Research ArticleChemical Composition and Seasonality of AromaticMediterranean Plant Species by NMR-Based Metabolomics

Monica Scognamiglio Brigida DrsquoAbrosca Assunta Esposito and Antonio Fiorentino

Department of Environmental Biological and Pharmaceutical Sciences and Technologies Second University of NaplesVia Vivaldi 43 81100 Caserta Italy

Correspondence should be addressed to Monica Scognamiglio monicascognamigliounina2it

Received 17 November 2014 Revised 28 January 2015 Accepted 30 January 2015

Academic Editor Gowda A Nagana Gowda

Copyright copy 2015 Monica Scognamiglio et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

An NMR-based metabolomic approach has been applied to analyse seven aromatic Mediterranean plant species used in traditionalcuisine Based on the ethnobotanical use of these plants the approach has been employed in order to study the metabolic changesduring different seasons Primary and secondary metabolites have been detected and quantified Flavonoids (apigenin quercetinand kaempferol derivatives) and phenylpropanoid derivatives (eg chlorogenic and rosmarinic acid) are the main identifiedpolyphenols The richness in these metabolites could explain the biological properties ascribed to these plant species

1 Introduction

Aromatic plants are widespread throughout the world andthey are extensively added to different food preparationsTheuse of these plants is very popular and has a long tradition inMediterranean area [1]

Plants in general have been shown to produce a widerange of chemicals traditionally categorized into primaryand secondary metabolites For the sake of simplicity pri-mary metabolites can be thought of to serve nutritionalpurposes while secondary metabolites are required by plantsas weapons against competitors herbivores or pathogensand so forth [2] However both classes of metabolites areimportant for the plant itself but also for their actions on plantconsumers and mainly in case of edible plants

Many aromatic plants are added to foods and eaten Thewhole plants or one or some of their components are used asfor example food preservatives flavour and additives Never-theless it has been shown that chemical composition of plantsis highly variable along the year Several analytical techniquesare available for studying plant metabolitesrsquo content Most ofthem are targeted techniques as an a priori knowledge on themetabolites to be analysed is required [2 3] Furthermore foraromatic plants a great effort has been devoted to the studyof essential oils [2 4]

Given this background a wider knowledge about theirwhole metabolite content is needed To this end a verypowerful approach ismetabolomics the comprehensive anal-ysis of the set of low molecular weight compounds of abiological system under a given condition [5] Analogouslyother related approaches like metabolic profiling [6] couldbe used

In particular NMR-based metabolomics has been shownto be very useful due to its untargeted and unbiased features[7] Furthermore it is highly reproducible it allows the con-temporary identification and quantification of a large numberof compounds and needs short times of analysis (includingthe extraction procedures) [6] The only limitation of NMRis its low sensitivity when compared to mass spectrometryalthough sensitivity has been drastically increasedwith recentadvances like higher magnetic fields and the introductionof microcryoprobes [5 8] On the other hand NMR allowsthe identification of unknown compounds in the analysedmixtures as it gives important structural information [8]

In order to demonstrate the potentiality of this approachit has been applied to seven aromatic plant species charac-teristic ofMediterranean garrigue the metabolitesrsquo content ofthese plants has been determined and the seasonality of theiraccumulation has been studied

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2015 Article ID 258570 9 pageshttpdxdoiorg1011552015258570

2 Journal of Analytical Methods in Chemistry

Table 1 Studied plants

Species and voucherspecimen Family Uses

Calamintha nepeta LCE236 Lamiaceae

Leaf used as food spice (usually added to meat fish and vegetable dishes mint aroma)and for medicinal purposes (antiseptic tonic antispasmodic diaphoretic expectorantetc) [25 26]

Helichrysum italicum GDon CE233 Asteraceae

Leaf used as food spice (also known as ldquocurry plantrdquo) and for medicinal purposes(anti-inflammatory and anti-infective antiallergic etc) essential oils used in cosmetics[16 27]

Foeniculum vulgareMillCE237 Apiaceae Leaf and fruits used to flavour several kinds of dishes Also used in cosmetics and

pharmaceutical products [28]Micromeria graeca LCE238 Lamiaceae Leaf used as food spice (added to meat and vegetables)

Origanum vulgare L CE239 LamiaceaeDried plant (epigeous part) used as food spice The most common spice inMediterranean cuisine Used since ancient times for medicinal purposes (antioxidantdigestive expectorant antiseptic antispasmodic etc) [29]

Satureja montana L CE234 LamiaceaeLeaf used as food spice (usually added to meat fish and vegetable dishes) Naturalfood preservative Savory honey is a very common ingredient in folk remedies Usedfor medicinal purposes [30]

Thymus longicaulis C PreslCE235 Lamiaceae Leaf used as food spice (usually added to meat fish and vegetable dishes) Natural

food preservative Used also for medicinal purposes [31]

2 Materials and Methods

21 Plant Material Sampling and Processing Seven plantspecies (Table 1) were collected in a garrigue on the calcareoushills of Durazzano (41∘31015840N 14∘271015840E southern Italy) in winter(February 2012) spring (May 2012) summer (July 2012)and autumn (October 2012) The plants were selected basedon their occurrence in the study site Origanum vulgaresamples were not available in autumn Plant leaf samples werecollected in the field always at the same moment of the dayin order to minimize differences due to metabolites changingbased on circadian clock

Three leaf samples (biological replicates) of each plantspecies were harvested and immediately frozen in liquid N

2

in order to avoid unwanted enzymatic reactions and stored atminus80∘C up to the freeze drying process Once freeze dried theywere powdered in liquid nitrogen and stored at minus20∘C Eachsample was extracted and analysed by NMR

Voucher specimens for all the plant species were depos-ited at the herbarium of the Second University of Naples(Table 1)

22 Metabolomics Analysis Freeze-dried plant material(50mg) was transferred to a 2mL microtube NMR sampleswere prepared in a mixture of phosphate buffer (FlukaChemika 90mM pH 60) in D

2O (Cambridge Isotope

Laboratories) containing 01ww trimethylsilylpropionic-2233-119889

4acid sodium salt (TMSP Sigma-Aldrich) and

methanol-1198894(Sigma-Aldrich) A volume of 15mL of phos-

phate buffer and methanol-1198894(1 1) was added to the plant

samples The mixture was vortexed at room temperature for1min ultrasonicated (Elma Transonic Digitals) for 40minand centrifuged (Beckman Allegra 64R) at 13000 rpm for10min An aliquot of 06mL was transferred to an NMR tubeand analysed by NMR [9] NMR spectra were recorded at

25∘Con a 30003MHz for 1Hand 7545MHz for 13Con aVar-ian Mercury Plus 300 Fourier transform NMR CD

3OD was

used as the internal lock Each 1HNMR spectrum consistedof 256 scans with the following parameters 016Hzpointacquisition time (AQ) = 10 s relaxation delay (RD) = 15 s90∘ pulse width (PW) = 138 120583s A presaturation sequencewas used to suppress the residual H

2O signal Free induction

decays (FIDs) were Fourier transformed with LB = 03Hzand the resulting spectra weremanually phased and baseline-corrected and calibrated to TMSP at 00 ppm using 1HNMRprocessor (MestReNova version 802)

23 Quantitative Analysis The main metabolites identifiedin plant extracts were analyzed by quantitative analysis 1H-NMR spectra were bucketed reducing it to integral segmentswith a width of 002 ppm with ACDLABS 120 1H-NMRprocessor (ACDLABS 120 Toronto Canada) Spectra werescaled to the internal standard (whose area from minus001 to001 ppmwas set equal to 1) For eachmetabolite buckets cor-responding to nonoverlapping signals were used to calculatethe relative amount as follows

Metabolite relative amount =SA times 119899HTMSP119899

119878

(1)

where SA is the metabolite signal area but it is also equal tothe signal areastandard area ratio as standard area is equalto 1 119899HTMSP is a constant equal to 9 (the number of protonsresponsible for the signal between minus001 and 001 ppm) and 119899

119904

is the number of protons of the metabolite signal area [10]

3 Results and Discussion

Recent research has shown culinary herbs and spices asa source of bioactive compounds [11] Although most ofthem have been extensively studied for their essential oil

Journal of Analytical Methods in Chemistry 3

composition far less information is available on their polarand semipolar chemical composition

Herewith seven Mediterranean plants (Calaminthanepeta Helichrysum italicum Foeniculum vulgare Microm-eria graeca Origanum vulgare Satureja montana and Thy-mus longicaulis) have been studied for their metabolite con-tent by NMR The identification of metabolites was carriedout by comparing NMR data with an in-house library withdatabases [12] and with some literature data [10 13ndash15]1H-NMR data and extract composition are given in Table 2and spectra are shown in Figure 1

Primary metabolites were easily identified based on dataextensively reported in literature of spectra acquired in thesame solvent mixture [10 12ndash15] Among free amino acidsalanine was observed in all of the plants while threonine wasonly detected inMicromeria graeca and Foeniculum vulgare

The sugar content was highly variable with glucose andsucrose as the main free carbohydrates detected

Finally some organic acids were identified Quinic acidwas present in all of the plants but Helichrysum italicum andSatureja montana while malic acid was clearly detected in allof the plants

Concerning the secondary metabolite content the anal-ysed Lamiaceae plants were all characterized by the presenceof high amounts of rosmarinic acid (with the exception ofC nepeta) along with analogous compounds Caffeic acidwas identified based on comparison of NMR data with theliterature [10 15] and confirmed by comparison with NMRspectra of an in-house library Rosmarinic acid was identifiedbased on the comparison with already reported data [10] andthe structure was confirmed by 2D NMR analysis Indeedthe olefinic proton at 120575 750 (H7) (showing HSQC correlationwith the carbon at 120575 1459) and that at 120575 630 (showingHSQC correlation with the carbon at 120575 1143) showed longrange correlations with a carbon at 120575 1684 (C9) This carbonwas in turn correlated with the proton at 120575 502 (H81015840)confirming the linkage between a caffeoylmoiety and the 34-dihydroxyphenyl lactic acid moiety Furthermore the formerwas identified based on the long range correlation of theH7 olefinic proton with the aromatic carbon at 120575 1264 (C1)showing further correlations with the signals belonging to anorthopara trisubstituted aromatic ring (Table 2) The latterwas identified as follows the proton H81015840 showed correlationswith a carboxylic carbon at 120575 1765 and with a methylenecarbon at 120575 369 (C71015840) showing HSQC correlations with thediastereotopic protons H71015840 (Table 2) The proton H81015840 alsoshowed long range correlation with a quaternary aromaticcarbon at 120575 1300 (C11015840) in turn correlated with the signalsbelonging to a second orthopara trisubstituted aromatic ring(Table 2)

Some phenylpropanoids in the extracts were not defini-tively characterized inasmuch as based on their scarceabundance andor strong signal overlapping they did notshow clear correlations in 2D NMR spectra However thecharacteristic signals and correlations of the trans-propenylicchain suggested their presence Indeed correlations wereobserved in the HSQC among the olefinic signals withcarbons at 140ndash145 ppm (for the proton at lower fields) and at114ndash120 ppm (for the proton at higher fields) and long range

correlations were shown with carbon resonances attributableto ester carboxyl carbons and with quaternary aromaticcarbons

Calamintha nepeta extracts were also rich in severalflavonoids and phenylpropanoids Unfortunately it was notpossible to definitely characterize these compounds but allof the flavonoids were identified as apigenin derivatives(Table 2) Indeed several sets of resonances attributable tometa coupled protons H7H8 (ring A) to proton H3 andto B ring ortho coupled protons were detected Interestinglythe compounds probably characterized by a different degreeof glycosylation showed a peculiar distribution along theseasons Two apigenin derivativeswere detected in spring andautumn (apigenin derivatives 1 and 2) while two differentcouples of these compounds were detected in summer (api-genin derivatives 3 and 4) and winter (apigenin derivatives 5and 6) samples Moreover apigenin derivatives 5 and 6 weredetected only in winter also in Satureja montana

Analogously as shown in Table 2 the presence of somephenylpropanoids was strongly dependent on the collectionseason (Table 2) phenylpropanoid 2 was detected only inwinter phenylpropanoid 5 only in spring phenylpropanoid6 only in summer and phenylpropanoid 7 only in autumnsamples

The most stable metabolome along the seasons wasdetected for Thymus longicaulis while Micromeria graecaand Origanum vulgare only changed for some metabolitesHowever differences in the amounts of the compounds wereobserved Indeed for all the Lamiaceae plants a higheramount of aromatic compounds (Table 2) was observedin spring and summer samples compared to autumn andwinter samplesHelichrysum italicum and Foeniculum vulgareextracts showed an analogous behaviour with changes ofmetabolites mainly on the quantitative point of view

Helichrysum italicum extracts besides chlorogenic acidsalso showed signals attributable to a 3-hydroxybenzofuranand an isobenzofuranone derivative Chlorogenic neochlo-rogenic and dicaffeoylquinic acids were identified basedon comparison of 1H-NMR data with the literature [1015] and with the in-house library The caffeoyl moiety wasclearly identified based on 1D and 2D NMR data and thelinkage(s) with the quinic acid moiety was confirmed bythe correlation observed in the long range spectrum The 3-hydroxybenzofuran and isobenzofuranone derivatives wereidentified based on comparison with the NMR spectrum ofthe compound previously isolated [16]

Finally Foeniculum vulgare was characterized by chloro-genic acids and flavonoids identified based on 1H-NMRdata as kaempferol and quercetin [17] Chlorogenic acid wasreported for the first time from this species to the best of ourknowledge

The identification of water soluble compounds in theseplants is very important as most of them are added to disheshence they might be eaten or however they could releasebioactive compounds into food In this framework it is worthto underlining that first of all the health promoting capacityof bioactive compounds could be dependent on synergismsSecondly these plants could also contain potential toxic

4 Journal of Analytical Methods in Chemistry

Table2Mainmetabolitesd

etectedin

plantextracts

1 H-N

MRdataarem

easuredin

ppm

andcoup

lingconstants(119869)inHertzR

elativea

mou

ntisexpressedas

them

eanvalue(119899=3)plusmn

SD

Forsom

emetabolitestheq

uantitativ

eanalysis

was

notp

ossib

ledu

etostr

onglyoverlapp

ingsig

nals

hencethe

presence

isindicatedby

ldquoXrdquo

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Calamintha

nepeta

Alanine

148(H

3d119869=72

)X

XX

XAp

igenin

deriv

ative1lowast

651

(H6d119869=21)670(H

3s)678(H

8d119869=21)710

(H31015840H

51015840d119869=87)794

(H21015840H

61015840d119869=87)

852plusmn10

8525plusmn373

Apigenin

deriv

ative2lowast

654

(H6d119869=21)667(H

3s)673(H

8d119869=21)707(H

31015840H

51015840d119869=87)790

(H21015840H

61015840d119869=87)

Apigenin

deriv

ative3lowast

650

(H6d119869=21)666(H

3s)678(H

8d119869=21)702(H

31015840H

51015840d119869=87)788

(H21015840H

61015840d119869=87)

781plusmn

324

Apigenin

deriv

ative4lowast

649

(H6d119869=21)661(H3s)669(H

8d119869=21)706(H

31015840H

51015840d119869=87)784

(H21015840H

61015840d119869=87)

Apigenin

deriv

ative5lowast

655

(H6d119869=21)665(H

3s)669(H

8d119869=21)712

(H31015840H

51015840d119869=87)795

(H21015840H

61015840d119869=87)

516plusmn059

Apigenin

deriv

ative6lowast

653

(H6d119869=21)665(H

3s)666(H

8d119869=21)709(H

31015840H

51015840d119869=87)792

(H21015840H

61015840d119869=87)

Citricacid

259

(H2ad119869

=176)272(H

2bd119869

=176)

838plusmn346

1366plusmn331

804plusmn017

1344plusmn002

Glucose

459

(H1120573

d119869

=78

)519

(H1120572

d119869

=38)

589plusmn233

687plusmn299

561plusmn028

564plusmn17

1Malicacid

239

(H3add119869=15693)278

(H3add119869=15636)431

(H2dd

119869=93

36)

2672plusmn1080

3757plusmn272

3145plusmn713

Phenylprop

anoid

1597

(H8d119869=159)74

3(H

7d119869=159)

304plusmn301

594plusmn045

305plusmn062

257plusmn067

Phenylprop

anoid

2617

(H8d119869=159)73

0(H

7d119869=159)

X

Phenylprop

anoid

3631

(H8d119869=159)752(H

7d119869=159)

102plusmn099

462plusmn19

9658plusmn15

1

Phenylprop

anoid

4645

(H8d119869=159)76

7(H

7d119869=159)

XX

XX

Phenylprop

anoid

5616

(H8d119869=159)72

5(H

7d119869=159)

X

Phenylprop

anoid

6611(H

8d119869=159)737(H

7d119869=159)

X

Phenylprop

anoid

7614

(H8d119869=159)739(H

7d119869=159)

X

Quinica

cid

187(H

2am

)19

6(H

6am

)201

(H2bm

)202

(H6bm

)340

(H4ov)400

(H3

ov)411(H

5ov)

4528plusmn665

5509plusmn483

6402plusmn416

7161plusmn

884

Sucrose

415

(H31015840d119869

=84)538(H

1d119869=36)

824plusmn363

709plusmn301

1551plusmn

284

1037plusmn054

Journal of Analytical Methods in Chemistry 5

Table2Con

tinued

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Helichrysum

Italicum

Alanine

SeeC

nepeta

124plusmn019

087plusmn060

091plusmn015

151plusmn

044

Chlorogenica

cid

184ndash

220

(H2andH6qu

inicacidm

)545

(H5m)637

(H81015840d119869

=159)690

(H51015840d119869

=81)707(

H61015840dd119869=8421)715

(H21015840d119869

=21)762(

H71015840d119869

=159)

831plusmn15

590

3plusmn12

4363plusmn259

326plusmn077

Dicaffeoylqu

inic

acid

630

(H81015840d119869

=162)648

(H810158401015840

d119869

=156)76

5(H

71015840d119869

=162)76

6(H

710158401015840

d119869

=156)

1086plusmn13

81088plusmn516

1153plusmn238

783plusmn207

Glucose

SeeC

nepeta

377plusmn174

356plusmn004

273plusmn059

378plusmn201

3-OHbenzofuran

518

(H2d119869=63)522(H

3d119869=63)690(H

7ov)804

(H6dd119869=8418

)406plusmn027

313plusmn13

9537plusmn18

9406plusmn14

7Isob

enzofurano

ne533

(H3s)673(H

4d119869=18

)684

(H6d119869=18

)603plusmn270

Malicacid

SeeC

nepeta

XX

XX

Neochlorogenic

acid

639

(H81015840d119869

=159)752(H

71015840d119869

=159)

XX

XX

Sucrose

SeeC

nepeta

1693plusmn855

915plusmn457

1024plusmn445

1014plusmn375

Foenicu

lum

vulga

re

Alanine

SeeC

nepeta

117plusmn005

090plusmn049

124plusmn025

109plusmn009

Caffeicacid

629

(H81015840d119869

=159)688

(H51015840d119869

=81)703(H

61015840dd119869=8421)712

(H21015840d119869

=21)752

(H71015840d119869

=159)

XX

XX

Chlorogenica

cid

SeeH

italicum

XX

XX

Dicaffeoylqu

inic

acid

SeeH

italicum

XX

X

Glucose

SeeC

nepeta

117plusmn002

555plusmn289

577plusmn16

4629plusmn13

8GABA

(120574-aminob

utyric

acid)

192(H

3m)236

(H2t119869=75

)301

(H4t119869=75

)X

XX

Kaem

pferol

635

(H6d119869=21)652

(H8d119869=21)700(H

21015840H

61015840d119869

=84)809(H

31015840H

51015840

d119869=84)

128plusmn017

178plusmn15

614

6plusmn044

118plusmn082

Malicacid

SeeC

nepeta

4468plusmn711

4740plusmn1023

16852plusmn1115

8662plusmn349

Quercetin

627

(H6d119869=21)648(H

8d119869=21)699(H

51015840d119869=85)759(H

61015840d119869=85

21)775(H

21015840d119869=21)

193plusmn036

555plusmn361

623plusmn12

5294plusmn209

Quinica

cid

SeeC

nepeta

2509plusmn262

3161plusmn

1748

3349plusmn673

2730plusmn225

Sucrose

SeeC

nepeta

4494plusmn228

3680plusmn1385

5436plusmn1186

3062plusmn90

3Th

reon

ine

132(H

4d119869=66)

XX

XX

Microm

eria

graeca

Alanine

SeeC

nepeta

093plusmn055

140plusmn088

209plusmn089

575plusmn404

Citricacid

SeeC

nepeta

1989plusmn94

41865plusmn15

61713plusmn684

1543plusmn510

Glucose

SeeC

nepeta

481plusmn249

687plusmn299

561plusmn028

564plusmn17

1Malicacid

SeeC

nepeta

XX

XX

Quinica

cid

SeeC

nepeta

3975plusmn1068

5509plusmn483

6402plusmn416

5741plusmn

1124

Rosm

arinicacid

300

(H71015840add

119869=141

96)315

(H71015840bdd

119869=141

36)502(H

81015840dd119869=100

33)630(H

8d119869=159)671(H61015840dd119869=78

21)681(H51015840d119869

=78

)682

(H5

d119869=81)689(H

21015840d119869

=21)700(H

6dd

119869=8118

)711(H2d119869=18

)75

0(H

7d119869=159)

102plusmn099

462plusmn19

8658plusmn15

115

4plusmn079

Sucrose

SeeC

nepeta

799plusmn260

709plusmn302

1551plusmn

284

822plusmn373

Threon

ine

SeeF

vulgare

X

6 Journal of Analytical Methods in Chemistry

Table2Con

tinued

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Orig

anum

vulga

re

Apigenin

deriv

ative2

SeeC

nepeta

432plusmn024

295plusmn091

mdash

Alanine

SeeC

nepeta

058plusmn012

090plusmn017

107plusmn014

mdashCh

oline

320

(s)

XX

mdashCitricacid

SeeC

nepeta

1474plusmn10

22292plusmn413

1554plusmn19

1mdash

Glucose

SeeC

nepeta

1659plusmn264

362plusmn288

439plusmn382

mdashLithosperm

icacid

300

(H71015840aa

ndbov)6

30(H

8d119869=159)78

2(H

7d119869=159)

XX

Xmdash

Malicacid

SeeC

nepeta

2759plusmn16

93240plusmn810

1474plusmn851

mdashQuinica

cid

SeeC

nepeta

3094plusmn286

4591plusmn

183

4312plusmn98

1mdash

Rosm

arinicacid

SeeM

graeca

1147plusmn611

1573plusmn482

3550plusmn591

mdashSucrose

SeeC

nepeta

1005plusmn15

8498plusmn045

1492plusmn436

mdash

Satureja

montana

Apigenin

deriv

ative5

SeeC

nepeta

X

Apigenin

deriv

ative6

SeeC

nepeta

X

Alanine

SeeC

nepeta

XX

XX

Choline

SeeO

vulgare

XX

XCh

lorogenica

cid

SeeH

italicum

947plusmn12

01501plusmn

051

1250plusmn554

366plusmn18

4Glucose

SeeC

nepeta

101plusmn

067

396plusmn15

5287plusmn14

7427plusmn15

4Malicacid

SeeC

nepeta

3932plusmn260

3240plusmn099

855plusmn331

4876plusmn678

Rosm

arinicacid

SeeM

graeca

715plusmn12

61090plusmn16

4884plusmn432

Sucrose

SeeC

nepeta

1009plusmn093

856plusmn442

1007plusmn12

593

7plusmn391

Thym

uslongica

ulis

Alanine

SeeC

nepeta

034plusmn024

105plusmn011

108plusmn022

128plusmn007

Citricacid

SeeC

nepeta

XX

XX

Malicacid

SeeC

nepeta

2692plusmn1544

6005plusmn328

3556plusmn680

5464plusmn1472

Quinica

cid

SeeC

nepeta

1985plusmn863

4116plusmn75

040

71plusmn

830

3196plusmn317

Glucose

SeeC

nepeta

306plusmn033

320plusmn083

265plusmn17

6336plusmn14

1Ph

enylprop

anoid

8613

(H8d119869=159)74

6(H

7d119869=159)

367plusmn113

565plusmn061

477plusmn12

419

0plusmn12

4

Rosm

arinicacid

SeeM

graeca

710plusmn370

778plusmn19

81012plusmn176

487plusmn214

Sucrose

SeeC

nepeta

710plusmn370

778plusmn19

81012plusmn176

487plusmn214

Sign

almultip

licity

indicatedas

follo

wsd=do

ubletdd

=do

ubleto

fdou

bletsm

=multip

letov

=overlap

pedq=qu

artets=

singletand

t=triplet

lowast

Apigenin

deriv

atives

1and

23and45and6wereq

uantified

together

duetooverlap

ping

signals

Journal of Analytical Methods in Chemistry 7

Calamintha nepetaWi

Sp

Su

Au

1

2323 23

4545 45

67 67 6788

9 9 10 10101819

19

11121314

11121314

16

17

Helichrysum italicumWi

Sp

Su

Au

202020 202020

Foeniculum vulgareWi

Sp

Su

Au

21 21 2122 22 23 2323 24

Micromeria graecaWi

Sp

Su

Au

252525

25 25

Origanum vulgare

Wi

Sp

Su

Satureja montana

Wi

Sp

Su

Au

Thymus longicaulisWi

Sp

Su

Au

100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm)

Figure 1 1H-NMR spectra of studied plants (Au = autumn Sp = spring Su = summer Wi = winter) The main resonances of the maincompounds are indicated on the spectra as follows 1 alanine 2 apigenin derivative 1 3 apigenin derivative 2 4 apigenin derivative 3 5apigenin derivative 4 6 apigenin derivative 5 7 apigenin derivative 6 8 citric acid 9 glucose 10 malic acid 11 phenylpropanoid 1 12phenylpropanoid 2 13 phenylpropanoid 3 14 phenylpropanoid 4 15 phenylpropanoid 5 16 phenylpropanoid 6 17 phenylpropanoid 7 18quinic acid 19 sucrose 20 chlorogenic acid 21 GABA 22 kaempferol 23 quercetin 24 threonine 25 rosmarinic acid

8 Journal of Analytical Methods in Chemistry

compounds as reported for several Lamiaceae [17] Never-theless the role in nutrition of primary metabolites is oftendisregarded [18] These considerations raise the attention tothe need for a comprehensive profiling of their metabolites

Furthermore the NMR-based metabolomic approachhere proposed due to the short time of analyses and to lowercosts compared to other analytical methods was very usefulfor the study of seasonal variation of metabolites

Indeed although it is clear that secondary metabolitesshow peculiar trends only fragmentary information is avail-able mainly because of the used approaches

The most common and easiest procedure was to performthe phytochemical study on samples collected in a specifictime of the year and then compare the other months (orseasons) by setting up a series of target analyses often byHPLC [19 20] or by less time and resource consumingapproaches based on colorimetric assays [21]

The improvement of analytical methods and espe-cially the availability of a high-throughput approach likemetabolomics [5] gives the chance to further explore theissue of seasonality [22] and to thoroughly study thesechanges

Concerning themetabolites identifiedwithin the extractsit is important to underline their biological activity

Phenols are well known for their antioxidant activity [11]As the studied plants are all very rich in phenolics they havea great antioxidant potential This could also explain the useof some of these herbs as natural food preservatives (Table 1)

Among the detected compounds rosmarinic acid is themost widespread and the most abundant A plethora ofbiological activities has been attributed to this compoundamong them adstringent antioxidative anti-inflammatoryantimutagen antibacterial and antiviral [23]

Many other phenylpropanoids and flavonoids were alsodetected Several properties have been reported for thesecompounds such as anti-inflammatory antimicrobial andantitumor activity [24]

The richness in these compounds of the studied plantssupports their traditional uses

However the study evidenced that qualitative and quan-titative variations of metabolites are observed along the year

This is to the best of our knowledge the first reportof metabolite content and seasonal qualitative and quanti-tative variation of this set of food spices Furthermore it isevidenced that a higher content of compounds known fortheir health promoting capacities can be found in spring andsummer samples of all the analysed species although season-specific compounds were also detected

4 Conclusions

NMR-based metabolomics has been applied to the study ofchemical composition of selected aromatic plants ofMediter-ranean vegetation

The method allowed determining the chemical compo-sition of plant extracts in terms of primary and secondarymetabolites The abundance of aromatic secondary metabo-lites suggested that the traditional uses of these plants mightbe supported by their chemical composition

Furthermore the seasonality of the accumulation of thesemetabolites was studied

Conflict of Interests

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

Acknowledgment

The authors would like to thank the anonymous reviewers fortheir valuable suggestions

References

[1] A M Scherrer R Motti and C S Weckerle ldquoTraditional plantuse in the areas of Monte Vesole and Ascea Cilento NationalPark (Campania Southern Italy)rdquo Journal of Ethnopharmacol-ogy vol 97 no 1 pp 129ndash143 2005

[2] T Efferth and H J Greten ldquoMedicinal and aromatic plantresearch in the 21st centuryrdquoMedicinal amp Aromatic Plants vol1 article e110 2012

[3] R Rodrıguez-Solana J M Salgado J M Domınguez andS Cortes-Dieguez ldquoComparison of soxhlet accelerated sol-vent and supercritical fluid extraction techniques for volatile(GCndashMS and GCFID) and phenolic compounds (HPLCndashESIMSMS) from Lamiaceae Species rdquo Phytochemical Analysisvol 26 no 1 pp 61ndash71 2015

[4] T Fornari G Vicente E Vazquez M R Garcıa-Risco andG Reglero ldquoIsolation of essential oil from different plants andherbs by supercritical fluid extractionrdquo Journal of Chromatogra-phy A vol 1250 pp 34ndash48 2012

[5] H K Kim Y H Choi and R Verpoorte ldquoNMR-based plantmetabolomics where do we stand where do we gordquo Trends inBiotechnology vol 29 no 6 pp 267ndash275 2011

[6] M Scognamiglio B DrsquoAbrosca A Esposito and A FiorentinoldquoMetabolomics an unexplored tool for allelopathy studiesrdquoJournal of Allelochemical Interactions vol 1 pp 9ndash23 2015

[7] J W Allwood D I Ellis and R Goodacre ldquoMetabolomictechnologies and their application to the study of plants andplant-host interactionsrdquo Physiologia Plantarum vol 132 no 2pp 117ndash135 2008

[8] R R Forseth and F C Schroeder ldquoNMR-spectroscopic analysisof mixtures from structure to functionrdquo Current Opinion inChemical Biology vol 15 no 1 pp 38ndash47 2011

[9] H K Kim Y H Choi and R Verpoorte ldquoNMR-basedmetabolomic analysis of plantsrdquo Nature Protocols vol 5 no 3pp 536ndash549 2010

[10] M Scognamiglio V Fiumano B DAbrosca et al ldquoChemicalinteractions between plants in Mediterranean vegetation theinfluence of selected plant extracts on Aegilops geniculatametabolomerdquo Phytochemistry vol 106 pp 69ndash85 2014

[11] A Vallverdu-Queralt J Regueiro M Martınez-Huelamo JF R Alvarenga L N Leal and R M Lamuela-RaventosldquoA comprehensive study on the phenolic profile of widelyused culinary herbs and spices rosemary thyme oreganocinnamon cumin and bayrdquo Food Chemistry vol 154 pp 299ndash307 2014

[12] Q Cui I A Lewis A D Hegeman et al ldquoMetabolite identifi-cation via the Madison Metabolomics Consortium DatabaserdquoNature Biotechnology vol 26 no 2 pp 162ndash164 2008

Journal of Analytical Methods in Chemistry 9

[13] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissusCarltonrdquoPhytochemistry vol 88 pp 43ndash53 2013

[14] R Verpoorte Y H Choi and H K Kim ldquoNMR-basedmetabolomics at work in phytochemistryrdquo PhytochemistryReviews vol 6 no 1 pp 3ndash14 2007

[15] J-L Wolfender S Rudaz Y H Choi and H K Kim ldquoPlantmetabolomics from holistic data to relevant biomarkersrdquo Cur-rent Medicinal Chemistry vol 20 no 8 pp 1056ndash1090 2013

[16] B DrsquoAbrosca E Buommino G DrsquoAngelo et al ldquoSpectroscopicidentification and anti-biofilm properties of polar metabolitesfrom the medicinal plant Helichrysum italicum against Pseu-domonas aeruginosardquo Bioorganic and Medicinal Chemistry vol21 no 22 pp 7038ndash7046 2013

[17] S Pacifico B DrsquoAbrosca M Scognamiglio et al ldquoNMR-basedmetabolic profiling and in vitro antioxidant and hepatotoxicassessment of partially purified fractions fromGolden german-der (Teucrium polium L) methanolic extractrdquo Food Chemistryvol 135 no 3 pp 1957ndash1967 2012

[18] R Scherer A C P Rybka C A Ballus A D Meinhart J TFilho and H T Godoy ldquoValidation of a HPLC method forsimultaneous determination of main organic acids in fruits andjuicesrdquo Food Chemistry vol 135 no 1 pp 150ndash154 2012

[19] J Liimatainen M Karonen J Sinkkonen M Helander andJ-P Salminen ldquoPhenolic compounds of the inner bark ofBetula pendula seasonal and genetic variation and inductionby woundingrdquo Journal of Chemical Ecology vol 38 no 11 pp1410ndash1418 2012

[20] K Hosni K Msaada M Ben Taarit and B Marzouk ldquoPhe-nological variations of secondary metabolites from Hypericumtriquetrifolium Turrardquo Biochemical Systematics and Ecology vol39 no 1 pp 43ndash50 2011

[21] F Brahmi B Mechri S Dabbou M Dhibi and M HammamildquoThe efficacy of phenolics compounds with different polaritiesas antioxidants from olive leaves depending on seasonal varia-tionsrdquo Industrial Crops and Products vol 38 no 1 pp 146ndash1522012

[22] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissus Carltonrdquo Phytochemistry vol 88 pp 43ndash53 2013

[23] M Petersen and M S J Simmonds ldquoRosmarinic acidrdquo Phyto-chemistry vol 62 no 2 pp 121ndash125 2003

[24] R Quirantes-Pine M Herranz-Lopez L Funes et al ldquoPhenyl-propanoids and their metabolites are the major compoundsresponsible for blood-cell protection against oxidative stressafter administration of Lippia citriodora in ratsrdquo Phytomedicinevol 20 no 12 pp 1112ndash1118 2013

[25] G Tibaldi E Fontana and S Nicola ldquoPostharvest managementaffects spearmint and calamint essential oilsrdquo Journal of theScience of Food and Agriculture vol 93 no 3 pp 580ndash586 2013

[26] K Hammer G Laghetti and K Pistrick ldquoCalamintha nepeta(L) Savi and Micromeria thymifolia (Scop) Fritsch cultivatedin ItalyrdquoGenetic Resources and Crop Evolution vol 52 no 2 pp215ndash220 2005

[27] G Appendino M Ottino N Marquez et al ldquoArzanol an anti-inflammatory and anti-HIV-1 phloroglucinol 120572-pyrone fromHelichrysum italicum ssp microphyllumrdquo Journal of NaturalProducts vol 70 no 4 pp 608ndash612 2007

[28] W-R Diao Q-P Hu H Zhang and J-G Xu ldquoChemicalcomposition antibacterial activity and mechanism of action ofessential oil from seeds of fennel (Foeniculum vulgare Mill)rdquoFood Control vol 35 no 1 pp 109ndash116 2014

[29] N Martins L Barros C Santos-Buelga M Henriques S Silvaand I C F R Ferreira ldquoDecoction infusion and hydroalcoholicextract of Origanum vulgare L different performances regard-ing bioactivity and phenolic compoundsrdquo Food Chemistry vol158 pp 73ndash80 2014

[30] T Mihajilov-Krstev D Radnovic D Kitic et al ldquoChemicalcomposition antimicrobial antioxidative and anticholinester-ase activity of Satureja Montana L ssp montana essential oilrdquoCentral European Journal of Biology vol 9 no 7 pp 668ndash6772014

[31] C Sarikurkcu M Sabih Ozer M Eskici B Tepe S Can andE Mete ldquoEssential oil composition and antioxidant activity ofThymus longicaulis C Presl subsp longicaulis var longicaulisrdquoFood andChemical Toxicology vol 48 no 7 pp 1801ndash1805 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

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Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

2 Journal of Analytical Methods in Chemistry

Table 1 Studied plants

Species and voucherspecimen Family Uses

Calamintha nepeta LCE236 Lamiaceae

Leaf used as food spice (usually added to meat fish and vegetable dishes mint aroma)and for medicinal purposes (antiseptic tonic antispasmodic diaphoretic expectorantetc) [25 26]

Helichrysum italicum GDon CE233 Asteraceae

Leaf used as food spice (also known as ldquocurry plantrdquo) and for medicinal purposes(anti-inflammatory and anti-infective antiallergic etc) essential oils used in cosmetics[16 27]

Foeniculum vulgareMillCE237 Apiaceae Leaf and fruits used to flavour several kinds of dishes Also used in cosmetics and

pharmaceutical products [28]Micromeria graeca LCE238 Lamiaceae Leaf used as food spice (added to meat and vegetables)

Origanum vulgare L CE239 LamiaceaeDried plant (epigeous part) used as food spice The most common spice inMediterranean cuisine Used since ancient times for medicinal purposes (antioxidantdigestive expectorant antiseptic antispasmodic etc) [29]

Satureja montana L CE234 LamiaceaeLeaf used as food spice (usually added to meat fish and vegetable dishes) Naturalfood preservative Savory honey is a very common ingredient in folk remedies Usedfor medicinal purposes [30]

Thymus longicaulis C PreslCE235 Lamiaceae Leaf used as food spice (usually added to meat fish and vegetable dishes) Natural

food preservative Used also for medicinal purposes [31]

2 Materials and Methods

21 Plant Material Sampling and Processing Seven plantspecies (Table 1) were collected in a garrigue on the calcareoushills of Durazzano (41∘31015840N 14∘271015840E southern Italy) in winter(February 2012) spring (May 2012) summer (July 2012)and autumn (October 2012) The plants were selected basedon their occurrence in the study site Origanum vulgaresamples were not available in autumn Plant leaf samples werecollected in the field always at the same moment of the dayin order to minimize differences due to metabolites changingbased on circadian clock

Three leaf samples (biological replicates) of each plantspecies were harvested and immediately frozen in liquid N

2

in order to avoid unwanted enzymatic reactions and stored atminus80∘C up to the freeze drying process Once freeze dried theywere powdered in liquid nitrogen and stored at minus20∘C Eachsample was extracted and analysed by NMR

Voucher specimens for all the plant species were depos-ited at the herbarium of the Second University of Naples(Table 1)

22 Metabolomics Analysis Freeze-dried plant material(50mg) was transferred to a 2mL microtube NMR sampleswere prepared in a mixture of phosphate buffer (FlukaChemika 90mM pH 60) in D

2O (Cambridge Isotope

Laboratories) containing 01ww trimethylsilylpropionic-2233-119889

4acid sodium salt (TMSP Sigma-Aldrich) and

methanol-1198894(Sigma-Aldrich) A volume of 15mL of phos-

phate buffer and methanol-1198894(1 1) was added to the plant

samples The mixture was vortexed at room temperature for1min ultrasonicated (Elma Transonic Digitals) for 40minand centrifuged (Beckman Allegra 64R) at 13000 rpm for10min An aliquot of 06mL was transferred to an NMR tubeand analysed by NMR [9] NMR spectra were recorded at

25∘Con a 30003MHz for 1Hand 7545MHz for 13Con aVar-ian Mercury Plus 300 Fourier transform NMR CD

3OD was

used as the internal lock Each 1HNMR spectrum consistedof 256 scans with the following parameters 016Hzpointacquisition time (AQ) = 10 s relaxation delay (RD) = 15 s90∘ pulse width (PW) = 138 120583s A presaturation sequencewas used to suppress the residual H

2O signal Free induction

decays (FIDs) were Fourier transformed with LB = 03Hzand the resulting spectra weremanually phased and baseline-corrected and calibrated to TMSP at 00 ppm using 1HNMRprocessor (MestReNova version 802)

23 Quantitative Analysis The main metabolites identifiedin plant extracts were analyzed by quantitative analysis 1H-NMR spectra were bucketed reducing it to integral segmentswith a width of 002 ppm with ACDLABS 120 1H-NMRprocessor (ACDLABS 120 Toronto Canada) Spectra werescaled to the internal standard (whose area from minus001 to001 ppmwas set equal to 1) For eachmetabolite buckets cor-responding to nonoverlapping signals were used to calculatethe relative amount as follows

Metabolite relative amount =SA times 119899HTMSP119899

119878

(1)

where SA is the metabolite signal area but it is also equal tothe signal areastandard area ratio as standard area is equalto 1 119899HTMSP is a constant equal to 9 (the number of protonsresponsible for the signal between minus001 and 001 ppm) and 119899

119904

is the number of protons of the metabolite signal area [10]

3 Results and Discussion

Recent research has shown culinary herbs and spices asa source of bioactive compounds [11] Although most ofthem have been extensively studied for their essential oil

Journal of Analytical Methods in Chemistry 3

composition far less information is available on their polarand semipolar chemical composition

Herewith seven Mediterranean plants (Calaminthanepeta Helichrysum italicum Foeniculum vulgare Microm-eria graeca Origanum vulgare Satureja montana and Thy-mus longicaulis) have been studied for their metabolite con-tent by NMR The identification of metabolites was carriedout by comparing NMR data with an in-house library withdatabases [12] and with some literature data [10 13ndash15]1H-NMR data and extract composition are given in Table 2and spectra are shown in Figure 1

Primary metabolites were easily identified based on dataextensively reported in literature of spectra acquired in thesame solvent mixture [10 12ndash15] Among free amino acidsalanine was observed in all of the plants while threonine wasonly detected inMicromeria graeca and Foeniculum vulgare

The sugar content was highly variable with glucose andsucrose as the main free carbohydrates detected

Finally some organic acids were identified Quinic acidwas present in all of the plants but Helichrysum italicum andSatureja montana while malic acid was clearly detected in allof the plants

Concerning the secondary metabolite content the anal-ysed Lamiaceae plants were all characterized by the presenceof high amounts of rosmarinic acid (with the exception ofC nepeta) along with analogous compounds Caffeic acidwas identified based on comparison of NMR data with theliterature [10 15] and confirmed by comparison with NMRspectra of an in-house library Rosmarinic acid was identifiedbased on the comparison with already reported data [10] andthe structure was confirmed by 2D NMR analysis Indeedthe olefinic proton at 120575 750 (H7) (showing HSQC correlationwith the carbon at 120575 1459) and that at 120575 630 (showingHSQC correlation with the carbon at 120575 1143) showed longrange correlations with a carbon at 120575 1684 (C9) This carbonwas in turn correlated with the proton at 120575 502 (H81015840)confirming the linkage between a caffeoylmoiety and the 34-dihydroxyphenyl lactic acid moiety Furthermore the formerwas identified based on the long range correlation of theH7 olefinic proton with the aromatic carbon at 120575 1264 (C1)showing further correlations with the signals belonging to anorthopara trisubstituted aromatic ring (Table 2) The latterwas identified as follows the proton H81015840 showed correlationswith a carboxylic carbon at 120575 1765 and with a methylenecarbon at 120575 369 (C71015840) showing HSQC correlations with thediastereotopic protons H71015840 (Table 2) The proton H81015840 alsoshowed long range correlation with a quaternary aromaticcarbon at 120575 1300 (C11015840) in turn correlated with the signalsbelonging to a second orthopara trisubstituted aromatic ring(Table 2)

Some phenylpropanoids in the extracts were not defini-tively characterized inasmuch as based on their scarceabundance andor strong signal overlapping they did notshow clear correlations in 2D NMR spectra However thecharacteristic signals and correlations of the trans-propenylicchain suggested their presence Indeed correlations wereobserved in the HSQC among the olefinic signals withcarbons at 140ndash145 ppm (for the proton at lower fields) and at114ndash120 ppm (for the proton at higher fields) and long range

correlations were shown with carbon resonances attributableto ester carboxyl carbons and with quaternary aromaticcarbons

Calamintha nepeta extracts were also rich in severalflavonoids and phenylpropanoids Unfortunately it was notpossible to definitely characterize these compounds but allof the flavonoids were identified as apigenin derivatives(Table 2) Indeed several sets of resonances attributable tometa coupled protons H7H8 (ring A) to proton H3 andto B ring ortho coupled protons were detected Interestinglythe compounds probably characterized by a different degreeof glycosylation showed a peculiar distribution along theseasons Two apigenin derivativeswere detected in spring andautumn (apigenin derivatives 1 and 2) while two differentcouples of these compounds were detected in summer (api-genin derivatives 3 and 4) and winter (apigenin derivatives 5and 6) samples Moreover apigenin derivatives 5 and 6 weredetected only in winter also in Satureja montana

Analogously as shown in Table 2 the presence of somephenylpropanoids was strongly dependent on the collectionseason (Table 2) phenylpropanoid 2 was detected only inwinter phenylpropanoid 5 only in spring phenylpropanoid6 only in summer and phenylpropanoid 7 only in autumnsamples

The most stable metabolome along the seasons wasdetected for Thymus longicaulis while Micromeria graecaand Origanum vulgare only changed for some metabolitesHowever differences in the amounts of the compounds wereobserved Indeed for all the Lamiaceae plants a higheramount of aromatic compounds (Table 2) was observedin spring and summer samples compared to autumn andwinter samplesHelichrysum italicum and Foeniculum vulgareextracts showed an analogous behaviour with changes ofmetabolites mainly on the quantitative point of view

Helichrysum italicum extracts besides chlorogenic acidsalso showed signals attributable to a 3-hydroxybenzofuranand an isobenzofuranone derivative Chlorogenic neochlo-rogenic and dicaffeoylquinic acids were identified basedon comparison of 1H-NMR data with the literature [1015] and with the in-house library The caffeoyl moiety wasclearly identified based on 1D and 2D NMR data and thelinkage(s) with the quinic acid moiety was confirmed bythe correlation observed in the long range spectrum The 3-hydroxybenzofuran and isobenzofuranone derivatives wereidentified based on comparison with the NMR spectrum ofthe compound previously isolated [16]

Finally Foeniculum vulgare was characterized by chloro-genic acids and flavonoids identified based on 1H-NMRdata as kaempferol and quercetin [17] Chlorogenic acid wasreported for the first time from this species to the best of ourknowledge

The identification of water soluble compounds in theseplants is very important as most of them are added to disheshence they might be eaten or however they could releasebioactive compounds into food In this framework it is worthto underlining that first of all the health promoting capacityof bioactive compounds could be dependent on synergismsSecondly these plants could also contain potential toxic

4 Journal of Analytical Methods in Chemistry

Table2Mainmetabolitesd

etectedin

plantextracts

1 H-N

MRdataarem

easuredin

ppm

andcoup

lingconstants(119869)inHertzR

elativea

mou

ntisexpressedas

them

eanvalue(119899=3)plusmn

SD

Forsom

emetabolitestheq

uantitativ

eanalysis

was

notp

ossib

ledu

etostr

onglyoverlapp

ingsig

nals

hencethe

presence

isindicatedby

ldquoXrdquo

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Calamintha

nepeta

Alanine

148(H

3d119869=72

)X

XX

XAp

igenin

deriv

ative1lowast

651

(H6d119869=21)670(H

3s)678(H

8d119869=21)710

(H31015840H

51015840d119869=87)794

(H21015840H

61015840d119869=87)

852plusmn10

8525plusmn373

Apigenin

deriv

ative2lowast

654

(H6d119869=21)667(H

3s)673(H

8d119869=21)707(H

31015840H

51015840d119869=87)790

(H21015840H

61015840d119869=87)

Apigenin

deriv

ative3lowast

650

(H6d119869=21)666(H

3s)678(H

8d119869=21)702(H

31015840H

51015840d119869=87)788

(H21015840H

61015840d119869=87)

781plusmn

324

Apigenin

deriv

ative4lowast

649

(H6d119869=21)661(H3s)669(H

8d119869=21)706(H

31015840H

51015840d119869=87)784

(H21015840H

61015840d119869=87)

Apigenin

deriv

ative5lowast

655

(H6d119869=21)665(H

3s)669(H

8d119869=21)712

(H31015840H

51015840d119869=87)795

(H21015840H

61015840d119869=87)

516plusmn059

Apigenin

deriv

ative6lowast

653

(H6d119869=21)665(H

3s)666(H

8d119869=21)709(H

31015840H

51015840d119869=87)792

(H21015840H

61015840d119869=87)

Citricacid

259

(H2ad119869

=176)272(H

2bd119869

=176)

838plusmn346

1366plusmn331

804plusmn017

1344plusmn002

Glucose

459

(H1120573

d119869

=78

)519

(H1120572

d119869

=38)

589plusmn233

687plusmn299

561plusmn028

564plusmn17

1Malicacid

239

(H3add119869=15693)278

(H3add119869=15636)431

(H2dd

119869=93

36)

2672plusmn1080

3757plusmn272

3145plusmn713

Phenylprop

anoid

1597

(H8d119869=159)74

3(H

7d119869=159)

304plusmn301

594plusmn045

305plusmn062

257plusmn067

Phenylprop

anoid

2617

(H8d119869=159)73

0(H

7d119869=159)

X

Phenylprop

anoid

3631

(H8d119869=159)752(H

7d119869=159)

102plusmn099

462plusmn19

9658plusmn15

1

Phenylprop

anoid

4645

(H8d119869=159)76

7(H

7d119869=159)

XX

XX

Phenylprop

anoid

5616

(H8d119869=159)72

5(H

7d119869=159)

X

Phenylprop

anoid

6611(H

8d119869=159)737(H

7d119869=159)

X

Phenylprop

anoid

7614

(H8d119869=159)739(H

7d119869=159)

X

Quinica

cid

187(H

2am

)19

6(H

6am

)201

(H2bm

)202

(H6bm

)340

(H4ov)400

(H3

ov)411(H

5ov)

4528plusmn665

5509plusmn483

6402plusmn416

7161plusmn

884

Sucrose

415

(H31015840d119869

=84)538(H

1d119869=36)

824plusmn363

709plusmn301

1551plusmn

284

1037plusmn054

Journal of Analytical Methods in Chemistry 5

Table2Con

tinued

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Helichrysum

Italicum

Alanine

SeeC

nepeta

124plusmn019

087plusmn060

091plusmn015

151plusmn

044

Chlorogenica

cid

184ndash

220

(H2andH6qu

inicacidm

)545

(H5m)637

(H81015840d119869

=159)690

(H51015840d119869

=81)707(

H61015840dd119869=8421)715

(H21015840d119869

=21)762(

H71015840d119869

=159)

831plusmn15

590

3plusmn12

4363plusmn259

326plusmn077

Dicaffeoylqu

inic

acid

630

(H81015840d119869

=162)648

(H810158401015840

d119869

=156)76

5(H

71015840d119869

=162)76

6(H

710158401015840

d119869

=156)

1086plusmn13

81088plusmn516

1153plusmn238

783plusmn207

Glucose

SeeC

nepeta

377plusmn174

356plusmn004

273plusmn059

378plusmn201

3-OHbenzofuran

518

(H2d119869=63)522(H

3d119869=63)690(H

7ov)804

(H6dd119869=8418

)406plusmn027

313plusmn13

9537plusmn18

9406plusmn14

7Isob

enzofurano

ne533

(H3s)673(H

4d119869=18

)684

(H6d119869=18

)603plusmn270

Malicacid

SeeC

nepeta

XX

XX

Neochlorogenic

acid

639

(H81015840d119869

=159)752(H

71015840d119869

=159)

XX

XX

Sucrose

SeeC

nepeta

1693plusmn855

915plusmn457

1024plusmn445

1014plusmn375

Foenicu

lum

vulga

re

Alanine

SeeC

nepeta

117plusmn005

090plusmn049

124plusmn025

109plusmn009

Caffeicacid

629

(H81015840d119869

=159)688

(H51015840d119869

=81)703(H

61015840dd119869=8421)712

(H21015840d119869

=21)752

(H71015840d119869

=159)

XX

XX

Chlorogenica

cid

SeeH

italicum

XX

XX

Dicaffeoylqu

inic

acid

SeeH

italicum

XX

X

Glucose

SeeC

nepeta

117plusmn002

555plusmn289

577plusmn16

4629plusmn13

8GABA

(120574-aminob

utyric

acid)

192(H

3m)236

(H2t119869=75

)301

(H4t119869=75

)X

XX

Kaem

pferol

635

(H6d119869=21)652

(H8d119869=21)700(H

21015840H

61015840d119869

=84)809(H

31015840H

51015840

d119869=84)

128plusmn017

178plusmn15

614

6plusmn044

118plusmn082

Malicacid

SeeC

nepeta

4468plusmn711

4740plusmn1023

16852plusmn1115

8662plusmn349

Quercetin

627

(H6d119869=21)648(H

8d119869=21)699(H

51015840d119869=85)759(H

61015840d119869=85

21)775(H

21015840d119869=21)

193plusmn036

555plusmn361

623plusmn12

5294plusmn209

Quinica

cid

SeeC

nepeta

2509plusmn262

3161plusmn

1748

3349plusmn673

2730plusmn225

Sucrose

SeeC

nepeta

4494plusmn228

3680plusmn1385

5436plusmn1186

3062plusmn90

3Th

reon

ine

132(H

4d119869=66)

XX

XX

Microm

eria

graeca

Alanine

SeeC

nepeta

093plusmn055

140plusmn088

209plusmn089

575plusmn404

Citricacid

SeeC

nepeta

1989plusmn94

41865plusmn15

61713plusmn684

1543plusmn510

Glucose

SeeC

nepeta

481plusmn249

687plusmn299

561plusmn028

564plusmn17

1Malicacid

SeeC

nepeta

XX

XX

Quinica

cid

SeeC

nepeta

3975plusmn1068

5509plusmn483

6402plusmn416

5741plusmn

1124

Rosm

arinicacid

300

(H71015840add

119869=141

96)315

(H71015840bdd

119869=141

36)502(H

81015840dd119869=100

33)630(H

8d119869=159)671(H61015840dd119869=78

21)681(H51015840d119869

=78

)682

(H5

d119869=81)689(H

21015840d119869

=21)700(H

6dd

119869=8118

)711(H2d119869=18

)75

0(H

7d119869=159)

102plusmn099

462plusmn19

8658plusmn15

115

4plusmn079

Sucrose

SeeC

nepeta

799plusmn260

709plusmn302

1551plusmn

284

822plusmn373

Threon

ine

SeeF

vulgare

X

6 Journal of Analytical Methods in Chemistry

Table2Con

tinued

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Orig

anum

vulga

re

Apigenin

deriv

ative2

SeeC

nepeta

432plusmn024

295plusmn091

mdash

Alanine

SeeC

nepeta

058plusmn012

090plusmn017

107plusmn014

mdashCh

oline

320

(s)

XX

mdashCitricacid

SeeC

nepeta

1474plusmn10

22292plusmn413

1554plusmn19

1mdash

Glucose

SeeC

nepeta

1659plusmn264

362plusmn288

439plusmn382

mdashLithosperm

icacid

300

(H71015840aa

ndbov)6

30(H

8d119869=159)78

2(H

7d119869=159)

XX

Xmdash

Malicacid

SeeC

nepeta

2759plusmn16

93240plusmn810

1474plusmn851

mdashQuinica

cid

SeeC

nepeta

3094plusmn286

4591plusmn

183

4312plusmn98

1mdash

Rosm

arinicacid

SeeM

graeca

1147plusmn611

1573plusmn482

3550plusmn591

mdashSucrose

SeeC

nepeta

1005plusmn15

8498plusmn045

1492plusmn436

mdash

Satureja

montana

Apigenin

deriv

ative5

SeeC

nepeta

X

Apigenin

deriv

ative6

SeeC

nepeta

X

Alanine

SeeC

nepeta

XX

XX

Choline

SeeO

vulgare

XX

XCh

lorogenica

cid

SeeH

italicum

947plusmn12

01501plusmn

051

1250plusmn554

366plusmn18

4Glucose

SeeC

nepeta

101plusmn

067

396plusmn15

5287plusmn14

7427plusmn15

4Malicacid

SeeC

nepeta

3932plusmn260

3240plusmn099

855plusmn331

4876plusmn678

Rosm

arinicacid

SeeM

graeca

715plusmn12

61090plusmn16

4884plusmn432

Sucrose

SeeC

nepeta

1009plusmn093

856plusmn442

1007plusmn12

593

7plusmn391

Thym

uslongica

ulis

Alanine

SeeC

nepeta

034plusmn024

105plusmn011

108plusmn022

128plusmn007

Citricacid

SeeC

nepeta

XX

XX

Malicacid

SeeC

nepeta

2692plusmn1544

6005plusmn328

3556plusmn680

5464plusmn1472

Quinica

cid

SeeC

nepeta

1985plusmn863

4116plusmn75

040

71plusmn

830

3196plusmn317

Glucose

SeeC

nepeta

306plusmn033

320plusmn083

265plusmn17

6336plusmn14

1Ph

enylprop

anoid

8613

(H8d119869=159)74

6(H

7d119869=159)

367plusmn113

565plusmn061

477plusmn12

419

0plusmn12

4

Rosm

arinicacid

SeeM

graeca

710plusmn370

778plusmn19

81012plusmn176

487plusmn214

Sucrose

SeeC

nepeta

710plusmn370

778plusmn19

81012plusmn176

487plusmn214

Sign

almultip

licity

indicatedas

follo

wsd=do

ubletdd

=do

ubleto

fdou

bletsm

=multip

letov

=overlap

pedq=qu

artets=

singletand

t=triplet

lowast

Apigenin

deriv

atives

1and

23and45and6wereq

uantified

together

duetooverlap

ping

signals

Journal of Analytical Methods in Chemistry 7

Calamintha nepetaWi

Sp

Su

Au

1

2323 23

4545 45

67 67 6788

9 9 10 10101819

19

11121314

11121314

16

17

Helichrysum italicumWi

Sp

Su

Au

202020 202020

Foeniculum vulgareWi

Sp

Su

Au

21 21 2122 22 23 2323 24

Micromeria graecaWi

Sp

Su

Au

252525

25 25

Origanum vulgare

Wi

Sp

Su

Satureja montana

Wi

Sp

Su

Au

Thymus longicaulisWi

Sp

Su

Au

100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm)

Figure 1 1H-NMR spectra of studied plants (Au = autumn Sp = spring Su = summer Wi = winter) The main resonances of the maincompounds are indicated on the spectra as follows 1 alanine 2 apigenin derivative 1 3 apigenin derivative 2 4 apigenin derivative 3 5apigenin derivative 4 6 apigenin derivative 5 7 apigenin derivative 6 8 citric acid 9 glucose 10 malic acid 11 phenylpropanoid 1 12phenylpropanoid 2 13 phenylpropanoid 3 14 phenylpropanoid 4 15 phenylpropanoid 5 16 phenylpropanoid 6 17 phenylpropanoid 7 18quinic acid 19 sucrose 20 chlorogenic acid 21 GABA 22 kaempferol 23 quercetin 24 threonine 25 rosmarinic acid

8 Journal of Analytical Methods in Chemistry

compounds as reported for several Lamiaceae [17] Never-theless the role in nutrition of primary metabolites is oftendisregarded [18] These considerations raise the attention tothe need for a comprehensive profiling of their metabolites

Furthermore the NMR-based metabolomic approachhere proposed due to the short time of analyses and to lowercosts compared to other analytical methods was very usefulfor the study of seasonal variation of metabolites

Indeed although it is clear that secondary metabolitesshow peculiar trends only fragmentary information is avail-able mainly because of the used approaches

The most common and easiest procedure was to performthe phytochemical study on samples collected in a specifictime of the year and then compare the other months (orseasons) by setting up a series of target analyses often byHPLC [19 20] or by less time and resource consumingapproaches based on colorimetric assays [21]

The improvement of analytical methods and espe-cially the availability of a high-throughput approach likemetabolomics [5] gives the chance to further explore theissue of seasonality [22] and to thoroughly study thesechanges

Concerning themetabolites identifiedwithin the extractsit is important to underline their biological activity

Phenols are well known for their antioxidant activity [11]As the studied plants are all very rich in phenolics they havea great antioxidant potential This could also explain the useof some of these herbs as natural food preservatives (Table 1)

Among the detected compounds rosmarinic acid is themost widespread and the most abundant A plethora ofbiological activities has been attributed to this compoundamong them adstringent antioxidative anti-inflammatoryantimutagen antibacterial and antiviral [23]

Many other phenylpropanoids and flavonoids were alsodetected Several properties have been reported for thesecompounds such as anti-inflammatory antimicrobial andantitumor activity [24]

The richness in these compounds of the studied plantssupports their traditional uses

However the study evidenced that qualitative and quan-titative variations of metabolites are observed along the year

This is to the best of our knowledge the first reportof metabolite content and seasonal qualitative and quanti-tative variation of this set of food spices Furthermore it isevidenced that a higher content of compounds known fortheir health promoting capacities can be found in spring andsummer samples of all the analysed species although season-specific compounds were also detected

4 Conclusions

NMR-based metabolomics has been applied to the study ofchemical composition of selected aromatic plants ofMediter-ranean vegetation

The method allowed determining the chemical compo-sition of plant extracts in terms of primary and secondarymetabolites The abundance of aromatic secondary metabo-lites suggested that the traditional uses of these plants mightbe supported by their chemical composition

Furthermore the seasonality of the accumulation of thesemetabolites was studied

Conflict of Interests

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

Acknowledgment

The authors would like to thank the anonymous reviewers fortheir valuable suggestions

References

[1] A M Scherrer R Motti and C S Weckerle ldquoTraditional plantuse in the areas of Monte Vesole and Ascea Cilento NationalPark (Campania Southern Italy)rdquo Journal of Ethnopharmacol-ogy vol 97 no 1 pp 129ndash143 2005

[2] T Efferth and H J Greten ldquoMedicinal and aromatic plantresearch in the 21st centuryrdquoMedicinal amp Aromatic Plants vol1 article e110 2012

[3] R Rodrıguez-Solana J M Salgado J M Domınguez andS Cortes-Dieguez ldquoComparison of soxhlet accelerated sol-vent and supercritical fluid extraction techniques for volatile(GCndashMS and GCFID) and phenolic compounds (HPLCndashESIMSMS) from Lamiaceae Species rdquo Phytochemical Analysisvol 26 no 1 pp 61ndash71 2015

[4] T Fornari G Vicente E Vazquez M R Garcıa-Risco andG Reglero ldquoIsolation of essential oil from different plants andherbs by supercritical fluid extractionrdquo Journal of Chromatogra-phy A vol 1250 pp 34ndash48 2012

[5] H K Kim Y H Choi and R Verpoorte ldquoNMR-based plantmetabolomics where do we stand where do we gordquo Trends inBiotechnology vol 29 no 6 pp 267ndash275 2011

[6] M Scognamiglio B DrsquoAbrosca A Esposito and A FiorentinoldquoMetabolomics an unexplored tool for allelopathy studiesrdquoJournal of Allelochemical Interactions vol 1 pp 9ndash23 2015

[7] J W Allwood D I Ellis and R Goodacre ldquoMetabolomictechnologies and their application to the study of plants andplant-host interactionsrdquo Physiologia Plantarum vol 132 no 2pp 117ndash135 2008

[8] R R Forseth and F C Schroeder ldquoNMR-spectroscopic analysisof mixtures from structure to functionrdquo Current Opinion inChemical Biology vol 15 no 1 pp 38ndash47 2011

[9] H K Kim Y H Choi and R Verpoorte ldquoNMR-basedmetabolomic analysis of plantsrdquo Nature Protocols vol 5 no 3pp 536ndash549 2010

[10] M Scognamiglio V Fiumano B DAbrosca et al ldquoChemicalinteractions between plants in Mediterranean vegetation theinfluence of selected plant extracts on Aegilops geniculatametabolomerdquo Phytochemistry vol 106 pp 69ndash85 2014

[11] A Vallverdu-Queralt J Regueiro M Martınez-Huelamo JF R Alvarenga L N Leal and R M Lamuela-RaventosldquoA comprehensive study on the phenolic profile of widelyused culinary herbs and spices rosemary thyme oreganocinnamon cumin and bayrdquo Food Chemistry vol 154 pp 299ndash307 2014

[12] Q Cui I A Lewis A D Hegeman et al ldquoMetabolite identifi-cation via the Madison Metabolomics Consortium DatabaserdquoNature Biotechnology vol 26 no 2 pp 162ndash164 2008

Journal of Analytical Methods in Chemistry 9

[13] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissusCarltonrdquoPhytochemistry vol 88 pp 43ndash53 2013

[14] R Verpoorte Y H Choi and H K Kim ldquoNMR-basedmetabolomics at work in phytochemistryrdquo PhytochemistryReviews vol 6 no 1 pp 3ndash14 2007

[15] J-L Wolfender S Rudaz Y H Choi and H K Kim ldquoPlantmetabolomics from holistic data to relevant biomarkersrdquo Cur-rent Medicinal Chemistry vol 20 no 8 pp 1056ndash1090 2013

[16] B DrsquoAbrosca E Buommino G DrsquoAngelo et al ldquoSpectroscopicidentification and anti-biofilm properties of polar metabolitesfrom the medicinal plant Helichrysum italicum against Pseu-domonas aeruginosardquo Bioorganic and Medicinal Chemistry vol21 no 22 pp 7038ndash7046 2013

[17] S Pacifico B DrsquoAbrosca M Scognamiglio et al ldquoNMR-basedmetabolic profiling and in vitro antioxidant and hepatotoxicassessment of partially purified fractions fromGolden german-der (Teucrium polium L) methanolic extractrdquo Food Chemistryvol 135 no 3 pp 1957ndash1967 2012

[18] R Scherer A C P Rybka C A Ballus A D Meinhart J TFilho and H T Godoy ldquoValidation of a HPLC method forsimultaneous determination of main organic acids in fruits andjuicesrdquo Food Chemistry vol 135 no 1 pp 150ndash154 2012

[19] J Liimatainen M Karonen J Sinkkonen M Helander andJ-P Salminen ldquoPhenolic compounds of the inner bark ofBetula pendula seasonal and genetic variation and inductionby woundingrdquo Journal of Chemical Ecology vol 38 no 11 pp1410ndash1418 2012

[20] K Hosni K Msaada M Ben Taarit and B Marzouk ldquoPhe-nological variations of secondary metabolites from Hypericumtriquetrifolium Turrardquo Biochemical Systematics and Ecology vol39 no 1 pp 43ndash50 2011

[21] F Brahmi B Mechri S Dabbou M Dhibi and M HammamildquoThe efficacy of phenolics compounds with different polaritiesas antioxidants from olive leaves depending on seasonal varia-tionsrdquo Industrial Crops and Products vol 38 no 1 pp 146ndash1522012

[22] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissus Carltonrdquo Phytochemistry vol 88 pp 43ndash53 2013

[23] M Petersen and M S J Simmonds ldquoRosmarinic acidrdquo Phyto-chemistry vol 62 no 2 pp 121ndash125 2003

[24] R Quirantes-Pine M Herranz-Lopez L Funes et al ldquoPhenyl-propanoids and their metabolites are the major compoundsresponsible for blood-cell protection against oxidative stressafter administration of Lippia citriodora in ratsrdquo Phytomedicinevol 20 no 12 pp 1112ndash1118 2013

[25] G Tibaldi E Fontana and S Nicola ldquoPostharvest managementaffects spearmint and calamint essential oilsrdquo Journal of theScience of Food and Agriculture vol 93 no 3 pp 580ndash586 2013

[26] K Hammer G Laghetti and K Pistrick ldquoCalamintha nepeta(L) Savi and Micromeria thymifolia (Scop) Fritsch cultivatedin ItalyrdquoGenetic Resources and Crop Evolution vol 52 no 2 pp215ndash220 2005

[27] G Appendino M Ottino N Marquez et al ldquoArzanol an anti-inflammatory and anti-HIV-1 phloroglucinol 120572-pyrone fromHelichrysum italicum ssp microphyllumrdquo Journal of NaturalProducts vol 70 no 4 pp 608ndash612 2007

[28] W-R Diao Q-P Hu H Zhang and J-G Xu ldquoChemicalcomposition antibacterial activity and mechanism of action ofessential oil from seeds of fennel (Foeniculum vulgare Mill)rdquoFood Control vol 35 no 1 pp 109ndash116 2014

[29] N Martins L Barros C Santos-Buelga M Henriques S Silvaand I C F R Ferreira ldquoDecoction infusion and hydroalcoholicextract of Origanum vulgare L different performances regard-ing bioactivity and phenolic compoundsrdquo Food Chemistry vol158 pp 73ndash80 2014

[30] T Mihajilov-Krstev D Radnovic D Kitic et al ldquoChemicalcomposition antimicrobial antioxidative and anticholinester-ase activity of Satureja Montana L ssp montana essential oilrdquoCentral European Journal of Biology vol 9 no 7 pp 668ndash6772014

[31] C Sarikurkcu M Sabih Ozer M Eskici B Tepe S Can andE Mete ldquoEssential oil composition and antioxidant activity ofThymus longicaulis C Presl subsp longicaulis var longicaulisrdquoFood andChemical Toxicology vol 48 no 7 pp 1801ndash1805 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

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Journal of

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Analytical ChemistryInternational Journal of

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Journal of

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Quantum Chemistry

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Organic Chemistry International

ElectrochemistryInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Analytical Methods in Chemistry 3

composition far less information is available on their polarand semipolar chemical composition

Herewith seven Mediterranean plants (Calaminthanepeta Helichrysum italicum Foeniculum vulgare Microm-eria graeca Origanum vulgare Satureja montana and Thy-mus longicaulis) have been studied for their metabolite con-tent by NMR The identification of metabolites was carriedout by comparing NMR data with an in-house library withdatabases [12] and with some literature data [10 13ndash15]1H-NMR data and extract composition are given in Table 2and spectra are shown in Figure 1

Primary metabolites were easily identified based on dataextensively reported in literature of spectra acquired in thesame solvent mixture [10 12ndash15] Among free amino acidsalanine was observed in all of the plants while threonine wasonly detected inMicromeria graeca and Foeniculum vulgare

The sugar content was highly variable with glucose andsucrose as the main free carbohydrates detected

Finally some organic acids were identified Quinic acidwas present in all of the plants but Helichrysum italicum andSatureja montana while malic acid was clearly detected in allof the plants

Concerning the secondary metabolite content the anal-ysed Lamiaceae plants were all characterized by the presenceof high amounts of rosmarinic acid (with the exception ofC nepeta) along with analogous compounds Caffeic acidwas identified based on comparison of NMR data with theliterature [10 15] and confirmed by comparison with NMRspectra of an in-house library Rosmarinic acid was identifiedbased on the comparison with already reported data [10] andthe structure was confirmed by 2D NMR analysis Indeedthe olefinic proton at 120575 750 (H7) (showing HSQC correlationwith the carbon at 120575 1459) and that at 120575 630 (showingHSQC correlation with the carbon at 120575 1143) showed longrange correlations with a carbon at 120575 1684 (C9) This carbonwas in turn correlated with the proton at 120575 502 (H81015840)confirming the linkage between a caffeoylmoiety and the 34-dihydroxyphenyl lactic acid moiety Furthermore the formerwas identified based on the long range correlation of theH7 olefinic proton with the aromatic carbon at 120575 1264 (C1)showing further correlations with the signals belonging to anorthopara trisubstituted aromatic ring (Table 2) The latterwas identified as follows the proton H81015840 showed correlationswith a carboxylic carbon at 120575 1765 and with a methylenecarbon at 120575 369 (C71015840) showing HSQC correlations with thediastereotopic protons H71015840 (Table 2) The proton H81015840 alsoshowed long range correlation with a quaternary aromaticcarbon at 120575 1300 (C11015840) in turn correlated with the signalsbelonging to a second orthopara trisubstituted aromatic ring(Table 2)

Some phenylpropanoids in the extracts were not defini-tively characterized inasmuch as based on their scarceabundance andor strong signal overlapping they did notshow clear correlations in 2D NMR spectra However thecharacteristic signals and correlations of the trans-propenylicchain suggested their presence Indeed correlations wereobserved in the HSQC among the olefinic signals withcarbons at 140ndash145 ppm (for the proton at lower fields) and at114ndash120 ppm (for the proton at higher fields) and long range

correlations were shown with carbon resonances attributableto ester carboxyl carbons and with quaternary aromaticcarbons

Calamintha nepeta extracts were also rich in severalflavonoids and phenylpropanoids Unfortunately it was notpossible to definitely characterize these compounds but allof the flavonoids were identified as apigenin derivatives(Table 2) Indeed several sets of resonances attributable tometa coupled protons H7H8 (ring A) to proton H3 andto B ring ortho coupled protons were detected Interestinglythe compounds probably characterized by a different degreeof glycosylation showed a peculiar distribution along theseasons Two apigenin derivativeswere detected in spring andautumn (apigenin derivatives 1 and 2) while two differentcouples of these compounds were detected in summer (api-genin derivatives 3 and 4) and winter (apigenin derivatives 5and 6) samples Moreover apigenin derivatives 5 and 6 weredetected only in winter also in Satureja montana

Analogously as shown in Table 2 the presence of somephenylpropanoids was strongly dependent on the collectionseason (Table 2) phenylpropanoid 2 was detected only inwinter phenylpropanoid 5 only in spring phenylpropanoid6 only in summer and phenylpropanoid 7 only in autumnsamples

The most stable metabolome along the seasons wasdetected for Thymus longicaulis while Micromeria graecaand Origanum vulgare only changed for some metabolitesHowever differences in the amounts of the compounds wereobserved Indeed for all the Lamiaceae plants a higheramount of aromatic compounds (Table 2) was observedin spring and summer samples compared to autumn andwinter samplesHelichrysum italicum and Foeniculum vulgareextracts showed an analogous behaviour with changes ofmetabolites mainly on the quantitative point of view

Helichrysum italicum extracts besides chlorogenic acidsalso showed signals attributable to a 3-hydroxybenzofuranand an isobenzofuranone derivative Chlorogenic neochlo-rogenic and dicaffeoylquinic acids were identified basedon comparison of 1H-NMR data with the literature [1015] and with the in-house library The caffeoyl moiety wasclearly identified based on 1D and 2D NMR data and thelinkage(s) with the quinic acid moiety was confirmed bythe correlation observed in the long range spectrum The 3-hydroxybenzofuran and isobenzofuranone derivatives wereidentified based on comparison with the NMR spectrum ofthe compound previously isolated [16]

Finally Foeniculum vulgare was characterized by chloro-genic acids and flavonoids identified based on 1H-NMRdata as kaempferol and quercetin [17] Chlorogenic acid wasreported for the first time from this species to the best of ourknowledge

The identification of water soluble compounds in theseplants is very important as most of them are added to disheshence they might be eaten or however they could releasebioactive compounds into food In this framework it is worthto underlining that first of all the health promoting capacityof bioactive compounds could be dependent on synergismsSecondly these plants could also contain potential toxic

4 Journal of Analytical Methods in Chemistry

Table2Mainmetabolitesd

etectedin

plantextracts

1 H-N

MRdataarem

easuredin

ppm

andcoup

lingconstants(119869)inHertzR

elativea

mou

ntisexpressedas

them

eanvalue(119899=3)plusmn

SD

Forsom

emetabolitestheq

uantitativ

eanalysis

was

notp

ossib

ledu

etostr

onglyoverlapp

ingsig

nals

hencethe

presence

isindicatedby

ldquoXrdquo

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Calamintha

nepeta

Alanine

148(H

3d119869=72

)X

XX

XAp

igenin

deriv

ative1lowast

651

(H6d119869=21)670(H

3s)678(H

8d119869=21)710

(H31015840H

51015840d119869=87)794

(H21015840H

61015840d119869=87)

852plusmn10

8525plusmn373

Apigenin

deriv

ative2lowast

654

(H6d119869=21)667(H

3s)673(H

8d119869=21)707(H

31015840H

51015840d119869=87)790

(H21015840H

61015840d119869=87)

Apigenin

deriv

ative3lowast

650

(H6d119869=21)666(H

3s)678(H

8d119869=21)702(H

31015840H

51015840d119869=87)788

(H21015840H

61015840d119869=87)

781plusmn

324

Apigenin

deriv

ative4lowast

649

(H6d119869=21)661(H3s)669(H

8d119869=21)706(H

31015840H

51015840d119869=87)784

(H21015840H

61015840d119869=87)

Apigenin

deriv

ative5lowast

655

(H6d119869=21)665(H

3s)669(H

8d119869=21)712

(H31015840H

51015840d119869=87)795

(H21015840H

61015840d119869=87)

516plusmn059

Apigenin

deriv

ative6lowast

653

(H6d119869=21)665(H

3s)666(H

8d119869=21)709(H

31015840H

51015840d119869=87)792

(H21015840H

61015840d119869=87)

Citricacid

259

(H2ad119869

=176)272(H

2bd119869

=176)

838plusmn346

1366plusmn331

804plusmn017

1344plusmn002

Glucose

459

(H1120573

d119869

=78

)519

(H1120572

d119869

=38)

589plusmn233

687plusmn299

561plusmn028

564plusmn17

1Malicacid

239

(H3add119869=15693)278

(H3add119869=15636)431

(H2dd

119869=93

36)

2672plusmn1080

3757plusmn272

3145plusmn713

Phenylprop

anoid

1597

(H8d119869=159)74

3(H

7d119869=159)

304plusmn301

594plusmn045

305plusmn062

257plusmn067

Phenylprop

anoid

2617

(H8d119869=159)73

0(H

7d119869=159)

X

Phenylprop

anoid

3631

(H8d119869=159)752(H

7d119869=159)

102plusmn099

462plusmn19

9658plusmn15

1

Phenylprop

anoid

4645

(H8d119869=159)76

7(H

7d119869=159)

XX

XX

Phenylprop

anoid

5616

(H8d119869=159)72

5(H

7d119869=159)

X

Phenylprop

anoid

6611(H

8d119869=159)737(H

7d119869=159)

X

Phenylprop

anoid

7614

(H8d119869=159)739(H

7d119869=159)

X

Quinica

cid

187(H

2am

)19

6(H

6am

)201

(H2bm

)202

(H6bm

)340

(H4ov)400

(H3

ov)411(H

5ov)

4528plusmn665

5509plusmn483

6402plusmn416

7161plusmn

884

Sucrose

415

(H31015840d119869

=84)538(H

1d119869=36)

824plusmn363

709plusmn301

1551plusmn

284

1037plusmn054

Journal of Analytical Methods in Chemistry 5

Table2Con

tinued

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Helichrysum

Italicum

Alanine

SeeC

nepeta

124plusmn019

087plusmn060

091plusmn015

151plusmn

044

Chlorogenica

cid

184ndash

220

(H2andH6qu

inicacidm

)545

(H5m)637

(H81015840d119869

=159)690

(H51015840d119869

=81)707(

H61015840dd119869=8421)715

(H21015840d119869

=21)762(

H71015840d119869

=159)

831plusmn15

590

3plusmn12

4363plusmn259

326plusmn077

Dicaffeoylqu

inic

acid

630

(H81015840d119869

=162)648

(H810158401015840

d119869

=156)76

5(H

71015840d119869

=162)76

6(H

710158401015840

d119869

=156)

1086plusmn13

81088plusmn516

1153plusmn238

783plusmn207

Glucose

SeeC

nepeta

377plusmn174

356plusmn004

273plusmn059

378plusmn201

3-OHbenzofuran

518

(H2d119869=63)522(H

3d119869=63)690(H

7ov)804

(H6dd119869=8418

)406plusmn027

313plusmn13

9537plusmn18

9406plusmn14

7Isob

enzofurano

ne533

(H3s)673(H

4d119869=18

)684

(H6d119869=18

)603plusmn270

Malicacid

SeeC

nepeta

XX

XX

Neochlorogenic

acid

639

(H81015840d119869

=159)752(H

71015840d119869

=159)

XX

XX

Sucrose

SeeC

nepeta

1693plusmn855

915plusmn457

1024plusmn445

1014plusmn375

Foenicu

lum

vulga

re

Alanine

SeeC

nepeta

117plusmn005

090plusmn049

124plusmn025

109plusmn009

Caffeicacid

629

(H81015840d119869

=159)688

(H51015840d119869

=81)703(H

61015840dd119869=8421)712

(H21015840d119869

=21)752

(H71015840d119869

=159)

XX

XX

Chlorogenica

cid

SeeH

italicum

XX

XX

Dicaffeoylqu

inic

acid

SeeH

italicum

XX

X

Glucose

SeeC

nepeta

117plusmn002

555plusmn289

577plusmn16

4629plusmn13

8GABA

(120574-aminob

utyric

acid)

192(H

3m)236

(H2t119869=75

)301

(H4t119869=75

)X

XX

Kaem

pferol

635

(H6d119869=21)652

(H8d119869=21)700(H

21015840H

61015840d119869

=84)809(H

31015840H

51015840

d119869=84)

128plusmn017

178plusmn15

614

6plusmn044

118plusmn082

Malicacid

SeeC

nepeta

4468plusmn711

4740plusmn1023

16852plusmn1115

8662plusmn349

Quercetin

627

(H6d119869=21)648(H

8d119869=21)699(H

51015840d119869=85)759(H

61015840d119869=85

21)775(H

21015840d119869=21)

193plusmn036

555plusmn361

623plusmn12

5294plusmn209

Quinica

cid

SeeC

nepeta

2509plusmn262

3161plusmn

1748

3349plusmn673

2730plusmn225

Sucrose

SeeC

nepeta

4494plusmn228

3680plusmn1385

5436plusmn1186

3062plusmn90

3Th

reon

ine

132(H

4d119869=66)

XX

XX

Microm

eria

graeca

Alanine

SeeC

nepeta

093plusmn055

140plusmn088

209plusmn089

575plusmn404

Citricacid

SeeC

nepeta

1989plusmn94

41865plusmn15

61713plusmn684

1543plusmn510

Glucose

SeeC

nepeta

481plusmn249

687plusmn299

561plusmn028

564plusmn17

1Malicacid

SeeC

nepeta

XX

XX

Quinica

cid

SeeC

nepeta

3975plusmn1068

5509plusmn483

6402plusmn416

5741plusmn

1124

Rosm

arinicacid

300

(H71015840add

119869=141

96)315

(H71015840bdd

119869=141

36)502(H

81015840dd119869=100

33)630(H

8d119869=159)671(H61015840dd119869=78

21)681(H51015840d119869

=78

)682

(H5

d119869=81)689(H

21015840d119869

=21)700(H

6dd

119869=8118

)711(H2d119869=18

)75

0(H

7d119869=159)

102plusmn099

462plusmn19

8658plusmn15

115

4plusmn079

Sucrose

SeeC

nepeta

799plusmn260

709plusmn302

1551plusmn

284

822plusmn373

Threon

ine

SeeF

vulgare

X

6 Journal of Analytical Methods in Chemistry

Table2Con

tinued

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Orig

anum

vulga

re

Apigenin

deriv

ative2

SeeC

nepeta

432plusmn024

295plusmn091

mdash

Alanine

SeeC

nepeta

058plusmn012

090plusmn017

107plusmn014

mdashCh

oline

320

(s)

XX

mdashCitricacid

SeeC

nepeta

1474plusmn10

22292plusmn413

1554plusmn19

1mdash

Glucose

SeeC

nepeta

1659plusmn264

362plusmn288

439plusmn382

mdashLithosperm

icacid

300

(H71015840aa

ndbov)6

30(H

8d119869=159)78

2(H

7d119869=159)

XX

Xmdash

Malicacid

SeeC

nepeta

2759plusmn16

93240plusmn810

1474plusmn851

mdashQuinica

cid

SeeC

nepeta

3094plusmn286

4591plusmn

183

4312plusmn98

1mdash

Rosm

arinicacid

SeeM

graeca

1147plusmn611

1573plusmn482

3550plusmn591

mdashSucrose

SeeC

nepeta

1005plusmn15

8498plusmn045

1492plusmn436

mdash

Satureja

montana

Apigenin

deriv

ative5

SeeC

nepeta

X

Apigenin

deriv

ative6

SeeC

nepeta

X

Alanine

SeeC

nepeta

XX

XX

Choline

SeeO

vulgare

XX

XCh

lorogenica

cid

SeeH

italicum

947plusmn12

01501plusmn

051

1250plusmn554

366plusmn18

4Glucose

SeeC

nepeta

101plusmn

067

396plusmn15

5287plusmn14

7427plusmn15

4Malicacid

SeeC

nepeta

3932plusmn260

3240plusmn099

855plusmn331

4876plusmn678

Rosm

arinicacid

SeeM

graeca

715plusmn12

61090plusmn16

4884plusmn432

Sucrose

SeeC

nepeta

1009plusmn093

856plusmn442

1007plusmn12

593

7plusmn391

Thym

uslongica

ulis

Alanine

SeeC

nepeta

034plusmn024

105plusmn011

108plusmn022

128plusmn007

Citricacid

SeeC

nepeta

XX

XX

Malicacid

SeeC

nepeta

2692plusmn1544

6005plusmn328

3556plusmn680

5464plusmn1472

Quinica

cid

SeeC

nepeta

1985plusmn863

4116plusmn75

040

71plusmn

830

3196plusmn317

Glucose

SeeC

nepeta

306plusmn033

320plusmn083

265plusmn17

6336plusmn14

1Ph

enylprop

anoid

8613

(H8d119869=159)74

6(H

7d119869=159)

367plusmn113

565plusmn061

477plusmn12

419

0plusmn12

4

Rosm

arinicacid

SeeM

graeca

710plusmn370

778plusmn19

81012plusmn176

487plusmn214

Sucrose

SeeC

nepeta

710plusmn370

778plusmn19

81012plusmn176

487plusmn214

Sign

almultip

licity

indicatedas

follo

wsd=do

ubletdd

=do

ubleto

fdou

bletsm

=multip

letov

=overlap

pedq=qu

artets=

singletand

t=triplet

lowast

Apigenin

deriv

atives

1and

23and45and6wereq

uantified

together

duetooverlap

ping

signals

Journal of Analytical Methods in Chemistry 7

Calamintha nepetaWi

Sp

Su

Au

1

2323 23

4545 45

67 67 6788

9 9 10 10101819

19

11121314

11121314

16

17

Helichrysum italicumWi

Sp

Su

Au

202020 202020

Foeniculum vulgareWi

Sp

Su

Au

21 21 2122 22 23 2323 24

Micromeria graecaWi

Sp

Su

Au

252525

25 25

Origanum vulgare

Wi

Sp

Su

Satureja montana

Wi

Sp

Su

Au

Thymus longicaulisWi

Sp

Su

Au

100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm)

Figure 1 1H-NMR spectra of studied plants (Au = autumn Sp = spring Su = summer Wi = winter) The main resonances of the maincompounds are indicated on the spectra as follows 1 alanine 2 apigenin derivative 1 3 apigenin derivative 2 4 apigenin derivative 3 5apigenin derivative 4 6 apigenin derivative 5 7 apigenin derivative 6 8 citric acid 9 glucose 10 malic acid 11 phenylpropanoid 1 12phenylpropanoid 2 13 phenylpropanoid 3 14 phenylpropanoid 4 15 phenylpropanoid 5 16 phenylpropanoid 6 17 phenylpropanoid 7 18quinic acid 19 sucrose 20 chlorogenic acid 21 GABA 22 kaempferol 23 quercetin 24 threonine 25 rosmarinic acid

8 Journal of Analytical Methods in Chemistry

compounds as reported for several Lamiaceae [17] Never-theless the role in nutrition of primary metabolites is oftendisregarded [18] These considerations raise the attention tothe need for a comprehensive profiling of their metabolites

Furthermore the NMR-based metabolomic approachhere proposed due to the short time of analyses and to lowercosts compared to other analytical methods was very usefulfor the study of seasonal variation of metabolites

Indeed although it is clear that secondary metabolitesshow peculiar trends only fragmentary information is avail-able mainly because of the used approaches

The most common and easiest procedure was to performthe phytochemical study on samples collected in a specifictime of the year and then compare the other months (orseasons) by setting up a series of target analyses often byHPLC [19 20] or by less time and resource consumingapproaches based on colorimetric assays [21]

The improvement of analytical methods and espe-cially the availability of a high-throughput approach likemetabolomics [5] gives the chance to further explore theissue of seasonality [22] and to thoroughly study thesechanges

Concerning themetabolites identifiedwithin the extractsit is important to underline their biological activity

Phenols are well known for their antioxidant activity [11]As the studied plants are all very rich in phenolics they havea great antioxidant potential This could also explain the useof some of these herbs as natural food preservatives (Table 1)

Among the detected compounds rosmarinic acid is themost widespread and the most abundant A plethora ofbiological activities has been attributed to this compoundamong them adstringent antioxidative anti-inflammatoryantimutagen antibacterial and antiviral [23]

Many other phenylpropanoids and flavonoids were alsodetected Several properties have been reported for thesecompounds such as anti-inflammatory antimicrobial andantitumor activity [24]

The richness in these compounds of the studied plantssupports their traditional uses

However the study evidenced that qualitative and quan-titative variations of metabolites are observed along the year

This is to the best of our knowledge the first reportof metabolite content and seasonal qualitative and quanti-tative variation of this set of food spices Furthermore it isevidenced that a higher content of compounds known fortheir health promoting capacities can be found in spring andsummer samples of all the analysed species although season-specific compounds were also detected

4 Conclusions

NMR-based metabolomics has been applied to the study ofchemical composition of selected aromatic plants ofMediter-ranean vegetation

The method allowed determining the chemical compo-sition of plant extracts in terms of primary and secondarymetabolites The abundance of aromatic secondary metabo-lites suggested that the traditional uses of these plants mightbe supported by their chemical composition

Furthermore the seasonality of the accumulation of thesemetabolites was studied

Conflict of Interests

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

Acknowledgment

The authors would like to thank the anonymous reviewers fortheir valuable suggestions

References

[1] A M Scherrer R Motti and C S Weckerle ldquoTraditional plantuse in the areas of Monte Vesole and Ascea Cilento NationalPark (Campania Southern Italy)rdquo Journal of Ethnopharmacol-ogy vol 97 no 1 pp 129ndash143 2005

[2] T Efferth and H J Greten ldquoMedicinal and aromatic plantresearch in the 21st centuryrdquoMedicinal amp Aromatic Plants vol1 article e110 2012

[3] R Rodrıguez-Solana J M Salgado J M Domınguez andS Cortes-Dieguez ldquoComparison of soxhlet accelerated sol-vent and supercritical fluid extraction techniques for volatile(GCndashMS and GCFID) and phenolic compounds (HPLCndashESIMSMS) from Lamiaceae Species rdquo Phytochemical Analysisvol 26 no 1 pp 61ndash71 2015

[4] T Fornari G Vicente E Vazquez M R Garcıa-Risco andG Reglero ldquoIsolation of essential oil from different plants andherbs by supercritical fluid extractionrdquo Journal of Chromatogra-phy A vol 1250 pp 34ndash48 2012

[5] H K Kim Y H Choi and R Verpoorte ldquoNMR-based plantmetabolomics where do we stand where do we gordquo Trends inBiotechnology vol 29 no 6 pp 267ndash275 2011

[6] M Scognamiglio B DrsquoAbrosca A Esposito and A FiorentinoldquoMetabolomics an unexplored tool for allelopathy studiesrdquoJournal of Allelochemical Interactions vol 1 pp 9ndash23 2015

[7] J W Allwood D I Ellis and R Goodacre ldquoMetabolomictechnologies and their application to the study of plants andplant-host interactionsrdquo Physiologia Plantarum vol 132 no 2pp 117ndash135 2008

[8] R R Forseth and F C Schroeder ldquoNMR-spectroscopic analysisof mixtures from structure to functionrdquo Current Opinion inChemical Biology vol 15 no 1 pp 38ndash47 2011

[9] H K Kim Y H Choi and R Verpoorte ldquoNMR-basedmetabolomic analysis of plantsrdquo Nature Protocols vol 5 no 3pp 536ndash549 2010

[10] M Scognamiglio V Fiumano B DAbrosca et al ldquoChemicalinteractions between plants in Mediterranean vegetation theinfluence of selected plant extracts on Aegilops geniculatametabolomerdquo Phytochemistry vol 106 pp 69ndash85 2014

[11] A Vallverdu-Queralt J Regueiro M Martınez-Huelamo JF R Alvarenga L N Leal and R M Lamuela-RaventosldquoA comprehensive study on the phenolic profile of widelyused culinary herbs and spices rosemary thyme oreganocinnamon cumin and bayrdquo Food Chemistry vol 154 pp 299ndash307 2014

[12] Q Cui I A Lewis A D Hegeman et al ldquoMetabolite identifi-cation via the Madison Metabolomics Consortium DatabaserdquoNature Biotechnology vol 26 no 2 pp 162ndash164 2008

Journal of Analytical Methods in Chemistry 9

[13] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissusCarltonrdquoPhytochemistry vol 88 pp 43ndash53 2013

[14] R Verpoorte Y H Choi and H K Kim ldquoNMR-basedmetabolomics at work in phytochemistryrdquo PhytochemistryReviews vol 6 no 1 pp 3ndash14 2007

[15] J-L Wolfender S Rudaz Y H Choi and H K Kim ldquoPlantmetabolomics from holistic data to relevant biomarkersrdquo Cur-rent Medicinal Chemistry vol 20 no 8 pp 1056ndash1090 2013

[16] B DrsquoAbrosca E Buommino G DrsquoAngelo et al ldquoSpectroscopicidentification and anti-biofilm properties of polar metabolitesfrom the medicinal plant Helichrysum italicum against Pseu-domonas aeruginosardquo Bioorganic and Medicinal Chemistry vol21 no 22 pp 7038ndash7046 2013

[17] S Pacifico B DrsquoAbrosca M Scognamiglio et al ldquoNMR-basedmetabolic profiling and in vitro antioxidant and hepatotoxicassessment of partially purified fractions fromGolden german-der (Teucrium polium L) methanolic extractrdquo Food Chemistryvol 135 no 3 pp 1957ndash1967 2012

[18] R Scherer A C P Rybka C A Ballus A D Meinhart J TFilho and H T Godoy ldquoValidation of a HPLC method forsimultaneous determination of main organic acids in fruits andjuicesrdquo Food Chemistry vol 135 no 1 pp 150ndash154 2012

[19] J Liimatainen M Karonen J Sinkkonen M Helander andJ-P Salminen ldquoPhenolic compounds of the inner bark ofBetula pendula seasonal and genetic variation and inductionby woundingrdquo Journal of Chemical Ecology vol 38 no 11 pp1410ndash1418 2012

[20] K Hosni K Msaada M Ben Taarit and B Marzouk ldquoPhe-nological variations of secondary metabolites from Hypericumtriquetrifolium Turrardquo Biochemical Systematics and Ecology vol39 no 1 pp 43ndash50 2011

[21] F Brahmi B Mechri S Dabbou M Dhibi and M HammamildquoThe efficacy of phenolics compounds with different polaritiesas antioxidants from olive leaves depending on seasonal varia-tionsrdquo Industrial Crops and Products vol 38 no 1 pp 146ndash1522012

[22] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissus Carltonrdquo Phytochemistry vol 88 pp 43ndash53 2013

[23] M Petersen and M S J Simmonds ldquoRosmarinic acidrdquo Phyto-chemistry vol 62 no 2 pp 121ndash125 2003

[24] R Quirantes-Pine M Herranz-Lopez L Funes et al ldquoPhenyl-propanoids and their metabolites are the major compoundsresponsible for blood-cell protection against oxidative stressafter administration of Lippia citriodora in ratsrdquo Phytomedicinevol 20 no 12 pp 1112ndash1118 2013

[25] G Tibaldi E Fontana and S Nicola ldquoPostharvest managementaffects spearmint and calamint essential oilsrdquo Journal of theScience of Food and Agriculture vol 93 no 3 pp 580ndash586 2013

[26] K Hammer G Laghetti and K Pistrick ldquoCalamintha nepeta(L) Savi and Micromeria thymifolia (Scop) Fritsch cultivatedin ItalyrdquoGenetic Resources and Crop Evolution vol 52 no 2 pp215ndash220 2005

[27] G Appendino M Ottino N Marquez et al ldquoArzanol an anti-inflammatory and anti-HIV-1 phloroglucinol 120572-pyrone fromHelichrysum italicum ssp microphyllumrdquo Journal of NaturalProducts vol 70 no 4 pp 608ndash612 2007

[28] W-R Diao Q-P Hu H Zhang and J-G Xu ldquoChemicalcomposition antibacterial activity and mechanism of action ofessential oil from seeds of fennel (Foeniculum vulgare Mill)rdquoFood Control vol 35 no 1 pp 109ndash116 2014

[29] N Martins L Barros C Santos-Buelga M Henriques S Silvaand I C F R Ferreira ldquoDecoction infusion and hydroalcoholicextract of Origanum vulgare L different performances regard-ing bioactivity and phenolic compoundsrdquo Food Chemistry vol158 pp 73ndash80 2014

[30] T Mihajilov-Krstev D Radnovic D Kitic et al ldquoChemicalcomposition antimicrobial antioxidative and anticholinester-ase activity of Satureja Montana L ssp montana essential oilrdquoCentral European Journal of Biology vol 9 no 7 pp 668ndash6772014

[31] C Sarikurkcu M Sabih Ozer M Eskici B Tepe S Can andE Mete ldquoEssential oil composition and antioxidant activity ofThymus longicaulis C Presl subsp longicaulis var longicaulisrdquoFood andChemical Toxicology vol 48 no 7 pp 1801ndash1805 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 Journal of Analytical Methods in Chemistry

Table2Mainmetabolitesd

etectedin

plantextracts

1 H-N

MRdataarem

easuredin

ppm

andcoup

lingconstants(119869)inHertzR

elativea

mou

ntisexpressedas

them

eanvalue(119899=3)plusmn

SD

Forsom

emetabolitestheq

uantitativ

eanalysis

was

notp

ossib

ledu

etostr

onglyoverlapp

ingsig

nals

hencethe

presence

isindicatedby

ldquoXrdquo

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Calamintha

nepeta

Alanine

148(H

3d119869=72

)X

XX

XAp

igenin

deriv

ative1lowast

651

(H6d119869=21)670(H

3s)678(H

8d119869=21)710

(H31015840H

51015840d119869=87)794

(H21015840H

61015840d119869=87)

852plusmn10

8525plusmn373

Apigenin

deriv

ative2lowast

654

(H6d119869=21)667(H

3s)673(H

8d119869=21)707(H

31015840H

51015840d119869=87)790

(H21015840H

61015840d119869=87)

Apigenin

deriv

ative3lowast

650

(H6d119869=21)666(H

3s)678(H

8d119869=21)702(H

31015840H

51015840d119869=87)788

(H21015840H

61015840d119869=87)

781plusmn

324

Apigenin

deriv

ative4lowast

649

(H6d119869=21)661(H3s)669(H

8d119869=21)706(H

31015840H

51015840d119869=87)784

(H21015840H

61015840d119869=87)

Apigenin

deriv

ative5lowast

655

(H6d119869=21)665(H

3s)669(H

8d119869=21)712

(H31015840H

51015840d119869=87)795

(H21015840H

61015840d119869=87)

516plusmn059

Apigenin

deriv

ative6lowast

653

(H6d119869=21)665(H

3s)666(H

8d119869=21)709(H

31015840H

51015840d119869=87)792

(H21015840H

61015840d119869=87)

Citricacid

259

(H2ad119869

=176)272(H

2bd119869

=176)

838plusmn346

1366plusmn331

804plusmn017

1344plusmn002

Glucose

459

(H1120573

d119869

=78

)519

(H1120572

d119869

=38)

589plusmn233

687plusmn299

561plusmn028

564plusmn17

1Malicacid

239

(H3add119869=15693)278

(H3add119869=15636)431

(H2dd

119869=93

36)

2672plusmn1080

3757plusmn272

3145plusmn713

Phenylprop

anoid

1597

(H8d119869=159)74

3(H

7d119869=159)

304plusmn301

594plusmn045

305plusmn062

257plusmn067

Phenylprop

anoid

2617

(H8d119869=159)73

0(H

7d119869=159)

X

Phenylprop

anoid

3631

(H8d119869=159)752(H

7d119869=159)

102plusmn099

462plusmn19

9658plusmn15

1

Phenylprop

anoid

4645

(H8d119869=159)76

7(H

7d119869=159)

XX

XX

Phenylprop

anoid

5616

(H8d119869=159)72

5(H

7d119869=159)

X

Phenylprop

anoid

6611(H

8d119869=159)737(H

7d119869=159)

X

Phenylprop

anoid

7614

(H8d119869=159)739(H

7d119869=159)

X

Quinica

cid

187(H

2am

)19

6(H

6am

)201

(H2bm

)202

(H6bm

)340

(H4ov)400

(H3

ov)411(H

5ov)

4528plusmn665

5509plusmn483

6402plusmn416

7161plusmn

884

Sucrose

415

(H31015840d119869

=84)538(H

1d119869=36)

824plusmn363

709plusmn301

1551plusmn

284

1037plusmn054

Journal of Analytical Methods in Chemistry 5

Table2Con

tinued

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Helichrysum

Italicum

Alanine

SeeC

nepeta

124plusmn019

087plusmn060

091plusmn015

151plusmn

044

Chlorogenica

cid

184ndash

220

(H2andH6qu

inicacidm

)545

(H5m)637

(H81015840d119869

=159)690

(H51015840d119869

=81)707(

H61015840dd119869=8421)715

(H21015840d119869

=21)762(

H71015840d119869

=159)

831plusmn15

590

3plusmn12

4363plusmn259

326plusmn077

Dicaffeoylqu

inic

acid

630

(H81015840d119869

=162)648

(H810158401015840

d119869

=156)76

5(H

71015840d119869

=162)76

6(H

710158401015840

d119869

=156)

1086plusmn13

81088plusmn516

1153plusmn238

783plusmn207

Glucose

SeeC

nepeta

377plusmn174

356plusmn004

273plusmn059

378plusmn201

3-OHbenzofuran

518

(H2d119869=63)522(H

3d119869=63)690(H

7ov)804

(H6dd119869=8418

)406plusmn027

313plusmn13

9537plusmn18

9406plusmn14

7Isob

enzofurano

ne533

(H3s)673(H

4d119869=18

)684

(H6d119869=18

)603plusmn270

Malicacid

SeeC

nepeta

XX

XX

Neochlorogenic

acid

639

(H81015840d119869

=159)752(H

71015840d119869

=159)

XX

XX

Sucrose

SeeC

nepeta

1693plusmn855

915plusmn457

1024plusmn445

1014plusmn375

Foenicu

lum

vulga

re

Alanine

SeeC

nepeta

117plusmn005

090plusmn049

124plusmn025

109plusmn009

Caffeicacid

629

(H81015840d119869

=159)688

(H51015840d119869

=81)703(H

61015840dd119869=8421)712

(H21015840d119869

=21)752

(H71015840d119869

=159)

XX

XX

Chlorogenica

cid

SeeH

italicum

XX

XX

Dicaffeoylqu

inic

acid

SeeH

italicum

XX

X

Glucose

SeeC

nepeta

117plusmn002

555plusmn289

577plusmn16

4629plusmn13

8GABA

(120574-aminob

utyric

acid)

192(H

3m)236

(H2t119869=75

)301

(H4t119869=75

)X

XX

Kaem

pferol

635

(H6d119869=21)652

(H8d119869=21)700(H

21015840H

61015840d119869

=84)809(H

31015840H

51015840

d119869=84)

128plusmn017

178plusmn15

614

6plusmn044

118plusmn082

Malicacid

SeeC

nepeta

4468plusmn711

4740plusmn1023

16852plusmn1115

8662plusmn349

Quercetin

627

(H6d119869=21)648(H

8d119869=21)699(H

51015840d119869=85)759(H

61015840d119869=85

21)775(H

21015840d119869=21)

193plusmn036

555plusmn361

623plusmn12

5294plusmn209

Quinica

cid

SeeC

nepeta

2509plusmn262

3161plusmn

1748

3349plusmn673

2730plusmn225

Sucrose

SeeC

nepeta

4494plusmn228

3680plusmn1385

5436plusmn1186

3062plusmn90

3Th

reon

ine

132(H

4d119869=66)

XX

XX

Microm

eria

graeca

Alanine

SeeC

nepeta

093plusmn055

140plusmn088

209plusmn089

575plusmn404

Citricacid

SeeC

nepeta

1989plusmn94

41865plusmn15

61713plusmn684

1543plusmn510

Glucose

SeeC

nepeta

481plusmn249

687plusmn299

561plusmn028

564plusmn17

1Malicacid

SeeC

nepeta

XX

XX

Quinica

cid

SeeC

nepeta

3975plusmn1068

5509plusmn483

6402plusmn416

5741plusmn

1124

Rosm

arinicacid

300

(H71015840add

119869=141

96)315

(H71015840bdd

119869=141

36)502(H

81015840dd119869=100

33)630(H

8d119869=159)671(H61015840dd119869=78

21)681(H51015840d119869

=78

)682

(H5

d119869=81)689(H

21015840d119869

=21)700(H

6dd

119869=8118

)711(H2d119869=18

)75

0(H

7d119869=159)

102plusmn099

462plusmn19

8658plusmn15

115

4plusmn079

Sucrose

SeeC

nepeta

799plusmn260

709plusmn302

1551plusmn

284

822plusmn373

Threon

ine

SeeF

vulgare

X

6 Journal of Analytical Methods in Chemistry

Table2Con

tinued

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Orig

anum

vulga

re

Apigenin

deriv

ative2

SeeC

nepeta

432plusmn024

295plusmn091

mdash

Alanine

SeeC

nepeta

058plusmn012

090plusmn017

107plusmn014

mdashCh

oline

320

(s)

XX

mdashCitricacid

SeeC

nepeta

1474plusmn10

22292plusmn413

1554plusmn19

1mdash

Glucose

SeeC

nepeta

1659plusmn264

362plusmn288

439plusmn382

mdashLithosperm

icacid

300

(H71015840aa

ndbov)6

30(H

8d119869=159)78

2(H

7d119869=159)

XX

Xmdash

Malicacid

SeeC

nepeta

2759plusmn16

93240plusmn810

1474plusmn851

mdashQuinica

cid

SeeC

nepeta

3094plusmn286

4591plusmn

183

4312plusmn98

1mdash

Rosm

arinicacid

SeeM

graeca

1147plusmn611

1573plusmn482

3550plusmn591

mdashSucrose

SeeC

nepeta

1005plusmn15

8498plusmn045

1492plusmn436

mdash

Satureja

montana

Apigenin

deriv

ative5

SeeC

nepeta

X

Apigenin

deriv

ative6

SeeC

nepeta

X

Alanine

SeeC

nepeta

XX

XX

Choline

SeeO

vulgare

XX

XCh

lorogenica

cid

SeeH

italicum

947plusmn12

01501plusmn

051

1250plusmn554

366plusmn18

4Glucose

SeeC

nepeta

101plusmn

067

396plusmn15

5287plusmn14

7427plusmn15

4Malicacid

SeeC

nepeta

3932plusmn260

3240plusmn099

855plusmn331

4876plusmn678

Rosm

arinicacid

SeeM

graeca

715plusmn12

61090plusmn16

4884plusmn432

Sucrose

SeeC

nepeta

1009plusmn093

856plusmn442

1007plusmn12

593

7plusmn391

Thym

uslongica

ulis

Alanine

SeeC

nepeta

034plusmn024

105plusmn011

108plusmn022

128plusmn007

Citricacid

SeeC

nepeta

XX

XX

Malicacid

SeeC

nepeta

2692plusmn1544

6005plusmn328

3556plusmn680

5464plusmn1472

Quinica

cid

SeeC

nepeta

1985plusmn863

4116plusmn75

040

71plusmn

830

3196plusmn317

Glucose

SeeC

nepeta

306plusmn033

320plusmn083

265plusmn17

6336plusmn14

1Ph

enylprop

anoid

8613

(H8d119869=159)74

6(H

7d119869=159)

367plusmn113

565plusmn061

477plusmn12

419

0plusmn12

4

Rosm

arinicacid

SeeM

graeca

710plusmn370

778plusmn19

81012plusmn176

487plusmn214

Sucrose

SeeC

nepeta

710plusmn370

778plusmn19

81012plusmn176

487plusmn214

Sign

almultip

licity

indicatedas

follo

wsd=do

ubletdd

=do

ubleto

fdou

bletsm

=multip

letov

=overlap

pedq=qu

artets=

singletand

t=triplet

lowast

Apigenin

deriv

atives

1and

23and45and6wereq

uantified

together

duetooverlap

ping

signals

Journal of Analytical Methods in Chemistry 7

Calamintha nepetaWi

Sp

Su

Au

1

2323 23

4545 45

67 67 6788

9 9 10 10101819

19

11121314

11121314

16

17

Helichrysum italicumWi

Sp

Su

Au

202020 202020

Foeniculum vulgareWi

Sp

Su

Au

21 21 2122 22 23 2323 24

Micromeria graecaWi

Sp

Su

Au

252525

25 25

Origanum vulgare

Wi

Sp

Su

Satureja montana

Wi

Sp

Su

Au

Thymus longicaulisWi

Sp

Su

Au

100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm)

Figure 1 1H-NMR spectra of studied plants (Au = autumn Sp = spring Su = summer Wi = winter) The main resonances of the maincompounds are indicated on the spectra as follows 1 alanine 2 apigenin derivative 1 3 apigenin derivative 2 4 apigenin derivative 3 5apigenin derivative 4 6 apigenin derivative 5 7 apigenin derivative 6 8 citric acid 9 glucose 10 malic acid 11 phenylpropanoid 1 12phenylpropanoid 2 13 phenylpropanoid 3 14 phenylpropanoid 4 15 phenylpropanoid 5 16 phenylpropanoid 6 17 phenylpropanoid 7 18quinic acid 19 sucrose 20 chlorogenic acid 21 GABA 22 kaempferol 23 quercetin 24 threonine 25 rosmarinic acid

8 Journal of Analytical Methods in Chemistry

compounds as reported for several Lamiaceae [17] Never-theless the role in nutrition of primary metabolites is oftendisregarded [18] These considerations raise the attention tothe need for a comprehensive profiling of their metabolites

Furthermore the NMR-based metabolomic approachhere proposed due to the short time of analyses and to lowercosts compared to other analytical methods was very usefulfor the study of seasonal variation of metabolites

Indeed although it is clear that secondary metabolitesshow peculiar trends only fragmentary information is avail-able mainly because of the used approaches

The most common and easiest procedure was to performthe phytochemical study on samples collected in a specifictime of the year and then compare the other months (orseasons) by setting up a series of target analyses often byHPLC [19 20] or by less time and resource consumingapproaches based on colorimetric assays [21]

The improvement of analytical methods and espe-cially the availability of a high-throughput approach likemetabolomics [5] gives the chance to further explore theissue of seasonality [22] and to thoroughly study thesechanges

Concerning themetabolites identifiedwithin the extractsit is important to underline their biological activity

Phenols are well known for their antioxidant activity [11]As the studied plants are all very rich in phenolics they havea great antioxidant potential This could also explain the useof some of these herbs as natural food preservatives (Table 1)

Among the detected compounds rosmarinic acid is themost widespread and the most abundant A plethora ofbiological activities has been attributed to this compoundamong them adstringent antioxidative anti-inflammatoryantimutagen antibacterial and antiviral [23]

Many other phenylpropanoids and flavonoids were alsodetected Several properties have been reported for thesecompounds such as anti-inflammatory antimicrobial andantitumor activity [24]

The richness in these compounds of the studied plantssupports their traditional uses

However the study evidenced that qualitative and quan-titative variations of metabolites are observed along the year

This is to the best of our knowledge the first reportof metabolite content and seasonal qualitative and quanti-tative variation of this set of food spices Furthermore it isevidenced that a higher content of compounds known fortheir health promoting capacities can be found in spring andsummer samples of all the analysed species although season-specific compounds were also detected

4 Conclusions

NMR-based metabolomics has been applied to the study ofchemical composition of selected aromatic plants ofMediter-ranean vegetation

The method allowed determining the chemical compo-sition of plant extracts in terms of primary and secondarymetabolites The abundance of aromatic secondary metabo-lites suggested that the traditional uses of these plants mightbe supported by their chemical composition

Furthermore the seasonality of the accumulation of thesemetabolites was studied

Conflict of Interests

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

Acknowledgment

The authors would like to thank the anonymous reviewers fortheir valuable suggestions

References

[1] A M Scherrer R Motti and C S Weckerle ldquoTraditional plantuse in the areas of Monte Vesole and Ascea Cilento NationalPark (Campania Southern Italy)rdquo Journal of Ethnopharmacol-ogy vol 97 no 1 pp 129ndash143 2005

[2] T Efferth and H J Greten ldquoMedicinal and aromatic plantresearch in the 21st centuryrdquoMedicinal amp Aromatic Plants vol1 article e110 2012

[3] R Rodrıguez-Solana J M Salgado J M Domınguez andS Cortes-Dieguez ldquoComparison of soxhlet accelerated sol-vent and supercritical fluid extraction techniques for volatile(GCndashMS and GCFID) and phenolic compounds (HPLCndashESIMSMS) from Lamiaceae Species rdquo Phytochemical Analysisvol 26 no 1 pp 61ndash71 2015

[4] T Fornari G Vicente E Vazquez M R Garcıa-Risco andG Reglero ldquoIsolation of essential oil from different plants andherbs by supercritical fluid extractionrdquo Journal of Chromatogra-phy A vol 1250 pp 34ndash48 2012

[5] H K Kim Y H Choi and R Verpoorte ldquoNMR-based plantmetabolomics where do we stand where do we gordquo Trends inBiotechnology vol 29 no 6 pp 267ndash275 2011

[6] M Scognamiglio B DrsquoAbrosca A Esposito and A FiorentinoldquoMetabolomics an unexplored tool for allelopathy studiesrdquoJournal of Allelochemical Interactions vol 1 pp 9ndash23 2015

[7] J W Allwood D I Ellis and R Goodacre ldquoMetabolomictechnologies and their application to the study of plants andplant-host interactionsrdquo Physiologia Plantarum vol 132 no 2pp 117ndash135 2008

[8] R R Forseth and F C Schroeder ldquoNMR-spectroscopic analysisof mixtures from structure to functionrdquo Current Opinion inChemical Biology vol 15 no 1 pp 38ndash47 2011

[9] H K Kim Y H Choi and R Verpoorte ldquoNMR-basedmetabolomic analysis of plantsrdquo Nature Protocols vol 5 no 3pp 536ndash549 2010

[10] M Scognamiglio V Fiumano B DAbrosca et al ldquoChemicalinteractions between plants in Mediterranean vegetation theinfluence of selected plant extracts on Aegilops geniculatametabolomerdquo Phytochemistry vol 106 pp 69ndash85 2014

[11] A Vallverdu-Queralt J Regueiro M Martınez-Huelamo JF R Alvarenga L N Leal and R M Lamuela-RaventosldquoA comprehensive study on the phenolic profile of widelyused culinary herbs and spices rosemary thyme oreganocinnamon cumin and bayrdquo Food Chemistry vol 154 pp 299ndash307 2014

[12] Q Cui I A Lewis A D Hegeman et al ldquoMetabolite identifi-cation via the Madison Metabolomics Consortium DatabaserdquoNature Biotechnology vol 26 no 2 pp 162ndash164 2008

Journal of Analytical Methods in Chemistry 9

[13] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissusCarltonrdquoPhytochemistry vol 88 pp 43ndash53 2013

[14] R Verpoorte Y H Choi and H K Kim ldquoNMR-basedmetabolomics at work in phytochemistryrdquo PhytochemistryReviews vol 6 no 1 pp 3ndash14 2007

[15] J-L Wolfender S Rudaz Y H Choi and H K Kim ldquoPlantmetabolomics from holistic data to relevant biomarkersrdquo Cur-rent Medicinal Chemistry vol 20 no 8 pp 1056ndash1090 2013

[16] B DrsquoAbrosca E Buommino G DrsquoAngelo et al ldquoSpectroscopicidentification and anti-biofilm properties of polar metabolitesfrom the medicinal plant Helichrysum italicum against Pseu-domonas aeruginosardquo Bioorganic and Medicinal Chemistry vol21 no 22 pp 7038ndash7046 2013

[17] S Pacifico B DrsquoAbrosca M Scognamiglio et al ldquoNMR-basedmetabolic profiling and in vitro antioxidant and hepatotoxicassessment of partially purified fractions fromGolden german-der (Teucrium polium L) methanolic extractrdquo Food Chemistryvol 135 no 3 pp 1957ndash1967 2012

[18] R Scherer A C P Rybka C A Ballus A D Meinhart J TFilho and H T Godoy ldquoValidation of a HPLC method forsimultaneous determination of main organic acids in fruits andjuicesrdquo Food Chemistry vol 135 no 1 pp 150ndash154 2012

[19] J Liimatainen M Karonen J Sinkkonen M Helander andJ-P Salminen ldquoPhenolic compounds of the inner bark ofBetula pendula seasonal and genetic variation and inductionby woundingrdquo Journal of Chemical Ecology vol 38 no 11 pp1410ndash1418 2012

[20] K Hosni K Msaada M Ben Taarit and B Marzouk ldquoPhe-nological variations of secondary metabolites from Hypericumtriquetrifolium Turrardquo Biochemical Systematics and Ecology vol39 no 1 pp 43ndash50 2011

[21] F Brahmi B Mechri S Dabbou M Dhibi and M HammamildquoThe efficacy of phenolics compounds with different polaritiesas antioxidants from olive leaves depending on seasonal varia-tionsrdquo Industrial Crops and Products vol 38 no 1 pp 146ndash1522012

[22] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissus Carltonrdquo Phytochemistry vol 88 pp 43ndash53 2013

[23] M Petersen and M S J Simmonds ldquoRosmarinic acidrdquo Phyto-chemistry vol 62 no 2 pp 121ndash125 2003

[24] R Quirantes-Pine M Herranz-Lopez L Funes et al ldquoPhenyl-propanoids and their metabolites are the major compoundsresponsible for blood-cell protection against oxidative stressafter administration of Lippia citriodora in ratsrdquo Phytomedicinevol 20 no 12 pp 1112ndash1118 2013

[25] G Tibaldi E Fontana and S Nicola ldquoPostharvest managementaffects spearmint and calamint essential oilsrdquo Journal of theScience of Food and Agriculture vol 93 no 3 pp 580ndash586 2013

[26] K Hammer G Laghetti and K Pistrick ldquoCalamintha nepeta(L) Savi and Micromeria thymifolia (Scop) Fritsch cultivatedin ItalyrdquoGenetic Resources and Crop Evolution vol 52 no 2 pp215ndash220 2005

[27] G Appendino M Ottino N Marquez et al ldquoArzanol an anti-inflammatory and anti-HIV-1 phloroglucinol 120572-pyrone fromHelichrysum italicum ssp microphyllumrdquo Journal of NaturalProducts vol 70 no 4 pp 608ndash612 2007

[28] W-R Diao Q-P Hu H Zhang and J-G Xu ldquoChemicalcomposition antibacterial activity and mechanism of action ofessential oil from seeds of fennel (Foeniculum vulgare Mill)rdquoFood Control vol 35 no 1 pp 109ndash116 2014

[29] N Martins L Barros C Santos-Buelga M Henriques S Silvaand I C F R Ferreira ldquoDecoction infusion and hydroalcoholicextract of Origanum vulgare L different performances regard-ing bioactivity and phenolic compoundsrdquo Food Chemistry vol158 pp 73ndash80 2014

[30] T Mihajilov-Krstev D Radnovic D Kitic et al ldquoChemicalcomposition antimicrobial antioxidative and anticholinester-ase activity of Satureja Montana L ssp montana essential oilrdquoCentral European Journal of Biology vol 9 no 7 pp 668ndash6772014

[31] C Sarikurkcu M Sabih Ozer M Eskici B Tepe S Can andE Mete ldquoEssential oil composition and antioxidant activity ofThymus longicaulis C Presl subsp longicaulis var longicaulisrdquoFood andChemical Toxicology vol 48 no 7 pp 1801ndash1805 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Analytical Methods in Chemistry 5

Table2Con

tinued

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Helichrysum

Italicum

Alanine

SeeC

nepeta

124plusmn019

087plusmn060

091plusmn015

151plusmn

044

Chlorogenica

cid

184ndash

220

(H2andH6qu

inicacidm

)545

(H5m)637

(H81015840d119869

=159)690

(H51015840d119869

=81)707(

H61015840dd119869=8421)715

(H21015840d119869

=21)762(

H71015840d119869

=159)

831plusmn15

590

3plusmn12

4363plusmn259

326plusmn077

Dicaffeoylqu

inic

acid

630

(H81015840d119869

=162)648

(H810158401015840

d119869

=156)76

5(H

71015840d119869

=162)76

6(H

710158401015840

d119869

=156)

1086plusmn13

81088plusmn516

1153plusmn238

783plusmn207

Glucose

SeeC

nepeta

377plusmn174

356plusmn004

273plusmn059

378plusmn201

3-OHbenzofuran

518

(H2d119869=63)522(H

3d119869=63)690(H

7ov)804

(H6dd119869=8418

)406plusmn027

313plusmn13

9537plusmn18

9406plusmn14

7Isob

enzofurano

ne533

(H3s)673(H

4d119869=18

)684

(H6d119869=18

)603plusmn270

Malicacid

SeeC

nepeta

XX

XX

Neochlorogenic

acid

639

(H81015840d119869

=159)752(H

71015840d119869

=159)

XX

XX

Sucrose

SeeC

nepeta

1693plusmn855

915plusmn457

1024plusmn445

1014plusmn375

Foenicu

lum

vulga

re

Alanine

SeeC

nepeta

117plusmn005

090plusmn049

124plusmn025

109plusmn009

Caffeicacid

629

(H81015840d119869

=159)688

(H51015840d119869

=81)703(H

61015840dd119869=8421)712

(H21015840d119869

=21)752

(H71015840d119869

=159)

XX

XX

Chlorogenica

cid

SeeH

italicum

XX

XX

Dicaffeoylqu

inic

acid

SeeH

italicum

XX

X

Glucose

SeeC

nepeta

117plusmn002

555plusmn289

577plusmn16

4629plusmn13

8GABA

(120574-aminob

utyric

acid)

192(H

3m)236

(H2t119869=75

)301

(H4t119869=75

)X

XX

Kaem

pferol

635

(H6d119869=21)652

(H8d119869=21)700(H

21015840H

61015840d119869

=84)809(H

31015840H

51015840

d119869=84)

128plusmn017

178plusmn15

614

6plusmn044

118plusmn082

Malicacid

SeeC

nepeta

4468plusmn711

4740plusmn1023

16852plusmn1115

8662plusmn349

Quercetin

627

(H6d119869=21)648(H

8d119869=21)699(H

51015840d119869=85)759(H

61015840d119869=85

21)775(H

21015840d119869=21)

193plusmn036

555plusmn361

623plusmn12

5294plusmn209

Quinica

cid

SeeC

nepeta

2509plusmn262

3161plusmn

1748

3349plusmn673

2730plusmn225

Sucrose

SeeC

nepeta

4494plusmn228

3680plusmn1385

5436plusmn1186

3062plusmn90

3Th

reon

ine

132(H

4d119869=66)

XX

XX

Microm

eria

graeca

Alanine

SeeC

nepeta

093plusmn055

140plusmn088

209plusmn089

575plusmn404

Citricacid

SeeC

nepeta

1989plusmn94

41865plusmn15

61713plusmn684

1543plusmn510

Glucose

SeeC

nepeta

481plusmn249

687plusmn299

561plusmn028

564plusmn17

1Malicacid

SeeC

nepeta

XX

XX

Quinica

cid

SeeC

nepeta

3975plusmn1068

5509plusmn483

6402plusmn416

5741plusmn

1124

Rosm

arinicacid

300

(H71015840add

119869=141

96)315

(H71015840bdd

119869=141

36)502(H

81015840dd119869=100

33)630(H

8d119869=159)671(H61015840dd119869=78

21)681(H51015840d119869

=78

)682

(H5

d119869=81)689(H

21015840d119869

=21)700(H

6dd

119869=8118

)711(H2d119869=18

)75

0(H

7d119869=159)

102plusmn099

462plusmn19

8658plusmn15

115

4plusmn079

Sucrose

SeeC

nepeta

799plusmn260

709plusmn302

1551plusmn

284

822plusmn373

Threon

ine

SeeF

vulgare

X

6 Journal of Analytical Methods in Chemistry

Table2Con

tinued

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Orig

anum

vulga

re

Apigenin

deriv

ative2

SeeC

nepeta

432plusmn024

295plusmn091

mdash

Alanine

SeeC

nepeta

058plusmn012

090plusmn017

107plusmn014

mdashCh

oline

320

(s)

XX

mdashCitricacid

SeeC

nepeta

1474plusmn10

22292plusmn413

1554plusmn19

1mdash

Glucose

SeeC

nepeta

1659plusmn264

362plusmn288

439plusmn382

mdashLithosperm

icacid

300

(H71015840aa

ndbov)6

30(H

8d119869=159)78

2(H

7d119869=159)

XX

Xmdash

Malicacid

SeeC

nepeta

2759plusmn16

93240plusmn810

1474plusmn851

mdashQuinica

cid

SeeC

nepeta

3094plusmn286

4591plusmn

183

4312plusmn98

1mdash

Rosm

arinicacid

SeeM

graeca

1147plusmn611

1573plusmn482

3550plusmn591

mdashSucrose

SeeC

nepeta

1005plusmn15

8498plusmn045

1492plusmn436

mdash

Satureja

montana

Apigenin

deriv

ative5

SeeC

nepeta

X

Apigenin

deriv

ative6

SeeC

nepeta

X

Alanine

SeeC

nepeta

XX

XX

Choline

SeeO

vulgare

XX

XCh

lorogenica

cid

SeeH

italicum

947plusmn12

01501plusmn

051

1250plusmn554

366plusmn18

4Glucose

SeeC

nepeta

101plusmn

067

396plusmn15

5287plusmn14

7427plusmn15

4Malicacid

SeeC

nepeta

3932plusmn260

3240plusmn099

855plusmn331

4876plusmn678

Rosm

arinicacid

SeeM

graeca

715plusmn12

61090plusmn16

4884plusmn432

Sucrose

SeeC

nepeta

1009plusmn093

856plusmn442

1007plusmn12

593

7plusmn391

Thym

uslongica

ulis

Alanine

SeeC

nepeta

034plusmn024

105plusmn011

108plusmn022

128plusmn007

Citricacid

SeeC

nepeta

XX

XX

Malicacid

SeeC

nepeta

2692plusmn1544

6005plusmn328

3556plusmn680

5464plusmn1472

Quinica

cid

SeeC

nepeta

1985plusmn863

4116plusmn75

040

71plusmn

830

3196plusmn317

Glucose

SeeC

nepeta

306plusmn033

320plusmn083

265plusmn17

6336plusmn14

1Ph

enylprop

anoid

8613

(H8d119869=159)74

6(H

7d119869=159)

367plusmn113

565plusmn061

477plusmn12

419

0plusmn12

4

Rosm

arinicacid

SeeM

graeca

710plusmn370

778plusmn19

81012plusmn176

487plusmn214

Sucrose

SeeC

nepeta

710plusmn370

778plusmn19

81012plusmn176

487plusmn214

Sign

almultip

licity

indicatedas

follo

wsd=do

ubletdd

=do

ubleto

fdou

bletsm

=multip

letov

=overlap

pedq=qu

artets=

singletand

t=triplet

lowast

Apigenin

deriv

atives

1and

23and45and6wereq

uantified

together

duetooverlap

ping

signals

Journal of Analytical Methods in Chemistry 7

Calamintha nepetaWi

Sp

Su

Au

1

2323 23

4545 45

67 67 6788

9 9 10 10101819

19

11121314

11121314

16

17

Helichrysum italicumWi

Sp

Su

Au

202020 202020

Foeniculum vulgareWi

Sp

Su

Au

21 21 2122 22 23 2323 24

Micromeria graecaWi

Sp

Su

Au

252525

25 25

Origanum vulgare

Wi

Sp

Su

Satureja montana

Wi

Sp

Su

Au

Thymus longicaulisWi

Sp

Su

Au

100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm)

Figure 1 1H-NMR spectra of studied plants (Au = autumn Sp = spring Su = summer Wi = winter) The main resonances of the maincompounds are indicated on the spectra as follows 1 alanine 2 apigenin derivative 1 3 apigenin derivative 2 4 apigenin derivative 3 5apigenin derivative 4 6 apigenin derivative 5 7 apigenin derivative 6 8 citric acid 9 glucose 10 malic acid 11 phenylpropanoid 1 12phenylpropanoid 2 13 phenylpropanoid 3 14 phenylpropanoid 4 15 phenylpropanoid 5 16 phenylpropanoid 6 17 phenylpropanoid 7 18quinic acid 19 sucrose 20 chlorogenic acid 21 GABA 22 kaempferol 23 quercetin 24 threonine 25 rosmarinic acid

8 Journal of Analytical Methods in Chemistry

compounds as reported for several Lamiaceae [17] Never-theless the role in nutrition of primary metabolites is oftendisregarded [18] These considerations raise the attention tothe need for a comprehensive profiling of their metabolites

Furthermore the NMR-based metabolomic approachhere proposed due to the short time of analyses and to lowercosts compared to other analytical methods was very usefulfor the study of seasonal variation of metabolites

Indeed although it is clear that secondary metabolitesshow peculiar trends only fragmentary information is avail-able mainly because of the used approaches

The most common and easiest procedure was to performthe phytochemical study on samples collected in a specifictime of the year and then compare the other months (orseasons) by setting up a series of target analyses often byHPLC [19 20] or by less time and resource consumingapproaches based on colorimetric assays [21]

The improvement of analytical methods and espe-cially the availability of a high-throughput approach likemetabolomics [5] gives the chance to further explore theissue of seasonality [22] and to thoroughly study thesechanges

Concerning themetabolites identifiedwithin the extractsit is important to underline their biological activity

Phenols are well known for their antioxidant activity [11]As the studied plants are all very rich in phenolics they havea great antioxidant potential This could also explain the useof some of these herbs as natural food preservatives (Table 1)

Among the detected compounds rosmarinic acid is themost widespread and the most abundant A plethora ofbiological activities has been attributed to this compoundamong them adstringent antioxidative anti-inflammatoryantimutagen antibacterial and antiviral [23]

Many other phenylpropanoids and flavonoids were alsodetected Several properties have been reported for thesecompounds such as anti-inflammatory antimicrobial andantitumor activity [24]

The richness in these compounds of the studied plantssupports their traditional uses

However the study evidenced that qualitative and quan-titative variations of metabolites are observed along the year

This is to the best of our knowledge the first reportof metabolite content and seasonal qualitative and quanti-tative variation of this set of food spices Furthermore it isevidenced that a higher content of compounds known fortheir health promoting capacities can be found in spring andsummer samples of all the analysed species although season-specific compounds were also detected

4 Conclusions

NMR-based metabolomics has been applied to the study ofchemical composition of selected aromatic plants ofMediter-ranean vegetation

The method allowed determining the chemical compo-sition of plant extracts in terms of primary and secondarymetabolites The abundance of aromatic secondary metabo-lites suggested that the traditional uses of these plants mightbe supported by their chemical composition

Furthermore the seasonality of the accumulation of thesemetabolites was studied

Conflict of Interests

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

Acknowledgment

The authors would like to thank the anonymous reviewers fortheir valuable suggestions

References

[1] A M Scherrer R Motti and C S Weckerle ldquoTraditional plantuse in the areas of Monte Vesole and Ascea Cilento NationalPark (Campania Southern Italy)rdquo Journal of Ethnopharmacol-ogy vol 97 no 1 pp 129ndash143 2005

[2] T Efferth and H J Greten ldquoMedicinal and aromatic plantresearch in the 21st centuryrdquoMedicinal amp Aromatic Plants vol1 article e110 2012

[3] R Rodrıguez-Solana J M Salgado J M Domınguez andS Cortes-Dieguez ldquoComparison of soxhlet accelerated sol-vent and supercritical fluid extraction techniques for volatile(GCndashMS and GCFID) and phenolic compounds (HPLCndashESIMSMS) from Lamiaceae Species rdquo Phytochemical Analysisvol 26 no 1 pp 61ndash71 2015

[4] T Fornari G Vicente E Vazquez M R Garcıa-Risco andG Reglero ldquoIsolation of essential oil from different plants andherbs by supercritical fluid extractionrdquo Journal of Chromatogra-phy A vol 1250 pp 34ndash48 2012

[5] H K Kim Y H Choi and R Verpoorte ldquoNMR-based plantmetabolomics where do we stand where do we gordquo Trends inBiotechnology vol 29 no 6 pp 267ndash275 2011

[6] M Scognamiglio B DrsquoAbrosca A Esposito and A FiorentinoldquoMetabolomics an unexplored tool for allelopathy studiesrdquoJournal of Allelochemical Interactions vol 1 pp 9ndash23 2015

[7] J W Allwood D I Ellis and R Goodacre ldquoMetabolomictechnologies and their application to the study of plants andplant-host interactionsrdquo Physiologia Plantarum vol 132 no 2pp 117ndash135 2008

[8] R R Forseth and F C Schroeder ldquoNMR-spectroscopic analysisof mixtures from structure to functionrdquo Current Opinion inChemical Biology vol 15 no 1 pp 38ndash47 2011

[9] H K Kim Y H Choi and R Verpoorte ldquoNMR-basedmetabolomic analysis of plantsrdquo Nature Protocols vol 5 no 3pp 536ndash549 2010

[10] M Scognamiglio V Fiumano B DAbrosca et al ldquoChemicalinteractions between plants in Mediterranean vegetation theinfluence of selected plant extracts on Aegilops geniculatametabolomerdquo Phytochemistry vol 106 pp 69ndash85 2014

[11] A Vallverdu-Queralt J Regueiro M Martınez-Huelamo JF R Alvarenga L N Leal and R M Lamuela-RaventosldquoA comprehensive study on the phenolic profile of widelyused culinary herbs and spices rosemary thyme oreganocinnamon cumin and bayrdquo Food Chemistry vol 154 pp 299ndash307 2014

[12] Q Cui I A Lewis A D Hegeman et al ldquoMetabolite identifi-cation via the Madison Metabolomics Consortium DatabaserdquoNature Biotechnology vol 26 no 2 pp 162ndash164 2008

Journal of Analytical Methods in Chemistry 9

[13] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissusCarltonrdquoPhytochemistry vol 88 pp 43ndash53 2013

[14] R Verpoorte Y H Choi and H K Kim ldquoNMR-basedmetabolomics at work in phytochemistryrdquo PhytochemistryReviews vol 6 no 1 pp 3ndash14 2007

[15] J-L Wolfender S Rudaz Y H Choi and H K Kim ldquoPlantmetabolomics from holistic data to relevant biomarkersrdquo Cur-rent Medicinal Chemistry vol 20 no 8 pp 1056ndash1090 2013

[16] B DrsquoAbrosca E Buommino G DrsquoAngelo et al ldquoSpectroscopicidentification and anti-biofilm properties of polar metabolitesfrom the medicinal plant Helichrysum italicum against Pseu-domonas aeruginosardquo Bioorganic and Medicinal Chemistry vol21 no 22 pp 7038ndash7046 2013

[17] S Pacifico B DrsquoAbrosca M Scognamiglio et al ldquoNMR-basedmetabolic profiling and in vitro antioxidant and hepatotoxicassessment of partially purified fractions fromGolden german-der (Teucrium polium L) methanolic extractrdquo Food Chemistryvol 135 no 3 pp 1957ndash1967 2012

[18] R Scherer A C P Rybka C A Ballus A D Meinhart J TFilho and H T Godoy ldquoValidation of a HPLC method forsimultaneous determination of main organic acids in fruits andjuicesrdquo Food Chemistry vol 135 no 1 pp 150ndash154 2012

[19] J Liimatainen M Karonen J Sinkkonen M Helander andJ-P Salminen ldquoPhenolic compounds of the inner bark ofBetula pendula seasonal and genetic variation and inductionby woundingrdquo Journal of Chemical Ecology vol 38 no 11 pp1410ndash1418 2012

[20] K Hosni K Msaada M Ben Taarit and B Marzouk ldquoPhe-nological variations of secondary metabolites from Hypericumtriquetrifolium Turrardquo Biochemical Systematics and Ecology vol39 no 1 pp 43ndash50 2011

[21] F Brahmi B Mechri S Dabbou M Dhibi and M HammamildquoThe efficacy of phenolics compounds with different polaritiesas antioxidants from olive leaves depending on seasonal varia-tionsrdquo Industrial Crops and Products vol 38 no 1 pp 146ndash1522012

[22] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissus Carltonrdquo Phytochemistry vol 88 pp 43ndash53 2013

[23] M Petersen and M S J Simmonds ldquoRosmarinic acidrdquo Phyto-chemistry vol 62 no 2 pp 121ndash125 2003

[24] R Quirantes-Pine M Herranz-Lopez L Funes et al ldquoPhenyl-propanoids and their metabolites are the major compoundsresponsible for blood-cell protection against oxidative stressafter administration of Lippia citriodora in ratsrdquo Phytomedicinevol 20 no 12 pp 1112ndash1118 2013

[25] G Tibaldi E Fontana and S Nicola ldquoPostharvest managementaffects spearmint and calamint essential oilsrdquo Journal of theScience of Food and Agriculture vol 93 no 3 pp 580ndash586 2013

[26] K Hammer G Laghetti and K Pistrick ldquoCalamintha nepeta(L) Savi and Micromeria thymifolia (Scop) Fritsch cultivatedin ItalyrdquoGenetic Resources and Crop Evolution vol 52 no 2 pp215ndash220 2005

[27] G Appendino M Ottino N Marquez et al ldquoArzanol an anti-inflammatory and anti-HIV-1 phloroglucinol 120572-pyrone fromHelichrysum italicum ssp microphyllumrdquo Journal of NaturalProducts vol 70 no 4 pp 608ndash612 2007

[28] W-R Diao Q-P Hu H Zhang and J-G Xu ldquoChemicalcomposition antibacterial activity and mechanism of action ofessential oil from seeds of fennel (Foeniculum vulgare Mill)rdquoFood Control vol 35 no 1 pp 109ndash116 2014

[29] N Martins L Barros C Santos-Buelga M Henriques S Silvaand I C F R Ferreira ldquoDecoction infusion and hydroalcoholicextract of Origanum vulgare L different performances regard-ing bioactivity and phenolic compoundsrdquo Food Chemistry vol158 pp 73ndash80 2014

[30] T Mihajilov-Krstev D Radnovic D Kitic et al ldquoChemicalcomposition antimicrobial antioxidative and anticholinester-ase activity of Satureja Montana L ssp montana essential oilrdquoCentral European Journal of Biology vol 9 no 7 pp 668ndash6772014

[31] C Sarikurkcu M Sabih Ozer M Eskici B Tepe S Can andE Mete ldquoEssential oil composition and antioxidant activity ofThymus longicaulis C Presl subsp longicaulis var longicaulisrdquoFood andChemical Toxicology vol 48 no 7 pp 1801ndash1805 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 Journal of Analytical Methods in Chemistry

Table2Con

tinued

Plantspecies

Metabolites

NMR

Wi

SpSu

Au

Orig

anum

vulga

re

Apigenin

deriv

ative2

SeeC

nepeta

432plusmn024

295plusmn091

mdash

Alanine

SeeC

nepeta

058plusmn012

090plusmn017

107plusmn014

mdashCh

oline

320

(s)

XX

mdashCitricacid

SeeC

nepeta

1474plusmn10

22292plusmn413

1554plusmn19

1mdash

Glucose

SeeC

nepeta

1659plusmn264

362plusmn288

439plusmn382

mdashLithosperm

icacid

300

(H71015840aa

ndbov)6

30(H

8d119869=159)78

2(H

7d119869=159)

XX

Xmdash

Malicacid

SeeC

nepeta

2759plusmn16

93240plusmn810

1474plusmn851

mdashQuinica

cid

SeeC

nepeta

3094plusmn286

4591plusmn

183

4312plusmn98

1mdash

Rosm

arinicacid

SeeM

graeca

1147plusmn611

1573plusmn482

3550plusmn591

mdashSucrose

SeeC

nepeta

1005plusmn15

8498plusmn045

1492plusmn436

mdash

Satureja

montana

Apigenin

deriv

ative5

SeeC

nepeta

X

Apigenin

deriv

ative6

SeeC

nepeta

X

Alanine

SeeC

nepeta

XX

XX

Choline

SeeO

vulgare

XX

XCh

lorogenica

cid

SeeH

italicum

947plusmn12

01501plusmn

051

1250plusmn554

366plusmn18

4Glucose

SeeC

nepeta

101plusmn

067

396plusmn15

5287plusmn14

7427plusmn15

4Malicacid

SeeC

nepeta

3932plusmn260

3240plusmn099

855plusmn331

4876plusmn678

Rosm

arinicacid

SeeM

graeca

715plusmn12

61090plusmn16

4884plusmn432

Sucrose

SeeC

nepeta

1009plusmn093

856plusmn442

1007plusmn12

593

7plusmn391

Thym

uslongica

ulis

Alanine

SeeC

nepeta

034plusmn024

105plusmn011

108plusmn022

128plusmn007

Citricacid

SeeC

nepeta

XX

XX

Malicacid

SeeC

nepeta

2692plusmn1544

6005plusmn328

3556plusmn680

5464plusmn1472

Quinica

cid

SeeC

nepeta

1985plusmn863

4116plusmn75

040

71plusmn

830

3196plusmn317

Glucose

SeeC

nepeta

306plusmn033

320plusmn083

265plusmn17

6336plusmn14

1Ph

enylprop

anoid

8613

(H8d119869=159)74

6(H

7d119869=159)

367plusmn113

565plusmn061

477plusmn12

419

0plusmn12

4

Rosm

arinicacid

SeeM

graeca

710plusmn370

778plusmn19

81012plusmn176

487plusmn214

Sucrose

SeeC

nepeta

710plusmn370

778plusmn19

81012plusmn176

487plusmn214

Sign

almultip

licity

indicatedas

follo

wsd=do

ubletdd

=do

ubleto

fdou

bletsm

=multip

letov

=overlap

pedq=qu

artets=

singletand

t=triplet

lowast

Apigenin

deriv

atives

1and

23and45and6wereq

uantified

together

duetooverlap

ping

signals

Journal of Analytical Methods in Chemistry 7

Calamintha nepetaWi

Sp

Su

Au

1

2323 23

4545 45

67 67 6788

9 9 10 10101819

19

11121314

11121314

16

17

Helichrysum italicumWi

Sp

Su

Au

202020 202020

Foeniculum vulgareWi

Sp

Su

Au

21 21 2122 22 23 2323 24

Micromeria graecaWi

Sp

Su

Au

252525

25 25

Origanum vulgare

Wi

Sp

Su

Satureja montana

Wi

Sp

Su

Au

Thymus longicaulisWi

Sp

Su

Au

100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm)

Figure 1 1H-NMR spectra of studied plants (Au = autumn Sp = spring Su = summer Wi = winter) The main resonances of the maincompounds are indicated on the spectra as follows 1 alanine 2 apigenin derivative 1 3 apigenin derivative 2 4 apigenin derivative 3 5apigenin derivative 4 6 apigenin derivative 5 7 apigenin derivative 6 8 citric acid 9 glucose 10 malic acid 11 phenylpropanoid 1 12phenylpropanoid 2 13 phenylpropanoid 3 14 phenylpropanoid 4 15 phenylpropanoid 5 16 phenylpropanoid 6 17 phenylpropanoid 7 18quinic acid 19 sucrose 20 chlorogenic acid 21 GABA 22 kaempferol 23 quercetin 24 threonine 25 rosmarinic acid

8 Journal of Analytical Methods in Chemistry

compounds as reported for several Lamiaceae [17] Never-theless the role in nutrition of primary metabolites is oftendisregarded [18] These considerations raise the attention tothe need for a comprehensive profiling of their metabolites

Furthermore the NMR-based metabolomic approachhere proposed due to the short time of analyses and to lowercosts compared to other analytical methods was very usefulfor the study of seasonal variation of metabolites

Indeed although it is clear that secondary metabolitesshow peculiar trends only fragmentary information is avail-able mainly because of the used approaches

The most common and easiest procedure was to performthe phytochemical study on samples collected in a specifictime of the year and then compare the other months (orseasons) by setting up a series of target analyses often byHPLC [19 20] or by less time and resource consumingapproaches based on colorimetric assays [21]

The improvement of analytical methods and espe-cially the availability of a high-throughput approach likemetabolomics [5] gives the chance to further explore theissue of seasonality [22] and to thoroughly study thesechanges

Concerning themetabolites identifiedwithin the extractsit is important to underline their biological activity

Phenols are well known for their antioxidant activity [11]As the studied plants are all very rich in phenolics they havea great antioxidant potential This could also explain the useof some of these herbs as natural food preservatives (Table 1)

Among the detected compounds rosmarinic acid is themost widespread and the most abundant A plethora ofbiological activities has been attributed to this compoundamong them adstringent antioxidative anti-inflammatoryantimutagen antibacterial and antiviral [23]

Many other phenylpropanoids and flavonoids were alsodetected Several properties have been reported for thesecompounds such as anti-inflammatory antimicrobial andantitumor activity [24]

The richness in these compounds of the studied plantssupports their traditional uses

However the study evidenced that qualitative and quan-titative variations of metabolites are observed along the year

This is to the best of our knowledge the first reportof metabolite content and seasonal qualitative and quanti-tative variation of this set of food spices Furthermore it isevidenced that a higher content of compounds known fortheir health promoting capacities can be found in spring andsummer samples of all the analysed species although season-specific compounds were also detected

4 Conclusions

NMR-based metabolomics has been applied to the study ofchemical composition of selected aromatic plants ofMediter-ranean vegetation

The method allowed determining the chemical compo-sition of plant extracts in terms of primary and secondarymetabolites The abundance of aromatic secondary metabo-lites suggested that the traditional uses of these plants mightbe supported by their chemical composition

Furthermore the seasonality of the accumulation of thesemetabolites was studied

Conflict of Interests

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

Acknowledgment

The authors would like to thank the anonymous reviewers fortheir valuable suggestions

References

[1] A M Scherrer R Motti and C S Weckerle ldquoTraditional plantuse in the areas of Monte Vesole and Ascea Cilento NationalPark (Campania Southern Italy)rdquo Journal of Ethnopharmacol-ogy vol 97 no 1 pp 129ndash143 2005

[2] T Efferth and H J Greten ldquoMedicinal and aromatic plantresearch in the 21st centuryrdquoMedicinal amp Aromatic Plants vol1 article e110 2012

[3] R Rodrıguez-Solana J M Salgado J M Domınguez andS Cortes-Dieguez ldquoComparison of soxhlet accelerated sol-vent and supercritical fluid extraction techniques for volatile(GCndashMS and GCFID) and phenolic compounds (HPLCndashESIMSMS) from Lamiaceae Species rdquo Phytochemical Analysisvol 26 no 1 pp 61ndash71 2015

[4] T Fornari G Vicente E Vazquez M R Garcıa-Risco andG Reglero ldquoIsolation of essential oil from different plants andherbs by supercritical fluid extractionrdquo Journal of Chromatogra-phy A vol 1250 pp 34ndash48 2012

[5] H K Kim Y H Choi and R Verpoorte ldquoNMR-based plantmetabolomics where do we stand where do we gordquo Trends inBiotechnology vol 29 no 6 pp 267ndash275 2011

[6] M Scognamiglio B DrsquoAbrosca A Esposito and A FiorentinoldquoMetabolomics an unexplored tool for allelopathy studiesrdquoJournal of Allelochemical Interactions vol 1 pp 9ndash23 2015

[7] J W Allwood D I Ellis and R Goodacre ldquoMetabolomictechnologies and their application to the study of plants andplant-host interactionsrdquo Physiologia Plantarum vol 132 no 2pp 117ndash135 2008

[8] R R Forseth and F C Schroeder ldquoNMR-spectroscopic analysisof mixtures from structure to functionrdquo Current Opinion inChemical Biology vol 15 no 1 pp 38ndash47 2011

[9] H K Kim Y H Choi and R Verpoorte ldquoNMR-basedmetabolomic analysis of plantsrdquo Nature Protocols vol 5 no 3pp 536ndash549 2010

[10] M Scognamiglio V Fiumano B DAbrosca et al ldquoChemicalinteractions between plants in Mediterranean vegetation theinfluence of selected plant extracts on Aegilops geniculatametabolomerdquo Phytochemistry vol 106 pp 69ndash85 2014

[11] A Vallverdu-Queralt J Regueiro M Martınez-Huelamo JF R Alvarenga L N Leal and R M Lamuela-RaventosldquoA comprehensive study on the phenolic profile of widelyused culinary herbs and spices rosemary thyme oreganocinnamon cumin and bayrdquo Food Chemistry vol 154 pp 299ndash307 2014

[12] Q Cui I A Lewis A D Hegeman et al ldquoMetabolite identifi-cation via the Madison Metabolomics Consortium DatabaserdquoNature Biotechnology vol 26 no 2 pp 162ndash164 2008

Journal of Analytical Methods in Chemistry 9

[13] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissusCarltonrdquoPhytochemistry vol 88 pp 43ndash53 2013

[14] R Verpoorte Y H Choi and H K Kim ldquoNMR-basedmetabolomics at work in phytochemistryrdquo PhytochemistryReviews vol 6 no 1 pp 3ndash14 2007

[15] J-L Wolfender S Rudaz Y H Choi and H K Kim ldquoPlantmetabolomics from holistic data to relevant biomarkersrdquo Cur-rent Medicinal Chemistry vol 20 no 8 pp 1056ndash1090 2013

[16] B DrsquoAbrosca E Buommino G DrsquoAngelo et al ldquoSpectroscopicidentification and anti-biofilm properties of polar metabolitesfrom the medicinal plant Helichrysum italicum against Pseu-domonas aeruginosardquo Bioorganic and Medicinal Chemistry vol21 no 22 pp 7038ndash7046 2013

[17] S Pacifico B DrsquoAbrosca M Scognamiglio et al ldquoNMR-basedmetabolic profiling and in vitro antioxidant and hepatotoxicassessment of partially purified fractions fromGolden german-der (Teucrium polium L) methanolic extractrdquo Food Chemistryvol 135 no 3 pp 1957ndash1967 2012

[18] R Scherer A C P Rybka C A Ballus A D Meinhart J TFilho and H T Godoy ldquoValidation of a HPLC method forsimultaneous determination of main organic acids in fruits andjuicesrdquo Food Chemistry vol 135 no 1 pp 150ndash154 2012

[19] J Liimatainen M Karonen J Sinkkonen M Helander andJ-P Salminen ldquoPhenolic compounds of the inner bark ofBetula pendula seasonal and genetic variation and inductionby woundingrdquo Journal of Chemical Ecology vol 38 no 11 pp1410ndash1418 2012

[20] K Hosni K Msaada M Ben Taarit and B Marzouk ldquoPhe-nological variations of secondary metabolites from Hypericumtriquetrifolium Turrardquo Biochemical Systematics and Ecology vol39 no 1 pp 43ndash50 2011

[21] F Brahmi B Mechri S Dabbou M Dhibi and M HammamildquoThe efficacy of phenolics compounds with different polaritiesas antioxidants from olive leaves depending on seasonal varia-tionsrdquo Industrial Crops and Products vol 38 no 1 pp 146ndash1522012

[22] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissus Carltonrdquo Phytochemistry vol 88 pp 43ndash53 2013

[23] M Petersen and M S J Simmonds ldquoRosmarinic acidrdquo Phyto-chemistry vol 62 no 2 pp 121ndash125 2003

[24] R Quirantes-Pine M Herranz-Lopez L Funes et al ldquoPhenyl-propanoids and their metabolites are the major compoundsresponsible for blood-cell protection against oxidative stressafter administration of Lippia citriodora in ratsrdquo Phytomedicinevol 20 no 12 pp 1112ndash1118 2013

[25] G Tibaldi E Fontana and S Nicola ldquoPostharvest managementaffects spearmint and calamint essential oilsrdquo Journal of theScience of Food and Agriculture vol 93 no 3 pp 580ndash586 2013

[26] K Hammer G Laghetti and K Pistrick ldquoCalamintha nepeta(L) Savi and Micromeria thymifolia (Scop) Fritsch cultivatedin ItalyrdquoGenetic Resources and Crop Evolution vol 52 no 2 pp215ndash220 2005

[27] G Appendino M Ottino N Marquez et al ldquoArzanol an anti-inflammatory and anti-HIV-1 phloroglucinol 120572-pyrone fromHelichrysum italicum ssp microphyllumrdquo Journal of NaturalProducts vol 70 no 4 pp 608ndash612 2007

[28] W-R Diao Q-P Hu H Zhang and J-G Xu ldquoChemicalcomposition antibacterial activity and mechanism of action ofessential oil from seeds of fennel (Foeniculum vulgare Mill)rdquoFood Control vol 35 no 1 pp 109ndash116 2014

[29] N Martins L Barros C Santos-Buelga M Henriques S Silvaand I C F R Ferreira ldquoDecoction infusion and hydroalcoholicextract of Origanum vulgare L different performances regard-ing bioactivity and phenolic compoundsrdquo Food Chemistry vol158 pp 73ndash80 2014

[30] T Mihajilov-Krstev D Radnovic D Kitic et al ldquoChemicalcomposition antimicrobial antioxidative and anticholinester-ase activity of Satureja Montana L ssp montana essential oilrdquoCentral European Journal of Biology vol 9 no 7 pp 668ndash6772014

[31] C Sarikurkcu M Sabih Ozer M Eskici B Tepe S Can andE Mete ldquoEssential oil composition and antioxidant activity ofThymus longicaulis C Presl subsp longicaulis var longicaulisrdquoFood andChemical Toxicology vol 48 no 7 pp 1801ndash1805 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Analytical Methods in Chemistry 7

Calamintha nepetaWi

Sp

Su

Au

1

2323 23

4545 45

67 67 6788

9 9 10 10101819

19

11121314

11121314

16

17

Helichrysum italicumWi

Sp

Su

Au

202020 202020

Foeniculum vulgareWi

Sp

Su

Au

21 21 2122 22 23 2323 24

Micromeria graecaWi

Sp

Su

Au

252525

25 25

Origanum vulgare

Wi

Sp

Su

Satureja montana

Wi

Sp

Su

Au

Thymus longicaulisWi

Sp

Su

Au

100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00100 90 80 70 60 50 40 30 20 10 00

100 90 80 70 60 50 40 30 20 10 00 100 90 80 70 60 50 40 30 20 10 00

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm) f1 (ppm)

f1 (ppm)

Figure 1 1H-NMR spectra of studied plants (Au = autumn Sp = spring Su = summer Wi = winter) The main resonances of the maincompounds are indicated on the spectra as follows 1 alanine 2 apigenin derivative 1 3 apigenin derivative 2 4 apigenin derivative 3 5apigenin derivative 4 6 apigenin derivative 5 7 apigenin derivative 6 8 citric acid 9 glucose 10 malic acid 11 phenylpropanoid 1 12phenylpropanoid 2 13 phenylpropanoid 3 14 phenylpropanoid 4 15 phenylpropanoid 5 16 phenylpropanoid 6 17 phenylpropanoid 7 18quinic acid 19 sucrose 20 chlorogenic acid 21 GABA 22 kaempferol 23 quercetin 24 threonine 25 rosmarinic acid

8 Journal of Analytical Methods in Chemistry

compounds as reported for several Lamiaceae [17] Never-theless the role in nutrition of primary metabolites is oftendisregarded [18] These considerations raise the attention tothe need for a comprehensive profiling of their metabolites

Furthermore the NMR-based metabolomic approachhere proposed due to the short time of analyses and to lowercosts compared to other analytical methods was very usefulfor the study of seasonal variation of metabolites

Indeed although it is clear that secondary metabolitesshow peculiar trends only fragmentary information is avail-able mainly because of the used approaches

The most common and easiest procedure was to performthe phytochemical study on samples collected in a specifictime of the year and then compare the other months (orseasons) by setting up a series of target analyses often byHPLC [19 20] or by less time and resource consumingapproaches based on colorimetric assays [21]

The improvement of analytical methods and espe-cially the availability of a high-throughput approach likemetabolomics [5] gives the chance to further explore theissue of seasonality [22] and to thoroughly study thesechanges

Concerning themetabolites identifiedwithin the extractsit is important to underline their biological activity

Phenols are well known for their antioxidant activity [11]As the studied plants are all very rich in phenolics they havea great antioxidant potential This could also explain the useof some of these herbs as natural food preservatives (Table 1)

Among the detected compounds rosmarinic acid is themost widespread and the most abundant A plethora ofbiological activities has been attributed to this compoundamong them adstringent antioxidative anti-inflammatoryantimutagen antibacterial and antiviral [23]

Many other phenylpropanoids and flavonoids were alsodetected Several properties have been reported for thesecompounds such as anti-inflammatory antimicrobial andantitumor activity [24]

The richness in these compounds of the studied plantssupports their traditional uses

However the study evidenced that qualitative and quan-titative variations of metabolites are observed along the year

This is to the best of our knowledge the first reportof metabolite content and seasonal qualitative and quanti-tative variation of this set of food spices Furthermore it isevidenced that a higher content of compounds known fortheir health promoting capacities can be found in spring andsummer samples of all the analysed species although season-specific compounds were also detected

4 Conclusions

NMR-based metabolomics has been applied to the study ofchemical composition of selected aromatic plants ofMediter-ranean vegetation

The method allowed determining the chemical compo-sition of plant extracts in terms of primary and secondarymetabolites The abundance of aromatic secondary metabo-lites suggested that the traditional uses of these plants mightbe supported by their chemical composition

Furthermore the seasonality of the accumulation of thesemetabolites was studied

Conflict of Interests

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

Acknowledgment

The authors would like to thank the anonymous reviewers fortheir valuable suggestions

References

[1] A M Scherrer R Motti and C S Weckerle ldquoTraditional plantuse in the areas of Monte Vesole and Ascea Cilento NationalPark (Campania Southern Italy)rdquo Journal of Ethnopharmacol-ogy vol 97 no 1 pp 129ndash143 2005

[2] T Efferth and H J Greten ldquoMedicinal and aromatic plantresearch in the 21st centuryrdquoMedicinal amp Aromatic Plants vol1 article e110 2012

[3] R Rodrıguez-Solana J M Salgado J M Domınguez andS Cortes-Dieguez ldquoComparison of soxhlet accelerated sol-vent and supercritical fluid extraction techniques for volatile(GCndashMS and GCFID) and phenolic compounds (HPLCndashESIMSMS) from Lamiaceae Species rdquo Phytochemical Analysisvol 26 no 1 pp 61ndash71 2015

[4] T Fornari G Vicente E Vazquez M R Garcıa-Risco andG Reglero ldquoIsolation of essential oil from different plants andherbs by supercritical fluid extractionrdquo Journal of Chromatogra-phy A vol 1250 pp 34ndash48 2012

[5] H K Kim Y H Choi and R Verpoorte ldquoNMR-based plantmetabolomics where do we stand where do we gordquo Trends inBiotechnology vol 29 no 6 pp 267ndash275 2011

[6] M Scognamiglio B DrsquoAbrosca A Esposito and A FiorentinoldquoMetabolomics an unexplored tool for allelopathy studiesrdquoJournal of Allelochemical Interactions vol 1 pp 9ndash23 2015

[7] J W Allwood D I Ellis and R Goodacre ldquoMetabolomictechnologies and their application to the study of plants andplant-host interactionsrdquo Physiologia Plantarum vol 132 no 2pp 117ndash135 2008

[8] R R Forseth and F C Schroeder ldquoNMR-spectroscopic analysisof mixtures from structure to functionrdquo Current Opinion inChemical Biology vol 15 no 1 pp 38ndash47 2011

[9] H K Kim Y H Choi and R Verpoorte ldquoNMR-basedmetabolomic analysis of plantsrdquo Nature Protocols vol 5 no 3pp 536ndash549 2010

[10] M Scognamiglio V Fiumano B DAbrosca et al ldquoChemicalinteractions between plants in Mediterranean vegetation theinfluence of selected plant extracts on Aegilops geniculatametabolomerdquo Phytochemistry vol 106 pp 69ndash85 2014

[11] A Vallverdu-Queralt J Regueiro M Martınez-Huelamo JF R Alvarenga L N Leal and R M Lamuela-RaventosldquoA comprehensive study on the phenolic profile of widelyused culinary herbs and spices rosemary thyme oreganocinnamon cumin and bayrdquo Food Chemistry vol 154 pp 299ndash307 2014

[12] Q Cui I A Lewis A D Hegeman et al ldquoMetabolite identifi-cation via the Madison Metabolomics Consortium DatabaserdquoNature Biotechnology vol 26 no 2 pp 162ndash164 2008

Journal of Analytical Methods in Chemistry 9

[13] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissusCarltonrdquoPhytochemistry vol 88 pp 43ndash53 2013

[14] R Verpoorte Y H Choi and H K Kim ldquoNMR-basedmetabolomics at work in phytochemistryrdquo PhytochemistryReviews vol 6 no 1 pp 3ndash14 2007

[15] J-L Wolfender S Rudaz Y H Choi and H K Kim ldquoPlantmetabolomics from holistic data to relevant biomarkersrdquo Cur-rent Medicinal Chemistry vol 20 no 8 pp 1056ndash1090 2013

[16] B DrsquoAbrosca E Buommino G DrsquoAngelo et al ldquoSpectroscopicidentification and anti-biofilm properties of polar metabolitesfrom the medicinal plant Helichrysum italicum against Pseu-domonas aeruginosardquo Bioorganic and Medicinal Chemistry vol21 no 22 pp 7038ndash7046 2013

[17] S Pacifico B DrsquoAbrosca M Scognamiglio et al ldquoNMR-basedmetabolic profiling and in vitro antioxidant and hepatotoxicassessment of partially purified fractions fromGolden german-der (Teucrium polium L) methanolic extractrdquo Food Chemistryvol 135 no 3 pp 1957ndash1967 2012

[18] R Scherer A C P Rybka C A Ballus A D Meinhart J TFilho and H T Godoy ldquoValidation of a HPLC method forsimultaneous determination of main organic acids in fruits andjuicesrdquo Food Chemistry vol 135 no 1 pp 150ndash154 2012

[19] J Liimatainen M Karonen J Sinkkonen M Helander andJ-P Salminen ldquoPhenolic compounds of the inner bark ofBetula pendula seasonal and genetic variation and inductionby woundingrdquo Journal of Chemical Ecology vol 38 no 11 pp1410ndash1418 2012

[20] K Hosni K Msaada M Ben Taarit and B Marzouk ldquoPhe-nological variations of secondary metabolites from Hypericumtriquetrifolium Turrardquo Biochemical Systematics and Ecology vol39 no 1 pp 43ndash50 2011

[21] F Brahmi B Mechri S Dabbou M Dhibi and M HammamildquoThe efficacy of phenolics compounds with different polaritiesas antioxidants from olive leaves depending on seasonal varia-tionsrdquo Industrial Crops and Products vol 38 no 1 pp 146ndash1522012

[22] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissus Carltonrdquo Phytochemistry vol 88 pp 43ndash53 2013

[23] M Petersen and M S J Simmonds ldquoRosmarinic acidrdquo Phyto-chemistry vol 62 no 2 pp 121ndash125 2003

[24] R Quirantes-Pine M Herranz-Lopez L Funes et al ldquoPhenyl-propanoids and their metabolites are the major compoundsresponsible for blood-cell protection against oxidative stressafter administration of Lippia citriodora in ratsrdquo Phytomedicinevol 20 no 12 pp 1112ndash1118 2013

[25] G Tibaldi E Fontana and S Nicola ldquoPostharvest managementaffects spearmint and calamint essential oilsrdquo Journal of theScience of Food and Agriculture vol 93 no 3 pp 580ndash586 2013

[26] K Hammer G Laghetti and K Pistrick ldquoCalamintha nepeta(L) Savi and Micromeria thymifolia (Scop) Fritsch cultivatedin ItalyrdquoGenetic Resources and Crop Evolution vol 52 no 2 pp215ndash220 2005

[27] G Appendino M Ottino N Marquez et al ldquoArzanol an anti-inflammatory and anti-HIV-1 phloroglucinol 120572-pyrone fromHelichrysum italicum ssp microphyllumrdquo Journal of NaturalProducts vol 70 no 4 pp 608ndash612 2007

[28] W-R Diao Q-P Hu H Zhang and J-G Xu ldquoChemicalcomposition antibacterial activity and mechanism of action ofessential oil from seeds of fennel (Foeniculum vulgare Mill)rdquoFood Control vol 35 no 1 pp 109ndash116 2014

[29] N Martins L Barros C Santos-Buelga M Henriques S Silvaand I C F R Ferreira ldquoDecoction infusion and hydroalcoholicextract of Origanum vulgare L different performances regard-ing bioactivity and phenolic compoundsrdquo Food Chemistry vol158 pp 73ndash80 2014

[30] T Mihajilov-Krstev D Radnovic D Kitic et al ldquoChemicalcomposition antimicrobial antioxidative and anticholinester-ase activity of Satureja Montana L ssp montana essential oilrdquoCentral European Journal of Biology vol 9 no 7 pp 668ndash6772014

[31] C Sarikurkcu M Sabih Ozer M Eskici B Tepe S Can andE Mete ldquoEssential oil composition and antioxidant activity ofThymus longicaulis C Presl subsp longicaulis var longicaulisrdquoFood andChemical Toxicology vol 48 no 7 pp 1801ndash1805 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

8 Journal of Analytical Methods in Chemistry

compounds as reported for several Lamiaceae [17] Never-theless the role in nutrition of primary metabolites is oftendisregarded [18] These considerations raise the attention tothe need for a comprehensive profiling of their metabolites

Furthermore the NMR-based metabolomic approachhere proposed due to the short time of analyses and to lowercosts compared to other analytical methods was very usefulfor the study of seasonal variation of metabolites

Indeed although it is clear that secondary metabolitesshow peculiar trends only fragmentary information is avail-able mainly because of the used approaches

The most common and easiest procedure was to performthe phytochemical study on samples collected in a specifictime of the year and then compare the other months (orseasons) by setting up a series of target analyses often byHPLC [19 20] or by less time and resource consumingapproaches based on colorimetric assays [21]

The improvement of analytical methods and espe-cially the availability of a high-throughput approach likemetabolomics [5] gives the chance to further explore theissue of seasonality [22] and to thoroughly study thesechanges

Concerning themetabolites identifiedwithin the extractsit is important to underline their biological activity

Phenols are well known for their antioxidant activity [11]As the studied plants are all very rich in phenolics they havea great antioxidant potential This could also explain the useof some of these herbs as natural food preservatives (Table 1)

Among the detected compounds rosmarinic acid is themost widespread and the most abundant A plethora ofbiological activities has been attributed to this compoundamong them adstringent antioxidative anti-inflammatoryantimutagen antibacterial and antiviral [23]

Many other phenylpropanoids and flavonoids were alsodetected Several properties have been reported for thesecompounds such as anti-inflammatory antimicrobial andantitumor activity [24]

The richness in these compounds of the studied plantssupports their traditional uses

However the study evidenced that qualitative and quan-titative variations of metabolites are observed along the year

This is to the best of our knowledge the first reportof metabolite content and seasonal qualitative and quanti-tative variation of this set of food spices Furthermore it isevidenced that a higher content of compounds known fortheir health promoting capacities can be found in spring andsummer samples of all the analysed species although season-specific compounds were also detected

4 Conclusions

NMR-based metabolomics has been applied to the study ofchemical composition of selected aromatic plants ofMediter-ranean vegetation

The method allowed determining the chemical compo-sition of plant extracts in terms of primary and secondarymetabolites The abundance of aromatic secondary metabo-lites suggested that the traditional uses of these plants mightbe supported by their chemical composition

Furthermore the seasonality of the accumulation of thesemetabolites was studied

Conflict of Interests

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

Acknowledgment

The authors would like to thank the anonymous reviewers fortheir valuable suggestions

References

[1] A M Scherrer R Motti and C S Weckerle ldquoTraditional plantuse in the areas of Monte Vesole and Ascea Cilento NationalPark (Campania Southern Italy)rdquo Journal of Ethnopharmacol-ogy vol 97 no 1 pp 129ndash143 2005

[2] T Efferth and H J Greten ldquoMedicinal and aromatic plantresearch in the 21st centuryrdquoMedicinal amp Aromatic Plants vol1 article e110 2012

[3] R Rodrıguez-Solana J M Salgado J M Domınguez andS Cortes-Dieguez ldquoComparison of soxhlet accelerated sol-vent and supercritical fluid extraction techniques for volatile(GCndashMS and GCFID) and phenolic compounds (HPLCndashESIMSMS) from Lamiaceae Species rdquo Phytochemical Analysisvol 26 no 1 pp 61ndash71 2015

[4] T Fornari G Vicente E Vazquez M R Garcıa-Risco andG Reglero ldquoIsolation of essential oil from different plants andherbs by supercritical fluid extractionrdquo Journal of Chromatogra-phy A vol 1250 pp 34ndash48 2012

[5] H K Kim Y H Choi and R Verpoorte ldquoNMR-based plantmetabolomics where do we stand where do we gordquo Trends inBiotechnology vol 29 no 6 pp 267ndash275 2011

[6] M Scognamiglio B DrsquoAbrosca A Esposito and A FiorentinoldquoMetabolomics an unexplored tool for allelopathy studiesrdquoJournal of Allelochemical Interactions vol 1 pp 9ndash23 2015

[7] J W Allwood D I Ellis and R Goodacre ldquoMetabolomictechnologies and their application to the study of plants andplant-host interactionsrdquo Physiologia Plantarum vol 132 no 2pp 117ndash135 2008

[8] R R Forseth and F C Schroeder ldquoNMR-spectroscopic analysisof mixtures from structure to functionrdquo Current Opinion inChemical Biology vol 15 no 1 pp 38ndash47 2011

[9] H K Kim Y H Choi and R Verpoorte ldquoNMR-basedmetabolomic analysis of plantsrdquo Nature Protocols vol 5 no 3pp 536ndash549 2010

[10] M Scognamiglio V Fiumano B DAbrosca et al ldquoChemicalinteractions between plants in Mediterranean vegetation theinfluence of selected plant extracts on Aegilops geniculatametabolomerdquo Phytochemistry vol 106 pp 69ndash85 2014

[11] A Vallverdu-Queralt J Regueiro M Martınez-Huelamo JF R Alvarenga L N Leal and R M Lamuela-RaventosldquoA comprehensive study on the phenolic profile of widelyused culinary herbs and spices rosemary thyme oreganocinnamon cumin and bayrdquo Food Chemistry vol 154 pp 299ndash307 2014

[12] Q Cui I A Lewis A D Hegeman et al ldquoMetabolite identifi-cation via the Madison Metabolomics Consortium DatabaserdquoNature Biotechnology vol 26 no 2 pp 162ndash164 2008

Journal of Analytical Methods in Chemistry 9

[13] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissusCarltonrdquoPhytochemistry vol 88 pp 43ndash53 2013

[14] R Verpoorte Y H Choi and H K Kim ldquoNMR-basedmetabolomics at work in phytochemistryrdquo PhytochemistryReviews vol 6 no 1 pp 3ndash14 2007

[15] J-L Wolfender S Rudaz Y H Choi and H K Kim ldquoPlantmetabolomics from holistic data to relevant biomarkersrdquo Cur-rent Medicinal Chemistry vol 20 no 8 pp 1056ndash1090 2013

[16] B DrsquoAbrosca E Buommino G DrsquoAngelo et al ldquoSpectroscopicidentification and anti-biofilm properties of polar metabolitesfrom the medicinal plant Helichrysum italicum against Pseu-domonas aeruginosardquo Bioorganic and Medicinal Chemistry vol21 no 22 pp 7038ndash7046 2013

[17] S Pacifico B DrsquoAbrosca M Scognamiglio et al ldquoNMR-basedmetabolic profiling and in vitro antioxidant and hepatotoxicassessment of partially purified fractions fromGolden german-der (Teucrium polium L) methanolic extractrdquo Food Chemistryvol 135 no 3 pp 1957ndash1967 2012

[18] R Scherer A C P Rybka C A Ballus A D Meinhart J TFilho and H T Godoy ldquoValidation of a HPLC method forsimultaneous determination of main organic acids in fruits andjuicesrdquo Food Chemistry vol 135 no 1 pp 150ndash154 2012

[19] J Liimatainen M Karonen J Sinkkonen M Helander andJ-P Salminen ldquoPhenolic compounds of the inner bark ofBetula pendula seasonal and genetic variation and inductionby woundingrdquo Journal of Chemical Ecology vol 38 no 11 pp1410ndash1418 2012

[20] K Hosni K Msaada M Ben Taarit and B Marzouk ldquoPhe-nological variations of secondary metabolites from Hypericumtriquetrifolium Turrardquo Biochemical Systematics and Ecology vol39 no 1 pp 43ndash50 2011

[21] F Brahmi B Mechri S Dabbou M Dhibi and M HammamildquoThe efficacy of phenolics compounds with different polaritiesas antioxidants from olive leaves depending on seasonal varia-tionsrdquo Industrial Crops and Products vol 38 no 1 pp 146ndash1522012

[22] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissus Carltonrdquo Phytochemistry vol 88 pp 43ndash53 2013

[23] M Petersen and M S J Simmonds ldquoRosmarinic acidrdquo Phyto-chemistry vol 62 no 2 pp 121ndash125 2003

[24] R Quirantes-Pine M Herranz-Lopez L Funes et al ldquoPhenyl-propanoids and their metabolites are the major compoundsresponsible for blood-cell protection against oxidative stressafter administration of Lippia citriodora in ratsrdquo Phytomedicinevol 20 no 12 pp 1112ndash1118 2013

[25] G Tibaldi E Fontana and S Nicola ldquoPostharvest managementaffects spearmint and calamint essential oilsrdquo Journal of theScience of Food and Agriculture vol 93 no 3 pp 580ndash586 2013

[26] K Hammer G Laghetti and K Pistrick ldquoCalamintha nepeta(L) Savi and Micromeria thymifolia (Scop) Fritsch cultivatedin ItalyrdquoGenetic Resources and Crop Evolution vol 52 no 2 pp215ndash220 2005

[27] G Appendino M Ottino N Marquez et al ldquoArzanol an anti-inflammatory and anti-HIV-1 phloroglucinol 120572-pyrone fromHelichrysum italicum ssp microphyllumrdquo Journal of NaturalProducts vol 70 no 4 pp 608ndash612 2007

[28] W-R Diao Q-P Hu H Zhang and J-G Xu ldquoChemicalcomposition antibacterial activity and mechanism of action ofessential oil from seeds of fennel (Foeniculum vulgare Mill)rdquoFood Control vol 35 no 1 pp 109ndash116 2014

[29] N Martins L Barros C Santos-Buelga M Henriques S Silvaand I C F R Ferreira ldquoDecoction infusion and hydroalcoholicextract of Origanum vulgare L different performances regard-ing bioactivity and phenolic compoundsrdquo Food Chemistry vol158 pp 73ndash80 2014

[30] T Mihajilov-Krstev D Radnovic D Kitic et al ldquoChemicalcomposition antimicrobial antioxidative and anticholinester-ase activity of Satureja Montana L ssp montana essential oilrdquoCentral European Journal of Biology vol 9 no 7 pp 668ndash6772014

[31] C Sarikurkcu M Sabih Ozer M Eskici B Tepe S Can andE Mete ldquoEssential oil composition and antioxidant activity ofThymus longicaulis C Presl subsp longicaulis var longicaulisrdquoFood andChemical Toxicology vol 48 no 7 pp 1801ndash1805 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Analytical Methods in Chemistry 9

[13] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissusCarltonrdquoPhytochemistry vol 88 pp 43ndash53 2013

[14] R Verpoorte Y H Choi and H K Kim ldquoNMR-basedmetabolomics at work in phytochemistryrdquo PhytochemistryReviews vol 6 no 1 pp 3ndash14 2007

[15] J-L Wolfender S Rudaz Y H Choi and H K Kim ldquoPlantmetabolomics from holistic data to relevant biomarkersrdquo Cur-rent Medicinal Chemistry vol 20 no 8 pp 1056ndash1090 2013

[16] B DrsquoAbrosca E Buommino G DrsquoAngelo et al ldquoSpectroscopicidentification and anti-biofilm properties of polar metabolitesfrom the medicinal plant Helichrysum italicum against Pseu-domonas aeruginosardquo Bioorganic and Medicinal Chemistry vol21 no 22 pp 7038ndash7046 2013

[17] S Pacifico B DrsquoAbrosca M Scognamiglio et al ldquoNMR-basedmetabolic profiling and in vitro antioxidant and hepatotoxicassessment of partially purified fractions fromGolden german-der (Teucrium polium L) methanolic extractrdquo Food Chemistryvol 135 no 3 pp 1957ndash1967 2012

[18] R Scherer A C P Rybka C A Ballus A D Meinhart J TFilho and H T Godoy ldquoValidation of a HPLC method forsimultaneous determination of main organic acids in fruits andjuicesrdquo Food Chemistry vol 135 no 1 pp 150ndash154 2012

[19] J Liimatainen M Karonen J Sinkkonen M Helander andJ-P Salminen ldquoPhenolic compounds of the inner bark ofBetula pendula seasonal and genetic variation and inductionby woundingrdquo Journal of Chemical Ecology vol 38 no 11 pp1410ndash1418 2012

[20] K Hosni K Msaada M Ben Taarit and B Marzouk ldquoPhe-nological variations of secondary metabolites from Hypericumtriquetrifolium Turrardquo Biochemical Systematics and Ecology vol39 no 1 pp 43ndash50 2011

[21] F Brahmi B Mechri S Dabbou M Dhibi and M HammamildquoThe efficacy of phenolics compounds with different polaritiesas antioxidants from olive leaves depending on seasonal varia-tionsrdquo Industrial Crops and Products vol 38 no 1 pp 146ndash1522012

[22] A Lubbe H Gude R Verpoorte and Y H Choi ldquoSeasonalaccumulation of major alkaloids in organs of pharmaceuticalcropNarcissus Carltonrdquo Phytochemistry vol 88 pp 43ndash53 2013

[23] M Petersen and M S J Simmonds ldquoRosmarinic acidrdquo Phyto-chemistry vol 62 no 2 pp 121ndash125 2003

[24] R Quirantes-Pine M Herranz-Lopez L Funes et al ldquoPhenyl-propanoids and their metabolites are the major compoundsresponsible for blood-cell protection against oxidative stressafter administration of Lippia citriodora in ratsrdquo Phytomedicinevol 20 no 12 pp 1112ndash1118 2013

[25] G Tibaldi E Fontana and S Nicola ldquoPostharvest managementaffects spearmint and calamint essential oilsrdquo Journal of theScience of Food and Agriculture vol 93 no 3 pp 580ndash586 2013

[26] K Hammer G Laghetti and K Pistrick ldquoCalamintha nepeta(L) Savi and Micromeria thymifolia (Scop) Fritsch cultivatedin ItalyrdquoGenetic Resources and Crop Evolution vol 52 no 2 pp215ndash220 2005

[27] G Appendino M Ottino N Marquez et al ldquoArzanol an anti-inflammatory and anti-HIV-1 phloroglucinol 120572-pyrone fromHelichrysum italicum ssp microphyllumrdquo Journal of NaturalProducts vol 70 no 4 pp 608ndash612 2007

[28] W-R Diao Q-P Hu H Zhang and J-G Xu ldquoChemicalcomposition antibacterial activity and mechanism of action ofessential oil from seeds of fennel (Foeniculum vulgare Mill)rdquoFood Control vol 35 no 1 pp 109ndash116 2014

[29] N Martins L Barros C Santos-Buelga M Henriques S Silvaand I C F R Ferreira ldquoDecoction infusion and hydroalcoholicextract of Origanum vulgare L different performances regard-ing bioactivity and phenolic compoundsrdquo Food Chemistry vol158 pp 73ndash80 2014

[30] T Mihajilov-Krstev D Radnovic D Kitic et al ldquoChemicalcomposition antimicrobial antioxidative and anticholinester-ase activity of Satureja Montana L ssp montana essential oilrdquoCentral European Journal of Biology vol 9 no 7 pp 668ndash6772014

[31] C Sarikurkcu M Sabih Ozer M Eskici B Tepe S Can andE Mete ldquoEssential oil composition and antioxidant activity ofThymus longicaulis C Presl subsp longicaulis var longicaulisrdquoFood andChemical Toxicology vol 48 no 7 pp 1801ndash1805 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of