色谱

色谱 ›› 2021, Vol. 39 ›› Issue (7): 708-714.DOI: 10.3724/SP.J.1123.2020.12009

• 研究论文 • 上一篇    下一篇

紫草油有效成分的高效液相色谱测定法及其在超临界流体萃取制备紫草油中的应用

沈洁1, 沈炜1, 蔡雪2, 王京霞1, 郑敏霞1,*()   

  1. 1.浙江中医药大学附属第一医院, 浙江 杭州 310006
    2.浙江工业大学分析测试中心, 浙江 杭州 310014
  • 收稿日期:2020-12-09 出版日期:2021-07-08 发布日期:2021-06-02
  • 通讯作者: 郑敏霞
  • 作者简介:*Tel:(0571)87073482,E-mail:slightsusu@163.com,zmxyxx@126.com.
  • 基金资助:
    浙江省中医药科技计划项目(2015ZB044);浙江省中医药科技计划项目(2016ZQ017);浙江省“十三五”中医药重点学科建设项目(2017-XK-B01);浙江省“十三五”中医药重点专科建设项目临床药学(浙卫办中医[2019] 1).

High performance liquid chromatographic method for determination of active components in lithospermum oil and its application to process optimization of lithospermum oil prepared by supercritical fluid extraction

SHEN Jie1, SHEN Wei1, CAI Xue2, WANG Jingxia1, ZHENG Minxia1,*()   

  1. 1. The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, China
    2. Analysis and Test Center, Zhejiang University of Technology, Hangzhou 310014, China
  • Received:2020-12-09 Online:2021-07-08 Published:2021-06-02
  • Contact: ZHENG Minxia
  • Supported by:
    Science and Technology Project of Traditional Chinese Medicine in Zhejiang Province(2015ZB044);Science and Technology Project of Traditional Chinese Medicine in Zhejiang Province(2016ZQ017);Key Discipline Construction Project of Traditional Chinese Medicine in Zhejiang Province During the 13th Five Year Plan(2017-XK-B01);Clinical Pharmacy of Key Specialty Construction Project of Traditional Chinese Medicine in the 13th Five Year Plan of Zhejiang Province (Zhejiang Healthy Office TCM [2019] 1).

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摘要：

紫草提取制备成的紫草油能够预防及治疗婴儿尿布疹、皮肤溃烂、湿疹等多种皮肤疾患,临床应用非常广泛,超临界流体萃取是紫草有效成分提取的优选方法。该文建立了紫草油有效成分的高效液相色谱(HPLC)测定方法,并以紫草油所含的有效成分含量为评价指标,采用三因素三水平正交试验法对紫草超临界流体萃取制备过程中的几个重要因素(萃取压力、萃取温度和CO2流量)进行考察,确定了紫草超临界流体萃取的最佳工艺流程。所建立的HPLC方法如下:Diamonsil C18色谱柱(250 mm×4.6 mm, 5 μm),流动相为含0. 1%甲酸的乙腈-含5 mmol/L甲酸铵的0.1%甲酸水溶液(75∶25, v/v),等度洗脱,流速为1 mL/min;进样量为15 μL;柱温为室温;二极管阵列检测器(PAD)检测波长为275 nm。该法可在30 min内同时测定紫草油中的有效成分紫草素、乙酰紫草素、β-乙酰氧基异戊酰阿卡宁、异丁酰紫草素、β,β-二甲基丙烯酰紫草素和2-甲基丁酰基紫草素的含量,方法精密度、准确度、重复性好。在萃取压力23 MPa、萃取温度40 ℃、CO2流量27 L/h这一优化工艺流程下得到的紫草油有效成分含量最高。所建立的HPLC-PAD法简便可行、准确可靠,可用于超临界流体萃取制备紫草油的工艺过程优化和质量控制,也可为紫草及其制剂的质量评价提供参考。优化后的工艺条件能够满足紫草油制备需求,也符合生产实际需求。

关键词: 高效液相色谱, 超临界流体萃取, 二极管阵列检测, 活性成分, 紫草油, 紫草, 工艺优化

Abstract:

Lithospermum erythrorhizon has the functions of cooling blood, activating blood, as well as detoxifying and penetrating rash. Lithospermum oil extracted from Lithospermum erythrorhizon can prevent and treat diaper rash, skin ulceration, eczema, and other skin diseases. Supercritical fluid extraction is the optimal method for the extraction of active components from lithospermum. In this study, an analytical method was established for simultaneously determination of six active components in lithospermum oil with high performance liquid chromatography (HPLC), and the contents of the active components as the evaluation index were used to investigate several important factors in the preparation of lithospermum oil by supercritical fluid extraction. The optimized HPLC conditions were as follows: separation column, Diamonsil C18 (250 mm×4.6 mm, 5 μm); mobile phases, acetonitrile containing 0.1% (v/v) formic acid-0.1% (v/v) formic acid aqueous solution containing 5 mmol/L ammonium formate (75∶25, v/v); flow rate, 1 mL/min; injection volume, 15 μL; room temperature; photodiode array detector (PAD); detection wavelength, 275 nm. The supercritical fluid extraction was optimized for ensuring stability of the amounts of effective components and the reliability of the quality of lithospermum oil. This will serve as the basis for preparation and quality control processes. Three factors and three levels orthogonal tests were adopted to investigate the important factors, viz. the pressure, temperature and CO2 flow rate in the preparation of lithospermum oil. The results showed that the developed HPLC-PAD method can be used for the simultaneous determination of shikonin, acetylshikonin, β-acetoxyisovaleryl akanin, isobutyryl shikonin, β,β-dimethylacryl shikonin, and 2-methylbutyryl shikonin in 30 min. The method has good precision, accuracy and repeatability. The contents of the active components were the highest when the extraction pressure, extraction temperature, and CO2 flow rate were 23 MPa, 40 ℃, and 27 L/h, respectively. The optimized conditions are suitable for the preparation and actual production of lithospermum oil. The HPLC-PAD method is simple, feasible, accurate, and reliable. It can be used for the preparation and quality control of lithospermum oil by supercritical fluid extraction. Thus, with this method, the stability of the contents of active ingredients and the reliability of the quality of lithospermum oil can be ensured; moreover, safe and effective drug use can be realized. The established method has obvious advantages over the traditional process and is a good candidate for widespread use.

Key words: high performance liquid chromatography (HPLC), supercritical fluid extraction (SFE), photodiode array detection (PAD), active components, lithospermum oil, Lithospermum erythrorhizon, process optimization

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