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. 2026 Apr 17;29(6):863–876. doi: 10.1007/s10123-026-00781-9

Integrating in vivo and in silico: a comprehensive study of Helix aspersa mucin’s therapeutic properties

Amina Mohamed Ibrahim 1,✉, Mostafa Y Morad 2, Rabab Kamel 3
PMCID: PMC13469375  PMID: 41996043

Abstract

Purpose

The current study aims to elucidate the use of a novel Helix aspersa snail mucin gel as an eco-friendly agent against certain bacterial strains and improve the wound repair process. Additionally, it aims to investigate its potential effects as a natural anticancer agent.

Methods

Gas chromatography-mass spectrometry (GC-MS) analysis was performed to detect mucin components. Twelve compounds were identified mostly phenol derivatives. Mucin gel inhibited the growth of various pathogenic Gram- positive and Gram-negative bacteria. The experimental mice’s backs were given an excisional wound.

Results

The treatment with mucin gel could hasten wound closure and decrease the time needed for full recovery. Only twelve days were needed to complete wound healing for the treated group with mucin gel, while fifteen days were required for full recovery in the Garamycin treated group. Mucin gel didn’t show any potential effect on the viability of the human melanoma cell line. The concentrations of IL-2 and TNF-α were significantly decreased upon the application of mucin gel. Also, the apoptosis effector caspase-3 has shown a decrease in its levels after the treatment. The molecular docking was conducted to predict the effect of 1,6-Octadien-3-ol, 3,7, - dimethyl- Linalyal acetate and Tricyclo[3.1.0.0(2,4)] hex-3-ene-3-carbonitrile compounds against the measured parameters. The In silico docking study showed an inhibitory effect for the tested components due to increasing its hydrophilicity and then, they could increase the pharmacological potential of snail mucin for use in wound-healing and antibacterial applications.

Conclusion

Helix aspersa mucin gel is a promising, inexpensive, eco-friendly agent against wounds and a highly effective antibacterial component.

Keywords: Brown garden snail, Mucin gel, Antibacterial activity, Wound healing, Molecular docking

Introduction

The skin, the body’s biggest organ, serves as an infection-prevention immunological barrier (Lopez-Ojeda et al. 2022; Sahu et al. 2020). Wound healing is an organized physiological mechanism that results in repairing the skin’s integrity after injury caused by sudden accidents, extrinsic factors, or medical surgery (Snedecor Wi, Cochran 1991). It consists of a number of predetermined processes, such as proliferative and inflammatory stages, followed by the return of normal skin structure (Kangal and Kopitnik 2025; Wernick et al. 2023). Chronic wounds and delayed acute wounds can both hinder the healing process and cause pathologic inflammatory reactions (Wernick et al. 2023). The microbial infection may appear after the wound, which makes wound healing more challenging and takes longer (Wong et al. 2025). Impaired healing wounds affect about millions of people around the world (Rashad et al. 2025). The good wound healing product should reduce the healing time, maintain a moist environment, be easily available, be non-allergic, and be inexpensive (Habibi et al. 2018; Kamel et al. 2019).

Mollusca is the second most widely distributed phylum after Arthropoda (Ibrahim et al. 2021). It can be found in all habitats (Tanideh et al. 2017). It served as a crucial food resource and was frequently employed in folk medicine (Naseer 2022). Class Gastropoda has gained great concern as a natural food supplement and in biomedical therapies (Muteeb et al. 2023). Many species among gastropods could secrete mucus from their pedal gland (Greistorfer et al. 2023). Mucopolysaccharides and glycosaminoglycans are the constituents of mucus (Harti et al. 2016; Velkova et al. 2024). Mucin was isolated from mucus, and new research has shown that it has excellent healing properties, particularly for burns, wounds, and skin disorders (Ali et al. 2018; Hatuikulipi et al. 2016; Ibrahim et al. 2022a, b).

The brown garden snail Helix aspersa (Müller 1774) is a member of the Phylum Mollusca, Class Gastropoda. Its pedal glands, located in the head-foot area, secrete mucus. Compounds formed from mucus have numerous medical uses, including antibacterial, antiviral, anti-inflammatory, and antioxidant properties (Atta et al. 2021; Azeem et al. 2020; Ibrahim et al. 2022a, b; Wallace et al. 2023). Mucus is widely used in treating skin disorders such as warts, abscesses and burns (Greistorfer et al. 2017) as well as in the pharmaceutical industry for wound repair and as an important constituent of cosmetics (Rathapon et al. 2005). In addition to many valuable constituents, such as vitamins A, C, E and amino acids, which have an effective role against inflammations (Ali et al. 2018; Hatuikulipi et al. 2016), allantoin and glycolic acid are recognized to be partially or entirely responsible for the primary functions of snail slime, including skin regeneration, moisturizing, anti-inflammatory, calming, and antioxidant properties (Bonafè et al. 2025). Despite the advances in antibiotic therapies, the susceptibility to bacterial infections is the main obstacle in wound management (Church et al. 2006; Nantarat et al. 2019). Accordingly, H. aspersa mucus exhibits antibacterial action against a few strains that have been examined (Ulagesan and Kim 2018).

So, the present study aimed to estimate the active component of H. aspersa snail mucin gel and study its role in the wound healing process and to assess its anti-cancer and antimicrobial activity against some tested microbes.

Materials and methods

Snails and mucin extraction

One hundred H. aspersa snails were gathered from Giza City and then transported to the Medical Malacology department, Theodor Bilharz Research Institute, Egypt. The laboratory was used to gently wash and acclimate the snails. The pure mucus was mechanically obtained from the head-foot region by using a sterile wooden rod that stimulated the pedal gland to secrete more slime (4 ml/ snail) with total yield 150 ml for all snails (Harti et al. 2016; Ibrahim et al. 2022a, b).

Chemical analysis of mucin by gas chromatography-mass spectroscopy

The phytochemical examination was performed by an Agilent 6890 gas chromatography system with an Agilent 5973 mass spectrometric detector. The retention indices and a comparison with the MS computer library (NIST Software Package, Finnigan) were used to identify the contents.

Preparation of mucin gel

To disperse carbopol 940 (Goodrich Co., Ltd., USA) (1% w/w) in distilled water, mechanical stirring was used continuously between 60 and 65 °C until a clear solution was obtained (Kamel et al. 2012; Rohan et al. 2024), then the mucin (25% w/w) as added (Ali et al. 2018). At room temperature, stirring continued until a homogenous preparation was formed. The final gel was left overnight to remove trapped air bubbles.

Antibacterial activity

The antibacterial activity of the prepared mucin gel was evaluated using a filter paper disc diffusion assay against some pathogenic gram-positive and gram-negative bacteria (S. faecalis, S. aureus, B. subtilis, E. coli, P. aeuroginosa, and N.gonorrhoeae). Nutrient agar media was used to cultivate the bacterial strains (HiMedia Laboratories Pvt. Ltd., Mumbai, India). The bacterial culture was incubated at 37 °C for 24 h, then a single colony was inoculated in nutrient broth (HiMedia Laboratories Pvt. Ltd., Mumbai, India) at 37 °C for 24 h to get count of around 108 cells/ml (Punjataewakupt et al. 2019).

100 µl of bacterial culture suspension was inoculated in nutrient agar medium. A filter paper disc (5 mm) was impregnated with 100 µl of mucin gel and placed on the centre of the agar plates inoculated with the tested bacteria. Agar plates inoculated with gentamicin (Memphis Pharmaceutical Company, Egypt) were the positive controls. The plates were incubated at 35–37 °C for 24–48 h, Then the inhibition zones were measured (Bauer et al. 1966).

Anticancer effect evaluation

Human melanoma was obtained from Nawah Scientific Inc. (Mokatam, Cairo, Egypt). In a humidified, 5% (v/v) CO2 environment, cells were kept in DMEM media supplemented with 100 mg/mL of streptomycin, 100 units/mL of penicillin, and 10% of heat-inactivated fetal bovine serum at 37 °C.

  • Sulforhodamine B (SRB) assay.

Cell viability was assessed by SRB assay. Aliquots of 100 µL cell suspension (5 × 10^3 cells) were in 96-well plates and incubated in complete media for 24 h. Another aliquot of 100 µL media containing medications at different concentrations was used to treat the cells. Cells were fixed by substituting medium with 150 µL of 10% trichloroacetic acid (TCA) after 72 h of drug treatment, and they were then incubated for one hour at 4 °C. After removing the TCA solution, distilled water was used to wash the cells five times. After adding aliquots of 70 µL SRB solution (0.4% w/v), the mixture was allowed to sit at room temperature for 10 min in a dark environment. After three rounds of washing with 1% acetic acid, the plates were left to air dry overnight. The protein-bound SRB stain was then dissolved with 150 µL of TRIS (10 mM); a BMG LABTECH®-FLUOstar Omega microplate reader (Ortenberg, Germany) was used to measure the absorbance at 540 nm.

Bioassay

Animals

Forty-five male mice (CD1), aged (6–8 weeks) were maintained in the animal house of Theodore Bilharz Research institute, Giza-Egypt (TBRI) (Ibrahim et al. 2019). Mice were put in plastic cages for 4 weeks in 20–25 °C temperature and were fed Purina chaw (20% protein) and drank tap water.

Experimental design and Preparation of wound site

Fifteen mice were randomly divided into three groups (n = 5 per group). Three replicates of each group were performed in this experiment. The mice hair was shaved on their back areas, and to ensure the purification wound area, we alternately used betadine scrub and 70% ethanol three times. All mice in each group were wounded with a sterilized razor blade (Glowania et al. 1987). A wound of 2 ± 0.03 cm was applied on the mice’s back, and then the wound length was immediately estimated and recorded. Applying the preparation to the wound was the first step in treatment after the incision.

The groups were divided into:

  • Group 1: was given phosphate buffer saline only (-ve control group).

  • Group 2: was treated topically with 1gm\cm2 Garamycin cream (Gentamicin 0.1 and is the +ve control group).

  • Group 3: was treated topically with 1gm\cm2 mucin gel spread on the wound once daily using a cotton bud.

The experiment was conducted for two weeks to make sure that all the mice wounds have been completely healed. The wound length of each animal was measured every day by using transparent tracing paper and the squares counted and the area recorded. The following formula was used to determine the percentage of wound closure:

graphic file with name d33e461.gif

Where: WA0 = starting wound area and WA = measured wound area in different days (Adikwu and Michael 2007). The treatment was done topically, and the wound areas were estimated on 0, 2, 5, 7, 9 and 11 days for all groups, using a transparency sheet and a permanent marker. A graph paper was used to measure the recorded wound areas.

Biochemical analysis

Mice were euthanized on day 15. Skin samples were taken from various groups, and homogenates (20% w/v) were made using a glass homogenizer (Laborzentrifugen, 2 k15, Sigma, Germany) in phosphate buffer (50 mM, pH 7.4). To get rid of any impurities, centrifugation was done to the homogenate at 1000 rpm at 4 °C for 10 min. The resultant supernatant was kept for further assays.

Enzyme- linked immunosorbent assay (ELISA)

Interleukin 2 (IL-2) concentration was measured using ELISA kit (OptEIA™ Kits; BD Biosciences) according to (Engvall and Perlmann 1971). With the use of the software Microplate Manager (Bio-Rad), a regression curve was created to determine the cytokine concentration. Caspase-3 levels were evaluated according to (Bonomini et al. 2004).Tumor necrosis factor-alpha (TNF-α) activity was estimated according to the ELISA kits purchased from BioSource, International (Camarillo, California, USA).

Docking analysis

Molecular docking was performed using the MOE 2015.10 (Sallam et al. 2009). The isolated compounds identified in the snail mucin (Table 1) were energy-minimized using Density Functional Theory (DFT) (Soonklang et al. 2015) at the B3LYP/6-311G level. The three-dimensional structures of the target proteins including IL-2 (PDB: 1M49) (Arkin et al. 2003), Caspase-3 (PDB: 6BDV) (Trapella et al. 2018), TNF-α (PDB: 3IT8) (Zhang et al. 2025), and DNA Gyrase (PDB: 6M1J) (Adikwu and Michael 2007), were obtained from the RCSB Protein Data Bank. Protein preparation was carried out using the MOE structure preparation tool, all water molecules and non-essential ions were removed, hydrogen atoms were added, and protonation states were assigned at pH 7.0 and 300 K using the AMBER12: EHT forcefield. Energy minimization of the protein structures was performed using the same forcefield until a root mean square gradient of 0.05 kcal·mol⁻¹·Å⁻² was reached. The binding site for each protein was defined based on the coordinates of the native co-crystallized ligand. Docking simulations were conducted using the Triangle Matcher placement method and scored using the London dG scoring function. For each compound, 10 docking poses were generated and retained. The resulting poses were refined using the Forcefield refinement protocol and re-scored using the GBVI/WSA dG scoring function. The docking protocol was validated by re-docking the native co-crystallized ligand into each protein’s binding site. The root-mean-square deviation (RMSD) between the docked pose and the original crystallographic pose was calculated and found to be less than 2.0 Å for all targets (Mentino et al. 2025), confirming the reliability of the docking methodology.

Statistical analysis

The data were reported as mean S.E., and the student’s t-test was used to compare the two means (Soga et al. 2007). The difference was considered significant when p ≤ 0.05.

Results

The metabolomic profile of H. aspersa mucin was performed using GC/MS analysis. Twelve compounds have been successfully identified as shown in Table 1. The higher area detected compounds were Phenol, 2,2’-methylenebis [6-(1,1-d imethylethyl)-4-methyl followed by 1,6-Octadien-3-ol, 3,7, - dimethyl- Linalyal acetate and Tricyclo [3.1.0.0(2,4)] hex-3-ene-3-carbonitrile.

Table 1.

list of identified phytochemical compounds by GC-Mass of Helix aspersa mucus

Compound Name Molecular Formula Molecular Wight (g/mol) Retention Time (min.) Area Peak %
1 Benzo[f] isoquinoline C13H9N 179.22 3.714 128,653 0.783747
2 Tricyclo[3.1.0.0(2,4)]hex-3-ene-3-carbonitrile C7H5N 103.12 4.806 4,036,461 24.58989
3 1,6-Octadien-3-ol, 3,7,- dimethyl- C10H18O 154.25 6.835 4,193,020 25.54364
4 Cyclopentasiloxane, decamethyl C10H30O5Si5 370.77 7.778 200,435 1.221039
5 Glycerol 1,2-diacetate C7H12O5 176.17 11.388 1,309,751 7.978929
6 Dodecane, 4-methyl C124H245N19 184.37 13.744 184,490 1.123903
7 Phenol, 2,5-bis(1,1-dimethylethyl) C34H54N2O2 522.799 14.58 587,269 3.57761
8 Tridecane, 2-methyl- C18H36S 284.5 16.883 178,458 1.087156
9 Dodecane, 2,6,10-trimethyl- C15H32 212.41 19.684 155,735 0.948729
10 Hexadecanoic acid, ethyl ester C18H36O2 284.5 20.544 443,770 2.703422
11 2,8,9-Trioxa-5-aza-1-silabicyclo[3.3.3]undecane, 1-ethyl- Indole-2-carboxylic acid C15H27NO5Si 329.46 22.215 552,954 3.368564
12 Phenol, 2,2’-methylenebis[6-(1,1-d imethylethyl)-4-methyl-Phenol C69H93O6P 1049.4 25.312 4,444,127 27.07337
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As shown in Table 2, the mucin gel has antibacterial activity against some bacterial strains. The inhibition zone diameter of three-gram positive bacteria S. faecalis, S. aureus and B. subtilis ranged from 30, 28, and 29, respectively while for the gram-negative bacteria E. coli, P.aeuroginosa and N.gonorrhoeae ranged from 29, 28, and 30, respectively.

Table 2.

Antimicrobial activity of mucin gel using agar well diffusion method. Values represent inhibition zone diameter (mm/ mg sample)

Sample S. faecalis S. aureus B. subtilis E. coli P. aeuroginosa N. gonorrhoeae
Gentamicin (+ ve control) 18 16 16 15 17 18
DMSO (-ve control) 0.0 0.0 0.0 0.0 0.0 0.0
Mucin gel 30 28 29 29 30 28
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The cytotoxic activity of mucin against human melanoma cells was determined using the SRB assay. This assay was performed with mucins' concentrations ranging from 0.03 to 300 µg/ml. Cell viability wasn’t affected by mucin increasing concentrations (Fig. 1).

Fig. 1.

Fig. 1

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“Viability of human melanoma cells (A375) after 72-hour treatment with Helix aspersa mucin, as assessed by SRB assay. Data points represent mean ± SEM of three independent experiments

The present study showed that the wound has been completely healed in the mucin gel treated mice group and with faster rates than the negative control group and even garamycin treated group (Fig. 2). In addition, mucin gel significantly decreased (P ≤ 0.05) the wound size compared to control animals (Table 3). The current results showed that the total days needed for complete healing in normal control group was 19 days, while group that treated topically with mucin gel was 12 days and that of garamycin cream group was 15 days (Fig. 3).

Fig. 2.

Fig. 2

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wound closure efficacy. After three days, (A) the control; (B) the garamycin group; and (C) the mucin gel. After 5 days; (D) control; (E) Garamycin; (F) mucin gel. After11days; (G) control; (H) Garamycin; (I) mucin gel. Scale bar = 2.5 cm

Table 3.

Effect of mucin gel on wound healing area as compared with untreated (-ve control) and Garamycin treated groups (+ve control). Values are represented in (cm)

Time (days) -Ve Control Garamycin (+ ve) Mucin gel
0 2 ± 0.03 2 ± 0.03 2 ± 0.03
2 1.8 ± 0.05 1.4 ± 0.03* 1.3 ± 0.04*
5 1.4 ± 0.02 * 1.1 ± 0.05* 1.0 ± 0.02*
7 1.2 ± 0.01* 0.8 ± 0.04 * 0.6 ± 0.01*
9 1 ± 0.03* 0.6 ± 0.07 * 0.3 ± 0.05*
11 0.8 ± 0.02* 0.3 ± 0.03* 0.1 ± 0.01*
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Values are mean ± SEM. * significance against control at p ≤ 0.05

Fig. 3.

Fig. 3

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The time needed for mucin gel to complete the wound healing compared to untreated and Garamycin treated groups. Values are mean ± SEM. * significance against control at p ≤ 0.05

Figure 4 illustrates that mucin gel treated mice significantly (P ≤ 0.05) decreased IL-2 levels (Fig. 4A). Also, caspase-3 activity was decreased after the topical treatment with mucin gel (Fig. 4B), while the proinflammatory cytokine TNF-α levels showed a significant decrease after the exposure to mucin gel or Garamycin (P ≤ 0.05) as compared with the wounded control group (Fig. 4C).

Fig. 4.

Fig. 4

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Histogram shows the effect of Garamycin and snail mucin loaded gel on (A) interleukin 2 (IL-2) levels; B) on caspase-3 activity. C) Tumor necrosis factor-alpha (TNF-α) concentration. Values are mean ± SEM. *significance against control at p ≤ 0.05; # significance against wounded group

To investigate the inhibition action of isolated compounds from snail, the docking study was applied against the IL-2, Capase-3, TNF-α and DNA Gyrase proteins (Figs. 5, 6, 7 and 8). The different docking energies were listed in Table 4. Compositions 2 and 3 showed the highest binding energy in (Kcal/mol.) as: (-6.84 & -6.24 against IL-2; 1M49), (-6.87&-6.72 against Caspase-3; 6BDv), (-5.73&-5.97 against TNF-α; 3IT8) and (-7.48&-7.63 against DNA Gyrase; 6M1J), respectively. The 4, 6–8 and 12 showed moderate binding efficiency against 6BDvin range (-5.01 to5.73 Kcal/mol). In 1M49 and 6M1J enzymes, 1, 4 and 7 compositions showed moderate binding efficacy, while in 3IT8 only compound 7 displayed the moderate interaction energy. All compounds stabilized in active binding sites for tested enzymes with the same manner for the reference inhibitors.

Fig. 5.

Fig. 5

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3D and 2D interaction for molecular docking of the active isolated compositions against (IL-2; 1M49)

Fig. 6.

Fig. 6

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3D and 2D interaction for molecular docking of the active isolated compositions against (Caspas-3; 6bdv)

Fig. 7.

Fig. 7

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3D and 2D interaction for molecular docking of the active isolated compositions against TNF-α (TNF; 3IT8)

Fig. 8.

Fig. 8

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3D and 2D interaction for molecular docking of the active isolated compositions against (DNA Gyrase; 6M1J)

Table 4.

Energetic score for Docking analysis for isolated structures

Cpd ΔΕ RMSD Econf Eplace E-Int. Cpd ΔΕ RMSD Econf Eplace E-Int.
(IL-2; 1M49) (TNF-α; 3IT8)
1 -5.11 1.63 -4.51 -19.99 -9.54 1 -4.53 1.58 -9.43 -1.60 -6.38
2 -3.85 1.96 38.52 -7.14 -8.96 2 -4.16 2.13 38.65 -3.61 -5.98
3 -5.55 1.62 -55.61 -14.70 -6.22 3 -4.62 2.79 -55.16 -9.28 -5.42
4 -5.55 2.05 17.69 -17.17 -6.03 4 -4.47 3.77 17.18 -7.87 -4.30
6 -4.78 1.55 -305.49 -9.58 -5.93 6 -3.37 2.23 -305.10 -5.51 -1.67
7 -5.38 2.94 8.97 -20.01 -5.99 7 -5.51 2.97 12.84 -1.62 -4.19
8 -4.91 1.91 -45.45 -9.15 -8.77 8 -4.59 1.62 -42.40 -2.90 -5.52
9 -6.84 1.75 34.68 -30.49 -7.29 9 -5.73 2.96 36.29 -9.77 -4.17
10 -6.24 1.66 11.22 -23.10 -7.40 10 -5.97 2.47 8.40 -4.82 -5.39
11 -4.51 2.17 -43.62 -24.13 -8.00 11 -4.12 1.84 -43.24 -12.85 -6.84
12 -4.28 1.34 215.00 -13.80 -6.83 12 -3.68 2.12 215.00 -4.15 -5.31
(Capes-3; 6BDv) (DNA Gyrase; 6M1J)
1 -4.59 1.66 -8.06 -10.71 -11.39 1 -5.58 2.21 -3.82 -17.18 -10.61
2 -4.30 1.34 42.00 -7.33 -11.99 2 -4.26 3.40 38.94 -10.85 -13.68
3 -5.72 1.52 -52.25 -16.42 -6.77 3 -5.30 2.30 -42.23 -13.90 -7.97
4 -5.51 1.07 25.33 -18.70 -7.12 4 -6.67 0.87 20.44 -23.26 -7.82
6 -5.69 1.48 10.45 -20.47 -9.55 6 -5.57 0.87 -300.38 -11.11 -4.11
7 -5.01 1.36 -40.90 -9.57 -10.06 7 -6.01 1.94 20.62 -14.83 -7.12
8 -5.39 3.45 37.90 -18.71 -7.59 8 -5.80 1.66 -43.74 -13.67 -9.65
9 -6.87 1.67 32.04 -24.69 -7.44 9 -7.48 2.85 38.98 -23.03 -7.45
10 -6.72 0.90 -42.95 -20.96 -9.58 10 -7.63 1.24 13.46 -22.23 -7.05
11 -4.16 1.90 214.93 -8.04 -7.39 11 -5.00 1.28 -42.82 -15.31 -10.73
12 -5.73 0.62 20.34 -19.54 -7.03 12 -4.43 1.82 215.04 -13.81 -7.50
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Where; ΔE, Econf and Eplace free binding energy for the ligand from a given pose, conformer and receptor. E-Int.: The ligand’s affinity binding energy with the receptor, E-ele: The Electrostatic interaction via receptor. RMSD; root-mean-square-deviation between docking pose and co-crystal ligand

Discussion

The high cost and inadequate treatment of dermal wounds represented a public health issue (Jain et al. 2025). Traditional natural medicines could be used as good alternatives for disease prevention and treatment (Ahmad et al. 2018; Smith et al. 2020). The current study revealed by GC-MS examination identified 12 compounds in H. aspersa mucin. The detected compounds were phenol derivatives, fatty acid esters, alcohol esters and fatty acids. These results were similar to findings of(Ibrahim et al. 2022a, b) who reported that GC-MS/MS examination of E. desertorum mucin resulted in ten compounds having been identified and being mainly quinolines, monoterpenes, alcohol esters, sesquiterpenoids, fatty acid esters, fatty acids, and phenol derivatives (Sarkar et al. 2025). reported that such compounds have antimicrobial, anticancer and antioxidant activities.

Snail mucin gel was used in this study, and its therapeutic activity on wounds has been evaluated. A cutaneous wound’s healing process involved collagenation, inflammation, coagulation, wound contraction, epithelialization, and remodeling (Shedoeva et al. 2019; Thomas et al. 2019).The mucus of the snail H. aspersa was widely used in many cosmetic products due to the presence of glycolic acid and allantoin in its structure (Ulagesan and Kim 2018). Besides, it has been used for wound healing for its anti-inflammatory, antiviral, and antioxidant activities (Atta et al. 2021). Moreover, it induced the formation of collagen and other dermal components that minimized the stress induced by the free radicals (Ellijimi et al. 2018). Additionally, it enhanced keratinocyte cell motility and the expression of adhesion molecules between cells and substrates (Ulagesan and Kim 2018). The present results showed that mucin gel has antibacterial activity against three-gram positive bacteria (S. faecalis, S. aureus, B. subtilis) and the three-gram negative bacteria (E. coli, P. aeuroginosa and N. gonorrhoeae. It was confirmed that snail mucous contained many proteins that have the ability to inhibit the growth of the skin-borne pathogens (S. epidermidis and P. aeruginosa) (Sousa 2024; Vinarski et al. 2020). Also, the antimicrobial activity of H. aspersa extract was confirmed against the resistant pathogens related to burn wound infection (EL-Zawawy and Mona 2021).

The topical application of mucin- loaded gel increased the rate of wound healing, while it reduced the size of the wound and the time needed for healing, taking only 12 days after using mucin gel as opposed to 19 days for the control. Also, the current findings demonstrated that only animals given mucin gel developed hair on their skin following healing (Adikwu and Okafor 2012). reported that treatment of the wound area in the experimental animals with the mucin and honey gels could decrease the wound radius and decrease the time for complete healing. Also, the authors confirmed that snail mucin promoted regeneration of hairs when combined with honey in the animal experiment. They elucidated the important role of the snail mucin in wound healing process as it enhanced the immune response and promoted the natural regenerative response of skin to form new tissues. Also, authors observed the presence of smoothness in the healing tissue and they reasoned this observation with that their preparation might prevent keloid formation. Also, it has been reported that the topical application of snail mucin to wounds could accelerate the superficial healing (Deng et al. 2023; Shedoeva et al. 2019) (Ali et al. 2018). recommended the use of the eel mucus ointment (Belutidine) in healing the wound, as it accelerated the healing time (9 day) and shorten the scar length. An efficient wound healing agent should reduce swelling, speed up the healing process, and leave the treated tissue with the fewest scars possible (Pfaller et al. 1988) (Etim et al. 2016). studied the antibacterial activity of the mucus secretions of Archachatina marginata snails compared to seven different antibiotics and concluded that Snail mucus could be used as antibacterial agents in wound treatment.

All phases of wound healing are regulated by cytokines, making them a prospective therapeutic target. Inflammatory wounds that do not heal can result from cytokine production malfunction. Whereas transforming growth factor-β (TGF-β), IL-4, and IL-13 suppress inflammation and promote fibroblast proliferation to initiate extracellular matrix (ECM) deposition, interleukin-1 (IL-1), IL-2, IL-6, IL-8, and tumor necrosis factor-α (TNF-α) aid in leukocyte recruitment and the removal of dead cells during the early stages of inflammation (João et al. 2016; Yang et al. 2009). The macrophage played a central role in the wound healing process where it secreted numerous enzymes and cytokines. These include collagenases, interleukins, and tumor necrosis factors (TNF). The major job of TNF is to stimulate fibroblasts that created collagen and increase angiogenesis and transforming growth (TGF) which stimulates keratinocytes (Adikwu and Michael 2007). IL-2 could be used as a target in enhancing the improvement of the wound healing quality and rate. Where it resulted in attracting immune mediators for initiating the healing process (Doersch et al. 2017). One of the caspases that carry out apoptosis is caspase-3, a death protease that is often activated and essential for the induction, transmission, and amplification of intracellular apoptotic signals. Caspases are cysteine proteases that are selective to aspartates and belong to the interleukin- family of 1β-converting enzymes (Oryan et al. 2016). Its overexpression in hypertrophic scars and keloid induced apoptosis of fibroblasts (Akasaka et al. 2000). The present work showed that high levels of IL-2 and caspase-3 activity and TNF-α in the wounded mice, while after the exposure of wound to mucin gel or Garamycin, there were marked decreases in IL-2, TNF-α levels and caspase-3 activity trying to reach the normal values. The explanation of these results might be due to the glycosaminogycans and amino acids in the snail mucin that is responsible for wound healing and repair (Harti et al. 2016). The mucus of H. aspersa snails contained vitamins A, C and E, which have anti-inflammatory and antioxidant effects. Also, it contained mucopolysaccharides, glycine, proline and glutamic acid, these components play a reliable role in repairing ulcers (Hatuikulipi et al. 2016) (EL-Zawawy and Mona 2021). reported that by the activation of fibroblast migration and boosting the production of genes linked to wound healing (TGF-1 and VEGF), E. desertorum mucin may hasten the healing of wounds.

The molecular docking identified the potency of each isolated compositions from snail mucin. 1,6-Octadien-3-ol, 3,7, - dimethyl- Linalyal acetate and Tricyclo[3.1.0.0(2,4)] hex-3-ene-3-carbonitrile are the most compositions shared in biological activity against IL-2, caspase-3, TNF- α and DNAgyrase, respectively. These compounds inhibited theses enzymes through interacted with vital amino acid residues as; (Arg38& Phe42 for IL-2), (Arg64, Gln161, Cys163& His121 for caspase-3), (Lys4& Asn6 for TNF-α) and (Arg78, Glu79&Thr167 for DNA Gyrase). It was anticipated that the inhibitory effectiveness for the tested compositions resulted from increasing their hydrophilicity based on the variation in the interaction mode between compositions and hydrophilic amino acids backbone [65]. Based on molecular docking studies evaluating the bioactive components of snail mucin (such as from Helix aspersa or Eremina desertorum), specific compounds have shown potential in inhibiting key inflammatory and bacterial targets. These substances enhance snail mucin’s pharmacological potential for use in antibacterial and wound-healing applications.

Conclusion

The findings revealed that a mucin gel made from H. aspersa could speed up wound healing and had antimicrobial properties. Mucin presents different advantages, as it is easily available, of low cost and has a natural origin. The formulated gel could ameliorate the quality of life of wounded patients. Molecular docking was used to determine the potency of each separated composition from the snail, which demonstrated an inhibitory effectiveness for the studied compositions against inflammatory indicators because of increasing its hydrophilicity. However, further clinical studies are needed to be conducted to apply it as a treatment regimen for skin wounds and disorders.

Acknowledgements

Not applicable.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Amina Ibrahim, Mostafa Y. Morad, and Rabab Kamel]. The first draft of the manuscript was written by [Amina Ibrahim, Mostafa Y. Morad, and Rabab Kamel] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

No funds, grants, or other support was received.

Data availability

Any enquiries for additional information are available upon request from the corresponding author.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Human Ethics

No experiments were done on human.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

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