The effect of ethanol extract of soursop leaf ( Annona muricata L . ) on adhesion of Streptococcus mutans ATCC 35668 to hydroxyapatite discs

The demineralization of dental hard tissues can be caused by dental plaque. Dental plaque contains various components, especially bacteria attached to the extracellular matrix. Streptococcus mutans (S. mutans) has extracellular glucan as adhesin that is important in the attachment mechanism of tooth surface. The natural substance can be used for preventing plaque formation by inhibiting the attachment of S. mutans. Soursop plant has been used in treating various diseases. The leaves of the soursop (Annona muricata L.) are used as a material to inhibit potential attachment of bacteria S. mutans. Common surfaces that is used in adhesion testing is hydroxyapatite (HA). The aim of this study was to evaluate the effect of ethanol extract of soursop leaf (EESL) on the adhesion of S. mutans ATCC 35668 to HA discs. Soursop leaves were extracted by the maceration method using 70% ethanol. The experiment was carried out by analyzing the inhibition adhesion of S. mutans ATCC 35668 on HA discs after incubation with different concentrations of soursop leaf extract. The concentrations of extract tested were: 150; 125; 100; 75; and 50 mg/ml. Chlorhexidine 0.2% was used as a positive control while DMSO 5% was used a negative one. Data were evaluated by One Way Anova. This study statistically showed significant differences of S. mutans colony count between groups (p<0.05).The results of a post hoc Dunnett T3 test showed that the 2 highest concentrations of extract (125 and 150 mg/ml) reduced S. mutans adhesion on HA discs.The obtained results showed that ethanol extract of soursop leave inhibits the adherence of S. mutans to the HA disc.


INTRODUCTION
Dental caries is a disease that destroys the tooth structure which is indicated with demineralization of dental hard tissue.This is view of the accumulation of plaque on the tooth surface. 1 Dental plaque is a bacterial deposit, and its product attaches to the tooth surface. 2 Streptococcus mutans (S. mutans) plays an important role in the formation of dental caries. 3It has surface protein antigen peptide (SpaP), Ag I/II family proteins, that will bind to receptors on the pellicle on the tooth surface.The adhesion of S. mutans to the tooth surface is also influenced by the enzyme glucosyltransferase (GTF) which is capable to convert sucrose into glucan.Glucan plays an important role in facilitating bacteria adherence on the tooth surface which is lead to the increasing accumulation of plaque and initiate caries on the tooth surface. 4e prevention of S. mutans attachment is an effort in reducing the incidence of caries.One approach to reducing the incidence of caries is to develop the therapeutic agents with antimicrobial and antiadherent properties to prevent the bacterial adhesion on the tooth surface. 5One of the medicinal plants used in several regions in Indonesia is soursop (Annona muricata L.) 6 in which its leaves are the part of the soursop plants which is widely used. 7n our previous studies, it showed that the ethanol extract of soursop leaf (EESL) contain secondary metabolites including saponins, terpenoids, steroids, flavonoids, tannins and alkaloids.The EESL inhibit the growth of S. mutans ATCC 35668 in a dosedependent manner. 8The inhibition of the adherence of S.mutans to the surface of the tooth is necessary for the prevention of plaque formation.Therefore, in this study, we investigated the inhibitory effect the EESL on the adhesion of S. mutans to HA discs which have been used as one of the experimental model systems.HA discs are the bacterial adhesion objects to simulated tooth enamel structure.Tooth enamel is composed of 99% inorganic compounds of HA. 9

MATERIALS AND METHODS
This was a laboratory study.This study has been approved by The Research Ethics Team of Faculty of Dentistry Universitas Gadjah Mada with the number: 00247/KKEP/FKG-UGM/EC/2015.
Soursop leaves were obtained from Balai Besar Penelitian dan Pengembangan Tanaman Obat dan Obat Tradisional (B 2 P 2 TOOT) Tawangmangu, Ministry of Health, Indonesia.The leaves have been identified in B 2 P 2 TOOT and extracted by the maceration method in Laboratorium Penelitian dan Pengujian Terpadu (LPPT) UGM.A reference stock of S. mutans (ATCC® 35668) was used as a standardized suspension.Mc Farland standard solution (1.5x10 8 CFU/ml) was used as the bacterial suspension turbidity standard.
HA discs were prepared as described by Guan. 10 HA discs (ϕ 10 mm X 2 mm) were made by pressing HA particles by Universal Testing Machine with 4 kgf pressure (Pharmacy Technology Laboratory of Faculty of Pharmacy UGM).HA discs were then sintered (120 min, 1300°C) through the heating of 5 °C/minute in air atmosphere to obtain a solid HA discs (Ceramics and Composite Technology Laboratory of Chemical Engineering Department of Faculty of Engineering UGM).HA discs were sterilized by autoclave (121 °C, 20 min).
Whole saliva was collected from a healthy volunteer (dental conditions free from cavities and good oral hygiene) based on methods described previously. 11Fresh saliva samples were collected without stimulation and obtained in the morning without stimulation.The samples were dispersed using vortex mixer for 60 min and centrifuged at 20,000 rcf for 60 min at 4 °C.The supernatant was filtered through two low-protein-binding filters (pore sizes of 0.45 μm and 0.22 μm).
The assay of S. mutans adhesion on HA discs.was done according to Lee,et.al. 12 S. mutans was cultured on BHI for 24 h at 37°C.A total of 14 discs HA were coated with saliva for 1 h at the room temperature.The saliva-coated HA discs (S-HA disc) were washed 3 times with potassium phosphate buffer (KPB) pH 7.0 and 2 HA discs were added to every tube.Everytube was filled with 0.5 ml EESL at different concentration (150; 125; 100; 75; 50 mg/ ml) and 0.5 ml of a suspension of bacteria S. mutans ATCC 35668 (1.5x10 7 CFU/ml).Positive control tube contained 0.5 ml Chlorhexidine 0.2% and 0.5 ml of a suspension of bacteria S. mutans (1.5x10 7 CFU/ml).Negative control tube contained 0.5 ml 5% DMSO and 0.5 ml of a suspension of bacteria S. mutans (1.5x10 7 CFU/ml).Based on a method as described by Anggraeni 13 tubes were incubated for 24 hours at 37°C.HA discs were washed with KPB and transferred into a tube containing KPB (pH 7.0).S. mutans adsorbed onto the HA discs was dispersed using vortex for 60 seconds, diluted and spread on trypticase soy-sucrose-bacitracin agars (TYS20B) plates.After incubation for 24 h at 37°C number of bacterial colonies were counted on each TYS20B plate.Five replicates were made for each concentration of test extracts, and the number of Colony Forming Unit (CFU) was calculated.

RESULTS
The bacterial adherence assay was performed to determine whether EESL inhibits S. mutans adherence to HA discs.As shown in Figure 1, the adhesion of S mutans to HA disc decreased following the increasing concentrations of EESL.ESSL inhibited the adherence of S. mutans completely at concentration 150 mg/ml.One Way Anova test showed a significant value of 0.000 (p <0.05) showing asignificant difference between groups.This showed that EESL has a significant effect on a number of colonies of S. mutans attaching on HA discs.The post hoc Dunnet T3 test was conducted to determine the differences among groups Table 1.Several concentrations of the extract were tested regarding their effect on adhesion of S. mutans on HA discs (Table 1).The results for 2 highest concentrations of extract that reduced S.mutans adhesion on HA discs.The results of a post hoc Dunnett T3 test showed that the group treated with 125 mg/ml significantly decreased in colony counts compared with the negative control and lower concentrations.The group treated with 150 mg/ml EESL showed no difference from positive control indicating that the concentration was effective in reducing the bacterial adherence to HA discs.

DISCUSSION
In the present study, the adhesion of S. mutans was evaluated using saliva-coated HA discs (S-HA disc).HA is common materials to analyze bacterial adherence. 14Saliva was used in this study for containing a glycoprotein that plays an important role in the bacterial adhesion in plaque formation. 15BHI was used as growth medium without any sucrose to allow the adhesion of S. mutans in the form of non-specific attachment followed by the specific attachment of interaction independent on sucrose through adhesion of S. mutans binding to salivary glycoprotein attaching to HA discs. 16ne-way analysis of variance (ANOVA), test on the effect of various concentrations of EESL on the adherence of S. mutans on the HA discs showed that EESL could significantly decrease the number of colonies.The group treated with 125 mg/ml showed a significant decrease in colony counts compared with the negative control and lower concentrations.The group treated with 150 mg/ml EESL showed no difference from positive control indicating that the concentration was effective in reducing bacterial adherence to HA discs.This might be the active compound in EESL reducing the bacterial colonies adhering to the HA discs.
The early phases of adhesion of S. mutans bacteria on the surface of the HA discs occur through non-specific attachment.One of the non-specific attachments is the hydrophobicity interaction between the cell and the adherence surface. The ability of the extract to prevent the adherence of S. mutans could be related to the effect of saponins and flavonoids components.Saponins are known to have a hydrophobic and hydrophilic action.Hydrophobicity of saponin allows binding to hydrophobic end of the bacterial cell membrane while the hydrophilic end is a free end and will bring a complex detergentprotein resulting in bacterial lysis. 19Flavonoids through their antibacterial action is to form a complex with proteins through nonspecific forces such as hydrogen bonding and hydrophobic effects, and covalent bond formation. 20In the next phase, adhesin-specific attachment of S. mutans that bind to glycoproteins of saliva adhering to HA discs.This specific attachment is thought to be inhibited by EESL through tannins and flavonoids.According to Agnol 21 tannins works by binding bacterial adhesin protein that binds to receptors on the surface of the host, Bennick 22 stated that the weakening bond with the bacterial cell surface protein on pellicle (host) would result in decreased bacterial adhesions.According to Kumar and Pandey 20 flavonoids can inactivate the bacterial adhesion and a disturbance in the bacterial adhesion will lead to a decrease in bacterial adhesion.In conclusion, this study showed the anti-adhesive properties of EESL inhibit adherence of S. mutans to the HA disc.

Figure 1 .
Figure 1.Effect of EESL on S. mutans adherence to salivacoated HA discs.The CFU of S. mutans to S-HA discs by various concentrations of EESL was determined.

Table 1 .
Post hoc analysis of S. mutans adherence to salivacoated HA discs among groups