Article pubs.acs.org/JAFC
Metabolite Profile, Antioxidant Capacity, and Inhibition of Digestive Enzymes in Infusions of Peppermint (Mentha piperita) Grown under Drought Stress Marely G. Figueroa-Pérez,† Nuria Elizabeth Rocha-Guzmán,‡ Iza F. Pérez-Ramírez,† Edmundo Mercado-Silva,† and Rosalía Reynoso-Camacho*,† †
Research and Graduate Studies in the Department of Food Science, School of Chemistry, Universidad Autónoma de Queretaro, Centro Universitario, Cerro de las Campanas S/N, Queretaro, Queretaro 76010, Mexico ‡ Department of Graduate Studies, Research, and Technology Development (UPIDET), Instituto Tecnologico de Durango, Boulevard Felipe Pescador 1830 Oriente, Durango, Durango 34080, Mexico ABSTRACT: Peppermint (Mentha piperita) infusions represent an important source of antioxidants, which can be enhanced by inducing abiotic stress in plants. The aim of this study was to evaluate the effect of drought stress on peppermint cultivation as well as the metabolite profile, antioxidant capacity, and inhibition of digestive enzymes of resulting infusions. At 45 days after planting, irrigation was suppressed until 85 (control), 65, 35, 24, and 12% soil moisture (SM) was reached. The results showed that 35, 24, and 12% SM decreased fresh (20%) and dry (5%) weight. The 35 and 24% SM treatments significantly increased total phenolic and flavonoid contents as well as antioxidant capacity. Coumaric acid, quercetin, luteolin, and naringenin were detected only in some drought treatments; however, in these infusions, fewer amino acids and unsaturated fatty acids were identified. The 24 and 12% SM treatments slightly improved inhibition of pancreatic lipase and α-amylase activity. Therefore, induction of moderate water stress in peppermint is recommended to enhance its biological properties. KEYWORDS: peppermint infusion, drought stress, phenolic compounds, antioxidant capacity, inhibition of enzyme activity
■
INTRODUCTION Herbal infusions are widely consumed because of the phenolic compounds that they contain. These compounds are considered the most abundant natural antioxidants in food and are recommended for inclusion in the diet because of the health benefits that they can produce. One of the most popular herbal preparations is peppermint (Mentha piperita) infusion. This plant has a phenolic compound content in leaves of approximately 19−23% dry weight, of which 12% are flavonoids, such as eriocitrin, rosmarinic acid, hesperidin, and luteolin.1 Approximately 75% of these compounds can be extracted in the preparation of an infusion, and many of them have been shown to have antioxidant, hypolipidemic, antidiabetic, and antitumoral properties.2,3 Other important components found in peppermint leaves are fatty acids, volatile compounds, chlorophyll, α- and γ-tocopherols, and ascorbic acid.1 Several studies have demonstrated that peppermint extracts decrease glucose, total cholesterol, triacylglycerols, very lowdensity lipoprotein (VLDL), and low-density lipoprotein (LDL) levels, thus decreasing the atherogenic index in diabetic rats.4,5 These health benefits can be enhanced using preharvest strategies to increase bioactive compounds in the peppermint leaves. In a wide variety of plant species, deficit irrigation has been shown to enhance the synthesis of several phytochemicals, including phenolic acids, flavonoids, and tannins, as a response to stress constraints.6 Under stress conditions, increased reactive oxygen species (ROS) production is observed in different cellular compartments, leading to the activation of the antioxidant system, which synthesizes phenolic compounds. © 2014 American Chemical Society
Nevertheless, the use of drought stress as a strategy to improve phytochemicals in plants should be carefully applied to avoid the detrimental effects of excessive ROS production, such as cellular damage and death.7 Therefore, the aim of this study was to cultivate peppermint (M. piperita) at different levels of drought stress and to evaluate the effect on plant growth as well as on the metabolite profile, antioxidant capacity, and inhibitory activity on digestive enzymes of resulting infusions.
■
MATERIALS AND METHODS
Reagents and Biological Materials. The peppermint plants were purchased from a local plant nursery, Floraplant S.A. de C.V. (Mexico) and taxonomically identified in the herbarium “Dr. Jerzy Rzedowski” of the Natural Science Department of Universidad Autónoma de Queretaro. 1,1-Diphenyl-2-picrylhydrazyl radical, 2,20-azinobis(3ethylbenzthiazoline-6-sulfonic acid), sodium nitroprusside, lipase from porcine pancreas (type II), 4-nitrophenyl butyrate, α-amylase, p-nitrophenyl-α-D-glucopyranoside, α-glucosidase, caffeic, coumaric, sinapic, and rosmarinic acids, eriocitrin, naringenin, rutin, vanillin, luteolin, quercetin, and hesperidin were purchased from Sigma-Aldrich (St. Louis, MO). Plant Growth Conditions and Measurement of Growth Parameters. The plants were grown in a greenhouse at the Universidad Autónoma de Queretaro in pots with a diameter of 40 cm, with irrigation every 3 days [85% soil moisture (SM)] during the first 45 days. Mean daily temperature inside the greenhouse was within optimal ranges for peppermint growth (19−25 °C).1 Fertilization was Received: Revised: Accepted: Published: 12027
July 30, 2014 November 12, 2014 November 22, 2014 December 2, 2014 dx.doi.org/10.1021/jf503628c | J. Agric. Food Chem. 2014, 62, 12027−12033
Journal of Agricultural and Food Chemistry
Article
carried out 15, 30, and 45 days after planting. Each pot was fertilized with a solution containing calcium nitrate (1.12 g), magnesium sulfate (0.50 g), potassium nitrate (0.36 g), monobasic potassium phosphate (0.30 g), iron chelate (0.07 g), and manganese sulfate (0.01 g). After 45 days, the plants were randomly subjected to different levels of water stress: 65, 35, 24, and 12% SM, by suppressing irrigation for a period of 7, 16, 21, and 26 days, respectively. For the same amount of time as for each treatment, a control was carried out (irrigation every 3 days, at 85% SM). Treatments and controls included 6 replicates, producing a total of 48 experimental units (pots). Plant response to drought stress was determined by the increase in shoot length (n = 12) as well as longitudinal growth and leaf expansion (n = 24), which were evaluated by measuring the length of the leaf from the base to the apex (longitudinal growth) and at the broadest part (transverse growth). All of these parameters were measured at day 45 and after 7, 16, 21, and 26 days without water added. Harvest and Infusion Preparation. Immediately after each drought treatment, after 7, 16, 21, and 26 days without water added, midlife leaves were collected from each experimental unit, dried at 45 °C for 24 h using a convection oven (Fisher Scientific, 650D, Waltham, MA), and milled in an herb grinder (Krups GX4100, Mexico) to a particle size of 0.7−1.0 mm. Infusions were prepared by adding 1 g of ground material to 100 mL of freshly boiled distilled water; the mixture was allowed to stand for 10 min and was then filtered using a 0.5 mm pore size filter. These conditions simulate the recommended preparation of commercial infusions. Total Phenolic and Flavonoid Contents. The total phenolic compound content of the peppermint infusions was determined according to the Folin−Ciocalteu colorimetric method.8 Briefly, 40 μL of the infusion diluted in 480 μL of water was mixture with 450 μL of a 1 M Folin−Ciocalteu reagent and 1250 μL of a sodium carbonate solution (5%, w/v). The tubes were left at room temperature for 120 min, and the absorbances were measured at 765 nm. Results are expressed as milligrams of gallic acid (GA) equivalents per milliliter of infusion (mg of GAE/mL). The flavonoid content was determined according to the method described by Liu et al.9 Briefly, 0.25 mL of each infusion were diluted with 1.25 mL of water and mixed with 75 μL of 5% NaNO2. After 6 min, 150 μL of a 10% AlCl3·6H2O solution and 500 μL of 1 M NaOH were added, and the reaction was adjusted to 2.5 mL with distilled water. The absorbances were measured at 510 nm, and the results are expressed as milligrams of (+)-catechin equivalents per milliliter of infusion (mg of CAE/mL). Identification and Quantification of Phenolic Compounds. For the identification of phenolic compounds by high-performance liquid chromatography with photodiode array detection and massspectrometric detection (HPLC/DAD−MSD) analysis, the extract was lyophilized and suspended in methanol at a concentration of 10 mg/mL. Separation and identification was carried out using an Agilent 1200 HPLC−DAD system connected to a SL quadrupole mass spectrometer Agilent 1100 equipped with an electrospray interface. The samples were injected into a reversed-phase column [Zorbax octadecylsilane (ODS-C18), 15 × 4.6 mm] operated at room temperature. A total of 10 μL of sample was injected, and the compounds were eluted at 1 mL/min using a linear gradient system consisting of two solvents: (A) 2:98 (v/v) acetic acid/water and (B) (2:30:68, v/v/v) acetic acid/acetonitrile/water. The ratios of mobile phases were 90% A and 10% B at t = 0 min and 0% A and 100% B at t = 30 min. The absorbance was set at λmax of 260, 280, and 320 nm. The mass spectrometer was operated in the negative ion mode, under the following conditions: capillary voltage, 4000 V; nebulizer pressure, 40 psi; drying gas flow rate, 10 L/min; gas temperature, 300 °C; skimmer voltage, 50 V; octapolerf, 150 V; and fragmentor voltage, 130 V. Liquid chromatography−mass spectrometry (LC−MS) accurate mass spectra were recorded across the range of m/z 50−1000. Quantification was carried out using phenolic acid and flavonoid standards (caffeic, coumaric, sinapic, and rosmarinic acids, eriocitrin, naringenin, rutin, vanillin, luteolin, quercetin, and hesperidin). Identification of Low-Molecular-Weight Metabolites. A sample solution (10 mg/mL) was prepared in methanol with
lyophilized infusions, which was concentrated with a nitrogen gas stream. Then, 50 μL of derivatizing agent, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS), was added and stirred for 2 min at room temperature. Finally, 1 μL was injected into an Agilent GC series 7890A (Wilmington, DE) coupled to an Agilent single quadrupole MS detector (Agilent 5975C), with electron energy set at 70 eV and mass range at m/z 50−700. A HP5MS capillary column (30 m × 0.25 mm inner diameter × 0.25 μm) was used. The injector temperature was set at 250 °C in splitless mode. The initial oven temperature was 100 °C, held for 1 min, and raised to 220 °C at 6 °C min−1, which was held for 1.23 min, then raised to 290 °C at 10 °C min−1, then raised to 310 °C at 40 °C/min, and held for 7.5 min. The flow rate of the carrier gas (helium) was maintained at 1 mL min−1. Gas chromatography−mass spectrometry (GC−MS) control and data processing were performed using ChemStation (Agilent Technologies) software. Free Radical Scavenging Assays. The capacity of peppermint infusions to bleaching diphenylpicrylhydrazyl (DPPH) radical was determinate by the method described by Brand-Williams et al.10 Aliquots of peppermint infusions were made to a total volume of 1 mL using methanol. A freshly prepared DPPH solution (98 μg/mL) was added, stirred, and left to stand at room temperature for 30 min in the dark. The inhibition capacity was determined by the decrease in absorbance at 517 nm. The 2,2′-azinobis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) radical scavenning assay was carried out as described by Re et al.11 ABTS• + was obtained by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulfate, and the mixture was left to stand in the dark at room temperature for 12−16 h before use. The ABTS• + solution was diluted with 5 mM phosphate-buffered saline (pH 7.4) to an absorbance at 730 nm of 0.70. After the addition of 10 μL of aliquots of sample to 1 mL of diluted ABTS• + solution, the absorbance was measured at 7 min. Furthermore, nitric oxide radical scavenging capacity was measured as described by Marcocci and Parker,12 with some modifications. Sodium nitroprusside (SNP, 5 mM) in phosphate-buffered saline was added to the sample at different concentrations to make up a volume of 3 mL and incubated at room temperature (27 °C) for 90 min. This incubated solution (1.5 mL) was added to 1.5 mL of Greiss reagent. The inhibition of nitrite formation was determined by the decrease in absorbance at 546 nm. All of these results were expressed as the half-maximal inhibitory concentration (IC50) and compared to a (+)-catechin standard. In Vitro Digestive Enzyme Inhibition Assays. The α-amylase activity was determined according to the methodology proposed by Kandra et al.13 Thus, 50 μL of the peppermint infusions and 50 μL of α-amylase from Bacillus subtilis (50 units/mg) were pre-incubated for 20 min in a water bath at 37 °C. The substrate was starch (1%) prepared in phosphate buffer at pH 7.0 with 38 mmol/L NaCl. After the addition of 100 μL of the substrate, the mixture was incubated for 2 h. The product (glucose) was quantified by the glucose oxidase− peroxidase method with a commercial kit (Spinreact) and measured at 540 nm. For determination of α-glucosidase inhibition, we used the method described by Apostolidis et al.,14 with some modifications, using 5 mmol/L p-nitrophenyl-α-D-glucopyranoside as the substrate in a 0.1 mol/L citrate−phosphate buffer at pH 7. In the assay, 50 μL of the infusion and 100 μL of enzyme (10 units/mg) were incubated in a water bath at 37 °C for 30 min after the addition of 50 μL of the substrate. The reaction was interrupted adding 1.000 μL of 0.05 mol/L NaOH, and the product was read in a spectrophotometer at 410 nm. Pancreatic lipase inhibition was measured according to the method described by McDougall et al.15 The mixture of 100 μL of porcine pancreatic lipase, 50 μL of the infusion, and 50 μL of 4 mmol/L pnitrophenyl laurate in 0.05 mmol/L Tris−HCl buffer at pH 8.0 containing 0.5% Triton X-100 was incubated for 30 min. The reaction was stopped, transferring the tubes to an ice bath and adding 1.000 μL of 0.05 mmol/L Tris−HCl buffer at pH 8.0. p-Nitrophenol, a product of the lipase action on p-nitrophenyl palmitate, was read in a spectrophotometer at 410 nm. 12028
dx.doi.org/10.1021/jf503628c | J. Agric. Food Chem. 2014, 62, 12027−12033
Journal of Agricultural and Food Chemistry
Article
Table 1. Changes in Fresh and Dry Weight of Leaves and Growth of Shoots and Leaves of Peppermint (M. piperita) Grown at Different Levels of SMa leaf growth (cm) soil water content control 65% control 35% control 24% control 12%
leaf width 2.8 2.7 2.6 2.1 2.7 2.3 2.8 2.2
± ± ± ± ± ± ± ±
0.1 0.1 0.2 0.1 0.0 0.1 0.1 0.0
a bb bb bb
leaf length 6.3 6.5 6.4 5.7 6.6 5.5 6.8 5.7
± ± ± ± ± ± ± ±
0.2 0.3 0.2 0.4 0.3 0.3 0.4 0.3
shoot growth (cm)
a bb bb bb
7.1 6.9 7.4 6.4 7.8 6.6 7.6 6.5
± ± ± ± ± ± ± ±
0.1 0.3 0.3 0.4 0.5 0.3 0.4 0.3
a bb bb bb
fresh weight (mg leaf−1) 135.5 122.8 147.2 133.5 164.8 126.9 171.0 138.3
± ± ± ± ± ± ± ±
8.1 6.3 7.2 5.6 7.2 6.3 8.2 6.1
b bb bb ab
dry weight (mg leaf−1) 39.8 38.2 43.2 41.2 48.5 45.2 50.3 46.9
± ± ± ± ± ± ± ±
2.0 1.8 2.1 1.7 2.3 1.6 2.3 1.7
b b ab ab
Results are the average of three independent determinations ± SE. Different letters indicate significant statistical differences between drought treatments (p < 0.05; Tukey’s test). bSignificant statistical differences between controls (plant growth of 85% SM over the same number of days of the respective treatment) and their respective treatment (p < 0.05; Dunnett’s test). a
The results of these determinations were expressed as a percentage of inhibition exhibited by a concentration of 10 mg/mL peppermint infusion. Statistical Analysis. All results were expressed as the mean ± standard error (SE). Data were analyzed by one-way analysis of variation (ANOVA), and differences among treatments were determined by a comparison of means using Tukey’s test or Dunnett’s test when was compared to a control. The level of statistical significance was considered at p < 0.05. Pearson correlation was performed to verify the association between measurements (p < 0.01).
treated plants, which may be of industrial importance, because peppermint infusions are prepared from dry material. Quantification of Total and Individual Phenolic Compounds. The total polyphenol and flavonoid contents as well as the phenolic profile of infusions of peppermint leaves cultivated under water stress are shown in Figure 1 and Table 2, respectively. Infusions prepared from peppermint grown at 35, 24, and 12% SM showed an increase of 32, 60, and 12% in total
■
RESULTS AND DISCUSSION Evaluation of Growth Parameters. Table 1 shows the effect of drought stress on the growth parameters of the plants. The plants grown at 35, 24, and 12% SM decreased in leaf width (20, 15, and 21%), leaf length (11, 16, and 17%), and shoot length (14, 15, and 14%) compared to the control plants, while the 65% SM treatment did not produce significant effects on growth parameters compared to the control. A similar trend was observed in the fresh weight of leaves (11, 23, and 20% for 35, 24, and 12% SM, respectively). However, the dry weight was less affected by drought treatments, resulting in decreases of approximately 5, 7, and 7% for 35, 24, and 12% SM, as compared to their respective controls. These effects were similar to those reported by Moeini et al.,16 who observed 2-, 6-, and 4-fold decreases in plant height, leaf area, and fresh yield, respectively, in basil (Ocimum basilicum) plants cultivated under constant water stress (at 60% of field capacity) for 6 weeks. The reduced growth of the aerial part of the plant is widely described as a consequence of water stress and depends upon the severity and duration of the stress period. The decrease in growth is mainly due to a loss of cell turgor (a physical process). Under normal conditions, water enters the cells because of a difference in potential, which is more negative inside, the cell swells, and the plasma membrane exerts turgor pressure against the cell wall. In drought conditions, the water potential is lower outside than inside the cell; therefore, the water tends to leave the cell, and this also causes the closure of stomata. In these situations, turgor pressure disappears and the plasma membrane detaches from the cell wall in some sections. The main consequence of this loss of turgor is the absence of cell growth and a reduction in plant biomass production.17,18 This may explain the decrease of fresh and dry weight observed in drought treatments. However, it is important to mention that the dry weight of leaves was not significantly affected in the
Figure 1. (a) Total phenolic and (b) flavonoid contents in infusions prepared from peppermint (M. piperita) leaves grown at different levels of SM. Results are expressed as milligrams of (a) gallic acid or (b) catechin equivalents per milliliter of peppermint infusion and are the average of three independent determinations ± SE. (∗) Significant statistical differences between controls (black bars, plant growth at 85% SM over the same number of days as the respective treatment in gray bars) and treatments (p < 0.05; Dunnett’s test). Different letters indicate significant statistical differences between treatments (p < 0.05; Tukey’s test). 12029
dx.doi.org/10.1021/jf503628c | J. Agric. Food Chem. 2014, 62, 12027−12033
Journal of Agricultural and Food Chemistry
Article
Table 2. Chromatographic Profile of Phenolic Compounds Identified in Infusions Prepared from Peppermint (M. piperita) Leaves Grown at Different Levels of SMa peak
compound
ion (m/z)
RT (min)
1 2 1 2 3 3 4 6 11 13 14
caffeic acid coumaric acid luteolin eriocitrin rutin sinapic acid rosmarinic acid hesperedin quercetin naringenin vanillin
179 146 285 597 609 207 359 609 301 271 285
12.7 16.6 17.3 19.4 20.0 21.6 22.5 23.1 28.4 31.4 33.4
control 0.2 ± 0.0 LDLb LDL 0.7 ± 0.0 15.2 ± 0.6 0.1 ± 0.0 51.6 ± 2.3 35.7 ± 2.1 LDL LDL LDL
65% 0.3 ± 0.0 LDL LDL 3.7 ± 0.1 31.5 ± 0.8 0.1 ± 0.0 51.3 ± 1.5 38.1 ± 3.5 0.4 ± 0.0 LDL LDL
c
d c b b d
35% b
0.8 2.9 1.5 7.5 50.8 2.2 78.1 34.9 0.8 0.8 0.2
c b b b c c
± ± ± ± ± ± ± ± ± ± ±
0.0 0.0 0.0 0.2 0.4 0.0 3.2 1.8 0.0 0.0 0.0
24% a a a a a a a d b a b
0.9 1.21 1.6 4.2 49.5 2.5 55.3 67.9 1.2 0.2 0.4
± ± ± ± ± ± ± ± ± ± ±
12%
0.0 0.0 0.0 0.1 0.2 0.0 1.8 4.5 0.0 0.0 0.0
0.3 ± 0.0 0.8 ± 0.0 LDL 6.0 ± 0.2 26.3 ± 0.3 2.3 ± 0.0 45.5 ± 3.1 45.8 ± 2.6 0.5 ± 0.0 LDL LDL
a b a c a a b a a b a
b c b b a c b c
Results are expressed as ng μL−1 of peppermint infusion and are the average of three independent determinations ± SE. Different letters indicate significant statistical differences for each compound (p < 0.05; Tukey’s test). bLDL = lower than the detection limit. a
Table 3. Low-Molecular-Weight Metabolites of Infusions Prepared from Peppermint (M. piperita) Leaves Grown at Different Levels of SMa area (%) nature of compound amino acid
proposed compound L-alanine L-leucine
carbohydrate organic acid
fatty acid
alcohol a
isoleucine L-proline L-serine threonine 4-aminobutiric acid 5-oxo-L-proline glutamine phenylalanine L-asparagine L-tyrosine tryptophan arabinose D-glucose lactic acid glycolic acid malonic acid succinic acid 2,3-hidroxipropionic acid fumaric acid malic acid threonic acid lauric acid tartaric acid citric acid myristic acid pantothenic acid palmitic acid linoleic acid oleic acid stearic acid α-linolenic acid glycerol tyrosol
RT (min)
control
65%
35%
24%
12%
4.1 6.8 7.2 7.3 8.5 9.0 11.7 11.6 13.4 13.5 14.4 19.0 23.8 18.7 20.0 3.6 3.8 5.6 7.5 7.9 8.1 11.0 12.2 13.8 14.0 17.2 17.3 19.9 20.5 23.3 23.4 24.0 23.5 6.9 12.4
0.73 1.47 2.54 7.35 1.49 0.90 0.82 10.79 0.02 0.11 0.01 0.04 0.03 1.83 0.03 1.87 1.15 0.71 9.34 1.34 0.75 16.26 0.30 0.02 0.01 LDL 0.02 0.01 0.15 0.02 LDL 2.69 0.08 18.01 0.25
0.51 0.15 0.70 7.14 LDL LDL LDL LDL 0.05 LDL LDL LDL LDL 2.85 8.26 15.67 6.07 0.99 5.51 4.22 0.61 2.20 0.45 4.01 2.02 LDL LDL LDL 16.19 LDL 5.43 4.37 LDL 17.88 1.89
LDLb LDL 0.46 6.68 LDL LDL LDL LDL 0.04 LDL LDL LDL LDL 2.96 27.10 19.54 2.45 0.18 6.41 3.64 0.53 1.27 LDL LDL 0.83 1.83 1.21 LDL 8.62 LDL 3.21 3.14 LDL 5.55 0.56
LDL LDL LDL 8.23 LDL LDL LDL LDL 0.02 LDL LDL LDL LDL 8.20 21.17 9.15 1.86 0.13 1.81 2.19 LDL 0.85 LDL 0.88 0.54 2.78 LDL LDL 4.50 LDL 1.99 1.04 LDL 13.08 0.41
LDL LDL 1.09 9.20 LDL LDL LDL LDL 0.03 LDL LDL LDL LDL 2.44 20.04 22.97 8.78 0.57 3.39 9.74 1.51 1.76 LDL LDL 2.12 9.27 1.53 LDL 8.87 LDL 5.35 LDL LDL 15.02 1.99
important ions in peaks (m/z) 218, 260, 260, 244, 306, 320, 304, 258, 363, 294, 351, 382, 357, 333, 435, 219, 205, 233, 247, 322, 245, 355, 409, 257, 423, 465, 303, 420, 331, 337, 339, 341, 335, 293, 282,
190, 232, 218, 216, 278, 291, 246, 230, 348, 266, 333, 354, 325, 305, 393, 191, 177, 204, 218, 292, 217, 307, 379, 229, 395, 375, 285, 363, 313, 309, 264, 315, 291, 263, 267,
172, 203, 158, 170, 260, 248, 216, 174, 320, 218, 258, 280, 291, 219, 345, 147, 121 117, 190, 265, 191, 265, 319, 201, 367, 305, 257, 330, 269, 262, 222, 297, 260, 205, 193,
116, 176, 149 142, 222, 218, 174 156, 246, 192, 188, 218, 251, 204, 305, 117 99 172 217, 171, 233, 292, 171, 333, 273, 201, 291, 171, 220, 185, 257, 213 177, 179
94 158, 133, 103 119, 100 204, 174 117 133 218, 174, 169, 179 202, 187, 231,
189, 115 189, 263, 145 292, 217, 171, 247, 145, 199 145, 227,
198, 174 100 116 147 147, 129 204
129, 103 171, 129 245, 177, 117 219 183, 129 145 219 117, 97 117 201
125, 103
Results are expressed as a percentage of the total area. bLDL = lower than the detection limit.
phenolic content and 71, 80, and 31% in total flavonoid content, respectively. However, the 65% SM treatment produced no significant effect on total phenolic and flavonoid
contents. An increase of 10 and 71% in total phenolic content after 4 and 8 days of water stress in different tea (Camellia sinensis) cultivars has been reported, as compared to control 12030
dx.doi.org/10.1021/jf503628c | J. Agric. Food Chem. 2014, 62, 12027−12033
Journal of Agricultural and Food Chemistry
Article
study, we found this amino acid in all water stress treatments; however, it is necessary to perform a quantitative study of proline to demonstrate the aforementioned effect. Despite the fact that amino acids are not the main bioactive compound in peppermint infusions and that these beverages are not consumed for nutritional purposes, these results showed that drought stress affected amino acid biosynthesis in the leaves. Carbohydrates, such as arabinose and D-glucose, were identified in the peppermint infusions; however, these compounds were found in all infusions evaluated, both control and treated. In addition, a similar trend was found for the majority of organic and fatty acids, in which no differences were found between the profiles of control and stressed plants. However, some of these compounds, such as citric and oleic acids, were only identified in infusions made from stressed plants. These compounds are primary metabolites and can be found in large amounts in all plants; in a manner similar to that of phenolic compounds, some organic and fatty acids may also play a protective role against various oxidative diseases because of their antioxidant properties,24 as is the case of citric acid because it has the ability to act as a singlet oxygen quencher and scavenger of OH• radicals.25 Moreover, these compounds are key components of some mechanisms used by plants for the management of nutrient deficiencies, plant−microbe interactions, and water stress, operating at the root−soil interface.26 Pantothenic, linoleic, and α-linolenic acids were found only in infusions made from control plants, suggesting that drought conditions decrease the amount of these compounds in leaves. Gigon et al.27 reported approximately 3-fold decreases of the total lipid content in Arabidopsis thaliana subjected to drought for 14 days. Similar results were found by Benhassaine-Kesri et al.,28 who reported decreases in linoleic and linolenic acid contents in rape leaves (Brassica napus) growing under water stress conditions for 6 days. Several plant species respond to environmental stresses by altering their membrane lipid composition.27 Indeed, membranes are primary targets of degradative processes induced by drought stress, and it has been shown that, under water stress, a decrease in the membrane lipid content is correlated with an inhibition of lipid biosynthesis and a stimulation of lipolytic and peroxidative activities, decreasing the lipid content of plant. However, the response may be different depending upon the class of lipid studied.27−29 Evaluation of Antioxidant Capacity. Antioxidant capacity is related to the presence of compounds with the ability to counteract the formation of free radicals by different mechanisms.30 Table 4 shows the antioxidant capacity of peppermint infusions as determined by DPPH, ABTS, and
plants, along with a decrease of about 75% in these compounds when the stress period was prolonged for 12 days.19 Plants exposed to drought present increased levels of abscicic acid (ABA) in leaves, shoots, and roots, which induces several responses in the plant, including the accelerated production of ROS in chloroplasts, mitochondria, and peroxisomes, which stimulates antioxidant enzyme activity and the synthesis of phenolic compounds. This mechanism is used in plants as an adaptation strategy to overcome oxidative stress.7 The phenolic profile of the different controls used for each treatment was determined, and no significant differences were found; hence, only the control corresponding to the 24% SM treatment is shown in Table 2 and in the following results. The main phenolic compounds identified in the peppermint infusions were phenolic acids and flavonoids. Most of these compounds were increased in infusions obtained from stressed plants, and this effect was greater in the 35 and 24% SM treatments. Among the compounds that were increased by drought treatment were rosmarinic acid (1.5-fold at 35% SM) and hesperidin (2-fold at 24% SM), which are the most abundant in this plant and have been related to the beneficial health effects of peppermint infusions.1 Furthermore, coumaric acid, luteolin, quercetin, naringenin, and vanillin were found in infusions of stressed leaves but not in controls. In a previous study, we treated peppermint plants with different concentrations of elicitors [salicylic acid (SA) or hydrogen peroxide] and found similar results, increasing the hesperidin and rosmarinic acid in leaves, among other phenolic compounds, and we also found some compounds, such as naringinin, in SAtreated plants but not in controls; these compounds are different in comparison to those found in this study, which suggests that different types of abiotic stress induce different pathways involved in phenolic compound synthesis.20 Identification of Low-Molecular-Weight Compounds. Table 3 shows the identification of low-molecular-weight polar compounds, such as amino acids, carbohydrates, organic acids, fatty acids, and alcohols, obtained by GC−MSD analysis. Drought stress led to important changes in the metabolite profile of infusions compared to the control. For instance, most of the amino acids identified in the control, such as Ser, Thr, Phe, and Asn, among others, were not detected in infusions prepared from stressed plants. Similar results were previously reported in tobacco (Nicotiana tabacum L.) leaves after 4 days of water deprivation, where decreases of approximately 40− 70% were observed in the concentrations of some amino acids, such as Asp, Glu, Ser, and Gly, as well as a decrease in NO3−. The low level of transpiration and turgor pressure because of water deficit leads to a decrease in root absorption of NO3− and transport of NO3− to the leaves, decreasing amino acid synthesis. This effect could explain the changes in the amino acid profile found in peppermint infusions made from stressed leaves, because some amino acids were not detected in plants grown under severe drought stress conditions.21 However, in several studies, the opposite effect has been shown for some amino acids, such as proline. Ghorbanli et al.22 found that severe drought stress (1/3 field capacity) on tomato cultivars growing under greenhouse conditions for 9 weeks resulted in a 6-fold increase of the amount of proline in the leaves. It has been shown that increased proline in droughtstressed plants may be an adaptation to stress conditions, because this amino acid has a scavenger function and acts as a supply of osmolytes and energy for the growth and survival of cells, thereby helping the plant to tolerate water stress.23 In this
Table 4. Capacity of Infusions Prepared from Peppermint (M. piperita) Leaves Grown at Different Levels of SM, for Inhibition of ABTS+, DPPH•, and NO• Radicalsa treatment control 65% 35% 24% 12%
IC50 DPPH• 61.2 58.2 24.5 32.2 53.4
± ± ± ± ±
1.4 1.2 1.5 1.2 1.3
a a d c b
IC50 ABTS+ 16.5 14.2 10.9 12.3 15.2
± ± ± ± ±
0.4 0.3 0.5 0.3 0.3
a b c bc a
IC50 NO• 48.5 52.3 35.3 32.8 51.2
± ± ± ± ±
0.9 1.2 0.8 1.6 1.8
a a b c a
a Results are expressed as μg/mL and reflect averages of triplicate assays ± SE. Different letters indicate significant statistical differences for each treatment (p < 0.05; Tukey’s test).
12031
dx.doi.org/10.1021/jf503628c | J. Agric. Food Chem. 2014, 62, 12027−12033
Journal of Agricultural and Food Chemistry
Article
nitric oxide scavenging assays, expressed as IC50 values. ABTS and DPPH are the main assays used for the evaluation of the ability of compounds to donate electrons or hydrogen atoms, respectively.30 We found that infusions made from control and stressed peppermint leaves reached a maximum inhibition percentage of approximately 92 and 94% for DDPH and ABTS, respectively (data not shown). However, the infusions obtained from peppermint subjected to water stress exhibited an important decrease in IC50 values, as compared to the control in both assays. SM of 35 and 24% had the greatest antioxidant activity, because IC50 values were reduced by 33 and 25% for ABTS and 60 and 47% for DPPH, respectively, as compared to the control infusion. These effects could be attributed to the increase of some phenolic compounds, such as coumaric acid, luteolin, rutin, rosmarinic acid, naringenin, and caffeic acid, in drought-stressed plants, because we found high correlation coefficients between these compound concentrations and the DPPH (r = 0.67−0.98) and ABTS (r = 0.80−0.98) scavenging capacities. On the other hand, we measure the ability of infusions to compete with oxygen to scavenge nitric oxide, which was generated from SNP. Results showed that infusions from all treatments exhibited a maximum inhibition of approximately 82%, while (+)-catechin reached a maximum of 91% (data not shown). Infusions obtained from stressed peppermint showed decreased IC50 values by approximately 31 and 37% for the 24 and 35% SM treatments, respectively, as compared to the control. Furthermore, we found a high correlation coefficient between the sinapic acid concentration and NO scavenging activity (r = 0.98). These results suggest that all infusions are likely to have a nitric oxide scavenging activity, which was monitored indirectly by the nitrite production. Nitrite is the final product of the reaction of nitric oxide with oxygen through reactive intermediates, such as NO2 and N2O3. Therefore, the inhibitory effect of peppermint infusions on nitrite production could be attributed to not only their NO scavenging activity but also their reaction with other nitrogen oxides.12,31 Inhibition of Digestive Enzymes. It has been demonstrated that peppermint infusions reduce the blood concentration of glucose and triglycerides; therefore, the evaluation of in vitro digestive enzyme inhibition represents a preliminary approach to the biological potential of peppermint infusions. Pancreatic α-amylase hydrolyzes starch to oligosaccharides, which are then hydrolyzed to glucose by intestinal αglucosidase. Pancreatic lipase hydrolyzes triglycerides to free fatty acids and is responsible for the hydrolysis of 50−70% of total dietary fats.32,33 Therefore, the inhibition of these digestive enzymes could reduce glucose and lipid absorption. It has been reported that some polyphenols may inhibit digestive enzymes involved in the breakdown of lipids and starches, leading to positive effects on obesity and blood glucose control.15,32 Therefore, we evaluated the capacity of the infusions for inhibition of α-amylase, α-glucosidase, and pancreatic lipase activity. Table 5 shows that infusions obtained with 35, 24, and 12% SM presented 4-, 24-, and 14-fold increases in α-amylase inhibition, as compared to the control; however, the maximum percentage of inhibition achieved was only 12% (with the 24% SM treatment), suggesting that peppermint infusions are not highly able to inhibit this enzyme. This inhibition was obtained with the concentration used to prepare this kind of beverage (10 mg/mL); however, the peppermint infusions presented a dose-dependent inhibitory capacity, and the maximum α-
Table 5. Inhibition of Digestive Enzyme Activity by Infusions Prepared from Peppermint (M. piperita) Leaves (10 mg mL−1) Grown at Different Levels of SMa treatment control 65% 35% 24% 12%
α-amylase 0.5 0.7 2.1 12.5 7.1
± ± ± ± ±
0.1 0.0 0.1 0.1 0.1
α-glucosidase d d c a b
7.6 8.1 7.5 7.2 7.9
± ± ± ± ±
1.0 1.1 0.6 0.5 1.2
a a a a a
lipase 45.6 49.0 48.7 53.5 55.1
± ± ± ± ±
4.2 3.6 4.1 4.9 6.5
b ab b a a
a Results are expressed as a percentage of enzyme inhibition and reflect averages of triplicate assays ± SE. Different letters indicate significant statistical differences for each treatment (p < 0.05; Tukey’s test).
amylase inhibition (45%) was achieved with 60−80 mg/mL (data not shown). McCue and Shetty34 reported that pure rosmarinic acid exhibits an inhibitory activity over pancreatic αamylase in a concentration-dependent manner, and these authors suggested that phenolic synergies in plant extracts may contribute to additional amylase inhibitory activity. Nevertheless, this is not in agreement with our results because we found a low correlation coefficient between the rosmarinic acid concentration and inhibition values to this enzyme (r = 0.19). However, high correlation coefficients were found between hesperidin (r = 0.95), quercetin (r = 0.77), and sinapic acid (r = 0.76) and the amylase inhibition. On the other hand, peppermint infusions (10 mg/mL) inhibited about 7−8% of the activity of α-glucosidase, finding the highest correlation with vainillin and luteolin (r = 0.86 and 0.82, respectively); however, no significant changes were found in infusions made from stressed leaves, as compared to the control. The maximum inhibitory capacity (24%) was reached at 50−65 mg/mL (data not shown). It has been reported that some phenolic compounds, such as quercetin, rutin, and chlorogenic acid, present in Gynuramedica leaf extracts have the capacity to inhibit α-glucosidase activity in vitro; however, we found low correlation coefficients between inhibition of this enzyme and rutin and quercetin concentrations (r = 0.54 and 0.60, respectively), which suggest that these inhibitions could be related to synergic effects between different compounds in plants.35 Control infusions inhibited approximately 45% of the pancreatic lipase activity, while infusions made from plants stressed with 24 and 12% SM showed inhibitions of 53.5 and 55.1%, respectively. However, the 65 and 35% SM treatments produced no significant changes, as compared to the control. It has been previously reported that hesperidin has the ability to inhibit pancreatic lipase activity in vitro, and this effect is associated with its chemical structure, specifically with the 7 position of rutinose on the C ring of the flavonoid structure.36 Furthermore, the highest correlation coefficient between phenolic compounds and lipase inhibition capacity was found for hesperidin (r = 0.68). The results of this study suggest that water deficit has an important effect on the synthesis of phenolic compounds in the peppermint plant, which is reflected in an increased in vitro antioxidant capacity and inhibition of digestive enzyme activity of the resulting infusions. However, drought stress negatively affected metabolites, such as amino acids and unsaturated fatty acids; therefore, further research is required to determine the biological significance of this effect in the peppermint plant and infusions. 12032
dx.doi.org/10.1021/jf503628c | J. Agric. Food Chem. 2014, 62, 12027−12033
Journal of Agricultural and Food Chemistry
■
Article
(18) Yordanov, I.; Velikova, V.; Tsonev, T. Plant responses to drought and stress tolerance. Bulg. J. Plant Physiol. 2003, 187−206. (19) Chakraborty, U.; Chakaborty, B. Response of tea plants to water stress. Biol. Plant. 2002, 45, 557−562. (20) Figueroa-Perez, M. G.; Rocha-Guzmán, N. E.; Mercado-Silva, E.; Loarca-Piña, G.; Reynoso-Camacho, R. Effect of chemical elicitors on peppermint (Mentha piperita) plants and their impact on the metabolite profile and antioxidant capacity of resulting infusions. Food Chem. 2014, 156, 273−278. (21) Ferrario-Mery, S.; Valadier, M. H.; Foyer, C. H. Overexpression of nitrate reductase in tobacco delays drought-induced decreases in nitrate reductase activity and mRNA. Plant Physiol. 1998, 117, 293− 302. (22) Ghorbanli, M.; Gafarabad, M.; Amirkian, T.; Allahverdi, B. Investigation of proline, total protein, chlorophyll, ascobate and dehydroascorbate under drought stress in Akria and Mobil tomato cultivars. Iran. J. Plant Physiol. 2011, 3, 651−658. (23) Sankar, B.; Jaleel, C. A.; Manivannan, P.; Kishorekumar, A.; Somasundaram, R.; Panneerselvam, R. Effect of paclobutrazol on water stress amelioration through antioxidants and free radical scavenging enzymes in Arachis hypogaea L. Colloids Surf., B 2007, 60, 229−235. (24) Oliveira, A.; Pereira, J.; Andradec, P.; Valentãoc, P.; Seabrac, R.; Silva, B. Organic acids composition of Cydonia oblonga Miller leaf. Food Chem. 2008, 111, 393−399. (25) Polumbryk, M.; Ivanov, S.; Polumbryk, O. Antioxidants in food systems. Mechanism of action. Ukr. J. Food Sci. 2013, 1, 15−40. (26) Lopez-Bucio, J.; Nieto-Jacobo, F.; Ramírez-Rodriguez, V.; Herrera-Estrella, L. Organic acid metabolism in plants: From adaptive physiology to transgenic varieties for cultivation in extreme soils. Plant Sci. 2000, 160, 1−13. (27) Gigon, A.; Matos, A. R.; Laffray, D.; Zuily-Fodil, Y. Effect of drought stress on lipid metabolism in the leaves of Arabidopsis thaliana (ecotype Columbia). Ann. Bot. 2004, 94, 345−351. (28) Benhassaine-Kesri, G.; Aid, F.; Demandre, C.; Kader, J. C.; Mazliak, P. Drought stress affects chloroplast lipid metabolism in rape (Brassica napus) leaves. Physiol. Plant. 2002, 115, 221−227. (29) Harwood, J. L. Recent advances in biosynthesis of plant fatty acids. Biophys. Acta 1996, 1301, 7−56. (30) Prior, R. L.; Wu, X.; Schaich, K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 2005, 53, 4290−4302. (31) Stamler, J. S.; Singel, D. J.; Loscalzo, J. Biochemistry of nitric oxide and its redox-activated forms. Science 1992, 258, 1898−1902. (32) McDougall, G.; Shpiro, F.; Dobson, P.; Smith, P.; Blake, A.; Stewart, D. Different polyphenolic components of soft fruits inhibit αamylase and α-glucosidase. J. Agric. Food Chem. 2005, 53, 2760−2766. (33) Shi, Y.; Burn, P. Lipid metabolic enzymes: Emerging drug targets for the treatment of obesity. Nat. Rev. Drug Discovery 2004, 3, 695−710. (34) McCue, P.; Shetty, K. Inhibitory effects of rosmarinic acid extracts on porcine pancreatic amylase in vitro. Asia Pac. J. Clin. Nutr. 2004, 13, 101−106. (35) Chao, T.; Qunxing, W.; Chunhua, L.; Sai, C.; Qianyuan, L.; Peng, Li Yeast α-glucosidase inhibitory phenolic compounds isolated from Gynura medica leaf. Int. J. Mol. Sci. 2013, 14, 2551−2558. (36) Kawaguchi, K.; Mizuno, T.; Aida, K.; Uchino, K. Hesperedin as an inhibitor of lipases from porcine pancreas and Pseudomonas. Biosci., Biotechnol., Biochem. 1997, 61, 102−104.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +52-442-1921300, ext. 5576. Fax: +52-4421921304. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) McKay, D. L.; Blumberg, J. B. A review of the bioactivity and potential health benefits of peppermint tea (Mentha piperita L.). Phytother. Res. 2006, 20, 619−633. (2) Mushtaq, N.; Schmatz, R.; Pereira, L.; Ahmad, M.; Stefanello, N.; Vieira, J. M.; Abdalla, F.; Rodrigues, M.; Baldissarelli, J.; Pelinson, L.; Dalenogare, D.; Reichert, K.; Dutra, E.; Mulinacci, N.; Innocenti, M.; Bellumori, M.; Morsch, V. M.; Schetinger, M. R. Rosmarinic acid prevents lipid peroxidation and increase in acetylcholinesterase activity in brain of streptozotocin-induced diabetic rats. Cell Biochem. Funct. 2013, 32, 287−293. (3) Bentli, R.; Ciftci, O.; Cetin, A.; Unlu, M.; Basak, N.; Cay, M. Oral administration of hesperidin, a citrus flavonone, in rats counteracts the oxidative stress, the inflammatory cytokine production, and the hepatotoxicity induced by the ingestion of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD). Eur. Cytokine Network 2013, 24, 91−96. (4) Barbalho, S.; Vazquez, S.; Machado, F.; Oshiiwa, M.; Abreu, M.; Tomazela, P.; Goulart, T. Investigation of the effects of peppermint (Mentha piperita) on the biochemical and anthropometric profile of university students. Cienc. Tecnol. Aliment. (Campinas, Braz.) 2011, 31, 584−588. (5) Mani, R.; Badal, D.; Badal, P.; Khare, A.; Shrivastava, J.; Kumar, V. Pharmacological action of Mentha piperita on lipid profile in fructose-fed rats. Iran. J. Pharm. Res. 2011, 10, 843−848. (6) Zingaretti, S. M.; Inácio, M. C.; de Matos Pereira, L.; Paz, T. A.; de Castro França, S. Water stress and agriculture. In Responses of Organisms to Water Stress, 1st ed.; Akinci, S., Ed.; InTech: Rijeka, Croatia, 2013; Chapter 7, pp 152−179. (7) Cruz, M. Drought stress and reactive oxygen species. Plant Signaling Behav. 2008, 3, 156−165. (8) Singleton, V. L.; Rossi, J. A. Colorimetry of total phenolics with phosphomolybdic and phosphotungstic acid reagents. Am. J. Enol. Vitic. 1965, 16, 144−153. (9) Liu, M.; Li, X. Q.; Weber, C.; Lee, C. Y.; Brown, J.; Liu, R. H. Antioxidant and antiproliferative activities of raspberries. J. Agric. Food Chem. 2002, 50, 2926−2930. (10) Brand-Williams, W.; Cuvelier, M.; Berset, C. Use of free radical method to evaluate antioxidant activity. Lebensm.-Wiss. Technol. 1995, 28, 25−30. (11) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med. 1999, 26, 1231− 1237. (12) Marcocci, T. K.; Parker, L. Antioxidant action of Ginkgo biloba extract EGB 761. Methods Enzymol. 1994, 234, 462−475. (13) Kandra, L.; Zajacz, A.; Remenyik, J.; Gyemant, G. Kinetic investigation of a new inhibitor for human salivary α-amylase. Biochem. Biophys. Res. Commun. 2005, 334, 824−828. (14) Apostolidis, E.; Kwon, Y.; Shetty, K. Inhibitory potential of herb, fruit, and fungal-enriched cheese against key enzymes linked to type 2 diabetes and hypertension. Innovative Food Sci. Emerging Technol. 2007, 8, 46−54. (15) McDougall, G.; Kulkarni, N.; Stewart, D. Berry polyphenols inhibit pancreatic lipase activity in vitro. Food Chem. 2009, 115, 193− 199. (16) Moeini, H.; Heidari, A.; Dizaji, A. Effect of water stress on some morphological and biochemical characteristics of purple basil (Ocimum basilicum). J. Biol. Sci. 2006, 6, 763−767. (17) Blum, A. Plant Breeding for Water-Limited Environments; Springer: New York, 2011. 12033
dx.doi.org/10.1021/jf503628c | J. Agric. Food Chem. 2014, 62, 12027−12033
