Extraction And Purification Prcoess,Contents Determination Of Water-soluble Alkaloids From Aconiti Lateralis Radix Praeparata And Comparative Analysis Of Chemical Components In Fresh Fuzi From Different Regions

Objective（1）Research the process of extraction and purification of water-soluble alkaloids of Aconiti Lateralis Radix Praeparata（Fuzi）.（2）Establish a quantitative method for simultaneous determination of the most five kinds of alkaloids contents in the extract of water-soluable alkaloids from Fuzi and Zheng fupian.（3）Compare and analyze the chemical constituents in different regions,to expolre the distribution law of the alkaloids and find the characteristic components which can discriminate these samples.Methods（1）Orthogonal design L₉（3⁴）and single factor experiments were employed to optimize the extraction conditions using UPLC-MS/MS.The total extraction yield of 14 alkaloids（karacoline,songorine,fuziline,neoline,talatisamine,mesaconine,aconine,hypaconine,benzoylmesaconine,benzoylaconine,benzoylhypaconine,mesaconitine,aconitine,hypaconitine）was used as an index.The absorption-desorption characteristics of five macroporous resins were evaluated to optimize purification conditions using contents of eight water-soluble alkaloids（aconine,mesaconine,hypaconine,fuziline,neoline,talatisamine,songorine,karacoline）as indexes.（2）HPLC-ELSD was adopted in the assay.The analysis was performed on an Agilent Extend C₁₈8 column（250 mm×4.6mm,5μm）,the mobile phase of acetonitrile（A）-0.5%（v/v）triethylamine solution（B）with gradient elution（0-8 min,10%A→20%A;8-15 min,20%A→25%A;15-25min,25%A→25%A;25-30 min,25%A→28%A;30-31 min,28%A→60%A;31-42 min,60%A-65%A）,the flow rate of 1.0 mL·min^-1,the column temperature of 35℃,and the evaporative light scattering detector（ELSD）was selected as the detector(the temperature of evaporation of 50℃,the flow rate of carrier gas of 1.5 L·min^-1,and the gain was 1).（3）HPLC coupled with ELSD was adopted to construct the HPLC-ELSD fingerprint of Fuzi from different regions,and quantitative analyze simultaneously of ester-alkaloids and water-soluable alkaloids,then,multivariative statistical analysis methods,including similarity analysis（SA）,principal hierarchical cluster analysis（HCA）,component analysis（PCA）,partial least-squares discriminate analysis（PLS-SD）and orthogonal partial least-squares discriminate analysis（OPLS-DA）,were used to compare and analyze the similarity and difference of 42 fuzi samples from four regions.Results（1）The optimized extraction and purification conditions were as follows:Zheng Fupian were decocted thrice with 10-fold pH 3.5 acidic water for two hour each time（maintaining pH 3.5）.The alkalinity of water decoction was adjusted to pH 10.0 with 20%NaOH and then heated for two hour.The supernatant was adjusted to pH 11.0 by 20%NaOH and then was subjected to macroporous resin HPD 300 whose relative adsorption amount was 2.5 g dried medicinal herb/mL resin,and then the resin was eluted with 6 BV of water and 4 BV of 80%ethanol respectively.The 80%ethanol fraction was evaporated under vacuum to give the water-soluble alkaloids extract.（2）Five alkaloids（mesaconine,hypaconine,fuziline,neoline,songorine）were completely separated under the above condition.Satisfactory linearities of mesaconine,hypaconine,fuziline,neoline,songorine were in the range of 50.01-500.08,30.00-300.02,30.01-300.13,50.01-500.07,and 15.00-150.04μg.mL^-1 respectively（r≥0.9991）,and the average recoveries（n=6）were 98.20%（RSD=1.92）,98.30%（RSD=1.58）,98.48%（RSD=1.26）,97.15%（RSD=1.28）and 97.72%（RSD=1.69）,respectively.（3）The typical characteristics of Fuzi from Jiangyou are that the contents of neoline and hypaconitine are relatively high,while the contents of fuziline and mesaconitine are relatively low.The typical characteristics of Fuzi from Yunan are that the contents of mesaconitine and fuziline are relatively high,while the contents of hypaconitine and neoline are relatively low.Results of SA suggested that 1)the similarity values of Fuzi samples from same product regions were above 0.78,2)Fuzi samples from Jiangyou have a relatively high degree of similarity with Shannxi,while a relatively low degree of similarity with Yunnan.Results of HCA suggested the tested samples were categorized into four groups.PLS-DA indicated that benozylmesaconine,mesaconitine,songorine and neoline are the characteristic components to distinguish the Fuzi samples from four regions.OPLS-DA demonstrated that neoline may be a key component to distinguish Fuzi of Jiangyou from other regions.Conclusion（1）The optimized extraction and purification process is stable and feasible,and this present study provides the reference for the research and application of water-soluble alkaloids in Fuzi.（2）The method is simple and accurate,and provides a reliable approach for the quality control and evaluation of water-soluble alkaloids extract from Fuzi.It is also a new reference method for the determination of water-soluble alkaloid components of Aconiti Lateralis Radix Praeparata slice.（3）There is a significant diffiernce in distridution law of alkaloids form four regions.HPLC-ESLD fingerprinting,coupled with determination of content and chemometric analysis,is a powerful and practical tool for full-scale quality analysis,species igentifiction of Fuzi.