基于UHPLC-DAD技术测定附子生品及炮制品酯类生物碱含量及急性毒性评价

Quantitative Analysis and Acute Toxicity Evaluation of Alkaloids in Aconiti lateralis radix.Praeparata.and It's Processed Products Based on UHPLC-DAD Technology

目的 利用UHPLC-DAD建立附子生品及炮制品中6种酯类生物碱成分定量的方法,并测定其在不同煎煮时间水提物中的含量,以便对中草药材附子的急性毒性进行监测和评估。方法 采用Waters ACQUITY BEH C18色谱柱(100 mm×2.1 mm, 1.7μm)来进行梯度洗脱,流动相A为5 mmol/L乙酸铵+0.1%甲酸水溶液,流动相B为乙腈:甲醇(v/v:1/3);流速为0.2 mL/min;检测波长235 nm;柱温35℃。结果 建立了附子生品及炮制品水提物在不同的煎煮时间内6种酯类生物碱成分含量同时测定的方法。6种酯类生物碱成分都表现出良好的线性(R2≥0.99),并且稳定性、精密度和重复性良好,RSD值均小于3.00%,回收率为94.8%~103.6%,并发现炮制后附子双酯型生物碱含量降低;之后采用急性毒性试验进行验证。对于KM小鼠,生附片水提物灌胃LD50为51.783 g生药/kg,蜜附片和黑顺片水提物MFD分别为80 g生药/kg和160 g生药/kg。结论 附子生品及各炮制品中6种成分含量均有所差异,对正常小鼠都具有一定的急性毒性,但炮制后毒性显著降低,其中用蜂蜜炮制能够降低附子的毒性,为附子临床用药及相关经典名方开发提供参考。

Objective To create a quantitative technique for 6 ester alkaloids in Aconiti lateralis radix.Praeparata.UHPLC-DAD prepared and raw products were examined to ascertain the contents at varying decocting times,as well as to contrast the acute toxicity of aconite.To evaluate and observe the acute toxicity of Chinese herbal medicine aconite,monitoring is essential.Methods The gradient elution was performed on a Waters ACQUITY BEH C18 column.The mobile phase A was 5 mmol/L ammonium acetate+0.1%formic acid solution aqueous,and the mobile phase B was acetonitrile:methanol(v/v:1/3).The flow rate was 0.2 mL/min,while the detection wavelength was set at 235 nm and the column temperature was 35℃.Results A method was developed for the simultaneous determination of the contents of six ester alkaloid components in the raw and concocted products of Aconiti lateralis radix.Praeparata.All six ester alkaloid components showed good linearity(R2≥0.99)and good stability,precision and reproducibility,with RSD values less than 3.00%and recoveries ranging from 94.8%~103.6%.The content of diester alkaloids in aconite was decreased after processing,and then verified by acute toxicity test.The LD50 of KM mice given water extract of Shengfupian was 51.783 g crude drug/kg,and the MFD of KM mice given water extract of Mifupian and Heishunpian were 80 g crude drug/kg and 160 g crude drug/kg,respectively.Conclusion There are some differences in the 6 components of Aconiti lateralis radix.Praeparata.raw and prepared products,and they all have certain acute toxicity to normal mice,and the toxicity is significantly reduced after processing,which indicates that processing with honey can reduce the toxicity of Aconiti lateralis radix.Praeparata.,and provide reference for the clinical use of Aconiti lateralis radix.Praeparata.and the development of related classic prescriptions.