Research Article Analysis of Phenolic Acids of Jerusalem ...downloads.hindawi.com/journals/tswj/2014/568043.pdf · Research Article Analysis of Phenolic Acids of Jerusalem Artichoke

Research ArticleAnalysis of Phenolic Acids of Jerusalem Artichoke(Helianthus tuberosus L) Responding to Salt-Stress by LiquidChromatographyTandem Mass Spectrometry

Fujia Chen1 Xiaohua Long1 Zhaopu Liu1 Hongbo Shao23 and Ling Liu1

1 Key Laboratory ofMarine Biology Jiangsu Province College of Resources andEnvironmental Sciences NanjingAgriculturalUniversityNanjing 210095 China

2 Key Laboratory of Coastal Biology amp Bioresources Utilization Yantai Institute of Coastal Zone ResearchChinese Academy of Sciences (CAS) Yantai 264003 China

3 Jiangsu Academy of Agricultural Sciences Nanjing 210014 China

Correspondence should be addressed to Hongbo Shao shaohongbochu126com and Ling Liu liulingnjaueducn

Received 24 May 2014 Accepted 6 July 2014 Published 5 August 2014

Academic Editor Marian Brestic

Copyright copy 2014 Fujia Chen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Plant phenolics can have applications in pharmaceutical and other industries To identify and quantify the phenolic compoundsin Helianthus tuberosus leaves qualitative analysis was performed by a reversed phase high-performance liquid chromatographycoupled with tandem mass spectrometry (HPLC-MSMS) and quantitative analysis by HPLC Ten chlorogenic acids (CGAs) wereidentified (3-o-caffeoylquinic acid two isomers of caffeoylquinic acid caffeic acid p-coumaroyl-quinic acid feruloylquinic acid34-dicaffeoyquinic acid 35-dicaffeoylquinic acid 15-dicaffeoylquinic acid and 45-dicaffeoylquinic acid) by comparing theirretention times UV-Vis absorption spectra and MSMS spectra with standards In addition four other phenolic compoundsincluding caffeoyl glucopyranose isorhamnetin glucoside kaempferol glucuronide and kaempferol-3-o-glucoside were tentativelyidentified in Helianthus tuberosus leaves for the first time The 3-o-caffeoylquinic acid (7752mgg DW) 45-dicaffeoylquinic acid(5633mgg DW) and 35-dicaffeoylquinic acid (4900mgg DW) were the major phenolic compounds in leaves of Helianthustuberosus cultivar NanYu in maturity The variations in phenolic concentrations and proportions in Helianthus tuberosus leaveswere influenced by genotype and plant growth stage Cultivar NanYu had the highest concentration of phenolic compounds inparticular 3-o-caffeoylquinic acid and 45-dicaffeoylquinic acid compared with the other genotypes (wild accession and QingYu)Considering various growth stages the concentration of total phenolics in cultivar NanYuwas higher at flowering stage (5270mggDW) than at budding and tuber swelling stages Cultivar NanYu of Helianthus tuberosus is a potential source of natural phenolicsthat may play an important role in the development of pharmaceuticals

1 Introduction

Helianthus tuberosus L (Jerusalem artichoke) Asteraceaefamily is a perennial herb originating from eastern NorthAmerica It has been introduced and cultivated widely inthe temperate areas for the edible tubers H tuberosus hastall stem large leaves bright yellow flowers resembling thoseof sunflowers and fleshy potato-like tubers As a source ofinulin the tubers have been used as a folk medicine for thetreatment of diabetes and rheumatism with a variety of phar-macological activities such as aperient cholagogue diureticspermatogenic stomachic and tonic [1] Additionally the

leaves of H tuberosus have been utilized as a folk medicinefor the treatment of bone fracture skin wounds swellingand pain [2 3] with antipyretic analgesic anti-inflammatoryand antispasmodic effects [4ndash6] Moreover the stalks andleaves of this plant were also found to possess antioxidantantimicrobial antifungal and anticancer activities [1 6 7]

The effective compounds in H tuberosus are coumarinsunsaturatedfatty acids polyacetylenic derivatives phenoliccompounds and sesquiterpenes [1] Recent studies haveshown that pharmacological characteristics of H tuberosuswere related to its phenolic compounds with antioxidantand radical-scavenging activity the main phenolic acids in

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 568043 8 pageshttpdxdoiorg1011552014568043

2 The Scientific World Journal

H tuberosus leaves were chlorogenic acids [6] Chlorogenicacids had inhibitory effects on carcinogenesis in the largeintestine liver and tongue and protective effects againstoxidative stress in vivo [8] More broadly phenolic acids arewidely distributed in plants as the secondary metabolites[9] some phenolic acids are allelochemicals used to controlbiological pests [10ndash12] plant pathogens [13] and weeds[14] The involvement of phenolics with plant protection andcommunication makes phenolics pivotal molecules in theresponses of plants to their ever-changing environment [15]

Previously it was demonstrated that the leaves of Htuberosus contained high concentration of phenolic com-pounds [5] Phenolics were separated and identified (such asferulic acids) from the tubers ofH tuberosus [16] However todate reports on analysis and identification of phenolic com-pounds from the leaves of H tuberosus are scarce and onlya few phenolics especially chlorogenic acid and isochloro-genic acids have been identified and qualitatively analysed[6]

Reversed phase high-performance liquid chromatogra-phy coupled to tandem mass spectrometry (HPLC-MSMS)has been extensively and successfully applied to the onlinestructure elucidation of phenolic compounds in foodstuffshaving advantages of high sensitivity speed and low sampleconsumption [17ndash24] In addition liquid chromatographycoupled to tandem mass spectrometry (LC-MSMS) tech-niques are useful for elucidating the structures of the activecompounds (eg nonvolatile phenolic compounds) and dis-tinguishing compounds with identicalmolecular weights [2325]

The objectives of the present work were to identify thephenolic compounds in H tuberosus leaves using HPLC-MSMS technique and to measure the concentration ofmain phenolics in H tuberosus leaves of different cultivarsat different sampling periods from budding stage to maturity(tuber swelling stage) using HPLC

2 Materials and Methods

21 Chemicals and Materials Gallic acid was obtained fromSinopharm Chemical Reagent Co Ltd (Shanghai China)and 3-o-caffeoylquinic acid was obtained from AladdinReagent Co Ltd (Shanghai China) Other standard sampleswere obtained from Yuanye Biological Technology Co Ltd(Shanghai China) All other analytical grade chemicals wereobtained from Shoude Experimental Equipment Co Ltd(Nanjing China)

The leaves of three H tuberosus cultivars (the wildaccession the southern cultivar NanYu [26] and QingYuoriginated from northern China) were collected fromDafengDistrict (Jiangsu China) in maturity at the end of October2011 Both cultivars NanYu and QingYu [27 28] are superiorvarieties in local areas which have obvious competitiveadvantages in yield quality saline-alkali tolerance and soon over the wild accession in Dafeng District The leaves ofcultivar NanYu were collected from August to October 2012at different growth stages including budding flowering andtuber swelling stages respectively

22 Extraction of Phenolic Compounds The air-dried (roomtemperature) andmilled [6] leaves (10 g) ofH tuberosuswererefluxedunder vacuumat 50∘Cusing 70 vv ethanol (EtOH)for three hours After evaporation under reduced pressurethe dry residue was redissolved in 25mL of methanol andused for colorimetric and chromatographic analyses ForHPLC analysis all samples were filtered through a 022120583mcellulose acetate filter (Millipore Corp Bedford MA USA)before injections

23 Measurement of Total Phenolic Concentration The totalphenolic concentration (TPC) was determined using theFolin-Ciocalteu reagent with gallic acid as standard [19 2930] The reaction mixture contained 05mL of test sample05mL of the Folin-Ciocalteu reagent freshly prepared in ourlaboratory 20mL of 10wv sodium carbonate solutionand 30mL of distilled water After 2 h of reaction underambient temperature in the dark the absorbance at 760 nmwas measured A calibration curve with equation 119910 =00029119909 + 00107 (1198772 = 09991) was constructed using gallicacid solutions in the range of 1470ndash294mgL Results wereexpressed in milligram gallic acid equivalents per gram ofdried sample

24 HPLC-MSMS Analysis HPLC-MSMS analysis of phe-nolics in H tuberosus extracts was performed using an Agi-lent 1200 series HPLC system (Agilent Technology Co LtdUSA) composed of a diode array detector and an Agilent6400 series triple quadrupole (QQQ) mass spectrometerequipped with an electrospray ionization (ESI) source Datawere collected and processed via a personal computer run-ning Agilent MassHunter workstation (Micromass Qualita-tive Analysis Version B0103 of Agilent Technology Co LtdUSA) A reverse-phase Eclipse XDB-C18 column (250mm times46mm 5120583m Agilent Technology Co Ltd USA) was usedfor separation The mobile phases consisted of methanol(A) and 10 vv formic acid aqueous solution (B) Gradientelution was started with 30 of A and ascended to 50 ofA in 45min The flow rate was kept at 08mLmin and thecolumn temperature was 30∘C Samples were filtered througha 022120583mfilter prior toHPLC injectionThe injection volumewas 5 120583L UV-Vis absorption spectra were recorded onlinefrom 200 to 600 nm during HPLC analysis Phenolics weredetected at the wavelength of 330 nm

Mass and MSMS spectra were achieved by electrosprayionization (ESI) in negative modes The voltages used were4000V for the source capillary and 10V for the extractioncone the source temperature was 150∘C and the desolvationtemperature was 350∘C The electrospray probe flow wasadjusted to 70mLmin The ESI-MS and ESI-MSMS spectrawere obtained by scanning from200 to 1200119898119911TheMSMSfragmentations were carried out with 10ndash50 energy

25 Identification and Quantification of Phenolic CompoundsThe phenolic compounds inH tuberosus leaves extracts wereidentified by comparing their UV-Vis absorption spectramatching their molecular ions (119898119911) obtained by ESI-MSand ESI-MSMS chromatographic characteristics with the

The Scientific World Journal 3

Table 1 Identification of phenolic compounds in H tuberosus leaves by HPLC-MSMS

Peaks number 119905119877

(min) UV 120582max (nm) MW MSminus MSMS Identification1 358 2460 2630 360 3594 2973 2816 2309 1352 Unknown2 436 2143 3234 354 3534 1911 1791 1611 1351 851 Caffeoylquinic acid (isomer of chlorogenic acid)3 578 2144 3270 354 3534 1911 1270 850 3-o-Caffeoylquinic acid (3-CQA)4 607 2379 3244 354 3534 1912 127 931 850 Caffeoylquinic acid5 857 3230 180 1791 1360 1079 Caffeic acid (CA)6 919 2391 3115 338 3373 1911 1730 930 p-Coumaroyl-quinic acid7 1012 2414 3258 368 3673 1910 1731 1340 930 Feruloylquinic acid8 1809 2438 3270 516 5155 3543 1911 1731 1791 1350 Dicaffeoylquinic acid (34-DiCQA)9 1903 2426 3270 516 5155 3541 1912 Dicaffeoylquinic acid (35-DiCQA)10 2004 327 342 3413 1791 1611 Caffeoyl glucopyranose11 2043 243 3294 516 5155 3541 1911 1791 1731 1351 Dicaffeoylquinic acid (15-DiCQA)12 2297 2533 3497 478 4774 3153 3001 2709 1802 Isorhamnetin glucoside13 2657 3270 516 5155 1911 1791 1731 1350 Dicaffeoylquinic acid (45-DiCQA)14 3020 2639 3413 462 4614 3152 2848 1610 1327 851 Kaempferol glucuronide15 3058 2630 3330 448 4474 2854 1908 1531 969 Kaempferol-3-o-glucoside

literature data reported [5 6] or with available referencestandards The external standard method was used forthe quantification of main phenolic acids Concentrationsof 3-o-caffeoylquinic acid caffeic acid 34-dicaffeoylquinicacid 35-dicaffeoylquinic acid 15-dicaffeoylquinic acid 45-dicaffeoylquinic acid and so on were calculated with theregression equations from the standard curves Concentra-tions were expressed as mgg dried weight sample (DW)

26 Statistical Analysis The TPC and concentration of phe-nolic compounds in H tuberosus leaves of different cultivarsand different growth stages were sources of variation Thesedata were reported as mean plusmn SD from triplicate determi-nations Statistical analysis was performed with analysis ofvariance (ANOVA) and statistical significance specified at119875 le 005

3 Results

31 Identification of the Chromatographic Peaks The exam-ination of the chromatograms in a full-scan mode revealedthe presence of several compounds which were positivelyidentified by comparison with available standards Figure 1showed the HPLC chromatogram of ethanol extract from Htuberosus leaves There were 15 phenolic peaks separated inextracts using the reversed phase C-18 column As shownin Table 1 peak identification was performed by comparingretention times (119905

119877) UV-Vis spectra mass and MSMS

spectra with those of reference standards or literature dataClassically chlorogenic acids (CGAs) are a family of

esters formed between quinic acid and certain trans-cinnamic acids most commonly caffeic p-coumaric andferulic acids [31] Fragment ions 119898119911 191 and 119898119911 179corresponding to deprotonated quinic acid and caffeic acidfragments were characteristics of the MSMS spectra ofquinic or caffeic acid derivatives [32]

17515125

107505025

0

0 2 4 6 8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

42

44

Response units versus acquisition time (min)

12

34

5 6 7 8

9 10

11

12

1314

15

times102

DAD1-A sig = 330 16 1d

Figure 1 HPLC chromatogram of the ethanol extract of Htuberosus leaves detected at 330 nm Peak numbers were consistentwith those shown in Table 1

Among all the peaks in the chromatogram (Figure 1)peak 9 was quite prominent indicating a predominant phe-nolic compound in H tuberosus leaves This peak presentedspectral characteristics of the dicaffeoylquinic acid [5 6] withUV 120582max at 2426 and 3270 nm and 119905

119877of 1903min The

ESI-MSMS spectra showed [M-H]minus at 119898119911 5155 fragmention [M-C

9H6O3]minus at 119898119911 3541 and fragment ion [M-

H-2C9H6O3]minus at 119898119911 1912 (Figures 2(c) 2(d) 2(e) and

2(f)) Compared with the standard this compound wasunambiguously identified as 35-dicaffeoylquinic acid Peaks8 11 and 13 had the same spectral characteristics as peak9 (Table 1) with UV 120582max at 2438 and 3270 nm (peak 8)2430 and 3294 nm (peak 11) and 3270 nm (peak 13) Basedon the MSMS analyses the caffeoylquinic acid 119898119911 3541ion further fragmented to form characteristic 119898119911 1731 [M-H-2C

9H6O3-H2O]minus 1350 [M-C

7H10O5-C9H6O3-COOH]minus

and 1790 [M-H-C9H6O3-C7H10O5]minus ions [33] Compared

with the standard these compounds were identified as34-dicaffeoylquinic acid 15-dicaffeoylquinic acid and 45-dicaffeoylquinic acid respectively [34]

The peaks 2 3 and 4 were identified as three isomers ofcaffeoylquinic acids (chlorogenic acid) based on the detailedfragmentation UV absorption and also [35] Previously


times101

4

3

2

1

0

50

60

70

80

90

100

110

120

130

140

150

160

10790

13600

Counts versus mass-to-charge (mz)

(a)

times103

5

4

3

2

1

0

60

80

100

120

140

160

180

200

220

240

260

280

300

320

850012700

19110


(b)

times103

17515125

107505025

0

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

13500

17310

17910

19110

35430


(c)

times103

3

25

2

15

1

05

0

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

450

19120

35410


(d)

times103

1614121

080604020

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

13510

17310

17910

19110

35410


(e)

times103

14121

080604020

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

13500

17310

17910

19110


(f)

Figure 2 HPLC-MS-MS spectra of phenolic acids (119898119911 179 of caffeic acid (a) 119898119911 353 of 3-o-caffeoylquinic acid (b) 119898119911 515 of 34-dicaffeoylquinic acid (c)119898119911 515 of 35-dicaffeoylquinic acid (d)119898119911 515 of 15-dicaffeoylquinic acid (e) and119898119911 515 of 45-dicaffeoylquinicacid (f))

the presence of cis derivatives of chlorogenic acids wasreported in coffee leaves Rudbeckia hirta Carlina acaulisH tuberosus Symphyotrichum novae-angliae mate tea (Ilexparaguariensis) and leaves of other Asteraceae plants [31 36ndash38] In a negative ion ESI mode the deprotonated molecule[M-H]minus at 119898119911 353 and fragment ion [M-H-C

9H6O3]minus

formed from deprotonated quinic acid at 119898119911 191 wereobserved (Figure 2(b)) Fragment ions 119898119911 85 and 119898119911 93characteristic of the quinic acid moiety of monoacyl anddiacyl chlorogenic acids defined the parent ions of putativechlorogenic acids [36] Other fragment ions with differentenergies such as the caffeic acid unit (119898119911 1791) and [M-H-C7H12O6]minus (119898119911 1611) were used to distinguish the three

isomers [38] Compared with the standard peak 3 wasidentified as 3-o-caffeoylquinic acid

The peak 5 was pseudomolecular ion [M-H]minus at 1198981199111791 and fragment ions at 119898119911 1360 [M-COO]minus and 1079[M-COO-CO]minus (Figure 2(a)) which were the typical masses

of caffeic acid in the negative mode [39] Fragment ions at119898119911 191 and 179 were also observed in ESI-MSMSminus spectraof peaks 6 7 and 10 (Table 1) indicating that they werederivatives of quinic acid or caffeic acid Peak 6 was eluted at919min (Figure 1) with the molecular ion at 119898119911 3373 [M-H]minus and themain fragment ions at119898119911 1911 [quinic acid-H]minus(UV 120582max at 2391 nm) and 1730 [quinic acid-H-H

2O]minus (UV

120582max at 3115 nm) this peak was identified as p-coumaroyl-quinic acid [31 36 40] Peak 7 (Table 1) was identified asferuloylquinic acid ([M-H]minus at119898119911 367 andUV 120582max at 2414and 3258 nm) [5 32]

The MSMS spectrum of peak 10 (Table 1) suggestedcaffeoyl glucopyranose that possesses both molecular ion119898119911 3413 and fragmental ion 119898119911 1791 formed by lossof one dehydrated molecule of glucose (Glc) [M-H-(Glc-H2O)]minus 1611 [(Glc-H

2O)-H]minus [33 41] To our knowledge

caffeoyl glucopyranose has not been previously reported inH tuberosus leaves


Table 2 Concentration of total phenolics and phenolic compoundsin H tuberosus leaves (cv NanYu)

Phenolic compounds Concentrationa (mgg dry weight)Caffeoylquinic acid (peak 2)b 0063 plusmn 0008d

3-o-Caffeoylquinic acid 7752 plusmn 2872b

Caffeoylquinic acid (peak 4)b 0538 plusmn 0081d

Caffeic acid 0098 plusmn 0052d

p-Coumaroyl-quinic acid 0153 plusmn 0061d

Feruloylquinic acid 0527 plusmn 0199d

34-Dicaffeoylquinic acid 0618 plusmn 0215d

35-Dicaffeoylquinic acid 4900 plusmn 1492c

Caffeoyl glucopyranosec 0001 plusmn 0319d


Isorhamnetin glucosided 0348 plusmn 0057d

45-Dicaffeoylquinic acid 5633 plusmn 2990bc

Kaempferol glucuronided 0186 plusmn 0034d

Kaempferol-3-o-glucosided 1020 plusmn 0379d

Total phenolicse 23570Total phenolicsf 30159 plusmn 4410aaValues are expressed as mean plusmn SD of triplicate measurements the meansin a column followed by the same letters represent values that are notsignificantly different according to Duncanrsquos test (119875 le 005) bquantified as3-o-caffeoylquinic acid cquantified as caffeic acid dquantified as glucosideesum of the individual phenolic compounds and fquantified as gallic acidequivalents

The MSMS analysis of peaks 12 14 and 15 (Table 1)showed fragment ions at119898119911 315 301 and 285 correspondingto methyl quercetin or methoxy kaempferol quercetin agly-cone and kaempferol suggesting that they were kaempferoland quercetin glycoside derivatives [33] Peak 12 had amolecular ion [M-H]minus at 119898119911 477 and fragment ions at119898119911 315 [M-H-(Glc-H

2O)]minus 3001 [M-H-(Glc-H

2O)-CH

3]minus

and 2709 [M-H-(Glc-H2O)-CH

3-CO]minus which proved to be

isorhamnetin glucoside [23 33] Peaks 14 and 15 were pos-sibly kaempferol glucuronide and kaempferol-3-o-glucosidewhich have similar fragment ion 285 [kaempferol-H]minus anddifferent parent ions 461 [M-H]minus and 447 [M-H]minus [2332 33 42ndash44] These kaempferol and quercetin glycosidederivatives (peaks 12 14 and 15) are also the first everreports in Helianthus tuberosus leaves Their exact structuresneed further confirmation and additional NMR data will berequired

Phenolics in peak 1 (Table 1) in the HPLC chromatogramwere not identified

32 Quantification of Phenolics Concentration of phenoliccompounds in H tuberosus leaves of cultivar NanYuwas determined by the HPLC method whereas theconcentration of total phenolics was calculated as the sum ofthe individual phenolic compounds and was also estimatedby using the Folin-Ciocalteu method (Table 2) The 3-o-caffeoylquinic acid (7752mggDW) 45-dicaffeoylquinicacid (5633mggDW) and 35-dicaffeoylquinic acid(4900mggDW) were the major phenolic compounds in Htuberosus leaves and their concentrations accounted for 33

24 and 21 of the total phenolics respectively Among allthe quantified phenolics chlorogenic acids (CGAs) including3-o-caffeoylquinic acid caffeoylquinic acids (peaks 2 and4) caffeic acid 119901-coumaroyl-quinic acid feruloylquinicacid 34-dicaffeoylquinic acid 35-dicaffeoylquinic acid 15-dicaffeoylquinic acid and 45-dicaffeoylquinic acid contri-buted to the total of 22015mggDW (93 of the totalphenolics)

As shown in Table 2 the content of total phenolicscalculated as the sum of the individual phenolic compoundswas 23570mggDW whereas the value obtained by theFolin-Ciocalteumethodwas 30159mggDWThe substantialdifference between the two values was likely due to the inter-ference of other reducing substances in phenolic extractsleading to overestimation of total phenolic contents in theFolin-Ciocalteu colorimetric analysis [45 46]

Concentration of six main phenolic compounds (peaks3 5 8 9 11 and 13 in Table 1) in leaves of different Htuberosus cultivars sampled at different periods frombuddingto tuber swelling stages was presented in Figure 3 Amongthe tested genotypes ofH tuberosus (Figure 3(a)) NanYu hadthe highest concentration of phenolic compounds in leaves(around 7-fold higher than the wild accession and 3-foldhigher than QingYu)

Caffeic acid was detected in low concentration in allgenotypes whereas concentrations of 3-o-caffeoylquinic acidand 45-dicaffeoylquinic acid were considerably higher in allcultivars investigated (Figure 3(a))

Concentration of total phenolics in leaves of cultivarNanYu was higher at flowering stage (5270mggDW) thanbudding and tuber swelling stages (Figure 3(b)) frombud-ding flowering to tuber swelling stages

4 Discussion

Phenolic acids are secondary metabolites that are commonlyfound in plant-derived foods They have attracted consid-erable interest due to their many potential health benefitswhich are powerful antioxidants and have been reported todemonstrate antibacterial antiviral anticarcinogenic anti-inflammatory and vasodilatory actions [47] As allelochemi-cals the phenolic acids might play an important role in plantdefense against pathogens [48] pests and weeds [14 49]Themechanism of a phenolic with defense communication andprotection roles was considered as a pivotal molecule inthe responses of plants to their ever-changing environment[15]

The variation in concentration of phenolic acids reportedhere and in the literature was probably due to the isomeri-sation of chlorogenic acids (CGAs) [50] and different Htuberosus parts considered (tubers leaves or whole plants)[6 9 31] In addition the phenolic profiles of Helianthustuberosus leaves of cultivar NanYu (Dafeng District JiangsuChina) were different from previous studies in which themajor phenolic compounds were 3-o-caffeoylquinic acid and15-dicaffeoylquinic acid [6] inH tuberosus leaves fromYulinDistrict (Shannxi China) probably due to different cultivarsof H tuberosus different sampling periods or differentorigins


35302520151050

TPC

3-C

QA CA

34

-DiC

QA

35

-DiC

QA

15

-DiC

QA

45

-DiC

QA

Con

cent

ratio

n

cb

a

c998400b998400

a998400

a998400998400 a998400998400 a998400998400 a1 a1 a1 C BA

C998400 A998400B998400 A998400

C998400998400 B998400998400A998400998400

Wild accessionQingYuNanYu

(mg

g)

(a)

TPC

3-C

QA CA

34

-DiC

QA

35

-DiC

QA

15

-DiC

QA

45

-DiC

QA

Con

cent

ratio

n

cb

a

c998400a998400

b998400b998400998400 a998400998400 b998400998400 b1

a1 b1 CBA

C998400A998400

B998400 c998400998400

A998400998400

B998400998400

76543210

Flowering stageBudding stageTuber swelling stage

(mg

g)

(b)Figure 3 Concentration of phenolics inH tuberosus leaves of different genotypes (a) in 2011 and growth stages of cultivar NanYu (floweringstage budding and tuber swelling stages) and (b) from August to October in 2012 Concentrations were in mgg dry weight of leaves Valuesare expressed as mean plusmn SD of triplicate measurements columns with the same letters are not significantly different according to Duncanrsquostest (119875 le 005)

However this was to be expected as there were so manyenvironmental factors such as pedoclimatic (soil type sunexposure and rainfall) and agronomic factors (growth ingreenhouses or fields biological culture hydroponic culturefruit yield per tree etc) that could affect phenolics concen-tration in plants [51] A degree of ripeness also considerablyaffected the concentrations and proportions of various phe-nolics [52] Thus cultivar NaYu can be a potential source ofnatural phenolics which could have multiple functions (egpharmaceuticals) and could play an important role in plantinteractions and ecosystem patterning [14]

5 Conclusions

Reversed phase high-performance liquid chromatographycoupled with tandem mass spectrometry (HPLC-MSMS)was successfully employed in the qualitative analysis ofphenolic compounds in H tuberosus leaves Ten chlorogenicacids (CGAs) were identified (3-o-caffeoylquinic acid twoisomers of caffeoylquinic acids caffeic acid p-coumaroyl-quinic acid feruloylquinic acid 34-dicaffeoylquinic acid35-dicaffeoylquinic acid 15-dicaffeoylquinic acid and 45-dicaffeoylquinic acid) and four others (caffeoyl glucopyra-nose isorhamnetin glucoside methoxy kaempferol gluco-side and kaempferol-3-o-glucoside) were tentatively identi-fied for the first time Quantitative analysis of phenolics indi-cated that 3-o-caffeoylquinic acid 45-dicaffeoylquinic acidand 35-dicaffeoylquinic acid were the three major phenoliccompounds in H tuberosus leaves The variation in phenolicconcentrations and proportions in H tuberosus leaves wascharacterised in different genotypes and at different samplingperiods from budding to tuber swelling stages H tuberosuscultivar NaYu had the highest concentration of total phe-nolics and might be a potential source of natural phenolicswhich could play an important role in the development ofpharmaceuticals

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publishing of this paper

Authorsrsquo Contribution

Fujia Chen and Xiaohua Long contributed to the work of thepaper equally

Acknowledgments

The authors are grateful for the financial support of JiangsuAgricultural Science and Technology Independent Inno-vation Fund Project (no CX(12)1005-6) National NaturalScience Foundation of China (no 31201692 41171216) theNational Key Projects of Scientific and Technical SupportPrograms funded by the Ministry of Science and Tech-nology of China (no 2011BAD13B09) the Ministry of Sci-ence and Technology of Jiangsu Province (no BE2011368)Fundamental Research Funds for Central Universities (noY0201100249) the CASSAFEA International PartnershipProgram for Creative Research Teams Yantai Double-hundred High-end Talent Plan (XY-003-02) and the Projectof a Special Fund for Public Welfare Industrial (Agriculture)Research of China (no 200903001-5)

References

[1] L Pan M R Sinden A H Kennedy et al ldquoBioactiveconstituents of Helianthus tuberosus (Jerusalem artichoke)rdquoPhytochemistry Letters vol 2 no 1 pp 15ndash18 2009

[2] H Baba Y Yaoita and M Kikuchi ldquoSesquiterpenoids from theleaves of Helianthus tuberosus Lrdquo Journal of Tohoku Pharma-ceutical University vol 52 pp 21ndash25 2005

[3] M Talipova ldquoLipids of Helianthus tuberosusrdquo Chemistry ofNatural Compounds vol 37 no 3 pp 213ndash215 2001

[4] R R Stange Jr S L Midland G J Holmes J J Sims and RT Mayer ldquoConstituents from the periderm and outer cortexof Ipomoea batatas with antifungal activity against Rhizopusstoloniferrdquo Postharvest Biology and Technology vol 23 no 2 pp85ndash92 2001

[5] X YuaxM Gao KWang H Xiao C Tan and Y Du ldquoAnalysisof chlorogenic acids in Helianthus tuberosus Linn leaves usinghigh performance liquid chromatography-mass spectrometryrdquo


Chinese Journal of Chromatography vol 26 no 3 pp 335ndash3382008

[6] X Yuan M Gao H Xiao C Tan and Y Du ldquoFree radicalscavenging activities and bioactive substances of Jerusalemartichoke (Helianthus tuberosus L) leavesrdquo Food Chemistry vol133 no 1 pp 10ndash14 2012

[7] M S Ahmed F S El-Sakhawy S N Soliman and D MR Abou-Hussein ldquoPhytochemical and biological study ofHelianthus tuberosus Lrdquo Egyptian Journal of Biomedical Sciencevol 18 pp 134ndash147 2005

[8] U Jin J Lee S Kang et al ldquoA phenolic compound 5-caffeoylquinic acid (chlorogenic acid) is a new type and strongmatrix metalloproteinase-9 inhibitor isolation and identifica-tion from methanol extract of Euonymus alatusrdquo Life Sciencesvol 77 no 22 pp 2760ndash2769 2005

[9] P Mattila and J Hellstrom ldquoPhenolic acids in potatoes vegeta-bles and some of their productsrdquo Journal of Food Compositionand Analysis vol 20 no 3-4 pp 152ndash160 2007

[10] M Friedman ldquoChemistry biochemistry and dietary role ofpotato polyphenols A reviewrdquo Journal of Agricultural and FoodChemistry vol 45 no 5 pp 1523ndash1540 1997

[11] G C Percival M S Karim and G R Dixon ldquoPathogenresistance in aerial tubers of potato cultivarsrdquo Plant Pathologyvol 48 no 6 pp 768ndash776 1999

[12] S L Sinden L L Sanford W W Cantelo and K L DeahlldquoBioassays of segregating plantsmdasha strategy for studying chem-ical defensesrdquo Journal of Chemical Ecology vol 14 no 10 pp1941ndash1950 1988

[13] A Wen P Delaquis K Stanich and P Toivonen ldquoAntilisterialactivity of selected phenolic acidsrdquo Food Microbiology vol 20no 3 pp 305ndash311 2003

[14] F Tesio L A Weston and A Ferrero ldquoAllelochemicals identi-fied from Jerusalem artichoke (Helianthus tuberosus L) residuesand their potential inhibitory activity in the field and labora-toryrdquo Scientia Horticulturae vol 129 no 3 pp 361ndash368 2011

[15] F Ferreres R Figueiredo S Bettencourt et al ldquoIdentificationof phenolic compounds in isolated vacuoles of the medicinalplant Catharanthus roseus and their interaction with vacuolarclass III peroxidase an H

2

O2

affairrdquo Journal of ExperimentalBotany vol 62 no 8 pp 2841ndash2854 2011

[16] M Tchone G Barwald G Annemuller and L G Fleis-cher ldquoSeparation and identification of phenolic compoundsin Jerusalem artichoke (Helianthus tuberosus L)rdquo Sciences desAliments vol 26 no 5 pp 394ndash408 2006

[17] C Alcalde-Eon G Saavedra S De Pascual-Teresa and J CRivas-Gonzalo ldquoIdentification of anthocyanins of pinta boca(Solanum stenotomum) tubersrdquo Food Chemistry vol 86 no 3pp 441ndash448 2004

[18] Z Charrouf M Hilali O Jauregui M Soufiaoui and D Guil-laume ldquoSeparation and characterization of phenolic com-pounds in argan fruit pulp using liquid chromatography-negative electrospray ionization tandem mass spectroscopyrdquoFood Chemistry vol 100 no 4 pp 1398ndash1401 2007

[19] Z Fang M Zhang and LWang ldquoHPLC-DAD-ESIMS analysisof phenolic compounds in bayberries (Myrica rubra Sieb etZucc)rdquo Food Chemistry vol 100 no 2 pp 845ndash852 2007

[20] L Longo and G Vasapollo ldquoExtraction and identification ofanthocyanins from Smilax aspera L berriesrdquo Food Chemistryvol 94 no 2 pp 226ndash231 2006

[21] R Pedreschi and L Cisneros-Zevallos ldquoPhenolic profiles ofAndean purple corn (Zea mays L)rdquo Food Chemistry vol 100no 3 pp 956ndash963 2007

[22] A P Rauter A Martins C Borges et al ldquoLiquid chromato-graphy-diode array detection-electrospray ionisation massspectrometrynuclear magnetic resonance analyses of the anti-hyperglycemic flavonoid extract of Genista tenera structureelucidation of a flavonoid-C-glycosiderdquo Journal of Chromatog-raphy A vol 1089 no 1-2 pp 59ndash64 2005

[23] N P Seeram R Lee H S Scheuller and D Heber ldquoIden-tification of phenolic compounds in strawberries by liquidchromatography electrospray ionization mass spectroscopyrdquoFood Chemistry vol 97 no 1 pp 1ndash11 2006

[24] Y Zu C Li Y Fu and C Zhao ldquoSimultaneous determinationof catechin rutin quercetin kaempferol and isorhamnetinin the extract of sea buckthorn (Hippophae rhamnoides L)leaves by RP-HPLC with DADrdquo Journal of Pharmaceutical andBiomedical Analysis vol 41 no 3 pp 714ndash719 2006

[25] P Hu Q Liang G Luo Z Zhao and Z Jiang ldquoMulti-component HPLC fingerprinting of Radix SalviaeMiltiorrhizaeand its LC-MS-MS identificationrdquo Chemical and Pharmaceuti-cal Bulletin vol 53 no 6 pp 677ndash683 2005

[26] X LONG J CHI L LIU Q LI and Z LIU ldquoEffect of seawa-ter stress on p hysiological and biochemical responses offiveJerusalem a rtichoke ecotypesrdquo Pedosphere vol 19 no 2 pp208ndash216 2009

[27] X H Long J Tian Q W Zhong Z R Huang L Li and ZP Liu ldquoStudy the cultivars comparison ofHelianthus TuberosusL and planting technology of low cost and high quality in non-infield of Qinghai and Xinjiang ProvincesrdquoChinese AgriculturalScience Bulletin vol 26 no 13 pp 354ndash358 2010

[28] Z Huang X Long LWang et al ldquoGrowth photosynthesis andH+-ATPase activity in two Jerusalem artichoke varieties underNaCl-induced stressrdquo Process Biochemistry vol 47 no 4 pp591ndash596 2012

[29] K Tawaha F Q Alali M Gharaibeh M Mohammad and TEl-Elimat ldquoAntioxidant activity and total phenolic content ofselected Jordanian plant speciesrdquo Food Chemistry vol 104 no4 pp 1372ndash1378 2007

[30] K Zhou and L Yu ldquoTotal phenolic contents and antioxidantproperties of commonly consumed vegetables grown in Col-oradordquo LWTmdashFood Science and Technology vol 39 no 10 pp1155ndash1162 2006

[31] R Jaiswal S Deshpande and N Kuhnert ldquoProfling the chloro-genic acids of Rudbeckia hirta Helianthus tuberosus Carlinaacaulis and symphyotrichum novae-angliae leavesby LC-MS119899rdquoPhytochemical Analysis vol 22 no 5 pp 432ndash441 2011

[32] L Bravo L Goya and E Lecumberri ldquoLCMS characterizationof phenolic constituents of mate (Ilex paraguariensis St Hil)and its antioxidant activity compared to commonly consumedbeveragesrdquo Food Research International vol 40 no 3 pp 393ndash405 2007

[33] X Wang W Sun H Sun et al ldquoAnalysis of the constituentsin the rat plasma after oral administration of Yin Chen HaoTang by UPLCQ-TOF-MSMSrdquo Journal of Pharmaceutical andBiomedical Analysis vol 46 no 3 pp 477ndash490 2008

[34] M Krizman D Baricevic and M Prosek ldquoDetermination ofphenolic compounds in fennel by HPLC and HPLCMS using amonolithic reversed-phase columnrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 43 pp 481ndash485 2007

[35] A Tolonen T Joustamo S Mattlla T Kamarainen and JJalonen ldquoIdentification of isomeric dicaffeoylquinic acids fromEleutheracoccus senticosus using HPLC-ESITOFMS and H-NMR methodsrdquo Phytochemical Analysis vol 13 no 6 pp 316ndash328 2002


[36] MNCliffordW Zheng andNKuhnert ldquoProfiling the chloro-genic acids of aster byHPLC-MSnrdquo Phytochemical Analysis vol17 no 6 pp 384ndash393 2006

[37] M N Clifford J Kirkpatrick N Kuhnert H Roozendaal andP R Salgado ldquoLC-MS119899 analysis of the cis isomers of chlorogenicacidsrdquo Food Chemistry vol 106 no 1 pp 379ndash385 2008

[38] R Jaiswal J Kiprotich and N Kuhnert ldquoDetermination ofthe hydroxycinnamate profile of 12 members of the Asteraceaefamilyrdquo Phytochemistry vol 72 no 8 pp 781ndash790 2011

[39] J Pan and Y Cheng ldquoIdentification and analysis of absorbedand metabolic components in rat plasma after oral adminis-tration of ldquoShuangdanrdquo granule by HPLC-DAD-ESI-MSMSrdquoJournal of Pharmaceutical and Biomedical Analysis vol 42 no5 pp 565ndash572 2006

[40] C Tian X Xu L Liao J Zhang J Liu and S Zhou ldquoSeparationand identification of chlorogenic acid and related impuritiesby high performance liquid chromatography-tandem massspectrometryrdquo Chinese Journal of Chromatography vol 25 no4 pp 496ndash500 2007

[41] L Zhang C Fan X Zhang Z Yin andW Ye ldquoA new steroidalglycoside from Lygodium japonicumrdquo Journal of China Pharma-ceutical University vol 37 no 6 pp 491ndash493 2006

[42] K Ablajan Z Abliz X Shang J He R Zhang and J ShildquoStructural characterization of flavonol 37-di-O-glycosides anddetermination of the glycosylation position by using negativeion electrospray ionization tandemmass spectrometryrdquo Journalof Mass Spectrometry vol 41 no 3 pp 352ndash360 2006

[43] P Kachlicki J Einhorn D Muth L Kerhoas and M StobieckildquoEvaluation of glycosylation and malonylation patterns inflavonoid glycosides during LCMSMS metabolite profilingrdquoJournal of Mass Spectrometry vol 43 no 5 pp 572ndash586 2008

[44] F Sanchez-Rabaneda O Jauregui I Casals C Andres-Lacueva M Izquierdo-Pulido and R M Lamuela-RaventosldquoLiquid chromatographicelectrospray ionization tandemmassspectrometric study of the phenolic composition of cocoa(Theobroma cacao)rdquo Journal of Mass Spectrometry vol 38 no1 pp 35ndash42 2003

[45] Z He andW Xia ldquoAnalysis of phenolic compounds in Chineseolive (Canarium album L) fruit by RPHPLC-DAD-ESI-MSrdquoFood Chemistry vol 105 no 3 pp 1307ndash1311 2007

[46] A Schieber P Keller and R Carle ldquoDetermination of phenolicacids and flavonoids of apple and pear by high-performanceliquid chromatographyrdquo Journal of Chromatography A vol 910no 2 pp 265ndash273 2001

[47] G G Duthie S J Duthie and J A M Kyle ldquoPlant polyphenolsin cancer and heart disease implications as nutritional antiox-idantsrdquo Nutrition Research Reviews vol 13 no 1 pp 79ndash1062000

[48] J H Doughari I S Human S Bennade and P A NdakidemildquoPhytochemicals as chemotherapeutic agents and antioxidantspossible solution to the control of antibiotic resistant verocyto-toxin producing bacteriardquo Journal of Medicinal Plants Researchvol 3 no 11 pp 839ndash848 2009

[49] C H Lu X G Liu J Xu et al ldquoEnhanced exudation of DIM-BOA and MBOA by wheat seedlings alone and in proximity towild oat (Avena fatua) and flixweed (Descurainia sophia)rdquoWeedScience vol 60 no 3 pp 360ndash365 2012

[50] K Schrader A Kiehne U H Engelhardt and H G MaierldquoDetermination of chlorogenic acids with lactones in roastedcoffeerdquo Journal of the Science of Food and Agriculture vol 71no 3 pp 392ndash398 1996

[51] C Manach A Scalbert C Morand C Remesy and L JimenezldquoPolyphenols food sources and bioavailabilityrdquo The AmericanJournal of Clinical Nutrition vol 79 no 5 pp 727ndash747 2004

[52] X Duan G Wu and Y Jiang ldquoEvaluation of the antioxidantproperties of litchi fruit phenolics in relation to pericarp brown-ing preventionrdquoMolecules vol 12 no 4 pp 759ndash771 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


H tuberosus leaves were chlorogenic acids [6] Chlorogenicacids had inhibitory effects on carcinogenesis in the largeintestine liver and tongue and protective effects againstoxidative stress in vivo [8] More broadly phenolic acids arewidely distributed in plants as the secondary metabolites[9] some phenolic acids are allelochemicals used to controlbiological pests [10ndash12] plant pathogens [13] and weeds[14] The involvement of phenolics with plant protection andcommunication makes phenolics pivotal molecules in theresponses of plants to their ever-changing environment [15]

Previously it was demonstrated that the leaves of Htuberosus contained high concentration of phenolic com-pounds [5] Phenolics were separated and identified (such asferulic acids) from the tubers ofH tuberosus [16] However todate reports on analysis and identification of phenolic com-pounds from the leaves of H tuberosus are scarce and onlya few phenolics especially chlorogenic acid and isochloro-genic acids have been identified and qualitatively analysed[6]

Reversed phase high-performance liquid chromatogra-phy coupled to tandem mass spectrometry (HPLC-MSMS)has been extensively and successfully applied to the onlinestructure elucidation of phenolic compounds in foodstuffshaving advantages of high sensitivity speed and low sampleconsumption [17ndash24] In addition liquid chromatographycoupled to tandem mass spectrometry (LC-MSMS) tech-niques are useful for elucidating the structures of the activecompounds (eg nonvolatile phenolic compounds) and dis-tinguishing compounds with identicalmolecular weights [2325]

The objectives of the present work were to identify thephenolic compounds in H tuberosus leaves using HPLC-MSMS technique and to measure the concentration ofmain phenolics in H tuberosus leaves of different cultivarsat different sampling periods from budding stage to maturity(tuber swelling stage) using HPLC

2 Materials and Methods

21 Chemicals and Materials Gallic acid was obtained fromSinopharm Chemical Reagent Co Ltd (Shanghai China)and 3-o-caffeoylquinic acid was obtained from AladdinReagent Co Ltd (Shanghai China) Other standard sampleswere obtained from Yuanye Biological Technology Co Ltd(Shanghai China) All other analytical grade chemicals wereobtained from Shoude Experimental Equipment Co Ltd(Nanjing China)

The leaves of three H tuberosus cultivars (the wildaccession the southern cultivar NanYu [26] and QingYuoriginated from northern China) were collected fromDafengDistrict (Jiangsu China) in maturity at the end of October2011 Both cultivars NanYu and QingYu [27 28] are superiorvarieties in local areas which have obvious competitiveadvantages in yield quality saline-alkali tolerance and soon over the wild accession in Dafeng District The leaves ofcultivar NanYu were collected from August to October 2012at different growth stages including budding flowering andtuber swelling stages respectively

22 Extraction of Phenolic Compounds The air-dried (roomtemperature) andmilled [6] leaves (10 g) ofH tuberosuswererefluxedunder vacuumat 50∘Cusing 70 vv ethanol (EtOH)for three hours After evaporation under reduced pressurethe dry residue was redissolved in 25mL of methanol andused for colorimetric and chromatographic analyses ForHPLC analysis all samples were filtered through a 022120583mcellulose acetate filter (Millipore Corp Bedford MA USA)before injections

23 Measurement of Total Phenolic Concentration The totalphenolic concentration (TPC) was determined using theFolin-Ciocalteu reagent with gallic acid as standard [19 2930] The reaction mixture contained 05mL of test sample05mL of the Folin-Ciocalteu reagent freshly prepared in ourlaboratory 20mL of 10wv sodium carbonate solutionand 30mL of distilled water After 2 h of reaction underambient temperature in the dark the absorbance at 760 nmwas measured A calibration curve with equation 119910 =00029119909 + 00107 (1198772 = 09991) was constructed using gallicacid solutions in the range of 1470ndash294mgL Results wereexpressed in milligram gallic acid equivalents per gram ofdried sample

24 HPLC-MSMS Analysis HPLC-MSMS analysis of phe-nolics in H tuberosus extracts was performed using an Agi-lent 1200 series HPLC system (Agilent Technology Co LtdUSA) composed of a diode array detector and an Agilent6400 series triple quadrupole (QQQ) mass spectrometerequipped with an electrospray ionization (ESI) source Datawere collected and processed via a personal computer run-ning Agilent MassHunter workstation (Micromass Qualita-tive Analysis Version B0103 of Agilent Technology Co LtdUSA) A reverse-phase Eclipse XDB-C18 column (250mm times46mm 5120583m Agilent Technology Co Ltd USA) was usedfor separation The mobile phases consisted of methanol(A) and 10 vv formic acid aqueous solution (B) Gradientelution was started with 30 of A and ascended to 50 ofA in 45min The flow rate was kept at 08mLmin and thecolumn temperature was 30∘C Samples were filtered througha 022120583mfilter prior toHPLC injectionThe injection volumewas 5 120583L UV-Vis absorption spectra were recorded onlinefrom 200 to 600 nm during HPLC analysis Phenolics weredetected at the wavelength of 330 nm

Mass and MSMS spectra were achieved by electrosprayionization (ESI) in negative modes The voltages used were4000V for the source capillary and 10V for the extractioncone the source temperature was 150∘C and the desolvationtemperature was 350∘C The electrospray probe flow wasadjusted to 70mLmin The ESI-MS and ESI-MSMS spectrawere obtained by scanning from200 to 1200119898119911TheMSMSfragmentations were carried out with 10ndash50 energy

25 Identification and Quantification of Phenolic CompoundsThe phenolic compounds inH tuberosus leaves extracts wereidentified by comparing their UV-Vis absorption spectramatching their molecular ions (119898119911) obtained by ESI-MSand ESI-MSMS chromatographic characteristics with the







3 Results





17515125

107505025

0

0 2 4 6 8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

42

44


12

34

5 6 7 8

9 10

11

12

1314

15

times102

DAD1-A sig = 330 16 1d














times101

4

3

2

1

0

50

60

70

80

90

100

110

120

130

140

150

160

10790

13600


(a)

times103

5

4

3

2

1

0

60

80

100

120

140

160

180

200

220

240

260

280

300

320

850012700

19110


(b)

times103

17515125

107505025

0

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

13500

17310

17910

19110

35430


(c)

times103

3

25

2

15

1

05

0

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

450

19120

35410


(d)

times103

1614121

080604020

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

13510

17310

17910

19110

35410


(e)

times103

14121

080604020

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

13500

17310

17910

19110


(f)



9H6O3]minus





2O]minus (UV
























2O)-CH

3]minus











4 Discussion




35302520151050

TPC

3-C

QA CA

34

-DiC

QA

35

-DiC

QA

15

-DiC

QA

45

-DiC

QA

Con

cent

ratio

n

cb

a

c998400b998400

a998400

a998400998400 a998400998400 a998400998400 a1 a1 a1 C BA

C998400 A998400B998400 A998400

C998400998400 B998400998400A998400998400


(mg

g)

(a)

TPC

3-C

QA CA

34

-DiC

QA

35

-DiC

QA

15

-DiC

QA

45

-DiC

QA

Con

cent

ratio

n

cb

a

c998400a998400

b998400b998400998400 a998400998400 b998400998400 b1

a1 b1 CBA

C998400A998400

B998400 c998400998400

A998400998400

B998400998400

76543210


(mg

g)



5 Conclusions






Acknowledgments


References


















2

O2















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







3 Results





17515125

107505025

0

0 2 4 6 8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

42

44


12

34

5 6 7 8

9 10

11

12

1314

15

times102

DAD1-A sig = 330 16 1d














times101

4

3

2

1

0

50

60

70

80

90

100

110

120

130

140

150

160

10790

13600


(a)

times103

5

4

3

2

1

0

60

80

100

120

140

160

180

200

220

240

260

280

300

320

850012700

19110


(b)

times103

17515125

107505025

0

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

13500

17310

17910

19110

35430


(c)

times103

3

25

2

15

1

05

0

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

450

19120

35410


(d)

times103

1614121

080604020

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

13510

17310

17910

19110

35410


(e)

times103

14121

080604020

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

13500

17310

17910

19110


(f)



9H6O3]minus





2O]minus (UV
























2O)-CH

3]minus











4 Discussion




35302520151050

TPC

3-C

QA CA

34

-DiC

QA

35

-DiC

QA

15

-DiC

QA

45

-DiC

QA

Con

cent

ratio

n

cb

a

c998400b998400

a998400

a998400998400 a998400998400 a998400998400 a1 a1 a1 C BA

C998400 A998400B998400 A998400

C998400998400 B998400998400A998400998400


(mg

g)

(a)

TPC

3-C

QA CA

34

-DiC

QA

35

-DiC

QA

15

-DiC

QA

45

-DiC

QA

Con

cent

ratio

n

cb

a

c998400a998400

b998400b998400998400 a998400998400 b998400998400 b1

a1 b1 CBA

C998400A998400

B998400 c998400998400

A998400998400

B998400998400

76543210


(mg

g)



5 Conclusions






Acknowledgments


References


















2

O2















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


times101

4

3

2

1

0

50

60

70

80

90

100

110

120

130

140

150

160

10790

13600


(a)

times103

5

4

3

2

1

0

60

80

100

120

140

160

180

200

220

240

260

280

300

320

850012700

19110


(b)

times103

17515125

107505025

0

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

13500

17310

17910

19110

35430


(c)

times103

3

25

2

15

1

05

0

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

450

19120

35410


(d)

times103

1614121

080604020

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

13510

17310

17910

19110

35410


(e)

times103

14121

080604020

50

75

100

125

150

175

200

225

250

275

300

325

350

375

400

425

13500

17310

17910

19110


(f)



9H6O3]minus





2O]minus (UV
























2O)-CH

3]minus











4 Discussion




35302520151050

TPC

3-C

QA CA

34

-DiC

QA

35

-DiC

QA

15

-DiC

QA

45

-DiC

QA

Con

cent

ratio

n

cb

a

c998400b998400

a998400

a998400998400 a998400998400 a998400998400 a1 a1 a1 C BA

C998400 A998400B998400 A998400

C998400998400 B998400998400A998400998400


(mg

g)

(a)

TPC

3-C

QA CA

34

-DiC

QA

35

-DiC

QA

15

-DiC

QA

45

-DiC

QA

Con

cent

ratio

n

cb

a

c998400a998400

b998400b998400998400 a998400998400 b998400998400 b1

a1 b1 CBA

C998400A998400

B998400 c998400998400

A998400998400

B998400998400

76543210


(mg

g)



5 Conclusions






Acknowledgments


References


















2

O2















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















2O)-CH

3]minus











4 Discussion




35302520151050

TPC

3-C

QA CA

34

-DiC

QA

35

-DiC

QA

15

-DiC

QA

45

-DiC

QA

Con

cent

ratio

n

cb

a

c998400b998400

a998400

a998400998400 a998400998400 a998400998400 a1 a1 a1 C BA

C998400 A998400B998400 A998400

C998400998400 B998400998400A998400998400


(mg

g)

(a)

TPC

3-C

QA CA

34

-DiC

QA

35

-DiC

QA

15

-DiC

QA

45

-DiC

QA

Con

cent

ratio

n

cb

a

c998400a998400

b998400b998400998400 a998400998400 b998400998400 b1

a1 b1 CBA

C998400A998400

B998400 c998400998400

A998400998400

B998400998400

76543210


(mg

g)



5 Conclusions






Acknowledgments


References


















2

O2















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


35302520151050

TPC

3-C

QA CA

34

-DiC

QA

35

-DiC

QA

15

-DiC

QA

45

-DiC

QA

Con

cent

ratio

n

cb

a

c998400b998400

a998400

a998400998400 a998400998400 a998400998400 a1 a1 a1 C BA

C998400 A998400B998400 A998400

C998400998400 B998400998400A998400998400


(mg

g)

(a)

TPC

3-C

QA CA

34

-DiC

QA

35

-DiC

QA

15

-DiC

QA

45

-DiC

QA

Con

cent

ratio

n

cb

a

c998400a998400

b998400b998400998400 a998400998400 b998400998400 b1

a1 b1 CBA

C998400A998400

B998400 c998400998400

A998400998400

B998400998400

76543210


(mg

g)



5 Conclusions






Acknowledgments


References


















2

O2















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













2

O2















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Analysis of Phenolic Acids of Jerusalem ...downloads.hindawi.com/journals/tswj/2014/568043.pdf · Research Article Analysis of Phenolic Acids of Jerusalem Artichoke