Leung Lai-Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 110-113). / Abstracts in English and Chinese. / Abstract English --- p.i / Chinese --- p.iii / Acknowledgments --- p.v / Table of contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Establishment of compound formulation --- p.4 / Chapter 2.1 --- Formulation research --- p.4 / Chapter 2.2 --- Aqueous extract preparation --- p.6 / Chapter 2.2.1 --- Materials and Methods --- p.6 / Chapter 2.2.2 --- Results --- p.7 / Chapter 2.2.3 --- Discussion --- p.9 / Chapter 2.3 --- Preliminary test --- p.10 / Chapter 2.3.1 --- Materials and Methods --- p.10 / Chapter 2.3.2 --- Results --- p.12 / Chapter 2.3.3 --- Discussion --- p.14 / Chapter Chapter 3 --- Quality Control --- p.15 / Chapter 3.1 --- HPLC standardization --- p.16 / Chapter 3.2 --- Materials and Methods --- p.19 / Chapter 3.3 --- Results --- p.20 / Chapter 3.4 --- Discussion --- p.28 / Chapter Chapter 4 --- Antioxidant study --- p.29 / Chapter 4.1 --- Red blood cell hemolysis model --- p.30 / Chapter 4.1.1 --- Materials and Methods --- p.30 / Chapter 4.1.2 --- Results --- p.30 / Chapter 4.1.3 --- Discussion --- p.32 / Chapter 4.2 --- Ischemia-Reperfusion on Langendorff --- p.33 / Chapter 4.2.1 --- Materials and Methods --- p.34 / Chapter 4.2.2 --- Results --- p.37 / Chapter 4.2.3 --- Discussion --- p.60 / Chapter Chapter 5 --- Vasodilation study --- p.61 / Chapter 5.1 --- Vasodilation in organ bath --- p.63 / Chapter 5.1.1 --- Materials and Methods --- p.63 / Chapter 5.1.2 --- Results --- p.65 / Chapter 5.1.3 --- Discussion --- p.79 / Chapter 5.2 --- Endothelium dependent vasodilation --- p.80 / Chapter 5.2.1 --- Materials and Methods --- p.80 / Chapter 5.2.2 --- Results --- p.83 / Chapter 5.2.3 --- Discussion --- p.95 / Chapter Chapter 6 --- Anti-platelet study --- p.96 / Chapter 6.1 --- CFU-MK plasma clot colony assay --- p.97 / Chapter 6.2 --- Materials and Methods --- p.97 / Chapter 6.3 --- Results --- p.100 / Chapter 6.4 --- Discussion --- p.103 / Chapter Chapter 7 --- Discussions and prospects --- p.104 / Chapter 7.1 --- Discussions --- p.104 / Chapter 7.2 --- Prospects --- p.107 / Chapter 7.2.1 --- TCM capsule with GMP --- p.107 / Chapter 7.2.2 --- Clinical Trial of the capsule --- p.109 / References --- p.110

影響全球數百萬的患者的慢性傷口，以其持續性過度發炎，纖維細胞增殖放緩，及血管生成受損為表徵。藥用草藥地黃已證明在大鼠糖尿足模型上有顯著傷口癒合作用。然而，關於地黃的炮製及其活性成分對此等傷口癒合的活動主要是未知的。 / 我們首先在地黃的炮製中，以抗一氧化氮（NO）和纖維細胞增殖實驗，確定了乾地黃表現出有效的傷口癒合活動。採用多方位生物活性導引分離（BGF），我們進一步研究乾地黃在抗炎，纖維細胞增殖，血管生成的活性成分，分別以抗NO產生，纖維細胞增殖，和TG(fli1:EGFP)y1/+(AB)斑馬魚芽血管生成模型為生物測定。此等具傷口癒合效果的活性成分將會作進一步研究。此外，我們會以電子細胞基質阻抗判斷(ECIS)的技術，對名為NF3(含RR的中草藥配方)在人類血管內皮細胞(HECV)上作體外血管生成及其信息的研究。 / 通過抗NO測定導引分離，我們證明活性萃取物C3比其粗提物擁有100倍更有效的抗炎效果。C3中含地黃苦甙元。地黃苦甙元可顯著地抑制NO的產生。C3中地黃苦甙元的存在可能是其具抗炎的原因。C3進一步以抑制一氧化氮合酶(iNOS)，環氧合酶-2(COX-2)和細胞白介素第六因子(IL-6)的基因，蛋白質，及/或中介物的表達，證明其舒緩炎症的作用。因此，C3可能對慢性傷口癒合中的炎症有治療作用。此外，我們發現先從水提的RR粗提物，再以乙酸乙酯提取的活性萃取物C2-B4，證明此萃取物在纖維細胞增殖中具最有效及劑量依存作用。 / 斑馬魚芽誘導模型導引分離顯示，C1-1萃取物在RR中具有最有效的血管生成作用。為斑馬魚芽誘導度身設計的30個血管生成相關基因顯示，C1-1廣泛地引發血管生成中的基因差異表達，特別在生長因子和血管穩定方面。就促進血管生成，C1-1進一步以體外人類微血管內皮細胞的血管生成檢測進行研究，結果發現C1-1具細胞移動及類血管生成能力。此外，降毛荚醛(norviburtinal)，在地黃提取物中首次被發現和具有血管生成作用。如此，C1-1和降毛荚醛為RR中血管生成作用的活性成分。 / 此外，應用ECIS技術，含RR配方的NF3能通過磷酯肌醇激酶(PI3K)及WiskottAldrich氏症候群神經元蛋白 (N-WASP) 路徑在人類血管內皮細胞（HECV）上誘導內皮細胞粘附，遷移及類血管生成。西方墨點法分析表明，NF3 在HECV上激活Akt和有絲分裂活化蛋白質激酶(MAPKs)的表達。這些誘導在各方面促進血管生成。因此，這顯示NF3能在分子及功能上激活血管生成作用的複雜性。此外，我們進一步支持ECIS在血管內皮細胞篩選傷口癒合劑中的高靈敏度。 / 總括而言，通過靶向抗炎，纖維細胞增殖增長和改善血管生成，各地黃生物活性導引化合物和萃取物，及其含RR的配方，可以在治療慢性傷口癒合上發揮功效。 / Chronic wounds, which influence millions of patients worldwide, are manifested with its sustained hyperinflammation, slackened fibroblast proliferation, and impaired angiogenesis. Agents retrieving these activities could facilitate the healing. Medicinal herb, Rehmanniae Radix (RR) demonstrated profound wound healing effect in rat diabetic foot model. However, the subtypes and the active components behind RR for such wound healing activities were largely unknown. / Here we firstly identified that dried RR, among its subtypes, exhibited potent wound healing activities through nitric oxide (NO) anti-inflammatory and fibroblast proliferation assays. Using multi-directional bioassay-guided fractionation (BGF), we further studied the active component(s) of dried RR in anti-inflammation, fibroblast proliferation, and angiogenesis, respectively, by anti-NO production, fibroblast proliferation, and TG(fli1:EGFP)[superscript y]¹/+(AB) zebrafish sprout angiogenesis model. Active component(s) of such wound healing effects were further characterized. Furthermore, with a RR-containing herbal formula, NF3, the in vitro angiogenic activities and its underlying signaling of NF3-treated human vascular endothelial cells (HECV) were studied using electric cell-substrate impedance sensing (ECIS) technology. / Via anti-NO assay-guided fractionation of dried RR, we demonstrated that the sub-fraction C3, possessed 100-fold more potent anti-inflammatory effect than that of the crude extract. Characterization of C3 showed that the anti-inflammatory activity could be partly due to the presence of rehmapicrogenin, which could significantly inhibit NO production. C3 was further demonstrated in blocking inflammation by inhibiting gene, protein, and/or mediator expression of inducible NO synthase, COX-2 and IL-6. Hence, C3 could be useful in treating inflammation in chronic wound healing. Additionally, we revealed that an active sub-fraction, C2-B4, from the ethyl acetate extract of the aqueous extract of RR, demonstrated the most potent and dose-dependent fibroblast proliferative effect. / Zebrafish sprout-inducing model-guided fractionation suggested C1-1 sub-fraction possessed the most potent angiogenesis effect in RR. A 30 tailor-made angiogenesis-associated gene panel designed for zebrafish sprout angiogenesis revealed that C1-1 triggered differential gene expression across wide angiogenic events, particularly concerned with those of growth factors and vessel stabilization. The pro-angiogenic activity was further supported by in vitro human microvascular endothelial cell-based angiogenesis assays, with C1-1 being pro-motogenic and tubule inducing. Also, norviburtinal was, for the first time, found in the extract of RR and possessed novel angiogenesis effect. Thus, C1-1 and norviburtinal were the active components responsible for the pro-angiogenesis effect of RR. / Moreover, RR-containing formula (NF3), which induced endothelial cell attachment, migration, and tubule formation in human vascular endothelial cell (HECV), could be mediated through PI3K and N-WASP pathways. Activated Akt and MAPK kinases expression in western blot analysis were also demonstrated in NF3-treated HECV. These inductions would promote angiogenesis at various levels. Hence, the complexity of angiogenesis effect activated by the NF3 treatment molecularly and functionally was shown, and we further supported the high sensitivity of ECIS in the screening of wound healing agents with endothelial cells. / In conclusion, through targeting anti-inflammation, elevated fibroblast proliferation and improved angiogenesis, our respective bioassay-guided active fractions and compounds in Rehmanniae Radix, and the RR-containing formula, could play beneficial uses in treating chronic wound healing. / 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. / Liu, Cheuk Lun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 221-249). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Table of Contents --- p.i / Abstract (in English) --- p.vi / Abstract (in Chinese) --- p.ix / Statement of Originality --- p.xiii / Acknowledgements --- p.xiv / Publications --- p.xiv / List of Tables --- p.xvii / List of Figures --- p.xviii / List of Abbreviations --- p.xxi / Chapter Chapter 1: --- Literature Review and Study Objectives / Chapter 1.1 --- Overview on wound healing / Chapter 1.1.1 --- Normal wound healing --- p.1 / Chapter 1.1.2 --- Chronic wound healing / Chapter 1.1.2.1 --- Venous ulcers --- p.8 / Chapter 1.1.2.2 --- Diabetic ulcers --- p.11 / Chapter 1.1.2.3 --- Mechanism of chronic wound healing --- p.13 / Chapter 1.1.2.4 --- Current treatments for chronic wound healing --- p.19 / Chapter 1.2 --- Rehmanniae Radix (RR) overview / Chapter 1.2.1 --- RR and its subtypes --- p.29 / Chapter 1.2.2 --- Chemistry of RR --- p.34 / Chapter 1.2.3 --- Pharmacology of RR --- p.38 / Chapter 1.2.3.1 --- RR and chronic wound healing / Chapter 1.3 --- Study objectives --- p.42 / Chapter Chapter 2: --- comparison of wound healing effect of the subtypes of rr / Chapter 2.1 --- Introduction --- p.43 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Preparation and authentication of subtypes of RR --- p.46 / Chapter 2.2.2 --- RAW 264.7 murine macrophage culture and sample treatment protocol, and cell viability test --- p.47 / Chapter 2.2.3 --- Nitric oxide inhibitory assay --- p.47 / Chapter 2.2.4 --- Hs27 human fibroblast culture and sample treatment protocol --- p.48 / Chapter 2.2.5 --- Fibroblast proliferation assay --- p.48 / Chapter 2.2.6 --- Statistical analysis --- p.49 / Chapter 2.3 --- Results --- p.63 / Chapter 2.3.1 --- Nitric oxide anti-inflammatory effect of the subtypes of RR --- p.49 / Chapter 2.3.2 --- Fibroblast proliferative effect of the subtypes of RR --- p.52 / Chapter 2.4 --- Discussion --- p.54 / Chapter Chapter 3: --- Fibroblast proliferative effect of RR / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.2 --- Methods / Chapter 3.2.1 --- Preparation of aqueous extracts of RR --- p.61 / Chapter 3.2.2 --- Hs27 human fibroblast culture and sample treatment protocol --- p.61 / Chapter 3.2.3 --- Hs27 human fibroblast proliferation assay --- p.61 / Chapter 3.2.4 --- Bioassay-guided fractionation of RR --- p.61 / Chapter 3.2.5 --- LC-MS analysis of bioassay-guided fraction, C2-B4 --- p.65 / Chapter 3.2.6 --- Statistical analysis --- p.66 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Fibroblast proliferative effect of RR aqueous crude extract and its bioassay-guided fractions --- p.67 / Chapter 3.3.2 --- Chemical structure of the isolated compounds --- p.70 / Chapter 3.3.3 --- LC-MS analysis of bioassay-guided fraction, C2-B4 --- p.72 / Chapter 3.4 --- Discussion --- p.73 / Chapter Chapter 4: --- Anti-inflammatory effect and its underlying mechanism of RR / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Methods / Chapter 4.2.1 --- Preparation of aqueous extracts of RR --- p.82 / Chapter 4.2.2 --- RAW 264.7 murine macrophage culture and sample treatment protocol, and cell viability test --- p.82 / Chapter 4.2.3 --- Assay for nitric oxide inhibitory effect using RAW264.7 cells --- p.83 / Chapter 4.2.4 --- Bioassay-guided fractionation of RR --- p.83 / Chapter 4.2.5 --- Ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS) analysis of sub-fraction, C3 --- p.85 / Chapter 4.2.6 --- Prostaglandin E2 (PGE2) and interleukin-6 (IL-6) assay --- p.87 / Chapter 4.2.7 --- Real-time PCR --- p.87 / Chapter 4.2.8 --- Western blot analysis --- p.89 / Chapter 4.2.9 --- Statistical analysis --- p.91 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Nitric oxide inhibitory effects of L-NMMA, RR aqueous crude extract and its bioassay guided fractions --- p.92 / Chapter 4.3.2 --- LC-MS analysis of bioassay-guided fraction, C3 --- p.96 / Chapter 4.3.3 --- Suppression of inflammation-related mRNA expression level in macrophages by C3 --- p.97 / Chapter 4.3.4 --- Suppression of protein expression of inducible nitric oxide synthase and COX-2 in macrophages by C3 --- p.99 / Chapter 4.3.5 --- Inhibition of release of inflammatory cytokine in macrophages by C3 --- p.102 / Chapter 4.4 --- Discussion --- p.103 / Chapter Chapter 5: --- Angiogenesis effect and its underlying mechanism of RR / Chapter 5.1 --- Introduction --- p.111 / Chapter 5.2 --- Methods / Chapter 5.2.1 --- Preparation of aqueous extracts of RR --- p.113 / Chapter 5.2.2 --- Zebrafish culture --- p.113 / Chapter 5.2.3 --- Collection of zebrafish embryos and herbal treatment protocol --- p.115 / Chapter 5.2.4 --- Microinjection of vascular endothelial growth factor (VEGF) to zebrafish embryos --- p.116 / Chapter 5.2.5 --- Screening of zebrafish embryos using fluorescence microscopy --- p.116 / Chapter 5.2.6 --- Bioassay-guided fractionation of RR / Chapter 5.2.7 --- Isolation and structure elucidation of compound C2A --- p.120 / Chapter 5.2.8 --- Gas chromatographymass spectrometry (GC-MS) analysis of bioassay-guided fraction of RR, C1-1 --- p.120 / Chapter 5.2.9 --- Detection of zebrafish mRNA expression level by real-time PCR (RT-PCR) --- p.122 / Chapter 5.2.10 --- Human microvascular endothelial cell (HMEC-1) culture and sample treatment protocol --- p.125 / Chapter 5.2.11 --- HMEC-1 proliferation assay --- p.125 / Chapter 5.2.12 --- HMEC-1 scratch assay --- p.126 / Chapter 5.2.13 --- HMEC-1 tubule formation assay --- p.126 / Chapter 5.2.14 --- Statistical analysis --- p.127 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Angiogenesis effects of RR aqueous crude extract, its bioassay-guided fractions and isolated compound, in zebrafish model --- p.128 / Chapter 5.3.2 --- Chemical structure and angiogenesis effect of the isolated compound C2A, norviburtinal --- p.133 / Chapter 5.3.3 --- Components of C1-1 from GC-MS analysis --- p.135 / Chapter 5.3.4 --- Angiogenesis effect of C1-1 in angiogenesis-related mRNA expression level in zebrafish --- p.137 / Chapter 5.3.5 --- Effect of endothelial cell proliferation of C1-1 in HMEC-1 cell --- p.142 / Chapter 5.3.6 --- Cell migration effect of C1-1 in HMEC-1 cell --- p.143 / Chapter 5.3.7 --- Tubule formation of C1-1 in HMEC-1 cell --- p.145 / Chapter 5.4 --- Discussion --- p.147 / Chapter Chapter 6: --- Angiogenesis effect and its underlying mechanism of RR AND AR-containing two-herbs formula, NF3 using ecis model / Chapter 6.1 --- Introduction --- p.165 / Chapter 6.2 --- Methods / Chapter 6.2.1 --- Preparation and authentication of aqueous extracts of NF3 --- p.176 / Chapter 6.2.2 --- Human vascular endothelial cells (HECV) culture --- p.177 / Chapter 6.2.3 --- HECV cell proliferation assay --- p.178 / Chapter 6.2.4 --- Scratch assay --- p.178 / Chapter 6.2.5 --- Tubule formation assay --- p.179 / Chapter 6.2.6 --- Electric cell-substrate impedance sensing (ECIS)-based cell attachment and motility assay --- p.180 / Chapter 6.2.7 --- Western blot analysis --- p.181 / Chapter 6.2.8 --- Statistical analysis --- p.182 / Chapter 6.3 --- Results / Chapter 6.3.1 --- LC-MS analysis of NF3 --- p.184 / Chapter 6.3.2 --- Effects of NF3, AR and RR on cell viability and migration of HECV --- p.185 / Chapter 6.3.3 --- Tubule formation effect of NF3, AR and RR in HECV cell --- p.188 / Chapter 6.3.4 --- Effects of NF3, AR, and RR on HECV cell attachment using ECIS model --- p.190 / Chapter 6.3.5 --- Effects of NF3, AR and RR on HECV cell migration using ECIS model --- p.191 / Chapter 6.3.6 --- Effects of NF3 on HECV for MAPK and Akt protein activation --- p.195 / Chapter 6.4 --- Discussion --- p.197 / Chapter Chapter 7: --- General Discussion and Conclusions / Chapter 7.1 --- General discussion and conclusions --- p.206 / Chapter 7.2 --- Limitation of the study --- p.215 / Chapter 7.3 --- Future work --- p.215 / Appendix --- p.218 / References --- p.221

丹參和葛根為我國民間常用的傳統藥材, 常用於心血管疾病的治療。本試驗主要研究丹參葛根複方(DG, 7:3)對大鼠基底動脈的舒張作用 及腦保護作用。 / 上述所有藥物對U46619預收縮的基底動脈環呈現濃度依賴性的舒張作用。一氧化氮合酶抑制劑L-NAME以及鳥苷酸環化酶抑制劑ODG部分抑制葛根素的舒張作用。在另一組去內皮試驗中, 腺苷酸環化酶抑制劑SQ22536, 鳥苷酸環化酶抑制劑ODG, 大電導鈣離子依賴型鉀通道抑制劑Iberiotoxin以及電壓門控型鉀通道抑制劑4-AP對所有藥物的舒張作用沒有影響。然而， ATP型鉀通道抑制劑格列本脲能夠抑制丹參葛根複方，丹參，葛根，丹參素，葛根素，大豆苷以及大豆苷元的最大舒張反應。內向整流型鉀通道抑制劑氯化鋇則降低丹參酚酸B和大豆苷元的最大反應值。非選擇性鉀通道抑制劑乙基氯化銨以及所有鉀通道抑制劑的混合物顯著抑制上述所有藥物的舒張作用。除了葛根素之外，所有的藥物動度依賴性的抑制氯化鈣所引起的血管收縮。 / 體內研究發現大鼠經歷10分鐘雙側頸總動脈夾閉合並低血壓，及24小時的複灌後，與假手術組動物相比，腦血流量顯著降低，氧化性損傷明顯可見。連續7天口服丹參葛根複方（0.3g/kg 和 3g/kg）, 丹參 (3g/kg)，或者葛根 (3g/kg)對血壓沒有影響。但是，高劑量的丹參葛根複方 (3g/kg) 能夠提高超氧化物歧化酶和過氧化氫酶的活性，抑制丙二醛和一氧化氮的產生。3g/kg的葛根可以提高超氧化物歧化酶的活性，3g/kg的丹參則能抑制一氧化氮的產生。在大鼠中動脈阻塞模型中，連續7天口服丹參葛根複方（3g/kg）能明顯降低腦部的梗死率，同時改善大鼠的神經行為學。 / 總體來說，研究發現丹參葛根複方，丹參，葛根，丹參酚酸B，大豆苷以及大豆苷元的血管舒張作用是通過開平滑肌細胞的通鉀離子通道以及抑制鈣離子內流而實現的。然而葛根素的血管舒張作用是內皮依賴性的，通過產生一氧化氮，開放平滑肌細胞的鉀離子通道而實現的。丹參葛根複方能起到一定的腦保護作用。總而言之，研究表明上述藥物可能會對阻塞性腦血管病的人群有益處。 / Danshen and gegen are used in traditional Chinese medicine for the treatment of cardiovascular diseases. In this study, the relaxant actions of a danshen and gegen formulation (DG; ratio 7:3) and its constituents were investigated on rat-isolated cerebral basilar artery. In addition, the neuroprotective effect of DG was explored in rats subjected to global and focal ischaemia. / DG and all its constituents produced concentration-dependent relaxation of the artery rings precontracted by U46619. Removal of the endothelium had no effect on their vasodilator actions except the maximum response (I[subscript max]) to puerarin was inhibited by 42%. The nitric oxide synthase (NOS) inhibitor L-NAME and guanylyl cyclase (GC) inhibitor ODQ but not the cyclo-oxygenase (COX) inhibitor flurbiprofen produced partial inhibition on the puerarin-induced effect. In a set of endothelium-denuded artery rings, adenylyl cyclase (AC) inhibitor SQ22536, GC inhibitor ODQ, KV channel inhibitor 4-aminopyridine (4-AP) and BK[subscript Ca) channel inhibitor iberiotoxin had no influence on their vasodilator actions. However, pretreatment with K[subscript ATP] channel inhibitor glibenclamide reduced Imax to DG, danshen, gegen, danshensu, puerarin, daidzein and daidzin. K[subscript IR] inhibitor barium chloride (BaCl₂) reduced II[subscript max] to salvianolic acid B and daidzein. The non-selective K⁺ channel inhibitor tetraethylammonium (TEA), or a combination of all the K⁺ channel inhibitors produced significant partial inhibitions on all the agents’ actions. Electrophysiological studies on smooth muscle cells isolated from rat basilar artery also confirmed that DG, danshen, gegen danshensu, puerarin, daidzein and daidzin elevated K[subscript ATP] currents. In addition, DG and all its constituents, except puerarin, produced concentration-dependent inhibition on CaCl₂-induced vasoconstrictions. These findings were confirmed by con-focal microscopy studies. / In vivo study on a rat model of global ischaemia showed that challenging the rats with 10 min bilateral common carotid artery occlusion combined with hypotension, and followed by 24 h reperfusion produced significant decrease in cerebral blood flow and oxidative damage compared to sham-operated animals. Administration of DG (0.3 g/kg and 3 g/kg, p.o.), danshen (3 g/kg, p.o.) or gegen (3 g/kg, p.o.) for 7 days had no effect on blood pressure. However, the 7 days treatment with DG (3 g/kg) restored superoxide dismutase (SOD) and catalase (CAT) activities, suppressed the production of maleic dialdehyde (MDA), and inhibited the production of nitric oxide (NO). In addition, gegen (3 g/kg) restored SOD enzyme activity, whereas, danshen (3 g/kg) inhibited NO production. In addition, treatment with DG (3 g/kg) showed a significant reduction in infarct weight and improved the neurological deficit in a rat model of focal cerebral ischaemia induced by middle cerebral artery occlusion (MCAO). / In conclusion, the vasorelaxant actions of DG, danshen, gegen, salvianolic acid B, danshensu, daidzein and daidzin were found to involve the opening of K⁺ channels and inhibition of Ca²⁺ influx in the vascular smooth muscle cells. In contrast, puerarin produced vasodilatation via an endothelium-dependent mechanism involving NO production and an endothelium-independent pathway mediated by the opening of K⁺ channels. DG may have some cerebro-protective effects. Overall, the present studies showed that DG and its constituents could be beneficial to patients with obstructive cerebrovascular diseases. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Deng, Yan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 164-178). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.v / 摘要 --- p.iviii / ACKNOWLEDGEMENTS --- p.x / PUBLICATIONS BASED ON THIS THESIS --- p.xii / ABBREVIATIONS --- p.xiv / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Chinese Medicines in treatment of cerebrovascular diseases --- p.2 / Chapter 1.2 --- Danshen --- p.4 / Chapter 1.2.1 --- Chemical constituents --- p.4 / Chapter 1.2.1.1 --- Hydrophilic compounds of danshen --- p.4 / Chapter 1.2.1.2 --- Lipophilic compounds of danshen --- p.5 / Chapter 1.2.1.3 --- Other compounds --- p.5 / Chapter 1.2.2 --- Pharmacological activities --- p.5 / Chapter 1.2.2.1 --- Vascular protection --- p.5 / Chapter 1.2.2.2 --- Anti-tumour --- p.7 / Chapter 1.2.2.3 --- Treatment of liver diseases --- p.8 / Chapter 1.2.2.4 --- Treatment of drug addiction --- p.9 / Chapter 1.2.2.5 --- Treatment of kidney diseases --- p.10 / Chapter 1.2.3 --- Pharmacokinetics --- p.10 / Chapter 1.3 --- Gegen --- p.12 / Chapter 1.3.1 --- Chemical constituents --- p.12 / Chapter 1.3.2 --- Pharmacology --- p.13 / Chapter 1.3.2.1 --- Vascular effects --- p.13 / Chapter 1.3.2.2 --- Anti-diabetes --- p.14 / Chapter 1.3.2.3 --- Anti-hypercholesterolaemia --- p.15 / Chapter 1.3.2.4 --- Anti-inflammation --- p.16 / Chapter 1.3.2.5 --- Anti-platelet aggregation --- p.17 / Chapter 1.3.3 --- Pharmacokinetics --- p.17 / Chapter 1.4 --- Danshen and gegen formulation --- p.19 / Chapter 1.5 --- Mechanisms of vasodilatation --- p.22 / Chapter 1.5.1 --- Endothelium derived relaxant factors (EDRFs) --- p.22 / Chapter 1.5.1.1 --- Nitric oxide (NO) --- p.22 / Chapter 1.5.1.2 --- Prostacyclin (PGI₂) --- p.23 / Chapter 1.5.1.3 --- Endothelium-derived hyperpolarizating factors (EDHFs)- --- p.23 / Chapter 1.5.2 --- Signal transduction pathways --- p.24 / Chapter 1.5.2.1 --- Guanylyl cyclase-cGMP pathway --- p.24 / Chapter 1.5.2.2 --- Adenylyl cyclase-cAMP pathway --- p.24 / Chapter 1.5.3 --- Ion channels --- p.25 / Chapter 1.5.3.1 --- Potassium channels (K⁺ channels) --- p.25 / Chapter 1.5.3.2 --- Calcium channel (Ca²⁺ channels) --- p.25 / Chapter 1.6 --- Aims of study --- p.27 / Chapter CHAPTER 2 --- Materials and method --- p.28 / Chapter 2.1 --- Herbal preparation --- p.28 / Chapter 2.1.1 --- DG, danshen and gegen preparation --- p.28 / Chapter 2.1.2 --- Identification and quantification of chemical markers in DG water extract --- p.29 / Chapter 2.2 --- Experiments on rat basilar artery --- p.30 / Chapter 2.2.1 --- Animals --- p.30 / Chapter 2.2.2 --- Chemicals --- p.30 / Chapter 2.2.3 --- Isolation and mounting of blood vessels --- p.33 / Chapter 2.2.4 --- Protocols --- p.34 / Chapter 2.2.4.1 --- Effects on U46619-precontracted tone --- p.34 / Chapter 2.2.4.2 --- Endothelium-dependent mechanism --- p.34 / Chapter 2.2.4.3 --- Endothelium-independent mechanism --- p.35 / Chapter 2.2.4.4 --- Calcium channels --- p.36 / Chapter 2.2.4.5 --- Positive control --- p.36 / Chapter 2.2.5 --- Statistical analysis --- p.37 / Chapter 2.3 --- Experiments on rat cerebral basilar artery smooth muscle cells K[subscript ATP] channals --- p.38 / Chapter 2.3.1 --- Animals --- p.38 / Chapter 2.3.2 --- Chemicals --- p.38 / Chapter 2.3.3 --- Isolation of rat cerebral vascular smooth muscle cells --- p.40 / Chapter 2.3.4 --- Whole cell patch-clamp electrophysiology --- p.40 / Chapter 2.3.5 --- Statistical analysis --- p.44 / Chapter 2.4 --- Experiments on rat cerebral basilar artery smooth muscle cells calcium channels --- p.45 / Chapter 2.4.1 --- Animals --- p.45 / Chapter 2.4.2 --- Chemicals --- p.45 / Chapter 2.4.3 --- Isolation of rat cerebral vascular smooth muscle cells --- p.47 / Chapter 2.4.4 --- Dye loading and determination of [Ca²⁺]i --- p.47 / Chapter 2.4.5 --- Statistical analysis --- p.48 / Chapter 2.5 --- In vivo study of global ischaemia --- p.49 / Chapter 2.5.1 --- Animals --- p.49 / Chapter 2.5.2 --- Drugs and chemicals --- p.49 / Chapter 2.5.3 --- Experimental protocols for global ischaemia --- p.49 / Chapter 2.5.4 --- Induction of global ischaemia --- p.50 / Chapter 2.5.5 --- Blood pressure measurement --- p.52 / Chapter 2.5.6 --- Measurement of cerebral blood flow --- p.52 / Chapter 2.5.7 --- Biochemical assessment --- p.53 / Chapter 2.5.7.1. --- Dissection and homogenization --- p.53 / Chapter 2.5.7.2. --- Measurement of malondialdehyde (MDA) --- p.53 / Chapter 2.5.7.3. --- Estimation of nitrite --- p.53 / Chapter 2.5.7.4 --- Superoxide dismutase activity (SOD) --- p.54 / Chapter 2.5.7.5 --- Reduced glutathione (GSH) --- p.54 / Chapter 2.5.7.6 --- Catalase (CAT) --- p.55 / Chapter 2.5.7.7 --- NOS activity --- p.55 / Chapter 2.5.7.8 --- Protein --- p.56 / Chapter 2.5.8 --- Statistical analysis --- p.56 / Chapter 2.6 --- In vivo study of focal ischaemia --- p.57 / Chapter 2.6.1 --- Animals --- p.57 / Chapter 2.6.2 --- Drugs and chemicals --- p.57 / Chapter 2.6.3 --- Experimental protocols for global ischaemia --- p.57 / Chapter 2.6.4 --- Focal cerebral ischaemia-reperfusion model --- p.57 / Chapter 2.6.5 --- Assessment of neurobehavioural changes --- p.59 / Chapter 2.6.6 --- Assessment of cerebral infarction --- p.60 / Chapter 2.6.7 --- Statistical analysis --- p.60 / Chapter CHAPTER 3 --- Results --- p.61 / Chapter 3.1 --- Identification and quantification of chemical markers in DG water extract --- p.61 / Chapter 3.2 --- Effects of DG and its constituents on rat cerebral basilar artery --- p.64 / Chapter 3.2.1 --- Investigations on endothelium-dependent mechanisms --- p.64 / Chapter 3.2.2 --- Investigations on endothelium-independent mechanisms --- p.68 / Chapter 3.2.3 --- Positive control --- p.86 / Chapter 3.2.3 --- Investigations on calcium channels --- p.88 / Chapter 3.3 --- Effects of DG and its constituents on rat cerebral basilar artery smooth muscle cells --- p.91 / Chapter 3.3.1 --- Effects of water crude-extracts of DG, danshen, and gegen on K[subscript ATP] channels --- p.91 / Chapter 3.3.2 --- Effects of active constituents of danshen hydrophilic fraction on K[subscript ATP] channels --- p.100 / Chapter 3.3.3 --- Effects of the major isoflavonoids of gegen on K[subscript ATP] channels --- p.105 / Chapter 3.4 --- Effects of DG and its constituents on calcium channels of basilar artery smooth muscle cells --- p.112 / Chapter 3.5 --- Effects of DG, danshen and gegen on rat global ischaemia --- p.117 / Chapter 3.5.1 --- Effects of DG, danshen and gegen on rats’ blood pressure and cerebral blood flow --- p.117 / Chapter 3.5.2 --- Effects of DG, danshen and gegen on lipid peroxidation --- p.120 / Chapter 3.5.3 --- Effects of DG, danshen and gegen on SOD activity --- p.120 / Chapter 3.5.4 --- Effects of DG, danshen and gegen on CAT activity --- p.120 / Chapter 3.5.5 --- Effects of DG, danshen and gegen on reduced GSH level --- p.121 / Chapter 3.5.6 --- Effects of DG, danshen and gegen on NOS system --- p.126 / Chapter 3.6 --- Effect of DG on rat focal ischaemia --- p.129 / Chapter 3.6.1 --- Effect of DG on cerebral infarction --- p.129 / Chapter 3.6.2 --- Effect of DG on neurological deficits --- p.129 / Chapter CHAPTER 4 --- Discussion --- p.132 / Chapter 4.1 --- Studies of DG and its constituents on rat cerebral basilar artery --- p.133 / Chapter 4.1.1 --- Constituents of DG on U46619-precontracted tone --- p.133 / Chapter 4.1.2 --- Investigations on endothelium-dependent mechanisms --- p.133 / Chapter 4.1.3 --- Investigations on endothelium-independent mechanisms --- p.136 / Chapter 4.1.4 --- Investigations on calcium channels --- p.139 / Chapter 4.2 --- Effects of DG and its constituents on rat cerebral basilar artery smooth muscle cell K[subscript ATP] channels --- p.143 / Chapter 4.3 --- Effects of DG and its constituents on calcium influx in rat basilar artery smooth muscle cells --- p.147 / Chapter 4.4 --- Effects of DG, danshen and gegen on rat transient global ischaemia --- p.150 / Chapter 4.4.1 --- Effects of DG, danshen and gegen on rats’ blood pressure and cerebral blood flow --- p.150 / Chapter 4.4.2 --- Effects of DG, danshen and gegen on lipid peroxidation, SOD and CAT activity, and GSH level --- p.152 / Chapter 4.4.3 --- Effects of DG, danshen and gegen on NOS system --- p.155 / Chapter 4.5 --- Effects of DG on rat focal ischaemia --- p.157 / Chapter 4.6 --- Further studies --- p.160 / Chapter 4.7 --- Conclusion --- p.162 / REFERENCES --- p.164

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.