Food Chemistry 197 (2016) 124–131

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Food Chemistry

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Variability of chemical composition and antioxidant activity of essentialoils between Myrtus communis var. Leucocarpa DC and var. MelanocarpaDC

http://dx.doi.org/10.1016/j.foodchem.2015.10.0560308-8146/� 2015 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: pintore@uniss.it (G. Pintore).

1 These authors contributed equally to the work.

Giacomo Luigi Petretto a,1, Mariateresa Maldini a,1, Roberta Addis a, Mario Chessa a, Marzia Foddai a,Jonathan P. Rourke b, Giorgio Pintore a,⇑aDepartment of Chemistry and Pharmacy, University of Sassari, 07100, ItalybDepartment of Chemistry, University of Warwick, Coventry CV4 7AL, UK

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 May 2015Received in revised form 10 September 2015Accepted 12 October 2015Available online 22 October 2015

Keywords:White berriesMyrtenyl acetateWild plantsCultivated plantsPCA

Essential oils (EOs) from several individuals of Myrtus communis L. (M. communis) growing in differenthabitats in Sardinia have been studied. The analyses were focused on four groups of samples, namely cul-tivated and wild M. communis var. melanocarpa DC, characterized by red/purple berries, and cultivatedand wild M. communis var. leucocarpa DC, characterized by white berries. Qualitative and quantitativeanalyses demonstrated different EO fingerprints among the studied samples: cultivated and wild leuco-carpa variety differs mainly from the melanocarpa variety by a high amount of myrtenyl acetate(>200 mg/mL and 0.4 mg/mL in leucocarpa and melanocarpa varieties respectively). Conversely, the wildgroup is characterized by a higher amount, compared with the cultivated species, of linalool (about110 mg/mL and 20 mg/mL respectively), linalyl acetate (about 24 mg/mL and about 6 mg/mL respec-tively) whereas EOs of the cultivated plants were rich in pinocarveol-cis compared with wild plants(about 2 mg/mL and about 0.5 mg/mL respectively). Principal component analysis applied to the chro-matographic data confirm a differentiation and classification of EOs from the four groups of M. communisplants. Finally, antioxidant activity of the studied EOs shows differences between the various categoriesof samples.

� 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Myrtus communis is a mediterranean shrub, belonging to thefamily of Myrtaceae, geographically distributed in South Europe,West Asia and North Africa. In coastal areas, it usually grows inassociation with Pistacia lentiscus L., Phillyrea spp. and Arbutusunedo L. It can reach 2 m in height and it usually grows in acidicsoil, sheltered from the winds. M. communis flowers are an impor-tant source of pollen for several insects, particularly for honey bees(Petretto, Cossu, & Alamanni, 2015) and it produces berry-fruitswhich have been recognized to be rich in polyphenol compounds(Barboni, Cannac, Massi, Perez-Ramirez, & Chiaramonti, 2010).

M. communis is a well-known medicinal plant (Alipour,Dashti, & Hosseinzadeh, 2014) that has been used as an antimicro-bial (Akin, Aktumsek, & Nostro, 2010; Deriu et al., 2007), ananti-proliferative (Cottiglia et al., 2012), an antinflammatory

(Hosseinzadeh, Khoshdel, & Ghorbani, 2011) and an antiviral agent(Moradi, Karimi, Rafiean, Kheiri, & Saedi, 2011). It is also used inthe cosmetic industry and last, but not least, it is an importantraw material for the production of a characteristic liqueur, pro-duced by hydro-alcoholic infusion of the berries or leaves (Mulas,2012).

The steam distillation of leaves gives a colorless EO in goodyield, characterized by an intense smell, which has been thor-oughly studied.

M. communis EO has been used as an anti-inflammatory, anti-mutagenic, anti-diabetic, anti-malarial and anti-protozoal agent.Furthermore, it has shown an insecticidal effect as well as antiox-idant activity in the preservation of edible oils (Alipour et al.,2014). The EO chemical composition shows variability related toseveral parameters, such as the geographical area it was grownin, the period of collection or other environmental variations.Chalchat, Garry, and Michet (1998) proposed a classification ofM. communis from the mediterranean littoral into two groups,based on the a-pinene amount in the EO: one group of EOs witha-pinene >50% and the second group of EOs with a-pinene <35%.

G.L. Petretto et al. / Food Chemistry 197 (2016) 124–131 125

More detailed classifications have been carried out by Bouzabataet al. (2013) who identified two groups and several subgroups ofAlgerian M. communis EO, mainly classified by the relative amountof a-pinene. Rahimmlaek, Mirzakhani, and Pirbalouti (2013), in astudy of M. communis from Iran, observed a strong chemicalpolymorphism among the EOs, identifying two main groups: onecharacterized by high a-pinene and eucalyptol and the other,‘‘limonene chemotype”, by high limonene content.

As well as the great number of chemo-types, the genetic(Melito, Chessa, Erre, Padoani, & Mulas, 2013) and the morpholog-ical characteristics of M. communis exhibit a great variability andseveral varieties are known (Picci & Atzei, 1996). Two mainsubspecies have been described: M. communis var. typica andM. communis var. tarentina each of which include several morevarieties. The M. communis var. typica and M. communis var.tarentina classification is based on the morphological characteris-tics of the leaves, stems and flowers (Fiori, 1925; Parlatore,1842). By contrast, another classification of M. communis plants isbased on the color of the berries: var. melanocarpa DC is character-ized by red-purple fruit and var. leucocarpa DC is characterized bywhite fruits (Mulas, 2013).

The majority of literature reports on M. communis are focusedon the more common melanocarpa DC variety and only a fewpapers describe the chemical and/or biological properties of theleucocarpa variety. Messaoud and Boussaid (2011) pointed out thatwhite berries contain unsaturated fatty acid and an EO rich inmyrtenyl acetate whilst red-purple fruits produce polypheols andan EO characterized by a high amount of a-pinene, linalool anda-terpineol. Haciseferogullari and Ozcan (2012) discriminate twomyrtle morphs from Turkey by comparison of the chemical,morphological and physical properties, highlighting no differencesin morphological characteristic and only sporadic differences in themineral content of the two varieties.

In Sardinia (Italy), M. communis plants grow wild and it is wellrepresented in all areas of the region. It is widespread in the twovarieties: melanocarpa variety and leucocarpa variety, and a largearea is dedicated to the cultivation of this plant (about 162 ha).Sardinia comes top in Italy in the production of liqueur byhydro-alcoholic infusion of M. communis (red berries for red liquorand leaves for white liquor) with over 2 million liters manufac-tured per year. The quality of the product is linked to the rawmaterial which, in some cases, due to the limited availability inSardinia, is imported from north African countries. Previous studiesonM. communis liquor has shown an in vitro and ex-vivo alleviationof oxidative stress and in vitro vasodilatory activity (Tuberoso,Boban, Bifulco, Budimir, & Pirisi, 2013).

Although there is a considerable amount of literature relating toM. communis, the published data do not reveal much about the EOextracted from the leaves of melanocarpa variety. Owing to thegreat interest in this plant and with our experience in the field,we now wish to report on a chemical investigation into the EOsfrom the two varieties, melanocarpa variety and leucocarpa varietyofM. communis, from selected Sardinian habitats. Furthermore, ourchromatographic data were subjected to a multivariate studydesigned to establish any chemical variability between the differ-ent varieties of M. communis.

2. Materials and methods

2.1. Plant material and extraction of EOs

Four samples of the aerial parts of wild M. communis var. mela-nocarpa DC (MRE), four samples of the aerial parts of cultivated M.communis var. melanocarpa DC (MRC), four samples of the aerialparts of wild M. communis var. leucocarpa DC (MBE) and four

samples of the aerial parts of cultivated M. communis var.leucocarpa DC (MBC) were collected in April from the Sassari area,Sardinia, Italy. Wild plants were grown in dim light, in calcareoussoil and with natural watering whilst cultivated plants were grownin full daylight, in clay soil and were periodically watered.

A sample weighing 50 g ofM. communis leaves was subjected tohydro-distillation using a Clevenger type apparatus for 2 h, accord-ing to the European Pharmacopoeia protocol (2002). The yield ofEOs ranged between 0.32% (v/w) and 0.52% (v/w) calculated fromthe dry weight.

The extraction was carried out in triplicate for each sample,and the obtained EOs were collected separately, dried over anhy-drous sodium sulfate (Na2SO4) and then stored under a nitrogenatmosphere at 4 �C in amber glass vials until analyzed.

2.2. Chemicals and reagents

Unless stated otherwise, all chemicals and reagents were sup-plied by Sigma (Dorset, UK). The following terpenoid compoundcommercial reference standards were used: a-pinene, b-pinene,myrcene, p-cymene, limonene, 1,8-cineole, c-terpinene, linalool,pinocarveol-cis, terpinen-4-ol, a-terpineol, neryl acetate, geranylacetate, a-humulene and caryophyllene oxide.

2.3. Gas chromatography–mass spectrometry (GC–MS) analysis

The GC–MS analysis was carried out using an Agilent 7890 GCequipped with a Gerstel MPS autosampler, coupled to an Agilent7000C MSD detector. The chromatographic separation wasperformed on a VF-Wax 60 m � 0.25 mm i.d., 0.5 lm film thick-ness column (Agilent), as well as on an HP-5MS capillary column(30 m � 0.25 mm, film thickness 0.17 lm), the following tempera-ture programme was used for the VF-Wax column: 40 �C held for4 min, then increased to 150 �C at a rate of 5.0 �C/min, held for3 min then increased to 240 �C at a rate of 10 �C/min, and finallyheld for 12 min. For the HP-5MS column the following temperatureprogram was used: 60 �C hold for 3 min, then increased to 210 �Cat a rate of 4 �C/min, then held at 210 �C for 15 min, before increas-ing to 300 �C at a rate of 10 �C/min, and finally held at 300 �C for15 min. Helium was used as the carrier gas at a constant flow of1 mL/min for both columns. The data was analyzed using aMassHunter Workstation B.06.00 SP1, with identification of theindividual components (Table 1) performed by comparison withthe co-injected pure compounds and by matching the MS fragmen-tation patterns and retention indices with the built in libraries orliterature data or commercial mass spectral libraries (NIST/EPA/NIH 2008; HP1607 purchased from Agilent Technologies).

2.4. Gas chromatography–flame ionization detector (GC–FID) analysis

The GC analysis of the EOs was carried out using an Agilent4890 N instrument equipped with an FID and an HP-5 capillarycolumn (30 m � 0.25 mm, film thickness 0.17 lm). The columntemperature was held at 60 �C for 3 min, then increased to210 �C at a rate of 4 �C/min and held at 210 �C for 15 min, thenincreased to 300 �C at a rate of 10 �C/min, and finally held at300 �C for 15 min. Injector and detector temperatures were250 �C. Helium was used as carrier gas at a flow rate of 1 mL/min. The compound quantification in the EOs was carried out usingthe internal standard method, injecting a solution of EOs in hexane(dilution ratio 1:200). A calibration curve was constructed for eachmatching standard compound in the EOs. When standards wereunavailable, quantification was performed with a calibration curveof a compound of the same classes of volatiles (monoterpenehydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocar-

Table 1Calibration equations and quantification of the volatiles in the EOs of M. communis plants. RI = retention index. The values are expressed as mean ± SD of three independentexperiments (⁄MRC vs MRE P < 0.05; ⁄⁄MRC vs MBE P < 0.01; �MBC vs MBE P < 0.05; ��MBC vs MBE P < 0.01; yMRC vs MBC P < 0.05; yyMRC vs MBC P < 0.01; eMRE vs MBE P < 0.05;eeMRE vs MBE P < 0.01).

MRC MRE MBC MBE

mg/mL SD mg/mL SD mg/mL SD mg/mL SD RIHP5 RIVF-WAX Quantitation curve R2

Propyl butanoate 3.6yy 0.6 35.1⁄⁄ 9.2 12.4�� 0.5 9.0ee 0.7 918.0 1101.4 Geranyl acetateThujene alfa 1.2yy 0.1 1.8 0.5 0.6�� 0.1 0.9e 0.1 927.7 1039.3 Pinene alphaPinene alfa 306.8yy 38.1 271.8 15.3 137.4 26.3 130.9ee 12.1 938.4 1035.0 y = 0.01115x + 0.034 0.991Pinene beta 3yy 0.2 3.3 1.0 1.9 0.3 1.6e 0.2 974.5 1126.4 y = 0.0126x + 0.038 0.99Myrcene 0.9yy 0.1 4.4⁄ 1.5 1.8�� 0.3 3.4 0.4 992.7 1176.1 y = 0.00094x � 0.029 0.999Propanoic acid 2 methyl 2 methylpropyl 13.1yy 0.3 6.8⁄⁄ 0.4 5.7�� 0.1 4.4ee 0.2 1005.2 1101.0 Geranyl acetateButanoic acid 2 methyl 2 methylpropyl 5.7yy 0.3 4.0⁄⁄ 0.4 7.8�� 0.2 5.6ee 0.1 1019.1 1187.0 Geranyl acetatep-Cymene 0.4 0.4 0.9 0.6 0.8� 0.1 0.4 0.2 1025.7 1294.0 y = 0.0157x � 0.003 0.995Limonene (overlapped 1,8-cineole) 1034.8 1219.0Cineole 1,8 320yy 27.7 235.3⁄ 31.4 229.8� 6.1 205.8 12.1 1040.2 1229.8 y = 0.01x + 0.071 0.99Ocimene beta (Z) 0.5 0.1 3.1 3.0 0.8�� 0.2 2.3 0.0 1049.7 1266.6 MyrceneOcimene beta (E) 1.3 0.2 3.6⁄ 0.9 0.9� 0.3 1.6e 0.2 1058.3 1248.3 MyrceneTerpinene gamma 0.3y 0.3 2.1 1.7 1.4�� 0.3 0.3 0.0 1087.3 1267.0 y = 0.0139x � 0.0058 0.999Terpinolene 2.4yy 0.2 2.9⁄ 0.1 1.0�� 0.0 1.9ee 0.3 1107.8 1306.8 Terpineol alphaLinalool 16.5yy 2.4 137.3⁄⁄ 26.7 26.1�� 1.9 106.1 3.7 1138.0 1557.8 y = 0.0105x � 0.024 0.998Pinocarveol-cis 2.0y 0.2 0.6⁄⁄ 0.2 2.4�� 0.1 0.5 0.1 1176.9 1692.2 y = 0.0116x � 0.0058 0.999Terpinen 4-ol 1.6 0.3 1.7 0.4 1.2 0.1 1.6 0.2 1192.2 1633.8 y = 0.0109x � 0.021 0.995Terpineol alfa 49.1 10.5 31.1 6.3 36.3 4.1 42.5e 2.5 1197.3 1725.3 y = 0.0082x � 0.0299 0.998Estragole 0.0yy 0.0 0.0 0.0 6.1 1.0 6.6ee 1.0 1201.1 1707.1 Terpinen 4-olLinalyl acetate 4.7y 1.1 24.5⁄⁄ 5.6 7.3� 0.9 24.4 8.7 1258.6 1573.9 Geranyl acetatePinocarvyl acetate 1.4yy 0.0 0.5⁄⁄ 0.1 5.9�� 0.2 4.7ee 0.3 1300.0 1685.5 Geranyl acetateMyrtenyl acetate 0.6yy 0.1 0.3⁄ 0.1 270.6� 18.8 212.4ee 14.6 1342.9 1726.9 Geranyl acetateTerpinyl acetate 12.5 1.5 10.9 2.3 14.4�� 0.9 9.9 1.2 1350.5 1731.4 Terpineol alphaNeryl acetate 1.9 0.2 14.0 19.4 1.8�� 0.4 3.7 0.1 1367.0 1747.3 y = 0.0092x � 0.0294 0.997Geranyl acetate 50.9yy 5.2 22.7 20.3 23.8� 3.7 35.4 2.8 1387.4 1776.3 y = 0.0097x � 0.0807 0.991Methyl eugenol 11.0 2.0 10.7 2.6 7.9 2.0 8.7 0.6 1408.0 2046.5 Cariophyllene oxideHumulene alfa 0.5yy 0.0 1,7⁄ 0.6 1.9 0.5 3.1e 0.7 1452.9 1719.0 y = 0.0111x + 0.0013 0.994Flavesone 3.5 0.6 2.4 1.0 2.4 0.4 2.8 0.6 1545.5 Cariophyllene oxideCaryophyllene oxide 1.3yy 0.2 1.4 0.5 3.6�� 0.2 2.3e 0.3 1583.7 1922.4 y = 0.0127x + 0.0104 0.998Humulene epoxide 3.6y 0.5 3.4 1.3 2.6� 0.3 6.0 2.0 1610.4 2122.0 Cariophyllene oxideLeptospremone (not identfied isomer) 3.6y 0.6 0.6⁄⁄ 0.3 1.8 0.8 1.1e 0.1 1620.9 2126.4 Cariophyllene oxideTotal 824.0 14.5 839.0 16.6 818.6 13.2 840.1 12.74

126 G.L. Petretto et al. / Food Chemistry 197 (2016) 124–131

bons, oxygenates sesquiterpenes) occurring in the EOs; resultswere expressed as mg per mL of distilled EO (Table 1).

2.5. Retention indexes

A hydrocarbon mixture of n-alkanes (C9-C22) was analyzedseparately under the same chromatographic conditions used onthe HP-5MS and the VF-Wax capillary columns in order to calcu-late the retention indexes with the generalized equation by Vandel Dool and Kartz (1963), Ix = 100[(tx � tn)/(tn+1 � tn) + n], wheret is the retention time, x is the analyte, n is the number of carbonsbelonging to the alkane that elutes before the analyte and n + 1 isthe number of carbons belonging to the alkane that elutes afterthe analyte.

2.6. Antioxidant activity

2.6.1. Antiradical activity by diphenyl-1-picarylhydrazyl (DPPH) assayThe radical scavenging activity assay was performed according

the method proposed by Prevc, Segatin, Ulrih, and Cigic (2013)with some modifications. Different concentrations of EOs, from0.1 mg and up to a maximum of 2 mg were added to a solutionof DPPH� (100 lM in ethyl acetate) to reach a final volume of1 mL. The mixtures were shaken and incubated at 25 �C in the dark.The reduction of the DPPH radical was determined by measuringthe absorbance of the solution at 517 nm using a 1 cm quartzcuvette on an Ultrospec 4300 pro UV–Vis spectrophotometer,equipped with a temperature controller set to 25 �C, atpre-chosen time intervals of 60, 180 and 300 min whereupon aconstant value was reached. A Trolox calibration curve in the range

0.25–7.5 lg/mL was used as the positive reference. The scavengingactivity of the DPPH radical was calculated as follows:

% scavenging of DPPH radical ¼ ½ðAblank � AsampleÞ=Ablank� � 100

where Ablank is the absorbance of the control reaction (containing allreagents except the test compound), and Asample is the absorbancein the presence of EOs.

Experiments were carried out in triplicate and results wereexpressed as mean ± SD.

2.6.2. Antioxidant capacity assayThe ABTS free radical-scavenging activity of each sample was

determined according to the method described by Re et al.(1999). It involves the direct production of the blue/green ABTS�+

chromophore through the reaction between ABTS and potassiumpersulfate. Addition of antioxidants to the preformed radical cationreduces it to ABTS, to an extent and on a timescale dependant onthe antioxidant activity, the concentration of the antioxidant, andthe duration of the reaction. ABTS was dissolved in water to givea final concentration of 7 mM. ABTS�+ was produced by reactingABTS stock solution with 2.45 mM potassium persulfate (final con-centration) and allowing the mixture to stand in the dark at roomtemperature for 12–16ch before use. The ABTS�+ solution wasdiluted with ethanol to an absorbance of 0.7 (±0.02) at 734 nm.In order to measure the antioxidant activity of the EOs, 100 lL ofEOs solution at various concentrations (0.1, 0.2, 0.5, 1.0, 2.0 mg/mL) was added to 900 lL of diluted ABTS�+ and the absorbancerecorded at time zero and after 50 min whereupon the absorbancewas stable. Each concentration was analyzed in triplicate. Thedecolouration percentage at 734 nm was calculated for each point;the antioxidant capacity of the test compound was expressed in

G.L. Petretto et al. / Food Chemistry 197 (2016) 124–131 127

percent inhibition (%), and the IC50 value was calculated fromregression analysis and expressed as the mean ± SD.

2.7. Statistical analysis

All experiments were repeated three times. All statistical anal-yses were performed by comparing EOs from MRE, MRC, MBEand MBC with unpaired Student’s t-test, when the data followeda normal distribution, using SigmaStat v 3.5 software. The distribu-tion of the sample was evaluated by the Kolmogorov–Smirnov andShapiro tests. Differences in antioxidant activity over time betweenMRE, MRC, MBE and MBC were assessed using linear regression inwhich the slope and intercept variations were compared with aglobal test of coincidence using GraphPad Prism 6 software. AP < 0.05 was considered statistically significant.

2.8. Principal component analysis procedure

The multivariate analysis of M. communis samples was carriedout by subjecting the quantitative chromatographic data to princi-pal component analysis (PCA).

The concentration of components in the EOs of theM. communisleaves were used to define an m � n matrix, where m are thesamples and n is the compounds in the EO. The data were centeredand autoscaled before the PCA.

All PCA analyses were performed with R-based chemometricsoftware designed by Chemometric group of Chemical ItalianSociety (Chemical Italian Society, 2014).

3. Results and discussion

3.1. Chemical composition of EOs

The steam distillation of the aerial parts of 16 individual plantsof M. communis growing in Sardinia gave colorless EOs in yieldsranging from 0.32% to 0.52% (v/w) calculated as a proportion oftheir dry-weight. Qualitative determination of the main con-stituents of the EOs was carried out by GC–MS techniques whereasquantitative analysis was performed by the internal standard(n-dodecane) method applied to a GC–FID technique. A total of31 components accounting for 92.2–95.6% (based on the FID packarea normalization) of the whole sample were identified. As shownin Fig. 1 and Table 1, the M. communis EOs compositions investi-gated show several similarities as well as some differences. Myrte-nyl acetate (retention time (RT) about 29 min, Fig. 1, Table 1) isfound to be characteristic of leucocarpa variety, where it was themajor component at 270 mg/mL and 212 mg/mL in cultivatedand wild plants respectively. In contrast, several literature reports(Bouzabata et al., 2013, 2015; Chalchat et al., 1998; Flamini, Cioni,Morelli, Maccioni, & Baldini, 2004; Rahimmlaek et al., 2013) showthat the chemical composition of the EO extracted from the leavesof M. communis is dominated by a-pinene, which is often detectedas the major compound. Conversely, only leaves of some samplesof M. communis from Morocco (Chalchat et al., 1998; Cherratet al., 2014), Greece (Chryssavgi, Vassiliki, Athanasios, Kibouris, &Michael, 2008) and Spain (Boelens & Jimenez, 1992) show an EOfingerprint similar to our results on leaf EOs of MBE and MBC.

On the other hand, linalool (RT about 18 min, Fig. 1, Table 1)could be considered a marker typical of our wild plants as it wasfound at a concentration of 137 and 106 mg/mL in MRE andMBE, respectively, and 16–26 mg/mL in the EOs extracted fromMBC and MRC. In addition, linalyl acetate has been found in higheramounts in EOs of wild plants compared with its concentration inthe EOs from cultivated plants. Our findings are in agreement witha previous classification carried out by Bouzabata et al. (2013) in

which the authors identified two main groups among the EOs ofseveral M. communis individuals collected in Algeria: the ‘‘GroupI” was characterized by a high amount of a-pinene and 1,8-cineole (like our cultivated plants), whereas the ‘‘Group II” wascharacterized by higher amount, in comparison with ‘‘Group I”,of linalool and linalyl acetate (like our wild plants). AlthoughBouzabata et al. (2013) did not find any correlation between pedo-climatic factors and chemical composition, Flamini et al. (2004)reported that M. communis growing in dim light and in siliceoussoil showed a volatiles fingerprint rich in linalool, linalyl acetateand oxigenate derivatives similar to our wild samples. Our wildsamples, in contrast to the cultivated plants, were grown in dimlight in calcareous soil with natural watering, thus it can behypothesized that the light plays a key role in the chemical compo-sition of M. communis.

3.2. Principal component analysis

Chromatographic data of the 16 EOs were used to construct am � n matrix with m =M. communis EOs and n = volatiles detectedin the EOs. The matrix was autoscaled and submitted to PCA to givea more detailed classification. PCA is a relevant technique for datadisplay, able to highlight relationships between variables and sam-ples examined by multivariate analysis instead of the commonlyused univariate approach. As shown in Fig. 2 the information con-tained in the variables is able to discriminate the samples into 4groups represented by the EOs extracted from MBE, MRE, MBCand MRC. The first principal component (PC1) explains 34% ofthe total variance and the second principal component (PC2)explains 28% of the total variance (Fig. 2).

By plotting the diagonal D2 through the score plane PC1–PC2displayed in Fig. 3A, it is possible to identify two groups whichare represented by MBC, MBE and MRC, MRE. By looking at theloadings plot Fig. 2B, as well as the overlay in Fig. 3A, it is possibleto identify which variables contribute most to the discriminationof MBC and MBE from the MRC and MRE. Along the directionthrough which is located the group composed of MBC and MBE(Fig. 3A), the variables furthest from the origin are mytenyl acetate,estragole, caryophyllene oxide and pinocarvyl acetate, meaningthat those variables are in higher concentration in MBC and MBEin comparison with the MRC and MRE. On the opposite side, inwhich the MRC and MRE are located, the furthest variables area- and b-pinene (Figs. 2B, 3A) meaning that in MRE and MRE thosevariables are in higher concentration than in MBE and MBC. Thusthe discrimination of the two groups is mainly due to mytenylacetate, estragole, caryophyllene oxide, pinocarvyl acetate anda- and b-pinene.

By plotting the diagonal D1 through the score plane PC1–PC2 inFig. 3B it is possible to identify two more main groups of EOs whichare represented by wild plants and cultivated plants. By examiningthe loadings plot in Fig. 2B, as well as the overlay in Fig. 3B, it ispossible discover which variables provide the main discriminationbetween the groups: myrcene, neryl acetate and linalool are thefurthest volatiles in the loading plot located in the directionthrough which is located the group made up of wild plants. Conse-quently those variables are highly represented in such groups andthus those variables provide the greatest information that allowsus to separate the two categories.

3.3. Antioxidant activity

The total antioxidant activity of the EOs was determined byDPPH� and ABTS�+ assays. In vitro assays of M. communis EOsrevealed moderate antioxidant activity (Table 2). This confirmsprevious results obtained from Iranian and Moroccan M. communisEOs (Yadegarina et al., 2006; Cherrat et al., 2014). However, if we

Fig. 1. Chromatograms of EOs from cultivated (MBC) and wild (MBE) leucocarpa variety, and cultivated (MRC) and wild (MRE) melanocarpa variety.

Fig. 2. Principal component analysis, (A) score plot and (B) loading plot.

128 G.L. Petretto et al. / Food Chemistry 197 (2016) 124–131

consider the difference between groups, the scavenging of theDPPH radical, obtained by plotting scavenging activity for eachsample against the concentration at 3 time points (60, 180,300 min), was significantly higher in MBC than MBE, as shown inTable 2. The comparison of slopes, (5.524 ± 0.5, 6.547 ± 0.5,9.065 ± 0.7) in MBC and (1.239 ± 0.2, 2.297 ± 0.3, 4.367 ± 0.5) inMBE at 60, 180, 300 min respectively, confirmed that the scaveng-ing of DPPH� was significantly higher in MBC (P < 0.01) than inMBE. Nevertheless the slopes of MRC (19.03 ± 10.6, 20.54 ± 11.5,

21.65 ± 11.8) and MRE (4.395 ± 0.4, 6.376 ± 0.5, 8.852 ± 0.8)were not significantly different. However the comparison of eleva-tions was significantly different between MRC (3.844 ± 11.8,3.671 ± 12.1) and MRE (�2.413 ± 0.5, �6.069 ± 0.8) at 180 minand 300 min when the reaction was stable. Conversely, if wild spe-cies were compared (MRE vs MBE) the antiradical activity of EOsfrom MRE was significantly higher. Furthermore the comparisonof the slopes of MRE (4.395 ± 0.4, 6.376 ± 0.5, 8.852 ± 0.8) vs MBE(1.239 ± 0.2, 2.297 ± 0.3, 4.367 ± 0.5), was significantly different

Fig. 3. Principal component analysis, overlaid plot. (A) differentiation between leucocarpa variety (MBC, MBE) and leucocarpa variety (MRC, MRE) of M. communis. (B)Differentiation between wild plants (MRE, MBE) and cultivated plants (MRC, MBC) of M. communis.

Table 2% scavenging of DPPH radical of EOs of M. communis. Data were collected at 3 timepoints (60, 180, 300 min) for each concentration in triplicate by MBE, MRE, MBC andMRC. Data are expressed as mean ± SD of three independent assays and the P valueindicates difference in regression-line slopes or intercepts.

% scavenging of DPPH radical

mg/mL MRC MRE MBC MBE P

60 min2 36.2 ± 39.3 7.9 ± 1.5 9.8 ± 0.3 0.5 ± 0.41 28.7 ± 35.0 3.9 ± 0.8 3.1 ± 1.4 �0.8 ± 0.50.5 18.9 ± 23.6 1.6 ± 0.7 1.4 ± 1.1 �1. 8 ± 0.4 MBC vs MBE

P < 0.010.2 1.3 ± 1.7 �0.2 ± 0.5 �0.8 ± 1.3 �2.2 ± 0.4 MRE vs MBE

P < 0.010.1 0.4 ± 0.8 �0.2 ± 0.4 �0.6 ± 0.5 �1.3 ± 0.4

180 min2 39.1 ± 39. 9 10.1 ± 1.9 13.4 ± 0.8 2.4 ± 0.71 33.±38.6 4.5 ± 1.2 4.9 ± 1 �0.3 ± 0.5 MRC vs MRE

P < 0.050.5 24.1 ± 29.4 0.9 ± 1.1 3.1 ± 0.5 �1.5 ± 0.6 MBC vs MBE

P < 0.010.2 1.3 ± 2.3 �1.8 ± 0.2 0.5 ± 0.4 �2.0 ± 0.4 MRE vs MBE

P < 0.010.1 �0.2 ± 0.9 �1.5 ± 0. 5 1.5 ± 1.8 �1.1 ± 0.0

300 min2 40.7 ± 38.8 11.1 ± 3.2 17.2 ± 1.9 5.8 ± 1.51 34.1 ± 38.7 4.0 ± 1.4 5.7 ± 1.2 0.4 ± 0.7 MRC vs MRE

P < 0.050.5 26.3 ± 31.6 �1.6 ± 0.6 3.8 ± 1.0 �1.6 ± 0.6 MBC vs MBE

P < 0.010.2 1.4 ± 2.6 �5.7 ± 1.0 0.8 ± 0. 9 �2.3 ± 0.4 MRE vs MBE

P < 0.010.1 �1.8 ± 1.4 �4.5 ± 0.4 �0.7 ± 2.0 �1.3 ± 0.0

G.L. Petretto et al. / Food Chemistry 197 (2016) 124–131 129

(P < 0.01), and corroborated the results. Finally, the comparisonof slope of cultivated species (MRC vs MBC) was similar. Takinginto account that MBE and MBC EOs differed most significantlyfrom MRE and MRC EOs in the content of estragole and

myrtenyl acetate, two of the most powerful scavenging com-pounds (Ruberto & Baratta, 2000), it is reasonable to assumethat these two compounds are involved in the differencesdescribed here.

Several studies on antioxidant abilities of volatiles proved thatcompounds with phenolic or aromatic moieties, such as estragoleor methyl eugenol, show the highest antioxidant ability (Bozin,Mimica-Dukic, Simin, & Anackov, 2006; Mimica-Dukic et al.,2010). Therefore, although estragole is found only in MBC andMBE EOs, methyl eugenol is well represented in all of the samplesand thus correlation between scavenging capacity and this class ofcompound cannot be inferred.

Another method often used to determine the ability of anantioxidant to scavenge free radicals is the ABTS�+ method, whichis highly sensitivity, fast and practical (Cano, Acosta, & Arnao,2000). Using this assay, antioxidant activity was also significantlyhigher in EOs from MBC when compared with MBE, while antiox-idant capacity was the same in EOs from MRC and MRE after50 min of reaction. The comparison of slope between MBC(�0.032) and MBE (�0.031) shows that the regression lines differfor the intercepts (P < 0.05) indicating that MBC has a higherantioxidant capacity than the wild type. The same results as thoseobtained in the DPPH� assay were revealed when we comparedwild species (MRE vs MBE) with a significant difference (P < 0.05)in the intercepts of regression lines. Although the antioxidantcapacities measured by the two assays were in line, the ABTS�+

method gives the best results (Fig. 4). The superiority of the ABTS�+

assay over DPPH� is explained because ABTS�+ is usable over a widerrange of pH, is inexpensive and the assay is more rapid than that ofthe DPPH� assay. Another important difference is that ABTS�+ can besolubilized in both aqueous and in organic media, so that theantioxidant activity of both hydrophilic and lipophilic media canbe measured. In contrast, DPPH� can only be dissolved in alcoholicmedia (Kuskoski, Asuero, Troncoso, Mancini-Filho, & Fett, 2005;Longhi, Perez, de Lima, & Cândido, 2011), thus limiting itsapplicability.

Fig. 4. Time-course of antioxidant activity of essential oils of M. communis evaluated by ABTS�+ assays. Free radical scavenging of 50% of ABTS� by MBE, MRE, MBC and MRC at2 time-points. The values are expressed as mean ± SD of three independent assays (+P < 0.05).

130 G.L. Petretto et al. / Food Chemistry 197 (2016) 124–131

4. Conclusions

A chemical study on sixteen individual samples of differentvarieties of M. communis growing in Sardinia was carried out andthe chemical composition of EO from leaves of M. communis var.melanocarpa DC is reported here for the first time. A multivariateapproach applied to the quantitative data of EOs from the leavesdifferentiated the samples by several factors. The chemical analy-ses displayed a typical fingerprint for MBE and MBC EOs, one thatis dominated by myrtenyl acetate. The presence of this compoundin M. communis EO was attributed by several authors (Bouzabataet al., 2013; Chryssavgi et al., 2008) to the geographical/environ-mental growing conditions whereas our results suggested thatthe presence of myrtenyl acetate was very likely to be related tothe variety, independent of other factors. In agreement with the lit-erature, no significant correlation between chemical compositionand pedoclimatic factors have been found (Bouzabata et al., 2013).

The evaluation of antioxidant activity, by DPPH� and ABTS�+

tests, of the EOs confirmed their ability to scavenge the radicalsof antioxidants assays in line with previous results (Bozin et al.,2006; Mimica-Dukic et al., 2010).

Funding

This research was supported by ‘‘Fondazione Banco di Sardegna– Italy, Prat.2014.0075”, P.O.R. SARDEGNA – ITALY F.S.E. 2007–2013—Obiettivo competitività regionale e occupazione, Asse IV Capi-tale umano, Linea di Attività l.3.1. and by ‘‘Parco Nazionale di LaMaddalena”.

References

Akin, M., Aktumsek, A., & Nostro, A. (2010). Antibacterial activity and compositionof the essential oils of Eucalyptus camaldulensis Dehn. and Myrtus communis L.growing in Northern Cyprus. African Journal of Biotechnology, 9, 531–535.

Alipour, G., Dashti, S., & Hosseinzadeh, H. (2014). Review of pharmacological effectof Myrtus communis L. and its active constituent. Phytoterapy Research, 28,1125–1136.

Barboni, T., Cannac, M., Massi, L., Perez-Ramirez, Y., & Chiaramonti, N. (2010).Variability of polyphenol compounds inMyrtus communis L. (Myrtaceae) Berriesfrom Corsica. Molecules, 15, 7849–7860.

Boelens, M. H., & Jimenez, R. (1992). The chemical composition of Spanish myrtleoils. Part II. Journal of Essential Oil Research, 4, 349–353.

Bouzabata, A., Cabral, C., Gonclaves, M. J., Cruz, M. T., Bighelli, A., Cavaleiro, C., ...Salgueiro, L. (2015). Myrtus communis L. as a source of bioactive and safeessential oil. Food and Chemical Toxicology, 75, 166–172.

Bouzabata, A., Castola, V., Bighelli, A., Abed, L., Casanova, J., & Tomi, F. (2013).Chemical variability of Algerian Myrtus communis L.. Chemistry & Biodiversity,10, 129–137.

Bozin, B., Mimica-Dukic, N., Simin, N., & Anackov, G. (2006). Characterization of thevolatile composition of essential oils of some Lamiaceae spices and theantimicrobial and antioxidant activities of the entire oils. Journal of Agricultureand Food Chemistry, 54, 1822–1828.

Cano, A., Acosta, M., & Arnao, M. B. (2000). A method to measure antioxidantactivity in organic media: Application to lipophilic vitamins. Redox Reports, 5,365–370.

Chalchat, J., Garry, R., & Michet, A. (1998). Essential oils from Myrtle (Myrtuscommunis L.) of the mediterranean littoral. Journal of Essential Oil Research, 10,613–617.

Chemical Italian Society. (2014). Website: <http://gruppochemiometria.it/gruppo-lavoro-r-in-chemiometria.html>.

Cherrat, L., Espina, L., Bakkali, M., Garcia-Gonzalo, D., Pagan, R., & Laglaoui, A.(2014). Chemical composition and antioxidant activity of Laurus nobilis L. andMyrtus communis L. essential oils from Morocco and evaluation of theirantimicrobial activity acting alone or in combined processes for foodpreservation. Journal of Science Food and Agriculture, 94(6), 1197–1204.

Chryssavgi, G., Vassiliki, P., Athanasios, M., Kibouris, T., & Michael, M. (2008).Essential oil composition of Pistacia lentiscus L. and Myrtus communis L.:Evaluation of antioxidant capacity of methanolic extract. Food Chemistry, 107,1120–1130.

Cottiglia, F., Casu, L., Leonti, M., Caboni, P., Floris, C., Busonera, B., ... Sanna, G. (2012).Cytotoxic phloroglucinols from the leaves of Myrtus communis. Journal ofNatural Products, 75(2), 225–229.

Deriu, A., Branca, G., Molicotti, P., Pintore, G., Chessa, M., Tirillini, B., ... Zanetti, S.(2007). In vitro activity of essential oil of Myrtus communis L. againstHelicobacter pylori. International Journal of Antimicrobial Agents, 30(6), 562–563.

Fiori, A. (1925). Myrtus L. In: Nuova flora analitica d’Italia. Firenze, Ricci. 2,3.Flamini, G., Cioni, P. L., Morelli, I., Maccioni, S., & Baldini, R. (2004). Phytochemical

typologies in some populations of Myrtus communis L. on Caprione promontory(East Liguria, Italy). Food Chemistry, 85, 599–604.

Haciseferogullari, H., & Ozcan, M. M. (2012). Biochemical compositional andtechnological characterization of black and white myrtle (Myrtus communis L.)fruits. Journal of Food Science & Technologies, 49(1), 82–88.

Hosseinzadeh, H., Khoshdel, M., & Ghorbani, M. (2011). Antinociceptive, anti-inflammatory effects and acute toxicity of aqueous extracts ofMyrtus communisL. aerial parts in mice. Journal Acupuncture Meridian Studies, 4, 242–247.

Kuskoski, M., Asuero, A. G., Troncoso, A. M., Mancini-Filho, J., & Fett, R. (2005).Aplicación de diversos métodos químicos para determinar actividad antioxidanteem pulpa de frutos. Ciência e Tecnologia de Alimentos, 25(4), 726–732.

Longhi, J. G., Perez, E., de Lima, J. J., & Cândido, L. M. B. (2011). In vitro evaluation ofMucuna pruriens (L.) DC. antioxidant activity. Brazilian Journal of PharmaceuticalSciences, 47, 3.

Melito, S., Chessa, I., Erre, P., Padoani, J., & Mulas, M. (2013). The genetic diversity ofSardinian myrtle (Myrtus communis L.) populations. Electronic Journal ofBiotechnology, 16, 6.

Messaoud, C., & Boussaid, M. (2011). Myrtus communis berry color morphs: Acomparative analysis of essential oil, fatty acid, phenolic compounds andantioxidant activities. Chemistry & Biodiversity, 2, 300–310.

Mimica-Dukic, N., Bugarin, D., Grbovic, S., Mitic-Culafic, D., Vukovic-Gacic, B., Orcic,D., ... Couladis, M. (2010). Essential oil of Myrtus communis L. as a potentialantioxidant and antimutagenic agents. Molecules, 15(4), 2759–2770.

Moradi, M. T., Karimi, A., Rafiean, M., Kheiri, S., & Saedi, M. (2011). The inhibitoryeffects of Myrtle (Myrtus communis) extract on Herpes simplex virus-1replication in baby Hamster Kidney cells. Journal of Shahrekord University ofMedical Sciences, 12, 54–61.

Mulas, M. (2012). The myrtle (Myrtus communis L.) case: From a wild shrub to a newfruit crop. ISHS Acta Horticulturae, 948, 235–242.

Mulas, M. (2013). Il mirto specie spontanea e coltivata, prodotto33. ISBN 978-88-6025-276-0. Editrice Democratica. Available at: <http://www.cersaa.it/project/pyrgi_progetto/Componente5/Componente5%20-%20prodotto33.pdf>.

Parlatore, F. (1842). Flora Italica. Ed. Le Monnier, Firenze, 5, 117–121.Petretto, G., Cossu, M., & Alamanni, M. (2015). Phenolic content, antioxidant and

physico-chemical properties of Sardinian monofloral honeys. InternationalJournal of Food Science and Technologies, 50, 482–491.

Picci, V., & Atzei, A. D. (1996). Ricerche su specie di interesse medicinale edaromatico della Sardegna. Ricerche morfo-sistematiche e biologiche sufinocchio, liquirizia, mirto, rosmarino e zafferano. Rivista Italiana EPPOS, 7(19),21–86.

G.L. Petretto et al. / Food Chemistry 197 (2016) 124–131 131

Prevc, T., Segatin, N., Ulrih, N. P., & Cigic, B. (2013). DPPH assay of vegetable oils andmodel antioxidants in protic and aprotic solvents. Talanta, 15(109), 13–19.

Rahimmlaek, M., Mirzakhani, M., & Pirbalouti, A. G. (2013). Essential oil variationamong 21 wild myrtle (Myrtus communis L.) populations collected from differentgeographical regions in Iran. Industrial Crops and Products, 51, 328–333.

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999).Antioxidant activity applying an improved ABTS radical cation decolorizationassay. Free Radical Biology and Medicine, 26(9/10), 1231–1237.

Ruberto, G., & Baratta, M. T. (2000). Antioxidant activity of selected essential oilcomponents in two lipid model systems. Food Chemistry, 68, 167–174.

Tuberoso, C. I. G., Boban, M., Bifulco, E., Budimir, D., & Pirisi, F. M. (2013).Antioxidant capacity and vasodilatory properties of Mediterranean food: Thecase of Cannonau wine, myrtle berries liqueur and strawberry-tree honey. FoodChemistry, 140, 686–691.

Van del Dool, H., & Kartz, P. D. A. (1963). Generalization of the retention indexsystem including linear temperature programmed gas–liquid partitionchromatography. Journal of Chromatography, 11, 463–471.

Yadegarinia, D., Gachkar, L., Rezaei, M. B., Taghizadeh, M., Astaneh, S. A., & Rasooli, I.(2006). Biochemical activities of IranianMentha piperita L. andMyrtus communisL. essential oils. Phytochemistry, 67, 1249–1255.