HS-SPME/GC-MS结合化学计量学鉴别不同菌种发酵虫草产品及挥发性成分分析
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摘要
目的:建立一种快速分析发酵虫草菌丝粉中挥发性成分的方法,并用于不同菌种发酵虫草产品的鉴别和特征性成分的寻找。方法:利用顶空固相微萃取结合气相色谱质谱联用(HS-SPME/GC-MS)技术测定发酵虫草菌丝粉的挥发性成分,在优化萃取头、萃取温度、吸附时间、预平衡时间和解吸时间等萃取条件的基础上,建立发酵虫草菌丝粉的HS-SPME/GC-MS测定方法,用峰面积归一化法计算各成分的相对含量,采用主成分分析(PCA)和偏最小二乘判别分析(PLS-DA)2种化学计量学方法对数据进行分析。结果:最佳萃取条件为DVB/CAR-PDMS萃取头、萃取温度90℃、吸附时间80 min、预平衡时间40 min、解析时间2 min。利用建立的HS-SPME/GC-MS方法从金水宝胶囊、百令胶囊、宁心宝胶囊、至灵胶囊和心肝宝胶囊5种发酵虫草制剂中分别鉴定出56、71、72、81和75个化合物,主要包括酯类、醇类、羧酸类、醛类、烃类、酮类、杂环类化合物等。通过化学计量学方法对HS-SPME/GC-MS数据进行处理,实现了不同发酵虫草制剂和伪品的鉴别,并寻找到5种发酵虫草产品中的特征挥发性成分,分别为马索亚内酯、吡嗪酰胺、2-吡咯烷酮、棕榈酸乙酯和棕榈酸。结论:HS-SPME/GC-MS方法可用于发酵虫草产品中挥发性成分的快速分析。结合化学计量学方法对不同发酵虫草制剂进行鉴别和差异性成分寻找,为特征性指标成分的确定提供了一条新思路,为发酵虫草制剂质量标准的制定和提升提供了科学依据。
Objective:To establish a rapid method for the analysis of volatile components in Cordyceps sinensis mycelium powder,and to distinguish the fermented Cordyceps products from different strains as well as to search the characteristic components.Methods:The volatile components in Cordyceps sinensis mycelium powder were determined by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry(HS-SPME/GC-MS).By optimizing the extraction conditions of extraction head,extraction temperature,adsorption time,adsorption time,pre-equilibration time and desorption time,the analytical method of volatile components in Cordyceps sinensis mycelium powder was established.The relative content of volatile components was calculated by peak area normalization,and the data of HS-SPME/GC-MS was further proceeded by chemometrics analyses of principal component analysis(PCA) and partial least squares-discriminant analysis(PLS-DA). Results:DVB/CAR/PDMS extraction head,extraction temperature of 90 ℃,adsorption time of 80 min,pre-equilibration time of 40 min and desorption time of 2 min were selected as the optimum SPME extraction conditions.56,71,72,81,and 75 of compounds were identified by the established method of HS-SPME/GC-MS in Jinshuibao capsules,Bailing capsules,Ningxinbao capsules,Zhiling capsules and Xinganbao capsules,respectively.They mainly included esters,alcohols,carboxylic acids,aldehydes,hydrocarbons,ketones,heterocyclic compounds.Different fermented Cordyceps preparations were distinguished and counterfeits were identified by chemometrics analysis based on the HS-SPME/GC-MS data.The characteristic volatiles of massoia lactone for Jinshuibao capsules,pyrazinamide for Bailing capsules,2-pyrrolidone for Ningxinbao capsules,ethyl palmitate for Zhiling capsules,and palmitic acid for Xinganbao capsules were also found by chemometrics analysis.Conclusion:HS-SPME/GC-MS can be used for rapid analysis of volatile components in fermented Cordyceps sinensis.Combined with chemometric methods for distinguishing different fermented Cordyceps sinensis and finding differential components,it gives us new ideas to search characteristic components,and provides us with scientific basis for the establishment and improvement of the quality standard of fermented Cordyceps preparations.
Objective:To establish a rapid method for the analysis of volatile components in Cordyceps sinensis mycelium powder,and to distinguish the fermented Cordyceps products from different strains as well as to search the characteristic components.Methods:The volatile components in Cordyceps sinensis mycelium powder were determined by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry(HS-SPME/GC-MS).By optimizing the extraction conditions of extraction head,extraction temperature,adsorption time,adsorption time,pre-equilibration time and desorption time,the analytical method of volatile components in Cordyceps sinensis mycelium powder was established.The relative content of volatile components was calculated by peak area normalization,and the data of HS-SPME/GC-MS was further proceeded by chemometrics analyses of principal component analysis(PCA) and partial least squares-discriminant analysis(PLS-DA). Results:DVB/CAR/PDMS extraction head,extraction temperature of 90 ℃,adsorption time of 80 min,pre-equilibration time of 40 min and desorption time of 2 min were selected as the optimum SPME extraction conditions.56,71,72,81,and 75 of compounds were identified by the established method of HS-SPME/GC-MS in Jinshuibao capsules,Bailing capsules,Ningxinbao capsules,Zhiling capsules and Xinganbao capsules,respectively.They mainly included esters,alcohols,carboxylic acids,aldehydes,hydrocarbons,ketones,heterocyclic compounds.Different fermented Cordyceps preparations were distinguished and counterfeits were identified by chemometrics analysis based on the HS-SPME/GC-MS data.The characteristic volatiles of massoia lactone for Jinshuibao capsules,pyrazinamide for Bailing capsules,2-pyrrolidone for Ningxinbao capsules,ethyl palmitate for Zhiling capsules,and palmitic acid for Xinganbao capsules were also found by chemometrics analysis.Conclusion:HS-SPME/GC-MS can be used for rapid analysis of volatile components in fermented Cordyceps sinensis.Combined with chemometric methods for distinguishing different fermented Cordyceps sinensis and finding differential components,it gives us new ideas to search characteristic components,and provides us with scientific basis for the establishment and improvement of the quality standard of fermented Cordyceps preparations.
引文
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[16]ZHANG Y,MITCHISON D.The curious characteristics of pyrazinamide:a review[J].Int J Tuberc Lung Dis,2003,7(1):6
[17]陈银基,鞠兴荣,周光宏.饱和脂肪酸分类与生理功能[J].中国油脂,2008,33(3):35CHEN YJ,JU XR,ZHOU GH.Classification and physiological function of saturated fatty acids[J].China Oil Fats,2008,33(3):35
[2]姚书文,李钰,陈丽华,等.冬虫夏草及各种发酵虫草菌粉产品药效学差异的文献研究[J].中国药房,2015,26(3):294YAO SW,LI Y,CHEN LH,et al.Pharmacodynamic differences in the literature research of Cordyceps sinensis and its fermented mycelium[J].China Pharm,2015,26(3):294
[3]黄慧莲,杨敏娟,管咏梅,等.近5年发酵虫草菌粉的化学成分和临床应用研究进展[J].世界科学技术-中医药现代化,2014,16(10):2242HUANG HL,YANG MJ,GUAN YM,et al.Review on chemical compositions and clinical application of cultured Cordyceps militaris in recent five years[J],World Sci Technol-Mod Tradit Chin Med Mater Med,2014,16(10):2242
[4]张萍,郑天骄,张文娟,等.5种发酵虫草菌丝类制剂的特征图谱及其模式识别研究[J].中国药学杂志,2015,50(4):293ZHANG P,ZHENG TJ,ZHANG WJ,et al.HPLC characteristic fingerprint and chemical pattern recognition of fermentation mycelium preparations[J].Chin Pharm J,2015,50(4):293
[5]PENG F,SHENG L,LIU B,et al.Comparison of different extraction methods:steam distillation,simultaneous distillation and extraction and headspace co-distillation,used for the analysis of the volatile components in aged flue-cured tobacco leaves[J].J Chromatogr A,2004,1040(1):1
[6]何颖.中药挥发油提取方法分析[J].天津药学,2015,27(1):47HE Y.The analysis of methods for extracting essential oils[J].Tianjin Pharm,2015,27(1):47
[7]ARTHUR CL,PAWLISZYN J.Solid phase microextraction with thermal desorption using fused silica optical fibers[J].Anal Chem,1990,62(19):2145
[8]ZHANG ZY,YANG MJ,PAWLISZYN J.Solid-phase microextraction.A solvent-free alternative for sample preparation[J].Anal Chem,1994,66(17):844A
[9]马继平,王涵文,关亚风.固相微萃取新技术[J].色谱,2002,20(1):16MA JP,WANG HW,GUAN YF.New technologies of solid-phase microextraction[J].Chin J Chromatogr,2002,20(1):16
[10]ARTHUR CL,POTTER DW,BUCHHOLZ KD,et al.Solid-phase microextraction for the direct analysis of water:theory and practice[J].LC GC,1992,10(9):656
[11]余泽红,贺小贤,丁勇,等.固相微萃取在食品挥发性组分测定方面研究进展[J].粮食与油脂,2010(7):44YU ZH,HE XX,DING Y,et al.Research progress on solid phase micro-extraction determination of volatile components in foods[J].Cereals Oils,2010(7):44
[12]乐之歆,庄伟强,黄健,等.固相微萃取/气-质联用分析猴头菇挥发性风味成分[J].食品工业,2015,36(9):278LE ZX,ZHUANG WQ,HUANG J,et al.Analysis of volatile flavors in hericium erinaceus by using solid phase microextraction combined with GC-MS[J].Food Ind,2015,36(9):278
[13]RIU-AUMATELL M,MIRO P,SERRA-CAYUELA A,et al.Assessment of the aroma profiles of low-alcohol beers using HSSPME–GC-MS[J].Food Res Int,2014,57:196
[14]KISHIMOTO N,SUGIHARA S,MOCHIDA KYO,et al.In vitro antifungal and antiviral activities ofγ-andδ-lactone analogs utilized as food flavoring[J].Biocontrol Sci,2005,10(1/2):31
[15]BARROS ME,FREITAS JC,OLIVEIRA JM,et al.Synthesis and evaluation of(-)-massoialactone and analogues as potential anticancer and anti-inflammatory agents[J].Eur J Med Chem,2014,76:291
[16]ZHANG Y,MITCHISON D.The curious characteristics of pyrazinamide:a review[J].Int J Tuberc Lung Dis,2003,7(1):6
[17]陈银基,鞠兴荣,周光宏.饱和脂肪酸分类与生理功能[J].中国油脂,2008,33(3):35CHEN YJ,JU XR,ZHOU GH.Classification and physiological function of saturated fatty acids[J].China Oil Fats,2008,33(3):35