| 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.
|
PDF Full Text Request