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An Aqueous Extract of Ilex paraguariensis Reduces CarrageenanInduced Edema and Inhibits the Expression of Cyclooxygenase-2 and Inducible Nitric Oxide Synthase in Animal Models of Inflammation

DOI 10.1055/s-0034-1382876 Planta Med 2014; 80: 961–968

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Publisher and Copyright: © 2014 by Georg Thieme Verlag KG Rüdigerstraße 14 70469 Stuttgart ISSN 0032‑0943 Reprint with the permission by the publisher only

Original Papers

961

Authors

Guillermo Schinella 1, 4, Elisa Neyret 2, Gloria Cónsole 3, 4, Horacio Tournier 1, 4, José M. Prieto 5, José-Luis Ríos 2, Rosa María Giner 2

Affiliations

The affiliations are listed at the end of the article

Key words " Ilex paraguariensis l " Aquifoliaceae l " mate l " anti‑inflammatory effects l " in vivo l

Abstract

received revised accepted

April 14, 2014 June 6, 2014 June 13, 2014

Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382876 Published online August 4, 2014 Planta Med 2014; 80: 961–968 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Guillermo Schinella Cátedra de Farmacología Básica Facultad de Ciencias Médicas Universidad Nacional de La Plata Av 60 and 120 1900 La Plata Argentina Phone: + 54 22 14 21 69 32 [email protected]

!

Mate (Ilex paraguariensis) is a highly popular herbal beverage in South America due to its high content of caffeine. Its hypolipidemic and antioxidant properties are of increasing interest in the treatment of cardiovascular disorders and for weight control. In the present study, we show for the first time both the local and systemic anti-inflammatory effects of an aqueous extract of mate in three classic in vivo models, namely acute and chronic 12-O-tetradecanoylphorbol 13-acetateinduced mouse ear edema and acute carrageenan-induced mouse paw edema. Caffeine, rutin, chlorogenic acid, 3,5-dicafeoyl quinic acid, and 4,5-dicafeoyl quinic acid, accompanied by a complex mixture of other simple phenolic acids, were identified in the extract by HPLC‑UV analyses. In the acute edema model, mate extract applied topically (1 mg/ear) halved the 12-O-tetradecanoylphorbol 13-acetate-induced acute edema (50%) and almost suppressed neutrophil infiltration (93%), while in the 12-O-tetradecanoylphorbol 13-acetate-induced subchronic inflammation, the edema was significantly reduced by 62 % (1 mg/ear/day × seven doses). The oral administration of the mate extract (250 mg/kg) significantly reduced the carrageenan-induced edema at all time points, an effect which was accom-

Introduction !

Ilex paraguariensis St. Hil. var. paraguariensis (Aquifoliaceae) is commonly known as “mate”, “yerba mate”, or “té del Paraguay”. Originally used for the preparation of psychostimulant beverages among native Guaranies, mate was incorporated into the colonizerʼs diet in different South American countries such as Paraguay, Uruguay, Northeastern Argentina, and Southern Brazil. Its high content of caffeine converted it into a highly pop-

panied by a 43% and 53 % reduction of the expression of cyclooxygenase-2 and inducible nitric oxide synthase, respectively. Histological analyses confirmed a reduction of epithelium thickness, dermis with mild inflammation, hair follicles with some secretory cells of sebaceous glands, and hypodermic adipocytes. In conclusion, mate is endowed with in vivo preventative or therapeutic anti-inflammatory effects in both local and systemic inflammatory processes.

Abbreviations !

ABTS•+: COX: FRAP: IL: iNOS: MPO: NF-κB: NO: NSAID: PG: PMN: TPA:

2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) cyclooxygenase ferric reducing ability of plasma interleukin inducible nitric oxide synthase myeloperoxidase nuclear factor-κB nitric oxide nonsteroidal anti-inflammatory drug prostaglandin polymorphonuclear neutrophil 12-O-tetradecanoylphorbol 13acetate

ular social beverage, successfully rivalling tea and coffee. Although incorporated very soon within the American and European phytotherapy systems [1, 2], it was only in recent years that its therapeutic potential, other than as a psychostimulant, became a global interest, mainly for its antioxidant, vasodilator, hypolipidemic, and weight control properties [1–3]. The upsurge of Korean, Japanese, Chinese, North American, and European research teams focusing on mate motivated the establishment of an international soci-

Schinella G et al. An Aqueous Extract …

Planta Med 2014; 80: 961–968

Electronic reprint for personal use

An Aqueous Extract of Ilex paraguariensis Reduces Carrageenan-Induced Edema and Inhibits the Expression of Cyclooxygenase-2 and Inducible Nitric Oxide Synthase in Animal Models of Inflammation

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Fig. 1 HPLC and UV profiles of the extract lyophilized from I. paraguariensis. The chromatogram was run in an RP-18 column in gradient mode as follows: 0 min 90 % A (H2O + acetic acid 0.2%, v/v) and 10 % B (methanol + acetic acid 0.2 %, v/v), 0–5 min 80% A, 5–65 min 50 % A, 65–75 min 20 % A; flow rate 0.8 mL/min. The chromatograms are representative of three different experiments. Identified compounds were chlorogenic acid (peak 3), caffeine (peak 4), 3,5-dicaffeoylquininic acid (peak 7), and 4,5-dicaffeoylquininic acid (peak 8). Peaks 1, 2, and 5 were identified according to the experimental values described by Bravo et al. [20] as a complex mixture of neochlorogenic acid (peak 1), dicaffeic acids (peak 1 + 2), and feruloyl acids (peak 5). (Color figure available online only.)

ety promoting active research on this plant through the organization of international symposia [1]. The phytochemistry of mate is characterized by the presence of xanthines (caffeine and theobromine) and polyphenols (chlorogenic acid, gallocatechin, 4,5-dicaffeoyl quinic acid, gallic acid, and epicathechin). The latter are endowed with pleiotropic pharmacological effects which have an impact on metabolism, inflammation, and mutagenesis as revealed in vitro and in vivo [1]. In particular, research using various mate extracts has shown that they can reduce LDL-cholesterol levels both in animals [4, 5] and in humans with dyslipoproteinemia [6]. Importantly, this effect is synergic with statin therapy, and could be used to reduce the dose of this drug [6]. Mate is also a potential therapeutic agent for obesity [7–9]. This effect could be associated with a decrease of lipid absorption, and, therefore, caloric intake [10, 11], the inhibition of pancreatic lipase activity [12], and the modulation of the expression of different genes implicated with obesity [13–15]. The systemic antioxidant properties of mate have been studied in both humans and animals. Reduced plasma oxidative stress parameters and reduced leukocyte antioxidant enzyme gene expression were achieved after consumption of mate tea by healthy young women [16], while mice exposed to cigarette smoke showed reduced acute lung inflammation with the same treatment (150 mg/kg, per os or i. p.) [17]. The contribution of mate polyphenols was preliminary demonstrated by Xu et al. [18]. Their research reports that different dicaffeoylquinic acid derivatives and flavonoids inhibit human neutrophil elastase with IC50 values ranging from 1.4 to 7.3 µM. Surprisingly, no studies on the effects of mate extracts on classic in vivo models used for the preclinical characterizsation of antiinflammatory effects have been reported to our knowledge. Only very recently, some preliminary evidence of the potential of mate in diminishing in vivo inflammatory parameters emerged from nutritional studies [19]. Therefore, the aim of this work was to study the properties of the traditional aqueous extract of mate on acute and chronic TPA-induced mouse ear edema and acute λ-carrageenan-induced mouse paw edema models, which are considered standard in vivo models of inflammation in natural product research. The biochemical and histological features of its anti-inflammatory mechanism were also studied in biopsies from the inflamed tissue.

Schinella G et al. An Aqueous Extract …

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Results !

The major identified compounds in the original aqueous extract (lyophilized), with the help of pure standards, were chlorogenic acid (peak 3), caffeine (peak 4), rutin (peak 6), 3,5-dicafeoyl quinic acid (peak 7), and 4,5-dicafeoyl quinic acid (peak 8), three of which are the major compounds present in ethyl acetate ex" Fig. 1). The unresolved peaks are typically composed of tract (l a complex mixture of neochlorogenic acid (peak 1), dicaffeic acids (peaks 1 and 2) and feruloyl acids (peak 5), with all of them showing the same UV typical spectra (λmax 324/326, 296 sh) as " Fig. 1). previously described by Bravo et al. [20] (l The value for total phenolics determined by the Folin-Ciocalteu method was 18 mg gallic acid equivalent per mg of extract. The antioxidant activity measured by the ABTS•+ assay was 6 mg gallic acid equivalent per mg of extract, and by the FRAP assay it was 33 mg gallic acid equivalent per mg of extract. In the carrageenan test, only the higher dose (250 mg/kg) of mate extract significantly reduced the edema by 29%, 53 %, and 31 %, at 1 h, 3 h, and 5 h, respectively, whereas indomethacin (10 mg/kg) reduced it by 53% and 38 %, at 3 h and 5 h, respectively, without " Table 1 A). The neutrophil infiltration was sigan effect at 1 h (l nificantly reduced by 24 % in both indomethacin and the mate ex" Table 1 B). Finally, a tract, but in this case only at a higher dose (l clear inhibition of the expressions of COX-2 and iNOS was observed in the treated groups in the carrageenan test, with a re" Fig. 2 A) and 53 % duction of about 43 % for COX-2 expression (l " Fig. 2 B). for iNOS expression (l In the acute TPA test, the mate extract (1 mg/ear) reduced the edema by 50% (6.48 ± 1.78, p < 0.01), whereas indomethacin at " Table 2 A). 0.5 mg/ear reduced it by 91 % (1.18 ± 0.42, p < 0.01) (l Neutrophil infiltration was significantly reduced by 93 %, where" Table 2 B). On the other as indomethacin reduced it by 100% (l hand, the subchronic inflammation induced by the multiple application of TPA was reduced by 62 % after repeated topical application of mate extract (1 mg/ear × 7 doses). In the case of the positive control, dexamethasone (at 0.025 mg/ear × 4 doses) signifi" Table 3 A). Neutrophil infilcantly reduced the edema by 83% (l tration was significantly reduced by 71 %, whereas dexametha" Table 3 B). sone reduced it by 99% (l " Fig. 3 A) with The histological study evidenced a control group (l an epithelium thickness of 5.1 ± 0.4 cells. Isolated fibroblasts and histiocytes were observed at the papillary dermis level, the fibrillar component predominated in the reticular dermis, and a row of adipocytes was identified in the hypodermis. The TPA-treated

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Table 1 Effect of the extract lyophilized from I. paraguariensis (125 and 250 mg/kg, p. o.) and indomethacin (10 mg/kg, p. o.) on carrageenan-induced mouse paw edema. A Footpad edema was induced 1 h later by subplantar injection of carrageenan (3% w/v in saline). Footpad volume was measured 1 h, 3 h, and 5 h after irritant injection. Paw edema is expressed as the mean of the increase of footpad volume in µL ± SD. B Neutrophil accumulation was assessed as MPO activity, and results are expressed as the mean of absorbance (A) at 630 nm ± SD and refers to 1 mg of protein. Statistically significant differences with respect to the control are expressed as **p < 0.01 (n = 6, Dunnettʼs t-test). Percentages of inhibition are expressed as the reduction of increase in ear weight with respect to the control group. A) 1 h (µL) ΔV ± SD 6.0 ± 2.7 6.5 ± 1.8

3 h (µL) %I – −8

5 h (µL) ΔV ± SD 12.4 ± 2.9 9.5 ± 1.6

4.2 ± 1.5**

29

5.8 ± 1.9**

53

7.8 ± 2.1**

31

7.2 ± 1.7

− 19

5.8 ± 1.3**

53

7.0 ± 1.5**

38

(A ± SD/mg protein) × 103 269.86 ± 83.24 739.45 ± 125.57 658.16 ± 70.98

% Inhibition

565.11 ± 60.45**

24

562.45 ± 109.03**

24

%I – 23

ΔV ± SD 11.3 ± 2.8 8.2 ± 1.5

%I – 28

– 11

Fig. 2 Inhibitory effect of the extract lyophilized from I. paraguariensis on cyclooxygenase-2 (A) and inducible nitric oxide synthase (B) expressions determined by densitometry analysis and normalized to β-actin content for each. A representative Western blot of each experiment is shown. Animals were sacrificed 5 h after irritant injection, and tissue proteins were extracted from the paw of the carrageenan test as indicated in the Materials and Methods section. Statistically significant differences with respect to the control were determined by Dunnettʼs t-test and expressed as *p < 0.05 and **p < 0.01 (n = 5). Blank (without treatment), Control (carrageenan group, without treatment), Ind (indomethacin-treated group), Mate (mate-treated group).

" Fig. 3 B) showed a hypertrophic and hyperplasic epithegroup (l lium thickness of 6.2 ± 0.6 cells. At the level of the dermis, a marked inflammatory infiltration with polymorphonuclear neutrophils, histiocytes, and numerous blood vessels was observed. Microphotography showed hair follicles and adipocytes in the " Fig. 3 C) hypodermis. The mate extract-treated samples (l showed a reduced epithelium thickness of 4.8 ± 0.3 cells, similar to that observed in the dexamethasone-treated group. The dermis presented blood capillaries and a mild inflammation with predominant lymphocytes. Hair follicles with some secretory cells of sebaceous glands and hypodermic adipocytes were also " Fig. 3 D) evidetected. The dexamethasone-treated group (l denced a lesser epithelium thickness of 4.3 ± 0.4 cells, numerous

blood capillaries, an absence of inflammatory infiltration, and a row of hypodermic adipose cells in the dermis.

Discussion !

We established here for the first time the potential of the lyophilized aqueous mate extract as a systemic and topical anti-inflammatory agent. It significantly inhibited the edema induced by different experimental edematogenic agents. For this, three in vivo models in mice were used: carrageenan-induced paw edema and chronic and acute TPA-induced ear edema. Edema, neutrophil infiltration, COX-2 and iNOS expressions, and histological changes were evaluated. The extract showed the

Schinella G et al. An Aqueous Extract …

Planta Med 2014; 80: 961–968

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Group Control (carrageenan) Extract lyophilized (125 mg/kg) Extract lyophilized (250 mg/kg) Indomethacin (10 mg/kg) B) Group Blank Control Extract lyophilized (125 mg/kg) Extract lyophilized (250 mg/kg) Indomethacin (10 mg/kg)

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Table 2 Effect of the extract lyophilized from I. paraguariensis (1 mg/ear, topically) and indomethacin (0.5 mg/ear, topically) on 12-O-tetradecanoylphorbol 13acetate-induced mouse acute ear edema. Ear edema was induced by topical application of TPA (2.5 µg/ear in acetone). A Ear weight was measured 4 h after the irritant application, and ear edema is expressed as the mean weight increase with respect to the blank (ΔW ± SD, mg). B Neutrophil accumulation was assessed as MPO activity, and results are expressed as the mean of absorbance (A) at 630 nm ± SD and refers to 1 mg of protein. Statistically significant differences with respect to the control group are expressed as **p < 0.01 (n = 6, Dunnettʼs t-test). Percentages of inhibition are expressed as the reduction of increase in ear weight with respect to the control group. A) Group Control (TPA) Extract lyophilized (1 mg/ear) Indomethacin (0.5 mg/ear) B) Group Blank Control (TPA) Extract lyophilized (1 mg/ear) Indomethacin (0.5 mg/ear)

ΔW ± SD (mg) 13.07 ± 4.08 6.48 ± 1.78** 1.18 ± 0.42**

% Inhibition – 50 91

(A ± SD/mg protein) × 103 190.00 ± 10.53 292.14 ± 106.13 197.05 ± 24.93** 187.21 ± 24.48**

% Inhibition – 93 100

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Table 3 Effect of the extract lyophilized from I. paraguariensis on 12-O-tetradecanoylphorbol 13-acetate-induced mouse subchronic ear edema. Inflammation was induced by repeated application of TPA (2 µg/ear in acetone). The extract (1 mg/ear, × 7 applications, twice/day) and dexamethasone (0.025 mg/ear, × 4 applications, once/day) were administered topically on days 8, 9, 10, and 11 after challenge. A) Ear edema was calculated as the increase in the ear weight in mg with respect to the blank group, which received acetone only (ΔW ± SD, mg). B) Neutrophil accumulation was assessed as MPO activity, and results are expressed as the mean of absorbance (A) at 630 nm ± SD and refers to 1 mg of protein. Statistically significant differences with respect to the control are expressed as *p < 0.05 and **p < 0.01 (n = 6, Dunnettʼs t-test). Percentages of inhibition are expressed as the reduction of increase in ear weight with respect to the control group. A) Group Control (TPA) Extract lyophilized (1 mg/ear) Dexamethasone (0.025 mg/ear) B) Group Blank Control (TPA) Extract lyophilized (1 mg/ear) Dexamethasone (0.025 mg/ear)

ΔW ± SD (mg) 14.37 ± 3.16 5.44 ± 1.48** 2.42 ± 1.04**

% Inhibition – 62 83

(A ± SD/mg protein)×103 282.29 ± 69.95 1585.94 ± 891.12 658.90 ± 141.46* 289.86 ± 85.38**

% Inhibition – – 71 99

Fig. 3 Histology of samples from the 12-O-tetradecanoylphorbol 13-acetate-induced ear edema test. A Blank (non-treated group): thickness of epidermis 50 µm (5.1 ± 0.4 cells); blood capillaries, histiocytes, and isolated fibroblasts in the papillary dermis; row of adipocytes in the hypodermis. B Control (TPA-group, without treatment): hypertrophic and hyperplasic epithelium thickness (6.2 ± 0.6 cells); a marked inflammatory infiltration with polymorphonuclear neutrophils, histiocytes, and numerous blood vessels were observed at the dermis level, and hair follicles and adipocytes in the hypodermis. C Problem (mate-treated group): reduced epithelium thickness 40 µm (4.8 ± 0.3 cells); blood capillaries and mild inflammation with predominant lymphocytes in the dermis; hair follicles with some secretory cells of sebaceous glands and hypodermic adipocytes. D Reference (dexamethasone-treated group): epithelium thickness of 40 µm (4.3 ± 0.4 cells); numerous blood capillaries, absence of inflammatory infiltration, and a row of hypodermic adipose cells in the dermis. (Color figure available online only.)

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Original Papers

cluding antioxidative and anti-inflammatory properties [29]. In addition, similar effects were described for other beverages rich in caffeine and polyphenols, such as green tea (Camellia sinensis, Theaceae), the most consumed beverage in the world. In this species, the catechins are the most relevant phenolics, and epigallocatechin gallate is the major compound present in the aqueous extract and is the most active form in a variety of biological activities [30, 31]. Recently, the anti-inflammatory activity of extracts assayed in different experimental models of acute inflammation, such as carrageenan, croton oil and dextran was demonstrated [32, 33], and similar results were obtained here for yerba mate extract. This also opens the door to epidemiological implications, as it is possible to infer that populations with a heavy intake of this popular beverage may be better protected against acute and chronic inflammatory processes. Moreover, the percentages of inhibition as well as the observed effects could make this plant of high interest for some posterior studies focusing on the possible activity against other local or systemic inflammatory processes as studied by Pimentel et al. [19]. Other pathologies potentially benefiting from the high consumption of mate may be inflammatory bowel disease, because the infusion reaches high concentrations in the intestines and could exert a topical effect on the intestinal cells followed by a systemic one after the absorption of its main principles, especially the caffeoyl quinic derivatives.

Materials and Methods !

Chemicals and biochemicals Unless otherwise specified, all chemical and biochemical reagents were purchased from Panreac and Sigma-Aldrich. Dexamethasone (≥ 99.0 %, HPLC) and indomethacin (≥ 97 %, TLC) from Sigma-Aldrich were used as reference drugs. Enhanced chemiluminescence detection reagents were obtained from Millipore Corporation. Precision plus protein standards were from BioRad. Polyvinylidene difluoride membrane was obtained from GE Healthcare. EDTA-free protease inhibitor cocktail tablets and DIG‑gel shift kit were purchased from Roche. Polyclonal antiCOX‑2 and anti-iNOS primary antibodies were purchased from Cayman. All other reagents were of analytical grade and purchased from Merck.

Animals Female CD1 mice, three-to-four weeks old, weighing 25–30 g (Harlan Interfauna Ibérica) were used for in vivo experiments. All animals were fed a standard diet ad libitum and housed under a 12-h light/dark cycle at 22 °C and 60 % humidity. Housing conditions and all in vivo experiments were approved by the Institutional Ethics Committee of the University of Valencia, Spain (no. 108 A1233134999884, 28–01–2009), according to the normative of October 2005 (RD1201/2005).

Plant material and extracts A sample of commercial mate (I. paraguariensis) produced in Las Marías (Corrientes, Argentina) was obtained for this study, and a voucher specimen was deposited in the herbarium of the Museo de Botánica y Farmacognosia ‘‘Carlos Spegazzini” (Universidad Nacional de La Plata, Argentina) under the number LPE 1005. The aqueous extract was prepared under similar conditions as those used traditionally. Briefly, the infusion was obtained from 10.0 g of dry powdered leaves soaked in 200 mL of boiling dis-

Schinella G et al. An Aqueous Extract …

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ability to reduce COX-2 and iNOS expressions, and therefore PGE2 and NO productions presumably will also be reduced. From a strictly methodological point of view, the measured effect in the classic carrageenan-induced inflammation model is the prevention of a chemically-induced inflammation by the test drug. It is considered a valid, standard screening method for the detection of active compounds, since the inflammation induced by carrageenan is highly predictive of anti-inflammatory drug activity in human inflammatory disease, and doses of nonsteroidal anti-inflammatory drugs are well correlated with an effective dose in patients. The inhibition of the edema induced by carrageenan was significant at all stages of the inflammatory process. During the first hour, the principal agents implicated in the edema formation are the bioamines histamine and serotonin, and the peptide bradykinin. These are followed in time by PGs, which are the principal mediators involved in the increased vascular permeability, together with NO, which is formed by leukocytes, endothelial cells, and sensory nerve cells at the site of inflammation. In the acute phase, the increase in vascular permeability produces cell infiltration, mainly neutrophils, increasing the inflammatory response after the production of oxygen-derived species [21]. In our experiments, the expression of proinflammatory enzymes was also reduced (43 % of COX-2 and 53% for iNOS) in the inflamed tissue. Our results demonstrate that oral administration of mate results in a reduced PMN infiltration. Since IL-1 and TNFα are likely released by macrophages present in the inflammatory site to induce accumulation of PMN cells into the inflammatory area, the quantification of these two important mediators does not appear to be so central in the characterization of the activity of drugs in the carrageenan model [21]. Therefore, one may assume a reduced released of IL-1 and/or TNFα. In the case of the acute TPA test, the extract inhibited both the edema (91 %) and leukocyte infiltration (93 %). In addition, it was also highly active in the subchronic test, significantly inhibiting both edema (62 %) and cell infiltration (71%). The edema induced by TPA is also due, in part, to the liberation of bioamines (histamine, serotonin, and others) and synthesis of prostaglandins after the activation of protein kinase C, and the model is also sensitive to histamine H1. H2 antagonists are active as well as nonsteroidal anti-inflammatory drugs. Contrarily, the topical inflammation induced by the multiple topical application of TPA is not responsive to antihistaminic or NSAIDs, the last class of anti-inflammatories usually aggravating the process. In this model, only corticoids have relevant effects on edema, cell infiltration, and epidermal thickness, whereas some lipoxygenase inhibitors could reduce cell infiltration but in a lesser measure than edema and epidermal thickness [22]. The histological study of the ears after the repeated application of TPA typically showed a reduction of epithelium thickness, with the presence of lymphocytes in the dermis and adipocytes in the hypodermis. Some of the principles present in the aqueous extract of mate have already been described as promising anti-inflammatory agents and may contribute to the therapeutic potential of mate. For example, caffeine and ferulic acid conjugates, in particular chlorogenic acids, are putative bioactive phytochemicals in coffee [23]. Chlorogenic acid has been highlighted as a bioactive compound of Artemisia capillaris (Asteraceae) [24], Scorzonera species (Asteraceae) [25], and Cymbopogon citratus (Poaceae) leaves [26]. Dicaffeoylquinic acids are the main phenolic components of Laggera species and are responsible for their anti-inflammatory [27] and antioxidative effects [28]. The common dietary flavonoid rutin exhibited significant pharmacological activities, in-

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%I Edema ¼

High-performance liquid chromatography-UV analysis

Mouse ear edema induced by multiple application of 12-O-tetradecanoylphorbol 13-acetate [22]

Equipment consisted on an Agilent 1200 series HPLC system with a UV‑VIS PDA detector (Agilent Technologies), Agilent ChemStation software, and a Phenomenex® C18 column (250 × 4.6 mm id, 5 µm). Solvents A (H2O + acetic acid 0.2 % v/v) and B (methanol + acetic acid 0.2 % v/v) were mixed in gradient mode as follows: 0 min 90 % A, 0–5 min 80 % A, 5–65 min 50% A, 65–75 min 20 % A; flow rate 0.8 mL/min. The injection volume and column temperature were set at 10 µL and 40 °C, respectively.

Determination of total phenols and antioxidant activity [35] Total phenols were determined by the Folin-Ciocalteu method. The antioxidant activity was established by ABTS•+ and FRAP assays.

Mouse paw edema induced by carrageenan [36]

Electronic reprint for personal use

Wcontrol  Wproblem  100 Wcontrol

tilled water. The infusion was cooled at room temperature until 40 °C before filtration and then lyophilized, kept at − 20 °C, and protected from light. The yield was 10 % (w/w) and the dry powder was solved in the corresponding aqueous vehicle immediately before its use [34].

Groups of six mice weighing 22–25 g were divided into four groups. The control group received only vehicle (Tween 80/ethanol/water 1 : 1 : 10, v/v), the reference group received indomethacin (10 mg/kg, reference group), and the problem groups received the extract lyophilized (125 mg/kg and 250 mg/kg, respectively). Both indomethacin and extracts were dissolved in the same excipient (Tween 80/ethanol/water 1 : 1 : 10, v/v). The vehicle and samples were administered orally (0.2 mL) through a gastric probe 1 h before carrageenan administration. Edema was induced on the right hind paw by subplantar injection of carrageenan 3% w/v in saline (0.05 mL), and the increase in volume was measured by a plethysmometer (Ugo Basile) 1 h, 3 h, and 5 h after edema induction. Edema was expressed as the increase in paw volume due to carrageenan injection, and edema inhibition was expressed as the reduction in volume with respect to the control group: %I Edema ¼

Vcontrol  Vproblem  100 Vcontrol

where V is the paw volume measured for the control and problem mice. After the last measurement (5 h), the mice were sacrificed by cervical dislocation, and the carrageenan-induced edema feet were dissected and frozen immediately with liquid nitrogen and stored at − 80 °C until their use for biochemical evaluation.

12-O-tetradecanoylphorbol 13-acetate-induced ear edema in mice [37] Edema was induced through the topical application of TPA (2.5 µg/ear in 20 µL of acetone, 10 µL on each side). At the same time, the extract lyophilized (1 mg/ear) and indomethacin (0.5 mg/ear) dissolved in EtOH : H2O (7 : 3) were also applied topically to the ears (20 µL/ear, 10 µL on each side). Blanks received only the vehicle (EtOH : H2O, 7 : 3). After 4 h, the mice were sacrificed by cervical dislocation, and right and left ear punches, 7 mm in diameter, were taken from each mouse. The swelling was assessed in terms of the mean weight increase of each ear, while inhibition of the swelling was expressed as weight reduction in comparison to the control group treated with TPA only:

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where W is the weight for the control and problem mice, respectively. Tissues were frozen and stored at − 80 °C until use.

Groups of six mice, weighing 22–25 g, were divided into four groups (blank, control, reference, and problem). Subchronic inflammation was induced through the topical application of five doses of TPA (2 µg/ear in 20 µL of acetone, 10 µL on each side), on alternate days (1, 3, 5, 8, and 10), to the inner and outer surfaces of each ear with the aid of a micropipette. The lyophilized extract (1 mg/ear), dissolved in EtOH : H2O (7 : 3), was administered twice daily for four consecutive days (8, 9, 10, and 11), 20 µL/ear (10 µL on each side, topically), while dexamethasone (0.025 mg/ear, in the same solvent (EtOH : H2O, 7 : 3), 20 µL/ear (10 µL in each side, topically), was applied only once a day in the morning for four consecutive days. On the last day, the test sample was applied only in the morning. The blank group was given topical applications of acetone in the same regimen as described above, and the control group was treated with TPA only. On day 11, the mice were sacrificed by cervical dislocation 6 h after the last application, and two ear punches from each animal were taken. Swelling was assessed in terms of the mean weight increase of each ear with respect to the blank, while inhibition of swelling was expressed as the weight reduction in comparison with the control group: %I Edema ¼

Wcontrol  Wproblem  100 Wcontrol

where ΔW is the increase in weight for the control and problem mice minus the blank. Ear samples were immediately frozen with liquid nitrogen and stored at − 80 °C until use.

Histological evaluation [37] Ear samples were fixed in 4 % neutral-buffered formalin. Each sample was cut longitudinally into equal halves. Half of each sample was embedded in paraffin, cut into 3–4 µm sections, and stained with hematoxylin-eosin. Epithelium thickness was evaluated using an objective × 100 and expressed as the mean ± SD of the number of epidermal layers from the basal to the granulous stratum, both included. The number of inflammatory cells, lymphocytes, macrophages, and neutrophils were counted in the papillary and in the reticular dermis/subcutis layers and conventionally expressed as 1–2–3 units, according to their relative abundance. Mastocytes were evaluated by a semiquantitative method and expressed as the mean ± SEM of the number of total cells in an area of 1000 µm2, on a toluidine blue stained slice.

Myeloperoxidase activity assay [38] Tissues obtained in the previous assays were thawed, grounded to powder in a mortar, weighed (about 40 mg of tissue for each sample), and then suspended (10% w/v) in 80 mM sodium phosphate buffer (pH 5.4) containing 0.5 % hexadecyltrimethylammonium bromide, sonicated for 30 s, and centrifuged at 11 000 × g for 20 min at 4 °C. After centrifugation, 100 µL of phosphate-buffered saline, 85 µL of 22 mM monopotassium phosphate/disodium phosphate buffer, and 15 µL of hydrogen peroxide 0.017 % were added to 30 µL of the supernatant. The enzymatic reaction began when adding 20 µL of tetramethylbencidine hydrochlor-

Original Papers

ide. After 3 min at 37 °C, the reaction was stopped. Absorbance was read at 630 nm. MPO activity is expressed as the amount of enzyme required to convert 1 µmol of hydrogen peroxide to water in 1 min, expressed per gram of wet weight of tissue.

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government for financial support (FEDER, SAF2009-10059-C0301).

Conflict of Interest Preparation of cytosolic fraction from paws [39]

!

Briefly, the skin was removed from the paws and tissues were then homogenized with a Polytron PT-2000 (Kinematica AG) tissue homogenizer in 2.5 mL of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2 mM phenylmethylsulfonyl fluoride, 100 mM EDTA, 0.32 M sucrose, 1 mM dithiothreitol, 2 mg/ mL aprotinin (all from Sigma-Aldrich), and a complete mini EDTA-free protease inhibitor cocktail. The mixture was sonicated (three cycles in 10 s) in a Branson sonifier 150 (Branson Ultrasonics Corporation) and centrifuged (Eppendorf centrifuge 5810R; Eppendorf) at 10 000 × g for 20 min at 4 °C.

The authors declare no conflict of interest.

The presence of proteins in the supernatants was determined by means of the Bradford method with bovine serum albumin as the standard [40]. After extraction, 30 µg of protein were loaded onto 10 % sodium dodecyl sulphate polyacrylamide electrophoresis gel (Sigma-Aldrich) and transferred onto polyvinylidene difluoride membranes at 125 mA for 90 min. Then, the membranes were blocked in phosphate-buffered saline-Tween 20 (Panreac) containing defatted milk (3 % w/v). For COX-2, the membranes were incubated with anti-COX-2 polyclonal antibody (1 : 1000) and for iNOS with anti-iNOS polyclonal antibody (1 : 1000). For β-actin, the membranes were incubated with anti-β-actin polyclonal antibody (1 : 10 000 dilution) from Sigma-Aldrich. The blots were washed and incubated with peroxidase-conjugate anti-rabbit or anti-mouse immunoglobulin G (1 : 12 000 dilution; Cayman). The immunoreactive bands were visualized with an enhanced chemiluminiscence system. Images for all Western blot experiments were acquired with the image analysis system LAS-3000 mini (Fujifilm). Digital images were processed and band density measurements were made with the aid of a Multi Gauge V3.0 software package (Fujifilm). To unify Western blot densitometry results in the processed images, data from the control group were taken as a reference and assigned the value 100. Relative percentages of the other groups were then calculated and represented graphically.

Statistical analysis Values are expressed as the means ± standard deviation (SD). Statistically significant differences between the groups were assessed by means of one-way analysis of variance (ANOVA) and Dunnettʼs t-test (* p < 0.05, ** p < 0.01). Data were evaluated with Graph Pad Prism version 5.01 software. Inhibition percentages (% I) were calculated from the differences between the drug-treated group and the control group, which was treated with the inflammatory agent only. For each group, n = 6 animals.

Acknowledgements !

G. S. thanks the Universitat de València for financial support (Convocatòria dʼEstades Temporals per a Investigadors Convidats, 2011). This work was partially supported by a grant from Universidad Nacional de La Plata (11/M162). J. L. R. thanks the Spanish

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Cátedra de Farmacología Básica, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, Argentina Departament de Farmacología, Universitat de València, Burjassot, Spain Cátedra de Citología, Histología y Embriología B, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, Argentina Comisión de Investigaciones Científicas (CIC), Provincia de Buenos Aires, La Plata, Argentina Centre for Pharmacognosy and Phytotherapy, Department of Pharmaceutical and Biological Chemistry, University College of London School of Pharmacy, London, United Kingdom

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