Structural characterization of a glycoprotein from white jade snails (Achatina Fulica) and its wound healing activity.

Snail mucus is rich in proteins and polysaccharides, which has been proved to promote wound healing in mice in our previous research. The aim of this study was to investigate the effective component in snail mucus that can exert the wound healing potential and its structural characterization. Here, the glycoprotein from the snail mucus (SM1S) was obtained by DEAE-Sepharose Fast Flow and Sephacryl S-300 columns. The structural characteristics of SM1S were investigated via chromatographic techniques, periodic acid oxidation, FT-IR spectroscopy and NMR spectroscopy. Results showed that SM1S was a glycoprotein with a molecular weight of 3.8 kDa (83.23 %), consists of mannose, glucuronic acid, glucose, galactose, xylose, arabinose, fucose at a ratio of 13.180:4.875:1043.173:7.552:1:3.501:2.058. In addition, the periodic acid oxidation and NMR analysis showed that SM1S contained 1,6-glycosidic bonds, and might also contain 1 â†’ 4 and 1 â†’ 2 glycosidic or 1 â†’ 3 glycosidic bonds. Furthermore, the migration experiment of human skin fibroblasts in vitro suggested that SM1S had a good effect to accelerate the scratch healing of cells. This study suggested that SM1S may be a prospective candidate as a natural wound dressing for the development of snail mucus products.

Snail Mucus-Enhanced Adhesion of Human Chondrocytes on 3D Porous Agarose Scaffolds.

This study reports the preparation of a novel porous 3D scaffold from agarose-snail mucus (AGSMu) for cartilage tissue repair applications. AG is reported for its unique thermal and mechanical properties, biocompatibility, and biodegradability, making it suitable for biomedical applications. Still, it lacks the cell adhesion properties required for tissue engineering applications. SMu is a complex substance identified to contain glycosaminoglycans (GAGs) and other bioactive molecules that promote wound healing and reduce cartilage deterioration and inflammation. Hence, porous 3D blend scaffolds containing AG and SMu were prepared by the freeze-drying method, characterized, and investigated for bioactive effects on human chondrocyte (C28/I2) cells. The scaffolds had a microporous structure with an average pore size of 245 µm. FTIR spectroscopy showed that SMu was successfully incorporated into the scaffolds. The SMu increased the mechanical strength of the composite scaffolds by more than 80% compared to the pristine AG scaffold. The scaffolds were found to be biocompatible with tunable degradation. The human chondrocyte cells attached and proliferated well on the 3D scaffolds in a few days, demonstrating a marked improvement in adhesion due to the presence of SMu. Enhanced cell adhesion and mechanical properties of 3D porous AG scaffolds could make them suitable for articular cartilage repair and regeneration.

Safety of HelixComplex snail mucus (HSM) as a novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on HelixComplex Snail Mucus (HSM) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF consists of snail mucus collected from Helix aspersa maxima and is proposed to be used by adults as a food supplement. The data provided by the applicant about the composition and stability of the NF together with the report of the subchronic toxicity study were overall considered unsatisfactory. The Panel noted inconsistencies in the reporting of the certificates of analysis and of the data on the subchronic toxicity provided by the applicant. Owing to these deficiencies, the Panel cannot establish a safe intake level of the NF. The Panel concludes that the safety of the NF has not been established.

Extraction, structure, pharmacological activities and applications of polysaccharides and proteins isolated from snail mucus.

Snail mucus had medical applications for wound healing as early as ancient Greece and the late Han Dynasty (China). A literature search found 165 modern research papers discussing the extraction methods, chemical compositions, pharmacological activities, and applications of snail mucus. Thus, this review summarized the research progress on the extraction, structure, pharmacological activities, and applications of polysaccharides and proteins isolated from snail mucus. The extraction methods of snail mucus include natural secretion and stimulation with blunt force, spray, electricity, un-shelling, ultrasonic-assisted, and ozone-assisted. As a natural product, snail mucus mainly comprises two polysaccharides (glycosaminoglycan, dextran), seven glycoproteins (mucin, lectin), various antibacterial peptides, allantoin, glycolic acid, etc. It has pharmacological activities that encourage cell migration and proliferation, and promote angiogenesis and have antibacterial, anti-oxidative and anticancer properties. The mechanism of snail mucus' chemicals performing antibacterial and wound-healing was proposed. Snail mucus is a promising bioactive product with multiple medical applications and has great potential in the pharmaceutical and healthcare industries. Therefore, this review provides a valuable reference for researching and developing snail mucus.

Evaluation of antioxidant, antityrosinase, and anticancer activity of mucus extract from both Egyptian land snails, Eremina desertorum and Helix aspersa, with emphasis on their chemical profiles.

The snail mucus provides several functions and is increasingly being exploited for medicinal and cosmetic purposes. This study aimed to determine the chemical profile of two snail mucus extracts: the garden snail (Helix aspersa) and the desert snail (Eremina desertorum). In addition, it elucidates the antityrosinase, antioxidant, and anticancer activities against the human cancer cell line epithelioid carcinoma (Hela). The mucus was extracted from the pedal glands of garden snails (H. aspersa) and desert snails (E. desertorum). 2,2-Diphenyl-1-picrylhydrazyl assay and the content of catalase, glutathione-S-transferase, superoxide dismutase, and reduced glutathione were utilized to assess the antioxidative screening activity of the mucus extracts. Besides a tyrosinase inhibitor assay, anticancer activity on cervical cancer cells (Hela) was studied. Additionally, the two mucus samples' total protein, total lipid, fatty acid, and amino acid profiles were compared. The mucus from both snails exhibited antioxidant activity. E. desertorum is more effective in inhibiting tyrosinase activity and has better scavenging activity than H. aspersa mucus extract. Both extracts revealed inhibitory activity against Hela cells, with insignificant differences. Moreover, the results indicated higher protein, lipids, and fatty acids mucus content of E. desertorum extract than those of H. aspersa mucus extract. Both snail slimes' obtained different biological activities, and amino acid contents could be related to their specific functions and beneficial for medical applications, especially antihyperpigmentation.

Biological properties of mucus from land snails (Lissachatina fulica) and freshwater snails (Pomacea canaliculata) and histochemical study of mucous cells in their foot.

Background: Mucus derived from many land snails has been extensively utilised in medicine and cosmetics, but some biological activities of the mucus need to be well documented. Nevertheless, most mucus is obtained from land snails, while mucus from freshwater snails has yet to be attended. Methods: This study aims to determine and compare mucus's antioxidant and anti-inflammatory activities from the land snail Lissachatina fulica and the freshwater snail Pomacea canaliculata. ABTS, DPPH, reducing power and total antioxidant activity assays were used to evaluate the antioxidant capacity. Inhibition of nitric oxide production in lipopolysaccharide-activated RAW 264.7 cells was performed to determine the anti-inflammatory activity. Additionally, the histochemical analysis of mucous cells in each snail foot was conducted to compare the distribution of mucous cells and types of mucins using periodic acid-Schiff and Alcian blue staining. Results: Mucus from L. fulica and P. canaliculata exhibited antioxidant and anti-inflammatory activities in different parameters. L. fulica mucus has higher total antioxidant (44.71 ± 2.11 mg AAE/g) and nitric oxide inhibitory activities (IC50 = 9.67 ± 0.31 µg/ml), whereas P. canaliculata mucus has better-reducing power activity (43.63 ± 2.47 mg AAE/g) and protein denaturation inhibition (IC50 = 0.60 ± 0.03 mg/ml). Histochemically, both species' dorsal and ventral foot regions contained neutral and acid mucins in different quantities. In the dorsal region, the neutral mucins level in L. fulica (16.64 ± 3.46%) was significantly higher than that in P. canaliculata (11.19 ± 1.50%), while the acid mucins level showed no significant difference between species. Levels of both mucins in the ventral foot region of L. fulica (15.08 ± 3.97% and 10.76 ± 3.00%, respectively) were significantly higher than those of P. canaliculata (2.25 ± 0.48% and 2.71 ± 0.56%, respectively). This study revealed scientific evidence of the biological capacity of mucus from L. fulica and P. canaliculata as well as provided helpful information on the region of the foot which produces effective mucus.

Standardized Extract from Wastes of Edible Flowers and Snail Mucus Ameliorate Ultraviolet B-Induced Damage in Keratinocytes.

Several studies have highlighted the ability of snail mucus in maintaining healthy skin conditions due to its emollient, regenerative, and protective properties. In particular, mucus derived from Helix aspersa muller has already been reported to have beneficial properties such as antimicrobial activity and wound repair capacity. In order to enhance the beneficial effects of snail mucus, a formulation enriched with antioxidant compounds derived from edible flower waste (Acmella oleracea L., Centaurea cyanus L., Tagetes erecta L., Calendula officinalis L., and Moringa oleifera Lam.) was obtained. UVB damage was used as a model to investigate in vitro the cytoprotective effects of snail mucus and edible flower extract. Results demonstrated that polyphenols from the flower waste extract boosted the antioxidant activity of snail mucus, providing cytoprotective effects in keratinocytes exposed to UVB radiation. Additionally, glutathione content, reactive oxygen species (ROS), and lipid peroxidation levels were reduced following the combined treatment with snail mucus and edible flower waste extract. We demonstrated that flower waste can be considered a valid candidate for cosmeceutical applications due to its potent antioxidant activity. Thus, a new formulation of snail mucus enriched in extracts of edible flower waste could be useful to design innovative and sustainable broadband natural UV-screen cosmeceutical products.

Snail Mucus from Achatina fulica as a Biomaterial Exhibits Pro-Survival Effects on Human Chondrocytes.

This study reports the novel use of Achatina fulica (A. fulica) mucus as a potential therapeutic repair agent in osteoarthritis and cartilage tissue repair in vitro. Snail mucus was isolated, sterilized, and characterized using FTIR, XPS, rheology, and LC-MS/MS. The GAGs, sugar, phenol, and protein contents were estimated using standard assays. The LC-MS/MS identified 6-gingerol and some other small molecules. The effects of the sterilized mucus were studied on human chondrocytes using the C28/I2 cell as a model for the in vitro assays. The MTT assay indicates that mucus extracted from the pedal of A. fulica is biocompatible with the cells up to a concentration of 50 µg/mL. The mucus promoted cell migration and proliferation and completely closed the wound within 72 h, as indicated in the in vitro scratch assay. In addition, the snail mucus reduced apoptosis significantly (p < 0.05) in the treated cells by 74.6%. It preserved the cytoskeletal integrity of the C28/I2 cells, attributed mainly to GAGs and 6-gingerol content of the mucus. In conclusion, this present study suggests that GAGs and 6-gingerol conferred wound-healing and antiapoptotic properties on the mucus secretion from A. fulica and can be explored for therapeutic repair and cartilage tissue engineering.

Purification and characterisation of heparin-like sulfated polysaccharides with potent anti-SARS-CoV-2 activity from snail mucus of Achatina fulica.

Heparin-like sulfated polysaccharide, acharan sulfate, was purified from the mucus of an African giant snail with unique sulfated glycosaminoglycans (GAGs). This study reported on finding novel and safe heparin resources from Achatina fulica for further use as well as easy isolation and purification of the active fraction from the initial raw material. Its structure was characterised by a strong-anion exchange combined with high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy. The results indicated that the potential acharan sulfate fraction is a glycosaminoglycan composed of several repeating disaccharide units, namely, of →4)-α-IdoA(2S)(1→4)-α-GlcNAc/GlcNAc(6S)/GlcNSO3(6S)(1→, and hence, presents heterogeneity regarding negative net charge density. Furthermore, the heparinase digests inhibit the binding of SARS-CoV-2 spike protein to the ACE2 receptor. In summary, the acharan sulfate presented in this work has shown its great potential for application in the preparation of sulfated polysaccharides as an alternative to heparin with important biological activity.

Prediction of Antibacterial Peptides against Propionibacterium acnes from the Peptidomes of Achatina fulica Mucus Fractions.

Acne vulgaris is a common skin disease mainly caused by the Gram-positive pathogenic bacterium, Propionibacterium acnes. This bacterium stimulates the inflammation process in human sebaceous glands. The giant African snail (Achatina fulica) is an alien species that rapidly reproduces and seriously damages agricultural products in Thailand. There were several research reports on the medical and pharmaceutical benefits of these snail mucus peptides and proteins. This study aimed to in silico predict multifunctional bioactive peptides from A. fulica mucus peptidome using bioinformatic tools for the determination of antimicrobial (iAMPpred), anti-biofilm (dPABBs), cytotoxic (ToxinPred) and cell-membrane-penetrating (CPPpred) peptides. Three candidate peptides with the highest predictive score were selected and re-designed/modified to improve the required activities. Structural and physicochemical properties of six anti-P. acnes (APA) peptide candidates were performed using the PEP-FOLD3 program and the four previous tools. All candidates had a random coiled structure and were named APAP-1 ori, APAP-2 ori, APAP-3 ori, APAP-1 mod, APAP-2 mod, and APAP-3 mod. To validate the APA activity, these peptide candidates were synthesized and tested against six isolates of P. acnes. The modified APA peptides showed high APA activity on three isolates. Therefore, our biomimetic mucus peptides could be useful for preventing acne vulgaris and further examined on other activities important to medical and pharmaceutical applications.

Microbial diversity of garden snail mucus.

The search for new natural compounds for application in medicine and cosmetics is a trend in biotechnology. One of the sources of such active compounds is the snail mucus. Snail physiology and the biological activity of their fluids (especially the mucus) are still poorly studied. Only a few previous studies explored the relationship between snails and their microbiome. The present study was focused on the biodiversity of the snail mucus used in the creation of cosmetic products, therapeutics, and nutraceuticals. The commonly used cultivation techniques were applied for the determination of the number of major bacterial groups. Fluorescence in situ hybridization for key taxa was performed. The obtained images were subjected to digital image analysis. Sequencing of the 16S rRNA gene was also done. The results showed that the mucus harbors a rich bacterial community (10.78 × 1010 CFU/ml). Among the dominant bacteria, some are known for their ability to metabolize complex polysaccharides or are usually found in soil and plants (Rhizobiaceae, Shewanella, Pedobacter, Acinetobacter, Alcaligenes). The obtained data demonstrated that the snail mucus creates a unique environment for the development of the microbial community that differs from other parts of the animal and which resulted from the combined contribution of the microbiomes derived from the soil, plants, and the snails.

Snail Mucus Enhances Chemosensitivity of Triple-negative Breast Cancer Via Activation of the Fas Pathway.

BACKGROUND/AIM: The poor prognosis and chemoresistance of patients with triple-negative breast cancer (TNBC) urge the development of new therapeutic strategies. Snail mucus has shown its ability against inflammation, a process closely related to tumorigenesis, suggesting a potential anti-cancer activity. MATERIALS AND METHODS: The effect and mechanisms of snail mucus on cell viability were determined by IncuCyte Live-cell analysis and molecular biological methods. The anti-cancer fractions of snail mucus were isolated and identified by medium pressure liquid chromatography (MPLC) and nuclear magnetic resonance (NMR) spectrometry analysis. RESULTS: Snail mucus significantly decreased the viability of TNBC cells with relatively lower cytotoxicity to normal breast epithelial cells and enhanced their response to chemotherapy through activation of Fas signaling by suppressing nucleolin. Two peptide fractions have been identified as the anti-cancer ingredients of the snail mucus. CONCLUSION: Snail mucus can induce programmed cell death via the extrinsic apoptotic pathway and has therapeutic potential by achieving a chemo-sensitizing effect in TNBCs.

GlicoPro, Novel Standardized and Sterile Snail Mucus Extract for Multi-Modulative Ocular Formulations: New Perspective in Dry Eye Disease Management.

This study aimed to evaluate the mucoadhesive and regenerative properties of a novel lubricating multimolecular ophthalmic solution (GlicoPro®) extracted from snail mucus and its potential anti-inflammatory and analgesic role in the management of dry eye disease (DED). GlicoPro bio-adhesive efficacy was assessed using a lectin-based assay, and its regenerative properties were studied in a human corneal epithelial cell line. In vitro DED was induced in human corneal tissues; the histology and mRNA expression of selected genes of inflammatory and corneal damage biomarkers were analyzed in DED tissues treated with GlicoPro. A higher percentage of bio-adhesivity was observed in corneal cells treated with GlicoPro than with sodium hyaluronate-based compounds. In the scratch test GlicoPro improved in vitro corneal wound healing. Histo-morphological analysis revealed restoration of cellular organization of the corneal epithelium, microvilli, and mucin network in DED corneal tissues treated with GlicoPro. A significant reduction in inflammatory and ocular damage biomarkers was observed. High-performance liquid chromatography-mass spectrometry analysis identified an endogenous opioid, opiorphin, in the peptide fraction of GlicoPro. In conclusion, GlicoPro induced regeneration and bio-adhesivity in corneal cells; moreover, considering its anti-inflammatory and analgesic properties, this novel ophthalmic lubricating solution may be an innovative approach for the management of DED.

Snail mucus from the mantle and foot of two land snails, Lissachatina fulica and Hemiplecta distincta, exhibits different protein profile and biological activity.

OBJECTIVE: Snails secrete different types of mucus that serve several functions, and are increasingly being exploited for medical and cosmetic applications. In this study, we explored the protein pattern and compared the biological properties of the mucus secreted from the mantle collar and foot of two snail species, Lissachatina fulica and Hemiplecta distincta. RESULT: Protein profile showed a different pattern between the two species and between the two secretory parts. The mantle-specific protein bands were further characterized and among them was an antibacterial protein, achacin. Accordingly, the mucus from the mantle exhibited the higher antibacterial activity than that from the foot in both snail species. The mucus from H. distincta, first reported here, also showed antibacterial properties, but with a lower activity compared to that for L. fulica. Snail mucus also exhibited anti-tyrosinase activity and antioxidant activity but with no significant difference between the foot and mantle mucus. These results indicate some different protein compositions and biological activities of snail slime from the mantle and foot, which might be associated with their specific functions in the animal and are useful for medical applications.

Novel drug-loaded film forming patch based on gelatin and snail slime.

Gelatin-based films enriched with snail slime are proposed as novel biodegradable and naturally bioadhesive patches for cutaneous drug delivery. Films (thickness range 163-248 µm) were stretchable and they adhered firmly onto the wetted skin, especially those with high amount (70% V/V) of snail slime extract. Fluconazole was selected as model drug and added to films containing the highest amount of snail slime. The presence of Fluconazole (4.53 ± 0.07% w/w) did not modify significantly the mechanical properties, the swelling degree and the bioadhesive performances of the films. Structural investigations demonstrated that the crystalline form III of the drug changed to the amorphous one, forming an amorphous solid dispersion. Moreover, snail slime prevented the drug recrystallization over time. In vitro permeation studies showed that film exhibited a cumulative drug concentration (over 60% in 24 h) similar to that of the control solution containing 20% w/V of ethanol. Fluconazole-loaded gelatin films proved to be effective towards clinical isolates of Candida spp. indicating that the drug maintained its remarkable antifungal activity once formulated into gelatin and snail slime-based films. In conclusion, snail slime, thanks to its peculiar composition, has proved to be responsible of optimal skin adhesion, film flexibility and of the formation of a supersaturating drug delivery system able to increase skin permeation.

Functional properties of chitosan films modified by snail mucus extract.

Snail mucus is an attractive natural substance, which is increasingly used in cosmetic creams and syrups thanks to its emollient, moisturizing, protective and reparative properties. The aim of the present study was to explore the physicochemical properties of chitosan-based films added with snail mucus extracted from Helix Aspersa Muller. To this aim, chitosan films at different content of snail mucus were fabricated by simple solvent casting technique. The results of X-ray diffraction analyses, tensile mechanical tests, Infrared spectroscopy and thermogravimetry demonstrated that snail mucus addition strongly modifies the properties of chitosan films. In particular, it acted like a plasticizer enhancing films extensibility up to ten times and strongly improving their water barrier and bioadhesion properties, with a trend depending on Snail mucus content. Furthermore, it provides the films with antibacterial properties and enhanced cytocompatibility, yielding materials with tailored properties for specific requirements.

Prediction of anticancer peptides against MCF-7 breast cancer cells from the peptidomes of Achatina fulica mucus fractions.

Several reports have shown antimicrobial and anticancer activities of mucous glycoproteins extracted from the giant African snail Achatina fulica. Anticancer properties of the snail mucous peptides remain incompletely revealed. The aim of this study was to predict anticancer peptides from A. fulica mucus. Two of HPLC-separated mucous fractions (F2 and F5) showed in vitro cytotoxicity against the breast cancer cell line (MCF-7) and normal epithelium cell line (Vero). According to the mass spectrometric analysis, 404 and 424 peptides from the F2 and F5 fractions were identified. Our comprehensive bioinformatics workflow predicted 16 putative cationic and amphipathic anticancer peptides with diverse structures from these two peptidome data. These peptides would be promising molecules for new anti-breast cancer drug development.

Simultaneous determination of allantoin and glycolic acid in snail mucus and cosmetic creams with high performance liquid chromatography and ultraviolet detection.

A new methodology for simultaneous quantitative analysis of allantoin and glycolic acid in snail mucus and cosmetic creams was developed. HPLC separation was achieved a Synergi-Hydro RP column within 7min using isocratic elution with potassium phosphate (pH 2.7; 10mM) at a flow rate of 0.7mL/min at 30°C. Sample pretreatment was performed by dilution of mucus or cosmetic cream in the elution buffer, heating at 60°C for 20min, adjusting the pH to 2.9 and purification with hexane extraction. Linearity was determined with spiked samples and the LLOQ values of 0.0125 and 0.2500mg/mL were determined for allantoin and glycolic acid, respectively. Accuracy and intra- and inter-day repeatability were studied at three levels of concentrations (0.04, 0.08 and 0.16mg/mL for allantoin and 0.1, 1.5 and 4.0mg/mL for glycolic acid) using spiked mucus and cream base samples; mean values of recovery were in the range of 96.81-102.42% in all matrices tested, whereas the respective RSDs (%Relative Standard Deviation) were less than 3.04% in all cases. Spiked mucus and cream samples were stable (RSD<4.16 and relative error<4.34%) at room temperature and at 4°C for 1 week and at -18°C for 6 months; samples were also stable after three freeze-thaw cycles. The method was applied to the analysis of different lots of snail mucus, and of three commercial creams containing snail mucus.