In Vivo Anti-inflammatory and Antiallergic Activity of Pure Naringenin

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

In Vivo Anti-inflammatory and Antiallergic Activity of Pure Naringenin, Naringenin Chalcone, and Quercetin in Mice Elvira Escribano-Ferrer,*,†,‡ Josep Queralt Regue,́ § Xavier Garcia-Sala,† Antoni Boix Montañeś ,† and Rosa M. Lamuela-Raventos‡,∥

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Department of Pharmacy and Pharmaceutical Technology and Physical-chemistry, Faculty of Pharmacy and Food Sciences, Institut of Nanoscience and Nanotechnology (IN2UB), University of Barcelona, E-08028 Barcelona, Spain ‡ CIBER Physiopathology of Obesity and Nutrition (CIBER-OBN), Instituto de Salud Carlos III, E-28029 Madrid, Spain § Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Sciences, University of Barcelona, E-08028 Barcelona, Spain ∥ Department of Nutrition, Food Sciences, and Gastronomy − INSA-UB, Faculty of Pharmacy and Food Sciences, University of Barcelona, E-08028 Barcelona, Spain ABSTRACT: Flavonoids, found in almost all fruits and vegetables, belong to a class of plant secondary metabolites with a polyphenolic structure and have properties with health-improving potential. However, few experimental studies on the effects of flavonoids have been carried out in vivo after external application and using pure compounds. Aiming to fill this gap, in this study we tested the topical anti-inflammatory and antiallergic activity of three flavonoids of high purity, naringenin, naringenin chalcone, and quercetin, in mouse models. The topical anti-inflammatory effects were assessed against arachidonic acid- (AA) and tetradecanoylphorbol-13-acetate- (TPA) induced ear edema. The anti-inflammatory effect of naringenin against ear edema was noticeable at a 1% dose in the AA model and at half this dose in the TPA model. Quercetin (1.3%) did not exert any topical anti-inflammatory activity in the AA model, but its inhibitory effect in the TPA model was similar to that of naringenin (2%); in contrast, naringenin chalcone was more active against the AA-induced than TPA-induced inflammation. The flavonoid effect on IgE-mediated passive cutaneous anaphylaxis was also studied in mice, both intravenously and topically. Naringenin, naringenin chalcone, and quercetin all showed strong antiallergic activity after intravenous dosing (0.02%) and when applied topically (2%). The results of this study suggest that the flavonoids naringenin, naringenin chalcone, and quercetin may be useful alternatives for the topical treatment of inflammatory and allergic skin disorders.

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vegetarian diet. However, the beneficial health properties of polyphenol-containing foods are difficult to demonstrate. The effects may vary according to whether the food is cooked or raw and depend on polyphenol bioavailability and metabolism and potential interactions with other dietary compounds.2 Regarding the clinical manifestations of type I allergies, even when the sensitization process has a systemic nature and leads to different clinical disease patterns,11 immediate hypersensitivity occurs as a local reaction (skin allergy, allergic rhinitis, and asthma, among others) or as a systemic disorder (anaphylactic shock).12 Polyphenols, particularly flavonoids, have been extensively studied for their anti-inflammatory4,5,13,14 and antiallergic activities,3,5,15,16 mainly in vitro. Numerous in vitro studies have shown that flavonoids inhibit the IgE-mediated release of histamine from mast cells,17−19 the formation of cytokines IL-3

lavonoids, a group of natural substances with variable phenolic structures, are found in fruits, vegetables, grains, bark, roots, stems, flowers, tea, and wine.1 They can act on several molecular targets simultaneously2 and have a wide range of biological effects, including anti-inflammatory, antimicrobial, antitumor, antioxidant, wound-healing, and immunomodulatory activities.3−5 Consequently, a diet rich in flavonoids is highly recommended to reduce the risk of cardiovascular disease, cancer, and other pathologies related with an inflammatory process.6 On the other hand, there is broad consensus that in the last few decades allergic diseases have become more prevalent in most industrialized countries, particularly type I allergies (immediate hypersensitivity). This increase has been attributed to different factors, including improved diagnostics, genetic susceptibility, psycho-social influences, allergen exposure, and reduced immune-system stimulation.7 Among the psycho-social factors, changes in dietary habits may play an important role in this tendency.8,9 In this context, Tanaka et al.10 reported that symptoms in patients with atopic dermatitis improved significantly after a two-month © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 8, 2018

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DOI: 10.1021/acs.jnatprod.8b00366 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

and IL-4, and the expression of cluster of differentiation 40 (CD40) required for IgE production and the appearance of allergic symptoms.20,21 To our knowledge, most of the in vivo studies in this field have used administration routes other than topical application. For example, allergy symptoms in animal models were found to decrease after oral12,22,23 and intraperitoneal17,24 administration of flavonoids. Only a few studies have evaluated the topical anti-inflammatory and/or antiallergic activity of flavonoids, and these have used the extracts of whole plants or fruits rather than the pure compound,18,24−26 which makes it difficult to attribute any effect to a particular component. Another advantage of applying the pure compound directly on the skin is an enhanced bioavailability of the unmetabolized drug at the target site and therefore greater effectiveness. The bioactive compounds naringenin (flavanone), naringenin chalcone (chalcone), and quercetin (flavonol) are the predominant polyphenols in grapefruit, red tomato skin, and onion, respectively. Quercetin (QUER) is the most studied compound of the three,27,28 and there is less information available on the biological/therapeutic activity of naringenin (NAR) and naringenin chalcone (NGC). NGC is an intermediate in flavonol biosynthesis and is spontaneously metabolized into NAR by chalcone isomerase.18 As an immunomodulatory agent, QUER has a strong affinity for mast cells and basophils. It tends to stabilize their cell membranes, preventing them from spilling their pro-inflammatory, allergy-symptom-causing mediators.19 In accordance with the above considerations, the aim of the present study was to assess the topical anti-inflammatory and antiallergic activity of pure NAR, NGC, and QUER in mice. We wanted to ascertain if the flavonoids were active when administered as isolated compounds and also without the metabolic transformation that occurs when they are taken orally. Different models are used to evaluate the topical antiinflammatory activity of drugs and phytochemicals,29 including mouse models of acute ear inflammation,30−32 particularly the short-lived edema response induced by arachidonic acid (AA) and the longer lasting edema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Comparing the inhibitory effects of the selected flavonoids on AA- and TPA-induced edema with those of drugs with well-known mechanisms of action may be a useful approach to identify or rule out potential flavonoid mechanisms.

Figure 1. Topical anti-inflammatory activity of flavonoids (naringenin, NAR; naringenin chalcone, NGC; and quercetin, QUER) comparatively with the standard drug disodium diclofenac (SDF) on AA-induced ear edema (A) and on TPA-induced ear edema (B) in mice. Each bar represents the mean ± SD for 5−8 animals. Different letters indicate significant differences between groups (p < 0.05, Scheffe’s multiple comparison test).

reduction of 27% was obtained with NGC 1%, whereas the effect of QUER 1.3% was comparable to NAR 2%. The antiinflammatory activity of NAR 1% was similar to that of NGC but lower than that of the standard drug SDF. No statistically significant differences (p > 0.05) between the activity of QUER and SDF were found. Co-involvement of prostaglandins (PGs) and leukotrienes (LTs) as mediators of both AA- and TPA-induced ear edema has been reported.31,33−36 However, AA seems to release mainly LTs, whereas the effect of TPA appears to be more dependent on PG production. Platelet activating factor (PAF) is implicated in both AA- and TPA-induced edemas.37 Tumor necrosis factor (TNFα),38 interleukin IL-6 and IL-1β,39 inducible NO synthase (iNOS),40 and 2-arachidonoylglycerol, the endogenous ligand of the cannabinoid CB2 receptor,41 are also involved in the generation of TPA-induced edema. NAR 0.5% and 1% had a greater anti-inflammatory effect on TPA-induced than on AA-induced ear edema (p < 0.05). Kim et al. (1993),42 using a dose 5-fold greater than here (2 mg/ear vs 0.4 mg/ear of a 2% solution), found no NAR activity against croton-oil-induced ear edema. This divergent result may be due to an irritant effect of the very high NAR dose used or to a different mechanism of action of the phlogogen agents in croton oil, which contains phorbol 12-myristate 13-acetate and several other components besides TPA.43 It is of interest to speculate on the working mechanisms of the tested flavonoids by comparing their effects with those obtained with standard drugs that have a well-defined mechanism of action. For

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RESULTS AND DISCUSSION Effects of Polyphenols on AA- and TPA-Induced Ear Edema. Since NAR has not been studied as extensively as QUER and less information about its biological activity is available, we focused particular attention on this flavonoid, testing its anti-inflammatory activity at three different concentrations (0.5%, 1%, and 2%), topically applied. As shown in Figure 1A and B, the topical treatment with NAR produced a dose-dependent inhibition of both AA- and TPA-induced ear edemas. In the AA model, ear swelling was reduced by 22% and 56% after the application of NAR 1% and 2%, respectively, and by 42% when using NGC 2%. The antiinflammatory activities of NAR 2% and NGC 2% were similar to that of the standard anti-inflammatory drug disodium diclofenac (SDF). At the QUER dose used, no antiinflammatory activity was observed in the AA model. In the TPA model, ear swelling was reduced by 32%, 56%, and 62% after the application of NAR 0.5%, 1%, and 2%, respectively; a B



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example, the phospholipase A2 (PLA2) inhibitors methylprednisolone34 and dexamethasone16,37 are more active against TPA-induced than AA-induced ear edema, as also are COX inhibitors, both COX-1 selective, such as indomethacin,32,37 and the COX-2 inhibitors flosulide and SC-57,666.44 According to our results, QUER anti-inflammatory activity, like that of NAR, was greater against TPA-induced than AAinduced edema (p < 0.05). Numerous studies have reported the PLA2 inhibitory activity of QUER45−48 and NAR45,46 as well as several other flavonoids.49,50 Additionally, the antiinflammatory effects of the polyphenol curcumin, which also shows more activity against TPA-induced than AA-induced ear edema, even at lower doses,51 have been attributed to PLA2 inhibition.52 On the basis of all these results, it can be speculated that NAR and QUER have a similar activity profile to PLA2 inhibitors. In addition, inhibitory COX-2 activity of QUER has been reported in Caco2 cells53 and in rat peritoneal macrophages.54 Wallace55 and Deng et al.56 describe QUER as a dual COX/LOX inhibitor, whereas its mechanisms of action according to González et al.5 are the inhibition of the transcription factor NF-κB signaling pathway and the downregulation of pro-inflammatory marker expression. The discrepancies in the elucidation of the mechanism of action of QUER reflect that the working mechanisms of flavonoids are still not properly understood and also that polyphenols may act on several molecular targets simultaneously.2 In the case of NGC, the anti-inflammatory activity was similar in both AA and TPA models (p > 0.05). Taking into account that the dual COX/LOX inhibitors phenidone and BW755C34 and the 5-LOX inhibitor zileuton44 show a higher anti-inflammatory activity against AA-induced than TPAinduced edema and that the anti-inflammatory activity of chalcones is reported to be partially mediated by LOX inhibition,57,58 we hypothesize a dual COX/LOX inhibitory activity for NGC. However, this would need to be confirmed by additional studies. Effect of Intravenous Flavonoids on IgE-Mediated Passive Cutaneous Anaphylaxis. Type 1 hypersensitivity reactions occur after allergen sensitization (IgE synthesis and attachment to mast cells). On exposure to a new allergen, inflammatory mediators are released after allergen−IgE crosslinking on the mast cell surface, causing clinical allergy symptoms. Inhibition of passive cutaneous anaphylaxis reactions is a widely used in vivo model for the screening of antiallergic compounds. In a preliminary acute study, we administered the three phytochemicals intravenously (i.v.) to achieve 100% bioavailability and maximum therapeutic efficacy. To the best of our knowledge, this is the first study to test the antiallergic activity of flavonoids after their i.v. administration. As shown in Figure 2, after i.v. administration, the three flavonoids all exhibited strong antiallergic activity, probably by blocking mast cell degranulation.17−19,59 At the dose tested, the allergic reaction was reduced by about 56%, 68%, and 80% after NAR, NGC, and QUER treatment, respectively, with no differences between groups. It should be noted that the tested flavonoids achieved the same antiallergic effect as the mast cell stabilizer disodium cromoglicate60 (DSCG) with only a tenth of the dose. It is well known that the antiallergic effects of QUER and DSCG are due to the stabilization of mast cells, QUER being even more effective than DSCG in inhibiting IL-8 in cultured mast cells.61 In our study, NAR, NGC, and QUER exhibited

Figure 2. Intravenous IgE-mediated passive cutaneous anaphylaxis of flavonoids (naringenin, NAR; naringenin chalcone, NGC; and quercetin, QUER) comparatively with the standard drug disodium cromoglycate (DSCG) in mice. Each bar represents the mean ± SD for 5 or 6 animals. Different letters indicate significant differences between groups (p < 0.05, Scheffe’s multiple comparison test).

similar levels of antiallergic activity. In contrast, in a previous study by Yamamoto et al.,18 after 5 days of oral administration of 0.8 mg/kg NGC, the allergic reaction in the picryl-chlorideinduced type I allergic model was reduced by approximately 47%, whereas NAR and QUER were less effective (∼15%). Iwamura et al.62 found that the repeated oral administration of NGC (0.8 mg/kg) attenuated allergic airway inflammation in sensitized mice. Kim et al.24 (2013) reported that NAR, administered daily by intraperitoneal (i.p.) injection for 7 days (50 and 100 mg/kg), inhibited atopic dermatitis induced by the hapten 2,4-dinitrofluorobenzene (DNFB). Park et al.63 (2005), after applying a single i.p. dose of NAR (5 mg/kg), noted a 70% reduction of passive cutaneous anaphylaxis compared to the control group. These studies, like the current work, all used an immediate hypersensitivity model and differ mainly in the type of hapten and method of compound administration (i.v. vs oral or i.p. and in single or repeated doses). In spite of these differences, all show antiallergic activity of NGC and NAR, in agreement with our results. Effect of Topical Flavonoids on IgE-Mediated Passive Cutaneous Anaphylaxis. The strong antiallergic effect of flavonoids observed after intravenous dosing encouraged us to study their topical activity. Positive results could lead to progress in the treatment of atopic dermatitis, which is highly prevalent in most industrialized countries. As shown in Figure 3, topically applied 2% solutions of NAR, NGC, and QUER reduced the allergic reaction by about

Figure 3. Topical IgE-mediated passive cutaneous anaphylaxis of flavonoids (naringenin, NAR; naringenin chlacone, NGC; and quercetin, QUER) comparatively with the standard drugs disodium cromoglycate (DSCG), zileuton, and methylprednisolone aceponate (MPA) in mice. Each bar represents the mean ± SD for 4−6 animals. Different letters indicate significant differences between groups (p < 0.05, Scheffe’s multiple comparison test). C



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inflammatory activity of NAR 1% was similar to that of the standard drug SDF 1% in the TPA model. After both intravenous and dermal application, NAR, NGC, and QUER all exhibited high levels of antiallergic activity. The topical administration of these flavonoids could be a solution to the low oral bioavailability of NAR, QUER, and NGC as dietary components, and they provide useful alternatives for the treatment of inflammatory and allergy skin disorders.

42%, 55%, and 34%, respectively. In contrast, topical application of DSCG 4% wt in an oil-in-water emulsion or a 2% (w/v) DSCG solution, applied twice, was not effective. As in the i.v. antiallergy test, DSCG was chosen as a control standard based on the results of Stainer et al.64 and Stevens and Edwards,65 who reported its activity against atopic dermatitis in children. DSCG has long been used to treat various allergic conditions as an ingredient of nasal sprays and eye drops. However, to our knowledge, no cream, gel, or lotion containing DSCG is commercially available for treating skin allergies, probably due to the contradictory reports about its clinical efficacy, which may be related to the different formulations used. The results from 17 trials on the topical efficacy and safety of DSCG, aiming to find the optimal formulation, have been summarized by Zur.66 The lack of activity of the DSCG application in our experimental conditions may be explained by the use of an unsuitable formulation or vehicle, which can influence skin penetration and subsequent effectiveness. Ariyanayagam et al.67 applied a 9-week treatment with DSCG topical cream (4%) and found very small (and highly variable) amounts of the drug in urine, indicating low percutaneous absorption. Interestingly, a slight but significant (p < 0.05) increase in vascular permeability was observed with the DSCG 4% emulsion but not the 2% (×2) solution (Figure 3), which could be attributed to the low irritant effect of the preservative sodium propyl paraben.68 There are no reports in the literature about skin irritation after topical application of DSCG, although a transient irritation was a major side-effect after mucosal dosing.69 The other positive controls used were the histamine H1 antagonist chlorpheniramine maleate (data not shown), the 5lipoxygenase inhibitor zileuton, and the PLA2 inhibitor methylprednisolone aceponate (MPA), a medium-potency corticosteroid of group III. Chlorpheniramine, applied topically at 1%, showed no antiallergic activity, although the same dose strongly reduced the histamine-induced increase in vascular permeability in mouse ears. Based on this result and those of previous reports, and following “the three R’s”70 on the ethical use of animals (the principle of reduction), the experiments with chlorpheniramine were discontinued. Using the same antiallergic test as in the current study, Katayama et al.71 found that antihistamine drugs did not significantly suppress the reactions. Inagaki et al.72 also observed that chlorpheniramine did not inhibit the immediate phase IgEmediated cutaneous reaction in mice. Moreover, Estelle and Simons73 report that only doses of H1-receptor antagonists three or more times higher than those required for H1 blockade had an antiallergic effect. The order of potency of the two other topically applied standard drugs was zileuton 5% > MPA 0.1%. However, since MPA was administered in the commercially available formulation Adventan cream, both the active agent and the excipients contributed to the results. Accordingly, it can be speculated that the role of histamine in the allergic response was negligible; in contrast, the antiallergic effect of zileuton 5% and MPA 0.1% wt. (Adventan cream) suggests that leukotriene synthesis and PLA2 were involved. Thus, the demonstrated activity of the tested flavonoids may be related to the inhibition of both PLA2 and leukotriene synthesis. In conclusion, topical NAR (1−2%), and to a lesser degree NGC 2%, had anti-inflammatory effects on both AA- and TPAinduced ear edemas, whereas QUER only inhibited the TPAinduced inflammation. It should be noted that the anti-

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EXPERIMENTAL SECTION

Materials. NAR, QUER, SDF, DSCG, chlorpheniramine maleate, zileuton, TPA, AA, Evans blue, monoclonal anti-dinitrophenyl antibody produced in mouse (anti-DNP IgE), and dinitrophenylbovine serum albumin (DNP-BSA) were provided by Sigma-Aldrich (Madrid, Spain). NGC was supplied by MicroCombiChem e.K. (Wiesbaden, Germany), MPA 0.1% wt. cream (Adventan) by Bayer (Milan, Italy), natrium chloride 0.9% (w/v) by Unolab Manufacturing, S.L. (Madrid, Spain), base Soft-care 1722 by Acofarma (Barcelona, Spain), and sodium propyl paraben by Fagron (Barcelona, Spain). Propylene glycol, ethanol, acetone, and formamide were ́ supplied by Panreac Quimica (Barcelona, Spain). Isoflurane was purchased from Laboratorios Esteve (Barcelona, Spain). Experimental Procedures. Animals. Six-week-old female CD-1 mice (Harlan Interfauna Ibérica, St. Feliu de Codines, Barcelona, Spain) were kept on a 12 h light/dark cycle at 22 ± 2 °C with 50 ± 10% relative humidity with free access to food and water. Mice were allowed to acclimatize for 1 week before the start of the allergy experiments. The studies were conducted following a protocol approved by the Animal Experimentation Ethics Committee of the University of Barcelona (Spain) and Generalitat de Catalunya (projects no. 7781 and 9397), and all the procedures relating to the animals and their care conformed to the International Guidelines for the Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985). AA- and TPA-Induced Ear Edema. Mice were divided into six different groups (n = 4−6 animals per group): control (vehicle) and five treatments. In different experiments AA (1 mg) or TPA (5 μg) in 20 μL of acetone was applied to the inner and outer surface of the left ear using a micropipette (volume range 10−100 μL, Nichiryo); the right ear received the vehicle. Immediately afterward, the following treatment solutions in ethanol were applied with a micropipette to the left ear surface (20 μL): 0.5, 1, and 2% NAR, 2% NGC, and 1.3% QUER. An ethanol solution of 1% SDF was used as a standard antiinflammatory drug. One hour or 4 h after the application of AA (maximum response) or TPA (no maximum response but edema response similar to AA at 1 h),33 the mice were killed by cervical dislocation prior to isoflurane anesthesia, and with a circular blade of 7 mm a pouch biopsy of the right and left auditory pinna was taken and weighed. The anti-inflammatory activity of the tested compounds was evaluated by the weight increase of ear samples expressed as a percentage according to the following formula: ([WL] − [WR])/ [WR] × 100, where [WL] and [WR] are the weights of the left and right ear discs, respectively. IgE-Mediated Passive Cutaneous Anaphylaxis. The antiallergic activity of flavonoids was tested both intravenously and topically. It was evaluated according to the method of Park et al.63 with some modifications. Briefly, control mice were sensitized with an i.v. injection of 100 μL of anti-DNP IgE solution (1 mg/mL) through the jugular vein (4 mg/kg BW). Before i.v. administration, a small incision was made in the neck to one side of the midline. For the injection, a 30 G needle (Becton Dickinson D Ultra-Fine U-100 insulin syringe, USA) was inserted into the vein through the pectoral muscle at an angle of about 10°, pointing toward the head. The muscle acts as a seal when the needle is withdrawn. To close the skin incision, one or two sterile silk suture 4/0 (Laboratorios Aragó, Barcelona, Spain) points were applied. Thirty minutes later, a cutaneous reaction was elicited by the application of 20 μL of DNP-BSA (12.5 mg/mL in saline/acetone, 1:1) to the left ear (the right ear acted as a control). Immediately afterward, 0.2 mL/25 g BW of Evans blue dye 1% (w/v) D



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saline solution was administered i.v. Thirty minutes later, the animals were euthanized by cervical dislocation, and both ears were removed by cutting close to, but not through, the ridge of the cartilage at the base of the ear. To test flavonoid intravenous activity, the test solutions (0.2 mL/ 20 g BW) were administered i.v. immediately after the i.v. sensitization. To test flavonoid topical activity, 15 min before and 15 min after the sensitization, 20 μL of the test solution was topically applied by means of a micropipette to the left ear of the mice. The right ear received the vehicle, delivered in the same manner. For intravenous administration, the following groups, with 5 or 6 animals each, were used: control group, NAR 0.02%, NGC 0.02%, QUER 0.02%, and DSCG 0.2% as the standard drug, all in physiological saline. The volume administered was 0.2 mL/20 g BW. For topical administration, the following treatment groups of 5 or 6 animals each were used: control, NAR 2%, NGC 2%, QUER 2%, zileuton 5%, and chlorpheniramine maleate 1% solutions in cold acetone, DSCG 2% (w/v) solution in water (pH 2.3)/acetone, 1:1, DSCG 4% wt in an oil-in-water emulsion, and MPA 0.1% wt. (Adventan cream). The DSCG 4% wt. emulsion contained 25% wt of base Soft-care 1722 and 5% wt of propylene glycol. Increased vascular permeability was tested by measuring the Evans blue extravasation. The leaked vessel dye was extracted from ears using the procedure described by Radu and Chernoff.74 Before the Evans blue extraction, ears were first washed by soaking them in warm formamide for 10 s and carefully dried with cotton paper. Then, ears were placed in Eppendorf tubes, 500 μL of formamide was added, and the tubes were transferred to a 65 °C water bath for 24 h to extract the dye from the ear tissue. The samples were allowed to cool and centrifuged at 8000g for 5 min. Their supernatants (200 μL) were transferred to the wells of a 96-well ELISA microplate. Absorbance readings were performed at 620 nm in a microplate spectrophotometer, BioTek (Winooski, USA), and the concentration of Evans blue/ear was calculated using a standard curve in the range of 25 and 1 μg/mL (six calibration standard points). The antiallergic activity of the compound was evaluated as the capacity to inhibit the vascular permeability. The increase in vascular permeability (%) was calculated as follows: ([BL] − [BR])/[BR] × 100, where [BL] and [BR] are the Evans blue concentration from the left and right ears, respectively. Statistical Analysis. The results are expressed by the mean ± standard deviation of the mean (SD). The mean increase in ear edema and vascular permeability (%) in the control and treatment groups was compared by the ANOVA test, followed by the post hoc Scheffe’s multiple comparison test, after verifying their normal distribution (Kurtosis normality of residuals) and the homogeneity of variance (modified-Levene equal-variance test). In addition, for NAR, NGC, and QUER, the results obtained with the two different models (AA- and TPA-induced edema) were compared using Student’s t test. NCSS software was used and p < 0.05 was considered significant.

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Elvira Escribano-Ferrer: 0000-0001-7837-9506 Notes

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ACKNOWLEDGMENTS

The authors acknowledge the financial support from the Spanish Ministry of Economy, Industry and Competitiveness within the projects AGL2013-49083-C3-1-R, Generalitat de Catalunya 2017 SGR196, and the Instituto de Salud Carlos III, ISCIII (CIBEROBN). E



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