Article pubs.acs.org/JAFC
Inoculation of the Nonlegume Capsicum annuum (L.) with Rhizobium Strains. 1. Effect on Bioactive Compounds, Antioxidant Activity, and Fruit Ripeness Luís R. Silva,*,† Jessica Azevedo,† Maria J. Pereira,† Lorena Carro,‡,§ Encarna Velazquez,‡,§ Alvaro Peix,§,∥ Patrícia Valentaõ ,† and Paula B. Andrade*,† †
REQUIMTE/Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, No. 228, 4050-313 Porto, Portugal ‡ Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de Unamuno, s/n, 37007 Salamanca, Spain § Unidad Asociada Grupo de Interacciones Planta-Microorganismo, Universidad de Salamanca-CSIC, Salamanca, Spain ∥ Instituto de Recursos Naturales y Agrobiología, IRNASA-CSIC, Cordel de Merinas, 40-52, 37008 Salamanca, Spain ABSTRACT: Pepper (Capsicum annuum L.) is an economically important agricultural crop and an excellent dietary source of natural colors and antioxidant compounds. The levels of these compounds can vary according to agricultural practices, like inoculation with plant growth-promoting rhizobacteria. In this work we evaluated for the first time the effect of the inoculation of two Rhizobium strains on C. annuum metabolites and bioactivity. The results revealed a decrease of organic acids and no effect on phenolics and capsaicinoids of leaves from inoculated plants. In the fruits from inoculated plants organic acids and phenolic compounds decreased, showing that fruits from inoculated plants present a higher ripeness stage than those from uninoculated ones. In general, the inoculation with Rhizobium did not improve the antioxidant activity of pepper fruits and leaves. Considering the positive effect on fruit ripening, the inoculation of C. annuum with Rhizobium is a beneficious agricultural practice for this nonlegume. KEYWORDS: Rhizobium spp., Capsicum annuum L., maturation, metabolites profile, bioactivity
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growth of phytopathogens.8 In the case of pepper, some bacteria have been reported as PGPR for different plants, as occurs with Pseudomonas and Pantoea.9 Nevertheless, not all bacterial species can be used in biofertilization schemes when the fruits are raw consumed, those used for decades as plant biofertilizers being preferred. Within them, Rhizobium, widely known for its ability to establish symbiotic relationships with legume plants,10 also has different plant growth-promoting mechanisms, being able to promote the development of nonlegumes.8,11,12 Some Rhizobium strains have a positive effect on pepper growth, since fruits from plants inoculated with them exhibited significantly higher fresh weight.12 Moreover, the number of mature fruits in inoculated plants was higher than that in noninoculated ones, which could suggest that these fruits reach the maturation stage earlier. This is an important factor for the composition of fruits, since several biochemical, physiological, and structural modifications occur during fruit ripening and these changes determine the attributes of the fruit.13 For example, a decline in phenolic compounds during maturation of Capsicum fruits has been reported.14 These losses during fruit maturation may reflect the metabolic conversion to secondary phenolic compounds and degradation via enzyme action and
INTRODUCTION Pepper (Capsicum annuum L.) is a member of the Solanaceae family, and its fruit is important as a vegetable food and spice.1 It is a good source of vitamins C and E, provitamin A, carotenoids, tocopherols, flavonoids, and phenolic acids, compounds with well-known antioxidant properties.1−3 Although flavonoids and phenolic acids are considered separately from capsaicinoids, which provide pungency or “heat” to hot peppers,4 it is known that the phenylpropanoid and capsaicinoid biosynthetic pathways may converge during pepper maturation.5 The intake of these compounds is an important health-protecting factor, since they have been considered as beneficial for the prevention of widespread human diseases, including cancer and cardiovascular diseases, when taken daily in adequate amounts.6 Pepper fruits are recognized for their economic importance and nutritional value, being the second most consumed vegetable worldwide. Nevertheless, information concerning byproducts resulting from its production, namely, leaves, is scarce. This material may also be an interesting source of bioactive phytochemicals. The increase of quality demanded by consumers requires specific agricultural practices, such as fertilization with products containing microorganisms.7 The inoculation with plant growth-promoting rhizobacteria (PGPR) reducing the chemicals added to crops can improve not only the growth but also the quality of plants, since these microorganisms can mobilize nutrients to the plants, influence the plant hormonal balance, or produce microbial inhibitory compounds that preclude the © 2014 American Chemical Society
Received: Revised: Accepted: Published: 557
October 16, 2013 December 31, 2013 January 1, 2014 January 9, 2014 dx.doi.org/10.1021/jf4046649 | J. Agric. Food Chem. 2014, 62, 557−564
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may indicate diversion of phenolic precursors from flavonoids to capsaicinoids.15 Organic acids also contribute to fruit maturation, since the concentration of individual acids changed in pepper contributing to pepper fruit flavor.16 Although some work has been carried out about the changes in the chemical composition of pepper fruits after PGPR inoculation,17 as far as we know there are no studies about the effect of rhizobial inoculation in the chemical composition of leaves and fruits of C. annuum, nor in their biological properties. Therefore the objectives of the present work were to compare the accumulation of bioactive compounds indicative of maturation, namely, phenolics and organic acids, in pepper leaves and fruits obtained after the inoculation with two PGPR Rhizobium strains with that of noninoculated material and its impact on their bioactivity.
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Hoagland’s solution every 8 days. The plants were maintained during 10 weeks in a greenhouse illuminated with natural light in summer (night temperature ranging from 15 to 20 °C and day temperature ranging from 25 to 35 °C) with humidity control. At the end of the experiment, flowers and fruits were counted, fruits were harvested, and their fresh weight was measured. Data related to the precocity of the pepper plants not published before are included in the present work. Five plants per treatment were randomly selected and used for the chemical and biological analysis. They were frozen, lyophilized, and powdered (mean particle size lower than 910 μm), being divided into three aliquots, extracted, and analyzed separately for chemical composition and biological activity. Phenolic Compounds. Extraction. C. annuum leaves and fruits (3 g) were extracted with 600 mL of boiling water during 15 min. The resulting extracts were filtered through a Büchner funnel, frozen, and lyophilized. The yield of extractions was 45.0 and 33.9% for fruits and leaves, respectively. The extracts were kept in a desiccator, in the dark, until analysis. Aqueous lyophilized extracts were redissolved in methanol and filtered through a PTFE membrane (0.45 μm). Analysis by HPLC-DAD. The phenolic compounds were analyzed using a previously described procedure.18 Briefly, each extract was analyzed on an analytical HPLC unit (Gilson), using a Spherisorb ODS2 (25.0 × 0.46 cm; 5 μm, particle size; Waters, Milford, MA) column. The solvent system used was a gradient of water:formic acid (19:1) (A) and methanol (B), starting with 5% methanol and installing a gradient to obtain 15% B at 3 min, 25% B at 13 min, 30% B at 25 min, 35% B at 35 min, 45% B at 39 min, 45% B at 42 min, 50% B at 44 min, 55% B at 47 min, 70% B at 50 min, 75% B at 56 min, and 80% B at 60 min, at a solvent flow rate of 0.9 mL/min. Detection was achieved with a Gilson diode array detector (DAD). Chromatograms were recorded at 280, 320, and 350 nm. The data were processed on Unipoint System software (Gilson Medical Electronics, Villiers-le-Bel, France). The compounds in each extract were identified by comparing their retention times and UV−vis spectra with those of authentic standards. Because standard was not commercially available, luteolin-7O-(2-apiosyl-6-malonyl)-glucoside was quantified as luteolin-7-Oglucoside. Phenolic acids were determined at 320 nm and flavonoids at 350 nm. This procedure was performed in triplicate. Capsaicinoids. Extraction. The method described by Estrada et al.19 was used, with some modifications: 1.0 g of fruits and leaves was extracted with 10 mL of acetonitrile at 80 °C, for 4 h. The extract obtained was centrifuged at 4000 rpm for 5 min, and the supernatant was filtered. A 20 μL aliquot was used for each HPLC-DAD injection. Analysis by HPLC-DAD. The samples were analyzed using an ACE3 C-18-AR column (150 × 4.6 mm, 3 μm particle size, Advanced Chromatography Technologies, Aberdeen, Scotland). The elution solvent was acetonitrile:water (60:40) at isocratic mode, flow rate of 1.0 mL/min, and a 35 min run. Detection was achieved with a Gilson DAD, and chromatograms were registered at 280 nm. Spectral data from peaks were accumulated in the 200−400 nm range. The data were processed on Unipoint System software (Gilson Medical Electronics, Villiers-le-Bel, France). The compounds in each extract were identified by comparing their retention times and UV−vis spectra with those of external authentic standards (capsaicin and dihydrocapsaicin). Capsaicinoids quantification was achieved by measuring the absorbance at 280 nm in the chromatograms relative to external standards. This procedure was performed in triplicate. Organic Acids. Extraction. Extraction of organic acids was performed according to a described procedure:18 0.1 and 1.0 g of lyophilized fruits and leaves, respectively, were mixed with 50 mL of H2SO4 0.01 N for 30 min under stirring (300 rpm). The obtained extracts were then filtered, evaporated to dryness under reduced pressure (40 °C), and redissolved in H2SO4 0.01 N. HPLC−UV Analysis. The separation and quantification of organic acids were carried out according to the procedure described by Silva et al.18 in an analytical HPLC unit (Gilson), using an ion exclusion Nucleogel Ion 300 OA (300 × 7.7 mm; Macherey-Nagel, Düren, Germany) column. Elution was performed in isocratic mode with
MATERIALS AND METHODS
Standards and Reagents. All chemicals used were of analytical grade. The standard compounds were purchased from various suppliers. 5-O-Caffeoylquinic acid, quercetin-3-O-rutinoside, quercetin-3-O-rhamnoside, luteolin-7-O-glucoside, myricetin-3-O-rhamnoside, and apigenin-7-O-glucoside were from Extrasynthese (Genay, France). Caffeic, ferulic, sinapic, oxalic, malic, quinic, succinic, fumaric, acetic, citric, and aconitic acids, capsaicin, and dihydrocapsaicin were obtained from Sigma-Aldrich (St. Louis, MO, USA). 1,1-Diphenyl-2picrylhydrazyl (DPPH •), β-nicotinamide adenine dinucleotide (NADH), phenazine methosulfate (PMS), nitrotetrazolium blue chloride (NBT), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), sulfanilamide, acetylcholinesterase (AChE) from electric eel (type VI-s, lyophilized powder), acetylthiocholine iodide (ATCI), sodium nitroprusside dehydrate (SNP), and methanol were purchased from SigmaAldrich (St. Louis, MO, USA). N-(1-Naphthyl)ethylenediamine dihydrochloride and sulfuric acid were obtained from Merck (Darmstadt, Germany). Water was deionized using a Milli-Q water purification system (Millipore, Bedford, MA). Plant Samples and Bacterial Strains. Samples of leaves and fruits used in this study were obtained from pepper var. “sweet Italian” plants inoculated with two fast-growing rhizobial strains, PETP01 and TVP08, and from uninoculated plants. These strains were isolated from Trifolium pratense and Phaseolus vulgaris nodules, respectively, presenting effective nodulation in their respective hosts.12 PETP01 and TVP08 strains were identified in a previous work as Rhizobium leguminosarum, and they belong to different subphyla within this species following the analysis of the core recA and atpD genes and of two different symbiovars (trifolii and phaseoli, respectively), according to the analysis of the symbiotic nodC gene.12 Inoculation assays were performed as was previously described:12 for plant inoculation, nonsterilized seeds were germinated in peat following the steps commonly used in the commercial process of seedling production and were irrigated from a bottom reservoir with water every 48 h and with commercial Hoagland’s solution (Sigma Co., USA) every 8 days. Once placed on peat, each seed was inoculated with 1 mL of a suspension (2 × 108 cells/mL) of 5-day-old Rhizobium strains TPV08 and PETP01 grown on yeast-mannitol agar (YMA). Then, they were covered with vermiculite and germinated in darkness in a growth chamber at 24 °C, for seven days. Afterward, they were maintained in a plant growth chamber with mixed incandescent and fluorescent lighting (400 microeinsteins m−2 s−1; 400 to 700 nm), programmed for a 16 h photoperiod, day−night cycle, with a constant temperature varying from 25 to 27 °C and 50−60% relative humidity. Uninoculated controls were kept under the same conditions. After four weeks, 25 plants per treatment were used for a microcosm experiment, which was performed in a clayey soil collected at Salamanca (Spain), with neutral pH, 1.5−1.8% of organic matter, and 0.09−0.1 of N content. The seedling roots were flooded in bacterial suspensions containing 2 × 108 cells/mL during 4 h. Each plant was transplanted to a pot containing 2 kg of soil. Plants were irrigated from a bottom reservoir with water every 48 h and with commercial 558
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Table 1. Phenolic Composition of C. annuum Fruits and Leaves Inoculated with Rhizobium (mg/kg Lyophilized Material)a fruits
leaves
phenolic compounds
control
PETP01
TVP08
control
PETP01
TVP08
5-O-caffeoylquinic acid caffeic acid sinapic acid ferulic acid luteolin-7-O-glucoside quercetin-3-O-rutinoside apigenin-7-O-glucoside myricetin-3-O-rhamnoside quercetin-3-O-rhamnoside luteolin-7-O-(2-apiosyl-6-malonyl)-glucoside total
6.0 ± 0.1 a 53.7 ± 1.4 a 132.2 ± 0.0 a nd 13.5 ± 1.1 a 55.4 ± 0.8 a nd 672.2 ± 6.7 a 721.1 ± 7.8 a 468.4 ± 11.8 a 2122.5
0.5 ± 0.0 b 44.2 ± 2.8 b 16.9 ± 0.9 b nd 1.3 ± 0.0 b 3.6 ± 0.3 b nd 228.2 ± 6.5 b 591.5 ± 11.5 b 56.6 ± 0.4 b 942.8
0.8 ± 0.0 b 14.0 ± 0.1 b 10.6 ± 0.7 b nd 2.5 ± 0.2 b nd b nd 25.6 ± 2.3 b 41.4 ± 2.0 b 10.8 ± 0.2 b 105.7
68.0 ± 0.2 a 49.2 ± 1.3 a nd 37.2 ± 2.0 a 897.3 ± 5.4 a 6.2 ± 0.8 a 1027.5 ± 18.4 a nd nd nd 2085.4
42.4 ± 0.1 b 80.5 ± 7.4 b nd 73.6 ± 5.9 b 810.3 ± 25.2 b 6.3 ± 0.5 a 1090.4 ± 58.7 a nd nd nd 2103.5
46.1 ± 0.1 b 64.9 ± 1.2 b nd 31.4 ± 2.9 a 779.1 ± 6.4 b 6.4 ± 0.6 a 1086.2 ± 2.0 a nd nd nd 2014.1
Values are expressed as mean ± standard deviation of three assays; nd, not detected. In the same line, different letters represent significant differences compared with the respective control (p < 0.05). a
H2SO4 (0.01 N), at a flow rate of 0.2 mL/min. Detection was achieved with a UV detector set at 214 nm. Quantification of organic acids was achieved by measuring the absorbance recorded in the chromatograms relative to external standards. This procedure was performed in triplicate. Antioxidant Activity. The aqueous lyophilized extracts prepared for phenolic compounds determination were used in the screening of the antioxidant activity. All determinations were made in a Multiskan Ascent plate reader (Thermo Electron Corporation). DPPH• Assay. For each extract, a dilution series (five different concentrations) was prepared in a 96-well plate. The reaction mixtures in the sample wells consisted of 25 μL of extract (redissolved in methanol) and 200 μL of 150 mM methanolic DPPH•. The plate was incubated for 30 min at room temperature after addition of DPPH• solution, and the absorbance was determined at 515 nm.20 Three experiments were performed in triplicate. Nitric Oxide Assay. Antiradical activity was determined spectrophotometrically in a 96-wells plate reader. The reaction mixtures in each well consisted on 100 μL of extract dissolved in buffer (KH2PO4 100 mM, pH 7.4) and 100 μL of SNP (20 mM). The plates were incubated at room temperature for 60 min, under light. Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine in 2% H3PO4) (100 μL) was then added, and 10 min later the absorbance of the chromophore formed during the diazotization of nitrite with sulfanilamide and subsequent coupling with naphthylethylenediamine was determined at 562 nm.20 Three experiments were performed in triplicate. Superoxide Radical Assay. Superoxide was generated by the NADH/PMS system, according to a described procedure: PMS reduced by NADH reacts with oxygen to produce O2•−; this radical then reduces NBT to a formazan blue dye, which has an absorption maximum at 560 nm. 20 The reaction mixtures in the wells consisted on 50 μL of extract dissolved in buffer (KH2PO4 19 mM, pH 7.4), 50 μL of NADH (166 μM), 150 μL of NBT (43 μM), and 50 μL of PMS (2.7 μM). The rate of the reaction was assessed at 560 nm, during 2 min after PMS addition.20 Three experiments were performed in triplicate. AChE Inhibitory Activity. The aqueous lyophilized extracts were also used to assess AChE inhibition properties. Spectrophotometric determinations at 405 nm were performed in a Multiskan Ascent plate reader (Thermo Electron Corporation), based on Ellman’s method, as previously described.20 In each well the mixture consisted of 25 μL of ATCI (15 mM), 125 μL of DTNB (3 mM), 50 μL of buffer (50 mM Tris-HCl, pH 8, containing 0.1% BSA), and 25 μL of sample dissolved in buffer with 10% of methanol. The absorbance was read at 405 nm during 2 min. After this step, 25 μL of AChE (0.44 U/mL) was added and the absorbance was read again. The rates of reactions were calculated by Ascent Software version 2.6 (Thermo Labsystems Oy). The rate of the reaction before adding the enzyme was subtracted from that obtained after adding the enzyme in order to correct eventual
spontaneous hydrolysis of substrate. 20 Three experiments were performed in triplicate. Statistical Analysis. All data were recorded as mean ± standard deviation of triplicate determinations. Mean values were compared using one-way analysis of variance (one-way ANOVA) (Graph Pad Prism Version 5.00, GraphPad Software, Inc., San Diego, CA). Differences were considered significant for p < 0.05.
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RESULTS AND DISCUSSION Effect of Rhizobium Strains in the Growth of Pepper Plants. The inoculated plants were more precocious than noninoculated ones, displaying more rapid flowering and fruiting when they were inoculated with the two Rhizobium strains. In this way, the percentage of plants with flowers three weeks after transplanting was 80 and 100%, for the plants inoculated with strains TVP08 and PETP01, respectively, whereas only 60% of the noninoculated plants had flowers at this time. Also, seven weeks after transplanting, the percentage of plants with fruits was 50 and 60% for the plants inoculated with strains TVP08 and PETP01, respectively, whereas only 35% of the noninoculated plants had fruits at this time. These results showed that the inoculation with Rhizobium increased the precocity of pepper plants since the fruit production began earlier in inoculated plants. Therefore, pepper plants inoculated with Rhizobium strains TVP08 and PETP01 not only yielded more fruits, which have higher weight, than noninoculated plants, as was described before,12 but also they are produced earlier. These results suggest that the inoculation has a direct effect on pepper fruit ripening, this being the first report about the ability of rhizobia to advance the fruit production of a nonlegume. Phenolic Compounds. Phenolic compounds are plant secondary metabolites, which are synthesized by plants as a result of their adaptation to biotic and abiotic stress conditions.1 They include flavonoids with antioxidant activity that may be beneficial for human health.21,22 The analysis of the aqueous extracts by HPLC-DAD allowed the identification of several phenolic compounds (Table 1). To evaluate the recovery of phenolic compounds, aliquots of known concentration of luteolin-7-O-glucoside standard solution were added to the samples and a recovery of 98% (±0.3) and 92% (±1.3) was obtained for leaves and fruits, respectively. In pepper leaves hydroxycinnamic acids (5-O-caffeoylquinic, caffeic, and ferulic acids) and flavonoids (luteolin-7-O-gluco559
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49, 52, and 54% of the total content in control sample and in the samples inoculated with strains PETP01 and TVP08, respectively. However, inoculation led to a significant increase of caffeic acid. Ferulic acid also increased significantly, but just in plants inoculated with the strain PETP01. On the contrary, the amounts of 5-O-caffeoylquinic acid and luteolin-7-Oglucoside were significantly higher in control leaves than in inoculated ones. Regarding the fruits, the phenolic content decreased with the inoculation of both Rhizobium strains, especially with strain TVP08 (Table 1). Quercetin-3-O-rhamnoside was the main phenolic compound in all fruits. Although the inoculation with Rhizobium strains led to a significant decrease of the amounts of this compound, its relative content was higher in fruits from plants inoculated with strains PETP01 (about 60%) and TVP08 (almost 40%) than in control leaves, in which it represented 34% of total phenolics. Several authors reported a decrease of phenolic compounds during ripening of several pepper varieties15,25,26 due to their enzymatic degradation in secondary metabolism15 and by the involvement in the structure of cell walls.1 The decrease of phenolics in the fruits of plants inoculated with Rhizobium strains supports that the ripeness of these fruits is higher when the plants were inoculated with Rhizobium strains, mainly when the strain TVP08 was used. This result contrasts with that obtained by Amor et al.,17 who did not find a decrease of phenolic compounds in pepper inoculated with Azospirillum and Pantoea, two bacteria considered as PGPR. Therefore, to our knowledge, this is the first report of a positive influence on fruit ripeness of the inoculation with Rhizobium PGPR strains of a nonlegume. Capsaicinoids. Capsaicinoids are a group of alkaloids responsible for the pungent sensation caused by pepper fruits, capsaicin and dihydrocapsaicin being responsible for about 90% of the spiciness.13 HPLC-DAD analysis allowed the identification of two capsaicinoids (Table 2). To evaluate the recovery of capsaicinoids, aliquots of known concentration of capsaicin standard solution were added to the samples, a recovery of 80% (±7.3) and 77% (±3.0) being obtained for fruits and leaves, respectively. In pepper leaves only capsaicin was detected (Table 2), ranging between 75.3 and 78.3 mg/kg. This contrasts with the results of Estrada et al.,19 who also detected dyhidrocapsaicin in the leaves of other varieties of pepper in which the content of capsaicin was lower, ranging from 1 to 20 μg/g. No significant differences were found in the amounts of capsaicin of leaves from uninoculated and inoculated plants. Besides capsaicin, dyhidrocapsaicin was also detected in the fruits. The total content of these capsaicinoids ranged between 275.2 and 309.2 mg/kg, capsaicin representing ca. 88.0% and dyhidrocapsaicin ca. 12.0%. As expected, these values are lower
side, quercetin-3-O-rutinoside, and apigenin-7-O-glucoside) were identified (Table 1 and Figure 1A). These flavonoids
Figure 1. HPLC phenolic profile of Capsicum annuum (A) leaf control and (B) fruit control. Detection at 320 nm. Peaks: (1) 5-Ocaffeoylquinic acid; (2) caffeic acid; (3) sinapic acid; (4) ferulic acid; (5) luteolin-7-O-glucoside; (6) quercetin-3-O-rutinoside; (7) apigenin7-O-glucoside; (8) myricetin-3-O-rhamnoside; (9) quercetin-3-Orhamnoside; (10) luteolin-7-O-(2-apiosyl-6-malonyl)-glucoside.
have already been described in pepper leaves by Reigosa et al.23 Several studies have already reported the fundamental role of luteolin and apigenin derivatives in the protection of cells (membranes, chlorophyll, and fragile organelles) from the damage caused by UV radiation.1 Pepper fruits showed a distinct phenolic profile, eight compounds being determined: 5-O-caffeoylquinic, caffeic, and sinapic acids, luteolin-7-O-glucoside, quercetin-3-O-rutinoside, myricetin-3-O-rhamnoside, quercetin-3-O-rhamnoside, and luteolin-7-O-(2-apiosyl-6-malonyl)-glucoside (Table 1 and Figure 1B). From these, only 5-O-caffeoylquinic acid and myricetin-3O-rhamnoside were not previously described in pepper fruits.1,2,15,24 Quantitatively, the sum of the identified phenolic compounds in the three leaves samples was similar (Table 1). The major compound was apigenin-7-O-glucoside, corresponding to ca.
Table 2. Capsaicinoids of C. annuum Fruits and Leaves Inoculated with Rhizobium (mg/kg Lyophilized Material)a fruits
leaves
capsaicinoids
RT (min)
control
PETP01
TVP08
control
PETP01
TVP08
capsaicin dihydrocapsaicin total
5.9 9.7
234.0 ± 17.0 a 41.2 ± 3.3 a 275.2
277.3 ± 4.8 a 31.9 ± 3.5 a 309.2
269.4 ± 9.0 a 33.2 ± 1.0 a 302.6
78.3 ± 2.9 a nd 78.3
75.7 ± 2.7 a nd 75.7
75.3 ± 3.3 a nd 75.3
Values are expressed as mean ± standard deviation of three assays; RT, retention time; nd, not detected. In the same line, different letters represent significant differences compared with the respective control (p < 0.05).
a
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Table 3. Organic Acids of C. annuum Fruits and Leaves Inoculated with Rhizobium (mg/kg Lyophilized Material)a fruits
leaves
organic acids
RT (min)
control
PETP01
TVP08
control
PETP01
TVP08
oxalic malic quinic succinic fumaric citric acetic aconitic total
20.2 36.4 37.6 47.4 61.6 30.1 51.5 24.3/43.8
53711.3 ± 512.8 a 21747.4 ± 1187.7 a 14349.1 ± 106.4 a 35197.2 ± 380.0 a 13341.7 ± 199.9 a 76786.0 ± 1701.3 a 55223.7 ± 8.9 a 1359.7 ± 78.5 a 271716.1
3169.8 ± 16.6 b 16780.1 ± 455.2 b 9182.8 ± 718.4 b 24509.1 ± 1954.7 b 6021.0 ± 112.8 b 95510.3 ± 378.2 b 58586.6 ± 1725.8 b 185.6 ± 6.4 b 213945.3
49257.6 ± 519.8 b 14785.6 ± 10.6 b 22680.2 ± 2638.0 b 21805.6 ± 397.0 b 6253.8 ± 170.7 b 99660.2 ± 46.1 b 34560.8 ± 1391.9 b 80.1 ± 3.3 b 249083.9
55292.7 ± 17.3 a 6438.7 ± 68.7 a 4664.0 ± 29.8 a 4892.9 ± 23.1 a 5826.2 ± 261.1 a 30048.2 ± 873.1 a 106.8 ± 0.5 a 16594.6 ± 47.2 a 123864.1
60640.7 ± 542.3 b 3194.8 ± 388.1 b 4923.1 ± 42.9 b 4746.3 ± 16.9 b 4656.0 ± 227.5 b 31754.8 ± 205.9 b 139.6 ± 0.8 b 16348.4 ± 83.4 b 126403.7
21542.2 ± 357.2 b 14785.6 ± 10.9 b 4015.6 ± 18.5 b 5307.4 ± 315.0 b 6951.9 ± 321.2 b 7576.4 ± 846.8 b 122.0 ± 4.8 b 16369.0 ± 1401.4 a 76670.1
a Values are expressed as mean ± standard deviation of three assays. RT, retention time. In the same line, different letters represent significant differences compared with the respective control (p < 0.05).
Figure 2. Effect of aqueous extracts of C. annuum leaves and fruits against (A, D) DPPH•, (B, E) superoxide radical (O2•−), and (C, F) nitric oxide (•NO), respectively. Values show mean ± SE of three experiments performed in triplicate.
The analysis of C. annuum fruits and leaves by HPLC−UV revealed a similar profile, composed of oxalic, malic, quinic, succinic, fumaric, citric, acetic, and aconitic acids (Table 3). To evaluate the recovery of organic acids, aliquots of a standard solution of fumaric acid with known concentration were added to the samples, 89 ± 0.9 and 81 ± 1.2% being recovered from fruits and leaves, respectively. The total amount of organic acids in the leaves from control plants was lower than those from plants inoculated with PETP01 strain, but higher than in those from plants inoculated with TVP08 strain (Table 3). In pepper leaves from all treatments oxalic acid was the main compound representing ca. 45, 48, and 28% from the total of the determined organic acids, in leaves of control, plants inoculated with strain PETP01, and those inoculated with strain TVP08, respectively (Table 3). The amounts of this acid together with quinic, citric, and acetic acids were significantly increased after the inoculation with strain PETP01, whereas it significantly decreased malic, succinic, fumaric, and aconitic acids. In the case of leaves from plants inoculated with strain TVP08 significant increases of malic, succinic, fumaric, and
than those found on mature fruits from the spicy variety Jalapeño, in which levels of capsaicin and dyhidrocapsaicin ranging from 499−899 mg/kg to 540−989 mg/kg of dried material, respectively, were reported.27 Menichini et al.13 observed that capsaicinoid amounts increase with ripening in Capsicum chinense Jacq. var. habanero, which is one of the most spicy varieties of pepper. In this study we also found a slight increase of capsaicin in peppers inoculated with Rhizobium. Nevertheless, the differences were not significant with respect to noninoculated ones, and in the case of dyhidrocapsaicin a decrease was even found. This was expected taking into account that a sweet variety (“sweet Italian”) was analyzed in this study. This is the first study about the effect on content of capsaicinoids in leaves and fruits of C. annuum after the inoculation with Rhizobium spp. showing that it has not a significant effect in sweet peppers. Organic Acids. Organic acids are primary metabolites, which can be found in great amounts in all plants, especially in fruits. Citric, malic, and tartaric acids are commonly found in fruits and berries, while oxalic acid is present in higher amounts in green leaves.28 561
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Table 4. IC Values (mg/mL) Found with Extracts of C. annuum Fruits (IC25) and Leaves (IC50) in the Assays against DPPH•, Superoxide (O2•−), and Nitric Oxide (•NO) Radicals and Acetylcholinesterase (AChE) Inhibitiona fruits
leaves
assay
control
PETP01
TVP08
control
DPPH• O2•− • NO AChE
1.047 (±0.040) a 0.020 (±0.002) a 0.010 (±0.002) a
0.991 (±0.051) a 0.033 (±0.003) a 0.039 (±0.005) b
1.345 (±0.076) b 0.075 (±0.005) a 0.013 (±0.001) a
0.234 0.080 0.377 2.570
PETP01
(±0.199) (±0.023) (±0.003) (±0.218)
a a a a
0.193 0.085 0.466 2.685
(±0.042) (±0.082) (±0.003) (±0.226)
TVP08 a a a a
0.294 0.093 0.806 3.492
(±0.013) (±0.057) (±0.001) (±0.044)
a b b b
a Values are expressed as mean + standard deviation of three assays. In the same line, different letters represent significant differences compared with the respective control (p < 0.05).
(chlorogenic acid), two compounds with antioxidant potential,32,33 were found in significant higher amounts in control leaves (Table 1). Although the contribution of other bioactive compounds not determined in the extracts cannot be discarded, the presence of higher amounts of luteolin-7-O-glucoside and 5-O-caffeoylquinic acid in control leaves may be linked to the higher antioxidant activities found in them. Comparing the results of the three assays performed, we can observe that the leaves’ extracts were more active against superoxide radical. The scavenging of this reactive oxygen species is of great importance due to its potential to originate other reactive species that could be extremely deleterious to the cells.34 The extracts obtained from fruits followed the same pattern as those from the leaves. The extract of fruits from plants inoculated with PETP01 strain was the most active against DPPH• radical and that of fruits from plants inoculated with TVP08 strain the less effective, the differences being significant when compared to control (Figure 2D, Table 4). The extract from control fruits also showed a tendency to be the most active against •NO and O2•− (Table 4). Conforti et al.25 observed that more immature pepper fruits showed better antioxidant activity, due to the presence of higher amounts of phenolic compounds. Since the inoculation of pepper with Rhizobium anticipated the fruit ripening, a decrease in the antioxidant activity of fruits inoculated with Rhizobium was expected. Nevertheless, the inoculation with the PETP01 strain significantly increased the activity against DPPH•, decreasing only with the inoculation of the strain TVP08, which was the strain that more significantly advanced the ripening of pepper fruits. The inoculation does not significantly affect the activity against •NO and O2•− radicals. Acetylcholinesterase (AChE) Inhibitory Activity. The search for natural matrices with capacity to inhibit AChE has increased due to their benefits in the treatment of Alzheimer’s disease and other forms of dementia.35 In this study the inhibitory activity of aqueous extracts of C. annuum leaves was evaluated against AChE and a concentration-dependent activity was obtained (data not shown). Extract from control leaves was the most active (Table 4), and this result can be explained by the presence of various bioactive compounds, namely, flavonoids.36 The capacity of fruits’ aqueous extracts to inhibit AChE was also assessed, but no effect was found for the tested concentrations. Despite the different composition, the same kind of phenolic compounds was found in both fruit and leaf extracts. As such, the absence of effect on AChE observed with the extracts prepared from fruits seems to suggest the interference of other nondetermined bioactive compounds. In summary, the most relevant result obtained in this study is a positive effect on fruit ripening of pepper plants inoculated with two Rhizobium strains. The fruits from inoculated plants
acetic acids were observed, while the content of oxalic, quinic, and citric acids was significantly reduced. Therefore, both strains had the same effect just in what concerns acetic acid content, which was significantly increased (Table 3). The increased levels of malic acid in leaves from plants inoculated with TVP08 strain are in agreement with the results found for rice leaves inoculated with the PGPR bacterium Herbaspirillum seropedicae by Curzi et al.29 This is the first report about the effect of the inoculation of Rhizobium in the organic acids composition of C. annuum leaves. The organic acids qualitative profile of C. annuum fruits was similar to that of the leaves (Table 3), citric acid being the major organic acid in all samples, representing ca. 28% in control fruits and 45 and 40% in fruits from plants inoculated with strains PETP01 and TVP08, respectively. Succinic, acetic, and aconitic acids are described for the first time in this matrix, while all the others were already reported.30 The inoculation with both Rhizobium strains led to the decrease of most of the organic acids in fruits, except citric acid; additionally, acetic and quinic acids increased when PETP01 and TVP08 strains were inoculated, respectively. These results are in agreement with those obtained with soybean, a legume, after the inoculation of Bradyrhizobium31 and in pepper fruits after inoculation with other PGPR bacteria, such as Azospirillum sp. and Pantoea sp.17 The concentrations of organic acids in sweet peppers change notably during ripening, a decrease of their total amounts being reported.6,15,16 These authors reported that the concentration of malic acid decreases while that of citric acid increases considerably during the ripeness process. The same results were found in our study when the plants were inoculated with the two Rhizobium, strengthen the results obtained for phenolic compounds and showing a more advanced degree of ripeness in peppers from plants inoculated with Rhizobium strains. Antioxidant Activity. Peppers are a good source of antioxidant compounds that can reduce harmful oxidation reactions in the human body, thus preventing various diseases associated with free radical oxidation, such as cardiovascular and neurological disorders and cancer.2 The antioxidant activity of the aqueous lyophilized extracts from C. annuum fruits and leaves was tested against DPPH•, superoxide (O2•−), and nitric oxide (•NO) radicals. A concentration-dependent potential was observed in all assays (Figure 2). As far we know, this is the first study about the antioxidant capacity of C. annuum leaves. The extract of leaves from plants inoculated with PETP01 strain revealed a tendency to be the most active against DPPH• radical (Table 4). In the assays against •NO and O2•− radicals the best results were obtained with the extract of leaves from control plants (Table 4). This result is in agreement with the phenolic composition of leaves, in which luteolin-7-O-glucoside and 5-O-caffeoylquinic acid 562
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had lower total levels of organic acids and phenolic compounds, pointing to a higher ripening stage than those from uninoculated ones. Data suggest that the application of nodulating rhizobacteria is a beneficial agricultural practice for this nonlegume, ensureing that they are optimal bacteria for biofertilization, with positive effects on production and fruit ripening, contributing to an increase in the consumption of this fruit, either as fruit or dietary supplement.
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AUTHOR INFORMATION
Corresponding Author
*Tel: + 351 220 428 654. Fax: +351 226 093 390. E-mail: lmsilva@ff.up.pt (L.R.S.) or pandrade@ff.up.pt (P.B.A.). Funding
The authors are grateful to Fundaçaõ para a Ciência e a Tecnologia (FCT) for Grant No. PEst-C/EQB/LA0006/2011. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED PGPR, plant growth-promoting rhizobacteria; DPPH•, 1,1diphenyl-2-picrylhydrazyl radical; O2•−, superoxide radical; • NO, nitric oxide; NADH, β-nicotinamide adenine dinucleotide; PMS, phenazine methosulfate; NBT, nitrotetrazolium blue chloride; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); AChE, acetylcholinesterase; ATCI, acetylthiocholine iodide; SNP, sodium nitroprusside dehydrate; HPLC, high-performance liquid chromatography; DAD, diode array detector
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REFERENCES
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