六味地黄丸药渣中4种活性成分含量测定及 抗氧化活性研究

梁诗瑶 刘倩倩 黄胜 颜冬兰 林丽美 吴萍

〔摘要〕 目的 采用HPLC法测定六味地黄丸药渣中丹皮酚、23-乙酰泽泻醇B、茯苓酸、熊果酸的含量，并评价六味地黄丸药渣及单味饮片（熟地黄、泽泻、牡丹皮、山茱萸）药渣的体外抗氧化活性。方法 Waters Athena C18色谱柱（4.6 mm×250 mm，5 μm），以乙腈-0.08%磷酸溶液为流动相进行梯度洗脱;柱温为35 ℃;流速1.0 mL/min;检测波长为210 nm;进样量10 μL。采用ABTS法、DPPH法以及FRAP法测定六味地黄丸药渣的抗氧化能力。结果 丹皮酚、23-乙酰泽泻醇B、茯苓酸、熊果酸分别在0.004～0.040、0.078～0.780、0.048～0.480、0.052～0.520 μg范围内线性关系良好;平均加样回收率分别为103.47%、97.14%、103.92%、100.18%（RSD<3.0%，n=6）。六味地黄丸药渣和单味饮片药渣对DPPH、ABTS自由基清除作用和Fe3+还原能力均较好;其中牡丹皮、山茱萸药渣的总体抗氧化效果优于其他药渣，二者在低浓度2 mg/mL时有一定的清除率，且随着浓度的增加，最高清除率可分别达到84.51%和76.39%。结论 该含量测定方法准确可靠，证实了药渣中还残留大量的活性成分，且具有良好的抗氧化生物活性。表明六味地黄丸药渣具有一定的营养价值，为药渣的综合开发利用提供依据。

〔關键词〕 六味地黄丸药渣;HPLC;丹皮酚;23-乙酰泽泻醇B;茯苓酸;熊果酸;含量测定

〔中图分类号〕R284.1       〔文献标志码〕A       〔文章编号〕doi：10.3969/j.issn.1674-070X.2021.05.010

Determination of Four Active Components in Liuwei Dihuang Pill Residue and Its

Antioxidant Activity

LIANG Shiyao1，2， LIU Qianqian1，2， HUANG Sheng2， YAN Donglan2， LIN Limei1，2*， WU Ping1，2*

（1. Key Laboratory of Hunan Province for Quality Evaluation of Bulk Medicinal MaterialsProduced in Hunan Province， Changsha， Hunan 410000， China; 2. Jiuzhitang Limited Company， Changsha， Hunan 410000， China）

〔Abstract〕 Objective To establish a HPLC method for the simultaneous determination of paeonol， 23-acetyl alisol B， poria acid and ursolic acid in Liuwei Dihuang Pill residue， and to evaluate the in vitro antioxidant activity of Liuwei Dihuang Pill residue and dregs of single decoction pieces [Shudihuang （Rehmanniae Radix Praeparata）， Zexie （Alismatis Rhizoma）， Mudanpi （Moutan Cortex）， Shanzhuyu （Corni Fructus）] Methods Waters Athena C18 column （4.6 mm×250 mm， 5 μm） was used. Acetonitrile and 0.08% phosphoric acid were used as mobile phase for gradient elution. The column temperature was 35 ℃， the flow rate was 1.0 mL/min， the detection wavelength was 210 nm， and the injection volume was 10 μL. The antioxidant capacity of Liuwei Dihuang Pill residue was determined by ABTS method， DPPH method and FRAP method. Results The linear relationship of paeonol， 23-acetyl alisol B， poria acid and ursolic acid was good in the range of 0.004～0.040， 0.078～0.780， 0.048～0.480， 0.052～0.520 μg， and the average recovery rate was 103.47%， 97.14%， 103.92% and 100.18% （RSD < 3.0%， n=6）. Liuwei Dihuang Pill residue and dregs of single decoction pieces had good scavenging effect on DPPH， ABTS free radical and Fe3+ reduction ability; the overall antioxidant effect of Mudanpi （Moutan Cortex） and Shanzhuyu （Corni Fructus） residue was better than other drug residues， and they had certain scavenging rate at low concentration （2 mg/mL）， and the highest scavenging rate could reach 84.51% and 76.39% respectively with the increase of concentration. Conclusion The method is accurate and reliable， which proves that there are a lot of active ingredients in the residues. The pharmacological experiments also further verify that the residues of Liuwei Dihuang Pill residue and dregs of single decoction pieces have good antioxidant biological activity. The residue of Liuwei Dihuang Pill have certain nutritional value， which provides the basis for the comprehensive development and utilization of the residue.

〔Keywords〕 Liuwei Dihuang Pill residue; HPLC; paeonol; 23-acetyl alisol B; poria acid; ursolic acid; content determination

随着中药产业的快速发展，中药药渣的排放量在逐年增加，形成了目前既浪费资源又污染环境的困境。六味地黄丸收载于《中华人民共和国药典》（2020年版）一部，主要由熟地黄、山药、泽泻、山茱萸、牡丹皮和茯苓6味药材经过提取浓缩而成，具有滋阴补肾之功效，是一个具有代表性的名优中成药大品种其药渣每年就可达3 000吨[1-3]。现代研究[4-5]表明，六味地黄丸主要含有环烯醚萜类、三萜类及其苷类、甾醇类及挥发性成分等，具有降血糖、降血脂、增强免疫、延缓衰老及防癌抗癌等作用。方中牡丹皮含有酚酸类代表性成分丹皮酚，具有抗炎、改善血液流变性、抗动脉粥样硬化、保护缺血组织等多种生物活性[6-7];泽泻中含有三萜类代表性成分泽泻醇，其中23-乙酰泽泻醇B对动脉粥样硬化、肾炎等疾病有一定疗……