蟾蜍类药材中吲哚生物碱和蟾毒配基类成分的含量

Content of indole alkaloids and bufadienolides contained in toad medicines

目的:建立蟾蜍类药材中吲哚生物碱和蟾毒配基类成分的HPLC含量测定法,并对蟾酥、蟾皮、蟾衣中2类成分进行分析。方法:吲哚生物碱类,Nucleosil C18柱,乙腈-0.5%磷酸二氢钾溶液(6∶94,磷酸调pH 3.2)为流动相,流速0.8 mL.min-1,检测波长275 nm,柱温30℃;蟾毒配基类,Alltima C18柱,乙腈(A)-0.3%乙酸溶液(B)为流动相,梯度洗脱(0～15 min,28%～54%A;15～35 min,54%A),流速0.6 mL.min-1,检测波长296 nm,柱温30℃。结果:五羟色胺、N-甲基五羟色胺、N,N-二甲基五羟色胺、N,N,N-三甲基五羟色胺、蟾蜍噻咛依次在0.079 6～0.796,0.097 2～1.945,0.074 4～0.744,0.103～2.05,0.067 2～0.672μg线性关系良好;五羟色胺和N-甲基五羟色胺的平均加样回收率依次为98.6%和91.3%;日蟾毒它灵、蟾毒它灵、蟾毒灵、华蟾酥毒基、脂蟾毒配基依次在0.004 83～0.614,0.007 9～1.006,0.007 95～1.016,0.009 7～1.24,0.009 6～1.22μg线性关系良好;平均加样回收率依次为101.6%,102.5%,101.0%,99.1%,98.9%。结论:蟾酥中吲哚生物碱和蟾毒配基类成分含量均较高,蟾皮中含量次之,蟾衣中含量最低甚至检测不到。

Objective： To kinds of establish a HPLC method for determining contents of indole alkaloids and bufadienolides contained in toad medicines, and analyze two kinds of components contained in toad venom, toad skin and toad periostracum. Meth- od： As for alkaloids, Nucleosil Cls column was adopted with acetonitrile and water containing 0. 5% potassium dihydrogen phosphate （6： 94, adjust pH to 3.2 with phosphate acid） as the mobile phase. The flow rate was 0. 8 mL · min-1 , the detection wavelength was 275 nm, and the column temperature was 30 ℃. As for bufadienolides, Alltima Cls column was adopted with acetonitrile and water containing 0. 3% acetic acid （B） as the mobile phase. The gradient process was as follows： a linear gradient from 28% to 54% aceto- nitrile in the first 15 rain, then kept at 54% for additional 20 min. The flow rate was 0. 6 mL · min-1 , the detection wavelength was 296 nm, and the column temperature was 30 ℃. Result： The linear ranges were 0. 079 6-0. 796 p.g for serotonin, 0. 097 2-1. 945 ug for N-methylserotonin, 0. 074 4-0. 744 ug for N, N-dimethylserotonin, 0. 103-2. 05ug for N, N, N-trimethylserotonin, and 0. 067 2- 0. 672 ug for bufothionine, respectively. The average recoveries of serotonin and N-methylserotonin were 98. 6% and 91.3%, respec- tively. The linear ranges of gamabufotalin, bufotalin, bufalin, cinobufagin and resibufogenin were 0. 004 83-0. 614, 0. 007 9-1. 006, 0. 007 95-1. 016, 0. 009 7-1.24 and 0. 009 6-1.22 p,g, respectively, and their average recoveries were 101.6% , 102. 5% , 101.0% , 99. 1% and 98. 9% , respectively. Conclusion： Toad venom has the highest contents of indole alkaloids and bnfadienolides, followed by toad skin, and toad periostracum showed the lowest contents and even no detection result.