{"paper_id":"2b606acd-49fd-4b91-9e3a-1dbd1da866fb","body_text":"Laevicaulis alte slug extract: Investigation of antibacterial and wound-healing properties, chemical profiling, and molecular docking insights | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Laevicaulis alte slug extract: Investigation of antibacterial and wound-healing properties, chemical profiling, and molecular docking insights Mostafa Y. Morad, Heba El-Sayed, Ahmed A. Elhenawy, Hana Sonbol, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7612415/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Wound infections, particularly those caused by bacteria like Staphylococcus aureus, present a significant challenge to effective healing, often leading to chronic inflammation and tissue damage. Finding naturally active, new substances and applying them as wound healing agents to decrease bacterial infection and accelerate the wound healing is among the most significant areas of research. This study explored the therapeutic potential of a chloroform extract from the terrestrial slug Laevicaulis alte as a multi-functional agent for wound care. The investigation inte-grated chemical profiling by gas chromatography-mass spectrometry (GC-MS), in vitro antibacterial evaluation, an in vivo excisional wound healing model, and in silico molecular docking to elucidate its mechanisms of action. GC-MS analysis identified twelve compounds, with tris(2,4-di-tert-butylphenyl) phosphate (TDTBPP) and cholesterol as major constituents. The extract exhibited potent antibacterial activity against S. aureus with a minimum inhibitory concentration (MIC) of 0.625 mg/mL. In vivo, topical application of the extract significantly accelerated wound closure, reduced the cutaneous bacterial load, and orchestrated a balanced host immune response by decreasing pro-inflammatory cytokines (TNF-α, IL-6) while increasing the an-ti-inflammatory cytokine IL-10. Molecular docking studies provided a strong mechanistic rationale for these observations, revealing that key compounds, particularly TDTBPP and a phenol phosphite derivative, showed high binding affinities for both bacterial DNA gyrase and the critical cytokine targets. These findings indicate that the L. alte extract is a promising, multi-functional therapeutic agent, whose efficacy stems from a synergistic chemical consortium that targets both the pathogen and the host's inflammatory response. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Computational biology and bioinformatics Biological sciences/Drug discovery Biological sciences/Microbiology slugs Laevicaulis alte Staphylococcus aureus wound healing antibacterial GC-MS in silico Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction The skin serves as a primary defense of the immune system. It can defend against bacterial and fungal invasion and perform defensive and protective roles [1]. Skin serves a number of purposes: it synthesizes vitamin D, maintains body temperature, improves metabolic processes, shields against various types of trauma, including thermal, chemical, and UV radiation, and keeps us in touch with our surroundings through a variety of nerve terminals [2]. Skin wounds are common and a serious public health concerndue to their harmful effects on patients' quality of life. Skin wounds do, in fact, hurt, impair function and mobility, and they can also have an adverse effect on mental health by creating social isolation, melancholy, anxiety, and embarrassment [3]. The skin may be infected with many pathogens such as viruses, bacteria, fungus, and parasites. Bacterial skin infections are frequently observed in tropical areas due to the humidity and ambient temperature, which can occasionally be linked to poor hygiene. Moreover, the rich supply of nutrients, water, and high temperature found in the skin make it a perfect medium for microbial growth [4]. A wide range of cellular activities support the complex, dynamic process of wound healing, which requires careful coordination to properly repair damaged tissue[5]. To complete the healing process, the skin passes through four stages: homeostatic, inflammatory, proliferative, and remodeling [4]. There are two main types of cutaneous wounds based on the degree of pathogenicity and consequences: acute and chronic. The immune system is crucial for initiating inflammation, cleaning the wound, and promoting tissue recovery in the early stages of wound healing. During the phase of wound healing, the immune response is an extremely important contributing factor by starting the inflammatory process, aiding in wound cleaning, and promoting subsequent tissue healing. Conversely in the course of wound healing, immune system dysregulation during the healing process can result in persistent inflammation and slowed healing, both of which can contribute to chronic wounds [6]. The second-largest phylum in the animal kingdom is called Mollusca. Gastropoda is the largest class and includes 80% species, which may be snails or slugs, within the phylum Mollusca. Throughout the Nile Delta Region and the North Coast belt of the Mediterranean Sea, large numbers of terrestrial gastropod species are economically significant as pests in horticulture and agriculture [7]. Several species of slugs are considered as pests in many agricultural fields causing huge loss in the environmental ecosystem in different parts of the world. Crops, such as Soybean and corn are damages by slugs due to their feeding behavior and the harvested plants can be contaminated with their bodies, eggs, slime or feces, leading to deterioration in the quality of the harvest and economic loss [8]. Furthermore, the slugs have the potential to restrict the plant's range of dispersion [9] and, by means of selective feeding, modify the composition and abundance of specific plant species [10]. Finding biologically active compounds originating from living animals and using them in biotechnological processes for the food, cosmetic, pharmaceutical, and other industries is one of the most important topics in global research. However, numerous scientific studies demonstrate snail and slug mucus’s cytotoxicity impact on a range of cell lines, but their body extract role in many applications hasn’t been elucidated. Therefore, this study aimed to evaluate the slug extract's effective role in promoting wound healing as well as its antimicrobial efficacy against Staphylococcus aureus through computational, in vivo and in vitro assessment . Results GC-MS results The slug sample's GC-MS results revealed the existence of many chemicals with various biological functions. Table 1 listed the compoundsꞌ molecular formula, molecular weight, retention time (RT), area sum (%), compound class, and biological activity. Between these compounds tris(2,4-di-tert-butylphenyl) phosphate; cholesterol; phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1); and 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione with area sums of 44.42, 37.4, 7.97, and 2.59%, respectively. Table 1. GC-MS analytical report of slug’s extract Compound name Molecular formula Molecular weight RT (min) Area sum% Compound class Biological activity Heptadecane, 2, 6, 10,15-tetra-methyl- C21H44 296.6 23.419 0.54 Long-chain alkane Metabolite observed in cancer metabolism. It has a role as a human metabolite [11] 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6, 9-diene-2, 8- dione C17H24O3 276.4 25.669 2.59 α, β - unsaturated ketone Pharmacological activities, including antineoplastic, antimicrobial and antiviral activities [12] Stearic acid C18H36O2 284.5 26.509 1.32 Long-chain fatty acid ethyl ester antioxidants, hypocholesterolemic, nematicide, and pesticide [13] 1-Octadecyne C18H34 250.5 28.067 0.62 Straight chain alkyne Antibacterial, antioxidant and anticancer [14] 2-Pentadecyn-1-ol C15H28O 224.3 28.118 0.81 Alcohol Octadecanoic acid, ethyl ester C20H40O2 312.5 28.361 1.27 long-chain fatty acid ethyl ester 1,11-Dodecadiyne C12H18 162.2 29.451 0.62 alkynes Oxirane, tetradecyl- C16H32O 240.4 29.806 1.07 Epoxides 1,5,9Undecatriene , 2, 6,10-trimethyl-, (Z)- C14H24 192.3 33.217 0.66 Terpens Cholesterol C27H46O 386.7 35.328 37.4 Cholestanoid (sterol) cardioprotective effect, anti-inflammatory, anticancer, antimicrobial, anti-psychotic, antioxidant activities and drug-delivery capability [15] Phenol,2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) C42H63O3P 646.9 36.921 7.97 Phenols antioxidant and anti-enterococcal properties [16] Tris(2,4-di-tert-butylphenyl) phosphate C42H63O4P 662.9 38.072 44.42 Aryl phosphate Anti-inflammatory [17] Analysis of the Chemical Profile of Laevicaulis alte Extract. The comprehensive chemical profiling of Laevicaulis alte slug chloroform extract via Gas Chromatography-Mass Spectrometry (GC-MS) provides critical insights into its remarkable therapeutic potential, as demonstrated by this study. The identification of 12 distinct compounds (Table 1), dominated by four major constituents comprising >90% of the total detected area, establishes a robust chemical foundation for the observed bioactivities. This analysis delves into the pharmacological significance of these compounds, their synergistic potential, and their mechanistic roles in antibacterial action and wound regeneration. The GC-MS chromatogram revealed a chemically diverse profile, with several compounds possessing well-documented biological activities relevant to the study's findings: Tris(2,4-di-tert-butylphenyl) phosphate (44.42%): This aryl phosphate ester was the most abundant compound. While direct studies on its biological activity are limited, structural analogues and related organophosphates demonstrate significant anti-inflammatory properties [17]. Its high abundance strongly correlates with the in vivo observation of drastically reduced pro-inflammatory cytokines (IL-6, TNF-α) and accelerated wound closure. Cholesterol (37.4%): The high concentration of this ubiquitous sterol is particularly noteworthy. Beyond its structural role in membranes, cholesterol possesses documented anti-inflammatory, antimicrobial, and wound-healing promoting activities [15]. Its presence likely contributes significantly to; membrane stabilization; enhancing skin barrier repair in the proliferative phase. Precursor function; which serving as a precursor for vitamin D synthesis (promoting keratinocyte differentiation) and steroid hormones involved in inflammation resolution. Growth factor modulation, that influencing signaling pathways crucial for cell migration and angiogenesis. Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) (7.97%), this phenolic antioxidant belongs to a class known for radical scavenging and anti-enterococcal properties [16]. Its role is crucial in mitigating oxidative stress at the wound site, a major impediment to healing [18]. By neutralizing reactive oxygen species (ROS) generated during inflammation, it protects cells and matrix components, facilitating a transition to the proliferative phase. Its antioxidant capacity complements the anti-inflammatory action of the dominant aryl phosphate. 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione (2.59%): This α, β-unsaturated ketone/spiro compound, though present in a smaller proportion, exhibited potent predicted antibacterial activity. The extract contained several minor components (<2% each) known for diverse bioactivities: Stearic acid (1.32%) and Octadecanoic a cid, Ethyl Ester (1.27%): These long-chain fatty acids/esters possess reported antioxidant, hypocholesterolemic, and antimicrobial activities [13,19]. 1-Octadecyne (0.62%): This alkyne has documented antibacterial, antioxidant, and anti-cancer properties [14]. Heptadecane derivatives, Terpenes (e.g., 1,5,9-Undecatriene, 2, 6,10-trimethyl-, (Z)-), and Epoxides : While their specific contributions require further study, these classes often exhibit antimicrobial or anti-inflammatory effects, potentially contributing to the overall extract efficacy through additive or synergistic effects. The chemical profile of L. alte extract reveals significant novelty: the high abundance of Tris(2,4-di-tert-butylphenyl) phosphate in a molluskan therapeutic extract is unusual and highlights its potential as a lead anti-inflammatory compound. The combination of a significant sterol (cholesterol) with potent phenolics and specific antimicrobials (Oxaspiro compound) differs from profiles reported in other snail/slug mucins or extracts, which often emphasize proteins, peptides, glycosaminoglycans, and hyaluronic acid [20–22]. This chloroform extract targets a distinct, lipid-soluble chemical space[23,24]. The study bridges traditional knowledge (use of slugs) with modern analytical (GC-MS) and computational (docking) techniques, providing mechanistic hypotheses for the observed efficacy. In vitro assessment of the slug extract's antibacterial activity against Staphylococcus aureus . 3.3.1. MIC determination The in vitro antibacterial activity of the L. alte chloroform extract was quantitatively assessed against the bacterium Staphylococcus aureus ATCC 6538 using a broth microdilution assay. The extract exhibited a clear dose-dependent inhibitory effect on the growth of S. aureus . A minimum inhibitory concentration (MIC) value of 0.625 mg/mL was determined. This value represents the lowest concentration of the extract that completely inhibited the visible growth of the bacteria after 24 hours of incubation at 37°C. As illustrated in the dose-response curve (Figure 1), a progressive reduction in bacterial cell viability was observed with increasing concentrations of the slug extract, with viability dropping from 100% in the untreated control to near-zero levels at, and above the determined MIC. This confirmed the potent antibacterial efficacy of the crude extract against this bacterial strain. Figure 1. Slug's MIC and antibacterial efficacy against S. aureus ATCC 6538. The obtained results were shown as the average (n = 3) and standard error (±5%). In -silico docking interaction results In Silico Docking Analysis against S. aureus DNA Gyrase To elucidate a potential molecular mechanism for the observed antibacterial activity of the L. alte extract, a molecular docking study was performed. All twelve major compounds identified via GC-MS were docked into the active site of the S. aureus DNA gyrase-DNA complex (PDB ID: 2XCT [23]), a validated target for antibacterial agents. The docking scores, including binding energy (B.E.), interaction energy (E_Int), and hydrogen bond energy (E_H_B), were presented in Figure 2. The study targeted the bacterial DNA gyrase, a type II topoisomerase that is essential for bacterial survival but absent in humans, making it an ideal and well-established target for antibacterial drugs, most notably the fluoroquinolone class [25]. DNA gyrase is responsible for introducing negative supercoils into DNA, a process critical for relieving torsional stress during DNA replication and transcription. Its inhibition leads to the disruption of these vital processes and ultimately results in bacterial cell death [26] The results revealed that several compounds from the extract exhibited strong predicted binding affinities for the DNA gyrase active site, with binding energies ranging from -5.203 to -7.523 kcal/mol. Notably, the organophosphorus compounds phenol,2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) ( compound 11 ) and tris(2,4-di-tert-butylphenyl) phosphate ( compound 12 ) emerged as the most potent binders, with binding energies of -7.523 kcal/mol and -7.509 kcal/mol, respectively. These compounds also demonstrated the most favorable interaction energies (-32.540 kcal/mol and -21.436 kcal/mol, respectively), indicating extensive and stable non-covalent interactions within the binding pocket. Other compounds also showed significant binding potential. Cholesterol ( compound 10 ) and octadecanoic acid, ethyl ester ( compound 6 ) displayed strong binding energies of -6.924 kcal/mol and -6.852 kcal/mol, respectively. Of particular note, compound 6 exhibited an exceptionally low RMSD value of 1.009 Å, suggesting a highly stable and well-defined binding pose. Visual inspection of the docking poses for the top three binders revealed key molecular interactions responsible for their high affinity (Figure 2). All three compounds positioned themselves within the enzyme's active site, interacting with key catalytic residues and the bound DNA. A critical interaction was observed with the residue arginine 458 (Arg458), as well as with the DNA bases deoxyguanosine 9 (DG9) and adenine 13 (A13). The bulky, hydrophobic tert-butyl groups of compounds 11 and 12 were stabilized by hydrophobic contacts within the pocket, while their phosphate/phosphite moieties formed strong hydrogen bonds and electrostatic interactions with the positively charged guanidinium group of Arg458. Similarly, the hydroxyl group of cholesterol ( 10 ) acted as a hydrogen bond donor/acceptor, while its rigid sterol backbone was anchored by extensive van der Waals forces and hydrophobic interactions with the active site and DNA backbone. These interactions effectively anchor the ligands in the active site, suggesting a potential mechanism for enzyme inhibition. Figure 2. (A) Docking poses of the top three binding ( 10-12 ) compounds from L. alte extract within the active site of S. aureus DNA gyrase (PDB: 2XCT). Dashed lines represent non-covalent interactions such as hydrogen bonds and hydrophobic contacts. (B) Molecular docking scores including, B.E. (Binding Energy), rmsd (Root Mean Square Deviation), E_Int (Interaction Energy), E_H_B (Hydrogen Bond Energy). All energy values are in kcal/mol. The impact of slug extract on wound healing and its antibacterial efficacy against S. aureus in vivo. Estimation of wound size at the macroscopic level The application of slug extract enhanced and accelerated the process of wound healing, as shown in Figure (3A), as after 14 days, the wound was completely healed after treatment with slug extract. Also, the induction of slug extract could decrease the wound diameter after 14 days (3A). The diameter of wound didn’t show significant changes after 7 days of treatment (Figure 3B1), while it significantly decreased from 1.76 mm in the negative control group to 0.47 mm in the slug treated group after 14 days of treatment (Figure 3B2). Figure 3. The effect of slug administration on the healing of S. aureus -infected wounds after 7 and 14 days. After 7 days, no significant changes in diameter were observed between the treated and control groups. However, after 14 days, notable changes (P<0.05) were observed between the two groups Bacterial load determination After seven days of wound therapy, the bacterial content in the tissue that received slug extract was the least comparing with that within the tissues of the positive and negative controls, as illustrated in Figure 4. On day zero, the total bacterial count was 4 × 10 4 CFU/mL, but after seven days of the experiment, the total count was uncountable, 16×10 2 , and 5×10 2 CFU/mL in the wounded tissues of the negative control (group I), positive control (group II), and treated with slug (group III), respectively. Figure 4. Bacterial load in wounded tissues after 7 days of in vivo experiment a) treated with slug, b) positive control, and c) negative control. Histological investigations According to the current findings, a skin segment from a normal mouse showed a typical structure, with an intact epidermis made up of two to three cell layers, followed by a layer of dermal connective tissue that contained hair follicles and sebaceous glands (Figure 5A). Vascular endothelial growth factor was moderately expressed in the cytoplasmic stain of the epidermis and dermis blood vessels in the sections of normal mice (Immunohistochemistry, VEGF, DAB) (Figure 5B). Numerous polymorpho-nuclear neutrophils and lymphocytes infiltrated the epidermis and dermis of the mice's skin sections with positive injuries, causing ulcer formation and indicating the presence of inflammatory cells. Furthermore, hair follicles and sebaceous glands were seen (Figure 5 C). Furthermore, the cytoplasmic staining of epidermal and dermis blood vessels revealed moderate to substantial expression of vascular endothelial growth factor (Figure 5D). Skin slices from the injured mice showed a healed lesion with intact epidermis and dermis after seven days of slug extract treatment. Sebaceous glands and hair follicles were not present in the healed ulcerated area, but polymorphonuclear neutrophils and lymphocytes moderately infiltrated the epidermis and dermis, indicating the existence of inflammatory cells (Figure 5E). Additionally, vascular endothelial growth factor was mildly expressed in the blood vessels of the epidermis and dermis, as indicated by cytoplasmic staining (Fig. 5F). Following 14 days of slug extract treatment, skin slices from the injured mice revealed an unbroken epidermis and a healed ulcer. A healed ulcer showed modest infiltration of neutrophils, lymphocytes, and inflammatory cells into the epidermis and dermis, along with degeneration of the sebaceous glands and hair follicles (Figure 5G). Additionally, vascular endothelial growth factor was moderately expressed in the blood vessels of the epidermis and dermis, as indicated by cytoplasmic staining (Figure 5H). Figure 5. A) a skin section from normal animal exhibited normal skin, intact epidermis formed of 2-3 cell layers) (black arrow), dermis (connective tissue) (yellow arrow), sebaceous glands (red arrow) and hair follicles (green arrow), (piliary canals), (H&E, x100). B) Showed moderate expression of vascular endothelial growth factor as cytoplasmic stain in epidermis and dermis blood vessels (Immunohistochemistry, VEGF, DAB, x100). C) A skin section from the injured mice in the positive control group displayed ulcer formation (black arrow), with nearby regions exhibiting normal skin distinguished by an epidermis formed of 2-3 cell layers) (red arrow), underlying with connective tissue (yellow arrow), and hair follicles (green arrow). Within the ulcerated region, infiltration of numerous polymorph neutrophils and lymphocytes into both the epidermis and dermis was evident (red head arrows), along with sebaceous glands and hair follicles (green arrow) (H&E, x100). D) Showed moderate- marked expression of vascular endothelial growth factor as cytoplasmic stain in epidermis and dermis blood vessels (black arrow) (Immunohistochemistry, VEGF, DAB, x100). E) a skin section from the treated group with slug mucin after 7 days showed healed ulcer (black arrow), intact epidermis (formed of 2-3 cell layers) (red arrow), dermis (layer of connective tissue) (yellow arrow), Healed ulcerated area, showed moderate infiltration of inflammatory cells, neutrophils, lymphocytes, and polymorphs into the epidermis and dermis; lack of sebaceous glands and hair follicles (H&E, x100). F) Showed mild expression of vascular endothelial growth factor as cytoplasmic stain in epidermis and dermis blood vessels (black arrow) (Immunohistochemistry, VEGF, DAB, x100). G) a skin section from the treated group with slug mucin for 14 days showed healed ulcer (black arrow), intact epidermis (external epithelium formed of 2-3 cell layers) (red arrow). Healed ulcerated area, showed mild infiltration of epidermis and dermis by polymorph, neutrophils and lymphocytes inflammatory cells (yellow arrows), degenerated of both sebaceous glands and hair follicles (black arrow) (H&E, x100). H) exhibited moderate expression of vascular endothelial growth factor as cytoplasmic stain in epidermis and dermis blood vessels (Immunohistochemistry, VEGF, DAB, x100). Pro-and anti-inflammatory cytokines detection: The slug extract showed a promising effect on both pro-inflammatory and anti-inflammatory cytokines in the wound of the skin of mice. There was a significant decrease in pro-inflammatory cytokines represented with IL-6 levels (from 389 to 277 pg/mL after 7 and 14 days of slug extract treatment) and TNF-α levels (from 432 to 350 pg/mL) for the same respective durations (Figure 6), compared to the negative controls. Conversely, the anti-inflammatory biomarker IL-10 levels were significantly increased as compared with negative controls after treating with the slug extract on 7- and 14-days post wound from 601to 626 pg/mL, respectively (Figure 6). Figure 6. Proinflammatory cytokine levels (IL-6 and TNF-α) and anti-inflammatory cytokine level (Il-10) after treatment with slug extract after 7 and 14 days. All values represent (mean± SD). * Significant (P<0.05) on compared with negative control. In -silico docking interaction results To investigate the molecular mechanisms underlying the observed immunomodulatory activity of the Laevicaulis alte extract, a comprehensive molecular docking study was performed. The twelve major compounds identified by GC-MS were docked against the binding sites of three key cytokines: the pro-inflammatory mediators including; Tumor Necrosis Factor-alpha (TNF-α, PDB: 2AZ5[27] and Interleukin-6 (IL-6, PDB: 1ALU [28]), and the anti-inflammatory cytokine Interleukin-10 (IL-10, PDB: 1Y6K [29]). The binding affinities and interaction parameters were represented in Figures 7 ,8, and 9. The docking results revealed that the compounds from the slug extract, particularly the organophosphorus derivatives and cholesterol, exhibited significant and differential binding affinities for the cytokine targets. Interaction with Pro-Inflammatory Cytokines: TNF-α (PDB: 2AZ5): The strongest predicted binding affinity was observed for compound ( 11 ), with a binding energy (B.E.) of -6.663 kcal/mol. This was closely followed by tris(2,4-di-tert-butylphenyl) phosphate (12) and cholesterol (10) with B.E. values of -6.373 kcal/mol and -5.693 kcal/mol, respectively. Visual analysis of the binding poses (Figure 7) revealed that compound 11 engaged in a crucial π-stacking interaction with the aromatic ring of Tyr119. Compounds 10 and 12 were stabilized by H-bonds with the backbone of Leu157 and electrostatic interactions with Lys11. Figure 7. (A) Docking poses of the top three binding ( 10-12 ) compounds from L. alte extract within the active site of TNF-α(PDB: 2AZ5). Dashed lines represent non-covalent interactions such as hydrogen bonds and hydrophobic contacts. (B) Molecular docking scores including, B.E. (Binding Energy), rmsd (Root Mean Square Deviation), E_Int (Interaction Energy), E_H_B (Hydrogen Bond Energy). All energy values are in kcal/mol. IL-6 (PDB: 1ALU): The binding affinities for IL-6 were generally more modest. The most favorable interaction was for tris(2,4-di-tert-butylphenyl) phosphate (12), with a B.E. of -5.163 kcal/mol. The interactions, shown in Figure 8, were primarily driven by π-π stacking with the indole ring of Trp157 and hydrophobic contacts with Gln156. Cholesterol (10) formed a key hydrogen bond with the side chain of Lys46. Figure 8. (A) Docking poses of the top three binding (10-12) compounds from L. alte extract within the active site of IL-6 (PDB: 1ALU). Dashed lines represent non-covalent interactions such as hydrogen bonds and hydrophobic contacts. (B) Molecular docking scores including, B.E. (Binding Energy), rmsd (Root Mean Square Deviation), E_Int (Interaction Energy), E_H_B (Hydrogen Bond Energy). All energy values are in kcal/mol. 3.5.2. Interaction with Anti-Inflammatory Cytokine: IL-10 (PDB: 1Y6K): The slug compounds demonstrated the strongest overall binding affinities for the anti-inflammatory cytokine IL-10. Compound 12 and 11 emerged as the most potent binders, with excellent B.E. values of -7.433 kcal/mol and -7.188 kcal/mol, respectively. Cholesterol (10) also showed a strong affinity with a B.E. of -5.997 kcal/mol. The binding modes for Compounds 11 and 12 were stabilized by extensive hydrophobic interactions with the aromatic ring of Phe37 (Figure 9). Figure 9. (A) Docking poses of the top three binding (10-12) compounds from L. alte extract within the active site of IL-10 (PDB: 1Y6K). Dashed lines represent non-covalent interactions such as hydrogen bonds and hydrophobic contacts. (B) Molecular docking scores including, B.E. (Binding Energy), rmsd (Root Mean Square Deviation), E_Int (Interaction Energy), E_H_B (Hydrogen Bond Energy). All energy values are in kcal/mol. Collectively, these results indicated that the lead compounds from the slug extract, particularly 10, 11, and 12, have the structural capacity to interact effectively with both pro- and anti-inflammatory cytokine targets through distinct molecular interactions. Discussion Searching for new, unconventional sources that have an antibacterial effect has become important nowadays. Although, snails and slugs perform multifunctional roles through the regulation of algae and fungi, endozoochory, litter decomposition, facilitation of nutrient recycling and by being prey to various invertebrates and vertebrates, snails and slugs are very destructive pests that eat the twigs of plants and crops, which hurts the economy [30]. To avoid serious harm of Laevicaulis alte slugs, we used in this study the roasted form of them as antibacterial agent against Staphylococcus aureas in vitro and in vivo . The obtained results indicated in vitro reduction of Staphylococcus aureas cell density with MIC value of 0.625 mg/mL. Additionally, the number of bacteria (bacterial load) was found to be lower during the in vivo trial, especially in the wounded tissue that was treated with slug comparing with negative and positive controls. Generally, insects like snails and slugs make slime, which is a mucus-like substance with a complex makeup. 90% to 99.7% of snail and slug slim is water by weight, with the remaining 0.3% to 10% being made up of enzymes, glycoproteins, proteoglycans such as hyaluronic acid, achacin, glycosaminoglycans, antimicrobial peptides, copper peptides, and metal ions[20]. Moreover, thousands of bioactive compounds, including polypropinate, terpenes, sterols, fatty acid derivatives, alkaloids, and nitrogenous substances, have been discovered to be present in mollusks. The antibacterial, cytotoxic, antitumor, anti-inflammatory, antileukemic, antineoplastic, and antiviral qualities of these substances in mollusks have been the subject of much research [22]. According to GC/Ms results, the current investigation showed that the slug extract included 13 different chemicals, including phenols, terpenes, cholesterol, and fatty acids. The current data were similar to findings of Ibrahim et al. [21] who reported that GC/MS examination of E. desertorum mucin resulted in ten compounds have been identified and were mainly quinolines, monoterpenes, alcohol esters, sesquiterpenoids, fatty acid esters, fatty acids, and phenol derivatives. Sallam et al. [31] reported that such compounds have antimicrobial, anticancer and antioxidant activities. But there’s was not any previous study regarding the slug extract identified compounds. The GC-MS investigation of the slug extract identified numerous compounds with exhibiting diverse biological functions. Between these compounds’ tris (2,4-di-tert-butylphenyl) phosphate; cholesterol; phenol, 2,4-bis (1,1-dimethylethyl)-, phosphite (3:1); and 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione with area sums of 44.42, 37.4, 7.97, and 2.59%, respectively. The GC-MS analysis of Laevicaulis alte chloroform extract was not merely a compositional list; it was the key to understanding its remarkable therapeutic potential. The profile revealed a sophisticated blend of bioactive lipids and phenolics, dominated by anti-inflammatory (tris(2,4-di-tert-butylphenyl) phosphate, cholesterol), antioxidant (phenol derivative), and antimicrobial (7,9-Di-tert-butyl-1-oxaspiro compound, fatty acids) compounds. In silico docking provided plausible molecular targets for these activities, corroborating the in vitro and in vivo results. The synergy between these components effectively combated the bacterial infection by S. aureus , resolved detrimental inflammation (reducing IL-6/TNF-α, elevating IL-10), promoted angiogenesis (moderate VEGF), and accelerated tissue regeneration, leading to complete wound closure within 14 days. This chemical blueprint positions L. alte slug extract as a highly promising natural source for developing novel therapeutic agents targeting infected and chronic wounds. Future research should focus on isolating the major active principles (particularly the aryl phosphate and oxaspiro compound), validating their individual and combined efficacies, and exploring formulation strategies for topical delivery. The docking results strongly suggested that multiple components of the slug extract can effectively occupy and interact with the enzyme's active site. The two most potent compounds 12 and 11 , displayed the highest predicted affinity. Their efficacy was likely derived from a combination of structural features. The bulky, lipophilic tert-butylphenyl groups can engage in extensive hydrophobic and van der Waals interactions within the binding pocket, while the central phosphate/phosphite group provided a critical anchor point. As seen in Figure 2, this polar moiety formed strong electrostatic interactions and H- bonds with Arg458. The positively charged guanidinium group of arginine residues was frequently involved in stabilizing the negatively charged phosphate backbone of DNA or interacting with small molecule inhibitors, making this interaction particularly significant for potent inhibition[32]. The strong binding of cholesterol (10) and ethyl ester of octadecanoic acid (6 ) further reinforced the hypothesis that the extract's activity stemmed from a multi-component and synergistic effect. While their binding energies were slightly less favorable than the top two organophosphates, their high abundance in the extract means they likely contribute significantly to the overall biological effect. Cholesterol's rigid, hydrophobic structure is well-suited for occupying hydrophobic pockets, while its single OH group provided specific H-bonding capacity. The long aliphatic chain of octadecanoic acid likewise provided hydrophobic anchoring. The remarkably low RMSD of compound 6suggested a particularly favorable and conformationally locked binding mode, which is often indicative of a high-affinity interaction. It was crucial to interpret these findings in the context of the whole extract. The observed MIC of 0.625 mg/mL was not the result of a single compound but rather the cumulative effect of this chemical consortium. The presence of multiple potential inhibitors, each with respectable binding affinities, presented a multi-pronged attack on the bacterial enzyme. This phenomenon of synergy, where the combined effect of compounds is greater than the sum of their individual effects, is a well-known advantage of natural product-based therapies [33]. This can also be a strategy to circumvent the development of drug resistance, as it is more difficult for a bacterium to simultaneously evolve resistance mechanisms against multiple active molecules. For medical applications, it is crucial to explore the possibility of discovering novel, potent bioactive compounds that can quicken wound healing by decreasing tissue fibrosis, re-epithelialization, and wound closure time. Cutaneous wound healing is a complex biological process. Restoring the injured epithelium's barrier function necessitates the synchronisation of time and spatially regulated cellular and molecular activities [34]. The histological sections of the wounded skin from the treated group with slug extract were demonstrated a recoveredulcer with slight infiltration of polymorphnuclear leukocytes, neutrophils, and lymphocytes, in the epidermis and dermis. Sebaceous glands and hair follicles were absent, while the epidermis and dermis remained intact. Similar outcomes were obtained by El-Sayed et al. [35] who found that selenium nanoparticles could heal the wound after 11 days. Additionally, Errajouani et al. [36] demonstrated that slime from the garden snail Cepaea hortensis effectively facilitated nearly complete tissue healing after 24 days of treatment on excision wounds in rabbits. The inflammatory phase is the most crucial stage in the healing process for wounds. Moreover, persistent inflammation results in significant healing disruptions, an increase in fibrosis and scarring, and elevated levels of cytokines, including IL-6, TNF-α, and IL-1. TNF-α is a cytokine that induces inflammation and is expressed in high levels during inflammation [18]. The multipurpose cytokine IL-6 also influences inflammation and haematopoiesis, and immune responses in pleiotropic ways [24]. The current study demonstrated that slug extract effectively reduced both cytokines, IL-6 and TNF-α levels after 7 and 14 days post wound exposure in comparing with the control group. Similarly, [37] reported that calophyllolidecompound produced from Calophyllum inophyllum has the ability to modulate inflammatory cytokines response by reducing levels of proinflammatory cytokines such as IL-1β, IL-6 and TNF-α. These suggests a potential anti-inflammatory effect associated with calophylloid. Additionally, [38], found that The mucus produced by the mollusks slug Arion subfuscus could lowered the expression of some proinflammatory cytokines like, Il-6 and TNF-α and IL-1β. The pleomorphic cytokine IL10 showed anti-inflammatory qualities and a different stimulated immunity cells may contribute to its secretion[19]. Its principal functions include anti-inflammatory, inhibitory, or self-regulatory. Interleukin-10 serves as a potent detrimental feedback regulator crucial for the management and resolution of inflammation via autocrine and paracrine mechanisms. IL-10 has two arms that driven a broad immunosuppressive effect, preventing dendritic cells from presenting antigens and preventing the activation of macrophages and their infiltration into the injured area, with the secondary target of reducing the expression of pro-inflammatory cytokines [39]. Additionally, HuR (human antigen R) is a messenger RNA (mRNA) stabilizing protein. It is thought that IL-10 functions as a posttranscriptional regulatory agent at the cellular level to repress HuR and encourage the destabilization of inflammatory cytokine mRNA [40]. The present study showed that slug extract could increase IL-10 levels after 7 and 14 days post wound exposure in comparing with the control. Comparable findings were noted by [41], who concluded that calophyllolide isolated from Calophyllum inophyllum Linn could up-regulate an anti-inflammatory cytokine, IL-10. Collectively, it is evident that slug extract promotes faster wound healing by anti-inflammatory actions, specifically by controlling inflammatory cytokines. The in vivo results of this study painted a clear picture of a sophisticated immunomodulatory effect: the slug extract concurrently suppressed the pro-inflammatory cytokines TNF-α and IL-6 while enhancing the anti-inflammatory cytokine IL-10. The molecular docking investigation provides a compelling, multivalent mechanistic framework that explains how this balanced immune response could be achieved at the molecular level. The findings suggest that the extract does not act as a blunt instrument but rather as a cocktail of regulatory molecules that interact differentially with key players in the inflammatory cascade. The predicted inhibition of the pro-inflammatory cytokines is primarily driven by the organophosphorus compounds 11 (phenol phosphite) and 12 (TDTBPP), with support from 10 (cholesterol). Compound 11 emerged as the most potent theoretical binder to TNF-α (B.E. -6.663 kcal/mol). Its ability to form a stable π-stacking interaction with Tyr119 was particularly significant, as this residue was known to be critical for the structural integrity and receptor-binding function of TNF-α [42]. By occupying this site, compound 11 could sterically hinder the trimerization of TNF-α or its binding to the TNFR1 receptor, effectively neutralizing its pro-inflammatory signaling. Similarly, the binding of compound 12 to IL-6 suggested a mechanism for disrupting the IL-6/IL-6R/gp130 signaling complex, which is central to chronic inflammation[43]. The ability of multiple major components of the extract to bind these targets suggests a synergistic blockade of pro-inflammatory pathways. In conclusion, this study revealed a sophisticated chemical strategy encoded within the slug extract. The organophosphorus compounds (11 and 12) appear to act as master immunomodulators, demonstrating the highest potential to both neutralize key pro-inflammatory signals (TNF-α) and stabilize the primary anti-inflammatory signal (IL-10). Cholesterol (10) acted as a robust supporting molecule, contributing to the overall effect. This multi-target approach, where the same set of molecules can engage with opposing sides of the immune response, provided a more nuanced \"re-tuning\" of the wound microenvironment than simple immunosuppression. It facilitated the resolution of inflammation while actively promoting an anti-inflammatory state, which is precisely the profile required for efficient and high-fidelity wound healing. The docking analysis provided strong theoretical evidence that the observed immunomodulatory properties of the L. alte extract were not coincidental but are based on specific, high-affinity molecular interactions between its major chemical constituents and key cytokine regulators. The data strongly implicates compounds 11, 12, and 10 as the primary mediators of this effect, laying a solid foundation for future experimental validation and the potential development of a novel, balanced anti-inflammatory therapy from this unique natural source. Materials and Methods Slug source and extract preparation Laevicaulis alte slugs (9- 10 mm) were reared in Invertebrate lab, Faculty of Science, Helwan University, Egypt. It was kept in 15 x 24 x 10 cm plastic boxes with oven dried lettuce leaves and dechlorinated tap water. Slugs were cleaned using distilled water and 70% ethyl alcohol. After complete drying, they were roasted at 37°C for 3 days in oven, later they were grinded with a 400 mm disc mill (EarthTechnica co.LTD, Japan,). The powdered slugs were submerged in chloroform solvent (Sigma-Aldrich, United States) (1:2) for two days. Subsequently, the slug particles were discarded using ultracentrifugation (Sigma, Germany) at a temperature of 4°C and a speed of 10,000 rpm for a duration of 10 min. Then, chloroform was evaporated using a rotary evaporator (IKA, Germany) and final extract was obtained. Slug extract analysis using GC-MS After the slug extract was prepared, it was dissolved in chloroform to be analyzed using a GC-MS instrument (Agilent Technologies, Santa Clara, California) at Central Laboratories Network, National Research Centre, Cairo, Egypt. The gas chromatography system was fitted with a 30-meter-long DB-5MS column that had an internal diameter of 0.25 mm and a film thickness of 0.25 μm. The used carrier gas was helium at a flow rate of 2.0 mL/min with a splitless injection volume of 1 µL. The followed program of temperature was 50°C for 5 min.; rising at 5°C/min. to 100°C and held for 0 min., and rising at 10°C/min. to 320°C and held for 10 min. Both detector and injector temperatures were maintained at 320°C and 280°C, respectively. Mass spectra were generated using electron ionization (EI) at 70 eV, covering a spectral range of m/z 25-800 with a solvent delay of 3 min. The mass temperature was set at 230°C, and Quad was set to 150°C. Identification of various constituents was accomplished by comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data. Antibacterial activity of slug extract against Staphylococcus aureus Broth microdilution assay and MICs determination The examination was carried out employing the broth microdilution method, which was slightly modified from original protocol [44,45]. To make stock extract, 2.5 mg of slug powder was added to 1 mL of sterile water. The initial column well of a microtiter plate was filled with 400 µL of the obtained stock extract, and each well between 2 and 10 received 200 µL of sterile tryptic soy broth (TSB). By moving 200 µL gently from the first well to the seventh, a two- fold dilution was created. Subsequently, 50 µL of the reference strain S. aureus ATCC 6538 with a concentration of 5×10 8 CFU/mL (OD~0.1) was applied to each well in a single raw, excluding the final well designated as a blank. Both chloramphenicol (1 mg/mL) and ciprofloxacin (1 mg/mL) were used as a positive control. The plate was kept in an incubator at 37 o C for 24 hours. Following incubation, the results were determined at 630 nm using ChroMate 4300, USA Elisa reader. Wound healing and antibacterial efficacy of slug extract against Staphylococcus aureus in vivo Animal ethics committee for mouse model of wound infection The care and utilization of research animals at Helwan Institution follow guidelines that authorized by committee of the university’s Animal Ethics, with each research project assigned a specific research ethics number [HU-IACUC/Z/MY3107-31]. This study was done in accordance with ARRIVE guidelines ( https://arriveguidelines.org ). Animals About six-week-old male mice (weighing around 30 g) were purchased from Theodor Bilharez Institute's animal house in Egypt and were acclimatized in the laboratory for two weeks. The animals received tap water to drink and Purina chaw (20 % protein). Developing excision wound Diethyl ether was used to anesthetize the mice before performing full thickness excisional skin wounds. The dorsal fur was subsequently cleansed, shaved, and disinfected in preparation for the experiment. An aseptic puncher was employed to produce an oval, full thickness wound (3 mm), on the upper back of the mice. At the end of the study, all euthanized mice were treated humanely and killed with cervical dislocation to allow for tissue and organ examination, prevent their reintroduction into other experiments, and eliminate potential biohazards if released. Treatment groups Three mice groups (n = 10) were housed in different cages, with free-flowing water and unlimited food. Group I (negative control) included mice with excisional skin wounds treated with phosphate buffer saline (PBS) only, group II (positive control) included mice with excisional skin wounds received gentamycin cream (0.1% gentamicin), and group III (treated) included mice with excisional skin wound received 20 µL slug extract (625 µg/mL), the treatments were topically administrated to the wounded skin once daily for 14 days. Macroscopic estimation of wound size Following topical application, daily photos of the wound were captured. The wound area was assessed by measuring the distance along the wound bed and delineating a circle around its perimeter [26]. To ascertain each image's magnification during measurement analysis, a fine ruler was positioned at the wound level to determine its magnification. Bacterial load estimation The following protocol was used for bacterial load determination in both treated (II and III) and untreated (I) groups, according to El-Sayed et al.[35]. The experiment was carried out on both the zero and seventh days of the in vivo experimentafter inoculating the wound of the experimental mice with bacteria, disposable medical scalpels were used to excise a 1 mm thick wound from each of the three groups. Then, samples were put into sterile 1X phosphate buffered saline (PBS), vortexed gently for 5 min., and then diluted one by one. 100 µL of each concentration was then put on Muller Hinton Agar (MHA) medium plates. Plates that were inoculated were then left at 37°C for 24 hours, followed by colony enumeration the subsequent day was carried out. Pathological investigations The samples were kept in 10% formalin for a full day after the skins of the treated and untreated mice were autopsied on the seventh day of the experiment and at the conclusion of the trial. Following a series of alcohol dilutions to dry them out, the samples were cleaned in xylene and then submerged in paraffin for an entire day in oven (56ºC). Tissue blocks with paraffin sliced to an overall thickness of 5 µm utilizing slide microtomes. Tissue specimens wesre removed, deparaffinized, and subjected to staining with Masson's trichrome, hematoxylin, and eosin (H&E) before being seen under a light microscope for histological examination [35,46]. Pro- and anti-inflammatory cytokine detection The enzyme-linked immunosorbent test (ELISA) was used. To eliminate all blood, a little portion of the damaged skin was taken out, and the area was cleaned with ice-cold PBS (0.01 M, and PH = 7.4). Using a glass homogenizer, pieces of frozen tissue were homogenized in PBS (1 g/9 mL), then the homogenate was subjected to ultrasonic cell disruption (BioLogics, Inc., 150V/T, Virginia, USA) to further disintegrate the cells. Finally, the homogenate was centrifuged for five min. at 5000 xg, and the resulting solution was preserved at -80°C for later testing. Using a commercial ELISA kit (Elabscience, USA), the levels of IL-6 and TNF-α cytokines were quantified. In short, following the addition of samples and standards to each well, biotinylated detection antibodies specific to both cytokines, along with an Avidin-Horseradish Peroxidase (HRP) complex, were introduced,and the plates were then incubated. The optical densities of IL-6 and TNF-α were determined using biotinylated detection antibodies. The concentrations of the two cytokines were determined based on their respective OD values. Additionally, ELISA kits from BioSource International (Camarillo, California, USA) and commercially available reagents were used to measure the IL-10 concentrations in mice. As directed by the manufacturer, cytokine concentrations were measured by referencing a standard curve derived from the known concentration of cytokine standards provided on each assay plate [47]. In Silico Molecular Docking Studies To elucidate the potential molecular mechanisms underlying the observed antibacterial and immunomodulatory activities of the L. alte extract, a comprehensive series of molecular docking simulations was conducted using the autodock software suite. Preparation of Receptor Structures The three-dimensional atomic coordinates of the four target proteins were retrieved from the RCSB Protein Data Bank (PDB). The selected targets were: Staphylococcus aureus DNA gyrase complexed with DNA (PDB ID: 2XCT), human Tumor Necrosis Factor-alpha (TNF-α) trimer (PDB ID: 2AZ5), human Interleukin-6 (IL-6) (PDB ID: 1ALU), and human Interleukin-10 (IL-10) (PDB ID: 1Y6K). Prior to docking, all protein structures were meticulously prepared. All water molecules, co-crystallized ligands, ions, and any other non-essential heteroatoms were removed from the PDB files. The structures were then subjected to the Protonate 3D tool to add H-atoms and assign appropriate ionization states at a simulated physiological pH of 7.4. Preparation of Ligand Structures The chemical structures of the twelve major compounds identified in the GC-MS analysis were constructed as 2D models using ChemDraw software. They were subsequently converted to 3D models and subjected to a rigorous energy minimization protocol using the Charm force field. The minimization was carried out until a root-mean-square gradient of <0.01 kcal/mol·Å was reached. This crucial step ensures that each ligand adopted a stable, low-energy conformation before being introduced to the protein's active site. The database of minimized ligands was then prepared for the docking procedure. Docking Simulation Protocol For each protein target, the active binding site was defined based on the location of the co-crystallized ligand in the original PDB file or identified using the Site Finder tool. A docking simulation was then performed for each of the twelve prepared ligands against each of the four protein targets. The 30 best poses from this initial placement were then subjected to refinement via forcefield energy minimization, allowing for flexible ligand-receptor interactions. The final poses were scored using the scoring function, which estimates the free energy of binding (B.E., in kcal/mol) of the ligand from a given pose. All 3D visualizations of the ligand-receptor complexes were generated using the built-in MOE visualizer and PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC). The binding energy (B.E.), root-mean-square deviation (rmsd), interaction energy (E_Int), and hydrogen bond energy (E_H_B) were tabulated for comparative analysis. Statistical data analysis The software used for analyzing data statistically was GraphPad Prism 9.0 (LLC). Tukey's post hoc test and one-way ANOVA were used to statistically compare the means. Data was provided in the form of mean ± standard deviation, with the threshold of significance set at less than 0.05. Abbreviations L. alte Laevicaulis alte GC-MS Gas chromatography-mass spectrometry TDTBPP Tris(2,4-di-tert-butylphenyl) phosphate MIC Minimum inhibitory concentration CFU Colony forming unit Declarations Author Contributions: Conceived and designed the study: H.E.-S., M.A.H., and M.Y.M.; methodology, H.E.-S., M.Y.M., M.A.H., A.F.A., H.S., O.A.H., A.A.E. and A.M.I.; collected the data: H.E.-S., M.Y.M., M.A.H., H.S. and O.A.H.; performed the analysis: H.E.-S., M.Y.M., A.A.E. and M.A.H.; funding acquisition, H.S.; wrote the paper: H.E.-S., M.Y.M., M.A.H., A.F.A., H.S., O.A.H., A.A.E. and A.M.I. All authors have read and agreed to the published version of the manuscript Funding: Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R83), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. Data Availability Statement: . The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgments: The authors are grateful to Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R83), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia Competing interests: The authors declare no competing interests. References Sounouvou, H. T. et al. Antimicrobial potentials of essential oils extracted from West African aromatic plants on common skin infections. Sci. African 11, e00706 (2021). Abdo, J. M., Sopko, N. A. & Milner, S. M. The applied anatomy of human skin: A model for regeneration. Wound Med. 28, 100179 (2020). Canchy, L., Kerob, D., Demessant, A. & Amici, J.-M. Wound healing and microbiome, an unexpected relationship. J. Eur. Acad. Dermatology Venereol. 37, 7–15 (2023). Van Hees, C. & Naafs, B. in Common Ski. Dis. Africa an Illus. Guid. 89 (2011). Wilkinson, H. N. & Hardman, M. J. Wound healing: cellular mechanisms and pathological outcomes. Open Biol. 10, 200223 (2020). Raziyeva, K. et al. Immunology of Acute and Chronic Wound Healing. Biomolecules 11, (2021). Gupta, N. K., Paul, P., Barman, H. & Aditya, G. The marsh slug, Deroceras laeve in Darjeeling Himalayas, India: First record and modelling of suitable habitats. Acta Ecol. Sin. 43, 432–438 (2023). Kumar, P. A Review—On Molluscs as an Agricultural Pest and Their Control. Int. J. Food Sci. Agric. 4, 383–389 (2020). Bruelheide, H. & Scheidel, U. Slug herbivory as a limiting factor for the geographical range of Arnica montana. J. Ecol. 87, 839–848 (1999). Hanley, M. E. Seedling herbivory, community composition and plant life history traits. Perspect. Plant Ecol. Evol. Syst. 1, 191–205 (1998). Reyes, V. M. H., Mart\\’\\inez, O. & Hernández, G. F. National center for biotechnology information. Plant Breeding. Univ. Autónoma Agrar. Antonio Narro, Calzada Antonio Narro (1923). Taskin, H. Detection of Volatile Aroma Compounds of Morchella by Headspace Gas Chromatography Mass Spectrometry (HS-GC/MS). Not. Bot. Horti Agrobot. Cluj-Napoca 41, 122–125 (2013). Siswadi, S. & Saragih, G. S. Phytochemical analysis of bioactive compounds in ethanolic extract of Sterculia quadrifida R. Br. in AIP Conf. Proc. 2353, (2021). Belakhdar, G., Benjouad, A., Abdennebi, E. H. & others. Determination of some bioactive chemical constituents from Thesium humile Vahl. J Mater Env. Sci 6, 2778–2783 (2015). Zhang, K. et al. Cholesterol: Bioactivities, Structural Modification, Mechanisms of Action, and Structure-Activity Relationships. Mini Rev. Med. Chem. 21, 1830–1848 (2021). TAHER, M. A. et al. Bioactive compounds extracted from leaves of G. cyanocarpa using various solvents in chromatographic separation showed anti-cancer and anti-microbial potentiality in in silico approach. Chinese J. Anal. Chem. 51, 100336 (2023). Vinuchakkaravarthy, T., Kumaravel, K. P., Ravichandran, S. & Velmurugan, D. Active compound from the leaves of Vitex negundo L. shows anti-inflammatory activity with evidence of inhibition for secretory phospholipase A2 through molecular docking. Bioinformation 7, 199 (2011). Zhao, Y. et al. Bletilla striata Polysaccharide Promotes Diabetic Wound Healing Through Inhibition of the NLRP3 Inflammasome. Front. Pharmacol. 12, (2021). Saraiva, M. & O’Garra, A. The regulation of IL-10 production by immune cells. Nat. Rev. Immunol. 10, 170–181 (2010). Rashad, M., Sampò, S., Cataldi, A. & Zara, S. Biological activities of gastropods secretions: snail and slug slime. Nat. Products Bioprospect. 13, (2023). Ibrahim, A., Morad, M., El-khadragy, M. F. & Hammam, O. The antioxidant and anti-inflammatory effects of Eremina desertorum snail mucin on experimentally-induced intestinal inflammation and testicular damage. Biosci. Rep. (2022). doi:10.1042/BSR20221020 Ulagesan, S. & Kim, H. J. Antibacterial and antifungal activities of proteins extracted from seven different snails. Appl. Sci. 8, (2018). Bax, B. et al. Type IIA topoisomerase inhibition by a new class of antibacterial agents. Nature 466, 935–940 (2010). Liu, C. et al. Mesenchymal stromal cells pretreated with proinflammatory cytokines enhance skin wound healing via IL-6-dependent M2 polarization. Stem Cell Res. Ther. 13, 414 (2022). Aldred, K. J., Kerns, R. J. & Osherof, N. Mechanism of Quinolone Action and Resistance. Biochemistry 53, 1565–1574 (2014). Pietrusiński, M. & Stączek, P. [Bacterial type II topoisomerases as targets for antibacterial drugs]. Postepy Biochem. 52, 271–282 (2006). He, M. M. et al. Small-Molecule Inhibition of TNF-α. Science (80-. ). 310, 1022–1025 (2005). Somers, W., Stahl, M. & Seehra, J. S. 1.9 Å crystal structure of interleukin 6: implications for a novel mode of receptor dimerization and signaling. EMBO J. 16, 989–997 (1997). Yoon, S. Il, Jones, B. C., Logsdon, N. J. & Walter, M. R. Same structure, different function: Crystal structure of the Epstein-Barr virus IL-10 bound to the soluble IL-10r1 chain. Structure 13, 551–564 (2005). Barman, H., Paul, P. & Aditya, G. Observations on the growth and life table estimates of the slug Mariaella dussumieri (L. Pfeiffer, 1855) (Gastropoda: Ariophantidae). Zool. Ecol. 32, 136–143 (2022). Sallam, A. A. A., El-Massry, S. A. & Nasr, I. N. Chemical analysis of mucus from certain land snails under Egyptian conditions. Arch. Phytopathol. Plant Prot. 42, 874–881 (2009). Lanz, M. A. & Klostermeier, D. The GyrA-box determines the geometry of DNA bound to gyrase and couples DNA binding to the nucleotide cycle. Nucleic Acids Res. 40, 10893–10903 (2012). Wagner, H. Synergy research: Approaching a new generation of phytopharmaceuticals. Fitoterapia 82, 34–37 (2011). Rhea, L. & Dunnwald, M. Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis. J. Vis. Exp. (2020). doi:10.3791/61616 El-Sayed, H. et al. Myco-Synthesized Selenium Nanoparticles as Wound Healing and Antibacterial Agent: An In Vitro and In Vivo Investigation. Microorganisms 11, 2341 (2023). Errajouani, F. et al. Exploring the Potential Anti-Inflammatory and Wound-Healing Proprieties of Cepaea hortensis Snail Mucin. Cosmetics 10, 170 (2023). Ferdosh, S. The Extraction of Bioactive Agents from Calophyllum inophyllum L., and Their Pharmacological Properties. Sci. Pharm. 92, (2024). Deng, T. et al. A natural biological adhesive from snail mucus for wound repair. Nat. Commun. 14, 396 (2023). Porro, C., Cianciulli, A. & Panaro, M. A. The Regulatory Role of IL-10 in Neurodegenerative Diseases. Biomolecules 10, (2020). Wills-Karp, M., Nathan, A., Page, K. & Karp, C. New Insights Into Innate Immune Mechanisms Underlying Allergenicity. Mucosal Immunol. 3, 104–110 (2010). Nguyen, V.-L. et al. Anti-inflammatory and wound healing activities of calophyllolide isolated from Calophyllum inophyllum Linn. PLoS One 12, e0185674 (2017). Van Ostade, X., Tavernier, J., Prangé, T. & Fiers, W. Localization of the active site of human tumour necrosis factor (hTNF) by mutational analysis. EMBO J. 10, 827–836 (1991). Hunter, C. & Jones, S. IL-6 as a keystone cytokine in health and disease. Nat. Immunol. 16, 448–457 (2015). CLSI. M100 Performance Standards for Antimicrobial Susceptibility Testing . (2021). Balouiri, M., Sadiki, M. & Ibnsouda, S. K. Methods for in vitro evaluating antimicrobial activity: A review. J. Pharm. Anal. 6, 71–79 (2016). Khalaf, A. A., Hassanen, E. I., Zaki, A. R., Tohamy, A. F. & Ibrahim, M. A. Histopathological, immunohistochemical, and molecular studies for determination of wound age and vitality in rats. Int. Wound J. 16, 1416–1425 (2019). Khalil, R. G., Ibrahim, A. M. & Bakery, H. H. Juglone: “A novel immunomodulatory, antifibrotic, and schistosomicidal agent to ameliorate liver damage in murine schistosomiasis mansoni”. Int. Immunopharmacol. 113, 109415 (2022). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 29 Jan, 2026 Editor assigned by journal 06 Jan, 2026 Editor invited by journal 23 Sep, 2025 Submission checks completed at journal 20 Sep, 2025 First submitted to journal 20 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7612415\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":583713769,\"identity\":\"ffcca7fa-3366-455f-a7c2-394bc3e391f4\",\"order_by\":0,\"name\":\"Mostafa Y. Morad\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Helwan University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mostafa\",\"middleName\":\"Y.\",\"lastName\":\"Morad\",\"suffix\":\"\"},{\"id\":583713771,\"identity\":\"74f5194f-fd84-4404-85e5-ab64c5747f3a\",\"order_by\":1,\"name\":\"Heba El-Sayed\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYDCCAzwMzEBKBszh+WEjBxZ8QIQWHjCHtyfNGCyYQLQWHrbDiQ0gBj4tfLfPHnxcUGPDI99+xvDBG57D6fPDDj8E2mInp9uAXYvkubxk4xnH0ngMzuQYG86xSM/deDvNAKgl2djsAHYtBmd4zKSB7uExYMgBMnisczfOTgBpOZC4DbcW8988//7zyPe/ATLYmNMNZ6d/IKTFjJm3DRgIN3LMmHnYnBPkpXPw2yJ5hsdYemZfMo/BjWfFknN70gw3SOcUHEgwwO0XvjM8hp8LvtnJyfcnb/zw5oeNvPzs9M0fPlTYyeHSggQ4DCBOBas0IKgcBNgfgCn5BqJUj4JRMApGwQgCAB/QYO870wZuAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"Helwan University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Heba\",\"middleName\":\"\",\"lastName\":\"El-Sayed\",\"suffix\":\"\"},{\"id\":583713772,\"identity\":\"16b7803d-75d5-45c5-af25-f9048fea992e\",\"order_by\":2,\"name\":\"Ahmed A. Elhenawy\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Al-Azhar University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ahmed\",\"middleName\":\"A.\",\"lastName\":\"Elhenawy\",\"suffix\":\"\"},{\"id\":583713774,\"identity\":\"06902bcf-31a1-44db-ba8c-d90725aae54d\",\"order_by\":3,\"name\":\"Hana Sonbol\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Princess Nourah bint Abdulrahman University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hana\",\"middleName\":\"\",\"lastName\":\"Sonbol\",\"suffix\":\"\"},{\"id\":583713775,\"identity\":\"a57bfda9-6b47-4a60-a476-f09612c40c39\",\"order_by\":4,\"name\":\"Olfat A. Hammam\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Theodor Bilharz Research Institute\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Olfat\",\"middleName\":\"A.\",\"lastName\":\"Hammam\",\"suffix\":\"\"},{\"id\":583713776,\"identity\":\"f9d50c47-75f9-4543-98b9-7ff373787284\",\"order_by\":5,\"name\":\"Asmaa F. Abdelmonem\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Helwan University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Asmaa\",\"middleName\":\"F.\",\"lastName\":\"Abdelmonem\",\"suffix\":\"\"},{\"id\":583713777,\"identity\":\"0450398e-393e-4cb4-b987-8576813787ae\",\"order_by\":6,\"name\":\"Amina M. Ibrahim\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Theodor Bilharz Research Institute\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Amina\",\"middleName\":\"M.\",\"lastName\":\"Ibrahim\",\"suffix\":\"\"},{\"id\":583713778,\"identity\":\"0a48bb45-5e9a-4750-82d2-bf43a5528138\",\"order_by\":7,\"name\":\"Marwa A. Hamada\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Helwan University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Marwa\",\"middleName\":\"A.\",\"lastName\":\"Hamada\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-09-14 11:53:20\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-7612415/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-7612415/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":101675626,\"identity\":\"1404d679-601d-4741-b1a7-2d83fa5a7001\",\"added_by\":\"auto\",\"created_at\":\"2026-02-02 13:30:57\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":20529,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSlug's MIC and antibacterial efficacy against \\u003cem\\u003eS. aureus\\u003c/em\\u003e ATCC 6538. The obtained results were shown as the average (n = 3) and standard error (±5%).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7612415/v1/da5f1602aeb92ec93e98a898.jpg\"},{\"id\":101675629,\"identity\":\"a4eb015e-ba83-4fdd-a394-25a118e7e52b\",\"added_by\":\"auto\",\"created_at\":\"2026-02-02 13:30:57\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":177641,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A) Docking poses of the top three binding (\\u003cstrong\\u003e10-12\\u003c/strong\\u003e) compounds from \\u003cem\\u003eL. alte\\u003c/em\\u003e extract within the active site of \\u003cem\\u003eS. aureus\\u003c/em\\u003eDNA gyrase (PDB: 2XCT). Dashed lines represent non-covalent interactions such as hydrogen bonds and hydrophobic contacts. (B) Molecular docking scores including, B.E. (Binding Energy), rmsd (Root Mean Square Deviation), E_Int (Interaction Energy), E_H_B (Hydrogen Bond Energy). All energy values are in kcal/mol.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7612415/v1/1ff9761e766c212c8aa72d90.jpg\"},{\"id\":101675625,\"identity\":\"5371961f-626d-41ac-b069-0ddd5b34caad\",\"added_by\":\"auto\",\"created_at\":\"2026-02-02 13:30:57\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":14799,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe effect of slug administration on the healing of \\u003cem\\u003eS. aureus\\u003c/em\\u003e-infected wounds after 7 and 14 days. After 7 days, no significant changes in diameter were observed between the treated and control groups. However, after 14 days, notable changes (P\\u0026lt;0.05) were observed between the two groups\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7612415/v1/a09a72d1f31fc37308ea859b.jpg\"},{\"id\":101754153,\"identity\":\"e7748c54-e7c2-4ff0-a3de-903a80fb0062\",\"added_by\":\"auto\",\"created_at\":\"2026-02-03 10:41:46\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":54492,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eBacterial load in wounded tissues after 7 days of \\u003cem\\u003ein vivo\\u003c/em\\u003e experiment a) treated with slug, b) positive control, and c) negative control.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7612415/v1/7e2a23f3d952b5c8ea386164.jpg\"},{\"id\":101675630,\"identity\":\"b75f9307-1a53-4559-9546-9a3e29e9a8b9\",\"added_by\":\"auto\",\"created_at\":\"2026-02-02 13:30:57\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":134919,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eA) a skin section from normal animal exhibited normal skin, intact epidermis formed of 2-3 cell layers) (black arrow), dermis (connective tissue) (yellow arrow), sebaceous glands (red arrow) and hair follicles (green arrow), (piliary canals), (H\\u0026amp;E, x100). B) Showed moderate expression of vascular endothelial growth factor as cytoplasmic stain in epidermis and dermis blood vessels (Immunohistochemistry, VEGF, DAB, x100). C) A skin section from the injured mice in the positive control group displayed ulcer formation (black arrow), with nearby regions exhibiting normal skin distinguished by an epidermis formed of 2-3 cell layers) (red arrow), underlying with connective tissue (yellow arrow), and hair follicles (green arrow). Within the ulcerated region, infiltration of numerous polymorph neutrophils and lymphocytes into both the epidermis and dermis was evident (red head arrows), along with sebaceous glands and hair follicles (green arrow) (H\\u0026amp;E, x100). D) Showed moderate- marked expression of vascular endothelial growth factor as cytoplasmic stain in epidermis and dermis blood vessels (black arrow) (Immunohistochemistry, VEGF, DAB, x100). E) a skin section from the treated group with slug mucin after 7 days showed healed ulcer (black arrow), intact epidermis (formed of 2-3 cell layers) (red arrow), dermis (layer of connective tissue) (yellow arrow), Healed ulcerated area, showed moderate infiltration of inflammatory cells, neutrophils, lymphocytes, and polymorphs into the epidermis and dermis; lack of sebaceous glands and hair follicles (H\\u0026amp;E, x100). F) Showed mild expression of vascular endothelial growth factor as cytoplasmic stain in epidermis and dermis blood vessels (black arrow) (Immunohistochemistry, VEGF, DAB, x100). G) a skin section from the treated group with slug mucin for 14 days showed healed ulcer (black arrow), intact epidermis (external epithelium formed of 2-3 cell layers) (red arrow). Healed ulcerated area, showed mild infiltration of epidermis and dermis by polymorph, neutrophils and lymphocytes inflammatory cells (yellow arrows), degenerated of both sebaceous glands and hair follicles (black arrow) (H\\u0026amp;E, x100). H) exhibited moderate expression of vascular endothelial growth factor as cytoplasmic stain in epidermis and dermis blood vessels (Immunohistochemistry, VEGF, DAB, x100).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7612415/v1/2f6b58eba6fc73682b43408c.jpg\"},{\"id\":101675627,\"identity\":\"9dda70ae-b8d5-40db-9b83-21fe74d29d7c\",\"added_by\":\"auto\",\"created_at\":\"2026-02-02 13:30:57\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":42403,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eProinflammatory cytokine levels (IL-6 and TNF-α) and anti-inflammatory cytokine level (Il-10) after treatment with slug extract after 7 and 14 days. All values represent (mean± SD). * Significant (P\\u0026lt;0.05) on compared with negative control.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7612415/v1/df604200d2b965da8dad19d5.jpg\"},{\"id\":101675628,\"identity\":\"451343fe-b8d5-4c22-8bad-7347ec42c5b9\",\"added_by\":\"auto\",\"created_at\":\"2026-02-02 13:30:57\",\"extension\":\"jpg\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":163567,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A) Docking poses of the top three binding (\\u003cstrong\\u003e10-12\\u003c/strong\\u003e) compounds from \\u003cem\\u003eL. alte\\u003c/em\\u003eextract within the active site of TNF-α\\u003cem\\u003e \\u003c/em\\u003e(PDB: 2AZ5). Dashed lines represent non-covalent interactions such as hydrogen bonds and hydrophobic contacts. (B) Molecular docking scores including, B.E. (Binding Energy), rmsd (Root Mean Square Deviation), E_Int (Interaction Energy), E_H_B (Hydrogen Bond Energy). All energy values are in kcal/mol.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture7.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7612415/v1/21c6662a50ddd5edc2bddd56.jpg\"},{\"id\":101675631,\"identity\":\"329887f7-c290-4d5b-95ef-0065eebfc1a2\",\"added_by\":\"auto\",\"created_at\":\"2026-02-02 13:30:57\",\"extension\":\"jpg\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":166626,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A) Docking poses of the top three binding (10-12) compounds from \\u003cem\\u003eL. alte\\u003c/em\\u003e extract within the active site of IL-6 (PDB: 1ALU). Dashed lines represent non-covalent interactions such as hydrogen bonds and hydrophobic contacts. (B) Molecular docking scores including, B.E. (Binding Energy), rmsd (Root Mean Square Deviation), E_Int (Interaction Energy), E_H_B (Hydrogen Bond Energy). All energy values are in kcal/mol.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture8.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7612415/v1/9641677b1d232cfac8097a0e.jpg\"},{\"id\":101753831,\"identity\":\"0d8af377-7f76-4a4b-b5a8-c32fd0625fd4\",\"added_by\":\"auto\",\"created_at\":\"2026-02-03 10:40:57\",\"extension\":\"jpg\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":176612,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A) Docking poses of the top three binding (10-12) compounds from \\u003cem\\u003eL. alte\\u003c/em\\u003e extract within the active site of IL-10 (PDB: 1Y6K). Dashed lines represent non-covalent interactions such as hydrogen bonds and hydrophobic contacts. (B) Molecular docking scores including, B.E. (Binding Energy), rmsd (Root Mean Square Deviation), E_Int (Interaction Energy), E_H_B (Hydrogen Bond Energy). All energy values are in kcal/mol.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture9.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7612415/v1/355ddc7233643c0aadbe8ca1.jpg\"},{\"id\":101755807,\"identity\":\"82e50a9c-926b-4960-b605-2e440271b92c\",\"added_by\":\"auto\",\"created_at\":\"2026-02-03 10:54:57\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2361915,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7612415/v1/16a2e325-0880-42e7-830c-19378a39989f.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Laevicaulis alte slug extract: Investigation of antibacterial and wound-healing properties, chemical profiling, and molecular docking insights\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eThe skin serves as a primary defense of the immune system. It can defend against bacterial and fungal invasion and perform defensive and protective roles [1]. Skin serves a number of purposes: it synthesizes vitamin D, maintains body temperature, improves metabolic processes, shields against various types of trauma, including thermal, chemical, and UV radiation, and keeps us in touch with our surroundings through a variety of nerve terminals [2].\\u003c/p\\u003e\\n\\u003cp\\u003eSkin wounds are common and a serious public health concerndue to their harmful effects on patients' quality of life. Skin wounds do, in fact, hurt, impair function and mobility, and they can also have an adverse effect on mental health by creating social isolation, melancholy, anxiety, and embarrassment [3]. The skin may be infected with many pathogens such as viruses, bacteria, fungus, and parasites. Bacterial skin infections are frequently observed in tropical areas due to the humidity and ambient temperature, which can occasionally be linked to poor hygiene. Moreover, the rich supply of nutrients, water, and high temperature found in the skin make it a perfect medium for microbial growth [4].\\u003c/p\\u003e\\n\\u003cp\\u003eA wide range of cellular activities support the complex, dynamic process of wound healing, which requires careful coordination to properly repair damaged tissue[5]. To complete the healing process, the skin passes through four stages: homeostatic, inflammatory, proliferative, and remodeling [4]. There are two main types of cutaneous wounds based on the degree of pathogenicity and consequences: acute and chronic. The immune system is crucial for initiating inflammation, cleaning the wound, and promoting tissue recovery in the early stages of wound healing.\\u003c/p\\u003e\\n\\u003cp\\u003eDuring\\u0026nbsp;the\\u0026nbsp;phase\\u0026nbsp;of\\u0026nbsp;wound\\u0026nbsp;healing,\\u0026nbsp;the\\u0026nbsp;immune\\u0026nbsp;response\\u0026nbsp;is\\u0026nbsp;an\\u0026nbsp;extremely\\u0026nbsp;important\\u0026nbsp;contributing\\u0026nbsp;factor by starting the inflammatory process, aiding in wound cleaning, and promoting subsequent tissue healing. Conversely in the course of wound healing, immune system dysregulation during the healing process can result in persistent inflammation and slowed healing, both of which can contribute to chronic wounds [6].\\u003c/p\\u003e\\n\\u003cp\\u003eThe second-largest phylum in the animal kingdom is called Mollusca. Gastropoda is the largest class and includes 80% species, which may be snails or slugs, within the phylum Mollusca. Throughout the Nile Delta Region and the North Coast belt of the Mediterranean Sea, large numbers of terrestrial gastropod species are economically significant as pests in horticulture and agriculture [7]. Several species of slugs are considered as pests in many agricultural fields causing huge loss in the environmental ecosystem in different parts of the world. Crops, such as Soybean and corn are damages by slugs due to their feeding behavior and the harvested plants can be contaminated with their bodies, eggs, slime or feces, leading to deterioration in the quality of the harvest and economic loss [8]. Furthermore, the slugs have the potential to restrict the plant's range of dispersion [9] and, by means of selective feeding, modify the composition and abundance of specific plant species [10].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eFinding biologically active compounds originating from living animals and using them in biotechnological processes for the food, cosmetic, pharmaceutical, and other industries is one of the most important topics in global research. However, numerous scientific studies demonstrate snail and slug mucus’s cytotoxicity impact on a range of cell lines, but their body extract role in many applications hasn’t been elucidated.\\u003c/p\\u003e\\n\\u003cp\\u003eTherefore, this study aimed to evaluate the slug extract's effective role in promoting wound healing as well as its antimicrobial efficacy against \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e through computational, \\u003cem\\u003ein vivo\\u0026nbsp;\\u003c/em\\u003eand \\u003cem\\u003ein vitro\\u003c/em\\u003e assessment\\u003cem\\u003e.\\u003c/em\\u003e\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eGC-MS results\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe slug sample\\u0026apos;s GC-MS results revealed the existence of many chemicals with various biological functions. Table 1 listed the compoundsꞌ molecular formula, molecular weight, retention time (RT), area sum (%), compound class, and biological activity. Between these compounds tris(2,4-di-tert-butylphenyl) phosphate; cholesterol; phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1); and 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione with area sums of 44.42, 37.4, 7.97, and 2.59%, respectively.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 1.\\u0026nbsp;\\u003c/strong\\u003eGC-MS analytical report of slug\\u0026rsquo;s extract\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"100%\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eCompound name\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eMolecular formula\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eMolecular weight\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eRT (min)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eArea sum%\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eCompound class\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eBiological activity\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003col\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003eHeptadecane, 2, 6, 10,15-tetra-methyl-\\u003c/strong\\u003e\\u003c/li\\u003e\\n \\u003c/ol\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eC21H44\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e296.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e23.419\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.54\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLong-chain alkane\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMetabolite observed in cancer metabolism. It has a role as a human metabolite [11]\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003col start=\\\"2\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003e7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6, 9-diene-2, 8- dione\\u003c/strong\\u003e\\u003c/li\\u003e\\n \\u003c/ol\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eC17H24O3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e276.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e25.669\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e2.59\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026alpha;, \\u0026beta; - unsaturated\\u003c/p\\u003e\\n \\u003cp\\u003eketone\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePharmacological activities, including antineoplastic, antimicrobial and antiviral activities [12]\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003col start=\\\"3\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003eStearic acid\\u003c/strong\\u003e\\u003c/li\\u003e\\n \\u003c/ol\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eC18H36O2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e284.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e26.509\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.32\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLong-chain fatty acid ethyl ester\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eantioxidants, hypocholesterolemic, nematicide, and pesticide [13]\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003col start=\\\"4\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003e1-Octadecyne\\u003c/strong\\u003e\\u003c/li\\u003e\\n \\u003c/ol\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eC18H34\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e250.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e28.067\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.62\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eStraight chain alkyne\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAntibacterial, antioxidant\\u0026nbsp;and anticancer [14]\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003col start=\\\"5\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003e2-Pentadecyn-1-ol\\u003c/strong\\u003e\\u003c/li\\u003e\\n \\u003c/ol\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eC15H28O\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e224.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e28.118\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.81\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAlcohol\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003col start=\\\"6\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003eOctadecanoic acid, ethyl ester\\u003c/strong\\u003e\\u003c/li\\u003e\\n \\u003c/ol\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eC20H40O2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e312.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e28.361\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003elong-chain fatty acid ethyl ester\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003col start=\\\"7\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003e1,11-Dodecadiyne\\u003c/strong\\u003e\\u003c/li\\u003e\\n \\u003c/ol\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eC12H18\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e162.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e29.451\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.62\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ealkynes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003col start=\\\"8\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003eOxirane, tetradecyl-\\u003c/strong\\u003e\\u003c/li\\u003e\\n \\u003c/ol\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eC16H32O\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e240.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e29.806\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.07\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eEpoxides\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003col start=\\\"9\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003e1,5,9Undecatriene\\u003c/strong\\u003e\\u003cstrong\\u003e, 2, 6,10-trimethyl-, (Z)-\\u003c/strong\\u003e\\u003c/li\\u003e\\n \\u003c/ol\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eC14H24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e192.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e33.217\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.66\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTerpens\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003col start=\\\"10\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003eCholesterol\\u003c/strong\\u003e\\u003c/li\\u003e\\n \\u003c/ol\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eC27H46O\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e386.7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e35.328\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e37.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCholestanoid (sterol)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ecardioprotective effect, anti-inflammatory, anticancer, antimicrobial, anti-psychotic, antioxidant activities and \\u0026nbsp; \\u0026nbsp; drug-delivery capability [15]\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003col start=\\\"11\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003ePhenol,2,4-bis(1,1-dimethylethyl)-, phosphite (3:1)\\u003c/strong\\u003e\\u003c/li\\u003e\\n \\u003c/ol\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eC42H63O3P\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e646.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e36.921\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e7.97\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePhenols\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eantioxidant and anti-enterococcal properties\\u003c/p\\u003e\\n \\u003cp\\u003e[16]\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003col start=\\\"12\\\"\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003eTris(2,4-di-tert-butylphenyl) phosphate\\u003c/strong\\u003e\\u003c/li\\u003e\\n \\u003c/ol\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eC42H63O4P\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e662.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e38.072\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e44.42\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAryl phosphate\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAnti-inflammatory [17]\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;Analysis of the Chemical Profile of\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003eLaevicaulis alte\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;Extract.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe comprehensive chemical profiling of\\u0026nbsp;\\u003cem\\u003eLaevicaulis alte\\u003c/em\\u003e slug chloroform extract via Gas Chromatography-Mass Spectrometry (GC-MS) provides critical insights into its remarkable therapeutic potential, as demonstrated by this study. The identification of 12 distinct compounds (Table 1), dominated by four major constituents comprising \\u0026gt;90% of the total detected area, establishes a robust chemical foundation for the observed bioactivities. This analysis delves into the pharmacological significance of these compounds, their synergistic potential, and their mechanistic roles in antibacterial action and wound regeneration.\\u003c/p\\u003e\\n\\u003cp\\u003eThe GC-MS chromatogram revealed a chemically diverse profile, with several compounds possessing well-documented biological activities relevant to the study\\u0026apos;s findings:\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eTris(2,4-di-tert-butylphenyl) phosphate\\u003c/em\\u003e\\u003c/strong\\u003e (44.42%): This aryl phosphate ester was the most abundant compound. While direct studies on its biological activity are limited, structural analogues and related organophosphates demonstrate significant anti-inflammatory properties [17]. Its high abundance strongly correlates with the in vivo observation of drastically reduced pro-inflammatory cytokines (IL-6, TNF-\\u0026alpha;) and accelerated wound closure.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eCholesterol\\u003c/em\\u003e\\u003c/strong\\u003e (37.4%): The high concentration of this ubiquitous sterol is particularly noteworthy. Beyond its structural role in membranes, cholesterol possesses documented anti-inflammatory, antimicrobial, and wound-healing promoting activities [15]. Its presence likely contributes significantly to; membrane stabilization; enhancing skin barrier repair in the proliferative phase. Precursor function; which serving as a precursor for vitamin D synthesis (promoting keratinocyte differentiation) and steroid hormones involved in inflammation resolution. Growth factor modulation, that influencing signaling pathways crucial for cell migration and angiogenesis.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003ePhenol, 2,4-bis(1,1-dimethylethyl)-, phosphite\\u003c/em\\u003e\\u003c/strong\\u003e \\u003cstrong\\u003e\\u003cem\\u003e(3:1)\\u003c/em\\u003e\\u003c/strong\\u003e (7.97%), this phenolic antioxidant belongs to a class known for radical scavenging and anti-enterococcal properties [16]. Its role is crucial in mitigating oxidative stress at the wound site, a major impediment to healing [18]. By neutralizing reactive oxygen species (ROS) generated during inflammation, it protects cells and matrix components, facilitating a transition to the proliferative phase. Its antioxidant capacity complements the anti-inflammatory action of the dominant aryl phosphate.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003e7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione (2.59%):\\u003c/em\\u003e\\u003c/strong\\u003e This \\u0026alpha;, \\u0026beta;-unsaturated ketone/spiro compound, though present in a smaller proportion, exhibited potent predicted antibacterial activity.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe extract contained several minor components (\\u0026lt;2% each) known for diverse bioactivities: \\u003cstrong\\u003eStearic acid\\u0026nbsp;\\u003c/strong\\u003e(1.32%) and \\u003cstrong\\u003eOctadecanoic\\u003c/strong\\u003e a\\u003cstrong\\u003ecid, Ethyl Ester\\u003c/strong\\u003e (1.27%): These long-chain fatty acids/esters possess reported antioxidant, hypocholesterolemic, and antimicrobial activities [13,19]. \\u003cstrong\\u003e1-Octadecyne\\u003c/strong\\u003e (0.62%): This alkyne has documented antibacterial, antioxidant, and anti-cancer properties [14]. \\u003cstrong\\u003eHeptadecane derivatives, Terpenes\\u003c/strong\\u003e (e.g., 1,5,9-Undecatriene, 2, 6,10-trimethyl-, (Z)-),\\u0026nbsp;and \\u003cstrong\\u003eEpoxides\\u003c/strong\\u003e: While their specific contributions require further study, these classes often exhibit antimicrobial or anti-inflammatory effects, potentially contributing to the overall extract efficacy through additive or synergistic effects.\\u003c/p\\u003e\\n\\u003cp\\u003eThe chemical profile of\\u0026nbsp;\\u003cem\\u003eL. alte\\u003c/em\\u003e extract reveals significant novelty: the high abundance of \\u003cstrong\\u003eTris(2,4-di-tert-butylphenyl) phosphate\\u003c/strong\\u003e in a molluskan therapeutic extract is unusual and highlights its potential as a lead anti-inflammatory compound. The combination of a significant sterol (cholesterol) with potent phenolics and specific antimicrobials (Oxaspiro compound) differs from profiles reported in other snail/slug mucins or extracts, which often emphasize proteins, peptides, glycosaminoglycans, and hyaluronic acid [20\\u0026ndash;22]. This chloroform extract targets a distinct, lipid-soluble chemical space[23,24]. The study bridges traditional knowledge (use of slugs) with modern analytical (GC-MS) and computational (docking) techniques, providing mechanistic hypotheses for the observed efficacy.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003e\\u0026nbsp; In vitro\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;assessment of the slug extract\\u0026apos;s antibacterial activity against \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e.\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e3.3.1. MIC determination\\u003c/p\\u003e\\n\\u003cp\\u003eThe \\u003cem\\u003ein vitro\\u003c/em\\u003e antibacterial activity of the \\u003cem\\u003eL. alte\\u0026nbsp;\\u003c/em\\u003echloroform extract was quantitatively assessed against the bacterium \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e ATCC 6538 using a broth microdilution assay. The extract exhibited a clear dose-dependent inhibitory effect on the growth of \\u003cem\\u003eS. aureus\\u003c/em\\u003e. A minimum inhibitory concentration (MIC) value of 0.625 mg/mL was determined. This value represents the lowest concentration of the extract that completely inhibited the visible growth of the bacteria after 24 hours of incubation at 37\\u0026deg;C. As illustrated in the dose-response curve (Figure 1), a progressive reduction in bacterial cell viability was observed with increasing concentrations of the slug extract, with viability dropping from 100% in the untreated control to near-zero levels at, and above the determined MIC. This confirmed the potent antibacterial efficacy of the crude extract against this bacterial strain.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 1.\\u003c/strong\\u003e Slug\\u0026apos;s MIC and antibacterial efficacy against \\u003cem\\u003eS. aureus\\u003c/em\\u003e ATCC 6538. The obtained results were shown as the average (n = 3) and standard error (\\u0026plusmn;5%).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eIn\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003e-silico\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;docking interaction results\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u0026nbsp;In Silico\\u003c/em\\u003e Docking Analysis against \\u003cem\\u003eS. aureus\\u003c/em\\u003e DNA Gyrase\\u003c/p\\u003e\\n\\u003cp\\u003eTo elucidate a potential molecular mechanism for the observed antibacterial activity of the \\u003cem\\u003eL. alte\\u003c/em\\u003e extract, a molecular docking study was performed. All twelve major compounds identified via GC-MS were docked into the active site of the \\u003cem\\u003eS. aureus\\u003c/em\\u003e DNA gyrase-DNA complex (PDB ID: 2XCT [23]), a validated target for antibacterial agents. The docking scores, including binding energy (B.E.), interaction energy (E_Int), and hydrogen bond energy (E_H_B), were presented in Figure 2. The study targeted the bacterial DNA gyrase, a type II topoisomerase that is essential for bacterial survival but absent in humans, making it an ideal and well-established target for antibacterial drugs, most notably the fluoroquinolone class [25]. DNA gyrase is responsible for introducing negative supercoils into DNA, a process critical for relieving torsional stress during DNA replication and transcription. Its inhibition leads to the disruption of these vital processes and ultimately results in bacterial cell death [26]\\u003c/p\\u003e\\n\\u003cp\\u003eThe results revealed that several compounds from the extract exhibited strong predicted binding affinities for the DNA gyrase active site, with binding energies ranging from -5.203 to -7.523 kcal/mol. Notably, the organophosphorus compounds phenol,2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) (\\u003cstrong\\u003ecompound 11\\u003c/strong\\u003e) and tris(2,4-di-tert-butylphenyl) phosphate (\\u003cstrong\\u003ecompound 12\\u003c/strong\\u003e) emerged as the most potent binders, with binding energies of -7.523 kcal/mol and -7.509 kcal/mol, respectively. These compounds also demonstrated the most favorable interaction energies (-32.540 kcal/mol and -21.436 kcal/mol, respectively), indicating extensive and stable non-covalent interactions within the binding pocket.\\u003c/p\\u003e\\n\\u003cp\\u003eOther compounds also showed significant binding potential. Cholesterol (\\u003cstrong\\u003ecompound 10\\u003c/strong\\u003e) and octadecanoic acid, ethyl ester (\\u003cstrong\\u003ecompound 6\\u003c/strong\\u003e) displayed strong binding energies of -6.924 kcal/mol and -6.852 kcal/mol, respectively. Of particular note, \\u003cstrong\\u003ecompound 6\\u0026nbsp;\\u003c/strong\\u003eexhibited an exceptionally low RMSD value of 1.009 \\u0026Aring;, suggesting a highly stable and well-defined binding pose.\\u003c/p\\u003e\\n\\u003cp\\u003eVisual inspection of the docking poses for the top three binders revealed key molecular interactions responsible for their high affinity (Figure 2). All three compounds positioned themselves within the enzyme\\u0026apos;s active site, interacting with key catalytic residues and the bound DNA. A critical interaction was observed with the residue arginine 458 (Arg458), as well as with the DNA bases deoxyguanosine 9 (DG9) and adenine 13 (A13). The bulky, hydrophobic tert-butyl groups of compounds \\u003cstrong\\u003e11\\u003c/strong\\u003e and \\u003cstrong\\u003e12\\u003c/strong\\u003e were stabilized by hydrophobic contacts within the pocket, while their phosphate/phosphite moieties formed strong hydrogen bonds and electrostatic interactions with the positively charged guanidinium group of Arg458. Similarly, the hydroxyl group of cholesterol (\\u003cstrong\\u003e10\\u003c/strong\\u003e) acted as a hydrogen bond donor/acceptor, while its rigid sterol backbone was anchored by extensive van der Waals forces and hydrophobic interactions with the active site and DNA backbone. These interactions effectively anchor the ligands in the active site, suggesting a potential mechanism for enzyme inhibition.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 2.\\u003c/strong\\u003e (A) Docking poses of the top three binding (\\u003cstrong\\u003e10-12\\u003c/strong\\u003e) compounds from \\u003cem\\u003eL. alte\\u003c/em\\u003e extract within the active site of \\u003cem\\u003eS. aureus\\u003c/em\\u003e DNA gyrase (PDB: 2XCT). Dashed lines represent non-covalent interactions such as hydrogen bonds and hydrophobic contacts. (B) Molecular docking scores including, B.E. (Binding Energy), rmsd (Root Mean Square Deviation), E_Int (Interaction Energy), E_H_B (Hydrogen Bond Energy). All energy values are in kcal/mol.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe impact of slug extract on wound healing and its antibacterial efficacy against\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003eS. aureus\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003ein vivo.\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp; \\u0026nbsp; Estimation of wound size at the macroscopic level\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe application of slug extract enhanced and accelerated the process of wound healing, as shown in Figure (3A), as after 14 days, the wound was completely healed after treatment with slug extract. Also, the induction of slug extract could decrease the wound diameter after 14 days (3A). The diameter of wound didn\\u0026rsquo;t show significant changes after 7 days of treatment (Figure 3B1), while it significantly decreased from 1.76 mm in the negative control group to 0.47 mm in the slug treated group after 14 days of treatment (Figure 3B2).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 3.\\u0026nbsp;\\u003c/strong\\u003eThe effect of slug administration on the healing of\\u0026nbsp;\\u003cem\\u003eS. aureus\\u003c/em\\u003e-infected wounds after 7 and 14 days. After 7 days, no significant changes in diameter were observed between the treated and control groups. However, after 14 days, notable changes (P\\u0026lt;0.05) were observed between the two groups\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp; \\u0026nbsp; Bacterial load determination\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAfter seven days of wound therapy, the bacterial content in the tissue that received slug extract was the least comparing with that within the tissues of the positive and negative controls, as illustrated in Figure 4. On day zero, the total bacterial count was 4 \\u0026times; 10\\u003csup\\u003e4\\u003c/sup\\u003e CFU/mL, but after seven days of the experiment, the total count was uncountable, 16\\u0026times;10\\u003csup\\u003e2\\u003c/sup\\u003e, and 5\\u0026times;10\\u003csup\\u003e2\\u003c/sup\\u003e CFU/mL in the wounded tissues of the negative control (group I), positive control (group II), and treated with slug (group III), respectively.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 4.\\u003c/strong\\u003e Bacterial load in wounded tissues after 7 days of \\u003cem\\u003ein vivo\\u003c/em\\u003e experiment a) treated with slug, b) positive control, and c) negative control.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp; \\u0026nbsp;Histological investigations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;According to the current findings, a skin segment from a normal mouse showed a typical structure, with an intact epidermis made up of two to three cell layers, followed by a layer of dermal connective tissue that contained hair follicles and sebaceous glands (Figure 5A). Vascular endothelial growth factor was moderately expressed in the cytoplasmic stain of the epidermis and dermis blood vessels in the sections of normal mice (Immunohistochemistry, VEGF, DAB) (Figure 5B). Numerous polymorpho-nuclear neutrophils and lymphocytes infiltrated the epidermis and dermis of the mice\\u0026apos;s skin sections with positive injuries, causing ulcer formation and indicating the presence of inflammatory cells. Furthermore, hair follicles and sebaceous glands were seen (Figure 5 C). Furthermore, the cytoplasmic staining of epidermal and dermis blood vessels revealed moderate to substantial expression of vascular endothelial growth factor (Figure 5D). Skin slices from the injured mice showed a healed lesion with intact epidermis and dermis after seven days of slug extract treatment. Sebaceous glands and hair follicles were not present in the healed ulcerated area, but polymorphonuclear neutrophils and lymphocytes moderately infiltrated the epidermis and dermis, indicating the existence of inflammatory cells (Figure 5E). Additionally, vascular endothelial growth factor was mildly expressed in the blood vessels of the epidermis and dermis, as indicated by cytoplasmic staining (Fig. 5F). Following 14 days of slug extract treatment, skin slices from the injured mice revealed an unbroken epidermis and a healed ulcer. A healed ulcer showed modest infiltration of neutrophils, lymphocytes, and inflammatory cells into the epidermis and dermis, along with degeneration of the sebaceous glands and hair follicles (Figure 5G). Additionally, vascular endothelial growth factor was moderately expressed in the blood vessels of the epidermis and dermis, as indicated by cytoplasmic staining (Figure 5H).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 5.\\u003c/strong\\u003e A) a skin section from normal animal exhibited normal skin, intact epidermis formed of 2-3 cell layers) (black arrow), dermis (connective tissue) (yellow arrow), sebaceous glands (red arrow) and hair follicles (green arrow), (piliary canals), (H\\u0026amp;E, x100). B) Showed moderate expression of vascular endothelial growth factor as cytoplasmic stain in epidermis and dermis blood vessels (Immunohistochemistry, VEGF, DAB, x100). C) A skin section from the injured mice in the positive control group displayed ulcer formation (black arrow), with nearby regions exhibiting normal skin distinguished by an epidermis formed of 2-3 cell layers) (red arrow), underlying with connective tissue (yellow arrow), and hair follicles (green arrow). Within the ulcerated region, infiltration of numerous polymorph neutrophils and lymphocytes into both the epidermis and dermis was evident (red head arrows), along with sebaceous glands and hair follicles (green arrow) (H\\u0026amp;E, x100). D) Showed moderate- marked expression of vascular endothelial growth factor as cytoplasmic stain in epidermis and dermis blood vessels (black arrow) (Immunohistochemistry, VEGF, DAB, x100). E) a skin section from the treated group with slug mucin after 7 days showed healed ulcer (black arrow), intact epidermis (formed of 2-3 cell layers) (red arrow), dermis (layer of connective tissue) (yellow arrow), Healed ulcerated area, showed moderate infiltration of inflammatory cells, neutrophils, lymphocytes, and polymorphs into the epidermis and dermis; lack of sebaceous glands and hair follicles (H\\u0026amp;E, x100). F) Showed mild expression of vascular endothelial growth factor as cytoplasmic stain in epidermis and dermis blood vessels (black arrow) (Immunohistochemistry, VEGF, DAB, x100). G) a skin section from the treated group with slug mucin for 14 days showed healed ulcer (black arrow), intact epidermis (external epithelium formed of 2-3 cell layers) (red arrow). Healed ulcerated area, showed mild infiltration of epidermis and dermis by polymorph, neutrophils and lymphocytes inflammatory cells (yellow arrows), degenerated of both sebaceous glands and hair follicles (black arrow) (H\\u0026amp;E, x100). H) exhibited moderate expression of vascular endothelial growth factor as cytoplasmic stain in epidermis and dermis blood vessels (Immunohistochemistry, VEGF, DAB, x100).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp; Pro-and anti-inflammatory cytokines detection:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe slug extract showed a promising effect on both pro-inflammatory and anti-inflammatory cytokines in the wound of the skin of mice. There was a significant decrease in pro-inflammatory cytokines represented with IL-6 levels (from 389 to 277 pg/mL after 7 and 14 days of slug extract treatment) and TNF-\\u0026alpha; levels (from 432 to 350 pg/mL) for the same respective durations (Figure 6), compared to the negative controls. Conversely, the anti-inflammatory biomarker IL-10 levels were significantly increased as compared with negative controls after treating with the slug extract on 7- and 14-days post wound from 601to 626 pg/mL, respectively (Figure 6).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 6.\\u003c/strong\\u003e Proinflammatory cytokine levels (IL-6 and TNF-\\u0026alpha;) and anti-inflammatory cytokine level (Il-10) after treatment with slug extract after 7 and 14 days. All values represent (mean\\u0026plusmn; SD). * Significant (P\\u0026lt;0.05) on compared with negative control.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eIn\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003e-silico\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;docking interaction results\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo investigate the molecular mechanisms underlying the observed immunomodulatory activity of the \\u003cem\\u003eLaevicaulis alte\\u003c/em\\u003e extract, a comprehensive molecular docking study was performed. The twelve major compounds identified by GC-MS were docked against the binding sites of three key cytokines: the pro-inflammatory mediators including; Tumor Necrosis Factor-alpha (TNF-\\u0026alpha;, PDB: 2AZ5[27] and Interleukin-6 (IL-6, PDB: 1ALU [28]), and the anti-inflammatory cytokine Interleukin-10 (IL-10, PDB: 1Y6K [29]). The binding affinities and interaction parameters were represented in \\u003cstrong\\u003eFigures 7\\u003c/strong\\u003e,8, and \\u003cstrong\\u003e9.\\u003c/strong\\u003e The docking results revealed that the compounds from the slug extract, particularly the organophosphorus derivatives and cholesterol, exhibited significant and differential binding affinities for the cytokine targets.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; Interaction with Pro-Inflammatory Cytokines:\\u003c/p\\u003e\\n\\u003cp\\u003eTNF-\\u0026alpha; (PDB: 2AZ5): The strongest predicted binding affinity was observed for compound (\\u003cstrong\\u003e11\\u003c/strong\\u003e), with a binding energy (B.E.) of -6.663 kcal/mol. This was closely followed by tris(2,4-di-tert-butylphenyl) phosphate (12) and cholesterol (10) with B.E. values of -6.373 kcal/mol and -5.693 kcal/mol, respectively. Visual analysis of the binding poses (Figure 7) revealed that compound 11 engaged in a crucial \\u0026pi;-stacking interaction with the aromatic ring of Tyr119. Compounds 10 and 12 were stabilized by H-bonds with the backbone of Leu157 and electrostatic interactions with Lys11.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 7.\\u003c/strong\\u003e (A) Docking poses of the top three binding (\\u003cstrong\\u003e10-12\\u003c/strong\\u003e) compounds from \\u003cem\\u003eL. alte\\u003c/em\\u003e extract within the active site of TNF-\\u0026alpha;(PDB: 2AZ5). Dashed lines represent non-covalent interactions such as hydrogen bonds and hydrophobic contacts. (B) Molecular docking scores including, B.E. (Binding Energy), rmsd (Root Mean Square Deviation), E_Int (Interaction Energy), E_H_B (Hydrogen Bond Energy). All energy values are in kcal/mol.\\u003c/p\\u003e\\n\\u003cp\\u003eIL-6 (PDB: 1ALU): The binding affinities for IL-6 were generally more modest. The most favorable interaction was for tris(2,4-di-tert-butylphenyl) phosphate (12), with a B.E. of -5.163 kcal/mol. The interactions, shown in Figure 8, were primarily driven by \\u0026nbsp; \\u0026nbsp;\\u0026pi;-\\u0026pi; stacking with the indole ring of Trp157 and hydrophobic contacts with Gln156. Cholesterol (10) formed a key hydrogen bond with the side chain of Lys46.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 8.\\u003c/strong\\u003e (A) Docking poses of the top three binding (10-12) compounds from \\u003cem\\u003eL. alte\\u003c/em\\u003e extract within the active site of IL-6 (PDB: 1ALU). Dashed lines represent non-covalent interactions such as hydrogen bonds and hydrophobic contacts. (B) Molecular docking scores including, B.E. (Binding Energy), rmsd (Root Mean Square Deviation), E_Int (Interaction Energy), E_H_B (Hydrogen Bond Energy). All energy values are in kcal/mol.\\u003c/p\\u003e\\n\\u003cp\\u003e3.5.2. Interaction with Anti-Inflammatory Cytokine:\\u003c/p\\u003e\\n\\u003cp\\u003eIL-10 (PDB: 1Y6K): The slug compounds demonstrated the strongest overall binding affinities for the anti-inflammatory cytokine IL-10. Compound 12 and 11 emerged as the most potent binders, with excellent B.E. values of -7.433 kcal/mol and -7.188 kcal/mol, respectively. Cholesterol (10) also showed a strong affinity with a B.E. of -5.997 kcal/mol. The binding modes for Compounds 11 and 12 were stabilized by extensive hydrophobic interactions with the aromatic ring of Phe37 (Figure 9).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFigure 9.\\u003c/strong\\u003e (A) Docking poses of the top three binding (10-12) compounds from \\u003cem\\u003eL. alte\\u003c/em\\u003e extract within the active site of IL-10 (PDB: 1Y6K). Dashed lines represent non-covalent interactions such as hydrogen bonds and hydrophobic contacts. (B) Molecular docking scores including, B.E. (Binding Energy), rmsd (Root Mean Square Deviation), E_Int (Interaction Energy), E_H_B (Hydrogen Bond Energy). All energy values are in kcal/mol.\\u003c/p\\u003e\\n\\u003cp\\u003eCollectively, these results indicated that the lead compounds from the slug extract, particularly 10, 11, and 12, have the structural capacity to interact effectively with both pro- and anti-inflammatory cytokine targets through distinct molecular interactions.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eSearching for new, unconventional sources that have an antibacterial effect has become important nowadays. Although, snails and slugs perform multifunctional roles through the regulation of algae and fungi, endozoochory, litter decomposition, facilitation of nutrient recycling and by being prey to various invertebrates and vertebrates, snails and slugs are very destructive pests that eat the twigs of plants and crops, which hurts the economy [30]. To avoid serious harm of \\u003cem\\u003eLaevicaulis alte\\u003c/em\\u003e slugs, we used in this study the roasted form of them as antibacterial agent against \\u003cem\\u003eStaphylococcus aureas\\u003c/em\\u003e \\u003cem\\u003ein vitro\\u0026nbsp;\\u003c/em\\u003eand \\u003cem\\u003ein vivo\\u003c/em\\u003e. The obtained results indicated \\u003cem\\u003ein vitro\\u003c/em\\u003e reduction of \\u003cem\\u003eStaphylococcus aureas\\u003c/em\\u003e cell density with MIC value of 0.625 mg/mL. Additionally, the number of bacteria (bacterial load) was found to be lower during the \\u003cem\\u003ein vivo\\u003c/em\\u003e trial, especially in the wounded tissue that was treated with slug comparing with negative and positive controls. Generally, insects like snails and slugs make slime, which is a mucus-like substance with a complex makeup. 90% to 99.7% of snail and slug slim is water by weight, with the remaining 0.3% to 10% being made up of enzymes, glycoproteins, proteoglycans such as hyaluronic acid, achacin, glycosaminoglycans, antimicrobial peptides, copper peptides, and metal ions[20]. Moreover, thousands of bioactive compounds, including polypropinate, terpenes, sterols, fatty acid derivatives, alkaloids, and nitrogenous substances, have been discovered to be present in mollusks. The antibacterial, cytotoxic, antitumor, anti-inflammatory, antileukemic, antineoplastic, and antiviral qualities of these substances in mollusks have been the subject of much research [22].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAccording to GC/Ms results, the current investigation showed that the slug extract included 13 different chemicals, including phenols, terpenes, cholesterol, and fatty acids. The current data were similar to findings of Ibrahim et al. [21] who reported that GC/MS examination of \\u003cem\\u003eE. desertorum\\u003c/em\\u003e mucin resulted in ten compounds have been identified and were mainly quinolines, monoterpenes, alcohol esters, sesquiterpenoids, fatty acid esters, fatty acids, and phenol derivatives. Sallam et al. [31] reported that such compounds have antimicrobial, anticancer and antioxidant activities. But there’s was not any previous study regarding the slug extract identified compounds.\\u003c/p\\u003e\\n\\u003cp\\u003eThe GC-MS investigation of the slug extract identified numerous compounds with exhibiting diverse biological functions. Between these compounds’ tris (2,4-di-tert-butylphenyl) phosphate; cholesterol; phenol, 2,4-bis (1,1-dimethylethyl)-, phosphite (3:1); and 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione with area sums of 44.42, 37.4, 7.97, and 2.59%, respectively. The GC-MS analysis of \\u003cem\\u003eLaevicaulis alte\\u003c/em\\u003e chloroform extract was not merely a compositional list; it was the key to understanding its remarkable therapeutic potential. The profile revealed a sophisticated blend of bioactive lipids and phenolics, dominated by anti-inflammatory (tris(2,4-di-tert-butylphenyl) phosphate, cholesterol), antioxidant (phenol derivative), and antimicrobial (7,9-Di-tert-butyl-1-oxaspiro compound, fatty acids) compounds.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eIn silico\\u003c/em\\u003e docking provided plausible molecular targets for these activities, corroborating the \\u003cem\\u003ein vitro\\u003c/em\\u003e and \\u003cem\\u003ein vivo\\u003c/em\\u003e results. The synergy between these components effectively combated the bacterial infection by \\u003cem\\u003eS. aureus\\u003c/em\\u003e, resolved detrimental inflammation (reducing IL-6/TNF-α, elevating IL-10), promoted angiogenesis (moderate VEGF), and accelerated tissue regeneration, leading to complete wound closure within 14 days. This chemical blueprint positions \\u003cem\\u003eL. alte\\u003c/em\\u003e slug extract as a highly promising natural source for developing novel therapeutic agents targeting infected and chronic wounds. Future research should focus on isolating the major active principles (particularly the aryl phosphate and oxaspiro compound), validating their individual and combined efficacies, and exploring formulation strategies for topical delivery. The docking results strongly suggested that multiple components of the slug extract can effectively occupy and interact with the enzyme's active site. The two most potent compounds\\u0026nbsp;\\u003cstrong\\u003e12\\u003c/strong\\u003e and\\u0026nbsp;\\u003cstrong\\u003e11\\u003c/strong\\u003e, displayed the highest predicted affinity. Their efficacy was likely derived from a combination of structural features. The bulky, lipophilic tert-butylphenyl groups can engage in extensive hydrophobic and van der Waals interactions within the binding pocket, while the central phosphate/phosphite group provided a critical anchor point. As seen in Figure 2, this polar moiety formed strong electrostatic interactions and H- bonds with Arg458. The positively charged guanidinium group of arginine residues was frequently involved in stabilizing the negatively charged phosphate backbone of DNA or interacting with small molecule inhibitors, making this interaction particularly significant for potent inhibition[32]. The strong binding of cholesterol (10) and ethyl ester of octadecanoic acid (6\\u003cstrong\\u003e)\\u003c/strong\\u003e further reinforced the hypothesis that the extract's activity stemmed from a multi-component and synergistic effect. While their binding energies were slightly less favorable than the top two organophosphates, their high abundance in the extract means they likely contribute significantly to the overall biological effect. Cholesterol's rigid, hydrophobic structure is well-suited for occupying hydrophobic pockets, while its single OH group provided specific H-bonding capacity. The long aliphatic chain of octadecanoic acid likewise provided hydrophobic anchoring. The remarkably low RMSD of compound 6suggested a particularly favorable and conformationally locked binding mode, which is often indicative of a high-affinity interaction. It was crucial to interpret these findings in the context of the whole extract. The observed MIC of 0.625 mg/mL was not the result of a single compound but rather the cumulative effect of this chemical consortium. The presence of multiple potential inhibitors, each with respectable binding affinities, presented a multi-pronged attack on the bacterial enzyme. This phenomenon of synergy, where the combined effect of compounds is greater than the sum of their individual effects, is a well-known advantage of natural product-based therapies\\u0026nbsp;[33]. This can also be a strategy to circumvent the development of drug resistance, as it is more difficult for a bacterium to simultaneously evolve resistance mechanisms against multiple active molecules.\\u003c/p\\u003e\\n\\u003cp\\u003eFor medical applications, it is crucial to explore the possibility of discovering novel, potent bioactive compounds that can quicken wound healing by decreasing tissue fibrosis, re-epithelialization, and wound closure time. Cutaneous wound healing is a complex biological process. Restoring the injured epithelium's barrier function necessitates the synchronisation of time and spatially regulated cellular and molecular activities [34]. The histological sections of the wounded skin from the treated group with slug extract were demonstrated a recoveredulcer with slight infiltration of polymorphnuclear leukocytes, neutrophils, and lymphocytes, in the epidermis and dermis. Sebaceous glands and hair follicles were absent, while the epidermis and dermis remained intact. Similar outcomes were obtained by El-Sayed et al. [35] who found that selenium nanoparticles could heal the wound after 11 days. Additionally, Errajouani et al. [36] demonstrated that slime from the garden snail \\u003cem\\u003eCepaea hortensis\\u003c/em\\u003e effectively facilitated nearly complete tissue healing after 24 days of treatment on excision wounds in rabbits. The inflammatory phase is the most crucial stage in the healing process for wounds. Moreover, persistent inflammation results in significant healing disruptions, an increase in fibrosis and scarring, and elevated levels of cytokines, including IL-6, TNF-α, and IL-1.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;TNF-α is a cytokine that induces inflammation and is expressed in high levels during inflammation [18]. The multipurpose cytokine IL-6 also influences inflammation and haematopoiesis, and immune responses in pleiotropic ways [24]. The current study demonstrated that slug extract effectively reduced both cytokines, IL-6 and TNF-α levels after 7 and 14 days post wound exposure in comparing with the control group. Similarly, [37] reported that calophyllolidecompound produced from \\u003cem\\u003eCalophyllum inophyllum\\u0026nbsp;\\u003c/em\\u003ehas the ability to modulate inflammatory cytokines response by reducing levels of proinflammatory cytokines such as IL-1β, IL-6 and TNF-α. These suggests a potential anti-inflammatory effect associated with calophylloid. Additionally,\\u0026nbsp;[38], found that The mucus produced by the mollusks slug \\u003cem\\u003eArion subfuscus\\u003c/em\\u003e could lowered the expression of some proinflammatory cytokines like, Il-6 and TNF-α and IL-1β.\\u003c/p\\u003e\\n\\u003cp\\u003eThe pleomorphic cytokine IL10 showed anti-inflammatory qualities and a different stimulated immunity cells may contribute to its secretion[19]. Its principal functions include anti-inflammatory, inhibitory, or self-regulatory. Interleukin-10 serves as a potent detrimental feedback regulator crucial for the management and resolution of inflammation via autocrine and paracrine mechanisms.\\u003c/p\\u003e\\n\\u003cp\\u003eIL-10 has two arms that driven a broad immunosuppressive effect, preventing dendritic cells from presenting antigens and preventing the activation of macrophages and their infiltration into the injured area, with the secondary target of reducing the expression of pro-inflammatory cytokines [39]. Additionally, HuR (human antigen R) is a messenger RNA (mRNA) stabilizing protein. It is thought that IL-10 functions as a posttranscriptional regulatory agent at the cellular level to repress HuR and encourage the destabilization of inflammatory cytokine mRNA [40]. The present study showed that slug extract could increase IL-10 levels after 7 and 14 days post wound exposure in comparing with the control. Comparable findings were noted by [41], who concluded that calophyllolide isolated from \\u003cem\\u003eCalophyllum inophyllum\\u003c/em\\u003e Linn could up-regulate an anti-inflammatory cytokine, IL-10. Collectively, it is evident that slug extract promotes faster wound healing by anti-inflammatory actions, specifically by controlling inflammatory cytokines.\\u003c/p\\u003e\\n\\u003cp\\u003eThe in vivo results of this study painted a clear picture of a sophisticated immunomodulatory effect: the slug extract concurrently suppressed the pro-inflammatory cytokines TNF-α and IL-6 while enhancing the anti-inflammatory cytokine IL-10. The molecular docking investigation provides a compelling, multivalent mechanistic framework that explains how this balanced immune response could be achieved at the molecular level. The findings suggest that the extract does not act as a blunt instrument but rather as a cocktail of regulatory molecules that interact differentially with key players in the inflammatory cascade.\\u003c/p\\u003e\\n\\u003cp\\u003eThe predicted inhibition of the pro-inflammatory cytokines is primarily driven by the organophosphorus compounds 11 (phenol phosphite) and 12 (TDTBPP), with support from 10 (cholesterol). Compound 11 emerged as the most potent theoretical binder to TNF-α (B.E. -6.663 kcal/mol). Its ability to form a stable π-stacking interaction with Tyr119 was particularly significant, as this residue was known to be critical for the structural integrity and receptor-binding function of TNF-α [42]. By occupying this site, compound 11 could sterically hinder the trimerization of TNF-α or its binding to the TNFR1 receptor, effectively neutralizing its pro-inflammatory signaling. Similarly, the binding of compound 12 to IL-6 suggested a mechanism for disrupting the IL-6/IL-6R/gp130 signaling complex, which is central to chronic inflammation[43]. The ability of multiple major components of the extract to bind these targets suggests a synergistic blockade of pro-inflammatory pathways.\\u003c/p\\u003e\\n\\u003cp\\u003eIn conclusion, this study revealed a sophisticated chemical strategy encoded within the slug extract. The organophosphorus compounds (11 and 12) appear to act as master immunomodulators, demonstrating the highest potential to both neutralize key pro-inflammatory signals (TNF-α) and stabilize the primary anti-inflammatory signal (IL-10). Cholesterol (10) acted as a robust supporting molecule, contributing to the overall effect. This multi-target approach, where the same set of molecules can engage with opposing sides of the immune response, provided a more nuanced \\\"re-tuning\\\" of the wound microenvironment than simple immunosuppression. It facilitated the resolution of inflammation while actively promoting an anti-inflammatory state, which is precisely the profile required for efficient and high-fidelity wound healing. The docking analysis provided strong theoretical evidence that the observed immunomodulatory properties of the \\u003cem\\u003eL. alte\\u003c/em\\u003e extract were not coincidental but are based on specific, high-affinity molecular interactions between its major chemical constituents and key cytokine regulators. The data strongly implicates compounds 11, 12, and 10 as the primary mediators of this effect, laying a solid foundation for future experimental validation and the potential development of a novel, balanced anti-inflammatory therapy from this unique natural source.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eSlug source and extract preparation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eLaevicaulis alte\\u003c/em\\u003e slugs (9- 10 mm) were reared in Invertebrate lab, Faculty of Science, Helwan University, Egypt. It was kept in 15 x 24 x 10 cm plastic boxes with oven dried lettuce leaves and dechlorinated tap water. Slugs were cleaned using distilled water and 70% ethyl alcohol. After complete drying, they were roasted at 37°C for 3 days in oven, later they were grinded with a 400 mm disc mill (EarthTechnica co.LTD, Japan,). The powdered slugs were submerged in chloroform solvent (Sigma-Aldrich, United States) (1:2) for two days. Subsequently, the slug particles were discarded using ultracentrifugation (Sigma, Germany) at a temperature of 4°C and a speed of 10,000 rpm for a duration of 10 min. Then, chloroform was evaporated using a rotary evaporator (IKA, Germany) and final extract was obtained.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp; Slug extract analysis using GC-MS\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAfter the slug extract was prepared, it was dissolved in chloroform to be analyzed using a GC-MS instrument (Agilent Technologies, Santa Clara, California) at Central Laboratories Network, National Research Centre, Cairo, Egypt. The gas chromatography system was fitted with a 30-meter-long DB-5MS column that had an internal diameter of 0.25 mm and a film thickness of 0.25 μm. The used carrier gas was helium at a flow rate of 2.0 mL/min with a splitless injection volume of 1 µL. The followed program of temperature was 50°C for 5 min.; rising at 5°C/min. to 100°C and held for 0 min., and rising at 10°C/min. to 320°C and held for 10 min. Both detector and injector temperatures were maintained at 320°C and 280°C, respectively. Mass spectra were generated using electron ionization (EI) at 70 eV, covering a spectral range of m/z 25-800 with a solvent delay of 3 min. The mass temperature was set at 230°C, and Quad was set to 150°C. Identification of various constituents was accomplished by comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;Antibacterial activity\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eof slug extract against \\u003cem\\u003eStaphylococcus aureus\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Broth microdilution assay and MICs determination\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe examination was carried out employing the broth microdilution method, which was slightly modified from original protocol [44,45]. To make stock extract, 2.5 mg of slug powder was added to 1 mL of sterile water. The initial column well of a microtiter plate was filled with 400 µL of the obtained stock extract, and each well between 2 and 10 received 200 µL of sterile tryptic soy broth (TSB). By moving 200 µL gently from the first well to the seventh, a two- fold dilution was created. Subsequently, 50 µL of the reference strain \\u003cem\\u003eS. aureus\\u003c/em\\u003e ATCC 6538 with a concentration of 5×10\\u003csup\\u003e8\\u003c/sup\\u003e CFU/mL (OD~0.1) was applied to each well in a single raw, excluding the final well designated as a blank. Both chloramphenicol (1 mg/mL) and ciprofloxacin (1 mg/mL) were used as a positive control. The plate was kept in an incubator at 37\\u003csup\\u003eo\\u003c/sup\\u003eC for 24 hours. Following incubation, the results were determined at 630 nm using ChroMate 4300, USA Elisa reader.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eWound healing and antibacterial efficacy of slug extract against\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003cem\\u003ein vivo\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; Animal ethics committee\\u0026nbsp;for mouse model of wound infection\\u003c/p\\u003e\\n\\u003cp\\u003eThe care and utilization of research animals at Helwan Institution follow guidelines that authorized by committee of the university’s Animal Ethics, with each research project assigned a specific research ethics number [HU-IACUC/Z/MY3107-31].\\u0026nbsp;This study was done in accordance with ARRIVE guidelines (\\u003ca href=\\\"https://arriveguidelines.org/\\\" target=\\\"_blank\\\"\\u003ehttps://arriveguidelines.org\\u003c/a\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003eAnimals\\u003c/p\\u003e\\n\\u003cp\\u003eAbout six-week-old male mice (weighing around 30 g) were purchased from Theodor Bilharez Institute's animal house in Egypt and were acclimatized in the laboratory for two weeks. The animals received tap water to drink and Purina chaw (20 % protein).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp; \\u0026nbsp;Developing excision wound\\u003c/p\\u003e\\n\\u003cp\\u003eDiethyl ether was used to anesthetize the mice before performing full thickness excisional skin wounds. The dorsal fur was subsequently cleansed, shaved, and disinfected in preparation for the experiment. An aseptic puncher was employed to produce an oval, full thickness wound (3 mm), on the upper back of the mice.\\u0026nbsp;At the end of the study, all euthanized mice were treated humanely and killed with cervical dislocation \\u0026nbsp; to allow for tissue and organ examination, prevent their reintroduction into other experiments, and eliminate potential biohazards if released.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;Treatment groups\\u003c/p\\u003e\\n\\u003cp\\u003eThree mice groups (n = 10) were housed in different cages, with free-flowing water and unlimited food. Group I (negative control) included mice with excisional skin wounds treated with phosphate buffer saline (PBS) only, group II (positive control) included mice with excisional skin wounds received gentamycin cream (0.1% gentamicin), and group III (treated) included mice with excisional skin wound received 20 µL slug extract (625 µg/mL), the treatments were topically administrated to the wounded skin once daily for 14 days.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp; \\u0026nbsp;Macroscopic estimation of wound size\\u003c/p\\u003e\\n\\u003cp\\u003eFollowing topical application, daily photos of the wound were captured. The wound area was assessed by measuring the distance along the wound bed and delineating a circle around its perimeter [26]. To ascertain each image's magnification during measurement analysis, a fine ruler was positioned at the wound level to determine its magnification.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp; Bacterial load estimation\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe following protocol was used for bacterial load determination in both treated (II and III) and untreated (I) groups, according to El-Sayed et al.[35]. The experiment was carried out on both the zero and seventh days of the in vivo experimentafter inoculating the wound of the experimental mice with bacteria, disposable medical scalpels were used to excise a 1 mm thick wound from each of the three groups. Then, samples were put into sterile 1X phosphate buffered saline (PBS), vortexed gently for 5 min., and then diluted one by one. 100 µL of each concentration was then put on Muller Hinton Agar (MHA) medium plates.\\u0026nbsp;Plates that were inoculated were then left at 37°C for 24 hours, followed by colony enumeration the subsequent day was carried out.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp; Pathological investigations\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe samples were kept in 10% formalin for a full day after the skins of the treated and untreated mice were autopsied on the seventh day of the experiment and at the conclusion of the trial. Following a series of alcohol dilutions to dry them out, the samples were cleaned in xylene and then submerged in paraffin for an entire day in oven (56ºC). Tissue blocks with paraffin sliced to an overall thickness of 5 µm utilizing slide microtomes. Tissue specimens wesre removed, deparaffinized, and subjected to staining with Masson's trichrome, hematoxylin, and eosin (H\\u0026amp;E) before being seen under a light microscope for histological examination\\u0026nbsp;[35,46].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; Pro- and anti-inflammatory cytokine detection\\u003c/p\\u003e\\n\\u003cp\\u003eThe enzyme-linked immunosorbent test (ELISA) was used. To eliminate all blood, a little portion of the damaged skin was taken out, and the area was cleaned with ice-cold PBS (0.01 M, and PH = 7.4). Using a glass homogenizer, pieces of frozen tissue were homogenized in PBS (1 g/9 mL), then the homogenate was subjected to ultrasonic cell disruption (BioLogics, Inc., 150V/T, Virginia, USA) to further disintegrate the cells. Finally, the homogenate was centrifuged for five min. at 5000 xg, and the resulting solution was preserved at -80°C for later testing. Using a commercial ELISA kit (Elabscience, USA), the levels of IL-6 and TNF-α cytokines were quantified. In short, following the addition of samples and standards to each well, biotinylated detection antibodies specific to both cytokines, along with an Avidin-Horseradish Peroxidase (HRP) complex, were introduced,and the plates were then incubated. The optical densities of IL-6 and TNF-α were determined using biotinylated detection antibodies. The concentrations of the two cytokines were determined based on their respective OD values. Additionally, ELISA kits from BioSource International (Camarillo, California, USA) and commercially available reagents were used to measure the IL-10 concentrations in mice. As directed by the manufacturer, cytokine concentrations were measured by referencing a standard curve derived from the known concentration of cytokine standards provided on each assay plate\\u0026nbsp;[47].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003e\\u0026nbsp;In Silico\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003eMolecular Docking Studies\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo elucidate the potential molecular mechanisms underlying the observed antibacterial and immunomodulatory activities of the \\u003cem\\u003eL. alte\\u003c/em\\u003e extract, a comprehensive series of molecular docking simulations was conducted using the autodock software suite.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;Preparation of Receptor Structures\\u003c/p\\u003e\\n\\u003cp\\u003eThe three-dimensional atomic coordinates of the four target proteins were retrieved from the RCSB Protein Data Bank (PDB). The selected targets were: \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e DNA gyrase complexed with DNA (PDB ID: 2XCT), human Tumor Necrosis Factor-alpha (TNF-α) trimer (PDB ID: 2AZ5), human Interleukin-6 (IL-6) (PDB ID: 1ALU), and human Interleukin-10 (IL-10) (PDB ID: 1Y6K). Prior to docking, all protein structures were meticulously prepared. All water molecules, co-crystallized ligands, ions, and any other non-essential heteroatoms were removed from the PDB files. The structures were then subjected to the Protonate 3D tool to add H-atoms and assign appropriate ionization states at a simulated physiological pH of 7.4.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp; \\u0026nbsp;Preparation of Ligand Structures\\u003c/p\\u003e\\n\\u003cp\\u003eThe chemical structures of the twelve major compounds identified in the GC-MS analysis were constructed as 2D models using ChemDraw software. They were subsequently converted to 3D models and subjected to a rigorous energy minimization protocol using the Charm force field. The minimization was carried out until a root-mean-square gradient of \\u0026lt;0.01 kcal/mol·Å was reached. This crucial step ensures that each ligand adopted a stable, low-energy conformation before being introduced to the protein's active site. The database of minimized ligands was then prepared for the docking procedure.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; Docking Simulation Protocol\\u003c/p\\u003e\\n\\u003cp\\u003eFor each protein target, the active binding site was defined based on the location of the co-crystallized ligand in the original PDB file or identified using the Site Finder tool. A docking simulation was then performed for each of the twelve prepared ligands against each of the four protein targets. The 30 best poses from this initial placement were then subjected to refinement via forcefield energy minimization, allowing for flexible ligand-receptor interactions. The final poses were scored using the scoring function, which estimates the free energy of binding (B.E., in kcal/mol) of the ligand from a given pose. All 3D visualizations of the ligand-receptor complexes were generated using the built-in MOE visualizer and PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC). The binding energy (B.E.), root-mean-square deviation (rmsd), interaction energy (E_Int), and hydrogen bond energy (E_H_B) were tabulated for comparative analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp; Statistical data analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; The software used for analyzing data statistically was GraphPad Prism 9.0 (LLC). Tukey's post hoc test and one-way ANOVA were used to statistically compare the means. Data was provided in the form of mean ± standard deviation, with the threshold of significance set at less than 0.05.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003e\\u003cem\\u003eL. alte \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; Laevicaulis alte \\u0026nbsp;\\u0026nbsp;\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eGC-MS \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; Gas chromatography-mass spectrometry\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTDTBPP \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; Tris(2,4-di-tert-butylphenyl) phosphate\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eMIC \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Minimum inhibitory concentration\\u003c/p\\u003e\\n\\u003cp\\u003eCFU \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Colony forming unit\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contributions:\\u0026nbsp;\\u003c/strong\\u003eConceived and designed the study: H.E.-S., M.A.H., and M.Y.M.; methodology, H.E.-S., M.Y.M., M.A.H., A.F.A., H.S., O.A.H., A.A.E. and A.M.I.; collected the data: H.E.-S., M.Y.M., M.A.H., H.S. and O.A.H.; performed the analysis: H.E.-S., M.Y.M., A.A.E. and M.A.H.; funding acquisition, H.S.; wrote the paper: H.E.-S., M.Y.M., M.A.H., A.F.A., H.S., O.A.H., A.A.E. and A.M.I. All authors have read and agreed to the published version of the manuscript\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding:\\u003c/strong\\u003e Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R83), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability Statement:\\u003c/strong\\u003e. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments:\\u003c/strong\\u003e The authors are grateful to Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R83), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests:\\u0026nbsp;\\u003c/strong\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eSounouvou, H. T. \\u003cem\\u003eet al.\\u003c/em\\u003e Antimicrobial potentials of essential oils extracted from West African aromatic plants on common skin infections. \\u003cem\\u003eSci. African\\u003c/em\\u003e\\u003cstrong\\u003e11,\\u003c/strong\\u003e e00706 (2021).\\u003c/li\\u003e\\n\\u003cli\\u003eAbdo, J. M., Sopko, N. A. \\u0026amp; Milner, S. M. The applied anatomy of human skin: A model for regeneration. \\u003cem\\u003eWound Med.\\u003c/em\\u003e\\u003cstrong\\u003e28,\\u003c/strong\\u003e 100179 (2020).\\u003c/li\\u003e\\n\\u003cli\\u003eCanchy, L., Kerob, D., Demessant, A. \\u0026amp; Amici, J.-M. Wound healing and microbiome, an unexpected relationship. \\u003cem\\u003eJ. Eur. Acad. Dermatology Venereol.\\u003c/em\\u003e\\u003cstrong\\u003e37,\\u003c/strong\\u003e 7\\u0026ndash;15 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eVan Hees, C. \\u0026amp; Naafs, B. in \\u003cem\\u003eCommon Ski. Dis. Africa an Illus. Guid.\\u003c/em\\u003e 89 (2011).\\u003c/li\\u003e\\n\\u003cli\\u003eWilkinson, H. N. \\u0026amp; Hardman, M. J. Wound healing: cellular mechanisms and pathological outcomes. \\u003cem\\u003eOpen Biol.\\u003c/em\\u003e\\u003cstrong\\u003e10,\\u003c/strong\\u003e 200223 (2020).\\u003c/li\\u003e\\n\\u003cli\\u003eRaziyeva, K. \\u003cem\\u003eet al.\\u003c/em\\u003e Immunology of Acute and Chronic Wound Healing. \\u003cem\\u003eBiomolecules\\u003c/em\\u003e\\u003cstrong\\u003e11,\\u003c/strong\\u003e (2021).\\u003c/li\\u003e\\n\\u003cli\\u003eGupta, N. K., Paul, P., Barman, H. \\u0026amp; Aditya, G. The marsh slug, Deroceras laeve in Darjeeling Himalayas, India: First record and modelling of suitable habitats. \\u003cem\\u003eActa Ecol. Sin.\\u003c/em\\u003e\\u003cstrong\\u003e43,\\u003c/strong\\u003e 432\\u0026ndash;438 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eKumar, P. A Review\\u0026mdash;On Molluscs as an Agricultural Pest and Their Control. \\u003cem\\u003eInt. J. Food Sci. Agric.\\u003c/em\\u003e\\u003cstrong\\u003e4,\\u003c/strong\\u003e 383\\u0026ndash;389 (2020).\\u003c/li\\u003e\\n\\u003cli\\u003eBruelheide, H. \\u0026amp; Scheidel, U. Slug herbivory as a limiting factor for the geographical range of Arnica montana. \\u003cem\\u003eJ. Ecol.\\u003c/em\\u003e\\u003cstrong\\u003e87,\\u003c/strong\\u003e 839\\u0026ndash;848 (1999).\\u003c/li\\u003e\\n\\u003cli\\u003eHanley, M. E. Seedling herbivory, community composition and plant life history traits. \\u003cem\\u003ePerspect. Plant Ecol. Evol. Syst.\\u003c/em\\u003e\\u003cstrong\\u003e1,\\u003c/strong\\u003e 191\\u0026ndash;205 (1998).\\u003c/li\\u003e\\n\\u003cli\\u003eReyes, V. M. H., Mart\\\\\\u0026rsquo;\\\\inez, O. \\u0026amp; Hern\\u0026aacute;ndez, G. F. National center for biotechnology information. \\u003cem\\u003ePlant Breeding. Univ. Aut\\u0026oacute;noma Agrar. Antonio Narro, Calzada Antonio Narro\\u003c/em\\u003e (1923).\\u003c/li\\u003e\\n\\u003cli\\u003eTaskin, H. Detection of Volatile Aroma Compounds of Morchella by Headspace Gas Chromatography Mass Spectrometry (HS-GC/MS). \\u003cem\\u003eNot. Bot. Horti Agrobot. Cluj-Napoca\\u003c/em\\u003e\\u003cstrong\\u003e41,\\u003c/strong\\u003e 122\\u0026ndash;125 (2013).\\u003c/li\\u003e\\n\\u003cli\\u003eSiswadi, S. \\u0026amp; Saragih, G. S. Phytochemical analysis of bioactive compounds in ethanolic extract of Sterculia quadrifida R. Br. in \\u003cem\\u003eAIP Conf. Proc.\\u003c/em\\u003e\\u003cstrong\\u003e2353,\\u003c/strong\\u003e (2021).\\u003c/li\\u003e\\n\\u003cli\\u003eBelakhdar, G., Benjouad, A., Abdennebi, E. H. \\u0026amp; others. Determination of some bioactive chemical constituents from Thesium humile Vahl. \\u003cem\\u003eJ Mater Env. Sci\\u003c/em\\u003e\\u003cstrong\\u003e6,\\u003c/strong\\u003e 2778\\u0026ndash;2783 (2015).\\u003c/li\\u003e\\n\\u003cli\\u003eZhang, K. \\u003cem\\u003eet al.\\u003c/em\\u003e Cholesterol: Bioactivities, Structural Modification, Mechanisms of Action, and Structure-Activity Relationships. \\u003cem\\u003eMini Rev. Med. Chem.\\u003c/em\\u003e\\u003cstrong\\u003e21,\\u003c/strong\\u003e 1830\\u0026ndash;1848 (2021).\\u003c/li\\u003e\\n\\u003cli\\u003eTAHER, M. A. \\u003cem\\u003eet al.\\u003c/em\\u003e Bioactive compounds extracted from leaves of G. cyanocarpa using various solvents in chromatographic separation showed anti-cancer and anti-microbial potentiality in in silico approach. \\u003cem\\u003eChinese J. Anal. Chem.\\u003c/em\\u003e\\u003cstrong\\u003e51,\\u003c/strong\\u003e 100336 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eVinuchakkaravarthy, T., Kumaravel, K. P., Ravichandran, S. \\u0026amp; Velmurugan, D. Active compound from the leaves of Vitex negundo L. shows anti-inflammatory activity with evidence of inhibition for secretory phospholipase A2 through molecular docking. \\u003cem\\u003eBioinformation\\u003c/em\\u003e\\u003cstrong\\u003e7,\\u003c/strong\\u003e 199 (2011).\\u003c/li\\u003e\\n\\u003cli\\u003eZhao, Y. \\u003cem\\u003eet al.\\u003c/em\\u003e Bletilla striata Polysaccharide Promotes Diabetic Wound Healing Through Inhibition of the NLRP3 Inflammasome. \\u003cem\\u003eFront. Pharmacol.\\u003c/em\\u003e\\u003cstrong\\u003e12,\\u003c/strong\\u003e (2021).\\u003c/li\\u003e\\n\\u003cli\\u003eSaraiva, M. \\u0026amp; O\\u0026rsquo;Garra, A. The regulation of IL-10 production by immune cells. \\u003cem\\u003eNat. Rev. Immunol.\\u003c/em\\u003e\\u003cstrong\\u003e10,\\u003c/strong\\u003e 170\\u0026ndash;181 (2010).\\u003c/li\\u003e\\n\\u003cli\\u003eRashad, M., Samp\\u0026ograve;, S., Cataldi, A. \\u0026amp; Zara, S. Biological activities of gastropods secretions: snail and slug slime. \\u003cem\\u003eNat. Products Bioprospect.\\u003c/em\\u003e\\u003cstrong\\u003e13,\\u003c/strong\\u003e (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eIbrahim, A., Morad, M., El-khadragy, M. F. \\u0026amp; Hammam, O. The antioxidant and anti-inflammatory effects of Eremina desertorum snail mucin on experimentally-induced intestinal inflammation and testicular damage. \\u003cem\\u003eBiosci. Rep.\\u003c/em\\u003e (2022). doi:10.1042/BSR20221020\\u003c/li\\u003e\\n\\u003cli\\u003eUlagesan, S. \\u0026amp; Kim, H. J. Antibacterial and antifungal activities of proteins extracted from seven different snails. \\u003cem\\u003eAppl. Sci.\\u003c/em\\u003e\\u003cstrong\\u003e8,\\u003c/strong\\u003e (2018).\\u003c/li\\u003e\\n\\u003cli\\u003eBax, B. \\u003cem\\u003eet al.\\u003c/em\\u003e Type IIA topoisomerase inhibition by a new class of antibacterial agents. \\u003cem\\u003eNature\\u003c/em\\u003e\\u003cstrong\\u003e466,\\u003c/strong\\u003e 935\\u0026ndash;940 (2010).\\u003c/li\\u003e\\n\\u003cli\\u003eLiu, C. \\u003cem\\u003eet al.\\u003c/em\\u003e Mesenchymal stromal cells pretreated with proinflammatory cytokines enhance skin wound healing via IL-6-dependent M2 polarization. \\u003cem\\u003eStem Cell Res. Ther.\\u003c/em\\u003e\\u003cstrong\\u003e13,\\u003c/strong\\u003e 414 (2022).\\u003c/li\\u003e\\n\\u003cli\\u003eAldred, K. J., Kerns, R. J. \\u0026amp; Osherof, N. Mechanism of Quinolone Action and Resistance. \\u003cem\\u003eBiochemistry\\u003c/em\\u003e\\u003cstrong\\u003e53,\\u003c/strong\\u003e 1565\\u0026ndash;1574 (2014).\\u003c/li\\u003e\\n\\u003cli\\u003ePietrusiński, M. \\u0026amp; Stączek, P. [Bacterial type II topoisomerases as targets for antibacterial drugs]. \\u003cem\\u003ePostepy Biochem.\\u003c/em\\u003e\\u003cstrong\\u003e52,\\u003c/strong\\u003e 271\\u0026ndash;282 (2006).\\u003c/li\\u003e\\n\\u003cli\\u003eHe, M. M. \\u003cem\\u003eet al.\\u003c/em\\u003e Small-Molecule Inhibition of TNF-\\u0026alpha;. \\u003cem\\u003eScience (80-. ).\\u003c/em\\u003e\\u003cstrong\\u003e310,\\u003c/strong\\u003e 1022\\u0026ndash;1025 (2005).\\u003c/li\\u003e\\n\\u003cli\\u003eSomers, W., Stahl, M. \\u0026amp; Seehra, J. S. 1.9 \\u0026Aring; crystal structure of interleukin 6: implications for a novel mode of receptor dimerization and signaling. \\u003cem\\u003eEMBO J.\\u003c/em\\u003e\\u003cstrong\\u003e16,\\u003c/strong\\u003e 989\\u0026ndash;997 (1997).\\u003c/li\\u003e\\n\\u003cli\\u003eYoon, S. Il, Jones, B. C., Logsdon, N. J. \\u0026amp; Walter, M. R. Same structure, different function: Crystal structure of the Epstein-Barr virus IL-10 bound to the soluble IL-10r1 chain. \\u003cem\\u003eStructure\\u003c/em\\u003e\\u003cstrong\\u003e13,\\u003c/strong\\u003e 551\\u0026ndash;564 (2005).\\u003c/li\\u003e\\n\\u003cli\\u003eBarman, H., Paul, P. \\u0026amp; Aditya, G. Observations on the growth and life table estimates of the slug Mariaella dussumieri (L. Pfeiffer, 1855) (Gastropoda: Ariophantidae). \\u003cem\\u003eZool. Ecol.\\u003c/em\\u003e\\u003cstrong\\u003e32,\\u003c/strong\\u003e 136\\u0026ndash;143 (2022).\\u003c/li\\u003e\\n\\u003cli\\u003eSallam, A. A. A., El-Massry, S. A. \\u0026amp; Nasr, I. N. Chemical analysis of mucus from certain land snails under Egyptian conditions. \\u003cem\\u003eArch. Phytopathol. Plant Prot.\\u003c/em\\u003e\\u003cstrong\\u003e42,\\u003c/strong\\u003e 874\\u0026ndash;881 (2009).\\u003c/li\\u003e\\n\\u003cli\\u003eLanz, M. A. \\u0026amp; Klostermeier, D. The GyrA-box determines the geometry of DNA bound to gyrase and couples DNA binding to the nucleotide cycle. \\u003cem\\u003eNucleic Acids Res.\\u003c/em\\u003e\\u003cstrong\\u003e40,\\u003c/strong\\u003e 10893\\u0026ndash;10903 (2012).\\u003c/li\\u003e\\n\\u003cli\\u003eWagner, H. Synergy research: Approaching a new generation of phytopharmaceuticals. \\u003cem\\u003eFitoterapia\\u003c/em\\u003e\\u003cstrong\\u003e82,\\u003c/strong\\u003e 34\\u0026ndash;37 (2011).\\u003c/li\\u003e\\n\\u003cli\\u003eRhea, L. \\u0026amp; Dunnwald, M. Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis. \\u003cem\\u003eJ. Vis. Exp.\\u003c/em\\u003e (2020). doi:10.3791/61616\\u003c/li\\u003e\\n\\u003cli\\u003eEl-Sayed, H. \\u003cem\\u003eet al.\\u003c/em\\u003e Myco-Synthesized Selenium Nanoparticles as Wound Healing and Antibacterial Agent: An In Vitro and In Vivo Investigation. \\u003cem\\u003eMicroorganisms\\u003c/em\\u003e\\u003cstrong\\u003e11,\\u003c/strong\\u003e 2341 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eErrajouani, F. \\u003cem\\u003eet al.\\u003c/em\\u003e Exploring the Potential Anti-Inflammatory and Wound-Healing Proprieties of Cepaea hortensis Snail Mucin. \\u003cem\\u003eCosmetics\\u003c/em\\u003e\\u003cstrong\\u003e10,\\u003c/strong\\u003e 170 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eFerdosh, S. The Extraction of Bioactive Agents from Calophyllum inophyllum L., and Their Pharmacological Properties. \\u003cem\\u003eSci. Pharm.\\u003c/em\\u003e\\u003cstrong\\u003e92,\\u003c/strong\\u003e (2024).\\u003c/li\\u003e\\n\\u003cli\\u003eDeng, T. \\u003cem\\u003eet al.\\u003c/em\\u003e A natural biological adhesive from snail mucus for wound repair. \\u003cem\\u003eNat. Commun.\\u003c/em\\u003e\\u003cstrong\\u003e14,\\u003c/strong\\u003e 396 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003ePorro, C., Cianciulli, A. \\u0026amp; Panaro, M. A. The Regulatory Role of IL-10 in Neurodegenerative Diseases. \\u003cem\\u003eBiomolecules\\u003c/em\\u003e\\u003cstrong\\u003e10,\\u003c/strong\\u003e (2020).\\u003c/li\\u003e\\n\\u003cli\\u003eWills-Karp, M., Nathan, A., Page, K. \\u0026amp; Karp, C. New Insights Into Innate Immune Mechanisms Underlying Allergenicity. \\u003cem\\u003eMucosal Immunol.\\u003c/em\\u003e\\u003cstrong\\u003e3,\\u003c/strong\\u003e 104\\u0026ndash;110 (2010).\\u003c/li\\u003e\\n\\u003cli\\u003eNguyen, V.-L. \\u003cem\\u003eet al.\\u003c/em\\u003e Anti-inflammatory and wound healing activities of calophyllolide isolated from Calophyllum inophyllum Linn. \\u003cem\\u003ePLoS One\\u003c/em\\u003e\\u003cstrong\\u003e12,\\u003c/strong\\u003e e0185674 (2017).\\u003c/li\\u003e\\n\\u003cli\\u003eVan Ostade, X., Tavernier, J., Prang\\u0026eacute;, T. \\u0026amp; Fiers, W. Localization of the active site of human tumour necrosis factor (hTNF) by mutational analysis. \\u003cem\\u003eEMBO J.\\u003c/em\\u003e\\u003cstrong\\u003e10,\\u003c/strong\\u003e 827\\u0026ndash;836 (1991).\\u003c/li\\u003e\\n\\u003cli\\u003eHunter, C. \\u0026amp; Jones, S. IL-6 as a keystone cytokine in health and disease. \\u003cem\\u003eNat. Immunol.\\u003c/em\\u003e\\u003cstrong\\u003e16,\\u003c/strong\\u003e 448\\u0026ndash;457 (2015).\\u003c/li\\u003e\\n\\u003cli\\u003eCLSI. \\u003cem\\u003eM100 Performance Standards for Antimicrobial Susceptibility Testing\\u003c/em\\u003e. (2021).\\u003c/li\\u003e\\n\\u003cli\\u003eBalouiri, M., Sadiki, M. \\u0026amp; Ibnsouda, S. K. Methods for in vitro evaluating antimicrobial activity: A review. \\u003cem\\u003eJ. Pharm. Anal.\\u003c/em\\u003e\\u003cstrong\\u003e6,\\u003c/strong\\u003e 71\\u0026ndash;79 (2016).\\u003c/li\\u003e\\n\\u003cli\\u003eKhalaf, A. A., Hassanen, E. I., Zaki, A. R., Tohamy, A. F. \\u0026amp; Ibrahim, M. A. Histopathological, immunohistochemical, and molecular studies for determination of wound age and vitality in rats. \\u003cem\\u003eInt. Wound J.\\u003c/em\\u003e\\u003cstrong\\u003e16,\\u003c/strong\\u003e 1416\\u0026ndash;1425 (2019).\\u003c/li\\u003e\\n\\u003cli\\u003eKhalil, R. G., Ibrahim, A. M. \\u0026amp; Bakery, H. H. Juglone: \\u0026ldquo;A novel immunomodulatory, antifibrotic, and schistosomicidal agent to ameliorate liver damage in murine schistosomiasis mansoni\\u0026rdquo;. \\u003cem\\u003eInt. Immunopharmacol.\\u003c/em\\u003e\\u003cstrong\\u003e113,\\u003c/strong\\u003e 109415 (2022).\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"slugs, Laevicaulis alte, Staphylococcus aureus, wound healing, antibacterial, GC-MS, in silico\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7612415/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7612415/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"Wound infections, particularly those caused by bacteria like Staphylococcus aureus, present a significant challenge to effective healing, often leading to chronic inflammation and tissue damage. Finding naturally active, new substances and applying them as wound healing agents to decrease bacterial infection and accelerate the wound healing is among the most significant areas of research. This study explored the therapeutic potential of a chloroform extract from the terrestrial slug Laevicaulis alte as a multi-functional agent for wound care. The investigation inte-grated chemical profiling by gas chromatography-mass spectrometry (GC-MS), in vitro antibacterial evaluation, an in vivo excisional wound healing model, and in silico molecular docking to elucidate its mechanisms of action. GC-MS analysis identified twelve compounds, with tris(2,4-di-tert-butylphenyl) phosphate (TDTBPP) and cholesterol as major constituents. The extract exhibited potent antibacterial activity against S. aureus with a minimum inhibitory concentration (MIC) of 0.625 mg/mL. In vivo, topical application of the extract significantly accelerated wound closure, reduced the cutaneous bacterial load, and orchestrated a balanced host immune response by decreasing pro-inflammatory cytokines (TNF-α, IL-6) while increasing the an-ti-inflammatory cytokine IL-10. Molecular docking studies provided a strong mechanistic rationale for these observations, revealing that key compounds, particularly TDTBPP and a phenol phosphite derivative, showed high binding affinities for both bacterial DNA gyrase and the critical cytokine targets. These findings indicate that the L. alte extract is a promising, multi-functional therapeutic agent, whose efficacy stems from a synergistic chemical consortium that targets both the pathogen and the host's inflammatory response.\",\"manuscriptTitle\":\"Laevicaulis alte slug extract: Investigation of antibacterial and wound-healing properties, chemical profiling, and molecular docking insights\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-02-02 13:30:40\",\"doi\":\"10.21203/rs.3.rs-7612415/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-01-29T08:02:06+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-01-06T15:10:59+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2025-09-23T17:14:52+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-09-20T06:57:28+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2025-09-20T06:53:35+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"2d40f72b-a63c-4358-9fc8-467526892265\",\"owner\":[],\"postedDate\":\"February 2nd, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[{\"id\":62101389,\"name\":\"Biological sciences/Biochemistry\"},{\"id\":62101390,\"name\":\"Biological sciences/Biotechnology\"},{\"id\":62101391,\"name\":\"Biological sciences/Computational biology and bioinformatics\"},{\"id\":62101392,\"name\":\"Biological sciences/Drug discovery\"},{\"id\":62101393,\"name\":\"Biological sciences/Microbiology\"}],\"tags\":[],\"updatedAt\":\"2026-02-02T13:30:40+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-02-02 13:30:40\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7612415\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7612415\",\"identity\":\"rs-7612415\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}