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
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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
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Analytical Methods in Chemistry
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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
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
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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
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Analytical Methods in Chemistry
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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
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
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Analytical ChemistryInternational Journal of
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Quantum Chemistry
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Organic Chemistry International
ElectrochemistryInternational Journal of
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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
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Analytical Methods in Chemistry
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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
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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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
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Analytical Methods in Chemistry
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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