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Antimicrobial and Wound Healing Potential of a New Chemotype from Piper cubeba L. Essential Oil and In Silico Study on S. aureus tyrosyl-tRNA Synthetase Protein

Piper cubeba is an important plant commonly known as cubeb or Java pepper, and it is cultivated for its fruit and essential oils, largely used to treat various diseases. Up to today, there was no scientific report on wound healing activity. Thus, this study was initiated to evaluate for the first time the antimicrobial activity and wound healing potential of a new chemotype from Piper cubeba essential oil (PCEO) from fruits. Thirteen microbial strains have been selected to investigate the antimicrobial potential of PCEO. For the evaluation of the wound healing potential, sixteen rats were excised on the dorsal back and divided into four groups. The effect of PCEO on the malondialdehyde (MDA) and superoxide dismutase (SOD) activities in the healed wound area of rats and the biochemical parameters and skin histological analysis were also assessed. Results: Data showed that PCEO exhibited a powerful antimicrobial potential especially against Listeria monocytogenes and Staphylococcus aureus. In addition, the topical application of PCEO cream appears to increase the SOD level, wound healing and contraction but reduced the MDA amount suggesting an impressive and a rapid cutaneous healing power. Additionally, histopathological analysis of the granulation tissue revealed that the derma is properly restored and arranged after treatment with PCEO. The docking analysis of PCEO constituents against S. aureus tyrosyl-tRNA synthetase enzyme showed binding energies values in the range of −7.2 to −4.8 kcal/mol. In conclusion, the topic use of PCEO healing cream showed significant effect in accelerating the healing process, which may be attributed to the synergetic effect of antioxidant and antimicrobial properties of PCEO volatile constituents, making it a relevant therapeutic agent for the management of wounds and therefore confirming the popular traditional uses of this plant.


Introduction
The oxidative stress (as the results of elevated intracellular levels of reactive oxygen species (ROS)) along with redox signaling play a substantial role in regulating normal wound healing under pathological conditions [1]. ROS are centrally involved and having a significant role in the orchestration of the normal wound-healing response acting as cellular messengers to many immunocytes and non-lymphoid cells [1,2]. They also directly damage microbes that invade tissues and are known to play a pivotal role in regulating healing at low concentrations [2]. The normal healing response begins when the tissue is injured. Afterwards, an inflammatory response manifests itself and the collagen level starts to increase via cells below the dermis. During the wound-healing response, a number of cells used these radicals, including platelets, macrophages, fibroblasts, endothelial cells and keratinocytes [3]. Wound healing is a complex biological process, which consists of a well-ordered progression of events allowing restoring the integrity of damaged tissue and homeostasis after injury via the inflammatory phase, proliferation stages and tissue remodeling. Thereby, redox signaling and increased oxidative stress are responsible for impaired wound repair. The hydrogen peroxide (H 2 O 2 ) produced by dismutation of superoxide acts as intracellular messengers with low concentrations (100-250 µM) by stimulating key phases of wound healing including cell recruitment, production of cytokines and angiogenesis, however excessive and uncontrolled amount of ROS generation have deleterious effects by accelerating the non-healing wounds [4][5][6][7][8]. The antioxidants have been applied to remove the deleterious detrimental effects of ROS, by donating their own electrons, and therefore preventing them from capturing electrons from other important molecules, such as DNA, proteins and lipids [1]. Thus, various medicinal herbs and plants have been reported to offer promising biosources of bioactive molecules with various biological properties such as antioxidant [9][10][11][12][13][14], antimicrobial [10,[12][13][14][15], antibiofilm [16,17], antiquorum sensing [11,17,18] and antidiabetic [19]. On the other hand, several treatments are available for the wound management such as analgesics, antibiotics and nonsteroidal anti-inflammatory drugs, but most of them produce numerous unwanted side effects [8,20]. Due to the emergence and spread resistance of microorganisms colonizing infections to antibiotics and synthetic drugs currently used in conventional wound healing cure, growing attention has been afforded to the search for natural wound healing agents. Plant-based bioactive compounds have therefore been a potential source of antioxidants and healing agents, which have received a great deal of attention due to the increased of oxidative stress [21]. Their role is to induce rapid re-epithelialization of wounds with minimal scarring associated with potent antimicrobial effects [22]. Consequently, there is a need to use essential oil containing many bioactive compounds to discover new treatment options able to stimulate healing with antimicrobial potential to overcome the antimicrobial resistance and side effects. Throughout the world, the estimated global pharmaceutical market in 2022 is $1.12 trillion, indicating the growing demand for pharmaceuticals by a huge percentage of population. The Saudi market offers many wound care treatments, including Piper cubeba, which has been used as a remedy by the local population for various medical purposes [23,24]. Therefore, in light of the above facts and in the continuation of our investigations into Piper cubeba essential oil (PCEO) with therapeutic properties and limited data on this spice, we have now studied for the first time the effects of its essential oil on wound healing and also its antimicrobial activities against a wide range of strains supported by in silico study.
Accordingly, the present study was designed to evaluate the antimicrobial activity of PCEO (new chemotype) from dry berries against thirteen pathogenic strains and to demonstrate for the first time their wound healing effects using an in vivo rat wound model based on a series of clinical, biochemical and histopathological assays. Furthermore, molecular docking studies were performed to investigate the potential binding mode of the selected PCEO phytocompounds against S. aureus TyrRS known as an attractive target enzyme for finding new antibacterial agents.

Antimicrobial Activity
Due to the increased severity of diseases caused by microbial pathogens, essential oils and their major chemical constituents are potential candidates as antimicrobial agents. According to their inhibition zone diameter (IZD), minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) values, the tested essential oil exhibited different degrees of antimicrobial activity against a panel of pathogenic microorganisms using the agar diffusion method. Depending on the susceptibility of the tested bacteria (Table 1), PCEO exhibited IZD values ranging from 13.0 ± 0.8 to 23.0 ± 0.8 mm with Listeria monocytogenes (19.0 ± 0.5 vs. 12.0 ± 2.0 mm) was found to be the most sensitive whereas B. cereus (15.0 ± 0.6 vs. 26.0±1.0 mm) was found to be the most resistant strains. Additionally, in the case of Gram-positive bacteria, S. aureus (19.5 ± 1.0 mm) possesses noteworthy value (p < 0.05) as compared to the positive control, chloramphenicol (16.5 ± 0.5 mm). Additionally, PCEO inhibited moderately the growth of the others bacteria. Due to the increased severity of diseases caused by microbial pathogens, essential oils and their major chemical constituents are potential candidates as antimicrobial agents. According to their inhibition zone diameter (IZD), minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) values, the tested essential oil exhibited different degrees of antimicrobial activity against a panel of pathogenic microorganisms using the agar diffusion method. Depending on the susceptibility of the tested bacteria (Table 1), PCEO exhibited IZD values ranging from 13.0 ± 0.8 to 23.0 ± 0.8 mm with Listeria monocytogenes (19.0 ± 0.5 vs. 12.0 ± 2.0 mm) was found to be the most sensitive whereas B. cereus (15.0 ± 0.6 vs. 26.0±1.0 mm) was found to be the most resistant strains. Additionally, in the case of Gram-positive bacteria, S. aureus (19.5 ± 1.0 mm) possesses noteworthy value (p < 0.05) as compared to the positive control, chloramphenicol (16.5 ± 0.5 mm). Additionally, PCEO inhibited moderately the growth of the others bacteria.
The MICs, MBCs and MFCs results of PCEO against microbial strains ranged from 1.56 to 12.5 mg/mL (MICs) and 3.12 to 25 mg/mL (MBCs) for bacterial strains, and from 3.12 to 6.25 mg/mL (MICs) and 12.5 to 25 mg/mL (MFCs) for fungal strains. We note the highest susceptibility of F. oxysporum and F. sp. to PCEO with MIC of 3.12 mg/mL and MFC of 12.5 mg/mL, respectively.
In addition, we determined the ratio MBC/MIC for bacteria MFC/MIC for fungi. When these ratios were less than or equal 2.0, the PCEO was considered as bactericidal or fungicidal, or otherwise bacteriostatic or fungistatic, respectively. If the ratio is greater than or equal 16.0, the extract was considered ineffective. Based on the results depicted in Table 1, it can be concluded that our oil exerted a bactericidal effect against B. subtilis, S. aureus, K. pneumoniae, S. enterica, S. typhimurium and E. coli. On the contrary, amongst the 15.0 ± 0.8 a 18.0 ± 1.5 b 3.12 12.5 4 The data are expressed as mean± S.D. (n = 3). Diameter of inhibition zones of extract including diameter of well 6 mm. No inhibition. Chloramphenicol was used as a standard antibiotic. Cycloheximide was used as a standard antibiotic. ± Standard deviation of three replicates. Values followed by the same letter under the same row are not significantly different (p < 0.05).

Wound Healing Activity
In order to develop our natural resources, we are interested in the experimental evaluation of healing properties in rats on a cutaneous mechanical wound of PCEO, which has not yet been the subject of a prior scientific evaluation.

General Characteristics of Animals
All rats were weighed before and after the experiment. Results are illustrated in Table 2. A slight increase (p < 0.05) in the weight of the rats in all groups was observed after treatment. However, for the groups 1 and 2, these variations did not show a significant difference in average body (p > 0.05) weight between the groups of rats at the end of the experimental period.  Wound photographs of a rat representative of each group are shown in Figure 3. All wounds showed a similar appearance with bright red coloring. Nevertheless, this staining persisted even at the 5th postoperative day in untreated rats and bleeding occurred during the change of compresses that tended to adhere. Until the third day, the aspects of the wounds started to differentiate: the group treated with the PCEO showed a brown color as for the wounds of the reference lot, the control lot and the batch with the cream (without PCEO) presented a perilesional redness.

Qualitative Study: Chromatic Assessment of Wounds
Wound photographs of a rat representative of each group are shown in Figure 3. All wounds showed a similar appearance with bright red coloring. Nevertheless, this staining persisted even at the 5th postoperative day in untreated rats and bleeding occurred during the change of compresses that tended to adhere. Until the third day, the aspects of the wounds started to differentiate: the group treated with the PCEO showed a brown color as for the wounds of the reference lot, the control lot and the batch with the cream (without PCEO) presented a perilesional redness. On the 7th day, an inflammatory nipple accentuated around the damaged skin of untreated wounds was noticed. However, a brown crust was observed in the treated groups (with PCEO and reference). It was thicker and rather hard in rats treated with the standard healing wound compared to the tested PCEO from fruit. This crust persisted in the group treated with PCEO until the 9th day of the experiment.
From the 10th day, the group treated with PCEO started to drop to reveal a pinkish colored tissue that characterizes an epithelialization ending after 13 days without sequelae and without residual tissue. However, some wounds in the control group remained slightly open even though all the rats in the reference batch presented scarred wounds at the end of the experiment. Table 3 illustrates the average wound areas of the four groups and shows the percentages of average contraction of the wound surfaces at the 1st, 3rd, 5th, 7th, 9th, 11th and 13th days of the study. Delayed scarring was observed in control rats compared to all rats in the other groups. At day 13 in contrast to other groups, contraction of wounds in rats treated with PCEO was complete.

Quantitative Study: Evaluation of Percentages of the Contraction of Wounds
This study aimed to evaluate the healing effect of PCEO in a rat model compared to a reference product, "Cicaflora ® ". Topical application of the tested PCEO appeared to accelerate wound healing and contraction of the skin margins compared to controls. A healing effect was observed with PCEO significantly better than the reference. It allowed complete healing after 13 days. On the 7th day, an inflammatory nipple accentuated around the damaged skin of untreated wounds was noticed. However, a brown crust was observed in the treated groups (with PCEO and reference). It was thicker and rather hard in rats treated with the standard healing wound compared to the tested PCEO from fruit. This crust persisted in the group treated with PCEO until the 9th day of the experiment.
From the 10th day, the group treated with PCEO started to drop to reveal a pinkish colored tissue that characterizes an epithelialization ending after 13 days without sequelae and without residual tissue. However, some wounds in the control group remained slightly open even though all the rats in the reference batch presented scarred wounds at the end of the experiment.
Quantitative Study: Evaluation of Percentages of the Contraction of Wounds Table 3 illustrates the average wound areas of the four groups and shows the percentages of average contraction of the wound surfaces at the 1st, 3rd, 5th, 7th, 9th, 11th and 13th days of the study. Delayed scarring was observed in control rats compared to all rats in the other groups. At day 13 in contrast to other groups, contraction of wounds in rats treated with PCEO was complete.
This study aimed to evaluate the healing effect of PCEO in a rat model compared to a reference product, "Cicaflora ® ". Topical application of the tested PCEO appeared to accelerate wound healing and contraction of the skin margins compared to controls. A healing effect was observed with PCEO significantly better than the reference. It allowed complete healing after 13 days. The values are expressed on average ±SD (n = 4/group). Group 1: control rats treated with physiological saline; Group 2: rats treated with cream without PCEO; Group 3: rats treated with healing cream; Group 4: rats treated with the reference "Cicaflora ® ". (±): Standard deviation of three replicates. Values with a different letter (a,b) within a row of the same group are significantly different (p < 0.05). Values with a different letter (A-C) within a column of the same day are significantly different (p < 0.05).

Histological Study
Histological sections of the scarred areas of representative rats in each lot were stained with hematoxylin-eosin and shown in Figure 4. Hematoxylin-eosin staining of PCEO biopsies revealed a complete epithelial regeneration with well-structured layers that cover the entire surface of the wound ( Figure 5). The treated groups showed a high density of fibroblasts and collagen with some macrophages. In the healed biopsies of the reference group, there was a thin epithelium with a moderate density of inflammatory nuclei. However, the biopsies of the control group showed incomplete epithelial regeneration (Group 1) and delayed healing characterized by a strong infiltration of inflammatory cells marked by small round nuclei and strongly colored purple. The dermis showed pronounced hyperemia with macrophage aggregation. The values are expressed on average ±SD (n = 4/group). Group 1: control rats treated with physiological saline; Group 2: rats treated with cream without PCEO; Group 3: rats treated with healing cream; Group 4: rats treated with the reference "Cicaflora®". (±): Standard deviation of three replicates. Values with a different letter (a,b) within a row of the same group are significantly different (p < 0.05). Values with a different letter (A-C) within a column of the same day are significantly different (p < 0.05).

Histological Study
Histological sections of the scarred areas of representative rats in each lot were stained with hematoxylin-eosin and shown in Figure 4. Hematoxylin-eosin staining of PCEO biopsies revealed a complete epithelial regeneration with well-structured layers that cover the entire surface of the wound ( Figure 5). The treated groups showed a high density of fibroblasts and collagen with some macrophages. In the healed biopsies of the reference group, there was a thin epithelium with a moderate density of inflammatory nuclei. However, the biopsies of the control group showed incomplete epithelial regeneration (Group 1) and delayed healing characterized by a strong infiltration of inflammatory cells marked by small round nuclei and strongly colored purple. The dermis showed pronounced hyperemia with macrophage aggregation. and highly potent compounds has been carried out to provide more insights into the interactions and examine their affinity with the catalytic domain of TyrRS from S. aureus. The outcomes summarized in Figure 5, outlined that out of the twenty four identified PCEO compounds, the best free binding energy was allowed for viridiflorol (−7.2 kcal/mol) and the lowest one for β-myrcene (−4.8 kcal/mol). The presented docking data revealed that all PCEO phytoconstituents occupy active pockets of the proteins of the target enzyme.

Receptor-Ligands Interaction Analysis
In order to get insight into the potent antibacterial activity of the PCEO against pathogenic strains, molecular docking of the predominant and the most potent constituents were performed on the into the active site of TyrRS from S. aureus binding model based on the TyrRS complex structure (pdb code: 1jij). All interactions between the receptor and the selected ligands are outlined in Figure 6 and Table 6, respectively.
β-caryophyllene oxide-TyrRS (1JIJ) interactions: Gly38, Asp40, Thr42, His47, Thr75, Asp80, Ser82, Lys84, Arg88, Tyr170, Gln174, Asp195 and Gln196 residues were involved The results of the present study show that treatment with PCEO for 13 days induced a significant decrease (p < 0.05) in the level of MDA in rats at the level of the skin (0.78 ± 0.18 nmoles of MDA/mg of protein) ( Table 4) and induction of the wound caused an increase in the level of MDA. Treatment did restore this rate, indeed, the MDA level of group (3) and group (4) were lower than that of group (1). MDA represents the product generated during the oxidation of polyunsaturated fatty acids. Oxidative stress is characterized by ROS that induce lipid peroxidation, which contains a series of free radical chain reaction processes and is associated with several types of biological damage. In the present study, an increase in MDA levels was noted after wound induction.

Effect of Treatment on Superoxide Dismutases (SODs) Activity
SODs are ubiquitous enzymes that catalyze disproportionation of superoxide anions to hydrogen peroxide and molecular oxygen. The SOD enzyme plays a role as a powerful antioxidant that provides the first line of defense against the harmful effects of lipid peroxidation, it confers the property of "scavenger". In healed rats compared with those without wounds, a significant decrease in dermal SOD activities was observed, from 148.58 ± 44.13 to 80.64 ± 9.01 unit/mg protein) (p < 0.05) ( Table 5). The activities of SOD dermal rats treated with PCEO and "Cicaflora ® " were comparable but did not reach activity in control rats.
For SOD: the induction of the wound causes a decrease of the SOD rate, and the treatment increases it. Group (3) and Group (4) SODs are good relative to Group (1). To rationalize the antibacterial potential of PCEO, the binding mode of their major and highly potent compounds has been carried out to provide more insights into the interactions and examine their affinity with the catalytic domain of TyrRS from S. aureus. The outcomes summarized in Figure 5, outlined that out of the twenty four identified PCEO compounds, the best free binding energy was allowed for viridiflorol (−7.2 kcal/mol) and the lowest one for β-myrcene (−4.8 kcal/mol). The presented docking data revealed that all PCEO phytoconstituents occupy active pockets of the proteins of the target enzyme.

Receptor-Ligands Interaction Analysis
In order to get insight into the potent antibacterial activity of the PCEO against pathogenic strains, molecular docking of the predominant and the most potent constituents were performed on the into the active site of TyrRS from S. aureus binding model based on the TyrRS complex structure (pdb code: 1jij). All interactions between the receptor and the selected ligands are outlined in Figure 6 and Table 6, respectively. Eugenol-TyrRS (1JIJ) interactions: The second major compound eugenol ( kcal/mol) was bounded only by hydrophobic interactions to Tyr36, Asp177, Gly Asn124, Thr75, Tyr170, Asp40, Gln174, Ile200, Gln196, Gly192, Val191, Gln190, Gly38 (v der Waals interactions) and to Cys37 (4.39) and Leu70 (4.84) (Alkyl/Pi-Alkyl).

Compounds
Interacting

Discussion
Studies of the PCEO are very limited with antimicrobial (against thirteen strains) and wound healing properties and molecular docking targeting TyrRS from S. aureus was reported here for the first time.

Antimicrobial Activity
Microbial pathogens are a major cause of a significant number of various illnesses around the world. Therefore, to overcome their virulence, essential oils derived from plants have been proposed and tested as powerful strategies in alternative medicine generally accompanied by reduced side effects. In a previous study, the inhibitory effects of essential oils are enhanced by the effect of monoterpene and sesquiterpenes constituents (representing 22.72% in our study) on the cell membrane [25]. In this study, the higher inhibitory action of the tested PCEO might be due to the presence of eugenol and methyleugenol in high quantity. In fact, phenolic -OH groups can easily bind the active site of enzymes and alter their metabolism via the presence of a double bond in the α,β positions of the side chain and to a methyl group located in the γ position [26]. We note that methyleugenol is the most sensitive against bacteria than eugenol because of its higher hydrophobicity. The lowest effect of eugenol is probably due the presence of hydroxyl group that is not involved for hydrogen bonding and to the presence of methoxy group in the ortho position (of eugenol) that produced steric hindrance. Hyldgaard et al. [27] reported that methyleugenol and eugenol show higher activity against Gram-negative bacteria than Gram-positive bacteria. Caryophyllene, as monoterpene exerted antimicrobial properties against some strains [28] and its mode suggest that diffuse into and damage the cell membrane structures by altering the membrane, affecting the transport of ions and ATP and therefore, changing the fatty acid profile of different bacteria and affects various bacterial enzymes, including ATPase, histidine, carboxylase, amylase and protease [29,30]. Lopez et al. [31] reported that natural methyl eugenol lacking of free hydroxyl groups attached to the benzene ring proved ineffective against the oral bacteria Streptococcus sobrinus. Recently, Nazzaro et al. [29] confirmed that the antimicrobial activity of eugenol could be ascribed to the presence of a free hydroxyl group in the molecule. Studying the mechanism action of eugenol against several S. aureus isolates. Devi et al. [32] demonstrate that eugenol first alters the Salmonella membrane permeability followed by the leakage of ions and extensive loss of other cellular contents, and ultimately results in cell death.
In addition to what was quoted above, the variation of the susceptibility of the tested microorganisms could be attributed to their intrinsic properties that are related to the permeability of their cell surface to the PCEO.

Wound Healing Activity
The process of healing was carried out by immunological activities of the victim itself generating various risk factors such as infection and week immunity delay in healing has brought attention to promote this process [33,34]. The body weights behavior of the rats during the experimental period indicate clearly that all groups were uniform suggesting that PCEO had no side effects on the rats. Topical application of the oil appears to accelerate wound healing and contraction of the skin banks compared to controls. The phytochemical profile revealed that PCEO contains a considerable phenylpropanoids, especially in methyeugenol and eugenol that present an important role in the inflammatory cascade (prostaglandins, thromboxanes and leukotrienes). These substances act as mediators of inflammation and accelerate the inflammatory process [35]. Thus, they increase local neovascularization, remodeling of the extracellular matrix, fibroblastic cell migration and differentiation, which accelerates the healing wounds [36]. As a result, healing can act by one or more mechanisms at a time. The proliferative phase was started from the 3rd day. This phase is characterized by the formation of granulation tissue that involves angiogenesis, migration of fibroblasts and collagen synthesis [37]. Morphological examination of wounds showed a more elaborate granulation tissue in the groups treated with the PCEO and the reference healing, which is composed of capillary neovessels and fibroblastic elements that elaborate the collagen and mononuclear cells. After 7 days, an increased inflammatory swelling was noted in all control wounds. This pronounced inflammatory reaction noted in this group, even at late stages of the experiment indicates a prolonged inflammatory behavior [38]. The latter predisposes to a higher risk of superinfection of the wound and delayed healing. In contrast, the wounds of rats treated with the oil showed faster healing dynamics with total contraction after 13 days of treatment. The granulation tissue thus formed was the bed of a rapid re-epithelialization observed with the oil and the reference-healing wound resulting in a total closure of the wound.
Amongst the tested strains, the Gram-positive bacterium S. aureus was known to be resistant to a large range of antibiotics acting by colonizing acne and burn wounds, which induces serious local and deep tissue infections. The potent antibacterial activity of PCEO against this bacterium reinforces the healing potential of the oil.
Our results showed also that the level of MDA, lipid peroxidation marker, increased after wound induction. In contrast, treatment with PCEO reduced this level. Lipid peroxidation is found to be an important pathophysiological event inducing also oxidative deterioration of poly unsaturated fatty acids, which leads to cellular injury and also promotes the generation of ROS in the tissue site, which damage cell structures [39]. On the other hand, the application of PCEO cream in rats to wound tissue increased the SOD level suggesting the protective effect of the PCEO against the oxidative damage induced by these wounds. Therefore, the enhanced wound healing by SOD repaired the tissue damage, which is a result of the increased antioxidant enzyme level in the granulation tissue due to the high free radical scavenging effect of PCEO [24,40]. This effect have been recently confirmed by our team revealing that PCEO exhibited a potent antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing/antioxidant power (FRAP) and β-carotene bleaching assays in comparison to the positive controls, butylated hydroxytoluene and ascorbic acid and the results have been well supported by molecular docking analysis [24].
Increased antioxidant activity can serve as an additional compensatory mechanism to maintain cell integration and protection against free radical damage. This can be attributed to the presence of several antioxidant compounds namely, polyphenols, flavonoids, tannins and vitamins [41,42]. Additionally, a significant wealth of tested antioxidant PCEO was described, especially in methyleugenol and eugenol, which are known for their powerful antiradical effect and therefore could promote the healing process of wounds [43]. Getie et al. [44] suggest that antioxidants prevent cell damage, promote DNA synthesis, increase vascularity and improve the strength of collagen fibers. Their effects related to the individual or the additive effect of different phytoconstituents associated with the antimicrobial activity of the oil that contributes synergistically to the healing process [45][46][47][48].
Consequently, the PCEO accelerated the healing of mechanical wounds with a complete re-epithelialization and better quality than those of the reference product and especially the control group due to the synergistically effect between antibacterial and antioxidant action. Antioxidants act by reducing oxidative stress via their ability to minimize lipid peroxidation and inflammatory damages, scavenge free radicals, promote the proliferation of fibroblast adhesion during wound healing, however antimicrobials induce the formation of a suitable environment for wound healing by preventing wound infections. Thus, the PCEO cream can be conveniently and safely used for the management of wound healing.

Molecular Docking Study vs. Antimicrobial Effect
The strongest activity of PCEO as new antimicrobial agents, prompted us to study the molecular docking inside the active site of S. aureus TyrRS to understand their interactions and predict the affinity and the activity of the potent bioactive molecules. Our docking results (e.g., for eugenol: Tyr36, Cys37, Gly38, Asp40, Leu70, Gly72, Thr75, Asn124, Tyr170, Gln174, Asp177, Gln190, Val191, Gly192, Gln196 and Ile200) seem to be in good agreement with those reported previously by Beg el al. [49]. In fact, these authors docked the bioactive compounds of the Calotropis gigantea flower extract against TyrRS and found that eugenol can interact with the following TyrRS residues Cys37, Leu70, Thr75, Asn124, Tyr170 and Ile 200. These interactions are in full accordance with the binding residues involved in the ternary structure of S. aureus TyrRS (Cys37, Gly38, Ala39, Asp40, His47, Gly49, His50, Leu70, Thr75, Gln174, Asp177, Gln190, Gly192, Asp195 and Pro222). Despite the antimicrobial contribution of eugenol and methyl eugenol (cited below), our docking results were well supported based on the literature survey. The sesquiterpene alcohol, spathulenol, displayed an inhibitory effect against C. neoformans and E. faecalis and against S. aureus, S. epidermidis and E. coli for Helichrysum fulgidum essential oil [50,51]. In Eugenia calycina leaf essential oil, the same compound present antimicrobial activity against anaerobic bacteria P. nigrescens and P. gingivalis [52]. In Salvia cilicica, it possesses an inhibitory effect against M. tuberculosis, M. gypseum, T. mentagrophytes and Candida spp. [53]. The sesquiterpene alcohol viridiflorol known for its lipophilicity founded into the phospholipid bilayers of bacterial membrane, inducing its disruption [54,55]. It interacts with the bacterial membrane and the consequences it bring out at the cellular level. Mechanistically, it interferes with the respiratory chain reaction and energy production [56]. It showed also antifungal activity against C. cucumerinum and P. oryzae [57,58]. The sesquiterpenes δ-cadinene presents a cadinane skeleton with farnesyl pyrophosphate as the precursor was demonstrated for its antimicrobial activity [59]. The isolation of δ-cadinene, sesquiterpenes from the essential oil obtained from the fruit of Schinus molle was found to be active against S. pneumoniae [60]. Additionally, caryophyllene oxide, an oxygenated terpenoid has been shown to exert significant antifungal activity [59]. It has been also reported to possess antimicrobial properties against a wide range of bacteria and fungi [61]. Silva et al. [62] have been showed that the antimicrobial activity of the essential oil of Psidium guajava leaves was related to its chemical constituents, and especially to the known caryophyllene oxide. Thus, our in vitro and in silico results are in agreement with the study of the literature thus confirming the significant antimicrobial effect of the main constituents of PCEO justifying its future use as an anti-infectious agent.

Plant Material and Extraction of Essential Oil
Cubeba pepper dry berries were purchased from a Saudi market (Baljurashi city, Albaha) and imported from India. The taxonomic position of this plant species was confirmed by Dr. Zouhair Noumi botanist in the Department of Life Sciences (Faculty of Sciences, University of Sfax, Sfax 3000, Soukra, Tunisia). Dried powder of the fruits of the Piper cubeba plant (800 g) was subjected to hydrodistillation for 4 h using a Clevenger type apparatus [13]. The obtained essential oils were dried using anhydrous sodium sulfate, and stored in sealed vials in the dark, at 4 • C for subsequent analysis.

Chemical Analysis GC/MS
The chemical composition of the PCEO have been identified using a GC/MS apparatus, on an Agilent 6890 series gas chromatograph interfaced with an Agilent 19091S-433 (Agilent Technologies, Santa Clara, CA, USA) (a detailed experiment was cited in reference [24]).

Microbial Strains Used
Antimicrobial assay was performed in in vitro cultures across 13 microbial strains, including 5 Gram-positive bacteria (Bacillus cereus (JN 934390) as the most important cause of food poisoning, Bacillus subtilis (JN 934392), which are endospore-forming, rod-shaped, facultative anaerobic bacteria that are widely distributed in nature, Staphylococcus aureus (ATCC 6538) as a sphere-shaped bacteria responsible of skin infections and can cause also pneumonia and heart valve infections, Listeria monocytogenes (ATCC 19115) as a food-borne pathogen responsible for listeriosis, a sickness with a high mortality rate, Micrococcus luteus (NCIMB 8166) is a non-motile, non-sporing cocci belonging to the Micrococcea family considered as a non-pathogenic saprophyte of human skin and eye), 4 Gram-negative bacteria (Klebsiella pneumonia (ATCC 10031) often causes bacterial pneumonia, or infection of the lungs, Salmonella enteric (ATCC 43972) and Salmonella typhimurium (ATCC 19430) are the dominant cause of non-typhoidal Salmonella infections and Escherichia coli (ATCC 25922) as a pathogenic bacteria predominately found in the intestinal lumen) and 4 fungi (Pythium catenulatum (AY598675) known to be plant pathogens of both crops and vegetables, Fusarium oxysporum (AB586994), Fusarium phyllophilum (AB587006) and Fusarium sp. (JX391934) frequently are responsible of skin infections and can produce mycotoxins, which cause various disorders, including cancer in animals and humans). The evaluation of the antimicrobial capacity was carried out based on the same protocol as displayed by Ghannay et al. [63,64]. The bacteria were seeded on petri dishes containing Mueller Hinton agar (MH) and incubated for 24 h. From these plates, bacterial culture was prepared in 3 mL MH broth for 24 h at 200 rpm and 37 • C. The optical density was read at 600 nm and adjusted at 0.08-0.10 density equivalent to 10 8 CFU/mL. 20 mL of (MH) agar were cast in Petri dishes 90 mm in diameter. After solidification, 100 µL of the bacterial suspension (10 8 CFU/mL) were spread on the surface of the agar using a sterile swab. After 5 min of contact, 6 mm diameter wells were dug with a sterile Pasteur pipette in MH and each well was filled with 75 µL of sample. Dimethylsulfoxide (DMSO) was served as a negative control. The Petri dishes thus prepared were incubated at +4 • C for 4 h to allow the diffusion of the active substance. Finally, the dishes were incubated in an oven at 37 • C or 30 • C, for 24 or 48 h depending on the microorganism tested. The tests were carried out in triplicate and the results are expressed in mm. The bacterial strains showing sensitivity to the EO were selected to determine the minimum inhibitory and bactericidal concentrations.
As for the fungal strains, the growth was carried out at 30 • C on Sabouraud agar until the mycelial growth covered the whole box. From these dishes, a spore suspension was obtained in 10 mL water containing sterile tween. Similarly, the optical density was adjusted to 0.08-0.10 in order to obtain a solution equivalent to 10 6 spores/mL.

Determination of MIC, MBC and MFC
The MIC is the lowest concentration in extract inhibiting any visible growth of microorganisms. It was determined by the microdilution method used by Felhi et al. [63]. Serial and semiserial dilutions were prepared to obtain a concentration range. Thus, 90 µL of MH broth for the bacteria were dispersed in each well by adding 10 µL of each inoculum already prepared. Of each dilution of the PCEO 100 µL was thus poured. The last well, containing the MH for bacteria without addition of the oil, was considered as a positive growth control. In addition, another well, containing DMSO without PCEO, was used as a negative control. The final volume in each well was equal to 200 µL.
After homogenization of the contents, the plates were incubated at 37 • C for 24 h for the bacterial strains. MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide) was used as an indicator of viability of microorganisms in each well and the mixture was incubated at 37 • C for 30 min. Wells, where microbial growth was inhibited, remained colorless after incubation with MTT while those with bacterial growth turned blue.
In contrast, MBC is the minimum concentration that inhibits any visible growth of microorganisms for 48 h at 37 • C. With regard to the fungal strains, after incubation, the first tubes of which inhibition is complete, relative to the controls, are seeded in plates containing Sabouraud medium without EO for 3-4 days at 30 • C. The concentration is called fungicide (MFC) when there is no resumption of fungal mycelium growth [64].
The bacterial and fungal strains showing sensitivity to the oil are selected to determine the ratios MBC/MIC and MFC/MIC. Indeed, an antibiotic is said bactericidal when the MBC/MIC ratio is less or equal to 2, bacteriostatic when it is between 4 and 16 and beyond 16 it is considered inactive [10].

Used Animals, Experimental Groups and Mechanical Wound Induction
Wistar albino rats (180-250 g) of both sexes were selected to carry out the experiment. Four rats were taken for each group. The rats were used after an acclimatization period of 7 days to the laboratory environment. They were housed in standard metal cages and provided with food and water ad libitum. The animals were treated in accordance with the current national guidelines on animal care and use. Four groups of animals containing 4 rats in each group were anaesthetized by the open mask method with anesthetic ether.
The evaluation of the wound healing effect was carried out on rats weighing 189.19 ± 12.35 g. These rats were divided into four groups of 4 rats each: Group 1: control rats receiving no treatment, but treated only by cleaning with physiological saline; Group 2: rats treated with cream without PCEO; Group 3: rats treated with healing cream (0.52 mg/mm 2 ) and Group 4: rats treated with the reference healing agent "Cicaflora ® " (0.13 mg/mm 2 ).
The rats were depilated on the back and a predetermined area of 500 mm 2 full thickness skin was excised in the dorsal. Rat's wounds were left undressed to the open environment. This model was used to monitor wound contraction and epithelialization time. The reference standard drug "Cicaflora ® " and cream including PCEO (prepared by mixing with petroleum jelly in a concentration of 5% w/w) were applied every day until the wound was completely healed. The progressive changes in wound area were monitored planimetrically by tracing the wound margin on a graph paper every alternate day. The changes in healing of the wound or the measurement of the wound area traced on graph paper were expressed as unit (mm 2 ). Wound contraction was expressed as a percentage reduction of original wound size.

Oxidative Stress
Oxidative status in skin tissue could be estimated from the concentration of lipid peroxidation and the activity of antioxidant enzymes, malondialdehyde (MDA) and superoxide dismutase (SOD). To measure these indicators organs tissues were grind into small pieces and inversed into 2 mL ice cold buffer (TBS, pH 7.4); the mixtures were homogenized on ice using an Ultra-Turraks homogenizer for 15 min and then filtered and centrifuged. Supernatants were collected and stored until use.

Histopathological Study
The skin tissues of each group of rats were processed for histopathological examinations and observed for the histological changes under microscope and the microscopic slides were photographed [22].

Molecular Docking Analysis
Interactions between natural compound and S. aureus tyrosyl-tRNA synthetase were assessed by in silico molecular docking in order to explore the preferred orientation of the ligands in the binding site of receptor. The crystal structures were obtained from the Protein Data Bank: S. aureus tyrosyl-tRNA synthetase (PDB code 1JIJ). All water molecules have been removed and the co-crystallized ligand from the structures. Polar hydrogens and Gasteiger charges were assigned with AutoDockTools1.5.2 (ADT) and the PDBQT file format was prepared [65]. ADT was used to select a docking grid. In 1JIJ, the grid box site was established in −10.908, 14.432 and 86.420 Å (x, y and z). The grid box size for the coordinates x, y and z was 25 Å, with a grid spacing of 0.375 Å.
The structures of the natural compounds were minimized using a conjugate gradient AMMP incorporated in VEGA ZZ. [66] The conversion of the file from PDB to PDBQT was done using ADT. We applied AutoDock Vina software [67] with an exhaustiveness parameter of 32 to perform the docking simulations. ADT was used for the docking conformation analysis. Receptor ligand interactions are visualized by Discovery Studio Visualizer [68].

Statistical Analysis
A one-way analysis of variance (ANOVA) and Tukey's post hoc test was performed to determine significant differences between the responses using SPSS 19 statistical package (SPSS Ltd., Woking, UK). Means and standard deviations were calculated [24].

Conclusions
In summary, PCEO from fruits showed potent antibacterial and antifungal activities against several pathogenic strains and antioxidant potential reinforcing the popular useful of this plant in traditional cure. The topical application of PCEO cream on rats exhibited significant wound healing potency by stimulating the wound contraction, increasing the antioxidant enzymes activities and regenerating skin tissues. In addition, computational study confirms the high potency of PCEO as an anti-infection agent. Therefore, the beneficial effects of PCEO cream on wound healing might be related to the dual antioxidant and antimicrobial properties of PCEO constituents able to prevent oxidative damage triggered by free radicals and to treat infections caused by pathogenic bacteria and fungi. Further studies with purified constituents are required to elucidate the optimal conditions, the prin-cipal bioactive compound and the mechanism of wound healing activity completed with clinical assays to assess the modalities of administration in practice.