The Global Burden of Disease Study reported that oral diseases afflicted about 2.3 billion people worldwide. Dental caries was the most common cause, accounting for 65% of infected permanent teeth and 15% of infected primary teeth1. Caries are caused by bacterial metabolism as a biofilm or plaque that changes sugars to acid, demineralizing the hard tissues of the teeth (enamel and dentine)2. Bacterial acid decreased the pH of tooth surfaces, causing demineralization from calcium ions and phosphate dentine release, which resulted in cavities3. The early stages of dental caries are frequently asymptomatic, but advanced stages can cause discomfort, infections, abscesses, and even sepsis. A severe condition in adults causes pain and infection, which may necessitate tooth extraction4. This caries might make children prone to infections in other parts of their bodies. The pain can make chewing meals excruciating, and getting enough nutrition can be problematic5.

The most prevalent bacteria that cause caries were Streptococci and Lactobacilli. Streptococci lead to caries, and Lactobacilli contribute to further infection6. Streptococcus mutans was not only the basic bacterium engaged in the development of plaque but also in the commencement of dental caries. Streptococcus mutans have been linked to other extraoral pathologies such as cerebral microbleeds, IgA nephropathy, and atherosclerosis as a human pathogens7,8.

Plaque treatment can be done mechanically or chemically. Brushing and flossing are mechanical techniques of plaque control, whereas mouthwash is a chemical plaque control treatment9,10. Mouthwash contains chemical compounds with antiseptic or antibacterial characteristics that aid in preventing plaque formation. On the other hand, regular utilization may cause tooth discoloration and temporary gustatory problems11. Pizzo et al.12 stated another negative impact was that it induces mouth dryness, burning, and is harmful if ingested. Hence, plant components can be used as antibacterial agents.

According to a World Health Organization (WHO) report, approximately 80% of the world's population was treated using plants or plant-derived products13. Plants have long been employed as the primary human therapy source and have helped address the world's healthcare demands14. Medicinal plants have proven to be a remarkable source of newer and potent therapeutic agents and have taken the central stage in most research centers worldwide15.

Colocasia esculenta, also known as taro (talas = Indonesian), was a nutritious plant widely consumed by the locals16. Colocasia esculenta tuber is used as ethnomedicine to treat diabetes, ringworm, cough, sore throat, wounds, asthma, arthritis, diarrhea, internal bleeding, neurological disorders, skin disorders, and it has been reported to have antihelminthic and anticancer properties17,18. Agyare et al.19 stated that a decoction of the leaves was drunk to promote menstruation and was used to treat stomach problems and cysts. In addition, C. esculenta leaf extract has anti-inflammatory and antioxidant properties.

Colocasia esculenta biological properties were derived from natural products such as alkaloids, flavonoids, tannins, phytates, and minerals17. Elmosallamy et al.20 showed that the significant constituents of C. esculenta leaves are flavonoids β-sitosterol and steroids. Anthocyanins, isoschaftoside, vicenin, apigenin, catechin, anthraquinones, β-amyrin, α-amyrin, riboflavin, and niacin were also reported in leaves21,22. The flavonoids in C. esculenta include orientin, isoorientin, vitexin, isovitexin, and luteolin-7-O-sophoroside23. Orientin flavonoids are reported to have antibacterial activity24.

Wang et al.25, in previous research, reported the presence of antimicrobial activity in tests using C. esculenta. The research of Singh et al.26 stated that the water extract of C. esculenta leaves showed the diameter of the inhibition zone of Staphylococcus aureus MTCC 96 and S. mutans MTCC 890 at a concentration of 400 mg/mL was 8.50±0.09 mm and 15.33±0.13 mm, respectively. A different solvent produced a more significant inhibition zone, ethanol extract of C. esculenta leaves against S. aureus of 14.3±1.45 mm at a similar concentration27.

The selection of the solvent and methods used for extraction was an important factor, so it must be fully considered28. The maceration extraction method has the advantage of being easy to use and requires less equipment. Since the maceration method was used at room temperature, thermolabile flavonoid compounds are appropriate29. Antimicrobial compounds are most commonly found in the phenolic compounds terpenoids but also found in flavonoids, saponins, and alkaloids classes30-32. Orientin was a flavonoid compound with low polarity, making it easily soluble in semipolar solvents. Since 96% ethanol was a semipolar solvent, it was chosen as the extraction solvent33. Based on the prior studies of 96% ethanol extract against S. mutans ATCC 31987, researchers intend to investigate the antibacterial activity of C. esculenta leaf ethanol extract against the growth of the primary bacteria responsible for dental caries.

Materials

The materials used in this study were old fresh C. esculenta leaves, collected from Balai Penelitian Tanaman Sayuran (Research Center of Agriculture Ministry) Lembang, West Java, Indonesia. Streptococcus mutans ATCC 31987 isolates from Biology Department Universitas Indonesia, 96% ethanol, dimethyl sulfoxide (DMSO), chlorhexidine, 0.9% sterile physiological NaCl, Mueller Hinton Agar (MHA), blood agar, H2SO4, BaCl2, 70% ethanol, HCl, acetic acid, n-hexane, Mg metal, Mayer reagent, Wagner reagent, aquadest, chloroform, FeCl3, and blank disc. The instruments used in this study were an autoclave (Hirayama®), incubator (Memmert®), blender (Philips®), vacuum rotary evaporator (Buchi®), biological safety cabinet (Thermo Scientific®), oven (Memmert®), microscope (Leica®), spectrophotometer (Shimadzu®), analytic scale (Mettler Toledo®), waterbath (Electro-mag®), micropipette (Effendorf®), refrigerator, caliper, inoculation loop, and laboratory glassware.

Methods

Preparation of ethanolic extraction

Preparations of simplicia adapted from Arnida et al.34 including wet sorting, washing, chopping, drying, and dry sorting (Figure 1). The drying was done in an oven at 50±2°C. Dry leaves or simplicia could be crushed by hand into small pieces with a water content of 10% or less. Gravimetric methods are used to determine the water content. Equation 1 was used to calculate the yield extract:

Water content (%) = … [1]

W0 = weight of container (g)

W1 = weight of container + weight of moist simplicia (g)

W2 = weight of container + weight of dry simplicia (g)

Simplicia was ground to a powder with a blender and sieved at 60 mesh. Colocasia esculenta leaves simplicia were macerated in 96% ethanol. Simplicia powder was soaked in ethanol (1 : 10) for 24 hours at room temperature, stirring occasionally. This process was repeated until the pellucidity was achieved. Following filtration of the suspension through filter paper, C. esculenta leaf extract was evaporated at 50°C using a rotary vacuum evaporator and a waterbath (Figure 1). The crude extract was kept at 40°C until it was analyzed. Equation 2 was used to calculate yield extract:

Yield extract (%) = … [2]

a b c

Figure1. Colocasia esculenta leaves before chopping (fresh) (a), drying process in the oven (b), and thickening extract process to evaporate the remaining solvent (c).

Phytochemical analysis35,36

Phytochemical compounds were identified by qualitative analysis. The extract's content of flavonoids, alkaloids, saponins, tannins, steroids, and terpenoids was determined qualitatively. The extraction solvent affects the secondary metabolite content of the extract.

Alkaloid (Wagner and Mayer test): About 5 mg extract was dissolved in chloroform, then 0.5 mL of 1 M sulfuric acid was added and slowly shaken. The mixture was allowed to stand for a few moments until two layers formed. The transparent top layer was divided into two parts, one receiving 2-3 drops of Wagner's and the other receiving 2-3 drops of Mayer's reagent. The brown precipitate indicated the alkaloid compounds by Wagner's and the white precipitate by Mayer's reagent.

Flavonoid (Wilstater test): About 5 mg of extract was dissolved in 5 mL of hot water, boiled for 5 minutes, and then filtered. The filtrate was mixed with Mg metal, 1 mL of concentrated sulfuric acid, and 2 mL of 70% ethanol. The mixture was shaken vigorously and set aside. The presence of flavonoid compounds was indicated by the formation of a red, yellow, or orange color on the ethanol layer.

Tannins and Polyphenol (FeCl3 test): About 0.5 g of the extract was dissolved in 2 mL of 70% ethanol, boiled in 10 mL of distilled water, and filtered in a test tube. Three drops of 0.1 % FeCl3 were added, and a brownish-green or blue-black color formed indicating the presence of tannins and phenols.

Saponin (Foaming test): Colocasia esculenta extract was dissolved in 10 mL of hot water and allowed to cool. Once cool, the mixture was vigorously shaken vertically for 10 seconds. The presence of saponin compounds was indicated by a stable foam as high as 1 cm if the foam remains stable after adding one drop of 1% HCl.

Triterpenoids and Steroids: In a test tube, 0.5 mg of extract was placed. The mixture was then treated with 2 mL of anhydrous acetic acid and 0.5 mL of concentrated sulfuric acid. A blue or green color indicated the presence of steroids, while a brownish or purple ring indicated the presence of triterpenoids.

Bacterial preparations

The bacteria were obtained, and then Gram stained to examine cell morphology. Gram staining was tested by dripping the reagents in a specific order. The first reagent was gentian violet as a primary stain, followed by iodine solution as a mordant, alcohol as a decolorizer, and safranin as a counterstain. Gram-positive bacteria retain the first color, making them appear violet under a 100x magnification microscope. The bacteria were recultured by streaking a loop of bacteria in blood agar media and incubating it at 37°C for 18-24 hours before using the bacteria to make suspensions.

Antibacterial activity

McFarland standard 0.5 was made by combining 0.05 mL of 1% BaCl2 with 9.95 mL of 1% H2SO4. The turbidity produced in the test tube was equivalent to 1.5x108 CFU/mL of bacteria. The mixture of the two solutions was attempted for no more than 15 minutes to serve as a standard37,39.

Recultured S. mutans in a sterile inoculation loop were suspended in a tube containing 10 mL of 0.9% physiological NaCl. The turbidity was then compared to McFarland 0.5 standard. Turbidity was measured with a spectrophotometer equal to 0.1 at 625 nm. The too high turbidity could be reduced by adding 0.9% physiological NaCl or bacterial colonies40.

The test bacteria were planted in the solidified MHA media using the spread plate method. The prepared bacterial suspension was taken 100 µL with a micropipette and dripped on the surface of the solidified MHA media. The suspension droplets were spread using a sterile L-rod, with repeated rotation of the petri dish, to ensure that the test bacteria spread evenly. Colocasia esculenta leaf ethanol extract was prepared in various concentrations of 10, 20, 30, 40, 50, 60, and 70% dissolved in DMSO. The positive control was chlorhexidine, the negative control was DMSO solution, and the growth control was suspension only. Blank discs with a diameter of 6 mm were immersed in that test solution for 10 minutes to make sample discs. Sample discs were given to solid media mixed with the test bacteria. The samples were incubated at 37ºC for 24 hours in an inverted position to prevent condensation from dripping onto the media41.

Furthermore, the presence or absence of the formed inhibition zone was observed. The apparent diameter of the clear zone around the disc was measured using a caliper diagonally, vertically, and horizontally and averaged. The diameter of the clear zone was reduced by the diameter of the disc37.

Statistical analysis

The inhibition zone values were calculated using IBM SPSS Statistic 25 for Windows and expressed as mean (n=3) per plate of three repetitions±standard deviations (SD). Suppose the data had a normal distribution (p <0.05), the normality test was performed using Analysis of Variance (ANOVA) and Kruskal-Wallis if not. Then a Post Hoc (p <0.05) analysis was performed.

Ethanol extract preparations

The water content of C. esculenta leaves was 7.07±0.05%, less than 10%. Microbes will easily overgrow in water with content greater than 10%. The simplicia was then mashed to facilitate the extraction process. A large surface area will increase the effectiveness of the solvent in breaking down plant cell walls, so it was expected that the extracted compounds would be maximal42.

Antibacterial compounds such as orientin in C. esculenta leaves were extracted with 96% ethanol. Other compounds such as luteolin, apigenin, isoorientin, vitexin, and isovitexin have similar solubility. Previous studies show ethanol was the best choice because it contains the tremendous variety and extent of bioactive components in C. esculenta extract20. The resulting yield extract was 20.53%, which was calculated to determine the adsorption value of the solvent in extracting the extract.

Phytochemical analysis

Qualitative identification of compound groups was performed to determine the content of compound groups in C. esculenta leaf extract that may have antibacterial properties. The presence of alkaloids, flavonoids, triterpenoids, and saponins was detected in the qualitative test on C. esculenta leaf extract shown in Table I. Eddy43 stated that the water extract of C. esculenta leaves contains saponins and tannins. Meanwhile, the ethanol extract of C. esculenta leaves, apart from containing saponins and tannins, also contains terpenoids, anthraquinones, flavonoids, and alkaloids. Different solvents can lead to the extraction of various phytochemical compounds. The precipitate formed in the alkaloid test was potassium-alkaloid because the alkaloid compound contains a nitrogen atom with a lone pair of electrons that can be used to form coordinate covalent bonds with metal ions. The alkaloids' nitrogen will react with the metal ions K+ from potassium tetraiodomercurate (II) to form a precipitated potassium-alkaloid complex44.

Flavonoid testing was carried out by adding HCl and Mg metal, which reduced the benzopyrone core contained in flavonoid compounds, resulting in the formation of red color in these compounds45. Meanwhile, the saponin test was performed by adding hot water to the ethanol extract. Saponins are polar in that they can dissolve in solvents such as water, though they are also non-polar in that they contain a hydrophobic group, an aglycone (sapogenin). The presence of glycosides, which can form foam in water and hydrolyze into glucose and other compounds, causes the foam produced in the saponin test46.

Triterpenoid testing was carried out by adding acetic acid and sulfuric acid, which can produce a color change reaction to a brownish or purple ring. This can occur due to condensation or release of water and incorporation with carbocations. This reaction begins with the acetylation of the hydroxyl group using anhydrous acetic acid. Furthermore, the release of the hydrogen group and its electrons causes the double bond to move and experience resonance which acts as an electrophile or carbocation. The presence of a carbocation causes electrophilic addition, followed by the release of hydrogen. Then the hydrogen group and its electrons are removed. As a result, the compound undergoes conjugation extension, which shows the appearance of a brownish ring47.

TableI. Phytochemical analysis of C. esculenta leaves extract

Note: (+) presence; (-) absence

Antibacterial activity

The extract diffuses on the agar and inhibits the test microbes' growth, resulting in a clear zone formation. The zone of inhibition can be seen as the formation of a clear zone around the disc, with the visible diameter of the zone concluding as the extract's inhibition zone against the test microbes. Outside the zone, turbidity indicates the growth of microbes that are not inhibited by the extract. Table II shows the results of testing a 96 % ethanol extract of C. esculenta leaves against the caries-causing S. mutans. Each variation of the concentration of 96% ethanol extract of C. esculenta leaves has antibacterial activity, and a clear zone around the disc indicates an inhibition zone.

Tests revealed that a concentration of 70% can provide inhibition of 21.11±0.46 mm, indicating that it has a very strong antibacterial ability. Ponce et al.48 classified the diameter of the inhibition zone into four categories: weak inhibition zone of 8 mm, moderate inhibition zone of 8-14 mm, strong inhibition zone of 15-19 mm, and very strong inhibition zone of >20 mm. The lowest concentration was in the weak category, and the highest was in the very strong category, according to the above categories. This could occur because the lower the concentration of an extract, the more dilute the extract produced and the smaller the inhibition zone formed to inhibit S. mutans.

The test revealed that chlorhexidine inhibited the microbes by 9.37±0.27 mm. Chlorhexidine was known to have bacteriostatic and bactericidal properties, and it can effectively inhibit and kill S. mutans. Chlorhexidine was an effective mouthwash in the treatment of dental caries49. The mechanism of chlorhexidine causes changes in the permeability of the bacterial cell membrane, resulting in the release of cytoplasm and cell components from within the cell through the cell membrane, resulting in bacterial death50. The 0.2% chlorhexidine concentration was chosen because it was the concentration commonly used as a mouthwash. Furthermore, increasing the concentration of chlorhexidine will make it toxic to dental cells.

The negative control showed no inhibition zones against the S. mutans. The negative control (10% DMSO), a solvent used in extract dilution, does not inhibit bacterial growth. DMSO was a neutral solvent capable of dissolving both polar and non-polar substances51. According to Trisia et al.52, who examined the antibacterial activity test of the ethanolic extract using the disc diffusion method, DMSO had no inhibitory effect on the growth of S. aureus (Kirby-Bauer).

Previous research by Dutta and Aich27 demonstrated that the diffusion of ethanol extract of C. esculenta leaves at a concentration of 400 mg/mL inhibited S. aureus by 14.3±1.45 mm. Our research yielded an inhibition of 7.33±0.64 mm at the same concentration (40%). Meanwhile, at a concentration of 20 mg/mL, the inhibition zones formed by C. esculenta leaves chloroform and methanol extract against S. aureus were 16.23±1.53 and 20±1.00 mm, respectively53. Singh et al.26 tested the antibacterial activity of C. esculenta leaves aqueous extract against S. mutans and found an inhibition zone of 15.33±0.10 mm at 400 mg/mL.

The extract's phytochemical content influenced the antibacterial activity of C. esculenta leaf extract. Flavonoids, saponins, alkaloids, and terpenoids are found in C. esculenta leaves ethanol extract. Alkaloid group compounds can function as antibacterials by interfering with the peptidoglycan components in bacterial cells, causing the cell wall layer not to form entirely and causing the bacterial cell to die54. Furthermore, flavonoid compounds inhibit cell membrane function, DNA gyrase, and bacterial metabolism55. Other groups of compounds that are positive in the extract and have an antibacterial function are saponins and triterpenoids. Saponins can work as an antibacterial by disrupting the permeability of cell membranes so that the membrane becomes unstable and causes lysis56. Meanwhile, the mechanism of action of triterpenoids by damaging the bacterial cell membrane. Cell membrane damage can occur when active antibacterial compounds react with the active site of the membrane54.

Table II showed that the test solution has a more significant zone of inhibition against S. mutans than the positive control (60 and 70%). The greatest concentration of 70% was twice as strong as the positive control, which had a concentration of 0.2%. According to the results of the tests, C. esculenta leaf extract can be utilized as an alternative to S. mutans inhibition.

TableII. Inhibition zone of C. esculenta leaves extract and control against S. mutans

Note: The diameter of the inhibition zone was reduced with the disc diameter (6 mm)

Statistical analysisis

All data on the antibacterial activity of the ethanol extract of C. esculenta leaves against S. mutans were analyzed using statistical tests. This study's variables were categorical and unpaired numeric variables and had more than two groups. Therefore, the statistical test that can be used is the ANOVA test with the condition that the data obtained must be normally distributed and have homogeneous data variances. The data obtained were first analyzed using the normality test, which aims to see whether the resulting data were normally distributed or not. The normality test results have two results: the Kolmogorov-Smirnov and the Shapiro-Wilk test. The normality value was seen in the Shapiro-Wilk test because the data obtained was less than 50. The normality value of each treatment was more than 0.05, so it can be concluded that the data obtained are normal.

After the normality test, the data from the antibacterial activity test results should be tested for homogeneity, which aims to see the homogeneity of the data variants. The homogeneity of variances test shows a p-value of >0.05. Therefore, it can be concluded that there are at least two groups that have significantly different data variances. Based on the normality and homogeneity test results, we used the ANOVA test because the data obtained were normally distributed, and the data's variance was homogeneous. Analysis was conducted Post hoc to find out which groups had significantly different data. Table III showed that the negative control significantly differed from the positive control. The overall activity data was significantly different from the negative control. Furthermore, the test group was significantly different from the positive control, significantly smaller or larger, with a p-value of <0.05.

TableIII. Statistical analysis of antibacterial activity of C. esculenta leaves extract

Note: (*) a significant difference with a 95% confidence level

The results showed that the best inhibition zone was 21.11±0.46 mm at a 70% concentration of C. esculenta, which was more significant than the control. Colosia esculenta leaves had very strong antibacterial activity against S. mutans.

The author would like to express gratitude to the Poltekkes Kemenkes Bandung for financial funding through the PDP research grant scheme.

Ayu Nala El Muna Haerussana: concept and design, research team leader and coordinator, validation, antibacterial assay, and article writing. Angreni Ayuhastuti: concept and design, validation, and article writing. Siti Fira Yuniar: sampling and simplicia preparation. Hana Alifah Bustami: extraction and phytochemical screening. Widyastiwi: data analysis.

None.

The authors declare that they have no conflicts of interest to report regarding the present study.

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