运用在线HPLC-UV-DPPH方法评价地黄药材的抗氧化成分及质量

目的：发展在线高效液相色谱-紫外-1,1-二苯基-2-三硝基苯肼（HPLC-UV-DPPH）方法快速筛选地黄药材的抗氧化成分并评价其活性,为中药质量控制提供新的研究思路和模式。方法：运用HPLC-UV-DPPH方法筛选13批地黄药材的抗氧化成分;以毛蕊花糖苷为阳性参比,计算主要抗氧化活性成分的相对活性及总活性;运用高效液相色谱-傅里叶变换质谱（HPLC-FTMS）方法对主要的抗氧化成分进行鉴定;13批地黄样品的总活性结果与《中国药典》方法中毛蕊花糖苷含量的检测结果进行比较分析。结果：HPLC-UV-DPPH方法学考察结果,精密度RSD<4.61%,稳定性RSD<4.25%,重复性RSD<5.28%;建立"量-效"曲线方程为y=0.000 6x⁴-0.024 1x³+0.404 1x²+1.299x+1.652 1（R²=1）;9个抗氧化成分中4个显示较强DPPH抑制峰的成分被鉴定为海胆苷（echinacoside）、焦地黄苯乙醇苷A1/A2（jionoside A1/A2）、毛蕊花糖苷（verbascoside）、异毛蕊花糖苷（isoverbascoside）;《中国药典》方法测得毛蕊花糖苷含量较高的4批样品其总活性也较高,而其他样品的结果不呈现正相关,表明除毛蕊花糖苷外的其他成分对总活性的贡献率不应被忽略,质量评价应考虑多成分的测定。结论：研究建立的HPLC-UV-DPPH方法具有良好的重复性和稳定性,化学和生物活性检测相结合为综合评价中药的质量提供了一种新的方法。

Evaluation of antioxidant ingredients and quality of Rehmanniae Radix by HPLC-UV-DPPH method

OBJECTIVE To develop an on-line HPLC-UV-DPPH method for quick screening of antioxidants and active evaluation of Rehmanniae Radix to provide a new research idea and mode for quality control of traditional Chinese medicine.METHODS HPLC-UV-DPPH was applied to screen antioxidants from 13 batches of Rehmanniae Radix samples. Verbascoside was regarded as a positive reference and used to calculate relative activities and total activities of the main antioxidants. The main antioxidants were identified by HPLC-FTMS. The results of total activities of 13 batches of samples were compared with the detection results of verbascoside content of Chinese Pharmacopoeia method.RESULTS The RSD values of precision, stability and repeatability of HPLC-UV-DPPH method were less than 4.61%, 4.25% and 5.28%, respectively. Equation formula of quantity-effect verbascoside was y=0.000 6x⁴+0.024 1x³+0.404 1x²+1.299x+1.652 1 (R²=1). Four antioxidants showing stronger DPPH inhibition peaks in nine antioxidants were identified as echinacoside, jionoside A1/A2, verbascoside, isoverbascoside. Total activities of 4 batches of samples with higher content of verbascoside were higher, while the results of other samples were not positively correlated. This indicated that the contributions of the other components beside of verbascoside should not be ignored. The quality control of Rehmanniae Radix should include the detection of multiple ingredients.CONCLUSION The proposed HPLC-UV-DPPH method is stable and repeatable. The combination of chemical and active detection will provide a new method for quality control of traditional Chinese medicine.