RP-HPLC法同时测定野葛的根、茎和叶中葛根素、大豆苷和大豆苷元的含量

Simultaneous RP-HPLC Determination of Puerarin, Daidzin and Daidzein in Roots, Stems and Leaves of Pueraria lobata (Wild) Ohwi

目的:建立RP-HPLC法同时测定野葛的根、茎及叶中葛根素、大豆苷和大豆苷元的方法。方法:样品前处理采用70%乙醇超声提取,HPLC直接测定,并与药典方法进行对比。结果:对照品葛根素、大豆苷和大豆苷元分别在11.87～73.91,1.00～6.48和0.28～1.53μg/ml范围内呈良好线性关系,相关系数r分别为0.9998,0.9997和0.9994,最低检出限分别为0.43、0.24、0.18μg/ml。平均回收率分别为99.78%、102.61%和105.57%,RSD分别为1.6%、2.8%和3.4%(n=5)。精密度、稳定性和重现性良好。超声提取法测得的葛根中葛根素、大豆苷和大豆苷元含量分别为3.98%、0.36%和0.039%,高于药典提取法的提取率,更适于同时提取葛根素、大豆苷和大豆苷元。葛茎中葛根素、大豆苷和大豆苷元含量分别为0.004%、0.0032%和0.0007%,葛叶中葛根素、大豆苷和大豆苷元含量分别为0.0016%、0.005%和0.0012%。结论:该方法准确、快速,操作简单,分离效果好,可用于野葛的根、茎和叶中葛根素、大豆苷和大豆苷元含量的同时测定;江苏茅山地区野葛茎和叶中葛根素、大豆苷和大豆苷元含量大大低于葛根中的含量。

A RP-HPLC method was established for the simultaneous determination of puerarin, daidzin and daidzein in roots, stems and leaves of Pueraria lobata （Wild） Ohwi. The procedure was based on extraction of test portions with 70% ethanol solution in an ultrasonic field for 20 min at 50 ℃ followed by direct HPLC analysis of the extracts. The chromatographic conditions were as follows： using a Hypersil ODS-2 column （4.6 mm× 250 mm, 5 μm） and a mobile phase composed of methanol and H2O, gradient elution [0-4 min 25：75 （methanol/water volume ratio）, 4-7 min 25：75 → 32：78, 7-9min 32：68, 9-11 min 32：68 → 50：50, 11-24 min 50：50, 24-30 min 50：50 → 25：75], column temperature 35 ℃, detection wavelength 250 nm, and flow rate of the mobile phase 0.8 ml/min. A good linear relationship between peak area and concentration was found to puerarin, daidzin and daidzein in the images of 11.87-73.91 （r = 0.9998）, 1.00-6.48 （r = 0.9997） and 0.28-1.53 μ g/ml （r = 0.9994）, respectively. The LODs were 0.43, 0.24 and 0.18 μ g/ml, respectively. Mean recoveries were 99.78%, 102.61% and 105.57% with the RSDs of 1.6%, 2.8% and 3.4% （n = 5）, respectively. The precision, stability and reproducibility of the method all were good. The contents of puerain, daidzin, and daidzein in roots of Pueraria lobata （wild） Ohwi determined by this method were 3.98%, 0.36% and 0.039% respectively, higher than 3.28%, 0.26% and 0.028% of those analyzed according to the method stipulated in the Chinese Pharmacopoeia 2005 respectively, suggesting high efficiency of ultrasonic-assisted extraction of the three analytes. The contents of puerain, daidzin, and daidzein in stems ofPueraria lobata （wild） Ohwi were 0.004%, 0.0032% and 0.0007% respectively, while those in leaves were 0.0016%, 0.005% and 0.0012%, respectively. This method is accurate, fast and simple and therefore can be used for the simultaneous determination of puerain, daidzin, and daidzein in roots, stems and leaves of Pueraria lobata （wild） Ohwi. Our results demonstrate that the contents of puerain, daidzin, and daidzein in stems and leaves of Pueraria lobata （wild） Ohwi growned in Maoshan area of Jiangsu province are obviously lower than those in the roots.