The Wound-Healing Potential of Olea europaea L. Cv. Arbequina Leaves Extract: An Integrated In Vitro, In Silico, and In Vivo Investigation

Olea europaea L. Cv. Arbequina (OEA) (Oleaceae) is an olive variety species that has received little attention. Besides our previous work for the chemical profiling of OEA leaves using LC–HRESIMS, an additional 23 compounds are identified. An excision wound model is used to measure wound healing action. Wounds are provided with OEA (2% w/v) or MEBO® cream (marketed treatment). The wound closure rate related to vehicle-treated wounds is significantly increased by OEA. Comparing to vehicle wound tissues, significant levels of TGF-β in OEA and MEBO® (p < 0.05) are displayed by gene expression patterns, with the most significant levels in OEA-treated wounds. Proinflammatory TNF-α and IL-1β levels are substantially reduced in OEA-treated wounds. The capability of several lignan-related compounds to interact with MMP-1 is revealed by extensive in silico investigation of the major OEA compounds (i.e., inverse docking, molecular dynamics simulation, and ΔG calculation), and their role in the wound-healing process is also characterized. The potential of OEA as a potent MMP-1 inhibitor is shown in subsequent in vitro testing (IC50 = 88.0 ± 0.1 nM). In conclusion, OEA is introduced as an interesting therapeutic candidate that can effectively manage wound healing because of its anti-inflammatory and antioxidant properties.

frost-resistant and pest-resistant [32]. Almost 78 percent of olive oil is grown on Arbequina rootstock [33]. As a result, Arbequina olives have one of the highest oil contents and are largely utilized in the production of olive oil [34]. Publicly, Olea europaea oil and polyphenol contents are fully documented to reduce oxidative stress, stimulate wound healing, and minimize inflammation [35][36][37][38][39][40]. However, the oil and polyphenol contents are generally affected by climatic conditions during ripening and the degree of maturation, especially the Arbequina types [41,42].
In an attempt to compare the cultivar olive with the wild types in the content and activities concerning this field, our previous work estimated the potential of OEA leaves (cultivated in Egypt) as an internal wound healer against gastric ulcers in a rat model compared to wild O. europaea [43]. The results showed that the crude extract of the OEA cultivar was richer with polyphenolic content using LC-HRESIMS. The ulcer index of the rat model was significantly decreased, and the mucosa from the lesions was protected [43].
To complement the previous work, we targeted the wound healing ability of OEA as an external wound healer by applying an excisional wound model. We focused on essential wound healing targets, encompassing transforming growth factor-beta (TGF-β), interleukin-1β (IL-1β) as well as tumor necrosis factor-α (TNF-α). Illustrating the mode of action of OEA using several in vitro and in silico assays. Additionally, we assessed the possible inhibitory effect of OEA on one of the key players in wound healing (i.e., MMP-1), as proposed by the in silico investigation. Figure 1 depicts the framework of the present investigation.
Olea europaea L. Cv. Arbequina, a cultivar of olives, is an olive variety species that has received little attention. Arbequina has lately become one of the world's most important olive cultivars, owing partly to extensive cultivation and "super-high-density" plantations [31]. Arbequina trees can fit a variety of climatic and soil conditions. Nonetheless, it flourishes in long, hot, and dry summers and grows best in alkaline soils. Nonetheless, it is frost-resistant and pest-resistant [32]. Almost 78 percent of olive oil is grown on Arbequina rootstock [33]. As a result, Arbequina olives have one of the highest oil contents and are largely utilized in the production of olive oil [34]. Publicly, Olea europaea oil and polyphenol contents are fully documented to reduce oxidative stress, stimulate wound healing, and minimize inflammation [35][36][37][38][39][40]. However, the oil and polyphenol contents are generally affected by climatic conditions during ripening and the degree of maturation, especially the Arbequina types [41,42].
In an attempt to compare the cultivar olive with the wild types in the content and activities concerning this field, our previous work estimated the potential of OEA leaves (cultivated in Egypt) as an internal wound healer against gastric ulcers in a rat model compared to wild O. europaea [43]. The results showed that the crude extract of the OEA cultivar was richer with polyphenolic content using LC-HRESIMS. The ulcer index of the rat model was significantly decreased, and the mucosa from the lesions was protected [43].
To complement the previous work, we targeted the wound healing ability of OEA as an external wound healer by applying an excisional wound model. We focused on essential wound healing targets, encompassing transforming growth factor-beta (TGF-β), interleukin-1β (IL-1β) as well as tumor necrosis factor-α (TNF-α). Illustrating the mode of action of OEA using several in vitro and in silico assays. Additionally, we assessed the possible inhibitory effect of OEA on one of the key players in wound healing (i.e., MMP-1), as proposed by the in silico investigation. Figure 1 depicts the framework of the present investigation. Figure 1. The general summary of the present research is as follows: Olea europaea leaves were extracted, chemical profiling was carried out, and finally the leaves were assessed for their potential wound healing activity by evaluating their antioxidant activity and ability to inhibit MMP1, upregulate TGF-β relative gene expression, and downregulate the relative gene expression of inflammatory cytokines (TNF-α and IL-β1). Figure 1. The general summary of the present research is as follows: Olea europaea leaves were extracted, chemical profiling was carried out, and finally the leaves were assessed for their potential wound healing activity by evaluating their antioxidant activity and ability to inhibit MMP1, upregulate TGF-β relative gene expression, and downregulate the relative gene expression of inflammatory cytokines (TNF-α and IL-β1).

Chemicals and Reagents
All reagents and compounds were obtained from Sigma-Aldrich unless otherwise stated (Germany, Biosystems SA Costa Brava 30, Barcelona, Spain).

Plant Material Extraction
Two kilograms of OEA leaves were collected, meticulously cleaned, and air-dried for 10 days in the shade. The leaves were ground with an OC-60B/60B herb grinding mill (60-120 mesh, Henan, China-Mainland). The powdered leaves were soaked in a huge, closed glass jar for intensive extraction with 70% EtOH (15 L X3) and concentrated under reduced pressure at 45 • C. Following these steps, 80 grams of dried residue was obtained. It was saved at 4 • C for further phytochemical and biological screenings [44,45].

Metabolomic Analysis
According to our previously reported protocol [44], LC-HRMS-assisted metabolomic analysis of OEA was carried out. The detailed method is described in the supplementary file.
2.5. In Vivo Wound Healing Activity 2.5.1. Animal Treatment Eighteen wholesome adult male New Zealand Dutch breed albino rabbits participated in this investigation (2.1-2.7 kg). Polypropylene cages were used for animal housing. The normal pellet feed and unlimited water were freely available during the experiment. The seven days leading up to the experiment were spent acclimating the animals to the lab environment. Animals were introduced to a home with adequate ventilation under conditions of 25 ± 2 • C and relative moisture of 44-55% with 12 h cycles of dark/light.

Samples Preparation for the Bioassay
In order to evaluate the effectiveness of OEA extract in healing wounds, excisional wound models were applied [42]. The extract was made for the wound models by dissolving OEA dry extract in carboxymethylcellulose (2 g of dried extract in 100 mL of 0.5% carboxymethylcellulose) and kept at 4 • C in the dark. Each test extract was applied topically to the wound site as soon as it was prepared [43].

Model for Circular Excision Wounds
Excisional wounds were induced in rabbits [46,47]. In summary, 0.01 mL Ketalar ® (Ketalar, Sankyo Lifetech Co., Ltd., Tokyo, Japan) was used to anesthetize the animals. The back hairs of the rabbits were carefully shaved. The circular incision of each animal was made with a 6-mm biopsy punch by only excising the skin on the dorsal interscapular area. A sterile cotton swab soaked in 0.9% saline was used to clean the wounds. The induced wounds were left undressed throughout the whole duration of the study.
18 rabbits were divided to 3 groups. Each group comprised 6 rabbits. Group 1 (control rabbit): ulcers treated with vehicle only twice daily; Group 2: ulcers treated topically with OEA 2% w/v extract twice a day for 14 days until the wounds were completely cured; and Group 3: ulcers were topically treated with MEBO ® (Julphar Gulf Pharmaceutical Industries, Ras Al Khaimah, United Arab Emirates) (a comparable market product) twice daily for 14 days until the wounds were mostly cured.

Collection of Tissue Samples
On days seven and fourteen, full-thickness skin biopsies of complete ulcers from each rabbit were taken under anesthesia. Tissue samples were sectioned into three halves. Most of the dissected wound tissue was utilized for gene expression research. For histological investigation, the remainder was kept in formalin [43].

Percentage Wound Closure Rate
A camera (Fuji, S20 Pro, Sendai, Japan) was used to monitor the development of the wound region every three days until it healed fully. Using Image J 1.49 v software from the National Institutes of Health in Bethesda, Maryland, the wound area was assessed, and the wound closure rate was reported as a percentage change in the original wound area using the following formula: Wound closure (%) = Wound area on day 0 − Wound area on day nth Area o f the wound on day 0 × 100 n: days numbers. Additionally, a wound aspect ratio was established to explain observed variations in the shape and angular direction of wound contraction between groups. Using Image J, the length to width ratio was calculated from measurements of the wound's length (measured from head to tail) and width.

Histological Study
Dorsal skin samples from all wounds were taken and fixed in buffered formalin before being treated with a graded series of alcohol and xylene and subsequently immersed in paraffin blocks. Tissue slices were cut at a thickness of 4 µm and discolored with hematoxylin/eosin dye. The Leica Application Suite (Leica Microsystems, Wetzlar, Germany, a light microscope) was applied to evaluate and photograph the mounted slides [47].

Gene Expression Analysis Total RNA Extraction
In 0.5 mL TRIzol reagent (RNA Isolation Reagent, Invitrogen-ThermoFisher Products & Kits, Amresco, LLC-Solon, Waltham, MA, USA), 50 mg of dorsal skin tissue was homogenized using an ultrasonic homogenizer (Sonics-Vibracell, Sonics and Materials Inc., Newtown, Fairfield County, CT, USA). According to the guidelines of the producer, total RNA was extracted from dorsal skin tissues, and the concentration of RNA yield and purity were calculated [48].
Real-Time qRT-PCR cDNA synthesis with a constant RNA concentration across all samples, the Rever-tAid H Minus First Strand cDNA synthesis kit was used as directed by the manufacturer (#K1632, Thermo Scientific Fermentas, St. Leon-Ro, Germany). SYBER Green (Thermo Scientific Fermentas St. Leon-Ro, Germany-Maxima SYBER Green qPCR Master Mix (2X)) was employed in real-time PCR using single-stranded cDNAs. A StepOne Real-Time PCR System (Applied Biosystems, Thermo Fischer Scientific, Waltham, MA, USA) was used to perform qRT-PCR. The set of primers used for real-time PCR is mentioned in Table 1 qRT-PCR was performed using 0.02 g RNA per reaction and 10 Pmol of particular primers for thirty cycles of 95 • C for ten seconds and 60 • C for one minute. After normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene, gene expression levels were obtained. To assess the relative quantities of RNA, the comparative Ct approach was utilized. Formula 2 (−∆∆Ct) was used to determine the relative expression [12].
2.6. Molecular Modeling 2.6.1. Prediction of the Molecular Targets of the Dereplicated Metabolites of OEA By carrying out docking against all protein structures saved in the PDB (https:// www.rcsb.org/) (accessed on 1 March 2022), several suggested targets for the OEA-isolated molecules were putatively determined. In this analysis, small overlapping grids are adaptively built to constrain the searching space on protein surfaces, allowing them to run many accurate dockings runs in a shorter time [49]. The information was compiled as a ranking of binding affinity scores. We utilized a binding affinity score threshold of 7 kcal/mol to recognize the ideal receptors for each OEA-isolated molecule. Molecular dynamics simulation and estimate of binding free energy were carried out as previously mentioned using Desmon software [50]. The complete technique is included in the supplemental file.

In Vitro MMP-1 Activity Assay
OEA was tested for its inhibitory activity against MMP-1. The enzyme inhibition assay was conducted in accordance with the company instructions (Abcam, Waltham, MA USA, Cat. No: ab118973).

Statistical Analyses
Data are reported as the mean ± standard deviation of the mean (n = 6). Tukey's test for multiple comparisons was applied after one-way analysis of variance (ANOVA). Calculations involving statistics were performed using Graph Pad Prism 8 (San Diego, CA, United States). The outcomes were deemed significant when the p value was less than 0.05 [12].

Wound Closure Rate
In the in vivo model, the rate of wound closure was improved in all studied groups in a time-dependent manner. On the third post-injury day, the rate of wound closure was approximately 10% in all groups, with the control group having the lowest and the treated group having the highest. There was no significant variation among groups (p < 0.05). On 7th and 10th post-excision days, the rates of wound closure in the OEA-treated group were 32% and 81%, respectively, which seemed to be statistically greater (p < 0.05) than those in the control group. The OEA group had faster wound closure than the MEBO ® -treated group on the seventh and tenth days postinjury. On the fourteenth post-excision day, the rabbits treated with OEA were completely recovered from their wounds, and the wound contraction reached 100% in the OEA group ( Figures 5 and 6A,B). 7th and 10th post-excision days, the rates of wound closure in the OEA-treated group were 32% and 81%, respectively, which seemed to be statistically greater (p < 0.05) than those in the control group. The OEA group had faster wound closure than the MEBO ®treated group on the seventh and tenth days postinjury. On the fourteenth post-excision day, the rabbits treated with OEA were completely recovered from their wounds, and the wound contraction reached 100% in the OEA group ( Figures 5 and 6A,B). A two-way ANOVA test was performed to evaluate whether there was a significant difference between the groups. The data is presented as the mean ± SD. * p < 0.05 compared to the untreated group on the same day, and # p < 0.05 compared to the MEBO® group on the same day. (B) To clarify observed differences in the shape and direction of wound contraction between groups, the wound aspect ratio was calculated (length: width).

Histopathological Study
Seven Days Post-Treatment The ordinary edge of the injury had a natural epidermis, normal hair follicles, well- A two-way ANOVA test was performed to evaluate whether there was a significant difference between the groups. The data is presented as the mean ± SD. * p < 0.05 compared to the untreated group on the same day, and # p < 0.05 compared to the MEBO®group on the same day. (B) To clarify observed differences in the shape and direction of wound contraction between groups, the wound aspect ratio was calculated (length: width).

Histopathological Study Seven Days Post-Treatment
• Group I (control group).
The ordinary edge of the injury had a natural epidermis, normal hair follicles, wellformed collagen bundle dermis, and healthy sebaceous glands. Alternatively, the injury was packed with blood clots, inflammatory cellular infiltration, extravasated RBCs, and sloughed granulation material, with collagen tissues compactly organized in a strange pattern. The striated muscle appeared to have necrotic myo-fibers in the deepest area. (Figure 7A).
formed collagen bundle dermis, and healthy sebaceous glands. Alternatively, the i was packed with blood clots, inflammatory cellular infiltration, extravasated RBCs sloughed granulation material, with collagen tissues compactly organized in a str pattern. The striated muscle appeared to have necrotic myo-fibers in the deepest ( Figure 7A).
The blood clot seen above the incision remained visible, with inadequate reepi zation, and the granulation tissue filling the defect from below was mostly cellul comparison to other treated groups, the heavy collagen was combined with fibers were compactly formed in an irregular manner, leading to apparent scarring ( Figure   • Group III (MEBO ® -treated).
Epidermal cells can be seen spreading around the edges of the incision. The re thelization was lacking, though. Approximately mirroring the nearby normal dermis lagen fibers and inflammatory cells infiltration were seen populating the damage space between them. The reticular dermis had a large number of active extended spi shaped fibroblasts, basophilic cytoplasm, and open-face oval nuclei. (Figure 7C). showing the typical wound margin with the normal epidermis (asterisk). The wound was clogged with blood clots and granulation tissue (curved arrow). Group II (B) blood clot seen above the incision visible (curved arrow). Compactly arranged dermal collagen fibers are seen (aster). Group III (C) scar tissue (curved arrow) blocked the incision, and collagen fibers (stars) resembled the neighboring normal dermis with inflammatory cellular activity, particularly macrophages (black arrows). (H & E stain ×200).
The blood clot seen above the incision remained visible, with inadequate reepithelization, and the granulation tissue filling the defect from below was mostly cellular. In comparison to other treated groups, the heavy collagen was combined with fibers that were compactly formed in an irregular manner, leading to apparent scarring ( Figure 7B).
Epidermal cells can be seen spreading around the edges of the incision. The reepithelization was lacking, though. Approximately mirroring the nearby normal dermis, collagen fibers and inflammatory cells infiltration were seen populating the damage with space between them. The reticular dermis had a large number of active extended spindleshaped fibroblasts, basophilic cytoplasm, and open-face oval nuclei. (Figure 7C).
The wound area was larger and heavily covered in granulation tissue, which was made up of several connective tissue layers in an acidophilic matrix and covered a significant amount of inflammatory cellular infiltration. The dermis has substantial neovascularization and was made of thin, disordered collagen ( Figure 8A).
The wound had become plugged by scar tissue that had shrunk. In the epidermis, only one to three rows of epithelial cells have recovered. The granulation tissue from below was primarily cellular and fibroblast packed. Collagen fibers were disorganized-dense and compactly oriented in the reticular layer ( Figure 8B).
The ordinary squamous keratinized epithelium was present in the skin tissue. The dermis was covered in thin scar tissue. There were numerous hair follicles, a dermal matrix with blood capillaries, and no inflammatory cellular infiltration. The collagen bundles of the papillary dermis were depicted as thin interweaving bundles, while the reticular dermis evolved into rough, wavy bundles that were established in diverse routes ( Figure 8C). larization and was made of thin, disordered collagen ( Figure 8A).
The wound had become plugged by scar tissue that had shrunk. In the epidermis, only one to three rows of epithelial cells have recovered. The granulation tissue from below was primarily cellular and fibroblast packed. Collagen fibers were disorganizeddense and compactly oriented in the reticular layer ( Figure 8B).
The ordinary squamous keratinized epithelium was present in the skin tissue. The dermis was covered in thin scar tissue. There were numerous hair follicles, a dermal matrix with blood capillaries, and no inflammatory cellular infiltration. The collagen bundles of the papillary dermis were depicted as thin interweaving bundles, while the reticular dermis evolved into rough, wavy bundles that were established in diverse routes ( Figure  8C).

Figure 8. Group I (A) had a wide wound area (curved arrow), heavy inflammatory cellular infiltration in an acidophilic matrix (tailed arrow), and normal skin (asterisk). Group II (B) had an epidermis formed of 1-3 rows of epithelial cells (asterisk) and collagen fibers compactly arranged with prominent fibroblasts (stars).
Group III (C) had typical epithelium, thin scar tissue extending into the dermis (black arrow), and reticular dermis with coarse, wavy collagen bundles arranged in different directions (star). Newly formed hair follicles (circles) (H&E stain ×200).

Effect of OEA Treatment on Relative Gene Expression of TGF-β in Excisional Wounds
The relative expression expression of TGF-β in skin tissues was significantly statistically higher in OEA-treated injuries on the seventh and fourteenth days than in individuals from the control group (p < 0.05). Furthermore, OEA-treated wounds had considerable development in the expression of the marker compared to MEBO ® -treated wounds (Figure 9).

Figure 8. Group I (A) had a wide wound area (curved arrow), heavy inflammatory cellular infiltration
in an acidophilic matrix (tailed arrow), and normal skin (asterisk). Group II (B) had an epidermis formed of 1-3 rows of epithelial cells (asterisk) and collagen fibers compactly arranged with prominent fibroblasts (stars). Group III (C) had typical epithelium, thin scar tissue extending into the dermis (black arrow), and reticular dermis with coarse, wavy collagen bundles arranged in different directions (star). Newly formed hair follicles (circles) (H&E stain ×200).

Effect of OEA Treatment on Relative Gene Expression of TGF-β in Excisional Wounds
The relative expression expression of TGF-β in skin tissues was significantly statistically higher in OEA-treated injuries on the seventh and fourteenth days than in individuals from the control group (p < 0.05). Furthermore, OEA-treated wounds had considerable development in the expression of the marker compared to MEBO ® -treated wounds (Figure 9).
Metabolites 2022, 12, x FOR PEER REVIEW 13 of 21 Figure 9. Gene expression in diverse categories of wounded tissues. qRT-PCR was used to examine gene expression in wound tissues. After being adjusted for GAPDH, the results displayed as fold change relative to the control group. The bars represent the mean ± SD. To determine whether there was a substantial difference between studied groups, a two-way ANOVA was performed, with * p < 0.05 comparing to the control group on a particular day and # p < 0.05 comparing to the MEBO ® group on the same day.

Effect of OEA on IL-1β, TNF-α and MMP-1 Gene Expression in Excisional Injuries
According to the analysis of mRNA levels on the seventh postinjury day, inflammatory mediators (IL-1β and TNF-α) as well as MMP-1 gene expression was dramatically downregulated in injuries treated with OEA or MEBO ® comparing to control injuries (Figure 10). Wounds treated with OEA had a much more significant decrease in inflammatory Figure 9. Gene expression in diverse categories of wounded tissues. qRT-PCR was used to examine gene expression in wound tissues. After being adjusted for GAPDH, the results displayed as fold change relative to the control group. The bars represent the mean ± SD. To determine whether there was a substantial difference between studied groups, a two-way ANOVA was performed, with * p < 0.05 comparing to the control group on a particular day and # p < 0.05 comparing to the MEBO ® group on the same day.

Effect of OEA on IL-1β, TNF-α and MMP-1 Gene Expression in Excisional Injuries
According to the analysis of mRNA levels on the seventh postinjury day, inflammatory mediators (IL-1β and TNF-α) as well as MMP-1 gene expression was dramatically downregulated in injuries treated with OEA or MEBO ® comparing to control injuries ( Figure 10). Wounds treated with OEA had a much more significant decrease in inflammatory markers (IL-1β and TNF-α) and MMP-1 than MEBO ® -treated wounds. Furthermore, compared to control wounds, OEA or MEBO ® therapy for fourteen days resulted in significantly lower TNF-α and IL-1β, MMP-1 relative gene expression (p < 0.05). Again, TNF-α, IL-1β, and MMP-1 expression were significantly lower in OEA-treated wounds than in MEBO ® -treated wounds. Figure 9. Gene expression in diverse categories of wounded tissues. qRT-PCR was used to examine gene expression in wound tissues. After being adjusted for GAPDH, the results displayed as fold change relative to the control group. The bars represent the mean ± SD. To determine whether there was a substantial difference between studied groups, a two-way ANOVA was performed, with * p < 0.05 comparing to the control group on a particular day and # p < 0.05 comparing to the MEBO ® group on the same day.

Effect of OEA on IL-1β, TNF-α and MMP-1 Gene Expression in Excisional Injuries
According to the analysis of mRNA levels on the seventh postinjury day, inflammatory mediators (IL-1β and TNF-α) as well as MMP-1 gene expression was dramatically downregulated in injuries treated with OEA or MEBO ® comparing to control injuries (Figure 10). Wounds treated with OEA had a much more significant decrease in inflammatory markers (IL-1β and TNF-α) and MMP-1 than MEBO ® -treated wounds. Furthermore, compared to control wounds, OEA or MEBO ® therapy for fourteen days resulted in significantly lower TNF-α and IL-1β, MMP-1 relative gene expression (p < 0.05). Again, TNF-α, IL-1β, and MMP-1 expression were significantly lower in OEA-treated wounds than in MEBO ® -treated wounds. Figure 10. Gene expression in wounded rabbit tissues from several groups. qRT-PCR was utilized to assess gene expression in wounded tissues. After normalization to GAPDH, the data represented as fold change relative to the control group. The bars represent the mean ± SD. To determine whether there was a significant variation between studied groups, a one-way ANOVA test was utilized, with * p < 0.05 comparing to the control group on that day and # p < 0.05 comparing to the MEBO ® treated group on the same day. Figure 10. Gene expression in wounded rabbit tissues from several groups. qRT-PCR was utilized to assess gene expression in wounded tissues. After normalization to GAPDH, the data represented as fold change relative to the control group. The bars represent the mean ± SD. To determine whether there was a significant variation between studied groups, a one-way ANOVA test was utilized, with * p < 0.05 comparing to the control group on that day and # p < 0.05 comparing to the MEBO ® treated group on the same day.

Predicted Targets for Chemical Compounds in OEA
The inverse docking approach was used to accurately propose the most likely molecular targets of all the annotated compounds in OEA using the idTarget docking platform (http://idtarget.rcas.sinica.edu.tw) (accessed on 1 March 2022).
Divide-and-conquer docking is a unique docking strategy in which small overlapping grids are adaptively generated by idTarget to confine the examination area on the protein surfaces. This procedure allows the execution of many accurate dockings in a brief period of time. The query molecule can be virtually docked to practically all available crystal protein structures in the PDB.
A list of binding affinity scores, ranging from the most negative to the least, was created from the combined results. For each identified molecule in OEA, the best targets were determined using a threshold value of 7 kcal/mol for binding affinity. As an essential protein in the wound healing process, MMP1 was retrieved as a target protein for compounds 2, 5, 8, and 12 with binding affinity scores of −9.3, −8.8, −8.1, and −12.7 kcal/mol, respectively. Several earlier studies have demonstrated that the healing of wounds is adversely affected by MMPs. Hence, using MMP inhibitors has been shown to result in outstanding outcomes in wound healing.
All molecules were then re-docked toward the active site of MMP1 using AutoDock Vina. It was also revealed by the Vina docking results that compounds 2, 5, 8, and 12 were the structures reaching the highest-ranking scores, with binding affinity scores between −7.9 and −10.3 kcal/mol. After that, the molecular dynamic simulation-derived binding free energies (∆Gs) of all compounds were also calculated to further support the docking results. Once again, compounds 2, 5, 8, and 12 were the top-scoring structures, with ∆G values between −7.1 and −8.9 kcal/mol ( Figure 11).
tively. Several earlier studies have demonstrated that the healing of wounds is adversely affected by MMPs. Hence, using MMP inhibitors has been shown to result in outstanding outcomes in wound healing.
All molecules were then re-docked toward the active site of MMP1 using AutoDock Vina. It was also revealed by the Vina docking results that compounds 2, 5, 8, and 12 were the structures reaching the highest-ranking scores, with binding affinity scores between −7.9 and −10.3 kcal/mol. After that, the molecular dynamic simulation-derived binding free energies (ΔGs) of all compounds were also calculated to further support the docking results. Once again, compounds 2, 5, 8, and 12 were the top-scoring structures, with ΔG values between −7.1 and −8.9 kcal/mol ( Figure 11). Figure 11. Docking scores of compounds 1-26 inside the active site of MMP1 along with their calculated ΔGs. The −7 kcal/mol score was set as a cutoff to select the best hits. CCl = co-crystalized ligand.

Binding Mode Investigation, Molecular Dynamics Simulation, and In Vitro Validation
The binding orientations of compounds 2, 5, 8, and 12 (common lignans in olive; [37]) inside the binding site of MMP1 were quite similar to that of the co-crystallized ligand [59] (Figure 12). LEU-81, ALA-82, and HIS-118 are critical residues involved in hydrogen bond interactions with the co-crystalized inhibitor ( Figure 12A), while VAL-115, HIS-118, and TYR-140 are critical residues implicated in hydrophobic interactions. The binding modes of compounds 2 and 8 were quite similar to that of the co-crystalized ligand, where they were H-bonded to LEU-81 and ALA-82. Moreover, they established additional H- Figure 11. Docking scores of compounds 1-26 inside the active site of MMP1 along with their calculated ∆Gs. The −7 kcal/mol score was set as a cutoff to select the best hits. CCl = co-crystalized ligand.

Binding Mode Investigation, Molecular Dynamics Simulation, and In Vitro Validation
The binding orientations of compounds 2, 5, 8, and 12 (common lignans in olive; [37]) inside the binding site of MMP1 were quite similar to that of the co-crystallized ligand [59] ( Figure 12). LEU-81, ALA-82, and HIS-118 are critical residues involved in hydrogen bond interactions with the co-crystalized inhibitor ( Figure 12A Additionally, they interacted with both PRO-138 and VAL-115. We exposed the selected docked compounds to 50 ns molecular dynamic simulations (MDS) to validate these binding poses. Compounds 2, 5, and 12 achieved stable binding throughout the course of MDS with low deviations from their original pose (average RMSD ranged from 1 Å for compound 12 to 3 Å for compound 2). On the other hand, compound 8 was far less stable during MDS, where it was highly fluctuating inside the binding pocket of MMP1, particularly at the beginning of the simulation until 19.2 ns, and its overall RMSD was relatively high (~6 Å).
As a validation step of the previous in silico and modeling outcomes, OEA was tested in vitro against MMP-1. The results showed that the OEA crude extract had MMP-1 inhibitory activity with IC 50 = 88 ± 0.1 µM. This potent activity could be attributed to compounds 2, 5, 8, and 12 being significant components in OEA (peak area > 10,000).
bonds with ALA-84, PRO-138, and TYR-140 ( Figure 12A,C). They also established hydrophobic interactions identical to those of the cocrystalized inhibitor. They took a slightly different orientation regarding compounds 5 and 12. Both established a network of strong H-bonds (<2 Å) with ALA-84, ARG-114, PRO-138, SER-139, and THR-141 ( Figure 12B,D). They established several hydrophobic interactions similar to those of the cocrystalized inhibitor. Additionally, they interacted with both PRO-138 and VAL-115. We exposed the selected docked compounds to 50 ns molecular dynamic simulations (MDS) to validate these binding poses. Compounds 2, 5, and 12 achieved stable binding throughout the course of MDS with low deviations from their original pose (average RMSD ranged from 1 Å for compound 12 to 3 Å for compound 2). On the other hand, compound 8 was far less stable during MDS, where it was highly fluctuating inside the
Wound healing is a network-dynamic procedure of bringing back tissue construction in damaged tissues close to its probable ordinary state [62]. This requires three phases: (1) an inflammatory stage that involves a section of proinflammatory mediators and suppression of the immune system, (2) a proliferative stage in which fibroblast proliferation, collagen aggregation, and new blood vessel formation occurs, and (3) a remodeling stage that involves wounded tissue regeneration and reconstruction [63,64]. For efficient treatment, remedies that hasten wound healing with potential involvement in all process phases, fewer side effects, and limited cost are needed.
In this work, a significant contraction in the wounded area of excision wounds of experimental animals was revealed compared to the control wounds in topical OEA treatment. These observations are due to the accelerated wound contraction rate in OEA-treated animals. To speed up and encourage wound closure, wound contraction is the centripetal movement of the wound's margins [65][66][67]. Wound contraction therefore indicates reepithelialization, keratinocyte differentiation, granulation, fibroblast proliferation, and proliferation [67]. The obtained results agree with other works [68][69][70][71].
Complex interactions between cells and numerous growth factors are required during wound healing processes [72]. Throughout the phases of wound healing, TGF-β has an extremely urgent role in the inflammation and hemostasis stage. Inflammatory cells like neutrophils and macrophages are attracted to and stimulated by TGF-β. At the same time, multiple cellular activities, such as angiogenesis, re-epithelialization, granulation tissue growth, and ECM deposition, are orchestrated by it during the proliferative phase [72]. Fibroblasts are encouraged to multiply and diversify into myofibroblasts that participate in the remodeling stage of wound contraction [73]. Pastar [76]. These notes are consistent with our observations, which concluded that enhanced TGF-β1 expression following OEA topical application hastens wound healing, as noticed by the gene expression and the accelerated wound healing in this study (Figure 9).
Proinflammatory cytokines like IL-1β and TNF-α are only important during the initial response of skin wound healing in the inflammatory phase. Adequate TNF-α and IL-1β expression is required for neutrophil recruitment and the cleanup of the wound area to get rid of bacteria and other contaminants. Additionally, these inflammatory mediators are considered potent MMP inducer in fibroblasts and inflammatory cells. The damaged ECM is degraded and eliminated by MMP to facilitate wound repair [77]. Nevertheless, prolonged inflammation stage results in a deformative healing process, as proteinases and cytokines damage the tissue, resulting in the progress of chronic wounds. TNF-α is a crucial proinflammatory cytokines secreted by macrophages, which, along with IL-1β, hinders collagen production and fibroblast proliferation [78]. TNF-α activates NF-κB, which in round activates a multitude of proinflammatory cytokines encompassing proteases, such as MMP and TNF-α itself, to give rise to free soluble TNF-α. As a result, these inflammatory cytokine effects are potentiated [79]. Thus, suppressing TNF-α and IL-1β by OEA could suppress prolonged inflammation and avoid defective wound healing ( Figure 10). These investigations made us assume that OEA quickens and improves the curing process.
Conversely, area, collagen disruption, and hence ECM destruction could be stimulated by reactive oxygen species at high levels in the wounded area. Events like angiogenesis and re-epithelization, which are crucial for wound healing, are reduced when the ECM is damaged [80,81]. Furthermore, inflammation can be induced by elevated ROS, increasing proinflammatory cytokines, and as result lengthening inflammation [82]. Due to its SOD activity and H 2 O 2 scavenging activity, OEA may have antioxidant properties. This was confirmed by in vitro antioxidant studies, potentially eliminating ROS, and enhanc-ing wound-healing properties. The phenolic contents of OEA are responsible for these antioxidant actions [43].
Additionally, persistent inflammation results in wound healing failure, and wounds typically start a pathological state that requires more aggressive treatments. The capability of an agent to deal with the inflammatory wound process will either improve or quicken the healing process [83]. As opposed to that, MMPs have been proven to have a crucial impact in wound healing, and their inhibition has been associated with significant positive outcomes. The ability of some lignan-related compounds (2, 5, 8, and 12) to interact with MMP-1 was revealed by an in silico investigation of the OEA primary metabolites. As a validation step, OEA was tested in vitro against MMP-1. The results showed that the OEA crude extract had MMP-1 inhibitory activity with IC 50 = 88 ± 0.1.

Conclusions
As presented here, the Arbequine olive leaves extracts, have a different phytochemical composition. Through an increased rate of wound closure, enhancement of TGF-1, and suppression of inflammatory markers (TNF-and IL-1), this extract demonstrated outstanding wound healing activity. The ability of some lignan-related molecules (2, 5, 8, and 12) to interact with MMP-1 was suggested by a series of virtual screening and physics-based experiments, which were conducted to explore the binding modes of molecules within the potential active site of each molecular target. This study reflected the potential of Arbequina cultivars as a source of polyphenolic wound healers, like wild cultivars.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/metabo12090791/s1, Supplementary file. References [84][85][86][87] are cited in the supplementary materials. Institutional Review Board Statement: The study was conducted according to Ethics Committee, Faculty of Pharmacy, Deraya University, which signed this study, indicating that animals should not compete during any stage of investigation, and they should be cared for in conformity with the Catalog for the Care and Use of Laboratory Animals (ethical approval number: 8/2021).

Data Availability Statement:
The data presented in this study are available in article and supplementary material.