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Atherosclerosis is the chief cause of acute coronary syndromes and may progress for many years before any noticeable clinical syndromes occur. Hyperlipidemia is the common clinical problem for people adopting a western style of living and it can initiate a series of vascular events that result in atherosclerosis. The pathological processes include the accumulation of modified lipid, mainly oxidized low-density lipoprotein (oxLDL), endothelial cell dysfunction and activation, increase in expression of adhesion molecules, activation and recruitment of inflammatory cells and induction of proliferation and migration of vascular smooth muscle cells. / Endothelial-monocyte adhesion is crucial process for the recruitment of monocyte into intima. DG (7:3), Danshen and SAB were found to inhibit TNF-alpha-induced endothelial-monocyte adhesion. They also showed inhibition on TNF-alpha induced production of chemokines, MCP-1 which promotes the transmigration of monocyte. However, it did not inhibit the production of cytokine, IL-6 which stimulates the expression of adhesion molecules such as VCAM-1 and ICAM-1. / For the in vivo study, DG (7:3) exhibited no anti-hyperlipidemic or hypolipidemic effect against diet-induced hyperlipidemia, nor did it lower cholesterol level in hamsters. Also, it did not inhibit HMG-CoA reductase activity or increase the total fecal sterols excretion. However, DG (7:3) exhibited hypocholesterolemic effect on diet-induced hyperlipidemia in the rabbit model, wherein it could lower plasma total cholesterol and liver cholesterol level. Moreover, it could significantly decrease the atheroma formation. / In the present study, the anti-oxidative effects of herbal extract/compound were measured by three in vitro assays, namely the inhibition of 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH)-induced red blood cells hemolysis, AAPH-induced cardiomyocyte (H9c2) cells death and Cu2+-induced low-density lipoprotein oxidation. The results showed that the aqueous extract of the compound formula Danshen (D) and Gegen (G) (7:3), abbreviated as DG (7:3), and an aqueous extract of Danshen as well as salvianolic acid B (SAB) exhibited anti-oxidant effect, but Gegen did not produce such effect. It was found that SAB showed a stronger anti-oxidant effect than that of ascorbic acid. / Proliferation and migration of vascular smooth muscle cells (vSMCs) are important pathological processes involved in the development of atherosclerosis. DG (7:3), Danshen and SAB were found to inhibit PDGF-induced vSMCs proliferation through G1/S cell cycle arrest. Cyclin D, a main component that governs the transition of G1 phase to S phase, was found to be down-regulated by DG (7:3), Danshen and SAB, as assessed by measurements of both protein and mRNA levels. Moreover, DG (7:3), Danshen and SAB showed anti-migratory effect against platelet-derived growth factor-induced vSMCs migration. / To summarize, DG (7:3) was found to have potential to produce anti-atherosclerotic effect by inhibiting the LDL oxidation, proliferation and migration of vascular SMC, thereby preventing the formation of atheroma plaque. / Koon Chi Man. / "March 2006." / Adviser: Kwok Pui Fung. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6324. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 246-264). / 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.

The isoflavones in kudzu roots, especially the C-glycosylated isoflavone puerarin, have been linked to many health benefits. Puerarin contains a carbon-carbon glycosidic bond that can withstand hydrolysis. The C-glycosylation reaction in the biosynthesis of puerarin has not been thoroughly investigated, with conflicting reports suggesting that it could take place on daidzein, isoliquiritigenin, or 2,7,4ʹ-trihydroxyisoflavanone. Kudzu species were identified for use in comparative transcriptomics. A non-puerarin producing kudzu was identified as Pueraria phaseoloides and a puerarin producing kudzu was identified as Pueraria montana lobata. Through the use of the plant secondary product glycosyltransferase (PSPG) motif, glycosyltransferases (UGTs) were identified from the transcriptomes. The UGTs that had higher digital expression in P. m. lobata were examined further using additional tools to home in on the UGT that could be responsible for puerarin biosynthesis. One of the UGTs identified, UGT71T5, had previously been characterized from kudzu as a C-glycosyltransferase involved in puerarin biosynthesis through in vitro enzyme activity (with daidzein) and a gain of function approach in soybean hairy roots. Previous studies have not supported the end-product of a pathway such as daidzein as the target for C-glycosylation, and no genetic analysis of UGT function had been conducted in kudzu. The activity of recombinant UGT71T5 with daidzein was confirmed in the present work. Following the development of a kudzu hairy root system, UGT71T5 expression was then knocked down by RNA interference (RNAi). When compared to control hairy roots there was a large reduction in puerarin content in the UGT71T5-RNAi roots, confirming the role of this enzyme in puerarin biosynthesis. Isotopic labeling of kudzu plants revealed that labeled daidzein could be directly incorporated into puerarin; however, the percent incorporation of daidzein was substantially lower than that of L-phenylalanine, a compound at the start of the pathway to isoflavone synthesis. The knockdown of 2-hydroxisoflavanone synthase (2-HIS) in kudzu hairy roots blocked formation of puerarin and daidzin (7-O-glycosyldaidzein), and was accompanied by accumulation of C-glycosylated isoliquiritigenin and C-glycosylated liquiritigenin. These compounds were found in low amounts in control hairy roots, but were virtually absent in UGT71T5 knockdown hairy roots. The knockdown of 2-hydroxyisoflavanone dehydratase (2-HID) in kudzu hairy roots resulted in a slight reduction in puerarin but no change to daidzin levels, suggesting that C-glycosylation might stabilize the substrate for 2-HID which can otherwise spontaneously dehydrate. Taken together these results reveal that UGT71T5 is likely the major C-glycosyltransferase involved in puerarin biosynthesis in kudzu. They also provide evidence for an alternative pathway to puerarin biosynthesis through the C-glycosylation of isoliquiritigenin or its immediate precursor. In one pathway, UGT71T5 acts as an operationally soluble enzyme that can directly C-glycosylate daidzein, and in the other pathway UGT71T5 acts as part of a metabolic channel for conversion of a C-glycosylated earlier precursor to puerarin. Other UGT enzymes identified in this work did not show C-glycosyltransferase activity; however, three enzymes showed activity in vitro that could be useful for introducing novel regiospecificity in biochemical synthesis of flavonoid glycosides.

Guan, Lei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 123-145). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / ABSTRACT --- p.II / LIST OF ABBREVIATIONS --- p.VII / TABLE OF CONTENT --- p.IIX / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Distribution and Origins --- p.1 / Chapter 1.3 --- History of Use --- p.2 / Chapter 1.4 --- Chemical Structure --- p.3 / Chapter 1.5 --- Physiologic Properties --- p.5 / Chapter 1.6 --- Absorption and Metabolism --- p.7 / Chapter 1.6.1 --- Concentration of Isoflavones in Plasma --- p.10 / Chapter 1.6.2 --- Urinary Excretion --- p.10 / Chapter 1.7 --- Healthy Effects --- p.11 / Chapter 1.7.1 --- Menopausal Symptom --- p.11 / Chapter 1.7.2 --- Cardiovascular Disease --- p.12 / Chapter 1.7.3 --- Osteoporosis --- p.13 / Chapter 1.7.4 --- Tumors --- p.14 / Chapter 1.7.4.1 --- Breast Cancer --- p.14 / Chapter 1.7.4.2 --- Prostate Cancer --- p.15 / Chapter 1.7.5 --- Alcohol Addiction --- p.16 / Chapter 1.7.6 --- Potential Adverse Effects --- p.16 / Chapter 1.8 --- Summary --- p.17 / Chapter Chapter 2 --- Determination of Isoflavones in Soybean and Gegen --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.1.1 --- Classification and Structure of Phytoestrogens --- p.19 / Chapter 2.1.2 --- Isoflavones in Soybeans and Gegen --- p.21 / Chapter 2.1.3 --- Methods of Determination --- p.26 / Chapter 2.1.3.1 --- Isolation and Purification of Isoflavones --- p.26 / Chapter 2.1.3.2 --- Analytical Methods for Isoflavones in Soybeans and Gegen --- p.26 / Chapter 2.2 --- Objective --- p.28 / Chapter 2.3 --- Materials and Methods --- p.29 / Chapter 2.3.1 --- Extraction and Isolation of Soybean and Gegen Isoflavone Extracts --- p.29 / Chapter 2.3.2 --- HPLC Analysis --- p.29 / Chapter 2.3.2.1 --- Sample Preparation for the HPLC Analysis --- p.29 / Chapter 2.3.2.2 --- HPLC Analysis --- p.30 / Chapter 2.3.3.3 --- Qualitative Analysis of the Isoflavones and Their Glycosides in Soybean and Gegen --- p.30 / Chapter 2.4 --- Results --- p.31 / Chapter 2.4.1 --- Isoflavone Identification of Soybean Extract --- p.31 / Chapter 2.4.2 --- Isoflavone Identification of Gegen Extract --- p.33 / Chapter 2.5 --- Discussion --- p.35 / Chapter Chapter 3 --- Hypocholesterolemic Effects of Soybean and Gegen Isoflavone Extracts in Ovariectomized，Intact Male and Castrated Golden Syrian Hamsters --- p.38 / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2 --- Objective --- p.41 / Chapter 3.3 --- Materials and Methods --- p.42 / Chapter 3.3.1 --- Preparation of Soybean and Gegen Isoflavone Extracts --- p.42 / Chapter 3.3.2 --- Animals and Diets --- p.42 / Chapter 3.3.3 --- Serum Lipid and Lipoprotein Determinations --- p.45 / Chapter 3.3.4 --- Determination of Cholesterol Concentration in the Organs --- p.45 / Chapter 3.3.5 --- Statistics --- p.46 / Chapter 3.4 --- Results --- p.48 / Chapter 3.4.1 --- Food Intake and Body and Relative Organ Weights --- p.48 / Chapter 3.4.2 --- Effects of Soybean and Gegen Isoflavone Extracts on Serum and Organ Cholesterol in Ovariectomized and Intact Male and Castrated Hamsters --- p.56 / Chapter 3.5 --- Discussion --- p.61 / Chapter Chapter 4 --- Possible Developmental and Reproductive Toxicity of Soybean Isoflavones on SD Rats --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Objective --- p.70 / Chapter 4.3 --- Materials and Methods --- p.71 / Chapter 4.3.1 --- Diet --- p.71 / Chapter 4.3.2 --- Animals --- p.73 / Chapter 4.3.3 --- Study Design --- p.73 / Chapter 4.3.4 --- Measurement of Reproductive Hormones --- p.74 / Chapter 4.3.5 --- Measurement of Sperm Number --- p.74 / Chapter 4.3.6 --- Statistics --- p.75 / Chapter 4.4 --- Results --- p.77 / Chapter 4.4.1. --- Food Intake and Food Efficiency Ratio --- p.77 / Chapter 4.4.2 --- Growth Trend --- p.79 / Chapter 4.4.3 --- Organ Weight --- p.82 / Chapter 4.4.3.1 --- Absolute Organ Weight --- p.82 / Chapter 4.4.3.2 --- Relative Organ Weight --- p.84 / Chapter 4.4.4 --- Reproductive Hormone Levels --- p.86 / Chapter 4.4.5 --- Epididymis Parameters of Male Rats --- p.88 / Chapter 4.5 --- Discussion --- p.90 / Chapter Chapter 5 --- Possible Developmental and Reproductive Toxicity of Gegen Isoflavones on SD Rats --- p.97 / Chapter 5.1 --- Introduction --- p.97 / Chapter 5.2 --- Objective --- p.99 / Chapter 5.3 --- Materials and Methods --- p.100 / Chapter 5.3.1 --- Animals and Diets --- p.100 / Chapter 5.3.2 --- Study Design --- p.100 / Chapter 5.3.3 --- Statistics --- p.101 / Chapter 5.4 --- Results --- p.103 / Chapter 5.4.1 --- Food Consumption and Food Efficiency Ratio --- p.103 / Chapter 5.4.2 --- Growth Trend --- p.105 / Chapter 5.4.3 --- Organ Weights --- p.108 / Chapter 5.4.3.1 --- Absolute Organ Weights --- p.108 / Chapter 5.4.3.2 --- Relative Organ Weight --- p.110 / Chapter 5.4.4 --- Reproductive Hormone Levels --- p.112 / Chapter 5.4.5 --- Epididymis Parameters of Male Rats --- p.114 / Chapter 5.5 --- Discussion --- p.116 / Chapter Chapter 6 --- Conclusions --- p.121 / References --- p.123

La nécrose de l'écorce (Trunk Phloem Necrosis : TPN) est la maladie de l'hévéa économiquement la plus importante or, à l'heure actuelle, son origine et les causes de son apparition sont mal connues. Cette étude vise à préciser les relations entre les activités biologiques du sol et la présence de TPN. Une première étude a été menée sur une parcelle d'hévéa très infestée par la TPN. L'analyse de la faune du sol montre que la présence de termites est fortement liée aux arbres malades. A l'inverse, les polysaccharidases sont moins actives sous les arbres affectés par la TPN alors que les activités liées au cycle de l'azote y sont plus élevées. La structure des communautés fongiques est différente dans les sols sous les arbres sains de sous les arbres malades. Une seconde étude s'est intéressée à l'influence de deux amendements, apport de matière organique seule et couverture de Pueraria + M.O., sur le fonctionnement biologique du sol. L'apport de M.O. entraîne une augmentation de la croissance des arbres et de la production de latex et préviennent aussi l'apparition de TPN. Dans ces sols, la densité de macrofaune est significativement plus faible dans les parcelles sans amendement mais la diversité y est comparable. L'ACP réalisée à partir des activités enzymatiques montre que la plupart des enzymes présentent des activités plus faibles dans les sols non amendés. Les sols des parcelles avec Pueraria ont les plus fortes xylanases alors que l'activité FDase est plus élevée dans les sols des parcelles n'ayant reçu que l'apport organique.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12807号 / 論農博第2792号 / 新制||農||1020(附属図書館) / 学位論文||H26||N4810(農学部図書室) / 80851 / (主査)教授 河田 照雄, 教授 金本 龍平, 教授 谷 史人 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Un dispositif agronomique pérennisé entre 1995 et 1999 avec des systèmes de culture de maïs à couvertures végétales a été installé dans la zone écologique à forêt semi-décidue de Côte d'Ivoire. L'offre en N d'origine organique est étudiée dans le cadre de systèmes de culture de maïs, avec une durée de jachère de 6 mois (SC6MJ) et 18 mois (SC18MJ). Les jachères sont occupées soit par Chromolaena odorata (subspontanée), soit par Pueraria phaseoloides (introduite). Ces quatre systèmes sont comparés à deux témoins, un sans Chromolaena pour les SC6MJ et un avec brûlis de Chromolaena pour les SC18MJ. Les systèmes à couvertures végétales sont gérés en couvertures vivantes avec des herbicides et leur pérennité est assurée par une reprise de croissance des espèces avant la récolte de la céréale. Les systèmes à SC6MJ bénéficient d'une fertilisation modérée en phosphore. L'étude réalisée pose comme hypothèses de départ que (i) les déterminants de l'offre à court terme (cycle cultural) et à moyen terme (effets cumulés) dépendront principalement des quantités et de la qualité des litières de surface d'origine des jachères (ii) ces effets se concentreront à la surface du sol. Le potentiel de minéralisation des litières au laboratoire est corrélé avec leur rapport C/N, plus faible pour Pueraria (C/N entre 20 à 23) que pour Chromolaena (C/N entre 25 à 43). En 1998 la vitesse de disparition des litières suivie avec des " sacs de décomposition " est beaucoup plus rapide qu'en 1999 en relation au début du cycle de décomposition avec le régime pluviométrique. Les deux années la quantité de litières présentes après la coupe détermine le taux de décomposition à moyen terme des litières. Ces dynamiques, modélisées sur deux années, permettent de distinguer un compartiment de litières à disparition rapide et un compartiment à disparition lente. La comparaison des pertes en C dans des " sacs de décomposition " à maille différente montre que l'activité de la mésofaune et de la macrofaune du sol pourrait être responsable de la disparition de 50 % des litières au début du processus de décomposition et de 35% à la fin du processus de décomposition. Leur action de fragmentation et d'incorporation au sol des litières (meules des termites champignonnistes situées à la surface du sol) permet une disparition comparativement plus rapide des litières de Chromolaena que celles de Pueraria. L'application des modèles aux données obtenues sur les mobilisations en N des jachères en relation avec les besoins de la culture en N indique (i) un risque " d'asynchronie " en début de cycle (ii) une offre insuffisante en fin de cycle pour les SC6MJ (iii) peu de différenciation entre l'offre de Chromolaena et celle de Pueraria. L'offre en N in situ du sol en surface (0-10 cm) en 1998 et 1999, estimée pendant des périodes de 90 jours durant le cycle du maïs par des mesures et des incubations de sol, est corrélée pour les SC6MJ avec les mobilisations en N du maïs dans les parties aériennes. C'est le traitement avec Pueraria qui les deux années satisfait le mieux les besoins en N de la culture et cette offre est comparable à celle des SC18MJ. L'offre en N varie peu selon les traitements des SC18MJ. Le régime pluviométrique intervient (i) à une échelle pluriannuelle, en déterminant, l'intensité du pic de minéralisation en N minéral du sol et les accumulations en N dans les parties aériennes des jachères (ii) à l'échelle de la campagne en interaction avec les litières en surface et l'activité des systèmes racinaires des couvertures végétales. La litière de Pueraria semble mieux conserver l'humidité du sol et le système racinaire de Chromolaena mobiliser de plus fortes quantités d'eau. En situations hydriques limitantes, la minéralisation nette est corrélée aux humidités de sol. Les litières semblent être à l'origine en 1998 de l'augmentation de la biomasse microbienne du sol en surface créant ainsi une source potentielle d'immobilisation temporaire de N. En 1998 l'offre en N estimée par minéralisation nette indique une meilleure synchronie avec les besoins de la culture malgré une pluviométrie plus abondante en début de cycle par rapport en 1999. Une étude utilisant l'abondance naturelle 15N permet d'estimer que la litière de Pueraria peut contribuer fortement à la nutrition azotée de la culture, de 30 à 41% en 1998 et de 64 à 87% en 1999 dans le cadre du système de culture à jachère de 6 mois. Les différenciations sur les matières organiques du sol (MOS) indiquent un début d'effet sur les stocks évalués sur 0-10 cm en moyenne à 2.34 t ha-1 N pour les SC6MJ et en moyenne à 2.83 t ha-1 N pour les SC18MJ. Pour les SC6MJ la différence obtenue entre les apports et les pertes sur quatre années suggère que Chromolaena conserve mieux les stocks en C et N que Pueraria. La comparaison avec une jachère forestière située hors dispositif indique que la disponibilité en matériaux facilement minéralisables dans nos agrosystèmes est (i) réduite en fin de saison des pluies (ii) diminuée par la pratique du brûlis (iii) peu augmentée par la pratique de la jachère. Les études sur des incubations de longue durée (106 et 85 jours) montrent des effets significatifs sur les modélisations faites par ajustement aux points expérimentaux sur les cinétiques de respiration et de minéralisation nette cumulées (exprimées en proportion par rapport au C et N total du sol). Elles permettent d'identifier d'importants facteurs de différenciation au niveau du compartiment des MOS à taux de renouvellement élevé. Pour les SC6JM ce compartiment représente une part plus importante avec Pueraria, certainement en relation directe avec des apports cumulés de plus grande qualité biochimique (rapport lignine / N). La durée de la jachère n'améliore pas la disponibilité en N facilement minéralisable indiquant ainsi un blocage de C et N au niveau du sol de nature physique et/ou biochimique.

Cheung, Wing Shing David. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 201-217). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Chan Yin Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 147-167). / Abstracts in English and Chinese. / ABSTRACT --- p.III / ACKNOWLEDGEMENT --- p.X / TABLE OF CONTENTS --- p.XI / ABBREVIATIONS --- p.XV / LIST OF FIGURES --- p.XVII / LIST OF TABLES --- p.XXI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Introduction to Cardiovascular Disease and Atherosclerosis --- p.1 / Chapter 1.1.1 --- Cardiovascular Disease --- p.1 / Chapter 1.1.2 --- A therosclerosis --- p.3 / Chapter 1.1.2.1 --- Structure of Arteries --- p.4 / Chapter 1.1.2.2 --- Pathophysiology of Atherosclerosis --- p.5 / Chapter 1.1.2.3 --- Endothelial Dysfunction --- p.8 / Chapter 1.1.3 --- Current Western Therapies --- p.11 / Chapter 1.1.3.1 --- Surgery --- p.11 / Chapter 1.1.3.2 --- Western Medications --- p.13 / Chapter 1.1.4 --- Traditional Chinese Medicine --- p.17 / Chapter 1.1.4.1 --- Long History --- p.17 / Chapter 1.1.4.2 --- As Alternative Medicine --- p.18 / Chapter 1.1.4.3 --- Modernization of Chinese Medicine --- p.19 / Chapter 1.2 --- Introduction and Selection of Chinese Medicine --- p.20 / Chapter 1.2.1 --- Selection ofTCM Formulation from Pharmacopoeia --- p.20 / Chapter 1.2.1.1 --- Compound Formulation --- p.20 / Chapter 1.2.2 --- Introduction to the Herbal Medicines --- p.21 / Chapter 1.2.2.1 --- Danshen (Salvia miltiorrhiza) --- p.21 / Chapter 1.2.2.2 --- Gegen (Puerariae thomsonii and Puerariae lobata) --- p.22 / Chapter 1.2.2.3 --- Yanhu (Corydalis yanhusuo) and its Exclusion --- p.24 / Chapter 1.2.3 --- Source and Authentication of the Herbal Medicines --- p.25 / Chapter CHAPTER 2 --- OPTIMIZATION OF DANSHEN-GEGEN FORMULA --- p.26 / Chapter 2.1 --- Project History --- p.26 / Chapter 2.2 --- aims for the present study --- p.27 / Chapter 2.3 --- Methods and Materials --- p.30 / Chapter 2.3.1 --- Extracts --- p.30 / Chapter 2.3.2 --- Extraction Process --- p.31 / Chapter 2.3.3 --- In vitro Antioxidation Model --- p.33 / Chapter 2.3.4 --- Ex vivo Vasodilation Model --- p.35 / Chapter 2.3.5 --- Statistical Analysis --- p.38 / Chapter 2.4 --- Results --- p.39 / Chapter 2.4.1 --- Vasodilation Results --- p.39 / Chapter 2.4.2 --- Antioxidation Results --- p.43 / Chapter 2.5 --- Discussion --- p.46 / Chapter 2.6 --- Further Modification of the Formula --- p.49 / Chapter 2.6.1 --- Extracts --- p.49 / Chapter 2.6.2 --- Results --- p.49 / Chapter 2.7 --- discussion --- p.52 / Chapter CHAPTER 3 --- MARKER CHEMICAL CONTENTS OF HERBAL EXTRACTS AND THEIR PHARMACOLOGICAL PROPERTIES --- p.56 / Chapter 3.1 --- HPLC Analysis of Marker Contents --- p.56 / Chapter 3.1.1 --- Methods --- p.57 / Chapter 3.1.2 --- Results --- p.58 / Chapter 3.1.2.1 --- HPLC Chromatograms --- p.59 / Chapter 3.1.2.2 --- Content Percentage of Marker Compounds --- p.63 / Chapter 3.1.3 --- Discussion --- p.64 / Chapter 3.2 --- Studies on Marker Compounds --- p.65 / Chapter 3.2.1 --- Introduction --- p.65 / Chapter 3.2.2 --- Methods and Materials --- p.67 / Chapter 3.2.2.1 --- Source of Pure Compounds --- p.67 / Chapter 3.2.2.2 --- Purification and Identification of SAB --- p.68 / Chapter 3.2.2.3 --- Vasodilation model --- p.70 / Chapter 3.2.2.4 --- Antioxidation Model --- p.71 / Chapter 3.2.2.5 --- Structures of Pure Compounds --- p.72 / Chapter 3.2.3 --- Results --- p.73 / Chapter 3.2.3.1 --- Vasodilation Results --- p.73 / Chapter 3.2.3.2 --- Antioxidation Results --- p.76 / Chapter 3.3 --- Discussion --- p.79 / Chapter 3.4 --- Synergistic Effect Study --- p.85 / Chapter 3.4.1 --- Introduction --- p.85 / Chapter 3.4.2 --- Methods --- p.85 / Chapter 3.4.3 --- Results --- p.86 / Chapter 3.4.4 --- Discussion --- p.88 / Chapter 3.5 --- STUDY ON 3'-HYDROXYPlIERARIN AND 3'-METHOXYPUERARIN PURIFIED FROM YFGE --- p.90 / Chapter 3.5.1 --- 3 '-hydroxypuerarin and 3'-methoxypuerarin --- p.90 / Chapter 3.5.2 --- Methods and Materials --- p.91 / Chapter 3.5.2.1 --- Purification by HPLC semi-preparation --- p.91 / Chapter 3.5.2.2 --- Bioassays --- p.93 / Chapter 3.5.3 --- Results --- p.94 / Chapter 3.5.3.1 --- Vasodilation Study --- p.94 / Chapter 3.5.3.2 --- Antioxidative Effect of Yege --- p.95 / Chapter 3.5.4 --- Discussion / Chapter CHAPTER 4 --- MECHANISTIC STUDY --- p.98 / Chapter 4.1 --- Introduction --- p.98 / Chapter 4.1.1 --- Nitric Oxide-mediated Vasodilation --- p.99 / Chapter 4.1.2 --- Prostacyclin-mediated Vasodilation --- p.100 / Chapter 4.1.3 --- EDHF-mediated Vasodilation --- p.101 / Chapter 4.1.4 --- Endothelium-dependent and -independent Vasodilations --- p.103 / Chapter 4.2 --- Methods and Materials --- p.104 / Chapter 4.3 --- Results --- p.107 / Chapter 4.3.1 --- Danshen-Gegen Formula (DY80) --- p.107 / Chapter 4.3.2 --- Salvianolic acid B --- p.112 / Chapter 4.3.3 --- Daidzein --- p.117 / Chapter 4.4 --- Discussion --- p.121 / Chapter CHAPTER 5 --- STUDY ON LIPID PEROXIDATION AND UPTAKE BY MACROPHAGES --- p.128 / Chapter 5.1 --- Study of DY 80 and SAB on Copper-ion induced Low Density Lipoprotein Oxidation --- p.128 / Chapter 5.1.1 --- Pathologic Role of oxidized Low Density Lipoprotein --- p.128 / Chapter 5.1.2 --- Antioxidants in Low Density Lipoprotein and Role of Transition Metals --- p.129 / Chapter 5.1.3 --- Methods and Materials --- p.130 / Chapter 5.1.4 --- Results --- p.131 / Chapter 5.1.5 --- Discussion --- p.133 / Chapter 5.2 --- Study of Scavenger Receptor Regulation in Macrophages --- p.135 / Chapter 5.2.1 --- Introduction --- p.135 / Chapter 5.2.2 --- Methods and Materials --- p.136 / Chapter 5.2.3 --- Results --- p.139 / Chapter 5.2.4 --- Discussions --- p.140 / Chapter CHAPTER 6 --- General Discussion --- p.143 / REFERENCES --- p.147

心臟病目前仍然是最普遍的威脅人類生命安全的三大病因之一。同西藥相比, 傳統中醫藥具有多靶點，協同作用及小副作用等特性。在中藥歷史中， 丹參和葛根這兩種草藥經常出現在中藥方劑用於治療心血管相關的疾病，已有幾千年的歷史。 我們實驗室發現了一個丹參葛根湯劑具有保護動脈粥樣硬化病人心臟功能的作用，並且可以使收縮的大鼠大動脈舒張的作用。 本研究主要通過舒張豬冠狀動脈，提高大鼠對抗過氧化和離子擾動能力以及提高血管增生四個方面探討丹參葛根複方水提物 (質量比7:3) (DG配方)對血管的作用以提供其治療心血管疾病的藥理基礎。 / 在本研究的第一部， 我們主要探討了DG配方對缺血再灌注損傷的心臟及其心肌細胞的保護作用。我們發現DG配方明顯抑制了心臟損傷相關的肌酸激酶和乳酸脫氫的釋放。同時DG配方顯著促進了再灌注後冠狀動脈內血流量速度和收縮力度的恢復。這些結果說明DG配方可以保護缺血再灌注心臟並且有效促進其功能恢復。我們還觀察了長期給大鼠用DG配方14天後其心臟在缺血再灌注中的表現。類似於再灌注時給藥的結果，DG配方同樣抑制了損傷酶的釋放並且有效促進了冠狀動脈內血流量速度和收縮力度的恢復。 / 同時，在缺氧再灌注離體細胞模型中，我們發現DG配方明顯抑制了缺氧再灌注損傷帶來的細胞死亡。流式細胞儀分析結果表明，藥物處理組中的凋亡類的細胞明顯比對照組中少主要通過抑制促凋亡的caspase3表達明以及促進抗凋亡的Bcl2表達升高。DG配方減少了心肌細胞內細胞色素c從線粒體中釋放明顯以及抑制了線粒體去極化。這說明DG配方也保護了線粒體的膜的完整性，從而確保線粒體功能進而保證細胞的能量系統穩定。最有意思的是DG配方可以直接抑制缺氧再灌注相關的兩條通路, 它不僅抑制活性氧化物質的釋放, 同時也抑制了再灌注後鈣離子的累積。總之，DG配方以抗氧化和抗離子擾動的方式保護了在缺血缺氧再灌注損傷中心臟和心肌細胞的結構和功能。 / 第二部分的研究是關於DG配方對從豬心臟上分離的左冠狀動脈前室間支 (左前降支) 血管的作用及其內在的機制，我們的結果表明對由U46619引起的冠狀動脈血管收縮DG配方表現了濃度依賴的舒血管作用。而該作用並非依賴于內皮細胞及其釋放的舒張血管因數一氧化氮和前列腺素類似物環素和大部分的鉀離子通道。其中只有內向整合鉀離子通道部分參與了舒血管的過程。肌球蛋白輕鏈的磷酸化明顯被DG配方抑制，但是RhoA 的活性並沒有受其影響。鈣離子引發的血管收縮則被DG配方濃度依賴性的受到抑制。這部分的研究證明瞭DG配方主要通過類似鈣離子通道拮抗劑作用抑制鈣離子進入到血管平滑肌細胞減少肌球蛋白磷酸化達到舒張血管的作用。結果說明DG配方可以作為一種安全的藥物用於治療心血管疾病特別是高血壓和心絞痛。 / 本研究的第三部分是關於DG配方的促血管增生的作用。我們發現DG配方可以明顯促進斑馬魚的腸下動脈的出芽並且促進血管增生相關基因的表達，血管內皮細胞生長因數及其受體的mRNA表達。內皮細胞是血管增生的基礎。所以我們利用人源微血管內皮細胞檢測了DG配方在細胞的增生，遷移，分化和形成血管方面的影響以解釋它在斑馬魚中促進血管增生的作用機理。結果發現，DG配方明顯促進了該種內皮細胞的增殖，遷移和形成管狀結構。 / 綜上所述，DG配方可以通過舒張血管，抗氧化，抗離子紊亂和促進血管增生提供心血管保護功能。DG配方通過螯合活性氧化物質和抑制鈣離子的累積保護了因缺血再灌注引起的心臟損傷，說明DG配方可以作為手術的輔助藥物減少心臟病人在缺血再灌注過程中受到的損傷。它以拮抗L型鈣離子通道方式減少鈣離子進入到血管平滑肌細胞來舒張收縮的冠狀動脈血管。說明DG配方可以用於治療高血壓和心絞痛等心臟病。另外DG配方也可以促進血管增生，可用于心肌梗死病人促進其心臟血管系統重建，本研究對於未來臨床實驗具有重要的參考價值。 / Coronary heart diseases (CHD) are one of the most prevalent causes of premature death all over the world. In contrast to western medicine, traditional Chinese medicine (TCM) has shown the benefit of multi-targeting and synergism to treat CHD. Two kinds of Chinese herbs, Danshen (Radix Salviae Miltiorrhiza) (D) and Gegen (Radix Puerariae Lobatae) (G) always present on the TCM formula for treating heart disease. We found a useful formula of Danshen and Gegen decoction with weight ratio of 7:3 (DG) exerting properties of improving the heart function in patient with atheroslcerosis and providing vasodiation and antioxidant protection on the rat cardiovascular system. The present study was designed to evaluate the effects of DG on the vascular activity by its properties on antioxidant and anti-ion stunning to inhibiting the ischemia and reperfusion injury, vasodilation effect on pig coronary artery and angiogenesis effect on zebrafish model. / In the first part of the study, we explored protective effect of DG on rat hearts and cardiomyocytes after ischemia-reperfusion and hypoxia-reoxygenation injury. Comparing to control group, the release of creatine kinase (CK) and lactate dehydrogenase (LDH) significantly decreased in the DG treated groups in a dose-dependent manner. The recovery percentage of coronary flow and contractile force in the DG was higher than that in the control group. These results suggested that DG dose-dependently improved the heart function after ischemia and reperfusion injury in a dose-dependent manner. We also examined chronic effect of DG (14 days pretreatment) on rat heart with ischemia and reperfusion injury. DG induced rat heart with high potential to deal with I/R injury, less damaged enzymes release and high recovery percentage of heart function recovery. / In the cell hypoxia and reoxygenation model, DG significantly inhibited the cell death after H/R treatment with bcl2 expression increase and caspase3 expression decrease. DG also reversed the H/R-induced mitochondrial depolarization and inhibited cytochrome c diffusing out of mitochondria, which confirmed DG anti-apoptosis activity. DG also was found to significantly decrease the intracellular calcium accumulation and reactive oxygen species release within H9c2. / In the second part of present study, results revealed that DG elicited a concentration-dependent relaxation of U46619-preconstricted porcine coronary artery. DG-induced relaxation responses were not altered by the presence of endothelium-related dilator inhibitors, most potassium channel blockers, GMP and AMP pathway inhibitors and endothelium removal. Ba²⁺ (an inward rectifier K⁺ channel blocker) slightly attenuated DG-induced relaxation. The protein expression of phosphorylated myosin light chain (MLC) was inhibited by DG in a concentration-dependent manner whereas the activity of RhoA was not modified. Ca²⁺-induced contraction of coronary artery was inhibited by DG in a concentration-dependent fashion. DG acted as an antagonist of calcium channel inducing the porcine artery dilation. / The third part of the present study is about the pro-angiogenic effect of DG. We found that DG dose-dependently induced zebrafish sub-intestinal vessel sprouting and increased the mRNA expression of vascular endothelial growth factor (VEGF) and its receptors. To explore the underlying mechanism, we also examined the proangiogenic effect of DG on the angiogenesis of endothelial cells. The results showed that DG induced the HMEC-1 proliferation, migration and forming tube. / In conclusion, we found that DG could provide cardiac and cardiovascular protection by its multiple targets. It prevented heart injuries after ischemia or hypoxia and reperfusion through scavenging ROS and inhibiting calcium accumulation. Moreover, it mainly acts as an antagonist of L-type calcium channel to relax the contracted LAD vessel. It also exerted property of inducing angiogenesis in vivo and in vitro. Therefore, DG would be useful for treating coronary artery disease depending on its multiple targets. / 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. / Hu, Fan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 170-215). / 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 --- Intorduction --- p.1 / Chapter 1.1 --- Cardiovascular system and coronary artery diseases --- p.1 / Chapter 1.1.1 --- The cardiovascular system --- p.1 / Chapter 1.1.2 --- Contraction and relaxation of the vascular myocyte in arteries --- p.4 / Chapter 1.1.2.1 --- Ultrastructure of the vascular myocyte --- p.4 / Chapter 1.1.2.2 --- Contraction mechanisms of vascular myocyte --- p.5 / Chapter 1.1.2.3 --- Relaxation mechanisms of vascular myocyte --- p.7 / Chapter 1.1.3 --- Chronic coronary heart disease --- p.9 / Chapter 1.2 --- The way to treat chronic CAD --- p.11 / Chapter 1.2.1 --- Angiogenesis --- p.11 / Chapter 1.2.2 --- Clinical surgery for treating CAD --- p.13 / Chapter 1.2.2.1 --- Three common surgeries for treating CAD --- p.13 / Chapter 1.2.2.2 --- Ischemia and reperfusion (I/R) injury in surgeries --- p.15 / Chapter 1.2.3 --- Drugs for treating CAD --- p.19 / Chapter 1.2.3.1 --- Western medicine therapy in CAD --- p.19 / Chapter 1.2.3.2 --- Traditional Chinese Medicine treatment in CAD --- p.20 / Chapter 1.3 --- Aims of studies --- p.28 / Chapter 2 --- Materials and Methods --- p.29 / Chapter 2.1 --- Solutions and Materials --- p.29 / Chapter 2.1.1 --- Solutions --- p.29 / Chapter 2.1.2 --- Chemicals and enzymes --- p.36 / Chapter 2.2 --- Methods --- p.38 / Chapter 2.2.1 --- Herbal preparation --- p.38 / Chapter 2.2.2 --- Identification and quantification of chemical markers in Danshen and Gegen decoction (DG) --- p.38 / Chapter 2.2.3 --- Assay development for the determination of the DG marker compounds in rat plasma --- p.40 / Chapter 2.2.4 --- Isolation of pig left anterior descending coronary artery --- p.44 / Chapter 2.2.5 --- Isometric tension measurement --- p.45 / Chapter 2.2.6 --- Langendorff related experiment --- p.50 / Chapter 2.2.7 --- Cell culture of H9c2 cells --- p.54 / Chapter 2.2.8 --- Cell viability assay (MTT assay) --- p.56 / Chapter 2.2.9 --- Cell proliferation measurement --- p.57 / Chapter 2.2.10 --- Hypoxia and reperfusion cell model (H9c2) --- p.58 / Chapter 2.2.11 --- Determination of cell apoptosis with Annexin VFITC and PI double staining --- p.59 / Chapter 2.2.12 --- Measurement of mitochondria depolarization --- p.61 / Chapter 2.2.13 --- Measurement of ROS release --- p.63 / Chapter 2.2.14 --- Measurement of calcium localization in H9c2 cells by fluo4 dye and confocal microscopy --- p.64 / Chapter 2.2.15 --- Extraction of proteins from tissue, cell and subcellular fractions --- p.65 / Chapter 2.2.16 --- Western blot assay --- p.67 / Chapter 2.2.17 --- Human microvascular endothelial cells (HMEC1) cell culture --- p.68 / Chapter 2.2.18 --- Cell cycle analysis by PI staining --- p.69 / Chapter 2.2.19 --- Scratch assay for HMEC1cells migration --- p.70 / Chapter 2.2.20 --- Tube formation assay --- p.71 / Chapter 2.2.21 --- Vessel sprouting of Zebrafish --- p.72 / Chapter 2.2.22 --- Real time PCR --- p.74 / Chapter 2.2.23 --- Statistical analysis --- p.76 / Chapter 3 --- Chapter 3 Cardiac protection of Danshen and Gegen decoction in hypoxia/ischemia and reperfusion induced injury --- p.77 / Chapter 3.1 --- Introduction --- p.77 / Chapter 3.2 --- Results --- p.81 / Chapter 3.2.1 --- Cytotoxicity of DG --- p.81 / Chapter 3.2.2 --- The morphology alteration of H9c2 after H/R treatment --- p.83 / Chapter 3.2.3 --- Effect on H H9c2 cell survival after H/R treatment --- p.84 / Chapter 3.2.4 --- Effect on membrane skeleton of H9c2 cells with H/R injury --- p.86 / Chapter 3.2.5 --- Effect on the apoptosis in H9c2 cells induced by H/R injury --- p.88 / Chapter 3.2.6 --- Effect on cytochrome c release from mitochondria of damaged H9c2 cells --- p.92 / Chapter 3.2.7 --- Effect on mitochondria depolarization of H9c2 after H/R treatment --- p.94 / Chapter 3.2.8 --- Effect on reactive oxidant species (ROS) release --- p.96 / Chapter 3.2.9 --- Effect on calcium accumulation within H9c2 in the reperfusion phase --- p.98 / Chapter 3.2.10 --- Effect on heart functions of rat hearts with I/R injury (acute effect) --- p.101 / Chapter 3.2.11 --- Effect on heart function in rats with I/R injury (chronic effect) --- p.107 / Chapter 3.3 --- Discussion --- p.113 / Chapter 4 --- Chapter 4 Vasodilation effects of Danshen and Gegen decoction in porcine coronary artery and its underlying mechanism --- p.118 / Chapter 4.1 --- Introduction --- p.118 / Chapter 4.2 --- Results --- p.121 / Chapter 4.2.1 --- Investigations of endothelium dependent and independent mechanisms --- p.121 / Chapter 4.2.2 --- Effects on cAMP and cGMP pathway --- p.121 / Chapter 4.2.3 --- Effects on potassium channel opening --- p.121 / Chapter 4.2.4 --- Effects on calcium induced contraction and calcium sensitization --- p.122 / Chapter 4.2.5 --- Effects on MLC phosphorylations --- p.123 / Chapter 4.3 --- Discussion --- p.132 / Chapter 5 --- Chapter 5 In vitro and in vivo angiogenic effects of DG --- p.138 / Chapter 5.1 --- Introduction --- p.138 / Chapter 5.2 --- Results --- p.140 / Chapter 5.2.1 --- Effect on subintestinal vessels sprouting in the zebrafish embryo --- p.140 / Chapter 5.2.2 --- Effect on the transcription and expression of VEGFA and VEGF receptors -- Flt1 and KDR/Flk2 --- p.143 / Chapter 5.2.3 --- Effect on HMEC1 proliferation --- p.145 / Chapter 5.2.4 --- Effect on cell cycle of HMEC1 --- p.148 / Chapter 5.2.5 --- Effect on cell migration of HMEC1 --- p.151 / Chapter 5.2.6 --- Effect on tube formation of HMEC1 --- p.154 / Chapter 5.3 --- Discussion --- p.157 / Chapter 6 --- Chapter 6 Conclusions and future work --- p.160 / Chapter 6.1 --- Cardiac protection of DG in the I/R and H/R injury --- p.160 / Chapter 6.2 --- Vasodilation effect of DG on the porcine coronary artery --- p.165 / Chapter 6.3 --- Angiogenic effect of DG in vivo and in vitro --- p.167 / Chapter 6.4 --- Overall conclusion of the study --- p.169 / Chapter 7 --- References --- p.170