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Carica Papaya

Carica Papaya

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JOURNAL OFFOOD COMPOSITIONAND ANALYSIS
Journal of Food Composition and Analysis 20 (2007) 584–590
Original Article
Gas chromatography–mass spectrometry analysis of phenoliccompounds from
Carica papaya
L. leaf 
Antonella Canini
a,
Ã
, Daniela Alesiani
a
, Giuseppe D’Arcangelo
b
, Pietro Tagliatesta
b
a
Department of Biology, University of Rome ‘‘Tor Vergata’’, Via della Ricerca Scientifica, 1-00133 Rome, Italy
b
Department of Sciences and Chemical Technologies, University of Rome ‘‘Tor Vergata’’, Via della Ricerca Scientifica, 1-00133 Rome, Italy
Received 2 October 2006; received in revised form 23 February 2007; accepted 8 March 2007
Abstract
Phenolic compounds are important components in vegetable foods, infusions and teas for their beneficial effects on human health. Thepresence of such compounds, evidenced for the first time in
Carica papaya
leaves, could partially explain the pharmacological propertiesof this plant and demonstrates its importance in alimentation and daily intake.
C. papaya
leaves were extracted with methanol in aSoxhlet apparatus and later with a liquid–liquid extraction (LLE) with the aim of identifying and quantifying secondary metabolitesfrom this plant, using gas chromatography–mass spectrometry (GC–MS) in the selected ion-monitoring (SIM) mode. Derivatizationprocedure of the extract was necessary to analyze the polar compounds in GC–MS. 5,7-Dimethoxycoumarin and polar molecules such asprotocatechuic acid,
p
-coumaric acid, caffeic acid, chlorogenic acid, kaempferol and quercetin were detected and identified in qualitativeanalysis. The quantitative analysis has shown the presence of phenolic acids as the main compound, while chlorogenic acid was found intrace amounts, compared to the flavonoids and coumarin compounds. The quantities detected were 0.25mg/g (dry leaf) for caffeic acid,0.33mg/g for
p
-coumaric acid and 0.11mg/g for protocatechuic acid. Kaempferol and quercetin were 0.03 and 0.04mg/g, respectively,while that for 5,7-dimethoxycoumarin was 0.14mg/g.
r
2007 Elsevier Inc. All rights reserved.
Keywords: Carica papaya
; 5,7-Dimethoxycoumarin; Protocatechuic acid;
p
-Coumaric acid; Caffeic acid; Chlorogenic acid; Kaempferol; Quercetin; Gaschromatography–mass spectrometry
1. Introduction
Carica papaya
(papaya) is a tree-like herbaceous plant, amember of the small family Caricaceae and widelycultivated for its edible fruits. It originates in the lowlandsof eastern Central America, from Mexico to Panama, andcan be found in all tropical countries and many sub-tropical regions of the world. Parts of the plant are used intropical diets as a fruit or vegetable; it is sometime used as atherapeutic remedy for several of its medicinal properties.Papaya fruit is thought to contain some immuno-stimulat-ing and anti-oxidant agents (Aruoma et al., 2006;Mehdipour et al., 2006): immature fruits and roots areused for their abortifacient activity (Cherian, 2000;Sarma and Mahanta, 2000); the seeds are now being used as apotential post-testicular anti-fertility drug (Lohiya et al.,2000); the pulp is used by African hospitals for treatingwounds and burns (Starley et al., 1999); the latex and theseeds are used in the care of gastrointestinal nematodeinfections and they have shown anthelmintic activity(Stepek et al., 2004); and the seeds and immature fruithave shown bacteriostatic activity against the humanenteric pathogens (Osato et al., 1993). The leaves are usedto relieve the symptoms of asthma and as a vermifuge, inthe treatment of gastric problems, fever and amoebicdysentery. Methanolic leaf extract demonstrated vasodila-tatory and anti-oxidant effects, both implicated in the
ARTICLE IN PRESS
www.elsevier.com/locate/jfca0889-1575/$-see front matter
r
2007 Elsevier Inc. All rights reserved.doi:10.1016/j.jfca.2007.03.009
Abbreviations:
BSTFA,
,
O
-Bis(trimethylsilyl)trifluoroacetamide; GC,gas chromatography; IS, internal standard; LLE, liquid–liquid extraction;MS, mass spectrometry;
m
/
z
, fragment mass/charge ratio;
r
2
, correlationcoefficients; rt, retention time; SIM, selected ion monitoring; TIC, totalions chromatogram; TMCS, trimethylchlorosilane
Ã
Corresponding author. Tel.: +390672594344; fax: +39062023500.
E-mail addresses:
 
reduction of cardiovascular risks (Runnie et al., 2004). Theaqueous extract showed beneficial effects for the accelera-tion of wound healing processes in rats (Mahmood et al.,2005).Very little has been published on the chemical composi-tion or biochemistry of 
C. papaya
. Chemical characteriza-tion of the metabolites extracted from the plant has shownthe presence of active compounds in the plant tissues, suchas cysteine endopeptidases, a class-II and class-III chit-inase, and glutaminyl cyclase in the latex (Azarkan et al.,2005), the linalool in fruit pulp (Winterhalter et al., 1986), the alkaloids carpaine, pseudocarpaine, dehydrocarpaine Iand II in the leaves (Khuzhaev and Aripova, 2000),kaempferol and quercetin in the shoots (Miean andMohamed, 2001), the cyanogenic compounds in leavesand roots and benzylglucosinolate and its breakdownproduct benzylisothiocyanate in all the tissues (Olafsdottiret al., 2002).The aim of this work was to carry out a phytochemicalanalysis of 
C. papaya
leaf extracts. Our analytical attentionwas focused on secondary metabolites, particularly phe-nolic compounds from papaya leaves, which have neverbefore been reported in the literature. These molecules playseveral roles in plant physiological processes, such asprotection from UV, defense against pathogens andphytophagous, pollination and dissemination, symbiosisand allelopathic interactions (Buchanan et al., 2000).Biological activity is the basis for traditional medicine,which uses the pharmacological efficacy of naturalcompounds present in herbal preparations for treatinghuman diseases. Furthermore, papaya leaves are used intropical alimentation cooked as a vegetable and inpreparation of teas and infusions. In connection with this,epidemiological evidence has supported the role that anti-oxidants, including phenolic compounds, play in theprevention of several chronic diseases such as cardiovas-cular disease, cancer, diabetes, bacterial and parasiticinfections (Murakami et al., 1994;Sherman and Billing, 1999). These reasons explain the importance of papayaplant for tropical and also non-tropical populations andthe scientific interest in knowing the chemical compositionof the extracts.Gas chromatography–mass spectrometry (GC–MS) wasselected as the method of chemical analysis to identify andquantify the metabolites in leaf extracts obtained usingliquid–liquid extraction (LLE). This extraction methodmade it possible to clean up the extracted sample beforederivatization reaction and chromatographic separationand identification. Such a method is extremely useful forthe purification of the samples resulting from the inter-ferences due to the biological matrix. An analyticalcomparison was even carried out between nonhydrolyzedand hydrolyzed extracts, to quantify free and boundphenolic compounds. In fact, the flavonoids occur innature as their glycoside derivatives in which one or moresugar groups are attached to the phenolic groups (Croft,1998;Zuo et al., 2002;Chen and Zuo, 2007). Such derivatives can be found especially for the phenolic acids,which occur mostly esterified, glycosylated or polymerized(Naczk and Shahidi, 2004). The extraction recovery of bound phenolics was evaluated using alkaline hydrolysis.
2. Materials and methods
 2.1. Reagents
,
O
-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) con-taining 1% trimethylchlorosilane (TMCS), pyridine,methanol and diethyl ether were purchased from Sigma-Aldrich (St. Louis, MO, USA). The standard referencecompounds were 5,7-dimethoxycoumarin, bergapten (5-methoxypsoralen), protocatechuic acid (3,4-dihydroxyben-zoic acid),
p
-coumaric acid (
trans
-4-hydroxycinnamic acid),caffeic acid (3,4-dihydroxycinnamic acid), chlorogenic acid[1,3,4,5-tetrahydroxycyclohexanecarboxylic acid 3-(3,4-di-hydroxycinnamate)], kaempferol (3,4
0
,5,7-tetrahydroxy-flavone), quercetin (3,3
0
,4
0
,5,7-pentahydroxyflavone) andgenistein (4
0
,5,7-trihydroxyisoflavone) and they were alsopurchased from Sigma-Aldrich. All solvents and reagentsused in this study were of analytical grade purity.
 2.2. Plant material 
Samples of 
Carica papaya
L. (Caricaceae) leaves (300g)were collected in West Cameroon (Africa) in March 2006and identified by one of us (A.C.). A voucher specimen of this raw material is deposited at Herbarium of Universityof Rome ‘‘Tor Vergata’’.
 2.3. Extraction of phenolic compounds
The extraction of phenolic compounds from
C. papaya
leaf was carried out as follows. Plant material (10g) wasreduced to a fine powder and extracted in a Soxhletapparatus (90
1
C for 24h) with 200ml of 70% aqueousmethanol (v/v) acidified to pH 2 with some drops of concentrated HCl.A part of the filtered extract was used for LLE of freephenolic compounds. After methanol evaporation in arotary evaporator, the resulting aqueous solution wasextracted three times with 30ml of diethyl ether in aseparatory funnel and the organic solutions were combinedand evaporated to dryness. Before GC injection, the driedsample was suspended in BSTFA+TMCS 1% for deriva-tization of the polar compounds. The remaining solutionwas evaporated to dryness and then submitted to alkalinehydrolysis by adding NaOH (5
M
, 30mL) under nitrogen atroom temperature for 4h, after which the sample wasconcentrated and the solution was acidified with HCl 12
M
(Germano `et al., 2006). The released phenolic compoundswere extracted with diethyl ether and dried for derivatiza-tion as reported above.
ARTICLE IN PRESS
A. Canini et al. / Journal of Food Composition and Analysis 20 (2007) 584–590
585
 
 2.4. Derivatization procedure
For GC–MS analysis of polar compounds, standardsand leaf extracts were derivatized in order to obtainthe alkyl silylated derivatives. The dried extracts weretreated by adding BSTFA+TMCS 1%/anhydrouspyridine (1.5mL, 1:1). The mixture was heated at 90
1
Cfor 1h and derivatized sample submitted to GC–MSanalysis.
 2.5. Preparation of the standard solutions
Solutions of 5,7-dimethoxycoumarin, bergapten, caffeicacid,
p
-coumaric acid, chlorogenic acid, protocathe-cuic acid, kaempferol, quercetin and genistein wereprepared for qualitative and quantitative analysis of leaf extracts. For the polar compounds, stock solutions wereprepared adding the compounds to BSTFA+TMCS1%/pyridine (50
m
L, 1:1): the mixture was heated at90
1
C for 1h and standard solutions at different concen-tration were prepared. For the apolar compounds, thederivatization was not necessary. In GC–MS analysis, 3
m
Lof each standard solution was injected into a capillarycolumn.
 2.6. Analytical protocol 
GC–MS analyses were performed using a VG QuattroMass spectrometer (VG Micromass, UK), equipped witha Supelco SPB-5 capillary column (length: 30m; ID:250
m
m; film thickness: 1.4
m
m). The helium carrier gaswas set to a column head pressure of 
¼
100kPa. Theinlet temperature was set at 280
1
C while the oventemperature was initially at 100
1
C (held for 3min) andthen increased to 315
1
C at 20
1
C/min. The mass spectro-meter was operated in the positive electron impact modewith an ionization energy of 70eV. The ion sourcetemperature was 190
1
C at a pressure of 10E-4Torr.Detection was performed in selective ion-monitoring(SIM) mode and peaks were identified and quantitatedusing target ions.
 2.7. GC–MS analysis
GC–MS analysis of 
C. papaya
extracts were carried outin SIM mode. The identification of each peak was achievedby comparing the retention time (rt) and mass spectra of the compounds in the extract to that of the standards,matching the area ratios of characteristic ions with those of respective standards.In quantitative analysis, a series of solutions containingthe standard molecules and relative internal standard (IS)in known concentrations was analyzed. Bergapten wasselected as IS for 5,7-dimethoxycoumarin due to itschemical similarity with it and, for the same reason,genistein for flavonoids and phenolic acids. The linearcalibration curves were traced to quantify the identifiedcompounds in the different extracts: each standard solutionwas analyzed to calculate the peak area ratio of moleculescompared to IS, then the response ratios (
 y
) and therelative molecule concentrations (
x
) were used for theconstruction of the calibration curve. Finally, each extractwith IS in the same concentration of standard solutionswas analyzed, and the peak area ratio was used to calculatethe concentration of the identified compounds by using thecurve equation. Three-microliter aliquots of each standardsolution and of the extracts were used for GC–MS analysisand triplicate injections were made for each sample. Ionswith the mass/charge (
m
/
z
) ratio shown inTable 1wereused in the SIM mode for quantification. For thequantitative study, three independent leaf samples wereextracted and analyzed independently and each sample wasanalyzed three times. Results are expressed as themean
7
standard deviation of three independent experi-ments and were evaluated by one-way analysis of variancein Microsoft Excel
s
2003.
3. Results and discussion
InTable 1, the rt, obtained in GC–MS system used inthis work, and the
m
/
z
ratio characteristic for each ion of all the analyzed standards are shown. The peak identifica-tion was obtained by GC–MS analysis of 
C. papaya
ARTICLE IN PRESS
Table 1Secondary metabolites detected using GC/SIM–MS conditions, with relative retention time (rt) and characteristic ions (
m
/
z
) in the mass spectra of silylatedderivatives and not in standard solutions and
C. papaya
extractsCompound MolecularweightTMSgroupsTMSderivatizedmolecular weightrt (min)
m
/
z
a
(relative intensity)
2
. Protocatechuic acid 154 3 370 11.02 370(100),355(9),193(2),73(1)
3
. p
-Coumaric acid 164 2 308 11.78 308(100),293(10),249(3),219(6)
4
. 5,7-Dimethoxycoumarin 206 0 11.89 206(100),191(23),178(40),163(28)
5
. Caffeic acid 180 3 396 12.97 396(100),381(2),219(4),73(1)
6
. Kaempferol 286 4 574 19.53 574(15),559(100),487(30),272(3)
7
. Quercetin 302 5 662 20.77 662(10),647(100),574(63),559(14)
8
. Chlorogenic acid 354 6 786 20.07 786(100),419(7),397(11),345(28)
a
Quantitation ions are underlined.
A. Canini et al. / Journal of Food Composition and Analysis 20 (2007) 584–590
586

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