HPLC法测定化风丹药母在发酵过程中6个酯型生物碱的含量

目的：为控制化风丹药母的稳定性，探究化风丹药母在发酵过程中6个酯型生物碱的含量变化。方法：采用高效液相色谱法，选用色谱柱：Wondasil^? C₁₈（4.6 mm×250 mm，5 μm），流动相：以0.1 mol·L^-1乙酸铵溶液-乙腈-四氢呋喃，梯度洗脱，流速1.0 mL·min^-1，检测波长235 nm，柱温30℃。结果：在此色谱条件下，6个酯型生物碱成分可达到较好分离，苯甲酰新乌头碱，苯甲酰乌头碱，苯甲酰次乌头碱，次乌头碱，新乌头碱，乌头碱进样量分别在0.086 48~1.383 68 μg、0.032 4~0.518 4 μg、0.027 44~0.439 04 μg、0.094 24~1.507 84 μg、0.084 48~1.351 68 μg、0.022 8~0.364 8 μg范围内呈现较好的线性关系，精密度、稳定性、重复性均较稳定，平均加样回收率分别为97.1%、96.3%、95.2%、94.4%、93.4%、94.4%。发酵6周后，单酯型总生物碱含量变化为4.52%~7.36%，双酯型总生物碱含量变化为12.40%~6.35%。结论：通过发酵后，单酯型生物碱含量有所增加，双酯型生物碱含量有所降低，化风丹药母毒性降低。

Objective: To study content change of six ester alkaloids in Huafengdan during fermentation in order to investigate the stability of Huafengdan Yaomu. Methods: HPLC was used to determine the contents of alkaloids. Analysis was performed on a Wondasil^? C₁₈ (4.6 mm×250 mm,5 μm) column with mobile phase consisting of 0.1 mol·L^-1 ammonium acetate solution,acetonitrile and tetrahydrofuran with gradient elution at a flow rate of 1.0 mL·min^-1. The detection wavelength was 235 nm and the column temperature was 30℃. Results: Six ester alkaloids could be separated well. The linear ranges of benzoylneoaconitine,benzoylaconitine,benzoylhypoaconitine,neoaconitine,aconitine and aconitine were 0.086 48-1.383 68 μg,0.032 4-0.518 4 μg, 0.027 44-0.439 04 μg,0.094 24-1.507 84 μg,0.084 48-1.351 68 μg and 0.022 8-0.364 8 μg,respectively. The precision,stability and repeatability were good,and the average recoveries were 97.1%,96.3%,95.2%,94.4%,93.4% and 94.4%,respectively. After 6 weeks' fermentation,the contents of monoester alkaloids and diester alkaloids changed from 4.52% to 7.36% and 12.40% to 6.35%,respectively. Conclusions: After fermentation,the contents of monoester alkaloids increased,and the contents of diester alkaloids decreased,which reduced the toxicity of the Yaomu.

[1] 徐文峰,李怡,金鹏飞,等.化风丹的研究进展[J].中南药学,2017,15(8):1100 XU WF,LI Y,JIN PF,et al.Research progress of Huafengdan[J].Cent South Pharm,2017,15(8):1100
[2] 曹国琼,张永萍,徐剑,等.化风丹中As₂S₂及As₂O₃的含量测定[J].中国实验方剂学杂志,2015,21(7):54 CAO GQ,ZHANG YP,XU J,et al.Determination of As₂S₂ and As₂O₃ in Huafengdan[J].Chin J Exper Form,2015,21(7):54
[3] 田兴中,付中祥,隋怡,等.化风丹对大鼠的亚慢性肝肾毒性研究[J].亚太传统医药,2016,12(19):5 TIAN XZ,FU ZX,SUI Y,et al.Subchronic hepatorenal toxicity of Huafengdan in rats[J].Asia-Pac Tradit Med,2016,12(19):5
[4] 曹国琼,张永萍,徐剑,等.化风丹药母干燥工艺的研究[J].中国民族医药杂志,2012,18(7):38 CAO GQ,ZHANG YP,XU J,et al.Study on drying technology of Huafengdan pharmaceutical mother[J].Chin J Ethn Med,2012,18(7):38
[5] 李智勇,邓亚利,孙冬梅.HPLC法测定癌痛巴布剂中乌头碱、次乌头碱、新乌头碱[J].中成药,2011,33(6):988 LI ZY,DENG YL,SUN DM.Determination of aconitine,hypaconitine and neoaconitine in Cantong cataplasm by HPLC[J].Chin Pat Med,2011,33(6):988
[6] 罗霄,彭善贵,文永盛,等.HPLC测定制川乌中的乌头碱、次乌头碱和新乌头碱[J].华西药学杂志,2010,25(4):472 LUO X,PENG SG,WEN YS,et al.Determination of aconitine,hypaconitine and neoaconitine in Radix Aconiti Preparata by HPLC[J].J West China Pharm,2010,25(4):472
[7] 曹国琼,张永萍,徐剑,等.高效液相色谱法测定化风丹药母中3种双酯型生物碱含量[J].贵州农业科学,2014,42(11):58 CAO GQ,ZHANG YP,XU J,et al.Determination of three diester alkaloids in Huafengdan pharmaceutical mother by high performance liquid chromatography[J].Guizhou Agr Sci,2014,42(11):58
[8] 丁文丽.HPLC法在中药饮片制川乌、乌头碱含量测定中的应用[J].西部中医药,2014,27(11):32 DING WL.Application of HPLC in the determination of aconitine and aconitine in the preparation of Chinese herbal pieces[J].West Chin Med,2014,27(11):32
[9] 刘双,杨静,江振作,等.中药"神曲"发酵工艺及质量标准研究进展[J].天津中医药,2015,32(5):318 LIU S,YANG J,JIANG ZZ,et al.Research progress on fermentation technology and quality standard of Chinese traditional medicine Shenqu[J].Tianjin Tradit Chin Med,2015,32(5):318
[10] 于定荣,麻印莲,顾雪竹,等.白芍发酵菌种及发酵工艺的优选[J].中草药,2014,45(20):2924 YU DR,MA YL,GU XZ,et al.Selection of fermentation strains and fermentation technology of Paeonia lactiflora[J].Chin Herb Med,2014,45(20):2924
[11] 杨清,徐立,王俊,等.桑红茶发酵工艺条件优化及活性成分含量的动力学研究[J].蚕业科学,2010,36(2):221 YANG Q,XU L,WANG J,et al.Study on optimization of fermentation conditions and kinetics of active ingredients in mulberry black tea[J].Sericul Sci,2010,36(2):221
[12] 孟金明,沈艳华,贾氏臣,等.液态发酵生产大麦酒发酵工艺的研究[J].酿酒科技,2015(9):86 MENG J,SHEN YH,JIA SC,et al.Study on fermentation technology of barley wine by liquid fermentation[J].Brewing technol,2015(9):86