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目的:建立松叶中槲皮素、山柰酚的HPLC含量测定方法,为松叶药材质量标准的建立提供参考。同时对不同产地、不同种属松叶进行比较研究。方法:采用高效液相色谱法,使用Agilent ZORBAX SB-C18色谱柱(4.6 mm×250 mm,5μm),以0.5%磷酸(A)-乙腈(B)为流动相,梯度洗脱(0~9 min,25%B→30%B;9~19 min,30%B;19~20 min,30%B→25%B;20~40 min,25%B),流速0.8 m L·min-1,检测波长380 nm,柱温30℃。通过优化参数(脱色、脱色溶剂、甲醇预提、盐酸水溶液浓度、溶剂用量、提取时间)确定槲皮素、山柰酚含量测定预处理的最佳条件,并测定不同产地马尾松及同一产地不同种属松叶中槲皮素、山柰酚的含量。结果:槲皮素、山柰酚的含量测定预处理方法为:石油醚(30~60℃)脱色,甲醇-盐酸水溶液(5.0→10)(4∶1)50 m L,回流提取110 min;松叶中槲皮素进样量在0.13~2.24μg范围内线性关系良好(r=0.9997),平均回收率为98.6%(RSD=1.6%);山柰酚进样量在0.20~1.04μg范围内线性关系良好(r=0.9999),平均回收率为100.5%(RSD=2.4%);两者方法精密度及重复性均良好。在所测的不同产地马尾松中槲皮素、山柰酚总含量为0.715%~0.371%,其中广西产马尾松最高,江苏产马尾松含量最低;在吉林产不同种属松叶中槲皮素、山柰酚的含量为0.846%~0.515%,以红松最高,樟子松最低。结论:本方法经过方法学考察证明可用于松叶药材中槲皮素、山柰酚的含量测定;不同产地马尾松和不同种属松叶中槲皮素、山柰酚含量有一定差异。
Objective: To establish a HPLC method for the determination of quercetin and kaempferol in pine needles, and to provide a reference for establishing the quality standard of pine needles. At the same time, different origin, different species of pine leaves for comparative study. Methods: High performance liquid chromatography (HPLC) was performed on an Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm) using 0.5% phosphoric acid (A) min, 25% B → 30% B; 9-19 min, 30% B; 19-20 min, 30% B → 25% B; 20-40 min, 25% B) at a flow rate of 0.8 m L · min -1 , Detection wavelength 380 nm, column temperature 30 ℃. The optimum conditions for the determination of quercetin and kaempferol were determined by optimizing the parameters (decolorization, decoloration solvent, methanol pre-extraction, aqueous hydrochloric acid concentration, solvent dosage and extraction time) Species pine leaf in quercetin, kaempferol content. Results: The pretreatment methods for the determination of quercetin and kaempferol were: decolorization of petroleum ether (30-60 ℃), extraction of 50 mL of methanol-hydrochloric acid solution (5.0 → 10) (4:1) The calibration curve of quercetin in pine needles was linear in the range of 0.13-2.24 μg (r = 0.9997), with an average recovery of 98.6% (RSD = 1.6%). The injection volume of kaempferol was 0.20-1.04 μg The linearity was good (r = 0.9999) and the average recovery was 100.5% (RSD = 2.4%). The precision and reproducibility of both methods were good. The content of quercetin and kaempferol in Pinus massoniana from different areas was 0.715% ~ 0.371%, among which the highest was Pinus massoniana in Guangxi and the lowest was in Masson pine in Jiangsu. Quercetin , Kaempferol content of 0.846% ~ 0.515%, the highest Korean pine, Pinus sylvestris lowest. CONCLUSION: The method has been proved by methodology to be used for the determination of quercetin and kaempferol in pine needles. The content of quercetin and kaempferol in pine needles and pine needles in different species have some differences.