Study On Preparation Technology And Pharmacodynamics Of Total Bakkenolides From Petasites Tricholobus Franch, Against Cerebral Ischemia

Petasites tricholobus Franch. is plant of Compositae,for our endemic species, widelydistributed in the southwest,northwest.Their rhizome has the effect of detoxification andremoves blood stasis,and was used as a folk medicine for treatment of fracture andsnake-bite.Its flower was utilized as the succedaneum of coltsfoot flower for treatment ofcough.BackgroundPetasites hybridus,a plant belonging to the same genus with Petasites tricholobusFranch.,have been used in abdominal colic and asthma and as mucolytic cough medicinefor hundreds years in Europe.The photochemical investigation of Petasites hybridusshowed the presence of sesquiterpene,pyrrolizidine alkaloids, essential oil and triterpenoid.Modern research confirms experimentally the effect of anti-inflammatory and spasmolyticof sesquiterpene-type compounds.So we presume that Petasites tricholobus Franch. shouldhave the similitude constituent and action.ObjectiveIt is the aim of this work to establishe standard of quality control and confirm theoptimal preparation technology of total bakkenolides from Petasites tricholobusFranch.(PTZNZ).Study the bioactivity on the total bakkenolides from Petasites tricholobusFranch. and specify its pharmacological activity and mechanism of its efficacy, in depth fordeveloping and utilizing the resources of Petasites tricholobus Franch. as well as laying thefoundation for the development of new drugs.Method and resultIn this study, we develop a specific and sensitive method for the analysis of the totalbakkenolides from Petasites tricholobus Franch. by using HPLC method and research thepreparation technology,which contains extractive technics and purification process bymacroporous resin.We confirm the optimal extractive technics as extraction by95%alcohol and absorption by macroporous,eluting with different density alcohol. We get thetotal bakkenolides from Petasites tricholobus Franch. directly by preparation technology. Itincludes bakkenolide-D,bakkenolide-B,bakkenolide-Ⅳa and bakkenolide-Ⅲa,which itscontent is65.9%and metastasis rate is89.3%.This method has the good effect on theseparation of bakkenolide-type compound.Firstly,we evaluate the effects of the bakkenolide-D, bakkenolide-B, bakkenolide-Ⅳa, bakkenolide-Ⅲa and total bakkenolides from the rhizome of Petasites tricholobus Franch.on a model of PC12cells suffered by hypoxia and glucose deprivation, by the methodMTT and LDH.The result of experiment shows that they have protective effect onPC12cells.Then we investigated its neuroprotection in a transient focal cerebralischemia–reperfusion model in rats,and in an in vitro model of ischemia. At doses of5,10and20mg/kg, PTZNZ administered immediately after reperfusion markedly reducedbrain infarct volume and neurological deficits. For primarily cultured neurons suffered1hhypoxia followed by24h reoxygenation, PTZNZsignificantly attenuated cell death andapoptosis. Morphological observation also directly confirmed its protective effect onneuron. These results provided strong pharmacological basis for its potential therapeuticrole in cerebral ischemic illness. Furthermore, we demonstrated that PTZNZ could rescuethe cell injury by Hypoxia via down-regulation NF-κB signaling pathway as well as Aktand ERK1/2activation.PTZNZ suppressed phosphorylation and nuclear translocationofNF-κB/p65, phosphorylation and degradation of inhibitor protein of kB(IκB) as well asthe phosphorylation of IkB-kinase complex (IκK) by western blotting or indirectimmunofluorescence assay. Besides, PTZNZalso inhibited the activation of Akt and theextracellular signal-regulated kinase1/2(ERK1/2).Preliminary acute toxicity test of the total bakkenolides. According to preliminaryexperiment results, we set7dose groups:3.4g/Kg、4g/Kg、4.8g/Kg、5.6g/Kg、6.6g/Kg、7.8g/Kg、9.2g/Kg,10mice per group, One-time oral administration.14days afteradministration, LD50is6.9g/Kg.DiscussionWe just get the total bakkenolides from Petasites tricholobus Franch. directly bypreparation technology. It includes bakkenolide-D,bakkenolide-B,bakkenolide-Ⅳa andbakkenolide-Ⅲa, which its content is65.9%and metastasis rate is89.3%.This method hasthe good effect on the separation of bakkenolide-type compoud.In summary, we demonstrated and confirmed the neuroprotective effect of totalbakkenolides.The neuroprotective mechanism is related with activation of the classicalNF-κB pathway.