Antibacterial effect of 70 % ethanol and water extract of cacao beans ( Theobroma cacao L . ) on Aggregatibacter actinomycetemcomitans

Background: Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is a Gram negative bacteria that form a subgingival plaque causing periodontitis. Nowadays, many natural resources can be used as a basic ingredient of drugs. One of the resources used as an antibacterial material is cacao bean. It contains of polyphenol flavonoids, such as catechin, epicatechin, anthocyanin, and proanthocyanidin. Chemical compounds contained in ethanol extract and water extract are different in quantity from those in cocoa beans. Purpose: This research aimed to find out difference in antibacterial activity between the 70% ethanol and water extract of cacao beans (Theobroma cacao L.) on A. actinomycetemcomitans. Method: This research was an in vitro laboratory experiment. The serial dilutions was performed on the 70% ethanol and water extract of cacao beans a concentration of 100% to 3.125%. At each concentration, the 70% ethanol and water extract of cacao beans were added with grown bacterial suspension of A. actinomycetemcomitans. After they were incubated for 24 hours, the bacteria grown on Luria Berthani media were observed. Bacteria colonies then were measured in CFU/ml. Result: There were significant differences in bacterial colonies grown at the concentrations of 6.25% and 3.125% between the 70% ethanol extract of cacao beans and the water extract of cacao beans as p-value = 0.000 (p<0.05). Conclusion: 70% Ethanol beans and water extract of cacao beans have antibacterial activity against A. Actinomycetemcomitans. The concentrations of MIC and MBC extracts were 6.25% and 12.5% respectively.

damage. A. actinomycetemcomitans are also frequently detected in periodontal tissues of healthy people and then can be grouped into a part of the normal flora in oral cavity. 5urrently, some herbal ingredients have been developed as an alternative to medicine ingredients, one of which is cocoa beans.Indonesia ranks on the third position in the world cocoa producers.Cocoa production in Indonesia has the potential to be improved, but it depends on political and local economic factors. 6The size of cocoa growing areas in Indonesia in 2002 has reached 776, 900 hectares scattered in all provinces, except in the capital city of Jakarta (DKI). 7ocoa is composed of stems, fruits and seeds, flowers, roots, bark seeds, and leaves.Cocoa (Theobroma cacao L.) has polyphenolic compounds, such as catechin, epicatechin, anthocyanin, proanthocyanidin, phenolic acid, condensed tannins, flavonoids, and other small compounds. 8Cocoa polyphenols are useful as an antioxidant, anticarcinogenic, anti-inflammatory, and antimicrobial. 9Generally, the majority of people consume cocoa only on the seeds alone.Several researches also have shown that cocoa bean skin and cocoa beans can be used as antibacterial ingredients. 7ocoa beans contain polyphenolic flavonoid of flavonol class, namely monomeric catechin and epicatechin flavonoids as well as proanthocyanidin polymerized flavonoids. 10Flavonols are classified into a flavonoid group composed of several molecules of phenols (polyphenols). 11lavonols contained in cocoa stimulate peripheral blood mononuclear cells to secrete interleukin-5 (IL-5 ) and stimulate immunoglobulin A (IgA) production that protects the oral cavity from Streptococcus mutans. 8ocoa beans can also inhibit the growth of Streptococcus mutans at an effective concentration of 12.5%. 12The extract water of cocoa beans and the 70% ethanol extract of cocoa beans even have antibacterial activity against Escherichia coli and Bacillus subtilis.A research on cocoa beans extracted with the ethanol 70% shows a higher antimicrobial activity against Streptococcus mutans and C. albicans bacteria than with water. 13ocoa, furthermore, will generate a different antibacterial activity when extracted with different polarities of its solvents. 14Extraction method varies depending on the samples to be tested.Several different solvents used include water, 70% ethanol, 70% methanol, acetonitrile, diethyl ether, and acetone.70% ethanol (ethyl alcohol) is a solvent which has a low boiling point and is widely used by industries.70% ethanol has a boiling point of 70° C so that extraction temperature is used to attract all the components in the basic materials. 1570% ethanol is widely used as a solvent of various chemical or natural materials devoted to drugs.In in vitro research, 70% ethanol can disrupt transport ions, namely Na +, K +, and ATP.The use of 70% ethanol in antibacterial material has a risk to human health, especially in liver because of the residual concentration and ethanol exposure. 16lyphenol level, furthermore, will be higher when extracted with a lower solvent polarity.Solvent water will tend to dissolve inorganic compounds and salts of acids or bases. 14The use of water compared to the 70% ethanol as cosolvent can generate higher residual polyphenols, so the solvent water is considered to be better because it can maintain a high quantity of beneficial compounds. 17his study aimed to determine whether there were differences in antibacterial activity between the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A. actinomycetemcomitan bacteria.

materials and method
This study was an in vitro laboratory experimental research using randomized post test only control group design.This study was conducted at the Laboratory of Microbiology, Faculty of Dental Medicine, Universitas Airlangga from June to October 2015.Samples were A. actinomycetemcomitans bacteria taken from the microbiology laboratory, Faculty of Dental Medicine, Universitas Airlangga, and then cultured in Brain Heart Infusion Broth media (BHIB).Minimal sample size eligible to be analyzed is determined by Lemeshow formula, as many as five.
Manufacture of 70% ethanol extract of cocoa beans and water extract of cocoa beans was conducted.The 70% ethanol extract of cocoa beans and the water extract of cocoa beans were obtained from Integrated Service Unit (UPT) Materia Medika Batu, East Java.Cocoa beans used were non-fermented cocoa beans and macerated using 70% ethanol and water solvent.Having obtained the 70% ethanol extract of cocoa beans and the water extract of cocoa beans, the preparation of A. actinomycetemcomitans bacteria then was performed by suspending the BHIB media until turbidity standard was equivalent to 0.5 Mc Farland (1.5 × 108 CFU/ ml).Manufacture of the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at various concentrations of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125% was performed using serial dilution method.
The determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A. actinomycetemcomitan bacteria was started from the preparation of the test tube as much as 9 tube.Six test tubes were filled with bacterial suspensions of A. actinomycetemcomitans as much as 0.05 ml that had been standardized to 0.5 Mc Farland and mixed with BHIB media as well as the extracts at the various concentrations (100%, 50%, 25%, 12.5%, 6.25 %, and 3.125).The test tube K + (as positive control) containing 0.05 ml bacterial suspension of A. actinomycetemcomitans were planted in the BHIB media, while the test tube K-(as negative control) contained only media BHIB and control extracts.Control is useful to make sure that no bacterial contamination in the media.
The reading of the results of antibacterial activity in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against the growth of A. actinomycetemcomitans bacteria was conducted by measuring the number of colonies grown on bacteria subculture as much as 0.1 ml of each tube as well as a positive control and a negative control on the Luria Berthani media.Planting on the Luria Berthani media was conducted using spreader, and incubated at 37 ° C for 24 hours.
The results showed 90% of A. actinomycetemcomitans bacterial growth was inhibited compared with the positive control used as MIC.The results also indicated 99.9% of A. actinomycetemcomitans bacterial death compared with the positive control was used as MBC.MIC and MBC were determined by measuring the number of colonies grown on the Luria Berthani media manually and expressed as CFU/ ml.The calculation was repeated five times by three different observers, and then the mean values were measured.
Processing data was conducted using a statistical analysis test for normality with Kolmogorov-Smirnov test to see whether the data obtained were normally distributed or not.Next, homogeneity test using Levene test was performed.A statistical test for antibacterial power difference in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at every concentration then was carried out using Independent t-test.

results
This research used A. actinomycetemcomitan bacteria incubated for 1 x 24 hours at 37° C in the BHIB media, synchronized with the 0.5 McFarland standard.Materials used were non-fermented cocoa beans from Kota Batu, Malang.Next, cocoa extracts were made in UPT Materia Medika Batu in East Java by maceration method using 70% ethanol and water.
Based on the analysis results of chemical compounds contained in the 70% ethanol extract of cocoa beans as well as in the water extract of cocoa beans, there were different dosages of flavonoids, catechins, epicatechin, anthocyanin, and proanthocyanidin contained (Table 1).Catechin, epicatechin, and anthocyanin contained in the 70% ethanol extract of cocoa beans were higher than in the water extract of cocoa beans.Meanwhile, flavonoids and proanthocyanidin contained in the water extract of cocoa beans were higher than in the 70% ethanol extract of cocoa beans.
Serial dilution method is a method usually used in a comparative research of MIC and MBC in both the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A.actinomycetemcomitan bacteria (Figure 1).
During the observation of MIC and MBC in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at every concentration, the results of serial     No.A B dilution were planted in Luria Berthani agar media using streak technique as a cross-check to see the growth of bacterial colonies.It was necessary to do because of dark-colored extract material and turbidity occured at any concentration.Furthermore, based on the observation results on the number of bacterial colonies grown in the Luria Berthani media expressed in colony forming units (CFU), 70% ethanol extract of cocoa beans and water extract of cocoa beans could be able to inhibit and kill A. actinomycetemcomitan bacteria (Table 2).
The comparative test on the MIC and MBC in the 70% ethanol extract cocoa beans and the water extract of cocoa beans was conducted using serial dilution method.The results showed that the extracts at the concentrations of 100%, 50%, 25%, and 12.5% contained no bacterial growth.Meanwhile, the mean bacterial growth in the 70% ethanol extract cocoa beans at the concentration of 6.25% was 11.4.On the other hand, the mean bacterial growth in the water extract of cocoa beans at the same concentration was 7.8 (Table 2).
Before the antibacterial power difference analysis test on the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A. actinomycetemcomitan bacteria was performed, the normality test using Kolmogorov-Smirnov test as well as the homogeneity test using Levene test were conducted in each group.The results of the normality and homogeneity tests showed that data obtained in the water extracts of cocoa beans at the concentrations of 6.25% and 3.125% were normally distributed and homogeneous (p>0.05).Whereas, data obtained in the 70% ethanol extract of cocoa beans at the concentration of 6.25% were not normally distributed (p <0.05), and at the concentration of 3.125% was not homogeneous (p<0.05).   A. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6.    A. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6.   Next, Kruskal-Wallis test was carried out to test significant differences in antibacterial activities between the 70% ethanol extract of cocoa beans and the water extract of cocoa beans since there were groups which were not normally distributed and not homogeneous.The results showed value of p was less than 0.05 (p = 0.000), indicating that there was a difference in antibacterial activity between the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at the concentrations of 6.25% and 3.125% (Table 3).

discussion
This research was an in vitro research aimed to see the difference in antibacterial power beween the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A. actinomycetemcomitan bacteria.Antibacterial agents used in this research were non-fermented cocoa beans since non-fermented cocoa beans actually have higher antibacterial effects than fermented ones.Fermentation and drying processes, however, can reduce the active components of natural materials, such as polyphenol. 18his research also used a serial dilution method with the concentrations of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%.A. actinomycetemcomitan bacteria grown on the BHIB media were considered as the positive control (+), whlie the bacteria grown on the BHIB media and the control extracts were considered as as the negative control (-).Serial dilution method was used because it can give the significant calculation results of microorganism colonies at small concentrations.Thus, if the serial dilution method is not conducted, there will be a number of colonies stacked so that the calculation of the colonies will be not accurate. 19Serial dilution method has widely been used for many years. 20n the antibacterial activity test, properties owned by the cell wall of bacteria may affect the ability of cocoa bean extracts to inhibit tested bacterial growth. 21.actinomycetemcomitan bacteria are gram-negative bacteria that have a cell wall in the form of peptidoglycan surrounded by a membrane structure, called as outer membrane.Outer membrane of Gram-negative bacteria has lipopolysaccharide or endotoxin components that can prevent an antibacterial agent to penetrate into the cells. 22,23n the layers of lipopolysaccharide, Gram-negative bacteria have a system of selection against strange substances so that Gram negative bacteria are more resistant to strange substances than Gram positive bacteria. 21The presence of polyphenols, such as flavonoids in cocoa beans can penetrate the membrane of Gram-negative bacteria to mediate the cell response. 24lavonoids have antibacterial activity through the barrier function of bacterial DNA gyrase so that replication capabilities and bacterial translation can be inhibited. 25lavonoids may cause damage to the permeability of bacterial cell wall, microsomes, and lysosomes as a result of the interaction of flavonoids with bacterial DNA. 26atechins, moreover, are natural polyphenolic compounds and secondary metabolites included in the constituent group of tannins. 21Catechins may damage cytoplasmic membrane causing the release of important metabolites that inactivate bacterial enzyme system.Proanthocyanidin, furthermore, is condensed tannins that can inhibit the formation of cell walls and lead to termination of the crosslinking peptide that combines the glycan chains of peptidoglycan in another chain, causing cell membrane damage. 28ow to cultivate antibacterial materials, thus, is a thing that needs attention.Antibacterial material can be extracted by using various methods, one of which is by solvent extraction (maceration).Solvents widely used for polyphenol extraction method are water and 70% ethanol.Cocoa, on the other hand, will yield a different antibacterial activity when extracted with a different solvent polarity. 14thanol is a polar solvent that dissolves polar compounds, while water is an inorganic solvent that dissolves inorganic materials.In addition, cacao bean is an inorganic material because it is derived from plants.
To dissolve the cocoa beans using ethanol, therefore, will take a long process because of evaporation process.The process of evaporation can affect the polyphenol content in the extract.Water, on the other hand, is a solvent that can maintain a high quantity of essential compounds contained in natural materials. 17Water solvent will dissolve inorganic compounds and salts of acids and bases. 14Cocoa beans diluted with water is more soluble and requires a shorter time than cocoa diluted with ethanol.The analysis results of chemical compounds in the 70% ethanol extract of cocoa beans contain flavonoids (1.32%), catechin (2.01%), epicatechin (1.36%), anthocyanin (1.74%), and proanthocyanidin (1.93%).The water extract of cocoa beans contain flavonoids (2.18%), catechin (1.22%), epicatechin (1.08%), anthocyanin (1.67%), and proanthocyanidin (2.51%).Proanthocyanidin contained in cocoa beans was 58%, catechin amounted to 37%, and anthocyanin amounted to 4%, thus indicating that the water extract of cocoa beans contain more useful chemical compounds than the ethanol extract of cocoa beans. 10n the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at the concentration of 6.25%, moreover, there was still bacterial colony growth on the Luria Berthani media after incubated for 1x24 hours.It means that not all bacteria die, or there are barriers to the growth of colonies by 90%. 28Nevertheless, in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at the concentration of 12.5%, there was no colony growth.It indicates that the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at this concentration could kill bacteria by 99.9% of the total average bacteria that managed to grow on the positive control and were considered as bactericide. 29,30t can be concluded that water and 70% ethanol extract of cocoa beans have antibacterial activities against A. actinomycetemcomitans.Both the extracts also have the same MIC and MBC at concentrations of 6.25 and 12.5%.

Figure 1 .
Figure 1.Serial dilution (a) the 70% ethanol extract of cocoa beans and (b) the water e againstA.actinomycetemcomitan bacteria at a concentration of 100% in tub 50% in tube 2; a concentration of 25% in tube 3; and a concentration o concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6.

Figure 2 .
Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. abacteria at any concentration as well as the controls grown in the Luria Bert results of the water extract of cocoa beans against A.actinomycetemcom concentration as well as the controls grown in the Luria Berthani media; c the 70% ethanol extract of cocoa beans against A. actinomycetemcom concentration as well as the controls grown in the Luria Berthani media; d the water extract of cocoa beans against A. actinomycetemcomitan bacteria a well as the controls grown in the Luria Berthani media.

Figure 1 .
Figure 1.Serial dilution (A) the 70% ethanol extract of cocoa beans and (B) the water extract of cocoa beans againstA.actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6.

Figure 1 .
Figure 1.Serial dilution (a) the 70% ethanol extract of cocoa beans and (b) the water extract of cocoa beans againstA.actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6.

Figure 2 .
Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) streak results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; c) replanting results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; d) replanting results of the water extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media.

Figure 1 .
Figure 1.Serial dilution (a) the 70% ethanol extract of cocoa beans and (b) the water extract of cocoa beans againstA.actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6.

Figure 2 .
Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) streak results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; c) replanting results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; d) replanting results of the water extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media.

Figure 2 .Table 2 .Table 3 .
Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) streak results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; c) replanting results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; d) replanting results of the water extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media.

Table 1 .
The analysis results of the chemical compounds contained in 70% ethanol extract of cocoa beans and the water extract of of cocoa beans

Table 1 .
The analysis results of the chemical compounds contained in 70% ethan and the water extract of of cocoa beans Streak results of the 70% ethanol extract of cocoa beans against A bacteria at any concentration as well as the controls grown in the Luria B results of the water extract of cocoa beans against A.actinomycetem concentration as well as the controls grown in the Luria Berthani media the 70% ethanol extract of cocoa beans against A. actinomycetem concentration as well as the controls grown in the Luria Berthani media the water extract of cocoa beans against A. actinomycetemcomitan bacter well as the controls grown in the Luria Berthani media.
Figure 1.Serial dilution (a) the 70% ethanol extract of cocoa beans and (b) the wa against A. actinomycetemcomitan bacteria at a concentration of 100% in 50% in tube 2; a concentration of 25% in tube 3; and a concentratio concentration of 6.25% in tube 5; and a concentration of 3.125% in tube Figure 2. a)