Phytochemical Characterization, Pharmacological Properties And Toxicity of Amazonian Fruit Cubiu (Solanum Sessiliorum Dunal)

The cubiu (Solanum sessiliorum) is a tropical fruit native to the Amazon region and widely used in medicine and cosmetics, despite the lack of research regarding the actual safety and effectiveness of its use for these purposes. This study aimed to evaluated the phytochemical characterization, pharmacological properties (skin wound healing, antimicrobial and anti-inammatory properties), and toxicity of cubiu extract. The cubiu antimicrobial capacity was determined against strains of Aeromonas caviae, Pseudomonas aeruginosa, and Sphingomonas paucimobilis. Additionally, cubiu toxicity (hemolysis, coagulation, cell viability, and genotoxicity tests), antioxidant activity (reactive oxygen species total levels), scratch assay (in vitro skin wound healing), and anti-inammatory activity against phytohemagglutinin and in the scratch assay (Interleukin 1, interleukin 6, interleukin 10, tumor necrosis factor-alpha, and interferon-gamma levels), were evaluated. Human broblast cells were used to evaluate skin wound healing, and human peripheral blood mononuclear cells were used for the other assessments. Our ndings showed that the cubiu extract is rich in phenolic compounds, the major compound being 5-caffeoylquinic acid. In addition, was effective against the three bacterial strains tested and inhibited and destroyed the biolm formed by Pseudomonas aeruginosa. The cubiu extract also was no toxicity, maintained the hemocompatibility parameters in the biological range, improved cell viability, decreased reactive oxygen species total levels and pro-inammatory cytokine levels, increased anti-inammatory cytokine levels, and accelerated the wound healing process. In conclusion, this is the rst research to prove that cubiu is an important compound for use in the skin diseases, promoting skin wound healing, antimicrobial and anti-inammatory activities.


Introduction
Solanum sessili orum Dunal (cubiu) is a tropical plant of the Solanaceae family and native to the Amazon region. Its fruit, cubiu, also known as "apple/peach tomato", has become an important raw material for the modern agro-industry, considering it comes from a highly productive and easily cultivated fruit that is widely consumed and nutritious with health-promoting components. In food its used in numerous ways such as juices, sweets, jellies and consumed in natura 1 .
Popularly cubiu is used as a medicine and cosmetic, albeit there is a lack of scienti c data con rming the safety and effectiveness of its use for these purposes. Hence, hypoglycemic, hypolipidemic, and antioxidant properties are attributed to the use of cubiu, which is also commonly used to treat diabetes, hypertension and several other pathologies 2 . These properties attributed to the cubiu are likely due to its phenolic-rich composition 3 , also, the cubiu has high iron, niacin, citric acid, and pectin content, favoring health as a whole 1 .
Furthermore, cubiu is popularly used as a treatment for skin diseases, mainly for infections and skin wound healing [4][5][6] , albeit without scienti c data con rming this activety so far. Regarding the skin wound healing, it is known that for a treatment to be effective, its must have antimicrobial and anti-in ammatory activities as its action mechanisms, which improve and accelerate the skin wound healing, keeping the skin intact and without infection 7,8 .
Given the above, this study aimed to determine the phytochemical characterization and pharmacological potential of cubiu, by investigating its skin wound healing, antimicrobial and anti-in ammatory properties, and investigating its toxicity and safety. Methodology

Cubiu extract preparation
The present research is part of a project previously authorized by the Brazil Environmental Ministry to assess the components of genetic patrimony in national territory (n° 010547/2013-4), according to Brazilian legislation (n° 2186-16). Cubiu samples, approximately 20 kg, was commercially acquired in the Municipal Market Adolfo Lisboa -Manaus city, Amazonas, Brazil. A botanic specialist Eduardo Vellez Marin (CRBio 09112-03) con rmed the fruits to be Solanum sessili orum Dunal. The material was registered in the Management of Genetic Patrimony Council, Brazil (CGEN, process number A6723EB).
To obtain the cubiu extract, the fresh fruits (147 ± 38 g) were washed, peeled and the pulp with small seeds was triturated using a mixer (particles ≤ 3mm) for approximately 5 min and placed into sealed amber glass containers with 70% absolute ethanol (Neon, commercial-03467; São Paulo, SP, Brazil), where they remained for seven days with solvent exchanged three times. After extraction, the product obtained was ltered, evaporated, and then lyophilized 9 . The use of fresh fruit followed institutional, national and international guidelines and legislation.

Cubiu extract phytochemical characterization
The phytochemical characterization of the cubiu extract was carried out by detecting the presence of phenolic and carotenoid compounds by high-performance liquid chromatography with a diode array detector (HPLC-DAD) according to Quatrin et al. 10 and Rosso and Mercadante 11 , respectively. The chromatograms for phenolic quanti cation purposes were obtained at 280 nm for hydroxybenzoates and tannins, 320 nm for hydroxycinnamates, 360 nm for avonols, and 450 nm for carotenoids. All compounds were identi ed by comparing with the retention time of authentic standards and the spectral data obtained from UV-visible absorption spectra.
Hydroxybenzoate derivatives were quanti ed as equivalent to gallic acid, hydroxycinnamate derivatives were quanti ed as equivalent to chlorogenic acid, and the results were expressed as mg per 100 g of dry sample weight (mean ± standard deviation).

Antimicrobial activity 2.3.1 Microbial strains and inoculum preparation
The antibacterial activity was evaluated against standard strains of Aeromonas caviae (ATCC 15468), Pseudomonas aeruginosa (PA01), and Sphingomonas paucimobilis (CCT 7809). These strains belong to the Laboratory of Oral Microbiology (LAPEMICRO) bacterial collection of the Federal University of Santa Maria (UFSM), which granted us permission to obtain the strains. The inoculum was prepared after bacterial growth (24 h and 35°C ± 2) on Mueller-Hinton agar. Colonies were suspended in sterile saline solution (0.85%) and adjusted to 0.5 on the McFarland scale (1 to 2 × 10 8 colony-forming units). For the broth microdilution assay, the standardized inoculum was diluted in a 1:10 ratio.

Minimum inhibitory concentration and minimum bactericidal concentration determination
To performed the antimicrobial and antibio lm activity tests, the lyophilized cubiu extract was diluted in Mueller Hinton broth. Cubiu extract activity was evaluated on a dose-response curve at the concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 30 mg/mL. Trials were performed in triplicate on three different days to ensure data reliability, and assays followed the standard protocols for each microbial group according to the Clinical and Laboratory Standards Institute (CLSI) M7-A9 (2015) 12 .
The minimum inhibitory concentration (MIC) was determined by the broth microdilution method using sterile 96-well plates. The plates were prepared and incubating at 37°C and, after the incubation period, the presence/absence of growth was observed. After the MIC reading, 10 µL were removed from the wells with the absence of growth, seeded, and reincubated at 37°C. The absence of growth characterized the minimum bactericidal concentration (MBC); positive and negative growth controls were performed in all tests.

Bio lm formation inhibition and bio lm destruction
According to Antunes et al. 13 , bio lm formation inhibition and bio lm destruction were analyzed by the crystal violet assay, using 96-well microtiter plates. The crystal violet retained in the adhered cells was dissolved with 200 µL of 95% absolute alcohol during 10 min and bacterial growth was quanti ed by measuring the optical density (OD) at 570 nm with a spectrophotometer (Spectra-max M2e Multimode Microplate Reader, Molecular Devices, USA). The results are demonstrated as absorbance values.

Experimental cell assays
In vitro experimental assays were conducted using peripheral blood mononuclear cells (PBMC) for toxicity assays (Cell viability evaluation using MTT assay, Fluorimetric DNA quanti cation assay using PicoGreen reagent, Reactive oxygen species quanti cation − 2'-7'dichloro uorescin diacetate assay, Nitric oxide determination) and erythrocytes for the Hemolytic activity and Coagulation tests. Also, PBMC were used in the evaluation of anti-in ammatory activity with phytohemagglutinin. Human broblast (HFF-1) cells were also used as an experimental model to investigate the potential skin wound healing effects of the cubiu extract.
The cubiu extract was tested at concentrations of 10 and 30 mg/mL since these were the concentrations chosen according to the concentration-effect curve of the previous assays. The extract was diluted in cell culture medium, and all treatments and assays were performed in triplicate. Hydrogen peroxide (H 2 O 2 ) at 100 µM was used as a positive control (PC) group in all tests, while just the cell group in the culture medium was used as a negative control (NC) group.

Cell culture
The PBMCs and erythrocytes derived from discarded total peripheral blood samples from healthy adults, with no identi cation data, were obtained from the Laboratory of Clinical Analysis of the Franciscan University (LEAC-UFN). This experimental protocol was approved by the Ethics Committee on Human Beings of the Franciscan University (CAAE 31211214.4.0000.5306), the ethics committee released this study from the presentation of informed consent, since these are discard samples and do not have identi cation data. All experiments were performed in accordance with the institutional, national and international guidelines and legislation, also the research was conducted in accordance with the 1964 Helsinki Declaration and its later amendments, and in agreement with national international and institutional rules.
Blood samples were initially processed to isolate the PBMCs using a density gradient difference protocol based on Ficcol Histopaque-1077® reagent (Sigma-Aldrich St Louis, MO, USA). After placing the blood in the reagent (1:1 v/v), the samples were centrifuged for 30 min at room temperature. The PBMCs were plated in 96-well plates with RPMI-1640 cell medium (Sigma-Aldrich St Louis, MO, USA) containing 10% fetal bovine serum and supplemented with 1% antibiotics. The cells were cultured at a concentration of 2x10 5 cells/mL per well 14 .
The Human broblasts HFF-1 cell line (ATCC®, CRL-2468TM), were obtained commercially from the Rio de Janeiro Cell Bank. The cells were thawed and maintained in polystyrene bottles in culture medium according to the American Type Culture Collection (ATCC), containing 10% fetal bovine serum (Invitrogen) inactivated at 56°C for 1 h, 100 U/mL penicillin (Invitrogen), and 100 U/mL streptomycin (Invitrogen) at 37°C in a humid atmosphere containing 5% CO 2 . Weekly replicates were performed in laminar ow in order for each bottle to receive 5 mL of medium with a xed amount of cells at the time of the replicate (2x10 5 cells/mL). After obtaining a satisfactory con uence for the experimental trials, the cells were seeded in 96-well plates (2x10 5 cells/mL per well), and the dilutions were made in a culture medium speci c for the cell line applied.  15 . Blood was added with a phosphate-buffered saline (PBS) 1X solution (1:1 v/v) and centrifuged for 15 min at 1000 rpm, and the supernatant was discarded (procedure performed three times). Afterward, 50 µL of washed red blood cells and 10 µL of treatment were added to microtubes containing 1 mL of PBS 1X at different pH (pH 7.2 to simulate sepsis due to metabolic acidosis, pH 7.4 to simulate the body's normal pH, and pH 7.5 to simulate alkalinity). The control groups were as follows: NC (red cells + 0.9% sodium chloride), PC (red cells + distilled water), and surfactants (red cells + surfactant mixture, Tween 60 + Span 60) at the same concentrations of the treatments. The tubes were incubated at room temperature and under rotation for 1 h. Subsequently, they were centrifuged for 15 min at 1000 rpm, followed by transferring 200 µL of the supernatant to a 96-well plate read on an ELISA reader at 540 nm. The results were expressed as a percentage of the negative control.

Coagulation test
The coagulation test was carried out according to Souza Filho et al. 15 . Total blood levels were collected in a citrate tube and centrifuged for 10 min at 2500 rpm. Then, 225 µL of plasma was placed in 96-well plates with 25 µL of treatments and incubated at 37°C for 30 min. Two independent experiments were performed in duplicate with different donors. Readings were carried out on the coagulometer Quick Timer II (Drake) that had been previously calibrated according to the manufacturer's recommendations for tests

Fluorimetric DNA quanti cation assay using PicoGreen reagent
The free DNA in the medium was quanti ed using the PicoGreen reagent (Invitrogen Life Technologies, Carlsbad, USA) and performed according to Sagrillo et al. 19 . The PicoGreen reagent was added to the sample in 96-well ELISA plates after being incubated in an oven at 37°C for 5 min, and uorescence reading was performed on a spectro uorometer (480 nm excitation and 520 nm emission). The results were expressed as a percentage of the negative control.

Reactive oxygen species quanti cation − 2'-7'dichloro uorescin diacetate assay
Reactive oxygen species (ROS) were quanti ed as described by Esposti et al. 20 , and the sample (50 µL), Tris buffer (60 µL), and 2',7'-dichloro uorescein diacetate reagent (10 µL; DCFH-DA) were added to the 96well ELISA plates. The plates were protected from light and incubated in an oven at 37°C for 1 h. Fluorescence reading was carried out on an ELISA reader (488 nm excitation and 525 nm emission). The results were expressed as a percentage of the negative control.

Nitric oxide determination
The nitric oxide (NO) levels were measured according to Choi et al. 21 and Noh et al. 22 . 50 µL of the sample was pipetted in a 96-well plate, followed by adding 50 µL of Griess reagent, and maintained for 15 min at room temperature and protected from light. After, the reading was performed in the spectrophotometer at 540 mn, and the results were expressed as a percentage of the negative control.

Evaluation of anti-in ammatory activity with phytohemagglutinin
The protocol was performed according to Maczynski et al. 23 . The PBMCs (2x10 5 cells/well) were plated in 96-well plates with 50 µL of phytohemagglutinin (PHA) and incubated at 37°C with 5% CO 2 for 48 h.
Then, the cells were washed with PBS (1X; Gibco) and the treatments were carried out and incubated once again for another 24 h. After this period, the plates were centrifuged and the supernatant was removed for MTT (as described in 2.4.3.1) and NO (as described in 2.4.3.4) assays. As a negative control, cells that were untreated or stimulated by PHA were used, and untreated and PHA-stimulated cells were used as a positive control.

In ammatory cytokines
In ammatory cytokine levels were analyzed in the anti-in ammatory activity with PHA (item 2.4.4) and in vitro skin wound healing scratch assay (item 2.4.6). Interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (INF-γ) levels were evaluated using a human ELISA kit (eBioscience, San Diego, USA) according to the manufacturer's instructions. Homocysteine levels were analyzed using an Immulite analyzer (Diagnostic Products Corporation, Los Angeles, USA). The results are expressed in pg/mL.

In vitro skin wound healing -scratch assay
The HFF-1 cells were used for the in vitro skin wound healing assay. A line was drawn in the middle of each well of the plate using a permanent marker to better standardize the scratch and measurements before plating the cells. This marking established a visual eld in the well, which was analyzed after making the scratch in the monolayer. The cells were plated in a 96-well plate and, after con uence/adhesion, the culture medium was removed. With the help of a needle, a continuous scratch was then made on the medial surface of each well, and this procedure led to a rupture of the contact between the cells and their removal from a certain region of the plate, thus forming a mechanical lesion in the cell monolayer. The wells were washed with PBS (Gibco) to remove the debrided cells according to the method of Seeliger et al. 24 , and the treatments were performed. Thereafter, the proliferation of adjacent cells towards the free space in the plate was followed at 48 h with the recording of photographic images.
To analyze the images of the skin wound healing process, was used a script in the Python programming language, according to rossato et al. 25 , that quanti es the number of cells present in the samples, and thus establishes the percentage of skin wound healing. The results are demonstrated as a percentage of the cells.

Statistical analysis
The GraphPad Prism (version 5.0; GraphPad Software, La Jolla, USA) software was used for statistical analyses and to create the gures. Data were expressed as mean ± standard deviation. The homogeneity of variances was veri ed with Levene's test and treatments were compared using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test, and p < 0.05 was considered signi cant.

Cubiu extract characterization
Cubiu extract used in this study had its composition evaluated by HPLC-DAD and the chromatographic pro le is shown in Fig. 1. Due to the polarity characteristics of the solvent (ethanol 70%) used during the extraction process, only phenolic compounds were found in the lyophilized extract. However, traces of lutein were observed, albeit in quantities below the LOQ (data shown in supplementary data S1 online). The total phenolic compound content in the cubiu extract was 456.31 ± 19.78 mg/100 g of lyophilized extract. Phenolic compounds found can be classi ed into two groups of phenolic acids: hydroxybenzoic (11.61 ± 3.50 mg/100 g) and hydroxycinnamic (444.70 ± 16.28 mg/100 g). The major compound identi ed was 5-caffeoylquinic acid, which had a concentration of 392.92 ± 13.25 mg/100 g of extract, corresponding to 84% of the compounds quanti ed.

Minimum inhibitory concentration and minimum bactericidal concentration determination
According to our ndings, the cubiu treatment acted as a bacteriostatic and bactericide against Aeromonas caviae, P. aeruginosa (PA01), and Sphingomonas paucimobilis strains. Assay susceptibility (mg/mL) of cubiu extract against standard strains (MIC and MBC values) are shown in Table 1.

Bio lm inhibition and bio lm destruction
For P. aeruginosa (PA01), which are bio lm-producing bacteria, the analysis of bio lm inhibition and destruction was performed, and it was possible to observe that all tested concentrations of the cubiu extract inhibited bio lm formation and destroyed the bio lm previously formed (Fig. 2). The treatment with cubiu extract, in the crystal violet assay, showed a decrease 36,53% and 35,06% the inhibited of bio lm formation when treated with 15 mg/mL and 30 mg/mL, respectively (Fig. 2a) when compared with positive control. Moreover, the destroyed of bio lm previously formad showed a decrease 49,64% and 42,17% when treated with 15 mg/mL and 30 mg/mL of cubiu extract, respectively (Fig. 2b) when compared as positive control.

Coagulation test
There were no results outside the biological range in the PT (Fig. 3d) and aPTT (Fig. 3e) tests.

Cell viability evaluation by MTT assay
Cubiu extract treatments at doses of 10 and 30 mg/mL improved cell viability compared to the PC group (Fig. 4a).

Fluorimetric DNA quanti cation assay using PicoGreen reagent
The results for detecting double-stranded DNA damage in cell culture are shown in Fig. 4b, in which no DNA damage was observed at any of the tested concentrations.

Reactive oxygen species quanti cation − 2'-7'dichloro uorescin diacetate assay
Treatments with 10 and 30 mg/mL of cubiu extract showed antioxidant activity, with lower total ROS levels than the PC group (Fig. 4c).

Nitric oxide determination
There were no changes in NO levels from the treatment (Fig. 4d).

Evaluation of anti-in ammatory activity with phytohemagglutinin
The anti-in ammatory activity with PHA of the cubiu extract was evaluated, and the results showed that anti-in ammatory activity at 10 and 30 mg/mL led to a sharp drop in IL-1, IL-6, TNF-α, and INF-γ levels compared to the PC, as well as an increase in IL-10 levels compared to the PC (Fig. 5a, 5b, 5c, 5d, 5e respectively). Furthermore, the MTT assay revealed that, through the treatments with 10 and 30 mg/mL, it was possible to resume the basal levels of cell viability, in addition to no alterations in NO levels ( Fig. 5f and 5g respectively).

In vitro wound skin wound healing -scratch assay
The cubiu extract (10 and 30 mg/mL) proved to have skin wound healing properties compared to the NC and PC groups (Fig. 6a). Moreover, the samples in both concentrations acted as anti-in ammatory agents during the skin wound healing process due to the signi cant decrease in pro-in ammatory cytokine levels (IL-1, IL-6, TNF-α and INF-γ) compared to the PC, as well as the increased anti-in ammatory cytokines (IL-10) compared to the PC (Fig. 6b, 6c, 6d, 6e, 6f respectively).

Discussion
This study aimed to investigate the pharmacological potential of cubiu extract, a tropical fruit of Solanum sessili orum and native to the Amazon region. Cubiu is rich in ber, minerals, iron, niacin, citric acid, and pectin 26 , and the presence of phenolic compounds stands out in its composition, including chlorogenic acid and, more speci cally, 5-caffeoylquinic acid as the major compound 3 . In agreement with the above, we also found phenolic compounds in the cubiu extract, with 5-caffeoylquinic acid being the major compound. Hence, it is seen that Solanaceae plants have caffeoylquinic acids in their composition to which are attributed several of their biological activities that bene t human health 27 .
We rst investigated the antimicrobial activity of the cubiu extract. From a curve with concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 30 mg/mL of cubiu extract, we showed that 10 and 30 mg/mL have bacteriostatic and bactericidal activity against Aeromonas caviae, Sphingomonas paucimobilis, P. aeruginosa (PA01), which are bacteria that affect various tissues, e.g., the skin, and cause mild and severe infections in hospitals 28-30 .
Furthermore, knowing that PA01 is a multidrug-resistant bio lm-producing bacterium 31 , we also tested the cubiu extract effectiveness in inhibiting bio lm formation and destroying this bio lm when previously formed by PA01. We evidenced that the cubiu extract was effectively in the inhibition bio lm formation by PA01 and destroy the bio lm previously formed by this bacterium at all concentrations tested (15mg/mL and 30mg/mL). Bio lm is mainly associated with prolonged infections and an important mechanism used by microorganisms to survive antibiotic treatments. Additionally, the bio lm formed by PA01 is closely related to infections in chronic skin wounds 31,32 .
Various conventional antimicrobial agents have become less effective against microorganisms as they have become increasingly resistant, making the search for new alternatives with antimicrobial activity indispensable. Thus, research with medicinal plants is a crucial alternative to treat different infectious diseases, as many of these plants have bioactive components with antimicrobial properties 33,34 . Therefore, plant extracts and their constituents are being used to combat numerous resistance mechanisms of microorganisms 35,36 .
Given this scenario, we can highlight the potential use of the cubiu extract as a bacteriostatic and bactericidal agent to prevent and treat bacterial bio lms, and the antimicrobial potential exerted by this fruit can be justi ed by the phenolic substances in its chemical matrix. Among the bioactive substances, phenolic substances are highly relevant concerning antimicrobial properties 37 . In a similar study, Rodrigues et al. 38 corroborate our ndings by demonstrating that pitanga (Eugenia uni ora L.) extract was effective against bacterial bio lm, attributing this result to the presence of phenolic substances in the fruit extract.
Evidence suggests that the possible mechanism of antimicrobial compounds is related to the cascade of reactions involving the bacterial cell 39,40 . These reactions may involve the antioxidant capacity of phenolic compounds, which may be important in the antimicrobial mode of action since these natural compounds can reduce the production of essential metabolites for microorganism survival under stress conditions. Hence, it is plausible that the cubiu extract attached and incorporate itself to the bio lm structure, impairing signaling pathways and inducing the disruption of cell membrane 41,42 .
Knowing that concentrations of 10 and 30 mg/mL of cubiu extract have promising biological activities, it is vital to verify if these concentrations are safe to use. Therefore, we followed up on the toxicity study of the cubiu extract using PBMC, and our ndings revealed that the cubiu extract did not present toxicity due to maintaining hemolysis and blood coagulation patterns within the biological range. In addition, the cubiu extract did not cause alterations or damage to double-stranded DNA, alter NO levels, and improved cell viability. Moreover, Hernandes et al. 43 reported that cubiu does not present cytotoxic or genotoxic effects, further echoing that its safe to consume.
Furthermore, the cubiu extract at the tested concentrations decrease ROS total levels, thus demonstrating antioxidant action. Kaunda et al. 44 also reported that different species of the genus Solanum exhibit antioxidant properties, and Morais et al. 27 described Solanaceae species as having antioxidant potential mainly due to their chemical matrices, including phenolic substances. These, in turn, are widely known for their antioxidant and chelating properties 45 .
High ROS levels, at the expense of the body's antioxidant capacity, are known to cause oxidative stress, promoting cellular damage in plasma membranes, lipids, and proteins and even culminating in cell death.
Moreover, oxidative stress is closely linked with the in ammatory response since lipid peroxidation causes in ammatory enzymes such as cyclooxygenase and lipoxygenase to be stimulated and induces leukocytes to release pro-in ammatory cytokines 46 . Cytokines are importants in ammatory mediators and can be pro-in ammatory, thus being responsible for manifesting and propagating in ammatory (e.g., IL-1, IL-6, TNF-α and INF-g) or anti-in ammatory (e.g., IL-10) responses and acting as an inhibitor of the in ammatory process 47,48 .
The phenolic substances present in Solanaceae plants also provide anti-in ammatory properties 27 , as demonstrated herein with the cubiu extract. Therefore, by using PBMCs, we aimed to verify the antiin ammatory properties of the cubiu extract against PHA, a natural agent inducer of in ammatory responses 23 . From this experiment, it was possible to evidence that the cubiu extract (10 and 30 mg/mL) has anti-in ammatory activity, restoring cell viability, maintaining NO levels unchanged, decreasing proin ammatory cytokine levels (IL-1, IL-6, TNF-a and INF-g), and increasing anti-in ammatory cytokine levels (IL-10).
Phenolic substances have anti-in ammatory activity given their ability to inhibit enzymes such as prostaglandin synthetase, lipoxygenase, and cyclooxygenase, which are involved in the in ammatory process. Moreover, it is also known that chlorogenic acid, which is present in the composition of cubiu, interferes with the response of leukocytes to chemokines, also preventing the interaction with adhesion molecules involved in cell migration during the in ammatory process 49 .
In addition, the anti-in ammatory activity of cubiu was also evidenced in the scratch assay using HFF-1 cells, as both cubiu extract concentrations decreased pro-in ammatory cytokine levels (IL-1, IL-6, TNF-a, and INF-g) and increased anti-in ammatory cytokine levels (IL-10). Furthermore, the scratch assay demonstrated the skin wound healing potential of the cubiu extract. It is known that phenolic substances present in natural extracts have skin wound healing properties 50,51 . The cubiu is popularly used for skin wound healing 4,5 , albeit without scienti c data so far. The present study is the pioneer to prove this activity, as shown herein, both concentrations (10 and 30 mg/mL) of the cubiu extract improved skin wound healing.
It is widely known that treatments that improve skin wound healing should promote antimicrobial, antiin ammatory, and antioxidant activities since infections, prolonged in ammatory processes, and oxidative stress delay the skin wound healing process 7,8 . As for the cubiu extract, we proved that all of these indispensable properties that enhance skin wound healing are found in it, therefore justifying the signi cant skin wound healing potential of this tropical fruit at the concentrations tested. This is the rst research to prove that cubiu extract is an important compound for use in the skin diseases, promoting skin wound healing, antimicrobial and anti-in ammatory activities.

Conclusions
Considering the main focus of this research, we seek to demonstrated cubiu extract phytochemical composition, increasing knowledge about the plant due to its importance in folk medicine. Additionally, we evaluated cubiu extract toxicity, its pharmacological properties in skin wound healing, antimicrobial activity against strains that affect the skin, anti-in ammatory and antioxidante activities. All data demonstrate that cubiu extract has no toxicity and its an important compound for use in the skin diseases, may be used for the treatment skin wound healing and skin infections, promoting also antiin ammatory and antioxidant activities. approved the manuscript. The authors declare that all datawere generated in-house and that no paper mill was used.  Representative chromatogram of cubiu extract (pink) and 5-Caffeoylquinic acid standard (gray).

Declarations
Chromatograms acquired at 320 nm.  Hemocompatibility assays. The hemolytic activity at pH 7.2, 7.4, and 7.5 are represented as the letters "a," "b," and "c," respectively. The results are expressed as a percentage of the negative control. For the coagulation tests, the prothrombin time test (PT) is reported as the letter "d", and the activated partial tromblopastin time test (aPTT) in the letter "e". For baseline PT values, the interval between 11 and 15 s was used, while for aPTT, the interval between 25 and 35 s was used as reference. Data were expressed as mean ± standard deviation (SD). Values with p<0.05 were considered statistically signi cant.
Values with p<0.05 were considered statistically signi cant. "*" indicates a difference of negative control (NC), and "#" indicates a difference of positive control (PC).

Figure 5
Evaluation of cubiu extract anti-in ammatory activity with phytohemagglutinin. The pro-in ammatory cytokines IL-1, IL-6, TNF-a and INF-g are demonstrated as the letters "a", "b", "c" and "d", respectively. The anti-in ammatory cytokine IL-10 is demonstrated as the letter "e", the results are expressed in pg/ml. "f" corresponds to the cellular viability by MTT assay (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoline bromide). "g" corresponds to the nitric oxide (NO) assay, the data are presented as % of the untreated control group. Data were expressed as mean ± standard deviation (SD). Values with p<0.05 were considered statistically signi cant. "*" indicates a difference of negative control (NC), and "#" indicates a difference of positive control (PC).

Figure 6
In vitro skin wound healing -scratch assay. "a" corresponds to skin wound healing activity, the results are demonstrated in % of cells. The pro-in ammatory cytokines IL-1, IL-6, TNF-and INF-g are demonstrated as the letters "b", "c", "d" and "e", respectively. The anti-in ammatory cytokine IL-10 is demonstrated as the letter "f". The results are expressed in pg/ml. Data were expressed as mean ± standard deviation (SD).
Values with p<0.05 were considered statistically signi cant. "*" indicates a difference of negative control (NC), and "#" indicates a difference of positive control (PC).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Supplementaldata.xlsx