Diabetic nephropathy is ameliorated with peppermint (Mentha piperita) infusions prepared from salicylic acid-elicited plants

Journal of Functional Foods 43 (2018) 55–61

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Diabetic nephropathy is ameliorated with peppermint (Mentha piperita) infusions prepared from salicylic acid-elicited plants

T

Marely G. Figueroa-Péreza, Iza F. Pérez-Ramírezb, José A. Enciso-Morenoc, ⁎ Marco A. Gallegos-Coronad, Luis M. Salgadoe, , Rosalía Reynoso-Camachob a

Universidad Tecnológica de Culiacán, Culiacán Rosales, Sin., Mexico Programa de Posgrado en Alimentos del Centro de la Republica (PROPAC), Centro Universitario, Universidad Autonoma de Queretaro, Mexico c Unidad de Investigación Biomédica, Instituto Mexicano del Seguro Social, Zacatecas, Mexico d Facultad de Medicina, Universidad Autónoma de Querétaro, Querétaro, Mexico e CICATA – Querétaro, Instituto Politécnico Nacional, Querétaro, Mexico b

A R T I C L E I N F O

A B S T R A C T

Keywords: Peppermint infusions Elicitor Diabetic nephropathy Oxidative stress Inflammation

Different alternatives are used to delay diabetic complications, and the functional foods have an important position due to their beneficial effects. For instance, herbal infusions are used widely to improve the health, and their favorable effects are enhanced by plant elicitation during cultivation. Peppermint (Mentha pipperita) infusions prepared from elicited plants (2 mM salicylic acid) increased their content of several compounds, principally p-hydroxybenzoic and rosmarinic acids, hesperidin, quercetin-3-O-glucoside, α-tocopherol, and βsitosterol. The administration of these infusions decreased microalbumin and urea in urine and serum uric acid levels, and the renal accumulation of 14 inflammation-related proteins. These proteins were associated with glomerular hypertrophy, tubular damage, expansion of mesangial matrix, and cell death. The application of 2 mM SA during peppermint cultivation improved the renoprotective properties of peppermint infusions.

1. Introduction Herbal infusions have been used widely to treat illness or to improve health, and their consumption has increased worldwide. Peppermint (Mentha piperita) infusion is a popular herbal preparation with several benefits on digestive, respiratory, immune, and cardiovascular systems (McKay & Blumberg, 2006; Badal et al., 2011). The beneficial effects of peppermint have been associated with its phytochemical content, mainly phenolic acids and flavonoids (Riachi & De Maria, 2015; Sun, Wang, Wang, Zhou, & Yang, 2014), which can be enhanced by elicitation during growth (Figueroa-Perez, Rocha-Guzmán, Mercado-Silva, Loarca-Piña, & Reynoso-Camacho, 2014). Diabetes and its complications are a leading cause of incapacity and dead worldwide, and the number of people affected is rising around the world. Among the complications, diabetic nephropathy is a major problem that develops in more than 40% of types 1 and 2 diabetic patients, and it is considered a major public health issue. Oxidative stress is considered a pathogenic factor for the development of diabetic nephropathy, causing severe damage to biomolecules, and promoting apoptosis of glomerular and tubular cells (Gross et al., 2005; Kashihara, Haruna, Kondeti, & Kanwar, 2010). It increases the synthesis of pro-

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inflammatory cytokines, and other proteins, which exacerbate renal injury (Tucker, Scanlan, & Dalbo, 2015). It has been demonstrated that aqueous peppermint extracts improve renal biochemical parameters as well as renal oxidative stress markers (Ullah, Khan, Khan, Asif, & Ahmad, 2014; Thangapandiyan, Sumedha, & Miltonprabu, 2013). We reported that infusions prepared using salicylic acid-elicited leafs exerted anti-hyperglycemic and hypolipidemic effects in diabetic-induced rats (Figueroa-Perez et al., 2014; FigueroaPérez, Gallegos-Corona, Ramos-Gomez, & Reynoso-Camacho, 2015). In this study we evaluated the effect of SA-elicited peppermint infusions on diabetes-induced renal damage. 2. Materials and methods 2.1. Plant material and infusion preparation Peppermint plants were purchased from plant nursery (Floraplant S.A. de C.V., Guanajuato, Mexico), and identified at the Natural Science Department of the UAQ. Plants were grown in a greenhouse facility at 25 °C and 80% RH in pots (40 cm Ø), with irrigation every three days. Fertilization and 2 mM salicylic acid (SA) application were carried out

Corresponding author at: Cerro Blanco 141, Col. Colinas del Cimatario, Querétaro, Qro. 76090, Mexico. E-mail address: [email protected] (L.M. Salgado).

https://doi.org/10.1016/j.jff.2018.01.029 Received 17 August 2017; Received in revised form 24 January 2018; Accepted 29 January 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.

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160–200 mg/dL were included in the experiment and divided into three groups; diabetic control, diabetic treated with control-peppermint infusion, and diabetic treated with 2 mM SA-elicited peppermint infusion. Peppermint infusions were administered ad libitum, and animals were fed with their respective diets during four weeks. Afterward, animals were placed in metabolic cages for urine collection. Then, animals were anesthetized to withdraw blood samples by cardiac puncture and sacrificed. Blood samples were centrifuged at 3000g at 4 °C for 10 min, to obtain serum, which was stored at −70 °C until analyses. Kidneys were removed and washed with PBS; a fraction was fixed in 10% neutral buffered formalin (pH 7.4) at room temperature, embedded in paraffin blocks and processed for subsequent H&E staining for histological analysis, whereas a kidney fraction was snap-frozen with liquid nitrogen and stored at −70 °C for biochemical analysis.

as previously reported (Figueroa-Perez et al., 2014). Leaves were collected and dried at 45 °C for 24 h in a convection oven (Fisher Scientific, 650D, USA), milling in a herb grinder (Krups GX4100, México) 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, allowed to stand for 10 min, and filtered using a 0.5 mm pore size filter to simulate the recommended preparation of commercial infusions. 2.2. Identification and quantitation of phytochemical compounds The analysis was carried out using an Agilent 1200 HPLC-DAD system connected to a single quadrupole mass spectrometer Agilent 1100, equipped with an electrospray interface, using a reverse-phase Phenomenex C18 column (250 × 4.6 mm, 5 μm). It was operated in negative ion mode, capillary voltage, 4000 V; nebulizer pressure, 40 psi; drying gas flow rate, 10 L/min; gas temperature, 300 °C; skimmer voltage, 50 V; octupole, 150 V; and fragment voltage, 130 V. LC-MS accurate mass spectra were recorded across the range of m/z 50–1200. Phytochemicals were identified according to their mass spectra as well as by comparison with the retention time of commercial standards. The quantitative analysis of the phytochemical profile was performed based on UV signals using the calibration curves of commercial standards. Three systems were used for the separation of phytochemicals compounds. System I (phenolic acids and flavonoids). Lyophilized samples (10 mg) were extracted with 1 mL of methanol:acetic acid 99:1. The mobile phase consisted of acetic acid-water 2:98 v/v (A) and acetic acid-water-acetonitrile 2:48:50 v/v/v (B) under gradient conditions (Lomas-Soria et al., 2015). Monitoring was performed at 280 nm (hydroxybenzoic acids and flavonols), 320 nm (hydroxycinnamic acids and stilbenes) and 370 nm (flavonols). System II (phytosterols). Lyophilized samples (0.5 g) were extracted with 4 mL of hexane during 6 h, and the mixture was centrifuged at 19,000g for 5 min. The hexane phase was recovered, evaporated to dryness, and dissolved in 200 μL of acetonitrile. The mobile phase consisted of acetonitrile-acetic acid-methanol-water 48:2:25:25 v/v/v/ v (A) and acetonitrile-acetic acid 98:2 v/v (B) under gradient conditions, monitoring was performed at 203 nm (Lomas-Soria et al., 2015). System III (Carotenoids). Lyophilized samples were extracted and analyzed as previously reported (Amaya-Cruz et al., 2015). The mobile phase consisted of methanol (A) and acetonitrile (B) in a ratio (A/B) of 55/45, with a total time of 30 min. Absorbance was measured at 290 and 450 nm.

2.5. Biochemical analysis Serum uric acid and urine urea were analyzed using commercial colorimetric-enzymatic kits (Spinreact, Girone, Spain), whereas urine microalbumin was quantified using an ELISA kit (ALPCO Diagnostics, NH, USA). 2.6. Histology analysis of renal tissues Renal tissues embedded in paraffin were sectioned at 5 µm and stained with Harris hematoxylin and eosin (H&E) solution. Samples were observed and photographed at 100X and 400X, analyzing five images per animal. 2.7. Oxidative and nitrosative stress parameters Lipid peroxidation was estimated in frozen renal homogenates by the thiobarbituric acid (TBA) assay. TBA-reactive substances (TBARS) were estimated with molar extinction coefficient ε = 1.56 × 105 1/ M cm, and results were expressed as nmol eq. TBARS/mg protein (Wright, Colby, & Miles, 1981). Protein oxidation was determined in frozen renal homogenates by protein carbonyl assay. Carbonyl content was calculated with ε = 22,000 1/M cm, and results were expressed as nmol eq. carbonyl groups/mg protein (Lenz, Costabel, Shatiel, & Levine, 1989). Protein nitrosylation level was determined by determining 3-nitrotyrosine levels using an ELISA kit (Alpco Diagnostics). Protein concentration was determined by Bradford assay (Bradford, 1976).

2.3. Animals

2.8. Protein isolation and microarray analysis

Male Wistar rats of 180–200 g were from the Institute of Neurobiology (UNAM, Juriquilla, Querétaro, México). Animals were housed at 23–24 °C with a 12/12 h light–dark cycle. Water and standard pellet diet (NIH-31, Zeigler Bros Inc., PA, USA) were administered ad libitum. Experiments on animals were performed in agreement with the Animal Care and Use protocol of the UAQ, based on the Guidelines for Animal Testing of the Mexican Official Norm (NOM-062-ZOO-1999).

Total protein was extracted from the renal tissue according to the method previously described (Sharma et al., 2014). Protein integrity was evaluated by SDS-PAGE electrophoresis, and total protein was estimated at 280 nm using a NanoDrop system (Thermo Scientific, Rockford, IL, USA). Thirty-four proteins were identified using an inflammation-related protein microarray (AAR-CYT-2, RayBiotech, GA, USA) according to the manufacturer procedure, using 500 mg of protein/blot, detected and quantified by chemiluminescence using horseradish peroxidase. Data were analyzed with the software Ingenuity Pathways Analysis (IPA Qiagen, CA, USA).

2.4. Diabetes induction and subchronic effect Thirty-two rats were divided into four groups of eight animals each. A commercial standard diet (6% fat, 22% protein and 70% carbohydrate) was administered ad libitum to the healthy control group, whereas a high-fat diet (32% fat, 18% protein and 48% carbohydrate) was given to the diabetic groups for five weeks. Afterward, diabetes was induced in overnight fasting animals by an intraperitoneal injection of streptozotocin (STZ) dissolved in 0.1 M citrate buffer (pH 4.5) at a dose of 30 mg/kg body weight (Qian, Zhu, Yu, Jiang, & Zhang, 2015). After one week, blood samples were taken from the tail vein, and glucose concentration was measured. Rats with blood glucose levels of

2.9. Statistical analyses All results were expressed as mean values ± standard error (SE). Data was analyzed by one-way ANOVA and differences among treatments were determined by comparison of means using Tukey’s test (P < .05). All statistical analyses were carried out with JMP software (v11.0, SAS Institute). 56

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3. Results

Table 1 Identification of phytochemical constituents in SA-elicited peppermint infusions by HPLCDAD-MSD. Proposed compound

Phenolic acids Chlorogenic acid Gallic acid Protocatechuic acid p-Hydroxybenzoic acid Caffeic acid Syringic acid p-Coumaric acid Sinapic acid Ferulic acid Rosmarinic acid Cinammic acid Ellagic acid Vanillic acid Flavonoids Catechin Epicatechin Hesperidin Gallocatechin gallate Epigallocatechin gallate Epicatechin gallate Quercetin-3-Ogalactoside Quercetin-3-Oglucoside Trans-resveratrol

Retention time (min)

[M-H]

Control peppermint

2 mM SA peppermint

3.8 5.5 12.6 17.7

100 169 153 137

3.1 ± 0.2a 7.3 ± 0.5b LDL 11.5 ± 0.7b

LDL 12.7 ± 0.9a 9.8 ± 0.5a 25.3 ± 1.3a

23.5 25.5 31.4 32.6 33.0 34.3 35.1 37.4 38.5

179 197 163 223 193 360 148 302 167

21.6 ± 1.2a LDL 2.1 ± 0.1a 2.9 ± 0.5b 15.7 ± 1.3a 40.2 ± 2.0b 8.9 ± 0.6a 26.1 ± 1.5a 5.3 ± 0.3a

29.4 ± 1.5a 1.5 ± 0.1a 2.6 ± 0.1a 5.1 ± 0.3a 17.4 ± 1.4a 92.3 ± 4.0a LDL 21.6 ± 1.3a LDL

17.6 25.6 37.6 38.5 39.9

289 289 610 457 457

10.5 ± 0.9a 7.9 ± 0.5b 53.4 ± 2.3b 1.4 ± 0.1a 4.8 ± 0.3a

12.1 ± 0.9a 12.8 ± 0.8a 104.1 ± 4.1a 1.9 ± 0.2a 2.9 ± 0.3a

41.5 49.6

441 463

6.5 ± 0.4a LDL

1.4 ± 0.1b 1.5 ± 0.2a

50.2

463

11.5 ± 0.7b

20.3 ± 1.3a

59.6

227

0.3 ± 0.0b

5.3 ± 0.6a

b

a

3.1. Phytochemical characterization of peppermint infusions Previously, we reported that the treatment with 2 mM SA significantly increased the content of rosmarinic acid, gallocatechin gallate, naringenin, and hesperidin (Figueroa-Perez et al., 2014). However, no strong association was identified between the phenolic compounds and the biological effects (Figueroa-Pérez et al., 2015). We extended the analysis of the polyphenolic profile using HPLCDAD-MSD, 13 phenolic acids, and 9 flavonoids were identified (Table 1). Protocatechuic and syringic acids, as well as quercetin-3-Ogalactoside, were only detected in SA-elicited peppermint infusions, whereas chlorogenic, cinnamic, and vanillic acids were only detected in the control infusion. Also, gallic, pehydroxybenzoic, sinapic, and rosmarinic acids, as well as epicatechin, hesperidin, quercetin-3-O-glucoside, and trans-resveratrol contents were significantly higher in SAelicited peppermint infusions compared to the control infusion (Table 1). The treatment with SA alters the profile of other bioactive compounds, such as carotenoids, chlorophylls, and phytosterols. We identified five carotenoids, two chlorophylls, and seven phytosterols (Table 1). SA-elicited peppermint infusions presented an increased amount of lutein, α-tocopherol, chlorophyll a, β-sitosterol, β-campesterol, and Δ5-avenasterol as compared to the control infusion, and violaxanthin, campestanol, and Δ7-avenasterol were only identified in the SA-elicited peppermint infusion.

3.2. Renal function parameters and morphology

Carotenoids Lutein Violaxanthin γ-Tocopherol α-Tocopherol Neoxanthin

3.1 5.8 6.4 6.7 10.3

550 601 417 430 601

0.7 ± 0.0 LDL 21.5 ± 1.2a 31.7 ± 1.6b 9.5 ± 0.6a

4.3 ± 0.5 6.9 ± 0.7a 22.8 ± 1.5a 61.8 ± 4.1a LDL

Chlorophylls Chlorophyll a Chlorophyll b

4.7 7.5

894 908

57.8 ± 2.1b 43.1 ± 1.6a

97.8 ± 4.1a 27.1 ± 1.4a

Phytosterols β-Sitosterol β-Campesterol Campestanol Δ5-Avenasterol Δ7-Stigmasterol Δ7-Avenasterol Stigmastanol

3.6 4.2 4.6 5.3 5.8 6.4 8.1

414 400 402 412 412 412 416

29.4 ± 1.5b 6.1 ± 0.4b LDL 0.5 ± 0.0b 35.7 ± 1.9a LDL 13.3 ± 0.8a

76.4 ± 3.0a 14.2 ± 1.1a 2.1 ± 0.2a 3.6 ± 0.3a 49.5 ± 3.7a 2.0 ± 0.2a 13.2 ± 1.1a

After the STZ injection, diabetic animals showed fasting blood glucose values of 168 ± 7.11 mg/dL and body weight of 220 ± 4.08 g (Data not shown). At the end of the experiment, fasting blood glucose increased to 196 ± 8.1 mg/dL in the diabetic control group, 180 ± 7.6 mg/dL in the control peppermint group, and 148 ± 6.2 in the 2 mM SA peppermint group (Figueroa-Pérez et al., 2015), whereas body weight increased to 462 ± 8.8 g without showing significant differences between the diabetic groups (Data not shown). In diabetic nephropathy, some metabolites are poorly filtrated in glomeruli or reabsorbed in tubules, and are used as markers for renal dysfunction. Table 2 shows that diabetic animals presented a significant increase of urine microalbumin, urine urea, and serum uric acid compared to healthy animals (1.4, 1.3, and 4.4-fold, respectively), suggesting the development of renal injury. Diabetic animals treated with peppermint infusions decreased the urine microalbumin, urine urea and serum uric acid levels as compared to the diabetic control. No microscopic differences were observed in either glomerular or tubular structures in any of the studied animals (Fig. 1). The absence of

Data are expressed as ng/mL. Results are the average of three independent determinations ± SD. a,bDifferent letters in the same row indicate significant difference (P < .05) by Tukey’s test. SA: salicylic acid, LDL: lower than the detection limit.

Table 2 Effect of SA-elicited peppermint infusions on renal function parameters and oxidative in high-fat diet and STZ-induced diabetic rats. Parameter

Healthy group

Diabetic group

Control peppermint

2 mM SA peppermint

Urine microalbumin§ Urine ureaω Serum uric acidω TBARS* Carbonyl residues* 3-nitrotyrosine*

94.0 ± 11.0b 303.0 ± 11.8b 2.9 ± 0.1c 1.59 ± 0.2b 2.43 ± 0.3b 1.4 ± 0.2b

133.9 ± 9.7a 387.7 ± 15.6a 12.7 ± 0.3a 2.39 ± 0.2a 3.78 ± 0.1a 2.6 ± 0.1a

113.83 ± 12.1ab 267.4 ± 19.0c 8.5 ± 0.3abc 2.31 ± 0.3a 3.10 ± 0.3ab 2.0 ± 0.0a

103.3 ± 11.3b 294.1 ± 19.3bc 5.4 ± 0.1bc 2.02 ± 0.2ab 2.55 ± 0.2b 1.8 ± 0.2b

Data are mean ± standard error for eight rats in each group. a,b,cDifferent letters in the same row indicate significant difference (P < .05) by Tukey’s test. §µg/(12 h), ωmg/dL, *nmol/mg of protein. SA: salicylic acid.

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Fig. 1. Renal histological analysis of (a) healthy group, (b) diabetic group, (c) control peppermint infusion group, and (d) 2 mM SA-elicited peppermint infusion group. Arrows indicate lumen area of renal arterioles.

Table 3 Effect of SA-elicited peppermint infusions on renal inflammation-related proteins in high-fat diet and STZ-induced diabetic rats. Protein

Healthy group

Diabetic groupa

Control peppermintb

Activin A Agrin B7-2/CD86 CINC-1 CINC-2 alpha CINC-3 CNTF Fas Ligand Fractalkine GM-CSF ICAM-1 IFN gamma IL-1 alpha IL-1 beta IL-1 R6 IL-2 IL-4 IL-6 IL-10 IL-13 Leptin LIX L-Selectin MCP-1 MIP-3 alpha MMP-8 NGF beta PDGF-AA Prolactin R RAGE Thymus Chemokine-1 TIMP-1 TNF alpha VEGF

35,924 63,447 27,171 57,125 84,708 43,292 51,806 23,978 64,990 8635 27,688 36,386 28,547 52,594 33,180 29,354 44,426 29,690 33,884 27,985 39,139 21,657 43,078 33,987 41,801 46,426 18,194 57,247 54,691 74,222 56,789 37,298 21,011 59,619

50,294 96,439 64,124 101,111 165,181 119,486 168,370 84,882 110,483 25,905 57,868 86,235 56,238 116,233 55,411 58,708 92,850 49,582 72,851 63,526 85,714 53,493 84,002 68,314 83,602 97,030 29,110 106,479 99,538 185,555 130,047 100,705 26,894 108,507

1.40 1.52 2.36* 1.77 1.95 2.76* 3.25* 3.54* 1.70 3.00* 2.09* 2.37* 1.97 2.21 1.67 2.00* 2.09* 1.67 2.15* 2.27* 2.19* 2.47* 1.95 2.01* 2.00* 2.09* 1.60 1.86 1.82 2.50* 2.29* 2.70* 1.28 1.82

2 mM SA peppermintb 38,687 91,847 41,105 62,414 93,322 71,123 111,503 80,840 79,484 12,514 36,859 50,430 36,757 74,508 42,954 46,227 73,111 44,270 68,727 61,083 60,362 37,671 51,535 41,655 51,606 67,382 15,735 74,461 73,189 86,708 99,272 73,507 16,104 59,948

1.30 1.05 1.56 1.62 1.77 1.68 1.51 1.05 1.39 2.07* 1.57 1.71 1.53 1.56 1.29 1.27 1.27 1.12 1.06 1.04 1.42 1.42 1.63 1.64 1.62 1.44 1.85 1.43 1.36 2.14* 1.31 1.37 1.67 1.81

33,529 74,184 41,911 47,920 82,590 62,232 88,152 58,138 64,990 8933 26,545 39,198 25,916 60,855 32,983 40,211 58,031 35,671 32,523 52,501 49,261 24,997 43,524 32,376 39,810 52,449 16,354 60,500 54,691 63,114 74,740 49,608 23,185 54,253

1.50 1.3 1.53 2.11* 2.00* 1.92 1.91 1.46 1.70 2.90* 2.18* 2.20* 2.17* 1.91 1.68 1.46 1.60 1.39 2.24* 1.21 1.74 2.14* 1.93 2.11* 2.10* 1.85 1.78 1.76 1.82 2.94* 1.74 2.03* 1.16 2.00*

Data are expressed in arbitrary density units (fold-change) (n = 4). SA: salicylic acid. a Fold change as compared to the healthy group. b Fold change as compared to the diabetic group. * Indicate significant fold-change (≥2.0).

nephropathy, characterized by the accumulation of serum proteins in the sub-endothelial space due to impaired autoregulation that increases the systemic pressure to the glomerulus and the risk of their injury (Olson, 2003). Interestingly, animals provided with SA-elicited peppermint infusions presented normal arterioles, similar to those from the healthy group (Fig. 1A vs. D). The renal arteriolar lumen/media ratio

morphological changes did not suggest the development of a renal injury. However, the diabetic group presented an augmented arterial wall diameter and a decreased lumen area as compared to the healthy group (Fig. 1A vs. B) and the renal arteriolar lumen/media ratio decreased up to 73% (data not shown). These alterations are distinctive of hyaline arteriolosclerosis, a vascular lesion commonly observed in diabetic 58

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inflammation-related proteins using a pathway-focused protein-microarray. The diabetic group presented a significant (≥2.0-fold) increase in 20 proteins as compared to the healthy group (Table 3), of which 13 have been previously related to kidney injury, such as tubule damage, expansion of mesangial matrix, glomerular growth, and cell death (Fig. 2). Diabetic animals treated with control peppermint infusions presented a decreased content, at least 2-fold, of GM-CSF, and RAGE as compared to the diabetic group (Table 3). SA-elicited peppermint infusions presented a significantly decreased content of 13 proteins, such as CINC-1, CINC-2 alpha, GM-CSF, ICAM-1, IFN-gamma, IL-1 alpha, IL10, LIX, MCP-1, MIP-3 alpha, RAGE, TIMP-1, and VEGF as compared to the diabetic group (2.0–2.9-fold) (Fig. 3).

for diabetic animals was 0.14 ± 0.02 arbitrary units. Meanwhile animal treated with control peppermint and SA-elicited peppermint showed values of 0.30 ± 0.07 and 0.53 ± 0.01 arbitrary units respectively (data not shown). 3.3. Renal oxidative stress Oxidative stress is a pathogenic factor related to renal injury. It promotes protein and lipid damage that can severely compromise cell health and viability, and induces detrimental cellular responses through the generation of secondary reactive species, leading to cell death (Dalle-Donne, Rossi, Colombo, Giustarini, & Milzani, 2006). The infusions of SA-elicited peppermint have a high content of antioxidant compounds, and their renoprotective effects could be associated with the amelioration of oxidative stress. As expected, the diabetic group increased presented an increased renal content of TBARS, carbonyl residues, and 3-nitrotyrosine as compared to the healthy group (1.5, 1.6, and 1.9-fold, respectively) (Table 2), indicating the development of oxidative and nitrossative stress. The administration of SA-elicited peppermint infusions significantly decreased carbonyl residues and 3-nitrotyrosine content, showing similar values as the healthy group (Table 2), whereas no beneficial effect was observed on renal TBARS since this group showed similar values as the diabetic control group. The animals treated with control peppermint infusions did not show reduction in oxidative products. These results support our hypothesis that the renoprotective effect of SA-elicited peppermint infusions could be related to the amelioration of oxidative and nitrossative damage.

4. Discussion Several efforts have been made for diabetes prevention and control. Different strategies to stop or delay their appearance are under development, and one alternative is the increase of functional food in the human diet. Numerous studies demonstrate the beneficial effects of functional nourishments trough their administration to animal models or controlled studies in humans. The consumption of these products increased in the last years, however limited information about their bioactive composition and their biological effects is available. Therefore, it is necessary to evaluate them to provide adequate information to consumers. Peppermint infusion has several reported benefits on digestive, respiratory, immune, and cardiovascular systems (McKay & Blumberg, 2006; Badal et al., 2011), associated with its phytochemical content, which can be enhanced by elicitation using 2 mM salicylic acid (SA) during its cultivation (Figueroa-Perez et al., 2014). SA is an endogenous plant growth regulator that plays an important role in several physiological processes, including photosynthesis and development of systemic acquired resistance (SAR). If applied exogenously, SA causes oxidative stress to plants, partly through the accumulation of hydrogen peroxide (Horvaáth, Szalai, & Janda, 2007). A

3.4. Renal inflammation Oxidative stress, triggered by diabetes, has been associated with the development of inflammation, characterized by the accumulation of pro-inflammatory cytokines, adhesion molecules and certain growth factors (Tucker, et al., 2015). Therefore, the effect of SA-elicited peppermint infusion was evaluated on the renal accumulation of thirty-four

Fig. 2. Network analysis of inflammation-related proteins in the kidney of STZ-induced diabetic rats.

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Fig. 3. Network analysis of inflammation-related proteins modulated in the kidney of STZ-induced diabetic rats treated with 2 mM SA-elicited peppermint infusions.

Olas, Nowak, Ponczek, and Wachowicz (2006) showed that resveratrol protects platelet proteins against oxidation by ONOO−, and it minimizes LDL modification by the inhibition of carbonyl groups formation (Brito, Almeida, & Dinis, 2002). Consequently, the improved phytochemical profile of infusions elaborated with elicited peppermint suggests that their infusions could produce a greater renoprotective effect than control peppermint infusions. Other compounds, not identified in this study, could be responsible, also, for the biological effects observed in this study. Furthermore, phytochemicals could produce synergic effects; for example, it has shown that the binary combination of gallic acid, rosmarinic acid, caffeic acid, chlorogenic acid and quercetin shows higher antioxidant capacity than their ternary combination or individual compounds (Hajimehdipoor, Shahrestani, & Shekarchi, 2014). Oxidative stress increases the production of inflammatory cytokines, which are involved in the development of renal damage (Chen et al., 2011), and the effect of peppermint has not been evaluated on renal inflammation and fibrosis-related proteins. In our study, several inflammatory proteins, adhesion molecules, and growth factors increased in kidneys from the diabetic animals. Those proteins were associated with tubule damage, expansion of mesangial matrix, glomerular growth, and cell death. Rats treated with infusions elaborated with control peppermint showed a decreased renal content of GM-CSF and RAGE, and those treated with SA-elicited peppermint infusions modulated the accumulation of those proteins and other eleven, including ICAM-1, IFN-gamma, IL-1 alpha, IL-10, MCP-1, MIP-3 alpha, RAGE, TIMP-1, and VEGF. Diabetes promotes the formation of advanced glycation ends (AGEs) and they have been widely linked to kidney damage (Sho-ichi & Takanori, 2010). AGEs induce apoptosis and VEGF expression in mesangial cells, increasing vascular permeability, and thus leading to the development of hyperfiltration and proteinuria (De Vriese, Flyvbjerg, Mortier, Tilton, & Lameire, 2003). Additionally, AGEs increase the activation of nuclear factor-κB (NF-κB), which increases the expression of several pro-inflammatory cytokines, such as interleukins 10 and 1β,

larger quantity of ROS is produced in the leaves, generating changes in the cellular redox homeostasis, which increases the biosynthesis of flavonoids and phenolic acids (Tajik et al., 2015). Cardiovascular diseases and diabetic nephropathy are leading causes of mortality among diabetic patients. Nephropathy is characterized by the increase of several metabolites, although other biochemical and morphological changes can be detected associated to the hyperglycemia-induced damage of the kidney. Diabetic animals treated with peppermint infusion showed improved biochemical parameters related to renal function as compared to the diabetic animals. Interestingly, rats treated with infusions prepared from elicited plants showed healthier parameters. These effects of peppermint had been reported, the administration of a peppermint extract for 21 days significantly decreased serum uric acid and urine protein (Ullah et al., 2014). Oxidative stress is involved in the development and progression of renal disease in diabetic patients (hojs, Ekart, Bevc, & Hojs, 2016). Diabetic animals showed an increased renal content of oxidized lipids and proteins. These oxidative processes were diminished with peppermint infusion, being more significantly the effect in animals treated with SA-elicited peppermint. Rosmarinic acid, hesperidin, trans-resveratrol and α-tocopherol were increased in 2 mM SA-elicited peppermint, and it has reported that these compounds ameliorate renal injury through the modulation of oxidative stress (Ozturk, Terzi, Ozgen, Duran, & Uygun, 2014; Balakrishnan et al., 2013; Tirkey, Pilkhwal, Kuhad, & Chopra, 2005; Palsamy & Subramanian, 2011) and, resveratrol exerts a renoprotective effect through the down-regulation of profibrotic proteins (Chen et al., 2011). On the other hand, α-tocopherol exerts protective effects against chromium-induced kidney damage through the inhibition of lipid peroxidation (Balakrishnan et al., 2013). Several phytosterols, such as βsitosterol have been reported to improve oxidative stress in STZ-induced diabetic rats (Gupta, Sharma, Dobhal, Sharma, & Gupta, 2011), whereas β-sitosterol, stigmasterol, and β-campesterol showed decreased in vitro lipid peroxidation (Yoshida & Niki, 2003). Otherwise, 60

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monocyte chemoattractant peptide 1 (MCP-1), and metalloproteinases exacerbating the inflammatory response and damage in glomeruli (Nowotny, Jung, Höhn, Weber, & Grune, 2015; Cornish et al., 2009). The modulation of these proteins (RAGE, IL10, IL1β) by SA-elicited peppermint infusion suggests a decreased glomerular hypertrophy and mesangial expansion matrix. Protocatechuic acid has shown to decrease the renal content of type-IV collagen, fibronectin, transforming growth factor-β1 (TGF- β1), and MCP-1, and glycated albumin in urine (Lin, Tsai, Huang, & Yin, 2011). Resveratrol and quercetin have been reported to suppress the activation of pro-inflammatory cytokines and iNOS genes, resulting in a decreased secretion of TNF-α, IL-1β, IL-6, and NO levels in murine macrophage cells (Qureshi et al., 2012). These results may be related to the decreased urine microalbumin and urea, and serum uric acid levels observed in rats treated with SA-elicited peppermint infusion.

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5. Conclusions The results obtained in this study demonstrated that the application of 2 mM salicylic acid during peppermint cultivation produces infusions with greater renoprotective effects in high-fat diet and STZ-induced diabetic rats. These renoprotective effects, which may be related to their high content of phenolic compounds, chlorophylls, carotenoids, and phytosterols, were associated with a decreased oxidative stress and the modulation of proteins related to tubular injury, glomerular hypertrophy, mesangial matrix expansion, and cell death. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported by Investigación Científica Básica CONACyT (222767). We are grateful to Ht. Evelyn Flores Hernández for the technical assistance in the histologic analysis. References Amaya-Cruz, D. M., Rodríguez-González, S., Pérez-Ramírez, I. F., Loarca-Piña, G., AmayaLlano, S., Gallegos-Corona, M. A., & Reynoso-Camacho, R. (2015). Juice by-products as a source of dietary fiber and antioxidants and their effect on hepatic steatosis. Journal of Functional Foods, 17, 93–102. Badal, R. M., Badal, D., Badal, P., Khare, A., Shrivastava, J., & Kumar, V. (2011). Pharmacological action of Mentha piperita on lipid profile in fructose-feed rats. Iranian Journal of Pharmaceutical Research, 10, 843–848. Balakrishnan, R., Satish-Kumar, C. S., Rani, M. U., Srikanth, M. K., Boobalan, G., & Reddy, A. G. (2013). An evaluation of the protective role of α-tocopherol on free radical induced hepatotoxicity and nephrotoxicity due to chromium in rats. Indian Journal of Pharmacology, 45, 490–495. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analitical Biochemistry, 7, 248–254. Brito, P., Almeida, L. M., & Dinis, T. C. (2002). The interaction of resveratrol with ferrylmyoglobin and peroxynitrite; protection against LDL oxidation. Free Radicicals Research, 36, 621–631. Chen, K. H., Hung, C. C., Hsu, H. H., Jing, Y. H., Yang, C. W., & Chen, J. K. (2011). Resveratrol ameliorates early diabetic nephropathy associated with suppression of augmented TGFβ/smad and ERK1/2 signaling in streptozotocin-induced diabetic rats. Chemical Biological Interactions, 190, 45–53. Cornish, T. C., Bagnasco, S. M., Macgregor, A. M., Lu, J., Selvin, E., & Halushka, M. K. (2009). Glomerular protein levels of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 are lower in diabetic subjects. Journal of Histochemistry and Cytochemistry, 57, 995–1001. Dalle-Donne, I., Rossi, R., Colombo, R., Giustarini, D., & Milzani, A. (2006). Biomarkers of oxidative damage in human disease. Clinical Chemistry, 52, 601–623. De Vriese, A. S., Flyvbjerg, A., Mortier, S., Tilton, R. G., & Lameire, N. H. (2003). Inhibition of the interaction of AGE–RAGE prevents hyperglycemia-induced fibrosis of the peritoneal membrane. Journal of the American Society of Nephrology, 14, 2109–2118. Figueroa-Perez, M. G., Rocha-Guzmán, N. E., Mercado-Silva, E., Loarca-Piña, F., &

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