlakke van verbindings dialliel disulfied, alliel-metiel-tri-sulfied, alliel-metieldisulfied en dimetiel-trisulfied is bespeur. Die aantal vrye sulfied en sulfied-verwante verbindings in die ekstrak kan moontlik ‘n verduideliking bied vir die anti-swam werking waargeneem gedurende hierdie studie. Ten slotte: knoffel toon ‘n anti-swam werking teenoor B. cinerea, P. expansum en N. alba. Die patogene in hierdie studie is nie met mekaar vergelyk nie, omdat elkeen uniek en uiteenlopend op knoffel reageer het. Alhoewel die huidige studie alreeds belowende resultate gelewer het, moet die ekstrak se effek op elke patogeen onderskeidelik nog in diepte ondersoek word, asook die wyse van die toediening in die na-oes praktyk voordat hierdie middel aanbeveel kan word vir gebruik.