Introduction

Skin wound healing consists of a series of orderly events in which cellular and molecular phenomena are established and act to recover this structure1. These events encompass an initial inflammatory phase, characterized by the release of cytokines such as tumor necrosis factor-α (TNF-α), interleukin- (IL-)¹, and IL-6, and the recruitment of neutrophils and macrophages to clear pathogens and debris. This is followed by a proliferative phase, involving fibroblast proliferation, collagen deposition, and angiogenesis to restore tissue structure. Ultimately, the process concludes with the remodeling phase, during which the extracellular matrix (ECM) is reorganized, and the wound undergoes contraction to regain strength and function^1–3. Given the need for coordinated and timely closure and the different levels of impairment that scar formation may have on patient’s quality of life, the search for more effective and innovative therapies aiming at improving the repair process is evident^4,5.

Several factors can affect the normal healing process, such as excessive or prolonged inflammation, infections or even elements related to the patient’s health status, such as obesity and metabolic disorders (including diabetes) and compromised immune response^6–9. These factors can lead to chronic wounds or delayed healing due to the persistent inflammatory response and insufficient cellular proliferation and ECM remodeling⁷. In this sense, the treatment regimens adopted in wound healing aim to enable the balanced performance of immune cells and fibroblasts, using substances capable of directly stimulating the healing process, either by providing favorable means for cell proliferation, migration, and enhancing matrix deposition and remodeling^10–12.

Some natural products have been indicated as effective in the treatment of wounds due to their antioxidant, anti-inflammatory, antimicrobial, or biostimulating properties, in addition to offering fewer adverse effects. Antioxidants protect cells from oxidative stress, which can exacerbate inflammation and tissue damage^13,14, while anti-inflammatory agents reduce the intensity of the inflammatory response, promoting a faster transition to the proliferative phase¹⁵. Compounds such as tannins, flavonoids, alkaloids, and phenolic acids are widely recognized for their potential beneficial effects on skin wound healing by stimulating fibroblast activity and enhancing ECM production^13–16.

The genus Solanum comprises almost half of the species belonging to the Solanaceae family, configuring a rich source for biotechnological and pharmaceutical production^17–20. In the search for effective formulations for wound healing and to confirm proposed activities, some species of this genus, including S. tuberosum²¹, S. diploconos (Mart.) Bohs²², S. incanum²³, and S. xanthocarpum²⁴, have been tested in different models regarding their efficacy. These studies have revealed effects, mainly related to phenolic phytoconstituents such as phenolic acids and flavonoids, which are known for their antioxidant and anti-inflammatory activities.

The species Solanum lycocarpum A. St. Hil. is commonly found in the Brazilian territory, in cerrado areas, and is often considered a weed popularly known as lobeira since its fruits are part of the diet of the maned wolf (from the Portuguese lobo-guará–Chrysocyon brachyurus)^25,26. This species is used in popular and traditional medicine in some regions of the country to control diabetes, cholesterol, in the treatment of inflammatory diseases such as asthma and ulcers^27–30.

For the species S. lycocarpum, antimicrobial^31,32, anti-inflammatory^31,33, antioxidant^31,32, anticancer^34,35, and antinociceptive³⁶ activities have been reported in several studies. Recent studies have demonstrated the reduction of acute inflammation stimulated by the ethanolic extract of the ripe fruit of S. lycocarpum in a carrageenan-induced paw edema model in mice³⁶, as well as the effects of different fractions on inflammation³⁷. These effects were especially correlated with the presence of alkaloids, such as solasodine and peiminine, and phenolic acids in the extract³⁶.

In this context and in view of the antioxidant, antimicrobial, and anti-inflammatory activities reported for the species and their interrelationship with the wound healing process, the aim of the present study was to evaluate the effect of the topical application of the ethanol extract (EESL) and the hydroethanolic fraction (HFSL) of ripe S. lycocarpum fruit on the healing of experimentally-induced wounds in mice.

Methods

Acquisition of the ethanol extract and hydroethanolic fraction of ripe Solanum lycocarpum A. St. Hil. fruit

The ripe fruits of S. lycocarpum were collected in São Sebastião do Oeste, West-Central Minas Gerais state, Brazil (20°14’38.96”S and 45°2’14.38”W) in March 2017 (SISBIO no. 30006). The material was collected, and the vouchers were identified by Dr. Alexandre Salino, and deposited in the Herbarium of the Biological Sciences Institute (BHCB 159397) of Universidade Federal de Minas Gerais. This study has access permission to the components of plant genetic heritage, and it is registered in the SisGen Platform (Register AEF6C95), according to Brazilian Biodiversity Law (no. 13,123/2015).

The ethanol extract of the ripe fruit of S. lycocarpum (EESL, 33.43 g) and its hydroethanolic fraction (HFSL, 13.00 g) were obtained based on the methodology previously described36,37. Basically, the fruits were dried in an oven at 40°C for six days and then ground in a knife mill. The ethanolic extract was obtained by the percolation method of the plant drug for 24 hours, using ethanol P.A. (99.5ºGL) as a solvent. The material was extracted for 15 days, and the percolate was concentrated in a rotary evaporator at 50ºC, under reduced pressure, for subsequent lyophilization and obtaining of the ethanolic extract and subsequent partition.

The two were analyzed by liquid chromatography coupled to a diode array detector and a mass spectrometer (LC-DAD-MS) to identify their constituents. The main compounds annotated in EESL were steroidal glycoalkaloids (such as robeneoside B or hydroxysolasonine isomers, khasianine or β₂-solanine isomer, and solanandaine isomers), the aglycone alkaloids peiminine and solasodine, di- and tri-O-caffeoylquinic acid derivatives, O-coumaroyl caffeoylquinic acid derivatives, N1, N10-bis-(dihydrocaffeoyl)spermidine, di-O-hexoside, and hexonic acid. For HFSL, compounds were previously noted and described, such as di-O-hexoside, hexonic acid, steroidal glycoalkaloid robeneoside B or hydroxysolasonine isomers, khasianine or β2-solanine isomer, and solanandaine isomers), mono-and tri-O-caffeoylquinic acid derivatives, O-coumaroyl dicaffeoylquinic acid derivatives, and O-caffeoyl dicoumaroylquinic acid^36,37.

For this study, EESL and HFSL were resuspended in saline solution at the concentration of 150 mg/kg for subsequent analysis.

Animals

All procedures were conducted according to the guidelines established in the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee for the Use of Animals of the Universidade Federal de São João del-Rei, Protocol No. 3128180521. During the experimental period, 72 male Swiss mice, aged 8 weeks old and approximately 40 g, obtained from the Central Animal Facility of the Tancredo Neves Campus, were maintained in cages containing six animals each, fed with balanced commercial feed and water ad libitum, and submitted to artificial light cycles (12 hours of light/12 hours of dark), room temperature between 21 and 22ºC, and 60 to 70% of relative humidity.

Excisional skin wound induction and topical application of the ethanol extract and hydroethanolic fraction

The animals were randomly distributed into three groups:

• A control group, which received topical application of physiological saline;

• A group treated with the ethanol extract (EESL Group);

• A group treated with the hydroethanolic fraction of ripe S. lycocarpum fruit (HFSL Group).

Each group was subdivided into four subgroups according to the treatment period adopted: one, five, seven, and 21 days, with n = 6 in each subgroup. Afterwards, the mice were anesthetized intraperitoneally with a solution of 10% ketamine and 2% xylazine at the dosage of 1 mL/kg (injectable Dopalen Sespo Indústria e Comércio, Brasil; and Xilazin, Syntec do Brasil) and had their dorsal hair trichotomized. Two excisional wounds were made on the dorsum of the mice according to the procedure previously described³⁸, with some modifications. Basically, the wounds were made with the aid of a circular metal punch measuring 7 mm in diameter, after anesthetizing the animals and performing the antisepsis of the site, removing the entire thickness of the skin until the exposure of the dorsal muscular fascia.

Topical application of the treatments and analysis of the injured areas

After the surgical procedures, the mice were provided with the respective treatments. In the animals from the treated groups, as of the first day, 40 μL of the EESL or HFSL solutions (standardized in all groups) were applied at the wound site at the concentration of 150 mg/kg, twice daily, using an automatic pipette with sterilized tips. This procedure was repeated for one, five, seven, or 21 days, depending on the subgroup. In the control group, the same volume of saline solution was applied.

The wound areas were measured at each time point after photographic recording with a digital camera fixed on a tripod. The images were imported into the ImageJ image analysis software, version 1.44 (Research Services Branch, U.S. National Institutes of Health, Bethesda, MD, United States of America), and the contours were manually traced to calculate the wound size in mm² at each time (one, five, seven, and 21 days after making the lesions).

Histopathological and histomorphometric analyses of the wounds

After the euthanasia of the animals, the wounds were collected and histologically processed, the fragments containing the surgical wound in all its length and depth were removed with the aid of a number 11 scalpel blade and fixed in a 10% buffered formalin solution for 24 hours, sectioned perpendicularly in its half, and the separated pieces were dehydrated in successive ethanol solutions and embedded in paraffin. The paraffin blocks were sectioned using a microtome, obtaining 4-µm sections, which were later stained with hematoxylin-eosin (HE), toluidine blue, and Gomori’s trichrome. The slides stained with HE were analyzed under an optical microscope for qualitative evaluation regarding the presence of inflammatory infiltrate, wound extension, re-epithelialization, and granulation tissue quality in the different observation periods. Those stained with Gomori’s trichrome were analyzed to assess matrix deposition after 21 days of treatment.

In the histological sections stained with HE, leukocytes were quantified at one, five, seven days, and fibroblasts at five and seven days. To this end, the slides were photographed using a digital camera (Moticam 3000) coupled to a conventional optical microscope. The photomicrographs were evaluated using the ImageJ software, version 1.44 (Research Services Branch, U.S. National Institutes of Health, Bethesda, MD, United States of America). Meanwhile, in the slides stained with toluidine blue, mast cells were quantified after five and seven days of treatment. The cells were identified based on their characteristic morphology in 10 fields per slide at 400x magnification for the slides stained with HE and in 15 randomly distributed fields in the slides stained with toluidine blue. Subsequently, the average number of leukocytes, mast cells and fibroblasts was presented.

Statistical analysis

The obtained data were expressed as the mean ± standard error of the mean of each experimental group, comparing the differences between the means of the control group and those observed in the treated groups. The GraphPad Prism 5 program (GraphPad Software, CA, United States of America) was used in the analysis. Three or more group comparisons were performed using one-way analysis of variance, followed by Tukey’s test. P < 0.05 were considered significant.

Results

Macroscopic area

In the macroscopic evaluation of the wounds, no significant differences were detected in the size of the scar areas after 21 days of treatment, as observed in Fig. 1a. After one day of treatment, the wounds of all groups appeared clean and lacked the presence of secretions. Five days later, the wounds presented with detaching crusts in the control group and dry edges in the EESL group, with a delay in detachment in the HFSL group. At seven days, 50% of the animals in the EESL group presented wounds with detached crusts, most of which were visibly smaller compared to the other groups. In the control group, most of the wounds exhibited intact crusts, as well as in the HFSL group, the latter being thicker and presenting a statistically larger area in relation to the control and EESL groups (p = 0.0027), as evidenced in Fig. 1b. After 21 days of treatment, as mentioned in the Methods section, the wound area was replaced by a small scar in all groups, without significant differences between areas.

Microscopic analysis

Histopathological and morphometric analysis of leukocytes

The predominant inflammatory pattern observed in the histopathological analysis after one day of treatment was polymorphonuclear, as shown in Fig. 2a, with the later presence of mononuclear cells at the wound site. A smaller number of leukocytes was detected in the groups treated with EESL and HFSL after one, five, and seven days when compared to the control group (p < 0.0001), as seen in Fig. 2b.

Histopathological and morphometric analysis of fibroblasts and mast cells

According to the histopathological analysis of the wounds as of five days of treatment in the slides stained with HE, the presence of fibroblasts in the wound bed was observed, identified based on the corresponding morphology, indicating the onset of the proliferative phase. In the slides stained with toluidine blue, it was possible to notice the presence of mast cells in the wound bed in all groups, as shown in Figs. 3a and 4a.

As for the effects of HFSL and EESL on fibroblasts and mast cells, the morphometric analysis did not evidence significant differences in the number of these cells after five and seven days of treatment, as it can be seen in Figs. 3b and 4b.

Histopathological analysis of matrix deposition and remodeling of the scar area

Based on the qualitative microscopic analysis of the wounds in the slides stained with HE after one, five, seven, and 21 days, the healing process was better in the group treated with the EESL of S. lycocarpum, with wounds presenting smaller areas, lower levels of inflammatory infiltrate, better tissue proliferation, and more advanced re-epithelialization compared to the control group and the group treated with the HFSL of S. lycocarpum (Fig. 5).

In all groups, deposition of the new collagen matrix was observed in the wound area, with the presence of fibroblasts, blood vessels, and some leukocytes in the formed scar tissue. Such deposition was more regular in the group treated with EESL, with intertwined and relatively thicker fibers than the control group. The group treated with HFSL exhibited a denser deposition of fibers with little intertwining between them, characterizing a scar (Fig. 6).

Discussion

The present study confirmed the effectiveness of both the EESL and the HFSL of S. lycocarpum in enhancing the wound healing process in mice, with the EESL showing more pronounced benefits in several aspects of tissue repair. Specifically, treatment with the EESL was associated with tissue filling in a shorter time, superior collagen deposition, and smaller scar areas after 21 days, compared to the HFSL. These findings highlight the potential of the EESL as a more effective treatment for promoting wound healing in comparison to the HFSL.

In the initial stages of healing, the EESL exhibited anti-inflammatory effects, which were critical for the acceleration of the repair process. The observed reduction in inflammation aligns with the role of phenolic compounds, including cinnamic, caffeic, and chlorogenic acids, in modulating the inflammatory response. These compounds, previously identified in extracts from ripe fruit of S. lycocarpum^34,36,37, are known for their potential to inhibit cyclooxygenase-2 activity^22,39,40, a key enzyme involved in the inflammatory cascade. The reduction of cyclooxygenase-2 activity may explain the diminished influx of leukocytes and the more controlled inflammatory environment conducive to faster tissue repair.

Furthermore, the present study revealed that the EESL not only reduced inflammation, but also promoted advanced re-epithelialization and tissue proliferation, evidenced by smaller crusts and enhanced wound closure compared to the hydroethanolic fraction. Wound closure is used to evaluate the evolution of the healing process²¹. These effects are likely attributable to the antioxidant and antimicrobial activities of phenolic acids and flavonoids present in the extract^21,31,36, which have been reported to play key roles in protecting tissue from oxidative stress and promoting collagen synthesis²¹.

The healing process has been also described as proportional to collagen biosynthesis24. The increased deposition of collagen, a hallmark of successful healing, was more prominent with the EESL treatment. This aligns with findings from other studies on related Solanum species, such as S. xanthocarpum²⁴ and Solanum incanum²³, in which phenolic compounds and flavonoids were linked to improved collagen biosynthesis in wound healing models. Oxidative stress is another problem to be overcome by cells and the natural antioxidant system during the healing process. The antioxidant properties of these phytoconstituents help neutralize free radicals, reducing tissue damage and further supporting the healing process¹³.

The advanced deposition of granulation tissue, observed after seven days in the present study, in parallel with a smaller and better scar area after 21 days of treatment, can therefore be associated with the synergistic action between the phytoconstituents present in the EESL. Alkaloids like solasodine and peiminine, combined with phenolic acids such as chlorogenic and caffeic acids^34,36, appear to contribute to the anti-inflammatory and wound healing properties seen in the ethanol extract.

The suppression of inflammatory response in therapies used for wound healing has been reported as an important alternative, as well as the control of oxidative stress^7,9,12,13. Here, we demonstrated that the S. lycocarpum species has a potential for healing, probably due to its anti-inflammatory and antioxidant effects already reported in the literature and in folk medicine. In this sense, the results provide a foundation for further research into the pharmacological properties of this species and its application in skin wound treatment. The findings suggest that mainly the EESL holds promise as a topical agent to enhance wound repair.

Conclusion

Based on the analysis of the effects of the HFSL and EESL on the healing of skin wounds in mice, we showed that this species has anti-inflammatory and healing potential through topical application. Anti-inflammatory effects were verified through the topical application of the EESL in the early stages of repair, possibly contributing to an apparently smaller scar area. However, for a comprehensive understanding of the underlying mechanisms and to evaluate its efficacy in different types of wounds and clinical scenarios, additional studies are needed, such as molecular and cellular investigation of anti-inflammatory effects, development of topical products with healing and/or cosmeceutical potential, or even combination in treatments using other approaches involving tissue engineering. In addition, the importance of valuing this cerrado species is highlighted, not only for its medicinal properties, but also as a resource for the conservation of the region’s biodiversity.

Acknowledgements

The authors are grateful to Universidade Federal de São João del-Rei by the support and to prof. dr. Alexandre Salino for the plant material identification.

Data availability statement

All data sets were generated or analyzed in the current study.

References

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