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Phytochemical Analysis

Phytochemical Analysis

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 Journal of Chromatography A, 1216 (2009) 2045–2062
Contents lists available atScienceDirect
 Journal of Chromatography A
 journal homepage:www.elsevier.com/locate/chroma
Review
Phytochemical analysis of traditional Chinese medicine using liquidchromatography coupled with mass spectrometry
Min Yang
a
, Jianghao Sun
b
, Zhiqiang Lu
a
, Guangtong Chen
a
, Shuhong Guan
a
,Xuan Liu
a
, Baohong Jiang
a
, Min Ye
b
, De-An Guo
a
,
b
,
a
Shanghai Research Center for TCM Modernization, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
b
The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China
a r t i c l e i n f o
 Article history:
Available online 3 September 2008
Keywords:
Phytochemical analysisTraditional Chinese medicineHPLC–MSTandem mass spectrometryQuality control
a b s t r a c t
Traditional Chinese medicine (TCM) is commonly considered to operate due to the synergistic effects of all the major and minor components in the medicines. Hence sensitive and comprehensive analyticaltechniques are needed to acquire a better understanding of the pharmacological basis of the herb and toenhance the product quality control. The present review mainly focuses on the phytochemical analysisof TCMs using high-performance liquid chromatography coupled with mass spectrometry (HPLC–MS).Atmosphericpressurechemicalionization(APCI)andelectrosprayionization(ESI)arethetwocommonlyused ion sources. Triple quadrupole, ion trap (IT), Fourier transform ion cyclotron resonance (FTICR) andtime-of-flight (TOF) mass spectrometers are used as on-line analyzer. The relationship between struc-tural features and fragmentation patterns should be investigated as thoroughly as possible and hence beapplied in the on-line analysis to deduce the structures of detected peaks. Characteristic fragmentationbehaviors of the reference standards, as well as information regarding polarity obtained from retentiontimedata,on-lineUVspectra,datafromtheliteratureandbio-sourcesofthecompoundsallowedtheiden-tification of the phytochemical constituents in the crude extracts. Although a mass spectrometer is nota universal detector, high-performance liquid chromatography coupled with multistage mass spectrom-etry (HPLC–MS
n
) technique was still proved to be a rapid and sensitive method to analyze the majorityof the many constituents in herbal medicines, particularly for the detection of those present in minor ortrace amounts. The methods established using HPLC–MS techniques facilitate the convenient and rapidqualitycontroloftraditionalmedicinesandtheirpharmaceuticalpreparations.However,thequantitativeanalysis is not the topic of this review.© 2008 Elsevier B.V. All rights reserved.
Contents
1. Introduction........................................................................................................................................ 20462. Identification of known and unknown constituents in the extracts of traditional Chinese medicines and their derived products............. 20472.1. Phenolic compounds (including avonoids)............................................................................................... 20472.1.1. Danshen: root of 
Salvia miltiorrhiza
............................................................................................... 20472.1.2. Dengzhanxixin: whole plant of 
Erigeron breviscapus
............................................................................ 20472.1.3. Rhubarb: root and rhizome of 
Rheum
species.................................................................................... 20482.1.4. Lianqiao: fruits of 
Forsythia suspense
............................................................................................. 20482.1.5. Huangqin: roots of 
Scutellaria baicalensis
........................................................................................ 20482.1.6. Maidong: tuber of 
Ophiopogon japonicus
......................................................................................... 20482.1.7. Ginger: rhizome of 
Zingiber officinale
............................................................................................ 20492.1.8. Propolis (Fengjiao)................................................................................................................ 20492.1.9. Kushen: roots of 
Sophora flavescens
.............................................................................................. 2050
Corresponding author at: Shanghai Research Center for TCM Modernization, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Guo Shoujing Road199, Zhangjiang, Shanghai 201203, China. Tel.: +86 21 50271516; fax: +86 21 50272789.
E-mail address:
gda5958@163.com(D.-A. Guo).0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2008.08.097
 
2046
M. Yang et al. / J. Chromatogr. A 1216 (2009) 2045–2062
2.1.10. Dandelion (Pugongying): whole plant of 
Taraxacum officinale
................................................................. 20502.1.11. Jiangxiang: heartwood of 
Dalbergia odorifera
................................................................................... 20502.1.12. Artichoke:
Cynara scolymus
..................................................................................................... 20502.1.13. Honghuayanhuangqi: roots of 
Hedysarum multijugum
......................................................................... 20502.1.14. Tusizi: seeds of 
Cuscuta chinensis
................................................................................................ 20502.1.15. Others............................................................................................................................ 20512.2. Saponins.................................................................................................................................... 20512.2.1. Ginseng: root and rhizome of 
Panax
spp.......................................................................................... 20512.2.2. Sanqi: root and rhizome of 
Panax notoginseng 
................................................................................... 20512.2.3. Ciwujia: roots of 
Acanthopanax senticosus
....................................................................................... 20522.2.4. Huashanfan: roots and leaves of 
Symplocos chinensis
........................................................................... 20522.2.5. Huangqi: roots of 
Astragalus
spp. ................................................................................................ 20522.2.6. Cynanchum: Radix Cynanchi atrati (Baiwei) and
Cynanchum chekiangense
(Manjiancao) ..................................... 20522.2.7. Chuanshanlong: rhizomes of 
Dioscorea nipponica
............................................................................... 20522.3. Alkaloids ................................................................................................................................... 20522.3.1. Aconite:
Aconitum sinomontanum
,
Aconitum kusnezoffii
,
Aconitum carmichaeli
................................................. 20532.3.2. Liangmianzhen: roots of 
Zanthoxylum nitidium
.................................................................................. 20532.3.3. Baibu: root tuber of 
Stemona tuberosa
........................................................................................... 20532.3.4. Lilu: root and rhizome o
Veratrum nigrum
...................................................................................... 20532.3.5. Others ............................................................................................................................ 20542.4. Monoterpene glycosides................................................................................................................... 20542.4.1. Shaoyao: dried roots of 
Paeonia lactiflora
........................................................................................ 20542.4.2. Zhizi: fruits of 
Gardenia jasminoides
............................................................................................. 20542.4.3. Jishiteng: whole plant of 
Paederia scandens
..................................................................................... 20542.4.4. Others ............................................................................................................................ 20542.5. Diterpenoids ............................................................................................................................... 20542.5.1. Danshen: roots of 
S. miltiorrhiza
.................................................................................................. 20542.6. Triterpenoids (aglycone)................................................................................................................... 20542.6.1. Lingzhi: fruit body of 
Ganoderma lucidum
....................................................................................... 20552.7. Steroids (aglycone)......................................................................................................................... 20552.7.1. Chansu: skin secretions of giant toads ........................................................................................... 20562.8. Others and miscellaneous natural products............................................................................................... 20562.8.1. Mudanpi (Cortex Moutan): root bark of 
Paeonia suffruticosa
.................................................................... 20562.8.2. Jinguolan: roots of 
Tinospora sagittata
and
Tinospora capillipes
................................................................. 20562.8.3. Shengma: rhizome and root of 
Cimicifuga
species............................................................................... 20582.8.4. Others ............................................................................................................................ 20592.9. Complex traditional Chinese medicine prescription ...................................................................................... 20602.9.1. Shuanghuanglian oral liquid ..................................................................................................... 20602.9.2. Shuangdan granule............................................................................................................... 20602.9.3. Qingkailing injection............................................................................................................. 20602.9.4. Compound Danshen Dripping Pill ............................................................................................... 20603. Conclusion......................................................................................................................................... 2061Acknowledgement................................................................................................................................. 2061References......................................................................................................................................... 2061
1. Introduction
Traditional Chinese medicine (TCM) played a significant rolein the health of the Chinese people for thousands of years. Overthe past decades, TCMs have always been the most importantresources for screening lead compounds. Fast analysis of natu-ral products in bioactive crude extracts attracted the attention of most investigators. Natural products gained prominence throughantibiotics earlier in the last century; today they have been devel-oped for a variety of medicinal uses such as immunosuppressiveagents, hypocholesterolemic agents, enzyme inhibitors, antimi-graneagents,herbicides,antiparasiticagentsandruminantgrowthpromoters, and bioinsecticides[1].Natural products are also the most important resources of anticancer agents, where over 60% of the approved and pre-new drug application candidates are eithernatural products or synthetic molecules based upon the naturalproduct molecular skeletons.Herbal medicines and their derived products are widely usedas therapeutic products in many countries. Their worldwide usehas increased in the last decade[2].Most herbal medicines and theirderivativeproductswereoftenpreparedfromthecrudeplantextracts, which comprise a complex mixture of different phyto-chemical constituents (plant secondary metabolites). There maybe hundreds of active components in these herbs. The chemicalfeatures of these constituents differ considerably among the dif-ferent species. Even the same herbal extracts may vary dependingupontheharvestseason,plantorigin,dryingprocessandotherfac-tors.Therefore,thequalitycontroloftheherbalmedicinesandtheirderived products is difficult. Recently, the chromatographic finger-printingofthecomponents,especiallybyhigh-performanceliquidchromatography–diode array detection (HPLC–DAD), is a power-ful and widely used technique to analyze plant extracts becausethistechniquecouldsystematicallyprofilethecompositionofsam-plesanditfocusesontheidentificationandconsistencyassessmentof the components[3].However, a valuable and convincing chro- matographic fingerprint should have most of its peaks assigned,especially those corresponding to the active constituents and toxicingredients. Unfortunately, such routine techniques as HPLC–DADcouldonlyprovideverylimitedstructuralinformationlikeUVspec-trum;standardcompounds,whicharecommerciallyunavailableinmost cases, are usually necessary for the characterization of indi-vidual constituents. As a result, the isolation and purification from
 
M. Yang et al. / J. Chromatogr. A 1216 (2009) 2045–2062
2047
crudeplantextractsofadequateamounts(atleast5–10mg)ofpurecompounds (>90% purity) for nuclear magnetic resonance (NMR)identification was needed before they could serve as referencecompounds. The whole process is tedious, laborious and expen-sive. Moreover, some constituents are only present in raw plantmaterials in very low amount or are quite unstable under normalconditions, and their enrichment and purification are laborious.The inherent variety of natural product extracts has shownsignificant challenges for separation and detection techniques toenable rapid characterization of the biologically active componentin the mixture. Except for paper chromatography and thin-layerchromatography,gaschromatography(GC)isapowerfulseparationtechnique that has been utilized since the 1960s for the analysis of volatile natural products or derivatives. However, the role that hasrecently been played by HPLC for the work of this nature has beeninvaluable, considering that approximately 80% of all known natu-ralcompoundsarenonvolatileorthermallyunstableandthereforeincompatible with GC. When coupled with a variety of detections,HPLCbegantoserveasapowerfultoolfortherapidcharacterizationofnaturalproductextracts.Inaddition,somedevelopingchromato-graphic techniques, such as capillary electrophoresis (CE), highspeed countercurrent chromatography (HSCCC), gradually foundtheir application in the separation of phytochemical constituentsof herbal medicines.Mass spectrometry (MS) is the most selective technique for therapid qualitative determination of known compounds as well astheidentificationofunknowncompoundsfromtheextractsofnat-ural products. The analysis of natural products has been especiallyeffectivefollowingthesuccessfulinterfacewithamassspectrome-terdetectionsysteminthechromatographicmethod.Atmosphericpressure ionization (API), which consists of atmospheric pressurechemical ionization (APCI) and electrospray ionization (ESI), is themost successful interface used in HPLC–MS configuration. There-fore, HPLC–MS combines the efficient separation capabilities of HPLCandthegreatpowerinstructuralcharacterizationofMS,andprovides a new powerful approach to identify the constituents inplantextractsrapidlyandaccurately.Otherwise,diodearraydetec-tion techniques could also be used in combination with HPLC–MS.DAD and MS can provide on-line UV and MS information at thesame time for each individual peak in a chromatogram. One cancharacterizesomeofthepeaksdirectlyon-linebycomparisonwithstandard compounds or with literature data. In the 1990s, someinvestigatorsmadesomepreliminaryattemptsonthecomprehen-siveanalysisofphytochemicalconstituentsinbotanicalextracts.Hedelivered a review in 2000[4].The present review focuses on the applicationofHPLC–MSintheanalysisofChineseherbalmedicinesafter 1999.Thementionedcompounds’numbersareonlyusedineachindi-vidual sub-section. Only qualitative analysis is discussed in thisreview.
2. Identification of known and unknown constituents inthe extracts of traditional Chinese medicines and theirderived products
The soft ionization methods [ESI, APCI, thermospray (TSP), etc.]do not typically produce many fragments. The collision-induceddissociation (CID) or collision activated dissociation (CAD) meth-ods can overcome this disadvantage. Quadrupole, ion trap (IT),Fourier transform ion cyclotron resonance (FTICR), time-of-flight(TOF) mass spectrometer are the frequently used detectors for thephytochemical analysis of herbal medicines. The multistage massspectra (MS
n
,
n
2) provided by IT-MS could confirm the relation-ship between precursor and daughter ions. This property is veryhelpful for the structural determination of unknown compounds.FTICR and TOF could give high-resolution mass spectra of all theions and allow the definition of the ions’ elemental composition.Here, a number of examples were used to describe how HPLC–MScould be effectively applied to perform component identificationin complex mixtures.
 2.1. Phenolic compounds (including flavonoids)
Phenolic compounds are the major bioactive constituents of the herbs. Nearly half of the literatures reported on the on-lineanalysis of phytochemical constituents of the bioactive pheno-lic compounds. The following sections are some of the examplestudies. Flavonoids appeared with very high frequency in thoseliteratures.
 2.1.1. Danshen: root of Salvia miltiorrhiza
The dried root of 
Salvia miltiorrhiza
Bunge (Chinese name ‘Dan-shen’)isoneofthemostwell-knowntraditionalChinesemedicines.It is widely used to treat coronary heart diseases, cerebrovasculardiseases,boneloss,hepatitis,hepatocirrhosisandchronicrenalfail-ure, dysmenorrheal and neurasthenic insomnia. Phenolic acids arethe water soluble active constituents of Danshen. Therefore, it hasincitedagreatdealofresearchontheanalysisofthephenolicacidsinthisherb[5–7].Electrosprayionizationinnegativeionmodewas adopted in all the reported studies.Zeng et al. have studied phenolic acids in
S. miltiorrhiza
byHPLC/ESI-MS/MS[7].They have applied triple quadrupole mass spectrometer as the detector. It was found that caffeic acid andits monomeric analogues containing a carboxyl group readily loseCO
2
,whiledimers,trimersandtetramersofcaffeicacideliminatedsuccessivelydanshensuorcaffeicacidortheiresters.Twenty-eightphenolic compounds in the extract of 
S. miltiorrhiza
were charac-terized and eight of them were positively identified by comparingwith the reference standards.The phenolic compounds in
S. miltiorrhiza
were investigated byLiu et al. using IT-MS[6],which has been considered more suit- able for qualitative analysis than quadrupole mass spectrometer.The major fragmentations were very similar to those reported byZengetal.[7].However,thegeneticrelationshipsbetweenthepre- cursor and daughter ions are more affirmative. Based on the MSfragmentation rules, the extract of Danshen was analyzed. In total,42 phenolic acids were identified or tentatively identified in oneHPLC–MS
n
run,and16ofthemwereidentifiedforthefirsttime.Inaddition,someisomersandcloseanaloguescouldbedistinguishedfrom each other by comparing their MS/MS and MS
n
spectra.In addition to IT, TOF was also employed for the study of phe-nolic compounds in
S. miltiorrhiza
by Zhu et al.[5].The accurate molecular weights of the constituents should be determined byHPLC/ESI-TOF-MS and most of the compounds in Danshen couldbe identified by TOF-MS from the formula database. HPLC–DAD,HPLC/ESI-TOF-MS and HPLC/ESI-MS
n
provided complementaryinformation for the identification of the constituents in Danshen.Usingtheestablishedmethods,22phenoliccompoundsand18tan-shinones were simultaneously characterized in 30min based ontheir negative and positive ion mass spectrometry. The accuratemolecularweightsobtainedbyon-lineTOFmassspectraareagreathelp for the determination of molecular formulas.
 2.1.2. Dengzhanxixin: whole plant of Erigeron breviscapus
The whole plant of 
Erigeron breviscapus
(Vant.) Hand.-Mazz.(Compositae), known as Dengzhanxixin, is an important herbaldrug in China for the treatment of cardiovascular and cerebral ves-seldiseases.Themainactivecomponentsidentifiedfromthisherbincluded flavonoids, coumarins, lignins, hydroxycinnamic acids,pyromeconic acids and erigesides.

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