An increasing number of studies show that nutritional antioxidants such as vitamin E and polyphenols are capable of blocking neuronal death in vitro and may have therapeutic properties in animal models of neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases. In the present study, the neuroprotective ability of one such polyphenolic antioxidant, curcumin, was investigated. Curcumin is the yellow curry spice derived from turmeric, and is widely used as a dietary component and herbal medicine in India. Most neurological disorders are postulated to have an oxidative or excitototoxic basis. Thus the effects of curcumin on oxidative stress in the rat brain were investigated. Curcumin, administered to the rat in vivo and in vitro, was able to exert protective effects on oxidative damage in the brain, induced by cyanide, a mitochondrial inhibitor. Curcumin also offered protection against quinolinic acid induced lipid peroxidation, and this protection was extended to lipid peroxidation induced by metals such as lead and cadmium in the rat brain. Experiments conducted on the pineal gland revealed an increased production of the neuroprotective hormone melatonin in presence of curcumin in vivo. The hippocampus is functionally related to vital behaviour and intellectual activities and is known to be a primary target for neuronal degeneration in the brains of patients with Alzheimer’s disease. Histological studies were undertaken to assess the effects of curcumin on lead induced toxicity on the rat hippocampus, the results of which show that curcumin affords significant protection to the hippocampus of the lead treated rats. This study also sought to elucidate possible mechanisms by which curcumin exerts its neuroprotective capabilities. Curcumin was found to inhibit the action of cyanide on the mitochondrial electron transport chain, one of the most common sources of free radicals. Electrochemical, UV/VIS and Infrared spectroscopy were used to characterise interactions between curcumin and the metals lead, cadmium, iron (II) and iron (III). Curcumin was shown to directly chelate these metals with the formation and isolation of two new curcumin complexes with lead, and one complex each with cadmium and iron (III). These results suggest chelation of toxic metals as a mechanism of neuroprotection afforded by curcumin. The need for neuroprotective agents is urgent considering the rapid rise in the elderly population and the proportionate increase in neurological disorders. The findings of this study indicate that curcumin, a well-established dietary antioxidant, is capable of playing a bigger role in neuroprotection, which needs to be further explored and exploited.

published_or_final_version / abstract / Chinese Medicine / Master / Master of Philosophy

 Squamous cell carcinoma of the tongue has a low survival rate, with cure rate reduced by half if cervical lymph node metastasis is present. Standard treatment regimen includes surgical resection of the tumor, radiotherapy and chemotherapy. These treatment modalities, however, can result in irreversible side effects including loss of form and function of the tongue. So far, there is no efficient treatment regime targeting migration and invasion of tongue carcinoma. Curcumin is a natural polyphenol extracted from Curcuma longa. Recent studies indicated that curcumin is a potential anti-cancer agent. The anticancer effects have been demonstrated in numerous cancers including lung cancer, liver cancer, breast cancer, prostate cancer and melanoma. In head and neck cancers, the number studies is limited and its inhibitory effects in migration and invasion is rarely explored. To explore the global expression changes in tongue cancer, we used microarray to evaluate the genes responsive to curcumin treatment and focused on genes related to migration and invasion of tongue cancer cell line HN21B. The genes down-regulated by curcumin were validated in HN21B and two other tongue cancer cell line CAL27 and HN96 using qRT-PCR, Western blotting and immunostaining. The identified genes were quantified in tongue carcinoma tissues to examine whether it was up-regulated in human tongue tissues. Scratch wound assay and radial-migration assay were used to assess the degree of inhibition on migration. Adhesion and invasion assays were also performed to assess the adhesion and invasion ability. Transcriptomic analyses showed that MMP-10 was 2.36 fold down-regulated in HN21B in response to curcumin. Curcumin treatment resulted in down-regulation of MMP-10 gene in all the 3 tongue carcinoma cell lines at mRNA and protein levels. Out of 24 tongue carcinoma cases, 55% tumor tissue had obvious up-regulation of MMP-10 expression in comparison with the normal counterpart. Adhesion, migration and invasion ability of tongue carcinoma cell lines was significantly reduced upon IC50 of curcumin treatment in all TSCC cell lines. In conclusion, our results indicated that curcumin could reduce migration, adhesion and invasion in tongue carcinoma cells partly through reducing MMP-10 expression. Further investigations are warranted to explore the potential therapeutic use of curcumin to inhibit migration and invasion of tongue carcinoma cells. / published_or_final_version / Surgery / Master / Master of Philosophy

背景及研究目的：據報導薑黃素（curcumin）在阿爾茨海默癥（老年癡呆癥）的動物模型中表現出有效性。這可能與其能阻抑β 澱粉樣蛋白的聚集有關，而β 澱粉樣蛋白正是老年癡呆癥中達成共識的神經毒性物質。然而，薑黃素的水溶性很差，這一弱點直接限制其在作為口服藥物的生物利用度。羥丙基-β-環糊精是一種由七個單糖分子鍵合成的環狀分子，該環具有親水性的外部和疏水性的內部，這一特點使得疏水性藥物可以裝入環糊精分子內部從而提高該藥物的親水性，進而提高藥物的口服吸收率。 / 方法：包合物在50 攝氏度下，由摩爾比為1:2 的薑黃素和羥丙基-β-環糊精經過6 小時的攪拌而形成。其后对包合物的物化性質與相同摩爾比的薑黃素與羥丙基-β-環糊精的混合物的物化特性进行了对比以证实包合物的形成，诸如水溶性，掃描電鏡下的形態以及傅利葉紅外圖譜特性。接著用經包合物和普通薑黃素粉末喂食的SD 大鼠做藥代動力學研究，取出大鼠血樣然之後用高效液相色譜-質譜聯用法對薑黃素進行定量，計算出達峰時間（T{U+2098}{U+2090}{U+2093} ）， 峰濃度（C{U+2098}{U+2090}{U+2093} ）以及 0 到4 小時藥時曲綫下面積 （AUC₀→₄{U+2095} ）從而比較包合物較普通薑黃素是否有優越之處。之後，用Tg2576 轉基因小鼠做短期研究，此种小鼠能過度表達β 澱粉樣蛋白，是一種被廣泛應用于老年癡呆癥相關科研的動物模型，經過連續7 日的喂食之後，對小鼠的β 樣澱粉蛋白進行觀察。進一步，將用能同時過度表達β 樣澱粉蛋白與神經纖維結（NTFs）的Tau/APP 轉基因小鼠做長達兩月的藥效學實驗，在實驗始末配以关联恐惧调件反应测试（CFC）和八臂迷宮（Radial Arm Maze）來對實驗小鼠的記憶失進行定量分析。處死小鼠之後，對β 樣澱粉蛋白與神經纖維結（NTFs）進行定量分析從而確定包合物在藥效學上的優勢。 / 結果：在包合的過程中，大部份的薑黃素被整合到包合物之中。通過傅利葉紅外圖譜和掃描電鏡照片都可以觀察到包合物與混合物的顯著不同。在藥代動力學研究中，普通薑黃素粉末的達峰時間為22 分鐘左右，而包合物是40 分鐘，同時，經過包合，峰濃度也提高了3 倍左右，藥時曲綫下面積提高了2 倍以上。在用Tg2576 進行的為期一周的短期實驗中，觀察不到包合物和普通薑黃素在β 樣澱粉蛋白清除方面有明顯差別，然後通過體內染色卻可以看到經包合物喂食的小鼠腦切片中可以觀察到更多的來自薑黃素的螢光信號。在2 個月的長期實驗中，就关联恐惧调件反应测试和八臂迷宮實驗的結果來看，可以觀察到包合物有更好延遲TAPP 小鼠記憶失過程的趨勢但無顯著性，除此之外，對處死后的小鼠腦部進行分析，其β 澱粉樣蛋白與神經纖維節的含量分析結果也和行為測試具有一致性。 / 結論：用羥丙基-β-環糊精對薑黃素進行包合確實可以通過增加薑黃素的水溶性從而提高其生物利用度，讓更多的薑黃素通過血腦屏障進入大腦進而與β 澱粉樣蛋白進行結合。然後短期實驗無法表明包合物具有β 澱粉樣蛋白清除效應。而長期實驗中行為實驗和處死後大腦分析顯示出較普通薑黃素而言包合物具有有限的優點，如果要證明這一優點確實存在，可能需要更長時間的喂食與另外劑量。 / Background: Curcumin is reported to be an effective treatment in animal models of Alzheimer’s disease (AD), possibly by inhibiting aggregation of amyloid-β peptides, which can be neurotoxic.However, curcumin is poorly soluble in water, limiting its oral bioavailability. Hydroxypropyl-β-cyclodextrin (HP-β-CD), a cyclic oligosaccharide made of seven sugar molecules bound togetherin a ring has a hydrophobic exterior and a hydrophobic interior, within which curcumin can reside, thus increasing the aqueous solubility of curcumin. This study aims to solve the problem of poor water-solubility of curcumin using HP-β-CD. / Method: The inclusion complexes were formed by stirring a suspension of curcumin and HP-β-CD at a molar ratio of 1:2 at 50°C for 6 hr. Physicochemical properties, including watersolubility,morphology under scanning electron microscopy (SEM) and the Fourier Transform Infrared (FTIR) spectrum, varied between the inclusion complex and a physical mixture of the two compounds. The inclusion complex and curcumin powder were fed to Sprague Dawley rats for pharmacokinetic studies, from which blood samples were analyzed using LC/MS/MS, and relevant parameters such as T{U+2098}{U+2090}{U+2093}, C{U+2098}{U+2090}{U+2093} and AUC₀→₄{U+2095} were calculated to study the effect of HP-β-CD on the bioavailability of curcumin. To evaluate the pharmacodynamic effects, Tg2576 mice, which over express amyloid-β, were treated for 7 consecutive days with curcumin powder or inclusion complexes. Further, to examine effects of long-term treatment, Tau/APP mice, a commonly used AD model producing both amyloid-β and mutant tau proteins, were treated for 2 months. Behavior tests were conducted at the beginning and end of the long-term treatment to quantify the memory loss of the mice, and post mortem analyses, including quantification of amyloid-β plaques and neurofibrillary tangles, were performed after sacrificing the mice. / Result: The majority of curcumin was integrated into complexes. FTIR profiles and SEM photographs of complexes displayed significant differences from the physical mixture. In the pharmacokinetic study, the concentration of curcumin in the control group peaked at around 22.5 min, while that of inclusion complexes peaked around 40 min. The maximum concentration of curcumin trebled and the area under the curve from 0 to 4 hours more than doubled. For shortterm treatment in Tg2576 mice, paraffin sections stained with Thioflavin T, a dye detecting amyloid-β plaques, showed no obvious difference between mice treated with curcumin powder or complexes; however brain sections from complex-treated mice had more fluorescence signal from curcumin than did mice treated with curcumin powder. For long-term treatment, in terms of the results of contextual fear conditioning and radial arm maze, there was a trend toward inclusion complexes delaying the memory loss of Tau/APP mice more effectively than curcumin powder. Compared to curcumin powder, complexes tended to reduce the number of plaques and neurofibrillary tangles. / Conclusion: Complexation with HP-β-CD can significantly enhance curcumin bioavailability by increasing its water-solubility, allowing more curcumin to penetrate the blood brain barrier to bind to amyloid-β plaques. However, short-term treatment showed no advantage of inclusion complexes in clearing amyloid-β plaques. The results of behavior tests and post-mortem studies from the 2-month long-term treatment indicated limited superiority of inclusion complexes over curcumin powder. A longer feeding period or altered dosage or both might be necessary to enhance the effect of curcumin inclusion complexes. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Hao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 104-117). / Abstracts also in Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview of Alzheimer’s disease --- p.1 / Chapter 1.1.1 --- The pathological mechanisms of Alzheimer’s disease --- p.2 / Chapter 1.1.2 --- Anti-Alzheimer’s disease drugs --- p.3 / Chapter 1.2 --- Curcumin as a potential anti-AD agent --- p.5 / Chapter 1.2.1 --- Actions of curcumin on Aβ --- p.6 / Chapter 1.2.2 --- Anti-inflammatory and anti-oxidant activities of curcumin in AD --- p.8 / Chapter 1.2.3 --- Activities of curcumin to combat metal toxicity --- p.9 / Chapter 1.3 --- The physicochemical properties of curcumin and limitations of curcumin in clinical usage --- p.12 / Chapter 1.4 --- Cyclodextrin in pharmaceutical usage --- p.18 / Chapter 1.5 --- Aims of the study --- p.23 / Chapter Chapter 2 --- Preparation and characterization of curcumin-HP-β-CD complexes --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Phase solubility study --- p.24 / Chapter 2.1.2 --- Preparation of solid complexes --- p.28 / Chapter 2.1.3 --- Characterization of solid complexes --- p.29 / Chapter 2.1.4 --- Previous work on the curcumin HP-β-CD inclusion complex --- p.31 / Chapter 2.2 --- Methodology --- p.33 / Chapter 2.2.1 --- Phase solubility test --- p.33 / Chapter 2.2.2 --- Preparation of curcumin-CD complexes --- p.33 / Chapter 2.2.3 --- Recovery --- p.35 / Chapter 2.2.4 --- Differential solubility --- p.36 / Chapter 2.2.5 --- SEM Studies --- p.37 / Chapter 2.2.6 --- Infrared --- p.37 / Chapter 2.3 --- Results and discussion --- p.38 / Chapter 2.3.1 --- Phase solubility analysis --- p.38 / Chapter 2.3.2 --- Recovery test --- p.40 / Chapter 2.3.3 --- Differential solubility --- p.42 / Chapter 2.3.4 --- SEM study --- p.44 / Chapter 2.3.5 --- Infrared study --- p.45 / Chapter Chapter 3 --- Staining of amyloid plaques in Tg2576 mice after oral administration of curcumin-HP-β-cyclodextrin inclusion complex --- p.47 / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- In vitro staining of amyloid plaques by thioflavin T and curcumin --- p.49 / Chapter 3.2.1 --- Thioflavin T and curcumin specifically binding to amyloid plaques --- p.49 / Chapter 3.2.2 --- Chemicals --- p.51 / Chapter 3.2.3 --- Method --- p.51 / Chapter 3.3 --- In vivo staining of amyloid plaques after oral administration of curcumin-HP-β-CD inclusion complex --- p.53 / Chapter 3.3.1 --- Animal treatment --- p.53 / Chapter 3.3.2 --- Method --- p.54 / Chapter 3.4 --- Results and discussion --- p.56 / Chapter 3.4.1 --- In vitro staining of amyloid plaques by thioflavin T and curcumin --- p.56 / Chapter 3.4.2 --- In vivo staining of amyloid plaques by curcumin --- p.57 / Chapter 3.4.3 --- Plaque removal effects after short-term feeding --- p.58 / Chapter Chapter 4 --- Pharmacokinetic study of curcumin in Sprague-Dawley (SD) rats after oral administration of curcumin-HP-β-cyclodextrin inclusion complex --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.1.1 --- Previous pharmacokinetic study of curcumin-HP-β-CD inclusion complex --- p.61 / Chapter 4.1.2 --- Previously established LC/MS/MS methods for quantification of curcumin in SD rat plasma sample --- p.62 / Chapter 4.2 --- Development and validation of LC/MS/MS assay --- p.63 / Chapter 4.2.1 --- Chemicals --- p.63 / Chapter 4.2.2 --- Instrumentation and chromatographic conditions --- p.63 / Chapter 4.2.3 --- Preparation of standard solutions --- p.63 / Chapter 4.2.5 --- Validation of the assay method --- p.64 / Chapter 4.3 --- Pharmacokinetic profile of curcumin in SD rats --- p.65 / Chapter 4.3.1 --- Animals --- p.65 / Chapter 4.3.2 --- Animal treatment and blood sampling --- p.65 / Chapter 4.3.3 --- Plasma sample analysis --- p.65 / Chapter 4.3.4 --- Data analysis --- p.66 / Chapter 4.4 --- Result and discussion --- p.67 / Chapter 4.4.1 --- Mass spectrum --- p.67 / Chapter 4.4.2 --- Chromatography and specificity --- p.69 / Chapter 4.4.3 --- Linearity and sensitivity --- p.71 / Chapter 4.4.4 --- Method validation---Precision and accuracy --- p.71 / Chapter 4.4.5 --- Method validation---Liquid-liquid extraction recovery --- p.72 / Chapter 4.4.6 --- Pharmacokinetics parameters --- p.73 / Chapter Chapter 5 --- Long-term effects of curcumin-HP-β-CD inclusion complex on Alzheimer’s disease model mice --- p.79 / Chapter 5.1 --- Introduction --- p.79 / Chapter 5.1.1 --- Tau/APP trangenic mouse --- p.79 / Chapter 5.1.2 --- Memory --- p.80 / Chapter 5.1.3 --- The role of the hippocampus in memory --- p.81 / Chapter 5.1.4 --- Memory task contextual fear conditioning (CFC) --- p.82 / Chapter 5.1.5 --- Memory task radial arm maze (RAM) --- p.83 / Chapter 5.2 --- Methods --- p.85 / Chapter 5.2.1 --- Animal treatment --- p.85 / Chapter 5.2.2 --- Contextual fear conditioning test --- p.85 / Chapter 5.2.3 --- Radial arm maze test --- p.87 / Chapter 5.2.4 --- Post-mortem analysis amyloid plaque removal effects of the curcumin-HP-β-CD inclusion complex --- p.88 / Chapter 5.2.5 --- Post-mortem analysis neurofibrillary tangle removal effects of the curcumin-HP-β-CD inclusion complex --- p.90 / Chapter 5.3 --- Results and discussion --- p.92 / Chapter 5.3.1 --- Mortality of the mice --- p.92 / Chapter 5.3.2 --- Contextual fear conditioning test --- p.93 / Chapter 5.3.3 --- Radial arm maze (RAM) test --- p.95 / Chapter 5.3.4 --- Post-mortem analysis amyloid plaque removal effects of the curcumin-HP-β-CD inclusion complex --- p.97 / Chapter 5.3.5 --- Post-mortem analysis neurofibrillary tangle removal effects of the curcumin-HP-β-CD inclusion complex --- p.99 / Chapter Chapter 6 --- Conclusions and future perspective --- p.100 / Chapter 6.1 --- Conclusions --- p.101 / Chapter 6.2 --- Future perspective --- p.103 / References --- p.104

Cheung, Kwok Kuen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 226-263). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Accumulating evidence suggests that anti-inflammatory agents and antioxidants have neuroprotective properties and may be beneficial in the treatment of neurodegenerative disorders. In the present study, the possible neuroprotective properties of tolmetin, sulindac and turmeric were investigated. The antioxidant effects of tolmetin and sulindac were determined by inducing free radical generation with quinolinic acid (QA), cyanide or iron (II) in rat brain homogenates or primary hippocampal neurons. Tolmetin and sulindac significantly reduce lipid peroxidation and scavenge the superoxide anion. Metal binding studies were conducted to determine whether metal chelation is a possible mechanism through which these agents reduce QA and iron (II)-induced lipid peroxidation. UV/VIS, infrared spectroscopy as well as electrochemical studies show that both agents bind to iron (II) and/or iron (III). Histological examination of the hippocampus showed that pre-treatment of animals with tolmetin or sulindac offers protection against intrahippocampal injections of QA. These agents also attenuate QA-induced apoptosis and reduce the loss of neurons in the hippocampus. The co-incubation of primary hippocampal neurons with the NSAIDS also enhanced cell viability which is significantly reduced by QA. Behavioural studies using a water maze showed that the treatment of animals after QA-induced neurotoxicity reduces QA-induced spatial memory loss. Tolmetin and sulindac also reduced glutathione depletion and protein oxidation in rat hippocampus. Both NSAIDS inhibit liver tryptophan 2,3-dioxygenase activity in vitro and in vivo and subsequently increased hippocampal serotonin levels. However, both NSAIDS also reduce dopamine levels in rat striatum. Tolmetin but not sulindac increased the synthesis of melatonin by the pineal gland. The active components of turmeric known as the curcuminoids were separated using preparative thin layer chromatography (TLC). The purity was confirmed by TLC, NMR and mass spectrometry. The environmental toxin lead, induces lipid peroxidation and reduces primary hippocampal neuronal viability. The co-incubation of the neurons with the curcuminoids significantly reduces lead-induced lipid peroxidation and enhances neuronal cell viability in the presence of lead. Lead-induced spatial memory deficit is also attenuated with curcumin, demethoxycurcumin but not bisdemethoxycurcumin. The curcuminoids also reduce lead-induced hippocampal glutathione depletion and protein oxidation. Metal binding studies show that the curcuminoids bind to lead and is another possible mechanism through which the curcuminoids reduce lead-induced neurotoxicity. The findings of this study indicate a possible role of tolmetin, sulindac and turmeric in neurodegenerative disorders such as Alzheimer’s disease. However, tolmetin and sulindac reduce dopamine levels.