published_or_final_version / Community Medicine / Master / Master of Public Health

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

and senkyunolide A were found as the three major compounds in all herbal samples investigated. In addition, great variations in both total and individual content of each of the ten main components investigated were observed in samples of different origins and those collected from a GAP developing base in the same or different years, suggesting the necessity of a thorough quality control for Rhizoma Chuanxiong. / Extraction of the main ingredients from Rhizoma Chuanxiong by supercritical fluid extraction using CO2 was investigated. An appropriate SCFE method for Chuanxiong was developed with the mild conditions for the extraction of the unstable components. The method provided a high recovery and adequate reproducibility, and may be suitable for large-scale industry extraction of Chuanxiong. / Firstly, a total of sixteen ingredients were identified from Chuanxiong by HPLC-UV-MS and HPLC-UV analyses. Among them, ten ingredients were determined to be the main components in Chuanxiong. A simple, sensitive and specific HPLC-UV method was developed, for the first time, to simultaneously qualitatively and quantitatively determine twelve ingredients, including the identified ten main ingredients, plus vanillin and tetramethylpyrazine (TMP), which although were not found in the present study, had also been reported to be present in Rhizoma Chuanxiong. The developed assay was fully validated and provided adequate accuracy and reproducibility for all compounds analyzed. It was applied successfully to simultaneously quantify all main constituents in different Chuanxiong samples. TMP and vanillin were not detected, while Z-ligustilide, coniferylferulate. / Furthermore, a comprehensive stability study was carried out for the first time with the three major components senkyunolide A, coniferylferulate, Z-ligustilide and the main ingredient 3-butylidenephthalide, in pure form or Chuanxiong extract obtained from supercritical fluid extraction using CO 2 (SCFE) under different conditions. Results showed that both sun light and elevated temperature led to degradations of these components to different extents. Owing to such thermal and light instability, post-harvest drying and processing procedures could significantly alter the chemical profile of Chuanxiong herb, and thus also need to be well controlled. / In conclusion, analytical and pharmacokinetic studies of the main chemical ingredients in Rhizoma Chuanxiong were systematically conducted. The results revealed, for the first time, that senkyunolide A, Z-ligustilide and 3-butylidenephthalide might be the primary chemical ingredients contributing to the beneficial effects of Chuanxiong. / Oral bioavailability was about 8%, 3% and 20% for senkyunolide A, Z-ligustilide and 3-butylidenephthalide, respectively. Instability in the gut mainly contributed to a low oral bioavailability of senkyunolide A. First-pass metabolism in the liver also contributed to the low oral bioavailability but to a much lower extent. For Z-ligustilide, extensive first-pass metabolism in the liver and degradation in the stomach only partly accounted for its poor oral bioavailability, while other gut factors involved are still unknown. In the case of 3-butylidenephthalide, its low oral bioavailability was attributed to extensive first-pass metabolism in both the gut and the liver. / Pharmacokinetic fates of the main ingredients in Chuanxiong SCFE extract were firstly evaluated in rats. After a single intravenous and oral administration, only senkyunolide A, Z-ligustilide and 3-butylidenephthalide were determined as the main herb related components in plasma. Coniferylferulate, although it is one of the abundant principles in the herb, was not detected in the plasma even immediately after dosing. / Pharmacokinetic profiles of senkyunolide A, Z-ligustilide and 3-butylidenephthalide were further elucidated individually in rats. All three compounds exhibited rapid absorption, extensive distribution, and rapid elimination. The pharmacokinetic profile of senkyunolide A followed a dose-independent pattern, whereas Z-ligustilide exhibited dose-dependent kinetics. 3-Butylidenephthalide underwent enterohepatic re-circulation. / Rhizoma Chuanxiong is derived from the dried rhizome of Ligusticum chuanxiong Hort. (Umbelliferae). In China, it has been widely prescribed for the treatment of cerebro- and cardio-vascular diseases for thousands of years. However, its chemical and pharmacological basis is poorly understood. In the present study, analytical methods for qualitative and quantitative determination of the main chemical components in Chuanxiong herb were developed. Furthermore, pharmacokinetic profiles of the main chemical ingredients in Chuanxiong were systematically investigated in rats for the first time. / The metabolic profiles of senkyunolide A, Z-ligustilide and 3-butylidenephthalide were investigated both in vivo and in vitro. Oxidation and hydration were found to be the main metabolic pathways for all three compounds. In addition, glutathione conjugation of senkyunolide A and Z-ligustilide also occurred in the rat. A novel metabolite 3-hydroxy-3-butylphthalide was identified as the major metabolite of 3-butylidenephthalide generated by a direct hydration, and was shown to have significantly higher plasma levels than those of the parent compound. Furthermore, the main metabolites detected in the plasma of rats administered with Chuanxiong extract were generated from senkyunolide A, Z-ligustilide and 3-butylidenephthalide. / Yan Ru. / "May 2005." / Adviser: Ge Lin. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1583. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 244-255). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Bi-directional transport studies in SimBioDASRTM and Caco-2 cells were employed to examine the transport profiles of Buph, Ligs and SenA. Apical to basolateral (A-B) transport studies of the tested compounds revealed high intestinal permeability and predicted human absorption of over 98%. Permeability ratio of B-A/A-B of Buph (0.7-1.3) and Ligs (0.8-1.2) indicated that they were transported by passive transcellular and paracellular pathways while the low B-A/A-B ratio of SenA may imply possible involvement of other transport mechanisms. One metabolite (M-1) generated from hydration of Buph was observed in Caco-2 cells and the fraction of metabolism was 12.5% (A-B). / In conclusion, Buph, Ligs and SenA were predicted to have good intestinal absorptions in human and rat. However, extensive hepatic and intestinal first-pass metabolism of Buph in rat and human were found to cause its low oral bioavailability. On the other hand, certain degree of hepatic first-pass metabolism of Ligs and SenA may account for the partial loss of drugs via oral administration to rat. Therefore, other routes of delivery, such as sublingual administration, are worth to be considered to improve the therapeutic effects of chuanxiong. / In the rat SPIP, permeability calculated from the appearance of Buph in mesenteric blood (Pblood) was 6.0+/-1.7 x 10-4 cm/s while the fraction of formation of M-1 was about 7.1%. Together with the in vitro results, it is proposed that first-pass metabolism of Buph was present in human and rat small intestine. Moreover, Ligs and SenA had high Pblood values of 4.2+/-1.2 x 10-3 cm/s and 3.8+/-2.8 x 10-3 cm/s, respectively, indicated that they were highly permeable across rat intestinal mucosa. No metabolism of Ligs was observed. But several metabolites of SenA were detected despite they were not quantified in the present study. / In vitro metabolic studies of Buph demonstrated that major metabolite M-1, which was also found in Caco-2 cells and SPIP, formed mainly in intestine and liver cytosol in rat and human. The intrinsic clearance (Vmax/Km) of Buph was extensive and similar in both organs, and its extent in human was comparable to that in rat. The sum of the estimated in vivo extraction ratio of Buph by liver (48.3%) and intestine (55.0%) was higher the loss via oral administration to rat (77%). On the other hand, several metabolites of Ligs and SenA were found in rat and human liver microsome but not in intestinal preparations. The estimated in vivo extraction ratio by liver of rat was 47.3% (Ligs) and 22.9% (SenA), respectively, which were less than the corresponding loss via oral administration to rat (Ligs: 92.2% and SenA: 97.7%), suggesting that first-pass effect other than metabolism of these two compounds in intestine also contributed to their low oral bioavailability. / Rhizoma chuanxiong is commonly prescribed orally for improving blood circulation and treating cardiovascular disorders in China. Like other traditional Chinese medicines, chuanxiong has been used for thousands of years in China but its chemical basis, pharmacological effects and pharmacokinetic fates of the active ingredients, especially absorption, are poorly understood. Recently, seventeen compounds such as 3-butylidenephthalide (Buph), Z-ligustilide (Ligs), senkyunolide A (SenA), vanillin (Vani), ferulic acid (Fera), senkyunolide I (SenI), senkyunolide H (SenH), coniferyl ferulate (ConFer), sedanolide (Sdan), riligustilide (Rili) and levistolide A (LevA) have been isolated and recognized as the main constituents of chuanxiong by our research team (Li et al., 2003). Moreover, it has been demonstrated that Buph, Ligs and SenA are bioactive components of chuanxiong for vasodilatation and anti-thromboembolism (Chan, 2005) though their oral bioavailability in rat are very low (2.3, 7.8 and 23% respectively) (Yan, 2005). Therefore, the present study aims at investigating the intestinal permeability of the major ingredients of chuanxiong and characterizing the intestinal absorption and first-pass metabolism of Buph, Ligs and SenA by in vitro Caco-2 cell monolayers, SimBioDASRTM , in situ single-pass intestinal perfusion (SPIP) in rat and in vitro metabolism using rat and human intestine and liver subcellular fractions respectively. / Using the in vitro cell monolayers of SimBioDAS RTM, the intestinal permeability of major components of chuanxiong ranged from 12.2+/-1.6 x 10-6 cm/s to 70.6+/-9.6 x 10-6 cm/s with a rank order of Fera &lt; Buph &lt; Ligs &lt; Sdan &lt; SenH &lt; SenI &lt; SenA &lt; Vani. They were predicted to have over 70% absorption in human. However, ConFer, Rili and LevA were estimated to have poor human oral absorption. / Ko Nga Ling. / "September 2005." / Adviser: Ge Lin. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1577. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 215-239). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

by Lam Fung Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 187-202). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.xiii / List of Tables --- p.xvii / List of Figures --- p.xviii / Publication --- p.xx / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of Diabetes Mellitus --- p.1 / Chapter 1.2 --- Definition of Diabetes Mellitus --- p.1 / Chapter 1.3 --- Glucose Homeostasis and Diabetes Mellitus --- p.2 / Chapter 1.4 --- Classification of Diabetes Mellitus --- p.6 / Chapter 1.4.1 --- Type 1 Diabetes Mellitus --- p.6 / Chapter 1.4.2 --- Type 2 Diabetes Mellitus --- p.7 / Chapter 1.4.3 --- Gestational Diabetes Mellitus --- p.8 / Chapter 1.4.4 --- Other specific types --- p.8 / Chapter 1.5 --- Diagnostic Criteria of Diabetes Mellitus --- p.9 / Chapter 1.6 --- Complications of Diabetes Mellitus --- p.11 / Chapter 1.7 --- Pharmacological Treatment of Diabetes --- p.12 / Chapter 1.7.1 --- Treatment for type 1 diabetes mellitus --- p.12 / Chapter 1.7.2 --- Treatment for Type 2 diabetes mellitus --- p.13 / Chapter 1.7.2.1 --- Sulfonylureas --- p.14 / Chapter 1.7.1.2 --- Meglitinides --- p.15 / Chapter 1.7.1.3 --- Biguanides --- p.15 / Chapter 1.7.1.4 --- Thazolidinediones --- p.16 / Chapter 1.7.1.5 --- α-Glucosidase inhibitor --- p.16 / Chapter 1.8 --- Diabetes and Traditional Chinese Medicine --- p.17 / Chapter 1.9 --- Objective of this project --- p.18 / Chapter Chapter 2 --- "Botanical, Preparation and Authentication of Traditional Chinese Herbs" --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.2 --- Herbal Materials --- p.22 / Chapter 2.3 --- Authentication of Herbal Material --- p.30 / Chapter 2.4 --- Extraction Method --- p.32 / Chapter 2.4.1 --- Material and Methods --- p.32 / Chapter 2.4.2 --- Results --- p.32 / Chapter 2.4 --- Discussion --- p.32 / Chapter Chapter 3 --- In vitro Studies on Selected Traditional Chinese Herbs --- p.35 / Chapter 3.1. --- Introduction --- p.35 / Chapter 3.2 --- Hepatic Gluconeogenesis Studies --- p.36 / Chapter 3.2.1 --- Introduction --- p.36 / Chapter 3.2.2 --- Material and Methods --- p.41 / Chapter 3.2.2.1 --- Cell Culture of H4IIE --- p.41 / Chapter 3.2.2.2 --- Glucose Production Assay --- p.42 / Chapter 3.2.2.3 --- Bicinchoninic Acid (BCA) Protein Assay --- p.43 / Chapter 3.2.3 --- Results --- p.44 / Chapter 3.3 --- Intestinal Glucose Absorption Studies --- p.46 / Chapter 3.3.1 --- Introduction --- p.46 / Chapter 3.3.2 --- Material and Methods --- p.48 / Chapter 3.3.2.1 --- Preparation of BBMV --- p.48 / Chapter 3.3.2.1.1 --- Chemicals --- p.48 / Chapter 3.3.2.1.2 --- Method --- p.48 / Chapter 3.3.2.2 --- Preparation of Herbal Extracts --- p.50 / Chapter 3.3.2.3 --- BBMV Glucose Uptake Assay --- p.51 / Chapter 3.3.2.4 --- Bicinchoninic Acid (BCA) Protein Assay --- p.54 / Chapter 3.3.3 --- Results --- p.54 / Chapter 3.4 --- Fibroblast Glucose Uptake Studies --- p.57 / Chapter 3.4.1 --- Introduction --- p.57 / Chapter 3.4.2 --- Material and Methods --- p.58 / Chapter 3.4.2.1 --- Cell Culture of Hs68 --- p.58 / Chapter 3.4.2.2 --- 2-Deoxy-D-glucose Uptake Assay --- p.59 / Chapter 3.4.2.3 --- Bicinchoninic Acid (BCA) Protein Assay --- p.60 / Chapter 3.4.3 --- Results --- p.60 / Chapter 3.5 --- Adipocyte Glucose Uptake Studies --- p.63 / Chapter 3.5.1 --- Introduction --- p.63 / Chapter 3.5.2 --- Material and Methods --- p.65 / Chapter 3.5.2.1 --- Cell Culture of 3T3-L1 --- p.65 / Chapter 3.5.2.2 --- Differentiation of 3T3-L1 --- p.65 / Chapter 3.5.2.3 --- 2-Deoxy-D-glucose Uptake Assay --- p.66 / Chapter 3.5.2.4 --- Bicinchoninic Acid (BCA) Protein Assay --- p.68 / Chapter 3.5.3 --- Results --- p.69 / Chapter 3.6 --- Glucose Transporter Type 4 (GLUT4) Expression Studies --- p.71 / Chapter 3.6.1 --- Introduction --- p.71 / Chapter 3.6.2 --- Material and Methods --- p.48 / Chapter 3.6.2.1 --- Cell Culture of 3T3-L1 --- p.71 / Chapter 3.6.2.2 --- Differentiation of 3T3-L1 --- p.71 / Chapter 3.6.2.3 --- GLUT4 Expression Assay --- p.72 / Chapter 3.6.2.4 --- Preparation of RNA --- p.72 / Chapter 3.6.2.5 --- RT-PCR --- p.73 / Chapter 3.6.2.6 --- PCR Analysis on GLUT4 Expression --- p.74 / Chapter 3.6.2.7 --- Real-time PCR --- p.75 / Chapter 3.6.3 --- Results --- p.77 / Chapter 3.7 --- Discussion --- p.81 / Chapter 3.7.1 --- Discussion of Hepatic Gluconeogenesis Studies --- p.81 / Chapter 3.7.2 --- Discussion of Intestinal Glucose Absorption Studies --- p.82 / Chapter 3.7.3 --- Discussion of Fibroblast Glucose Uptake Studies --- p.83 / Chapter 3.7.4 --- Discussion of Adipocyte Glucose Uptake Studies --- p.84 / Chapter 3.7.5 --- Discussion of Glucose Transporter Type 4 (GLUT4) Expression Studies --- p.86 / Chapter 3.7.6 --- Conclusion --- p.87 / Chapter Chapter 4 --- Purification of Cortex Moutan --- p.90 / Chapter 4.1 --- Introduction --- p.90 / Chapter 4.1.1 --- Phytochemical Studies of Cortex Moutan --- p.90 / Chapter 4.2 --- Organic Extraction of Cortex Moutan --- p.93 / Chapter 4.2.1 --- Extraction Material and Methods --- p.93 / Chapter 4.2.2. --- Results --- p.93 / Chapter 4.3 --- BBMV Glucose Uptake Assay with Cortex Moutan Organic Extract (CM-C and CM-D) --- p.96 / Chapter 4.3.1 --- Material and Methods --- p.48 / Chapter 4.3.2 --- Results --- p.96 / Chapter 4.4 --- Fractionation of CM-C and CM-D --- p.98 / Chapter 4.4.1 --- Material and Methods --- p.98 / Chapter 4.4.1.1 --- Chemicals --- p.98 / Chapter 4.4.1.2 --- Methods --- p.98 / Chapter 4.4.2 --- Results --- p.100 / Chapter 4.5 --- BBMV Glucose Uptake Assay of CM-C and CM-D Sub-fractions --- p.105 / Chapter 4.5.1 --- Results --- p.105 / Chapter 4.6 --- Sulfonylation of CM-D1 --- p.107 / Chapter 4.6.1 --- Material and Methods --- p.107 / Chapter 4.6.1.1 --- Chemicals --- p.107 / Chapter 4.6.1.2 --- Methods --- p.107 / Chapter 4.6.2 --- Structure Elucidation of CM-D1s --- p.108 / Chapter 4.6.2.1 --- 1H-NMR Analysis --- p.108 / Chapter 4.6.3 --- BBMV Glucose Uptake Assay of CM-D1s --- p.108 / Chapter 4.6.4 --- Results --- p.108 / Chapter 4.7 --- "Structural Elucidation of CM-D3, CM-D4 and CM-D5" --- p.112 / Chapter 4.7.1 --- Material and Methods --- p.112 / Chapter 4.7.1.1 --- Mass Spectrometry --- p.112 / Chapter 4.7.1.2 --- 1H-NMR Analysis --- p.112 / Chapter 4.7.2 --- Results --- p.113 / Chapter 4.8 --- "BBMV Glucose Uptake Assay of Acetovallione, CM-D3，CM-D4 and CM-D5" --- p.116 / Chapter 4.8.1 --- Results --- p.116 / Chapter 4.9 --- Synthesis of CM-D3s --- p.118 / Chapter 4.9.1 --- Material and Methods --- p.118 / Chapter 4.9.1.1 --- Chemicals --- p.118 / Chapter 4.9.1.2 --- Methods --- p.118 / Chapter 4.9.2 --- Structure Elucidation of synthesized product --- p.119 / Chapter 4.9.3 --- Results --- p.119 / Chapter 4.10 --- Discussion --- p.121 / Chapter Chapter 5 --- In vivo Studies on Selected Herbs --- p.123 / Chapter 5.1 --- Introduction --- p.123 / Chapter 5.1.1 --- Diabetic Animal Models --- p.123 / Chapter 5.1.2 --- Neonatal Streptozotocin-induced Diabetic Rat Model --- p.125 / Chapter 5.2 --- Oral Glucose Tolerance Test (OGTT) --- p.126 / Chapter 5.2.1 --- Animal --- p.126 / Chapter 5.2.2 --- Rat Induction Material and Methods --- p.126 / Chapter 5.2.3 --- Testing Method for diabetic condition of rats --- p.127 / Chapter 5.3.4 --- Results --- p.128 / Chapter 5.3 --- Basal Glycaemia Test --- p.138 / Chapter 5.3.1 --- Animal --- p.138 / Chapter 5.3.2 --- Rat Induction Material and Methods --- p.138 / Chapter 5.3.3 --- Testing Method --- p.138 / Chapter 5.3.4 --- Results --- p.140 / Chapter 5.4 --- Discussion --- p.143 / Chapter Chapter 6 --- General Discussion --- p.147 / Chapter 6.1 --- Introduction --- p.147 / Chapter 6.2 --- Summary of Research Findings --- p.151 / Chapter 6.3 --- Result Interpretation --- p.152 / Chapter 6.3.1 --- Result Interpretation of In Vitro Studies --- p.152 / Chapter 6.3.2 --- Result Interpretation of Cortex Moutan Purification --- p.154 / Chapter 6.3.3 --- Result Interpretation of In Vivo Studies --- p.157 / Chapter 6.4 --- Limitations and Improvements --- p.161 / Chapter 6.5 --- Future Directions --- p.163 / Chapter 6.6 --- Conclusions --- p.169 / Appendices --- p.170 / References --- p.187

In a mouse model of pulmonary thromboembolism induced by a collagen-adrenaline mixture, the SFE extract and ligustilide reduced the paralysis-death ratio, and the anti-thrombotic response of senkyunolide A was more pronounced. The effect of BDPH was not significant. Neither the SFE extract nor the three phthalides prolonged bleeding time in tail-transected mice. / In a rat myocardial ischemia-reperfusion model involving coronary artery ligation, 7-day pre-treatment with the SFE extract and ligustilide reduced ventricular arrhythmias in isolated hearts. BDPH and senkyunolide A were without significant effects. / In rat platelet-rich plasma, platelet aggregation induced by collagen and U46619 but not by adenosine diphosphate was inhibited by the SFE extract. Ligustilide inhibited the responses of all three agonists, while BDPH and senkyunolide A inhibited the collagen response only. / Raw Rhizoma Chuanxiong herb and its crude extract as obtained by supercritical fluid extraction (SFE) comprised mainly phthalides. The SFE extract and three representative phthalides, butylidenephthalide (BDPH), ligustilide and senkyunolide A, were studied on vasorelaxation, myocardial ischemia, platelet aggregation and thrombosis. The mechanisms underlying BDPH-mediated vasorelaxation were also explored. / Rhizoma Chuanxiong, the dried rhizome of Ligusticum chuanxiong Hort., is a common traditional Chinese medicine used for the treatment of cardiovascular diseases. Surprisingly, the scientific basis of its action is poorly understood. The current study aims to establish the pharmacological basis of the cardiovascular effects of Rhizoma Chuanxiong and its active constituents by examining their effects in several cardiovascular domains. / The current study demonstrated various cardiovascular actions of Rhizoma Chuanxiong, and thereby established the pharmacological basis of the effects of the herb. Phthalides, in particular BDPH, ligustilide and senkyunolide A, were important contributors to such actions. Future investigation of the SFE extract and/or individual phthalides related to the progression from in vitro and in vivo effectiveness to clinical efficacy is much anticipated. / The SFE extract, BDPH, ligustilide and senkyunolide A produced vasorelaxation on isolated preparations of rat aorta, rat saphenous vein and pig coronary artery. BDPH-mediated relaxation appeared to involve both extracellular Ca 2+-dependent (L-type voltage-operated, receptor-operated and store-operated Ca2+ channels) and independent (NO modulation, Ca2+ release from internal stores and Ca2+ desensitization) mechanisms. BDPH was also observed to augment relaxation induced by sodium nitroprusside and forskolin through mechanisms that remain undefined. / Chan Sun Kin Sunny. / "July 2005." / Advisers: G. Lin; R. L. Jones. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1575. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 190-209). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

differential scanning calorimetric profile and the generation of much less intense and broader peaks in the powder X-ray diffraction pattern compared to beta-CD. FTIR analysis revealed significant physical interactions between CX oil and beta-CD in the granules, possibly due to complexation. Results from phase solubility measurements and proton nuclear magnetic resonance ( 1H-NMR) analysis of pure 3-butylidenephthalide (3-BDPH), a representative CX component, lend some support for the formation of a 1:1 stoichiometric inclusion complex between 3-BDPH and beta-CD. / Rhizoma chuanxiong (CX), the dried rhizome of Ligusticum Chuangxiong Hort. (Umbelliferae), has been extensively used in mainland China as a traditional herbal medicine for treating cardio-/cerebrovascular diseases and gynecological disorders. However, the active components in CX, which are predominantly essential oils, generally exhibit poor stability (mostly photo-oxidation), high volatility, low aqueous solubility, and extensive gut/hepatic metabolism, all of which can significantly reduce their oral bioavailability and therapeutic efficacy. The present project has investigated the feasibility of utilizing three formulation approaches to circumvent the aforementioned problems associated with the peroral delivery of CX (as crude oil mixture or individual components). / The first approach involved inclusion of CX oil in beta-cyclodextrin (beta-CD) as solid granules using a coprecipitation method optimized with the aid of an orthogonal study design. The resulting CX oil granules were colorless and odorless with a median particle size of 11.38mum; were stable to heat, light and moisture, and readily soluble in simulated gastric and intestinal fluids. The granules were largely amorphous, as evidenced by an absence of the melting endotherm for beta-CD in the formulation could be largely explicated by the complexation behavior and hydration properties of the two polymers blended in different weight percentages, as substantiated by turbidity measurement, viscosity determination and FTIR analysis of the pure polymer mixtures as well as swelling measurement of the formulated tablets. The sustained release behavior of 3-BDPH from the tablet was dependent on the relative proportion of the two polymers present, and could be similarly explained by the changes in hydration and complexation behavior of the polymers during the penetration of aqueous fluid into the tablet matrix. / The second approach involved incorporation of CX oil into surfactant micelles and liquid crystals as a self-emulsifying drug delivery system (SEDDS). An optimal formulation was developed through a judicial choice of excipients (lipids and surfactants/cosurfactant) and their proper combination in the correct proportions, as determined by the spontaneity of the emulsification process and the change in emulsion droplet size. The formulation was readily dispersible in water upon mild agitation, free from unpleasant odor, and stable in soft gelatin capsules for a storage period of at least 12 months under ambient condition. The optimal utilization of the lipid and surfactant blends in defined proportions in the formulation was further substantiated by interfacial tension determination and equilibrium phase analysis. / The third approach involved formulation of 3-BDPH (or crude CX oil) into a sustained-release buccoadhesive tablet, based on a systematic evaluation of the adhesive properties of two polymers (Carbopol 974P and hydroxypropyl methylcellulose K4M) used in the formulation. The adhesive properties of the formulation could be largely explicated by the complexation behavior and hydration properties of the two polymers blended in different weight percentages, as substantiated by turbidity measurement, viscosity determination and FTIR analysis of the pure polymer mixtures as well as swelling measurement of the formulated tablets. The sustained release behavior of 3-BDPH from the tablet was dependent on the relative proportion of the two polymers present, and could be similarly explained by the changes in hydration and complexation behavior of the polymers during the penetration of aqueous fluid into the tablet matrix. / Gao Yuan. / "April 2005." / Adviser: Albert H. L. Chow. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1585. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 193-223). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Xian, Jiawen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 201-230). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Law, Wai Tak. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 107-123). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgements --- p.vi / Publications --- p.viii / Table of Contents --- p.ix / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Wound healing --- p.1 / Chapter 1.1.1 --- Physiology of wound healing --- p.1 / Chapter 1.1.2 --- Three phases of wound healing --- p.3 / Chapter 1.1.3 --- Angiogenesis in wound healing --- p.10 / Chapter 1.2 --- Delayed wound healing --- p.11 / Chapter 1.2.1 --- Chronic ulcers --- p.11 / Chapter 1.2.2 --- Examples of ulcers --- p.12 / Chapter 1.3 --- Traditional Chinese medicine (TCM) --- p.16 / Chapter 1.3.1 --- Principles of TCM --- p.16 / Chapter 1.3.2 --- TCM and chronic ulcers --- p.16 / Chapter 1.4 --- Objectives of study --- p.19 / Chapter Chapter 2 --- Materials and Methods --- p.21 / Chapter 2.1 --- Selection of traditional Chinese herbs --- p.21 / Chapter 2.2 --- Authentication of TCM --- p.22 / Chapter 2.3 --- Preparation of TCM --- p.23 / Chapter 2.4 --- In vitro studies on the effects of TCM on wound healing --- p.23 / Chapter 2.4.1 --- Angiogenesis study by using human umbilical vein endothelial cell (HUVEC) --- p.25 / Chapter 2.4.2 --- Granulation study by using human fibroblast cell line (CRL) --- p.32 / Chapter 2.4.3 --- Preparation of cell culture conditions --- p.35 / Chapter 2.5 --- In vivo study on the effects of TCM on wound healing by using diabetic mice --- p.38 / Chapter 2.5.1 --- Diabetic mice model --- p.38 / Chapter 2.5.2 --- Diabetic mice wound induction --- p.41 / Chapter 2.5.3 --- "Measurement of body weight, blood glucose level and ulcer area" --- p.43 / Chapter Chapter 3 --- Results / Chapter 3.1 --- The percentage yield of each herbs --- p.48 / Chapter 3.2 --- pH value of all the effective treatment concentration --- p.49 / Chapter 3.3 --- Selection of traditional Chinese herbs --- p.53 / Chapter 3.4 --- Effect of selected TCM on the proliferation of HUVEC --- p.55 / Chapter 3.5 --- Effect of selected TCM on the migration of HUVEC --- p.61 / Chapter 3.6 --- Effect of selected TCM on the proliferation of CRL --- p.63 / Chapter 3.7 --- "Effect of Radix Rehmanniae (selected TCM) on the change in body weight, blood glucose level and ulcer area" --- p.66 / Chapter Chapter 4 --- Discussions --- p.75 / Chapter Chapter 5 --- How does my study contribute towards the modernisation of Chinese medicine? --- p.100 / References --- p.107 / Appendix --- p.124

Chen Feng. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 166-178). / 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.