Kurniati N. Inhibitory Power of Papaya Leaves to the Adhesion of Streptococcus Mutans Bacteria to Neutrophils. Biomed Pharmacol J 2021;14(3).

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Manuscript received on :11-08-2021
Manuscript accepted on :08-09-2021
Published online on: 23-09-2021

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Reviewed by: Dr. Salman Ahmed
Second Review by: Dr. Mirza Mienur Meher
Final Approval by: Dr. Fai poon

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Nining Kurniati

Poltekkes Kemenkes Banten

Corresponding Author E-mail: niningkurniatipolkesten@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/2240

Abstract

Papaya leaves contain several active substances that are thought to inhibit bacterial adhesion. This study aims to determine the inhibitory power of papaya leaf extraction and papaya leaf infusion to the adhesion of Streptococcus mutans bacteria to neutrophils and differences in inhibition in various concentrations. The method used is an in vitro experimental laboratory with a post-test-only control group design. The sample consisted of several concentrations of papaya leaf extraction and papaya leaf infusion (control, 25%, 50%, 75%, and 100%). The treatment group was added with neutrophil isolates and incubated for 3 hours, then exposed to Streptococcus mutans for 2.5 hours. Knowing the adhesion index can be done by calculating the average number of Streptococcus mutans attached to 100 neutrophils. The results showed successively with rounding in control, 25%, 50%, 75%, 100% papaya leaf infusion, adhesion of 9237, 8929, 7436, 3870, 3162 bacteria to neutrophils occurred. The minimum concentration of 25% papaya leaf extraction and 50% papaya leaf infusion can inhibit the adhesion of Streptococcus mutans to neutrophils. There is a difference in the ability between papaya leaf extraction and papaya leaf infusion in inhibiting the adhesion of Streptococcus mutans to neutrophils as follows: papaya leaf extraction 25% to papaya leaf infusion 25% obtained sig value ≥0.001* with a mean square of -16.38, papaya leaf extraction 50% to Papaya leaf infusion 50% got a sig value of ≥0.001* with a mean square of -42.91, 75% papaya leaf extraction to 75% papaya leaf infusion got a sig value of ≥0.001* with a mean square of -28.85 and 100% papaya leaf extraction to 100% papaya leaf infusion obtained a sig value of ≥0.001* with a mean square of -26.11.

Keywords

Adhesion; Papaya leaf juice; Papaya leaf infusion; neutrophils; Streptococcus mutans

Introduction

Microbial normal flora is a group of microorganisms that naturally live on the skin and mucous membranes (mucosa) in normal and healthy humans. Flora in the human body can be permanent or temporary. Normal microbes that persist can not cause disease and may be beneficial in the absence of abnormal conditions. Various things affect the presence of microorganisms in the human body, including nutrition, personal hygiene, living conditions, and changes in diet. Streptococcus mutans is one of the normal flora microbial bacteria that can cause disease. ^1,2

Streptococcus mutans is a Gram-positive bacterium that causes dental caries. Dental caries is an infectious disease and is a progressive demineralization process in the hard tissues of the tooth surface from foods containing sugar. Dental caries is the most common disease in the oral cavity, so it is a significant problem for oral health.³ Disease prevention through plants is one of the uses of natural resources in Indonesia.⁴ Indonesia has many types of medicinal plants. The types of plants that are included in the therapeutic plant group reach more than 1000 species. One of them is Papaya (Carica papaya L.).⁴ Papaya leaf extract contains antibacterial compounds such as flavonoids, karpain alkaloids, papain enzymes, and tannins.⁵

Lipophilic flavonoids will damage the membrane so that the permeability will increase and interfere with bacterial metabolism.^6–8 Alkaloids can interfere with the formation of the constituent components of peptidoglycan in bacteria so that the cell wall layer is not fully formed and causes the death of bacteria.⁹ In addition, papain, a proteolytic enzyme, also has a bactericidal and bacteriostatic effect, thus inhibiting the growth of both Gram-positive and Gram-negative bacteria.¹⁰ The antimicrobial effect of tannins is in the form of inactivating microbial adhesins and inactivating hydrolytic enzymes such as proteases and carbohydrolases, as well as inhibiting enzymes in envelope transport proteins. ^11,12

In a study on the inhibitory power of papaya leaf extract (Carica papaya L.) to the adhesion of bacteria Porphyromonas gingivalis to neutrophils, the higher the concentration of papaya leaf extract, the less the adhesion of bacteria to neutrophils.¹³ The presence of antibacterial in papaya leaves may inhibit the adhesion of Streptococcus mutans to neutrophils because the development of Streptococcus mutans is disrupted and dies before adhesion to neutrophils. In proving the ability of papaya leaves to inhibit the adhesion of Streptococcus mutans to neutrophils, it is necessary to study the optimum concentration by comparing in the form of juice and infusion.

Material and Methods

This study is a laboratory experiment by determining the inhibition of papaya leaf extract and infusion on the adhesion of Streptococcus mutans to neutrophils with concentrations of 25%, 50%, 75%, and 100%. The repetition in the study was carried out four times.. The control was divided into two parts: giving neutrophil cells 100% infusion without going through incubation and exposure to Streptococcus mutans and 100% infusion control. The research materials consisted of papaya leaf extract and infusion of 100, 75, 50 and 25%, pure strain of Streptococcus mutans bacteria, neutrophil isolate, absolute methanol Giemsa dye.

Processing Papaya Leaf Extract

150 g of papaya leaves that have been washed, dried, and extracted using a juicer into an extract of about 40 mL. The juice specified as a concentration of 100% was accommodated in a sterile Erlenmeyer. Dilution using sterile distilled water to obtain concentrations of 24%, 50%, and 75%.

Processing Papaya Leaf Infusion

100 g of clean and dry papaya leaves were cut about 1.5 cm, then put into a sterile 1 L beaker, and 100 mL of sterile distilled water were added. Mixing using a beaker with heating using a water bath for 15 minutes starting at 90 0C while stirring occasionally. Strain the papaya leaf extract using a sterile flannel cloth into a sterile Erlenmeyer glass to obtain an infusion of about 80 mL. Defined as a 100% concentration infused. Infuses with concentrations of 75, 50, and 25% were made using sterile distilled water from 100%.

Treatment for neutrophil cell control without papaya leaf extract or infusion

A total of 100 L of neutrophil isolate with 100 L sterile physiological NaCl was homogenized by gently shaking. Added 200 L of Streptococcus mutans isolate by shaking gently. The mixture was incubated at 37 0C incubator for 3 hours, including controls without incubation. The results of the mixture of each treatment made smear preparations. The preparations were fixed using absolute methanol for 5 minutes and stained with Giemsa stain for 30 minutes. The preparations were rinsed with distilled water, then drained. Preparations are ready to be observed under a microscope with a magnifying 1000x.

Bacterial antiadhesion treatment against neutrophil cells from papaya leaf extract

A total of 100 L of papaya leaf juice, each concentration of 25%, 50%, 75%, and 100% with 100 L of the neutrophil isolate, was homogenized by gently shaking. The mixture was incubated at 37 ⁰C incubator for 2.5 hours. Each treatment was exposed to/added bacterial isolates. Streptococcus mutans as much as 200 L, homogenized by shaking slowly. The mixture was incubated at 37 ⁰C incubator for 3 hours. The results of the mixture from each treatment were made smear preparations. The preparations were fixed using absolute methanol for 5 minutes. The preparations were stained with Giemsa dye for 30 minutes, rinsed with distilled water, and then drained. Preparations are ready to be observed under a microscope with a magnifying 1000x.

Bacterial antiadhesion treatment against neutrophil cells from papaya leaf infusion

A total of 100 L of papaya leaf infusion, each with a concentration of 25%, 50%, 75%, and 100% with 100 L of neutrophil isolates, were homogenized by gently shaking. The mixture was incubated at 37 ⁰C incubator for 2.5 hours. Each treatment added 200 IU of Streptococcus mutans isolate by shaking gently. Then the mixture was incubated at 37 ⁰C for 3 hours. The results of the mixture of each treatment made smear preparations. The preparations were fixed with absolute methanol for 5 minutes. The preparations were stained with Giemsa dye for 30 minutes, rinsed with distilled water, and then drained. Preparations are ready to be observed under a microscope with 1000x magnification.

Data analysis

Data processing to test the strength of anti-adhesion between the concentrations between the juice and the infusion used the Anova statistical test to distinguish the strength between the variations in the concentration of the extract and the infusion followed by multiple comparisons analysis (MCA) with Bonferroni.

Results and Discussion

Treat With Papaya Leaf Extract

Treatment of control and papaya leaf extract with concentration variations of 25%, 50%, 75%, and 100%, which were contacted with neutrophil cells before being exposed to Streptococcus mutans showed differences in the inhibition of adhesion to neutrophils at each concentration (figure1).

Figure 1: The number of Streptococcus mutans in neutrophils in contact with papaya leaf juice Click here  to view figure

Table 1: The number of Streptococcus mutans in neutrophils in contact with papaya leaf  juice

^aOne-way ANOVA, α = 5%.
*Significant (p ≤ 0,05).

Post hocBonferroni: control vs 25%: ≥0.001 ;25% vs 50%: ≥0.001  ; 25% vs 75%: ≥0.001 ; 25% vs 100%: ≥0.001  ; control vs 50%:≥0.001 50% vs 75%: ≥0.001; 50% vs 100%: ≥0.001;control vs 75%:≥0.001; 75% vs 100%: 1.00; control vs 100%: ≥0.001

Table 1 shows the statistical test results of differences in the inhibitory ability of papaya leaf extract in inhibiting the adhesion of mutant streptococci to neutrophils (p≥0.001). Papaya leaf extract 25% had the lowest effectiveness in inhibiting the adhesion of mutant streptococci to neutrophils. The higher concentration of papaya leaf extract indicates the effectiveness of the adhesion inhibition of mutant streptococci against neutrophils is getting better with an optimum dose of 75%.

Control and treatment with papaya leaf infusion with concentration variations of 25%, 50%, 75%, and 100%, which contacted with neutrophil cells before being exposed to Streptococcus mutans, showed differences in inhibition of adhesion to neutrophils at each concentration, as seen from the number of bacteria attached, on neutrophil cells (figure 2). The confirmation test was carried out using the ANOVA statistical test followed by the multiple comparisons test, Bonferroni.

Figure 2: Streptococcus mutans counts in neutrophils contact with a papaya leaf infusion Click here to view figure

Table 2: Streptococcus mutans counts in neutrophils contact with a papaya leaf infusion

^aOne-way ANOVA, α = 5%.
*Significant (p ≤ 0,05).

Post hoc Bonferroni: control vs 25%: ≥ 1.00 ;25% vs 50%: ≥0.001  ; 25% vs 75%: ≥0.001 ; 25% vs 100%: ≥0.001  ; control vc 50%: ≥0.001 50% vs 75%: ≥0.001; 50% vs 100%: ≥0.001; control vs 75%: ≥0.001; 75% vs 100%: 0.077; control vs 100%: ≥0.001

Table 2 shows the statistical test results that there are differences in papaya leaf infusion’s ability to inhibit the adhesion of mutant streptococci to neutrophils (p ≥ 0.001). Papaya leaf infusion 25% had the lowest effectiveness in inhibiting the adhesion of mutant streptococci to neutrophils (p ≥ 0.001). The higher concentration of papaya leaf infusion indicates that the inhibition of adhesion inhibition of mutant streptococci against neutrophils is getting better. The post hoc test results showed that the optimum dose of papaya leaf infusion in inhibiting the adhesion of mutant streptococci to neutrophils was at an amount of 75%. The results of this study indicate that papaya leaves contain chemical compounds tocophenol, carpain alkaloids, and flavonoids as antibacterial.¹⁴

Tocophenol is a phenolic compound found in papaya plants. Phenol compounds are acidic alcohols, so they are also called carbolic acids. Acid conditions due to the presence of phenol can affect the growth of Streptococcus mutans bacteria. Phenol compounds can break the peptidoglycan bond in the bacterial cell wall by damaging the hydrophobic bonds of cell membrane components such as proteins and phospholipids and the dissolution of hydrophobic components, resulting in increased membrane permeability, causing leakage and release of cell contents.¹⁵

Carpain alkaloids have primary groups that can react with bacterial DNA and damage bacterial DNA, which causes damage to the bacterial cell nucleus. Cell damage makes the bacteria unable to metabolize, so that it undergoes lysis. Thus the bacteria become inactive and destroyed.¹⁶ Flavonoids inhibit the growth of Streptococcus mutans bacteria by inhibiting the glucosyltransferase enzyme, a compound that catalyzes sucrose into adesin material in the form of glucans in Streptococcus mutans bacteria. By inhibiting the glucosyltransferase enzyme, the bacterial cell membrane becomes lysed.¹⁷

The mechanism of action of saponins as an antibacterial is by lowering the surface tension, resulting in increased permeability and cell leakage so that intracellular compounds will come out. These compounds will diffuse through the outer membrane and vulnerable cell walls, then bind to the cytoplasmic membrane and reduce the stability of the membrane. Thus causing leakage in the cytoplasm, which causes cell death.^18,19annins work by inactivating bacterial adhesion and inactivating hydrolytic enzymes such as proteases and carbohydrolases, as well as inhibiting enzymes in envelope transport proteins so that bacterial cells cannot form. Tannins inhibit Streptococcus mutans bacteria by inhibiting the glucosyltransferase enzyme and disrupting the bacterial cell membrane so that the bacterial cell membrane becomes lysed.^10,20

This study proves that papaya leaf extract (Carica papaya L.) has antibacterial effectiveness in inhibiting the growth of Streptococcus mutans bacteria in the oral cavity in vitro. This result is the first step in the possibility of using papaya leaf extract as an alternative material in dentistry, requiring a series of tests such as clinical trials, toxicity, and side effects so that the community can utilize this research.

Conclusion

There are differences in the ability of papaya leaves in the form of extracts and infusions to inhibit the adhesion of Streptococcus mutans to neutrophils. The minimal concentration of papaya leaves in the form of extract and infusion can inhibit the adhesion of Streptococcus mutans to neutrophils at a concentration of 25% and 50%, with an optimum concentration of 75%, respectively.

Based on the results obtained, it is recommended to conduct further research on the inhibition of the attachment of Streptococcus mutans bacteria with variations in contact time between papaya leaf infusion and infusion to neutrophil cells and determine the minimum inhibitory concentration of papaya leaf infusion and anti-adhesion infusion to neutrophil cells.

Acknowledgement

Thank you very much to all laboratory staff and technical personnel who have helped in this research

Conflicts of Interest

There are no conflicts of interest.

Funding Source

There are no funding sources.

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