摘要
目的:应用超高效液相色谱-四极杆串联飞行时间质谱法(UPLC-Q TOF/MS法)研究马钱子砂烫炮制前后化学差异,并初步评价生品及炮制品的药效及毒性。方法:采用Agilent ZORBAX SB C色谱柱(100 mm×2.1 mm, 1.8μm);以0.1%甲酸水溶液(A)-乙腈溶液(B)为流动相,梯度洗脱;流速0.2 mL·min^(-1);柱温30℃;质谱采用电喷雾离子源,正/负离子模式,信息关联采集模式扫描;扫描范围m/z 0~1 000。应用PeakView软件的Formula Finder、Mass Calculators等功能,结合SciFinder、Reaxys、ChemSpider等在线数据库进行定性分析和差异性分析;利用SIMCA-P软件构建正交偏最小二乘判别分析(OPLS-DA)统计学模型,进行Scatter及S-plot分析;运用MultiQuant软件,采用外标法对马钱子碱、士的宁进行定量分析;最后采用二甲苯耳廓致炎及不同剂量小鼠灌胃实验以对比评价药效毒性。结果:结果显示马钱子的化学成分主要包括生物碱、糖苷、脂肪酸酯和醇类,其中已知成分有29个,首次报道的化合物达20个。OPLS-DA分析中差异最显著的成分是士的宁、1-(4-联苯羰基)-N-(2,3-二氢-1,4-苯并二恶英-6-基)-4-哌啶甲酰胺、绿原酸和油酸。差异性分析中离子强度变化最显著的成分是3,6,9-三恶-12-四氮杂多糖-1-醇、1-脱氧-1-{[1,3-二羟基-2-(羟甲基)-2-丙基]氨基}己醇和甲基苯丙氨酸。定量分析结果显示马钱子碱在砂烫品、生品中含量分别为1.17%、1.21%,士的宁在砂烫品、生品中含量分别为1.35%、1.52%,表明砂烫炮制后马钱子碱和士的宁的含量有所降低,且士的宁的下降幅度略大于马钱子碱,能达到2020年版《中华人民共和国药典》马钱子项下要求(士的宁应为1.20%~2.20%,马钱子碱不得少于0.80%)。初步毒性及药效学实验结果显示当灌胃剂量在0.5 g·kg^(-1)时,砂烫品组无死亡,生品组死亡率为20%;当灌胃剂量在1.0 g·kg^(-1)时,砂烫品组30%死亡,而生品组50%死亡;当灌胃剂量在1.5 g·kg^(-1)时,砂烫品和生品组均100%死亡。采用t检验计算砂烫品组与生品组所测耳肿胀率的P均>0.05,表明砂烫品毒性低于生品,抗炎效果无显著差异。结论:本研究将化学差异和药效毒性差异相结合,证明炮制方法、减毒效果及化学成分间存在显著相关性,探讨认为砂烫法可以起到减毒存效作用,同时有必要对新鉴定出的差异性成分进行分析验证。本研究结果为马钱子炮制机理解释及药效物质基础阐明提供新的思路和数据参考。
Objective: To study the chemical differences on Semen Strychni before and after stir-baking in sandby UPLC-Q TOF/MS, and to evaluate the efficacy and toxicity of raw products and processed products. Methods: Separation was performed on an Agilent ZORBAX SB Ccolumn(100 mm×2.1 mm, 1.8 μm), using 0.1% formic acid aqueous solution(A)-acetonitrile solution(B) as mobile phase in gradient elution at a flow rate of 0.2 mL·min^(-1)and a column temperature of 30 ℃. Electrospray ion source, positive/negative ion modes and information correlation acquisition mode were used for mass spectrometry in ascanning range of m/z 0-1 000. The functions of Formula Finder and Mass Calculators of Peak View software were applied to conduct qualitative analysis and difference analysis in combination with online databases such as SciFinder, Reaxys and Chem Spider. The statistical model of orthogonal partial least squares discriminant analysis was constructed by SIMCA-Psoftware, and Scatter and S-plot analysis were carried out. Brucine and strychnine were quantitatively analyzed by external standard method with Multi Quant software. Finally, auricle inflammation induced by xylene and intragastric administration of different doses of mice were used to compare and evaluate the efficacy and toxicity. Results:The results showed that the chemical constituents of Semen Strychni mainly include alkaloids, glycosides, fatty acid esters and alcohols. Among them, there were 29 known constituents and 20 compounds were reported for the first time. The most significant differences in OPLS-DA analysis were strychnine, 1-(4-biphenylylcarbonyl)-N-(2, 3-dihydro-1, 4-benzodioxin-6-yl)-4-piperidi-necarboxamide, chlorogenic acid and oleic acid. The components with the most significant changes in ionic strength in the difference analysis were 3, 6, 9-trioxa-12-azatetracosan-1-ol, 1-deoxy-1-{[1, 3-dihydroxy-2-(hydroxyl-methyl)-2-pro-panyl]amino}hexitol and methylphenylalanine. The results of quantitative analysis showed that the contents of brucine in sand-baked products and raw products were 1.17% and 1.21% respectively, and the contents of strychnine in sand-baked products and raw products were 1.35% and 1.52% respectively, indicating that the contents of brucine and strychnine decreased after sand baking, and the decrease of strychnine was slightly greater than that of brucine, It could meet the requirements of Semen Strychni in Pharmacopoeia of the people’s Republic of China(2020)(strychnine should be 1.20%-2.20%, and strychnine should be not less than 0.80%).The results of preliminary toxicity and pharmacodynamic tests results showed that when the intragastric dose was 0.5 g·kg^(-1), there was no death in the sand-baked group, and the death rate in the raw product group was 20%. When the intragastric dose was 1.0 g·kg^(-1), 30% of the sand-baked group died, while 50% of the raw product group died. When the intragastric dose was 1.5 g·kg^(-1), 100% of the sand-baked group and the raw product group died.T-test on ear swelling rate measured in sand-baked group and raw product group was performed and the P value was above 0.05. The results showed that the toxicity of sand-baked product was lower than that of raw product, and there was no significant difference in anti-inflammatory effect. Conclusion: This study combineschemical differences with differences in pharmacodynamics and toxicity, and proves that there is a significant correlation between processing methods, attenuation effects and chemical components. The sand-baked method can play a role in attenuating toxicity, and it is necessary to analyze and verify the newly identified differential components. The results of this study provide new ideas and data references for the explanation of the processing mechanism of Semen Strychni and the elucidation of the effective material basis.
作者
秦伟瀚
阳勇
刘翔
李卿
张小梅
胡娟
QIN Wei-han;YANG Yong;LIU Xiang;LI Qin;ZHANG Xiao-mei;HU Juan(Chongqing lnstiute of Traditional Chinese Medicine,Chonging400065,China)
出处
《药物分析杂志》
CAS
CSCD
北大核心
2022年第7期1159-1169,共11页
Chinese Journal of Pharmaceutical Analysis
基金
重庆市卫生健康委项目-马钱子炮制新工艺用于类风湿、骨折的药效学评价研究(2021ZY023651)。
