Effect of pequi tree Caryocar coriaceum Wittm . leaf extracts on different mouse skin inflammation models : inference with their phenolic compound content

Departamento de Química Biológica, Programa de Pós-Graduação em Bioprospecção Universidade Regional do Cariri, 63105-000, Crato, CE, Brazil. Unidade Acadêmica de Serra Talhada, Universidade Federal Rural de Pernambuco, 56900-000, Serra Talhada, PE, Brazil. Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. Centro de Ciências da Saúde, Departamento de Farmácia Industrial, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. Centro de Ciências da Saúde, Departamento de Fisiologia e Farmacologia, Universidade Federal de Pernambuco, Recife, PE, Brazil. Faculdade de Medicina Estácio de Sá – Juazeiro do Norte, Juazeiro do Norte CE, Brazil.


INTRODUCTION
The skin is a large and complex organ that is able to provide a set of defense mechanisms in response to a variety of external stimuli such as physical injury, microbial invasion, environmental pollutants or solar irradiation (Freinkel and Woodley, 2000).These defense mechanisms are normally supposed to repair the damaged tissue or destroy the hazard agent (Lawrence and Gilroy, 2007).However, a misdirected or inappropriate immune activity can lead to a set of different inflammatory skin disorders, including atopic dermatitis, eczema and psoriasis (Novak and Leung, 2011;Stevenson and Lebwohl, 2011).
Skin inflammation is produced and maintained by the interaction of various inflammatory cell populations that migrate to the injury site in response to a variety of released pro-inflammatory mediators such as cytokines, interleukins, neuropeptides, histamine, serotonin, prostaglandins, leukotrienes, reactive oxygen species, among others (Sacca et al., 1997).Hence, the modulation on the production of these pro-inflammatory mediators has been applied in the treatment of cutaneous disorders.
For this purpose, drugs including glucocorticoids, antihistamines and non-steroidal antiinflammatory agents are employed.However, these drugs have demonstrated some limitations since they can exhibit some undesirable side effects and cannot be effective in all cases (Kupper and Fuhlbrigge, 2004;Schoepe et al., 2006).
Additionally, a steadily growing interest has been noticed in skin protection from excessive inflammatory insults by phenolic compounds, since their benefits are largely attributed to their classical chain-breaking antioxidant or free radical scavenging activities (Ismail et al., 2004;Nazaruk, 2008;Potapovich et al., 2011).
Caryocar coriaceum Wittm.(Caryocaraceae) is a tree widely found in Cerrado (savannah) areas from Araripe plateau, Southern Ceará State, Northeastern Brazil (Costa et al., 2004).Its fruit, popularly known as "pequi", is used as food by local population and it is a source of essential nutritional components including antioxidant vitamins (A and E) and unsaturated fatty acids (De Oliveira et al., 2010;Sena Jr. et al., 2010).Previous preclinical studies evidenced that the fixed oil from "pequi" pulp fruit presents gastroprotective, wound healing (Quirino et al., 2009) and topical anti-inflammatory activities in vivo (Saraiva et al., 2011a) and potential synergistic antibacterial effects when combined with antibiotic drugs (aminoglycosides) in vitro (Saraiva et al., 2011b).
Taking into account the ethnopharmacological evidences in relation to the use of pequi tree leaves as an herbal drug, we were encouraged to investigate the potential therapeutic use of C. coriaceum leaves hydroethanolic extract (CCHE) and methanolic fraction (CCMF) by evaluating their topical anti-inflammatory effect on different acute and chronic cutaneous inflammation models in mice; besides, to unveil the possible mechanisms of action involved in their activity.

Animals
Swiss mice (Mus musculus) from both sexes (25 to 35 g), with free access to water and food (Rodent Chow Labina, Brazil) and housed in standard polypropylene cages under controlled conditions of temperature (22 ± 2°C) and 12 h light/dark cycle, were used in this study.All the protocols concerned in this research were previously evaluated and approved by Research Ethics Committee from Fortaleza University (UNIFOR, Brazil), under number 10 to 020.

Extraction and fractionation procedures
The extract from C. coriaceum leaves was first obtained by CCHE, in contact with a solution of ethanol and water at ratio 1:1, for a period of72 h (room temperature).After that, CCHE was filtered, and the solvent was evaporated in vacuum at 50°C.CCMF was obtained from CCHE, using 90% methanol extraction and then evaporated in vacuum.After that, the residues was freeze-dried by liophylization and stored in a desiccator in the dark until further analysis.

Identification of phenolic compounds by HPLC-DAD
The procedures on the determination of phenolic compounds by HPLC-DAD were executed according to Laghari et al. (2011).Reverse phase chromatographic analyses were carried out under gradient conditions using C 18 column (4.6 × 250 mm) packed with 5 μm diameter particles; the mobile phase contained 2% acetic acid (A) and methanol (B), and the composition gradient was: 5% (B) for 2 min; 25% (B) until 10 min; 40, 50, 60, 70 and 80% (B) every 10 min.
All the samples and mobile phase were filtered through 0.45 μm membrane filter (Millipore) and then degassed by ultrasonic bath prior to use.Stock solutions of standards references were prepared in the HPLC mobile phase at a concentration range of 0.050 to 0.250 mg/ml for quercetin and rutin, and 0.020 to 0.200 mg/ml for gallic, chlorogenic and caffeic acids.Quantification was carried out by integration of the peaks using the external standard method, at 257 nm for gallic acid, 325 nm for chlorogenic and caffeic acids, and 365 nm for quercetin and rutin.The flow rate was 0.8 ml/min and the injection volume was 40 μl.The chromatography peaks were confirmed by comparing their retention time and Diode-Array-UV spectra with those of the reference standards.The quantification of quercetin, rutin, gallic acid, caffeic acid and chlorogenic acid by HPLC-DAD were based on references standards calibration curves.Calibration curves for each polyphenol were calculated as follows: gallic acid: y = 25681x -1536.2(r = 0.9971); chlorogenic acid: y = 27235x -1604.6 (r = 0.9799); caffeic acid: y = 23674x -1288.4(r = 0.9993); rutin: y = 29767x -1258.7 (r = 0.9989) and quercetin: y = 28077x -1741.5 (r = 0.9965).All chromatography operations were carried out at 25°C and in triplicate.

DPPH free radical scavenging assay
The antioxidant activity of the extracts was evaluated by monitoring their ability in quenching the stable free radical 2,2-diphenyl-1pikrylhydrazyl (DPPH • ), according to Choi et al. (2002), with minor modifications.CCHE or CCMF at concentrations ranging from 0.78 to 100 µg/ml diluted in ethanol vehicle were incubated in absence (blank) or presence of 0.3 mmol/L DPPH • ethanol solution in the dark and at room temperature.Vitamin C (L-ascorbic acid) and the flavonoid rutin at concentrations ranging from 0.78 to 100 µg/ml were used as positive controls.After 30 min, the absorbance was measured at 518 nm.
The concentration of DPPH • free radicals ([DPPH • ], in the percent of control) in the absence or presence of compounds was calculated using the following equation: where Abs sample is the absorbance obtained in the presence of different CCHE or CCMF concentrations in the presence of DPPH • ethanol solution, Abs blank is the absorbance obtained in the presence of different CCHE or CCMF concentrations in the absence of DPPH • ethanol solution (blank) and Abs control is that obtained in the absence of extracts and in the presence of DPPH • ethanol solution without any extract or positive control.The free radical scavenging capacity of CCHE and CCMF was calculated as their EC 50 values (the concentration necessary to inhibit 50% radical formation), using non-linear regression fit of plots where the abscissa axis represented the logarithm of concentration of tested plant extracts and the ordinate axis of the mean percentage of concentration of free radicals DPPH • in the well after 30 min.Tests were carried out in quadruplicate.

Arachidonic acid, croton oil and phenol single applicationinduced mouse ear edema
In order to evaluate the topical anti-inflammatory effect of CCHE and CCME with 1 and 2 mg/ear or 50 and 100 mg/ml, different acute models of cutaneous inflammation in vivo (Gábor, 2003;Ferreira et al., 2010) were performed.Swiss mice (n = 7/group) received 20 μl of arachidonic acid 0.1 mg/µl, 5% croton oil (v/v) and 10% phenol (v/v) diluted in acetone onto the inner and outer surfaces of the right ears previously pre-treated with a topical application of CCHE or CCMF diluted in ethanol (20 µl/ear), ethanol (negative control, 20 µl/ear), indomethacin (positive control for arachidonic acid, 2 mg/ear) or dexamethasone (positive control for phenol and croton oil, 0.08 mg/ear).The left ear received 20 μl of acetone (vehicle for irritant agents).The ear edema was evaluated 1 h after arachidonic acid, 1 h after phenol and 6 h after croton oil single application.
At the end of the stipulated period of exposure to each of the irritant substances, the mice were killed by cervical dislocation.Sixmillimeter diameter sections of the right and left ears were removed using a circular punch and weighed on a precision balance.The extent of the edema was expressed as the difference between the weight (in mg) of the section removed from the right ear (which received the irritant agent) and the weight (in mg) of the section removed from the left ear (which received vehicle acetone).The percentage of antiedematous effect (%) was calculated using the following formula.
where "A" is the mean of edema weight (mg) of the group treated with the drug (CCHE, CCMF, indomethacin or dexamethasone) and "B" is the mean of edema weight in the negative control.

Histamine subcutaneous application-induced mouse ear edema
In this model, mice (n = 7/group) were previously anesthetized with ketamine 20 mg/kg intraperitoneally (i.p.) and xylazine 10 mg/kg i.p.After that, their right ears were treated topically with ethanol (20 µl/ear, negative control), dexamethasone (0.08 mg/ear, positive control), CCHE or CCMF diluted in ethanol (1 and 2 mg/ear).Fifteen minutes later, the edema was induced on the right ear by intradermal application of 5 µl of histamine dihydrochloride 0.1 mg/ µl using a syringe with a 29G-hypodermical needle, while the left ear received 5 µl of saline solution by the same procedure described (Sham).The ear edema was evaluated and measured 2 h after the histamine solution application by the same procedures described in section 2.7 (Brand et al., 2002).

Croton oil multiple application-induced mouse ear edema
The following model is representative of a chronic cutaneous inflammation (Stanley et al., 1991), with duration of 9 days (days 0 to 8).Croton oil 5% (v/v) in acetone (20 µl/ear) was applied on the right ears and acetone on the left ears of Swiss mice (n = 6/group) with a micropipette on alternate days (days 0, 2, 4, 6 and 8).On days 4 to 7, the mice were treated on the inner and outer surfaces of the right ear with CCHE or CCMF 1 mg/ear, saline solution (NaCl 0.9%, 20 µl/ear, negative control) or dexamethasone (0.08 mg/ear, positive control) twice a day.The ear edema was evaluated 4 h after the first application of croton oil solution and daily by measuring the ear thickness with a digital caliper.The digital caliper was applied near the tip of the ear just distal to the cartilaginous ridges and the thickness was recorded in micrometer.

Statistical analysis
The results are expressed as mean ± standard error of mean (SEM).The comparison between groups was assessed by one-way analysis of variance (ANOVA) followed by Student-Newmann-Keuls test or by two-way ANOVA followed by Bonferroni test (repeated measures) when appropriated.Values of p < 0.05 were accepted as statistically significant.

Effect of CCHE and CCME on arachidonic acid, croton oil, phenol and histamine single applicationinduced ear edema
The topical single application of arachidonic acid, croton oil, phenol and histamine caused a significant inflammatory response in mouse ears in comparison to ears that received the correspondent vehicle of their respective irritant agent, as determined by the increase of ear weight or thickness.When used as positive control, both the corticosteroid dexamethasone (DEX) and the non-steroidal anti-inflammatory drug indomethacin (IND) significantly reduced the mouse ear edema when compared with vehicle-treated group (Figures 4 and 5).
As shown in Figure 4 and Table 2, both CCHE and CCME at concentrations 1 and 2 mg/ear exhibited significant topical anti-inflammatory activity that reduced inflammation edema in mouse ears caused by arachidonic acid (Figure 4A), phenol (Figure 4C) and histamine (Figure 4D).Although the comparison between the anti-inflammatory effect of both CCHE and CCMF extracts at 1 and 2 mg/ear did not significantly differ on arachidonic acid and phenol-induced edema models (Figure 4A and C), CCHE had better antiedematous effect when compared with CCMF in histamine-induced ear edema model (Figure 4D).In contrast, CCHE and CCME did not reduce the croton oil-induced edema as compared to negative control group, however, this is almost certainly experimental variation between protocols, mice or specific croton oil sample (or all three factors) (Figure 4B).The anti-inflammatory effect of both CCHE and CCME has demonstrated a profile of action similar to that observed using the control IND what can suggest that the participation of anti-inflammatory activity involves the path of prostaglandins and cytokines.

Effect of CCHE and CCMF on croton oil multiple application-induced ear edema
The alternate application of 5% croton oil on right ears of mice during the days 0 to 4 significantly increased the ear thickness in comparison to left ears of mice (treated with acetone), maintaining the inflammatory process during all the procedure.It was observed that the application of CCMF on days 4 to 8 (in an established inflammatory process by previous application of croton oil solution) caused a significant reduction (but slight) in ear thickness when compared with negative control group 48 h after the first treatment with CCMF and subsequent days (days 6 to 8) (Figure 5).Dexamethasone was effective in significantly reducing the established edema 24 h after its first application (on days 5 to 8) (Figure 5).The CCHE did not decrease the ear edema as compared to the negative control (Figure 5).The effect following topic administration did not exhibit anti-inflammatory activity, because tannins are capable of coagulate with lipid-protein present in the skin leading to complexes formations.Tannins also aid vasoconstriction, reducing vascular permeability; these facts can be responsible for decreased dermal absorption that leads to the low antiinflammatory action.On the other hand, this result corroborated with tannin levels present in the CCHE and CCMF (Table 1) since the levels of tannins present CCHE 30% higher than as shown by CCMF.

DISCUSSION
The screening of possible natural therapeutic agents from plants in animal models has been used as an efficient strategy in the discovery of novel and safe antiinflammatory drugs.The croton oil-induced ear edema has been widely used as acute or chronic skin inflammation models in vivo (Stanley et al., 1991;Gábor, 2003).A single application is able to activate protein kinase C (PKC), which in turn activates other enzymatic cascades, such as mitogen activated protein kinases (MAPK) and phospholipase A 2 (PLA 2 ), leading to release of platelet activation factor (PAF) and arachidonic acid (Gábor, 2003).Concomitantly, also induces the activation of several inducible nitric oxide synthase (iNOS)dependent intracellular signaling pathways that present a key role in the control of inflammatory response in the skin (Medeiros et al., 2009).As consequence, this set of events leads to vascular permeability, vasodilation, polymorphonuclear leukocytes migration, release of histamine and serotonine, and moderate synthesis of eicosanoids (Murakawa et al., 2006).
The use of repeated croton oil application (chronic model) is also associated with intense neutrophil and macrophage infiltration, T cells (CD 4+ and CD 8+ ) migration and hyperproliferative epidermis (acanthosis) (Stanley et al., 1991).Corticosteroids and 5-LOX inhibitors decrease the ear edema in these models while anti-histamines and cyclooxygenase (COX) inhibitors show little or no effect (Green and Shuster, 1987).CCMF and CCHE did not have anti-edematous effect against croton oil single application-induced ear edema at concentrations tested.However, CCMF exhibited a slight but significant anti-edematous effect on croton oil multiple applicationinduced ear edema (Figure 5).
It is accepted that the inflammatory process also leads to a situation of oxidative stress, where reactive oxygen species (ROS) such as superoxide anion (O 2 •− ), hydroxyl (OH • ) and peroxyl (ROO • ) radicals are generated by neutrophil and macrophage cellular infiltration (Khodra and Khalila, 2001).The mechanism of inflammation injury was attributed by damage of macromolecules and lipid peroxidation of cell membranes (Parejo et al., 2003).In addition, ROS can also stimulate the release of cytokines such as interleukin (IL)-1, tumor necrosis factor (TNF)-α, and interferon (IF)-γ, which stimulate the recruitment of additional neutrophils and macrophages.
HPLC analysis identified in both extracts the presence of chlorogenic acid, rutin, quercetin and lower concentrations of gallic acid and caffeic acid (Figure 1 and Table 1).The beneficial effect of phenolic compounds on skin has been largely attributed to their classical antioxidant properties, which could decrease the levels of ROS during an inflammatory process (Geronikaki and Gavalas, 2006).Furthermore, several anti-inflammatory drugs have been shown to have an antioxidant and/or radical scavenging mechanism as part of their activity (Sosa et al., 2002;Fu et al., 2010).According to DPPH free radical scavenging activity in vitro, both CCHE and CCMF exhibited strong antioxidant activity as compared to known antioxidants rutin (found in both extracts) and vitamin C (Figure 3).
Arachidonic acid is a precursor of inflammatory eicosanoids such as prostaglandin E 2 (PGE 2 ) and leukotrienes via COX-2 and 5-lipoxygenase (5-LOX) enzymes, respectively.Gallic acid alone is considered a contact skin sensitizer (Basketter et al., 1999), recent studies demonstrated that gallic acid can inhibit COX-2 activity in vitro and decrease PGE 2 and TxB 2 levels at 100 and 200 μmol/L, while increase TNF-α and IL-1β levels only at 200 μmol/L (Del Bufalo et al., 2011).In both extracts used in this study, the concentration of gallic acid is lower in comparison with other phenolic compounds (Figure 1 and Table 1).
Quercetin at 2 mg/ear showed a higher activity against arachidonic acid-induced ear edema than croton oilinduced edema by topical application in vivo (Yasukawa et al., 1989;Kim et al., 1993).COX and LOX inhibitors, leukotriene antagonists, anti-histamines and immunophillin-ligands are active in reducing the arachidonic acid-induced ear edema, whereas corticosteroids present slow effect in this model (Tramposch, 1999).Quercetin is pointed as a dual COX/LOX inhibitor (Laughton et al., 1991;You et al., 1999) and can also inhibit the histamine release in basophils activated with anti-IgE or with the calcium ionophore A23187 (Trinh et al., 2010).Caffeic acid is considered a classical 5-LOX inhibitor (Boudreau et al., 2012).Both CCHE and CCMF were able to decrease the arachidonic acid-induced ear edema when compared with negative control group (Figure 4A).
Histamine is a vasoactive amine involved in immediate type-hypersensitivity reactions and also plays a key role in human allergic reactions (Lee et al., 2012).It is released by mast cells activated by C3a and C5a protein complements, IgE-activated lymphocytes and arachidonic acid, which increases vascular permeability and vasodilation (Brand et al., 2002).Recent in vitro studies have demonstrated that chlorogenic acid blunted LPSinduced nitric oxide (NO), PGE 2 , and intracellular ROS production in RAW 264.7 cells (Yu et al., 2009) as well as inhibit compound 48/80-induced systemic anaphylactic shock in mice and anti-dinitrophenyl (DNP) IgE-mediated passive cutaneous anaphylaxis, besides to reduce histamine, cytokines from allergy and TNF-α release from RBL-2H3 cells activated by anti-DNP IgE (Qin et al., 2010;Trinh et al., 2010).Antihistamines and corticosteroids (dexamethasone) can decrease the ear edema in this model (Brand et al., 2002).Both extracts showed a significant antiedematous effect against histamine induced ear edema in mice (Figure 4D).
Phenol is an irritant agent able to mimic pathophysiological conditions similar to contact dermatitis in mice (Lim et al., 2004).A single application of a 10% phenol solution on skin is able to disrupt keratinocyte membranes, leading to release of cytokines such as interleukin (IL)-1α, tumor necrosis factor (TNF)-α and IL-8 via protein kinase C (PKC)-independent mechanism, which in turn release other inflammatory mediators, such as histamine, arachidonic acid metabolites and ROS (Wilmer et al., 1994;Murray et al., 2007).Both CCHE and CCMF decreased the phenol-induced ear edema (Figure 4C).
Taking together the results presented here, our data give important support that CCHE and CCMF could be used as topical anti-inflammatory herb by possibly exerting modulation on production of inflammatory  mediators such as histamine, reactive oxygen species and arachidonic acid metabolites.The continual efforts on understanding of the potential use of C. coriaceum leaves as herb will provide new insight into the antiinflammatory activity of its extracts, and eventually lead to development of safe and effective treatment on cutaneous inflammation.

Figure 3 .
Figure 3. DPPH • free radical scavenging activity of CCHE, CCMF, and the positive controls rutin (RUT) and L-ascorbic acid (VITC).The X-axis presents the logarithm of concentration of tested extracts or positive controls (ranging from 0.78 to 100.00 µg/ml) and the Y-axis the mean percentage of concentration of free radicals DPPH • in the well after 30 min (absorbance read at 518 nm wavelength).The absence of extracts or positive controls (negative control) represents 100% of concentration (maximum concentration) of DPPH • .The assay was conducted in quadruplicate.

Figure 4 .
Figure 4. Effect of CCHE, CCMF, indomethacin (IND) or dexamethasone (DEX) on mice ear edema induced by the arachidonic acid (A), croton oil (B), phenol (C) and histamine (D) single application.Ears from control group received ethanol vehicle as treatment.Each point represents the mean ± SEM of 7 mice.Means with different lowercase letters differ significantly at least at p < 0.05 (One-Way ANOVA followed by Student-Newman-Keuls test).

Figure 5 .
Figure 5.Effect of CCHE, CCMF and dexamethasone (DEX) expressed as time-response curve on croton oil multiple application-induced ear edema from days 0 to 8. A solution of 5% croton oil in acetone was applied on alternate days(0, 2, 4, 6 and 8).The thickness of the ears was measured daily, using a digital caliper.On days 4 to 7, DEX 0.08 mg/ear, CCHE 1 mg/ear or CCMF 1 mg/ear were administered (arrows indicate the days when the treatment occurred).The effect of the compounds was examined by changes in ear thickness, calculated as the difference between the initial and final thickness.The points represent the mean of 6 animals and vertical bars SEM (Two-way ANOVA followed by Bonferroni test; *p < 0.05, **p < 0.01, and ***p < 0.001 compared to salinetreated group).

Table 1 .
Phenolic compounds detected in CCHE and CCMF by HPLC-DAD.Results are expressed as mean ± SEM of three determinations.RT = retention time.