Comparative Studies on the Antioxidant, Antifungal, and Wound Healing Activities of Solenostemma arghel Ethyl Acetate and Methanolic Extracts

: Various herbal compounds are used for medical purposes due to their safety, as there are no or minimal side effects. This study was performed to assess the wound healing and antioxidant activities of ethyl acetate (EtOAc) and methanolic extract (MeoH) of Solenostemma arghel ( S. arghel ). Their antifungal activities were also evaluated against isolated swabs of equine wounds. They underwent GC-MS analysis for the characterization of both extracts. For wound healing evaluation, forty-ﬁve male albino rats were divided into three groups; the control group was treated with normal saline, and the other two groups were treated with S. arghel EtOAc and MeoH extract gels, respectively. The wounds were examined clinicopathologically and immunohistochemistry on the 3rd, 7th, and 14th days post-wounding. GC-Ms analysis of S. arghel recorded ﬁfty-one volatile organic compounds (VOCs) within EtOAc extraction and thirty VOCs in MeoH extract. VOCs represented in EtOAc extract showed higher antioxidant activity and better and faster wound healing than VOCs of MeOH extract. The treated groups showed improved wound healing clinically and pathologically in comparison with the control group as they decreased the wound surface area (WSA) and percent (WSA%) and increased the wound contraction percent (WC%), epithelization, ﬁbroblast proliferation with neovascularization, and reduced the inﬂammatory reaction. Moreover, the treated groups showed higher expression of vascular endothelial growth factor (VEGF) compared with the control. The EtOAc extract showed higher antifungal activity against Penicillium funiculosum , P. jensenii , M. cinctum , and Candida albicans , which were isolated from infected clinical equine wounds, than MeOH extract. The treated groups showed improved wound healing clinically and pathologically in comparison with the control group as they decreased the wound surface area (WSA) and percent (WSA%) and increased the wound contraction percent (WC%), epithelization, ﬁbroblast proliferation with neovascularization, and reduced the inﬂammatory reaction. Moreover, the treated groups showed higher expression of vascular endothelial growth factor (VEGF) compared with the control. Additionally, the two extract gels showed promising healing of equine wounds. In conclusion, the study recommended the use of S. arghel EtOAc extract as it was proven to promote wound healing compared with MeoH extract.


Introduction
The wound is the disruption of cellular and anatomical continuity of living tissue produced by physical, chemical, electrical, or microbial insults to the tissue [1]. The subcutaneous tissue loses its integrity when wounded and colonized by microbes. These microorganisms involve potential pathogens, which interrupt the wound healing process in the injured tissues [2,3]. Wound infection is a major factor in the progression of chronicity, delaying wound healing [4].
The development of wound infection is an ongoing problem for many patients. Infected wounds can cause great distress in terms of associated morbidity and mortality, increased length of hospital admission, delayed wound healing, and increased discomfort, and have long been known to increase healthcare costs significantly, as the exudate levels are relatively higher in infected wounds. Pus and odor may indicate that the patient is pyrexial or has an increased neutrophil count [5].
Most chronic wound microbiome research is focused on bacteria, but contamination of the wound by fungi needs more elucidation. Ref. [6] classified seventeen fungal species from different wounds, which obstruct the healing of these wounds.
The yeasts of the genus Candida were the most numerous fungus. In particular, the high relative abundance of fungus within the polymicrobial ecology of wound infections, when present, shows that the fungus can be a major contributor to the biofilm of wounds. In addition, the diversity of fungal genera and species found in and among such wounds is more than previously thought or recorded [7]. The vulnerability to fungal wound infection is also increasing in geographical locations with a warm and humid climates [8].
The link between microbial aggregates and wound healing has been studied using a number of animal models. In order to imitate wound healing, most research used small rodents. However, the architecture of these species' skin and healing patterns differ significantly from those of humans [9]. Horses have thick, tight skin, similar to humans [10]. In humans and horses, wound edge contraction has a minor role (20-25%) in extremity/limb healing, in contrast to the situation in loose-skinned animals such as rodents [11].
Horses appear to be valid model animals for wound healing in humans because of their similar skin architecture and the relative role of contraction and epithelialization in healing [12,13]. Because of their surroundings, horses' wounds have a significant risk of infection, as the infected wounds house a variety of microorganisms. However, these germs can be challenging to identify and not react to antibiotic treatment, resulting in chronic non-healing wounds [14]. Therefore, in the study, we selected some cases of equine wounds for clinical application of the gel and to find a resolution for the problem of equine wounds through the investigation of new wound-healing agents.
The present study was designed to compare the antifungal, antioxidant, and wound healing activities of EtOAc and MeoH S. arghel extracts in albino rats besides the clinical application of the extracts for investigation of wound healing in equines.

Plant Materials
The vegetative part of S. arghel was collected from the extremely arid desert region of Southern Egypt, Aswan University campus, Aswan. The plant was washed with tap water, leaves and flowers were separated from the stem, air-dried at room temperature, followed by grinding into a powder with a grinder (mortar and pestle).

Preparation of Plant Extract
Powdered leaves and flowers (100 gm) were extracted with two organic solvents (EtOAc and MeoH (0.1 gm/mL)). Extraction was incubated overnight at room temperature and repeated using an ultrasonic homogenizer. The extracts were decanted, filtered, and the residue was re-extracted again under the same experimental conditions to ensure full extraction. The clear filtrates were air-dried using a rotary evaporator at 45 • C [21].

Collection and Preservation of Material from Injured Parts for Investigation of Yeast and Fungal Infections
By using sterile swabs, samples were collected from 12 equine species (a horse, a mare, 5 donkeys, and 5 female donkeys), which were admitted to the Brooke Hospital in Aswan, Egypt, suffered from traumatized wounds. Swabs were placed in a sterile cryotube and stored at −40 • C. These samples were soon cultured on Sabroud media for isolation of fungi and yeast, which were morphologically identified using a light microscope, and the isolated fungi were purified and kept in slants.

Antifungal Activity Assay
For screening of antifungal activity of Arghel plant extracts, a food technique was followed [22]. Sabroud medium was prepared and sterilized; then, the medium was supplemented with EtOAc and MeoH plant extracts at two concentrations (5 mg/mL and 10 mg/mL), mixed well, and kept at 4 • C overnight. The mycelial disc (0.5 cm in diameter) was deposited in the center of the plate (5.0 cm in diameter) according to the poisoned food method, which was used to evaluate the antifungal effect against pathogens [23]. After further incubation for 7 days at 28 ± 2 • C for the fungal strains tested, the diameters of fungal growth for control (fungus in media without any extraction and containing only solvent) and treated plates were measured with a calculation of the inhibition percentage [24].

DPPH Antioxidant Activity
The DPPH solution (0.5 mmol/L) was prepared in 95% methanol, and different concentrations (50, 100, and 150 µg) of Ethyl acetate and Methanol plant extracts were used. A total of 2 mL of DPPH solution (0.5 mnol/L) was added to (1 mL) of the test sample and incubated for 30 min at room temperature in darkness. After 30 min, the absorbance was measured at 517 nm. Ascorbic acid (AA) served as a positive control at a concentration of 10 µg. The percentage of the DPPH radical scavenging was calculated as: Inhibition of DPPH radical (%) = [(control absorbance (Ai) − extract absorbance (At))/(control absorbance (Ai))] × 100

GC-MS Analysis of Released VOCs from S. arghel EtOAc and MeoH Extracts
The extracted VOCs from the S. arghel were analyzed and identified by gas chromatography-mass spectrometry (GC/MS). The GC-MS analysis was carried out by using a TRACE GC Ultra Gas Chromatograph (THERMO Scientific Corp, Carlsbad, CA, USA), which connected with a Thermo mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer). The GC-MS system was equipped with a TR-5 MS column (30 m × 0.32 mm i.d., 0.25 µm film thickness). Analysis was achieved using helium gas at a flow rate of 1.0 mL/min and a split ratio of 1:10 using the following temperature program: 60 • C for 1 min; increasing 4.0 • C/min to 240 • C and held for 1 min. Both injector and detector were held at 210 • C, and diluted samples (1:10 hexane, v/v) of 1 µL of the mixtures were injected. Mass spectra were obtained by electron ionization (EI) at 70 eV, using a spectral range of m/z 40-450. The chemical constituents of the essential oil (Eos) identification was de-convoluted using (AMDIS version 2.70, http://www.amdis.net/, accessed on 11 April 2022) and identified by its retention indices (relative to n-alkanes C8-C22) mass spectrum matching to authentic standards (when available) or Wiley spectral library collection, and NSIT library database.

Preparation of S. arghel EtOAc and MeoH Extracts 2% Gels
The two gels were prepared according to [25]. In a mixture containing 100 mL D. W, 1.5 g of Carbopol 940 ® was added slowly and mixed well at a higher speed. Followed by the addition of extract until gel formation with 2% concentration.

Animals
Forty-five (45) male albino rats aged 2-3 months and with 150-200 g body weight were purchased from Abou-Rawah farm, Egypt. Rats were housed in plastic cages with balanced diet ad libitum, with clean water readily accessible to the caged rats in clean bottles under constant environmental conditions (temperature of 25 • C, normal daylight, and relative humidity of 45-55%). The rats were kept a week before the experiment for Acclimatization.

Excisional Wound Model
After the adaptive period, the rats were anesthetized with an intraperitoneal (IP) injection of ketamine (70 mg/kg) and xylazine (7 mg/kg) combination, and the hair on the back was shaved and disinfected with ethyl alcohol (70%). A 1.5 cm wound was created with the aid of toothed tissue forceps, surgical blades, and pointed scissors to keep the skin tension constant during wounding. The hemostasis was achieved by compression with sterile gauze, and wounds were left untreated for 24 h. After wound creation, the rats were housed individually, each rat in a separate cage. The wound site was cleaned with a tampon soaked in alcohol. The rats were randomized into 3 groups (15 rats each). The excisional cutaneous wounds of the control group (Group I) were treated topically with 0.9% normal saline once daily, and the excisional wounds of the other two groups (Group II, Group III) were treated topically with S. arghel EtOAc and MeoH extracts 2% gels, respectively. The two herbal formulations were applied to the wounds once daily for 14 days starting from the second day of wounding.

Assessment of Wound Diameter
The wound size was measured and photographed on 0, 3, 7, and 14 days posttreatment for visual comparison before and after clearance of the wound area. The wound surface area (WSA), wound surface area percent (WSA%), and wound contraction percent (WC%) were calculated according to the following formulae:

Histopathology
On the 3rd, 7th, and 14th days: 5 rats from each group were euthanized, and the wounds with part of normal skin were preserved in 10% neutral buffered formalin. The tissues were subjected to different concentrations of ethyl alcohol (70-80-90-100%), embedded in paraffin wax at 40-60 • C, sectioned into 5 µm thickness, and stained with hematoxylin-eosin stain [26].
2.9.6. Immunohistochemistry Immunohistochemistry assessment was performed according to [27]. Retrieval of antigen was performed by de-waxing and immersion of skin sections in a citrate buffer solution (0.05 M and pH 6.8). These sections were then treated with H 2 O 2 (0.3%) and protein block. They were then incubated with polyclonal rabbit VEGF (BioGenix, 49026 Milmont Drive, Fremont, CA 94538 Emergo Europe, Molenstraat 15, NL-2513 BH The Hague, The Netherlands, cat. No. AR483-5R, dilution 1/100). The slides were washed up with PBS and then incubated with a goat anti-rabbit secondary antibody (cat. no. K4003, EnVision+™ System Horseradish Peroxidase Labelled Polymer; Dako, Glostrup, Denmark) for 30 min at room temperature. Eventually, slides were visualized with a DAB kit and stained with Mayer's hematoxylin as a counterstain was evaluated under the light microscope. The staining intensity was assessed and reported as a percentage of positive cells in approximately 8 high power fields.

Clinical Study
The two prepared gels were topically applied on 5 equines (3 horses and 2 donkeys) that suffered from recent and old, traumatized contaminated wounds; some wounds were treated before without any tendency to wound healing.

Treatment Procedures
Before the gel application, the wound area was prepared according to the type of wound (old or recent); in a recent wound, hemorrhages were controlled by packing, and the wound area was aseptically prepared. In an old wound, unhealthy tissues were surgically debrided and freshened, and hemorrhages were controlled by packing.

Statistical Analysis
All statistical analyses were carried out by computer software (SPSS version 16.0, Chicago, IL, USA) using One-Way Analysis of Variance (ANOVA), and the data are presented as mean ± standard deviation (SD) values. The differences between mean values were considered significant at p < 0.05. The graphs were performed using Microsoft Excel 2010.3.

Antioxidant Activity of S. arghel
Both EtOAc and MeoH extracts showed recognized antioxidant activity. This activity increased with increasing the concentration of both extracts from 50 µg to 150 µg. While EtOAc extract showed higher antioxidant activity (19.21-58.23%) than methanol extract (13.71-44.82%) (Figure 2).

Contaminated Wounds by Fungi and Yeast
Exposed subcutaneous tissue provides a favorable substratum for fungi to contaminate and colonize the wounds. In this study, variable animals' wounds were contaminated by three fungal species and one species of yeast. These contaminants were identified as P. funiculosum, P. jensenii, M. cinctum, and C. albicans. The most abundant species in wounds was C. albicans (18.0 colonies), which was isolated from all wounds of horses (7.0 colonies), mare (2.0 colonies), donkeys (3.0 colonies), and female donkeys (6.0 colonies). Myrothecium cinctum (12.0 colonies) and P. funiculosum (8.0 colonies) colonized horse (5.0 colonies and 3.0 colonies), mare (one colony and 2.0 colonies), three donkeys (3.0 colonies and 1.0 colony) and four female donkeys (3.0 colonies and 2.0 colonies), respectively. While, P. jensenii (5.0 colonies) was isolated from a horse (one colony), two female donkeys, and two donkeys (two colonies each).

Antifungal Activity of S. arghel VOCs
Generally, VOCs represented in S. arghel EtOAc extract showed higher antifungal activity than MeOH extract (Figure 3a,b). All fungal species were inhibited by S. arghel secondary metabolites extracted by MeoH and EtOAc in both concentrations (5 mg and 10 mg/mL). Penicillium funiculosum was the most sensitive to both extracts, with concentrations 5 mg and 10 mg/mL, where the inhibition percentage was high and ranged between 54.05 and 61.35%. Interestingly, the reaction of M. cinctum to S. arghel differed from other studied fungi, where this fungus recorded a higher inhibition percentage with MeoH extract (44.54% and 52.23%) than EtOAc (29.15% and 48.18%) with 5 mg and 10 mg/mL, respectively. Pencillium jensenii and C. albicans showed clear inhibition with 10 mg EtOAc (37.14% and 33.57%, respectively), while MeoH extract in both concentrations showed less inhibition percentage.

Clinical Observations
On the clinical assessment of wounds (Figure 4), it was observed that on the day of wounding, the wound edges showed hyperemia, edema, and swelling of the tissues around the wound margins. On the 3rd day post wounding, wounds of GI showed yellowish exudate with shrinkage of the wounds, which was accompanied by hyperemia and swelling of the wound edges. While in GII, the wounds were covered with brownish scabs with shrinkage and significantly decreased wounds size compared with the control. Additionally, the wounds of GIII were non-significantly narrower than the wounds size of GI, with brownish scab covering the wound surface, which sloughed from some of them. On the 7th day post-treatment, the wounds of GI were covered with thick moist scabs with significantly less wound contraction in comparison with GII and GIII. While the wounds size of GII was non-significantly narrower than that of GIII. On the 14th day post-treatment, the wounds contraction of GII and GIII were significantly higher than GI. The wounds of GII showed complete healing with scab formation in comparison with the wound of GIII. Meanwhile, the wounds of GI showed incomplete healing. The WSA and WSA% (Tables 3 and 4) on the 3rd and 7th day post-treatment significantly decreased in GII compared with GI, while the WC% (Table 5) significantly increased in GII compared with GI when p was ≤0.05. On the 14th day post-treatment, the WSA and WSA% significantly decreased in GII and GIII compared with GI; furthermore, the WC% significantly increased in GII and GIII compared with GI when p was ≤0.05.

Histopathological Findings
As shown in (Figure 5), on the 3rd day post-treatment, the microscopic examination of wounds in the GI revealed ulcerated and desquamated necrotic mass of tissue, inflammatory reaction with intense polymorph nuclear cells infiltration and dilated blood vessels with hemorrhage and edema of the underlying dermis. While in wounds of GII, the dermal blood vessels were congested in addition to fibroblast cells proliferation and with the presence of mononuclear cells infiltration. GIII showed congested blood vessels, hemorrhage, and increased inflammatory reaction. On the 7th day, GI showed fewer polymorph-nuclear cells in the dermis but more macrophages infiltration, less edema, and hemorrhages compared with the 3rd day of the experiment. While, GII showed moderate fibroblast proliferation, mild collagen deposition, and increased angiogenesis in the upper layer of the granulation tissue. The pathological examination of wounds in GIII revealed little fibroblast proliferation with increased inflammatory cells proliferation. On the 14th day, there was enhanced repair and remodeling of the wound area. The wounds of GI showed complete epithelization but with increased necrotic tissues and inflammatory cells. Moreover, GII revealed complete epithelization with few polymorph-nuclear cells in the dermal tissue with full skin thickness and healing near to the normal skin. Wounds of GIII were characterized by complete epithelization with intense polymorph-nuclear cells infiltration in the dermal tissue and with increased necrotic tissue. Figure 5. Photomicrograph of the control and treated groups on the 3rd, 7th, and 14th days posttreatment where GI at 3rd day showed ulcerated and desquamated necrotic tissue with inflammatory cells infiltration (arrows), while at 7th day, there were dilated blood vessels with epithelization. On 14th day, increased mononuclear inflammatory cells infiltration with necrotic tissue mass at the wound area was shown. While GII on the 3rd day showed inflammatory cells infiltration and mild angiogenesis, and on the 7th day, fibroblast proliferation with neovascularization and granulation tissue formation was shown. Moreover, on the 14th day, full skin thickness and healing near to normal skin were shown. GIII showed congested blood vessels with mild fibroblast proliferation on the 3rd day, while on the 7th day, increased fibroblast proliferation and neovascularization were shown. On the 14th day post-treatment, complete epithelization of wounds with mononuclear inflammatory cells infiltration was shown.

Immunohistochemistry Findings
Microscopic examination of the wounds stained with vascular endothelial growth factor immune-staining on the 3rd, 7th, and 14th days post-treatment, where GI showed mild expression of VEGF within the endothelial cells of blood vessels and fibroblast at 3rd, 7th, and 14th days. While, GII showed mild VEGF on the 3rd day within endothelial cells of blood vessels, which increased on the 7th and 14th days within the fibroblast cells and endothelial cells of blood vessels besides epithelial cells of granulation tissue. In GIII, on the 3rd day of the experiment, VEGF was expressed mild reaction within the endothelial cells of blood vessels, and increased on the 7th and 14th days and appeared within the endothelial cells of blood vessels, fibroblast cells, and epithelial cells ( Figure 6). Figure 6. Immunohistochemical expression of VEGF in control and treated wounds. GI showed mild expression of VEGF within the endothelial cells of blood vessels and fibroblast on the 3rd, 7th, and 14th days. GII showed mild VEGF on the 3rd day within endothelial cells of blood vessels, which then increased on the 7th and 14th days within the fibroblast cells and endothelial cells of blood vessels besides epithelial cells of granulation tissue. In GIII, on the 3rd day, VEGF was mild within the endothelial cells of blood vessels, and increased on the 7th and 14th days and appeared within the endothelial cells of blood vessels, fibroblast cells, and epithelial cells.

Clinical Study
Wound management strategies applied, such as the surgical debridement in the case of the old wounds, which were considered very important for the removal of dead and necrotized tissues that provide a suitable environment for the action of the gel for good and clean healing. Any hemorrhage that could have resulted from surgical debridement was arrested immediately and controlled by packing after clearance of the wound.
By application of the herbal formulations to the equines wounds, it was observed that wounds treated with S. arghel EtOAc gel showed rapid wound epithelization compared with S. arghel MeoH extract gel, and the S. arghel EtOAc gel was easily applied on wound surface compared with S. arghel MeoH extract gel (Figures 7-9).

Discussion
The wound induces loss of the integrity of the skin resulting in functional imbalance, possibly accompanied by disability or even death [28]. Therefore, the wound healing process is one of the utmost medical issues in human and veterinary medicine, which explains the urgent need for developing new agents that possess wound healing activities [25].
Recently medicinal plants' antioxidant and antimicrobial activity attracted many researchers. Almost all these phytochemicals activities are emulated with artificial antimicrobials and antioxidants [29]. The current study was conducted to evaluate the antifungal, antioxidant, and efficiency of topical S. arghel EtOAc and MeoH extracts for the cutaneous wounds treatment in experimentally induced wounds and equines suffered from traumatized wounds. Biomembrane-forming yeasts and cutaneous opportunistic pathogens were most strongly associated with wound necrosis and poor outcome [6]. VOCs extracted from S. arghel by EtOAc and MeoH in this study match VOCs identified by many previous studies with qualifications of biological and medical activities ( Table 6). These activities were confirmed in this study by limiting the growth of fungal isolates from clinical cases of equines wounds due to active compounds of arghel EtOAc and MeoH extracts (Tables 1 and 2) (Figure 3a,b). Both extracts inhibited the growth of the filamentous fungi (P. funiculosum, P. jensenii, and M. cinctum) and yeast (C. albicans) isolated from wounds. C. albicans is the most common human pathogen responsible for painful mucosal infection [30]. Therefore, inhibition of yeast cells is needed to limit the rapid reproduction of cells and reduce the pain. Interestingly, C. albicans colonized all studied wounds while filamentous fungi (P. funiculosum, P. jensenii, and M. cinctum) were irregularly distributed in the studied animals' wounds. These results confirmed the hypothesis that infected skins serve as a portable for filamentous fungi to enter the host body [31]. Both extracts inhibited the sporulation of these fungi, which supports wound recovery. Fungal spores supported fungi in wound colonization and prolonged the wound's inflammation [6]. Thus, limiting fungal contamination is an important step for wound treatment. Moreover, the antioxidant characteristics of S. arghel (Figure 2) encouraged the inhibition of fungi and wound healing. Table 6. Activities of some VOCs emitted from S. arghel extractions (EtAoH and MeoH).

Heptacosane
Anti-corrosive and antioxidant [69] Sulfurous acid, butyl heptadecyl ester Antimicrobial [70] Dibutyl phthalate Antimicrobial [71] It is known that S. arghel is used in traditional medicine as an antispasmodic, antiinflammatory, and anti-rheumatic herbal [72]. The obtained results revealed that the surgically induced cutaneous rats' wounds showed accelerated wound closure with minimal scare formation after treatment with S. arghel EtOAc extract gel in comparison with that treated with S. arghel MeoH extract gel. Ref. [73] reported that there are chemical compounds that occur naturally in plants and possess important wide applications in medicine, and so the wound healing activity of S. arghel is attributed to the phytochemical components of EtOAc extract, which have anti-inflammatory and antioxidant effects ( Table 6).
The S. arghel EtOAc extract has good analgesic action due to its phytochemical ingredients as Neophytadiene, Digitoxin, and Vitamin E (alpha-tocopherol), which were recorded for its analgesic action ( Table 6). As a result, topical use of S. arghel has a good effect in reducing the pain.
Equines are known for their tendency to wounding, probably due to their inquisitive nature, large size, and confining in areas with potential obstacles such as metal or wire and their known difficulties with healing. The present study showed the excellent beneficial use of S. arghel in the treatment of recent and old traumatized wounds in equines. Extract application resulted in relieving edema and inflammation around the wounds, disappearance of infection, and observable decreased wound surface. After extract application, the wounds were not covered with bandages due to the authors of [74] reporting that the bandaged wound took a longer time to heel than unbandaged wounds. The application of the extracts absorbing exudate, keeping the wound moist, which helps epithelization, prevent contamination of wound from flies due to presence of Oxiranepent Hexadecanoic Anoic Acid, 3-UndecyL-, Methyl Ester, Trans, which has pesticide activity as recorded by [44]. Ref. [75] reported that delayed healing associated with distal limb wounds is a particular problem in equine clinical practice. In this study, the topical extract of S. arghel, especially EtOAc extract, showed accelerated the wound healing of the distal limb wound.

Conclusions
From the aforementioned results, it can be concluded that S. argel possesses antifungal, antioxidant, and wound healing activities. These findings were well appreciated, with histopathological studies suggesting that S. argel can serve as a good therapeutic agent for the treatment of cutaneous wounds as well as beneficial effects for the treatment of equine wounds, especially those related to the distal limbs.

Data Availability Statement:
The data presented in this study are available in this manuscript.