Regulation Of Cerebral Permeation Activated By Photosensitizers From TCM And Analgesia Mechanism Of Peptides From Snake Venom

With high biological activity and good central effect, venom neurotoxin is an importantprobe to study neurotransmitter’s generation and molecular mechanisms of its transmissionprocess in nervous system. However，venom neurotoxin are macromolecules and cannot passthrough blood-brain barrier（BBB）, then the expected effect cannot be achieved by peripheraladministration. Therefore, it has important clinical value and practical significance thatno-invasive change permeability of central drugs. Guangdong is located in south China withabundant resources and high yields of snakes and this project will not only open up newresources for analgesic drugs，but also promote the development of the pharmaceuticalindustry in Guangdong.In this experiment，neurotoxin was obtained by separation and purification from crudevenom of Chinese cobra，which was selected as raw material. And traditional Chinesemedicine photosensitizer was used to mediate BBB to open for neurotoxin entering the centraltissues to promote its absorption. Its transmission and distribution were researched，and itsanalgesic mechanism was discussed. Methods and results were as follows：(1) Pheophorbide a extracted from traditional Chinese medicine silkworm excrementwith acetone. This method has the advantages of simple operation，low cost, high extractionrate and high product purity. The operation process of extraction was as follows：Thesilkworm excrement was extracted by acetone and the concentrate was transferred to dissolvein ether, and then separated by30%hydrochloric acid to make chlorophyll removemagnesium. Pheophorbide a was obtained by precipitation under condition of the pH2.5to3.0.The extraction rate was0.66%and the purity was97.9%.(2) Separation and purification of crude venom from Chinese cobra were carried out byCM-Agrose ion exchange chromatography，gel filtration chromatography Sephadex G-50andSephadex G-25column, including gathering chromatographic peaks and freeze-drying. Thepurified product was identified as neurotoxin (CNT) by toxicity test, and the extraction ratewas3.51%. Its molecular weight was7989Da determinated by SDS-PAGE. Analgesic activityof CNT was studied with acetic acid writhing method and hot plate method，and the resultsshowed that CNT had the analgesic effects， and especially the groups of CNT and pheophorbide a with illumination had better analgesic effects.(3) Central pain animal model was established by excitotoxic spinal cordinjury(ESCI)induced by quisqualic acid. Changes of reactive oxygen species(ROS) indifferent brain regions were determined after intraperitoneal injection with pheophorbide acoordinated with CNT in weak laser irradiation. The results indicated that neurotoxin did haveeffects on the brain regions; under light excitation, pheophorbide a promoted the neurotoxintowork on the brain.（4）In this research，CNT isolated by ion exchange, gel filtrate ion chromatographyfrom Chinese cobra has the significant analgesic effect. By ESCI model experiments，pheophorbide a is confirmed to promote CNT brain permeability and analgesic effects oncentral tissues. Its analgesic mechanism may be related to ROS.