【作者】 徐静华；

【导师】 于庆海； 渡边裕司；

【作者基本信息】 沈阳药科大学 ， 药理学， 2000， 博士

【摘要】 黄连解毒汤是清热解毒方中的经典方剂，由黄连、黄芩、黄柏、栀子四味生药组成。临床上，该方剂已被广泛用于治疗慢性炎症及一些溃疡性疾病。对其进行的药理学研究也多集中于其所具有的清热解毒、抗炎、抗溃疡等作用，而对其促智作用的药理学研究却罕见报道。 本文采用三维高效液相色谱（Three-dimensional high performance liquid chromatography,3D-HPLC）法定性分析了黄连解毒汤提取物中所含的化学成分，采用HPLC法定量测定了其中小檗碱、巴马亭、黄芩苷以及栀子苷的含量。对黄连解毒汤提取物的药理学研究并没有局限于其所具有的清热解毒作用，而是采用现代药理学实验方法研究了其对脑缺血动物的促智作用，并对其作用机制进行了探讨。 化学成分测定结果表明，黄连解毒汤提取物中含有小檗碱、巴马亭、木兰花碱、表小檗碱、药根碱、黄连碱、非洲防己碱等异喹啉类生物碱；含有黄芩苷、黄芩素、汉黄芩素、千层纸素A、千层纸素A 7-O-葡萄糖醛酸苷、黄芩新素Ⅱ等黄酮类化合物；此外，还含有栀子苷这一环烯醚萜类化合物。其中小檗碱含量为81.6 mg／g、巴马亭含量为22.2 mg／g、黄芩苷含量为37.0 mg／g、栀子苷含量为46.3 mg／g。与黄连解毒汤传统水煎剂及日本东洋黄连解毒汤颗粒剂相比，黄连解毒汤提取物较大程度地富集了组方生药所含的已知主要成分。 药理学研究结果表明，黄连解毒汤提取物灌胃（ig）给药2.0g／kg、4.0g／kg、8.0g／kg均不影响脑缺血小鼠在新异环境中的自主活动和探究行为，对中枢神经系统无兴奋和抑制作用。在跳台实验中，黄连解毒汤提取物（ig）2.0g／kg、4.0g／kg、8.0g／kg均能显著延长脑缺血小鼠跳台潜伏期，4.0 g／kg、8.0 g／kg能显著减少脑缺血小鼠跳台错误次数；在避暗实验中，黄连解毒汤提取物（ig）2.0 g／kg、4.0 g／kg、8.0 g／kg均能显著减少脑缺血小鼠避暗错误次数；在水迷宫实验中，黄连解毒汤提取物（ig）2.0 g／kg、4.0 g／kg、8.0 g／kg均能显著缩短脑缺血小鼠逃避平台潜伏期，4.0g／kg、8.0g／kg能够显著增加脑缺血小鼠跨越原平台所在区域百分率。结果表明，黄连解毒汤提取物能够改善脑缺血引起的小鼠学习记忆障碍。 此外，黄连解毒汤提取物（ig）能够明显延长双侧颈总动脉结扎小鼠及NaNO₂（800 mg／kg,ip）致缺氧小鼠存活时间；对KCN（4.5mg／kg,ip）所致缺氧小鼠昏睡潜伏期无明显影响，但能明显缩短小鼠昏睡时间；不能影响常压密闭状态下小鼠耗氧速度，但可以通过提高小鼠对氧的利用能力延长小鼠存活时间。结果表明，黄连 摘要解毒汤提取物具有一定的抗缺血缺氧作用。 黄连解毒汤提取物 125 mg／kg、250 mg／kg、500 mg／kg股静脉给药能够明显增加大鼠大脑皮质和海马组织脑血流量。 黄连解毒汤提取物Og)2．0 hg、4刀叭g、8刀叭g能够明显减少KCN致脑缺氧小鼠大脑皮质丙H醛（MDA）含量，4刀吵g、8．0 g／kg能够明显减少 KCN致脑缺氧小鼠海马组织中 MDA含量。黄连解毒汤提取物门g)＊刀 g／kg、4刀 g／kg、8刀 g／kg还能减少脑缺血小鼠全脑、大脑皮质及海马组织中MDA含量，与脑缺血模型组比较具有统计学意义。结果表明，黄连解毒汤提取物具有较强的抗氧化作用。进一步的研究表明，黄连解毒溅取物门g)40 g／kg、8刀 g／kg能够明显提高脑缺血小鼠大脑皮质和海马组织中超氧化物歧化酶活力；2刀 g／kg能明显提高脑缺血小鼠大脑皮质过氧化氢酶（CAT）活力，4刀 g／kg、8刀 g／kg能明显提高脑缺血小鼠海马组织中 CAT活力；2刀 g／kg、4刀 g／kg、8刀 g／kg能明显提高脑缺血小鼠海马组织中谷眈甘肽过氧化物酶活力；黄连解毒汤提取物门… 2刀 g／kg。4刀 g／kg、8刀 g／kg能明显增加脑缺血小鼠大脑皮质谷眯甘肽（GSH）含量，2．0 g／kg能明显增加脑缺血小鼠海马组织中GSH含量。结果表明，黄连解毒汤提取物能够通过提高脑缺血动物与学习记忆密切相关脑区的抗氧化酶活力、增加其低分子抗氧化剂含量而发挥抗氧化作用。 本文还考察了黄连解毒汤提取物对脑缺血小鼠脑内与学习记忆密切相关的神经递质——乙酚胆碱（ACh）含量的影响。脑缺血小鼠大脑皮质、海马及纹状体中ACh含量的测定采用 HPLC法进行。结果表明，黄连解毒汤提取物Og)2刀 g／kg、8刀 g／kg能明显提高脑缺血小鼠大脑皮质ACh含量，4刀Wg、8刀Wg能明显提高脑缺血小鼠海马组织中 ACh含量，2刀叭g、4刀 g／kg、8刀 g／kg能明显提高脑缺血小鼠纹状体中 ACh含量。进一步的研究表明，黄连解毒汤提取物Og)2刀 g／kg、4刀 g／kgJ刀 g／kg能够显著延长东莫若碱致学习记忆障碍小鼠跳台潜伏期、减少跳台错误次数，结果提示黄连解毒汤提取物能够增强中枢胆碱能系统的功能。 综合本文实验结果，我们可以肯定黄连解毒汤这一传统复方具有改善脑缺血动物学习记忆障碍的作用。其作用机制涉及抗脑缺血缺氧作用、增加脑血流量、抗自由基毒性作用及增加脑内ACh含量等诸多方面，通过防止脑缺血所引起的一系列病理改变易化和提高中枢神经系统的功能来维持一个正常的学习?更多还原

【Abstract】 Oren-gedoku-to (Huanglian-Jie-Du-Tang, OGT), a classical Traditional Chinese Medicine prescription, consists of four herbs, namely, Coptidis rhizoma, Scutellariae radix, Phellodendri cortex and Gardeniae fructus. It has been clinically used to treat chronic inflammatory and ulcerative diseases for many years. Its pharmacological actions such as anti-inflammatory and anti-ulcer had been studied. However, sufficient experimental effects of OGT on improving learning and memory have not yet been done.In this paper, chemical components in OGT extract were qualitatively analyzed by three-dimensional high performance liquid chromatography (HPLC). And the content of Berberine, Palmatine, Baicalin and Geniposide was quantitatively determined by HPLC. In the present pharmacological research, improving effects of OGT extract on learning and memory in animals subjected to cerebral ischemia were investigated. Furthermore, the mechanisms of OGT extract on improving learning and memory were studied.The analytical results of components showed that there were at least fourteen components such as Berberine, Palmatine, Magnoflorine, Epiberberine, Jateorrhizine, Coptisine, Columbamine, Baicalin, Baicalein, Wogonin, Oroxylin A, Oroxylin A 7-O-glucuronide, Skullcapflavone II and Geniposide in OGT extract. The content of Berberine, Palmatine, Baicalin gnd Geniposide in OGT extract was 81.6 mg/g, 22.2 mg/g, 37.0 mg/g and 46.3 mg/g, respectively.The pharmacological research showed that spontaneous locomotor activity and explorative activity in novel environment in mice subjected to cerebral ischemia were not affected by OGT extract (2.0, 4.0 and 8.0 g/kg, ig). In step-down task, the step-down latency in cerebral ischemic mice was significantly prolonged by OGT extract (2.0, 4.0 and 8.0 g/kg, ig) and the step-down errors were markedly decreased by OGT extract (4.0 and 8.0 g/kg, ig). In step-through task, the step-through errors in cerebral ischemic mice were notably decreased by OGT extract (2.0, 4.0 and 8.0 g/kg, ig). In Morris water maze test, the latency of escaping onto platform in training trial in cerebral ischemic mice was remarkably shortened by OGT (2.0, 4.0 and 8.0 g/kg, ig). The percentage of crossing theformer platform quadrant in probe trial was significantly increased by OGT extract (4.0 and 8.0 g/kg, ig). The experimental results suggested that OGT extract could improve the learning and memory deficits induced by cerebral ischemia in mice.In addition, the survival time in mice subjected to bilateral common carotid artery occlusion or treated with NaNO2 (800 mg/g, ip) was significantly prolonged by OGT extract. The sleeping latency induced by KCN (4.5 mg/kg, ip) in mice was not affected by OGT extract (2.0, 4.0 and 8.0 g/kg, ig), but the sleeping duration was significantly shortened by OGT extract (2.0, 4.0 and 8.0 g/kg, ig). The speed of consuming oxygen in mice under the condition of closed normobaric hypoxia was not affected by OGT extract (2.0, 4.0 and 8.0 g/kg, ig), while the survival time in mice was significantly prolonged by OGT extract due to improving the ability of using oxygen. The results suggested that OGT had effects against the acute hypoxia and cerebral ischemia to a certain extent.The cerebral blood flow (CBF) of cortex and hippocampus in rat was significantly increased after rat was administered OGT extract (125, 250 and 500 mg/kg) by fermoral intravenous injection.The malondialdehyde (MDA) content of cortex and hippocampus in hypoxia mice induced by KCN (6.0 mg/kg, ip) was significantly reduced by OGT extract. In addition, the MDA content of the whole brain, cortex and hippocampus in mice subjected to cerebral ischemia was reduced by OGT extract. The experimental results suggested that OGT had strong anti-oxidation effects.Furthermore, the activity of antioxidase in cortex and hippocampus in cerebral ischemic mice, including superoxide dismutase, catalase and glutathione peroxidase was significantly raised by OGT extract. The glutathione content of cortex and hippocampus in cerebral更多还原