Preparation Of Anhydrotetrodotoxin And Its Effects On Excitability Of Nerve Trunks

Anhydrotetrodotoxin（anhydroTTX）, the most abundant tetrodotoxin（TTX） analog, is a nature alkaloid active compound, which is closely similar to TTX in structur and pharmacological properties. The main function of anhydroTTX is to block sodium channels and inhibit the propagation of action potential, but anhydroTTX is much weak than TTX and more better bio-security. However there were few repots about the study on pharmacological action of anhydroTTX at home and abroad, one of the reasons is that it is hard to obtain pure and sufficient anhydroTTX for the difficult separation. In present thesis, anhydroTTX and TTX were separated by preparative high performance liquid chromatography-mass spectrometry （Prep-HPLCoMS）, the effects of different chromatogram conditions on the separation of anhydroTTX and TTX were studied, and the best liquid preparative method of anhydroTTX with high yield and high purity was explored ; then the electrophysiological parameters of compound action potential of sciatic nerve isolated from frog were examined so as to compare the effects of anhydroTTX on excitability of nerve trunks with TTX and discuss the pharmacological mechanism of anhydroTTX, which might provided experimental basis for the application of anhydroTTX on Neurophysiology and Biomedicine. The studies in this thesis were summarized as follows:In the liquid preparative experiment, the effects of different revered-phase columns, mobile phases of acetic acid solution in different concentrations, different flow rates of mobile phase, and different sample sizes loaded on the separation of anhydroTTX and TTX were investigated. The optimal chromatogram condition: C₈ column （5μm, 20.0×300mm）; mobile phase: an aqueous solution with 0.02% HAc; flow rate: 40mL/min; sample size: 5mg. The anhydroTTX obtained was identified by MS, NMR and IR spectra. 9.4mg anhydroTTX was separated from 100mg crude toxin by applying this method. Its average yield was 73.8% with purity of 98.3%. In the other part of this thesis, the effects of anhydroTTX and TTX in different concentrations on excitability of nerve trunks isolated from frogs in vitro were studied. As a result, both anhydroTTX and TTX could significantly inhibit action potential; amplitude of first phase, amplitude of second phase, upward slope and downward slope decreased with exposure time or increasing concentration of anhydroTTX and TTX, but latent period extended.These effects of anhydroTTX and TTX on action potential show stime-dependent and dose-dependent. However the toxicity of anhydroTTX was about 0.4%～1% of TTX, and anhydroTTX took effect more rapidly than TTX on action potential.