Metabolomics and TCM

Metabolomics and traditional Chinese medicineChunxiu Hu Guowang Xu Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics Chinese Academy of Sciences 457 Zhongshan RoadDalian 116023 China

A R T I C L E I N F O

KeywordsAnalytical characterizationBioactive componentBiochemistryChinese herbal medicineChromatographic fingerprintingHolistic bioactivityMetabolomicsMulti-component mixtureTCM syndromeTraditional Chinese medicine

A B S T R A C T

Traditional Chinese medicine (TCM) has attracted increasing interest throughout the world because ofits potential as complementary therapy of choice and a source for discovery of new drugs However theanalytical characterization of TCM is still in its infancy due to its chemical nature of multi-componentmixtures that often possess their own inherent holistic bioactivities Metabolomics incorporates state-of-the-art approaches enabling systemic molecular characterization in complex samples and providesthe option for an integrated view of the biochemistry in TCM In this review we present currentmetabolomics technologies in research on TCM related to chemical composition bioactive componentsand efficacy We also highlight the potential role of metabolomics technologies in evidence-based studiesof TCM syndromes

Contents

1 Introduction 2072 Advances in metabolomics technologies 2083 Applications of metabolomics in systems-based TCM 208

31 Chemical characterization of CHM 20932 Bioactive component screening of CHM 20933 Efficacy evaluation of CHM 210

4 Evidence-based studies in TCM syndromes 21141 TCM pattern diagnosis 21142 TCM pattern intervention 212

5 Future perspectives 212Acknowledgments 213References 213

1 Introduction

Interest in study of traditional Chinese medicines (TCMs) or socalled Chinese herbal medicines (CHMs) recently became wide-spread due to their potential as a source of new drug discovery andcomplementary therapy of choice although the history of TCM canbe dated back to thousands of years ago TCM holds a holistic healthcare philosophy (ie looking at the behavior of the system as awhole) It regards disease as energetic imbalance and adheres tothe concept of cure instead of treatment The components of CHMare very complex CHMs usually consist of hundreds or even thou-sands of different compounds The therapeutic effects of CHM canusually be attributed to the combination of multiple elements

Abbreviations APCI Atmospheric pressure chemical ionization APPI Atmo-spheric pressure photoionization CE Capillary electrophoresis CHM Chinese herbalmedicine DART-MS Direct analysis in real time mass spectrometry EI Electron ion-ization ESI Electrospray ionization FAB Fast atom bombardment FT-ICR Fouriertransform ion cyclotron resonance GC Gas chromatography GC times GC Comprehen-sive two-dimensional gas chromatography GK Goto-Kakizaki HILIC Hydrophilicinteraction chromatography IT Ion trap LC Liquid chromatography LC times LC Com-prehensive two-dimensional liquid chromatography MALDI Matrix-assisted laserdesorptionionization MS Mass spectrometry NMR Nuclear magnetic reso-nance OPLS-DA Orthogonal partial least squares discriminant analysis PCA Prin-cipal component analysis Q Quadrupole QQQ Triple quadrupole TCM traditionalChinese medicine TOF Time-of-flight UPLC Ultra-performance liquid chromatog-raphy WM Western medicine

Corresponding author Tel +86 411 84379530 Fax +86 411 84379559E-mail address xugwdicpaccn (G Xu)

Trends in Analytical Chemistry 61 (2014) 207ndash214

Contents lists available at ScienceDirect

Trends in Analytical Chemistry

journal homepage wwwelseviercom locate t rac

(ie multi-component mixture) of the herb or the formula actingtogether

Traditionally a TCM is administered as an individually pre-pared water decoction containing multiple herb materials in certainproportions Notably the final item that is taken orally by a patientis an extract of herbs that is influenced by not only the botanicalorigin of its herbal components but also the procedure that is usedto treat the herbal material [1] Any minor changes in these twoaspects will greatly affect the therapeutic efficacy of the productsSuch complexity in TCM administration brings a great challenge inestablishing suitable analytical approaches for the development ofmodernization and standardization of TCMs and their natural prod-ucts Besides TCM possesses two unique features ndash multidimen-sional pharmacology ie synergetic effects [2] and combinatorialeffects and opposing principles [3] and personalized or individu-al treatment Such features of TCM determine that the traditionalanalysis based on major components is not sufficient to provide in-formation on bioactivity of herbal products Analytical character-ization of TCM is still in its infancy due to the chemical natureof multi-component mixtures that often possess inherent holisticbioactivities

To meet this challenge metabolomics which aims at compre-hensive characterization of the total metabolome in a biologicalsystem and dynamic metabolomics responses to alterations of innerandor external factors [4] is in great demand As an emerging fieldof science in the post-genomics era metabolomics has been broadlyapplied in many fields eg toxicological survey [5] functional ge-nomics [67] clinical diagnostics [8] plant and microbes [9ndash11] andnutritional biochemistry [1213] The most commonly used two tech-nologies in metabolomics applications are nuclear magnetic reso-nance (NMR) [14] and mass spectrometry (MS) [15] Although itssensitivity is less than that of MS NMR is still popular in many ap-plications due to its rapidity high throughput easy operation beingnon-destructive of samples and quantitation of metabolites fromdifferent groups However in most cases MS is preferred becauseof its advantages of unparalleled sensitivity super resolution andstructural specificity In practical applications chromatographic sep-aration technologies [eg gas chromatography (GC) liquid chro-matography (LC) and capillary electrophoresis (CE)] are often coupledto MS depending on the properties of the analytes being measured

As the data matrix generated in metabolomics studies is usuallyvery large data processing and data analysis are crucial for theirfinal outcome Many available software packages eg JDAMP [16]MetAlign [17] MSFACTs [18] Mzmine [19] and XCMS [20] weredeveloped for processing raw data from metabolomics On this basismultivariate (eg PCA PLS-DA PLS and OPLS) and univariate (egT-test and ANOVA) analyses were carried out for classification anddifferential metabolite discovery In combination with advanced bio-informatics tools many studies demonstrated that metabolomicsincorporating state-of-the-art analytical approaches enables sys-temic molecular characterization of complex samples [2122] andprovides an integrated biochemical view of TCM

In this review we summarize metabolomics methods based onGC-MS and LC-MS and applied in research on TCM We highlightthe subjects related to chemical composition bioactive compo-nents and the efficacy of CHM and metabolomics applications inevidence-based studies of TCM syndromes

2 Advances in metabolomics technologies

As early as the 1970s the embryonic form of metabolomics (atthat time called ldquometabolic profilerdquo) was coming into being A typicalexample is that GC-MS became feasible to measure qualitatively andquantitatively metabolic profiles of body fluids and tissue ex-tracts Concurrently rapid improvement was achieved in NMR spec-troscopy due to the increasing strengths of magnetic field and magic

angle spinning which remarkably improved the sensitivity and madeNMR a leading analytical tool to detect metabolites of many dif-ferent classes in biological samples

The initiation of study on MS-based metabolomics in a real sensewas carried out by van der Greef et al using field desorption andfast atom bombardment (FAB)-MS for profiling complex non-volatile metabolites in body fluids to study gender difference [23]They pioneered the utilization of pattern-recognition techniques toMS-based datasets Meanwhile the NMR-based metabonomics ap-proach was pioneered in the study of vertebrate blood and plasmaby Nicholson et al [24]

Although there were some sporadic applications the limita-tion in early analytical technologies (eg low sensitivity low speci-ficity and low throughput) made it a big challenge to qualify andto quantify complex metabolites in biological samples Analyticaltechniques with super sensitivity and selectivity high specificitygood reliability and easy automation are required to detect and toidentify metabolites in complex biological systems The revolu-tion in soft-ionization technologies for MS included matrix-assisted laser desorptionionization (MALDI) [25] and electrosprayionization (ESI) [26] as well as atmospheric pressure chemical ion-ization (APCI) [27] Metabolomics has largely been driven by thesetechnologies as they possess better resolution and sensitivity andhigher specificity and mass accuracy than earlier technologies TheseMS instruments are still undergoing rapid advances in terms of sen-sitivity resolution mass accuracy and dynamic range

As complexity increases in the composition of metabolome ndash thewhole set of endogenous and exogenous metabolites with small mo-lecular weight in an individual organism ndash modern technologies thatallow for qualitative and quantitative measurement of a vast numberof metabolites in complex biological systems are required formetabolomics studies Modern metabolomics platforms usually com-prise approaches using chromatography-based technologies suchas GC and LC including high-performance LC (HPLC) ultra-performance LC (UPLC) hydrophilic interaction chromatography(HILIC)) and CE coupled to MS or MSMS Notably frequently usedion sources are for GC-related techniques electron ionization (EI)and for LC- and CE-based MS systems ESI Occasionally APCI andatmospheric pressure photoionization (APPI) are coupled to LC fordetection of non-polar compounds Frequently used mass analyz-ers in current metabolomics include quadrupole (Q) ion trap (IT)time-of-flight (TOF) Fourier transform ion cyclotron resonance (FT-ICR) orbital trap and a combination of them [eg triple quadru-pole (QQQ) Q-TOF IT-TOF IT-FT-ICR]

Besides NMR is also frequently applied in modern metabolomicsstudies In recent decades many studies using NMR for metabolomicswere carried out by Nicholson and his group The relatedmetabolomics studies generated many valuable findings rangingfrom early diagnosis to disease treatment drug toxicity gene func-tion microbial physiological and epidemiological aspects [628ndash31]

3 Applications of metabolomics in systems-based TCM

Differing from Western medicine (WM) that is based on theconcept of ldquoone size fits allrdquo (ie 1 disease ndash 1 target ndash 1 drug) TCMfollows the principle of multiple components ndash multiple target in-teractions ndash and has been developed into a holistic healthcare system[32] Although attractive worldwide as a rich source of leading mol-ecules for complementary and alternative medicine TCM suffers frominextricable obstacles in modern CHM research [33] In CHM mostof the active components are not well defined The constituents andtheir related concentrations in each herbal medicine can vary greatlyeg depending on species [34] plant-growth conditions and pro-cessing procedure [35] Metabolomics provides a valuable chancefor studying TCM in terms of characterizing the chemical compo-nents defining the multiple active components and evaluating the

208 C Hu G XuTrends in Analytical Chemistry 61 (2014) 207ndash214

efficacy Fig 1 shows the workflow of MS-based metabolomics insystems-based TCM

31 Chemical characterization of CHM

The targets in TCM research no matter what the crude materi-als formulas or body fluids from different TCM syndromes are verycomplex samples Metabolomics incorporating advanced analyti-cal instruments is capable of simultaneously determining andidentifying thousands of metabolites in complex samplesChromatography-MS-based technologies are the mainstream foranalysis of endogenous and exogenous metabolites in complexsamples

Volatile oils are one of the main pharmacologically active com-ponents of many herb plants GC- or comprehensive two-dimensionalGC (GC times GC)-MS-based technologies are preferred for their anal-ysis GC-MS in selected ion mode and GC times GC-TOFMS were appliedfor qualitative and quantitative analysis of volatile compounds ofCyperus rotundus [36] The yield and the quality of three extrac-tion methods [ie hydrodistillation pressurized liquid extraction(PLE) and supercritical fluid extraction (SFE)] were compared by thechemical constituents of different extracts It was found that PLEhad the highest extraction efficiency whereas SFE had the best se-lectivity for extraction of two compounds (β-cyperone andα-cyperone) In another study Qiu et al characterized and quan-tified chemical compositions of volatile oils in radix ginseng at dif-ferent growth ages using GC times GC-TOFMS metabolomics combinedwith an unsupervised pattern-recognition method [37] Chemicalcompositions of samples were clearly discriminated among the threegrowth ages and a large number of compounds were found to con-tribute to the differentiation It was found that volatile com-pounds of α-cadinol α-bisabolol thujopsene and n-hexadecanoicacid showed positive correlation with the growth of the age of panaxginseng These studies demonstrate that coupled GC with achemometric method can be an ideal tool for characterization ofcertain specific bioactive components of a CHM

LC is a separation technology that is most commonly used in CHMresearch due to its universal adaptability to most compounds withoutneed to take the volatility or the stability of the sample into con-sideration Monolithic column LC capillary LC and UPLC were de-veloped to enhance the separation efficiency In a newly publishedreview on untargeted metabolomics to determine the composi-tion of herbal products Commisso et al summarized LC-MS-based untargeted metabolomics approaches in comprehensiveanalysis of complex plant tissues unraveling environment-dependentplant cell composition and analysis of natural remedies [38] Wang

et al combined the technologies of MALDI-MS with LC-MS to semi-quantitate and to identify the alkaloid in a Chinese herbal medi-cine Fuzi the processed lateral roots of Aconitum carmichaeli Debx(A carmichaeli) [39] The contents of alkaloids were found signifi-cantly different in 14 batches of Fuzi analyzed and decreased withthe length of the processing period In another study a UPLC-QTOFMS-based metabolic profiling approach with pattern-recognitionanalysis successfully discriminated Fuzi from its different prepara-tions by comprehensive chemical composition analysis of Fuzi andits processed products [40] Besides metabolomics analytical tech-nology by direct analysis in real time mass spectrometry (DART-MS) in conjunction with multivariate analysis was a novel rapidmethod for monitoring batch-to-batch reproducibility of 12 testedbatches of Danshen alkaline precipitations [41] Artificial varia-tions (eg starting material change and process fault) were suc-cessfully identified under this method In other applications themetabolomics technologies of DART-MS and HPLC-DAD-ESIMS wereapplied for monitoring processing procedures of herbs [42ndash44]

The studies discussed above demonstrate that MS-basedmetabolomics technologies can be applied for characterization ofchemical components of CHM from different growth conditionsgrowth ages batches and processing procedures

32 Bioactive component screening of CHM

A particular feature of TCM is holistic interactions (ie the syn-ergetic interactions of the bioactive multi-components of the herbor the formula) As bioactive components in CHM generally havelow abundance how to rapidly screen the bioactive components ata systematic level is key in modernizing TCM

Traditionally procedures to find out bioactive components are

(1) to extract the effective compound groups from the complexmixture of CHMs

(2) to purify the compounds from the extractions and(3) to perform animal pharmacological experiments on the

bioactivity of the purified compounds singly

Such a screening method for single target constituents of CHMsis not only inefficient but also does not match the holistic conceptof multi-targets and systems of TCM theory [45] Metabolomics ashas been described can provide an optimal platform for achiev-ing this aim by incorporating state-of-the-art technologies

A GC-MS metabolomics approach (Fig 2) has been developedfor screening bioactive components from zedoary turmeric oiland glucose injection using extracts from living cells [46] Elemene

Fig 1 Workflow for mass spectrometry-based metabolomics in research on systems-based traditional Chinese medicine

209C Hu G XuTrends in Analytical Chemistry 61 (2014) 207ndash214

emulsion injection a representative TCM with known bioactive com-ponents was used to validate the method by interacting with themurine ascites hepatocarcinoma cell strain with high metastatic po-tential It was concluded that curzerene and β-elemene were twomajor bioactive compounds in zedoary turmeric oil and glucose in-jection Metabolomics utilizing HPLC-TOFMS screened the poten-tial bioactive constituents in a combined prescription of DangguiBuxue decoction using cell extraction [47] Validation of the methodwas achieved using HL-7702 cells RAW 2647 cells and Caco-2 cellsthat were incubated together with the extract of TCM The resultsdemonstrated that 9 7 and 13 potential bioactive compounds werefound in these three cell extractions respectively

LC-MS-based metabolomics has also been widely applied inscreening bioactive components in CHM An LC-ESI-MS(n) system wasused for systematic screening and characterization of the majorbioactive compounds of Poria cocos and their metabolites in rats[48] A total of 34 compounds were detected and identified fromPoria cocos extract In vivo seven compounds were observed in raturine while one was detected in rat plasma

Another study employed a UPCL-Q-TOFMS analytical methodand macrophage cell extraction to screen and to analyze the po-tential bioactive components from Reduning Injection [49] Chlo-rogenic acid 34-dicaffeoylquinic acid 35-dicaffeoylquinic acid and45-dicaffeoylquinic acid were identified as potentially bioactive com-ponents from TCM injection In addition LCDAD-ESIMS was usedin screening bioactive components of Baihe Zhimu Tang [50] HPLC-Q-TOFMS and human umbilical vein endothelial cell were used forscreening the potentially bioactive components from a Shaofu Zhuyudecoction [51] and HPLC-ESI-MSMS was used for screening andanalyzing the multiple absorbed bioactive compounds and me-tabolites of Jitai tablets [52] These studies demonstrated that LC-MS-based metabolomics can be a valuable tool in screening andidentifying potentially bioactive components accountable for ther-apeutic effects of TCM further promoting the development of TCMand facilitating modern drug discovery

33 Efficacy evaluation of CHM

TCM is a complex and interacted system which is frequently usedin the form of formula (the combination of several different herbs)The strategy of TCM is to utilize a system therapy to support theself-healing capabilities of the body to regain its resilience from an

imbalanced state [53] Such system therapy possesses multi-component and multi-target characteristics in the treatment of dis-eases as each herbal drug comprises multiple bioactive constituentsthat have concerted actions [54] To date TCM has been an empir-ical healthcare system with a long documented history Such a tra-ditional medical system can provide only a certain type of evidenceto support its alleged healthcare benefits By introducing system-based metabolomics into TCM research we expect that there is abetter chance of evaluating the efficacy of TCM

Many applications using metabolomics have been reported tobe revealing the efficacy of CHMs in the treatment of diseases Twoapproaches to LC-MS-based metabolomics combined with mea-suring biochemical parameters were used to study the biologicaleffects of Panax ginseng upon type 2 diabetes [55] Panax ginsengroots harvested after being grown for 3ndash6 years were selected totreat Goto-Kakizaki (GK) rats with spontaneous type 2 diabetes fora nine-week period Plasma lipid profiling of all studied GK rats andginsenoside profiling of the root extracts of Panax ginseng with dif-ferent growth ages were obtained on LC-IT-TOFMS and UPLC-QTOFMS systems respectively Compared to the untreated controlspatients treated with 4- and 6-year-old ginseng showed a signifi-cantly beneficial effect on glucose disposal and on regulation oftriacylglycerol lipid species while 5-year-old ginseng significantlyimproved the level of high-density lipoprotein cholesterol Besides6-year-old ginseng significantly decreased the level of very lowdensity lipoprotein cholesterol and improved plasma-glycated he-moglobin in type 2 diabetic GK rats Such results demonstrated thatginseng roots have growth age-dependent therapeutic effects on twomajor issues in diabetic GK rats ndash hyperlipidemia and hyperglyce-mia Ginseng roots ge 4-year-old contain bioactive natural compo-nents that may be valuable in drug discovery and dietarysupplements After combining the results of the treatment effectsof ginseng with the profiles of ginseng extracts it can be arguedthat the age-dependent therapeutic effects be correlated with thevariation in the contents of several ginsenosides in ginseng rootsat different growth ages Fig 3 shows the therapeutic effects ofginseng observed [55] and the correlated ginsenoside biologicaleffects reported in literature [56ndash59]

In another case UPLC-MS-based metabolomics was applied witha statistical technique to evaluate the therapeutic effects ofEpimedium brevicornum Maxim a herbal medicine used for streng-thening bones and toning kidney on rats with kidney-deficiency

Fig 2 Flow scheme for screening bioactive components in traditional Chinese medicines based on cell extract and GC-MS [46] [Reproduced by permission of Elsevier (LicenseNumber 3406820710544)]

syndromes [60] The chemical profile of Epimedium brevicornumMaxim and plasma metabolic profiles of rats from three differenttreatments (pre- and post-hydrocortisone intervention and treat-ment with Epimedium brevicornum Maxim) were obtained by UPLC-MS PCA was used for comparison of the profiles Four compoundsthat were regarded as the active constituents of the herb were foundin the plasma of kidney-deficient rats and two of them were alsoobserved in urine

In other examples a metabolomics study based on UPLC-QTOFMS was used to evaluate the therapeutic effect and possible actionmechanism of a TCM preparation Xindi soft capsule on a rat modelwith acute blood stasis [61] Urinary metabolomics study of myo-cardial ischemia and treatment effects of compound Danshen Tabletsin rats was conducted on a UPLC-QTOFMS system [62] The detailsof these examples and several selected metabolomics applica-tions in the study of the therapeutic effects of TCM are summa-rized in Table 1 [63ndash69]

All of these studies illustrate that MS-based metabolomics is verypowerful in exploring the therapeutic effects of TCM and providesinsights into the potential action mechanism of TCM

4 Evidence-based studies in TCM syndromes

Therapy with syndrome differentiation is the essence of a TCMcure As the physiopathological changes of a disease usually en-gender a corresponding syndrome in the body the differences ofdisease symptoms are sure to be reflected by the metabolome inthe body system As an approach of studying the metabolome in a

holistic context metabolomics allowing a systemic study of all sortsof small molecular metabolites matches well with the holistic natureof TCM treatment so it has potential as a useful tool for studyingTCM syndromes [4570]

41 TCM pattern diagnosis

Taking the application of metabolomics on diagnosis and treat-ment of psoriasis patients with blood stasis syndrome in TCM asan example an HPLC-MS metabolomics method in combination withOPLS-DA and molecular docking analysis was applied to investi-gate the biological characterization of the patient urine and the ther-apeutic metabolomics mechanism of the optimized Yinxielingformula that was taken orally by patients after 0 4 8 and 12 weeks[71] A total of 60 subjects (ie 41 patients versus 19 healthy vol-unteers) participated in this study Three potential biomarkers in-volved in glucose metabolism eight involved in fat metabolism andfour involved in amino-acid and nucleotide metabolism were iden-tified from LC-MS metabolomics data The analyzed results suggestthat ldquothe deficiency of bile acids and the consequent cytokine releaseand endotoxin translocation might play a role in the pathogenesisof psoriasisrdquo [71]

Wei et al used a GC-MS urine metabolomics approach with PCAand CM diagnosis to identify diagnostic biomarkers for pre-diabetic sub-types [72] Three pre-diabetic groups including ldquoQi-Yin deficiencyrdquo and ldquoQi-Yin deficiency with dampnessrdquo (sub-typeA) and ldquoQi-Yin deficiency with stagnationrdquo (sub-type B) were dis-tinctly separated The majority of metabolites (ie sugars and amino

Fig 3 Observed ginseng therapeutic effects and the correlated ginsenoside biological effects [55] [Reproduced by permission of the Royal Society of Chemistry(httppubsrscorgencontentarticlelanding2011mbc1mb05254c)]

acids) were at higher levels in urine of sub-type B as compared tosub-type A indicating more disorders in carbohydrate metabo-lism and renal function in sub-type B versus sub-type A These studiesdemonstrated that metabolomics technology combined with mul-tivariate statistical analysis is helpful in identifying potential diag-nostic biomarkers for diseases enhancing our understanding of thebiological response to TCM syndrome and the action mechanismof CHM

42 TCM pattern intervention

A good example of metabolomics applications in TCM syn-drome is on the study of pyretic pulmonary syndrome model in-terfered by Radix Scutellariae using a UPLC-Q-TOFMS-basedapproach and PCA method to analyze the urinary metabolome ofWistar rats treated differently [73] Radix Scutellariae is a well-known medicinal herb in China and can alleviate heat syndromeSix specific biomarkers were determined that can represent Strep-tococcus pneumonia-induced pyretic pulmonary syndrome in ratsThe results of statistical analysis showed that the Chinese herb couldsubstantially make the disease-related increased biomarkers comeback to normal levels

More specifically a UPLC-MS-based metabolomics strategy wassuccessfully employed to characterize the kidney-yang deficiency(LYD) syndrome induced by high dose of hydrocortisone and theefficacy of Rhizoma Drynariae [74] PCA of urine metabolomics dataof rats from hydrocortisone-induced group and the pre-dose groupshowed that there are distinct metabolic patterns between themThe time-dependent recovery effect of Rhizoma Drynariae on KYDsyndrome rats was observed from day 3 to day 15 after therapy Themetabolites that were considered to contribute to the differentia-tion between the two groups were found to be involved in energy

metabolism amino acid metabolism and gut microflora Such find-ings provide valuable information for illustrating the therapeuticmechanism of Rhizoma Drynariae

Moreover Kuang et al studied Cold and Hot syndromes (ie fourproperties such as cold-cool and hot-warm) in TCM using a UPLC-QTOFMS-based metabolomics strategy in combination with bio-informatics tools [75] Some 56 male Sprague-Dawley rats (n = 8 pergroup) received the extracts of six herbs for 30 days respectivelyOne group without treatment was used as the controls Unsuper-vised PCA and supervised orthogonal partial least squares discrimi-nant analysis (OPLS-DA) multivariate data analyses of the global urineprofiles of rats on the 30th day from each group were performed toevaluate sample clustering according to the group information andobtain discrimination between different groups as well as find thesignificantly differential metabolites that might be potentialbiomarkers The Cold and Hot medication-treated rats were dis-tinguished clearly by the differences in the metabolic profiles provingthe existence of the four properties of TCM

Taken together the findings of these studies show that MS-based metabolomics strategy is very promising in studying theessence of TCM syndrome For CHM and TCM studies a better way[537677] is utilizing the conception of systems biology to combinethe chemical fingerprint biological response and biologicalbiomarkers together to control quality to define active compo-nents to shed light on active mechanisms and to evaluate phar-maceutical effects (Fig 4)

5 Future perspectives

Obviously the applications of MS-based metabolomics (eg GC-MS or LC-MS) have made a significant impact on TCM researchin characterizing herb chemical compositions screening and

Table 1Selected metabolomics applications in evaluating the therapeutic effects of traditional Chinese medicine (TCM)

Metabolomics technique Samples TCM Disease Findings Ref

GC-MS Rat plasma Compound Danshen drippingpills

Myocardial infarction Compound Danshen dripping pills showtherapeutic efficacy in the regulation of energymetabolism glycolysis and lipid metabolismdamaged by myocardial ischemia

UPLC-QTOF-MS Rat urine A TCM preparation Xindi softcapsule

Acute blood stasis The TCM preparation showed a tendency torecover acute blood stasis to healthy status

UPLC-QTOFMS Rat plasma Compound Danshen tablets Myocardial ischemia Compound Danshen Tablets exhibitedprotective effects on myocardial ischemia byreversing potential biomarkers close to normallevels especially for the four metabolites inthe pathway of purine metabolism

UPLC-QTOFMS andUPLC-SQ-MS

Human serum Berberine Type 2 diabetes Berberine treated type 2 diabetes by down-regulating the high levels of free fatty acidsand mediating glucose and lipid metabolism inpatient serum

LC-MS Rat urine Rehmanniae glutinosa Kidney yin deficiency 5-hydroxymethyl-2-furfural was the mainchemical marker of Rehmanniae glutinosa inthe treatment of type 2 diabetes

UPLC-QTOFMS Rat urine and serum Yunnan Baiyao Rheumatoid arthritis Yunnan Baiyao exhibit therapeutic effect onrheumatoid arthritis by suppressing some AA-derived proinflammatory mediator excretionfrom osteoblasts

LC-IT-TOFMS Rat urine Tongxinluo Endothelial dysfunction Tongxinluo exerted protective effect onendothelial dysfunction by adjusting pathwaysof fatty acid oxidation proteolysis of collagenadenine and phenylalanine and porphyrinmetabolites close to their normal state

LC-FTMS Mouse plasmaand liver

A multi-componentspreparation of eight naturalingredients

Early stage of obesity The multi-component preparation producedanti-atherogenic effects by regulating lipidmetabolism in mice in the early stage ofobesity

HILICRPLC-MS Rat urine Ginsenoside Rg3 Liver tumor Rg3 in a beta-cyclodextrin-based formulationinduced considerable change in the urinemetabolic pattern in cancer rats

identifying potential bioactive components and evaluating thera-peutic effects of CHM and its products Tremendous advances inrecent hybrid analytical techniques with unparalleled sensitivity res-olution reliability and specificity will further extend metabolomicsapplications in TCM research domains such as sample processingbatch-to-batch variation quality control safety toxicity stability etcGrowing evidence from recent publications indicates that by com-bining advanced bioinformatics tools MS-based metabolomics hasthe opportunity to examine the synergetic effects of CHM usinganimal experiments preclinical or clinical trials However many chal-lenges exist in metabolomics applications to study TCM due to theadverse factors including (1) the bioactive multi-components ofCHM are not well defined (2) the action mechanisms of TCM remainunclear (3) quality control of TCM lacks unified criteria (4) safetyof TCM is mainly empirical (5) the multi-component interactionsof TCM are obscure and (6) the scientific basis of symptom clas-sification is ambiguous Accordingly crucial core technologies (eghybrid MS instruments equipped with two or more mass analyz-ers used for detection comprehensive bidimensional or multi-dimensional chromatographic techniques used for separation) arerequired for analytical characterization of TCM in relation to theseaspects

Acknowledgments

The authors gratefully acknowledge support from the China In-ternational Science and Technology Cooperation Program(2009DFA41250) funded by the Ministry of Science and Technol-ogy of the Peoplersquos Republic of China and the Creative ResearchGroup Project (No 21321064) of the National Natural Science Foun-dation of China

References

[1] H Sheridan L Krenn R Jiang I Sutherland S Ignatova A Marmann et alThe potential of metabolic fingerprinting as a tool for the modernisation ofTCM preparations J Ethnopharmacol 140 (2012) 482ndash491

[2] FR Stermitz P Lorenz JN Tawara LA Zenewicz K Lewis Synergy in amedicinal plant antimicrobial action of berberine potentiated by 5rsquo-methoxyhydnocarpin a multidrug pump inhibitor Proc Natl Acad Sci USA97 (2000) 1433ndash1437

[3] S Sengupta SA Toh LA Sellers JN Skepper P Koolwijk HW Leung et alModulating angiogenesis the yin and the yang in ginseng Circulation 110(2004) 1219ndash1225

[4] JK Nicholson JC Lindon E Holmes lsquoMetabonomicsrsquo understanding themetabolic responses of living systems to pathophysiological stimuli viamultivariate statistical analysis of biological NMR spectroscopic dataXenobiotica 29 (1999) 1181ndash1189

[5] DG Robertson PB Watkins MD Reily Metabolomics in toxicology preclinicaland clinical applications Toxicol Sci 120 (Suppl 1) (2011) S146ndashS170

[6] JK Nicholson J Connelly JC Lindon E Holmes Metabonomics a platformfor studying drug toxicity and gene function Nat Rev Drug Discov 1 (2002)153ndash161

[7] R Baran NN Ivanova N Jose F Garcia-Pichel NC Kyrpides M Gugger et alFunctional genomics of novel secondary metabolites from diverse cyanobacteriausing untargeted metabolomics Mar Drugs 11 (2013) 3617ndash3631

[8] MV Milburn KA Lawton Application of metabolomics to diagnosis of insulinresistance Annu Rev Med 64 (2013) 291ndash305

[9] MA Farag HA Gad AG Heiss LA Wessjohann Metabolomics driven analysisof six Nigella species seeds via UPLC-qTOF-MS and GC-MS coupled tochemometrics Food Chem 151 (2014) 333ndash342

[10] G Xie S Zhang X Zheng W Jia Metabolomics approaches for characterizingmetabolic interactions between host and its commensal microbesElectrophoresis 34 (2013) 2787ndash2798

[11] V Arbona M Manzi C Ollas A Gomez-Cadenas Metabolomics as a tool toinvestigate abiotic stress tolerance in plants Int J Mol Sci 14 (2013) 4885ndash4911

[12] DS Wishart Metabolomics applications to food science and nutrition researchTrends Food Sci Tech 19 (2008) 482ndash493

[13] G Astarita J Langridge An emerging role for metabolomics in nutrition scienceJ Nutrigenet Nutrigenomics 6 (2013) 181ndash200

[14] CA Daykin F Wulfert NMR Spectroscopy based metabonomicscurrent technology and applications Front Drug Des Discov 2 (2006)151ndash173

[15] K Dettmer PA Aronov BD Hammock Mass spectrometry-basedmetabolomics Mass Spectrom Rev 26 (2007) 51ndash78

[16] M Sugimoto A Hirayama T Ishikawa M Robert R Baran K Uehara et alDifferential metabolomics software for capillary electrophoresis-massspectrometry data analysis Metabolomics 6 (2010) 27ndash41

[17] A Lommen MetAlign interface-driven versatile metabolomics tool forhyphenated full-scan mass spectrometry data preprocessing Anal Chem 81(2009) 3079ndash3086

[18] AL Duran J Yang L Wang LW Sumner Metabolomics spectral formattingalignment and conversion tools (MSFACTs) Bioinformatics 19 (2003) 2283ndash2293

[19] T Pluskal S Castillo A Villar-Briones M Oresic MZmine 2 modular frameworkfor processing visualizing and analyzing mass spectrometry-based molecularprofile data BMC Bioinformatics 11 (2010) 395

[20] CA Smith EJ Want GO Maille R Abagyan G Siuzdak XCMS processingmass spectrometry data for metabolite profiling using nonlinear peakalignment matching and identification Anal Chem 78 (2006) 779ndash787

[21] A Zhang H Sun G Yan P Wang Y Han X Wang Metabolomics in diagnosisand biomarker discovery of colorectal cancer Cancer Lett 345 (2014) 17ndash20

[22] L Yan W Nie T Parker Z Upton H Lu MS-based metabolomics facilitatesthe discovery of in vivo functional small molecules with a diversity of biologicalcontexts Future Med Chem 5 (2013) 1953ndash1965

[23] J van der Greef AC Tas J Bouwman MC Ten Noever de Brauw WHPSchreurs Evaluation of field-desorption and fast atom-bombardment massspectrometric profiles by pattern recognition techniques Anal Chim Acta 150(1983) 45ndash52

[24] JK Nicholson MJ Buckingham PJ Sadler High resolution 1H NMR studiesof vertebrate blood and plasma Biochem J 211 (1983) 605ndash615

[25] F Hillenkamp M Karas RC Beavis BT Chait Matrix-assisted laser desorptionionization mass spectrometry of biopolymers Anal Chem 63 (1991) 1193Andash1203A

[26] DY Chang CC Lee J Shiea Detecting large biomolecules from high-saltsolutions by fused-droplet electrospray ionization mass spectrometry AnalChem 74 (2002) 2465ndash2469

[27] KW Siu GJ Gardner SS Berman Atmospheric pressure chemical ionizationand electrospray mass spectrometry of some organoarsenic specie RapidCommun Mass Spectrom 2 (1988) 69ndash71

Fig 4 Core of Herbal Medicine research on the basis of metabolomics

[28] JK Nicholson Global systems biology personalized medicine and molecularepidemiology Mol Syst Biol 2 (2006) 52

[29] E Holmes ID Wilson JK Nicholson Metabolic phenotyping in health anddisease Cell 134 (2008) 714ndash717

[30] E Holmes J Nicholson Variation in gut microbiota strongly influencesindividual rodent phenotypes Toxicol Sci 87 (2005) 1ndash2

[31] JK Nicholson E Holmes ID Wilson Gut microorganisms mammalianmetabolism and personalized health care Nat Rev Microbiol 3 (2005) 431ndash438

[32] J Van der Greef Perspective all systems go Nature 480 (2011) 1[33] E Ernst Obstacles to research in complementary and alternative medicine Med

J Aust 179 (2003) 279ndash280[34] L Duan T Chen M Li M Chen Y Zhou G Cui et al Use of the metabolomics

approach to characterize chinese medicinal material huangqi Mol Plant 5(2012) 376ndash386

[35] WT Chang U Thissen KA Ehlert MM Koek RH Jellema T Hankemeier et alEffects of growth conditions and processing on Rehmannia glutinosa usingfingerprint strategy Planta Med 72 (2006) 458ndash467

[36] CU Tam FQ Yang QW Zhang J Guan SP Li Optimization and comparisonof three methods for extraction of volatile compounds from Cyperus rotundusevaluated by gas chromatography-mass spectrometry J Pharm Biomed Anal44 (2007) 444ndash449

[37] Y Qiu X Lu T Pang C Ma X Li G Xu Determination of radix ginseng volatileoils at different ages by comprehensive two-dimensional gas chromatographytime-of-flight mass spectrometry J Sep Sci 31 (2008) 3451ndash3457

[38] M Commisso P Strazzer K Toffali M Stocchero F Guzzo Untargetedmetabolomics an emerging approach to determine the composition of herbalproducts Comput Struct Biotechnol J 4 (2013) e201301007

[39] J Wang R van der Heijden G Spijksma T Reijmers M Wang G Xu et alAlkaloid profiling of the Chinese herbal medicine Fuzi by combination ofmatrix-assisted laser desorption ionization mass spectrometry with liquidchromatography-mass spectrometry J Chromatogr A 1216 (2009) 2169ndash2178

[40] H Sun B Ni A Zhang M Wang H Dong X Wang Metabolomics study onFuzi and its processed products using ultra-performance liquid-chromatographyelectrospray-ionization synapt high-definition massspectrometry coupled with pattern recognition analysis Analyst 137 (2012)170ndash185

[41] S Zeng T Chen L Wang H Qu Monitoring batch-to-batch reproducibility usingdirect analysis in real time mass spectrometry and multivariate analysis a casestudy on precipitation J Pharm Biomed Anal 76 (2013) 87ndash95

[42] S Zeng L Wang T Chen Y Wang H Mo H Qu Direct analysis in real timemass spectrometry and multivariate data analysis a novel approach to rapididentification of analytical markers for quality control of traditional Chinesemedicine preparation Anal Chim Acta 733 (2012) 38ndash47

[43] L Wang S Zeng T Chen H Qu Direct analysis in real time mass spectrometrya process analytical technology tool for real-time process monitoring inbotanical drug manufacturing J Pharm Biomed Anal 91 (2014) 202ndash209

[44] M Liu YG Li F Zhang L Yang GX Chou ZT Wang et al Chromatographicfingerprinting analysis of Danshen root (Salvia miltiorrhiza Radix et Rhizoma)and its preparations using high performance liquid chromatography with diodearray detection and electrospray mass spectrometry (HPLC-DAD-ESIMS) J SepSci 30 (2007) 2256ndash2267

[45] H Sun A Zhang X Wang Potential role of metabolomic approaches for Chinesemedicine syndromes and herbal medicine Phytother Res 26 (2012) 1466ndash1471

[46] HY Zhang CX Hu CP Liu HF Li JS Wang KL Yuan et al Screening andanalysis of bioactive compounds in traditional Chinese medicines using cellextract and gas chromatographyndashmass spectrometry J Pharm Biomed Anal43 (2007) 151ndash157

[47] X Zhang LW Qi L Yi P Li XD Wen QT Yu Screening and identification ofpotential bioactive components in a combined prescription of Danggui Buxuedecoction using cell extraction coupled with high performance liquidchromatography Biomed Chromatogr 22 (2008) 157ndash163

[48] Y Ling M Chen K Wang Z Sun Z Li B Wu et al Systematic screeningand characterization of the major bioactive components of Poria cocos andtheir metabolites in rats by LC-ESI-MS(n) Biomed Chromatogr 26 (2012)1109ndash1117

[49] Y Li P Wang W Xiao L Zhao Z Wang L Yu Screening and analyzing thepotential bioactive components from reduning injection using macrophagecell extraction and ultra-high performance liquid chromatography coupled withmass spectrometry Am J Chin Med 41 (2013) 221ndash229

[50] K Qin H Cai X Liu T Lu Q Fang Z Yao et al Screening and analysis of themultiple absorbed bioactive components and metabolites of Baihe Zhimu Tangby the metabolic fingerprinting technique and liquid chromatographydiodearray detection-electrospray ionization-mass spectrometry Pharmacogn Mag7 (2011) 177ndash185

[51] SL Su L Yu YQ Hua JA Duan HS Deng YP Tang et al Screening andanalyzing the potential bioactive components from Shaofu Zhuyu decoctionusing human umbilical vein endothelial cell extraction and high-performanceliquid chromatography coupled with mass spectrometry Biomed Chromatogr22 (2008) 1385ndash1392

[52] SP Wang L Liu LL Wang P Jiang JQ Zhang WD Zhang et al Screeningand analysis of the multiple absorbed bioactive components and metabolitesin rat plasma after oral administration of Jitai tablets by high-performance liquidchromatographydiode-array detection coupled with electrospray ionizationtandem mass spectrometry Rapid Commun Mass Spectrom 24 (2010)1641ndash1652

[53] J Wang R van der Heijden S Spruit T Hankermeier K Chan J van der Greefet al Quality and safety of Chinese herbal medicines guided by a systemsbiology perspective J Ethnopharmacol 126 (2009) 31ndash41

[54] K Chan Chinese medicinal materials and their interface with Western medicalconcepts J Ethnopharmacol 96 (2005) 1ndash18

[55] C Hu H Wei H Kong J Bouwman V Gonzalez-Covarrubias R van der Heijdenet al Linking biological activity with herbal constituents by systems biology-based approaches effects of Panax ginseng in type 2 diabetic Goto-Kakizakirats Mol Biosyst 7 (2011) 3094ndash3103

[56] L Li L Jiao BH Lau Protective effect of gypenosides against oxidative stressin phagocytes vascular endothelial cells and liver microsomes Cancer Biother8 (1993) 263ndash272

[57] MG Wulffele A Kooy D de Zeeuw CD Stehouwer RT Gansevoort The effectof metformin on blood pressure plasma cholesterol and triglycerides in type2 diabetes mellitus a systematic review J Intern Med 256 (2004) 1ndash14

[58] C-Y Yang J Wang Y Zhao L Shen X Jiang Z-G Xie et al Anti-diabetic effectsof Panax notoginseng saponins and its major anti-hyperglycemic componentsJ Ethnopharmacol 130 (2010) 231ndash236

[59] EJ Kim HI Lee KJ Chung YH Noh Y Ro JH Koo The ginsenoside-Rb2 lowerscholesterol and triacylglycerol levels in 3T3-L1 adipocytes cultured under highcholesterol or fatty acids conditions BMB Rep 42 (2009) 194ndash199

[60] F Li X Lu H Liu M Liu Z Xiong A pharmaco-metabonomic study on thetherapeutic basis and metabolic effects of Epimedium brevicornum Maxim onhydrocortisone-induced rat using UPLC-MS Biomed Chromatogr 21 (2007)397ndash405

[61] X Zhao Y Zhang X Meng P Yin C Deng J Chen et al Effect of a traditionalChinese medicine preparation Xindi soft capsule on rat model of acute bloodstasis a urinary metabonomics study based on liquid chromatography-massspectrometry J Chromatogr B Analyt Technol Biomed Life Sci 873 (2008)151ndash158

[62] Y Lv X Liu S Yan X Liang Y Yang W Dai et al Metabolomic study ofmyocardial ischemia and intervention effects of Compound Danshen Tabletsin rats using ultra-performance liquid chromatographyquadrupole time-of-flight mass spectrometry J Pharm Biomed Anal 52 (2010) 129ndash135

[63] X Xin H Zou N Zheng X Xu Y Liu X Wang et al Metabonomic strategyto the evaluation of chinese medicine compound danshen dripping pillsinterfering myocardial ischemia in rats Evid Based Complement Alternat Med2013 (2013) 718305

[64] Y Gu Y Zhang X Shi X Li J Hong J Chen et al Effect of traditional Chinesemedicine berberine on type 2 diabetes based on comprehensive metabonomicsTalanta 81 (2010) 766ndash772

[65] J Wang H Kong Z Yuan P Gao W Dai C Hu et al A novel strategy to evaluatethe quality of traditional Chinese medicine based on the correlation analysisof chemical fingerprint and biological effect J Pharm Biomed Anal 83 (2013)57ndash64

[66] H He X Ren X Wang X Shi Z Ding P Gao et al Therapeutic effect of YunnanBaiyao on rheumatoid arthritis was partially due to regulating arachidonic acidmetabolism in osteoblasts J Pharm Biomed Anal 59 (2012) 130ndash137

[67] W Dai C Wei H Kong Z Jia J Han F Zhang et al Effect of the traditionalChinese medicine tongxinluo on endothelial dysfunction rats studied by usingurinary metabonomics based on liquid chromatography-mass spectrometryJ Pharm Biomed Anal 56 (2011) 86ndash92

[68] H Wei C Hu M Wang AM van den Hoek TH Reijmers S Wopereis et alLipidomics reveals multiple pathway effects of a multi-components preparationon lipid biochemistry in ApoE3LeidenCETP mice PLoS ONE 7 (2012) e30332

[69] Y Wang J Wang M Yao X Zhao J Fritsche P Schmitt-Kopplin et alMetabonomics study on the effects of the ginsenoside Rg3 in a β-cyclodextrin-based formulation on tumor-bearing rats by a fully automatic hydrophilicinteractionreversed-phase column-switching HPLC ndash ESI-MS Approach AnalChem 80 (2008) 4680ndash4688

[70] P Liu S Liu D Tian P Wang The applications and obstacles of metabonomicsin traditional chinese medicine Evid Based Complement Alternat Med 2012(2012) 945824

[71] C Lu J Deng L Li D Wang G Li Application of metabolomics on diagnosisand treatment of patients with psoriasis in traditional Chinese medicineBiochim Biophys Acta 1844 (2014) 280ndash288

[72] H Wei W Pasman C Rubingh S Wopereis M Tienstra J Schroen et al Urinemetabolomics combined with the personalized diagnosis guided by Chinesemedicine reveals subtypes of pre-diabetes Mol Biosyst 8 (2012) 1482ndash1491

[73] S Liu C Liu J Zu J Li N Wang F Lu Researches on pyretic pulmonarysyndrome model interfered by Radix Scutellariae based on variation ofbiomarker Zhongguo Zhong Yao Za Zhi 36 (2011) 1212ndash1216

[74] X Lu Z Xiong J Li S Zheng T Huo F Li Metabonomic study on lsquoKidney-YangDeficiency syndromersquo and intervention effects of Rhizoma Drynariae extractsin rats using ultra performance liquid chromatography coupled with massspectrometry Talanta 83 (2011) 700ndash708

[75] H Kuang Y Wang Q Wang B Yang Y Xia Metabonomics research of the fourproperties in traditional Chinese medicine based on UPLC-QTOF-MS systemChin Med 11 (2011) 13ndash28

[76] M Wang RJ Lamers HA Korthout JH van Nesselrooij RF Witkamp Rvan der Heijden et al Metabolomics in the context of systems biology bridgingtraditional Chinese medicine and molecular pharmacology Phytother Res 19(2005) 173ndash182

[77] J van der Greef Systems biology connectivity and the future of medicine SystBiol (Stevenage) 152 (2005) 174ndash178

Metabolomics and traditional Chinese medicine

Introduction

Advances in metabolomics technologies

Applications of metabolomics in systems-based TCM

Chemical characterization of CHM

Bioactive component screening of CHM

Efficacy evaluation of CHM

Evidence-based studies in TCM syndromes

TCM pattern diagnosis

TCM pattern intervention

Future perspectives

Acknowledgments

References

(ie multi-component mixture) of the herb or the formula actingtogether

Traditionally a TCM is administered as an individually pre-pared water decoction containing multiple herb materials in certainproportions Notably the final item that is taken orally by a patientis an extract of herbs that is influenced by not only the botanicalorigin of its herbal components but also the procedure that is usedto treat the herbal material [1] Any minor changes in these twoaspects will greatly affect the therapeutic efficacy of the productsSuch complexity in TCM administration brings a great challenge inestablishing suitable analytical approaches for the development ofmodernization and standardization of TCMs and their natural prod-ucts Besides TCM possesses two unique features ndash multidimen-sional pharmacology ie synergetic effects [2] and combinatorialeffects and opposing principles [3] and personalized or individu-al treatment Such features of TCM determine that the traditionalanalysis based on major components is not sufficient to provide in-formation on bioactivity of herbal products Analytical character-ization of TCM is still in its infancy due to the chemical natureof multi-component mixtures that often possess inherent holisticbioactivities

To meet this challenge metabolomics which aims at compre-hensive characterization of the total metabolome in a biologicalsystem and dynamic metabolomics responses to alterations of innerandor external factors [4] is in great demand As an emerging fieldof science in the post-genomics era metabolomics has been broadlyapplied in many fields eg toxicological survey [5] functional ge-nomics [67] clinical diagnostics [8] plant and microbes [9ndash11] andnutritional biochemistry [1213] The most commonly used two tech-nologies in metabolomics applications are nuclear magnetic reso-nance (NMR) [14] and mass spectrometry (MS) [15] Although itssensitivity is less than that of MS NMR is still popular in many ap-plications due to its rapidity high throughput easy operation beingnon-destructive of samples and quantitation of metabolites fromdifferent groups However in most cases MS is preferred becauseof its advantages of unparalleled sensitivity super resolution andstructural specificity In practical applications chromatographic sep-aration technologies [eg gas chromatography (GC) liquid chro-matography (LC) and capillary electrophoresis (CE)] are often coupledto MS depending on the properties of the analytes being measured

As the data matrix generated in metabolomics studies is usuallyvery large data processing and data analysis are crucial for theirfinal outcome Many available software packages eg JDAMP [16]MetAlign [17] MSFACTs [18] Mzmine [19] and XCMS [20] weredeveloped for processing raw data from metabolomics On this basismultivariate (eg PCA PLS-DA PLS and OPLS) and univariate (egT-test and ANOVA) analyses were carried out for classification anddifferential metabolite discovery In combination with advanced bio-informatics tools many studies demonstrated that metabolomicsincorporating state-of-the-art analytical approaches enables sys-temic molecular characterization of complex samples [2122] andprovides an integrated biochemical view of TCM

In this review we summarize metabolomics methods based onGC-MS and LC-MS and applied in research on TCM We highlightthe subjects related to chemical composition bioactive compo-nents and the efficacy of CHM and metabolomics applications inevidence-based studies of TCM syndromes

2 Advances in metabolomics technologies

As early as the 1970s the embryonic form of metabolomics (atthat time called ldquometabolic profilerdquo) was coming into being A typicalexample is that GC-MS became feasible to measure qualitatively andquantitatively metabolic profiles of body fluids and tissue ex-tracts Concurrently rapid improvement was achieved in NMR spec-troscopy due to the increasing strengths of magnetic field and magic

angle spinning which remarkably improved the sensitivity and madeNMR a leading analytical tool to detect metabolites of many dif-ferent classes in biological samples

The initiation of study on MS-based metabolomics in a real sensewas carried out by van der Greef et al using field desorption andfast atom bombardment (FAB)-MS for profiling complex non-volatile metabolites in body fluids to study gender difference [23]They pioneered the utilization of pattern-recognition techniques toMS-based datasets Meanwhile the NMR-based metabonomics ap-proach was pioneered in the study of vertebrate blood and plasmaby Nicholson et al [24]

Although there were some sporadic applications the limita-tion in early analytical technologies (eg low sensitivity low speci-ficity and low throughput) made it a big challenge to qualify andto quantify complex metabolites in biological samples Analyticaltechniques with super sensitivity and selectivity high specificitygood reliability and easy automation are required to detect and toidentify metabolites in complex biological systems The revolu-tion in soft-ionization technologies for MS included matrix-assisted laser desorptionionization (MALDI) [25] and electrosprayionization (ESI) [26] as well as atmospheric pressure chemical ion-ization (APCI) [27] Metabolomics has largely been driven by thesetechnologies as they possess better resolution and sensitivity andhigher specificity and mass accuracy than earlier technologies TheseMS instruments are still undergoing rapid advances in terms of sen-sitivity resolution mass accuracy and dynamic range

As complexity increases in the composition of metabolome ndash thewhole set of endogenous and exogenous metabolites with small mo-lecular weight in an individual organism ndash modern technologies thatallow for qualitative and quantitative measurement of a vast numberof metabolites in complex biological systems are required formetabolomics studies Modern metabolomics platforms usually com-prise approaches using chromatography-based technologies suchas GC and LC including high-performance LC (HPLC) ultra-performance LC (UPLC) hydrophilic interaction chromatography(HILIC)) and CE coupled to MS or MSMS Notably frequently usedion sources are for GC-related techniques electron ionization (EI)and for LC- and CE-based MS systems ESI Occasionally APCI andatmospheric pressure photoionization (APPI) are coupled to LC fordetection of non-polar compounds Frequently used mass analyz-ers in current metabolomics include quadrupole (Q) ion trap (IT)time-of-flight (TOF) Fourier transform ion cyclotron resonance (FT-ICR) orbital trap and a combination of them [eg triple quadru-pole (QQQ) Q-TOF IT-TOF IT-FT-ICR]

Besides NMR is also frequently applied in modern metabolomicsstudies In recent decades many studies using NMR for metabolomicswere carried out by Nicholson and his group The relatedmetabolomics studies generated many valuable findings rangingfrom early diagnosis to disease treatment drug toxicity gene func-tion microbial physiological and epidemiological aspects [628ndash31]

3 Applications of metabolomics in systems-based TCM

Differing from Western medicine (WM) that is based on theconcept of ldquoone size fits allrdquo (ie 1 disease ndash 1 target ndash 1 drug) TCMfollows the principle of multiple components ndash multiple target in-teractions ndash and has been developed into a holistic healthcare system[32] Although attractive worldwide as a rich source of leading mol-ecules for complementary and alternative medicine TCM suffers frominextricable obstacles in modern CHM research [33] In CHM mostof the active components are not well defined The constituents andtheir related concentrations in each herbal medicine can vary greatlyeg depending on species [34] plant-growth conditions and pro-cessing procedure [35] Metabolomics provides a valuable chancefor studying TCM in terms of characterizing the chemical compo-nents defining the multiple active components and evaluating the

Acknowledgments

References

Introduction

Future perspectives

Acknowledgments

References

Acknowledgments

References

Introduction

Future perspectives

Acknowledgments

References

Acknowledgments

References

Introduction

Future perspectives

Acknowledgments

References

Acknowledgments

References

Introduction

Future perspectives

Acknowledgments

References

Acknowledgments

References

Introduction

Future perspectives

Acknowledgments

References

Acknowledgments

References

Introduction

Future perspectives

Acknowledgments

References

Introduction

Future perspectives

Acknowledgments

References

Documents

Metabolomics and TCM