中药白芷中欧前胡素、异欧前胡素体外经皮渗透特性的串联质谱研究
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摘要
欧前胡素和异欧前胡素属于6,7-呋喃香豆素类化合物,是中药白芷的主要药效成分。为了考察二者的经皮渗透特性,采用改进型的Franz扩散池,以大鼠离体皮肤作为渗透屏障,以高效液相色谱-串联质谱(HPLC-MS/MS)法测定白芷提取液的经皮渗透液中欧前胡素和异欧前胡素的含量,据此来考察白芷提取液中欧前胡素和异欧前胡素的经皮渗透特性。实验结果表明,欧前胡素和异欧前胡素在48h内,单位面积的累积渗透量分别为116.11μg/cm~2和140.84μg/cm~2,累积渗透百分率分别为37.75%和71.86%。欧前胡素的稳态渗透速率为26.5μg/h·cm~2,其体外经皮渗透符合Higuchi方程。本研究阐明了欧前胡素的体外经皮渗透机制,可为白芷作为外用经皮给药制剂的发展提供实验依据和理论基础。
Imperatorin and isoimperatorin are 6,7-furocoumarin compounds,which are the major medicinal effective ingredients of Radix Angelicae Dahuricae.Coumarin com-pounds are widely used in topical formulations because it has many properties,such as anti-bacterial,anti-inflammatory,antipyretic and analgesic effects.At present,the chemical components of traditional Chinese medicine are mainly analyzed by thin layer chromatography(TLC),gas chromatography(GC)and high performance liquid chromatography(HPLC).However,because there are some difficulties in Chinese medicine analysis,such as complex composition,difficulty in separation and low content,traditional methods(TLC,HPLC,etc.)were difficult to determine and analyze the composition sometimes.HPLC/MS is a comprehensive technique,it has become an important modern separation and analysis technique because of its high separation ability,high sensitivity,wide application range and strong specificity.Tandem mass spectrometry(MS/MS)has higher sensitivity and selectivity than unipolar mass spectrometry and can obtain more structural information.In this study,the in vitro percutaneous permeability of imperatorin and isoimperatorin in Radix Angelicae Dahuricael extracting solution was evaluated by the modified Franz diffusion cell with isolated rat skin.The content of imperatorin and isoimperatorin was determined by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).The chromatographic conditions were as follows:Dimonsil C18column(250mm×4.6mm,5μm),the temperature of column is 35℃,mobile phase was methanol-water(80∶20,V/V)and flow rate was 0.8mL/min.A tandem mass spectrometer coupled with positive electro-spray ionization(ESI)source was used for detection.The quantitative and qualitative analysis were performed on selective ion chromatograms acquired by a multiple reaction monitoring(MRM)mode of following transitions:m/z 271.1→ m/z203.04,m/z 271.1→m/z 147.05.The results showed that the limits of detection(LODs)of imperatorin and isoimperatorin were 0.01 and 1μg/L,and the limits of quantification(LOQs)of imperatorin and isoimperatorin were 0.25 and 2μg/L,respectively.Calibration curves were linear over the range of 0.001-1 mg/L for imperatorin and isoimperatorin.Both the inter-day and intra-day reproducibility(RSDs)were lower than 4%,the precision and stability of the method were lower than 2%,which indicated that the method has good precision,and the test solution could keep steady within 24 h.The mean recoveries of imperatorin and isoimperatorin were 98.09%and 100.60%.The cumulative permeation amounts of imperatorin and isoimperatorin were 116.11μg/cm~2 and 210.06 μg/cm~2 and the percentage of cumulative permeation were 37.75% and71.86%,respectively.The steady state permeation rate of imperatorin was 26.5μg/h·cm~2and its in vitro percutaneous permeabilities was conformed to Higuchi equation.This study clarified the percutaneous permeation mechanism of imperatorin in vitro and provided a theoretical basis for the development of Radix Angelicae Dahuricae as a topical transdermal preparation.
Imperatorin and isoimperatorin are 6,7-furocoumarin compounds,which are the major medicinal effective ingredients of Radix Angelicae Dahuricae.Coumarin com-pounds are widely used in topical formulations because it has many properties,such as anti-bacterial,anti-inflammatory,antipyretic and analgesic effects.At present,the chemical components of traditional Chinese medicine are mainly analyzed by thin layer chromatography(TLC),gas chromatography(GC)and high performance liquid chromatography(HPLC).However,because there are some difficulties in Chinese medicine analysis,such as complex composition,difficulty in separation and low content,traditional methods(TLC,HPLC,etc.)were difficult to determine and analyze the composition sometimes.HPLC/MS is a comprehensive technique,it has become an important modern separation and analysis technique because of its high separation ability,high sensitivity,wide application range and strong specificity.Tandem mass spectrometry(MS/MS)has higher sensitivity and selectivity than unipolar mass spectrometry and can obtain more structural information.In this study,the in vitro percutaneous permeability of imperatorin and isoimperatorin in Radix Angelicae Dahuricael extracting solution was evaluated by the modified Franz diffusion cell with isolated rat skin.The content of imperatorin and isoimperatorin was determined by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).The chromatographic conditions were as follows:Dimonsil C18column(250mm×4.6mm,5μm),the temperature of column is 35℃,mobile phase was methanol-water(80∶20,V/V)and flow rate was 0.8mL/min.A tandem mass spectrometer coupled with positive electro-spray ionization(ESI)source was used for detection.The quantitative and qualitative analysis were performed on selective ion chromatograms acquired by a multiple reaction monitoring(MRM)mode of following transitions:m/z 271.1→ m/z203.04,m/z 271.1→m/z 147.05.The results showed that the limits of detection(LODs)of imperatorin and isoimperatorin were 0.01 and 1μg/L,and the limits of quantification(LOQs)of imperatorin and isoimperatorin were 0.25 and 2μg/L,respectively.Calibration curves were linear over the range of 0.001-1 mg/L for imperatorin and isoimperatorin.Both the inter-day and intra-day reproducibility(RSDs)were lower than 4%,the precision and stability of the method were lower than 2%,which indicated that the method has good precision,and the test solution could keep steady within 24 h.The mean recoveries of imperatorin and isoimperatorin were 98.09%and 100.60%.The cumulative permeation amounts of imperatorin and isoimperatorin were 116.11μg/cm~2 and 210.06 μg/cm~2 and the percentage of cumulative permeation were 37.75% and71.86%,respectively.The steady state permeation rate of imperatorin was 26.5μg/h·cm~2and its in vitro percutaneous permeabilities was conformed to Higuchi equation.This study clarified the percutaneous permeation mechanism of imperatorin in vitro and provided a theoretical basis for the development of Radix Angelicae Dahuricae as a topical transdermal preparation.
引文
[1]白洁,陆洋,杜守颖,等.欧前胡素体外经皮渗透研究[J].中国中药杂志,2013,38(16):2 605.BAI Jie,LU Yang,DU Shouying,et al.Study on in vitro penetration of imperatorin[J].China Journal of Chinese Materia Medica,2013,38(16):2 605(in Chinese).
[2]连其深,胡晓,上官珠,等.蛇床子素镇静作用的研究[J].中药新药与临床药理,2000,11(4):244.LIAN Qishen,HU Xiao,SHANGGUAN Zhu,et al.Study on sedative effect of Osthole[J].Traditional Chinese Drug Research and Clinical Pharmacology,2000,11(4):244(in Chinese).
[3]杨小花,胡晓.欧前胡素与异欧前胡素的药理学研究进展[J].南昌大学学报:医学版,2012,52(3):95-100.YANG Xiaohua,HU Xiao.Pharmacology research progress of imperatorin and isoimperatorin[J].Journal of Nanchang University(Medical Science),2012,52(3):95-100(in Chinese).
[4]NGWENDSON J N,BEDIR E,EFANGE S M,et al.Constituents of Peucedanum zenkeri seeds and their antimicrobial effects[J].Pharmazie,2003,58(8):587-589.
[5]王梦月,贾敏如,马逾英,等.白芷中四种线型呋喃香豆素类成分药理作用研究[J].天然产物研究与开发,2010,22(3):485-489.WANG Mengyue,JIA Minru,MA Yuying,et al.Pharm acological effect of four linear furocoum arins in Radix Angelicae Dahuricae[J].Natural Product Research and Development,2010,22(3):485-489(in Chinese).
[6]贾小靖,杨月青.白芷散敷贴治疗足跟痛56例临床观察[J].临床医药实践,2012,21(7):557-558.JIA Xiaojing,YANG Yueqing.Heel pain 56cases of Angelica dahurica powder sticking treatment clinical observation[J].Proceeding of Clinical Medicine,2012,21(7):557-558(in Chinese).
[7]樊东升,曹学东,刘春泉.东风痤康凝胶剂的质量控制[J].中国药物与临床,2014,14(7):909-910.PAN Dongsheng,CAO Xuedong,LIU Chunquan.The quality control of Dongfeng Cuokang gel[J].Chinese Remedies and Clinics,2014,14(7):909-910(in Chinese).
[8]王文凯,袁宏伟.白芷烧伤酊的研制与应用[J].河南中医,2000,20(5):61.WANG Wenkai,YUAN Hongwei.The development and application of Angelica dahurica burn tincture[J].Henan Traditional Chinese Medicine,2000,20(5):61(in Chinese).
[9]周典,张建华,李华,等.白芷不同提取物对黑色素影响的比较研究[J].上海中医药大学学报,2007,21(5):72-75.ZHOU Dian,ZHANG Jianhua,LI Hua,et al.Comparative study on effects of different extracts from Angelica dahurica on melanin[J].Acta Universitatis Traditionis Medicalis Sinensis Pharmacologiaeque Shanghai,2007,21(5):72-75(in Chinese).
[10]张莉,张丽萍,孙成春.液-质联用技术在中药研究中的应用[J].解放军药学学报,2010,26(6):558.ZHANG Li,ZHANG Liping,SUN Chengchun.Application of liquid-mass spectrometry in traditional Chinese medicine research[J].Pharmaceutical Journal of Chinese People’s Liberation Army,2010,26(6):558(in Chinese).
[11]陈晓水,侯宏卫,边照阳,等.气相色谱-串联质谱(GC-MS/MS)的应用研究进展[J].质谱学报,2013,34(5):308-320.CHEN Xiaoshui,HOU Hongwei,BIAN Zhaoyang,et al.Research progress on application of GC-MS/MS[J].Journal of Chinese Mass Spectrometry Society,2013,34(5):308-320(in Chinese).
[12]刘聪敏,白洁,杜守颖,等.止痛凝胶膏剂体外透皮行为研究[J].中国中药杂志,2013,38(16):2 609-2 613.LIU Congmin,BAI Jie,DU Shouying,et al.Study on transdermal behavior of Zhitong cataplasm[J].China Journal of Chinese Materia Medica,2013,38(16):2 609-2 613(in Chinese).
[13]卢闻,贺浪冲.白芷香豆素类有效部位体外透皮特性研究[J].中国药学杂志,2003,38(12):45-47.LU Wen,HE Langchong.Study on the transdermal permeability of coumarins in Radix Angelica Dahurica in vitro[J].China Pharmaceutical Journal,2003,38(12):45-47(in Chinese).
[14]章烨雯,王琼,于竞新,等.止痛微乳凝胶膏的制备及体外评价[J].北京中医药大学学报,2015,38(4):253-259,291.ZHANG Yewen,WANG Qiong,YU Jingxin,et al.Preparation and in vitro evaluation of analgesic microemulsion cataplasm[J].Journal of Beijing University of Traditional Chinese Medicine,2015,38(4):253-259,291(in Chinese).
[2]连其深,胡晓,上官珠,等.蛇床子素镇静作用的研究[J].中药新药与临床药理,2000,11(4):244.LIAN Qishen,HU Xiao,SHANGGUAN Zhu,et al.Study on sedative effect of Osthole[J].Traditional Chinese Drug Research and Clinical Pharmacology,2000,11(4):244(in Chinese).
[3]杨小花,胡晓.欧前胡素与异欧前胡素的药理学研究进展[J].南昌大学学报:医学版,2012,52(3):95-100.YANG Xiaohua,HU Xiao.Pharmacology research progress of imperatorin and isoimperatorin[J].Journal of Nanchang University(Medical Science),2012,52(3):95-100(in Chinese).
[4]NGWENDSON J N,BEDIR E,EFANGE S M,et al.Constituents of Peucedanum zenkeri seeds and their antimicrobial effects[J].Pharmazie,2003,58(8):587-589.
[5]王梦月,贾敏如,马逾英,等.白芷中四种线型呋喃香豆素类成分药理作用研究[J].天然产物研究与开发,2010,22(3):485-489.WANG Mengyue,JIA Minru,MA Yuying,et al.Pharm acological effect of four linear furocoum arins in Radix Angelicae Dahuricae[J].Natural Product Research and Development,2010,22(3):485-489(in Chinese).
[6]贾小靖,杨月青.白芷散敷贴治疗足跟痛56例临床观察[J].临床医药实践,2012,21(7):557-558.JIA Xiaojing,YANG Yueqing.Heel pain 56cases of Angelica dahurica powder sticking treatment clinical observation[J].Proceeding of Clinical Medicine,2012,21(7):557-558(in Chinese).
[7]樊东升,曹学东,刘春泉.东风痤康凝胶剂的质量控制[J].中国药物与临床,2014,14(7):909-910.PAN Dongsheng,CAO Xuedong,LIU Chunquan.The quality control of Dongfeng Cuokang gel[J].Chinese Remedies and Clinics,2014,14(7):909-910(in Chinese).
[8]王文凯,袁宏伟.白芷烧伤酊的研制与应用[J].河南中医,2000,20(5):61.WANG Wenkai,YUAN Hongwei.The development and application of Angelica dahurica burn tincture[J].Henan Traditional Chinese Medicine,2000,20(5):61(in Chinese).
[9]周典,张建华,李华,等.白芷不同提取物对黑色素影响的比较研究[J].上海中医药大学学报,2007,21(5):72-75.ZHOU Dian,ZHANG Jianhua,LI Hua,et al.Comparative study on effects of different extracts from Angelica dahurica on melanin[J].Acta Universitatis Traditionis Medicalis Sinensis Pharmacologiaeque Shanghai,2007,21(5):72-75(in Chinese).
[10]张莉,张丽萍,孙成春.液-质联用技术在中药研究中的应用[J].解放军药学学报,2010,26(6):558.ZHANG Li,ZHANG Liping,SUN Chengchun.Application of liquid-mass spectrometry in traditional Chinese medicine research[J].Pharmaceutical Journal of Chinese People’s Liberation Army,2010,26(6):558(in Chinese).
[11]陈晓水,侯宏卫,边照阳,等.气相色谱-串联质谱(GC-MS/MS)的应用研究进展[J].质谱学报,2013,34(5):308-320.CHEN Xiaoshui,HOU Hongwei,BIAN Zhaoyang,et al.Research progress on application of GC-MS/MS[J].Journal of Chinese Mass Spectrometry Society,2013,34(5):308-320(in Chinese).
[12]刘聪敏,白洁,杜守颖,等.止痛凝胶膏剂体外透皮行为研究[J].中国中药杂志,2013,38(16):2 609-2 613.LIU Congmin,BAI Jie,DU Shouying,et al.Study on transdermal behavior of Zhitong cataplasm[J].China Journal of Chinese Materia Medica,2013,38(16):2 609-2 613(in Chinese).
[13]卢闻,贺浪冲.白芷香豆素类有效部位体外透皮特性研究[J].中国药学杂志,2003,38(12):45-47.LU Wen,HE Langchong.Study on the transdermal permeability of coumarins in Radix Angelica Dahurica in vitro[J].China Pharmaceutical Journal,2003,38(12):45-47(in Chinese).
[14]章烨雯,王琼,于竞新,等.止痛微乳凝胶膏的制备及体外评价[J].北京中医药大学学报,2015,38(4):253-259,291.ZHANG Yewen,WANG Qiong,YU Jingxin,et al.Preparation and in vitro evaluation of analgesic microemulsion cataplasm[J].Journal of Beijing University of Traditional Chinese Medicine,2015,38(4):253-259,291(in Chinese).