Antifungal Activity of Soft Tissue Extract from the Garden Snail Helix aspersa (Gastropoda, Mollusca)

Gastropods comprise approximately 80% of molluscans, of which land snails are used variably as food and traditional medicines due to their high protein content. Moreover, different components from land snails exhibit antimicrobial activities. In this study, we evaluated the antifungal activity of soft tissue extracts from Helix aspersa against Candida albicans, Aspergillus flavus, and Aspergillus brasiliensis by identifying extract components using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Two concentrations of three extracts (methanol, acetone, and acetic acid) showed antifungal activity. Both acetone (1 g/3 mL) and acetic acid extracts (1 g/mL) significantly inhibited C. albicans growth (p = 0.0001, 5.2 ± 0.2 mm and p = 0.02, 69.7 ± 0.6 mm, respectively). A. flavus and A. brasiliensis growth were inhibited by all extracts at 1 g/mL, while inhibition was observed for acetic acid extracts against A. brasiliensis (p = 0.02, 50.3 ± 3.5 mm). The highest growth inhibition was observed for A. flavus using acetic acid and acetone extracts (inhibition zones = 38 ± 1.7 mm and 3.1 ± 0.7 mm, respectively). LC-MS-MS studies on methanol and acetone extracts identified 11-α-acetoxyprogesterone with a parent mass of 372.50800 m/z and 287.43500 m/z for luteolin. Methanol extracts contained hesperidin with a parent mass of 611.25400 m/z, whereas linoleic acid and genistein (parent mass = 280.4 and 271.48900 m/z, respectively) were the main metabolites.


Introduction
For hundreds of years, land snails have been used as a food source and for medical treatments. Land snails are pharmacologically and medicinally important [1] as they are high in protein, which they use to combat different environmental conditions [2]. Previously, seven crude proteins were extracted from different snails. The most active crude proteins identified in the land snail, Cryptozona bistrialis, were active against different bacterial and fungal pathogens [3]. Therefore, snails should be considered important bioactive compound sources with safe pharmaceutical applications as polypeptides, proteins, and glycans from snail mucus could function as promising candidates for some dermal diseases [4]. Similarly, protein and peptide components from the hemolymph and mucus of garden snails showed antimicrobial activity [5]. Slime from the African giant land snail Archachatina marginata demonstrated antibacterial activity against Escherichia coli (inhibition zone = 15.2 mm), Klebsiella sp. (inhibition zone = 14.2 mm), Pseudomonas aeruginosa (13.0 mm), and Proteus mirabilis (13.3 mm) [6].
Three protein fractions from the marine snail Rapana venosa were effective against Aspergillus niger, Botrytis cinerea, and Candida albicans [7].
Two protein fractions from the mucus of Helix aspersa (>20 kDa) and a peptide fraction from the hemolymph of Helix lucorum (<10 kDa) were isolated, and their antibacterial activities against E. coli and Brevindomonas diminita were characterized. Their minimum inhibitory concentration values ranged from 145 to 682.5 µg/mL for E. coli and B. diminuta [8]. In hemolymph from H. lucorum, nuclear magnetic resonance metabolic analysis and tandem mass spectrometry (MS-MS) were used to detect metabolites (<1 kDa and <3 kDa) with antioxidant and antimicrobial activities [9]. Ref. [10] isolated a 1485. 26 Da peptide (Cm-p1, sequence = SRSELIVHQR) from a crude extract of the marine snail, Cenchritis muricatus, which demonstrated antifungal activity against filamentous fungi and yeast. Moreover, the antifungal activity of a crude methanol extract from Cypraea spp. Against C. albicans and A. niger was similarly demonstrated [11]. In other work, ethanol, acetone, and methanol crude extracts from Babylonia spirata were effective against A. flavus and C. albicans [12].
Fungal infections may occur in the hair, nails, and skin and may cause serious diseases [13]. The total number of individuals who experience different fungal infections is approximately 1,000,000,000. Fungal diseases may kill more than 1.5 million and affect over a billion people. The health consequences of serious fungal infections include asthma, serious chronic illness, blindness and corticosteroid therapies. The modern global estimates have found~700,000 cases of invasive candidiasis and 3,000,000 cases of chronic pulmonary aspergillosis [14]. C. albicans causes superficial, deep tissue, and invasive candidiasis [15], and its mortality rate is 35-60% due to disseminated candidiasis [16]. Fungal diseases such as aspergillosis cause high mortality rates due to respiratory disorders [17]. Bronchopulmonary aspergillosis caused by A. fumigatus, A. flavus, and A. niger colonies in the lung mucosa spread infection, with a mortality rate of 50-90% [18]. Therefore, antifungal properties from bioactive compounds in invertebrates could provide new directions for medical treatments and scientific research.
A novel neurotoxin (conotoxin TVIIA) was extracted from the sea snail Conus tulipa [19] and was identified as belonging to the conotoxin family, which is composed of six-cysteine/ four-loop structures with pharmacological activities. The molecules block ionic calcium, sodium, and potassium channels [20,21]. The contryphan-Vn peptide was extracted from the Mediterranean snail, Conus ventricosus; it contained a D-tryptophan residue and maintained its five-residue intercystine loop. The peptide was bound to potassium channels and displayed distinct molecular targets [22].
In this study, we isolated soft tissue extracts from H. aspersa and identified antifungal activities against C. albicans, A. flavus, and A. brasiliensis. Extract components were identified using liquid chromatography-mass spectrometry (LC-MS-MS).

Results
Two concentrations, 1 g/mL and 1 g/3 mL, of three crude extracts (methanol, acetone, and acetic acid extracts) from the soft body (viscera) of H. aspersa were used to test for antifungal effects against C. albicans, A. flavus, and A. brasiliensis.
The antifungal drug fluconazole (25 µg/mL) inhibited A. brasiliensis and A. flavus growth at 17 ± 0.4 mm and 14 ± 0.1 mm, respectively. The solvents had no effects on fungal growth except for acetic acid (inhibition zone = 15 ± 0.1 mm and 10 ± 0.5 mm for A. brasiliensis and A. flavus, respectively) ( Figure 1). Both A. flavus and A. brasiliensis growth was inhibited by all extracts at 1 g/mL. Still, inhibition was significant for the acetic acid extract against A. brasiliensis (ANOVA, p = 0.02, 50.3 ± 3.5 mm) (Figures 1 and 3).

Results
Two concentrations, 1 g/mL and 1 g/3 mL, of three crude extracts (methanol, acetone, and acetic acid extracts) from the soft body (viscera) of H. aspersa were used to test for antifungal effects against C. albicans, A. flavus, and A. brasiliensis.
The most significant growth inhibition was observed for A. flavus against acetic acid and acetone extracts (inhibition zones = 38 ± 1.7 mm and 3.1 ± 0.7 mm, respectively).  flavus growth with methanol viscera extract (1), acetone viscera extract (2) and acetic acid viscera extract (3). Arrowheads are pointing to the inhibition zone. The antifungal drug fluconazole (25 µg/mL) inhibited A. brasiliensis and A. flavus growth at 17 ± 0.4 mm and 14 ± 0.1 mm, respectively. The solvents had no effects on fungal growth except for acetic acid (inhibition zone = 15 ± 0.1 mm and 10 ± 0.5 mm for A. brasiliensis and A. flavus, respectively) ( Figure 1). Both A. flavus and A. brasiliensis growth was inhibited by all extracts at 1 g/mL. Still, inhibition was significant for the acetic acid extract against A. brasiliensis (ANOVA, p = 0.02, 50.3 ± 3.5 mm) (Figures 1 and 3).
The most significant growth inhibition was observed for A. flavus against acetic acid and acetone extracts (inhibition zones = 38 ± 1.7 mm and 3.1 ± 0.7 mm, respectively).  The most signifi and acetone extracts (  The antifungal drug fluconazole (25 µg/mL) inhibited A. brasiliensis and A. flavus growth at 17 ± 0.4 mm and 14 ± 0.1 mm, respectively. The solvents had no effects on fungal growth except for acetic acid (inhibition zone = 15 ± 0.1 mm and 10 ± 0.5 mm for A. brasiliensis and A. flavus, respectively) ( Figure 1). Both A. flavus and A. brasiliensis growth was inhibited by all extracts at 1 g/mL. Still, inhibition was significant for the acetic acid extract against A. brasiliensis (ANOVA, p = 0.02, 50.3 ± 3.5 mm) (Figures 1 and 3).
The most significant growth inhibition was observed for A. flavus against acetic acid and acetone extracts (inhibition zones = 38 ± 1.7 mm and 3.1 ± 0.7 mm, respectively).  The most significant growth inhibition was observed for A. flavus against acetic acid and acetone extracts (inhibition zones = 38 ± 1.7 mm and 3.1 ± 0.7 mm, respectively).

Discussion
Our data indicated that all extracts from the snail Helix aspersa have bioactive ingredients which exerted antifungal activities against C. albicans, A. flavus, and A. brasiliensis in a concentration-dependent manner. Furthermore, our results were supported by [12], who extracted bioactive compounds from the marine snail, B. spirata. Moreover, the antibacterial activity of crustacean and molluscan methanol extracts was higher when compared with water extracts [3].
The antifungal activity of extracts may have been related to their metabolite components, as demonstrated by LC-MS-MS; the most common component was hesperidin, with a parent mass = 611.25400. This flavanone glycoside induced apoptosis and cell cycle arrest [27,28] and showed antifungal, antiviral, antihelminthic, antioxidant, and molluscicidal activities toward Schistosoma mansoni. Genistein is also present in plants and humans and is an isoflavone with antihelminthic qualities [29]. Genistein was speculated to inhibit fungal growth due to its apoptosis-inducing characteristics [30]. Genistein also induced apoptosis in human cancer cells by triggering both caspase-3 and caspase-9 activity, causing cell death by inhibiting NF-κB signaling and altering levels of the antiapoptotic protein Bcl-2 and proapoptotic protein. In addition, genistein excited oxidative stress-induced apoptosis by increasing nitric oxide production and its bioavailability [31][32][33]. Furthermore, genistein is an estrogenic isoflavone found in the molluscan tissues, and its receptors were isolated from the cerebral ganglia of the gastropod Thais clavigera [34].
The mass of 11-α-acetoxyprogesterone was 372.50800. Progesterone is a steroid hormone that functions as a substantial metabolic intermediate during corticosteroid and sex hormone production and also functions as a neurosteroid [35,36]. The antifungal effects of this compound are facilitated by the presence of high-affinity progesterone-binding sites in the plasma membrane, as confirmed by [37] in Rhizopus nigricans. A corticosteroid-binding protein and steroid receptor were identified by [38] in C. albicans [39] as well as other fungal species. Progesterone is considered an effective fungal growth inhibitor [40]. Its growth inhibition is related to reduced intracellular cyclic adenosine monophosphate(cAMP) levels that are crucial arbiters of fungal growth and responses to nutritional stress [41].
Luteolin is a tetrahydroxyflavone and exerts antifungal effects against C. albicans [42]. The fungal growth inhibition was reported whereby plasma membrane disruption led to membrane permeability changes and excess reactive oxygen species (ROS) production [43].
The ROS effects are mainly directed toward membrane lipids in C. albicans, which generate lipid hydroperoxides and thus lipid peroxidation [44]. The induction of mitochondrial dysfunction via inhibited mitochondrial electron transport chain reduces ATP synthesis and causes cell death by inhibiting cell wall formation, cell division, and RNA and protein synthesis [45].
Linoleic acid is a polyunsaturated essential fatty acid that reduces biomass production in Rhizoctonia solani, Pythium ultimum, Pyrenophora avenae, and Crinipellis perniciosa, while 1000 µM linoleic acid reduces mycelial growth in R. solani, P. ultimum, and P. avenae [46]. Linoleic acid is a structural component related to membrane fluidity functions and directly interacts with fungal cell membranes by entering lipid bilayers, increasing membrane fluidity, disorganizing cell membranes, and causing cell disintegration [47]. Antifungal fatty acids can replace synthetic agrochemicals that control fungal pathogens [48]. The antifungal activity of linoleic acid was reported against Aspergillus amylovorus (NRRL 5813) and A. flavus (NRRL 3518) [49,50]) reported its antifungal activity against C. albicans and Candida parapsilosis [50].
In conclusion, soft tissue extract metabolites from H. aspersa are promising antifungal agents.

Sample Collection and Care
Wild H. aspersa were collected from infested ornamental fruit and grass plants in Menoufia governorate, Egypt. Snails were maintained under the following laboratory conditions: 12 h:12 h light/dark photoperiod, room temperature, and relative humidity, 85%. Approximately 10-15 individuals (average weight = 4 g) were placed in glass containers (15 × 15 × 22 cm) filled with moist soil (sandy loam) and covered in muslin for ventilation. Fresh lettuce leaves (Lactuca sativa) and water for soil humidity were supplied daily. Waste food and fecal matter were removed at the end of every other day. Snails were acclimatized under laboratory conditions for at least 4 weeks.

Tissue Extraction
Snails were rinsed, and the shells removed. The soft body (viscera) was cut into small pieces and homogenized in different solvents, methanol, acetone, and acetic acid [3,12]. Solvent tissue homogenization was performed on ice. Two concentrations of the three solvents (methanol, acetone and acetic acid) were used: 1 and 3 mL for one gram of tissue (viscera). Homogenates were centrifuged at maximum speed in a refrigerated centrifuge, added to clean tubes, and maintained on ice till the inculcation of samples. Crude extracts were used for antifungal assays against the fungal pathogens, C. albicans, A. flavus, and A. brasiliensis.

Antifungal Assays
Two concentrations (1 g/mL and 1 g/3 mL) of the three crude extracts (methanol, acetone and acetic acid extracts) were prepared to test the growth inhibition of C. albicans, A. flavus, and A. brasiliensis (ATCC 16404) clinical isolates.
Antifungal activity was determined using the agar well diffusion method [51]. Nutrient agar plates were aseptically spread with 24 h cultures from respective pathogens and incubated for 15 min in a laminar chamber to facilitate absorption. Next, 5 mm wells were aseptically cut in the agar for visceral extract addition-100 µL of each extract was added, and plates were left for 1 h for infusion. Then, plates were incubated at 37 • C for 24 h, after which extract inhibition zone diameters were measured in mm. Fluconazole (25 µg/mL) was used as a positive control. All tests were performed in triplicate, and mean values were recorded for statistical analysis.

Chemical Analysis of Extracts
Methanol and acetone extracts were analyzed using LC-MS-MS. A Shimadzu LC-10 high-performance liquid chromatography instrument with a Grace Vydac Everest Narrowbore C18 column (100 mm × 2.1 mm i.d., 5 µm, 300 Å) was connected to an LCQ electrospray ion trap MS (Thermo Finnigan, San Jose, CA, USA). Raw data files were converted to mzXML format using MSConvert from the ProteoWizard suite (http: //proteowizard.sourceforge.net/tools.shtml, (accessed on 1 March 2022).). A molecular network was created using the GNPS online workflow. Spectra in the network were then searched against GNPS spectral libraries and published data [52].

Statistical Analysis
Data were expressed as the mean ± standard deviation and analyzed using Statgraphics Centurion XVI (Stat-Point Technologies Inc., Warrenton, VA, USA). Statistical analysis was conducted using a two-way analysis of variance to identify differences between tissue extracts and solvents and tissue extracts and concentrations. A probability p ≤ 0.05 level was accepted as significant.