Inhibition effect of cashew stem bark extract ( Anacardium Occidentale L . ) on biofilm formation of Streptococcus sanguinis

Background: Biofilm is communities of microorganisms attached to solid surface and enclosed in extracellular matrix that protected microorganisms from antibacterial agents and host defense. One of bacteria might have a role in initial colonization of biofilm formation is Streptococcus sanguinis (S. sanguinis). Previous studies showed that cashew stem bark extract (Anacardium occidentale L.) can inhibit the growth of Streptococcus strains. Purpose: The purpose of this study was to determine the inhibition effect of cashew (Anacardium occidentale L.) stem bark ethanol extract on biofilm formation of S. sanguinis. Methods: Streptococcus sanguinis grown in Brain Heart Infusion (BHI) + 2% sucrose medium by using microplate polystyrene 96 wells. The samples were divided into 3 groups, 5% polyethyleneglycol (PEG) as negative control, cashew stem bark extract (concentration 3.125 mg/ml, 6.25 mg/ml, 9.375 mg/ml, and 12.5 mg/ml), and 0.12% chlorhexidine (as positive control). Biofilm was stained by 1% crystal violet. Afterwards, optical density (OD) of samples were measured by microplate reader λ 595 nm. The data of biofilm formation inhibition percentage were analyzed by one way ANOVA and then continued by Least Significant Difference (LSD) test. results: The result of one way ANOVA showed that there were significant differences in inhibition of S. sanguinis biofilm formation (p<0.05). LSD test showed that concentration extract 3.125 mg/ml had significant difference with concentration 9.375 mg/ml and 12.5 mg/ml. Reciprocally, concentration 6.25 mg/ml had significant difference with concentration 9.375 mg/ml and 12.5 mg/ml. Conclusion: Cashew stem bark extract was able to inhibit biofilm formation of S. sanguinis.


Kata kunci: Daya hambat pembentukan biofilm, Streptococcus sanguinis, kulit batang jambu mete
Correspondence: Rizni Amaliah, c/o: Bagian Biomedik, Fakultas Kedokteran Gigi Universitas Gadjah Mada.Jl.Denta, Sekip Utara, �ogyakarta 55281, Indonesia.Email address: rizni.amaliah@gmail.com2] Since 1996, dental plaque is not only recognized as the etiologic factor of periodontal disease, but also considered as a biofilm. 3Biofilms are a community of bacteria that have extracellular matrix, circulation, and communication system.3] The formation of biofilm begins with the attachment of bacteria such as Neisseria and Streptococcus, at over tooth surface which dominated by mitis groups such as S. sanguinis. 4The bacteria in biofilm will grow and become maturate, then it form microcolonies. 2,5 An extracellular polymeric matrix is a thick layer surrounded the cells that form biofilm. 4 This layer is a biofilm barrier against antibiotics, antimicrobials, and immunity cells.The main component is exopolysaccharides (EPS). 67] This bacteria plays an important role in initial colonization of biofilm formation. 6S. sanguinis have some adhesin that bind to tooth surface that was layered by saliva.Beside that, IgA1 protease that is produced by colony of S. sanguinis, enable this bacteria grow and proliferate over tooth surface. 4he cashew excessively have been widely used such as from its wood, bark, leaf, fruit, and seed. 8Cashew was reported might have antidiabetic, antibacterial, and antiinflammation. 9Cashew's leaf could have inhibited bacterial growth such as Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis, Salmonella typhy, Escherichia coli, and Candida albicans. 10The stem bark was also used for medication.From previously report, the stem bark could inhibit Staphylococcus aureus growth. 11he aim of the research was to determine the effect of cashew stem bark ethanol extract (Anacardium occidentale L.) on inhibition biofilm formation of S. sanguinis.

materials and methods
Cashew stem bark (Anacardium occidentale L.) was taken from cashew trees at Kasiutri Plantation, Imogiri.Plant identification and extraction processed at Unit II laboratory of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada.One kilogram of cashew stem bark was dried using a drying cabinet at 40-50 0 C for 48 hours.After drying, the bark was made into powder using machine pollinators.The 300g of powdered cashew stem bark was then extracted using 3L of 70% ethanol by maceration method then stirred for 30 minutes and allowed to stand for 24 hours.Powdered bark that was extracted then filtered using a Buchner funnel.Ethanol in filtrate was evaporated using stove for 3-4 hours until the stiff extract obtained.
Bacteria were prepared in McFarland V suspension (15 x 10 8 cfu/ml) using Densicheck.Plate was divided into two, one test plate and one blank plate.The test plate was containing extract solution/PEG and BHI + 2% sucrose with the addition of 10% v/v suspension of bacteria, while the blank plate containing extract solution/PEG and BHI + 2% sucrose with the addition of 10% v/v saline.According to Pereira et al., 14 cashew stem bark extract was starting inhibit S. sanguinis growth in concentration 3.12% (mg/ml), the antimicrobial activity was carried out on solid media plates by a diffusion method for the screening and determination of minimum inhibitory concentration (MIC) of the extract on bacterial.Whereas in this study, biofilm inhibition assay did with microdilution method.Extract concentrations was 3.125 mg/ml, 6.25 mg/ml, 9.375 mg/ml, and 12.5 mg/ml and BHI + 2% sucrose added to the polystyrene U bottom microplate with a total volume of each well 90 μL.
Chlorhexidine is the most effective antibacterial agent for oral use. 4 As a positive control using 0.12% chlorhexidine (v/v) each well while the negative control using 5% PEG.Afterward, the microplate was incubated for 24 hours at 37 0 C.After the incubation, the microplate was washed with water three times.Then a solution of 1% crystal violet in 125 μL each well was added, and incubated at room temperature for 15 minutes, and then microplate was washed with water three times.Two hundred μL 96% ethanol added to each well using a 50-200 μL micropippet and incubated at room temperature for 15 minutes.Furthermore, as each 150 μL solution was transferred into a microplate flat bottom polystyrene 96 wells.OD readings  The data of biofilm inhibition percentage assay were tested for normality and homogenity.To determine the mean difference between groups statistically analyzed using one way ANOVA test and then proceed using LSD test.

results
The inhibition of biofilm formation was done by calculating the percentage of inhibition of S. sanguinis biofilm formation after adding cashew stem bark extract.Biofilms will absorb color crystal violet.The percentage of inhibition of biofilm formation values obtained with the four values substituted into the equation OD percentage inhibition.The results of the calculation of the percentage of inhibition of biofilm formation can be seen in Table 1.
Table 1 showed that 5% PEG did not inhibit biofilm formation because it had a negative mean.As the increasing concentrations of cashew stem bark extract, the inhibition of biofilm formation also increased.Cashew stem bark extract concentration 12.5 mg/ml was the highest average inhibition biofilm formation if compared with concentration 3.125 mg/ml, 6.25 mg/ml, 9.375 mg/ml and 0.12% chlorhexidine.To make clearing up the results in Table 1, the mean of each group would presented in Figure 1.

0.12% Chlorhexidine
Based on one way ANOVA result, the treatment between groups had significant difference (p<0.05).Furthermore, to find a group that has a significant difference, the LSD test was using with a level of 95%.Least Significant Difference (LSD) analysis results can be seen in Table 2.
Based on LSD test, the inhibition of formation 5% PEG groups had a significant differences with the cashew stem bark extract concentration of 3.125 mg/ml, 6.25 mg/ml, 9.375 mg/ml, and 12.5 mg/ml.This was due to cashew stem bark extract had substances that act as an antibacterial, while PEG did not have antibacterial substances.The results were consistent with studied of Pereira et al., 14 who showed that cashew stem bark extract had antibacterial power against Streptococcus mutans, S. sanguinis, and Streptococcus mitis.discussion Biofilm is a bacterial community that attach to surface that covered by extracellular matrix.This matrix has a main component, that is exopolysaccharides (EPS).Exopolysaccharides is formed by bacterial that produce GTF such as S. sanguinis. 6,15This study used BHI with 2% sucrose as growth medium.Addition 2% sucrose in BHI because of sucrose is substrate that will breakdown by S. sanguinis to form EPS. 6 In this study, biofilm of S. sanguinis attached to wall of microplate polystyrene wells.According to Ge et al., 16 this surface is one of abiotic surface that biofilm can attach.Biofilm that have attached, was stained with crystal violet.Assessment of crystal violet-biofilm bonding quantitatively was using an ELISA reader (microplate reader). 17he result showed that cashew (Anacardium occidentale L.) stem bark extract capable of inhibiting biofilm formation of S. sanguinis.Anacardium occidentale L. stem bark extract had antibacterial activity against S. sanguinis. 14nacardium occidentale L. had some antibacterial agent such as tannin and flavonoid that can disturb metabolism of S. sanguinis.Beside that, these bacterial agent can also to deactivated enzyme. 18,19he high concentration of Anacardium occidentale L. extract more inhibit biofilm formation than others.This is due to the high concentration has more antibacterial agent, so that make an effect to number of percentage of inhibiting biofilm formation.Increase in the concentration of cashew stem bark extract will increase the antibacterial of S. sanguinis. 14n this study, chlorhexidine had antibacterial power greater than the concentration of 3.125 mg/ml, 6.25 mg/ml, and 9.375 mg/ml and lower than the cashew stem bark extract concentration 12.5mg/ml.Chlorhexidine against some bacterials and fungi because of this agent increased cell membrane permeability then followed by cytoplasm macromolecule coagulating.Chlorhexidine against some bacterials and fungi because of this agent increased cell membrane permeability then followed by cytoplasm macromolecule coagulating. 20However, the results of our study showed that chlorhexidine 0.12% inhibition of biofilm formation was lower than the cashew stem bark extract concentration of 12.5 mg/ml.This might be due to the concentration of 12.5 mg/ml was too thick so it settles on the surface of the base well and stained with crystal violet.This caused the difference between the OD test well (12.5 mg/ml + BHI + sucrose 2% + S. sanguinis) to the OD blank sample well (extract 12.5 mg/ml + BHI + sucrose 2% + saline) very small compared with the difference in OD control vehicle (2.5% PEG).Therefore, the inhibition of biofilm formation was intense.In addition, the antibacterial microdilution test required agent in small volume. 21Cashew stem bark extract concentration 12.5 mg/ml with a volume 2.5 mL per well seem too many that are less appropriate to be used in the microdilution test.
Broadly, cashew bark extract can inhibit the formation of biofilms that was formed by S. sanguinis.This might be caused by the phenolic compounds such as tannins and flavonoids contained in cashew stem bark extract.Phenolic compounds can provide an antibacterial effect by disrupting the cell wall and membrane, precipitate proteins, and deactivate the enzyme. 22One of enzyme activity might be inhibited by phenolic compounds was GTF enzymes.Some phenolic compounds could inhibit enzyme activity GTF. 23f the GTF enzyme activity was interrupted, as a matrix of EPS biofilm formation also declined.Thus, the formation of biofilm S. sanguinis can be inhibited.In conclusion, cashew stem bark extract (Anacardium occidentale L.) can inhibit Streptococcus sanguinis biofilm formation.