Schinus terebinthifolius Raddi (Aroeira) leaves oil attenuates inflammatory responses in cutaneous wound healing in mice1

Purpose: To investigated the inflammatory, angiogenic and fibrogenic activities of the Schinus terebinthifolius Raddi leaves oil (STRO) on wound healing. Methods: The excisional wound healing model was used to evaluate the effects of STRO. The mice were divided into two groups: Control, subjected to vehicle solution (ointment lanolin/ vaseline base), or STROtreated group, administered topically once a day for 3, 7 and 14 days post-excision. We evaluated the macroscopic wound closure rate; the inflammation was evaluated by leukocytes accumulation and cytokine levels in the wounds. The accumulation of neutrophil and macrophages in the wounds were determined by assaying myeloperoxidase and N-acetyl-β-D-glucosaminidase activities. The levels of TNF-α, CXCL-1 and CCL-2 in wound were evaluated by ELISA assay. Angiogenesis and collagen fibers deposition were evaluated histologically. Results: We observed that macroscopic wound closure rate was improved in wounds from STRO-group than Control-group. The wounds treated with STRO promoted a reduction in leucocyte accumulation and in pro-inflammatory cytokine. Moreover, STRO treatment increased significantly the number of blood vessels and collagen fibers deposition, as compared to control group. Conclusion: Topical application of STRO display anti-inflammatory and angiogenic effects, as well as improvement in collagen replacement, suggesting a putative use of this herb for the development of phytomedicines to treat inflammatory diseases, including wound healing.

understanding of the effects of Schinus terebinthifolius Raddi in wound healing may provide futures insights to develop new strategies therapeutic approaches for the management fibroproliferative disorders.

Collection, identification, processing, and extraction of plant material
Leaves of the pepper tree (Schinus terebinthifolius Raddi, Anacardiaceae) were collected in the morning of September 2010 on the campus of Universidade Federal Rural de Pernambuco (UFRPE). The plant was identified by comparison with a previously identified specimen and deposited in the Herbarium Vasconcelos Sobrinho UFRPE under the number 49259.
To obtain the essential oil, fresh leaves (200g) were crushed and subjected to hydrodistillation technique in a modified Clevenger apparatus. After two hours of hydrodistillation the oil obtained was separated from water by density difference and excess moisture was removed with anhydrous sodium sulfate (Na 2 SO 4 ). The total oil amount was calculated based on the weight of fresh leaves. The oil was stored in amber glass container, tightly closed kept in freezer, until the experiment, at a temperature of -20°C. The study of the leaves oil had its chemical profile reported by Silva et al. 16 , which identified thirty-three components, representing 95.5% of the oil. Among the majority of this oil compounds are p-Cymen-7-ol (22.5%), 9-epi-(E)-cariophyllene (10.1%), carvone (7.5%) and Verbenone (7.4%).

■ Introduction
The Brazilian pepper tree (Schinus terebinthifolius Raddi) is native to the Northeast, Central-West, Southeast and South regions of Brazil known as aroeira. It has been used in folk medicine as teas, infusions or tinctures as an anti-inflammatory, febrifuge, analgesic, and depurative agent; also, it has been used to treat skin wounds, repair injury in respiratory, digestive and genitourinary systems 1-6 . Previous reports have demonstrated that S. terebinthifolius extracts or fractions is rich in polyphenols, which display antioxidant, antibacterial, antifungal and anti-allergic activities in different experimental models [7][8] .
Skin wound healing is a dynamic process that involves cellular, molecular and humoral components to tissue restore after injury 9 . In adult mammals, it involves an early inflammatory stage characterized by the infiltration of neutrophil and macrophages, with the subsequent formation of granulation tissue. In addition, keratinocytes from the borders of the legion proliferate and migrate to restore the epidermis of the injured skin 10 .
However, an impaired inflammatory response in the early stage of skin wound healing may compromise the quality of repair 11 . Thus, the development of therapies based on the attenuation of inflammation response during wound healing will lead to the development of efficient therapeutic approaches to improve the quality of repair [12][13] .
Many new therapies that target various aspects of wound repair are emerging in recent years using natural components derived from plants [14][15] . However, the healing effects from Schinus terebinthifolius Raddi are not completely understood.
Therefore, we hypothesized that the leaves oil of Schinus terebinthifolius Raddi (STR) would improve wound healing by modulating inflammation, angiogenesis and collagen deposition in wound site. A better from Pharma Special, solid vaseline and BHT acquired from DEG) 16 .

Experimental animal
The project proposal was approved by the Animal Ethics Committee of the Universidade Federal Rural University de Pernambuco (license n o 123/2016).
Male C57BL/6 mice 7-8 weeks (25-30 g body weight) (n=30), were provided by the Centro de Bioterismo (CEBIO) of Universidade Federal de Minas Gerais (UFMG)-Brazil. The animals were housed individually and provided with chow pellets and water ad libitum. The light/dark cycle was 12:12 h with lights on at 7:00 am and lights off at 7:00 pm. The experiment was conducted in the animal colony at the Animal Morphology Department, UFRPE. The animals were acclimatized for 2 weeks in a well-ventilated room, and standard pellet feed and tap water were provided ad libitum throughout the experimentation period. At the end of the experiment the animals were euthanized, prepared and discarded complied with the guidelines established by our local Institutional Animal Welfare Committee.

Wounding, experimental groups and measurement of wound area
The mice were divided into two groups (control and treated) of fifteen animals each, and all animals were anesthetized with a combination of 2% xylazine hydrochloride (10 mg/Kg) and 10% ketamine hydrochloride (60 mg/Kg), administered, intraperitoneally, prior to infliction of skin wounds. The animal's furry thoracic region was shaved with an electrical clipper and antisepsis was performed with 0.5% topical alcoholic chlorhexidine. Four excisional wounds were made on the dorsum of mice with the aid of a sterile 5-mm circular punch, removing the entire thickness of the skin. Wound excision was made carefully to avoid muscle incision. Immediately after surgical excision the control group (n=15), received a daily topical application of the ointment lanolin and vaseline base (Vehicle group) -and treated group (n=15), received a daily topical application of the ointment containing 10% aroeira Leaf oil (STRO group). The mice were then divided into three groups of five animals each according to the time of application of the ointment, as described: G3 -three days application; G7 -seven days of application and G14 -14 days of application. After surgery, mice were kept in individual cages.
The area of wounds was measured with a digital caliper, on days 0, 3, 7 e 14 after surgery and the results were expressed as percentage closure, relative to original size (wound contraction = original wound area -present wound area / total wound area x 100) 1 . No signs of infection were detected in the wounds area.

Quantification of neutrophil and macrophage tissue accumulation
The extent of neutrophil accumulation in the wound tissue as measured by assaying myeloperoxidase (MPO) activity, as described previously 17 . On days 3, 7 and 14 P.O, after euthanasia with an overdose of anesthetic (Pentobarbital sodium 100mg/kg, intraperitoneally), the wound and surrounding skin area were removed and frozen in liquid nitrogen. On thawing and processing, the tissue (100 mg tissue/1 ml buffer) was homogenized in 0.02 mol/l NaPO 4 buffer (pH 4.7) containing 0.015 mol/l Na-EDTA, centrifuged at 10.000 g for 10 min, and the pellet was submitted to hypotonic lyses. After a further centrifugation, the pellet was resuspended in 0.05 mol/l NaPO 4 buffer (pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide and rehomogenized. Suspensions were then submitted to three freeze-thaw cycles using liquid nitrogen and centrifuged for 15 min at . Results were expressed as the relative unit that denotes activity of MPO. The extent of macrophages accumulation in the wound tissue was measured by assaying N-acetylb-D-glucosaminidase (NAG) activity, as described previously. Briefly, the tissue (100 mg tissue/1 ml buffer) was homogenized in 0.9% saline containing 0.1% Triton X-100 (v/v) and centrifuged at 4°C for 10 min at 3000 3g; supernatants were used for NAG assay. The assay was performed by measuring the change in OD at 405 nm using p-nitrophenyl-Nacetyl-β-D-glucosaminide (Sigma-Aldrich) and 0.2 M glycine buffer (pH 10.6). Results were expressed as the relative unit that denotes activity of NAG.

Measurement of cytokine/chemokine concentrations in the wounds
On thawing and processing, the tissue was homogenized in extraction solution (100mg tissue/1ml), containing 0.4MNaCl, 0.05% Tween 20, 0.5% BSA, 0.1mM PMSF, 0.1 mM benzethoniumchloride, 10 mM EDTA, and 20 KI aprotinin, using Ultra-Turrax. The suspension was then spin at 10.000 3g for 10 min at 4°C. The supernatants from wounds were used to examine the levels of tumor necrosis factor (TNFα), chemokine (C-C motif) ligand 2 (CCL-2) and chemokine (C-X-C motif) ligand 1 (CXCL1) by ELISA assay, performed using kits from R&D Systems (Minneapolis, MN) , according to the manufacturer's instructions. All samples were assayed in duplicate. The threshold of sensitivity for each cytokine/chemokine was 7.5 pg/ml.

Histological assessment
On days 3, 7 and 14 of the experiment, skin samples were collected and fixed in 10% buffered formalin. Sections (5μm) were stained with hematoxylin and eosin (H.E) and picrosirius red and processed for lightmicroscopic studies. The images were digitized with Olympus BX43 microscope with an Olympus Q-Color 5 micro camera. Angiogenesis were determined by counting blood vessels in the entire area of wound tissue on H&E stained sections, cross sections images were obtained from 6 sequential fields from each wound and observed with an objective (x40) in light microscopy (final magnification = x400) 1 .
For collagen analysis, images were obtained from 5 fields from each wound and observed with an objective x20 (final magnification = x200) under polarized light (Olympus). Morphometric and digital analysis were performed using ImageProPlus 7.0 Software. All histomorphometric analysis were performed as blind study.

Statistical analysis
Statistical analysis were performed using GraphPad Prism 5.0 software. Results were presented as the mean ± SEM. Comparisons between two groups were carried out using Student t test for unpaired data. Two-way ANOVA was used for graph lines to verify the interaction between the independent variables time and strain followed by Bonferroni post test (p < 0.05).

Wound closure is improved in STRO-treated group
The STRO-treated group exhibited a significant increase in the percentage of wound closure after day 3 post-wounding, Schinus terebinthifolius Raddi (Aroeira) leaves oil attenuates inflammatory responses in cutaneous wound healing in mice Estevão LRM et al. when compared with vehicle-treated group; approximately 37% in STRO-treated group versus 21% in vehicle-treated group (p<0.05) (Figure 1 A-B).

Leukocyte accumulation is reduced in STROtreated group
We observed a reduction in the leukocyte accumulation of wounds from STROtreated group, when compared to Vehicletreated group, as assayed by MPO (Figure 2A

Wound angiogenesis is increased in STRO-treated group
At day 7 after wounding, the number of vessels in wounds from STRO-treated group increased significantly when compared with Vehicle-treated group (Figure 4 A-B, p<0.05).

Collagen deposition is increased in STRO-treated group
Collagen deposition increased significantly in wounds from STRO-treated group when compared with Vehicle-treated group ( Figure 5A

■ Discussion
Here, we investigated the effects of Schinus terebinthifolius Raddi oil leaves (STRO) on wound closure, inflammation, angiogenesis and collagen deposition in skin wound healing after excision in mice. This experimental model has been used to study the biological mechanisms involved skin wound healing 1,2 . STRO was applied topically as natural compounds with healing potential can be tested directly in wound bed 18 .
It has been reported that topical application of Schinus terebinthifolius Raddi extracts exerts angiogenic 1 and pro-healing 4 effects in vivo. Accordingly, our study demonstrated that topical application of STRO improved wound closure and displayed angiogenic and fibrogenic effects. Interesting, we observed that STRO treatment also promoted anti-inflammatory responses in wounds mice.
The inflammatory response following tissue injury plays important roles both in normal and pathological healing processes 19 . It provides protection against invading pathogens and remove damaged tissues 20 . However, prolonged inflammatory response lead to a delay in the progress through the physiological stages of wound healing 12 .
Natural products with antiinflammatory activity have long been used as a folk remedy for inflammatory conditions in wound, such as Curcuma longa 21 , Ranunculus pedatus 22 .
Here, we observed that STRO treatment was able to decrease the markers of inflammation in wounds. A significant reduction in TNF-α, CXCL-1 and CCL-2 levels as well as in neutrophil and macrophage accumulation, as measured by MPO and NAG activities, respectively, were found in the STRO treated group. Indeed, the reduction in these cytokines levels is usually associated with reduced inflammation 23,24 . Our study is the first to demonstrate that STRO has antiinflammatory effects on skin wound healing. There is considerable evidence to suggest that angiogenesis and inflammation are codependent 25 . Angiogenesis is critical to wound repair. The process is important to provide nutrition and oxygen to growing tissues 26 . A wide variety of plant-based compounds have been reported to act in angiogenesis process; these compounds include polyphenols, alkaloids, terpenoids and tannins 27 . We also observed an increased wound angiogenesis in STRO-treated mice when compared with Vehicle-treated group. In accordance, our group reported the angiogenic effects by Schinus terebinthifolius Raddi in wound repair 1,28 . However, further studies are necessary to determine possible mechanisms.
In addition to its inflammatory and angiogenic activity, we observed that STRO was able to increase collagen deposition in the skin wound. Consistent with this finding, it has been reported that STRO induces collagen deposition in tissue repair in rats 1 . The improved wound closure observed in STRO treated group may have been, at least in part, due to its fibrogenic effect.
Our present results have identified and confirmed the effects of Schinus terebinthifolius Raddi, on some critical features of wound healing processes (angiogenesis, inflammation and collagen deposition). These results the range of Schinus terebinthifolius Raddi actions and provides future insights in therapeutic of wound healing.

■ Conclusions
Topical application of STRO display anti-inflammatory and angiogenic effects, as well as improvement in collagen replacement, suggesting a putative use of this herb for the development of phytomedicines to treat inflammatory diseases, including wound healing.