Article pubs.acs.org/JAFC
Chemical and Bioactive Quality Traits During Fruit Ripening in Eggplant (S. melongena L.) and Allied Species Giuseppe Mennella,*,† Roberto Lo Scalzo,‡ Marta Fibiani,‡ Antonietta D’Alessandro,† Gianluca Francese,† Laura Toppino,§ Nazzareno Acciarri,∥ Adelia Emilia de Almeida,⊥ and Giuseppe Leonardo Rotino§ †
Consiglio per la Ricerca e la Sperimentazione in Agricoltura, CRA-ORT Centro di Ricerca per l’Orticoltura, via Cavalleggeri 25, 84098 Pontecagnano-Faiano (Salerno), Italy ‡ Consiglio per la Ricerca e la Sperimentazione in Agricoltura, CRA-IAA Unità di Ricerca per i Processi dell’Industria Agroalimentare, via Venezian 26, 20133 Milan, Italy § Consiglio per la Ricerca e la Sperimentazione in Agricoltura, CRA-ORL Unità di Ricerca per l’Orticoltura, via Paullese 28, 26836 Montanaso Lombardo (Lodi), Italy ∥ Consiglio per la Ricerca e la Sperimentazione in Agricoltura, CRA-ORA Unità di Ricerca per l’Orticoltura, via Salaria 1, 63030 Monsampolo del Tronto (Ascoli Piceno), Italy ⊥ Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Fármacos e Medicamentos, Faculdade de Ciências Farmacêuticas - Rodovia Araraquara - Jaú, km 1, Araraquara SP, Brazil S Supporting Information *
ABSTRACT: A chemical and bioactive quality evaluation of phytochemicals content of 10 eggplant lines and three allied species (S. sodomaeum, S. aethiopicum and S. integrifolium) was performed. The eggplant lines were divided into the two subgroups of delphinidin-3-rutinoside (D3R) and nasunin (NAS) typologies, on the basis of the anthocyanin detected in their fruit skin. The allied species had higher glycoalkaloids content, lower soluble solids and PPO activity and absence of anthocyanins compared to the eggplant lines; S. sodomaeum stood out for high phenols content. Orthogonal contrast revealed a higher sugar content and low PPO activity in NAS- compared to D3R-typologies, whereas higher chlorogenic acid and anthocyanin contents were present in D3R-typologies. The main effect of the ripening was a decrease in phenols and in the PPO activity, not evidenced in S. sodomaeum, and an increase of glycoalkaloids in overripe fruits. A good relationship was found between superoxide anion scavenging capacity and chlorogenic acid. This study highlighted the pattern of accumulation, also evidencing variations, of several phytochemicals during the eggplant fruit development and ripening. KEYWORDS: glycoalkaloids, phenols, antioxidants, nasunin, delphinidin 3-rutinoside, S. aethiopicum, S. integrifolium, S. sodomaeum
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INTRODUCTION The improvement of quality traits in fruits/vegetables, among other purposes, is aimed at producing fruits/vegetables rich in compounds important for human health by the evaluation and manipulation of particular phytochemical components and, consequently, of their biological activity.1,2 These molecules, such as the phenolics considered “nutraceuticals”, may have an important role in the resistance/tolerance to biotic3 and/or abiotic stresses.4 Eggplant (S. melongena L.) is an interesting fruit/vegetable in this respect because it is well-known to contain phytochemicals, mainly phenolics and steroids, which are considered to have, respectively, beneficial effects and toxic/ medicamentous properties according to their dosage.5−8 Eggplant berries have a high antioxidant capacity mainly due to chlorogenic acid7,9,10 and the anthocyanin pigments delphinidin-3-rutinoside (D3R) and/or nasunin.11,12 Eggplant lines and relatives have shown interesting variations in their chemical composition with regard to phenolic and steroid compounds.9,13,14 Employment of exotic eggplant germplasm in the breeding programs may represent a valid option to manipulate the content of such compounds. Moreover, the use of allied species allowed successful © 2012 American Chemical Society
incorporation of disease resistance/tolerance traits into the eggplant gene pool15 and the introgression lines developed showed a wide range of variation in their phytochemical content.14 The knowledge of the phytochemical variations during eggplant fruit development could greatly help the improvement of the commercial and nutritional value of the fruit. In fact, the physiological ripeness of the eggplant fruit is not coincident with the commercial ripeness and, furthermore, overripe fruits are unmarketable because of the presence of mature seed, and also of modifications in taste and firmness. During the eggplant fruit development and ripening, relevant changes occur in the color of the skin and flesh, in the fruit firmness and texture, and in seed maturation. Very little information is available about the correspondent modification in the biochemical composition and molecular aspects of the transition of eggplant fruit from commercial to physiological ripeness. Received: Revised: Accepted: Published: 11821
August 29, 2012 November 6, 2012 November 8, 2012 November 8, 2012 dx.doi.org/10.1021/jf3037424 | J. Agric. Food Chem. 2012, 60, 11821−11831
Journal of Agricultural and Food Chemistry
Article
powdered and held at −80 °C. All results were referred to as dry weight (dw).
This work is aimed at the evaluation of the quality traits regarding the presence of the phytochemicals and their antioxidant capacity in 10 eggplant lines from two different fruits typologies and in three allied genotypes (S. linnaeanum = S. sodomaeum, S. aethiopicum gr. gilo and S. aethiopicum gr. aculeatum = S. integrifolium). These allied species have been employed in an introgression breeding program mainly devoted to development of fungal wilt resistant eggplant lines.16 The 10 eggplant genotypes were divided into two groups according to their anthocyanin form, one containing delphinidin-3-rutinoside (D3R) and the other containing nasunin, delphinidin-3-(pcoumaroylrutinoside)-5-glucoside (NAS), which correspond to a previous subdivision made by Azuma et al.11 in Japanese (NAS) and non-Japanese (D3R) eggplant types. The NAS- and D3R-containing eggplant breeding lines have been employed in our introgression breeding programs as recurrent genotypes and, among other traits, according to local Southern Italy lore they differ each other by a sweeter taste and a lower tendency to brown after cutting in NAS-type eggplants with respect to D3R-types. The fruits of all the genotypes were collected during three phenological stages (unripe, commercially ripe and physiologically ripe fruit), to monitor the evolution of phytochemicals of interest for their human-health properties. Information about a different pattern of accumulation of healthrelated compounds might be efficiently exploited to breed eggplants with superior nutritional value.
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Table 1. Average Moisture Content (%) of the Assayed Solanum spp. Fruits before Freeze-Dried Process moisture content (%) genotype Allied S. integrifolium S. aethiopicum S. sodomaeum D3R-Containing Typology S. melongena 1 S. melongena 2 S. melongena 3 NAS-Containing Typology S. melongena 4 S. melongena 5 S. melongena 6 S. melongena 7 S. melongena 8 S. melongena 9 S. melongena 10
stage A
stage B
stage C
86.2 88.3 82.1
85.0 85.8 79.6
83.0 87.3 79.0
87.7 92.1 92.1
91.0 91.8 92.5
91.8 91.9 89.3
92.2 92.8 93.8 92.1 92.3 93.3 92.6
92.1 90.8 93.4 91.9 90.5 91.4 92.6
91.8 90.0 92.6 91.7 91.3 91.4 91.3
Chemical Quality Traits. The extraction and the analysis of anthocyanins was carried out on 200 mg of lyophilized and powdered peel, diluted in 10 mL of methanol containing 3% trifluoroacetic acid (TFA), as previously reported.8 RP-HPLC analysis was performed through a Waters E-Alliance HPLC system constituted by a 2695 separations module with quaternary pump, autosampler, and a 2996 photodiode array detector; data were acquired and analyzed with Waters Empower software on a PC. The chromatographic separations were performed at a flow rate of 0.8 mL/min and at 0.1 AUFS. Purified D3R (Polyphenols Laboratories AS, Sandnes, Norway) was used as external standard in RP-HPLC analyses, with a different retention time (23.9 min) compared to nasunin, that was eluted at a longer retention time (25.8 min for cis-nasunin and 26.1 min for transnasunin, respectively; see Supporting Information). As for nasunin quantification, a partially purified standard was used according to Lo Scalzo et al.17 The results were expressed as μmol/100 g of peel dw; the limit of detection was 2.00 μmol/100 g of peel dw. Soluble refractometric residue (SRR) was measured on the vortexed and centrifuged extract of 30 mg of powdered flesh with 1 mL of 1 mM HCl (1 + 5 min at room temperature), and it was expressed as percent substance on dw by refractometry (°Bx). Glycoalkaloids, solamargine and solasonine, were extracted from 0.5 g samples of lyophilized and powdered flesh tissue by 95% ethanol as described by Birner18 with some modifications. The analyses were performed by means of RP-HPLC according to Kuronen et al.,6 using partially purified solasonine and solamargine as the external standard. The data were expressed as mg/100 g dw; the limit of detection was 0.03 mg/100 g of dw. Phenolic acids were extracted and analyzed according to Whitaker and Stommel7 with minor modifications. A binary mobile phase gradient of methanol in 0.01% aqueous phosphoric acid was used according to this procedure: 0−15 min, linear increase from 5 to 25% methanol; 15−28 min, linear increase from 25 to 50% methanol; 28− 30 min, linear increase from 50 to 100% methanol; 30−32 min, 100% methanol; 32−36 min, linear decrease from 100 to 5% methanol; 36− 43 min, 5% methanol. The flow rate was 0.8 mL/min. Quantification of chlorogenic acid (CA), carried out after a RP-HPLC separation, was based on absorbance at 325 nm relative to the sesamol internal standard and an external standard of authentic CA (Sigma-Aldrich, St. Louis, MO). The results were expressed as mmol/100 g of dw. Total phenols index (TP) was assayed through a spectrophotometric method. TP was evaluated by a modified Folin-Ciocalteu
MATERIALS AND METHODS
Plant Material. Fruits from eggplant (Solanum melongena L., S. mel) and from three allied species S. sodomaeum (S. sod), S. aethiopicum gr. gilo (S. aeth) and S. integrifolium (S. int) were collected from 18 plants for each genotype, grown during 2006 season in an experimental field located in Montanaso Lombardo (Lodi, Italy) at the Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Unità di Ricerca per l’Orticoltura. Three D3R-containing eggplant lines (S. mel 1−3) exhibiting fruits with a long shape and a deep purple color and seven NAS-containing lines (S. mel 4−10) with spherical pale purple (violet) fruits color belonging to “Tunisina” typology were evaluated. The fruit peels of the allied parents did not exhibit detectable levels of these anthocyanin pigments. The samplings were carried out at three different fruit ripening stages, unripe [A, approximately 21 days after flowering (DAF)], commercial (B, approximately 38 DAF) and physiological ripening (C, approximately 60 DAF). The fruits at the stage A were actively growing, presumably because of cellular enlargement, close to half of the final size, the skin color was glossy, the calyx and peduncle were quite tender and flexible, the flesh appeared still soft and greenish in the D3R-types and white in the NAS-types, the seeds had not reached final size and they displayed a white tegument. The fruit at the stage B had reached their final size, the skin color became less brilliant and in some lines was a little dull; the fruit, calyx, and peduncle had the typical commercial firmness, the flesh became less greenish in the DR3-types, which showed the characteristic green ring next to the skin, and the seeds had almost reached their final size but were still immature. The fruits harvested at the stage C had an increased firmness, the calyx and peduncle were quite lignified, the peel color turned brownish, the flesh became spongy and thready with a whiteyellowish color containing visible hard mature seeds showing a brown tegument. Fruits of the ripening stages A and B were simultaneously harvested. The experimental sample was constituted by portions obtained from 5−8 fruits, sampled in duplicate. Flesh cubes from peeled fruits and peel slices of about 1.5−2 cm, made within 2 h after harvest, were immediately frozen in liquid N2 and lyophilized. The weight of the fresh flesh and peel samples and the correspondent weight at the end of drying process was detected for the conversion to fresh weight of the compounds analyzed (Table 1). The freeze-dried tissue was 11822
dx.doi.org/10.1021/jf3037424 | J. Agric. Food Chem. 2012, 60, 11821−11831
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Figure 1. Level of anthocyanins in single D3R-containing and NAS-containing eggplant lines during three ripening stages (A, B, and C). The values of allied species have not been shown because of their absence. The respective significance was obtained by orthogonal contrasts between values of D3R- vs NAS-typologies in each ripening stage. Stage A, F = 734.06**; stage B, F = 944.52**; stage C, F = 14.98**. (** significant at the 0.01 level of probability). For each ripening stage, different lower-case letters indicate significantly different means among different lines at p ≤ 0.05 (Tukey HSD test). For each line, different capital letters indicate significantly different means among different ripening stages at p ≤ 0.05 (Tukey HSD test).
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method19 on a McIlvaine buffer (pH 3.0, 1 mL) extract of 30 mg of lyophilized and powdered flesh tissue. Results were expressed as mmoles CA per 100 g of dw. CA was used because it is the main phenolic compound in the eggplant fruit. Biological Quality Traits. The PPO activity was assayed following the Fujita and Tono20 method, using 30 mg of lyophilized flesh, extracted with 1 mL of McIlvaine buffer (pH 5.0). Results were expressed as U/100 mg of dw, with 1U = 0.01 absorbance unit variation/min, using CA as substrate at 420 nm. The assays of antiradical activity were performed on superoxide anion and hydroxyl radical by electron spin resonance spectrometry at 25 °C, following the method used by Privat et al.21 and by Valavanidis et al.,22 with some modifications. The free radical generation (2.8 mM KO2-crown-ether-18−6 1:1 in dimethylsulfoxide for superoxide anion; 2 mM Fenton system in 0.1 M phosphate buffer pH 7.4 for hydroxyl radical) was followed by spin trapping with 5,5-dimethyl-1-pyrrolin-Noxide 25 mM and 10 mM dissolved in 0.1 M phosphate buffer pH 7.4 for superoxide anion and hydroxyl radical, respectively. The reaction was elapsed for exactly 1 min, after this time the spectra were recorded in presence and absence of eggplant extract (supernatant of 30 mg of flesh powder with 1 mL HCl 1 mM), respectively. In order to calculate the scavenging index, the main band amplitude measure was used applying the equation I = 100-(Ix/I0 × 100), where Ix is the spectrum amplitude in presence of eggplant extract and I0 is the spectrum amplitude in its absence. The results were expressed as mmol CA equivalents per 100 g of dw, by interpolating the data from flesh extracts with the scavenging index of chlorogenic acid solutions at known concentrations. Data Analysis. All the determinations were carried out at least three times. Data were subjected to ANOVA performed by JMP (SAS Institute, Cary, NC) according to a completely randomized design. Means were compared by using Tukey HSD test (p ≤ 0.05). For each parameter considered, orthogonal contrasts were used to determine significant differences between the two subgroups of S. melongena L. (i.e., D3R- vs NAS-type). For each genotype and parameter studied, the three stages of ripening were analyzed by ANOVA and means compared through Tukey HSD test (p ≤ 0.05). The correlation indexes (rxy) were calculated by simple linear regression analysis.
RESULTS AND DISCUSSION Genotypic Difference. The first approach for the eggplant quality analysis was the pigment evaluation in the fruit peel. The allied species do not have the typical anthocyanins from S. melongena, represented by D3R and/or nasunin,11 therefore eggplant lines were defined as D3R-types due to the presence of delphinidin-3-rutinoside or NAS-type, containing nasunin. However, as eggplants have a huge morphological diversity in shape and size of fruit further investigations, using other eggplant types belonging to both D3R- and NAS-type, are needed in order to verify if the differences observed are maintained. For each cultivar (except S. mel 10), the anthocyanin content was significantly higher in the stage A and B with respect to the stage C. The fruits of D3R typologies had significantly higher anthocyanin levels than NAS-containing fruits in stage A and B as confirmed by the orthogonal contrast, the averages being 2069 vs 587 and 2252 vs 563 μmol/100 g of peel dw, respectively. On the contrary, the NAS-type showed a mean value significantly higher than D3R one (233 vs 181 μmol/100 g of peel dw) in the stage C (Figure 1). The highest amount of anthocyanin was detected in S. mel 2 line (D3R-type), both in the stage A and B, 2198 and 3080 μmol/100 g of peel dw, respectively. Among the NAS-type, a noticeable level of the pigment in the three ripening stages for S. mel 9 (1425, 849, and 256 μmol/100 g of peel dw, respectively) was found. The lowest amounts of the pigment, for the three ripening stages, were detected in the lines belonging to NAS-type [stage A, S. mel 8 (194 μmol/100 g); stage B, S. mel 7 (387 μmol/100 g); stage C, S. mel 4 (37 μmol/ 100 g of peel dw)] (Figure 1). The allied species (except S. aethiopicum in the stages A and C) showed a significantly lower SRR amount than eggplant lines (Table 2). The two eggplant subgroups had a significant difference in the SRR content, on the average the NAS-types were significantly higher than D3R ones in all ripening stages 11823
dx.doi.org/10.1021/jf3037424 | J. Agric. Food Chem. 2012, 60, 11821−11831
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the levels sometimes reached and exceeded the reference level (lines S. mel 9, 6, 7, and 1). Interestingly, in some lines such as S. mel 4 and S. mel 10, the solasonine level resulted
