Chinese traditional medicine

Journal of Pharmaceutical and Biomedical Analysis 83 (2013) 34–42

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical

Analysis

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j p b

a

Chemical differentiation of Da-Cheng-Qi-Tang,a Chinese medicine formula,prepared by traditional and modern decoction methods using UPLC/Q-TOFMS-based metabolomics approach

Jian-Bo Wan a ,?,1,Xu Bai b ,1,Xiu-Jiang Cai a ,Yi Rao c ,Yue-Sheng Wang c ,Yi-Tao Wang a ,??

a

State Key Laboratory of Quality Research in Chinese Medicine,Institute of Chinese Medical Sciences,University of Macau,Taipa,Macao,PR China b

Waters Technologies (Shanghai)Ltd.,Shanghai,PR China c

National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine,Jiangxi University of Traditional Chinese Medicine,Nanchang,PR China

a r t i c l e

i n f o

Article history:

Received 19February 2013

Received in revised form 16April 2013Accepted 16April 2013

Available online 22 April 2013

Keywords:

Da-Cheng-Qi-Tang UPLC/Q-TOFMS Metabolomics

Chemical consistency

Traditional decoction method

a b s t r a c t

In order to evaluate chemical consistency between traditional and modern decoctions of Da-Cheng-Qi-Tang (DCQT),a classical Chinese medicine formula commonly used in the treatment of digestive diseases,an ultra performance liquid chromatography-electrospray ionization-quadrupole time-of-?ight mass spectrometry (UPLC-ESI-Q-TOFMS)combined with multivariate statistical analysis was established to globally characterize the chemical pro?le and discover differentiating chemical markers.Two kinds of decoctions,namely traditional decoction (multi-step decoction of constituent herbs),and modern decoc-tion (one-step decoction of all herbs),were prepared and subjected to UPLC-MS analysis,the datasets of t R –m /z pairs,ion intensities and sample codes were processed with supervised orthogonal partial least squared discriminant analysis (OPLS-DA)to comprehensively compare the chemical difference between these two kinds of decoction samples.The global chemical difference was found between traditional and modern decoctions,and rhein,sennoside A/B,diosmetin,magnoloside B and naringin were the compo-nents contributing most to these differences.Based on the fact that traditional decoction of DCQT presents the higher concentration of rhein and sennoside A/B,mainly contributed to laxative activity of DCQT,the purgative effect of traditional decoction might be more potent,compared with modern decoction.How-ever,the comparative study on purgative effect of traditional and modern DCQT remains to be further investigated using pharmacological approaches.Our ?ndings also provide the early scienti?c evidence of traditional decoction method of DCQT.

? 2013 Elsevier B.V. All rights reserved.

1.Introduction

Traditional chinese medicine (TCM)has been widely used to treat a variety of diseases and conditions in oriental countries since the ancient times.In clinical practice,the herbalist rarely describes a single herb to treat a clinical condition,instead they create composite formula,which might produce the synergistic action and/or reduce the possible adverse effect.According to the TCM theory,the decoction process of Chinese medicine formula should be in harmony with the characteristics of each individual herb and its role in the formula.For some formulas,the part of constituent herbs are ?rstly boiled with water for the speci?ed time,then put the other herbs to get decoction.This traditional

?Corresponding author.Tel.:+868533974873;fax:+8685328841358.??Corresponding author.Tel.:+868533974691;fax:+86853841358.

E-mail addresses:jbwan@umac.mo ,wjbcpu@https://www.360docs.net/doc/f04990033.html, (J.-B.Wan),ytwang@umac.mo (Y.-T.Wang).1

The authors contributed equally to this work.

multi-step decoction method has been applied through the cen-turies since the formula was created.The major disadvantage of this traditional method is time-consuming with high labor inten-sity,and dif?cult to control the boiling time and amount of water,leading to vary the quality of decoction [1,2].Currently,many phar-macies and hospitals provide the cooking service of TCM formula for patients using automatic boiling machine.All herbs in the for-mula are mixed together in the container of machine and one-step boiled at a speci?c temperature or pressure,which has the advan-tages of better quality control and easier to consumption.However,there has been a debate on the ef?cacy equivalence of decoction using two different approaches.The focus of this debate is that the chemical components and their dissolution rate of one-step decoction might be different from those of traditional multi-step decoction.It is possible that diversely chemical and physical interactions between decoction components might occur during different decoction.Therefore,chemical differentiation in Chinese medicine formula prepared by traditional and modern decoction approaches should be evaluated before we choose the decoction method.

0731-7085/$–see front matter ? 2013 Elsevier B.V. All rights reserved.https://www.360docs.net/doc/f04990033.html,/10.1016/j.jpba.2013.04.019

J.-B.Wan et al./Journal of Pharmaceutical and Biomedical Analysis83 (2013) 34–4235

Metabolomics is an emerging new platform that originally allows for the assessment of global metabolic pro?les in a sys-tem under a given set of condition[3].The metabolomics have been also extensively used for quality control and discrimi-nation of TCM[4–11].It could be readily applied to monitor the changes in the pro?le of chemical components and their content in the Chinese medicines that derived from different origin[4,6],processing method[7,8],cultivation age[9]and grown locations[11].Ultra performance liquid chromatography (UPLC)coupled to quadrupole,hybrid orthogonal acceleration time-of-?ight tandem mass spectrometer(Q-TOFMS/MS),a newly developed hyphenated technique,has been increasingly used in the metabolomics studies of Chinese medicinal https://www.360docs.net/doc/f04990033.html,ing small particle size chromatographic columns(less than2?m), UPLC enhance chromatographic resolution,increase retention time reproducibility,improve sensitivity and shorten operation time far more.A Q-TOF-MS/MS could provide accurate mass value of the analyte and the high energy collision-induced dissoci-ation(CID),which made UPLC/Q-TOFMS/MS to be a powerful tool for mapping the chemical pro?ling of Chinese medicine formula.

Da-Cheng-Qi-Tang(DCQT),a famous TCM formula commonly used in the treatment of digestive diseases,is composed of Radix et Rhizoma Rhei(Polygonaceae),Cortex Magnoliae Of?cinalis(Mag-noliaceae),Fructus Aurantii Immaturus(Rutaceae)and Mirabilitum (crystals of sodium sulfate)[12].As?rstly recorded in‘Shang-Han-Lun’,a classical treatise of febrile diseases by a physician Zhong-Jing Zhang(150–219A.D.)in Eastern Han Dynasty,DCQT has been used as a representative purgative to ameliorate constipation and to purge internal heat in the stomach and intestine[13].Clinically, DCQT was widely prescribed to promote the recovery of gastroin-testinal motility after abdominal surgery[14],and to treat acute abdominal diseases,such as acute pancreatitis[15],adhesive bowel obstructions[16]and acute appendicitis[17].According to the pro-cedure of traditional decoction method,DCQT is prepared by three steps:boiling Cortex Magnoliae Of?cinalis and Fructus Aurantii Immaturus,then adding Radix et Rhizoma Rhei,?nally dissolving with Mirabilitum.In modern decoction method,four crude mate-rials are mixed together and boiled with water to get decoction. To our knowledge,the global chemical comparison between tradi-tional and modern decoction methods has not reported yet.In the present study,therefore,an UPLC/Q-TOFMS-based metabolomics approach was established to evaluate the chemical consistency between traditional and modern decoctions of DCQT.

2.Experimental

2.1.Chemicals and herbal medicines

HPLC-grade acetonitrile and analytical grade formic acid(purity of above99.5%)were purchased from J.T.Baker,Inc.(Phillipsburg, NJ,USA)and Sigma–Aldrich(St.Louis,MO,USA),respectively.Other chemicals and solvents were of analytical grade.Ultra high purity water was prepared by a Millipore SAS–67120(Molsheim,Cedex, France).

Radix et Rhizoma Rhei,Cortex Magnoliae Of?cinalis and Fruc-tus Aurantii Immaturus were purchased from the Beijing Yanjing Shuangqiao Herbal Medicine Factory(Beijing,China).Mirabilitum was purchased from the Jiangxi Jiangzhong Pharmaceutical Co. Ltd.(Nanchang,China).The botanical origin of all materials was authenticated by Dr.Yi Rao,a pharmacognosist from National Phar-maceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine,Nanchang,China.The voucher specimens were deposited at Institute of Chinese Medical Sciences,University of Macau.2.2.Sample preparation of traditional and modern decoctions

Traditional decoction was prepared as described in“Shang-Han-Lun”with some modi?cations.The pulverized samples of Cortex Magnoliae Of?cinalis(1.2g)and Fructus Aurantii Immaturus(2.4g) were accurately weighed and extracted with36mL of boiling water for30min.After cooling and?ltering,Radix et Rhizoma Rhei(1.2g) was added in the extract and sequentially boiled for20min.The aqueous extract was separated by?ltration(100mesh),in which 0.9g of Mirabilitum was dissolved to get the traditional decoction. For modern decoction,Radix et Rhizoma Rhei(1.2g),Cortex Mag-noliae Of?cinalis(1.2g),Fructus Aurantii Immaturus(2.4g)and Mirabilitum(0.9g)were mixed together and extracted with36mL of boiling water for50min.The traditional and modern decoction were concentrated by removing a portion of water in vacuum at 60?C,and the residue was transferred into a25ml volumetric?ask which was brought up to its volume with water.Prior to analysis,all samples were?ltered through a0.22?m polytetra?uoroethylene ?lter(Whatman,Florham Park,NJ,USA).

2.3.Liquid chromatography

Chromatography was performed on an ACQUITY UPLC HSS T3 C18column(100mm×2.1mm i.d.,1.7?m)using an ACQUITY UPLC system(Waters Corp.,Milford,MA,USA)equipped with a binary solvent delivery system,an auto-sampler,and high temper-ature(HT)column oven.A binary gradient elution system consisted of0.1%aqueous formic acid(A)and acetonitrile containing0.1% formic acid(B),and separation was achieved using the following gradient program:isocratic1%B(0–1min),linear gradient from 1%to50%B(1–10min),50%to90%B(10–12min),isocratic90%

B for1min and then back to1%B in4min.The?ow rate was

0.45mL/min,and6?L aliquot of each sample was subjected to the column that was maintained at45?C.A“purge–wash–purge”cycle was employed on the auto-sampler with90%aqueous formic acid used for the wash solvent and0.1%aqueous formic acid used as the purge solvent.This process ensured that the carry-over between injections was minimized.

2.4.Mass spectrometry

The eluent was introduced to a SYNAPT G2-S high-de?nition mass spectrometer(Waters Corp.,Milford,MA,USA)equipped with an electrospray ionization(ESI)source.The optimal conditions of analysis were as follows:negative-ion mode,source temperature of120?C,desolvation gas?ow of800L/h at temperature of450?C, cone gas?ow of20L/h,the sampling cone voltage of30.0V,extrac-tion cone voltage of4.0V,capillary voltage of2500V,the TOF acquisition rate of0.2s/scan with0.01s inter-scan delay.Data were collected in centroid mode from50to1200Da in full scan during 0–12min.For accurate mass acquisition,a lock-mass calibrant of leucine-enkephalin at a concentration of200ng/mL was continu-ously introduced to mass spectrometer via a lock spray interface at a?ow-rate of50?L/min,generating a reference ion for negative ion mode([M–H]?=554.2615)to ensure accuracy during the MS analysis.

2.5.Data processing and pattern recognition analysis

The raw UPLC/Q-TOFMS data of all samples were processed using the MarkerLynx application manager software(version4.1, Waters Corp.,Milford,MA,USA).This application manager incor-porates a peak deconvolution package that allows detection of the mass,retention time and intensity of the peaks eluting in each chromatogram,the parameters were set as follows:mass toler-ance of0.01Da;retention time tolerance of0.1min and noise

36J.-B.Wan et al./Journal of Pharmaceutical and Biomedical Analysis83 (2013) 34–42

elimination of level5.After being recognized and aligned,the inten-sity of each peak was normalized with respect to the total ion intensity of each chromatogram.For peak integration,peak width at5%of the height was1s,peak-to-peak baseline noise was0.1, and peak intensity threshold was1000.Thus,a three-dimensional data matrix composing of the retention time,m/z value,and the normalized peak area was generated and introduced to EZinfo2.0 software for unsupervised principal component analysis(PCA)and supervised orthogonal partial least squared discriminant analysis (OPLS-DA).From the S-plot of OPLS-DA,various chemical com-ponents could be extracted as being mainly responsible for the discrimination between traditional and modern decoctions,and were therefore considered as potential chemical markers.These markers were chosen according to their contribution to the varia-tion and correlation within the data set,and subjected to further structural identi?cation.

3.Results and discussion

3.1.Method development and validation

DCQT,consists of three herbal medicines and one mineral medicine,is a very complex matrix that contains numerous com-pounds.The major components including Polyphenol acid and anthraquinones originated from Radix et Rhizoma Rhei,lignans and phenylethanoid glycosides from Cortex Magnoliae Of?cinalis and?avonoids from Fructus Aurantii Immaturus could be detected in the DCQT decoction(Fig.1)[18,19].Therefore,simultaneous determination of these different types of compounds in DCQT encounters the great challenge in short analytic time using UPLC. To achieve the higher resolution and faster separation,the UPLC chromatographic conditions,such as column and mobile phase, were optimized in the pilot study.Three ACQUITY UPLC columns, including BEH C18column(100mm×2.1mm i.d.,1.7?m),BEH HILIC column(50mm×2.1mm i.d.,1.7?m)and HSS T3C18column (100mm×2.1mm i.d.,1.7?m),were tested.The results indicated that ACQUITY HSS T3C18column was the most suitable for the anal-ysis of DCQT due to the most peak capacity and the best resolution of major components.Different kinds of mobile phases,such as acetonitrile–water system and methanol–water system with vari-ous modi?ers,were compared to obtain the chromatograms with the best resolution.It was found that0.1%aqueous formic acid–acetonitrile containing0.1%formic acid was the optimum choice, which not only could simultaneous separation of four types of major component in DCQT,but also be compatible to MS analysis due to the volatile property of formic acid.For the mass detec-tion,both positive and negative ion modes were compared to achieve maximum signal,and the results showed that the higher sensitivities and more straightforward structural information were obtained in negative mode.Thus,under the optimized chromato-graphic and MS conditions,the major components in DCQT were well-separated and detected in12min.The typical base peak inten-sity(BPI)chromatograms of the DCQT prepared by traditional and modern decoction methods were shown in Fig.2.

The robustness or ruggedness of instrumental analysis should be evaluated to guarantee statistical difference is not derived from analytical drift in a chemometric study[20].One DCQT sample was chosen as the quality control(QC)sample,and three representa-tive peaks(peak No.1,12and22)in UPLC chromatograms and their molecular weights of[M–H]?were selected for method vali-dation.The repeatability of UPLC/Q-TOFMS was evaluated as the relative standard deviations(RSD,%)of retention times,peak areas in UPLC chromatograms,and molecular weight of[M–H]?using three replicates of the QC sample at different time intervals(0h, 6h and12h).The RSD of peak areas and m/z of[M–H]?of selected peaks were less than3.32%and0.01%,respectively,and their reten-tion times remained precisely the same,which demonstrated that the established method was robust with excellent repeatability and stability.

3.2.Multivariate statistical analysis and chemical consistency evaluation

Unsupervised principal component analysis(PCA),a non-biased statistical technique,was?rstly applied to investigate whether traditional decoction and modern decoction of DCQT could be sep-arated according to their differences in the chemical compositions. After Pareto scaling and mean-centering,all data were displayed as scores and loadings in a coordinate system of principal com-ponents resulting from data dimensionality reduction.As shown in Fig.3A,two-dimensional PCA score plots showed a tendency to separate the traditional and modern decoctions of DCQT.R2X and Q2(cum)are usually used for evaluation of PCA model,and values of these parameters close to1.0indicating a good?tness for the constructed model[21].A validation method using sevenfold cross-validation,the parameters for the classi?cation from the software were R2X=0.407and Q2(cum)=0.265,indicating the low modeling quality of PCA.

In order to further characterize the differences in chemical com-positions between traditional and modern decoction,OPLS-DA,a supervised multivariate statistical method to sharpen an already established weak separation between the groups of observations plotted in PCA,was performed to obtain better discrimination between the two groups.After Pareto scaling with mean-centering, all data from negative ion mode were displayed as scores in a coor-dinate system of latent variables.The scores plots analysis of the chromatographic data demonstrated that all tested samples were clearly classi?ed into two clusters,i.e.traditional decoction and modern decoction samples,according to the differences in their global pro?les(Fig.3B),suggesting that there is global chemical dif-ference in the composition and/or content of components between two kinds of DCQT decoctions.All the observations fell within the Hotelling T2(0.95)ellipse,where the model?t parameters were 0.995of R2Y(cum)and0.982of Q2Y(cum),indicating that OPLS-DA model established in this study has good?tness and prediction.

In order to reveal potential chemical components contributing most to the differences between traditional and modern decoc-tions,an S-plot was constructed following the OPLS-DA,in which the X-axis and Y-axis represent variable contribution and variable con?dence,respectively.The further the ion RT–m/z pair point departs from zero of X-axis and Y-axis,the more the ion con-tributes to the difference between the two groups and the higher the con?dence level of the ion is for the difference,respectively [2].Therefore,the points at the two ends of“S”represent poten-tial chemical markers with the high con?dence.As shown in the S-plot(Fig.4),three ions(a–c)at the top-right corner and three ions(e–g)at the bottom-left corner of“S”end were regarded to be the components which contribute most to discrimination between traditional and modern decoctions of DCQT.The trends of their ion intensities in all tested samples were shown in Fig.5,the ions a (t R9.77min,m/z283.0239),b(t R7.91min,m/z299.1278)and c (t R4.28min,m/z333.1335)in all18traditional decoctions were found to present the higher intensity than that in all18modern decoctions.On the contrary,the intensities of ions e(t R4.10min, m/z312.1594),f(t R4.40min,m/z785.2504)and g(t R5.83min,m/z 579.1711)were the lower in traditional decoctions,compared with modern decoctions.In addition,sennoside A/B(d,t R5.89min,m/z 861.1885),a major active component contributing to the purgative action of Radix et Rhizoma Rhei,was also tracked and shown in Fig.5d,its corresponding ion was detected with the higher inten-sity in16traditional decoction samples than that in all18modern

J.-B.Wan et al./Journal of Pharmaceutical and Biomedical Analysis83 (2013) 34–42

37

Fig.1.Chemical structures of major components identi?ed from Da-Cheng-Qi-Tang.The number of compound represents in the same manner as in Table1.

decoctions.Therefore,the components that correlate to ions a–g could be considered as potential chemical markers for the discrim-ination of traditional decoction from modern decoction of DCQT.

3.3.Identity assignment and con?rmation of chemical markers

Under the current chromatographic and MS conditions,nearly twenty-three peaks were found in both of two kinds of DCQT decoc-tions.TOFMS data provides accurate molecular mass of the parent ion,which allows to deduce the exact molecular formula of corre-sponding component,MS/MS data highlights fragment ions,which are structural characteristics of component.The MassFragment TM application manager software(Waters corp.,Milford,MA,USA)was applied to facilitate the structural assignment of fragment ion.Eighteen of these major peaks in both traditional and mod-ern decoctions of DCQT were tentatively assigned by matching the molecular formula with that of the published literatures [19,22–24]or interpreting with available biochemical databases, such as ChemBank(https://www.360docs.net/doc/f04990033.html,/),PubChem (https://www.360docs.net/doc/f04990033.html,/),MassBank(http://www. massbank.jp/),HMDB(http://www.hmdb.ca/spectra/ms/search) and METLIN(https://www.360docs.net/doc/f04990033.html,/).As summarized in Table1, the molecular mass accuracy of all assigned components detected was less than5ppm by comparing with the theoretical exact mass provided by authoritative websites mentioned above.At least4major types of compounds,including polyphenol acids,

38

J.-B.Wan et al./Journal of Pharmaceutical and Biomedical Analysis 83 (2013) 34–42

Fig.2.Representative base peak intensity (BPI)chromatograms of Da-Cheng-Qi-Tang prepared by traditional (A)and modern (B)decoction methods.BPI chromatograms were monitored in negative ion mode.

-30

-20-100102030-70-60-50-40-30-20-10010203040506070

t [2]

t[1]

A

-30

-20-10

010

2030-70 -60 -50 -40 -30 -20 -10

0 10

20

30

40

50

60

70

t [2]O

t[1]P

B

Fig.3.PCA/Scores plot (A)and OPLS-DA/Scores plot (B)based on the global chemical pro?ling of Da-Cheng-Qi-Tang prepared by traditional and modern decoction methods.(?)traditional decoction;( )modern decoction.

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39

Table 1

Components identi?ed from Da-Cheng-Qi-Tang.

Peak no.

t R (min)

Assigned identity

Molecular formula

[M–H]?/(m /z )Mean measured mass (Da)

Theoretical exact mass (Da)

Mass accuracy (ppm)

1 2.11Gallic acid

C 7H 6O 5169.0132169.0142?4.72 2.84Caffeoyl-O-glu-galloyl C 19H 26O 15493.1220493.1198 4.53 3.28Unknown C 20H 29O 13477.1603477.1608?1.04 4.04Catechin C 15H 14O 6289.0709289.0718?3.15 4.10Unknown C 19H 23NO 3312.1994312.1905 2.86 4.28Unknown

C 17H 20O 4333.1335333.1344?2.77 4.40Magnoloside B C 35H 46O 20785.2504785.2510?0.88 4.66Unknown

C 35H 46O 20785.2500785.2510?1.39 4.89Magnoloside A C 29H 36O 15623.1974623.1981?1.110 5.83Naringin

C 27H 32O 14579.1711579.1719?1.411 5.89Sennoside A/B

C 42H 38O 20861.1885861.18840.112 6.16Hesperidin/Neohesperidin

C 28H 34O 15609.1816609.1819?0.513 6.51Caffeoyl-dihydroxy benzoic acid-O-rha C 22H 22O 11461.1077461.1089?2.6147.26Poncirin

C 28H 34O 14593.1880593.18760.7157.46Emodin-8-O-?-D-glucopyranoside C 21H 20O 10431.0975431.0978?0.7167.91Diosmetin

C 16H 12O 6299.1278299.1288?3.3178.01Aloe-emodin-O-glu(OAc)

C 23H 22O 11473.1081473.1089?1.7188.163,4-dihydroxy benzoic acid-O-glu C 18H 20O 5315.1229315.1238?2.9198.81Unknown

C 18H 34O 5329.2320329.2333?3.9208.8811-O-acetyl-aloe-emodin-O-glu-xyl C 31H 26O 13605.1291605.12950.7218.99Chrysophanol C 15H 10O 4253.0498253.0506?3.2229.77Rhein

C 15H 8O 6283.0239283.0248?3.223

10.27

Acetyl-chrysophanol-O-glu-xyl

C 31H 26O 12589.1343

589.1346

?0.5

anthraquinones,phenylethanoid glycosides and ?avonoids were found in DCQT prepared by traditional and modern decoction methods.The representative mass spectra of chemical marker a (peak 22)was shown in Fig.6A,the accurate deprotonated molecular weight (m /z 283.0239)in negative mode suggested that its empirical molecular formula is most likely C 15H 8O 6,which matched that of rhein.The mass differences between the parent ion and three main fragment ions (m /z 239.0343,211.0382and 183.0441)were 44Da,72Da and 90Da,respectively,which corresponded to successive loss of the CO 2,CO and CO units.Thus,the assignment of rhein was further con?rmed by its fragment ions.

Similarly,in the mass spectrum of ion d (peak 11),deproto-nated molecular ion (m /z 861.1885)suggested empirical molecular formula as C 42H 38O 20,which matched that of sennoside A or B.The fragment ion at m /z 699.1426exhibited 162Da of mass difference with parent ion,indicating the loss of glucosyl unit.After cleavage of 10-10 bond of sennoside,the charac-terized fragment ion at m /z 430.1725was formed,and ions of m /z 386.1029and 225.0438indicated further successive losses

of CO 2and glucosyl units.In addition,the signal of glucosyl residue m /z 161.0583was also observed.The proposed frag-mentation pathway was shown in Fig.6B.However,the mass spectra could not discriminate the steric conformation of hydrogen atoms in 10-10 bond,and no corresponding authentic standard was used herein,the ion d were tentatively assigned as sen-noside A or its isomer sennoside B.According to the protocol above,total 18components were identi?ed,including 5chem-ical markers that contributed most to the difference between two kinds of https://www.360docs.net/doc/f04990033.html,ponents b,f and g were identi-?ed to be diosmetin,magnoloside B and naringin,respectively.Unfortunately,the identity of chemical markers c and e corre-sponding to the ions m /z 333.1335and m /z 312.1594could not be elucidated.

https://www.360docs.net/doc/f04990033.html,parison of the purgative effect of traditional and modern decoctions

Obviously,the signi?cant chemical differences exist between traditional decoction and modern decoction of DCQT,rhein,

-1.

-0.

-0.2-0.1-0.00.10.20.3

V a r i a b l e c o n f i d e n c e

Variable contribution

Fig.4.OPLS-DA/S-Plot of traditional and modern decoctions of Da-Cheng-Qi-Tang obtained using Pareto scaling with mean centering.(a)t R 9.77min,m /z 283.0239;(b)t R

7.91min,m /z 299.1278;(c)t R 4.28min,m /z 333.1335;(d)t R 5.89min,m /z 861.1885;(e)t R 4.10min,m /z 312.1594;(f)t R 4.40min,m /z 785.2504;(g)t R 5.83min,m /z 579.1711.

40J.-B.Wan et al./Journal of Pharmaceutical and Biomedical Analysis 83 (2013) 34–

42

Fig.5.Selected ion intensity trend plots.(a)t R 9.77min,m /z 283.0239;(b)t R 7.91min,m /z 299.1278;(c)t R 4.28min,m /z 333.1335;(d)t R 5.89min,m /z 861.1885;(e)t R 4.10min,m /z 312.1594;(f)t R 4.40min,m /z 785.2504;(g)t R 5.83min,m /z 579.1711.(?)traditional decoction;( )modern decoction.

sennoside A/B,diosmetin,magnoloside B and naringin are the components contributing most to these differences.Among these chemical markers,the contents of magnoloside B and naringin in traditional decoction is the lower than that in modern decoc-tion.However,to our knowledge,currently,no evidence indicates that they have a laxative effect.On the contrary,rhein,senno-side A/B and diosmetin in traditional decoction present the higher concentration than that in modern decoction.Perhaps the most

plausible underlying mechanisms for these changes are that Radix et Rhizoma Rhei suffers from the longer heating time and the higher ionic strength provided by mirabilitum (Sodium Sulfate)in modern decoction,comparing to traditional multi-step decoc-tion.Boiling Radix et Rhizoma Rhei for more than 15min could decrease the content of total anthraquinones [25],and the presence of mirabilitum could reduce the solubility of anthraqunones during the decoction [26].The purgative effect of DCQT mainly attributes

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41

Fig.6.Mass spectra and proposed fragmentation pathway of(A)rhein and(B)sennoside A/B.

to anthraquinones originated from Radix et Rhizoma Rhei[18]. Actually,both rhein and sennoside A/B are the inactive,and could be transformed to an active metabolite,rhein anthrone,by intesti-nal bacteria[27,28],that might be associated with inhibition of Na+-K+-ATPase,activation of calcium channel[29]and stimulation of PGE-like material production[30]in the colon.Due to the higher content of rhein anthrone sources,rhein and sennoside A/B,the purgative effect of traditional decoction might be the more effec-tive.

4.Conclusion

In the present study,an UPLC/Q-TOFMS based chemical pro?ling followed by multivariate statistical analysis was proposed to eval-uate the chemical consistency of DCQT prepared by traditional and modern decoction methods.In contrast to the conventional phyto-chemical and chromatographic approaches,the proposed method could globally characterize the chemical pro?les of traditional and modern decoctions,and rapidly reveal the differentiating chemi-cal markers.Our?ndings demonstrated that there were obvious chemical differences between traditional decoction and modern decoction of DCQT,and rhein,sennoside A/B,diosmetin,magnolo-side B and naringin were revealed as chemical markers contributing most to these differences.Based on the fact that traditional decoc-tion of DCQT presents the higher concentration of rhein and sennoside A/B,the purgative effect of traditional decoction may be more potent than that of modern decoction.However,it remains to be further investigated by comparative pharmacological and clinic studies.

Acknowledgments

We are grateful to Mr.Renbo Ding from our institute for his expert technical assistance.This research was supported by the grants from the Research Committee of the University of Macau (SRG009-ICMS12and MYRG123-ICMS12to J.B.Wan)and National Basic Research Program of China(973program,2010CB530600). References

[1]F.C.Zhang,M.Chen,Y.D.Yan,Modern decoction method vs traditional decoc-

tion method,Zhong Guo Yao Fang19(2008)478–480(Chinese).

[2]S.L.Li,J.Z.Song,C.F.Qiao,Y.Zhou,H.X.Xu,UPLC-PDA-TOFMS based chemical

pro?ling approach to rapidly evaluate chemical consistency between tradi-tional and dispensing granule decoctions of traditional medicine combinatorial formulae,J.Pharm.Biomed.Anal.52(2010)468–478.

[3]S.Rochfort,Metabolomics reviewed:a new“omics”platform technology for

systems biology and implications for natural products research,J.Nat.Prod.68 (2005)1813–1820.

[4]Z.Xiang,X.Q.Wang,X.J.Cai,S.Zeng,Metabolomics study on quality control and

discrimination of three curcuma species based on gas chromatograph-mass spectrometry,Phytochem.Anal.22(2011)411–418.

[5]X.Wang,H.Sun, A.Zhang,W.Sun,P.Wang,Z.Wang,Potential role of

metabolomics apporoaches in the area of traditional Chinese medicine:as pil-lars of the bridge between Chinese and Western medicine,J.Pharm.Biomed.

Anal.55(2011)859–868.

[6]E.J.Lee,R.Shaykhutdinov,A.M.Weljie,H.J.Vogel,P.J.Facchini,S.U.Park,

Y.K.Kim,T.J.Yang,Quality assessment of ginseng by(1)H NMR metabo-lite?ngerprinting and pro?ling analysis,J.Agric.Food Chem.57(2009) 7513–7522.

[7]E.C.Chan,S.L.Yap,https://www.360docs.net/doc/f04990033.html,u,P.C.Leow,D.F.Toh,H.L.Koh,Ultra-performance

liquid chromatography/time-of-?ight mass spectrometry based metabolomics of raw and steamed Panax notoginseng,Rapid Commun.Mass Spectrom.21 (2007)519–528.

[8]D.F.Toh,L.S.New,H.L.Koh, E.C.Chan,Ultra-high performance liquid

chromatography/time-of-?ight mass spectrometry(UHPLC/TOFMS)for time-dependent pro?ling of raw and steamed Panax notoginseng,J.Pharm.Biomed.

Anal.52(2010)43–50.

[9]S.O.Yang,Y.S.Shin,S.H.Hyun,S.Cho,K.H.Bang,D.Lee,S.P.Choi,H.K.Choi,

NMR-based metabolic pro?ling and differentiation of ginseng roots according to cultivation ages,J.Pharm.Biomed.Anal.58(2012)19–26.

[10]N.Kim,K.Kim,D.Lee,Y.S.Shin,K.H.Bang,S.W.Cha,J.W.Lee,H.K.Choi,B.Y.

Hwang,Nontargeted metabolomics approach for age differentiation and struc-ture interpretation of age-dependent key constituents in hairy roots of Panax ginseng,J.Nat.Prod.75(2012)1777–1784.

42J.-B.Wan et al./Journal of Pharmaceutical and Biomedical Analysis83 (2013) 34–42

[11]J.Sun,P.Chen,Differentiation of Panax quinquefolius grown in the USA and

China using LC/MS-based chromatographic?ngerprinting and chemometric approaches,Anal.Bioanal.Chem.399(2011)1877–1889.

[12]W.F.Tang,Q.Yu,M.H.Wan,F.Qin,Y.G.Wang,G.Y.Chen,M.Z.Liang,X.Huang,

Simultaneous determination and pharmacokinetic studies of aloe emodin and chrysophanol in rats after oral administration of Da-Cheng-Qi decoction by high-performance liquid chromatography,Biomed.Chromatogr.21(2007) 701–707.

[13]M.Katakai,T.Akamaru,T.Tani,An analysis of the frequency of formulations and

crude drugs described in Shan-Han-Lun,Yakushigaku Zasshi37(2002)28–35.

[14]Q.H.Qi,J.Wang,G.G.Liang,X.Z.Wu,Da-Cheng-Qi-Tang promotes the recovery

of gastrointestinal motility after abdominal surgery in humans,Dig.Dis.Sci.52 (2007)1562–1570.

[15]H.Chen,F.Li,J.G.Jia,Y.P.Diao,Z.X.Li,J.B.Sun,Effects of traditional Chinese

medicine on intestinal mucosal permeability in early phase of severe acute pancreatitis,Chin.Med.J.(Engl.)123(2010)1537–1542.

[16]R.Q.Wang,J.Z.Chen,G.Y.Ren,Intraperitoneal perfusion of compound injection

of salvia miltiorrhiza with dachengqi decoction in treating adhesive intestinal obstruction,Zhongguo Zhong Xi Yi Jie He Za Zhi14(1994)595–597(Chinese).

[17]J.Hu,Modi?ed DaChaiHu tang combined with DaChengQi tang decoction on

32cases of acute cholecystitis treatment,Gan Su Zhong Yi Yao Za Zhi24(2011) 25–26(Chinese).

[18]R.F.Xie,X.Zhou,Z.N.Shi,Y.M.Li,Z.C.Li,Study on spectrum–effect

relationship of Rhizoma Rhei,Cortex Magnoliae Of?cinalis,Fruc-tus Aurantii Immaturus and their formula,J.Chromatogr.Sci.(2012), https://www.360docs.net/doc/f04990033.html,/10.1093/chromsci/bms172.

[19]Q.Yu,J.Xiang,W.Tang,M.Liang,Y.Qin,F.Nan,Simultaneous determination

of the10major components of Da-Cheng-Qi decoction in dog plasma by liquid chromatography tandem mass spectrometry,J.Chromatogr.B Analyt.Technol.

Biomed.Life Sci.877(2009)2025–2031.[20]X.J.Zhang,L.L.Huang,X.J.Cai,P.Li,Y.T.Wang,J.B.Wan,Fatty acid variability in

three medicinal herbs of Panax species,Chem.Cent.J.7(2013)12.

[21]Z.H.Su,S.Q.Li,G.A.Zou,C.Y.Yu,Y.G.Sun,H.W.Zhang,Y.Gu,Z.M.Zou,Urinary

metabonomics study of anti-depressive effect of Chaihu-Shu-Gan-San on an experimental model of depression induced by chronic variable stress in rats,J.

Pharm.Biomed.Anal.55(2011)533–539.

[22]F.Xu,Y.Liu,Z.Zhang,R.Song,H.Dong,Y.Tian,Rapid simultaneous quan-

ti?cation of?ve active constituents in rat plasma by high-performance liquid chromatography/tandem mass spectrometry after oral administration of Da-Cheng-Qi decoction,J.Pharm.Biomed.Anal.47(2008)586–595.

[23]F.Xu,Y.Liu,R.Song,H.Dong,Z.Zhang,Constituents of Da-Cheng-Qi decoction

and its parent herbal medicines determined by LC-MS/MS,https://www.360docs.net/doc/f04990033.html,mun.

5(2010)789–794.

[24]H.Y.Zhao,M.X.Fan,X.Wu,H.J.Wang,J.Yang,N.Si,B.L.Bian,Chemical pro?ling

of the chinese herb formula Xiao-Cheng-Qi decoction using liquid chromatog-raphy coupled with electrospray ionization mass spectrometry,J Chromatogr.

Sci.51(2013)273–285.

[25]K.Kisa,K.Sasaki,K.Yamauchi,S.Kuwano,Potentiating effect of sennoside C on

purgative activity of sennoside A in mice,Planta Med.42(1981)302–303. [26]K.Takayama,H.Tsutsumi,T.Ishizu,N.Okamura,The in?uence of rhein8-O-

beta-D-glucopyranoside on the purgative action of sennoside A from rhubarb in mice,Biol.Pharm.Bull.35(2012)2204–2208.

[27]K.Yamauchi,T.Yagi,S.Kuwano,Suppression of the purgative action of rhein

anthrone,the active metabolite of sennosides A and B,by calcium channel blockers,calmodulin antagonists and indomethacin,Pharmacology47(1993) 22–31.

[28]T.Yagi,Y.Miyawaki,T.Nishikawa,K.Yamauchi,S.Kuwano,Involvement of

prostaglandin E-like material in the purgative action of rhein anthrone,the intraluminal active metabolite of sennosides A and B in mice,J.Pharm.Pharma-col.40(1988)27–30.