Cytotoxicity Test of Active Compounds Natural Ingredients of Snail Mucus ( Achatina fulica ) Against BHK-21 Fibroblast Cells

Snails are unpleasant yet beneficial. Rural people have used one to treat illnesses like toothache for years. We will test snail's mucus Achatina fulica's cytotoxic activity against Baby Hamster Kidney (BHK-21) fibroblast cells at 12.5%, 25%, 50%, 100% and its resistance to Phorpyromonas gingivalis, Fusobacterium nucleatum, E. Faecalis, and S. aureus using Microtetrazolium (MTT) assay. The test and comparison solution was incubated with 5x103/100 l cells in 96-well plates. 5 mg/mL MTT completed the solution's incubation. ELISA readers measured purple color intensity. The formula transformed absorbance data at 595 nm into percent alive cells. ELISA readers read data. ANOVA, parametric Kolmogorov-Smirnov data normality test were performed. The cytotoxicity statistical test shows the following results: 12.5 % (0.76875 ±0.01117), 25% (0.49350 ±0.004796), 50% (0.30250 ±0.006658) and 100% (0.171 ±0.10488). The lowest cytotoxicity of Achatina fulica snail mucus is 12.5% with an average of 0.768. Achatina fulica snail mucus resists Phorpyromonas gingivalis, Fusobacterium nucleatum, E. Faecalis, and S. aureus at 12.5%.

Periodontal diseases involve a wide variety of infections and chronic inflammatory conditions which affect the structures of the teeth (the gingiva, bone, and periodontal ligament), and affect eating, aesthetics, and speaking in particular. Periodontal disease is prevalent in adults, but aggressive periodontitis may occur in children. 1,2 People with periodontal diseases suffer from tooth loss and higher risk for systemic inflammation 3 . The loss of the tooth influences mastication, and subsequently ruin nutrition and diet 4,5 . Periodontal diseases are caused by bacteria in sub gingival dental plaque. 6,7 Among the gramnegative bacteria causing periodontal infections are Porphyromonas gingivalis 8,9 , Fusobacterium nucleatum as one of the most abundant gram-negative bacteria in periodontitis 10, 11 , Enterococcus faecalis [12][13] and Staphylococcus aureus which is associated with the aggressive periodontitis. 14,15 Localized periodontitis can be treated with mechanical debriment and good oral hygiene. 16,17 Meanwhile, generalized periodontitis requires antibiotic therapy. 18 This is because generalized periodontitis affects more than 30% of sites of the teeth. 19 Therefore, new treatment modalities such as antimicrobial therapy for tissue repair and regeneration are indispensable. 20 Snails' mucus is a natural ingredient can be used as a traditional medicine for curing minor wounds and dental diseases. 21,22 In tropical country, various species of snails can be found, including land snail or Achatina fulica. However, the biological compound of the mucus is necessary to examine in order to determine whether a compound has the potential to be toxic to biological organisms, and if it so, to what extent. 23 The examination to determine such necessity is through cytotoxicity test. 24,25 A cytotoxicity test is a biological evaluation of natural substances and is required for standard screening procedures. 26 The purpose of a cytotoxicity test is to determine the toxic effect of a substance directly on tissue culture. 27 Wounds healing process includes homeostasis and inflammation, proliferation and maturation phases. 28,29 The proliferative phase increases the number of cells and wound healing factors, one of which is the proliferation of fibroblasts. Fibroblasts are the most common cells found in connective tissue. 30 It is used to synthesize several components of the extracellular matrix (collagen, elastin, reticular), several anionic macromolecules (glycosaminoglycans, proteoglycans) and multi-adhesive glycoproteins, laminins, and fibronectins that can promote cell attachment to substrates. 31,32 They also secrete cytokines and several growth factors, which can stimulate cell proliferation and inhibit the differentiation process. 33, 34 The proliferation of fibroblasts determines the final outcome of wound healing. 35 Fibroblasts will produce collagen which will link the wound, and fibroblasts will also affect the re-epithelialization process which will make a wound closure. 36,37 Snail mucus contains beta agglutinins (antibodies) in plasma (serum), achasin protein, glycoconjugates and acharan sulfate which play a role in the wound healing process by helping the blood clotting process and fibroblast cell proliferation. 38,39 In measuring the number of cells for proliferation and cytotoxicity assays, the MTT (Microtetrazolium) assay is one of the best methods to use. MTT assay is included to colorimetric procedure. Colorimetric procedures are considered economical, rapid, and able to measure multiple samples simultaneously. 40 Besides, the colorimetric procedures can be automated, and preferred for evaluating the physiological state of microbes. 41,42 The MTT assay method usually uses a 96well microplate so that many samples can be studied simultaneously. A material can be said biocompatible when the material does not cause irritation to the tissue life, does not cause a toxic response, free of ingredients that can trigger an allergic reaction, and not has carcinogenic potential. Deciding biocompatible of a material can be carried out through a biocompatibility test or test toxicity. 43,44 The first level of the biocompatibility test of a material can be done through in-vitro, namely the material to be tested contacted outside the body of microorganisms such as cell culture. 45 The previous work related to snails' mucus quantitative researches have been done to assess the composition of the snail. 46 46 The results show that the proteins from Achatina Fullica is weighted 83.67 kDa (achasin), 50.81 kDa, 15 kDa, 11.45 kDa and 9.7 kDa (mytimacin-AF). 46 The isolation and characterization of the protein was conducted using SDS-PAGE method, electro-elution, and dialysis. 46 Another research by Noothuan, et al (2021) about a different snail mucus from Lissachatina fulica and Hemiplecta distincta was examined and proved that they have exhibits different pattern of protein. The protein concentration was determined using Bradford assay and the protein pattern of the two snails analyzed by 12.5% SDS-PAGE. 47 The Lissachatina fulica mucus showed major bands at about 13, 37, 70, and > 200 kDa, whereas Hemiplecta Distincta showed major bands at approximately 11,12,14,25, and 120 kDa. 47 The snail mucus also exhibits various biological activities such as antimicrobial, antioxidant, antityrosinase and antitumoral activities. 48 Research by Trapella, et al (2018) about chemical composition and biological effect of the snail mucus has been conducted with snail species of Helix aspersa muller. 49 The method used in vitro experimental model. The results show that snail mucus exhibits glycolic acid and allantoin. It is also found that the mucus is lacked of cytotoxicity and induce cell proliferation. 49 Most of the researches show the composition of the snail's mucus. 46,47 However, the qualitative-quantitative research about comprehensive chemical compounds from snail mucus, and their molecular formulas have not been widely studied, especially for periodontal disease.
Considering recent studies of developing medical substances from environment as an alternative to prevent further infections or wounds, it is worth to evaluate the comprehensive chemical compounds extracted fromsnail's mucus, especially for peridontitis. Mammalian cell lines encompass 51% of approved biologics in industrial cell lines and the most used mammalian cell lines includes Baby Hamster Kidney (BHK) cells. 50 BHK-21 has found applications in vaccines against mouth diseases and heterologous protein production and is the most important cell, the largest component of the dental pulp, periodontal ligament and gingiva. 51,52 This study aims to analyze the cytotoxic activity of snail mucus Achatina fulica against BHK-21 fibroblast cells, carried out with various concentrations of the snail mucus at 12.5%, 25%, 50%, 100% to periodontitis bacteria of Porphyromonas gingivalis, Fusobacterium nucleatum, Enterococcus faecalis and Staphylococcus aureus. The percentage of concentrations used in this research is based on the research by Daud, et al (2018) which shows that at 11%, the snail mucus has the antibacterial activity. 53 Meanwhile, at concentration 30% and 60%, snail mucus reduces the cell viability. 54 The concentrations of 12.5%, 25%, 50% and 100% are also used in analyzing mucus antibacterial activity. 47 Porphyromonas gingivalis contributes to chronic periodontitis. This bacterium creates virulence factors causing destruction to periodontal tissue. 55 Research by Hendrawati, et al (2019) shows that 20% snail mucus gel can enhance the osteoblasts in rats suffering from periodontitis. 56 Fusobacterium nucleatum causes lesions in periodontal disease, halitosis, dental pulp infection, oral cancer and systemic disease. 57 Enterococcus faecalis appear more frequent in subgingival samples with periodontitis than from periodontally healthy one. 58,59

Material and Methods
The process of preparation to the generation of snail mucus is shown in Figure 1. The process is detailed according to the research design, preparation and generation of the snail mucus.

research design
The method used in this study is a qualitative-quantitative method with a "True Experiment Laboratory" research approach.
The method was carried out by laboratory analysis of snail mucus (Achatina fulica) to obtain snail's various chemical elements. This qualitative analysis is carried out using 16opicastandard testing techniques in the laboratory to identify the protein, antibacterial power or inhibition against Porphyromonas gingivalis, Fusobacterium nucleatum, Enterococcus faecalis and Staphylococcus aureus, and detect the antiinflammation (Acharan sulfat). Quantitative research is carried out to determine the concentration of a compound in the sample, which can be in the form of moles, or percentages in grams. This technique requires high accuracy because errors in measurement will result in data errors in research. Quantitative analysis is generally carried out after qualitative analysis. The research was carried out at the Oral Biology Laboratory, Airlanggga University, Indonesia.

Preparation
The snails were obtained from community plantations in Nyalian village, Banjar Angkan District, Klungkung, Bali, Indonesia. The type of snail we used is Achatina fulica. The snail is weighted 200-250 grams each. The total snails we used were 25 snails. The object of the research is the chemical compound in the snail. Snail mucus was taken, then analyzed to obtain the active compound or chemical compounds contained in it. The tip of a syringe was used to stimulate the secretion of mucus. 60,61

Generation of the snail's Mucus
A snail produces approximately 3-5 cc mucus. Snail mucus was collected in a bottle, then centrifuged. Then the snail mucus was tested for cytotoxicity using BHK-21 fibroblast cells, using the MTT Assay method. Cells in the number (5x103/100 l) were distributed to a 96-well plate and incubated with test and comparison solutions of various concentration series of 100 l for 24 hours in a CO 2 incubator with 5% percentage at 37p C. After incubation, 100 l of solution with 5 mg/mL of MTT was added to each well. The reaction was stopped by adding a 10% SDS stopper into 0.1N HCl after 6 hours. After that, it was incubated at room temperature in a dark room overnight. The intensity of the purple color formed was measured with an Enzyme-linked immunosorbent (ELISA) reader or also known as microplate reader at a wavelength of 595 nm 60 . The absorbance data obtained were converted into percent Live Cells with the formula 62 : Live Cell (%) = ((Absorbance treatment-Absorbance control media)/((Absorbance control cell-Absorbance control media)) x 100% ...(1) Figure 2 shows the process where the first step is disinfected culture shock, then stock planting on RPMI media, centrifuge stock cell media RPMI. Next, is the picture of cell culture well plate 96 and then got the sample of 100%, 50%, 25% and 12.5%. At last, the data were read on ELISA reader.

inhibition test
The data collected is primary data in the form of zone diameter. The diameter of the inhibition zone of snail mucus against Porphyromonas gingivalis, Fusobacterium nucleatum, Enterococcus faecalis and Staphylococcus aureus is the diameter of the clear zone that appears on the disk diffusion as measured by a caliper (in millimeters) to determine the inhibition power. The qualitative analysis comprises the diameter of bacterial inhibition power that was categorized according to Davis and Stout (1971): very strong (clear zone >20mm), strong (10-20mm clear zone), moderate (5-10mm clear zone), weak (<5mm). 63

statistical analysis
The analysis of normality test data used Kolmogorov-Smirnov. It was performed using SPSS version 25 and Program R. If the data are normally distributed, the test to determine significance is followed by a parametric statistical test using one-way ANOVA. If the data are not normally distributed, then the significance test is carried out with a non-parametric statistical test using Kruskal-Wallis.

results and discussion inhibition of Porphyromonas gingivalis
Porphyromonas gingivalis is known as major etiological agent in periodontal diseases which resulted in gingival inflammation 9 . Tabel 1 shows the results of the examination of the inhibition of Achatina fulica against Porphyromonas gingivalis bacteria (four repetitions). Table 2 shows the results of the examination of the inhibition diameter of the snail's mucus against Porphyromonas gingivalis. The results of the inhibition of snail mucus against Phorpyromonas gingivalis is the largest in the treatment group with a concentration of 100%, with an area of 21.35 mm, and the smallest at a concentration of 12.5%. This indicates that the higher the concentration of snail mucus material, the wider the inhibition. So, in conclusion, the inhibition power of Achatina fulica to Porphyromonas gingivalis is very strong with 21.35 mm at 100% concentration and weak at 12.5% concentration. Analysis of differences in inhibition power between the treatment groups of Phorpyromonas gingivalis is given in Table 3. It presents that there is significant difference in the treatment group, where the difference between the 12.5% treatment group and the control group is the highest.

inhibition of Fusobacterium Nucleatum
The results of inhibition power of Fusobacterium Nucleatum in four repetitions are presented in Table 4. Meanwhile, table 5 shows the inhibition zone diameter of Fusobacterium Nucleatum. From table 5, it can be that the largest average diameter of the inhibition zone in the treatment group with various variations was at a concentration of 100%, with a diameter of 19.60 mm, while at the smallest 12.5% there is no inhibition zone. The table is revealing that the highest average at 100% concentration is categorized as strong diameter inhibition power. Whereas, the lowest concentration at 12.5% has weak diameter of bacterial inhibition power. From the category, it is noted that the inhibition power of Achatina fulica to Fusobacterium Nucleatum is categorized strong at 100% concentration and weak at 12.5% concentration. The statistical test on significance of differences in in inhibition is shown in table 6, where it notes that there is significant difference of inhibition power in each treatment group. Figure 3 shows the inhibition zone of the snail mucus against Fusobacterium nucleatum. inhibition of E. Faecalis Table 7 presents the inhibition zone results in four repetitions on E. Faecalis in the treatment group. Table 8 indicates that the largest inhibition zone in the treatment group is with a concentration of 100%, namely 21.93 mm, while the smallest   Table 10 shows the inhibition power of the snail mucus of Achatina fulica against S. aureus bacteria in four repetitions. From table 10, it can be processed to analyze the inhibition zone diameter on S. aureus bacteria as presented in table 11. The results show that the average inhibition power in the treatment group with four repetitions with an inhibition zone area of 23.15 mm is at 100%  Table 13 details the chemical compounds of snail's mucus obtained from Achatina fulica. The content of active chemical compounds from snail slime was tested by GCMS (Gas Chromatography-Mass Spectrometry) which is to identify the active substance from snail's mucus. It is noted that the most compound found is Achasin protein with an average of 102.20 mg/100g, and the least compound contained in the mucus is glycoconjugate in as  Table 14 shows the highest toxicity to lowest toxicity respectively at the concentration of 12.5% (mean: 0.768), concentration of 25%   Table 14 also shows clearly that there are differences in the control media, control cell and various concentration of Achatina fulica in which the least mean is at concentration of 12.5%, and the highest mean is at concentration of 100%. Table 15 shows the statistical test with Kolmogorov-Smirnov data that is normally distributed. Since all data were normally distributed, statistical tests to determine which group has the most significance were tested using the one-way ANOVA parametric statistical test. Subsequently, it is to determine which group has the most significance by using the one-way ANOVA parametric statistical test as shown in Table 16. Table 16 shows a significant difference between groups with various concentration and between treatment groups, where the significance is < 0.05. Table 17 indicates a significant difference the increase in snail mucus (%) to the number of living cells (cytotoxicity) with p < 0.05. From the tables above, it can be concluded that the minimum cytotoxicity value of snail mucus is at a concentration of 12.5%. This means that the increase in concentration above the concentration of 12.5% is cytotoxic to the number of cells.

cytotoxicity to Fibroblast BhK 21
The use of traditional medicinal snail mucus must be carried out by knowledge of the safety level of preparations obtained through toxicity test so as not to cause harmful effects. 64,65 Various ingredients or active chemical substances are found in the snails' mucus, such as antibacterial and anti-inflammatory. The cytotoxicity test in this study was snail's mucus of Achatina fulica against BHK-21 fibroblast cells. The use of cultured cells of BHK-21 is the most important cell and the largest component of the dental pulp, periodontal ligament and gingiva. 51 This study used 12.5%, 25%, 50%, and 100% concentrations. This is to determine the potential toxicity of the active compound of snail's mucus (Achatina fulica) against BHK-21 mice fibroblast cells in-vitro, using the MTT Assay method. Formazan can be generated even in cell-free conditions: MTT can be reduced by some particular compounds present in culture media such as polyphenols. 66 The results of the snail's mucus cytotoxicity test show that the average mean of four repetitions at a concentration of 12.5% is 0.768, while at a concentration of 50% is 0.493, the concentration of 25% was 0.302, while the concentration of 100% is 0.17. So at a concentration of 12.5 % is not toxic because it has more than 50% number of fibroblasts, while at a concentration of 25-100% it is toxic, this is due to the protein content of acharan sulfate. 67

Fibroblast Proliferation
Snail mucus causes faster proliferation. 39 These contents play an important role in cells and matrix cells interactions associated with normal and pathological conditions of cell recognition, adhesion, migration, and cell growth, and these active substances can also chemically stimulate the process of fibroplasia in the wound area. Increased proliferation of fibroblast cells can be used as a biological marker of the wound healing process, namely by the presence of a high percentage of increased fibroblast proliferation. The presence of a high percentage of live cells in BHK-21 fibroblast cells means that snail mucus has the effect of increasing fibroblast cell proliferation so that it can accelerate the wound healing process. 68,69 The reduction of yellow MTT salt to purple formazan is performed by tetrazolium succinate reductase, which is included in the respiratory chain in the mitochondria of living cells. 43,70 In this study, the average optical density of formazan in snail mucus with increasing concentrations of 12.5%, 25%, 50%, 100% showed a decrease (Table 14) due to the ability of living cells to reduce MTT salts. The principle of this assay is the breakdown of the yellow MTT tetrazolium ring (3-4-5-dimethylthiazol-2-yl)-2-5 diphenyl tetrazolium bromide) by the presence of dehydrogenase in the active mitochondria, resulting in an insoluble purplish-blue formazan product. 71 The mechanism is that the yellow tetrazolium salt will be reduced in cells with metabolic activity, which has an important role, in this case, in the mitochondria of living cells that produce dehydrogenase. If dehydrogenase is inactivated due to cytotoxic effects, formazan will not be formed. In table 16, statistical calculations using One Way ANOVA followed by LSD with a significance level of 5% showed that the higher the snail mucus concentration, the lower the formazan density value was significant. Natural materials such as snails' mucus, before being used as a medicine, must perform an enzymatic test process, not irritating, and have biocompatibility, or the material produced must not have a detrimental effect on the biological environment local and systemic. 46 The basis of the MTT enzymatic test is to measure the ability of living cells based on mitochondrial activity from cell cultures. 43 For this reason, natural ingredients have now been developed which can be used as alternative ingredients for healing inflammation. 72 In the present study, the higher concentration, decreasing percentage rate of fibroblast cells. This is in accordance with the research conducted by (Apriasari, et al., 2014) on the toxicity of Mauli banana stem extract against BHK-21 fibroblast cells which proved that the higher the concentration of the extract, the lower the viability of fibroblast cells. 73 Antimicrobial susceptibility testing (AST) performance of bacterial pathogens is an essential procedure to ensure and determine the susceptibility to antimicrobial agents and to analyse the resistance. 74,75 Disk diffusion has been the pledge for antimicrobial susceptibility testing. 76 On a larger scale, the AST helps in the evaluation of treatment services provided by hospital, clinics, and health programs to control and prevent infectious diseases. 77,78 The determination of susceptibility and its resistance is by categorized the results of zone diameter of inhibition. 74 The diameter of the inhibition zone around each antibiotic disk is measured in milimeters. 79,80 The results of zone diameter of inhibition of Phorpyromonas gingivalis, E. Faecalis , and S. aureus show at concentration 100%, the zone diameter of inhibition are categorized very strong in 21.35, 21.93, 23.15 mm respectively. Meanwhile, the result of zone diameter of inhibition of Fusobacterium nucleatum is categorized strong with 19.7 mm diameter. The large zone diameter of inhibition indicates that the organism is susceptible, while the small or no zone inhibition shows resistance. 76,81 So, it can be drawn in this study, that the snail's mucus from Achatina fulica is resistant with concentration 12.5%.

conclusion
This study conducted cytotoxicity test of snail mucus Achatina fulica with various concentration against BHK-21 fibroblast cells in mice. The concentration we used are 12.5%, 25%, 50% and 100%. Based on the results obtained, it can be concluded that the active compound of snails' mucus in various concentrations has the highest cytotoxicity activity at a concentration of 12.5%, with an average of 0.768. After analyzing the active substances or chemical compounds of snail mucus, the antibacterial content is Achatin and acharan sulfate as antibacterial and painkillers, and for the anti-inflammation, it is obtained Heparan sulfate. The inhibition test of snail mucus against bacteria (Phorpyromonas gingivalis, Fusobacterium nucleatum, S. aureus, and E. foecalis, has a very strong category of inhibition. The results of the snail slime cytotoxicity test showed that more than 50% of the number of fibroblasts in BHK21 cells was at a concentration of 12.5%, meaning that the snail mucus at that concentration is not toxic. The future work of this research can be performed in the analysis of histological and clinical research to establish the snail mucus from Achatina fulica to be used for periodontitis therapy.