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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.iviiiACKNOWLEDGEMENTS --- p.xPUBLICATIONS BASED ON THIS THESIS --- p.xiiABBREVIATIONS --- p.xivChapter CHAPTER 1 --- Introduction --- p.1Chapter 1.1 --- Chinese Medicines in treatment of cerebrovascular diseases --- p.2Chapter 1.2 --- Danshen --- p.4Chapter 1.2.1 --- Chemical constituents --- p.4Chapter 1.2.1.1 --- Hydrophilic compounds of danshen --- p.4Chapter 1.2.1.2 --- Lipophilic compounds of danshen --- p.5Chapter 1.2.1.3 --- Other compounds --- p.5Chapter 1.2.2 --- Pharmacological activities --- p.5Chapter 1.2.2.1 --- Vascular protection --- p.5Chapter 1.2.2.2 --- Anti-tumour --- p.7Chapter 1.2.2.3 --- Treatment of liver diseases --- p.8Chapter 1.2.2.4 --- Treatment of drug addiction --- p.9Chapter 1.2.2.5 --- Treatment of kidney diseases --- p.10Chapter 1.2.3 --- Pharmacokinetics --- p.10Chapter 1.3 --- Gegen --- p.12Chapter 1.3.1 --- Chemical constituents --- p.12Chapter 1.3.2 --- Pharmacology --- p.13Chapter 1.3.2.1 --- Vascular effects --- p.13Chapter 1.3.2.2 --- Anti-diabetes --- p.14Chapter 1.3.2.3 --- Anti-hypercholesterolaemia --- p.15Chapter 1.3.2.4 --- Anti-inflammation --- p.16Chapter 1.3.2.5 --- Anti-platelet aggregation --- p.17Chapter 1.3.3 --- Pharmacokinetics --- p.17Chapter 1.4 --- Danshen and gegen formulation --- p.19Chapter 1.5 --- Mechanisms of vasodilatation --- p.22Chapter 1.5.1 --- Endothelium derived relaxant factors (EDRFs) --- p.22Chapter 1.5.1.1 --- Nitric oxide (NO) --- p.22Chapter 1.5.1.2 --- Prostacyclin (PGI₂) --- p.23Chapter 1.5.1.3 --- Endothelium-derived hyperpolarizating factors (EDHFs)- --- p.23Chapter 1.5.2 --- Signal transduction pathways --- p.24Chapter 1.5.2.1 --- Guanylyl cyclase-cGMP pathway --- p.24Chapter 1.5.2.2 --- Adenylyl cyclase-cAMP pathway --- p.24Chapter 1.5.3 --- Ion channels --- p.25Chapter 1.5.3.1 --- Potassium channels (K⁺ channels) --- p.25Chapter 1.5.3.2 --- Calcium channel (Ca²⁺ channels) --- p.25Chapter 1.6 --- Aims of study --- p.27Chapter CHAPTER 2 --- Materials and method --- p.28Chapter 2.1 --- Herbal preparation --- p.28Chapter 2.1.1 --- DG, danshen and gegen preparation --- p.28Chapter 2.1.2 --- Identification and quantification of chemical markers in DG water extract --- p.29Chapter 2.2 --- Experiments on rat basilar artery --- p.30Chapter 2.2.1 --- Animals --- p.30Chapter 2.2.2 --- Chemicals --- p.30Chapter 2.2.3 --- Isolation and mounting of blood vessels --- p.33Chapter 2.2.4 --- Protocols --- p.34Chapter 2.2.4.1 --- Effects on U46619-precontracted tone --- p.34Chapter 2.2.4.2 --- Endothelium-dependent mechanism --- p.34Chapter 2.2.4.3 --- Endothelium-independent mechanism --- p.35Chapter 2.2.4.4 --- Calcium channels --- p.36Chapter 2.2.4.5 --- Positive control --- p.36Chapter 2.2.5 --- Statistical analysis --- p.37Chapter 2.3 --- Experiments on rat cerebral basilar artery smooth muscle cells K[subscript ATP] channals --- p.38Chapter 2.3.1 --- Animals --- p.38Chapter 2.3.2 --- Chemicals --- p.38Chapter 2.3.3 --- Isolation of rat cerebral vascular smooth muscle cells --- p.40Chapter 2.3.4 --- Whole cell patch-clamp electrophysiology --- p.40Chapter 2.3.5 --- Statistical analysis --- p.44Chapter 2.4 --- Experiments on rat cerebral basilar artery smooth muscle cells calcium channels --- p.45Chapter 2.4.1 --- Animals --- p.45Chapter 2.4.2 --- Chemicals --- p.45Chapter 2.4.3 --- Isolation of rat cerebral vascular smooth muscle cells --- p.47Chapter 2.4.4 --- Dye loading and determination of [Ca²⁺]i --- p.47Chapter 2.4.5 --- Statistical analysis --- p.48Chapter 2.5 --- In vivo study of global ischaemia --- p.49Chapter 2.5.1 --- Animals --- p.49Chapter 2.5.2 --- Drugs and chemicals --- p.49Chapter 2.5.3 --- Experimental protocols for global ischaemia --- p.49Chapter 2.5.4 --- Induction of global ischaemia --- p.50Chapter 2.5.5 --- Blood pressure measurement --- p.52Chapter 2.5.6 --- Measurement of cerebral blood flow --- p.52Chapter 2.5.7 --- Biochemical assessment --- p.53Chapter 2.5.7.1. --- Dissection and homogenization --- p.53Chapter 2.5.7.2. --- Measurement of malondialdehyde (MDA) --- p.53Chapter 2.5.7.3. --- Estimation of nitrite --- p.53Chapter 2.5.7.4 --- Superoxide dismutase activity (SOD) --- p.54Chapter 2.5.7.5 --- Reduced glutathione (GSH) --- p.54Chapter 2.5.7.6 --- Catalase (CAT) --- p.55Chapter 2.5.7.7 --- NOS activity --- p.55Chapter 2.5.7.8 --- Protein --- p.56Chapter 2.5.8 --- Statistical analysis --- p.56Chapter 2.6 --- In vivo study of focal ischaemia --- p.57Chapter 2.6.1 --- Animals --- p.57Chapter 2.6.2 --- Drugs and chemicals --- p.57Chapter 2.6.3 --- Experimental protocols for global ischaemia --- p.57Chapter 2.6.4 --- Focal cerebral ischaemia-reperfusion model --- p.57Chapter 2.6.5 --- Assessment of neurobehavioural changes --- p.59Chapter 2.6.6 --- Assessment of cerebral infarction --- p.60Chapter 2.6.7 --- Statistical analysis --- p.60Chapter CHAPTER 3 --- Results --- p.61Chapter 3.1 --- Identification and quantification of chemical markers in DG water extract --- p.61Chapter 3.2 --- Effects of DG and its constituents on rat cerebral basilar artery --- p.64Chapter 3.2.1 --- Investigations on endothelium-dependent mechanisms --- p.64Chapter 3.2.2 --- Investigations on endothelium-independent mechanisms --- p.68Chapter 3.2.3 --- Positive control --- p.86Chapter 3.2.3 --- Investigations on calcium channels --- p.88Chapter 3.3 --- Effects of DG and its constituents on rat cerebral basilar artery smooth muscle cells --- p.91Chapter 3.3.1 --- Effects of water crude-extracts of DG, danshen, and gegen on K[subscript ATP] channels --- p.91Chapter 3.3.2 --- Effects of active constituents of danshen hydrophilic fraction on K[subscript ATP] channels --- p.100Chapter 3.3.3 --- Effects of the major isoflavonoids of gegen on K[subscript ATP] channels --- p.105Chapter 3.4 --- Effects of DG and its constituents on calcium channels of basilar artery smooth muscle cells --- p.112Chapter 3.5 --- Effects of DG, danshen and gegen on rat global ischaemia --- p.117Chapter 3.5.1 --- Effects of DG, danshen and gegen on rats’ blood pressure and cerebral blood flow --- p.117Chapter 3.5.2 --- Effects of DG, danshen and gegen on lipid peroxidation --- p.120Chapter 3.5.3 --- Effects of DG, danshen and gegen on SOD activity --- p.120Chapter 3.5.4 --- Effects of DG, danshen and gegen on CAT activity --- p.120Chapter 3.5.5 --- Effects of DG, danshen and gegen on reduced GSH level --- p.121Chapter 3.5.6 --- Effects of DG, danshen and gegen on NOS system --- p.126Chapter 3.6 --- Effect of DG on rat focal ischaemia --- p.129Chapter 3.6.1 --- Effect of DG on cerebral infarction --- p.129Chapter 3.6.2 --- Effect of DG on neurological deficits --- p.129Chapter CHAPTER 4 --- Discussion --- p.132Chapter 4.1 --- Studies of DG and its constituents on rat cerebral basilar artery --- p.133Chapter 4.1.1 --- Constituents of DG on U46619-precontracted tone --- p.133Chapter 4.1.2 --- Investigations on endothelium-dependent mechanisms --- p.133Chapter 4.1.3 --- Investigations on endothelium-independent mechanisms --- p.136Chapter 4.1.4 --- Investigations on calcium channels --- p.139Chapter 4.2 --- Effects of DG and its constituents on rat cerebral basilar artery smooth muscle cell K[subscript ATP] channels --- p.143Chapter 4.3 --- Effects of DG and its constituents on calcium influx in rat basilar artery smooth muscle cells --- p.147Chapter 4.4 --- Effects of DG, danshen and gegen on rat transient global ischaemia --- p.150Chapter 4.4.1 --- Effects of DG, danshen and gegen on rats’ blood pressure and cerebral blood flow --- p.150Chapter 4.4.2 --- Effects of DG, danshen and gegen on lipid peroxidation, SOD and CAT activity, and GSH level --- p.152Chapter 4.4.3 --- Effects of DG, danshen and gegen on NOS system --- p.155Chapter 4.5 --- Effects of DG on rat focal ischaemia --- p.157Chapter 4.6 --- Further studies --- p.160Chapter 4.7 --- Conclusion --- p.162REFERENCES --- p.16