关防风HPLC特征指纹图谱研究及4个色原酮类成分一测多评法分析研究

目的：建立指纹图谱及一测多评法测定关防风中4个化学成分含量。方法：采用Agilent ZORBAXEclipse XDB C₁₈（2）色谱柱（4.6 mm×250 mm，5 μm），以甲醇（A）-水（B）为流动相，梯度洗脱（0~10min，20% A → 40% A；10~30 min，40% A → 65% A；30~40 min，65% A；40~60 min，65% A → 80% A；60~70min，80% A），流速1.0 mL·min^-1，检测波长254 nm，柱温30℃；采用高效液相色谱法测定12批关防风的指纹图谱，建立指纹图谱共有模式。以5-O-甲基维斯阿米醇苷为内参物，建立升麻素、升麻素苷、亥茅酚苷的相对校正因子，并进行含量计算，实现一测多评。同时采用外标法测定12批关防风中4个色原酮类成分的含量，比较计算值与实测值的差异，验证一测多评法的准确性。结果：12批关防风指纹图谱标定了共20个共有峰，指认了其中7个化学成分。12批样品指纹图谱与对照指纹图谱的相似度均大于0.90。建立的相对校正因子重现性良好，采用校正因子计算的含量值与实测值之间无显著差异（P>0.05）；通过聚类分析，栽培品和野生药材能有效区分。结论：所建立的方法准确、可行，可用于关防风中4个化学成分的定性定量分析。

Objective: To establish fingerprint and quantitative analysis of multi-components by single marker (QAMS) method for 4 components of Radix Saposhnikoviae from northeast. Methods: The analysis was carried out on an analytical column Agilent ZORBAX Eclipse XDB C₁₈ (2) (4.6 mm×250 mm,5 μm) with gradient elution by methanol (A) -water (B) (0-10 min, 20%A → 40%A; 10-30 min, 40%A → 65%A; 30-40 min, 65%A; 40-60 min, 65%A → 80%A;60-70 min, 80%A), at the detection wavelength of 254 nm and a flow rate of 1.0 mL·min^-1;the column temperature was 30℃;12 batches of Radix Saposhnikoviae from northeast were determined by HPLC and a common mode of fingerprint has been established. A QAMS method was developed to determine prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisammioside and sec-O-glucosylhamaudol. 4'-O-β-D-glucosyl-5-O-methylvisamminol was selected as internal reference;the relative correction factors (RCF) of other three components to the internal reference were calculated. 12 batches of Radix Saposhnikoviae from northeast were determined by both external standard method and QAMS. Results: There were 20 common peaks in fingerprints of 12 batches of Radix Saposhnikoviae from northeast and seven of them were identified. The similarities of fingerprints of 12 batches of samples were above 0.90. The established correcting factors had a good reproducibility. No significant differences (P>0.05) were found between the quantitative results of external standard method and QAMS. The cultivated could be distinguished effectively from wild medicinal materials by cluster analysis. Conclusion: The developed method is accurate, feasible, and can be used for the qualitative and quantitative analysis of Radix Saposhnikoviae from northeast.

[1] 中华人民共和国药典2015年版.一部[S]. 2015:149 ChP 2015. Vol Ⅰ[S]. 2015:149
[2] 杨景明,姜华,孟祥才.中药防风质量评价的现状与思考[J].中药材, 2016, 39(7):1678 YANG JM, JIANG H, MENG XC. Status and thoughts on quality assessment of Saposhnikovia divaricata[J]. Chin Med Mater J, 2016, 39(7):1678
[3] 薛宝云,李文,李丽,等.防风色原酮甙类成分的药理活性研究[J].中国中药杂志, 2000, 25(5):41 XUE BY, LI W, LI L, et al. A Pharmacodynamic research on chromone glucosides of Fangfeng[J]. China J Chin Mater Med, 2000,25(5):41
[4] 李悦悦,王慧,陈俊,等. RRLC-TOF/MS鉴别防风血浆、尿液中成分及代谢产物[J].第二军医大学学报, 2010, 31(7):760 LI YY,WANG H,CHEN J,et al. RRLC-TOF/MS in identification of constituents and metabolites of Radix Saposhnikoviae in rat plasma and urine[J]. Acad J Second Mil Med Univ,2010,31(7):760
[5] 罗超,沈立,吕昌.亥茅酚苷对脂多糖诱导巨噬细胞一氧化氮和白介素6表达的影响[J].中国免疫学杂志, 2015, 31(11):1486 LUO C, SHEN L, LÜ C. Effects of sec-O-glucosylhamaudol on expressions of nitric oxide and interleukin-6 in ipopolysaccharide stimulated murine macrophage[J]. Chin J Immunol, 2015, 31(11):1486
[6] ZHAO B, YANG XB, YANG XW, et al. Intestinal permeability of the constituents of the roots of Saposhnikovia divaricata in the human Caco-2 cell monolayer model[J]. Plant Med,2011, 77(13):1531
[7] 杨妮娜,杨天梅,赵应红,等. HPLC指纹图谱技术在傣药灯台叶资源评价中的应用[J].药物分析杂志, 2016, 36(3):473 YANG NN, YANG TM, ZHAO YH, et al. Application of HPLC fingerprint technology in the resource evaluation of Alstonia scholaris (L.)R. Br.[J]. Chin J Pharm Anal,2016, 36(3):473
[8] 张春泥,王英姿,孙欣光,等. HPLC-CAD结合化学计量学的川楝子饮片指纹图谱研究[J].药学学报, 2017, 52(3):456 ZHANG CN, WANG YZ, SUN XG, et al. Chromatographic fingerprint analysis of Toosendan Fructus by HPLC-CAD coupled with chemometrics methods[J]. Acta Pharm Sin, 2017, 52(3):456
[9] 潘伟东,杜义龙,赵胜男.等.韩信草的高效液相色谱指纹图谱及化学模式识别[J].药物分析杂志,2015, 35(2):250 PAN WD,DU YL,ZHAO SN,et al. HPLC fingerprint and chemical pattern recognition of Scutellaria indica[J]. Chin J Pharm Anal, 2015, 35(2):250
[10] 杨宁,刘艳妮,康杰芳.山茱萸药材HPLC指纹图谱的优化研究[J].药物分析杂志, 2014, 34(11):1989 YANG N,LIU YN,KANG JF. Optimization of the HPLC fingerprint for Cornus officinalis[J]. Chin J Pharm Anal, 2014, 34(11):1989
[11] 王智民,高慧敏,付雪涛,等."一测多评"法中药质量评价模式方法学研究[J].中国中药杂志,2006, 31(23):1925 WANG ZM,GAO HM,FU XT,et al. Multi-components quantitation by one marker new method for quality evaluation of Chinese herbal medicine[J]. China J Chin Mater Med,2006, 31(23):1925
[12] 赵博,杨鑫宝,杨秀伟,等. HPLC法同时测定防风中6个主要成分的含量[J].药物分析杂志,2013, 33(3):382 ZHAO B,YANG XB,YANG XW,et al. Simultaneous determination of six major constituents in the roots of Saposhnikovia divaricata by HPLC[J]. Chin J Pharm Anal,2013, 33(3):382
[13] 王晓燕,霍甜甜,李振国.一测多评法同时测定杞菊地黄口服液中4种有效成分的含量[J].药物分析杂志,2017, 37(2):290 WANG XY, HUO TT, LI ZG. Simultaneous determination of 4 active components in Qijudihuang oral liquid by QAMS method[J]. Chin J Pharm Anal,2017, 37(2):290