Effectivity of Irrigation Solution from Stevia rebaudiana Bertoni Leaf Extract on the Growth of Enterococcus faecalis Bacteries

ABSTRACT


INTRODUCTION
One part of the cleaning and shaping stage is the use of irrigation solutions to disinfect the root canal system which plays an important role in achieving successful endodontic treatment.The biological purposed of using an irrigation solution in root canal treatment, related to the antimicrobial effect and the capacity of the irrigation solution to get rid or lessen bacteria in the root canal effectively.The basic principles for achieving the biological objectives of a root canal irrigation solution are that it must have "high efficacy against anaerobic and facultative microorganisms in their planktonic state and biofilms, inactive endotoxin, be nontoxic when they come in contact with vital tissues, and not cause anaphylactic reaction." 1 The regular used solution for root canal irrigation is NaOCl solution with 0.5% to 5.25% concentration and is the gold standard "because of its antibacterial capacity and the ability to dissolve necrotic tissue, vital pulp tissue and organic components of dentin and biofilms." in the root canal system. 1,2,3,4However, NaOCl irrigation solution still has disadvantages until now, namely it is cytotoxic to intra oral soft tissues and peri-radicular tissues, has an unpleasant taste and odor, does not have the ability to dissolve inorganic materials in the root canal, can cause corrosion of metal objects and can cause hypersensitivity reactions 1,3,4 Through root canal disinfection is still a challenge because of root canal anatomy complexity. 5he bacterium Enterococcus faecalis, which is known to cause 80-90% of cases of endodontic secondary infection (infection after endodontic treatment failure), is known to evade the action of endodontic instruments and irrigation solutions during the chemomechanical preparation stage of root canal treatment. 1,4Related to the obstacles that are still found in NaOCl irrigation solutions and in order to find alternative irrigation solutions that are effective against Enterococcus faecalis bacteria, research on alternative irrigation solutions sourced from herbal ingredients, one of which is from Stevia rebaudiana Bertoni leaf extract, is now starting to be developed. 6Shrub plant named Stevia rebaudiana Bertoni is originating from South American and has antibacterial activity. 7Research by Deviyanti S et al in 2022 has proven that all test solutions of 100%, 50%, 25%, 12.5%, 6.25%, 3.125% and 1.56% concentrations of extract Stevia rebaudiana Bertoni leaf have ability to form inhibition zone towards Enterococcus faecalis ATCC 29212 with the largest inhibition zone diameter of 23.07 mm at 100% Stevia leaf extract concentration and the smallest inhibition zone diameter of 10.34 mm at 1.56% Stevia leaf extract concentration. 6The inhibition activity to this bacteria growth seems to be related to the phytochemical content of flavonoids, steroids, tannins, alkaloids and saponins which are proven to be contained in this extract. 6This in vitro research was purposed to evaluate the effectiveness of irrigation solution from Stevia rebaudiana Bertoni leaf extract on the growth of Enterococcus faecalis bacteria by evaluating the MIC (Minimum inhibitory Concentration), MBC (Minimum Bactericide Concentration) and measuring the colony count of Enterococcus faecalis bacteria.

Plant extract
The dried Stevia leaves were minced using a blender and sieved with a 100 mesh sieve.A total of 150 g of Stevia leaf fine powder (simplisia) was put into 3 Duran bottles containing 50 g in each bottle and 1200 ml of 96% ethanol solvent was added which was divided into three Duran bottles so that each bottle contained 400 ml.The Duran bottles were then tightly closed and shaken until well mixed.and left for 3 days at 27°C.During the maceration process, each Duran bottle was jiggled in 15 minutes with interval 8 hours every day until the soluble ingredients could be dissolved. 10he sample was filtered through Whatman paper in a glass funnel and evaporated using a rotary evaporator at 72°C until a thick Stevia leaf extract was obtained, then stored in centrifuge tubes at -20°C for later use.

Bacterial strain culture
Reference strain of Enterococcus faecalis (ATCC 29212) was used.To culture E. faecalis strain ATCC 29212, 20 µl of bacteria were added to 5 ml of Nutrient broth medium (Sigma-Aldrich, St. Louis, USA) and incubated at 37°C for 24 hours.The optical density (OD) of the bacterial suspension was determined at 600 nm using a microplate reader (MP 96 SAFAS, Monaco), and adjusted to match the McFarland standard of 1.5x10 -8 (CFU/ml) Preparing irrigation solution with Stevia rebaudiana Bertoni leaf extract.Eppendorf tubes were used to prepare 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, and 1.56% Stevia leaf extract irrigation solutions using the serial dilution method.To prepare a 100% Stevia leaf extract solution in tube 1, 1 g Stevia leaf extract was dissolved in 1 ml of sterile distilled water.To make a 50% Stevia leaf extract solution, 500 µl of extract was transferred from tube 1 to tube 2 with 500 µl of sterile distilled water.Similar technique was repeated until a solution with 1.625% Stevia leaf extract was achieved.

Media preparation
Nutrient Agar (NA) media was prepared by weighing 2.6 g of Bacteriological Agar No.1 powder (Oxoid) plus 3 g of Nutrient broth No.3 powder (Sigma-Aldrich, St.Louis, USA) then dissolved with 200 ml of sterile distilled water in an erlenmeyer tube.Boiled and stirring using hot plate until dissolved homogeneous.The surface of the erlenmeyer tube was then tightly encased in aluminum foil and subjected to sterilization via autoclaving at 121°C for 15 minutes then stirred again and poured as much as 20 µl into each petri dish (petri disc).After media cool and solidify.thenincubated for 24 hours at 37°C to ensure the media was not contaminated before being used for the total plate count assay.

MIC (Minimal Inhibitory Concentration) and MBC (Minimal Bactericide Concentration) Determination
The MIC and MBC values determinated to see the antibacterial effectiveness of E. faecalis from Stevia rebaudiana Bertoni leaf extract irrigation solution in this in vitro research was carried out based on the broth microdilution method using 96-well microtitration plates followed by the total plate count method on Nutrient Agar medium in petri dishes. 11,12,13,16The broth microdilution procedure began by inserting 100 µl of each concentration of Stevia rebaudiana Bertoni leaf extract irrigation solution tested (100%, 50%, 25%, 12.5%, 6.25%, 3.125% and 1.56%) and 100 µl NaOCL 2.5% and 100 µl of distilled water into the wells of 96-well microtitration plate in the 1st row in columns 1, 2, 3, 4, 5, 6, 9 and 11 respectively.A total of 100 µl of E. faecalis bacterial suspension that has been equalized with Mc Farland standard 0.5 was then inoculated into each well of 96-well microtitration plate that has contained each concentration of Stevia leaf extract irrigation solution tested as well as into the control group solution, then incubated at 37°C for 24 hours.Blank solution (serial dilutions of Stevia leaf extract irrigation solution and control solution without E. faecalis) in the 5th row in columns 1, 2, 3, 4, 5, 6, 9 and 11.Three replications (triplo) were performed for all measurements.Optical Density (OD) measurement using a microplate reader at 600 nm, after 24 hours incubation for turbidity assessment.Dilution using sterile PBS (Phosphate Buffer Saline-Oxoid) up to 10 -9 was performed on each test concentration of Stevia rebaudiana Bertoni leaf extract irrigation solution as well as the control group on 96-well microtitration plate after incubation at 37℃ for 24 hours.A total of 9 Petri dishes containing Nutrient Agar media were prepared and confirmed to be free of contaminants after previous incubation for 24 hours.Each Petri dish was divided into 3 parts for one type of concentration of Stevia leaf extract irrigation solution tested as well as for each positive and negative control tested.The results of 10 -9 dilutions of each sample concentration of Stevia rebaudiana Bertoni leaf extract irrigation solution tested and the control solution were then vortexed until homogeneous and taken as much as 5 µl to be dripped (subculture) onto each part of the Nutrient Agar surface in a petri dish and then spreaded on the surface of the Nutrient Agar medium in a petri dish.All Petri dishes were then incubated for 24 hours in an anaerobic jar at 37℃ and the number of bacterial colonies that grew was counted.The formula for calculating the number of colonies (CFU or colony forming unit) of the sample: 13

Stevia rebaudiana Bertoni Leaves Maceration Extraction Results
The maceration of 150 grams of Stevia rebaudiana Bertoni leaf simplisia resulted in a total of 22.47 grams of thick extract, which was then filtered and evaporated for 1.5 hours using a rotary vacuum evaporator.Effectivity of Stevia Leaf Extract Irrigation solution against Colony Count of E.faecalis Bacteries The total plate count Stevia rebaudiana Bertoni method results of leaf extract irrigation solution concentrations of 100%, 50%, 25%, 12.5%, 6.25%, 3.125% and 1.56% and the control group against E. faecalis bacteria (Figure 1A to 1I).The mean colony count of E.faecalis bacteria (CFU/ml) in the Stevia rebaudiana Bertoni leaf extract irrigation solution group in various test concentrations as well as the control group can be seen in table 1.

5%) and K-(aquadest) groups
The results of the Shapiro-Wilk normality test (n<50) for 25%, 12.5%, 6.25%, 3.125% and 1.56% Stevia rebaudiana Bertoni leaf extract irrigation solution showed that the data were normally distributed (p>0.05),except for 100% and 50% Stevia rebaudiana Bertoni leaf extract irrigation solution, positive control and negative control (p<0.05).The results of the Kruskal-Wallis test of various concentrations of Stevia leaf extract and the control group on the mean colony count of E.faecalis bacteria, obtained a value of p=0.001 (p < 0.05), indicating that there is a difference in the mean colony count of E.faecalis bacteria at various concentrations of Stevia leaf extract irrigation solution and the control group so that it is continued with the Mann-Whitney post hoc test at the 95% confidence level.The results of the Mann-Whitney post hoc test showed that the 100% and 50% Stevia leaf extract irrigation solution groups did not show a significant difference in mean colony count of E. faecalis bacteria with the positive control group (p>0.05).The 25%, 12.5%, 6.25%, 3.125% and 1.56% Stevia leaf extract irrigation solution groups showed significant differences in mean colony count of E. faecalis bacteria with the positive control group (p<0.05).The 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, 1.56% Stevia leaf extract irrigation solution groups and the positive control group showed significant differences in mean colony count of E.faecalis bacteria with the negative control group (p<0.05).
The percentage value of E.faecalis growth inhibition can be seen in table 2. inhibition of E.faecalis bacteria at a concentration of 6.25% there is the first negative value which means that growth inhibition (death of bacteria) begins to occur.Sample irrigation solution of Stevia leaf extract with the smallest concentration that is able to kill 99.9% of E.faecalis bacteria on Nutrient Agar in petri dishes (from the results of total colony count assay) in this study, designated as MBC. 11,14,15he average number of colony growth of E.faecalis bacteria in the Stevia leaf extract irrigation solution group with a concentration of 6.25% as the MIC value in this study by turbidimetry is 1.17 x 10 8 CFU/ml and 1.56% as the MIC value by total plate count is 4.93 x 10 7 .Based on the results of the Mann-Whitney post hoc test analysis, the average number of colony growth of E.faecalis bacteria in the Stevia leaf extract irrigation solution group with 6.25% concentration have no significant difference with the Stevia leaf extract irrigation solution groups with 25%, 12.5%, 3.125% and 1.56% concentration.Similarly, the average number of colony growth of E.faecalis bacteria in the Stevia leaf extract irrigation solution group with 1.56% concentration have no significant difference with the Stevia leaf extract irrigation solution groups with 25%, 12.5%, 6.25% and 3.125% concentration, although there were differences in the average number of colony growth of E.faecalis bacteria in the five groups of Stevia leaf extract irrigation solution.(table 1 and figure 2).In contrast to the negative control group which showed the highest growth of E.faecalis bacterial colonies, namely 2.000 x 10 9 CFU/ml, the Stevia leaf extract irrigation solution groups with concentrations of 25%, 12.5%, 6.25%, 3.125% and 1.56% have shown bacteriostatic activity which causes a significant decrease in the number of E.faecalis bacterial colony growth (table 1 and figure 2).Stevia leaf extract irrigation solution with concentrations of 100% and 50% showed the best antibacterial effectiveness equivalent (p>0.05) to the positive control group (NaOCL 2.5%) because it has the ability to kill bacteria (bactericidal) so that no growth of E.faecalis bacterial colonies was found from the total plate count assay results.The total plate count assay was used in this study because it is the most sensitive way to calculate the number of viable live bacteria in the range of 30-300 CFU/ml. 16,17The choice of dilution factor in this study was carried out based on considerations in order to obtain calculation results according to the standard range of the number of viable bacterial colonies.Measurement of the number of bacterial colonies in each petri dish in this study was replicated 3 times (triplo) to see the accuracy of the calculation results. 18he difference of MIC value from those two methods are caused by the number of bacterial cells counted.Turbidimetry method will include every bacterial cell in medium, viable or not viable cell.In contras only viable cell of bacteria will count in the total plate count method. 16,17e ability of Stevia leaf ethanol extract in various test concentrations to inhibit or kill the growth of E. faecalis bacterial colonies in this study can occur due to the content of bioactive substances with antibacterial activity in the form of phytochemical flavonoids, tannins, saponins, steroids, terpenoids, and alkaloids. 6,19"Flavonoids cause damage to bacterial cell wall permeability and inhibit bacterial motility" 20 Tannins are known to cause bacterial cell wall damage through the mechanism of attacking bacterial cell wall polypeptides. 20Saponins have hydrophobic and hydrophilic molecules that can reduce cell surface tension which results in the destruction of bacterial cells. 20Steroids can cause leakage in liposomes resulting in a decrease in bacterial cell membrane integrity and changes in bacterial cell membranes that cause bacterial cells to become brittle and lysed. 20Terpenoid phytochemicals exhibit antibacterial action, according to Griffin SG et al cited by Mahizan MA et al 21 happens through inhibition of the oxygen uptake process that limits bacterial respiration and inhibition of the oxidative phosphorylation process in bacterial cellular respiration.Alkaloid phytochemicals exhibit antibacterial action via the destruction of bacterial cell membranes. 22he results of this Stevia leaf extract irrigation solution research, however, cannot be compared with the results of other studies because there is no recent research that discusses the MIC and MBC values of Stevia rebaudiana Bertoni leaf extract irrigation solution with 96% ethanol solvent against Enterococcus faecalis ATCC 29212.Recent research related to the effectiveness of Stevia against the growth of Enterococcus faecalis bacteria, has been conducted by Badra H et al in 2023 in vitro, but evaluated the effect of intracanal medicinal use of Stevia gel mixed with chitosan on observation for 24 hours, 48 hours, 72 hours and after one week through the diffusion test method.His study reported evidence that Stevia gel mixed with chitosan medicated was able to improve antimicrobial activity (in forming a zone of bacterial growth inhibition) against Enterococcus faecalis ATCC 29212 when compared to Stevia gel medicated and chitosan solution medicated in single dosage form. 23he bactericidal ability NaOCl 2.5% irrigation solution as a positive control against Enterococcus faecalis bacteria in this study, can occur through the mechanism of action of saponification reaction, neutralization reaction, hypochlorous acid formation, solvent action and high pH. 1 The content of hypochlorous acid (HOCl) in NaOCl irrigation solution which acts as an oxidizer, plays a role in activating bacteria because it causes degradation of amino acid and hydrolysis.1,3 Mode of action from NaOCl which is able to release chlorine, is also known to inhibit crucial bacterial enzymes through irreversible oxidation of sulfhydryl (SH) groups 1 Hydroxyl ion action from NaOCl solution as a strong base with high pH (pH>11), is also effective as an antimicrobial.

Effectivity of Irrigation Solution from Stevia rebaudiana Bertoni Leaf Extract on the Growth of Enterococcus faecalis Bacteries
The diagram of mean colony count of E. faecalis bacteria (CFU:Colony Form Unit/ml) in the treatment groups of Stevia rebaudiana Bertoni leaf extract irrigation solution in various concentrations tested as well as positive control group (NaOCL 2.5%) and negative control (aquadest) can be seen in figure2.

Table 2 . Percentage Value Of Growth Inhibition Of E.Faecalis Bacteries
K: concentration of Stevia leaf extract irrigation solution; % KB: percentage of growth inhibition of bacteriaDISCUSSIONThe utilization of irrigation solutions from biological resources of Stevia rebaudiana Bertoni leaf extract as an effort to find alternative materials for dental root canal irrigation solutions against Enterococcus faecalis bacteria in this study, has proven effective in inhibiting and killing the growth of Enterococcus faecalis strain ATCC 2921 bacteria with MIC (Minimal Inhibitory Concentration) value at a concentration of 6.25% by turbidimetry method using microplate reader and MIC value at a concentration of 1.56% by total plate count method using nutrient agar and MBC (Minimal Bactericide Concentration) value at a concentration of 50%.The determination of the MIC value is obtained from the smallest concentration of Stevia leaf extract irrigation solution samples that are able to show inhibition of the growth of E.faecalis bacterial colonies , which gives a negative value from the calculation of the percentage formula of inhibition of E.faecalis bacterial growth in turbidimetry methode and which exhibited the lowest total average of colony forming unit in total plate count methode.Results on Table2showed the percentage of growth Effectivity