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

Fahad Alminderej 1, Sana Bakari 2, Tariq I. Almundarij 3, Mejdi Snoussi 4,5 , Kaïss Aouadi 1,6 and Adel Kadri 2,7,*

1 Department of Chemistry, College of Science, Qassim University, Buraidah 51452, Saudi Arabia; [email protected] (F.A.); [email protected] (K.A.) 2 Department of Chemistry, Faculty of Science of Sfax, University of Sfax, B.P. 1171, Sfax 3000, Tunisia; [email protected] 3 Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University, PO Box 6622, Buraidah 51452, Saudi Arabia; [email protected] 4 Department of Biology, College of Science, Hail University, P.O. Box 2440, Ha’il 2440, Saudi Arabia; [email protected] 5 Laboratory of Genetics, Biodiversity and Valorization of Bio-Resources (LR11ES41), University of Monastir, Higher Institute of Biotechnology of Monastir, Avenue Tahar Haddad, BP74, Monastir 5000, Tunisia 6 Faculty of Sciences of Monastir, University of Monastir, Avenue of the Environment, Monastir 5019, Tunisia 7 Faculty of Science and Arts in Baljurashi, Albaha University, P.O. Box (1988), Albaha 65527, Saudi Arabia * Correspondence: [email protected]; Fax: +216-74-27-44-37

Abstract: Piper cubeba is an important commonly known as cubeb or pepper, and it is cultivated for its 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 Citation: Alminderej, F.; Bakari, S.; essential oil (PCEO) from . Thirteen microbial strains have been selected to investigate the Almundarij, T.I.; Snoussi, M.; Aouadi, antimicrobial potential of PCEO. For the evaluation of the wound healing potential, sixteen rats were K.; Kadri, A. Antimicrobial and excised on the dorsal back and divided into four groups. The effect of PCEO on the malondialde- Wound Healing Potential of a New hyde (MDA) and superoxide dismutase (SOD) activities in the healed wound area of rats and the Chemotype from Piper cubeba L. biochemical parameters and skin histological analysis were also assessed. Results: Data showed Essential Oil and In Silico Study on that PCEO exhibited a powerful antimicrobial potential especially against Listeria monocytogenes and S. aureus tyrosyl-tRNA Synthetase Staphylococcus aureus. In addition, the topical application of PCEO cream appears to increase the SOD Protein. Plants 2021, 10, 205. https:// doi.org/10.3390/plants10020205 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

Received: 29 November 2020 revealed that the derma is properly restored and arranged after treatment with PCEO. The docking Accepted: 15 January 2021 analysis of PCEO constituents against S. aureus tyrosyl-tRNA synthetase enzyme showed binding Published: 22 January 2021 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 Publisher’s Note: MDPI stays neutral synergetic effect of antioxidant and antimicrobial properties of PCEO volatile constituents, making it with regard to jurisdictional claims in a relevant therapeutic agent for the management of wounds and therefore confirming the popular published maps and institutional affil- traditional uses of this plant. iations. Keywords: Piper cubeba L.; dry ; volatile oil; antimicrobial; healing effect; biochemical parame- ters; molecular docking

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and The oxidative stress (as the results of elevated intracellular levels of reactive oxygen conditions of the Creative Commons species (ROS)) along with redox signaling play a substantial role in regulating normal Attribution (CC BY) license (https:// wound healing under pathological conditions [1]. ROS are centrally involved and having creativecommons.org/licenses/by/ 4.0/). a significant role in the orchestration of the normal wound-healing response acting as

Plants 2021, 10, 205. https://doi.org/10.3390/plants10020205 https://www.mdpi.com/journal/plants Plants 2021, 10, 205 2 of 20

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 (H2O2) produced by dismutation of superoxide acts as intracellular messengers with low concentrations (100–250 µM) by stim- ulating 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 [4–8]. 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 and plants have been reported to offer promising biosources of bioactive molecules with various bio- logical properties such as antioxidant [9–14], antimicrobial [10,12–15], 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 an- tibiotics 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 op- tions 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 , 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.

2. Results 2.1. Phytochemical Descriptions Continuous to our previous work on the effectiveness of PCEO, GC–MS analyses have been already carried out recently by our team and here we are only able to present the structure of the identified compounds, which will be used for biological activities and Plants 2021, 10, 205 3 of 20

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thethe molecular molecular docking docking study study [24]. [24]. The The chemical chemical structures structures of of the the twenty-four twenty-four compounds compounds identifiedidentified in in the the essential essential oil oil tested tested using using the the GC–MS GC–MS technique technique are are listed in Figure 1 1..

FigureFigure 1. ChemicalChemical structure structure of of the the 24 24 compounds compounds identified identified in in the the essential essential oil oil extracted extracted from from cubeba cubeba pepper dry berries byby using using hydrodistillation hydrodistillation technique. technique. Legend: Legend: (1) β (1)-myrceneβ-myrcene 1.23%, 1.23%,(2) D-limonene (2) D-limonene 0.12%, (3) 1,8-cineole 0.12%, (3) 2.94%, 1,8-cineole (4) β-ocimene 2.94%, 0.30%, (4) β- (5) α-terpinolene 1.41%, (6) linalool 0.22%, (7) terpinen-4-ol 1.80%, (8) p-cymene-8-ol 3.50%, (9) α-terpineol 0.96%, (10) estragole 0.15%, α p α (11)ocimene citronellol 0.30%, 0.10%, (5) (12)-terpinolene (E)-geraniol 1.41%, 0.19%, (13) (6) linalooleugenol 33.95%, 0.22%, (14) (7) terpinen-4-olβ-elemene 0.66%, 1.80%, (15) me (8)thyleugenol-cymene-8-ol 41.31%, 3.50%, (16) β (9)--terpineol 5.65%,0.96%, (17) (10) α-caryophyllene estragole 0.15%, 1.14%, (11) (18) citronellol Germacrene 0.10%, D 0.15%, (12) (19) (E)-geraniol α-selinene 0.47%, 0.19%, (20) (13) δ-cadinene eugenol 0.19%, 33.95%, (21) (14) spathulenolβ-elemene 0.18%, 0.66%, (22) β(15)-caryophyllene methyleugenol oxide 0.96%, 41.31%, (23) (16) viridiflβ-caryophylleneorol 0.39% and (24) 5.65%, isocembrol (17) α -caryophyllene0.16%. 1.14%, (18) Germacrene D 0.15%, (19) α- selinene 0.47%, (20) δ-cadinene 0.19%, (21) spathulenol 0.18%, (22) β-caryophyllene oxide 0.96%, (23) viridiflorol 0.39% and (24) isocembrol 0.16%. Figure 2 summarized the main compounds identified. Results revealed the richness of this oil in methyleugenol (41.31%) and eugenol (33.95%) followed by β-caryophyllene (5.65%), p-cymene-8-ol (3.50%) and 1,8-cineole (2.94%).

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Figure2 summarized the main compounds identified. Results revealed the richness of this oil in methyleugenol (41.31%) and eugenol (33.95%) followed by β-caryophyllene Plants 2020, 9, x FOR PEER REVIEW (5.65%), p-cymene-8-ol (3.50%) and 1,8-cineole (2.94%). 4 of 21

FigureFigure 2.2. ChromatogramChromatogram showingshowing thethe mainmain identifiedidentified compoundscompounds inin PiperPiper cubebacubeba essentialessential oiloil (PCEO):(PCEO): methyleugenolmethyleugenol (41.31%),(41.31%), eugenoleugenol (33.95%),(33.95%), ββ-caryophyllene-caryophyllene (5.65%),(5.65%), pp-cymene-8-ol-cymene-8-ol (3.50%)(3.50%) andand 1,8-cineole1,8-cineole (2.94%).(2.94%).

2.2. Biological Properties 2.2.2.2.1. Biological Antimicrobial Properties Activity 2.2.1. Antimicrobial Activity Due to the increased severity of diseases caused by microbial pathogens, essential oils andDue their to the major increased chemical severity constituents of diseases are potential caused candidates by microbial as pathogens,antimicrobial essential agents. oilsAccording and their to majortheir chemicalinhibition constituents zone diameter are potential (IZD), minimum candidates inhibitory as antimicrobial concentration agents. According(MIC), minimum to their bactericidal inhibition zoneconcentration diameter (MBC) (IZD), and minimum minimum inhibitory fungicidal concentration concentra- (MIC),tion (MFC) minimum values, bactericidal the tested concentrationessential oil exhi (MBC)bited and different minimum degrees fungicidal of antimicrobial concentration ac- (MFC) values, the tested essential oil exhibited different degrees of antimicrobial activity tivity against a panel of pathogenic microorganisms using the agar diffusion method. De- against a panel of pathogenic microorganisms using the agar diffusion method. Depending pending on the susceptibility of the tested bacteria (Table 1), PCEO exhibited IZD values on the susceptibility of the tested bacteria (Table1), PCEO exhibited IZD values ranging ranging from 13.0 ± 0.8 to 23.0 ± 0.8 mm with Listeria monocytogenes (19.0 ± 0.5 vs. 12.0 ± 2.0 from 13.0 ± 0.8 to 23.0 ± 0.8 mm with Listeria monocytogenes (19.0 ± 0.5 vs. 12.0 ± 2.0 mm) mm) was found to be the most sensitive whereas B. cereus (15.0 ± 0.6 vs. 26.0±1.0 mm) was 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, found to be the most resistant strains. Additionally, in the case of Gram-positive bacteria, S. S. aureus (19.5 ± 1.0 mm) possesses noteworthy value (p < 0.05) as compared to the positive 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 control, chloramphenicol (16.5 ± 0.5 mm). Additionally, PCEO inhibited moderately the growth of the others bacteria. growth of the others bacteria. As shown in Table1, Fusarium sp. (15.0 ± 0.8 vs. 18.0 ± 1.5 mm) and F. oxysporum Table 1. Antimicrobial parameters, inhibition zones, minimum inhibitory concentration (MIC), minimum bactericidal (17.0 ± 0.1 vs. 20.0 ± 2.0 mm) were found to be the most sensitive fungi to PCEO, whereas concentration (MBC), minimum fungicidal concentration (MFC) (mg/mL), MBC/MIC and MFC/MIC of PCEO. no activity was observed against F. phyllophilum. The MICs, MBCsInhibition and MFCsZones resultsDiameter of (mm) PCEO against microbialPCEOstrains ranged from 1.56 to 12.5 mg/mL (MICs) and 3.12 to 25 mg/mL (MBCs)MIC for bacterial strains, and from Microorganisms Tested MBC MBC/MIC 3.12 to 6.25 mg/mLPCEO (MICs) andChloramphenicol 12.5 to 25 mg/mL (MFCs)mg/m for fungal strains. We note the mg/mL Ratio highest susceptibility of F. oxysporum and F. sp. to PCEOL with MIC of 3.12 mg/mL and Gram-positive bacteriaMFC of 12.5 mg/mL, respectively. In addition, we determined the ratio MBC/MIC for bacteria MFC/MIC for fungi. Bacillus cereus (JN 934390) 15.0 ± 0.6 a 26.0 ± 1.0 b 3.12 12.5 4 When these ratios were less than or equal 2.0, the PCEO was considered as bactericidal a b Bacillus subtilis (JN 934392)or fungicidal, or otherwise16.0 ± 0.7 bacteriostatic 24.0 ± or0.0 fungistatic, 6.25 respectively. 12.5 If theratio 2 is greater b a Staphylococcus aureus (ATCCthan or 6538) equal 16.0,19.5 the ± extract 1.0 was considered 16.5 ± 0.5 ineffective.1.56 Based3.12 on the results 2 depicted in b a Listeria monocytogenes (ATCCTable1 19115), it can be concluded 19.0 ± 0.5 that our 12.0 oil ± exerted 2.0 a bactericidal1.56 effect3.12 against 2 B. subtilis, S. a b Micrococcus luteus (NCIMBaureus 8166), K. pneumoniae 16.0, ±S. 0.8 enterica , S.typhimurium 20.0 ± 0.0 and E.1.56 coli . On6.25 the contrary, 4 amongst the Gram-negative bacteria Klebsiella pneumoniae (ATCC 10031) 13.0 ± 0.8 a 22.0 ± 1.0 b 3.12 6.25 2 Salmonella enterica (ATCC 43972) 23.0 ± 0.8 b 16.0 ± 0.0 a 12.5 25 2 Salmonella typhimurium (ATCC 19430) 13.5 ± 1.5 a 17.0 ± 1.0 b 6.25 12.5 2 Escherichia coli (ATCC 25922) 21.0 ± 0.8 a 23.5 ± 0.5 b 3.12 6.25 2 MFC/MIC Fungal strains PCEO Cycloheximide MIC MFC Ratio

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tested fungi, P. catenulatum, F. oxysporum and F. sp. showed a fungistatic effect however, F. phyllophilum remains ineffective.

Table 1. Antimicrobial parameters, inhibition zones, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum fungicidal concentration (MFC) (mg/mL), MBC/MIC and MFC/MIC of PCEO.

Inhibition Zones Diameter (mm) PCEO Microorganisms Tested MIC MBC MBC/MIC PCEO Chloramphenicol mg/mL mg/mL Ratio Gram-positive bacteria Bacillus cereus (JN 934390) 15.0 ± 0.6 a 26.0 ± 1.0 b 3.12 12.5 4 Bacillus subtilis (JN 934392) 16.0 ± 0.7 a 24.0 ± 0.0 b 6.25 12.5 2 Staphylococcus aureus (ATCC 6538) 19.5 ± 1.0 b 16.5 ± 0.5 a 1.56 3.12 2 Listeria monocytogenes (ATCC 19115) 19.0 ± 0.5 b 12.0 ± 2.0 a 1.56 3.12 2 Micrococcus luteus (NCIMB 8166) 16.0 ± 0.8 a 20.0 ± 0.0 b 1.56 6.25 4 Gram-negative bacteria Klebsiella pneumoniae (ATCC 10031) 13.0 ± 0.8 a 22.0 ± 1.0 b 3.12 6.25 2 Salmonella enterica (ATCC 43972) 23.0 ± 0.8 b 16.0 ± 0.0 a 12.5 25 2 Salmonella typhimurium (ATCC 19430) 13.5 ± 1.5 a 17.0 ± 1.0 b 6.25 12.5 2 Escherichia coli (ATCC 25922) 21.0 ± 0.8 a 23.5 ± 0.5 b 3.12 6.25 2 MFC/MIC Fungal strains PCEO Cycloheximide MIC MFC Ratio Pythium catenulatum (AY598675) 13.0 ± 0.5 a 17.5 ± 1.5 b 6.25 25 4 Fusarium oxysporum (AB586994) 17.0 ± 0.1 a 20.0 ± 2.0 b 3.12 12.5 4 Fusarium phyllophilum (AB587006) 0.0 ± 0.0 a 14.5 ± 0.5 b --- Fusarium sp. (JX391934) 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).

2.2.2. 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 Table2. 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.

Table 2. Mean weights before and after treatment of rats with a mechanical wound.

Weights (g) Test Groups Before Treatment After Treatment (Day 13) Group 1: control rats treated with physiological saline 188.4 ± 9.6 aA 207.0 ± 8.8 bA Group 2: rats treated with cream without PCEO 189.6 ± 10.4 aA 205.7 ± 8.0 bA Group 3: rats treated with PCEO 188.8 ± 13.5 aA 226.2 ± 7.9 bC Group 4: rats treated with the reference “Cicaflora®” 187.3 ± 9.5 aA 215.0 ± 5.8 bB The values are represented as mean ±SD (n = 4/group). (±): 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 treatment are significantly different (p < 0.05). Plants 2021, 10, 205 6 of 20

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2.2.2.2.Qualitative Qualitative Study: Study: Chromatic Chromatic Assessment Assessment of Wounds of Wounds WoundWound photographs photographs of of a a rat rat representative representative of of each each group group are are shown shown in in Figure Figure 33.. All woundswounds showed showed a a similar similar appearance appearance with with brig brightht red red coloring. coloring. Nevertheless, Nevertheless, this this staining staining persistedpersisted even even at at the the 5th 5th postoperative postoperative day day in in untreated untreated rats rats and and bleeding bleeding occurred occurred during during thethe change change of of compresses compresses that that tended tended to to ad adhere.here. Until Until the the third third da day,y, the the aspects aspects of of the the woundswounds started started to differentiate: the groupgroup treatedtreated withwith the the PCEO PCEO showed showed a a brown brown color color as asfor for the the wounds wounds of of the the reference reference lot, lot, the the controlcontrol lotlot andand the batch with the the cream cream (without (without PCEO)PCEO) presented presented a a perilesional perilesional redness. redness.

FigureFigure 3. 3. PhotographPhotograph of of mechanical mechanical wounds wounds of of the the four four groups groups during during the the treatment treatment period. period.

OnOn the the 7th 7th day, day, an an inflammatory inflammatory nipple nipple accentuated accentuated around around the the damaged damaged skin skin of of untreateduntreated wounds wounds was was noticed. noticed. However, However, a abrown brown crust crust was was observed observed in in the the treated treated groupsgroups (with (with PCEO PCEO and and reference). reference). It It was was thicke thickerr and and rather rather hard hard in in rats rats treated treated with with the the standardstandard healing healing wound wound compared compared to to the the tested tested PCEO PCEO from from fruit. fruit. This This crust crust persisted persisted in in thethe group group treated treated with with PCEO PCEO until until the the 9th 9th day day of of the the experiment. experiment. FromFrom the the 10th 10th day, day, the the group group treated treated with with PCEO PCEO started started to to drop drop to to reveal reveal a a pinkish pinkish coloredcolored tissue tissue that that characterizes characterizes an an epithelialization epithelialization ending ending after after 13 13 days days without without sequelae sequelae andand without residualresidual tissue. tissue. However, However, some some wounds wounds in the in control the control group group remained remained slightly slightlyopen even open though even though all the ratsall the in therats reference in the reference batch presented batch presented scarred scarred wounds wounds at the end at theof theend experiment. of the experiment. Quantitative Study: Evaluation of Percentages of the Contraction of Wounds 2.2.2.3. Quantitative Study: Evaluation of Percentages of the Contraction of Wounds Table3 illustrates the average wound areas of the four groups and shows the percent- Table 3 illustrates the average wound areas of the four groups and shows the per- ages of average contraction of the wound surfaces at the 1st, 3rd, 5th, 7th, 9th, 11th and centages of average contraction of the wound surfaces at the 1st, 3rd, 5th, 7th, 9th, 11th 13th days of the study. Delayed scarring was observed in control rats compared to all rats and 13th days of the study. Delayed scarring was observed in control rats compared to all in the other groups. At day 13 in contrast to other groups, contraction of wounds in rats ratstreated in the with other PCEO groups. was complete.At day 13 in contrast to other groups, contraction of wounds in rats treatedThis study with PCEO aimed was to evaluate complete. the healing effect of PCEO in a rat model compared to aThis reference study product, aimed to “Cicaflora evaluate ®the”. Topicalhealing applicationeffect of PCEO of the in testeda rat model PCEO compared appeared to to ® aaccelerate reference woundproduct, healing "Cicaflora and". contraction Topical application of the skin of marginsthe tested compared PCEO appeared to controls. to ac- A celeratehealing wound effect was healing observed and contraction with PCEO of significantly the skin margins better compared than the reference. to controls. It allowedA heal- ingcomplete effect was healing observed after 13with days. PCEO significantly better than the reference. It allowed com- plete healing after 13 days.

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Table 3. Evolution of the average wound surfaces (cm2) of the four groups.

Day Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13

Group 1 3.70 ± 0.48 2.69 ± 0.15 eAB 2.21 ± 0.26 dA 1.46 ± 0.25 0.46 ± 0.08 bAB 0.26 ± 0.08 abA 0.19 ± 0.04 ab

eC cA

Plants 2021,Group10, 205 2 3.11 ± 0.84 2.63 ± 0.52 eAB 2.15 ± 0.80 dA 1.45 ± 0.28 0.37 ± 0.14 bA 0.25 ± 0.06 abA 0.17 ± 0.04 Ab 7 of 20 fA cA

Group 3 3.43 ± 0.64 2.55 ± 0.36 cA 2.59 ± 0.38 cb 2.44 ± 0.98 0.60 ± 0.14 bB 0.22 ± 0.05 abA 0.11 ± 0.02 aA Table 3. Evolution of the average wound surfaces (cm2) of the four groups. cB cC Day Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13 Group 4 3.70 ± 0.37 2.80 ± 0.39 eB 2.52 ± 0.40 Db 1.80 ± 0.41 0.48 ± 0.12b 0.34 ± 0.20 aB 0.28 ± 0.18 aC Group 1 3.70 ± 0.48 eC 2.69 ± 0.15 eAB 2.21 ± 0.26 dA 1.46 ± 0.25 cA 0.46 ± 0.08 bAB 0.26 ± 0.08 abA 0.19 ± 0.04 ab fC cB AB Group 2 3.11 ± 0.84 fA 2.63 ± 0.52 eAB 2.15 ± 0.80 dA 1.45 ± 0.28 cA 0.37 ± 0.14 bA 0.25 ± 0.06 abA 0.17 ± 0.04 Ab GroupThe 3 values3.43 are± 0.64exprecB ssed2.55 on ±average0.36 cA ±SD2.59 (n =± 4/group).0.38 cb Group2.44 ± 0.981: controlcC 0.60rats ±trea0.14tedbB with0.22 physiological± 0.05 abA saline;0.11 ± Group0.02 aA 2: Grouprats 4 treated3.70 with± 0.37 creamfC without2.80 ± 0.39 PCEO;eB Group2.52 ± 3:0.40 ratsDb treated1.80 wi± 0.41th healingcB 0.48 cream;± 0.12b GroupAB 4:0.34 rats± treated0.20 aB with0.28 the± reference0.18 aC "Cicaflora®".The values are expressed(±): Standard on average deviation±SD of (n =three 4/group). replicates. Group Values 1: control with rats a treated different with letter physiological (a,b) within saline; aGroup row of 2: the rats same treated group arewith significantly cream without different PCEO; Group (p < 3:0.05). rats treatedValues with with healing a different cream; Groupletter 4:(A–C) rats treated within with a column the reference of the “Cicaflora same day®”. ( ±are): Standardsignificantly differentdeviation of(p three< 0.05). 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).

2.2.2.4. Histological Study Histological Study HistologicalHistological sections sections of theof scarredthe scarred areas ofareas representative of representative rats in each rats lot in were each stained lot were withstained hematoxylin–eosin with hematoxylin–eosin and shown and in Figureshown4 .in Hematoxylin–eosin Figure 4. Hematoxylin–eosin staining of PCEOstaining of biopsiesPCEO biopsies revealed revealed a complete a epithelialcomplete regeneration epithelial regeneration with well-structured with well-structured layers that cover layers thethat entire cover surface the entire of the surface wound of (Figure the wound5). The (F treatedigure 5). groups The treated showed groups a high densityshowed of a high fibroblastsdensity of andfibroblasts collagen and with collagen some macrophages. with some macrophages. In the healed biopsiesIn the healed of the biopsies reference of the group,reference there group, was a thinthere epithelium was a thin with epithelium a moderate with density a moderate of inflammatory density nuclei.of inflammatory How- ever,nuclei. the However, biopsies of the the biopsies control group of the showed control incomplete group showed epithelial incomplete regeneration epithelial (Group regener- 1) andation delayed (Group healing 1) and characterizeddelayed healing by acharacterized strong infiltration by a strong of inflammatory infiltration cells of inflammatory marked bycells small marked round by nuclei small andround strongly nuclei coloredand strongly purple. colored The dermispurple. showedThe dermis pronounced showed pro- hyperemianounced hyperemia with macrophage with ma aggregation.crophage aggregation.

Figure 4. Photomicrographs of histological sections of cutaneous scars mechanical wounds (40) at day 13 of treatment (hematoxyline–osin staining). EP: epidermis, Der: dermis, (↑): inflammatory cells, (*): blood vessels.

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2.3. Molecular Docking Study 2.3.1. Binding Energies vs. PCEO Compounds 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 in- teractions 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 Plants 2021, 10, 205 kcal/mol) and the lowest one for β-myrcene (−4.8 kcal/mol). The presented docking data8 of 20 revealed that all PCEO phytoconstituents occupy active pockets of the proteins of the tar- get enzyme.

FigureFigure 5. Binding energies energies of of the the identified identified PCEO PCEO co compoundmpound docked docked with with TyrRS TyrRS receptor receptor (PDB: (PDB: 1JIJ). 1JIJ). Malonedialdehyde (MDA) Level 2.3.2.Malonedialdehyde Receptor–Ligands Interaction (MDA) is Analysis considered as one of the degradation products of lipid peroxidationIn order to reactions, get insight it is into formed the potent by reactive antibacterial oxygen activity species of during the PCEO the against attack of path- polyun- saturatedogenic strains, fatty acids.molecular Lipid docking peroxidation of the waspredominant evaluated and by thethe measurementmost potent constituents of thioarbituric acidwere reactiveperformed substances on the into (TBARS) the active including site of TyrRS aldehydes, from MDAS. aureus and binding hydroperoxide model based lipids. on theThe TyrRS results complex of the structure present (pdb study code: show 1jij). thatAll interactions treatment between with PCEO the receptor for 13 daysand in- ducedthe selected a significant ligands are decrease outlined (p in< 0.05)Figure in 6 the and level Table of 6, MDA respectively. in rats at the level of the skin (0.78Viridiflorol-TyrRS± 0.18 nmoles of MDA/mg(1JIJ) interactions: of protein) It can (Table be suggested4) and induction from our molecular of the wound docking caused anresults increase tabulated in the in level (Table of MDA.6) that Treatmentviridiflorol didexhibiting restore the this highest rate, indeed, binding the energy MDA (-7.2 level of groupkcal/mol). (3) andThis group compound (4) were was lower nicely than bound that to of TyrRS group with (1). van der Waals interactions with Tyr36, Cys37, Gly38, Ala39, Asp40, Gly72, Thr75, Asp80, Asn124, Tyr170, Gln174, Table 4. MalonedialdehydeAsp177, (MDA) Gln190, level of Val191, the different Glu192, groups Gln196 of rats, and expressed Ile200 residues in nmoles and of MDA/mgAlkyl interactions of protein. with only Leu70 (4.71) (5.33). δ-cadinene-TyrRSWith (1JIJ) Wounds interactions: δ-cadinene (-7.1 kcal/mol) formed Without van Wounds der Group 1Waals interaction Group with 2 Gly38, Asp40, Group Asp80, 3 Tyr170, GroupGln174, 4 Thr75, Gln190, Group Gly192, A NormalGly193, CreamGln196 ( −and) PCEO Ile200 and CreamAlkyl/Pi-Alkyl (+) PCEO interactions Reference with Tyr36 (4.63), NotCys37 Treated (4.65), Ala39 (4.35) and Leu70 (3.99) (5.18), respectively. MDA ± d 1.19 ± 0.41 c ± b ± b 0.43 ± 0.28 a 1.67 0.56 Spathulenol-TyrRS (1JIJ) interact0.78 ions:0.18 Spathulenol0.61 (-6.6 0.24kcal/mol) was able to build ± Standard deviation of three replicates.van der ValuesWaals followed interactions by the same with letter Cys37, under Al thea39, same Gly38, row, for Asp40, each parameter, Thr75, are Asp80, not significantly Asn124, different (p < 0.05). Asp177, Gln190, Gly 192, Gln196, Asn199 and Ile200, C-H bond interactions with Gln174 (1.81)MDA and Alkyl/Pi-Alkyl represents the interactions product generated with Tyr36 during (4.88) the (5.35), oxidation Leu70 of (4.24) polyunsaturated (5.28) and Tyr fatty acids.170 (5.31). Oxidative stress is characterized by ROS that induce lipid peroxidation, which β-caryophyllene oxide-TyrRS (1JIJ) interactions: Gly38, Asp40, Thr42, His47, Thr75, contains a series of free radical chain reaction processes and is associated with several Asp80, Ser82, Lys84, Arg88, Tyr170, Gln174, Asp195 and Gln196 residues were involved 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) (Table5). 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). Plants 2021, 10, 205 9 of 20

Table 5. Effect of treatment on SOD activity, expressed as (unit/mg protein).

With Wounds Without Wounds Group 1 Group 2 Group 3 Group 4 Group A Normal Cream (−) PCEO Cream (+) PCEO Reference Not Treated SOD 80.64 ± 9.01 a 121.37 ± 21.77 b 148.58 ± 44.13 c 144.67 ± 38.87 c 165.87 ± 8.99 d ± Standard deviation of three replicates. Values followed by the same letter under the same row, for each parameter, are not significantly different (p < 0.05).

2.3. Molecular Docking Study 2.3.1. Binding Energies vs. PCEO Compounds 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. ThePlants outcomes 2020, 9, x FOR summarized PEER REVIEW in Figure5, outlined that out of the twenty four identified PCEO 10 of 21 − compounds, the best free bindingin the stability energy of TyrRS- was allowedβ-caryophyllene for viridiflorol oxide (−6.5 ( kcal/mol)7.2 kcal/mol) by van der and Waals interac- the lowest one for β-myrcenetions, ( −Alkyl/Pi-Alkyl4.8 kcal/mol). interaction The presented with Ala39docking (5.12) anddata His50 revealed (4.78) and thatonly one H-bond all PCEO phytoconstituentswith occupy His50 (2.19). active pockets of the proteins of the target enzyme. Methyleugenol-TyrRS (1JIJ) interactions: The first PCEO major compound methyleu- 2.3.2. Receptor–Ligands Interactiongenol (−5.8 kcal/mol) Analysis established several van der Waals interactions with the following TyrRS residues named Gly38, Asp40, Leu70, Thr75, Asp80, Asn124, Tyr170, Gln174, Gly In order to get insight into the potent antibacterial activity of the PCEO against 192, Gly193 and Gln196, one H-bond interaction with Asp177 (3.77), one C-H bond inter- pathogenic strains, molecularaction docking with Pro45 of the (3.45) predominant and also one alkyl and theinteraction most potent with Cys37 constituents (4.75). were performed on the into theEugenol-TyrRS active site of (1JIJ) TyrRS interactions: from S. aureus The bindingsecond major model compound based on eugenol (6.3 the TyrRS complex structurekcal/mol) (pdb code:was bounded 1jij). All only interactions by hydrophobic between interactions the receptor to Tyr36, and theAsp177, Gly72, selected ligands are outlinedAsn124, in Figure Thr75,6 Tyr170, and Table Asp40,6, Gln174, respectively. Ile200, Gln196, Gly192, Val191, Gln190, Gly38 (van der Waals interactions) and to Cys37 (4.39) and Leu70 (4.84) (Alkyl/Pi-Alkyl).

Eugenol-1JIJ

Methyleugenol-1JIJ

Figure 6. Cont.

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Plants 2020, 9, x FOR PEER REVIEW 11 of 21

β-caryophyllene oxide-1JIJ

δ-cadinene-1JIJ

Germacrene D-1JIJ

Figure 6. Cont.

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Plants 2020, 9, x FOR PEER REVIEW 12 of 21

Viridiflorol-1JIJ

Spathulenol-1JIJ Figure 6. Two dimensional (2D) and three dimensional (3D) residual interactions network of selected PCEO with the active Figure 6. site of tyrosylTwo tRNA dimensional synthetase (2D)(PDB: and 1JIJ). three dimensional (3D) residual interactions network of selected PCEO with the active site of tyrosyl tRNA synthetase (PDB: 1JIJ). Table 6. Molecular docking result analysis of major and potent bioactive compounds from essential oil of P. cubeba against tyrosyl-tRNAViridiflorol-TyrRS synthetase from (1JIJ)Staphylococcus interactions: aureus (PDB It Id: can 1JIJ) be enzyme. suggested from our molecular dock- ing results tabulated in (Table6) thatInteracting viridiflorol Residues exhibiting the highestBinding binding Energy energy Compounds (−7.2 kcal/mol). This compound wasReceptor nicely vs bound. Targets to 1JIJ TyrRS with van der(kcal/mol) Waals interac- tions with Tyr36, Cys37, Gly38, Ala39,van der Asp40,Waals: Gly38, Gly72, Asp40, Thr75, Leu70, Asp80, Asn124, Tyr170, Gln174, Thr75, Asp80, Asn124, Tyr170, Gln174, Asp177,Methyleugenol Gln190, Val191, Glu192, Gln196 and Ile200 residues and Alkyl interactions with Gly 192, Gly193, Gln196. H-bond: −5.8 only Leu70(Main compound (4.71) (5.33). 1) δ-cadinene-TyrRS (1JIJ) interactions:Asp177 (3.77). C-Hδ-cadinene bond: Pro45 (− (3.45).7.1 kcal/mol) formed van der Alkyl: Cys37 (4.75) Waals interaction with Gly38, Asp40, Asp80, Tyr170, Gln174, Thr75, Gln190, Gly192, van der Waals: Tyr36, Gly38, Asp40, Gly193, Gln196 and Ile200 andGly72, Alkyl/Pi-Alkyl Thr75, Asn124, interactions Tyr170, Gln174, with Tyr36 (4.63), Cys37 (4.65), Eugenol Ala39 (4.35) and Leu70 (3.99) (5.18),Asp177, respectively. Gln190, Val191, Gly192, −6.3 (Main compound 2) Spathulenol-TyrRS (1JIJ) interactions:Gln196, Ile200. SpathulenolAlkyl/Pi-Alkyl: (− Cys376.6 kcal/mol) was able to build van der Waals interactions with Cys37, Ala39,(4.39), Leu70 Gly38, (4.84) Asp40, Thr75, Asp80, Asn124, Asp177, Gln190, Gly 192, Gln196, Asn199van and der Ile200,Waals: Gly38, C-H bond Asp40, interactions Asp80, with Gln174 (1.81) and Alkyl/Pi-Alkylδ-cadinene interactions withTyr170, Tyr36 Gln174, (4.88) Thr75, (5.35), Gln190, Leu70 Gly192, (4.24) (5.28) and Tyr7.1 170 (5.31). β-caryophyllene oxide-TyrRSGly193, (1JIJ) Gln196, interactions: Ile200. Alkyl/Pi-Alkyl: Gly38, Asp40, Thr42, His47, Thr75, Asp80, Ser82, Lys84, Arg88, Tyr170, Gln174, Asp195 and Gln196 residues were involved in the stability of TyrRS-β-caryophyllene oxide (−6.5 kcal/mol) by van der Waals interactions, Alkyl/Pi-Alkyl interaction with Ala39 (5.12) and His50 (4.78) and only one H-bond with His50 (2.19). Methyleugenol-TyrRS (1JIJ) interactions: The first PCEO major compound methyleugenol (−5.8 kcal/mol) established several van der Waals interactions with the following TyrRS residues named Gly38, Asp40, Leu70, Thr75, Asp80, Asn124, Tyr170, Gln174, Gly 192, Plants 2021, 10, 205 12 of 20

Gly193 and Gln196, one H-bond interaction with Asp177 (3.77), one C-H bond interaction with Pro45 (3.45) and also one alkyl interaction with Cys37 (4.75). Eugenol-TyrRS (1JIJ) interactions: The second major compound eugenol (6.3 kcal/mol) was bounded only by hydrophobic interactions to Tyr36, Asp177, Gly72, Asn124, Thr75, Tyr170, Asp40, Gln174, Ile200, Gln196, Gly192, Val191, Gln190, Gly38 (van der Waals interactions) and to Cys37 (4.39) and Leu70 (4.84) (Alkyl/Pi-Alkyl).

Table 6. Molecular docking result analysis of major and potent bioactive compounds from essential oil of P. cubeba against tyrosyl-tRNA synthetase from Staphylococcus aureus (PDB Id: 1JIJ) enzyme.

Interacting Residues Binding Energy Compounds Receptor vs. Targets 1JIJ (kcal/mol) van der Waals: Gly38, Asp40, Leu70, Thr75, Asp80, Asn124, Tyr170, Methyleugenol Gln174, Gly 192, Gly193, Gln196. H-bond: Asp177 (3.77). C-H bond: Pro45 −5.8 (Main compound 1) (3.45). Alkyl: Cys37 (4.75) van der Waals: Tyr36, Gly38, Asp40, Gly72, Thr75, Asn124, Tyr170, Gln174, Eugenol Asp177, Gln190, Val191, Gly192, Gln196, Ile200. Alkyl/Pi-Alkyl: Cys37 −6.3 (Main compound 2) (4.39), Leu70 (4.84) van der Waals: Gly38, Asp40, Asp80, Tyr170, Gln174, Thr75, Gln190, δ-cadinene Gly192, Gly193, Gln196, Ile200. Alkyl/Pi-Alkyl: Tyr36 (4.63), Cys37 (4.65), 7.1 Ala39 (4.35), Leu70 (3.99) (5.18) van der Waals: Gly38, Asp40, Thr42, His47, Thr75, Asp80, Ser82, Lys84, Arg88, Tyr170, Gln174, Asp195, Gln196. H-bond: His50 (2.19). β-caryophyllene oxide −6.5 Alkyl/Pi-Alkyl: Ala39 (5.12), His50 (4.78)

van der Waals: Tyr36, Cys37, Gly38, Ala39, Asp40, Gly72, Thr75, Asp80, Viridiflorol Asn124, Tyr170, Gln174, Asp177, Gln190, Val191, Glu192, Gln196, Ile200. −7.2 Alkyl: Leu70 (4.71) (5.33) van der Waals: Cys37, Ala39, Gly38, Asp40, Thr75, Asp80, Asn124, Spathulenol Asp177, Gln190, Gly 192, Gln196, Asn199, Ile200. C-H bond: Gln174 (1.81). −6.6 Alkyl/Pi-Alkyl: Tyr36 (4.88) (5.35), Leu70 (4.24) (5.28), Tyr 170 (5.31)

3. 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.

3.1. 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 and constituents (represent- ing 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 Plants 2021, 10, 205 13 of 20

strains [28] and its mode suggest that diffuse into and damage the cell membrane struc- tures 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.

3.2. 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 phytochem- ical 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 angio- genesis, 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 per- oxidation is found to be an important pathophysiological event inducing also oxidative deterioration of poly unsaturated fatty acids, which leads to cellular injury and also pro- motes 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 Plants 2021, 10, 205 14 of 20

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 power- ful 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 antimi- crobial activity of the oil that contributes synergistically to the healing process [45–48]. Consequently, the PCEO accelerated the healing of mechanical wounds with a com- plete re-epithelialization and better quality than those of the reference product and espe- cially the control group due to the synergistically effect between antibacterial and antioxi- dant 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.

3.3. 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 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 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 Plants 2021, 10, 205 15 of 20

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.

4. Materials and Methods 4.1. Used Products For antimicrobial tests: Mueller Hinton agar and potato dextrose agar (PDA) were purchased from Bio-Rad (Bio-Rad, France). For the wounds healing evaluation: Bal- ance rocking, scissors, razor blade, knife blade, pliers, cotton, syringe, beaker, spatula, “Cicaflora®”, physiological serum, HCl (2%) and ether were used too.

4.2. Plant Material and Extraction of Essential Oil Cubeba pepper dry berries were purchased from a Saudi market (Baljurashi city, Albaha) and imported from . 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.

4.3. 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]).

4.4. Antimicrobial Activity 4.4.1. 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 vegeta- bles, 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 108 CFU/mL. 20 mL of (MH) agar were cast in Petri dishes 90 mm in diameter. After solidification, 100 µL of the bacterial suspension (108 CFU/mL) were spread on the surface of the agar using a sterile swab. After 5 min of contact, 6 mm Plants 2021, 10, 205 16 of 20

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 106 spores/mL.

4.4.2. Determination of MIC, MBC and MFC The MIC is the lowest concentration in extract inhibiting any visible growth of mi- croorganisms. 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].

4.5. Healing Activity 4.5.1. 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/mm2) and Group 4: rats treated with the reference healing agent “Cicaflora®” (0.13 mg/mm2). The rats were depilated on the back and a predetermined area of 500 mm2 full thick- ness skin was excised in the dorsal. Rat’s wounds were left undressed to the open environ- ment. This model was used to monitor wound contraction and epithelialization time. The reference standard drug “Cicaflora®” and cream including PCEO (prepared by mixing with Plants 2021, 10, 205 17 of 20

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 (mm2). Wound contraction was expressed as a percentage reduction of original wound size.

4.5.2. 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 super- oxide 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.

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

4.6. 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].

4.7. 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].

5. 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 benefi- cial 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- Plants 2021, 10, 205 18 of 20

cipal bioactive compound and the mechanism of wound healing activity completed with clinical assays to assess the modalities of administration in practice.

Author Contributions: Conceptualization, A.K., and F.A.; methodology, T.I.A., M.S., and S.B.; soft- ware, S.B.; investigation, T.I.A., K.A., and S.B.; writing—original draft preparation, A.K., F.A., and S.B.; writing—review and editing; A.K., F.A., and M.S.; visualization, K.A., and F.A.; supervision, F.A., and A.K.; project administration, F.A.; funding acquisition, F.A. All authors have read and agreed to the published version of the manuscript. Funding: The authors gratefully acknowledge Qassim University, represented by the Deanship of Scientific Research, on the financial support for this research under the number 5481-cos-2019-2-2-I during the academic year 1440 AH / 2019 AD. Institutional Review Board Statement: The experimental protocols were conducted in accordance with the guide for the care and use of laboratory animals issued by the University of Sfax, Tunisia and approved by the Committee of Animal Ethics (Protocol no. 94-1939). Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available because this work is funded by Qassim University, represented by the Deanship of Scientific Research. Conflicts of Interest: Authors declare that there are no conflicts of interest.

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