Anti-Inflammatory Profile of Jungia sellowii Less. by Downregulation of Proinflammatory Mediators and Inhibition of NF-κB and p38 Pathways.

Jungia sellowii Less. (Asteraceae) is a native plant found in Southeast Brazil used traditionally to treat inflammatory diseases. This study was conducted (1) to investigate the toxicity of the crude extract (CE) and (2) to investigate the mechanism of the anti-inflammatory action of J. sellowii L. roots. The potential acute toxicity of CE was performed by administration of only different doses of CE (500, 1,000, and 2,000 i.p.) on mice for 14 days. The anti-inflammatory effect was evaluated using carrageenan-induced acute pleural cavity inflammation in a mouse model, evaluated through the following inflammatory variables: leukocyte, protein concentrations of the exudate, myeloperoxidase (MPO), adenosine deaminase (ADA), nitric oxide metabolites (NOx), and proinflammatory cytokine (tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin- (IL-) 6, and IL-12) levels in mouse pleural fluid leakage. The p65 protein phosphorylation of nuclear factor NF-kappa B (p65 NF-κB) and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation were analyzed in lung tissue. Our results demonstrated that the administration of CE up to 2,000 mg/kg did not present a toxic effect. In addition, the pretreatment of mice with CE; its derived fractions (aqueous fraction (AqF), butanol fraction (BuOHF), and ethyl acetate fraction (EtOAcF)); and isolated compounds (curcuhydroquinone O-β-glucose (CUR) and α and β piptizol (Pip)) reduced the following inflammatory variables: neutrophils, protein concentrations of the exudate, MPO, ADA, NOx, and proinflammatory cytokine (TNF-α, IFN-γ, IL-6, and IL-12) levels in mouse pleural fluid leakage. The compounds CUR and Pip also decreased the p65 protein phosphorylation of NF-kappa B and p38 (MAPK) in lung tissue. J. sellowii L. has important anti-inflammatory activity with potential applications in drug development against inflammatory disorders. These effects found can be attributed to the ability of the new isolated compounds CUR and Pip to suppress p65 NF-κB and p-p38 MAPK pathways.


Introduction
The control of mediators released in the inflammatory process ensures the anti-inflammatory effectiveness. The limitations of current therapies with uncontrolled or unresolved inflammation result in tissue damage with eventual loss of organ function, characteristic of chronic inflammatory diseases [1,2].
The mouse model of pleurisy induced by carrageenan (Cg) is a well-characterized inflammation model which allows the quantification of different proinflammatory mediators released into the pleural fluid leakage of the pleural cavity [3]. This model is characterized by infiltration of neutrophils 4 h after pleurisy induction, followed by lung injury by reactive oxygen species (ROS) and reactive nitrogen species (RNS) released from active neutrophils such as hydrogen peroxide (H2O2), superoxide anion (O2−), hydroxyl radical (OH−), and peroxynitrite (ONOO−) [4].
Although a broad spectrum of anti-inflammatory drugs is currently available, a new strategy for the identification of new targets and discovery of new molecules, particularly to treat diseases resistant to conventional treatment with an inflammatory nature, must be done [5,6].
Natural products remain an important source for drug discovery and structural diversity of new substances or prototypes with relevant therapeutic potential, and many antiinflammatory drugs have their origin in extracts or compounds from plants [6].
Jungia (Asteraceae) is a perennial herb that comprises shrubs, lianas, and herbs recorded from Mexico to the Andes and also present in Brazilian dry forests [7]. In traditional Brazilian folk medicine, in Andean communities at the middle north of Perú, and in traditional medicine of Ecuador, species of the genus Jungia (J. paniculata and J. rugosa) are used in tea form (decoction) to treat inflammation of the liver and kidneys and of the digestive system, to treat urinary tract inflammations, and also as an aseptic agent for treating wounds [8,9].
Moreover, pharmacological studies have reported the anti-inflammatory and antioxidant properties of various species of Jungia. Recently, analysis of Wilches et al. [10] demonstrated the potential anti-inflammatory of methanolic extract from J. rugosa leaves, using an acute model of croton oil ear edema and carrageenan-induced paw edema in rats. Also, the anti-inflammatory activity was demonstrated in a chronic inflammation model using cotton pellet-induced granuloma in mice.
However, there are few past studies concerning the antiinflammatory effect of Jungia sellowii Less. Thus, the present study is aimed at investigating the toxicity of J. sellowii L. roots using in vivo and in vitro assays and at analyzing the anti-inflammatory effect of J. sellowii L. roots using the classical in vivo experimental model of acute inflammation. In this protocol, we evaluated the mechanism of the antiinflammatory action of the crude extract (CE), its derived fractions, and isolated compounds by evaluating several proinflammatory variables such as leukocyte influx, protein concentrations of the exudate, myeloperoxidase and adenosine deaminase, nitric oxide metabolite (NO x ) levels, and Th1 polarization immune response through analysis of the level of proinflammatory cytokines: tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-(IL-) 6, and IL-12 cytokines. Furthermore, the effect of the newly isolated compounds (curcuhydroquinone O-β-glucose (CUR) and α and β piptizol (Pip)) upon p65 protein phosphoryla-tion of nuclear factor-kappa B (p65 NF-κB) and p38 of mitogen-activated protein kinase MAPK (p38 MAPK) phosphorylated levels in the lungs was also investigated. l for 3 d in the absence of light. This procedure was repeated three times at room temperature (approximately 25°C) for obtainment of the crude extract (CE). Subsequently, the CE was dissolved in H 2 O (1/3) and subjected to liquid-liquid partitioning with the solvents hexane, dichloromethane, ethyl acetate, and butanol. The resulting fractions were concentrated to dryness in a rotary evaporator to obtain hexane (HexF) (3 g), dichloromethane (DCMF) (1.2 g), ethyl acetate (EtOAcF) (2.9 g), butanol (BUOHF) (16 g), and aqueous (AqF) (94 g) fractions. The isolated compounds (CUR and Pip) were obtained from the ethyl acetate (2.9 g) and butanol (16 g) fractions, respectively.

Structural
2.6. In Vivo Study 2.6.1. Animals. Female Swiss mice weighing 20-22 g were used for all experiments. The animals were kept in a room at a constant temperature of 22 ± 2°C with alternating 12 h periods of light and darkness at 50-60% humidity. The mice were kept in plastic cages with water and food provided ad libitum before the experiments. The present study was approved by the Committee for Ethics in Animal Research at the Federal University of Santa Catarina, Brazil (License No. 4720140616). All animal experiments were conducted in accordance with the International guidelines (NIH publication #85-23, revised in 1985), as well as the guidelines of the Brazilian College of Animal Experimentation (CONCEA).
2.6.2. Pleurisy Induced by Carrageenan. Carrageenaninduced pleurisy in mice is a useful model, for testing natural products with potential anti-inflammatory activity and for further understanding their mechanism of action.
Pleurisy was induced by a single intrapleural (i.pl.) injection of 0.1 mL Carrageenan (Cg, 1%), as previously described by Saleh et al. [3]. After 4 h, the animals were killed with an overdose of ketamine and xylazine administered by intraperitoneal route (i.p.), the thorax was opened, and the pleural cavity was washed with 1.0 mL of sterile phosphatebuffered saline (PBS, pH 7.6, composition: NaCl, 130 mmol; Na 2 HPO 4 , 5 mmol; and KH 2 PO 4 , 1 mmol) in distilled water containing heparin (20 IU/mL). Samples of the mice pleural fluid leakage were collected to analyze leukocyte influx, protein concentration of the exudate, myeloperoxidase (MPO), adenosine deaminase (ADA), metabolites of nitric oxide (NO x ), and cytokine (TNF-α, IFN-γ, IL-6, and IL-12) levels in mouse pleural fluid leakage. Lung tissue samples were collected for determination of the total/phosphorylated levels of p65 NF-κB and p38 MAPK.
2.6.3. Acute Toxicity. The acute toxicity test for the hydroalcoholic extract of J. sellowii L. roots was conducted to evaluate any possible toxicity. In this protocol, four groups of five female mice were divided into a control group and test groups. The negative group received an injection of sterile saline solution (NaCl, 0.9% i.p.) (vehicle), and the second to fourth group received different doses of CE of J. sellowii L. (500, 1,000, or 2,000 mg/kg, i.p.) This was considered the start of the test (day 0). The general behavior and condition of the animals (tremors, convulsions, diarrhea, sleep, coma, mortality, and weight lost) were observed and registered from 0.25 to 3 h after the herb injection and daily for 14 d. On day 14, the animals were sacrificed and vital organs such as the kidney, spleen, liver, and lungs were isolated and weighed (humidity weight).
To determine the time course profile, different groups of animals were pretreated at different time points (0.5 to 2 h) with only one dose of CE (lower doses that inhibited leukocytes and/or protein concentrations of the exudate).

Mediators of Inflammation
Inflammation was analyzed 4 h after Cg administration. The optimal time of pretreatment was the lower time of pretreatment required to inhibit leukocytes and/or protein concentrations of the exudate. This period of pretreatment applied to the CE was extended to the fractions and isolated compounds.
Thus, based on the results of these experiments the following doses were chosen: 50 mg/kg for CE, 25 mg/kg for AqF, 10 mg/kg for BuOHF, 10 mg/kg for EtOAcF, 2.5 mg/kg for CUR, and 1 mg/kg for Pip, all administered 0.5 h before Cg induction. These doses were selected to analyze their effects upon other proinflammatory variables such as MPO, ADA, and NO x and the levels of cytokines (TNF-α, IFN-γ, IL-6, and IL-12) in the mouse pleural fluid leakage. The phosphorylation of p65 NF-κB and p38 MAPK levels was analyzed in the mouse lung tissues.
Dexamethasone (0.5 mg/kg, i.p.) administered 0.5 h before pleurisy induction was used as the reference antiinflammatory drug.

Quantification of Cell Migration and Protein
Concentrations of the Exudate. The total leukocyte counts from mouse pleural fluid leakage were performed using a veterinarian automatic counter (Mindray, BC-2800 Vet, Nanshan, Shenzhen, China), and the cytospin preparations were stained with May-Grünwald-Giemsa for the differential leukocyte count. The protein concentrations of the exudate were indirectly determined by the quantity of Evans blue dye extravasation in the mouse pleural fluid leakage [3]. Thus, in each experimental group, the animals were challenged 0.5 h before the Cg injection with a solution of Evans blue dye (25 mg/kg) administered intravenously (i.v.) to evaluate the protein concentrations of the exudate. The amount of dye in the pleural wash aliquot was estimated via colorimetric measurements using an enzyme immune assay (EIA) plate reader (Organon Teknika, Roseland, NJ, USA) at 620 nm by interpolation from a standard curve of Evans blue dye (in the range of 0.01-50 μg/mL).
2.6.6. Quantification of Myeloperoxidase (MPO) Levels. Inhouse assays for the MPO were employed according to the methodology described in the literature by Rao et al. [13]. The colorimetric analysis was performed using an EIA plate reader (Organon Teknika, Roseland, NJ, USA) at 450 nm and interpolated from a standard curve of MPO from human neutrophils (Sigma: M6908, St. Louis, MO, USA) ranging from 0.7 to 140 mU/mL. The results were expressed in mU/mL.

Quantification of Adenosine-Deaminase (ADA) Levels.
A 20 μL sample from the mouse pleural fluid leakage was used to determine the ADA levels using a previously described methodology by Giusti and Galanti [14]. This analysis was performed calorimetrically using an EIA plate reader (Organon Teknika, Roseland, NJ, USA) at 630 nm and estimated by an ammonium sulfate solution, measured in triplicate as a standard point of 20 U/L on a colorimetric plate reader (Organon Teknika). The results were expressed in U/L.

Quantification of Nitric Oxide Metabolite (NO X ) Levels.
Quantification of the nitric oxide product (nitrite (NO 2 − ) and nitrate (NO 3 − )) levels was determined according to the methodology of Griess [15]. Levels of nitrite were estimated by interpolation from a standard curve of sodium nitrite (0-150 μM) by colorimetric measurements at 540 nm in an EIA plate reader (Organon Teknika, Roseland, NJ, USA). The results were expressed as μM.
2.6.9. Quantification of the Levels of TNF-α, IFN-γ, IL-6, and IL-12. The determination of the TNF-α, IFN-γ, IL-6, and IL-12 levels was performed with aliquots of the washed mouse pleural fluid leakage using a commercial kit for flow cytometric CBA (cytometric bead array) mouse inflammation (BD Biosciences Pharmingen, San Diego, CA, USA), according to manufacturer's instructions. The measurements were performed in a FACSVerse flow cytometer (BD FACSVerse™, San Jose, CA, USA), and the data were analyzed in FCAP Array software version 3.0, from the same manufacturer. The limits of detection were 7.3 pg/mL, 2.5 pg/mL, 5 pg/mL, and 10.7 pg/mL for TNF-α, IFN-γ, IL-6, and IL-12, respectively. The intra-assay precision was 3-4%, 3-5%, 4-5%, and 4-15% for TNF-α, IFN-γ, IL-6, and IL-12, respectively. The interassay precision was 6-11%, 5-8%, 8-11%, and 3-9% for TNF-α, IFN-γ, IL-6, and IL-12, respectively. The values were expressed in pg/mL. 2.6.10. Quantification of Total p65 Protein and Phosphorylated (p-p65 NF-κB) Levels. The evaluation of p65 protein (NF-κB) was performed in the lung tissue. Samples were collected and the total protein concentration was adjusted to 60 μg using the method described by Lowry and Lewis (1951). In this protocol, commercial kits were used with monoclonal antibodies specific for total and phosphorylated p65 (PathScan®-NF-κB p65 (Ser536)) EIA Kit (Cell Signaling Technology, Inc., Danvers, Massachusetts, USA) following the instructions of the manufacturer. The absorbance of the samples was measured calorimetrically using an EIA plate reader (Organon Teknika, Roseland, NJ, USA) at 450 nm. The results were expressed as the relative fold change in comparison with the negative control group, which represented the basal level of p65 phosphorylation.
2.6.11. Evaluation of the Total and Phosphorylated p38 protein (P-p38 MAPK). The evaluation of the p38 protein (p38 MAPK) was performed in the lung tissue. The total protein concentrations of the lung samples were adjusted to 60 μg according to method developed by Lowry and Lewis (1951). In this experimental protocol, commercial kits with monoclonal antibodies specific for total and phosphorylated p38 were used (Instant One® Phospho-p38 MAPK (Tyr180/Tyr182) and eBioscience®, San Diego, CA, USA) using EIA assays following the instructions of the manufacturer. From the obtained absorbance, a relative analysis comparing the results with the negative control group was performed, which represents the baseline concentrations of phosphorylated p38.

Statistical Analysis.
To assess data normality and the homogeneity of variances, the Shapiro-Wilk test was used. 4 Mediators of Inflammation The normal distributions of variables were summarized as means ± standard error of the mean (SEM), and categorical variables were expressed as percentage of five animals per group. For the statistical analyses of the results (animals treated with phlogistic agent vs. animals treated with J. sellowii L.), a test of analysis of variance (ANOVA) was used followed by a Newman-Keuls post hoc test. For all analyses, p < 0:05 was considered significant, and all data were analyzed by the statistical program GraphPad version 5.0 (San Diego, CA, USA).  (Figure 1(a)). The ethyl acetate fraction is shown in the first peak CUR with a retention time of 1.38 min and the second peak the Pip with a retention time of 3.50-3.61 (Figure 1(b)). The identified pure substances CUR and Pip in the butanol and ethyl acetate fractions were performed by comparison of retention time with known standards (Figures 2(a) and 2(b)).

Cytotoxicity
In Vitro Test. Initially, a concentrationresponse evaluation of the cytotoxic effects of J. sellowii L. was performed. Murine macrophage J774 cell line was incubated with indicated concentrations of CE (1, 0.1, 0.01, and 0.001 mg/mL) for 24 h. Results of the MTT-based assay of control DMSO vehicle-treated cells were defined as 100% of viable cells, and cells incubated with J. sellowii L. were presented as a relative decrease on viable cells with increasing concentration (Figure 3(a)). Based on the results, the cytotoxic concentration was calculated, which is the concentration able to kill 50% of cells (CC 50 ). For the CE of J. sellowii L., the CC 50 was 580 μg/mL.

Acute Toxicity
Test. The CE of J. sellowii L. (i.p.) administered in doses of 500 mg/kg, 1,000 mg/kg, or 2,000 mg/kg did not cause any mortality up to 2,000 mg/kg (Figure 3(b)). Signs and symptoms which occurred in response to a high dose of the extract were a decrease in motor activity and weight loss with diarrhea. After two days of CE administration, the mice that received the high dose (2,000 mg/kg) returned to normal without demonstrating any signs and symptoms of toxicity.    Table 1).
HexF only inhibited the protein concentrations of the exudate at a dose of 25 mg/kg (p < 0:05), and the dichloromethane fraction (DCMF) did not inhibit the studied inflammatory variables (p > 0:05). Therefore, the effect of these fractions upon other inflammatory variables was not studied (results not shown).
3.6. Effects of Extract, Fractions, and Compounds of J. sellowii L. upon NO X Concentrations. Because J. sellowii L. caused a significant decrease of the protein concentrations of the exudate, we investigated whether this inhibition could be associated with a decrease in NO x levels. The results showed that CE; its derived fractions AqF (25 mg/kg), BuOHF (10 mg/kg), and EtOAcF (10 mg/kg); and the isolated compounds CUR (2.5 mg/kg) and Pip (1 mg/kg) significantly decreased NO x levels (p < 0:01) ( Table 2). Dex also reduced this proinflammatory variable (p < 0:01) ( Table 2).   (Table 3). Dex also reduced these cytokine levels (p < 0:01) ( Table 3).

Discussion
Several important in vivo and in vitro models have been developed with an experimental value and clinical payoffs. Cytotoxicity in vitro assay showed a high CC 50 value (580 μg/mL) of CE of J. sellowii L. for murine macrophage J774 cell line, demonstrating a good result to this study, since the intention was not to kill macrophages but just to modulate the inflammation, as they play an important role in inflammation and in the presentation of antigens to lymphocytes [16]. Thus, the CE of J. sellowii L. supposedly proved promising for inflammatory in vivo study evaluation.
Acute toxicity studies in vivo were evaluated by the organ weight, by changes in behavior, and by the index of physiological status of the animal, considering increase organ, organomegaly, or inflammation as a strong indicator. Furthermore, together with hematological variables, these are considered sensitive parameters evaluated in studies with toxic plants [17,18]. Based on the results, roots of J. sellowii L. extract in acute treatment by i.p. route at a dose of up to 2000 mg/kg did not produce deaths during 14 d of observation ensuring the doses in experiments to evaluate the anti-inflammatory activity.
Based on these previous studies that do not report evidence of toxicity, we evaluated the effect of CE, fractions obtained from CE (AqF, BuOHF, EtOAcF, HEX-F, and DMC-F), and isolated compounds (CUR and Pip) on the levels of several proinflammatory mediators to investigate the anti-inflammatory mechanism of action of J. sellowii L. using a typical mouse model of pleurisy induced by carrageenan.
Many of the classic experiments in the field of inflammation have been performed using murine [19]. Carrageenaninduced pleurisy in mice is a useful model, for testing natural products with potential anti-inflammatory activity and also for further understanding their mechanism of action [3]. The results of our study show that J. sellowii L. demonstrated a pronounced inhibition on leukocyte migration due in part to CE and polar fractions AqF, BuOHF, and EtOAcF and compounds CUR and Pip have the ability to reduce neutrophil migration.
The amplitude of leukocyte activation and migration can be modulated by the proinflammatory enzymes such as MPO and ADA and mediators such as NO x and proinflammatory cytokine levels (TNF-α, IFN-γ, IL-6, and IL-12) important in the context perpetuation of inflammatory response.
MPO is a hemoprotein located in azurophil granules of neutrophils and has been used as a biochemical marker for neutrophil infiltration into tissues and tissue damage. MPO activity is also directly related to the amount of leucocyte infiltration, which is indicative of ROS and RNS release from  Figure 4: (a) Effects of the isolated compounds curcuhidroquinone O-β-glucose (CUR: 2.5 mg/kg) and piptizol (Pip: 1 mg/kg) from J. sellowii L. roots administered 0.5 h prior to carrageenan (1% Cg, i.pl.) on total and phosphorylated protein (p-p65 of NF-κB) in the lungs; (b) effects of the isolated compounds curcuhidroquinone O-β-glucose (CUR: 2.5 mg/kg) and piptizol (Pip: 1 mg/kg) from J. sellowii L. roots administered 0.5 h prior to carrageenan (1% Cg, i.pl.) on total and phosphorylated protein (p-p38 MAPK) in the lungs. Sal: negative control group, animals treated only with sterile saline (NaCl, 0.9%); Cg: positive control group, animals treated only with 1% Cg; Dex: animals pretreated with dexamethasone (0.5 mg/kg). The results were expressed in relative fold change in comparison to Sal, which represents the basal level of p65 and p38 MAPK phosphorylation. Bars represent the mean ± SEM of five animals. ANOVA: Newman-Keuls test. 9 Mediators of Inflammation activated neutrophils and the hypochlorous acid (HOCL) production, which in large quantities leads to the injury of the host cells [20].
Corroborating our results, Wilches et al. [10] observed that leaves of J. rugose showed an anti-inflammatory effect by reducing the MPO levels, as well as neutrophil accumulation at doses of 125 to 500 mg/kg on the croton oil-induced model in mice.
Moreover, ADA is another molecule that has emerged for the management of several inflammatory conditions in preclinical and clinical settings. The adenosine system plays a crucial part in the regulation of immune homeostasis, acting as a self-limiting signal aimed to preserve the host integrity through immune cell activation and promoting the resolution of inflammation [21].
The inhibition of the ADA enzyme promotes the increase of extracellular adenosine from activated cells such as neutrophils and macrophages. This adenosine binds to the A2A receptors in an autocrine/paracrine manner, thereby promoting the inhibition of intercellular adhesion molecule 1 (ICAM-1) and preventing cell infiltration, proinflammatory cytokine release, neutrophil degranulation, and oxidative stress in the lungs [22].
The formation of protein of the exudate is another important sign of inflammation, and the metabolites of NO play a role in the exudative phase during the inflammatory process. Interferons (IFNs), cytokines, and microbial products are prototypic transcriptional inducers of NOS 2 which effectively stimulate the release of high amounts of nitric oxide. The released nitrite/nitrate plays an important role as a vasodilator and contributes to increase of edema, thereby contributing to the formation of (ONOO−) and promoting important tissue injury [23].
Furthermore, the proinflammatory cytokines IL-6, TNFα, IFN-γ, and IL-12 play such an important inductor of inflammation in disorders such as severe/uncontrolled asthma, Alzheimer's disease, and rheumatoid arthritis and may be considered a predictive factor for treatment failure [24]. Thus, we investigated if J. sellowii L. could modulate the Th1 immune response by analysis of certain cytokines released into the inflamed mouse pleural fluid leakage. The results indicate an increase of these cytokines induced by carrageenan administration that was significantly inhibited by the CE, AqF, BuOHF, and EtOAcF of J sellowii L.
The effects on cytokine levels involve a complex intracellular process. NF-κB is an important self-control of cytokine expression. The nuclear transcription factor NF-κB plays a role as a principal mediator to physiological processes and in response to pulmonary injury, and several inflammation processes regulate the innate immunity and inflammation. NF-κB controls the expression of proinflammatory cytokines and other effector proteins and enzymes involved in immune response such as inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) [25].
The classical pathway of NF-κB activation depends on the activation of the IκB kinase (IKK) complex, formed by two catalytic subunits IKKα and IKKβ and a regulatory unit NEMO. IKKβ promotes the phosphorylation of IκBα. Activation of the classical NF-κB pathway depends on the p65/p50 heterodimer, in which the p65 subunit phosphorylation leads to the transmigration of the NF-κB activated form to the nucleus, where the gene expression of proinflammatory mediators is triggered [26].
In addition, p38 MAPK is considered an important regulator of multiple responses during the inflammatory process. The superfamily of highly conserved kinases regulates cell growth, differentiation, and stress responses. During inflammation, p38 MAPK regulates distinctly different functions, including the chemotaxis of neutrophils through chemoattractants (CXCL8, CXCL2, and LTB4), molecules of endothelial and leukocyte-associated adhesion, activation of NF-κB, synthesis and release of ROS, and cytokines such TNFα when they are induced by carrageenan [27].
Thus, based on our results, we showed for the first time that the compounds isolated from the polar fractions, CUR and Pip, were effective in reducing the influx of neutrophils and production of proinflammatory mediators in vivo, at least in part, due to the ability to reduce the phosphorylation of p65 NF-κB subunit and p38 MAPK.
The compound curcuhydroquinone O-β-glucose is a new molecule described as being similar to bisabolane sesquiterpene isolated from the rhizome of the plant Curcuma xanthorrhiza. This molecule shows anti-inflammatory and antioxidant proprieties preventing erythema, edema, and skin changes mediated by ultraviolet light B (UVB) by reduction of metalloproteinase-1 (MMP-1) messenger RNA (mRNA) expression in human keratinocytes [28,29].
Piptizol is other molecule from the ethyl acetate fraction of J. sellowii L. roots, described such as an epimeric mixture of two isomers, α and β piptizol. There are relatively few approaches to α and β piptizol, but this mixture is considered the product of thermal rearrangement of perezone. For the Nassauvieae tribe, the first isolation report was from the roots of Perezia cuernavacana; however, there is currently no biological approach related to activity data for this compound in the literature [30,31].

Conclusions
In summary, this study suggests that J. sellowii L. has an important anti-inflammatory propriety by inhibiting leukocyte influx, protein concentration of the exudate, and the liberation of several proinflammatory mediators. The results showed that J. sellowii L. was effective in inhibiting acute inflammation by downregulation of NF-κB and p-p38 MAPK activation pathways by the isolated compounds (Pip and CUR). In addition, CE did not show a significant toxic activity, supporting its use in Brazilian folk medicine for treating inflammatory disorders.

Data Availability
The data used to support the findings of this study are included in the article.

Conflicts of Interest
The authors declare that they have no conflicts of interest. 10 Mediators of Inflammation