Bacterial inhibition test of methanolic extracts of strawberry ( Fragraia x ananassa Duchesne ) , lime ( Citrus aurantifolia ) , and radish ( Raphanus sativus L . ) , towards Streptococcus sanguis ATCC 10556

Introduction: Caries are initiated by the plaque formation on the tooth surface, due to the interaction between food debris and bacteria in the mouth. The pioneer bacterium of plaque formation is Streptococcus sanguis. The strawberry fruit, lime fruit, and radish root are part of the plant that contains antibacterial substances. Flavonoid, tannin, saponin, alkaloid, polyphenol, terpenoid and quinon as antibacterial substances. This study was aimed to evaluate strawberry fruit, lime fruit, and radish root methanol extract can inhibit Streptococcus sanguis ATCC 10556. Therefore, strawberry, lime, and radish can be made into mouthwash, bubble gum, or toothpaste to prevent plaque formation. Methods: This study was using a Kirby-Bauer diffusion test as the inhibition test. Results: The result of this study that the biggest inhibition zone was lime methanol extract 20,000 ppm, and the smallest one was radish methanol extract 5,000 ppm. Conclusion: Strawberry fruit, lime fruit peel, and radish root methanol extract and their combination had the effect of inhibiting bacterial growth synergistically against Streptococcus sanguis ATCC 10556.


INTRODUCTION
The most encountered dental health problem is teeth caries.Nearly every individual in Indonesia has caries. 1 According to Indonesian Basic Health Research (Riskesdas) in 2013, caries prevalence in Indonesia was 46.5% and 72.1%.Caries is a progressive dental disease, developed from bacterial carbohydrate fermenting activity in plaque biofilm on teeth surface.Initially, caries is formed by plaque biofilm due to acquired pellicle and bacterial colony interaction on enamel surface until the bacteria co-aggregated. 2,3acultative anaerobic gram positive bacteria such as Actinomyces viscous and Streptococcus sanguis is the dominant pioneer bacteria adhere to pellicle and formed plaque biofilm. 4laque biofilm process can be interrupted by doing plaque control. 4Mechanically, it can be done with brushing and chemically with mouthwash containing antibacterial agent, either synthetic chemical or natural. 2,4Recently, people take interest in utilizing natural substance as there is no adverse effect of long term use as it is in synthetic chemical.Some of the natural substances we can utilize are strawberry, lime, and radish.
6][7] It has flavonoid active agent as antibacterial, denaturing bacterial protein. 8Besides, study reported that strawberry can inhibit bacterial growth with MIC at 12.5% concentration. 7][9][10][11] It has antibacterial properties due to essential oil content consisting of active agent : flavonoid, polyphenol, and saponin. 12hese compounds can denature bacterial proteins thereby inhibiting bacterial growth.Recent study showed that lime fruit peel can inhibit MRSA (Methicillin Resistant Staphylococcus aureus). 13adish (Raphanus sativus L) has plenty benefits, used to deal insomnia, pharyngitis, sinusitis, heart cirrhosis, as a diuretic, antibacterial, and toxin detoxification. 7Radish roots and leaves contain active agents such as flavonoid, polyphenol, and saponin.Recent study conducted by Takaya et  al. in 2009 revealed that radish has antibacterial substances which can inhibit E. coli growth. 14ntibacterial agents in strawberry, lime, and radish are expected to be utilized as a teeth plaque formation inhibitor.It can be made into different forms: mouthwash, gum, or dentifrice.This study is conducted to evaluate how strawberry fruit, lime fruit and radish root extract and their combination in inhibiting Streptococcus sanguis ATCC 10556.
Inhibition test using combination of three extract purpose was to know whether the combination interacted and affected antibacterial effect.Interaction result can be synergist or antagonist, both is pharmacodynamic interaction.Synergist effect is the effect which cause one substance enhances the other, while antagonist is the effect which cause one substance nullifies the other. 15

METHODS
Method used in this study was Kirby Bauer with two repetitions and bacterial number was adjusted according to McFarland standard 0.5.Extracts concentration tested made adjusted to 1,000 ppm, 5,000 ppm, 10,000 ppm, and 20,000 ppm.
Extracts were made with maceration method using 1 kg of strawberry fruit, lime fruit peel, and radish.It was done for 72 hours at room temperature.Extract was stored in sterile bottle at freezer temperature.Then, it was dissolved in DMSO (dimethyl sulphoxide) to achieve desired concentration.
Subcultured and standardized bateria in Muller Hinton Broth medium was moved by swabbing it to Muller Hinton Agar (MHA) medium.Each medium filled with the extract and DMSO as a negative control.Medium was incubated for 24-48 hours at 37°C in anaerobic facultative environment.Inhibition zone was measured using caliper.

RESULTS
Initial test of the antibacterial activity of 100% exract of strawberry fruit, lime fruit peel, and radish root, showed that inhibition was marked with clear area as inhibition zone around medium.Inhibition zone diameter measurement at 20,000 ppm, 10,000 ppm, 5,000 ppm, and 1,000 ppm concentration in strawberry extract as well as lime fruit peel extract and radish root extract showed the smaller the concentration, the smaller the inhibition zone formed (Table 2).Combination of strawberry fruit, lime fruit peel, and radish root extract showed a synergist and antagonist effect, which was marked with an increase and decrease of inhibition zone diameter compared with the previous test (Table 2).

DISCUSSION
Phytochemical screening showed that strawberry has alkaloid, polyphenol, tanin, and quinon. 16Lime contains alkaloid, phenol, flavonoid, saponin, and terpenoid. 12Radish has polyphenol, flavonoid, and saponin. 17lkaloid in strawberry fruit and lime fruit peel can inhibit bacterial growth by inhibit peptidoglican formation hence, cell wall was not intact and leads to cell death.Nitrogen in alkaloid will react with amino acid which forms cell wall and bacteria DNA, thus DNA balance is impaired and leads to bacterial lysis. 8lavonoid and phenol in strawberry, lime, and radish, as well as terpenoid in lime denaturate bacterial protein and form hydrogen bond with protein damaging the protein structure.It leads to decreasing of cell wall and cytoplasm membrane permeability, furthermore creates macromolecule and micromolecule imbalance and cell lysis.Phenol, flavonoid, and terpenoid are either bactericidal or bacteriostatic depends on concentration given.Flavonoid also forms a nucleate acid-base bond which caused bacteria inhibition of DNA/RNA synthesis and metabolism. 18,19ponin in lime and radish can lower cytoplasm membrane surface tension, making the nutrient and other substances cannot through inside the cell.Thus, bacteria cell metabolism is inhibited and ATP is decreased.It will make bacteria growth and development is disturbed.Last, tanin and quinon in strawberry will inhibit bacterial growth in similar way.It will inactivate bacterial adhesin, interrupt bacterial enzyme function and protein transport in cell layer. 20ecent study revealed that both extract has antibacterial effect, showed by clear zone around medium.The biggest inhibition zone diameter average is resulted by lime methanol extract (Table 1).Inhibition zone diameter size formed by lime methanol extract because of more light texture compared with the others.Besides, antibacterial compound in lime methanol extract is various and it can work optimally to inhibit bacterial growth.Therefore, the inhibition zone is bigger.Inhibition zone diameter measurement result showed that the smaller the concentration given, the smaller the inhibition diameter zone formed (Table 2).It is in agreement with existing theory that concentration change in a drug will caused change in response or drug effect. 15,21xtract with small concentration has a small amount of antibacterial substance; hence it cannot inhibit bacterial growth optimally.
Lime fruit peel and strawberry fruit extract formed relatively big inhibition zone and radish root extract formed a relatively small inhibition zone (Table 2).For example in 20000 ppm concentration, inhibition zone formed by radish root was 22.7 mm, strawberry fruit 27.465 mm, and lime fruit peel 28.8 mm.It can be caused of texture difference, radish had the most viscous texture and lime had the most light texture.Extract texture affected diffusion into the agar medium, thus the bacteria inhibition distance was restricted.It is in agreement with existing theory that inhibition zone diameter size is affected by agent ability to diffuse, the worse the agent solubility, the smaller the inhibition zone formed compared with more soluble agent. 22ll three extracts at 1000 ppm concentration showed no inhibition zone formed however, combination of strawberry fruit and lime fruit peel extract and lime and radish methanol extract at 1000 ppm concentration showed inhibition zone formed.It implies that particular substances in the extract alone at 1000 ppm concentration not show antibacterial effect except combined.Inhibition ability at 1000 ppm concentration was rise with relation of lime fruit peel extract combination.It means that particular substance in lime is the most dominant.
Other than that, antibacterial effects become more vary if combined, mainly radish root methanol extract which have only 3 antibacterial agents alone.However, in this study combination of the three extract at 1000 ppm concentration couldn't inhibit bacteria growth due to antibacterial effect at 1000 ppm concentration of each extract may be was too small to work optimally.
Size of inhibition zone formed from extract combination can be affected by solution diffuse factor to MHA medium.Viscous extract in certain concentration combined with light extract at the same concentration will improve viscous extract diffuse ability to MHA medium yet decrease light extract ability to diffuse to MHA medium. 23

CONCLUSION
Strawberry fruit, lime fruit peel, and radish root methanol extract and their combination had the effect of inhibiting bacterial growth synergistically against Streptococcus sanguis ATCC 10556.

Table 1 .
Inhibition zone measurement of strawberry fruit, lime fruit peel, and radish roots methanol extract against Streptococcus sanguis ATCC 10556 at 100% concentration

Table 2 .
Inhibition zone measurement of strawberry fruit, lime fruit peel, and radish roots methanol extract against Streptococcus sanguis ATCC 10556 at 100% concentration

Table 3 .
The effect of strawberry methanol, lime methanol, and radish methanol extract combination on inhibition diameter zone average against Streptococcus sanguis ATCC 10556