【作者】 彭绍忠；

【导师】 丁平；

【作者基本信息】 广州中医药大学 ， 中药学， 2011， 博士

【摘要】 背景：流行性感冒是指由流感病毒感染引起的急性上呼吸道传染病,具有传染性强、传播快、人群反复易感等特点。目前防治流感的主要方法是注射疫苗和服用抗病毒药物,但由于流感病毒惊人的变异性和耐药性,使人类社会面临严峻的考验。广东地处岭南是温病多发地区,也是世界流感流行的疫源地之一。岭南医家在长期防治瘟疫过程使用本地区的地产药材治疗流感,疗效确切。但其有效成分和作用机制尚不明确,阻碍了岭南地产药材的进一步推广和应用。本研究在国家自然科学基金(项目编号：U0732004)的资助下,按照该项目的研究思路,进行岭南地产药材广藿香抗流感病毒的有效成分筛选和作用机制的初步探讨。目的：1.对岭南地产药材广藿香进行体内抗流感病毒药效实验以明确其抗流感病毒作用,并进一步确定药材的有效部位、有效成分；2.研究广藿香抗病毒有效成分对流感病毒感染小鼠肺炎、细胞因子、免疫细胞、过氧化损伤等方面的影响,探讨其作用机制；3.研究有效成分在抗炎、免疫调节、镇痛、镇咳等方面的作用,并开展急性毒性试验,明确其在缓解流感症状方面的作用和毒性；4.利用分子模拟的手段,预测有效成分的类药性,并为其作用靶点研究提供参考。方法和结果：1.广藿香体内抗流感病毒筛选研究根据岭南医家临床用药规律和文献记载,选择单味中药广藿香为研究对象。为避免加热使中药的成分发生变化,我们采用二氧化碳超临界萃取、超声提取技术提取各其不同极性部位,并把上述各极性提取物根据药材提取部位的提取得率换算,重新组合成各提取部位混合物,采用更符合中药作用特点的体内抗病毒实验对药材各提取部位混合物进行药效实验,确定其抗病毒作用。实验结果显示,广藿香能显著抑制甲型流感病毒鼠肺适应株(FM1)病毒引起的小鼠肺炎和延长病毒感染的小鼠的生存时间,具有抗甲型流感病毒作用。在此基础上,本课题对广藿香不同提取部位进行体内抗病毒筛选,确定广藿香二氧化碳超临界萃取部位是其抗流感病毒的有效部位。对广藿香的有效部位进行成分分离,得到8种单体化合物,并鉴定了它们的结构分别为：表木栓醇(1)、β-谷甾醇(2)、广藿香醇(3)、齐墩果酸(4)、3,3’,4’,7-四甲氧基-5-羟基黄酮(5)、芹菜素(6)、广藿香酮(7)和5-羟基糠醛(8)。结合体内、外抗流感病毒实验,本课题上述各单体化合物中,广藿香醇具有体内抗流感病毒作用,是中药广藿香抗流感的有效成分,但该作用并非通过直接抑制病毒而实现。2.广藿香抗流感病毒有效成分—广藿香醇作用机制初步探讨为验证其抗流感药效并观察量效关系,我们采用不同剂量的广藿香醇重复体内抗流感药效实验。同时,从调节T淋巴细胞亚群、调节炎症因子、抗氧化等方面,对广藿香醇的作用机制进行初步的探讨。实验结果显示广藿香醇能有效对抗流感病毒的致病作用,减轻流感病毒所致的小鼠肺炎,延长流感病毒感染小鼠的生存时间,并出显示一定的剂量依赖关系。在免疫调节方面,广藿香醇可抑制流感病毒感染小鼠脾和胸腺的缩小,并能提高机体细胞免疫功能,调节流感病毒感染小鼠的免疫功能状态；广藿香醇对流感病毒感染小鼠肺炎的治疗作用与其抑制血清中TNF-α的表达和提高IL-10、IFN-γ的表达有着密切关系。在抗氧化方面,广藿香醇给药5天后小鼠肺组织中SOD活力上升,MDA明显下降,表明小鼠肺组织脂质过氧化损伤明显减轻,抗氧自由基损伤的能力明显增强。说明广藿香醇能够有效减轻脂质过氧化损伤,增强机体抗氧化能力。3.广藿香醇治疗流感相关药效学评价为考察广藿香醇能否具有缓解流感症状的作用,本部分将进行其抗炎、调节免疫、镇痛、镇咳等方面的药效学实验,并开展其急性毒性试验。在抗炎作用方面,广藿香醇能有效抑制二甲苯引起的耳肿胀及角叉菜胶所致大鼠足趾肿胀,且呈一定的剂量依赖关系,说明广藿香醇具有抗急性炎症的作用。致炎大鼠给予广藿香醇后,足趾中的NO、PGE-2、TNF-α含量均有所下降,其中高剂量组三种炎症因子含量较模型组明显降低(P<0.05),说明广藿香醇抗炎作用是通过抑制炎症介质和促炎症因子的分泌而实现的。在免疫调节方面,免疫功能低下小鼠给予广藿香醇后,胸腺指数、脾指数、血清溶血素含量和廓清指数均较模型组升高,说明广藿香醇具有调节免疫功能低下小鼠体液免疫功能和单核巨噬细胞吞噬功能的作用。在镇痛和镇咳方面,广藿香醇并不能显示出镇痛、镇咳作用。急性毒性试验结果显示,广藿香醇灌胃给药的LD50为4.523g/Kg即4523mg/Kg,是抗流感药效学最高给药剂量的56.5倍；腹腔注射的LD50为2.974g/Kg。两者均大于2000mg/Kg,提示本品的安全性较高,为低毒药物。4.广藿香醇的计算机分子模拟研究采用分子模拟的方法对广藿香抗流感有效成分广藿香醇进行类药性预测和相关作用靶点的分子对接研究,为其进一步开发提供参考。ADME预测结果表明,广藿香醇的吸收水平、血浆结合水平、血脑屏障穿透水平均较高,同时其肝毒性低,具有较好的类药性。分子对接结果显示,广藿香醇与乙酰胆碱酯酶的分子对接的分值较高,提示其对乙酰胆碱酯酶有一定的抑制作用,能通过抑制乙酰胆碱酯酶,提高体内乙酰胆碱含量,激活胆碱能抗炎通路而实现抗炎作用。结论：由上述实验结果可知,广藿香各提取部位混合物具有体内抗流感病毒的作用,二氧化碳超临界萃取部位是其抗病毒的有效部位。运用硅胶柱层析等手段从对该部位进行化学成分研究,得到广藿香醇等8个单体化合物。体外细胞抗病毒筛选实验结果证明广藿香有效部位及其主要成分并非通过直接抑制病毒而实现对流感病毒感染小鼠的保护作用。对广藿香醇等单体成分进一步体内筛选,确定广藿香醇具有抑制流感小鼠所致肺炎,延长病毒感染小鼠生存时间的作用,是广藿香抗流感病毒的有效成分。运用ELISA等技术,通过比较给药组与模型组小鼠感染病毒后免疫器官、免疫细胞、细胞因子、SOD和MDA含量变化探讨其作用机理,提示广藿香醇通过调节机体免疫功能、调控体内细胞因子水平、抗氧化等方面减轻免疫损伤,从而达到抗病毒的作用。相关药效学研究表明,广藿香醇具有较强的抗炎、免疫调节作用,而镇痛、镇咳方面的作用不明显。急性毒性实验和ADME预测结果表明广藿香醇的毒性较低、类药性良好。分子对接提示其能通过抑制乙酰胆碱酯酶,提高体内乙酰胆碱含量,激活胆碱能抗炎通路而实现抗炎作用。上述实验结果同时说明了《中国药典》选用广藿香醇作为广藿香质量标准指标性成分的科学依据。本研究以体现中医药作用特点的体内抗病毒实验为主,结合中药化学分离手段,开展岭南地产中药广藿香抗流感病毒有效成分筛选实验。从直接抑制病毒、整体调节、抗氧化等角度探讨有效成分的作用机制,采用计算机模拟的方法推测药物作用的靶点,并开展有效成分对症治疗的药效学研究,为其进一步开发奠定基础,并为中药有效成分的发现和评价研究提供借鉴。更多还原

【Abstract】 Influenza is a kind of acute upper respiratory tract epidemic caused by influenza virus infection, which is characteristized by strong infectivity, fast transmission, repeatedly susceptibility. Nowadays, flu vaccines and antiviral drugs treatments are the two main methods against influenza. However, due to the flu virus’amazing variability and drug resistance, the human society is facing serious threat. Since located in LingNan, Guangdong is not only a high-incidence region of WenBing, but also one of the headstream of the epidemic influenza plague around the world. Therefore physicians of these region have found many effective genuine regional in during the hundreds-of-years plague prevention. Nevertheless, it is the unclear effective components and mechanism of thees LingNan medicinal materials underlying their antivirus activity that has been hindering their promotion and application.Based on the support of National Nature Science Foundation of China (Project No.U0732004), this study was conducted to screen the effective composition that is responsible for Pogostemon cabling’s antivirus activity as well as to explore its mechanism.Objects:1 To verify the anti-influenza property of Pogostemon cablin by antivirus experiment in vivo, thereby finding out the effective part as well as effective compound.2 To investigate the antivirus mechanism of the above effective components in the aspects of cytokine, immunity, and peroxidation damage on the mouse pneumonia model caused by influenza virus.3 To explore the medical properties of the above effective components in aspects of anti inflammation, immunogical regulation, analgesia and cough relieving. Moreover, acute toxicity tests was carried out to find out toxic dose.4, To predict drug likeness of the above effective components by molecular simulation and to prepare the necessary foundation for the drug target searching. Methods and Results:1.Effective components screening of Pogostemon cablin by antivirus experiment in vivoAccording to the clinical medical history and documents of physicians in Lingnan, Pogostemon cablin was selected for screening. Briefly, we prepared different parts of Pogostemon cablin of different polarity by supercritical CO2 fluid extraction and ultrasonic extraction to avoid any change caused by extraction with heating. Together with the all-polarity extrat, which was regroup by the above crude extrat according to their extraction rate, all the above extrats were verified their anti-influenza activity by antivirus experiment in vivo. Results indicated that Pogostemon cablin could inhibit mice pneumonia caused by influenza virus FM1 and prolong the survival time of the infected mice, and the supercritical CO2 fluid extract of Pogostemon cablin was proved to be responsible for this activity.By component separation chromatography,8 compounds were obtained from the supercritical CO2 fluid extract. After structure identification, these compounds were affirmed as epifriedelinol,β-rhamno, patchouli alcohol, oleanolic acid, 3,3’,4’,7-tetramethoxy-5- hydroxyflavone, pelargidenon,patchoulenone and 5 hydroxy-furfural. Although none of the above extracts or components exhibited virus inhibiton effect on influenza virus FM1 directly, patchouli alcohol was proved to have an excellent anti-influenza character according to the in vivo experiment.All the results suggested that patchouli alcohol is the effective compound of Pogostemon cablin, but its anti-virus activity has nothing to do with the direct inhibition on virus.2. Exploration on anti-virus mechanism of Patchouli alcoholIn order to verify the anti-influenza efficacy of patchouli alcohol and to observe the dose-effect relationship, we conducted antivirus experiment in vivo with different doses of patchouli alcohol. Besides, we investigated patchouli alcohol’s anti-virus mechanism in aspects of T lymphocyte subsets regulation, inflammatory factors regulation and anti-oxidation etc.Results showed that patchouli alcohol could be effective against influenza virus pathogenicity, relieving mice’s pneumonia caused by influenza virus and prolonging the survival time of the infected mice with a dose dependent manner. In terms of immune regulation, patchouli alcohol could inhibit the spleen and thymus depauperation of influenza virus infected mice, enhance cellular immunity function. And it was showed that anti-virus efficacy of patchouli alcohol on mice pneumonia caused by influenza virus was closely related with effects of inhibiting the expression of TNF-a at the same time enhancing expression of IL-10, IFN-y in serum. In terms of anti-oxidation, when mice were administered patchouli alcohol for 5 days, activity of SOD was raised while that of MDA was decreased in infected mice’s lung tissue, which meant that the lipid peroxidational damnification was mitigated markedly and the capacity of antioxidant free radical damage was markedly increased. These suggested that patchouli alcohol could effectively reduce lipid peroxidation and enhance antioxidant capacity.3. Pharmacodynamic evaluation on patchouli alcohol in anti-influenza applicationTo investigate whether Patchouli alcohol can alleviate the symptoms of influenza, the pharmacodynamic experiment on anti-inflammation, immunoregulation, analgesia, cough relieving and the acute toxicity experiment were conducted in this part.In aspect of anti-inflammatory activity, patchouli alcohol could inhibit the xylene induced ear swelling and carrageenan induced rat paw edema, and a dose dependent manner was observed, indicating that patchouli alcohol owning the anti-acute inflammation property. Contents of NO, PGE2, TNF-a in edematous paw tissued decreased when comparing with that of the model group. And the contents of three inflammatory factors in high dose group markedly decreased, indicating that patchouli alcohol’s anti-inflammatory effect could be attributed to its inhibition on inflammatory medium and promotion of the inflammatory factors. In aspect of immunoregulation, mice’s under immunity had higher thymus index, spleen index, phasin content and clearance index than model groups after administration of patchouli alcohol, showing humoral immune function and the function of mononuclear phagocyte could be restored by patchouli alcohol treatment. However, patchouli alcohol did not have medical effect on analgesia and cough relieving. The acute toxicity test showed that the LD50 of patchouli alcohol’s intragastric administration was 4.523g/Kg, which was 56.5 times of the hightest administration dosage of anti-influenza virus. The LD50 of intraperitoneal injections is 2.974g/Kg. Both of the LD50 are more than 2000mg/Kg, indicating that patchouli alcohol had high security with low toxicity.4. Computer molecular simulation of patchouli alcoholDrug likeness of patchouli alcohol was predicted by molecular simulation and the molecular docking study provided us basic information for its further development. ADME predicted results showed that the absorption level, plasma combining level, and blood brain barrier penetration level of patchouli alcohol were high, while its liver toxicity was low, suggesting that it has good drug likeness. Molecular docking results show that molecular docking score of patchouli alcohol-cholinesterase was high, suggesting that it has certain inhibiton on cholinesterase, which can inhibit cholinesterase, thereby uplifting acetylcholine content and activating the choline anti-inflammatory pathways to exhibite its anti-inflammation effect.Conclusion:From the above experimental results, it is confirmed that the all polarity extract of patchouli has anti-influenza virus effect, and supercritical fluid CO2 extract is the effective part of its anti-virus property. Among the 8 mocompounds separated by using silica gel chromatography, patchouli alcohol is proved to be the major active ingredient responsible for Pogostemon cablin’s anti-virus activity, which could inhibits pneumonia caused by influenza virus and prolong their survival time, though it couldn’t inhibite the influenza virs directly.After that, we explored the mechanism of Patchouli alcohol by using technologies such as ELSIA and comparing the difference of treatment group and model group mice infected with virus, including immune organs, immune cells, cytokines, SOD and MDA content, which suggested that patchouli alcohol displayed anti-virus activity by regulating immune function, body cytokine levels and antioxidant to reduce the immune damage. Relevant pharmacodynamic studies showed that patchouli alcohol had a strong effect of anti-inflammatory and immune regulation, but effects on analgesic and cough relieving were not obvious. Acute toxicity and ADME prediction results showed that patchouli alcohol is low toxic and have nice chemiotropism. Molecular docking study suggested that it may act as an anti-iflammation drug by inhibiting acetylcholinesterase, increasing acetylcholine levels and activating cholinergic anti-inflammatory pathway.更多还原