Baccharis dracunculifolia DC (Asteraceae) Root Extract and Its Triterpene Baccharis Oxide Display Topical Anti-Inflammatory Effects on Different Mice Ear Edema Models

B. dracunculifolia is popularly used to treat skin diseases. This work aimed to evaluate the topical anti-inflammatory properties of B. dracunculifolia root extract (BdR) and its major compound baccharis oxide (BOx) on mice ear edema models. BdR was analyzed by GC-MS, and BOx was isolated by chromatographic fractionation. Topical anti-inflammatory activities were determined by using the croton oil, capsaicin, histamine, and phenol-induced mouse ear edema models. N-acetyl-β-D- glucosaminidase (NAG) and myeloperoxidase (MPO) activities, as well as NO dosage and histopathological analyses, were also evaluated. Phytochemical analysis of BdR showed BOx as one of the major constituents. BdR and BOx (both at 0.1, 0.5, and 1.0 mg/ear) significantly reduced croton oil, histamine, and phenol-induced ear edema, while only BOx was effective in reducing capsaicin-induced edema. MPO and NAG activities, as well as NO production, were significantly inhibited by BdR and BOx. Histopathological analysis confirmed the topical anti-inflammatory properties of BdR and BOx. Our findings showed that BdR and BOx demonstrated significant topical anti-inflammatory effects in mouse ear edema induced by different agents, suggesting their possible application on skin inflammatory diseases.


Introduction
Infammatory skin conditions, like psoriasis and dermatitis, are one of the major causes of disability due to their negative efects in patients, mainly pain and psychological impact [1]. Tese skin conditions can be occasional (such as redness) or chronic (such as acne, dermatitis, rosacea, and psoriasis) [2]. Contact dermatitis is an infammatory response that arises due to the contact with physical or chemical agents that cause skin damage and cellular alterations, as well as releasing of proinfammatory mediators [3]. Additionally, skin infammatory disorders are related to the stimulation of infammatory pathways with involvement of many chemical mediators, substrates, and enzymes, which may serve as potential targets for novel anti-infammatory drugs [4]. Nowadays, topical corticosteroids are used in the treatment of cutaneous infammatory diseases [5]. However, these medications may cause pruritus, telangiectasia, stomach irritation, and ulceration, among other side efects [6]. Terefore, these drawbacks emphasize the need for discovering new and efcient therapeutic options to treat skin disorders [5]. In this regard, natural products, such as plant extracts and their compounds, can be used as an attractive strategy for treating skin infammatory illnesses [7].
Plants of the genus Baccharis are used in South America as anti-infammatory to treat skin disorders [8]. Among them, Baccharis dracunculifolia De Candole (Asteraceae), known as "alecrim do campo," is a plant popularly used for the treatment of infammations and skin diseases [9][10][11]. Also, the economic and commercial interests of B. dracunculifolia have increased in the last years, since this species is one of the most important plant sources of the Brazilian green propolis [8]. B. dracunculifolia leaf extracts displayed analgesic and anti-infammatory efects in mice [10], also exhibiting immunomodulatory [12,13], antimicrobial [14], and hepatoprotective [15] activities. Te antiinfammatory efects of B. dracunculifolia leaf extract have been associated with some phenolic acids, mainly artepillin C, as well as favonoids, such as isosakuranetin [12]. In addition, it was demonstrated an interesting antiinfammatory efect of B. dracunculifolia essential oil on skin infammation [9]. Additionally, the previous study showed that the B. dracunculifolia root extract exhibits in vitro immunomodulatory action in murine macrophages [13]. However, the potential of the B. dracunculifolia root extract and its compounds as topical anti-infammatory agents has not yet been explored.
Ten, in this study, it was evaluated the topical antiinfammatory properties of the B. dracunculifolia root extract and its major compound baccharis oxide on skin infammation through mice ear edema models.

Preparation of the Crude Plant Extract.
Tis research was registered (Number #AE32DB3) at SisGen, the National System for the Management of Genetic Heritage and Associated Traditional Knowledge. After plant authentication (Dr P.L. Viana; voucher specimen CESJ 47482), B. dracunculifolia roots were collected in August 2017 at the campus of the Federal University of Juiz de Fora (UFJF). Roots (2300 g) were dried, pulverized, and extracted, by maceration, employing n-hexane (5.6 L) as solvent. Next, nhexane was removed via rotary evaporation to yield the nhexane extract of B. dracunculifolia roots (BdR, 18.5 g).

GC-MS Analysis.
BdR and its isolated compound BOx were analyzed by GC-MS (Shimadzu QP2010 Plus with a Restek RTX-5MS column). Temperature was programed to increase from 120 to 240°C (4°C/min) with an injector temperature of 150°C. Also, an ion-source temperature of 280°C with He (1.0 mL/min) as carrier gas, a split ratio of 1 : 10, and an injection volume of 8 μL were used. Te electron ionization mode (70 eV) was used, and the spectra were carried with a scan interval of 0.5 s over the mass range of 40-600 Da. Identifcation of BdR compounds was achieved by a comparison of their mass spectral fragmentation patterns with those of spectral libraries (NIST 08 and Wiley 7), as well as by their retention indices obtained with reference to a homologous series of n-alkanes (C 7 -C 30 ) [17].

Animals.
Male Swiss mice (30-40 g) were kept in groups of eight animals (n � 8) and maintained in plastic cages (47 × 34 × 18 cm) under a 12/12 h light-dark cycle, in controlled temperature (22 ± 2°C), with food and water ad libitum. Mice were acclimated in the experimentation room for 24 h before the experiments. Te euthanasia of the animals was performed with anesthetic overdose (300 mg/kg ketamine and 30 mg/kg xylazine). All experimental procedures were approved by the Ethics Committee on Animal Use/UFJF and conducted in accordance with the guidelines of the COBEA (Brazilian College of Animal Experimentation-Protocol no 022/2018, approved in 08/21/ 2018). All experiments were performed according to the experimental design ( Figure 1).

Croton Oil-Induced Ear Edema Model.
Ear edema was produced with croton oil (2.5% v/v in acetone, 20 µL), which was applied on the surface of the right ear, while the left ear received only acetone (20 µL) [7,18]. Te right ear was treated with BdR (0.1, 0.5 and 1.0 mg/ear), BOx (0.1, 0.5 and 1.0 mg/ear), dexamethasone (0.1 mg/ear), and 0.9% NaCl (negative control group). Te ear thickness (µm) was measured after 6 and 24 h [7,9], using a digital micrometer, to evaluate edema development. Edema was measured by the diference in thickness (µm) between the right and left ears [7,9,18]. Ear fragments were also used in histopathological analysis and for determination of infammatory markers.
To assess NAG activity [9,20], quadruplicates of 100 μL of the supernatant were placed on a 96-well plate in which were added NAG (200 µL of 2.24 mM in citrate bufer 0.1 M, pH 4.5) and 50 µL of p-nitrophenyl-N-acetyl-β-D-glucosamine. After incubation of microplates (37°C, 10 min), the reaction was stopped by addition of glycine bufer (30 µL of 0.2 M, pH 10.6). A microplate reader, at 405 nm, was used to determine the colorimetric enzymatic activity. Results were expressed as optical density/mg of protein (mOD/mg of protein) [9,20].

Nitric Oxide Assay.
Nitrite, the stable product of nitric oxide (NO), was measured [21]. In this assay, the homogenate was prepared from three ear fragments obtained from the croton oil-induced model. Ten, PBS (3 mL, pH 7.4) was added to each ear fragment, which was crushed for 1 min. After, the obtained homogenate was transferred to test tubes and centrifuged (7000 RPM, 15 min). Next, 150 µL of each supernatant was mixed with 150 µL of Griess reagent, and microplates were incubated at room temperature for 30 min. Te measurements of absorbance were made in a microplate reader at 540 nm. Nitrite levels were performed in triplicate using a standard curve of nitrite (NaNO 2 ) prepared in PBS (pH 7.4). Results were expressed in µM, and experiments were made in triplicate [21].

Phenol-Induced Ear Edema
Model. Phenol (10% in acetone, 20 µL) was applied in the right ears of mice, while in the left ears, only acetone was applied [22,23]. After 15 min, BdR and BOx (both at 0.1, 0.5, and 1.0 mg/ear), as well as dexamethasone (0.1 mg/ear, positive control), were dissolved in acetone and topically administered (20 µL) to the right ears. Saline (20 µL) was used as a negative control. Te thickness (µm) and weight (mg) were evaluated after 2 h of treatment, and edema was determined by the diference between the right and left ears [22,23].  Evidence-Based Complementary and Alternative Medicine 2.11. Capsaicin-Induced Ear Edema Model. BdR (0.1, 0.5, and 1.0 mg/ear), BOx (0.1, 0.5, and 1.0 mg/ear), and dexamethasone (0.1 mg/ear, used as a positive control) were dissolved in acetone and administered topically (20 µL) to the right ears of mice, also using saline (20 µL) as a negative control [7,22]. After 1 h, capsaicin (20 µL of 0.01 mg/µL v/v in acetone) was applied to the inner surface of the right ears of mice, while in the left ears, only acetone was received (20 mL). Ten, after 30 min [7], the animals were euthanized, and the measure of edema (thickness) was calculated by the diference between the right and left ears [7,22].

Histamine-Induced Ear Edema
Model. BdR (0.1, 0.5 and 1.0 mg/ear), BOx (0.1, 0.5 and 1.0 mg/ear), and dexchlorpheniramine (0.1 mg/ear) were dissolved in acetone and topically administered (20 µL) to the right ears of mice, using saline (20 μL) as a negative control [24]. After 30 min of treatment, animals were anesthetized (10 mg/kg xylazine and 100 mg/kg ketamine), and histamine (1 µg in 10 µL of saline) was applied intradermally to the right ear, while in the left ear, saline was administered (10 µL). Tickness (mm) of the ears was evaluated after 90 min, and edema was determined by the diference between the right and left ears [24].

Cell Viability.
Cell viability of BdR and baccharis oxide (BOx) was determined in RAW264.7 cells (murine macrophage cell line), using the MTT assay [25] and three independent experiments in duplicate.
2.14. Statistical Analysis. Diferences between groups were assessed by analysis of variance (ANOVA) followed by post-hoc Student-Newman-Keuls using GraphPad Prism® 7.0 program. Data are presented as the mean ± S.E.M. Statistical diferences were considered signifcant at P < 0.05.

Isolation and Identifcation of Baccharis
Oxide. BOx (Figure 2) was obtained from BdR followed by few steps of chromatographic fractionation. GC-MS analysis ( Figure S1 in supplementary information) and 13 C NMR ( Figure S2 in supplementary information) estimated the purity of BOx to be greater than 95%. Using GC-MS ( Figure S1 in supplementary information), as well as 13 C and 1 H NMR data analysis ( Figures S2-S3 in supplementary information), the identifcation of BOx was confrmed in comparison to the literature [16]. Figures 3(a) and  3(b), the application of croton oil signifcantly increased ear thickness at both 6 and 24 h after its administration. All tested BdR concentrations reduced croton edema after 6 h, while after 24 h, only at 1.0 mg/mL, BdR signifcantly reduced edema formation in 33.0%. On the other hand, when BOx was applied topically, it was able to reduce the croton oil edema at all tested doses (0.1, 0.5, and 1.0 mg/mL) after both 6 h and 24 h administration. At 1.0 mg/ear, BOx exhibited the highest inhibitory activities in ear edema formation of 78.3% ± 2.6 and 70.6% ± 9.0, after 6 and 24 h, respectively, while dexamethasone (at 0.1 mg/ear) reduced the ear thickness in 75.7% ± 3.2 and 57.3% ± 4.9, after 6 and 24 h, respectively. Te left ears, which received only vehicle, did not display quantifed edema.

Anti-Infammatory Efects of BdR and BOx on Edema Induced by Croton Oil. As demonstrated in
Also, the enzymatic activities of MPO and NAG, as well as the activity of NO, were measured in the ears after 24 h of croton oil administration. Topical administration of croton oil on mice ears promoted signifcant increases in the enzymatic activities of MPO and NAG in comparison with the naive group (Figures 3(c)-3(e)). In contrast, the increase in MPO activity was inhibited by both BdR and BOx at all tested doses. Te maximum inhibition of BdR was 57.7% ± 3.7 at 0.5 mg/ear, while BOx showed its maximum inhibition of 37.70% ± 4.3 at 0.1 mg/ear. Similarly, dexamethasone (at 0.1 mg/ear) reduced MPO activity by 57.5% ± 1.3 when compared to the control group (Figure 3(c)).
In addition, both BdR and BOx, at all tested doses, were able to reduce the increase in NAG activity caused by croton oil (Figure 3(d)). At 0.1 mg/ear, BdR and BOx inhibited the NAG activity in 61.1% ± 0.8 and 53.0% ± 1.6, respectively, while dexamethasone (at 0.1 mg/ear) showed an inhibition of 53.8% ± 0.4.
Additionally, as shown in Figure 3(e), croton oil caused an increase in NO production. In contrast, treatment with BdR or BOx reduced NO production at all tested doses, with a maximum inhibition of 48.7% ± 0.2 by BdR (at 0.5 mg/ear) and 53.5% ± 0.2 by BOx (at 0.1 mg/ear), while dexamethasone (at 0.1 mg/ear) reduced NO production by 40.0% ± 0.1 compared to the control group.
As evidenced by ear histopathological analysis (Figure 4), croton oil promoted typical infammatory efects associated with vasodilatation, edema, marked infltration of  Histopathological analysis also showed that dexamethasone inhibited edema (Figure 4).

Cell Viability.
Te MTT assay demonstrated that BdR and BOx are not cytotoxic to the RAW 264.7 cell lines at the tested concentrations (10-100 µg/mL) ( Figure 6).

Discussion
Te economic, commercial, and anti-infammatory potential of B. dracunculifolia makes this species a prospective source of bioactive compounds for the development of pharmaceutical products to treat infammatory illnesses [12]. In this regard, this study assessed the topical anti-infammatory efects of BdR and its triterpene BOx in mice ear models using diferent infammatory agents. Although few reports have been published about the composition of B. dracunculifolia roots, previous phytochemical studies showed the presence of triterpenes, mainly BOx and friedelanol [11,13]. In our present study, the phytochemical analysis of BdR by GC-MS allowed the identifcation of BOx as one of the major constituents. Among the identifed triterpenes in BdR, friedelin and friedelanol are well known for their anti-infammatory activity in the cutaneous infammatory processes [26,27]. In contrast, there is no previous report about the anti-infammatory activity of BOx. Because BOx is one of the major constituents and might be related to the activities  Evidence-Based Complementary and Alternative Medicine of BdR, we have isolated it by chromatographic fractionation. Before topical applications on mice ear edema models, BOx and BdR were in vitro evaluated in a MTT assay, showing no cytotoxicity against mammalian cells when tested up to 100 µg/mL.
Ten, the anti-infammatory properties of BdR and BOx were frst analyzed on the skin infammation model using croton oil as phlogistic agent on mice ears. Te doses used of BdR and BOx were established in accordance with the previous protocols [7,22]. Te considerable increase in ear thickness and edema formation promoted by croton oil was inhibited by the application of BdR and BOx. Croton oil contains a series of phorbol-12, 13-diesters, which activates protein kinase C and other infammatory mediators, promoting irritant and cutaneous signs similar to psoriasis [28,29]. Also, topical application of croton oil triggers local infammation, inducing edema and erythema, also increasing vascular permeability, leukocytes infltration, synthesis of eicosanoids, and liberation of histamine and serotonin [19]. In addition, croton oil induces skin infammation, activating an enzymatic cascade, including Evidence-Based Complementary and Alternative Medicine phospholipase A 2 (PLA 2 ), which further induces the releasing of arachidonic acid (AA), prostaglandins, and platelet activation factor [30,31]. Corticosteroids, such as dexamethasone, present signifcant anti-infammatory effects in this croton oil model, as well as COX and 5-LOX inhibitors and leukotriene B4 (LTB4) antagonists [32,33]. Tus, the inhibition of croton oil infammation by both BdR and BOX suggests that they may interfere in relevant steps throughout the infammatory cascade triggered in this model. To evaluate the efects of BdR and BOx in the infltration of leukocytes, the MPO activity was analyzed in ears samples. MPO is an enzyme found in intracellular granules of neutrophils [34] that are an indicator of the leucocyte chemiotaxis process and a marker of the presence of polymorphonuclear cells in the infammatory exudate [35]. Based on results of MPO activity, BdR and BOx produced important inhibitions of cell migration and neutrophil accumulation, which may avoid an amplifcation of the infammatory process [3].
Additionally, the infltration of mononuclear cells in ears after administration of croton oil was also assessed by NAG activity, since NAG is an enzyme produced by activated macrophages [36]. Although the elevation of NAG release by mononuclear cells is typical of chronic infammation, acute skin infammation also may evidence an increment of NAG activity [36]. In the same manner of MPO activity, the treatment with BdR and BOx also promoted a decrease in NAG activity. Also, it was observed that both BdR and BOx decrease NO concentration after croton oil exposure. NO is a mediator involved principally in vascular homeostasis, and in excess, NO can initiate several infammatory diseases/processes, such as septic shock, psoriasis, and systemic lupus [37,38].
Additionally, histological analyses were consistent with the inhibitory efects of BdR and BOx on cellular infltration and on ear edema caused by croton oil. Histological analysis of the ears, exposed with croton oil and treated with BdR and BOx, showed infltration of infammatory cells similar to dexamethasone-treated ears. Also, BdR and BOx reduced all evaluated infammatory parameters, such as monocytes/ macrophages and neutrophils migration, as well as edema and epidermis thickness. Taken together, these data suggested that both BdR and BOx may decrease the infammatory response, inhibiting edema and chemotaxis of infammatory cells and decreasing vascular permeability in croton oil edema.
Moreover, in the phenol-induced edema model, the direct contact of phenol with the skin may cause rupture of the keratinocyte membranes, resulting in releasing of many cytokines, such as IL-8, IL-1α, and TNF-α, which leads to the release of other infammatory mediators, like AA metabolites and ROS [39]. Ten, phenol is considered a suitable infammatory agent for simulating dermatitis in mice [40,41]. In this model, both BdR and BOx demonstrated signifcant reduction in ear edema, with noticeable potential in the treatment of contact dermatitis, suggesting that their anti-infammatory activities may also be related to a possible decrease in the production of AA metabolites and/or with some antioxidant likely properties, which should be further explored in the future.
Additionally, capsaicin is an alkaloid found in hot peppers (Capsicum sp), which in contact with epidermis activates vanilloid receptor subtype 1 (TRPV1), elicits a quick response via the release of neuropeptides (such as substance P and tachykinins), and monoamines (serotonin and histamine) [32]. Dexamethasone and antihistamines present signifcant antiedematous efect in this model [32]. Interestingly, BOx was able to reduce edema on the capsaicin model, while the topical application of BdR did not present any signifcant decrease, suggesting that BOx may also interfere in mediators or receptors involved in capsaicinactivated infammatory pathways.
Histamine, an amine released by activated mast cells, increases vasodilation and vascular permeability and is involved in the pathogenesis of many allergic diseases, such as allergic asthma, atopic dermatitis, and allergic rhinitis [24,32]. Intriguingly, our fndings demonstrated that both BdR and BOx reduce histamine-induced ear edema, suggesting that BdR and BOx may also interfere with histamine release.
Furthermore, considering the anti-infammatory efects observed by BdR, although a signifcant anti-infammatory contribution of BOx is present, we cannot discard the efects of other active compounds in the extract, such as friedelin and friedelanol. In this regard, previous studies have shown that friedelin possess anti-infammatory properties [42], while friedelanol inhibits NO production by murine macrophage RAW264.7 cell [26]. Tus, the topical antiinfammatory efects of BdR may be due to not only to BOx but also to a combined efect of all its active compounds, resulting in a fnal and efective anti-infammatory action. Finally, BdR and BOx may act at diferent points of infammation, which may contribute to their topical antiinfammatory efcacy. Although the precise antiinfammatory mechanisms of BOx and BdR should be further clarifed, BOx presents comparable topical efectiveness to corticosteroid dexamethasone in reversing the infammatory process of croton ear edema.

Conclusions
Tis work evidenced, for the frst time, that B. dracunculifolia root extract (BdR) and baccharis oxide (BOx) demonstrated signifcant anti-infammatory efects in ear edema caused by diferent agents, suggesting their possible application on infammatory skin conditions, such as psoriasis and dermatitis. Finally, although remarkable anti-infammatory efects were observed, the precise mechanisms and safety profles of BdR and BOx should be further investigated.

Data Availability
Te data used to support data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that there are no conficts of interest. 8 Evidence-Based Complementary and Alternative Medicine

Supplementary Materials
Te supplementary information consists of the GC-MS, 13