The enhanced healing effect of Anadenanthera colubrina ethanolic extract on excisional skin wounds in mice

doi:10.21203/rs.3.rs-5326954/v1

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The enhanced healing effect of Anadenanthera colubrina ethanolic extract on excisional skin wounds in mice

https://doi.org/10.21203/rs.3.rs-5326954/v1

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Background: Anadenanthera colubrina has been widely used in traditional medicine as a healing agent. However, the body of evidence supporting the use of this extract as a wound healer is still limited. Therefore, the study aimed to investigate the effect of the ethanolic extract of Anadenanthera colubrina (EEAc) on the viability of L929 fibroblasts and its antioxidant potential in vitro, as well as its effect on the healing of excisional skin wounds in mice.

Methods: The extract was analyzed for its major compound using HPLC, as well as for the total phenolic content. In vitro antioxidant activity was evaluated through radical scavenging assays, such as ABTS, DPPH, and FRAP. The cell viability of L929 fibroblasts treated with EEAcwas determined using the MTT assay. Skin excision was performed in female swiss mice and distributed groups: control (saline), vehicle (2% of DMSO in PG) or EEAc (5%). Treatment was administered daily, and the progress of wound healing was monitored on days 0, 3, 7 and 14. The wounds was collected on days 1, 3, 7 and/or 14 for histological analysis. The concentration of cytokines TNF-α and IL-10, MPO and NAG activity enzymes were assessed in wounds, at various time points.

Results: HPLC analysis showed that EEAc has caffeic acid as its main compound. EEAcexhibited a high total phenolic content and good antioxidant capacity against the evaluated free radicals. Furthermore, it was not cytotoxic to L929 fibroblasts. In mice, EEAc reduced the wound area on the 3rd and 7th days and increased collagen deposition on the 14th day. Additionally, EEAc reduced MPO activity on the 3rd day and NAG activity on the 7th day, in contrast to the increase in IL-10 cytokine concentration on the 7th day.

Conclusion: EEAc showed good antioxidant potential and does not present cytotoxicity in L929 cells. Taken together, our data demonstrate, for the first time, that EEAcimproved skin wound healing by modulating the inflammatory response during this phase of the repair process. This effect possibly led to efficient healing with increased collagen deposition, revealing its potential application as a healing agent.

skin wound

lesion

inflammation

repair

traditional medicine

Skin wounds are characterized by interruption of the skin's integrity due to tissue damage [1] initiating a complex wound healing process. This process involves several cellular and molecular interactions to restore the skin and form a scar. The wound healing process typically involves four overlapping phases: hemostasis, inflammation, proliferation, and remodeling [2]. The inflammatory phase, in particular, has been considered a fundamental event for the repair of skin lesions. However, the exacerbated and persistent inflammatory response can impair healing, resulting in complications such as chronic wounds [3, 4].

Wound treatment is commonly performed due to the possibility of complications such as exacerbated pain, hypertrophic scars, keloids, infections, amputations, sepsis, and death [5, 6]. Chronic wounds, in particular, pose significant economic burdens, with the treatment in the United States alone costing approximately $25 billion annually [7]. While several conventional wound treatment methods are available, they often face limitations such as limited accessibility, high costs, and concerns regarding safety and efficacy [8, 9]. As a result, the development of cost-effective alternative therapies has become increasingly important, particularly when related to the use of herbal therapies [10].

Anadenanthera colubrina, commonly known as Angico, is a plant highly regarded for its medicinal properties in regions where it is native [11]. It has been extensively used in traditional medicine, particularly for its purported wound healing and anti-inflammatory effects [12]. Among its various parts, the bark is the most utilized, known for its rich content of tannins and flavonoids [13, 14]. Studies conducted by Pessoa et al.,[15] have identified several bioactive compounds in the bark of A. colubrina, including condensed proanthocyanidins, leucoanthocyanidins, reducing sugars, flavonoids (such as quercetinic glycosides), saponins, triterpenes, and steroids. These metabolites are associated with a range of beneficial properties, including anti-inflammatory,[16] antioxidant,[17] and antimicrobial activities [18]. According to Weber et al.,[19] A. colubrina has great therapeutic potential and, although it is widely explored from an ethnobotanical and economic point of view, it remains little investigated pharmacologically.

The healing effects of A. colubrina bark extract on excisional wounds in rats have been previously examined [15, 20]. However, there is a scarcity of studies focusing on its healing properties in skin repair, and further exploration of the mechanisms is warranted. Therefore, there is potential to safely demonstrate the effectiveness of A. colubrina in wound healing, thus validating its use in traditional medicine [12, 21] and prompting the development of a more effective alternative therapy with anti-inflammatory mechanisms for the repair process [16]. Given these considerations, investigating the effects of the ethanolic extract of A. colubrina on the healing of excision cutaneous wounds induced in mice is justified.

Therefore, we hypothesize that the ethanolic extract of A. colubrina can promote excisional wound repair in mice, particularly those with anti-inflammatory mechanisms that can enhance the healing process. In summary, the investigation of the wound-healing effects of EEAc on excisional wounds in mice represents a valuable contribution to both traditional medicine validation and the development of alternative therapies for wound care.

Plant material

The bark of an adult tree of Anadenanthera colubrina (Vell.) Brenan var. cebil (Griseb.) Altschul (Fabaceae) were collected in November 2018, during the dry season, in the city of Monte Alegre de Sergipe, Sergipe, Brazil (10° 2' 44” S and 37° 35' 4” W). The species was authenticated by phD Marcus Vinicius Meiado and deposited at the Federal University of Sergipe Herbarium (ASE nº 42361).

Preparation of plant extract

The bark was dried at 40°C (± 2) for 72 hours, ground, and weighed, yielding 1.1 kg of powder. This material underwent exhaustive maceration in absolute ethanol for 72 hours, a process repeated three consecutive times. The resulting liquid was filtered and subjected to rotary evaporation at 45°C (Fisatom®, São Paulo/SP, Brazil). The resulting dry extract (338 g) was stored at 2–8°C. For formulation purposes, 5 g of the extract were diluted in 100 mL of a vehicle composed of 2% Dimethylsulfoxide (DMSO; Merck, Brazil) in Propyleneglycol (PG),[22] resulting in the Ethanolic Extract of Anadenanthera colubrina (EEAc) at 5% [15, 20].

Characterization of EEAc by HPLC

The EEAc was diluted with methanol at a 5 mg/ml concentration. Both extract solutions were submitted to sonication for 30 minutes in an ultrasonic bath and were filtered on membrane filters (PTFE – 0.45 µm), before High-performance Liquid Chromatography coupled to Diode-Array Ultraviolet Detector (HPLC-DAD-UV) injection.

The HPLC-DAD-UV analyses were performed using a high-performance liquid chromatography system that consisted of a DGU-20A3 degasser, two LC-20AD pumps, an SIL-20A HT auto-injector, a CTO-20A column oven, an SPDM20Avp photodiode array detector (DAD) and a CBM-20A system controller (Shimadzu Co., Kyoto, Japan). The chromatographic separation was performed using a Shimadzu ODS analytical column 180 × 4.6 mm (5 µm particle size), equipped with a Shimadzu ODS guard cartridge system 20 x 4 mm (5 µm particle size). The mobile phase consisted of (A) ultrapure water with acetic acid (1.0%) and (B) methanol. The flow rate was 0.8 ml/min and the sample injection volume was 20 µL. The elution gradient started with 5% B (0.01–5 min); 5–15% B (5–12 min); 15–28% B (12–13 min); 28–28% B (12–13 min); 28–30% B (13–14 min); 30% B (14–22 min); 30– 31% B (23–25 min); 31–32% B (25–35 min); 32% B (35–39 min); 33–34% (40–45 min); 35–40% (50–52 min). Finalizing the analysis and returning to the initial conditions. The photodiode array detector was set at 280 nm for acquiring chromatograms. Compound identification was based on comparisons of absorption spectra and by co-injection with standard substances (based on retention times). Caffeic acid (C₉H₈O₄), chlorogenic acid (C₁₆H₁₈O₉), gallic acid (C₇H₆O₅), ferulic acid (C₁₀H₁₀O₄), myricetin (C₁₅H₁₀O₈) and quercetin (C₁₅H₁₀O₇) standards were obtained by Sigma-Aldrich® and prepared at a final concentration of 0.1 mg/mL with acetonitrile.

Quantification of total phenolics

The methodology for total phenolics content determination was based on the protocol proposed by Swain and Hillis (1959), with modifications adapted for a microplate format. A 12 µL aliquot of the extract was pipetted in quadruplicate into a 96-well plate. Subsequently, 12 µL of Folin-Ciocalteu reagent and 200 µL of distilled water were added to each well. After a 3-minute reaction time, 25 µL of saturated sodium carbonate solution (Na₂CO₃) was introduced. The plate was then kept at room temperature, and shielded from light. After one hour, the absorbance was measured using a spectrophotometer at 720 nm. Results were expressed as milligrams of gallic acid per gram of dry extract, determined using a standard curve with concentrations ranging from 25 to 200 µg/mL (y = 0.0092x + 0.0385; R² = 0.9976).

In vitro experiments

Antioxidant assays

In vitro tests were performed in triplicate, to determine the antioxidant capacity of EEAc. The extract concentrations used were 25, 50, 100, and 200 µg/mL, and the concentration required to inhibit half of the maximum evaluated parameter (IC₅₀) was calculated. Trolox (Sigma Aldrich, USA) was used as a standard of the antioxidant. The antioxidant capacity against the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH; Sigma-Aldrich, USA) was measured by adding 50 µL of extract to 150 µL of DPPH at a concentration of 400 µM/L in a microplate. After 30 minutes of rest, protected from light, the reduction of the DPPH radical was measured at 515 nm using a plate reader (Biotek, Synergy Mx).

The ABTS radical assay [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] was performed adding 30 µL of the sample in a microplate and was added to 300 µL of the ABTS radical solution (1,25 mL of a 7 mM ABTS solution in 22 µL of a 140 mM potassium persulfate (K₂S₂O₈) solution). For these tests, the results were expressed as a percentage of inhibition [% inhibition = [(control − test)/control] × 100], based on the absorbance values, from which the IC₅₀ values were calculated.

The ferric-reducing antioxidant power (FRAP) assay was conducted in a dark environment by adding 9 µl of each extract concentration in triplicate to a microplate., followed by 27 µl of distilled water, and 270 µl of the FRAP reagent, consisting of TPTZ, ferric chloride solution, and acetate buffer (0.3 M, pH 3.6) in a 1:1:10 ratio. The microplate was then incubated at 37°C for 30 minutes. After incubation, absorbance was measured using a spectrophotometer at 595 nm. Results were expressed in µM Trolox equivalents, calculated using the equation y = 0.0008x + 0.102.

Cytotoxicity in fibroblasts

Cell viability of the fibroblast cell line, L929, was assessed using the methylthiazolyl tetrazolium bromide (MTT) colorimetric assay. Cells were cultured in DMEM (Sigma Aldrich, USA) containing 10% fetal bovine serum (Life Technologies, India), 1% penicillin (Life Technologies, India)/streptomycin (Life Technologies, India), and maintained in an atmosphere of 5% CO₂ at 37°C. Fibroblasts were seeded in 96-well culture plates (2 × 10⁴ cells/well) and treated with EEAc at concentrations of 12.5, 25, 50, 100, and 200 µg/mL for 24 hours. Then, MTT (0.5 mg/mL in phosphate-buffered saline (PBS)) was added to the cells and incubated at 37°C for 3 hours. After MTT removal, DMSO was added to solubilize the formazan crystals, and the absorbance was measured at 570 nm. The tests were conducted in triplicate and normalized by considering the control absorbance as 100% viability.

Wound healing in vivo

Swiss albino mice (Mus musculus), female, nulliparous, healthy, weighing between 25–30, aged 8–12 weeks. Were kept individually in polypropylene cages, with 21 ± 2°C temperature, 60 ± 5% humidity, and a 12-hour light / dark cycle, in addition to food and water ad libitum.

Excision of skin wounds and topical treatments

Before the induction of the lesions, the animals underwent a seven-day acclimatization period. Ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) were used as anesthetic medications. Following anesthesia, the dorsothoracic region was shaved, and antisepsis was performed using 0.5% alcoholic chlorhexidine solution. Circular cutaneous excision was performed with a 6 mm metallic punch [23]. The animals were randomly assigned to three groups: control (treated with sterile saline 0.9%), vehicle (2% DMSO in PG), or EEAc. Treatments were administered immediately after wound induction and repeated every 24 hours for 14 days. Each wound received 30 µL of the respective treatment (equivalent to 1.5 mg of EEAc per wound per day).

Wound contraction

Wounds were measured on days 0 (initial wound), 3, 7, and 14 using a digital caliper (n = 5–7/group). The wound area was calculated according to the equation: Wound area (mm²) = π.R.r, where π = 3.1416; R = larger radius and r = smaller radius. Wound closure was determined using the equation: Wound closure (%) = Wi-Wd/Wi x 100, where Wi equals the initial wound area and Wd the wound area on the measured days [24]. After obtaining this parameter, the % wound area remaining open was calculated by reducing the % closure value by 100.

Tissue collection

The animals were euthanized using an overdose of ketamine (300 mg/kg, i.p.) and xylazine (30 mg/kg, i.p.). Skin fragments, including both the intact area and the wound site, were collected using an 8 mm punch. The collection was performed at different time points depending on the healing progression, including days 1, 3, 7, and/or 14 post-injury.

Histopathological analysis

Animals designated for wound closure analysis (n = 5/group) underwent histological evaluation on the 14th day post-injury. Tissues were sectioned in half into two fragments, cut in a microtome (5 µm), and stained with Hematoxylin & Eosin (H&E) or Masson's Trichrome (Masson’s). Subsequently, the tissue sections were examined (10 different areas) under an optical microscope (Nikon, Japan), and images were captured using a digital camera (Coolpix 4500, Roper Scientific, Japan). Analysis was conducted by two researchers independently, in a blinded manner. A scale ranging from 0 to 3 was utilized to assess various parameters including leukocyte infiltrate, vascularization, re-epithelialization, and collagen deposition. The scoring system was as follows: 0 for absent, 1 for mild, 2 for moderate, and 3 for accentuated [25].

Inflammatory essays

Myeloperoxidase and N-acetyl-β-D-glycosaminidase activity

Quantification of myeloperoxidase (MPO) activity was performed for indirect assessment of neutrophil infiltrate on days 1, 3, and 7 of healing (n = 5–7/group/time). The tissues were homogenized in PBS and Hexadecyltrimethylammonium Bromide 0.5% (HTAB 0.5%), in the proportion of 1mL/100mg of tissue. Following homogenization, the samples were centrifuged for 2 minutes at 14000 rpm. A volume of 20 µL of supernatant was used for the reaction with 200 µL of O-dianisidine solution and, finally, the absorbance of the samples was determined at 460 nm [26].

For the indirect determination of the macrophage infiltrate in the lesions, N-acetyl-β-D-glycosaminidase (NAG) activity was quantified on days 1, 3, and 7 of the repair process (n = 5–7/group/time). Tissue samples were homogenized in cytokine extraction buffer (PBS containing protease inhibitor cocktail, Phenylmethylsulfonyl fluoride (PMSF), Sodium chloride (NaCl), Ethylenediamine Tetra-acetic acid (EDTA) and Tween 20) and centrifuged. The pellet was resuspended in Buffer containing 0.2% NaCl and 1.6% NaCl with 5% glucose, posteriorly. Subsequent steps involved homogenization, centrifugation, and resuspension of the pellet in NaCl 0.9% and Triton x-100 0.1% (1:1). The supernatant was mixed with 0.767 mg/ml of p-nitrophenyl-N-acetyl-β-D-glycosaminide for the NAG assay. The plate was incubated at 37ºC for 30 minutes and the reaction was stopped with buffer containing 0.2 M glycine. The samples were analyzed at 405 nm. Finally, the result was normalized by the weight of the tissue and expressed in Optical Density (OD)/ 100 mg of the tissue [27].

Quantification of cytokines

The tissue samples on days 1 and 3 of repair (n = 5–7/group/time) were subjected to quantification of cytokines TNF-α (Tumor Necrosis Factor–α) and IL-10 (Interleukin 10) using ELISA kits according to the manufacturer's protocol (Peprotech®, USA).

Statistical analysis

The data were submitted to the Shapiro-Wilk normality test. According to the data normality, a one-way or two-way ANOVA test was performed with Bonferroni post-test, or the Kruskal-Wallis test was used with Dunn's post-test. Data were expressed as mean ± SEM or median and interquartile range for scores of histological parameters. Statistical analysis was performed using the GraphPad Prisms® 8.0 software, with p < 0.05 being considered significant.

Phytochemical analysis

The chromatographic profile of the EEAc, besides the result showing that there are various compounds in the extract, was specifically identified as the presence of caffeic acid (Fig. 1).

The total phenolic content of EEAc was estimated using gallic acid as the standard. We found that at a concentration of 500 µg/mL, the total phenolic content was 168.02 ± 3.2 µg of gallic acid equivalent/ mg of dry extract, calculated according to the standard curve described in the methodology (Fig. 1S).

Antioxidant potential of the EEAc

To evaluate the antioxidant capacity of EEAc, we conducted the DPPH, ABTS, and FRAP tests (Fig. 2).

In the DPPH radical scavenging assay, which is based on the hydrogen donor acceptance by the DPPH compound from the antioxidant molecule, EEAc showed an increase in the percentage of radical scavenging proportional to the concentration increase. At a concentration of 100 µg/mL, EEAc presented a scavenging percentage similar to the standard, and at the highest concentration, it showed a higher scavenging percentage than the standard (p = 0.0007). However, all concentrations of EEAc and the standard (Trolox) had a greater antioxidant capacity than the system (p < 0.0001) (Fig. 2a).

In the ABTS radical scavenging assay, EEAc showed greater antioxidant activity starting from the concentration of 100 µg/mL of the extract, when compared to the standard (Trolox) (p = 0.0002). Similarly to the DPPH assay, all concentrations and the standard had higher antioxidant activity than the system (p < 0.0001) (Fig. 2b).

Regarding FRAP, it was observed that the extract presented iron reduction activity in a concentration-response manner, reducing iron from its ferric form to the ferrous form (Fig. 2c).

EEAc does not show significant cytotoxicity in L929

The MTT assay was performed to evaluate the potential of EEAc to support cell proliferation. Thus, in the assay, when metabolic activity decreases, it eventually leads to apoptosis or necrosis, representing a reduction in cell viability. Thus, in all tested concentrations there was viability 72% for the fibroblasts L929 (Fig. 3).

EEAc decreases the area of excisional skin wounds

The impact of EEAc on wound healing progress was initially assessed by monitoring the percentage of open wound areas. On the 3rd and 7th days, a significant reduction in the percentage of the wound area that remained open was observed in the animals treated with EEAc, in contrast to the control or vehicle groups (p < 0.01 on the 3rd day and p < 0.001 on the 7th day) (Fig. 4a, b).

To further elucidate these differences between the groups, the results were expressed as the area under the curve (AUC). Notably, treatment with EEAc led to a reduction in the open wound area, facilitating closure. This was evidenced by the lower AUC observed exclusively in the treated group compared to the control (p = 0.0132) or vehicle (p = 0.0068) (Fig. 4c).

EEAc reduced MPO and increased NAG in healing wounds

MPO is an enzyme released by neutrophils, which can serve as a marker of the content of these cells in the skin tissue [25]. Our study revealed that animals treated with EEAc exhibited reduced MPO activity on the 3rd day compared to the control and vehicle groups (p <0.01 and p <0.001) (Fig. 5a).

The macrophage infiltrate was quantified by measuring the lysosomal enzyme NAG, highly expressed in activated macrophages [28]. The increase in NAG was observed on the 7th day, in the EEAc group when compared to the control (p= 0.0001) and vehicle (p=0.0009) groups (Fig. 5b).

EEAc has anti-inflammatory potential

No significant difference was found in the TNF concentration among the groups (Fig. 5c). Despite this, the concentration of IL-10 increased in the group that received the EEAc treatment, observed after 3 days, compared to the control and vehicle groups (p <0.01) (Fig. 5d).

EEAc improves collagen deposition in skin lesions

Figure 6 (a) shows histological images of the wound, stained with H&E and Masson's Trichrome. The parameters of re-epithelialization, collagen deposition, leukocyte infiltration, and vascularization (Fig. 6 b-e) were evaluated using a scoring system ranging from 0 to 3 to assess wound quality. We found an increase in the score only in collagen deposition in the EEAc group compared to the control (p=0.0039) and vehicle (p=0.0039) groups.

The healing effects of medicinal plants are closely associated with the presence of secondary compounds produced by the plant's metabolism, which typically induce an anti-inflammatory response [29, 30]. For A. colubrina, its healing effect can be mainly attributed to the abundant presence of flavonoids and tannins in the bark [12–14]. In this study, the amount of total phenolics and the predominant presence of caffeic acid may be directly associated with the observed healing effects. Plants rich in polyphenols are closely associated with the inhibition of ROS (Reactive Oxygen Species) generation, serving as important antioxidants and modulating the release of pro-inflammatory cytokines and interleukins during the tissue repair process [31].

The EEAc demonstrated a potential effect in reducing free radicals, such as DPPH and ABTS, either by donating hydrogen and/or electrons, which may reflect anti-inflammatory effects in vivo. Additionally, iron-reducing capacity can enhance antioxidant effects, an activity related to flavonoid content [32]. Indeed, phenolic compounds present in plants, such as caffeic acid and the phenethyl ester derivative of caffeic acid, have been associated with various biological properties, including antibacterial and anti-inflammatory effects [33, 34, 35]. Moreover, the extract promoted good viability in L929 fibroblasts. Together, these data indicate a potential for EEAc to treat skin wounds.

The excisional wound model used in this study evaluated the impact of EEAc on acute lesions, allowing for the determination of the degree of closure through secondary intention healing [36, 37]. In our investigation, EEAc effectively reduced the area of excisional wounds during the healing periods of 3 and 7 days. However, it did not accelerate the repair process at the end of the experimental period. This suggests that EEAc predominantly exerted its activity during the inflammatory phase, probably due to the antioxidant and anti-inflammatory properties of A. colubrina [16]. However, it is essential to note that although wound area measurement is a common practice, this parameter alone does not fully determine the efficacy of healing agents. As mentioned earlier, phenolic compounds and flavonoids have high antioxidant capacity due to their redox properties, which can play an important role in the absorption and neutralization of free radicals, culminating in their antiproliferative and anti-inflammatory properties [38].

Therefore, the quality of wound repair at the end of the experiment (14th day) was assessed through histological analysis, revealing an increase in collagen deposition. However, there were no differences observed for the parameters of leukocyte infiltrate, vascularization, and re-epithelialization. Previous research conducted by Pessoa et al. [36], evaluated the effect of A. colubrina bark extract on the healing of excisional cutaneous wounds of rats and did not observe differences in the area, but similarly found an increase in collagen deposition, consistent with the findings of the present study. In this context, A. colubrina seems to be involved in the optimization of the initial stages of the repair process, facilitating a smooth transition between the phases, resulting in the formation of an improved scar.

Considering the macroscopic and microscopic effects observed for the EEAc, the decision was made to measure the cell biomarkers MPO and NAG to assess the accumulation of neutrophils and macrophages, respectively. Neutrophils are associated with uncontrolled inflammation and impaired tissue repair [37] making them dispensable and unfavorable to the repair process [38]. In contrast, macrophages play an indispensable role in healing, being observed that depletion of macrophages in mice has been associated with reduced contraction, deficient re-epithelialization, collagen deposition, and angiogenesis as well as increased neutrophils presence [39, 40]. It is essential for tissue resolution to maintain the M2 macrophage profile [41].

Moura et al.,[42] in a study on the healing activity of the hydroethanolic extract of Maytenus ilicifolia leaves, observed a similar reduction in MPO activity on the 3rd day, while there was an increase in NAG on the 7th day. The authors suggested that this occurred due to a reduction in neutrophils and a simultaneous increase in macrophages over time, thus favoring the progression of the repair process.

Numerous natural products exhibit anti-inflammatory activity that supports the healing process, such as TNF-α inhibition and/or IL-10 stimulation [30]. TNF-α potentiates inflammation by activating the Nuclear Transcription Factor-κB and producing pro-inflammatory mediators such as IL-1β, IL-6 and TNF-α itself [43]. Consequently, the inhibition of TNF-α promotes wound closure while its elevation hampers healing [44].

Alternatively, IL-10 has been linked to skin regeneration, by assisting in the reduction of inflammation and encouraging M2 polarization [45, 46]. Similar to the findings of the present study, Amorim et al., [47] demonstrated that Copaifera paupera oleoresin enhances wound closure, boosts collagen deposition, and elevates IL-10 levels in diabetic excisional wounds of mice [25]. Although our study did not explore the correlation between IL-10 release and wound quality, there is an increasing amount of research being conducted. on the "non-classical" role of IL-10 concerning wound repair [48]. Its antifibrotic properties activate the signal transduction and activator of Transcription 3 (STAT3), thereby augmenting the hyaluronan (HA) content, prompting fibroblasts to synthesize a pericellular matrix abundant in HA. Consequently, this process facilitates the repair of epithelial regenerative wounds [49].

We demonstrated that EEAc has good antioxidant potential and is not toxic to fibroblasts. Moreover, the EEAc facilitates efficient wound healing in mice by reducing wound area and neutrophil infiltration, while increasing macrophage infiltration, IL-10 levels, and collagen deposition in the lesions. Therefore, we suggest that A. colubrina optimizes the skin wound healing process, mainly by attenuating the inflammatory response. However, our study has limitations that need to be approached, as it remains crucial to assess how sexual dimorphism may influence the treatment response to this extract. Nevertheless, the study provides valuable insights to the scientific literature on the promising activity of the extract in wound healing.

EEAc- Anadenanthera colubrina

HPLC-DAD-UV - Diode-Array Ultraviolet Detector

IL-10- Interleukin-10

IL-17- Interleukin-17

IL-6- Interleukin-6

M1- classically activated macrophage

M2- alternatively activated macrophage

MPO- myeloperoxidase

NAG- N-acetyl-β-D-glycosaminidase

ROS- Reactive Oxygen Species

STAT3- Signal Transduction and Activator of Transcription 3

TNF- α- Tumor Necrosis Factor –α

Availability of data and materials

The authors declare that data pertaining to this research will be accessible upon request from the editors.

Competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This research was supported by CNPq- Conselho Nacional de Desenvolvimento Científico e Tecnológico under grant universal 01/16 (nº 429179/2016-2).

Authors’ contributions

WSN designed and conducted all in vivo experiments, interpreted the results, and wrote the manuscript. JMDAA participated in the experiments, conducted the analysis of cytokine, MPO, and NAG concentrations, and prepared the manuscript for publication. ACSN assisted with the in vivo experiments, reviewed and prepared the manuscript for publication. SSM prepared the extract and performed characterization by HPLC. MCD supervised and evaluated the HPLC results. DAS conducted assessments of the extract's antioxidant capacity, as well as total phenolics. JMFN assisted in the preparation of histological slides. JFS conducted in vitro cytotoxicity experiments. CBC, supervised and evaluated the results of the histopathological analyses. VSMJ assisted with the drafting of the first version of the manuscript, prepared the figures, and provided resources for the in vivo experiments. EAC assisted with discussions on experimental design, supervised and provided facilities and materials for the in vivo research. RG was involved in the conception and design of the study and secured funding for the research. All authors edited and reviewed the manuscript and approved the submitted version.

Ethic approval and consent to participate

The experimental procedures were carried out in accordance with the guidelines and standards established by the Animal Research Ethics Committee. The protocol received approval from the Animal Research Ethics Committee (CEPA) of the Federal University of Sergipe on March 25, 2019, under protocol number 07/2019.

Acknowledgements

The authors would like to acknowledge Federal University of Sergipe for providing the laboratory facility to carry out the research.

Author details

¹Graduate Program in Physiological Sciences, Department of Physiology, Federal University of Sergipe, Brazil; ²Graduate Program in Physiological Sciences, Department of Morphology, Federal University of Sergipe, Brazil; ³Graduate Program in Pharmaceutical Sciences, Department of Pharmacy, Federal University of Sergipe, Brazil; ⁴Department of Morphology, Federal University of Sergipe, Brazil. ⁵Gonçalo Moniz Institute – Fiocruz, Waldemar Falcão Street, Brazil.

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No competing interests reported.

Fig.1S.tif
Supplementary information Fig. 1S. Standard curve of gallic acid.The standard curve was determined using concentrations of 12.5, 25, 50, 100, and 200 µg/ml of gallic acid, and the linear equation was used to calculate the gallic acid equivalent (GAE) of the dry extract
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The enhanced healing effect of Anadenanthera colubrina ethanolic extract on excisional skin wounds in mice

Status:

Version 1

Abstract

Figures

BACKGROUND

MATERIAL AND METHODS

Plant material

Preparation of plant extract

Quantification of total phenolics

Antioxidant assays

Cytotoxicity in fibroblasts

Excision of skin wounds and topical treatments

Wound contraction

Tissue collection

Histopathological analysis

Inflammatory essays

Myeloperoxidase and N-acetyl-β-D-glycosaminidase activity

Quantification of cytokines

Statistical analysis

RESULTS

DISCUSSION

CONCLUSION

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

The enhanced healing effect of Anadenanthera colubrina ethanolic extract on excisional skin wounds in mice

Status:

Version 1

Abstract

Figures

BACKGROUND

MATERIAL AND METHODS

Plant material

Preparation of plant extract

Quantification of total phenolics

Antioxidant assays

Cytotoxicity in fibroblasts

Excision of skin wounds and topical treatments

Wound contraction

Tissue collection

Histopathological analysis

Inflammatory essays

Myeloperoxidase and N-acetyl-β-D-glycosaminidase activity

Quantification of cytokines

Statistical analysis

RESULTS

DISCUSSION

​

CONCLUSION

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1