Introduction
Inflammation is defined as immunological, biochemical, and cellular changes in response to molecular patterns associated to pathogens or cell, tissue damage (Rudrapal et al.
2022; Upadhyay and Dixit
2015). The main clinical signs of this response are pain, heat, and redness, which are associated with the development of edema, vasodilation, and leukocyte migration to the injury site. If this process is not controlled, it leads to increased tissue damage, which worsens the loss of tissue function and drives to chronic inflammatory process (Herrero-Cervera et al.
2022).
Cells, such as macrophages and fibroblasts, are activated locally and systemically, inducing the release of mediators in the inflammatory condition (Upadhyay and Dixit
2015). Among these mediators, pro-inflammatory cytokines such as interleukins (IL-6) and the tumor necrosis factor-α (TNF-α) play a key role in the inflammatory response (Hirano
2020). In addition, several inflammatory stimuli, such as the excess of reactive oxygen (ROS) and nitrogen (RNS) species, contribute to the inflammatory process cascade (Tanabe et al.
2022).
During inflammation, there is a greater formation of ROS and RNS. The imbalance between the production of these species and the antioxidant defense mechanisms leads to oxidative stress, which plays an important role in inflammatory conditions (Doktorovova et al.
2014; Gutteridge and Halliwell
2018).
Several drugs are used to treat inflammation, especially, non-steroidal anti-inflammatory drugs and corticosteroids (Juthani et al.
2017). Despite the wide variety of anti-inflammatory drugs on the market, the adverse effects contribute to the continued need for more research to discover isolated molecules or mixtures of compounds such as those presented in medicinal plants that can serve as therapeutic alternatives (Souza et al.
2015).
Derivatives of herbal products are important sources for the discovery of new drugs (Amaral et al.
2020; Matsuo et al.
2011; Santos et al.
2021). For example, polyphenols are a group of metabolites found in parts of plants that have a series of biological activities, such as anti-inflammatory and antioxidant properties (Durazzo et al.
2019; Pimentel-Moral et al.
2018). There is great interest in the search and identification of secondary metabolites, such as polyphenols and other compounds from plant-based natural sources, since they can have valuable therapeutic potential.
One of the promising medicinal plants to treat inflammation is
S. terebinthifolius Raddi (Anacardiaceae), known as Brazilian pepper tree. In folk medicine, the bark and leaves are used in as infusions and tinctures, to treat bacterial infections (Martínez et al.
1996), or to promote healing, anti-inflammatory and anti-ulcerogenic effect (Martorelli et al.
2011). The leaves can even be used for the green synthesis of silver nanoparticles (de Oliveira et al.
2021). Phytochemical studies of this species have resulted in the isolation of terpenes, monoterpenes, sesquiterpenes, and flavonoids (El-Massry et al.
2009; Matsuo et al.
2011).
A previous study showed that the acetate fraction of
S. terebinthifolius leaves has anti-allergic activity when administered orally (Cavalher-Machado et al.
2008b). Although this represents consistent evidence of anti-inflammatory effect after oral administration of a
S. terebinthifolius leaves fraction, there are no detailed reports on the effect of preparations from
S. terebinthifolius leaves in a model of skin inflammation after topical application. Thus, in this study, we prepared the ethyl acetate extract from the leaves of
S. terebinthifolius (EAELSt) and tested the effect of this extract in selected in vitro models regarding its antioxidant effects and cytotoxicity activity, and in an in vivo model of skin inflammation and oxidative stress.
Discussion
In the present study, we show results about the in vitro antioxidant effect and in vivo anti-inflammatory and antioxidant effect of EAELSt in a model of skin inflammation, which seems to correlate with the composition of the extract.
The chemical characterization of the components presented in the EAELSt showed a high concentration of phenolic compounds and total flavonoids, which may have a greater correlation with pharmacological effects. Similarly, El-Massry et al. (
2009) observed the presence of a high concentration of phenolic compounds in the ethanolic extract of the leaves of
S. terebinthifolius, however, using the maceration technique for extraction.
The analysis of the chemical composition of the EAE by LC–MS/MS confirmed the presence of phenolic compounds and their derivatives with a total of 43 compounds identified. Among them, the major peaks area in the chromatogram were for myricetin-O-pentoside, quercetin-O –rhamnoside, and kaempferol-O-rhamnoside. Rosas et al. (
2015), using the hydroalcoholic extract of the leaves of
S. terebinthifolius, identified the presence of polyphenols such as gallic acid, methyl gallate, and penta-galloyl glucose. These data partially corroborate our findings, since these compounds were also identified in EAE, but to a lesser extent. In a study by Uliana et al. (
2016), ferulic and caffeic acids, and quercetin were the major components identified by mass spectroscopy in the extracts.
In this study, it was possible to verify an antioxidant potential by reducing the free radical DPPH. The fact that EAE reduced the amount of this radical in all concentrations tested suggests that the chemical constituents of EAE may act as donors of H
+ which indicates a mechanism for reducing the DPPH free radical (Floegel et al.
2011; Shahidi and Zhong
2015). In the study by El-Massry et al. (
2009), a greater antioxidant activity was observed in the ethanolic extract than in the methanolic or dichloromethane extracts from the leaves of
S. terebinthifolius. These data corroborate our study, considering that EAE presented a high concentration of total phenols and flavonoids, associated with antioxidant capacity. Flavonoids can act directly or indirectly as antioxidants (Jucá et al.
2020), so that the antioxidant activity is related to the amount of hydroxyl group in its structure (Havsteen
2002).
To complement the evaluation of antioxidant activity in vitro, the evaluation method by inhibiting lipoperoxidation in a biological matrix consisting of rat brain homogenate was used. The results obtained indicate that there was a protective effect for the formation of MDA for all evaluated EAELSt concentrations. MDA is formed during oxidative degeneration as one of the products of free radicals and serves as a marker of lipoperoxidation (Alam et al.
2013). Based on the study by Lesjak et al. (
2018), it is possible to suggest that EAELSt effect is related to the presence of phenolic compounds, such as quercetin and its derivatives, which has already been shown to have inhibitory effects on MDA (Lu et al.
2018; Tian et al.
2021).
Before the study in a model of skin inflammation in vivo, a cytotoxicity test with L929 fibroblasts was carried out, to verify whether EAELSt presented any cellular toxicity. Using the MTT test, we showed that EAELSt did not have a cytotoxic effect until the concentration of 100 µg/mL in this cell line. There is no information on the toxicity of
S. terebinthifolius leaves in in vitro studies. However, data from other authors showed that the ethanolic extract of the bark of this plant did not produce acute or subacute toxicity (45 days of administration) in Wistar rats of both sex, indicating that the oral pretreatment does not cause cytotoxic effect (Lima et al.
2009).
Despite the ethnobotanical suggestions of this species having an anti-inflammatory effect, few studies have investigated its chemical composition and its association with anti-inflammatory and antioxidant activity in vivo
. For this purpose, the TPA-induced ear inflammation model was used to evaluate the topical anti-inflammatory effect of EAELSt in vivo. The time point of 6 h was chosen because it is the peak time of edema formation, and it was previously reported to show infiltration of neutrophils, according to reference study we used to perform this assay (De Young et al.
1989).
In the present study, it was shown that the topical application of EAELSt reduced the edema caused by TPA in the range doses of 0.3–3 mg/ear. These data indicate that EAE influences the vascular component of the inflammatory response that contributes to edema formation, suggesting a topical anti-inflammatory effect. Accordingly, histological analysis reinforced that treatment with EAELSt at the high dose used reduced edema.
The mechanism for the formation of edema induced by TPA is not completely understood. However, evidence indicates that treatment with TPA activates protein kinase C, promotes release of eicosanoid mediators, such as prostaglandins and leukotrienes, increased expression of cyclooxygenase-2, migration of leukocytes, and increased concentrations of cytokines IL-1β and TNF-α (Carlson et al.
1985; Oliveira et al.
2017), which suggests the involvement of these pathways in the anti-inflammatory effect of EAELSt. In agreement with our data, Fedel-Miyasato et al. (
2014) showed that oral treatment with the methanolic extract of the leaves of
S. terebinthifolius reduced the edema induced by Croton oil in mice ear, similar to that observed in the present study, but these authors did not report any other inflammatory markers in their model.
In addition to the anti-edematogenic effect, a similar effect was observed for doses of EAELSt on MPO activity, which reflects the inhibition of neutrophil migration to the inflamed site. Neutrophil infiltration, characteristic of acute inflammation, was assessed indirectly through the activity of MPO, an enzyme located in neutrophil azurophil granules (Jorch and Kubes
2017). The recruitment of neutrophils occurs through the stimulation of cytokines and chemokines, which, in turn, initiates a series of interactions between different types of leukocytes and endothelial cells (Timmerman et al.
2016).
The study by Rosas et al. (
2015) corroborates the reduction in neutrophil migration observed in our study. These authors showed that the oral pretreatment with the hydroethanolic extract obtained from the leaves of
S. terebinthifolius inhibited the migration of neutrophils in a model of pleurisy induced by zymosan. In the same study, using the zymosan-induced arthritis model, there was also a reduction in joint edema and inhibition of neutrophil migration to the joint.
Our data raised the possibility that components of the EAE may act to reduce the migration of neutrophils. In the study by Rosas et al. (
2015), gallic acid, another component found in EAE, reduced the in vitro migration of isolated human neutrophils stimulated with N-formylmethionyl-leucyl-phenylalanine. These data reinforce that the phenolic compounds identified in EAELSt may be responsible for the anti-inflammatory effects shown in the present study.
Increased concentration of IL-6 and TNF-α is associated with cutaneous inflammatory response, as well as with other pro-inflammatory cytokines (Murakawa et al.
2006; Scheller et al.
2011). Accordingly, in the study by Blaser et al. (
2016), it was shown that the use of a TNF-α antagonist inhibited both edema and TPA-induced concentrations of TNF-α. We chose to measure TNF-alfa and IL-6 because these are cytokines involved since the initial stage of the inflammatory response. Many other inflammatory mediators might also be reduced by the administration of the extract. However, as we worked with the crude extract, the exact mechanism of action cannot be fully disclosed, provided that a variety of compounds that can contribute to the beneficial effects have been identified.
We showed a reduction of IL-6 and TNF-α in the ears treated with EAELSt, which corroborates our data on the reduction of edema and MPO. It is interesting that all doses of EAELSt reduced the concentration of IL-6, but only the highest dose of this extract decreased the concentration of TNF-α, which suggests differential modulation between these cytokines in the evaluated time point.
Other authors observed that the treatment with hydroethanolic extract of the leaves of
S. terebinthifolius caused a reduction in the concentration of IL-6 and TNF-α in a model of arthritis induced by zymosan in mice (Rosas et al.
2015), which corroborates the effect observed in our study. It is also interesting that the treatment with the ethyl acetate fraction of the leaves of
S. terebinthifolius decreased the concentrations of chemokines with a Th2 profile, namely eotaxin and CCL5/RANTES in ovalbumin-induced allergic pleurisy in rats (Cavalher-Machado et al.
2008a).
Considering the compounds presented in EAELSt, it is well described that flavonoids can reduce the formation of pro-inflammatory cytokines (Maleki et al.
2019). Thus, it is possible that the anti-edematogenic effect and the reduction in the concentrations of IL-6 and TNF-α by EAELSt, in part, are attributed to the presence of phenolic compounds in this extract, such as quercetin, which can act solely or synergistically, most likely by modulating intracellular signaling pathways such as phosphatidylinositol-3-kinase or other tyrosine kinase proteins (Lolli et al.
2012; Yokoyama et al.
2015) or transcription factors like the nuclear factor κB (Peng et al.
2018).
In the present study, the effect of EAELSt on IL-10 concentrations was also evaluated. The pretreatment with EAELSt only in the lowest doses (0.3 and 1.0 mg/ear) prevented the reduction of IL-10 levels produced by TPA. IL-10 is a cytokine that plays an important role in maintaining homeostasis and in responding to inflammatory stimuli by suppressing pro-inflammatory cytokines (Ouyang and O’Garra
2019). Considering this fact, it is possible to speculate that the concentrations of IL-10 in the ears would be linked to the concentrations of TNF-α. Thus, at the lowest doses of EAELSt, the increased concentrations of IL-10 would be compensating for the lack of reduction in TNF-α concentrations, which did not occur for the highest dose of the extract. Anyway, the results obtained indicate that the treatment with EAELSt modulated this anti-inflammatory cytokine, which confirms the action of this extract in the cutaneous inflammatory response induced by TPA.
These protective actions of EAELSt may be related to the compounds presented in this extract. These phenolic compounds, in addition to being able to modulate signaling pathways and transcription factors (Lolli et al.
2012; Peng et al.
2018; Yokoyama et al.
2015), are known for their antioxidant effects, which could contribute to the action on the inflammatory response. Thus, we also investigated whether EAELSt could alter the oxidative stress that accompanies the induction of skin inflammation induced by TPA.
In fact, treatment with EAELSt promoted modulation of the formation of hydroperoxides, sulfhydryl groups, and the potential to reduce iron. Our data indicate that there was an inhibitory effect on oxidative stress markers (by reducing total hydroperoxides and by increasing the sulfhydryl groups). The formation of hydroperoxides denotes initial stages of lipid peroxidation, since these species are primary products of lipoperoxidation (Esterbauer
1993). In turn, it is known that the sulfhydryl groups are present in the constitution of several proteins and oxidative stress causes oxidation in these groups, resulting in malfunction of the cellular structures (Santos et al.
2011). Thus, it is most likely that the phenolic compounds in EAELSt reduced the formation of hydroperoxides and preserved the sulfhydryl groups from possible changes induced by oxidative stress induced by TPA. Taken together, these parameters show the decrease in the lipid peroxidation, and increase in sulfhydryl groups and in the Fe
2+/Fe
3+ rate, which strongly indicates the antioxidant effect of the extract in mice ears.
Additionally, it was observed that the highest dose of EAE increased the reduction potential indicating antioxidant effect through the FRAP method in vivo. It is known that, during oxidative stress, Fe
3+ reacts with O
2− becoming Fe
2+. This occurs through the Fenton reaction, which leads to the formation of hydroxyl radical which is highly reactive (Shahidi and Zhong
2015). The data found suggest that the reducing potential of EAELSt possibly occurs by the action of polyphenolic compounds identified in this plant, as proven in other studies (Jeyadevi et al.
2013). In fact, the antioxidant activity of polyphenolic compounds such as quercetin identified in EAELSt is directly related to the amount of hydroxyl group, position, and glycosylation (Cai et al.
2006).
We also found that treatment with this extract increased the activity of SOD activity at 3.0 mg/ear. Since SOD is responsible for the conversion of ·O
2− to H
2O
2 and water, this data shows that there was modulation of this enzyme to protect the tissue against oxidative stress. It has been documented in the literature that SOD can contribute to the resolution of inflammation through apoptosis of neutrophils, regulated by the H
2O
2 (Yasui and Baba
2006). For the CAT enzyme, treatment with 0.3 and 1 mg EAE/ear increased the activity of this antioxidant enzyme. CAT catalyzes the conversion of H
2O
2 into H
2O, which indicates that the increased activity of this enzyme results in the detoxification of free radicals.
Another interesting finding was related to the effect induced by the dose of 3 mg EAE/ear, which, despite not modulating CAT activity, reduced the formation of hydroperoxides, preserved the sulphidryl groups and increased SOD activity. One possibility would be that the GPx activity acts in a compensatory way at the different doses of the EAE; however, the GPx activity remained unchanged in all the doses evaluated. In this context, it can be suggested that, at the different doses of EAE, there was a compensatory effect between the activities of CAT and SOD that acted primarily in the detoxification process.
In the literature, studies involving oxidative stress in the model of ear edema used in the present study are still seldomly described. To our knowledge, this is the first study to demonstrate the effect of
S. terebinthifolius on antioxidant markers and enzymes. It is important to highlight the involvement of oxidative stress in the inflammatory process, as tissue damage during this situation leads to an excess of oxygen and nitrogen reactive species (Hussain et al.
2016) and several transcription factors involved in inflammation, such as the nuclear factor-κB, are activated by ROS (Li et al.
2002). Thus, it is plausible to suggest that the anti-inflammatory effect of EAELSt is associated, in part, with protection against oxidative damage. Besides, it is also possible to assume that the anti-inflammatory effect is not solely due to the antioxidant activity, since the anti-inflammatory effect was detected even when using a lower dose, when the antioxidant effect was not fully achieved. Despite these facts, our data suggest that the EAELSt can be promising in the search for alternatives for the treatment of inflammatory conditions for topical use. Furthermore, this study showed that EAELSt is promising for the treatment of skin inflammation. Finally, this study can serve as a basis for future studies to better understand the pharmacological action and its possible mechanisms of action.