广藿香抗甲型流感病毒有效成分筛选及评价研究
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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。
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 vivo
According 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 alcohol
In 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 application
To 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 alcohol
Drug 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.
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。
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 vivo
According 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 alcohol
In 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 application
To 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 alcohol
Drug 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.
引文
[1]雷达,艾立新,邓红樱,等.流感大流行特征启示甲型H1N1流感防控策略[J].中国国境卫生检疫杂志,2009,32(3)：145-150.
[2]李龙芸,王孟昭.流感的流行特征[J].中国临床医学,2001,8(5)：580-581.
[3]许晶瑶.流行性感冒、人禽流感10年回顾[J].传染病信息,2008,21(1)：7-9.
[4]董志珍,姚登福.流感病毒致病机制的研究进展[J].现代医药卫生,2003,19(4)：406.
[5]Leahy MB, Dobbyn HC, Brownlee GG. Hairpin loopstructure in the 3 arm of the influenza A virus virionRNA pmmoter is required for endonuclease activity[J]. J Virol,2001,75:7042.
[6]李智,但汉雄,邴飞虹.中医药抗流感病毒研究概况与展望[J].湖北中医杂志,2009,31(5)：60-62
[7]Yoshikawa T,Suzuki Y,Nomoto A,et al.Antibody responses and protection against influenza virus infection in different congenic strains of mice immunized intranasally with adjuvant combined A/Beijing/262/95(H1N1)virus hemagglutinin or neuraminidase[J].Vaccine,2002,21(1):60-66.
[8]闫若潜,杜向党.流感病毒基因组结构及其编码蛋白研究进展[J].动物医学进展,2004,25(1)：32-35
[9]张钫,张舒.甲型流感病毒的变异及其跨物种传播[J].国外医药(抗生素分册),2010,31(2)：63-67
[10]蒲秀瑛.贯叶连翘提取物对甲型流感病毒的作用及免疫调节机制的研究[D].甘肃农业大学,2010
[11]Glaser L,Stevens J,Zamarin D,et al.A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity[J].J Virol,2005,79:11533-11536.
[12]Fang R,Min J W,Huylebroeck,et al.Complete structure of A/duck/Ukraine/63 influenza hemagglutinin gene:animal virus as progenitor of human H3 Hong Kong 1968 influenza hemagglutinin[J].Cell,1981,25:315-323.
[13]Kawaoka Y,Krauss S, Webster R G.Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics[J].J Virol,1989,63:4603-4608.
[14]Scholtissek C,Rohde W.On the origin of the human influenza virus subtypes H2N2 and H3N2[J]. Virology,1978,87:13-20.
[15]张文彤,姜庆五.全球历年人甲型流感病毒H3A1抗原的分子进化研究[J].中华流行病学杂志,2005,26(11)：843-847.
[16]肖东楼.中国流感与人禽流感预防控制[J].中华流行病学杂志,2005,26(11)：837-838.
[17]Katz JM.The impact of avian influenza viruses on public health [J].Avian Dis,2003,47(Suppl3):S914-S920.
[18]潘曌曌,王雪峰.流感病毒感染机制及中西医防治研究现状[J].中医儿科杂志2010,20(24)：40474-4048
[19]Stenbauer D A.Role of bemagglutinin cleavage for the pathogenicity of influenza virus[J]. Virol, 1999,258:1-20.
[20]Skebel J J.Wileg D C.Receptor binding and membrane fusion in virus entry:the influenza bemagglutinin [J]. Annu Rev Biochem,2000,69:531-569.
[21]金奇.分子生物学[M].北京：科学出版社,2001：633-656.
[22]Takizawa T, Matsukawa S, Higuchi Y, et al. Induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells [J]. J Gen Virol,1993,74(11):2350-2355.
[23]Earnshaw W C, Martins L M, Kaufmann S H.Mammalian caspases:structure, activation, substrates, and function during apoptosis [J]. Annu Rev Biochem,1999,68:383-424.
[24]Lu Y,Wambach M, Katze M G, et al.Binding the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the eIF-2 translation initiation factor [J].Virology,1995,214(1):222-228.
[25]杨鹏辉,杨潞芳,王希良.流感病毒呼吸道黏膜感染免疫防御机制的研究进展[J].国际免疫学杂志.2006,29(5)：305-309
[26]Rimmelzwaan GF, Boon AC, Geelhoed2MierasMM, et al. Human airway ep ithelial cells p resent antigen to influenza virus2specific CD8+CTL inefficiently after incubation with viral p rotein together with ISCOMATR IX. Vaccine,2004,22 (21-22):2769-2775.
[27]Robinson K, Chamberlain LM, Lopez MC, et al. Mucosal and cellular immune responses elicited by recombinant Lactococcus lactis strains exp ressing tetanus toxin fragment C. Infect Immun,2004, 72(5):2753-2761.
[28]Mutsch M, Zhou W, Rhodes P, et al. Use of the inactivated intranasal influenza vaccine and the risk of Bellps palsy in Switzerland. N Engl J Med,2004,350 (9):8962903.
[29]Greenbaum E, Engelhard D, Levy R, et al. Mucosal (SIgA) and serum (IgG) immunologic responses in young adults following intranasal administration of one or two doses of inactivated, trivalent anti-influenza vaccine. Vaccine,2004,22 (20):2566-2577.
[30]巴德年主编.当代免疫学技术与应用[M].北京医科大学、北京协和医科大学联合出版社,1998,第一版,49-71.
[31]毕爱华主编.免疫分子(三)-细胞因子,医学免疫学[M].人民军医出版社,1995,73.
[32]李洪霞.白介素-10对急性肺损伤的保护作用[J].国外医学呼吸系统分册,2000,20(2)：94-96.
[33]Pajkrt D,Camoglio L,Tiel-van Buul MC,et al.Attenuation of proinflammatory response by recombinnant human IL-10 in human endotoxemia:effect of timing of recombinant human IL-10 administration[J].J Immunol,1997,158:3971.76
[34]洪新,郭振辉,毛宝龄.白细胞介素-10和地塞米松对人外周血单个核细胞核因子-κ B活化及肿瘤坏死因子产生的影响[J].解放军医学杂志,1999,24(5)：317-320.
[35]De Waal Malefyt R,Abrams J,Bennett B,et al.Interleukin 10(IL-10)inhibits cytokine synthesis by human macrophage:an autoregulatory role of IL-10 produced by monocytes[J].J Exp Med,1991,174:1209-1220.
[36]Hurme M,Lahdenpohja N,Santtila S.Gene polymorphisms of interleukins 1 and 10 in infectious and autoimmune diseases[J].Ann Med,1998,30:469-473.
[37]Salom D。, Hill Bo Ro,lear J。 D。,et al.,pH-Dependent tetranmerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus[J].Bicochemistry 2000, 39(46):14160-14170.
[38]Saito Ro, Sakai T。, Sato I。 et al. Frequency of amantadine-resistant influenza A viruses during two seasons featuring co-circulation of H1N1 and H3N2 [J].J Clin Micrubiol,2003, 41(5):2164-2165.
[39]Astrahan P。, Kass I。 Cooper M。 A。, et al.。 A novel method of resistance for influenza against a channel--blocking antiviraldrug[J]. Proteins,2004,55(2):251-257
[40]Englund, Janet A.。 Antiviral therapy of influenza[J].Semin Pediatr Infect Dis,2002,13(2): 120-128.
[41]徐颂,贺星,田红,等.抗流感病毒药物研究进展[J].现代药物与临床,2009,24(2)：65-70.
[42]刘颂.抗流感病毒药物的研究进展[J].国外医学药学分册,2005,32(2)：111-115
[43]何明俭.病毒唑治疗病毒性上呼吸道感染30例疗效观察[J].广州医药,1996,27：62
[44]吴俊东,朱凤才.流感疫苗研究的进展[J].江苏预防医学,2008,19(2)：83-86
[45]王婷,林绍强,姚崇舜,等.莪术油制剂研究进展[J].医药导报,2008,27(5)：578-580
[46]严银芳,陈晓,杨小清,等.石香薷挥发油对流感A3病毒的抑制作用[J].微生物学杂质,2002,122(1)：32-33.
[47]赵文华,石任兵,刘斌,等.连翘病毒清胶囊抗病毒有效部位化学成分的研究[J].中成药,2005,27(4)：44.
[48]冯书晓,陈文,冯俊阳,等.抗流感病毒植物药有效部位筛选研究概况[J].中国中医药信息,2005,12(11)：101～102
[49]冯煦,王鸣,赵友谊,等.北柴胡茎叶总黄酮抗流感病毒的作用[J].植物资源与环境学报,2002,11(4)：15～18.
[50]Utsunomiya T,Kobayashi M,Pollard RB,et al. Glycyrrhizin, anactive component of licorice roots,reduces morbidity and mortality of mice infected with lethal doses of influenza virus [J]. Antimicrob Agents Chemother,1997,41(3):551.
[51]樊宏伟.苦参碱类生物碱的体外抑菌、抑病毒及诱生干扰素的实验研究[J].中医药信息,2002,(4)：75～76.
[52]姚月梅,张英艳,陈国静,等.人参果汁对小鼠免疫功能的影响[J].中医药学报,2002,30(4)：142.
[53]李丽娅,凌秋,崔洪波,等.黄芪多糖抗流感病毒的实验研究[J].中国中医药科技,2002,9(6)：354～355.
[54]肖美添,杨军玲,林海英,等.紫菜多糖的提取及抗流感病毒活性研究[J].福州大学学报(自然科学版),2003,31(5)：631-635
[55]胡兴昌,郑伟强.板蓝根粗提液抑制流感病毒的实验研[J].上海师范大学学报(自然科学版),2003,32(1)：2.
[56]张大志,易井海.板蓝根提取液抗流感病毒的药效学试[J].药学研讨,2001,10(5)：51.
[57]刘思贞,祝希娴,邵玉芹,等.板蓝根抗流感病毒有效部的筛选[J].中草药,1999,30(9)：650.
[58]谭家风,黄薇薇,李三红,等.抗病毒口服液药效学研究[J].中国药科大学学报,2001,32(5)：388.391
[59]石钺,石任兵,刘斌,等.银翘散抗流感病毒有效部位群化学成分的分离与鉴定[J].中国中药,2003,28(1)：43.
[60]朱明,王桂珍,董洁,等.双黄口服液抗流感病毒的实验研究[J].中药,1995,17(9)：32-34.
[61]段泾云,于利森,陈瑞明,等.柴连口服液药理作用研究[J].中国实验方剂学杂志,1998,4(3)：22-25
[62]李建国小儿热速清口服液对FMl病毒感染小鼠的保护性实验及呼吸、免疫器官病理观察[J].77河南中医,1991,(2)：35-38.
[63]张丽,郑虎占,郭元吉,等.黄芪赤风口服液在狗肾传代细胞(MDCK)上抗流感病毒活性的研究[J].北京中医药大学学报,1998,21(1)：28-29.
[64]刘新春,王连山,梁济乐,等.退热Ⅲ号的抑菌、抗病毒药效学研究[J].成都中医药大学学报,2000,23(4)：35-36.
[65]谢鸣,郭元吉,安乃莉.中药咳宁冲剂抗流感病毒作用的实验研究[J].中药新药与临床药 理,1997,8(3)：144-146.
[66]龚镇奎,林乔,冯玲玲,等.肺炎喘嗽1号口服液抗病毒效果实验研究[J].湖北预防医学杂志,1996,7(2)：3-4.
[67]欧敏,杜怀棠,郭元吉,等.清肺饮抗小鼠流感病毒性肺炎的实验研究[J].中华实验和临床病毒学杂志,1996,10(4)：381-383.
[68]欧敏,董建华,段蕴铀.清肺饮对流感病毒感染小鼠免疫功能的调节作用[J].北京中医药大学学报,1998,21(6)：19-22.
[69]刘菲菲,何士勤.穿琥宁抑制H3N2病毒诱导细胞凋亡的研究[J].江西医药,2001,36(3)：185-87.
[70王彦云,陈朝,李多娇,等.流感病毒对鼠肺免疫细胞释放活性氧的影响及药物的调节作用[J].中国中医药科技,2001,8(4)：215-216.
[71]邹政权.牛蒡子甙元复方体内抗流感病毒作用及其机制研究[D].广州中医药大学：广州中医药大学,2007.
[72]王飞.感毒清口崩片防治呼吸道病毒感染药效及免疫机理研究[D].成都中医药大学：成都中医药大学,2006.
[73]张艳丽.毒热平注射液体外抗流感病毒作用机制的实验研究[D].北京中医药大学：北京中医药大学,2008.
[74]庞佶.抗流感病毒天然产物的筛选及其抗病毒活性的评价[D].中国人民解放军军事医学科学院：中国人民解放军军事医学科学院,2009.
[75]潘沅.蒿芩清胆汤抗流感病毒性肺炎湿热证及其信号转导通路的研究[D].南方医科大学：南方医科大学,2009.
[76]陈元生.珍稀南药广藿香的研究进展[J].广东农业科学,2007,1：109-112
[77]国家药典委员会.中华人民共和国药典[M],一部2005年版,北京：化学工业出版社
[78]中国医学科学院药用植物资源开发研究所.中药志第四册(第二版)[M].,北京：人民卫生出版社,1998：128.
[79]徐鸿华,詹若挺.广东中药材生产的规范化、产业化建设[J].世界科学技术一中医药现代化,2003,5(2)：50-54.
[80]徐颂军,王晓峰,徐祥浩,等.药用植物广藿香的品种分类探讨[J].华南师范大学学报(自然科学版),2003(1)：82-86.
[81]李薇,潘超美,宋力飞,等.不同产地广藿香花的观察比较[J].中药材,2003,26(2)：79-81.
[82]罗集鹏,刘玉萍,冯毅凡,等.广藿香的两个化学型及产地与采收期对其挥发油成分的影响[J].药学学报,2003,38(4)：307-310.
[83]李薇,魏刚,潘超美,等.广藿香药材挥发油及主要成分含量影响因素的考察[J].中国中药杂志,2004,29(1)：28-31.
[84]冯承浩,姚辉,吴鸿,等.广藿香药用部位成熟结构及有效成分分布研究[J].中草药,2003,34(2)：174-176.
[85]罗集鹏,冯毅凡,郭晓玲.石牌藿香的挥发油成分分析[J].中草药,2001,32(4)：299-302.
[86]罗集鹏,冯毅凡,郭晓玲.广藿香根与根茎挥发油成分研究[J].天然产物研究与开发,2000,12(4)：66-70.
[87]关玲,权丽辉,徐丽珍,等.广藿香化学成分的研究[J].中国中药杂志,1994,19(6)：355-356.
[88]张强,李章万,朱江粤,等.广藿香挥发油成分的分析[J].华西药学杂志,1996,11(4)：249-250.
[89]刘廷礼,邱琴,崔兆杰,等.气相色谱一质谱联用分析广藿香挥发油成分含量[J].中草 药,1999,30(12)：903-904.
[90]黄丽莎,吴惠勤,张桂英,等.广藿香超临界032萃取产物的Gc—MS分析[J].分析测试学报,2001,20(4)：79-81.
[91]张广文,马祥全,苏镜娱,等.广藿香中的黄酮类化合物[J].中草药,2001,32(10)：871-874.
[92]罗集鹏,吴忠.不同产地广藿香宏量与微量元素分析[J].中药材,2001,24(12)：869-870.
[93]张家明,郑学勤,孙雪飘.广藿香体细胞培养植株再生的研究[J].热带作物学报,1994,15(1)：73-77.
[94]肖省娥,贺红,徐鸿华.广藿香组织培养与植株再生研究[J].中药材,2001,24(6)：391-392.
[95]杜勤,王振华,徐鸿华,等.石牌广藿香试管苗的研制[J].中国中药杂志,2002,27(3)：179-181.
[96]张家明,孙雪飘,郑学勤.抗菌肽B、D双基因表达载体的构建及转化广藿香的研究[J].热带作物学报,1997,18(1)：50-57.
[97]潘超美,李薇,贺红,等.不同栽培居群广藿香的种内遗传多样性研究[J].中国中药杂志,2006,31(9)：723-726.
[98]曹柳英,李劲平,梁瑞燕,等.不同产地广藿香的RAPD分析[J].中药新药与临床药理,2006,17(3)：209-211.
[99]罗集鹏,曹晖,刘玉萍,等.广藿香与土藿香的DNA序列分析及其分子鉴别[J].药学学报,2002,37(9)：739-742.
[100]孔赓星.广藿香酮分离方法的改进[J].中草药,1984,15(3)：26.
[101]黄静.藿香正气颗粒剂的薄层色谱检测[J].中国药学杂志,1997,32(6)：365.
[102]魏刚,符红,工淑英,等.GC-MS法建立广藿香挥发油指纹特征图谱研究[J].中成药,2002,24(6)：407-410.
[103]张铭光,袁敏,袁鹏,等.广藿香裂解色谱指纹图谱及其聚类分析[J].中草药,2003,34(8)：749-752.
[104]袁旭江,霍务贞,朱盛山,等.广藿香HPLC指纹图谱的初步研究[J].中药新药与临床药理,2006,17(3)：195-197.
[105]刘玉萍,罗集鹏,冯毅凡,等.广藿香的基因序列与挥发油化学型的相关性分析[J].药学学报,2002,37(4)：304-308
[106]陈小夏,何冰,李显奇,等.广藿香三种提取物对肠道功能作用的比较[J].中药药理与临床,1998,14(2)：31-33.
[107]陈小夏,何冰,李显奇,等.广蕾香胃肠道药理作用[J].中药材,1998,21(9)：465.467.
[108]杜一民,陈汝筑,胡本荣.广藿香的化学成分及其药理作用研究进展[J].中药新药与Ⅰ临床药理,1998,9(4)：238
[109]朱金照,冷恩仁,桂先勇,等.白术、藿香等中药对胃排空、肠推进影响的实验研究[J]中国中医基础医学杂志,2000,6(1)：21.23.
[110]陆茵,陈文星,孟政杰,等.加味藿香正气软胶囊调节胃肠运动功能[J].中药新药与临床药理,2003,14(6)：381-383.
[111]杨国汉,胡德耀,戴裕光,等.藿香正气液及其组方药物对大鼠胃排空和肠推进作用的影响[J],实用中医药杂志,2005,21(9)：521-523.
[112]朱金照,张捷,张志坚.藿香提取液对大鼠小肠一氧化氮合酶分布的影响[J].福建医药杂志,2002,24(3)：99.100.
[113]Ichikawa K, Kinoshita T, Sankawa U.The screening of Chinese crude drugs for Ca "antagonist activity;identifcationof active principles from the aerial part of Pogostemo cablinand the fruits of Prunusmume[J].Chem Pharm Bull,1989,37(2):345-348.
[114]谢肆聪,唐方.藿香对肢体缺血一再灌注模型大鼠肠屏障功能保护作用的机理研究[J].中国中药杂志,2004,29(5)：61-63.
[115]刘琥琥,罗集鹏,赖沛炼.广东高要与吴川产广蕾香提取物对肠道致病菌抗菌作用的比较研究[J].中药材,1999,22(8)：408.410.
[116]罗超坤.广藿香水提物的抗菌实验研究[J].中药材,2005,28(8)：700—-701.
[117]苏镜娱,张广文,李核,等.广藿香精油化学成分分析与抗菌活性研究[J],中草药,2001,32(3)：204.205.
[118]杨得坡.藿香和广藿香挥发油对皮肤癣菌和条件致病真菌的抑制作用[J].中国药学杂志,2000,35(1)：10-11.
[119]刘爱如,于宗渊,吕丽莉,等.广藿香挥发油对青蒿酯钠抗伯氏疟原虫的增效作用和对抗青蒿酯钠伯氏疟原虫的逆转抗性作用[J].中国寄生虫学与寄生虫病杂志,2000,18(2)：76.78.
[120]任守忠,靳德军,张俊清,刘明生.广藿香药理作用研究进展[J].中国现代中药,2006,(08).
[121]刘世增,杨吉成,顾振纶.香菊感冒冲剂的药理作用研究[J].中成药,1997,19(3)：25.
[122]高相雷,熊盛,王一飞,等.广藿香三种有效部位体外抗柯萨奇病毒B3作用的初步研究[J].中成药,2009,32(5)：261-264
[123]江苏新医学院.中药大词典[M].上海：上海人民出版社,1977：2701.2715.
[124]莫小路,严振,王玉生,等.广藿香精油对植物病原真菌的抑茵活性研究[J].中药材,2004,27(11)：805.807.
[125]王洋.广藿香中新的具抗锥虫活性的倍半萜氢过氧化物[J].国外医学·中医中药分册,2005,27(6)：1495-1496.
[126]解字环,沈映君,纪广亮,等.香附、藿香挥发油抗炎、镇痛、解热作用的实验研究[J].四川生理科学杂志,2005,27(3)：37-39.
[127]齐珊珊,胡丽萍,陈文娜,等.广藿香叶挥发油对小鼠免疫调节作用的实验研究[J].中医药学刊,2009,4：
[128]吴周和,刘建成,吴小刚,等.藿香中天然防腐剂的提取方法及其抑菌作用研究[J]中国调味品,2004,8：18
[129]罗集鹏,冯毅凡,郭晓玲.不同采收期对广藿香产量及挥发油成分的影响[J].中药材.2001,24(5)：316-317.
[130]刘青,江波,于宗渊.气相色谱法测定广藿香挥发油中广藿香醇的含量[J].山东医药.2007,47(2)：3-3.
[131]唐克轩,王明月,唐岳立.利用pts基因和RNA干扰ads基因提高青蒿广藿香醇含量的方法：中国；200910050782.9.2009-12-09.
[132]黄晓舞,白林,徐风华,吴永杰.广藿香醇对β-淀粉样蛋白神经毒性的抑制作用[J].解放军药学学报.2008,24(4)：338-340.
[133]黄晓舞,栾复新,徐风华.百秋李醇在制备防治老年性痴呆症的药物中的用途：中国；200710176344.8.2008-06-11.
[134]陈立敏,吕秋军,Satoshi Inoue,卞广兴,陈振花,温利青.基于报告基因的雌激素受体β亚型激动剂细胞筛选模型的建立[J].药学学报,2006,(08).
[135]Mirko Weide,Anja Schloesser,Bockmuehl,Andreas Bolte,Roland Breves.Agents agains microorganisms containing patchouli oil.,patchouli alcohol and/or the derivatives thereof:United States;US 2006/0134239A1.2006-6-22.
[136]Kongkathip,N.;Sam-ang, P.;Kongkathip, B. Pankaew, Y.;Tanasombat, M.;Udomkusonsri, P. Development of Patchouli Extraction with Quality Control and Isolation of Active Compounds with Antibacterial Activity. Kasetsart J. (Nat. Sci.) 2009,43,519-525.
[137]米尔科·魏德,安雅·施洛瑟,迪尔克·博克米尔,安德烈亚斯·伯尔特,罗兰德·布雷韦斯.含有广藿香油,广藿香醇和/或其衍生物的抗微生物试剂：德国；20048002000.2.2006-08-23.
[138]王洋.广藿香中新的具抗锥虫活性的倍半萜氢过氧化物[J].国外医学中医中药分册.2005,27(6)：358-358.
[139]任守忠,张俊清,刘明生.广藿香醇的急性毒性实验.[J]海南医学院学.2009,15(12)：1503-1504.
[140]Bang;Guy Ourisson.Hydroxylation of patchoulol by rabbits,hemi-synthesis of nor-patchoulenol, the odour carrier of patchouli oil. Tetrahedron letter 1975,26,2211-2214.
[141]杨甫传,徐丽珍,邹忠梅,杨世林.广藿香醇及广藿香油中广藿香醇在大鼠体内药代动力学比较.药学学报.2004,39(9)：726-729.
[142]黄烈军,穆淑珍,张建新,邓彬,宋智琴,郝小江.中药广藿香非挥发性化学成分的研究[J].中国中药杂志.2009,34(4)：410-413
[143]Fattorossi A,Battaglia A,Buzzonetti A,et al. Thymopoiesis,Reg-ulatory T Cells,and TCR-beta Expression in Thymoma With andWithout Myasthenia Gravis,and Modulatory Effects of SteroidTherapy[J] J Clin Immunol,2008,28 (2):194-206.
[144]黄晓舞,刘睿婷,吕秋军.广藿香醇对东莨菪碱致记忆障碍小鼠学习记忆功能的影响[J].中草药,2009,40(9)：1431-1433
[145]孙汶生.医学免疫学[M].北京：人民卫生出版社.2005
[146]Pajkrt D, Camoglio L, Tiel-van Buul M C, et al. Attenuation of proinflammatory response by recombinnant human IL-10 in human endotoxemia:effect of timing of recombinant human IL-10 administration.Immunol,1997,158:3971.
[147]Waal Malefyt R, Abrams J, Bennett B, et al. Interleukin 10 (IL-10) inhibits cytokine synthesis by human macrophage:an autoregulatory role of IL-10 produced by monocytes J Exp Med,1991, 174:1209.
[148]周炳荣.IFN-γ及TNF调节单核吞噬细胞超氧阴离子及在抗感染中的作用[J].国外医学(免疫学分册),1994,(02).
[149]Oda T, Akaike T, Namamoto T, et al. Oxygen radicals in influenza-induced pathogenesis and treatment with pyran polymer-conjugated SOD.Science,1989,244:974-976.
[150]Akaike, T, Noguchi Y, Ijiri S, et al. Pathogenesis of influenza virus-inrusinduced pneumonia: involvement of both nitric oxide and oxygen radicals.Proc Nalt Acad Sci U S A,1996,93:2488.
[151]陈奇,主编.中药药理研究方法学[M].北京：人民卫生出版社,1993.
[152]Oprea T I. Virtual screening in lead discovery:Aviewpoint[J].Molecules,2002,7(1):51.
[153]Schneider G. Virtual screening:an endless staircase[J].Nature Reviews Drug Discovery,2010, (9):273.
[154]Valentin A. Pavlov. Brain acetylcholinesterase activity controls systemic cytokine levels through the cholinergic anti-inflammatory pathway. Brain, Behavior, and Immunity 23 (2009):41-45
[155]朱伟,姚丽梅.肿瘤坏死因子-α中药小分子抑制剂的计算机虚拟筛选[J].中国实验方剂学杂志.2010,16(13)：199-202.
[156]马岳峰.胆碱酯酶抑制剂加兰他敏对脓毒症炎症反应的作用及其机制研究[J].浙江大学,浙江大学,2009
[2]李龙芸,王孟昭.流感的流行特征[J].中国临床医学,2001,8(5)：580-581.
[3]许晶瑶.流行性感冒、人禽流感10年回顾[J].传染病信息,2008,21(1)：7-9.
[4]董志珍,姚登福.流感病毒致病机制的研究进展[J].现代医药卫生,2003,19(4)：406.
[5]Leahy MB, Dobbyn HC, Brownlee GG. Hairpin loopstructure in the 3 arm of the influenza A virus virionRNA pmmoter is required for endonuclease activity[J]. J Virol,2001,75:7042.
[6]李智,但汉雄,邴飞虹.中医药抗流感病毒研究概况与展望[J].湖北中医杂志,2009,31(5)：60-62
[7]Yoshikawa T,Suzuki Y,Nomoto A,et al.Antibody responses and protection against influenza virus infection in different congenic strains of mice immunized intranasally with adjuvant combined A/Beijing/262/95(H1N1)virus hemagglutinin or neuraminidase[J].Vaccine,2002,21(1):60-66.
[8]闫若潜,杜向党.流感病毒基因组结构及其编码蛋白研究进展[J].动物医学进展,2004,25(1)：32-35
[9]张钫,张舒.甲型流感病毒的变异及其跨物种传播[J].国外医药(抗生素分册),2010,31(2)：63-67
[10]蒲秀瑛.贯叶连翘提取物对甲型流感病毒的作用及免疫调节机制的研究[D].甘肃农业大学,2010
[11]Glaser L,Stevens J,Zamarin D,et al.A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity[J].J Virol,2005,79:11533-11536.
[12]Fang R,Min J W,Huylebroeck,et al.Complete structure of A/duck/Ukraine/63 influenza hemagglutinin gene:animal virus as progenitor of human H3 Hong Kong 1968 influenza hemagglutinin[J].Cell,1981,25:315-323.
[13]Kawaoka Y,Krauss S, Webster R G.Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics[J].J Virol,1989,63:4603-4608.
[14]Scholtissek C,Rohde W.On the origin of the human influenza virus subtypes H2N2 and H3N2[J]. Virology,1978,87:13-20.
[15]张文彤,姜庆五.全球历年人甲型流感病毒H3A1抗原的分子进化研究[J].中华流行病学杂志,2005,26(11)：843-847.
[16]肖东楼.中国流感与人禽流感预防控制[J].中华流行病学杂志,2005,26(11)：837-838.
[17]Katz JM.The impact of avian influenza viruses on public health [J].Avian Dis,2003,47(Suppl3):S914-S920.
[18]潘曌曌,王雪峰.流感病毒感染机制及中西医防治研究现状[J].中医儿科杂志2010,20(24)：40474-4048
[19]Stenbauer D A.Role of bemagglutinin cleavage for the pathogenicity of influenza virus[J]. Virol, 1999,258:1-20.
[20]Skebel J J.Wileg D C.Receptor binding and membrane fusion in virus entry:the influenza bemagglutinin [J]. Annu Rev Biochem,2000,69:531-569.
[21]金奇.分子生物学[M].北京：科学出版社,2001：633-656.
[22]Takizawa T, Matsukawa S, Higuchi Y, et al. Induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells [J]. J Gen Virol,1993,74(11):2350-2355.
[23]Earnshaw W C, Martins L M, Kaufmann S H.Mammalian caspases:structure, activation, substrates, and function during apoptosis [J]. Annu Rev Biochem,1999,68:383-424.
[24]Lu Y,Wambach M, Katze M G, et al.Binding the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the eIF-2 translation initiation factor [J].Virology,1995,214(1):222-228.
[25]杨鹏辉,杨潞芳,王希良.流感病毒呼吸道黏膜感染免疫防御机制的研究进展[J].国际免疫学杂志.2006,29(5)：305-309
[26]Rimmelzwaan GF, Boon AC, Geelhoed2MierasMM, et al. Human airway ep ithelial cells p resent antigen to influenza virus2specific CD8+CTL inefficiently after incubation with viral p rotein together with ISCOMATR IX. Vaccine,2004,22 (21-22):2769-2775.
[27]Robinson K, Chamberlain LM, Lopez MC, et al. Mucosal and cellular immune responses elicited by recombinant Lactococcus lactis strains exp ressing tetanus toxin fragment C. Infect Immun,2004, 72(5):2753-2761.
[28]Mutsch M, Zhou W, Rhodes P, et al. Use of the inactivated intranasal influenza vaccine and the risk of Bellps palsy in Switzerland. N Engl J Med,2004,350 (9):8962903.
[29]Greenbaum E, Engelhard D, Levy R, et al. Mucosal (SIgA) and serum (IgG) immunologic responses in young adults following intranasal administration of one or two doses of inactivated, trivalent anti-influenza vaccine. Vaccine,2004,22 (20):2566-2577.
[30]巴德年主编.当代免疫学技术与应用[M].北京医科大学、北京协和医科大学联合出版社,1998,第一版,49-71.
[31]毕爱华主编.免疫分子(三)-细胞因子,医学免疫学[M].人民军医出版社,1995,73.
[32]李洪霞.白介素-10对急性肺损伤的保护作用[J].国外医学呼吸系统分册,2000,20(2)：94-96.
[33]Pajkrt D,Camoglio L,Tiel-van Buul MC,et al.Attenuation of proinflammatory response by recombinnant human IL-10 in human endotoxemia:effect of timing of recombinant human IL-10 administration[J].J Immunol,1997,158:3971.76
[34]洪新,郭振辉,毛宝龄.白细胞介素-10和地塞米松对人外周血单个核细胞核因子-κ B活化及肿瘤坏死因子产生的影响[J].解放军医学杂志,1999,24(5)：317-320.
[35]De Waal Malefyt R,Abrams J,Bennett B,et al.Interleukin 10(IL-10)inhibits cytokine synthesis by human macrophage:an autoregulatory role of IL-10 produced by monocytes[J].J Exp Med,1991,174:1209-1220.
[36]Hurme M,Lahdenpohja N,Santtila S.Gene polymorphisms of interleukins 1 and 10 in infectious and autoimmune diseases[J].Ann Med,1998,30:469-473.
[37]Salom D。, Hill Bo Ro,lear J。 D。,et al.,pH-Dependent tetranmerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus[J].Bicochemistry 2000, 39(46):14160-14170.
[38]Saito Ro, Sakai T。, Sato I。 et al. Frequency of amantadine-resistant influenza A viruses during two seasons featuring co-circulation of H1N1 and H3N2 [J].J Clin Micrubiol,2003, 41(5):2164-2165.
[39]Astrahan P。, Kass I。 Cooper M。 A。, et al.。 A novel method of resistance for influenza against a channel--blocking antiviraldrug[J]. Proteins,2004,55(2):251-257
[40]Englund, Janet A.。 Antiviral therapy of influenza[J].Semin Pediatr Infect Dis,2002,13(2): 120-128.
[41]徐颂,贺星,田红,等.抗流感病毒药物研究进展[J].现代药物与临床,2009,24(2)：65-70.
[42]刘颂.抗流感病毒药物的研究进展[J].国外医学药学分册,2005,32(2)：111-115
[43]何明俭.病毒唑治疗病毒性上呼吸道感染30例疗效观察[J].广州医药,1996,27：62
[44]吴俊东,朱凤才.流感疫苗研究的进展[J].江苏预防医学,2008,19(2)：83-86
[45]王婷,林绍强,姚崇舜,等.莪术油制剂研究进展[J].医药导报,2008,27(5)：578-580
[46]严银芳,陈晓,杨小清,等.石香薷挥发油对流感A3病毒的抑制作用[J].微生物学杂质,2002,122(1)：32-33.
[47]赵文华,石任兵,刘斌,等.连翘病毒清胶囊抗病毒有效部位化学成分的研究[J].中成药,2005,27(4)：44.
[48]冯书晓,陈文,冯俊阳,等.抗流感病毒植物药有效部位筛选研究概况[J].中国中医药信息,2005,12(11)：101～102
[49]冯煦,王鸣,赵友谊,等.北柴胡茎叶总黄酮抗流感病毒的作用[J].植物资源与环境学报,2002,11(4)：15～18.
[50]Utsunomiya T,Kobayashi M,Pollard RB,et al. Glycyrrhizin, anactive component of licorice roots,reduces morbidity and mortality of mice infected with lethal doses of influenza virus [J]. Antimicrob Agents Chemother,1997,41(3):551.
[51]樊宏伟.苦参碱类生物碱的体外抑菌、抑病毒及诱生干扰素的实验研究[J].中医药信息,2002,(4)：75～76.
[52]姚月梅,张英艳,陈国静,等.人参果汁对小鼠免疫功能的影响[J].中医药学报,2002,30(4)：142.
[53]李丽娅,凌秋,崔洪波,等.黄芪多糖抗流感病毒的实验研究[J].中国中医药科技,2002,9(6)：354～355.
[54]肖美添,杨军玲,林海英,等.紫菜多糖的提取及抗流感病毒活性研究[J].福州大学学报(自然科学版),2003,31(5)：631-635
[55]胡兴昌,郑伟强.板蓝根粗提液抑制流感病毒的实验研[J].上海师范大学学报(自然科学版),2003,32(1)：2.
[56]张大志,易井海.板蓝根提取液抗流感病毒的药效学试[J].药学研讨,2001,10(5)：51.
[57]刘思贞,祝希娴,邵玉芹,等.板蓝根抗流感病毒有效部的筛选[J].中草药,1999,30(9)：650.
[58]谭家风,黄薇薇,李三红,等.抗病毒口服液药效学研究[J].中国药科大学学报,2001,32(5)：388.391
[59]石钺,石任兵,刘斌,等.银翘散抗流感病毒有效部位群化学成分的分离与鉴定[J].中国中药,2003,28(1)：43.
[60]朱明,王桂珍,董洁,等.双黄口服液抗流感病毒的实验研究[J].中药,1995,17(9)：32-34.
[61]段泾云,于利森,陈瑞明,等.柴连口服液药理作用研究[J].中国实验方剂学杂志,1998,4(3)：22-25
[62]李建国小儿热速清口服液对FMl病毒感染小鼠的保护性实验及呼吸、免疫器官病理观察[J].77河南中医,1991,(2)：35-38.
[63]张丽,郑虎占,郭元吉,等.黄芪赤风口服液在狗肾传代细胞(MDCK)上抗流感病毒活性的研究[J].北京中医药大学学报,1998,21(1)：28-29.
[64]刘新春,王连山,梁济乐,等.退热Ⅲ号的抑菌、抗病毒药效学研究[J].成都中医药大学学报,2000,23(4)：35-36.
[65]谢鸣,郭元吉,安乃莉.中药咳宁冲剂抗流感病毒作用的实验研究[J].中药新药与临床药 理,1997,8(3)：144-146.
[66]龚镇奎,林乔,冯玲玲,等.肺炎喘嗽1号口服液抗病毒效果实验研究[J].湖北预防医学杂志,1996,7(2)：3-4.
[67]欧敏,杜怀棠,郭元吉,等.清肺饮抗小鼠流感病毒性肺炎的实验研究[J].中华实验和临床病毒学杂志,1996,10(4)：381-383.
[68]欧敏,董建华,段蕴铀.清肺饮对流感病毒感染小鼠免疫功能的调节作用[J].北京中医药大学学报,1998,21(6)：19-22.
[69]刘菲菲,何士勤.穿琥宁抑制H3N2病毒诱导细胞凋亡的研究[J].江西医药,2001,36(3)：185-87.
[70王彦云,陈朝,李多娇,等.流感病毒对鼠肺免疫细胞释放活性氧的影响及药物的调节作用[J].中国中医药科技,2001,8(4)：215-216.
[71]邹政权.牛蒡子甙元复方体内抗流感病毒作用及其机制研究[D].广州中医药大学：广州中医药大学,2007.
[72]王飞.感毒清口崩片防治呼吸道病毒感染药效及免疫机理研究[D].成都中医药大学：成都中医药大学,2006.
[73]张艳丽.毒热平注射液体外抗流感病毒作用机制的实验研究[D].北京中医药大学：北京中医药大学,2008.
[74]庞佶.抗流感病毒天然产物的筛选及其抗病毒活性的评价[D].中国人民解放军军事医学科学院：中国人民解放军军事医学科学院,2009.
[75]潘沅.蒿芩清胆汤抗流感病毒性肺炎湿热证及其信号转导通路的研究[D].南方医科大学：南方医科大学,2009.
[76]陈元生.珍稀南药广藿香的研究进展[J].广东农业科学,2007,1：109-112
[77]国家药典委员会.中华人民共和国药典[M],一部2005年版,北京：化学工业出版社
[78]中国医学科学院药用植物资源开发研究所.中药志第四册(第二版)[M].,北京：人民卫生出版社,1998：128.
[79]徐鸿华,詹若挺.广东中药材生产的规范化、产业化建设[J].世界科学技术一中医药现代化,2003,5(2)：50-54.
[80]徐颂军,王晓峰,徐祥浩,等.药用植物广藿香的品种分类探讨[J].华南师范大学学报(自然科学版),2003(1)：82-86.
[81]李薇,潘超美,宋力飞,等.不同产地广藿香花的观察比较[J].中药材,2003,26(2)：79-81.
[82]罗集鹏,刘玉萍,冯毅凡,等.广藿香的两个化学型及产地与采收期对其挥发油成分的影响[J].药学学报,2003,38(4)：307-310.
[83]李薇,魏刚,潘超美,等.广藿香药材挥发油及主要成分含量影响因素的考察[J].中国中药杂志,2004,29(1)：28-31.
[84]冯承浩,姚辉,吴鸿,等.广藿香药用部位成熟结构及有效成分分布研究[J].中草药,2003,34(2)：174-176.
[85]罗集鹏,冯毅凡,郭晓玲.石牌藿香的挥发油成分分析[J].中草药,2001,32(4)：299-302.
[86]罗集鹏,冯毅凡,郭晓玲.广藿香根与根茎挥发油成分研究[J].天然产物研究与开发,2000,12(4)：66-70.
[87]关玲,权丽辉,徐丽珍,等.广藿香化学成分的研究[J].中国中药杂志,1994,19(6)：355-356.
[88]张强,李章万,朱江粤,等.广藿香挥发油成分的分析[J].华西药学杂志,1996,11(4)：249-250.
[89]刘廷礼,邱琴,崔兆杰,等.气相色谱一质谱联用分析广藿香挥发油成分含量[J].中草 药,1999,30(12)：903-904.
[90]黄丽莎,吴惠勤,张桂英,等.广藿香超临界032萃取产物的Gc—MS分析[J].分析测试学报,2001,20(4)：79-81.
[91]张广文,马祥全,苏镜娱,等.广藿香中的黄酮类化合物[J].中草药,2001,32(10)：871-874.
[92]罗集鹏,吴忠.不同产地广藿香宏量与微量元素分析[J].中药材,2001,24(12)：869-870.
[93]张家明,郑学勤,孙雪飘.广藿香体细胞培养植株再生的研究[J].热带作物学报,1994,15(1)：73-77.
[94]肖省娥,贺红,徐鸿华.广藿香组织培养与植株再生研究[J].中药材,2001,24(6)：391-392.
[95]杜勤,王振华,徐鸿华,等.石牌广藿香试管苗的研制[J].中国中药杂志,2002,27(3)：179-181.
[96]张家明,孙雪飘,郑学勤.抗菌肽B、D双基因表达载体的构建及转化广藿香的研究[J].热带作物学报,1997,18(1)：50-57.
[97]潘超美,李薇,贺红,等.不同栽培居群广藿香的种内遗传多样性研究[J].中国中药杂志,2006,31(9)：723-726.
[98]曹柳英,李劲平,梁瑞燕,等.不同产地广藿香的RAPD分析[J].中药新药与临床药理,2006,17(3)：209-211.
[99]罗集鹏,曹晖,刘玉萍,等.广藿香与土藿香的DNA序列分析及其分子鉴别[J].药学学报,2002,37(9)：739-742.
[100]孔赓星.广藿香酮分离方法的改进[J].中草药,1984,15(3)：26.
[101]黄静.藿香正气颗粒剂的薄层色谱检测[J].中国药学杂志,1997,32(6)：365.
[102]魏刚,符红,工淑英,等.GC-MS法建立广藿香挥发油指纹特征图谱研究[J].中成药,2002,24(6)：407-410.
[103]张铭光,袁敏,袁鹏,等.广藿香裂解色谱指纹图谱及其聚类分析[J].中草药,2003,34(8)：749-752.
[104]袁旭江,霍务贞,朱盛山,等.广藿香HPLC指纹图谱的初步研究[J].中药新药与临床药理,2006,17(3)：195-197.
[105]刘玉萍,罗集鹏,冯毅凡,等.广藿香的基因序列与挥发油化学型的相关性分析[J].药学学报,2002,37(4)：304-308
[106]陈小夏,何冰,李显奇,等.广藿香三种提取物对肠道功能作用的比较[J].中药药理与临床,1998,14(2)：31-33.
[107]陈小夏,何冰,李显奇,等.广蕾香胃肠道药理作用[J].中药材,1998,21(9)：465.467.
[108]杜一民,陈汝筑,胡本荣.广藿香的化学成分及其药理作用研究进展[J].中药新药与Ⅰ临床药理,1998,9(4)：238
[109]朱金照,冷恩仁,桂先勇,等.白术、藿香等中药对胃排空、肠推进影响的实验研究[J]中国中医基础医学杂志,2000,6(1)：21.23.
[110]陆茵,陈文星,孟政杰,等.加味藿香正气软胶囊调节胃肠运动功能[J].中药新药与临床药理,2003,14(6)：381-383.
[111]杨国汉,胡德耀,戴裕光,等.藿香正气液及其组方药物对大鼠胃排空和肠推进作用的影响[J],实用中医药杂志,2005,21(9)：521-523.
[112]朱金照,张捷,张志坚.藿香提取液对大鼠小肠一氧化氮合酶分布的影响[J].福建医药杂志,2002,24(3)：99.100.
[113]Ichikawa K, Kinoshita T, Sankawa U.The screening of Chinese crude drugs for Ca "antagonist activity;identifcationof active principles from the aerial part of Pogostemo cablinand the fruits of Prunusmume[J].Chem Pharm Bull,1989,37(2):345-348.
[114]谢肆聪,唐方.藿香对肢体缺血一再灌注模型大鼠肠屏障功能保护作用的机理研究[J].中国中药杂志,2004,29(5)：61-63.
[115]刘琥琥,罗集鹏,赖沛炼.广东高要与吴川产广蕾香提取物对肠道致病菌抗菌作用的比较研究[J].中药材,1999,22(8)：408.410.
[116]罗超坤.广藿香水提物的抗菌实验研究[J].中药材,2005,28(8)：700—-701.
[117]苏镜娱,张广文,李核,等.广藿香精油化学成分分析与抗菌活性研究[J],中草药,2001,32(3)：204.205.
[118]杨得坡.藿香和广藿香挥发油对皮肤癣菌和条件致病真菌的抑制作用[J].中国药学杂志,2000,35(1)：10-11.
[119]刘爱如,于宗渊,吕丽莉,等.广藿香挥发油对青蒿酯钠抗伯氏疟原虫的增效作用和对抗青蒿酯钠伯氏疟原虫的逆转抗性作用[J].中国寄生虫学与寄生虫病杂志,2000,18(2)：76.78.
[120]任守忠,靳德军,张俊清,刘明生.广藿香药理作用研究进展[J].中国现代中药,2006,(08).
[121]刘世增,杨吉成,顾振纶.香菊感冒冲剂的药理作用研究[J].中成药,1997,19(3)：25.
[122]高相雷,熊盛,王一飞,等.广藿香三种有效部位体外抗柯萨奇病毒B3作用的初步研究[J].中成药,2009,32(5)：261-264
[123]江苏新医学院.中药大词典[M].上海：上海人民出版社,1977：2701.2715.
[124]莫小路,严振,王玉生,等.广藿香精油对植物病原真菌的抑茵活性研究[J].中药材,2004,27(11)：805.807.
[125]王洋.广藿香中新的具抗锥虫活性的倍半萜氢过氧化物[J].国外医学·中医中药分册,2005,27(6)：1495-1496.
[126]解字环,沈映君,纪广亮,等.香附、藿香挥发油抗炎、镇痛、解热作用的实验研究[J].四川生理科学杂志,2005,27(3)：37-39.
[127]齐珊珊,胡丽萍,陈文娜,等.广藿香叶挥发油对小鼠免疫调节作用的实验研究[J].中医药学刊,2009,4：
[128]吴周和,刘建成,吴小刚,等.藿香中天然防腐剂的提取方法及其抑菌作用研究[J]中国调味品,2004,8：18
[129]罗集鹏,冯毅凡,郭晓玲.不同采收期对广藿香产量及挥发油成分的影响[J].中药材.2001,24(5)：316-317.
[130]刘青,江波,于宗渊.气相色谱法测定广藿香挥发油中广藿香醇的含量[J].山东医药.2007,47(2)：3-3.
[131]唐克轩,王明月,唐岳立.利用pts基因和RNA干扰ads基因提高青蒿广藿香醇含量的方法：中国；200910050782.9.2009-12-09.
[132]黄晓舞,白林,徐风华,吴永杰.广藿香醇对β-淀粉样蛋白神经毒性的抑制作用[J].解放军药学学报.2008,24(4)：338-340.
[133]黄晓舞,栾复新,徐风华.百秋李醇在制备防治老年性痴呆症的药物中的用途：中国；200710176344.8.2008-06-11.
[134]陈立敏,吕秋军,Satoshi Inoue,卞广兴,陈振花,温利青.基于报告基因的雌激素受体β亚型激动剂细胞筛选模型的建立[J].药学学报,2006,(08).
[135]Mirko Weide,Anja Schloesser,Bockmuehl,Andreas Bolte,Roland Breves.Agents agains microorganisms containing patchouli oil.,patchouli alcohol and/or the derivatives thereof:United States;US 2006/0134239A1.2006-6-22.
[136]Kongkathip,N.;Sam-ang, P.;Kongkathip, B. Pankaew, Y.;Tanasombat, M.;Udomkusonsri, P. Development of Patchouli Extraction with Quality Control and Isolation of Active Compounds with Antibacterial Activity. Kasetsart J. (Nat. Sci.) 2009,43,519-525.
[137]米尔科·魏德,安雅·施洛瑟,迪尔克·博克米尔,安德烈亚斯·伯尔特,罗兰德·布雷韦斯.含有广藿香油,广藿香醇和/或其衍生物的抗微生物试剂：德国；20048002000.2.2006-08-23.
[138]王洋.广藿香中新的具抗锥虫活性的倍半萜氢过氧化物[J].国外医学中医中药分册.2005,27(6)：358-358.
[139]任守忠,张俊清,刘明生.广藿香醇的急性毒性实验.[J]海南医学院学.2009,15(12)：1503-1504.
[140]Bang;Guy Ourisson.Hydroxylation of patchoulol by rabbits,hemi-synthesis of nor-patchoulenol, the odour carrier of patchouli oil. Tetrahedron letter 1975,26,2211-2214.
[141]杨甫传,徐丽珍,邹忠梅,杨世林.广藿香醇及广藿香油中广藿香醇在大鼠体内药代动力学比较.药学学报.2004,39(9)：726-729.
[142]黄烈军,穆淑珍,张建新,邓彬,宋智琴,郝小江.中药广藿香非挥发性化学成分的研究[J].中国中药杂志.2009,34(4)：410-413
[143]Fattorossi A,Battaglia A,Buzzonetti A,et al. Thymopoiesis,Reg-ulatory T Cells,and TCR-beta Expression in Thymoma With andWithout Myasthenia Gravis,and Modulatory Effects of SteroidTherapy[J] J Clin Immunol,2008,28 (2):194-206.
[144]黄晓舞,刘睿婷,吕秋军.广藿香醇对东莨菪碱致记忆障碍小鼠学习记忆功能的影响[J].中草药,2009,40(9)：1431-1433
[145]孙汶生.医学免疫学[M].北京：人民卫生出版社.2005
[146]Pajkrt D, Camoglio L, Tiel-van Buul M C, et al. Attenuation of proinflammatory response by recombinnant human IL-10 in human endotoxemia:effect of timing of recombinant human IL-10 administration.Immunol,1997,158:3971.
[147]Waal Malefyt R, Abrams J, Bennett B, et al. Interleukin 10 (IL-10) inhibits cytokine synthesis by human macrophage:an autoregulatory role of IL-10 produced by monocytes J Exp Med,1991, 174:1209.
[148]周炳荣.IFN-γ及TNF调节单核吞噬细胞超氧阴离子及在抗感染中的作用[J].国外医学(免疫学分册),1994,(02).
[149]Oda T, Akaike T, Namamoto T, et al. Oxygen radicals in influenza-induced pathogenesis and treatment with pyran polymer-conjugated SOD.Science,1989,244:974-976.
[150]Akaike, T, Noguchi Y, Ijiri S, et al. Pathogenesis of influenza virus-inrusinduced pneumonia: involvement of both nitric oxide and oxygen radicals.Proc Nalt Acad Sci U S A,1996,93:2488.
[151]陈奇,主编.中药药理研究方法学[M].北京：人民卫生出版社,1993.
[152]Oprea T I. Virtual screening in lead discovery:Aviewpoint[J].Molecules,2002,7(1):51.
[153]Schneider G. Virtual screening:an endless staircase[J].Nature Reviews Drug Discovery,2010, (9):273.
[154]Valentin A. Pavlov. Brain acetylcholinesterase activity controls systemic cytokine levels through the cholinergic anti-inflammatory pathway. Brain, Behavior, and Immunity 23 (2009):41-45
[155]朱伟,姚丽梅.肿瘤坏死因子-α中药小分子抑制剂的计算机虚拟筛选[J].中国实验方剂学杂志.2010,16(13)：199-202.
[156]马岳峰.胆碱酯酶抑制剂加兰他敏对脓毒症炎症反应的作用及其机制研究[J].浙江大学,浙江大学,2009
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