Antimicrobial effect of Stem cells from human exfoliated deciduous teeth metabolites combined with Epigallocatechin-3-gallate: As In vitro study

Sidarningsih¹, Yuliati¹, Deny Saputra^2,3, Nastiti Faradila Ramadhani³, Sasha Aprilia Rochmat⁴,

Bunga Fauzia^5,6, Mohammed Ahmed Aljunaid^7,8, Huda Rashad Qaid^2,8,

Rini Devijanti Ridwan¹*, Shuhdi Gamal Alaghbari⁷, Mogeb Al-Nahari ⁹, Habib Thabet ^10,11

¹Department of Oral Biology Faculty of Dental Medicine Universitas Airlangga, Surabaya.

²Doctoral Program of Dental Medicine, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.

³Department of Dento-maxillofacial Radiology,

Faculty of Dental Medicine Universitas Airlangga, Surabaya, Indonesia.

⁴Undergraduate Student, Faculty of Dental Medicine Universitas Airlangga, Surabaya, Indonesia.

⁵Magister of Dental Health, Faculty of Dental Medicine Universitas Airlangga Surabaya, Indonesia.

⁶Faculty of Dental Medicine Hang Tuah University, Surabaya, Indonesia.

⁷Department of Oral and Dental Medicine, Faculty of Medicine, Taiz University, Taiz, Yemen.

⁸Faculty of Oral and Dental Medicine, AL-Saeed University, Taiz, Yemen.

⁹Pharmacy Department, Faculty of Medicine and Health Sciences, Taiz University

¹⁰Department of food and Science and Technology, Ibb University.

¹¹Department of Therapeutic Nutrition, Al-Saeed University.

*Corresponding Author E-mail: rini-d-r@fkg.unair.ac.id

ABSTRACT:

Background/purpose: According to The Global Burden of Disease Study in 2016, oral health problems, especially periodontal disease, become the 11th most common globally, including dental cavities, periodontal disease, and oral fungal infection. Epigallocatechin-3-gallate (EGCG), the composition of green tea, has excellent benefits and plays a vital role in health, especially in regulating oral pathogenic bacteria activity that causes oral inflammation. Stem cells of human exfoliated deciduous teeth (SHED) metabolites also have great benefits and play an essential role in the health sector, especially as an alternative to regenerative therapy with its many bioactive activities. This study aims to prove the antimicrobial power of the SHED metabolite combined with EGCG against Fusobacterium nucleatum (F. nucleatum), Lactobacillus acidophilus (L. acidophilus), and Candida albicans (C. albicans). Materials and methods: The six different passages of SHED were prepared in Dulbecco’s Modified Eagle medium and added with EGCG. After 48 hours of incubation, the Mueller-Hinton agar medium diffusion method measured the inhibition zone. The research data was analyzed statistically. Results: The SHED metabolite was antibacterially active combined with EGCG against L. acidophilus and F. nucleatum bacteria, but there was no antifungal activity against the fungus C. albicans. Conclusion: The SHED metabolite combined with EGCG had antibacterial properties against L. acidophilus and F. nucleatum bacteria but did not have antifungal properties against C. albicans fungi.

KEYWORDS: Antimicrobial, Candida albicans, EGCG, Fusobacterium nucleatum, Lactobacillus acidophilus, Medicine, SHED.

INTRODUCTION:

Based on The Global Burden of Disease Study in 2016, oral and dental health problems, especially periodontal disease, have become the 11^th most common disease in the world.¹

In Indonesia, the results of the RISKESDAS in 2018 held by the Ministry of Health stated that the prevalence of people with oral and dental problems was 57.6%²_.Lactobacillus acidophilus (L. acidophilus) is one of the most pathogenic bacterial species out of 200 bacterial species isolated from dental plaque. It is a cariogenic bacterium that produces organic acids, which can cause a drastic decrease in the pH of the oral cavity, causing demineralization of tooth enamel_.^3,4

Dental and oral health problems experienced by most of the Indonesian population aredisorders of the tooth-supporting tissues (periodontal), such as swollen gums and abscesses, by 14%.⁵Disorders of the tooth-supporting tissue begin with gingivitis, which, if left untreated, will become periodontitis.⁶One of the most common species in the human gingival sulcuswas Fusobacterium nucleatum (F. nucleatum). Its prevalence increases with the severity of periodontal disease and the development of inflammation.⁷The treatment phase of the periodontal disease includes antimicrobial therapy in the form of antibiotics^8,9. However, widespread use of antibiotics results in strong and consistent selective pressure so that resistant bacteria survive and spread, resulting in antibiotic resistance.^10,11

In Indonesia, fungal infections, including candidiasis, are also common. Fungal infections occur in 20-25% of the world's population and are a common daily infection problem.¹²The prevalence of fungal infections has increased since 1980 in various patient groups.¹³Candidiasis can occur on the tongue and another oral mucosa; this candidiasis is oral candidiasis¹⁴. To a large degree, Candida albicans (C. albicans) was the primary causative agent of oral candidiasis in up to 95% of cases.¹⁵The research conducted by Rezeki et al. (2017) revealed that the treatment of oral candidiasis generally by the use of synthetic antifungal drugs, namely nystatin, amphotericin B, ketoconazole, fluconazole, clotrimazole, itraconazole, miconazole, and voriconazole, but these drugs have some side effects such as nausea, vomiting, chills, and fever.^16,17

Traditional medicine and treatment are increasingly gaining recognition in the health sector, with health research concluding that various plants benefit health.¹⁸Green tea has many benefits and is essential in the health sector.¹⁹One of the most abundant compositions in green tea was the Epigallocatechin-3-gallate (EGCG), which represents around 59% of the total catechins.^20–22

Based on research by Anita et al., EGCG in green tea has antibacterial activity against L. acidophilus bacteria, which leads to the destruction of bacteria²³. Moreover, the study of Lagha et al. (2017) revealed that the EGCG has the potential to be an anti-biofilm agent and weakens the adhesion ability of F. nucleatum.²⁴Furthermore, the research conducted by Murtiastutik et al. revealed that the EGCG has antifungal activity, which can destroy biofilms, increase the number of neutrophils, and reduce the number of cells infected by C. albicans.²⁵

Stem cells (SCs) are the unspecialized cells of the body. They can differentiate into any cell type and regenerate themselves.²⁶The potential of oral mesenchymal stem cells, including periodontal ligament stem cells,²⁷ gingiva stem cells (GMSCs),²⁸apical papilla stem cells²⁹, and Human Exfoliated Deciduous teeth stem cells (SHED).^30–32

Currently, Stem cells from SHED are getting a lot of attention because SHED has the potential to be widely used in the field of regenerative medicine^33,34. The tendency of lineage-specific differentiation, especially endothelial and nerve differentiation possessed by SHED, can be beneficial because nerve innervation and blood vessel formation are crucial in tissue regeneration.³⁵SHED metabolites can express Human β defensin 4 (HBD4), which has antimicrobial properties.^36,37Due to the various properties of EGCG, especially the anti-bacterial and antifungal properties and the wide use of SHED as regenerative medicine, this study aimed to prove the antimicrobial power of the combination of SHED metabolite with EGCG against F. nucleatum, L. acidophilus, and C. albicans.

MATERIALS AND METHODS:

Ethical clearance:

The study protocolwas approved by ethical clearances No.550/HRECC.FODM/VIII/2022 (Date approval: 15 August 2022) from the Faculty of Dentistry Research Ethics Commission, Airlangga University, Surabaya, Indonesia.

Study design:

The study was an experimental laboratory. This study was divided into three groups: a positive control group with doxycycline, a negative control group with distilled water, and SHED metabolite combined with EGCG solution as a treatment group.

Preparation of F. nucleatum and L. acidophilus culture bacteria:

Preparation of F. Nucleatum(ATCC 25586) and L. acidophilus culture begins by adding one-ose of F. nucleatum and L. acidophilus bacteria into a test tube containing 2ml of BHI broth (Oxoid, Thermo Scientific) into a test tube by passing it over a spirit lamp, then incubating it in an incubator at 37℃ for 2x24 hours. The incubated F. nucleatum and L. acidophilus cultures were vibrated with thermolyne, and the absorbance was measured according to 0.5 McFarland standards using a spectrophotometer with a wavelength of 560nm. Bacterial growth is characterized by turbidity in the media. The bacteria were obtained from the Research Center Laboratory of the Faculty of Dental Medicine, Airlangga University.³⁸

Preparation of C. albicans culture fungi:

Preparation of C. albicans culture begins by adding oneose of C. albicans fungi into a test tube containing 2 ml of Sabouraud Dextrose Broth (SDB) into a test tube by passing it over a spirit lamp, then incubating it in an incubator with a temperature of 37℃ for 2x24 hours. The incubated C. albicans culture was vibrated with thermolyne, and the absorbance was measured according to 0.5 McFarland standard using a spectrophotometer with a wavelength of 560nm. Fungal growth is characterized by turbidity in the media. The fungiwere obtained from the Research Center Laboratory of the Faculty of Dental Medicine, Airlangga University.³⁸

Preparation SHED metabolites:

SHED metabolites are purified from the SHED provided by the Research Centre Faculty of Dental Medicine Universitas Airlangga. The SHED was cultured from passages 1 to 6 in Dulbecco’s modified Eagle medium. SHED culture medium was purified using the dialysis method to remove waste products of metabolism that were not useful, resulting in beneficial results of metabolites that contained several cytokines, growth factors, and exosomes.

Preparation of EGCG combined with SHED:

The EGCG was produced by ChemFaces (C. Faces). The combination of SHED and EGCG metabolites was initiated by adding 0.5 mg/ml of EGCG solution with 0.5ml of SHED metabolite taken using a micropipette for each sterile microtube above the spirit lamp to obtain a 1mg/ml dose.³⁹

Disc Diffusion Method:

The disc diffusion method was conducted to measure the inhibition zone. The disk diffusion (Kirby-Bauer) method was used for susceptibility testing following the standards established by CLSI in 2012. The MHA surface-containing plates were evenly seeded with tested microorganisms and then placed on the bench to allow the extra fluid to be absorbed. Disc papers with a diameter of 5mm that had been dripped with 10μl of the 0, 5mg/ml EGCG combined with 0.5ml of SHED metabolite passages 1-6 were placed on the surface of the media as a treatment group. The positive control was equally dripped with doxycycline disc paper, while distilled water was used as a negative control. The plates were incubated anaerobically for 48 hours at 37°C, and then the diameter of the inhibition zone was measured in mm using a caliper.^40,41

Statistical Analysis:

Data, which is the result of inhibition zone measurements, were analyzed using SPSS software version 25. The data were analyzed for normality, Homogeneity, and parametric tests (One-way ANOVA).

RESULTS:

Regarding the disc diffusion method that was conducted to measure the inhibition zone. The antimicrobial activity of the combination of SHED metabolites with EGCG solution against L. Acidophilus, F. nucleatum bacteria, and C. albicans fungi was expressed as Mean (mm) ±standard deviation (Mean±SD) of inhibition zones diameter as described in Table 1.2.3.

Table 1. Average Results of Antimicrobial Activity of the SHED Metabolites combined with EGCG on the Growth of L. acidophilus Bacteria.

Group	*L.acidophilus* n=4	P
Group	Mean + SD	0,000*
C +	24,28 + 0,12
C -	0,00 + 0,00
P1	13,40 + 0,11
P2	14,19 + 0,17
P3	15,04 + 0,17
P4	15,94 + 0,13
P5	16,95 + 0,11
P6	18,34 + 0,29

Table 2. Average Results of Antimicrobial Activity of the SHED Metabolites combined with EGCG on the Growth of F. Nucleatum Bacteria.

Group	*F. nucleatum* n=4	P
Group	Mean + SD	0,000*
C +	21,71 + 0,09
C -	0,00 + 0,00
P 1	9,63 + 0,17
P 2	10,55 + 0,25
P 3	10,98 + 0,27
P 4	11,63 + 0,20
P 5	12,81 + 0,15
P 6	13,59 + 0,17

Table 3. Average Results of Antimicrobial Activity of the SHED metabolites combined with EGCG on the Growth of C. albicans Fungi.

Groups	*C. albicans* n=4	P
Groups	Mean + SD	0,000*
C+	10,13 + 0,06
C-	0,00 + 0,00
P1	0,00 + 0,00
P2	0,00 + 0,00
P3	0,00 + 0,00
P4	0,00 + 0,00
P5	0,00 + 0,00
P6	0,00 + 0,00

Table 1 and Graph 1 show the antimicrobial activity of the combination of SHED metabolites with EGCG solution, positive control group, and negative control group on L. acidophilus bacteria—no inhibition zones formed in control negative groups (0 mm). In contrast, the inhibition zone diameter of a combination of SHED metabolites with EGCG solution was shown higher in passage six than in passage 5 to passage 1. Doxycycline antibiotic formed the most significant inhibition zone diameter Figure 1,2.

Graph 1. The average inhibition of the growth of L. acidophilus bacteria based on the SHED metabolites combined with EGCG

The results of the one-way ANOVA test revealed a significance value of 0.001(p<0.05), which ensured a significant difference in inhibition zones. In other words, there was a significant difference in the antimicrobial activity of the combination of metabolites SHED with EGCG and doxycycline patches against the L. acidophilus bacteria.

Table 2 and Graph 2 show the antimicrobial activity of the combination of SHED metabolites with EGCG solution, positive control group, and negative control group on F. nucleatum bacteria—no inhibition zones formed in negative control groups (0 mm). In contrast, the inhibition zone diameter of a combination of SHED metabolites with EGCG solution was higher in passage six than in passage 5 to passage 1. Doxycycline antibiotic formed the most significant inhibition zone diameter Figure 3,4.

The results of the one-way ANOVA test revealed a significance value of 0.000 (p < 0.05), which ensured a significant difference in inhibition zones. In other words, there was a significant difference in the antimicrobial activity of the combination of metabolites SHED and doxycycline patches against the C. albicans fungi.

DISCUSSION:

Regarding the inhibition quality, the criteria for antibacterial activity in the study, the L. acidophilus, was in a strong category because the average diameter was 13.40mm + 0.11mm - 18.34mm + 0.29mm of the inhibition zone. Meanwhile, the F. nucleatum is included in the medium category because the average diameter of the inhibition zone obtained ranges from 9.63mm + 0.17mm - 13.59mm + 0.17mm. This result is similar to the study of Surjowardojo et al., which revealed that the inhibition zone diameter of <10mm was a moderate inhibition zone response, and the inhibition zone diameter of 11-20mm was a strong inhibition zone response.⁴²

The highest results of the inhibition zone of growth diameter in L. acidophilus bacteria were obtained by combining metabolites SHED passage 6 with EGCG, which showed an average of 18.34 + 0.29. This aligned with a study conducted by Anita et al., who stated that EGCG has antibacterial activity against L. acidophilus by causing disturbances in cell membranes and preventing DNA super-coiling, which leads to the destruction of L. acidophilus bacteria.²³

Moreover, the F. nucleatum bacteria inhibition zone diameter of the combined metabolite SHED passage six and EGCG was obtained with an average of 13.59 + 0.17. This is appropriate because EGCG also has antibacterial activity against F. nucleatum by inducing the secretion of human β-defensins by oral keratinocytes, where the antimicrobial peptide is active against F. nucleatum bacteria.²⁴

Based on the inhibition zone diameter average obtained, the inhibition of the combination of SHED and EGCG metabolites is more potent in inhibiting the growth of L. acidophilus bacteria.^43,44The inhibition zone diameter for the development of Gram-negative bacteria F. nucleatum, which was lower than the growth inhibition zone for Gram-positive bacteria L. acidophilus, was caused by differences in the composition of the cell wall between the two tested bacteria, resulting in different responses to the composition of the SHED metabolite with EGCG.⁴⁵The sensitivity of Gram-positive and Gram-negative bacteria to antibacterial substances depends on differences in the structure of their cell walls.⁴⁶Different from Gram-positive bacteria, Gram-negative bacteria have a peptidoglycan layer that is located on the periplasmic membrane, with the outside composed of a thick layer of lipopolysaccharide (LPS) as a form of defense for Gram-negative bacteria against foreign substances, including antibacterial compounds^47,48. This causes the Gram-negative bacteria F. nucleatum more resistant to combining SHED metabolites with EGCG than the Gram-positive bacteria L. acidophilus.

In terms of the inhibition quality, no antifungal power was found in this study, or it could not inhibit C. albicans because the average diameter of the inhibition zone in C. albicans was 0.00mm + 0.00mm. The results of this study, when compared with the results of previous studies, showed inappropriate results. In a study conducted by Murtiastusik et al., EGCG had antifungal activity against all strains of Candida isolates, whereas, in this study, the combination of SHED metabolites did not have antifungal activity against C. Albicans. ²⁵

In this study, procedures have been carried out according to several controlled aspects such as incubation time of 2x24 hours, incubation temperature of 37°C, sterility of tools, bacterial growth media, namely MHA, fungal growth media, namely SDA, and suspensions that are by the standard McFarlands 0.5. Due to the things mentioned, the factor that might cause the lack of antifungal power in this study is the presence of the C. albicans defense system. C. albicans has walls that provide protection against several physical and chemical threats and determine the morphology of cell shape. The cell wall has a dynamic structure, so it has excellent plasticity to allow changes in cell morphology, molecular remodelling, and cell wall composition.⁴⁹

In this study, all the highest results were obtained in combinations using the metabolite SHED passage 6. This aligned because a study by Cong et al. stated that in passage 3, there was an increase in differentiation ability and expression of cell surface markers. Then, in passage 9, the resulting expression rate decreased.⁵⁰ This may occur because increased passage in culture can affect cellular physiology and cell morphology, especially cell surface molecules. As passage increases in culture, cells lose the ability to differentiate, doubling cells' capacity and telomere length.⁵¹

The present study provides a novel antimicrobial treatment using a combination of SHED metabolites with EGCG. They have high antibacterial properties against L. acidophilus bacteria and moderate against F. nucleatum. Although the finding of this study provides a promising effect, its limitation lies in the fact that all conclusions were under idealistic conditions in vitro, and whether the same results can be obtained clinically is still being determined. For that reason, it is necessary to do further in vitro research on other bacteria and fungi that cause oral diseases. Moreover, both in-vivo and clinical research confirm the utilization of EGCG combined with SHED metabolite, especially regarding the optimization of dosage, toxicity, and side effects in the case of oral and dental health problems, especially periodontal disease.

CONCULSION:

In this study, it can be concluded that the combination of SHED metabolites with EGCG has antimicrobial properties in the form of high antibacterial properties against L. acidophilus bacteria and moderate antibacterial properties against F. nucleatum. However, it does not have antimicrobial properties in the form of antifungal properties against C. albicans.

CONFLICTS OF INTEREST:

No conflict of interest.

ACKNOWLEDGMENTS:

We want to thank Faculty of Dental Medicine, Universitas Airlangga Surabaya, Indonesia and the Directorate General of Higher Education, Ministry of Education Culture, Research and Technology, Indonesia.

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Received on 25.12.2023 Revised on 12.04.2024

Accepted on 13.06.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):885-892.

DOI: 10.52711/0974-360X.2025.00130

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.