Professional Documents
Culture Documents
7597
DOI:10.1021/jf101004z
CRA-ORT, Agricultural Research Council, Research Center for Vegetable Crops, via Cavalleggeri 25,
84098 Pontecagnano-Faiano (Salerno), Italy, CRA-ORL, Agricultural Research Council, Research Unit
for Vegetable Crops, via Paullese 28, 26836 Montanaso Lombardo (Lodi), Italy, CRA-IAA, Agricultural
Research Council, Food Technology Research Unit, via Venezian 26, 20133 Milan, Italy, and #CRA-ORA,
Agricultural Research Council, Research Unit for Vegetable Crops, via Salaria 1, 63030 Monsampolo del
Tronto (Ascoli Piceno), Italy
The purpose of the present study was to investigate the levels of either the nutraceutical and healthpromoting compounds or the antioxidant properties of innovative eggplant (Solanum melongena L.)
genotypes tolerant and/or resistant to fungi, derived from conventional and non-conventional breeding
methodologies (i.e., sexual interspecific hybridization, interspecific protoplast electrofusion, androgenesis, and backcross cycles) in comparison with their allied and cultivated parents. Chemical measures
of soluble refractometric residue (SRR), glycoalkaloids (solamargine and solasonine), chlorogenic acid
(CA), delphinidin 3-rutinoside (D3R), total phenols (TP), polyphenoloxidase (PPO) activity, antiradical
activity on superoxide anion and hydroxyl radical were carried out in raw fruit and peel of 57 eggplant
advanced introgression lines (ILs), of three eggplant recurrent genotypes and of three allied species
during 2005 and 2006. The majority of the ILs, obtained after several backcross cycles, showed
positive characteristics with respect to the allied parents such as good levels of SRR, CA, D3R, TP,
PPO activity, the scavenging activity against superoxide anion and hydroxyl radical and, in particular,
significantly (p e 0.05) reduced concentrations of the toxic steroidal glycoalkaloids, solasonine and
solamargine. These results showed the possibility to obtain new eggplant genotypes bearing useful
traits derived from the allied parents (i.e., resistance/tolerance to plant pathogen fungi) together with
nutraceutical and antioxidant properties typical of the cultivated species.
KEYWORDS: Eggplant breeding; disease resistance; solanaceous species; glycoalkaloids; polyphenols;
antioxidants
INTRODUCTION
pubs.acs.org/JAFC
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Mennella et al.
obtained from 5-8 fruits. Flesh cubes and peel slices of about 1.5-2
cm, made within 2 h after harvest, were immediately frozen in liquid N2
and lyophilized. The freeze-dried tissue was powdered and held at -80 C.
All results were referred to as dry weight (dw).
Extractions and Analytical Conditions. The lyophilized and powdered eggplant tissue was treated in different ways for each performed
assay.
Soluble refractometric residue (SRR) was measured on the centrifuged
extract of 30 mg of eggplant powder with 1 mL of 1 mM HCl (5 min at
room temperature), and it was expressed as percent substance on dw.
Glycoalkaloids, solamargine and solasonine, were extracted from 0.5 g
samples of lyophilized fruit tissue by 95% ethanol as described by
Birner (44) with some modifications. The analyses were performed by
means of RP-HPLC according to Kuronen et al. (45), using partially purified solasonine and solamargine (kindly provided by Prof. Adelia Emilia
de Almeida), as the external standard. The data were expressed as mg/
100 g of dw (46, 47).
Phenolic acids were extracted and analyzed according to Whitaker and
Stommel (48) with minor modifications. The analyses were 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. A binary mobile phase gradient of methanol in 0.01%
acqueous phosphoric acid was used according to this procedure: 015 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 chlorogenic acid (Sigma-Aldrich, St. Louis, MO). The results were expressed as
mmol/100 g of dw.
The fruits of the recurrent eggplant parents analyzed in the present
study only had delphinidin-3-rutinoside (D3R) as peel anthocyanin,
whereas the fruit peels of the allied parents did not exhibit detectable
levels of D3R; hence, the ILs were exclusively analyzed for their D3R content. The extraction and the analysis of D3R were carried out on 200 mg of
lyophilized and powdered peel, diluted in 10 mL of methanol containing
3% trifluoroacetic acid (TFA), as reported in Ichiyanagi et al. (34). RPHPLC analysis was performed through the same Waters HPLC system as
for CA analysis at a flow rate of 0.8 mL/min. Purified D3R (Polyphenols
Laboratories AS, Sandnes, Norway) was used as the external standard in
RP-HPLC separations. The results were expressed as mg/100 g of peel dw;
the limit of detection was 1.3 mg/100 g of peel dw.
Total phenols (TP) and polyphenoloxidase (PPO) activity were assayed
through spectrophotometric methods. TP was evaluated by a modified
Folin-Ciocalteu method (49) on a McIlvaine buffer (pH 3.0, 1 mL) extract
of 30 mg of lyophilized tissue. Results were expressed as mmol of CA per
100 g of dw. Chlorogenic acid was used because it is the main phenolic
compound in the eggplant fruit.
The PPO activity was assayed following the Fujita and Tono (50)
method, using 30 mg of lyophilized fruit extracted with 1 mL of McIlvaine
buffer (pH 5.0). Results were expressed as U/100 mg of dw, with 1 U=0.01
absorbance unit variation/min, using chlorogenic acid as the substrate at
420 nm (30, 51).
The assays of antiradical activity were performed on superoxide anion
and hydroxyl radical by electron spin resonance (ESR) spectrometry,
following the method used by Privat et al. (52) and by Valavanidis
et al (53), 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-N-oxide 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 ESR spectra were recorded in the presence
and absence of eggplant extract (supernatant of 30 mg of eggplant powder
with 1 mL of HCl 1 mM), respectively. In order to calculate the scavenging
index, the main band amplitude measure was used applying this equation:
I=100 - (Ix/I0 100), where Ix is the spectrum amplitude in the presence
of eggplant extract and I0 is the spectrum amplitude in its absence.
Article
7599
Figure 1. Soluble refractometric residue (SRR, % dw) in fruits of the three Solanum spp., the pools of the recurrent eggplant genotypes and ILs analyzed.
Different letters indicate significantly different means at p e 0.05 (Tukey HSD test). Bars indicate the min-max values within each pooled genotype.
The results were expressed as mmol of CA per 100 g of dw, by interpolating
the data from eggplant extracts with the scavenging index of chlorogenic
acid solutions at known concentrations.
Statistical Data Treatment. For each year, all the determinations
were carried out at least three times. The statistical analyses here presented
were performed using the pooled average values of the ILs introgressed
from the same donor species, the average value of the three recurrent
eggplant genotypes, and the values of the allied donor species. Data
were subjected to ANOVA according to a completely randomized design.
Means were compared by using Tukey HSD test (p e 0.05). To show a
better picture of the data gathered, the maximum and minimum values
within the recurrent eggplant genotypes and the ILs have been also shown.
Moreover, ANOVA statistical analyses and Tukey HSD test were
performed separately for each year using the data of the single genotypes
employed (the results of this statistical analysis are presented as supporting
online information). The correlation indexes (rxy) were measured by
simple linear regression analysis.
RESULTS AND DISCUSSION
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Mennella et al.
Table 1. Solasonin, Solamargin, Chlorogenic Acid (CA), Delphinidin 3-Rutinoside (D3R), and Total Phenols (TP) Content in Fruits of the Three Solanum spp., the
Pools of the Recurrent Eggplant Genotypes, and ILs Analyzeda
genotype
solasonineb
(mg/100 g of dw)
solamargineb
(mg/100 g of dw)
CAb
(mmol/100 g of dw)
D3Rb
(mg/100 g of peel dw)
TPb
(mmol of CA eq/100 g of dw)
<1.3c i
<1.3c i
<1.3c i
200.5(81.6-406.7) e
36.6 (<1.3c-88.7) g
65.4 (<1.3c-295.2) f
20.6 (<1.3c-62.9) h
1.64 d
2.09 d
12.83 a
5.45 (4.40-7.42) bc
5.13 (3.75-6.93) bc
4.52 (3.10-6.24) c
6.21 (4.27-9.10) bc
<1.3c i
<1.3c i
<1.3c i
555.7(394.2-811.8) a
269.7(1.8-474) d
295.8(1.8-568.7) c
424.9(212.6-752.8) b
2.11 d
1.90 d
11.86 a
6.43 (5.56-7.90) bc
5.50 (4.47-5.93) bc
5.58 (3.06-7.16) bc
6.68 (5.51-6.94) b
2005
S. integrifolium
S. aethiopicum
S. sodomaeum
recurrent eggplant genotypes
ILs from S. integrifolium
ILs from S. aethiopicum
ILs from S. sodomaeum
68.1 de
178.5 c
610.5 b
10.9 (9.2-12.5) f
11.0 (1.9-33.7) f
6.2 (2.7-10.2) f
9.6 (1.7-59.1) f
80.9 cd
107.2 c
769.6 a
26.7 (22.9-29.9) e
15.3 (5.1-31.2) e
12.5 (2.6-23.1) e
29.2 (7.3-92.3) e
1.28 f
0.59 g
6.22 b
4.37 (3.16-6.76) c
2.29 (1.62-3.91) e
2.28 (0.83-2.94) e
3.60 (1.94-5.78) d
2006
S. integrifolium
S. aethiopicum
S. sodomaeum
recurrent eggplant genotypes
ILs from S. integrifolium
ILs from S. aethiopicum
ILs from S. sodomaeum
a
96.2 d
91.3 d
690.5 a
3.6 (0.0-10.7) f
15.3 (0.0-65.7) ef
8.1 (0.0-26.4) f
35.2 (0.0-85.8) ef
102.1 c
44.8 de
185.0 b
13.9 (0.0-23.4) e
30.3 (3.3-99.6) e
14.0 (0.0-54.7) e
35.1 (7.3-84.6) e
0.22 g
0.45 g
9.22 a
4.58 (3.87-5.92) c
2.20 (0.52-3.07) e
3.32 (1.19-5.07) d
4.84 (2.42-6.34) c
Different letters indicate significantly different means at p e 0.05 (Tukey HSD test). b Mean values (concentration range). c Limit of detection.
Article
7601
Figure 2. Polyphenoloxidase (PPO, U/100 mg of dw) activity in fruits of the three Solanum spp., the pools of the recurrent eggplant genotypes and ILs
analyzed. Different letters indicate significantly different means at p e 0.05 (Tukey HSD test). Bars indicate the min-max values within each pooled genotypes.
Figure 3. Superoxide anion scavenging (mmol of CA eq/100 g of dw) capacity in fruits of the three Solanum spp., the pools of the recurrent eggplant
genotypes and ILs analyzed. Different letters indicate significantly different means at p e 0.05 (Tukey HSD test). Bars indicate the min-max values within
each pooled genotype.
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Mennella et al.
Figure 4. Hydroxyl radical scavenging capacity (mmol of CA eq/100 g of dw) in fruits of the three Solanum spp., the pools of the recurrent eggplant genotypes
and ILs analyzed. Different letters indicate significantly different means at p e 0.05 (Tukey HSD test). Bars indicate the min-max values within each pooled genotypes.
quality traits (SRR, CA, D3R, PPO, TP, superoxide anion, and
hydroxyl radical scavenging) analyzed in the ILs displayed,
similarly to glycoalkaloids, a variability that can allow selection
of ILs enhanced for qualitative traits (e.g., TP content or reduced
PPO activity). However, no clear link can be now gathered
between the variation in the glycoalkaloids, phenols profile and
antioxidant activity and the enhanced response to fungal wilts
observed in the improved ILs. Further molecular and biochemical
dissection of the traits studied may confirm whether the emerged
variation is associated with the introgression into the eggplant
genome, of other exotic genes from the allied species in addition to
the resistance/tolerance ones.
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ACKNOWLEDGMENT
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7603
Received for review March 16, 2010. Revised manuscript received May
17, 2010. Accepted May 25, 2010. This research was partially supported
by the Italian Ministry of Agricultural, Alimentary, and Forest Politics,
with funds released by CIPE (resolution 17/2003), in the framework of
the PROM project.