Study On The Hydrosoluble Constituents Of Fuzi And The Cardiotoxicity On Zebrafish

By means of recrystallization,silica gel column chromatography,Sephadex LH-20,MCI column chromatography,C₁₈ reversed phase column chromatography,preparative thin layer chromatography,C₁₈ reversed phase semi-preparative high performance liquid chromatography and other chromatographic separation methods,16 compounds were isolated and purified from Radix Aconiti Praeparata（Fuzi）.13 of them have been identified and presented as follows:Songorine（1）,Hetisine（2）,Neoline（3）,Karacoline（4）,BeiwucineIII（5）,N-（2’-β-D-glucopyranosyl-5’-hydroxysalicyl）-4-hydroxyanthranilic acid methyl ester（6）,Adenosine（7）,Adenine（8）,Carnine（9）,Hypoxanthine（10）,Pennogenin（11）,2,3-Furandiol（12）,Benzoic acid（13）.Compounds 14,15,16 were primarily identified as 2 lignans and 1 aromatic glycoside and conjectured as 3 new compounds which are being determined by 2D-NMR spectroscopy.The structure of compound 11 was isolated from Fuzi for the first time and compound 12 was isolated from Fuzi.Alkaloids in seven processing products of Fuzi and the decoction were analyzed by acid dye colorimetry and high performance liquid chromatography.The detoxification characteristics of processing and decocting were expressed,and the impact on alkaloids in Fuzi with different processing methods were analyzed.Results demonstrated the content of diester alkaloids in Fuzi could be significantly reduced by different processing products and the destruction rate was over 95%.The alkaloids in Fuzi may run off after bittern processing and rinsing while the products without bittern processing and rinsing contained more alkaloids.Furthermore,the toxicities of decoction,total alkaloids in decoction and single alkaloids were analyzed on zebrafish（clmc:GFP）.Results demonstrated decoction caused arrhythmia and death on zebrafish,but no significant morphological damage.In same does,the toxicities of total alkaloids were slight lower,causing bradycardia,slight malformation,pericardiac cyst and congestion.The toxicities of single alkaloids were diester diterpene alkaloids>monoester ones≈non-ester ones.The toxicity of diester diterpene alkaloids,mesaconitine>aconitine>hypaconitine,showed significant toxicities on zebrafish,causing serious arrhythmia,and the zebrafish was near death in the highest dose group treated for 24 h.Besides,serious morphological damges were also found in diester diterpene alkaloids groups,including malformation,pericardiac cyst,congestion,atrioventricular disorder and ventricular arrest.On the contrary,monoester ones and non-ester ones showed lower toxicities,mainly causing bradycardia without any morphological damage on zebrafish.Inovations:（1）16 compounds were isolated and purified from the n-butanol part of Fuzi water decoction.13 of them were identified and another 3 were conjectured as new compounds which are being determined by 2D-NMR spectroscopy.The structure of compound 11 was isolated from Fuzi for the first time and compound 12 was isolated from Fuzi.（2）The detoxification characteristics of processing and decocting for a long time were clarified,and the key process influencing the content of alkaloids were considered as bittern processing and rinsing.（3）The cardiotoxicity of decoctions of different processing products of Fuzi and their total alkaloids on zebrafish were reported for the first time.（4）Thecardiotoxicitiesofaconitine,mesaconitine,hypaconitine,14-benzoylaconi-tine,14-benzoylmesaconitine,14-benzoylhypaconitine,Songorine,and Karacoline on zebrafish were reported for the first time.