Fitoterapia 84 (2013) 286–294

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Fitoterapia

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Discrimination of raw and vinegar-processed Genkwa Flosusing metabolomics coupled with multivariate data analysis

A discrimination study with metabolomics coupled with PCA

Lulu Geng a, Haoyang Sun b, Yang Yuan a, Zhenzhen Liu a, Yan Cui a, Kaishun Bi a, Xiaohui Chen a,⁎a School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 103 Wenhua Road, 110016, P.R. Chinab Health-food and cosmetics inspect department, Shenzhen institute for drug control, Shenzhen, 518057, P.R. China

a r t i c l e i n f o

Abbreviations: BPI, Base peak intensity; CID, CollisioESI, Electrospray ionization; PCA, Principal componenUltra performance liquid chromatography tandemmas⁎ Corresponding author at: School of Pharmacy, She

University, Wenhua Road 103, Shenyang 110016, P.R24 23986259.

E-mail address: cxh_syphu@yahoo.com.cn (X. Che

0367-326X/$ – see front matter © 2012 Elsevier B.V.http://dx.doi.org/10.1016/j.fitote.2012.12.003

a b s t r a c t

Article history:Received 3 September 2012Accepted in revised form 30 November 2012Available online 10 December 2012

In this paper, a novel approach using UPLC–MS (Ultra performance liquid chromatography tandemmass spectrometry) coupledwithmultivariate statistic analysiswas established for the profiling anddiscrimination of raw and processed herb using Genkwa Flos as a model herb. A batch of raw andprocessed samples was analyzed, and the datasets of tR–m/z pairs, ion intensities and sample codeswere subjected to the principal component analysis (PCA). Raw and processed herb showed a clearclassification of the two groups on the score plot. Loading plot was performed, and the chemicalmarkers having great contributions to the differentiation were screened out. The identities of thechemical markers were identified by comparing the mass spectra and retention times with those ofreference compounds and/or tentatively assigned by matching empirical molecular formulae andmass fragments with those of the known compounds published in the literatures. Based on theproposed strategy, yuanhuacine, genkwadaphnin, genkwanin-5-O-β-D-primeveroside, genkwanineN, genkwanin, 3′-hydroxy-genkwanin and apigenin were explored as representative markers indistinguishing the raw from the processed herbs. The method has been successfully applied in thedistinguishing raw from processed herbs. Furthermore, the underlying detoxification mechanism oftraditional processing procedure on the herb was predicted, and was related to the changes in themetabolic profiling. This research could be valuable to explore the chemical markers, investigate themechanism underlying the processing procedure, and promote the quality control and safetyapplication of traditional Chinese herbs.

© 2012 Elsevier B.V. All rights reserved.

Keywords:Chemical markerGenkwa FlosHerb processingMultivariate statistical analysisUltra performance liquid chromatographytandem mass spectrometry

1. Introduction

The processing of traditional Chinese herbs is a commonprocedure and usually applied before the herb was pre-scribed. There are quite amount of traditional ways forprocessing herbs, such as frying with sand or oil, sauteingwith wheat bran, steaming with water, and braising with rice

n induced dissociation;t analysis; UPLC–MS,s spectrometry.nyang Pharmaceutical. China. Tel./fax: +86

n).

All rights reserved.

wine, rice vinegar or licorice liquids, etc. [1]. Under theguidance of the traditional theory of processing, it has beenproved that the processing procedure could reduce thetoxicity and/or enhance the curative effect after beingproperly processed with parching, steaming and soaking,etc. [2]. A variety of effective herbs have demonstratedserious toxic side effects, such as Radix Aconiti LateralisPreparata [3], Radix Phytolaccae [4] and Semen Strychni [5],though they could be used in safety after the toxicity wasremoved by processing. One typical example is that thetoxicity of Radix Aconiti Lateralis Preparata, the root ofAconitum Carmicadi Debx, is removed by processing, and ithas been proved by modern toxicological and pharmacolog-ical researches [3].

287L. Geng et al. / Fitoterapia 84 (2013) 286–294

Genkwa Flos, the flower buds of Daphne genkwa Sieb. etZucc. (Thymelaeaceae), is a traditional oriental herb widelydistributed in China, and it has been employed for diuretic,antitussive, expectorant, abortifacient, antileukemia, and anti-tumor purpose. The main components in this herb are fla-vonoids and diterpenes, and they are proved to have definitebioactive effect for the treatment of edema, ascites, suddencough, asthma, and cancer [6]. Genkwa Flos classified as“Xiapin” (low grade) in Shen Nong's Herbal Classic whichmeans mild toxicity existed. There is evidence that excessiveand chronic use of the raw herb will finally result in seriousdamage to kidney, liver andheart [7], and itwas publicly knownto have irritation to mucous and skin. It is widely recognizedthat raw Genkwa Flos should not be taken unless the toxicity isremoved [8]. Vinegar-processing for Genkwa Flos is applied toweaken the toxicity and to relieve the symptom of decanta andcelialgia. It has been practiced for hundreds of years, and isdocumented in the Chinese Pharmacopoeia [9]. Two differentforms of Genkwa Flos are employed, namely, the raw andvinegar-processed forms. As raw and processed herbs areboth available on the market, the quality control and theidentification of raw and processed herbs are particularlyessential for the safe and effective use of medicinal herbs inclinical practice.

While the underlying mechanisms of herb processingwere found mainly related to the changes in the compositionand/or activity of the components in the herbs [10], it isessential to screen out those components greatly changed aschemical markers. Chemical markers are crucial as they couldbe used to differentiate raw from processed herbs for thequality control and safety application in clinical practice. Inthe past few decades, a large number of researches have beenconcentrated on the quality control of raw herbs, but littleprogress was made on the processed herb. The main reasonscould be concluded as follows: there is no appropriatecomponent as characteristic marker of the processed herb,and no comprehensive research was made on the metabolicchange in the progress of processing.

Metabolomics, a valuable approach for the metabolicprofiling in complex matrix, with the help of multivariatestatistic analysis, could extensively assess complex herb orprescription while classify the samples of various origin,diverse status, different qualities. Metabolomics coupledwith principal component analysis (PCA) was applied bymany investigators to discriminate between young andmature of the herb [11], dig out the characteristic markersremarkably changed in the processing procedure [12], andmonitor the chemical consistency of different preparations[13]. It has great advantage over the traditional phytochem-ical methods seeking for chemical markers, leaving out thetime-consuming extraction, isolation, purification and iden-tification process.

Therefore, an effective metabolomics study based on UPLC-MS along with multivariate statistic analysis was developedand applied for the discrimination of raw and processed herbsusing Genkwa Flos as a model herb. Chemical markers largelycontributed to the classification were screened out andtentatively identified. The underlying mechanism of vinegar-processing procedure was elucidated. This approach could be ahelpful tool for the discrimination between raw and processedherbs.

2. Experimental

2.1. Chemicals and herbal materials

Benzoic acid, genkwanin-5-O-β-D-primeveroside, genkwanin-5-O-β-D-glucoside, syringaresinol, tiliroside, luteolin, kaempherol,apigenin, isodaphnoretin, 3′-hydroxy-genkwanin, genkwanin,genkwadaphnin, yuanhuadine and yuanhuacine were previous-ly isolated and identified from raw Genkwa Flos. Their identitieswere elucidated by UV, MS, NMR analysis, and the purities wedetermined to be more than 98% by HPLC–DAD (Shimadzu,Tokyo, Japan).

Genkwa Flos was collected from Shanxi Province, one of thenative cultivating provinces in China. A total of 20 batches ofherbs were collected. Eight commercial available bathes of raw(GF-001-01 to GF-001-07, GF-001-09) and processed GenkwaFlos (GF-002-01 to GF-002-07, GF-002-09) samples werepurchased from different herbal shops all over China. The timeand locations of collectionwere listed in Supplementarymaterial1. In addition, two batches of raw Genkwa Flos (GF-001-08 andGF-001-10) were obtained by sun-drying the fresh flower budsofDaphne genkwa Sieb. et Zucc. (Thymelaeaceae). The processedGenkwa Flos (GF-002-08 and GF-002-10) was produced by theprocessing method described in Chinese Pharmacopoeia asfollows: the dried and powdered GF (1 kg) was evenly mixedwith 0.3 kg vinegar diluted in 0.6 kg water and moistened in aclosed container for one night, then put into a drug-parchingmachine to get nearly dry with gentle heat and taken outtimely [9].

Raw Genkwa Flos (GF-001-01 and GF-001-08) and pro-cessed Genkwa Flos (GF-002-01 and GF-002-08) were appliedto develop themethod, and the other two batcheswere used toverify the established approach.

Methanol of HPLC grade was supplied by Fisher Corporation(Pittsburgh, PA, USA), and formic acid of HPLC grade waspurchased from Sigma-Aldrich (St. Louis, MO, USA). Distilledwater was purified using a Milli-Q system (Millipore, Bedford,MA, USA). Other chemicals and solvents were of analyticalgrade.

2.2. Sample preparation

The powdered samples of Genkwa Flos were accuratelyweighed (approximately 2.5 g), and was ultrasonic-extractedwith 20.0 mL methanol for 30 min. The resulting solutionswere centrifuged at 3000 r/min, the supernatant was filteredthrough a 0.2 μm PTFE syringe filter, and an aliquot (10 μL) ofeach filtrate was subjected to UPLC–MS analysis. Blankmethanol (10 μL) was injected between selected analysis tovalidate inter-sample cross-talking effect.

2.3. Ultra performance liquid chromatography tandem massspectrometry (UPLC–MS)

UPLC analysis was performed using ACQUITY™ Ultra Perfor-mance Liquid Chromatography system (Waters, Milford, MA,USA), equipped with a binary solvent delivery system, auto-sampler. The separation was achieved on an Agilent TC-C18(4.6 mm×250 mm, 5 μm, Agilent, Palo Alto, CA, USA). Themobile phase consisted of (A) water containing 0.1% formic acidand (B) methanol containing 0.1% formic acid. The gradient

288 L. Geng et al. / Fitoterapia 84 (2013) 286–294

elution condition was optimized as follows: linear gradientfrom 85% to 55% A (0–5 min), 55% to 30% A (5–10 min), 30% to10% A (10–25 min), 10% to 5% A (25–30 min), 5% to 0% A(30–40 min), and then back to 85% A in 1 min. The flow ratewas set at 0.8 mL/min (25% of the eluent was splitted into theinlet of the mass spectrometer). The column and auto-samplerwere maintained at 35 °C and 4 °C, respectively. A “purge–wash–purge” cycle was employed on the auto-sampler, with90% aqueous formic acid used for the wash solvent and 0.1%aqueous formic acid used as the purge solvent to ensure thecarry-over between injections was minimized.

Micromass Quattromicro™APImass spectrometer (Waters,Milford, MA, USA) was equipped with electrospray ionization(ESI) interface operating in both positive and negative ionmode. The source temperaturewas set to 120 °Cwith a cone gasflow of 50 L/h, a desolvation temperature of 350 °C, and adesolvation gas flow of 600 L/h. A scan time of 1 s with aninter-scan delay of 0.1 s was used for the analysis. The capillaryvoltage and cone voltage were set to 3200 V and 30 V. Thecollision induced dissociation (CID) experiment was performedto get the fragments of putative chemical biomarkers withargon as collision gas, while collision energy varied from 10 eVto 30 eV to get abundant fragment information. The mass wascorrected with NaCsI before the study. Data were collected incentroid mode. The mass spectrometric data was collected infull scan mode.

2.4. Compound database establishment and peak assignment

The compound database of Genkwa Flos was establishedby searching database such as Pubmed, SciFinder, METLINand Chinese National Knowledge Infrastructrue (CNKI) ofTsinghua University, summarizing the components reportedin literatures of Genkwa Flos and others from Thymelaeaceae,including the name, molecular formula, quasi-molecular ion,chemical structure [Supplementary material 2], the potentialMS/MS fragments and literatures of each published knowncompounds.

Meanwhile, chromatographic peaks were also assigned bycomparing the accuratelymeasuredmass value to the theoreticalexact mass value on Bruker's ESI-QTOF-MS (Bruker-FranzenDaltonics, Bremen, Germany) under the same liquid chromatog-raphy condition.

2.5. Multivariate data processing

The UPLC–MS data of all determined samples wereanalyzed by MarkerLynx software (Waters, Milford, MA, USA)to identify the potential chemical markers for discriminationand quality control of raw and processed Genkwa Flos. For datacollection, the parameters were set as follows: retention timeranging from 0 to 40 min, mass ranging from 100 to 1000 Da,noise elimination level was set at 5.

For data analysis, a list of the identities of the detected peakswas generated using retention time (tR)–mass data (m/z) pairsas the identifier of each peak. A numberwas assigned to each tR–m/z pair in the order of their LC elution. The informationcollected for each tR–m/z pair was listed in Table 1. The ionintensity for each detected peak was normalized against thesum of the peak intensity within that sample. Ions of differentsamples were considered to be the same ion when they

demonstrated the same tR (tolerance of 0.01 min) and m/zvalue (tolerance of 0.1 Da). If a peak was not detected in asample, the ion intensity was recorded as zero. The resultingthree-dimensional data comprising of peak number (tR–m/zpair), sample name and normalized ion intensity were analyzedby unsupervised PCA using the MarkerLynx software.

3. Results and discussion

3.1. Ultra performance liquid chromatography tandem massspectrometry (UPLC–MS)

The constituents of Genkwa Flos are mainly consisted offlavonoids, lignans and diterpenes. Earlier studies primarilyfocused on the determination of flavonoids in the herb [14] andthe quantification of four diterpenes [15]. It was a challenge tosimultaneously determine the different types of bioactivecomponents in the same run using chromatographic analysisdue to their differences in polarity and in content.

In the present study, a UPLC–MS method was developed,and the chromatographic and mass conditions were optimizeddue to the poor UV absorption and low content of diterpenes inGenkwa Flos. Different kinds of mobile phases, such asacetonitrile and methanol with a variety of modifiers, weretested. It was found that a mixture of methanol and 0.1% formicacid was a suitable mobile phase which can simultaneouslyseparate the different types of major components in GenkwaFlos. ESI+ was selected as the ionization mode for theexperiments since our pre-test demonstrated that the flavo-noids, lignans and diterpenes were detected with greater ionsensitivities using the positive ionization mode and henceprovided richer information. [M+H]+ or [M+Na]+ waseventually used as the target ions for data analyses since theygenerally showed good abundance and ion count consistency.There are few peaks detected in ESI- mode, and most of themwere identified as flavonoids. Thus the negative mode wasutilized to aid the identification process of chemical markers.Component was identified by comparing its retention time,MSMS fragmentation and accurately measured mass value ofthe extracted ion peak to that of its reference standard.While forthose lack of reference standards, the identificationwas realizedby comparing the accurately measured mass value and MSMSfragments with the reference or database. Under the optimizedchromatographic and MS conditions, the major components inGenkwa Flos were well separated and detected within 40 min.The representative positive base peak intensity (BPI) chromato-grams of raw and processed Genkwa Flos extracted sampleswere shown in Fig. 1.

3.2. Method validation

The applied method was validated for sample preparationrepeatability, within-day stability and precision of injectionprior to the analysis of experimental samples. The extractedion chromatographic peaks of eight ions (2.7–343.2, 6.8–120.3, 14.6–873.2, 18.3–911.2, 21.6–487.1, 28.1–587.4, 34.2–609.2, 35.2–279.3) were selected for validating the method.The selected ions were evenly distributed in the analysis timein the range of m/z 100–1000, taking the large as well assmall peaks into consideration. Precision of injection wasvalidated by the continuous detection of six injections of the

Table 1Components identified from Genkwa Flos.

Peakno.

tR(min)

Assigned identity Molecularformula

Meanmeasuredmass(Da)

Massaccuracy(ppm)

Theoreticalexact mass(Da)

Quasi-molecularion

Changetrend afterprocessing

Reference

1 4.71 Unknown C17H15O6N2 343.0944 5.8 343.0925 [M+H]+ –

2a 5.16 Benzoic acid C7H6O2 123.0438 −2.4 123.0441 [M+H]+ – [17]3 6.17 Unknown C12H16O4 224.1065 9.5 224.1043 [M+H]+ –

4 6.76 Unknown5 7.87 Unknown C10H52O1 188.4027 7.6 188.4013 [M+H]+ ↑⁎⁎

6 8.43 Unknown C7H12ON7 210.1109 5.3 210.1098 [M+H]+ ↓7 8.87 Unknown C22H15O4 343.0943 −6.3 343.0965 [M+H]+ ↓8 10.67 Unknown C13H24O1Na 219.1711 −3.8 219.1719 [M+Na]+ –

9 11.54 Kaempferol-3-O-β-D-glucoside C21H20O11 449.1111 7.2 449.1078 [M+H]+ ↓ [18]10 12.05 Unknown C22H23O11 463.1224 −2.3 463.1235 [M+H]+ ↓11a 12.43 Genkwanin-5-O-β-D-primeveroside C27H30O14 579.1715 1.1 579.1708 [M+H]+ ↓⁎ [19]12a 12.69 Genkwanin-5-O-β-D-glucoside C22H22O10 447.1255 −6.5 447.1286 [M+H]+ ↓ [20]13a 13.13 Syringaresinol C22H26O8 419.1738 9.0 419.1700 [M+H]+ – [21]14 13.60 Dihydrosesamin C20H20O6 357.1354 6.0 357.1333 [M+H]+ ↑ [22]15a 14.02 Tiliroside C30H26O13 595.1457 1.8 595.1446 [M+H]+ ↓ [23]16a 14.60 Luteolin C15H10O6 287.0540 −3.4 287.0550 [M+H]+ ↑⁎ [24]17 14.92 Genkwanine P C27H36O9 527.2248 −0.7 527.2252 [M+Na]+ – [25]18 15.21 8-methoxykaempferol C16H12O7 317.0653 −0.8 317.0656 [M+H]+ ↑ [20]19a 15.42 Kaempherol C15H10O6 287.0537 −4.4 287.0550 [M+H]+ ↑⁎ [23]20a 15.83 Apigenin C15H10O5 271.0616 5.5 271.0601 [M+H]+ ↑⁎ [19]21 15.90 Genkwanine O C27H36O9 505.2429 −0.6 505.2433 [M+H]+ – [26]22 16.55 Aurantiamide C25H26O3N2 403.2005 −2.8 403.2016 [M+H]+ ↓ [27]23 16.85 Genkdaphin C20H16O7 391.0766 −5.6 391.0788 [M+Na]+ ↓ [28]24a 16.95 Isodaphnoretin C19H12O7 353.0639 −4.8 353.0656 [M+H]+ ↓⁎ [29]25a 17.60 3′-Hydroxy-genkwanin C16H12O6 301.0733 4.1 301.0707 [M+H]+ ↑⁎⁎ [30]26 18.36 Aurantiamide acetate C27H28O4N2 445.2092 −6.7 445.2122 [M+H]+ ↑27 18.82 4′, 5-Dihydroxy-3′, 7-dimethoxy flavone C17H14O6 315.0846 −5.3 315.0863 [M+H]+ – [18]28 19.30 Yuanhuaoate F/Yuanhuafine C29H32O10 541.2055 −2.4 541.2068 [M+H]+ – [26]29a 19.54 Genkwanin C16H12O5 285.0758 0.1 285.0757 [M+H]+ ↑⁎⁎ [30]30 20.11 Yuanhuapine/Yuanhuaoate H C29H34O10 543.2224 −0.1 543.2225 [M+H]+ ↑ [31]31 20.62 Yuanhuaoate A C30H34O10 555.2233 1.5 555.2225 [M+H]+ – [32]32 21.28 Asperphenamate C32H30O4N2 507.2249 −5.8 507.2278 [M+H]+ ↓ [33]33 21.29 5-Hydroxy-3′,4′,7-trimethoxyflavone C18H16O6 329.0997 −7.0 329.1020 [M+H]+ –

34 21.69 Orthobenzoate 2 C27H34O8 487.2303 −4.7 487.2326 [M+H]+ – [34]35 21.88 Genkwanine Q C27H36O9 527.2231 −3.8 527.2252 [M+Na]+ –

36 22.75 4′, 7-Dimethylapigenin C17H14O5 299.0915 0.4 299.0914 [M+H]+ ↑ [20]37 22.92 Genkwanine R C27H36O9 505.2431 −0.1 505.2432 [M+H]+ ↑38 23.05 Genkwanine A C27H36O9 505.2407 −4.9 505.2432 [M+H]+ – [6]39a 23.31 Genkwadaphnin C34H34O10 603.2225 0.1 603.2225 [M+H]+ ↓⁎ [35]40 24.12 Yuanhuaoate B C34H36O11 621.2335 0.8 621.2330 [M+H]+ – [32]41 24.41 Yuanhuatine C34H36O10 605.2333 −7.0 605.2381 [M+H]+ – [26]42 24.99 4′,5,7-Trimethoxyflavone C18H16O5 313.2208 −1.3 313.2212 [M+H]+ ↑ [20]43 25.43 Genkwanine I C29H34O9 527.2230 −8.6 527.2276 [M+H]+ – [36]44 25.95 12-O-(2′E,4′

E-decadienoyl)-4-hydroxyphorbol-13-acetylC32H44O8 557.3081 −4.9 557.3109 [M+H]+ ↓ [37]

45 25.96 Genkwanine M C34H38O9 591.2550 −6.6 591.2589 [M+H]+ –

46 26.14 Yuanhuaoate C C32H44O11 605.2904 −8.6 605.2956 [M+H]+ – [32]47 27.46 Yuanhuaoate G C34H36O10 605.2327 −8.9 605.2381 [M+H]+ –

48 27.48 Genkwanine N C34H38O9 591.2542 −8.0 591.2589 [M+H]+ ↑⁎⁎ [26]49a 28.15 Isoyuanhuadine/Yuanhuadine C32H42O10 587.2833 −3.0 587.2851 [M+H]+ – [31]50 29.29 Unknown C37H39O4 547.2844 0.1 547.2843 [M+H]+ ↓⁎⁎

51 29.82 Genkwanine U C37H4O10 655.3445 −4.8 655.3477 [M+H]+ ↑52 30.24 Unknown ↑⁎

53a 30.78 Yuanhuacine C37H44O10 649.2960 −7.3 649.3007 [M+H]+ ↓⁎⁎ [31]54 32.19 Daphnane-type diterpene ester-7/

Yuanhuaoate EC20H22O6 667.3103 −1.5 667.3113 [M+H]+ – [20]

55 33.01 Genkwanine T C37H50O10 655.3433 −6.6 655.3477 [M+H]+ ↑56 33.36 Genkwanine H C34H40O10 609.2638 −9.3 609.2694 [M+H]+ – [6]57 33.58 Genkwanine F C37H50O10 655.3433 −6.6 655.3477 [M+H]+ – [6]58 34.23 Genkwanine D C34H40O10 609.2642 −8.6 609.2694 [M+H]+ ↓ [6]59 35.30 Unknown C22H37O3N3 391.2800 −7.6 391.2829 [M+H]+ ↑60 36.73 Unknown C41H36O4 593.2716 5.0 593.2686 [M+H]+ –

⁎pb0.1; ⁎⁎pb0.05; ⁎⁎⁎pb0.01 “–”: no significant change happened.↑increased after processing procedure; ↓ decreased after processing procedure.

a Identified with reference standards.

289L. Geng et al. / Fitoterapia 84 (2013) 286–294

Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

%

0

100

Time

%

0

100

1

2

3

4

216

10

12

15

16 17

1819

22

7 811

913

1423

24

25

2627

2829

30 323334 36

37383940

41

42

20

3531 43

44 45

46

48

49 50515253 54

5556

57

58

59

60

5

47

A

B

Fig. 1. The representative positive base peak intensity (BPI) chromatograms of Genkwa Flos extracted samples. A: raw Genkwa Flos; B: processed Genkwa Flos.

290 L. Geng et al. / Fitoterapia 84 (2013) 286–294

same sample. Within-day stability was investigated by sixinjections of the same sample in 12 h with an interval of 2 h.Sample preparation repeatability was evaluated by analyzingsix aliquots of a random sample. The RSDs of retention timefor sample preparation repeatability, within-day stability andprecision of injection were estimated to be less than 0.9%;while the RSDs for intensity were no more than 13.5%. Theresults indicated large-scale sample analysis did not influ-ence the reliability of data, and the method could be appliedto analyze of experimental samples.

3.3. Multivariate statistical analysis and chemicalmarkers exploring

To compare the differences between raw and processedGenkwa Flos, unsupervised principal component analysis (PCA)were performed. After Pareto scaling with mean centering, thedata were displayed as score plot (Fig. 2A). Pareto scalingtechnique is applied because it enhances the contribution oflower concentration metabolites without amplifying noise andartifacts commonly present in themetabolomics data sets,whichis positive for themetabolomics study [16]. The score plot shows

that the experimental samples clearly clustered into two groups,i.e. the raw and the processed Genkwa Flos, indicating that theprocessing procedures caused changes in the composition and/or content of components in the herb. To find the potentialchemical markers for the discrimination between raw andprocessed Genkwa Flos, extended statistical analysis wasperformed to generate a loading plot (Fig. 2B). In the loadingplot, each point represents a variable including its intensity andits corresponding tR–m/z pair. Chemical markers were selectedaccording to the distance from the origin on the loading plot. Thedistances of a series of ions stand for the contribution of thevariables in discrimination of the two groups on the PCAcomponents. The ions far away from the mean center wereselected as potential chemical markers for the discrimination ofthe groups.

According to the loading plot, ten ions (a) 29.3–547.3; (b)30.8–649.3; (c) 23.3–603.2; (d) 17.0–353.1; (e) 12.4–579.2;(f) 7.9–188.4; (g) 27.5–591.3; (h) 19.5–285.1; (i) 17.6–301.1; (j) 15.8–271.1 distributed far from the center are theions contributed most to differentiation between raw andprocessed herb. References [6,17–37] were searched, and the

Scores: Component 1 - Component 2

Component 1-0.200 0.000 0.200

Com

pone

nt 2

-0.40

-0.20

0.00

0.20

0.40

0.60

Loadings: Component 1 - Component 2

Component 1-0.200 -0.100 0.000 0.100 0.200

-0.100

-0.050

0.000

0.050

0.100

0.150

0.200

Scores: Component 1 - Component 2

Component 1-0.200 0.000 0.200

Com

pone

nt 2

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pone

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pone

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-0.10

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Scores: Component 1 - Component 2

Component 1-0.200 0.000 0.200

-0.30

-0.20

-0.10

-0.00

0.10

0.20

0.30

0.40

0.50

0.60

a

b

c

e

h

f

g

dj

i

A B

C D

Fig. 2. PCA score plot and the resulting loading plot of raw and processed Genkwa Flos samples obtained using Pareto scaling with mean centering (a) tR 29.3 min,m/z 547.3; (b) tR 30.8 min, m/z 649.3; (c) tR 23.3 min, m/z 603.2; (d) tR 17.0 min, m/z 353.1; (e) tR 12.4 min, m/z 579.2; (f) tR 7.9 min,m/z 188.4; (g) tR 27.5 min,m/z 591.3; (h) tR 19.5 min, m/z 285.1; (i) tR 17.6 min, m/z 301.1; (h) tR 15.8 min, m/z 271.1. Raw Genkwa Flos; □ Processed Genkwa Flos; Raw Genkwa Flos(GF-001-09 and GF-001-10); Processed Genkwa Flos (GF-002-09 and GF-002-10).

291L. Geng et al. / Fitoterapia 84 (2013) 286–294

ion intensity change trends of these selected ions in processedsamples compared with the raw ones were listed in Table 1.

3.4. Chemical markers identification

Under the present chromatographic and spectrometricconditions, a total of 60 major peaks were detected from rawand processed Genkwa Flos, and a number of 48 peaks wereidentified by comparing accurately measured mass value withthe theoretical exact mass. The identities of ten most changedcomponents together with othermajor peakswere identified ortentatively assigned by comparison with the reference com-pounds or matching the molecular weight with those of thepublished compounds of Genkwa Flos and Daphne genus. Thedetails of the identified components were summarized, wecould conclude fromTable 1 that themajor types of componentsincluding flavonoids, lignans and diterpenes from Genkwa Floswere detected and identified.

Benzoic acid (2), Genkwanin-5-O-β-D-primeveroside (11),genkwanin-5-O-β-D-glucoside (12), syringaresinol (13),tiliroside (15), luteolin (16) kaempherol (19), apigenin(20), isodaphnoretin (24), 3′-hydroxy-genkwanin (25),genkwanin (29), genkwadaphnin (39), yuanhuadine (49) andyuanhuacine (53) were further confirmed by comparing theretention time,mass spectra and the accuratelymeasuredmassvalue with those of reference compounds. When the referencestandard was unavailable, it was a challenge to identify thechemical marker. Then CID experiment was performed to getthe MS/MS spectra, together with the accurately measuredmass value, the compounds were tentatively identified.

Here, we take 30.8–649.3 (53) as an example to illustratethe identification process. The chemical marker with tR–m/zpairs of 30.8–649.3 (53) in positive ion mode was identified asyuanhuacine. Besides the base peak ion at m/z 649, the ion atm/z 671was found. Thus, we infer that the quasi-molecular ionis m/z 649 ([M+H]+) and the ion atm/z 671 is the adduction

292 L. Geng et al. / Fitoterapia 84 (2013) 286–294

([M+Na]+). Then CID experiment was performed to get theMS/MS spectra, and was compared with the spectra presentedin the literature and was searched in the in-house chemicaldatabase of this herb. Three major fragment ions of the parention [M+H]+ atm/z 649 were found [Supplementary material3A]. They were ions at m/z 509, 341 and m/z 323, and thepossible MS fragmentation mechanism was elucidated [Sup-plementary material 3B]. Then the reference [31] was searchedbased on the clues obtained above. After comparing theretention time andmass spectra with the reference compound,it was tentatively identified as yuanhuacine.

When several components found in the in-house databasematch the same molecular weight and fragment information,those isomeric components previously reported from the herbwould be selected as the putative components in preference. ThetR–m/z pair of 28.2–587.3 (49) was identified as isoyuanhuadineand/or yuanhuadine due to the samemolecularweight andmassfragments [37].

In this paper, it was found that ions a 29.3–547.3 and b30.8–649.3 were detected with higher intensity in raw sam-ples, but was almost undetectable in all processed samples. Ionf 7.9–188.4 and g 27.5–591.3 were detectable with higherintensity in processed samples, but were much lower in rawsamples. It was also found that ion c-e were detected withhigher intensity in rawGenkwa Flos than in processed samples,and ion h–j were detected with higher intensity in processedherbs than in raw samples. These ions could be used aspotential characteristic markers to distinguish processedGenkwa Flos from raw samples.

In particular, the components that correlates to ion a, b, fand gwould be the most suitable chemical markers to identifyprocessed Genkwa Flos. The changes induced by the progressof vinegar-processing are both in content and in amount.Therefore, the ions of c–e and h–j which significantly changedin amount in the processing procedure could also serve aschemical markers in differentiation.

Ions of b–e, g–j were identified as yuanhuacine (53),genkwadaphnin (39), isodaphnoretin (24), genkwanin-5-O-β-D-primeveroside (11), genkwanine N (48), genkwanin (29),3′-hydroxy-genkwanin (25) and apigenin (20), respectively.The identification of a and f are still in progress. These com-ponents can be used as potential chemical markers to dis-criminate between raw and processed Genkwa Flos.

3.5. Application

The developed method was successfully used to discrimi-nate the available Genkwa Flos samples. It is demonstrated inFig. 2C that two batches of experimental raw Genkwa Flos areclustered with the raw herbs in the score plot. Furthermore,both the herbs processed by us and commercial available onesshowed tight cluster with the processed group in Fig. 2D. Itcould be concluded that samples of raw and processed GenkwaFlos could easily be identified by the established approach.

3.6. Preliminary study of vinegar-processing mechanism

Studies have shown that processed Genkwa Flos was foundto contain more 3′-hydroxy-genkwanin and genkwanin thanthe raw herb [38], while the content of yuanhuacine dropped

after the vinegar-processing procedure, which accounted forthe toxicity of this herb [39].

Flavonoids are the major pharmacologically active compo-nents in Genkwa Flos. The general trend of flavonoids aftervinegar-processing is a significant increase. Our finding isconsistent with the previous studies, i.e. genkwanin (29),3′-hydroxy-genkwanin (25) and apigenin (20) are higher incontent in the processed samples. This might be due to the acidhydrolysis of flavonoid glycoside in the procedure of vinegar-processing. It is reported that the glycosidic bond is apt to behydrolyzed in the acidic conditions or on heat. It is observed thatgenkwanin-5-O-β-D-primeveroside (11), genkwanin-5-O-β-D-glucoside (12) decreased, and thismight be a reflection of its acidhydrolysis to genkwanin. Vinegar-processing may promote thehydrolyzation from flavonoid glycoside to its correspondingaglycone which made the aglycone accumulated. Besides, it isreported that subacid flavonoids are easily extracted at thecondition of acid which will be another explanation for theincrease in flavonoids after vinegar-processing procedure.

On the other hand, the processing procedure only hasgreat effect on the content of a few diterpenes, rather thanmost diterpenes in the herb. The content of yuanhuacine(53) and genkwadaphnin (39) decreased, while the contentof genkwanine N (48) increased. Yuanhuacine (53) andgenkwadaphnin (39), the representatives of benzoyl-deterpenes, which mainly accounted for the toxicity ofthis herb, significantly decreased after the processing proce-dure, and it may be an explanation for the detoxification ofprocessing procedure. Yuanhuacine and genkwadaphnin aretypical bioactive constituents in the herb [6,21,23,25,31], whilethey are the most toxic components [7,8]. The content of thesetwo components decreased after vinegar-processing proce-dure. It is probable that the prototypes of diterpenes weredestroyed, and new compound-hydrolytic products yielded[40] which were less toxic or neutralize the toxicity of theprototype. The content of genkwanine N (48) is elevated afterprocessing. While there is limited information on the pharma-cological activity and toxicity of this compound, further re-search is required to investigate the underlying transformationin the duration of vinegar-processing.

Isodaphnoretin, a dicoumarin separated from Genkwa Flos[29], decreased in the amount in the acidic environment duringthe vinegar-processing procedure. During vinegar processingprocedure, it would combine with other compounds due to itsalkalic property which may be an explanation for the down-regulation of isodaphnoretin.

Under the guidance of the theory of traditional Chinesemedicine (TCM) science, processing procedures have beenproved to be important in reducing the toxicity or enhancingthe curative effect. Thousands of years of history on herb practicehas proved the effectiveness and rationality of traditional herb-processing procedure. The effect of vinegar processing procedureonTCMhadbeen confirmed, such as promoting the circulation ofthe drug, avoiding the adverse effects and adjusting the un-pleasant flavor [2]. In this paper, the content of apigenin,3′-hydroxy-genkwanin and genkwanin increased, and thesecomponents mainly accounted for the pharmacologicaleffects of this herb. The elevation of these constituents maypromote the potency of Genkwa Flos after the vinegarprocessing procedure. On the other hand, genkwadaphninand yuanhuacine, the representative toxic component of the

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herb, dropped significantly after vinegar processing. To sumup, through the vinegar processing procedure, the pharma-cological activities could be enhanced while the side effectsalleviated. All these findings meet the aims, and basicallyconfirmed the scientific rationality of traditional processingprocedure

Based on these findings, the fact that it is the vinegar-processing procedure which caused changes to the metabolicprofiling of Genkwa Flos is confirmed. The exact mechanism ofprocessing is still unclear and inconclusive. Therefore, moreresearch is essential in the future. It is critical to study thedifferences between raw and processed Genkwa Flos usingmetabolomics as a tool to investigate their metabolic profilesand relating them to their different toxic effects. It is beneficialto the quality control of the herb and the administration ofcorrect form of the herb.

4. Conclusion

A novel strategy to screen out the potential chemicalmarkers to discriminate raw and processed herb by UPLC–MScoupled with multivariate statistical analysis was developed.Genkwa Floswas selected as amodel herb to study the changesof metabolic profiling and detoxification of the traditionalprocessing method. A batch of samples was processed bystatistical analysis, ten ion pairs were identified as chemicalmarkers which could be representative in distinguishing theraw from processed herbs. The strategy was successfullyapplied in the identification of raw and processed GenkwaFlos, and it could be applied in the research about the chemicaltransformation in content and/or in amount in the processingprocedure.

Acknowledgements

This work was financially supported by National KeyScientific Project for New Drug Discovery and Development ofChina (grant no: 2009ZX09301-012). The authorswould like tothank Dr. Yue Yu for the valuable suggestions and supportduring the experiment.

References

[1] Jin SY, Wang Q. Studies on processing of Chinese Medicinal Yinpian andits clinical application. Beijing: Press of Chemistry Industry; 2004.

[2] Ye DJ, Yuan ST. Dictionary of Chinese herbal processing science.Shanghai: Shanghai Science and Technology Press; 2005.

[3] Singhuber J, Zhu M, Prinz S, Kopp B. Aconitum in Traditional ChineseMedicine—a valuable drug or an unpredictable risk. J Ethnopharmacol2009;126:18–30.

[4] Chen L, Wu H,WangM, Shi R. Comparative study of mucosa irritation ofcrude and processed Radix Phytolaccae. Zhongguo Zhong Yao Za Zhi2011;36:859.

[5] Cai B, Nagasawa T, Kadota S, Hattori M, Namba T, Kuraishi Y. Processingof nux vomica. VII. Antinociceptive effects of crude alkaloids from theprocessed and unprocessed seeds of Strychnos nux-vomica in mice.Biol Pharm Bull 1996;19:127.

[6] Zhan ZJ, Fan CQ, Ding J, Yue JM. Novel diterpenoids with potentinhibitory activity against endothelium cell HMEC and cytotoxicactivities from a well-known TCM plant Daphne genkwa. Bioorg MedChem 2005;13:645–55.

[7] Yang ZL, Wang YZ. Toxicity experiment on Sargassum, radix Knoxiae,radix Ransul and flos Genkwa against radix Glycyrrhizae in theantagonism of 18 Chinese drugs. Zhongguo Zhong Yao Za Zhi 1989;2:48–50.

[8] Che JH, KangBC, Kwon E, KimYS, Kim SH, You JR, et al. P27: thirteen-weekrepeated dose toxicity and genetic toxicity studies of processed genkwaflos extract. Exp Toxicol Pathol 2009;61:294.

[9] The State Pharmacopoeia Commission of PR China. Pharmacopoeia ofPR China. Beijing: Chemical Industry Press; 2010.

[10] Cai BC, Gong QF. Processing of Chinese medicinal herbs. Beijing: PeopleHealth Press; 2009.

[11] Shuib NH, Shaari K, Maulidiani AK, Kneer R, Zareen S, Raof SM, et al.Discrimination of young and mature leaves of Melicope ptelefolia using1H NMR and multivariate data analysis. Food Chem 2011;126:640–5.

[12] Toh DF, New LS, Koh HL, Chan ECY. Ultra-high performance liquidchromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) fortime-dependent profiling of raw and steamed Panax notoginseng.J Pharm Biomed Anal 2010;52:43–50.

[13] Li SL, Song JZ, Qiao CF, Zhou Y, Xu HX. UPLC–PDA–TOFMS based chemicalprofiling approach to rapidly evaluate chemical consistency betweentraditional and dispensing granule decoctions of traditional medicinecombinatorial formulae. J Pharm Biomed Anal 2010;52:468–78.

[14] PangNN, Bi KS, Yan BQ, ChenXH. RP-HPLC simultaneous determination ofseven flavonoids in Flos Genkwa. Chinese J Pharm Anal 2010;30:633–6.

[15] Wang MZ, Liu JS, Song LL, Xiang BR, An DK. Application of orthogonalfunction spectrophotometry to the determination of total diterpeneorthoesters in yuanhua (Daphne genkwa Sieb. et Zucc.) root injection.Yao Xue Xue Bao 1986;21:119–23.

[16] Wiklund S, Johansson E, Sjöström L, Mellerowicz EJ, Edlund U, ShockcorJP, et al. Visualization of GC/TOF-MS-based metabolomics data foridentification of biochemically interesting compounds using OPLS classmodels. Anal Chem 2008;80:115–22.

[17] Lin JH, Lin YL, Huang YJ, Wen KC, Chen RM, Ueng TH, et al. Isolation andcytotoxicity of the flavonids from Daphnis genkwae flos. J Food DrugAnal 2001;9:6–11.

[18] Zeng YM, Xiao J, Li X, Wang JH. Isolation and identification of flavanoidsfrom buds of Daphne genkwa Sieb.et Zucc.processed by rice vinegar.J Shenyang Pharm Univ 2009;5:353–6.

[19] Park BY, Min BS, Oh SR, Kim JH, Bae KH, Lee HK. Isolation of flavonoids,a biscoumarin and an amide from the flower buds of Daphne genkwaand the evaluation of their anti-complement activity. Phytother Res2006;20:610–3.

[20] Li LZ, Gao PY, Li FF, Huang XX, Peng Y, Song SJ. Research progress in thechemical constituents and pharmacological activities of Daphne genkwaSieb. et Zucc. J Shenyang Pharm Univ 2010;27:699–703.

[21] Park BY, Oh SR, Ahn KS, Kwon OK, Lee HK. (−)-Syringaresinol inhibitsproliferation of human promyelocytic HL-60 leukemia cells via G1arrest and apoptosis. Int Immunopharmacol 2008;8:967–73.

[22] Ruan Z, Chen RJ, Yu YY, Cao SW. Research progresses on chemicalconstituents of genus daphne genus and their bioactivities. Food Sci2009;30:249–58.

[23] Nikaido T, Ohmoto T, Sankawa U. Inhibitors of adenosine 3′, 5′-cyclicmonophosphate phosphodiesterase in Daphne genkwa Sieb. et Zucc.Chem Pharm Bull 1987;35:675.

[24] Noro T, Oda Y, Miyase T, Ueno A, Fukushima S. Inhibitors of xanthineoxidase from the flowers and buds of Daphne genkwa. Chem Pharm Bull1983;31:3984.

[25] Khong A, Forestieri R, Williams DE, Patrick BO, Olmstead A, Svinti V,et al. A Daphnane Diterpenoid isolated from Wikstroemia polyanthainduces an inflammatory response and modulates miRNA activity. PLoSOne 2012;7:e39621, http://dx.doi.org/10.1371/journal.pone.0039621.

[26] Li LZ, Gao PY, Peng Y, Wang LH, Yang JY, Wu CF, et al. Daphnane-typediterpenoids from the flower buds of Daphne genkwa. Helv Chim Acta2010;93:1172–9.

[27] Banerji A, Ray R. Aurantiamides: a new class of modified dipeptidesfrom Piper aurantiacum. Phytochemistry 1981;20:2217–20.

[28] Wang MT, Li YZ, Zhao TZ, Ji CR, FengWS, Liu YZ. A new lignanolide fromthe leaves of Daphne genkwa. Yao Xue Xue Bao 1990;25:866–8.

[29] Zheng WF, Shi F. Isolation and identification of a new dicoumarin fromthe roots of Daphne genkwa. Yao Xue Xue Bao 2004;39:990–2.

[30] Li YN, Yin LH, Xu LN, Peng JY. A simple and efficient protocol for large‐scale preparation of three flavonoids from the flower of Daphne genkwaby combination of macroporous resin and counter‐current chromatog-raphy. J Sep Sci 2010;33:2168–75.

[31] Zhang S, Li X, Zhang F, Yang P, Gao X, Song Q. Preparation of yuanhuacineand relative daphne diterpene esters from Daphne genkwa and structure–activity relationship of potent inhibitory activity against DNA topoisom-erase I. Bioorg Med Chem 2006;14:3888–95.

[32] Zeng YM, Zuo WJ, Sun MG, Meng H, Wang QH, Wang JH, et al. Threenew Daphne Diterpenes from the buds of Daphne genkwaSieb. etZucc.processed by rice vinegar. Helv Chim Acta 2009;92:1273–81.

[33] Smedsgaard J, Hansen ME, Frisvad JC. Classification of TerverticillatePenicillia by electrospray mass spectrometric profiling. Stud Mycol2004;49:243–51.

294 L. Geng et al. / Fitoterapia 84 (2013) 286–294

[34] Li LZ, Gao PY, Peng Y, Wang L, Song SJ. A novel daphnane-type diterpenefrom the flower bud ofDaphne genkwa. ChemNat Compd 2010;46:380–2.

[35] Kasal R, Lee KH, Huang HC. Genkwadaphnin, a potent antileukemicditerpene from Daphne genkwa. Phytochemistry 1981;20:2592–4.

[36] Li LZ, Gao PY, Peng Y, Wang LH, Song SJ. A novel daphnane-typediterpene from the flower bud of Daphne Genkwa. Chem Nat Compd2010;46:380–2.

[37] Xia SX, Li LZ, Li FF, Peng Y, Song SJ, Gao PY, et al. Two novel diterpenesisolated from the flower buds of Daphne genkwa. Acta Chim Sin 2011;69:2518–22.

[38] Wang HZ, Liu J. Determination of genkwanin and hydrogenkwanin of rawand processed Genkwa Flos. Zhongguo Zhong Yao Za Zhi 1989;14:24–6.

[39] Yuan ST, Zhang BX, Xia K. The effect of vinegar-processing on the contentof yuanhuacine by HPLC. Zhongguo Zhong Yao Za Zhi 1995;5:280–2.

[40] Yuan S, Zhang B, Xia K. HPLC analysis of mimic vinegar-processedyuanhuacine. Zhongguo Zhong Yao Za Zhi 1996;21:469–71.