Chemical composition , antioxidant and topical anti-inflammatory activities of Croton cordiifolius Baill . ( Euphorbiaceae )

Chemical composition, antioxidant and topical antiinflammatory activities of Croton cordiifolius Baill. (Euphorbiaceae) Iasmine Andreza Basilio dos Santos Alves, Simone Maria dos Santos, Raudiney Frankilin Vasconcelos Mendes, José Wellinton da Silva, Maria de Fátima Rodrigues, Bárbara de Azevedo Ramos, Márcia Vanusa da Silva, Maria Tereza dos Santos Correia, Fernanda das Chagas Angelo Mendes Tenório, Roberta Jeane Bezerra Jorge, René Duarte Martins, Julianna Ferreira Cavalcanti de Albuquerque, Karina Perrelli Randau and Rafael Matos Ximenes

type apparatus, yielding 1.56% of C. cordiifolius essential oil (CcEO).The essential oil was subsequently dried over anhydrous sodium sulfate, protected from light and frozen under -20ºC until use.Fresh plant material was also extracted with ethanol (1:10, w/v) by dynamic maceration for 3 h.After filtration, the solvent was evaporated using a rotary evaporator (RV10, IKA, Germany), yielding 6.1% of C. cordiifolius ethanolic extract (CcEE).

Essential oil composition
Gas chromatography and mass spectrometry were carried out using a Shimadzu™ model 7A/QP 5050A equipped with a capillary non-polar DB-5 column (30 m x 0.25 mm x 0.25 µm).The oven temperature was programmed at 70ºC with an increase of 4ºC/min until 280ºC, and maintained for 15 min.The carrier gas was helium, with a constant flow of 1.4 ml/min.The temperature of the ionization source was maintained at 280ºC, the ionization energy at 70 eV, and the ionization current at 0.7 kV.Mass spectra were recorded from 30 to 450 m/z.Individual components were identified by matching their 70 eV mass spectra with those of the spectrometer data base using the Wiley L-Built and NIST libraries and by comparing their retention indices with those reported in the literature (Adams, 2001).The retention indices were compared with those of obtained by Craveiro et al. (1981) for other Euphorbiaceae species.

Phytochemical screening, total phenolic and flavonoid content
The ethanolic extract of C. cordiifolius stem back was analyzed by thin-layer chromatography (TLC) using Si gel F 254 plates (ALUGRAM ® 818131, Macherey-Nagel, Germany) and different solvent systems.It was verified the presence or absence of flavonoids, phenylpropanoids, terpenoids, steroids, saponins, alkaloids, coumarins, proanthocyanidins and quinones as described in Table 1.
Total phenolic content was determined by the Folin-Ciocalteu method (Li et al., 2008).Briefly, an aliquot of 200 µL of the extracts diluted in methanol (1 mg/mL) was added to 1 mL of the 10-fold diluted Folin-Ciocalteu reagent.After 4 min, 2.5 mL of sodium carbonate solution (0.7 M) was added.The test tubes were incubated for 30 min at room temperature, protected from light, after which the absorbance was read at 765 nm.Gallic acid was used for standard curve calibration (0 -100 mg/L).The results were obtained from the regression equation: y = 0.0121x + 0.032, R 2 = 0.9967 and expressed as milligram of gallic acid equivalent (mgGAE)/g of extract.The experiments were performed in triplicate.
The determination of flavonoids was made by the method described by Woisky and Salatino (1998).An aliquot of 0.5 mL of the extracts diluted in methanol (1 mg/mL) was added to 0.5 mL of 2% AlCl 3 in methanol.The test tubes were incubated for 30 min at room temperature, protected from light, after which the absorbance was read at 420 nm.Quercetin was used for standard curve calibration (0 -100 mg/L).The results were obtained from the regression equation: y = 0.0262x -0.0892, R 2 = 0.9982 and expressed as milligram of quercetin equivalent (mgQE)/g of extract.
The experiments were performed in triplicate.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License phosphomolybdate method using ascorbic acid as standard (PIETRO et al., 1999).A 0.1 mL aliquot of extract diluted to 1 mg/mL in methanol was added to 1 mL of solution containing 600 mM sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate.Test tubes were capped and incubated in a boiling water bath at 95ºC for 90 min.Samples were then cooled to 25ºC at room temperature and absorbance relative to a blank (1 ml of reagent solution and 0.1 mL of methanol) was measured at 695 nm.Total antioxidant activity (TAC) was expressed relative to ascorbic acid and calculated by the following formula, Where Babs is the absorbance of the blank, Sabs is the absorbance of the sample (extracts) and AAabs is the absorbance of ascorbic acid.

1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging activity
DPPH free radical-scavenging activity of CcEE was determined using 96-well microplates as described by Brand-Williams et al. (1995).Briefly, 0.04 mL of CcEE or gallic acid diluted in methanol (31.25, 62.5, 125, 250, 500, 1000 µg/ml) and 0.25 ml of 1 mM DPPH in methanol was added to each well.Microplate was kept for 30 min at 25ºC, protected from light.After this time, absorbance was measured at 517 nm.The free radical-scavenging activity of CcEE was expressed as percentage of inhibition of DPPH, calculated by the following formula, Where Babs is the absorbance of the blank (0.04 mL of methanol and 0.25 mL of 1 mM DPPH) and Sabs is the absorbance of the sample (CcEE or gallic acid).

2,2-azino-bis-(3 ethylbenzothiazoline)-6-sufonic acid (ABTS) radical cation scavenging activity
ABTS (7 mM) was oxidized by the addition of 140 Mm potassium persulphate.The solution was kept at 25ºC for 16 h, in the dark.Before use, the ABTS+ solution was diluted with ethanol to an absorbance of 0.70 ± 0.02 units at 734 nm.For the determination of ABTS+ radical scavenging activity of CcEE, 0.01 mL of CcEE (31.25,62.5,125,250,500,1000 µg/ml) diluted in methanol was added to 1.0 mL of ABTS+ solution.After 6 min, the absorbance was measured at 734 nm.ABTS radical cation scavenging activity of CcEE was expressed as percentage of inhibition of ABTS+, calculated by the following formula, Where Babs is the absorbance of the blank (0.01 mL of methanol and 1.0 mL of ABTS+) and Sabs is the absorbance of CcEE.

Animals
Male Swiss or BALB/c mice (20-30, n = 6) were provided by the Animal Facility of the Universidade Federal de Pernambuco.The animals were housed in a room with controlled temperature (23 ± 2°C) under a 12/12 h light/dark cycle with food and water ad libitum.Experiments were carried out according to the Guide for the Care and Use of Laboratory Animals of the US Department of Health and Human Services (NIH publication number 85-23, revised in 1985).The project received prior approval from the Animal Ethics Committee of Universidade Federal de Pernambuco (protocol number 23076.016724/2016-62).

Ear edema measurement
For evaluation of ear weight, animals were euthanized and samples of 6 mm diameter were taken from both ears using a biopsy punch (Richter®, Brazil).Each biopsy was weighed on a semi-micro analytical balance (AUW-D 220, Shimadzu, Japan).Ear edema (EE) was expressed as the increase in ear sample weight, using the following formula: Where wRE is the weight obtained from the right ear sample (inflamed ear) and wLE is the weight obtained from the left ear sample (noninflamed ear).
For all treatments, animals were anesthetized with 1% halothane.Right ears were then challenged with different phlogistic agents diluted in acetone (20 µL).CcEE and CcEO were applied topically in 20 µL acetone.Dexamethasone or indomethacin (0.1 and 0.5 mg/ear, respectively) was used topically as a positive control.Ruthenium red (3 mg/kg, s.c.) was used as a positive control for capsaicin-induced ear edema.

PMA-induced ear edema
Ear edema was induced by topical application of 20 µL of PMA (2.5 µg/ear) in acetone on both sides of the right ear.Immediately after, CcEE (0.1, 0.3, and 1.0 mg/ear), CcEO (1, 3 and 10 µL/ear) or dexamethasone (0.1 mg/ear) was topically applied on both sides of the right ear, while the left ear received 20 µL of acetone.After 6 h, animals were euthanized for ear edema measurement (Carlson et al., 1985).

Histological analysis
After weighing, ear samples were fixed in buffered formalin for 24 h.Fixed tissued were then dehydrated by adding increasing concentrations of ethanol (70-100%), and processed for embeddging in paraffin.The resulting blocks were sliced into 4 µm thick sections, stained with hematoxylin and eosin and observed under a light microscope (Nikon, Japan).

Statistical analysis
Data are expressed as mean ± S.E.M. and analyzed by ANOVA followed by Bonferroni post-test using GraphPad Prism 5.0 with significance * set at p < 0.05.
Total antioxidant activity was determined with the phosphomolybdenum method, which is based on reduction of molybdenum VI to molybdenum V, in the presence of antioxidants.This reduction results in the formation of a green complex.According to this assay, total antioxidant activity in CcEE was 26.88 ± 2.83% of the activity of the ascorbic acid control.Free radical scavenging activity by DPPH and ABTS+ assays were also measured, with EC 50 values > 1,000 µg/mL of CcEE for both assays.

PMA-induced ear edema
CcEE (0.1, 0.3 and 1 mg/ear) significantly inhibited PMAinduced ear edema at all tested doses as seen in Figure 1A.By contrast, CcEO (1, 3 and 10 µL/ear) showed less promising results, with significant activity only at the highest dose (Figure 1B).Histological analysis of ear samples aimed to evaluate the following parameters: congested vessels (CV); polymorphonuclear cells infiltration (PCI) and/or edema (E).Total ear and epidermis thickness was also evaluated relative to the negative control group.PMA-induced ear edema produced CV filled with white blood cells, intense PCI and edema.Dexamethasone (0.1 mg/ear) and CcEE at 0.3 and 1 mg/ear yielded similar results.CV were present and filled with red blood cells and discrete PCI and E. Ears treated with CcEE at 0.1 mg/ear and CcEO at all doses tested (1, 3 and 10 µL/ear) had CV filled with red and white blood cells and moderate PCI and E (Figure 2A-H).Based on these results, CcEE at 1.0 mg/ear was chosen for elucidation of CcEE's mechanism of topical anti-inflammatory activity using other phlogist agents.

DISCUSSION
The Croton genus is characterized by a large chemical diversity.Based on the existing descriptions of chemical composition of many Croton species, this genus can be characterized as a promising source of bioactive molecules with a remarkable potential for discovery of novel biological activities.Growing interest in studying Croton species has resulted in isolation and identification of more than 100 currently known chemical compounds in Brazil (Angélico, 2011;Song et al., 2015).
Although not common in the Euphorbiaceae family, some Croton species should be highlighted for the production of different alkaloid classes (Salatino et al., 2007).Identification of this group of secondary metabolites by TLC in C. cordiifolius corroborates several studies in other species of the same genus.Salatino et al. (2007) described 35 alkaloid types from 14 Croton species.Among the species present in Brazilian caatinga, β-carboline alkaloids 2ethoxycarbonyltetrahydroharman and 6-hydroxy-2methyltetradydroharman have been isolated from C. moritibensis (Araújo-Júnior et al., 2004); anabasine and the novel guaiane-type alkaloids muscicapines A, B and C were isolated from roots of C. muscicapa (Araújo-Júnior et al., 2005).Alkaloids were also identified in C. campestris (Brito-Júnior et al., 2014) and C. rhamnifolius roots, and C. rhamnifolioides leaves, stem and roots (Randau et al., 2004); however, no compounds have been isolated.
Flavonoids are a class of polyphenols widely distributed in plants, including the genus Croton.Different studies have reported antioxidant, analgesic, antibacterial, antifungal, anti-inflammatory, antiviral, antitumor, antiallergic, and antiparasitic activities for these metabolites (Coellho et al., 2016).Among Croton species, it has not yet been possible to establish a standard for flavonoids.Although some compounds have already been isolated from hexanic, ethanolic and methanolic extracts of Croton spp., most investigations are not directed at the characterization of the flavonoid profile presented by the plant.A standardized analysis of the flavonoids present in different species of Croton may be an auxiliary tool in the differentiation of this genus subgroups.
Although tannins are ubiquitous polyphenols in plants, only proanthocyanidins have been reported for Croton species, specifically those containing red latex such as C. urucurana, C. lechleri and C. panamensis.These metabolites were not found in Caatinga species (Salatino et al., 2007).
Terpenes and steroids are chemical constituents of interest owing to their anti-inflammatory, analgesic, antimicrobial, antiviral, hepatoprotective, and hormonal activities (Gupta et al., 1969;Mahato et al., 1988).Clerodane diterpenes are frequently found in the stem bark of Croton species, such as C. cajucara, C. urucurana, C. lechleri, but few were found in caatinga species, such as C. brasiliensis, from which clerodanes diterpenes crotobrasilins A and B were isolated from leaves and stem bark (Palmeira-Júnior et al., 2005).Diterpenes bearing the novel skeleton sarcopetalane, besides labdane, trachylobane, cembranoid, and halimane skeletons are not found in species from Caatinga.
Coumarins, which are rare in the Euphorbiaceae family and Croton genus, were found in C. cordiifolius in this study.This result concurs with those reported by Lima and collaborators (2010), who related the presence of a coumarin in C.adenocalyx, representing an important role in the chemotaxonomy differentiation of these species.Essential oils have been described for various species of this genus (Almeida et al., 2015;LIMA et al., 2010;LEITE et al., 2015;Melo et al., 2013;Neves and Camara, 2012;Ramos et al., 2013;Turiel et al., 2016).Nogueira et al. (2015) described the constituents of the essential oil of C. cordiifolius leaves, which is rich in mono-and sesquiterpenes and whose main constituents were the monoterpenes 1,8-cineole and α-phellandrene.By contrast, the major constituents of the essential oil of Croton cordiifolius stem bark were the monoterpenes αand β-pinene.α-pinene was also a major compound of the essential oil of C. argyrophylloides aerial parts (Morais et al., 2006), C. conduplicatus stem bark (Almeida et al., 2015), and C. adenocalyx leaves (Lima et al., 2010).Some authors have speculated that the cooccurrence of αand β-pinene is a characteristic of the genus Croton.Others claim that β-caryophyllene and linalool are equally frequent major constituents (Almeida et al., 2015).
In addition to differences in essential oil constitution among distinct species of the genus Croton, several authors have demonstrated distinct essential oil compositions of different tissues from the same species (Brasil et al., 2009;Lima et al., 2010;Morais et al., 2006;Neves and Camara, 2012).In general, the essential oils of Brazilian Croton species present mono and sesquiterpenes as major components, with the exception of C. zehntneri, in which the phenylpropanoids eugenol and anetol predominate (Craveiro et al., 1981).
Most studies of the chemical constitution of the essential oil of Croton species refer to the leaves or aerial parts.Among the studies that report the constitution of the stem essential oil, it can be highlighted the one realized by Neves and Camara (2012) for C. pulegioides, with α-calacorene as the major compound, and for C. heliotropiifolius, with guaiol as the major compound; by Almeida et al. (2015) for C. conduplicatus, with α-pinene as the major compound; and by Suarez et al. (2005) for C. molambo with methyleugenol as the major compound.
The chemical composition of Croton species may be correlated with their geographical distribution.An example of this is the labdane and caurane diterpenes, which have not yet been reported for New World species; alkaloids, which were only found in American species; and the aromatic species, which are also prevalent in America (Salatino et al., 2007).Moreover, the presence of compounds found only in Brazilian caatinga species could indicate the existence of unique chemotypes within species of Croton found only in that biome (Angélico, 2011).
C. cordiifolius, C. heliotropiiflolius, and C. conduplicatus are popularly known as "quebra-faca" due to the rigidity of their stem (Randau et al., 2004).These species also present similar popular uses: C. cordiifolius is used to treat general inflammation, pain, and gastrointestinal disturbances (Nogueira et al., 2015); C. heliotropiiflolius is used as folk remedy for the treatment of wounds, inflammation, fever, cancer, stomach pain, vomiting, bloody diarrhea and to alleviate fever (Neves and Camara, 2012;Randau et al., 2004); the decoction of C. conduplicatus leaves and stem bark is used in folk medicine to treat influenza, headache, indigestion, stomach problems and stomachache (Almeida et al., 2015).
In this context, the differentiation of their chemical composition and pharmacological activities, together with their botanical characterization, constitute a diagnostic tool for these species.Neves and Camara (2012) studied the chemical composition of the essential oil of C. heliotropiiflolius stem bark, observing a characteristic profile of the genus by the presence of β-caryophyllene as one of the major constituents.It differs from C. cordiifolius stem bark essential oil by the presence of sesquiterpenes and phenylpropanoid derivatives.Almeida et al. (2015) described for the first time the chemical constitution of the essential oil of C. conduplicatus stem bark, whose main compounds were α-pinene, β-pinene, camphor and (E)-caryophyllene.The chemical composition of C. cordiifolius stem bark essential oil is very similar, with α-and β-pinene as major constituents.The major difference is the presence of the monoterpene camphene as the third major constituent in C. cordiifolius, which can be converted into camphor in vivo.Randau et al. (2004) performed a phytochemical study of the extract of different parts of C. rhamnifilolioides.It was observed the presence of alkaloids, triterpenes and steroids, sugars, flavonoids and phenylpropanoglycosides.Therefore, the presence of coumarins in C. cordiifolius bark extract can be considered a useful marker in the differentiation of these species.Despite the existence of some studies that point to chemical patterns in the genus, given its variability and the small sampling of species chemically studied, definitive conclusions can not be drawn about chemical relationships among Croton species (Salatino et al., 2007).
Many Croton species studies have reported outstanding antioxidant activities, which set a precedent for the results of this study of C. cordiifolius.Morais and collaborators (2006)  Chronic inflammation mediates the development of various diseases, and increased levels of free radicals have been found in many pathological conditions other than inflammation, including cancer, ischemic disorders, and dementia (Ramos et al., 2013).The search for medicinal plants with anti-inflammatory and antioxidant activities is fundamental to direct the development of pharmaceutical products designed to treat major diseases.
Mechanisms of anti-inflammatory activity can be elucidated through research with animal models in which edema is induced by different phlogistic agents with different well-understood mechanisms.To identify topical anti-inflammatory mechanisms of C. cordiifolius, we used five phlogistic agents with distinct mechanisms in a mouse model of topical inflammation.
Ear edema induced by PMA is primarily mediated by prostaglandin E2 and protein kinase C.This model is useful for screening topical active extracts and/or compounds, and anti-inflammatory efficacy implies the presence of prostaglandin and leukotriene biosynthesis inhibitors (Silva et al., 2005).In this model, CcEE probably inhibited edema by directly interfering with prostaglandins' action or by inhibiting enzymes such as cyclooxygenase or phospholipase A2 (PLA2).According to Nair and collaborators (2006), the flavonoid quercetin decreased production and expression of proinflammatory cytokine TNF-α in human mononuclear cell cultures stimulated by phorbol myristate acetate (PMA).This effect was associated with inhibition of NF-kB.Other flavonoids, such as santin and ermanin, also inhibited PMA-induced ear edema in mice, being more active than the positive controlindomethacin at identifcal concentrations (3 mg/ear) (Martinez et al., 1997).Thus, the presence of flavonoids demonstrated in phytochemical screening and quantified by spectrophotometry could contribute to this activity.
The inflammatory process instigated by AA is mediated by production of metabolites such as prostaglandin E2 (PGE2), leukotriene C4 (LTC4), and leukotriene D4 (LTD4) (Humes et al., 1986).The ear edema induced by AA is characterized by sensitivity to inhibitors of cyclooxygenase and lipoxygenase enzymes.Therefore, AA is commonly used to validate substances capable of inhibiting the action of mediators of edema (Crummey et al., 1987;Opas et al., 1986;Humes et al., 1986).This model is not sensitive to PLA2 inhibitors such as glucocorticoids.CcEE probably inhibit edema in this model by preventing the metabolism of arachidonic acid to prostaglandins and leukotrienes or by inhibiting the action of its metabolites.In the literature, there is no evidence of topical anti-inflammatory activity in that model from species of the genus Croton.
Because ethyl phenylpropiolate (EPP) is responsible for release of inflammatory mediators such as histamine and serotonin (Nualkaew et al., 2009), CcEE may interfere with the release of these inflammatory mediators.Phenol-induced ear edema mimics contact dermatitis processes visualized in humans (Lim et al., 2004).The intense irritation resulting from application of this phlogistic agent is a consequence of disruption of keratinocytes plasma membranes, with release of preformed IL-1α, IL-8, TNF-α, free radicals, and inflammatory mediators such as AA downstream metabolites.
Although this process occurs independently of AA metabolites, the mentioned cytokines (IL-1α, IL-8, TNF-α) can induce production and release of AA and reactive oxygen species (Lim et al., 2004;Murray et al., 2007;Wilmer et al. 1994).In contrast to PMA, phenol activates nuclear transcription factors through protein kinase C dependent pathways to induce inflammatory cytokines.Despite using different routes, both models produce arachidonic acid metabolites and reactive oxygen species in the inflammatory response (Wilmer et al., 1994).In this context, the performance of CcEE in reducing production of inflammatory mediators and cytokines through protein kinase C dependent pathways can be highlighted.
Capsaicin, an alkaloid obtained from species of the genus Capsicum, mediates a neurogenic inflammatory response characterized by vasodilation-induced edema with increased blood flow, and plasma extravasation, in addition to sensitized nociceptors responsible for contact induced-pain sensation in skin or mucous membranes (Zegarska et al., 2006).In this process, TRPV-1 receptors are activated, resulting in the release of neuropeptides, such as substance P, which are responsible for erythema and papule formation and macrophage degranulation, followed by release of substances such as histamine and serotonin (Inoue et al., 1993).
Topical application of capsaicin promotes the release of pro-inflammatory mediators which result in an immediate vasodilation and erythema response followed by edema, which peaks within 30 minutes after administration (Gabor, 2000;Zegarska et al., 2006).CcEE did not reduce capsaicin-induced edema, suggesting that it does not act on TRPV-1 receptors, and therefore does not inhibit mast cell degranulation or release of histamine and serotonin, mediators responsible for increasing cell permeability and edema (Inoue et al., 1993).
The anti-inflammatory activity of C. cordiifolius found in this study corroborates the ethnobotanical survey conducted by Nogueira and collaborators (2015), which showed the highest number of "quebra-faca" citations for inflammation and pain treatment.

Conclusions
The first of its kind phytochemical survey of Croton cordiifolius stem bark extract and the characterization of its essential oil provide important information for directing future research aimed at isolating chemical constituents of this species, which is widely used in Northeast Brazilian folk medicine.
Our in vivo anti-inflammatory activity tests showed that C. cordiifolius essential oil is not effective at the doses tested.In contrast, the extract contained anti-inflammatory compounds with different mechanisms of action because it showed significant activity in four of the five assays.Our results suggest that the extract can interfere with AA metabolism and prostaglandin biosynthesis, release of inflammatory mediators such as histamine and serotonin, and inhibition of preformed cytokine release.No inhibition of TRPV-1 receptors or mast cells degranulation was observed.Additionally, the observed antioxidant activity of C. cordiifolius stem bark extract could be, in part, responsible for its anti-inflammatory effects.

Table 1 .
Solvent systems and spray reagents used for thin-layer chromatography analysis of Croton cordiifolius Baill.(Euphorbiaceae) stem bark ethanolic extract.

Table 2 .
Chemical composition of the essential oil of Croton cordiifolius Baill.(Euphorbiaceae) stem bark.