Next Article in Journal
Effect of Naturally Occurring Compounds on Fumonisin Production and fum Gene Expression in Fusarium verticillioides
Next Article in Special Issue
Preliminary Predictive Model of Termiticidal and Repellent Activities of Essential Oil Extracted from Ocotea quixos Leaves against Nasutitermes corniger (Isoptera: Termitidae) Using One-Factor Response Surface Methodology Design
Previous Article in Journal
The Effect of Gaseous Ozone Generated by Surface Dielectric Barrier Discharge on the Decay and Quality of Stored Onion Bulbs
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 11
Issue 6
10.3390/agronomy11061059
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Quantification of Total Phenols, Tannins, Anthocyanins Content in Myrtus communis L. and Antioxidant Activity Evaluation in Function of Plant Development Stages and Altitude of Origin Site

by
Silvia Medda
1,
Angela Fadda
2,
Leonarda Dessena
1 and
Maurizio Mulas
1,3,*
1
Department of Agricultural Science, University of Sassari, Via De Nicola 9, 07100 Sassari, Italy
2
Institute of Sciences of Food Production, National Research Council, Traversa La Crucca, 3 Loc. Baldinca, Li Punti, 07100 Sassari, Italy
3
Centre for Conservation and Evaluation of Plant Biodiversity, University of Sassari, Via De Nicola 9, 07100 Sassari, Italy
*
Author to whom correspondence should be addressed.
Agronomy 2021, 11(6), 1059; https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy11061059
Submission received: 26 April 2021 / Revised: 21 May 2021 / Accepted: 22 May 2021 / Published: 25 May 2021
(This article belongs to the Special Issue Feature Papers on Medicinal and Aromatic Plants)
Browse Figures
Versions Notes

Abstract

:
Most of the biological properties of myrtle (Myrtus communis L.) are linked to the antioxidant activity of the phenolic compounds present in the extracts. In this study, the content of total phenols, tannins, and anthocyanins of acidified ethanol extracts of berries and leaves of five myrtle cultivars was quantified during the whole fruit development. In some development stages, the antioxidant activity (AA) of both tissues was evaluated with FRAP, ABTS, DPPH, β-carotene assays, and spectroscopic method (EPR). The trend of AA in leaf and berry extracts was different among genotypes and evaluation assays. In general, leaves showed with DPPH assay, the highest AA during autumn season, while with FRAP and β-carotene was higher at 120 DAF (days after flowering) corresponding to summer season. In berries, the AA with DPPH, ABTS, and EPR assays was the highest for all cultivars at 150 DAF and 210 DAF, while with β-carotene assay was higher in the first development stage. Both in leaf and berry, the total phenols and tannins contents influence the AA depending on the assay used, while the berries anthocyanins seem to play a minor role. The altitude of the site of origin differently affected the AA of myrtle leaf and berry. The study showed the potentiality to use the studied myrtle genotypes for further evaluation on biological applications of antioxidant activity.

1. Introduction

Myrtle (Myrtus communis L.), belonging to the Myrtaceae family, is present on the coasts of Portugal, southern France, Spain, southern Turkey, southern Italy, and other areas like Greece, Israel, Lebanon, Iran, Libya, Morocco, Algeria, and Tunisia. In Sardinia (Italy) myrtle grows spontaneously in neutral or sub-acidic soils and areas below 800 m of altitude [1]. Traditionally, Mediterranean folk medicine uses this plant for its carminative and astringent functions, as an antifertility agent, to treat respiratory diseases, for intestinal inflammation and menstrual problems [2]. This medicinal and aromatic plant received great attention in the last years for the large appreciation of the liqueurs produced by its berries (red liqueur) and leaves (white liqueur). The myrtle industry encouraged a domestication process of the species to integrate the harvests of biomasses from wild plants with those from selected cultivars with known chemical composition and quality attributes [1]. The availability of myrtle cultivars may be interesting for nutraceutical and medicinal industries.
Plant extracts, including those from myrtle berries and leaves, attract consumers and researchers for the high concentration of phenolic compounds involved in the scavenging of ROS (reactive oxygen species) and RNS (reactive nitrogen species) [3,4] which have been associated to Parkinson’s [5], Alzheimer’s [6], cardiovascular diseases [7], and diabetes [8]. Moreover, and as other foods rich in natural antioxidants, they are the object of numerous research [9,10,11,12,13].
The antioxidant properties of food are associated to the action of single bioactive compounds, to their interactions and to the interactions among antioxidants and other components of the food matrix [14]. Moreover, the type of chemical extraction largely affected the evaluation of both total antioxidant activity of the matrices, and of the role of the extractable and non-extractable biomasses fractions [14,15].
Myrtle leaves are rich in phenolic acids (gallic, caffeic, and ellagic acids), flavonol derivatives (myricetin, quercetin, and kaempferol derivatives), and ellagitannins (such as galloyl hexose derivatives) [16]. Berries contain anthocyanins (principally in berry skin and, partially, in the pulp) mainly delphinidin3-O-glucoside, petunidin3-O-glucoside, and malvidin 3-O-glucoside [17], flavonols, flavonols glycosides, phenolic acids, and flavanols [18]. Plant genotype, growing site, climatic conditions, maturation stage, tissue nature control the quantitative and qualitative composition of phenolic compounds and their antioxidant activity [19].
In previous studies, the antioxidant activity of myrtle, red and white myrtle liqueurs, and other derived myrtle products, was measured with ABTS (2,2’-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid), DPPH (1,1-diphenyl-2-picrylhydrazyl), FRAP (ferric reducing antioxidant power), LDL (low-density lipoproteins) oxidation, β-carotene-bleaching methods, and the spin trapping method coupled with EPR (Electron Paramagnetic Resonance) [20,21,22,23].
These methods, based on different antioxidant mode of action, highlight the high antioxidant activity of myrtle plant tissues and its derivate food products [24,25,26].
Moreover, it is worth noticing that most of the biological properties of myrtle extracts are associated to their antioxidant activity [27,28]. Some of these demonstrated biological properties include neuro-protective effects [29], antimutagenic [30], and anti-hepatic ischemia [31].
According to Cruciani et al., [4] myrtle extracts controlled the ageing process in cells exposed to oxidative stress, suggesting their use in the prevention of senescence-associated diseases. Similarly, the extracts obtained from liqueur industry by-products counteract cells stress conditions and control cell senescence [28]. These studies suggest the use of myrtle extracts in nutraceutical formulations.
For this reason, it is essential to identify the best harvest time to maximize the antioxidant activity of myrtle berries and leaves [32]. Indeed, antioxidant activity and its development may vary among genotypes according to the leaf age, as well as to the polymorphism of myrtle fruit, i.e., the presence of pigmented and unpigmented genotypes. The unpigmented berry, although lacking anthocyanins, has an antioxidant power equal to that of the pigmented berry, due to the high content of gallic and ellagic acids [24].
The main objective of this study was to evaluate the influence of tissue developmental stages on the antioxidant activity of myrtle berry and leaf ethanol extracts, using different genotypes and assays in vitro. Furthermore, total phenolic compounds, total tannins, and anthocyanins accumulation in the plant, as well as the altitude of the site of origin of the genotypes were correlated to the antioxidant activities. Five myrtle cultivars were analysed, one with unpigmented fruits, to assess differences in antioxidant activity according to fruit polymorphism. Cultivars analysed were selected during the myrtle domestication program [1], and the obtained data should be considered in the light of a further breeding objective for the selection of plus variant cultivars with high antioxidant value to destine to pharmacological and food uses.

2. Materials and Methods

2.1. Chemicals

Trolox (6-hydroxy-2,5,7,8-tetramethyl chromane-2-carboxylic acid), ABTS (2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), DPPH (2,2-diphenyl-1-picrylhydrazyl), TPTZ (2,4,6-Tri(2-pyridyl)-s-triazine), quinolinic acid (pyridine-2,3-dicarboxylic acid), Iron (II) sulphate hexahydrate and Iron (III) chloride hexahydrate were purchased from Merck (Darmastadt, Germany) Sigma. DMPO (5,5 dimethyl-1-pyrroline N-oxide) was purchased from Enzo Life Sciences (Milan, Italy). All other reagents and standards used in this study were purchased from Carlo Erba reagents S.r.l (Milan, Italy).

2.2. Plant Material Sampling and Extracts Preparation

The leaves and the berries of five myrtle cultivars (’Giovanna’, ‘Grazia’, ‘Maria Antonietta’, ‘Maria Rita’, ‘Sofia’) were used to prepare the extracts. One of the cultivars (‘Grazia’) had white berries while the others had pigmented fruit. Myrtle plants were grown at the experimental orchard located at the “Antonio Milella” station of the University of Sassari (Central Western Sardinia 39°54′12″ N, 8°37′19″ E), but were originally collected in Sardinian sites at different altitude as reported in Table 1.
Table 2 reports some agrometeorological parameters of the meteorological station closest to the experimental field recorded during the harvest years.
The samplings were carried out during eight stages of development, at 0, 30, 60, 90, 120, 150, 180, 210, 225 days after flowering (DAF) that correspond to the sampling of June, July, August, September, October, November, early December, and late December of 2019. The berries and leaves sampling were done randomly on a plot of 20 plants for each cultivar. A graphical representation of sampling times of samples is shown in Figure 1. The first and last plant of the plot were excluded by the sampling as border in the row, and the remaining 18 plants were arranged in three subplots of six plants each, which represented the three biological replications. All the 18 plants were sampled at each time.
Acidified ethanol was used as extraction solvent. Ethanol is one of the most employed solvents for the extraction of phenolic compounds; moreover, a hydroalcoholic extract is the base for myrtle liqueur production [33,34,35]. Acidification allows avoiding oxidation and increases extraction of compounds.
The berries (peel and 1 mm of pulp) and leaves were ground with liquid nitrogen. Ground tissue (0.6 g) was extracted with 15 mL of acidified ethanol (0.1% HCl) for 12 h at room temperature in the dark. Afterwards, the infusion was filtered with filter paper (Whatman No. 4) and stored at −20 °C before analysis. Each extract has been replicated three times. For each tissue sample, the dry matter was determined, after drying of 1 g of sample at 100 °C for 24 h.
For the evaluation of antioxidant activity, the analyses were carried out at selected stages: for berries extracts at 60, 150, 210, and 225 DAF; 0 and 120 DAF for leaves extracts. Leaf’s sampling dates were chosen according to the completed development stage of the spring shoots (0 DAF) and autumnal maturity of the same shoots (120 DAF). Each chemical determination was carried out in triplicate.

2.3. Total Phenols, Anthocyanins, and Tannins Content

The determination of total phenols was carried out with the colorimetric method of Folin–Ciocalteu [36]. An aliquot (0.5 mL) of diluted ethanol extract was mixed with deionized water (35 mL) and 2.5 mL of Folin–Ciocalteu reagent; after 3 min of incubation, a sodium carbonate solution (20% in water) (5 mL) was added. The solution was incubated at 70 °C for 20 min and was brought to volume (50 mL) with deionized water. Absorbance was read at 750 nm with UV–VIS spectrophotometer CARY 50 VARIAN (Amsterdam, The Netherlands). The results were expressed as mg gallic acid equivalent per gram of dry weight (mg GAE/g DW). For calibration curve, gallic acid concentrations were used between 0.05–0.5 mg/mL (R2 = 0.99).
The total anthocyanin content was determined following the method described by Lee et al. [37]; the diluted extract was mixed with pH 1 buffer solution (potassium chloride, 0.025 M). The blank was prepared by adding to the same extract a pH 4.5 buffer solution (sodium acetate, 0.4 M). After 30 min of incubation at room temperature, the absorbance was read at 520 and 700 nm against a blank. For concentrations determination, the equation reported by Lee et al. was used [37]. Results were expressed in mg of cyanidin 3-glucoside equivalent (C3G)/g DW.
Tannins were determined according to Fadda and Mulas [38]. Four mL of the diluted extract was mixed with 2 mL of ethanol and 4 mL of the vanillin solution (1% vanillin in 70% sulphuric acid). After 30 min of incubation at room temperature, the absorbance was read at 500 nm. The results were expressed as mg of catechin equivalent (CE)/g DW based on the calibration curve (0.05–0.5 mg/mL of catechin, R2 = 0.99).

2.4. Antioxidant Activity (AA) Assays

2.4.1. ABTS Assay

The method described by Re et al. [39] with some modifications was used to determine ABTS radical scavenging activity. The ABTS+ cation radical was produced by mixing ABTS stock solution (7 mM in water) and 2.45 mm potassium persulfate in a ratio of 1:0.5 respectively and was stored in the dark at room temperature for at least 16 h before use. The ABTS radical was diluted with water until the absorbance measured at 734 nm reached the value of about 0.7. The ABTS solution was prepared fresh before each analysis.
Fruit extract (5 μL) and leaf extract (2.5 μL) was mixed with 1000 μL of ABTS diluted. The reaction mix was incubated for 6 min in the dark at room temperature and the absorbance was immediately read at 734 nm using CARY 50 Scan Uv143 Vis VARIAN spectrophotometer (Amsterdam, The Netherlands). The calibration curve was prepared using a range of 0–4 mM (R2 = 0.98) of Trolox reagent. Results were expressed as mmol of Trolox equivalent/g dry weight (mM TE/g DW).

2.4.2. FRAP Assay

The FRAP method has been carried out according to Benzie and Strain [40]. The FRAP reagent was prepared with 2.5 mL of 10 mM TPTZ, 2.5 mL of 20 mM FeCl3- 6H2O and 25 mL of 0.3 mM acetate buffer pH 3.6. The FRAP reagent was prepared fresh each time and warmed at 37 °C. A volume of 5 μL of berries or leaves ethanolic extract or ethanol (for the blank) was mixed with 900 μL of FRAP reagent and 115 μL of distilled water. The mix was incubated at 37 °C for 30 min and the absorbance was read at 595 nm using Cary 50 spectrophotometer. The calibration curve was prepared whit Trolox reagent at different concentrations (0.1, 0.4, 1, 2, 3 mM; R2 = 0.99). The results were expressed in mmol of Trolox equivalent/g dry weight (mM TE/g DW).

2.4.3. DPPH Assay

The DPPH assay was carried out following the procedure reported by Sanna et al. [22], with some modifications. Briefly, a volume of 100 μL of ethanol acidified extract, properly diluted, was mixed with 100 μL of DPPH 1 mM solution (prepared in ethanol) and 1800 μL of ethanol. The mix was incubated at room temperature in the dark for 30 min, and then, the absorbance was read using Cary 50 spectrophotometer at 517 nm. The blank was prepared with 100 μL of DPPH solution and 1900 μL of ethanol. Trolox was used as a standard for calibration curve (range between 6–21 μM; R2 = 0.97). The results were expressed in micromole of Trolox equivalent/g of dry weight (µmol TE/g DW).

2.4.4. β-Carotene Bleaching Assay

To provide a comprehensive view of the antioxidant potential of myrtle berries and leaves, the peroxyl radical (LOO•)-inhibiting capacity of the extracts was evaluated. Consequently, the β-carotene bleaching assay was performed following the method described by Aidi Wannes et al. [20] with some modifications. This assay was chosen to evaluate Two mg of β-carotene were dissolved in chloroform; one aliquot of this solution (4 mL) was mixed with 40 mg of linoleic acid and 400 mg of Tween 40. The chloroform in the solution was evaporated under a rotary vacuum at 50 °C, and then 100 mL of deionized water was added and mixed well. Three mL of this emulsion were added to 0.2 mL of sample extract. The control contained 0.2 mL of ethanol 3 mL of emulsion. BHT was used for comparison. The absorbance was immediately read (t = 0) at 470 nm and, a second time, after incubation in a water bath at 50 °C for 120 min (t = 120).
The antioxidant activity was expressed as antioxidant activity coefficient (CAA) calculated according to the equation reported by Klančnik et al. [41]:
CAA = 1 − (As(t = 0) − As(t = 120)/Ac(t = 0) − Ac(t = 120))
  • As = Absorbance of sample extract
  • Ac = Absorbance of control

2.4.5. Spin Trapping Assay of the •OH Radical

The •OH radical scavenging activity was determined with the spin trapping method coupled with Electron Paramagnetic Resonance Spectroscopy according to Fadda et al. [21].
The Fenton reaction was used to produce hydroxyl radicals using, as Fe (II) source a Fe (II) quinolic acid complex, prepared by mixing an aqueous FeSO4 solution with quinolinic acid in order to have a final concentration of 0.1 mM and a ligand to metal ratio of 5:1. DMPO (261 µM) was used to entrap the hydroxyl radicals produced by the reaction of Fe (II) quinolinic acid complex with hydrogen peroxide (0.03%). Ten µL of the extracts properly diluted were used to evaluate the hydroxyl radicals scavenging activity.
The experiments were carried out at room temperature with a Bruker EMX spectrometer operating at the X-band (9.4 GHz) and a Bruker Aqua X capillary cell. EPR spectra were recorded immediately after the preparation of the reaction mixture; the concentration of the DMPO-OH adduct was estimated by the double integration of spectra. Results were expressed as µmol of gallic acid equivalents (mmols GAE/g DW) based on a calibration curve (gallic acid 80–200 mM, R2 = 0.99). Three replications were performed for each extract. The EPR instrument was set under the following conditions: modulation frequency 100 kHz, modulation amplitude 1 G, receiver gain 1 × 105, microwave power 20 mW.

2.4.6. Data Treatment and Statistical Analysis

Results of phenolic compounds determination and AA are expressed as the means and standard deviations of three biological replicates. The results of the phenolic compounds were submitted to a one-way ANOVA comparing the significance of the differences at different stages inside the same cultivar. A two-way analysis was instead applied to evaluate the difference among cultivars and development stages to the results of the antioxidant tests. Mean separations were performed by application of the Multiple Range Test of Duncan (p < 0.01). The correlation coefficients (r) between parameters were calculated using Pearson’s coefficient at p ≤ 0.01 and p ≤ 0.05. For all analysis, the MSTAT-C software was used.

3. Results

3.1. Dry Matter of Samples

The percentage of berry and leaf dry matter of the cultivars analysed at each sampling time (from 0 to 225 DAF) are reported in Table 3 and Table 4.

3.2. Total Phenols, Anthocyanins, and Tannins Content

Berry total phenols content was higher during fruit set (0 DAF) than in ripe berry for all cultivars (Figure 2a–e). The following values were observed at 0 DAF: 173.92 mg GAE g−1 DW for ‘Giovanna’, 171.33 mg GAE g−1 DW for ‘Grazia’, 141.23 mg GAE g−1 DW for ‘Maria Rita’, 139.77 mg GAE g−1 DW for ‘Maria Antonietta’, and 145.53 mg GAE g−1 DW for ‘Sofia’. During berries development, the content of total phenols decreased, with differences among cultivars. The ‘Sofia’ cultivar showed the lowest content during 225 DAF with 35.45 mg GAE g−1 DW (Figure 2f). In the other cultivars, the minimum content was 63.91 mg GAE g−1 DW for ‘Giovanna’ at 210 DAF, 33.46 mg GAE g−1 DW for ‘Grazia’ at 225 DAF, 57.32 mg GAE g−1 DW for ‘Maria Rita’ at 150 DAF, and 52.89 GAE g−1 DW for ‘Maria Antonietta’ at 30 DAF.
In leaf extracts, total phenols content showed during development different trends in the cultivars and a higher content as compared to fruit. ‘Maria Antonietta’ (Figure 2d) and ‘Sofia’ (Figure 2e) showed the maximum content at 120 DAF (respectively, with 144.22 and 111.25 mg GAE g−1 DW), while the leaves of the Maria Rita cultivar (Figure 2c) reached the maximum content at 225 DAF.
Tannin’s content was higher in berries than in leaves. In berries (Figure 2f–l), tannins tended to increase during the first development stages and then decrease in the last stage of ripening in all cultivars, with values between 0 and 22.45 mg CE g−1 DW. In ‘Giovanna’ (Figure 2f) cultivar, the tannins content increases from 60 DAF reaching the maximum content (15.24 mg CE g−1 DW) and then decreases to the values of 1.68 mg CE g−1 DW in the last stage of ripening. In addition, in ‘Grazia’ (Figure 2g) and Maria Antonietta (Figure 2i), the maximum values were found at 90 DAF and the minimum content during the overripe stage (225 DAF). Indeed, the ‘Sofia’ and ‘Maria Rita’ cultivars reached the highest content of total tannins (Figure 2h, l).
Leaf extracts contained low levels of total tannins during all stages of development with respect to tannins of the berries (Figure 2g–m), with values including 0.28 mg for ‘Sofia’ and 4.63 mg CE g−1 DW for ‘Maria Antonietta’.
Anthocyanins were only detected in the berries of the pigmented fruit cultivars. Anthocyanins increased from 180 DAF to 225 DAF (Figure 2m–p). ‘Giovanna’ (Figure 2m) was the cultivar with the highest content (34.20 mg C3G g−1 DW at 225 DAF). In the other cultivars, the anthocyanins content at 225 DAF were 14.02, 21.64, and 15.28 mg C3G g−1 DW, respectively for ‘Maria Antonietta’, ‘Maria Rita’, and ‘Sofia’ (Figure 2n–p).

3.3. Antioxidants Activity (AA) with Colorimetric Assays

The results of AA of leaf extracts detected by colorimetric assays are reported in Table 5. The highest AA with DPPH assay was found in ‘Maria Antonietta’ cultivar at 0 DAF (Table 5) while the lowest activity was for ‘Grazia’ unpigmented cultivar in the same stage. Regarding the trend of AA during the two development stages, all cultivars except ‘Maria Antonietta’ showed the highest AA in the leaves sampled at 120 DAF.
Determining AA with ABTS assay (Table 5), significant variations between the two stages analysed were found for ‘Maria Antonietta’, ‘Maria Rita’, and ‘Sofia’, detecting the maximum AA at 120 DAF with respect to 0 DAF.
The results with β-Carotene bleaching assay (Table 5) showed that the leaves of all cultivars have a higher AA at 120 DAF. The same trend was found in the cultivars with AA detected with FRAP assay (Table 5), except for ‘Sofia’ which shows no significant differences between the two stages.
In Table 6, the AA of berry extracts detected with colorimetric assays are reported. With DPPH assay (Table 6), all cultivars showed the higher AA in the colour-breaking and ripe fruit (210 and 225 DAF). The white ‘Grazia’ cultivar showed the highest AA with respect the other cultivars, with values of 30.07 e 32.74 µmol TE g−1 DW. ‘Sofia’ cultivar maintains during all the development period the lowest AA without significant differences.
The results with FRAP showed a different trend in the cultivars among the analysed stages (Table 6). ‘Grazia’ and ‘Maria Rita’ cultivar reached the maximum AA at 225 DAF. In ‘Maria Antonietta’, the maximum was reached in concomitance with anthocyanins accumulation (150 DAF), while ‘Sofia’ cultivar reached the highest AA one month after and then decrease with ripening.
The AA detected with ABTS ranged between 45.27 and 196.52 mmol TE g−1 DW (Table 6) and was higher at 150 and 210 DAF, except for ‘Sofia’ cultivar that showed low AA in all stages.
The AA detected with β-Carotene bleaching (Table 6) followed the same trend in all cultivars: values decreased from 60 DAF to ripe stage. In this case, the highest AA was found for ‘Maria Antonietta’ that showed an antioxidant coefficient (CAA) of 1.08 at 60 DAF.

3.4. Spin Trapping Assay of the •OH Radical

DMPO traps the hydroxyl radicals produced in the Fenton reaction and forms a DMPO-OH adduct, which has a four-line signal with the following hyperfine splitting constants aN = aH = 14.9 G (Figure 3). When the extract is present in the reaction mixture, the intensity of the DMPO-OH adduct’ signal decreases due to competition, for the hydroxyl radical, between the trapping agent and the antioxidants present in the extract (Figure 3).
Leaf extracts own a lower hydroxyl radical scavenging activity than berries extracts. At 0 DAF, the hydroxyl radical scavenging activity of leaf extracts ranged between 121 and 152 mmol GAE/g D.W, and no statistical differences were observed among cultivars. During leaf development, it slightly increased in all cultivars, but in ‘Sofia’, only the increase was statistically significant.
At 60 DAF myrtle berries belonging to the five cultivars showed quite similar hydroxyl radical scavenging activities, ranging from 0.05 ± 0.01 (Maria Rita) to 0.16 ± 0.01 (Grazia) (Table 6). At the beginning of fruit development, no differences were observed between pigmented and white cultivars. The berries hydroxyl radical scavenging activity increased during development reaching its highest values at 210 DAF in all cultivars. At this developmental stage, the non-pigmented cultivar ‘Grazia’ showed a hydroxyl scavenging activity higher than the other pigmented cultivars. At the last sampling 225 DAF, the scavenging activity drastically dropped in all cultivars and reached values similar to those observed at the beginning of fruit development.

3.5. Correlation between Antioxidant Activity, Phenolic Compounds Content, and Altitude of Site of Origin

The analysis of correlation among the AA detected with the different methods, the content of total phenols, anthocyanins and tannins, and the altitude of the site origin of the cultivar in both tissues (berry and leaves) was carried out.
In leaves extracts (Table 7), the total phenols content was correlated with AA found with DPPH and β-Carotene while the tannins content is involved positively with AA detected with β-Carotene and EPR methods.
The total phenols and that of tannins were strictly interrelated in myrtle leaves. With regard to the influences of altitude on AA, the results showed that this parameter influenced positively the antioxidant activity detected with ABTS and FRAP.
Moreover, the AA with DPPH assay is negatively correlated with AA of FRAP, β-Carotene, and EPR assays. The AA detected with ABTS and FRAP assays were correlated positively, as well as the EPR and β-Carotene assays.
Moreover, in berry extracts (Table 8), the total phenols are correlated on the AA (with ABTS and DPPH assays). As found for leaves (Table 7), total phenols were correlated with tannins content in the berry.
The results showed that the tannins contribute more to AA than anthocyanins content, although the coefficient of r was not high. Total tannins were correlated with AA detected with DPPH and β-Carotene assays.
The anthocyanins content was weakly correlated to ABTS and β-Carotene. The altitude affected the AA of berries in different manner with respect to myrtle leaves; in fact, the AA (detected with DPPH and ABTS assays) were negatively correlated with the altitude of site of origin, except for the AA with β-Carotene that was weakly positively correlated. Moreover, also the content of total phenols and tannins in myrtle berry decrease with high altitude.

4. Discussion

4.1. Differences in Phenolic Compounds Determined by Biological Diversity of Samples

The recent health claims encourage the consumption of nutraceutical and functional food like myrtle due to the role that phenolic compounds have on the reduction of degenerative diseases [42,43,44,45,46].
Myrtle is a natural source of bioactive compounds [47] exploitable for medical and pharmaceutical purposes. The domestication process of Sardinian myrtle genotypes provided cultivars with high biomass production; these were partially studied for leaf and fruit phenolic profile; however, little is known about the evolution of these compounds during fruit and leaf development [1,16,22,48].
The results of this paper widen the information on myrtle plant antioxidant properties and suggest some criteria for the selection of biomasses with high medical and pharmaceutical potentialities.
The concentration of the main phenolic sub-groups in myrtle leaves and berries was evaluated during fruit and shoot development. As previously observed, the total phenolic content of myrtle berries decreased during fruit maturation [33,38]. This was observed both in berries of ‘Giovanna’ (pigmented) and ‘Grazia’ (unpigmented), two cultivars with the same site of origin.
As found by Snoussi et al. [49], the total phenols of ethanol myrtle extracts were higher in berry than leaf. The changes of phenolic concentration during leaf development were different among cultivars, but the final value was similar to other small berries [50].
Pigmented berries synthetized anthocyanins from colour-break stage (150 DAF) until full ripe according to other authors [38]. In all small fruit, the high anthocyanin concentration is associated to favourable effects on human health [51].
Myrtle pigmented berries are a good anthocyanin source, and particularly ‘Giovanna’ showed the highest concentration. Some authors indicated an anthocyanin content of 8.4–41.1 mg C3G/g DW in haskap berries [52], 15.4 mg of cyanidin in the cultivar ‘Red thorn’ of blackberry at ripe stage [53], and 14.5 mg of cyanidin in rabbiteye blueberry fresh berries [54].
The unripe myrtle berry has an astringent taste due to the high tannin concentration, which decreased when anthocyanin storage began.
In our previous study, we observed a genotype dependent decrease of tannin concentration; moreover, the highest tannin concentration was detected at 30 DAF and suddenly disappeared at the subsequent stages, contrary to what was observed in this work [38].
In this paper, myrtle leaves had less tannins than berries, contrary to what was observed by other authors [55]. Both berries and leaf tannins concentrations were positively correlated to total phenols (respectively, r = 0.698 and r = 0.719).
The correlation analysis revealed no effect of altitude on leaf total phenols, whereas a negative correlation was calculated with berries total phenols and tannins concentrations.
These results suggest the involvement of other factors associated to altitude, thermic conditions or increased exposition to solar radiation, in the accumulation of secondary metabolites. These factors may induce plant stress with a consequent decrease of tannins and total phenols.

4.2. AA Observed According to Biological and Sampling Variables

The leaf AA analysed at two development stages showed a high variability among genotypes, tissue development, and method used to evaluate AA.
The results of the DPPH, Frap, and β-carotene-bleaching methods showed an increase of the leaf extracts AA in all cultivars (‘Maria Antonietta’ is the only exception) during leaf development. Instead, with ABTS method, the AA of leaf extracts reached the maximum AA in autumn season (0 DAF).
A previous study [32] on the AA of Algerian myrtle (M. communis var. italica L.) leaves and berries at two stages of development (September and December) revealed a higher free radical scavenging in tissues sampled in September.
In addition to the colorimetric assays, the spin trapping method coupled with EPR spectroscopy was used to estimate the antioxidant activity of both leaves and berries extracts. In a previous paper, this methodology was employed to study the hydroxyl radical scavenging activity of myrtle berries hydroalcoholic extracts used for the production of the typical liqueur [34]. The results indicated that the higher the ethanol concentration in the extraction medium, the higher the hydroxyl radical scavenging activity of the hydroalcoholic extracts.
The concentration of total phenols and tannins affected the AA of myrtle leaves. Total phenols were correlated to AA detected with ABTS and β-Carotene methods while tannins content influenced β-Carotene and hydroxyl radical scavenging activity results. According to Sanna et al. [34] the high hydroxyl radical scavenging activity of myrtle hydroalchoolic extracts was likely due to the high concentration of ellagic acid in the extracts even after 10 months of storage. The results presented in this paper seem to confirm this hypothesis since the hydroxyl radical scavenging activity was positively correlated to tannins concentration in myrtle leaf extracts. Moreover, no differences were observed between the sampling dates in leaf extracts, with the only exception of the cultivar ‘Sofia’.
Amensour et al. [18] and Pereira et al. [56] reported a higher antioxidant activity in leaf extracts than berries ones, suggesting that leaf phenolic composition has a higher antioxidant power than anthocyanins, principally present in berries.
The AA of the berries was assessed during four development stages, from unripe (60 DAF) to overripe fruit stage (225 DAF). The 150 DAF correspond to fruit colour-break. AA results showed a high variability according to genotype and fruit development. The ABTS and DPPH radical scavenging activities were highest, in all cultivars at 150 DAF and 210 DAF (ripe stage fruit). Only, the AA of ‘Sofia’, the cultivar originated from the locality with highest altitude, showed any variation during the development stages of fruit.
Only with the β-carotene assay, the AA was higher in the fruit when is unripe with respect the ripe stages. The ability of scavenging the OH radicals, evaluated by EPR, indicated higher values at 150 and 210 DAF for all berry cultivars.
In black chokeberry (Aronia melanocarpa) the antiradical activity was greater in unripe fruit compared to ripe one [57]. In sweet cherry, the evolution of antioxidant activity during 14 developmental stages showed an increase alongside anthocyanins accumulation. In strawberry fruit, the antioxidant activity at extra-early period during was lower of 17.8% than early and ripe stages [58].
In this study, the unpigmented genotype had higher DPPH and ABTS radical scavenging activities than pigmented ones. Messaoud and Boussaid [59], comparing white and pigmented myrtle Tunisian genotypes, found a greater AA in pigmented berries than in white ones. These authors calculated an IC50 value of 2.1 and 2.8 mg/mL for pigmented and white genotype with DPPH assay, and the value of 2.7 mmol Fe2+/g for pigmented berries and 2.1 mmol Fe/g for white berries.
The content of phenolic compounds, anthocyanins, and tannins affected the AA of myrtle berries in a different way. The total phenol content influenced the AA with ABTS (r = 0.546) and DPPH (r = 0.588) assays, while the tannins are positively involved in AA with β -carotene (r = 0.307), and negatively with AA with FRAP assay (r = −0.411). Dudonne et al. [60], analysing aqueous extract of 30 medicinal plants, observed a correlation between the antioxidant activity obtained with DPPH, ABTS, and FRAP assays and total phenol content.
The anthocyanins content in this study appears to play a more marginal role in the AA of myrtle fruit than the total phenols and tannins. It is correlated with ABTS (r = 0.258) and negatively with β -carotene (r = −0.527). Contrary to our results, anthocyanins of grape extract were highly correlated with FRAP, DPPH, and ABTS methods [61]. In other fruit, high amounts of anthocyanins were correlated with AA obtained with DPPH assay [62]. Arnous et al. [62] observed a greater impact on antioxidant activity of total phenols and total flavanols than anthocyanins in Greek red wines.

4.3. AA as Influenced by the Altitude of the Site of Origin of Cultivars

Another objective of this study was to evaluate the influence of altitude, and consequently related environmental parameters, of the site of origin of cultivars on AA despite the actual cultivation in the same field of comparison. We found a different influence of the altitude on AA according to the tissue of plant. The altitude was positively correlated with AA (detected with FRAP and ABTS) of leaf extracts; however, different correlations between AA (with ABTS and DPPH) were found for berry extracts. In this case, the assay used for AA evaluating strongly affected the results, thus confirming the previously observed variability. Various climatic factors depend on altitude like visible radiation, UV radiation and air temperature [63]. Altitude affects the natural selection of plants genotype, of bioactive compounds, plants’ physic-chemical characteristics, and sensory properties [64]. There is no agreement in the literature on the effects of altitude on antioxidant activity. Most of the papers deal with the influence on phenotypic expression rather than on antioxidant activity. Gündüz and Özbay [65] observed in some strawberry genotypes an increase of antioxidant activity with increasing altitude, although the greater variability in this activity was due to the genotype. Another study on this species indicated a lower AA in the cultivar grown at 1500 m above sea level site than 1100 and 1200 m sites, due to the lower accumulation in anthocyanins at high altitude [66]. The ability of radical scavenging in Calluna vulgaris was higher with rising altitude due to the higher flavonoid’s accumulation.
As a completion of our study, the promotion of a further experiment with the same genotypes growing at different altitudes appears logical, including the localities of origin of the cultivars. This additional experiment will be expected to show how the genetic differences may express some eventual plus variance that the actual location of the field of comparison is unable to stress.

4.4. Information Provided by the Application of Different AA Methods

The methods we have used allow us to assess the overall antioxidant activity of plant tissues, considering some of the main phenolic chemical families, without evaluating the contribution of each individual compound (e.g., delphinine or gallic acid) to this activity. This is undoubtedly a possible perspective to improve the information obtained by our experiment, because the AA is dependent on each individual compound role within the main group (e.g., flavonoids, tannin families) and by their chemical modifications [67]. For example, the glycosylation of cyanidin anthocyanin increases its antioxidant activity while in malvidin anthocyanins, the antioxidant activity decreases after glycosylation [68]. Arnous et al. [62] suggest that a low correlation between total content of anthocyanins and antioxidant activity may be justified by both monomeric and polymeric anthocyanins, characterized by different antioxidant activities.
However, the results of the antioxidant activity presented in this paper provide complete information about the ability of myrtle extracts to counteract the damaging effects of free radicals. Several authors reported the importance of the use of different methodologies to determine the antioxidant properties of plant extracts since each method reflects different mode of action of the antioxidant molecules and may provide different information about the extract’s activity and use.
Myrtle extracts used as food additives protect food from lipid oxidation while as food ingredient; they protect cells and tissues from oxidative damage. In this paper, we compared the results of the antioxidant activities measured with different methods with the aim to provide a wide spectrum of information on myrtle extracts’ antioxidant activity. The methods used in this paper were based on the antioxidants’ reaction mechanisms: DPPH (free radical scavenging activity), ABTS (radical cations reducing activity), FRAP (metal ions reducing activity), the β- carotene bleaching method (peroxyl radical scavenging activity), and spin trapping method coupled with EPR (hydroxyl radical scavenging activity).
DPPH and ABTS are the most widely used methods to determine the antioxidant activity of plant extracts and often used for comparison purposes. In myrtle, leaf extracts DPPH and ABTS were used to study the radical scavenging properties of Sicilian myrtle populations to provide a chemical characterization of the leaves for future domestication programmes [69]. It is not always easy to compare results from different laboratories since they are often reported with different units of measurement. The cultivars analysed in this paper presented a high variability of the radical scavenging activities measured with DPPH and ABTS methods, and on the contrary, a moderate variability was observed among Sicilian populations.
Regarding ABTS method, the ability of the antioxidants to reduce the radical cation ABTS+ depends on their reduction potential. Only the antioxidants with a redox potential lower than that of the couple ABTS•+/ABTS (E0 = 0.68 V) are able to reduce the ABTS•+ radical [70]. Similarly, the FRAP method measures the reducing power of the extracts. It is based on the ability of the extracts to reduce the yellow ferric tripyridyltriazine complex (Fe (III)-TPTZ) to the blue ferrous complex (Fe (II)- TPTZ). This method provides similar results to ABTS assay since the redox potential of the couple Fe (III)/Fe (II) is 0.7 V similar to that of ABTS•+/ABTS couple. The lack of significant correlation observed in this paper between results obtained from ABTS and FRAP methods may be due to the effect of a different reaction environment on the extracts: in particular, the pH value. The FRAP assay was carried out under acidic conditions (pH 3.6), while ABTS under neutral conditions (pH 7.4). Moreover, in the extracts, other electron donating substances with an E0 lower than that of the couple Fe (III)/Fe (II) may cause an overestimation of FRAP results.
The β- carotene bleaching method estimates the ability of plant extracts to scavenge peroxyl radicals, which are the major causes of food quality deterioration. It is based on the ability of the antioxidants present in the extracts to react with the peroxyl radical thus preventing the β-carotene bleaching. In the case of myrtle, this test may provide a good indication of the evolution of leaf tissue that in the same season showed a natural increase of the chlorophyll and related carotenoid content, which can contribute to the observed increase of the AA from springtime to autumn.
The spin trapping method coupled with EPR is the most specific and reliable method for the estimation of biological relevant radicals like hydroxyl and superoxide radicals. The hydroxyl radical is one of the most dangerous radicals produced in cells; the spin trapping method provides useful information about the biological relevance of extracts. In food chemistry, the spin trapping method has been used to evaluate the antioxidant activity of food, beverages, and plant extracts [21,22,34].

5. Conclusions

Numerous studies highlight the potential use of the myrtle plant for medicinal purposes. Clinical studies carried out so far show a key role for the AA of phenolic compounds of myrtle on these health-promoting properties.
In this study, the influence of the genotype, biodiversity of the samples, sampling times, altitude of site of origin on the AA in myrtle berry and leaf extracts were evaluated. We used both spectrophotometric and spectroscopic methods to evaluate the AA of the extracts with different mechanism of action.
With respect to development stages of samples, the trend of AA in leaf and berry extract was different among the cultivars and the method used for the AA evaluation. The evaluation of development stage of vegetal material is necessary to identify the best harvest time to maximize the AA.
In leaf extracts, the total phenols content was correlated to the AA detected with ABTS and β-Carotene assays while the content of tannins content was correlated to β-Carotene and EPR. In the berry extracts, the content of phenolic compounds, anthocyanins, and tannins affected differently the AA. The total phenols content influenced the AA with ABTS and DPPH, while the tannins are positively involved in AA with β-Carotene and negatively to AA detected with FRAP assay. Instead, the anthocyanins of the berries were correlated positively with the AA with ABTS and negatively with the AA with β -carotene, showing a lower contribution to the AA of myrtle berries. The role of other flavonoids compounds such as tannins should be studied in depth.
The altitude of the site of origin of cultivars affected positively the AA in leaves and negatively the AA in berries but with a high variability in the results due to the type of assay used. Subsequent studies on a larger number of cultivars and other climatic parameters, as temperatures and rainfalls, would be useful to better understand both the mechanism of phenolic compound accumulation and antioxidant activity. In addition, it would be interesting to evaluate the behaviour of these cultivars with respect to these traits in their areas of origin to evaluate the differences with the same growing conditions.
In conclusion, these genotypes have shown good antioxidant activity and will subsequently be assessed for their biological properties. In particular, these genotypes will be tested for their possible use in the formulation of food supplements or pharmaceutical products, using processing plant residues as a source of bioactive compounds.

Author Contributions

Conceptualization, S.M. and M.M.; methodology, S.M., A.F., L.D., and M.M.; software, S.M. and A.F.; validation, A.F.; formal analysis, S.M. and A.F.; investigation, S.M., A.F., and L.D.; resources, M.M.; data curation, S.M.; writing—original draft preparation, S.M. and A.F.; writing—review and editing, M.M.; visualization, S.M.; supervision, M.M.; project administration, M.M.; funding acquisition, M.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by a grant of the Regione Autonoma della Sardegna P.O.R. program, by the FAR2020MULAS found, and by the project SYSTEMIC “An integrated approach to the challenge of sustainable food systems adaptive and mitigatory strategies to address climate change and malnutrition”, Knowledge hub on Nutrition and Food Security, in a joint action of JPI HDHL, JPI OCEANS and FACCE JPI launched in 2019 under the ERA NET ERA HDHL (n 696295).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Mulas, M. The myrtle (Myrtus communis L.) case, from a wild shrub to a new fruit crop. Acta Hortic. 2012, 948, 235–242. [Google Scholar] [CrossRef]
  2. Özkan, A.M.G.; Güray, Ç.G. A mediterranean: Myrtus communis L.(myrtle). Plants Cult. Seeds Cult. Herit. Eur. Edipuglias 2009, 1, 163. [Google Scholar]
  3. Franco, A.M.; Tocci, N.; Guella, G.; Dell’Agli, M.; Sangiovanni, E.; Perenzoni, D.; Manca, G. Myrtle Seeds (Myrtus communis L.) as a rich source of the bioactive ellagitannins oenothein B and eugeniflorin D2. ACS Omega 2019, 4, 15966–15974. [Google Scholar] [CrossRef] [Green Version]
  4. Cruciani, S.; Garroni, G.; Ginesu, G.C.; Fadda, A.; Ventura, C.; Maioli, M. Unravelling Cellular Mechanisms of Stem Cell Senescence: An Aid from Natural Bioactive Molecules. Biology 2020, 9, 57. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Dias, V.; Junn, E.; Mouradian, M.M. The role of oxidative stress in Parkinson’s disease. J. Parkinson’s Dis. 2013, 3, 461–491. [Google Scholar] [CrossRef] [Green Version]
  6. Emerit, J.; Edeas, M.; Bricaire, F. Neurodegenerative diseases and oxidative stress. Biomed. Pharmacother. 2004, 58, 39–46. [Google Scholar] [CrossRef]
  7. Ravarotto, V.; Simioni, F.; Pagnin, E.; Davis, P.A.; Calò, L.A. Oxidative stress–chronic kidney disease–cardiovascular disease: A vicious circle. Life Sci. 2018, 210, 125–131. [Google Scholar] [CrossRef]
  8. Forbes, J.M.; Coughlan, M.T.; Cooper, M.E. Oxidative stress as a major culprit in kidney disease in diabetes. Diabetes 2008, 57, 1446–1454. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Olas, B. Berry phenolic antioxidants–implications for human health? Front. Pharmacol. 2018, 9, 78. [Google Scholar] [CrossRef]
  10. Giampieri, F.; Alvarez-Suarez, J.M.; Battino, M. Strawberry and human health: Effects beyond antioxidant activity. J. Agric. Food Chem. 2014, 62, 3867–3876. [Google Scholar] [CrossRef]
  11. Durazzo, A.; Lucarini, M.; Novellino, E.; Daliu, P.; Santini, A. Fruit-based juices: Focus on antioxidant properties—Study approach and update. Phytother. Res. 2019, 33, 1754–1769. [Google Scholar] [CrossRef] [PubMed]
  12. Mazzoni, L.; Perez-Lopez, P.; Giampieri, F.; Alvarez-Suarez, J.M.; Gasparrini, M.; Forbes-Hernandez, T.Y.; Battino, M. The genetic aspects of berries: From field to health. J. Sci. Food Agric. 2016, 96, 365–371. [Google Scholar]
  13. Junqueira-Gonçalves, M.P.; Yáñez, L.; Morales, C.; Navarro, M.; Contreras, R.A.; Zúñiga, G.E. Isolation and characterization of phenolic compounds and anthocyanins from murta (Ugni molinae Turcz.) fruits. Assessment of antioxidant and antibacterial activity. Molecules 2015, 20, 5698–5713. [Google Scholar] [CrossRef] [PubMed]
  14. Durazzo, A. Study Approach of Antioxidant Properties in Foods: Update and Considerations. Foods 2017, 6, 17. [Google Scholar] [CrossRef] [Green Version]
  15. Kristl, J.; Slekovec, M.; Tojnko, S.; Unuk, T. Extractable antioxidants and non-extractable phenolics in the total antioxidant activity of selected plum cultivars (Prunus domestica L.): Evolution during on-tree ripening. Food Chem. 2011, 125, 29–34. [Google Scholar] [CrossRef]
  16. D’Urso, G.; Sarais, G.; Lai, C.; Pizza, C.; Montoro, P. LC-MS based metabolomics study of different parts of myrtle berry from Sardinia (Italy). J. Berry Res. 2017, 7, 217–229. [Google Scholar] [CrossRef]
  17. Scorrano, S.; Lazzoi, M.R.; Mergola, L.; Di Bello, M.P.; Del Sole, R.; Vasapollo, G. Anthocyanins profile by Q-TOF LC/MS in Myrtus communis berries from Salento Area. Food Anal. Methods 2017, 10, 2404–2411. [Google Scholar] [CrossRef]
  18. Barboni, T.; Cannac, M.; Massi, L.; Perez-Ramirez, Y.; Chiaramonti, N. Variability of polyphenol compounds in Myrtus communis L. (Myrtaceae) berries from Corsica. Molecules 2010, 15, 7849–7860. [Google Scholar] [CrossRef] [Green Version]
  19. Hennia, A.; Miguel, M.G.; Nemmiche, S. Antioxidant activity of Myrtus communis L. and myrtus nivellei batt. & trab. extracts: A brief review. Medicines 2018, 5, 89. [Google Scholar]
  20. Aidi Wannes, W.; Saidani Tounsi, M.; Marzouk, B. Morphological and chemical characterization of two wild Tunisian myrtle (Myrtus communis L.) populations. Trends Phytochem. Res. 2019, 3, 231–242. [Google Scholar]
  21. Fadda, A.; Barberis, A.; Sanna, D. Influence of pH, buffers and role of quinolinic acid, a novel iron chelating agent, in the determination of hydroxyl radical scavenging activity of plant extracts by Electron Paramagnetic Resonance (EPR). Food Chem. 2018, 240, 174–182. [Google Scholar] [CrossRef] [PubMed]
  22. Sanna, D.; Delogu, G.; Mulas, M.; Schirra, M.; Fadda, A. Determination of free radical scavenging activity of plant extracts through DPPH assay: An EPR and UV–Vis study. Food Anal. Methods 2012, 5, 759–766. [Google Scholar] [CrossRef]
  23. Amensour, M.; Sendra, E.; Abrini, J.; Pérez-Alvarez, J.A.; Fernández-López, J. Antioxidant activity and total phenolic compounds of myrtle extracts actividad antioxidante y contenido de compuestos fenólicos totales en extractos de myrtus. CyTA J. Food 2010, 8, 95–101. [Google Scholar] [CrossRef]
  24. Serreli, G.; Jerković, I.; Gil, K.A.; Marijanović, Z.; Pacini, V.; Tuberoso, C.I.G. Phenolic compounds, volatiles and antioxidant capacity of white myrtle berry liqueurs. Plant Foods Hum. Nutr. 2017, 72, 205–210. [Google Scholar] [CrossRef]
  25. Dairi, S.; Galeano-Díaz, T.; Acedo-Valenzuela, M.I.; Godoy-Caballero, M.P.; Dahmoune, F.; Remini, H.; Madani, K. Monitoring oxidative stability and phenolic compounds composition of myrtle-enriched extra virgin olive during heating treatment by flame, oven and microwave using reversed phase dispersive liquid–liquid microextraction (RP-DLLME)-HPLC-DAD-FLD method. Ind. Crops Prod. 2015, 65, 303–314. [Google Scholar] [CrossRef]
  26. Montoro, P.; Tuberoso, C.I.; Piacente, S.; Perrone, A.; De Feo, V.; Cabras, P.; Pizza, C. Stability and antioxidant activity of polyphenols in extracts of Myrtus communis L. berries used for the preparation of myrtle liqueur. J. Pharm. Biomed. Anal. 2006, 41, 1614–1619. [Google Scholar] [CrossRef]
  27. Jabri, M.A.; Hajaji, S.; Rtibi, K.; Sebai, H. Role of Anti-Inflammatory, Reactive Oxygen Species Scavenging Activity and Nematicidal Properties of Myrtle Berry Seeds on Helminthiasis Treatment. J. Med. Food 2020, 4, 15966–15974. [Google Scholar] [CrossRef]
  28. Cruciani, S.; Santaniello, S.; Fadda, A.; Sale, L.; Sarais, G.; Sanna, D.; Mulas, M.; Ginesu, G.C.; Cossu, M.L.; Serra, P.A.; et al. Extracts from Myrtle Liqueur Processing Waste Modulate Stem Cells Pluripotency under Stressing Conditions. BioMed Res. Int. 2019, 2019. [Google Scholar] [CrossRef] [Green Version]
  29. Tumen, I.; Senol, F.S.; Orhan, I.E. Inhibitory potential of the leaves and berries of Myrtus communis L.(myrtle) against enzymes linked to neurodegenerative diseases and their antioxidant actions. Int. J. Food Sci. Nutr. 2012, 63, 387–392. [Google Scholar] [CrossRef] [PubMed]
  30. Mimica-Dukić, N.; Bugarin, D.; Grbović, S.; Mitić-Ćulafić, D.; Vuković-Gačić, B.; Orčić, D.; Couladis, M. Essential oil of Myrtus communis L. as a potential antioxidant and antimutagenic agents. Molecules 2010, 15, 2759–2770. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  31. Ferchichi, H.; Salouage, I.; Bacha, S.; Said, D.B.; Gaies, E. Effect of Myrtus Communis L. Extracts on Attenuation of Liver Normothermic Ischemia-Reperfusion Injury. J. Transplant. Technol. Res. 2011, 3, 2161-0991. [Google Scholar]
  32. Babou, L.; Hadidi, L.; Grosso, C.; Zaidi, F.; Valentão, P.; Andrade, P.B. Study of phenolic composition and antioxidant activity of myrtle leaves and fruits as a function of maturation. Eur. Food Res. Tech. 2016, 242, 1447–1457. [Google Scholar] [CrossRef]
  33. Medda, S.; Dessena, L.; Mulas, M. Monitoring of the PAL Enzymatic Activity and Polyphenolic Compounds in Leaves and Fruits of Two Myrtle Cultivars during Maturation. Agriculture 2020, 10, 389. [Google Scholar] [CrossRef]
  34. Sanna, D.; Mulas, M.; Molinu, M.G.; Fadda, A. Oxidative stability of plant hydroalcoholic extracts assessed by EPR spin trapping under forced ageing conditions: A myrtle case study. Food Chem. 2019, 271, 753–761. [Google Scholar] [CrossRef] [PubMed]
  35. Tuberoso, C.I.; Barra, A.; Cabras, P. Effect of different technological processes on the chemical composition of myrtle (Myrtus communis L.) alcoholic extracts. Eur. Food Res. Technol. 2008, 226, 801–808. [Google Scholar] [CrossRef]
  36. Singleton, V.L.; Rossi, J.A. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 1965, 16, 144–158. [Google Scholar]
  37. Lee, J.; Durst, R.W.; Wrolstad, R.E. Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: Collaborative study. J. AOAC Int. 2005, 88, 1269–1278. [Google Scholar] [CrossRef] [Green Version]
  38. Fadda, A.; Mulas, M. Chemical changes during myrtle (Myrtus communis L.) fruit development and ripening. Sci. Hort. 2010, 125, 477–485. [Google Scholar] [CrossRef]
  39. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 1999, 26, 1231–1237. [Google Scholar] [CrossRef]
  40. Benzie, I.F.; Strain, J.J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Anal. Biochem. 1996, 239, 70–76. [Google Scholar] [CrossRef] [Green Version]
  41. Klančnik, A.; Guzej, B.; Kolar, M.H.; Abramovič, H.; Možina, S.S. In vitro antimicrobial and antioxidant activity of commercial rosemary extract formulations. J. Food Prot. 2009, 72, 1744–1752. [Google Scholar] [CrossRef] [PubMed]
  42. Annunziata, A.; Vecchio, R. Consumer perception of functional foods: A conjoint analysis with probiotics. Food Qual. Prefer. 2013, 28, 348–355. [Google Scholar] [CrossRef]
  43. Urala, N.; Lähteenmäki, L. Consumers’ changing attitudes towards functional foods. Food Qual. Prefer. 2007, 18, 1–12. [Google Scholar] [CrossRef]
  44. Dai, J.; Mumper, R.J. Plant phenolics: Extraction, analysis and their antioxidant and anticancer properties. Molecules 2010, 15, 7313–7352. [Google Scholar] [CrossRef]
  45. Gothai, S.; Ganesan, P.; Park, S.Y.; Fakurazi, S.; Choi, D.K.; Arulselvan, P. Natural phyto-bioactive compounds for the treatment of type 2 diabetes: Inflammation as a target. Nutrients 2016, 8, 461. [Google Scholar] [CrossRef]
  46. Direito, R.; Rocha, J.; Sepodes, B.; Eduardo-Figueira, M. Phenolic Compounds Impact on Rheumatoid Arthritis, Inflammatory Bowel Disease and Microbiota Modulation. Pharmaceutics 2021, 13, 145. [Google Scholar] [CrossRef] [PubMed]
  47. González de Peredo, A.; Vázquez-Espinosa, M.; Espada-Bellido, E.; Jiménez-Cantizano, A.; Ferreiro-González, M.; Amores-Arrocha, A.; Barbero, G. Development of new analytical microwave-assisted extraction methods for bioactive compounds from myrtle (Myrtus communis L.). Molecules 2018, 23, 2992. [Google Scholar] [CrossRef] [Green Version]
  48. D’Urso, G.; Montoro, P.; Lai, C.; Piacente, S.; Sarais, G. LC-ESI/LTQOrbitrap/MS based metabolomics in analysis of Myrtus communis leaves from Sardinia (Italy). Ind. Crop. Prod. 2019, 128, 354–362. [Google Scholar] [CrossRef]
  49. Snoussi, A.; Hayet, B.H.K.; Essaidi, I.; Zgoulli, S.; Moncef, C.M.; Thonart, P.; Bouzouita, N. Improvement of the composition of Tunisian myrtle berries (Myrtus communis L.) alcohol extracts. J. Agric. Food Chem. 2012, 60, 608–614. [Google Scholar] [CrossRef]
  50. Pavlović, A.V.; Papetti, A.; Zagorac, D.Č.D.; Gašić, U.M.; Mišić, D.M.; Tešić, Ž.L.; Natić, M.M. Phenolics composition of leaf extracts of raspberry and blackberry cultivars grown in Serbia. Ind. Crop. Prod. 2016, 87, 304–314. [Google Scholar] [CrossRef]
  51. de Pascual-Teresa, S.; Sanchez-Ballesta, M.T. Anthocyanins: From plant to health. Phytochem. Rev. 2008, 7, 281–299. [Google Scholar] [CrossRef]
  52. Auzanneau, N.; Weber, P.; Kosińska-Cagnazzo, A.; Andlauer, W. Bioactive compounds and antioxidant capacity of Lonicera caerulea berries: Comparison of seven cultivars over three harvesting years. J. Food Compos. Anal. 2018, 66, 81–89. [Google Scholar] [CrossRef] [Green Version]
  53. Araya, M.; Carvajal, Y.; Alvarez, V.; Orozco, R.; Rodriguez, G. Polyphenol characterization of three varieties of Blackberry fruit (Rubus adenotrichos), cultivated in Costa Rica. J. Berry Res. 2017, 7, 97–107. [Google Scholar] [CrossRef]
  54. Somsong, P.; Srzednicki, G.; Konczak, I.; Lohachoompol, V. Effects of preconditioning on quality of dried blueberries. Jul. Kühn Arch. 2010, 425, 264. [Google Scholar]
  55. Amessis-Ouchemoukh, N.; Madani, K.; Falé, P.L.; Serralheiro, M.L.; Araújo, M.E.M. Antioxidant capacity and phenolic contents of some Mediterranean medicinal plants and their potential role in the inhibition of cyclooxygenase-1 and acetylcholinesterase activities. Ind. Crop. Prod. 2014, 53, 6–15. [Google Scholar] [CrossRef]
  56. Pereira, P.; Cebola, M.J.; Oliveira, M.C.; Bernardo-Gil, M.G. Supercritical fluid extraction vs. conventional extraction of myrtle leaves and berries: Comparison of antioxidant activity and identification of bioactive compounds. J. Supercrit. Fluids 2016, 113, 1–9. [Google Scholar] [CrossRef]
  57. Gralec, M.; Wawer, I.; Zawada, K. Aronia melanocarpa berries: Phenolics composition and antioxidant properties changes during fruit development and ripening. Emir. J. Food Agric. 2019, 31, 214–221. [Google Scholar]
  58. Ariza, M.T.; Martínez-Ferri, E.; Domínguez, P.; Medina, J.J.; Miranda, L.; Soria, C. Effects of harvest time on functional compounds and fruit antioxidant capacity in ten strawberry cultivars. J. Berry Res. 2015, 5, 71–80. [Google Scholar] [CrossRef]
  59. Messaoud, C.; Boussaid, M. Myrtus communis berry colour morphs: A comparative analysis of essential oils, fatty acids, phenolic compounds, and antioxidant activities. Chem. Biodivers. 2011, 8, 300–310. [Google Scholar] [CrossRef]
  60. Dudonne, S.; Vitrac, X.; Coutiere, P.; Woillez, M.; Mérillon, J.M. Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J. Agric. Food Chem. 2009, 57, 1768–1774. [Google Scholar] [CrossRef]
  61. Kähkönen, M.P.; Heinämäki, J.; Ollilainen, V.; Heinonen, M. Berry anthocyanins: Isolation, identification and antioxidant activities. J. Sci. Food Agric. 2003, 83, 1403–1411. [Google Scholar] [CrossRef]
  62. Arnous, A.; Makris, D.P.; Kefalas, P. Correlation of pigment and flavanol content with antioxidant properties in selected aged regional wines from Greece. J. Food Compos. Anal. 2002, 15, 655–665. [Google Scholar] [CrossRef]
  63. Spinardi, A.; Cola, G.; Gardana, C.S.; Mignani, I. Variation of anthocyanins content and profile throughout fruit development and ripening of highbush blueberry cultivars grown at two different altitudes. Front. Plant Sci. 2019, 10, 1045. [Google Scholar] [CrossRef]
  64. Zeng, Q.; Dong, G.; Tian, L.; Wu, H.; Ren, Y.; Tamir, G.; Yu, H. High Altitude Is Beneficial for Antioxidant Components and Sweetness Accumulation of Rabbiteye Blueberry. Front. Plant Sci. 2020, 11, 1492. [Google Scholar] [CrossRef]
  65. Gündüz, K.; Özbay, H. The effects of genotype and altitude of the growing location on physical, chemical, and phytochemical properties of strawberry. Turk. J. Agric. For. 2018, 42, 145–153. [Google Scholar] [CrossRef]
  66. Guerrero-Chavez, G.; Scampicchio, M.; Andreotti, C. Influence of the site altitude on strawberry phenolic composition and quality. Sci. Hort. 2015, 192, 21–28. [Google Scholar] [CrossRef]
  67. Kähkönen, M.P.; Hopia, A.I.; Heinonen, M. Berry phenolics and their antioxidant activity. J. Agric. Food Chem. 2001, 49, 4076–4082. [Google Scholar] [CrossRef] [PubMed]
  68. Kähkönen, M.P.; Heinämäki, J.; Heinonen, M. Antioxidant activity of anthocyanins and their aglycons. J. Agric. Food Chem. 2003, 51, 628–633. [Google Scholar] [CrossRef]
  69. Melito, S.; La Bella, S.; Martinelli, F.; Cammalleri, I.; Tuttolomondo, T.; Leto, C.; Mulas, M. Morphological, chemical, and genetic diversity of wild myrtle (Myrtus communis L.) populations in Sicily. Turk. J. Agric. For. 2016, 40, 249–261. [Google Scholar] [CrossRef]
  70. Müller, L.; Fröhlich, K.; Böhm, V. Comparative antioxidant activities of carotenoids measured by ferric reducing antioxidant power (FRAP), ABTS bleaching assay (αTEAC), DPPH assay and peroxyl radical scavenging assay. Food Chem. 2011, 129, 139–148. [Google Scholar] [CrossRef]
Agronomy 11 01059 g001 550
Figure 1. Graphical representation of the timing of sampling of myrtle berries and leaves.
Figure 1. Graphical representation of the timing of sampling of myrtle berries and leaves.
Agronomy 11 01059 g001
Agronomy 11 01059 g002 550
Figure 2. Content of total phenols (a–e), tannins (f–i,l), and anthocyanins (m–p) in leaf and berry extracts of the different cultivars. The ‘Grazia’ cultivar give white fruits. Data are the mean and standard deviation of three replicates. Data labelled with the same letters are not significantly different for components of the berry tissues (lowercase) and leaf tissues (capital) letters according to Duncan’s Multiple Range Test at p ≤ 0.01 level.
Figure 2. Content of total phenols (a–e), tannins (f–i,l), and anthocyanins (m–p) in leaf and berry extracts of the different cultivars. The ‘Grazia’ cultivar give white fruits. Data are the mean and standard deviation of three replicates. Data labelled with the same letters are not significantly different for components of the berry tissues (lowercase) and leaf tissues (capital) letters according to Duncan’s Multiple Range Test at p ≤ 0.01 level.
Agronomy 11 01059 g002
Agronomy 11 01059 g003 550
Figure 3. EPR signal intensity of the DMPO-OH radical adduct measured without extract or black line (A), with ‘Grazia’ berry extract (0 DAF) (B), and ‘Grazia’ leaves extract (O DAF) (C).
Figure 3. EPR signal intensity of the DMPO-OH radical adduct measured without extract or black line (A), with ‘Grazia’ berry extract (0 DAF) (B), and ‘Grazia’ leaves extract (O DAF) (C).
Agronomy 11 01059 g003
Table
Table 1. Analysed five cultivars and its sites of origin. The altitudes and coordinate of the in sites of origin are reported.
Table 1. Analysed five cultivars and its sites of origin. The altitudes and coordinate of the in sites of origin are reported.
CultivarSite of OriginAltitude (m Above Sea Level)
‘Giovanna’, ‘Grazia’Rumanedda (40°33′36″ N 8°18′54″ E)164
‘Maria Antonietta’Cuglieri (40°11′20″ N 8°34′02″ E)248
‘Sofia’Laconi (39°51′09.71″ N 9°03′07.37″ E)637
‘Maria Rita’Capoterra (39°10′30.67″ N 8°58′15.07″ E)54
Table
Table 2. Meteorological parameters recorded in the experimental field (Fenosu site) during 2019. T = air temperature; RH = relativity humidity.
Table 2. Meteorological parameters recorded in the experimental field (Fenosu site) during 2019. T = air temperature; RH = relativity humidity.
Meteorological RecordsMonths
JFMAMJJASOND
T max (°C)17.118.821.624.826.839.237.238.334.230.923.121.1
T min (°C)0.00.54.13.75.812.317.215.914.211.45.92.5
T mean (°C)8.49.512.714.116.024.126.126.023.019.614.213.1
Rainfall (mm)0.091.016.216.658.455.41.82.417.234.846.2290.4
RH max (%)100.092.092.092.093.091.092.092.093.092.093.093.0
RH min (%)43.022.030.033.030.024.028.025.028.039.055.042.0
RH mean (%)82.075.071.073.073.064.067.068.072.074.082.079.0
Table
Table 3. Dry weight expressed in percentage of myrtle leaf extracts.
Table 3. Dry weight expressed in percentage of myrtle leaf extracts.
Dry Weight of Leaf Samples (%)
DAFGiovannaGraziaMaria RitaMaria AntoniettaSofia
040.7628.8042.9444.8835.09
3037.0040.2043.1143.3044.19
6041.2541.0043.1743.3548.32
9045.2048.0646.5546.1143.94
12044.0246.9046.4332.9450.61
15039.5542.8440.8342.4445.46
21040.5943.4644.2142.7744.77
22552.1938.3937.3043.7342.94
Table
Table 4. Dry weight expressed in percentage of myrtle berry extracts.
Table 4. Dry weight expressed in percentage of myrtle berry extracts.
Dry Weight of Berry Samples (%)
DAFGiovannaGraziaMaria RitaMaria AntoniettaSofia
029.2239.5829.5530.8540.20
3034.2028.8532.0336.4639.45
6037.2136.7136.4438.7740.41
9035.5234.8835.3339.3632.99
12038.1834.4232.9441.2134.45
15021.1720.5323.6027.6223.65
21027.0422.6728.6925.6128.32
22536.2631.9236.3931.3334.06
Table
Table 5. Antioxidant activity (AA) in myrtle leaves cultivar evaluated by DPPH, FRAP, ABTS, β-Carotene and EPR assays. Data are the mean and standard deviation of three replicate. Values in the same column labelled with the same letters are not significantly different according to Duncan’s multiple range test at p ≤ 0.01 level.
Table 5. Antioxidant activity (AA) in myrtle leaves cultivar evaluated by DPPH, FRAP, ABTS, β-Carotene and EPR assays. Data are the mean and standard deviation of three replicate. Values in the same column labelled with the same letters are not significantly different according to Duncan’s multiple range test at p ≤ 0.01 level.
CultivarDAFDPPH (µmol TE/g DW)FRAP (mmol TE/g DW)ABTS (mmol TE/g DW)β-Carotene (CAA)•OH Scavenging Activity(mmol GAE/g DW)
Giovanna025.36 ± 0.21B53.63 ± 3.09c171.55 ± 0.57d0.68 ± 0.22b1.52 ± 0.10abc
12021.34 ± 0.33De83.24 ± 5.46a173.91 ± 3.74d1.02 ± 0.42a1.99 ± 0.04a
Grazia025.16 ± 0.70B61.97 ± 3.38bc174.2 ± 12.83d0.60 ± 0.01b1.25 ± 0.35bc
12018.88 ± 0.04F88.87 ± 5.46a188.17 ± 5.83d1.15 ± 0.15a1.72 ± 0.20abc
Maria Antonietta021.93 ± 0.14Cd71.09 ± 2.41b211.50 ± 0.03c0.63 ± 0.00b1.35 ± 0.50bc
12028.29 ± 0.64A84.53 ± 1.97a242.6 ± 10.93b1.02 ± 0.01a1.50 ± 0.06abc
Maria Rita023.80 ± 0.86Bc64.38 ± 6.87b182.73 ± 2.70d0.59 ± 0.01b1.35 ± 0.03bc
12019.94 ± 0.63Ef84.58 ± 3.53a135.7 ± 10.21e1.01 ± 0.00a1.71 ± 0.04abc
Sofia022.94 ± 0.62Cd93.25 ± 4.45a294.25 ± 8.16a0.68 ± 0.06b1.21 ± 0.04c
12019.06 ± 0.66F84.98 ± 8.08a245.03 ± 1.21b1.05 ± 0.00a1.81 ± 0.06ab
Table
Table 6. Antioxidant activity (AA) in berries of myrtle cultivars evaluated by DPPH, FRAP, ABTS, β-Carotene, and EPR assays. Data are the mean and standard deviation of three replicates. Values in the same column labelled with the same letters are not significantly different according to Duncan’s Multiple Range Test at p ≤ 0.01 level.
Table 6. Antioxidant activity (AA) in berries of myrtle cultivars evaluated by DPPH, FRAP, ABTS, β-Carotene, and EPR assays. Data are the mean and standard deviation of three replicates. Values in the same column labelled with the same letters are not significantly different according to Duncan’s Multiple Range Test at p ≤ 0.01 level.
CultivarDAFDPPH (µmol TE/g DW)FRAP (mmol TE/g DW)ABTS (mmol TE/g DW)β-Carotene (CAA)•OH Scavenging Activity (mmol GAE/g DW)
Giovanna6020.44 ± 0.65fg110.77 ± 8.46d–h65.54 ± 3.69i1.01 ± 0.07b0.12 ± 0.04d
15029.66 ± 1.00b119.28 ± 9.05b–e148.32 ± 8.19c0.38 ± 0.02j3.89 ± 0.72c
21024.97 ± 0.06de114.29 ± 1.11c–f121.85 ± 10.5ef0.77 ± 0.02de4.67 ± 1.00bc
22522.96 ± 0.73ef118.97 ± 5.92b–e132.40 ± 1.53de0.50 ± 0.01i0.13 ± 0.02d
Grazia6019.45 ± 0.73g100.80 ± 0.01hi109.58 ± 8.79fg0.90 ± 0.03c0.16 ± 0.01d
15030.07 ± 1.80ab109.17 ± 2.53e–h118.91 ± 11.7f0.43 ± 0.01ij4.63 ± 0.77bc
21032.74 ± 0.33a119.79 ± 1.58bcd176.61 ± 7.27b0.73 ± 0.02def6.64 ± 1.77a
22522.07 ± 1.65fg127.12 ± 1.70ab71.68 ± 0.87i0.74 ± 0.00def0.26 ± 0.11d
Maria Antonietta6021.83 ± 0.23fg92.29 ± 0.34ij62.42 ± 4.30ij1.08 ± 0.02a0.15 ± 0.04d
15026.16 ± 1.75cd123.95 ± 5.08abc111.39 ± 0.15fg0.71 ± 0.03efg3.75 ± 0.28c
21031.12 ± 0.81ab101.74 ± 3.48gh173.94 ± 9.58b0.66 ± 0.02fgh5.32 ± 0.82b
22528.90 ± 1.16bc88.62 ± 7.38j137.46 ± 2.74cd0.67 ± 0.04fg0.67 ± 0.15d
Maria Rita6023.15 ± 0.65ef110.35 ± 0.82d–h107.80 ± 2.30fg0.80 ± 0.05d0.05 ± 0.01d
15025.36 ± 2.36de106.64 ± 1.27f–h101.71 ± 11.4g0.69 ± 0.02efg4.76 ± 0.28bc
21026.37 ± 0.36cd105.51 ± 1.14f–h196.52 ± 3.01a0.68 ± 0.01fg5.16 ± 0.24b
22520.42 ± 0.01fg130.26 ± 9.17a108.60 ± 1.40fg0.59 ± 0.02h0.31 ± 0.06d
Sofia6013.76 ± 1.34h110.53 ± 1.00d–h88.57 ± 2.84h0.96 ± 0.01b0.08 ± 0.02d
15015.72 ± 0.88h112.98 ± 2.15def45.27 ± 5.10k0.91 ± 0.02c4.22 ± 0.27 bc
21014.72 ± 0.97h128.77 ± 0.28ab50.14 ± 0.33jk0.73 ± 0.01def4.84 ± 0.57bc
22513.37 ± 1.12h111.71 ± 1.08d–g49.60 ± 0.90jk0.64 ± 0.02gh0.26 ± 0.05d
Table
Table 7. Correlation coefficient (r) calculated between the phenolic compounds content analysed, antioxidant activity (AA) obtained with the different assays, developmental stages (DAF), and altitude in myrtle berries in leaf extracts. Significance: ** = p ≤ 0.01; * = p ≤ 0.05; ns = not significant.
Table 7. Correlation coefficient (r) calculated between the phenolic compounds content analysed, antioxidant activity (AA) obtained with the different assays, developmental stages (DAF), and altitude in myrtle berries in leaf extracts. Significance: ** = p ≤ 0.01; * = p ≤ 0.05; ns = not significant.
Total PhenolsTotal TanninsDPPHFRAPABTSβ-CaroteneEPR
Total tannins0.719 **
DPPH0.507 **0.288 ns
FRAP0.042 ns0.177 ns−0.436 **
ABTS0.122 ns−0.128 ns0.157 ns0.457 *
β-Carotene0.402 **0.564 **−0.443 *0.631 **−0.047 ns
•OH scavenging0.203 ns0.368 **−0.462 *0.310 ns−0.278 ns0.673 **
Altitude0.041 ns−0.319 ns−0.139 ns0.462 **0.881 **0.059 ns−0.092 ns
Table
Table 8. Correlation coefficient (r) calculated between the phenolic compounds content analysed, antioxidant activity (AA) obtained with the different assays, developmental stages (DAF), and altitude in myrtle berries in berry extracts. Significance: ** = p ≤ 0.01; * = p ≤ 0.05; ns = not significant.
Table 8. Correlation coefficient (r) calculated between the phenolic compounds content analysed, antioxidant activity (AA) obtained with the different assays, developmental stages (DAF), and altitude in myrtle berries in berry extracts. Significance: ** = p ≤ 0.01; * = p ≤ 0.05; ns = not significant.
Total PhenolsTotal TanninsTotal AnthocyaninsDPPHFRAPABTSβ-CaroteneEPR
Total tannins0.698 **
Total anthocyanins0.131 ns−0.347 **
DPPH0.588 **0.375 **0.105 ns
FRAP−0.542 ns−0.411 **0.111 ns−0.180 ns
ABTS0.546 **0.228 ns 0.258 *0.784 **−0.147 ns
β-Carotene−0.047 ns0.307 **−0.527 **−0.452 **−0.315 *−0.464 **
•OH scavenging−0.100 ns0.126 ns0.058 ns0.493 **0.127 ns0.452 **−0.317 *
Altitude−0.571 **−0.360 *0.021 ns−0.647 **0.037 ns−0.566 **0.264 *−0.035 ns
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Medda, S.; Fadda, A.; Dessena, L.; Mulas, M. Quantification of Total Phenols, Tannins, Anthocyanins Content in Myrtus communis L. and Antioxidant Activity Evaluation in Function of Plant Development Stages and Altitude of Origin Site. Agronomy 2021, 11, 1059. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy11061059

AMA Style

Medda S, Fadda A, Dessena L, Mulas M. Quantification of Total Phenols, Tannins, Anthocyanins Content in Myrtus communis L. and Antioxidant Activity Evaluation in Function of Plant Development Stages and Altitude of Origin Site. Agronomy. 2021; 11(6):1059. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy11061059

Chicago/Turabian Style

Medda, Silvia, Angela Fadda, Leonarda Dessena, and Maurizio Mulas. 2021. "Quantification of Total Phenols, Tannins, Anthocyanins Content in Myrtus communis L. and Antioxidant Activity Evaluation in Function of Plant Development Stages and Altitude of Origin Site" Agronomy 11, no. 6: 1059. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy11061059

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop