Anti-Inflammatory and Healing Activity of the Hydroalcoholic Fruit Extract of Solanum diploconos (Mart.) Bohs.

BACKGROUND: Solanum diploconos (Mart.) Bohs is a native Brazilian plant belonging to the Solanaceae family, popularly known as "tomatinho do mato" and poorly investigated. Herein, we presented for the first time evidence for the anti-inflammatory and wound healing activities of S. diploconos fruit hydroalcoholic extract. Material and Methods. In vitro fMLP-induced chemotaxis, LPS-induced inflammatory mediator levels (cytokines by ELISA and NO release by Griess reaction), and adhesion molecule expression (CD62L, CD49d, and CD18, by flow-cytometry) were assessed in neutrophils treated with different concentrations of the extract. Inflammation resolution was measured by the efferocytosis assay and the healing activity by in vivo and in vitro assays. The air pouch model of carrageenan-induced inflammation in Swiss mice was used to investigate the in vivo anti-inflammatory effects of the extract. Leukocyte influx (by optical microscopy) and cytokine release were quantified in the pouch exudates. Additionally, the acute and subacute toxic and genotoxic effects of the extract were evaluated.
RESULTS: In vitro, the extract impaired neutrophil chemotaxis and its ability to produce and/or release cytokines (TNFα, IL-1β, and IL-6) and NO upon LPS stimuli (p < 0.01). LPS-treated neutrophils incubated with the extract presented increased CD62L expression (p < 0.01), indicating a reduced activation. An enhanced efferocytosis of apoptotic neutrophils by macrophages was observed and accompanied by higher IL-10 and decreased TNFα secretion (p < 0.01). In vivo, similar results were noted, including reduction of neutrophil migration, protein exudation, and cytokine release (p < 0.01). Also, the extract increased fibroblast proliferation and promoted skin wound healing (p < 0.01). No signs of toxicity or genotoxicity were observed for the extract.
CONCLUSION: S. diploconos fruit extract is anti-inflammatory by modulating neutrophil migration/activation as well macrophage-dependent efferocytosis and inflammatory mediator release. It also indicates its potential use as a healing agent. Finally, the absence of acute toxic and genotoxic effects reinforces its possible use as medicinal product.

1. Introduction

Inflammation is a physiological response to injury characterized by complex processes which are aimed at restoring tissue homeostasis. Its first stage comprises the quick activation and migration of immune cells to the injury site to prevent the invasion of microorganisms and damage by hazardous substances in the absence of tissue integrity [1]. In this context, neutrophils play an important role, arriving at the site of inflammation in a multistep controlled process which encompasses marginalization, slow rolling, adhesion, and transendothelial or abluminal migration [2, 3]. All these processes are dependent on the specific interactions between proteins expressed on endothelial cells and leukocytes, such as integrins and selectins [2, 4]. The CD62L selectin is expressed on the neutrophil membrane and mediates its binding to endothelial cell adhesion molecules (P and E-selectins and integrins); it is also responsible for the neutrophil rolling behaviour [3, 5]. On the other hand, integrins such as CD18 and CD49a, are involved in the neutrophil adhesion to the endothelium, allowing its transmigration to the injury site [6].

Once at the injury site, neutrophils carry out an efficient local response, marked by the production of nitric oxide (NO), reactive oxygen species (ROS), and cytokines; release of cytotoxic granular contents; formation of neutrophil extracellular traps; and phagocytosis. Neutrophil migration can become continuous in some cases, contributing to an uncontrolled inflammation, thus, leading to tissue damage and/or loss of function and chronification of the injury [1].

In this context, neutrophil clearance is an important step of the resolution of inflammation and ultimate tissue repair [7]. This process comprises the limitation of neutrophil migration, downregulation of proinflammatory mediators, induction of neutrophil apoptosis, and its subsequent phagocytosis (efferocytosis) by resident macrophages [7, 8]. Following efferocytosis, the macrophages switch from the inflammatory to the anti-inflammatory phenotype, characterized by the release of anti-inflammatory cytokines and growth factors. In turn, these changes contribute towards angiogenesis, wound healing, tissue repair, and homeostasis [7].

Natural products have been used as sources of biological active substances and even of new drugs [9]. The Solanum genus comprises approximately 1500 species, making this genus the largest and most complex of the Solanaceae family. It is distributed in temperate and tropical regions, and it presents several classes of compounds such as alkaloids, triterpenes, flavonoids, and saponins [10]. Some compounds found in this genus have toxic effects; therefore, toxicological studies are needed to ensure safe usage [11].

S. diploconos (Mart.) Bohs is a poorly investigated Brazilian native plant, popularly known as “tomatinho-do-mato” which belongs to the same genus of important food crops such as tomato (S. lycorpersicum) and potato (S. tuberosum). Its fruits are edible and slightly acidic, consumed either in natura or as a juice [12]. Despite its consumption as food, the biological activities of S. diploconos fruits remain unclear. Phenolic compounds, carotenoids, tocopherols, and ascorbic acid were the main classes of secondary metabolites found in the freeze-dried pulp and peel and in the extract obtained from the whole fruit of S. diploconos. Furthermore, the whole fruit extract presented antioxidant actions when assessed in vitro against ROS and reactive nitrogen intermediates [13].

Data from the literature indicate a great therapeutic potential for the Solanum genus, highlighting the anti-inflammatory activity of extracts [14, 15] and some of the compounds also identified in S. diploconos [16], including wound healing activity [17]. In this context, this study was aimed at investigating the anti-inflammatory effects, wound healing activity, and the acute toxicity profile of the hydroalcoholic extract obtained from S. diploconos fruits using in vivo and in vitro protocols.

2. Material and Methods

2.1. Plant Material and Phytochemical Analysis

S. diploconos (Mart) Bohs fruits were harvested from Itaiópolis, SC, Brazil (latitude 60° 66′ 27″ S and longitude 70° 68′ 53″ W, elevation: 800.33 m; SisGen protocol A478BBA) in April 2014, and voucher specimens were deposited in the Herbarium Barbosa Rodrigues (HBR55276) and the Herbarium Dr. Roberto Miguel Klein (FURB49359). Whole fresh ripe fruits (peel and pulp with seeds) were ground and subjected to extraction by dynamic maceration with 95% ethanol in the proportion 1 : 10 (m/v), for 6 h at room temperature (24-28°C). The extract was concentrated and dried under reduced pressure on a rotary evaporator with temperature below 50°C.

The chromatographic analysis was carried out on a HPLC Shimadzu® LC 20-AT system, consisting of a quaternary pump and a Shimadzu SPD-M20A photodiode array detector. A SIL-20A HT autosampler with a Shimadzu CTO-10AS VT column oven equilibrated at 35°C and software LC Solution were used. The chromatographic column used was Phenomenex® C18 (250 × 4.6 mm) with particles of 5 μm. For the method development, different solvent systems were assayed in gradient conditions using acetonitrile (A) and ultrapure water acidified with acetic acid (pH 2.5; B). The chosen gradient was (A : B): 05 : 95 at 0-10 min, 15 : 85 at 10-20 min, 30 : 70 at 20-30 min, 40 : 60 at 30-40 min, 55 : 45 at 40-45 min, and 05 : 95 at 4550 min. The flow rate was 1.0 mL/min. The detection of the compounds was carried out at 320 nm. This method was chosen for the analysis of phenolic compounds as previously established by Ribeiro et al. [13].

2.2. Animals

Experiments were performed in male Swiss mice and female Wistar rats (exclusively for the toxicological evaluation). All animals were obtained from the Central Animal Facility of the Universidade do Vale do Itajaí (UNIVALI) and kept in a climate-controlled room at 22 ± 2°C, under light/dark (12 : 12 h) cycle, with water and food ad libitum. All experiments were approved by the local ethics committee (CEUA/UNIVALI: 028/16 and 019/18).

2.3. In Vitro Anti-Inflammatory Effects

2.3.1. Neutrophil Isolation

For this, male Swiss mice received an intraperitoneal (i.p.) injection of 1% sterile oyster glycogen in phosphate-buffered saline (PBS; 3 mL). After 4 h, the animals were culled with an overdose of ketamine (80 mg/kg, i.p.) and xylazine (8 mg/kg, i.p.), and their peritoneal cells were collected by washing the peritoneal cavity with 3 mL of sterile PBS. The number of viable cells (98%) was counted in a Neubauer chamber using a light microscope (Nikon, Japan) by the trypan blue exclusion test.

2.3.2. Lipopolysaccharide-Stimulated Neutrophils

Neutrophils (1 × 106 cells per well) were incubated with RPMI medium (10% foetal bovine serum (FBS)) in the presence or absence of lipopolysaccharide (LPS; 5 μg/mL) and simultaneously treated with S. diploconos extract (1, 10, or 100 μg/mL) for 18 h, at 37°C in a 5% CO2 atmosphere. Then, the supernatant was collected for the subsequent analysis of cytokine and nitrite (NO2−) levels. Neutrophil viability was assessed by the trypan blue exclusion test. None of the tested extract concentrations produced cell toxicity (data not shown).

2.3.3. Expression of Adhesion Molecules on Neutrophils

Neutrophils (1 × 106 cells per well) stimulated or not with LPS (5 μg/mL) were coincubated with either vehicle (cell culture medium) or the extract (1, 10, or 100 μg/mL). After 1 h, the cells were collected and incubated with monoclonal anti-CD62L (FITC), anti-CD18 (PE), or anti-CD49d (APC) antibodies in the dark for 20 minutes, at 4°C. Cells were then analysed on an Accuri C6 Flow Cytometer (BD Bioscience), and data from 10,000 events was obtained. The results are expressed as mean fluorescence intensity (MFI) [18].

2.3.4. In Vitro Neutrophil Chemotaxis

Neutrophil chemotaxis was evaluated as described by Nelson et al. [19]. Briefly, neutrophils (1 × 107 cells/mL) treated or not with S. diploconos extract (1, 10, or 100 μg/mL, for 1 h) were placed into peripheral wells (10 μL) made in an agarose gel solidified in a Petri dish. The chemotactic factor (N-Formyl-Met-Leu-Phe (fMLP); 0.1 μM, 10 μL) was added to the central well, and the plates were incubated for 4 h, at 37°C and 5% CO2. After incubation, the number of cells that migrated towards the central well containing fMLP was counted.

2.3.5. Efferocytosis Assay

Macrophages were obtained by washing the mouse medullary cavity with sterile PBS. The cell suspension (1 × 106 cells/well) was incubated in a 24-well plate onto a round glass coverslip at 37°C and 5% CO2 for up to 2 h to allow macrophage adherence. After this period, the wells were washed three times with sterile PBS to remove nonadherent cells and then, the remaining ones were incubated with S. diploconos extract diluted in DMEM (1, 10, or 100 μg/mL). After 1 h, senescent mouse neutrophils (1 × 106 cells) were added to each well and 20 min later, the supernatant was collected for cytokine analysis. The remaining cells on the slides were analysed by microscopy with immersion objective (100x). One hundred macrophages were observed, and the phagocytosis of senescent neutrophils was expressed as the percentage (%) of macrophages containing neutrophils [20].

2.3.6. Cytokine and Nitrite Analysis

The levels of IL-1β, TNFα, IL-6, and IL-10 were analysed in the supernatants obtained from the cell cultures, including those from the efferocytosis assay, or in exudate samples from the air pouch model. One hundred μL of each sample was used for the assays performed in accordance to the manufacturer's instructions (DuoSet R&D Systems, Minneapolis, MN, USA). NO levels were indirectly quantified by the detection of nitrite (NO2−) using the Griess reaction [21]. For this, samples (100 μL/well) were incubated for 10 min at 37°C, with 100 μL of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride in 5% phosphoric acid). The absorbances were then determined at 550 nm. All cytokine results are expressed in picograms per milliliter, and NO levels as micromolar of NO2−.

2.4. In Vivo Anti-Inflammatory Effects

2.4.1. In Vivo Leukocyte Migration: Air Pouch Model

Air pouches were produced on the back of the mice as previously described [22, 23]. Briefly, the animals were fasted for 4 h and then orally treated by gavage with S. diploconos extract (100 mg/kg, n = 6), indomethacin (30 mg/kg, positive control, n = 6), or vehicle (PBS; 10 mL/kg; n = 6). After 1 h, carrageenan (1%; 3 mL/cavity) was injected directly into the air pouch chamber. Four hours later, a small incision was made in the air pouch. The cavity was washed with 5 mL of PBS, and the inflammatory infiltrate collected for analysis. Total and differential cell counts were performed, and the rest of the exudate was kept for the further quantification of cytokines.

The air pouch lining tissue was collected for histological analysis and preserved in formaldehyde solution until the blades were made. The preparation consisted of the insertion of the tissue fragment into a cassette. The sample then underwent a 1 h dehydration process in increasing concentrations of ethanol (70%, 96%, and absolute ethanol). Subsequently, each sample was placed in xylol followed by paraffin bath. Then, the cassettes containing samples were placed in a microtome where serial sections of 3 or 4 μm were made. The sections were placed onto slides and stained with haematoxylin and eosin. Samples were analysed by microscopy under the 4, 10, and 40x objectives.

2.5. Wound Healing Assessment

2.5.1. In Vitro Scratch Assay

Murine L929 cells were obtained from the Bank of Cells of Rio de Janeiro (Rio de Janeiro, RJ, Brazil) and maintained in DMEM supplemented with 10% FBS, 100 μg/L streptomycin, and 100 IU/mL penicillin, at 37°C in a 5% CO2 atmosphere. Briefly, L929 cells (5 × 104 cells/mL) were seeded into each well of a 24-well plate and incubated at 37°C with 5% CO2. After cell confluence, the culture medium was removed and a continuous scratch was made in the medial surface of each well with a 200 μL tip. Then, the wells were washed with PBS to remove cell debris. The remaining cells were incubated with DMEM containing S. diploconos fruit extract (1, 10, or 100 μg/mL). The analysis was performed by measuring the scratched area in square millimeter at 0 (immediately after mechanical trauma) and 24 h after incubation with the extract, by microscopy (Olympus CKX 41), using ImageJ1.46r software. The results are expressed as the percent of the coverage area at 24 h in relation to time 0 [24].

2.5.2. In Vivo Wound Healing Assay

For topical application, a semisolid formulation of the S. diploconos extract was made. An oil in water (O/W) emulsion-based cream was prepared by mixing Sepigel 305, vaseline, crodalan, isopropyl myristate, BHT, cetostearyl alcohol, Phenonip®, EDTA, and water with slight warming. The oil-soluble components were dissolved in the oil phase and heated to 75°C. The water-soluble components were dissolved in the aqueous phase and heated to 75°C. After heating, the aqueous phase was added in portions to the oil phase with continuous stirring until cooling took place. Then, the hydroalcoholic extract obtained from S. diploconos fruits (1%) was incorporated to the base cream. The formulation was packed in aluminium tubes, with a seal and polypropylene cover.

Mice were divided in two groups: group I (base cream, n = 6) and group II (1% S. diploconos, n = 6). The animals were anaesthetized, and their backs were shaved with an electric clipper. Then, a surgical excision of the skin (1.5 cm of diameter) was made on the dorsal area of each mouse, as previously described by Tonin et al. [25]. The extract was topically applied to each wound (once a day, for 7 days). The wounds were photographed daily with a scale, and the wounded areas were measured and calculated in square millimeter by using the ImageJ1.46r software.

2.6. Toxicity Evaluation

2.6.1. Cytotoxicity Assays

Possible toxic effects of the extract were investigated in L929 cells. For this, the cells were cultured in DMEM (containing 10% FBS, 100 μg/L streptomycin, and 100 IU/mL penicillin) at 37°C in a 5% CO2 atmosphere and incubated with either S. diploconos extract (0.1, 1, 10, or 100 μg/mL) or vehicle (RPMI medium). After 24 h, the cell viability was evaluated by the MTT method [26]. The results are expressed as percent of viable cells in relation to the control (vehicle-treated wells).

2.6.2. Pathophysiological Analysis

In the acute toxicity assay, rats (n = 5/group) received either S. diploconos extract (2 g/kg) or vehicle (distilled water, 10 mL/kg) by oral route (gavage). The animals were then observed at every 30 min during the first 4 h followed by a daily observation for 14 days. The cumulative weight change was calculated as percent to baseline. At the end of the observation period, all survivors were autopsied. The blood was collected for biochemical (aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea, and creatinine) and hematological (total and differential cell, erythrogram, and platelet counts) analysis [27].

In the repeated dose 28-day oral toxicity (subacute) study, female Wistar rats were divided into different groups (n = 5/group) and orally treated by gavage with S. diploconos extract (30, 100, or 300 mg/kg) or vehicle (distilled water, 10 mL/kg). The extract and vehicle were administered once a day, for 28 days. At the end of the observation period, the macroscopic characteristics of the lungs, kidneys, liver, spleen, and heart were observed. In addition, the blood was collected for analysis of biochemical and hematological parameters.

2.6.3. Micronucleus Test

The micronucleus test was performed 24 h after S. diploconos extract (2 g/kg, by gavage) administration to the mice. Vehicle- (10 mL/kg) and methyl methanesulfonate- (MMS; 50 mg/kg, i.p.) treated animals were used as negative and positive controls, respectively. After treatments, the mouse right femurs were collected, and the epiphyses cut. The medullary cavity of each femur was washed using a syringe containing FBS (3 mL). The lavage fluid was centrifuged at 1000 × g, for 10 min at 5°C; the cells were obtained; and their viability was assessed by using 0.4% trypan blue dye. Then, 50 μL of the cell suspensions was placed onto glass slides, fixed with cold methanol, and stained with Giemsa for 10 min. The slides were washed and dried for examination under a light microscope (1000x). One thousand polychromatic cells were assessed per slide. The ratio between monochromatic and polychromatic erythrocytes was established and analysed [28].

2.7. Statistics

Data is expressed as mean ± standard error of the mean (SEM). The percentages of inhibition were individually calculated and expressed as mean ± SEM%. Statistical differences between groups were assessed by Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's post hoc test, as appropriate. Values of p < 0.05 were considered as significant.

3. Results

3.1. Chemical Profile of Solanum diploconos Fruit Extract

The yield of the whole fresh ripe fruit hydroalcoholic extract was 11.49%. Its chemical composition (main classes of metabolites) was analysed by HPLC with a photodiode detector. The HPLC/UV profiles of the extract are shown in Figure 1. Six distinct absorption profiles were detected. The first profile demonstrates a band with a maximum absorption at 210 nm (peaks 2-4 and 6-10) which indicates compounds with chromophore groups that present π − π∗ electronic transition such as those with olefinic groups. Many classes of compounds are absorbed in this region; making it difficult to determine the major one. Other five profiles indicate bands with a maximum absorption between 270 and 330 nm (other peaks). This is a region in which n − π∗ transitions are observed. Of note, carbonyl compounds as well as aromatic groups present this transition (Figure 1).

Figure 1

Chromatogram of the S. diploconos fruit extract at λ = 320 nm (a) and respective UV absorption spectra (b).

3.2. S. diploconos Fruit Extract Affects the Secretion of Proinflammatory Cytokines and Nitric Oxide by Neutrophils

In order to evaluate the direct effects of the fruit extract on inflammatory mediator production (TNFα, IL-6, IL-1β, and NO2− levels), the supernatants from the neutrophil culture were assessed. Data depicted in Figures 2(a)–2(d) demonstrate that LPS induces the production of inflammatory mediators by neutrophils. Treatment with S. diploconos extract significantly reduced NO2− (maximum inhibition of 63.5 ± 7.3%), TNFα (maximum inhibition of 54.2 ± 2.1%), IL-1β (maximum inhibition of 68.9 ± 3.1%), and IL-6 (maximum inhibition of 73.6 ± 0.9%) levels in comparison with LPS-treated neutrophils (Figures 2(a)–2(d)).

Figure 2

Effects of the S. diploconos fruit extract on neutrophils. Neutrophils obtained from the peritoneal cavity of Swiss mice (1% oyster glycogen) were incubated in the presence or absence of LPS (5 μg/mL) and with S. diploconos (1, 10, or 100 μg/mL). Nitrite levels were measured by the Griess reaction (a). The levels of (b) TNFα, (c) IL-1β, and (d) IL-6 were determined by ELISA. For the chemotaxis assay (e), a neutrophil suspension (1 × 107) was incubated with different concentrations of S. diploconos (1, 10, or 100 μg/mL) for 15 minutes and placed in front of the fMLP (1 μM) on an agarose plate. Quantification was made from the margin of the peripheral perforations towards the chemotactic agent (central perforation). Neutrophils (1 × 106) were incubated in the presence or absence of LPS (5 μg/mL) along with S. diploconos (1, 10, or 100 μg/mL) for evaluation of the neutrophil adhesion molecules. Adhesion molecule expression was assessed by labeling with (f) CD62L, (g) CD18, and (h) CD49 detection antibodies by flow cytometry. Values express the mean ± SEM of tests performed with cells obtained from 8 animals per group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001 vs. the LPS group significantly different from the basal group ####p < 0.0001 (one-way ANOVA followed by Turkey's post hoc test).

3.3. S. diploconos Fruit Extract Impairs Neutrophil Migration

Figure 2 also shows the effect of S. diploconos extract on neutrophil migration. Vehicle-treated neutrophils presented a significant migration towards fMLP, whilst those treated with S. diploconos exhibited impaired chemotaxis (maximum inhibition of 78.1 ± 1.2%; Figure 2(e)).

Once leukocyte migration is mediated by adhesion molecules, the expression of L-selectin (CD62L), β2-integrin (CD18), and CD49d on neutrophils was evaluated by flow cytometry. LPS triggered the activation of neutrophils as they presented shedding of CD62L and increased expression of CD18 and CD49b on their surface. Incubation of LPS-treated neutrophils with S. diploconos extract impaired neutrophil activation, denoted by increased expression of CD62L (1.7-fold increase for the concentrations of 10 and 100 μg/mL), i.e., increased MFI in comparison with LPS controls (Figures 2(f) and 2(g)).

3.4. S. diploconos Fruit Extract Induces Efferocytosis of Apoptotic Neutrophils

All tested concentrations of the S. diploconos fruit extract (1, 10, or 100 μg/mL) were able to significantly increase the efferocytosis process (maximum increase of 2.0-fold; Figure 3(a)). The extract was also able to reduce TNFα (maximum inhibition of 80.7 ± 1.2%; Figure 3(b)) and increase IL-10 (maximum increase of 11.5-fold; Figure 3(c)) levels in the supernatant samples obtained from the efferocytosis assay.

Figure 3

Effects of S. diploconos on efferocytosis. Quantification of efferocytosis (a) and supernatant levels of TNFα (b) and IL-10 (c). Data is expressed as a mean ± SEM of cells obtained from four animals. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 vs. basal.

3.5. S. diploconos Fruit Extract Presents In Vivo Anti-Inflammatory Activity

The in vivo effects of S. diploconos fruit extract on neutrophil migration were evaluated in the air pouch model, using carrageenan as a phlogistic agent. The histological analysis (Figure 4) of air pouch tissue sections from naive animals indicated that they present an air pouch wall with connective tissue, rich in blood vessels, and with relatively low cellular content (Figures 4(a) and 4(b)). Their cell lining was primarily formed by fibroblasts, and, in some areas, scarce macrophages were observed within the air pouch walls (Figures 4(b) and 4(c)).

Figure 4

S. diploconos actions in mouse air pouches challenged with carrageenan. Air pouch was induced in the dorsal subcutaneous tissue of Swiss mice. The animals were treated orally one hour before the injection of 2 mL of carrageenan (1%). Representative H.E. histological sections of skin biopsies obtained from mice with air pouches (40, 100, and 400x). Black arrows indicate PMN leukocytes, dashed black arrows indicate macrophages, and arrowheads indicate fibroblasts (a-l). Air space pouch “a”; pouch membrane “pm.” The lavage of the inflammatory infiltrate was collected 4 hours after the injection of carrageenan into the air pouch. The determination of the (m) total number of exudate cells was performed on a Neubauer chamber, and the (n) differential value was performed. (o) The protein concentration was measured in a spectrophotometer at 590 nm using the Bradford reagent. The levels of (P) TNFα, (q) IL-1β, and (r) IL-6 were determined by the ELISA method. Values express the mean ± SEM of tests performed with the inflammatory exudate obtained from 6 animals per group. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001, vs. the control group significantly different from the naïve group ####p < 0.0001 (one-way ANOVA followed by Tukey's post hoc test).

Vehicle-treated animals challenged with carrageenan exhibited typical morphological changes of inflammation including increased thickness of the air pouch membrane and intense oedema, with fibrin-haemorrhagic exudate, leukocyte accumulation, and tissue injury (Figures 4(d) and 4(e)). Most of the inflammatory cells were neutrophils, as identified by their lobulated nuclei. These cells were extensively infiltrated in the damaged tissue and distributed within the adjacent muscular fibres. Macrophages with a foamy appearance were also observed, possibly due to the phagocytosis of carrageenan (Figures 4(e) and 4(f)).

Indomethacin treatment reduced the thickness of carrageenan-challenged air pouch membranes in comparison with vehicle-injected mice (Figures 4(g) and 4(h)). Oedema was restricted to limited areas, and there was also an evident reduction of the neutrophil infiltrate. Few normal and foamy macrophages were seen in the pouch membrane, along with fibroblasts and neutrophils (Figures 4(h) and 4(i)).

In the S. diploconos-treated group, the thickness of the air pouches challenged with carrageenan was visibly reduced, and the walls were formed by loose connective tissue with newly formed blood vessels. No signs of oedema were observed (Figures 4(j) and 4(k)). Neutrophil and macrophage numbers were largely diminished in comparison with the negative control group, and a lining of fibroblast-like cells with no directional orientation was observed. Additionally, under the hair follicles, there was a layer of fibroblasts on an augmented deposit of collagen (Figures 4(k) and 4(l)). These histological findings suggest that the acute inflammatory response caused by carrageenan in the pouch wall is significantly suppressed by the treatment with S. diploconos fruit extract.

The data presented in Figure 4(m) demonstrate that similarly to indomethacin, the fruit extract at 100 mg/kg was able to reduce cell counts in the exudate in comparison with the control group. Inhibitions were of 73.5 ± 2.9 and 95.4 ± 0.8% for S. diploconos and indomethacin, respectively. Differential cell analysis indicates that both S. diploconos fruit extract and indomethacin affect mainly neutrophils (Figure 4(n)).

The fruit extract also reduced by nearly half the exudate protein concentration (Figure 4(o)). Additionally, carrageenan-challenged mice treated with S. diploconos exhibited lower levels of TNFα (Figure 4(p), 76.0 ± 2.7%), IL-1β (Figure 4(q), 63.3 ± 2.6%), and IL-6 (Figure 4(r), 25.3 ± 3.1%) in comparison with vehicle controls. Indomethacin-treated animals presented similar results; however, this compound did not affect IL-6 production (Figure 4(r)). Indomethacin diminished TNFα and IL-1β concentrations by 50.4 ± 6.2% and 63.8 ± 3.3%, respectively.

3.6. S. diploconos Fruit Extract Presents Wound Healing Activity

Data obtained from the in vitro scratch model using L929 cells show that S. diploconos fruit extract is able to promote an increase of fibroblast migration at all tested concentrations (maximum increase of 2.6-fold) in comparison with the control group (Figures 5(a) and 5(b)). Additionally, when assessed in vivo as a semisolid cream, S. diploconos extract (1%) promoted a faster healing of skin wounds in comparison with the control group (base cream). This is denoted by a significant reduction (~35%) of the wounded area as demonstrated in Figures 5(c) and 5(d).

Figure 5

Analysis of the effects of the S. diploconos fruit extract in fibroblast migration and wound healing. (a) Representative image of the in vitro scratch migration assay in fibroblasts (L929) and (b) percentage of fibroblast migration. Skin ulcers were treated individually with S. diploconos 1% cream or vehicle (base cream). Progression of wound healing area over time after extract treatment (c) and area under the curve (AUC) (d). Values express the mean ± SEM from 6 animals per group. Statistical analysis was performed using one-way ANOVA followed by Tukey's test (cell migration assay) with ∗∗p < 0.01 vs. basal and t-test (AUC assessment) ∗∗∗∗p < 0.0001 vs. base cream.

3.7. S. diploconos Fruit Extract Does Not Present Acute or Subacute Toxicity

The fruit extract did not present genotoxic effects since no significant differences in the ratio of polychromatic and monochromatic erythrocytes were observed. On the other hand, MMS induced a significant increase in the ratio of polychromatic to monochromatic erythrocytes (36.0-fold increase, Table 1). Additionally, the extract did not promote cell cytotoxicity in the MTT assay.

Table 1

Percentage of polychromatic erythrocyte (PCE) micronuclei (PCEMN) in animals treated with S. diploconos fruit extract. Percentage of polychromatic erythrocyte (PCE) micronuclei (PCEMN) in the micronucleus assay performed in erythrocytes obtained from the bone marrow (the values are expressed as mean ± SEM (n = 5). ∗∗∗p < 0.001 vs. the vehicle group).

Group	Dose (mg/kg)	PCEMN/2000 PCE (%)
Vehicle	—	0.33 ± 0.33
MMS	50	11.90 ± 1.09∗∗∗
S. diploconos	2000	1.75 ± 0.52

In the acute toxicity test, animals received a single administration of S. diploconos fruit extract at the dose of 2 g/kg. The extract did not induce animal death or alterations in the following parameters: vocalization, behaviour, grip, body tonus, and twisting. Ataxia, tremors, convulsions, tail Straub sign, hypnosis, anaesthesia, ptosis, lacrimation, piloerection, alterations of the respiratory rate, and cyanosis were not observed. The characteristics of the skin, fur, and eyes remained normal and unchanged during the observation period (12 h). Accordingly, no alterations were seen in the food/water intake or body weight of S. diploconos-treated animals in comparison with vehicle-treated rats.

Similarly, S. diploconos administration did not cause any alterations to internal organs (Table 2). Additionally, the histological analysis of the liver and kidney demonstrated normal and conserved architecture, without degenerative or inflammatory lesions in S. diploconos and vehicle groups.

Table 2

Effects of Solanum diploconos extract treatment on the organ weights of mice following the acute oral toxicity experiments (values expressed as mean ± SEM (n = 5)).

Organ		Acute toxicity		Subacute toxicity
		Vehicle	2000 (mg/kg)	Vehicle	30 (mg/kg)	100 (mg/kg)	300 (mg/kg)
Heart	Absolute (g)	0.853 ± 0.071	0.763 ± 0.053	0.676 ± 0.027	0.625 ± 0.012	0.623 ± 0.031	0.679 ± 0.039
	Relative (%)	0.326 ± 0.013	0.307 ± 0.017	0.351 ± 0.008	0.319 ± 0.008	0.335 ± 0.015	0.352 ± 0.020
Lung	Absolute (g)	1.191 ± 0.097	1.098 ± 0.124	0.975 ± 0.023	0.936 ± 0.032	0.941 ± 0.020	0.962 ± 0.024
	Relative (%)	0.455 ± 0.029	0.442 ± 0.041	0.506 ± 0.007	0.477 ± 0.016	0.506 ± 0.012	0.499 ± 0.017
Liver	Absolute (g)	9.182 ± 0.522	8.123 ± 0.840	5.300 ± 0.248	5.364 ± 0.141	5.071 ± 0.155	5.202 ± 0.122
	Relative (%)	3.515 ± 0.256	3.273 ± 0.316	2.751 ± 0.098	2.734 ± 0.058	2.725 ± 0.741	2,699 ± 0.070
Spleen	Absolute (g)	0.635 ± 0.047	0.593 ± 0.056	0.542 ± 0.038	0.554 ± 0.033	0.480 ± 0.008	0.496 ± 0.022
	Relative (%)	0.242 ± 0.012	0.238 ± 0.014	0.280 ± 0.015	0.282 ± 0.017	0.258 ± 0.003	0.257 ± 0.012
Left kidney	Absolute (g)	0.998 ± 0.076	0.914 ± 0.057	0.765 ± 0.034	0.862 ± 0.038	0.737 ± 0.017	0.779 ± 0.024
	Relative (%)	0.381 ± 0.024	0.368 ± 0.013	0.397 ± 0.012	0.439 ± 0.017	0.396 ± 0.008	0.404 ± 0.014
Right kidney	Absolute (g)	1.021 ± 0.071	0.910 ± 0.100	0.802 ± 0.034	0.836 ± 0.043	0.753 ± 0.012	0.756 ± 0.029
	Relative (%)	0.391 ± 0.038	0.366 ± 0.030	0.416 ± 0.013	0.426 ± 0.019	0.405 ± 0.007	0.392 ± 0.015

No significant hematological parameter changes were found. In the platelet count, the high SEM presented for the extract-treated group refers to just one animal. Biochemical analysis indicated that the extract promotes a decrease of 39.8% in glucose and an increase of 1.3-fold in urea levels when compared with the vehicle group (Table 3).

Table 3

Biochemical markers and hematological parameters following the acute administration of the S. diploconos fruit extract (values expressed as mean ± SEM (n = 5)). ∗p < 0.05 vs. the vehicle group.

Parameters	Acute toxicity		Subacute toxicity
	Vehicle	2000 (mg/kg)	Vehicle	30 (mg/kg)	100 (mg/kg)	300 (mg/kg)
Biochemical parameters
Glucose (mg/dL)	261.60 ± 16.93∗	157.30 ± 16.23	131.6 ± 6.30	148.20 ± 11.10	125.20 ± 10.54	125.80 ± 3.48
AST (U/L)	91.60 ± 5.97	128.81 ± 14.45	108.21 ± 10.79	100.69 ± 7.02	104.23 ± 6.16	89.22 ± 7.97
ALT (U/L)	56.40 ± 5.14	38.61 ± 9.69	39.89 ± 4.39	37.86 ± 2.11	37.36 ± 1.89	33.07 ± 2.09
ALP (U/L)	61.20 ± 7.94	73.80 ± 15.65	53.12 ± 4.30	60.59 ± 6.01	56.68 ± 2.06	56.30 ± 6.25
Urea (mg/dL)	41.60 ± 1.21	53.50 ± 3.03∗	35.00 ± 1.70	32.60 ± 1.50	36.80 ± 2.78	36.80 ± 1.39
Creatinine (mg/dL)	0.53 ± 0.02	0.39 ± 0.053	0.30 ± 0.01	0.28 ± 0.01	0.25 ± 0.01	0.29 ± 0.02
Hematological parameters
Leukocyte (mil/mm3)	1.120 ± 0.149	0.800 ± 0.152	3.425 ± 0.118	2.900 ± 0.207	4.020 ± 0.497	2.520 ± 0.323
Neutrophil (%)	10.600 ± 1.805	13.333 ± 2.333	2.750 ± 0.250	2.400 ± 0.245	2.400 ± 0.245	3.600 ± 0.678
Lymphocyte (%)	87.400 ± 2.014	84.667 ± 2.403	95.500 ± 0.288	95.800 ± 0.374	96.200 ± 0.374	94.400 ± 1.886
Monocytes (%)	2.000 ± 0.707	1.333 ± 0.333	1.750 ± 0.250	1.800 ± 0.374	1.400 ± 0.245	2.000 ± 1.265
Eosinophil (%)	0.000 ± 0.000	0.666 ± 0.333	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000
Basophil (%)	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000
Erythrocyte (millions/mm3)	5.562 ± 0.067	4.960 ± 0.496	7.050 ± 0.131	7.000 ± 0.092	7.146 ± 0.129	6.698 ± 0.113
Hemoglobin (g/dL)	11.540 ± 0.153	10.266 ± 1.185	14.050 ± 0.342	13.940 ± 0.150	13.960 ± 0.265	13.220 ± 0.587
Hematocrit (%)	31.100 ± 0.389	27.533 ± 2.936	40.600 ± 0.849	40.220 ± 0.414	40.560 ± 0.867	38.120 ± 0.587
Platelets (103/mm3)	408.400 ± 34.993	293.000 ± 142.815	702.250 ± 46.424	657.400 ± 44.800	651.800 ± 39.328	715.600 ± 69.974

The data obtained in the subacute toxicity test demonstrate that S. diploconos extract does not induce any alteration in the biochemical and hematological parameters at the highest evaluated dose (300 mg/kg). In this case, a no observed adverse effect level (NOAEL) was established at 300 mg/kg.

4. Discussion

Neutrophils are cells with multiple functions which modulate inflammation from its initial phase to tissue repair. Neutrophils interact with other immune cells, directly or via inflammatory mediator production, affecting the course of both innate and adaptive immune responses [29]. We showed for the first time that S. diploconos fruit extract is able to modulate neutrophil functions by reducing chemotaxis, cytokine, and NO release and also promotes resolution of inflammation and wound healing. Additionally, the assays performed indicate that the extract may directly affect intracellular neutrophil pathways.

Several inflammatory mediators are synthesized and secreted during an inflammatory response with neutrophils responding directly to these signals and producing different inflammatory mediators such as cytokines that, in turn, modulate inflammation and drive the immune response [29, 30]. In this context, it is well known that cytokines such as TNFα and IL-6 are increased in inflammation and may contribute to excessive neutrophil accumulation and subsequent tissue damage [31]. Previous studies demonstrated that some Solanum species are able to reduce inflammatory mediator secretion by immune cells; however, their effects on neutrophils were unknown [18, 32]. In this context, our data clearly show the direct inhibitory actions of S. diploconos fruit extract on LPS-induced neutrophil-mediated secretion of cytokines and NO.

Herein, the obtained phytochemical profile was compared to that presented by Ribeiro et al. [13]. The authors identified derivatives of caffeic, ferulic and coumaric acids in the freeze-dried pulp and peel of S. diploconos fruits, as well as in an extract obtained from its whole fruit using HPLC-DAD-ESI-MS/MS [13]. Some phenolic compounds detected by the authors presented a similar UV absorption profile also observed in this study for the peaks 5, 11-13, and 18-24.

The anti-inflammatory effect observed for S. diploconos may be due to the content of caffeic acid, lutein, and β-carotene present in the extract [13], which were previously shown to reduce the translocation of the transcription factor NF-ĸB [16, 33, 34] necessary for the transcription of inflammatory mediators such as cytokines [35].

We also found that S. diploconos fruit extract markedly reduces fMLP-induced neutrophil chemotaxis in vitro. Interestingly, S. diploconos extract increased the expression of CD62L on LPS-stimulated neutrophils without altering CD18 and CD49d profiles. It is important to highlight that CD62L mediates neutrophil rolling [36], whilst CD49d and CD18 mediate adhesion to the endothelium [6, 37]. CD62L becomes highly expressed on the cell membrane of leukocytes when haemodynamic changes or production of inflammatory mediators occurs [3]. After the activation process, however, CD62L is cleaved by the action of membrane metalloproteinases, allowing the activation of CD18 which is responsible for the strong adhesion of inflammatory cells to endothelial cells [6, 36]. It is possible, thus, that the extract prevents L-selectin from being cleaved, allowing leukocytes to remain in rolling behaviour instead of adhering to the endothelium and migrating to the inflammatory focus.

S. diploconos inhibitory effects on neutrophil migration were confirmed in vivo. Our data demonstrate that the oral treatment with S. diploconos decreases the production of inflammatory mediators (TNFα, IL-6, and IL-1β) which may explain the reduced leukocyte recruitment and protein levels in the air pouch exudates obtained from mice challenged with carrageenan. A similar effect on neutrophils was also observed in the skin tissue.

Neutrophils can indirectly contribute to tissue restoration by modulating the functions of other cells. In this context, macrophages are moved to a state of tissue remodelling by performing efferocytosis (phagocytosis of apoptotic neutrophils). Indeed, when neutrophils become apoptotic, they trigger a phagocytic activity in macrophages and stimulate their differentiation into M2 macrophages which are involved in tissue repair [38, 39]. The increased production of TGF-β and IL-10 is suggested to underlie these responses [7, 38, 40]. Herein, macrophages treated with S. diploconos presented increased efferocytosis, paralleled to decreased TNFα and enhanced IL-10 production.

These data indicate that S. diploconos may favour the resolution of inflammation. In order to confirm this evidence, we assessed the effects of the extract in a scratch assay with L929 cells and in vivo, in animals with skin wounds. S. diploconos induced in vitro fibroblast proliferation and accelerated the healing of skin wounds in mice. Similar proliferative and healing actions were previously observed for other Solanum species, such as S. incanum and S. xanthocarpum, which presented healing activities in burn models and in the treatment of diabetic skin ulcers [32, 41].

Both the anti-inflammatory and healing activities of S. diploconos may be correlated to the presence of caffeic, ferulic, and coumaric acids in its extract. It is known that caffeic acid presents anti-inflammatory and wound healing effects in different experimental models [42, 43], and the same was observed for ferulic acid [44, 45].

Finally, an analysis of the possible toxic effects of the extract was performed. No important toxic effects were observed in vitro (in L929 cells) or in vivo, apart from a reduction of glucose and an increase in urea levels in the acute but not in the subacute toxicity assay. Normal urea levels indicate that the metabolism of ammoniac to urea by the liver is not altered and that the detoxification and excretion liver functions are preserved [45]. Despite observing increased urea levels, the histopathological analysis showed no alterations in the renal tissue. These data indicate that the acute and subacute oral treatments with S. diploconos fruit extract may be safe. Nonetheless, further studies are necessary to address possible toxic effects of this extract when administered repeatedly.

5. Conclusion

Taken together, the data demonstrate the anti-inflammatory and prohealing activities of the S. diploconos fruit extract. These effects were linked to its ability to modulate neutrophil functions including the production of inflammatory mediators and chemotaxis, in addition to promoting efferocytosis and the resolution of acute inflammation. The absence of important acute and subacute toxic effects suggests the promising application of the extract as a therapeutic aid for acute inflammatory reactions and wound healing.