189 Dental Journal (Majalah Kedokteran Gigi) 2024 September; 57(3): 189–194 Original article Antibacterial assessment of Ziziphus mauritiana Lam on inhibition of the growth and biofilm of Streptococcus mutans Dharli Syafriza1, Uswatun Hasanah2, Taufiqi Hidayatullah1, Ayudia Rifki1, Basri A. Gani3 1Department of Pediatric Dentistry, Faculty of Dentistry, Universitas Syiah Kuala, Banda Aceh, Indonesia 2Undergraduate Student, Faculty of Dentistry, Universitas Syiah Kuala, Banda Aceh, Indonesia 3Department of Oral Biology, Faculty of Dentistry, Universitas Syiah Kuala, Banda Aceh, Indonesia ABSTRACT Background: Streptococcus mutans (S. mutans) causes dental caries. S. mutans biofilms are inhibited by the antibacterial properties of Ziziphus mauritiana Lam (Z. mauritiana Lam). Purpose: Evaluating the potential of Z. mauritiana Lam in inhibiting the growth and biofilm formation of S. mutans ATCC 25175 in vitro. Methods: This study used the ethanolic extract of Z. mauritiana Lam as the test material and S. mutans as the research subject. Spectrophotometry (620 nm) was used to assess the growth of S. mutans, the inhibition of S. mutans biofilm using a 1% crystal violet staining was measured by spectrophotometry (520 nm), and the visualization of the biofilm mass was conducted with an electric microscope (200x). Results: At all concentrations, Z. mauritiana Lam displayed excellent growth inhibition of S. mutans 0.04–0.09 (< 300 CFU/mL) and was able to inhibit the formation of S. mutans biofilm with a strong scale of optical density (OD) 0.4 at 24 hours incubation time. At the same time, the incubation time of 48 and 72 hours tended to have moderate-scale biofilm inhibition (OD 0.2–3.9). At a concentration of 25%, the biofilm mass decreased by a relatively small size, the same as the positive control group. At 50%, 12.5%, and 6.25%, it was seen that the S. mutans biofilm mass experienced a dominant loss. Conclusion: Z. mauritiana Lam can be bacteriostatic against the growth of S. mutans and can inhibit the formation of biofilms by degrading the structure and mass of S. mutans biofilms. Keywords: biofilm; dental caries; growth; Streptococcus mutans; Ziziphus mauritiana Lam Article history: Received 1 August 2023; Revised 14 November 2023; Accepted 13 December 2023; Published 1 September 2024 Correspondence: Dharli Syafriza, Department of Pediatric Dentistry, Faculty of Dentistry, Universitas Syiah Kuala, Jl. Hamzah Fansuri Kopelma Darussalam, Banda Aceh, Aceh, Indonesia, 23111. Email: dharli.usk@gmail.com INTRODUCTION Streptococcus mutans (S. mutans) has been reported as the primary agent in caries pathogenesis. These bacteria can form biofilms and produce acid from carbohydrate fermentation. A biofilm is a collection of microorganisms that can adhere to solid surfaces, such as tooth enamel, tooth roots, or dental implants.1 Microorganisms in biofilms can produce an extracellular matrix and live surrounded by this matrix. The process of attachment of bacteria to the tooth surface occurs in the early stages of biofilm formation. Specific and nonspecific interactions control bacterial attachment to the tooth surface.2 Glucans mediate particular interactions, while nonspecific interactions involve hydrophobic interactions. This interaction activity can increase the or biofilm formation.3 The increased biofilm formation by S. mutans on the dental pellicle may make it easier to enhance the stability of quorum-sensing formation with other oral bacteria.4 This property can interfere with the mucosal defense system or the activity of the tooth pellicle protein to stem the adhesion of S. mutans, thus making it easier for S. mutans to increase its growth rate.5 Biofilm eradication and inhibiting the growth of S. mutans is one strategy to reduce dental caries infection.6 Efforts to eliminate S. mutans as normal flora in the mouth with antibiotics are considered inappropriate because they can disrupt the ecological balance of the oral cavity.7 The extended use of antibiotics tends to increase the resistance of S. mutans, lead to an increase in biofilm formation and growth. It will be a severe problem in the treatment of dental caries.8 Karikalan and Mohankumar9 Copyright © 2024 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v57.i3.p189–194 mailto:dharli.usk@gmail.com https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v57.i3.p189-194 190Syafriza et al. Dent. J. (Majalah Kedokteran Gigi) 2024 September; 57(3): 189–194 demonstrated that prolonged exposure to antibiotics leads to the development of resistance in S. mutans against ampicillin and Penicillin G. Throughout history, humans have used plants as anti-microbial agents to prevent tooth infections and dental caries.10 One plant whose properties may help prevent biofilm formation and reduce the growth of S. mutans by Z. mauritiana Lam.11 Z. mauritiana Lam is reported to have antifungal and antibacterial properties. Its high antioxidant content allows this plant to neutralize bacterial virulence activities, including inhibiting the growth and formation of S. mutans biofilm as a prerequisite for dental caries.11 In addition, Z. mauritiana Lam contains secondary metabolites such as flavonoids, saponins, and tannins that aid in reducing bacterial growth and removing dental plaque.12 This study aims to evaluate the antibacterial properties of Z. mauritiana Lam in inhibiting the growth and formation of S. mutans biofilm in vitro. MATERIALS AND METHODS This study acquired ethical clearance under No. 324.KE/ FKG/2021 from the Faculty of Dentistry, Universitas Syiah Kuala, Banda Aceh, Indonesia. The research was conducted in vitro, using Z. mauritiana Lam as a test material to evaluate its antibacterial potential for inhibiting the growth and formation of biofilm S. mutans ATCC 25175. The concentration and incubation time were used as a reference to create an overview of the optimum inhibition of each concentration and incubation time. Z. mauritiana Lam was obtained from Aceh Besar District, Province of Aceh, Indonesia (5.570704102637194, 95.35980633540842). The extraction of assay materials and Gas chroma- tography/mass spectrometry (GCMS) analysis of chemical compounds were adopted from Soraya et al.2 One kilogram of Z. mauritiana Lam leaves was chopped and macerated in five liters of 96% ethanol for 24 hours while stirring every four hours, after which it was decanted and filtered. The resulting residue was macerated for 48 hours with fresh 96% ethanol. The filtrate was then evaporated using a rotary vacuum evaporator to obtain a concentrated extract, which was then heated to 45 0C any remaining ethanol. The spectrophotometric assessment of S. mutans showed its growth was initiated by preparing Z. mauritiana Lam extract at various concentrations. Each well of the 96-well plate received 50 µL of Tryptic Soy Broth (TSB) medium, was incubated for 15 minutes, and then washed twice with (Phosphate-buffered saline) BS (pH 7.0). The S. mutans was then placed in a 25 µL well in the medium and incubated for 15 minutes at room temperature (27°C). Each well of 100 µL contained a predetermined concentration of test material (1:4), which was then incubated in an anaerobic atmosphere for 24, 48, and 72 hours. Using spectrophotometry (Bio-Rad, USA) with an optical density (OD) of 620 nm, the quantity of S. mutans was measured based on turbidity.13 S. mutans biofilm was measured using the 1% violet crystal method, according to Gani et al 14. The Z. mauritiana Lam was extracted at 50%, 25%, 12.5%, 6.25%, and 3.125%. They were coated on a 96-well plate with 100 µL of TSB medium, allowed to incubate for 15 minutes, and rinsed with PBS (pH 7.0), after which 25 µL of S. mutant was added to each well, then allowed to incubate for 15 minutes at room temperature. S. mutans and assay material were added to each well containing a different test (triple serial), homogenized at 1,000 xg for five minutes, and incubated anaerobically for 24, 48, and 72 hours. The formation of S. mutans biofilms was evaluated by removing all solutions from the wells and washing them with PBS in a dishwasher at 1,000 xg for five minutes (repeated twice). Then, 150 µL of 1% violet crystal was injected into each well of the plate. The crystalline violet dye and biofilm protein were then homogenized for 10 minutes using a shaker at 100 xg. Each well was rinsed with 150 µL of PBS for five minutes, after which the solution was discarded and replaced with 150 µL of 70% ethanol for one minute. Following marking the 96-well plates containing biofilms based on the absorption of violet crystalline dyes, the plates were incubated at room temperature for 15 minutes. At 520 nm, the biofilm mass of S. mutans was measured using a spectrophotometer. Anti-biofilm evaluation was based on OD spectrophotometry: OD ≥ 0.4 (strong); OD = 0.2–0.39 (moderate); OD = 0.05–0.19 (low); OD ≤ 0.05 (weak, no biofilm formation).13 The differences in the growth of S. mutans with biofilm formation and its correlation to incubation time were analyzed with the Kruskal-Wallis test with p < 0.05 as a significant reference. Table 1. Growth inhibition of S. mutans after being influenced by Z. mauritiana Lam Z. mauritiana Lam N Growth Inhibition of S. mutans (OD 620 nm) *p-value24 h 48 h 72 h Mean S.Devt Scale Mean S.Devt Scale Mean S.Devt Scale 50% 3 0.099 0.005 10% 0.0890 0.002646 9% 0.0900 0.002 13% 0.004 25% 3 0.084 0.005 9% 0.0917 0.012423 9% 0.0870 0.06811 13% 12.5% 3 0.058 0.060 6% 0.0943 0.112401 9% 0.0461 0.0461 7% 6.25% 3 0.047 0.027 5% 0.0963 0.126255 10% 0.0941 0.0941 14% 3.125% 3 0.055 0.017 6% 0.0853 0.078647 9% 0.0625 0.0625 9% CHX 3 0.081 0.005 8% 0.0837 0.007095 8% 0.0850 0.0850 13% S. mutans 3 0.539 0.114 56% 0.4627 0.019296 46% 0.2050 0.2050 31% *p-value 0.02 0.189 0.188 *Kruskal-Wallis Test Copyright © 2024 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v57.i3.p189–194 https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v57.i3.p189-194 191 Syafriza et al. Dent. J. (Majalah Kedokteran Gigi) 2024 September; 57(3): 189–194 Table 2. The influence of Z. mauritiana Lam on the inhibition of S. mutans biofilm formation Z. mauritiana Lam N Inhibition of S. mutans biofilm (520 nm) 24 h 48 h 72 h *p-value Mean S.Devt Scale Mean S.Devt Scale Mean S.Devt Scale 50% 3 0.53 0.58 14% 0.365 0.481 18% 0.457 0.397 17% 0.017 25% 3 0.48 0.69 13% 0.225 0.151 11% 0.352 0.216 13% 12.5% 3 0.87 0.59 23% 0.271 0.052 13% 0.461 0.461 17% 6.25% 3 0.67 0.11 18% 0.303 0.037 15% 0.376 0.376 14% 3.125% 3 0.66 0.10 17% 0.352 0.026 17% 0.406 0.406 15% CHX 3 0.55 0.40 15% 0.545 0.399 26% 0.619 0.619 23% *p-value 0.02 0.061 0.172 *Kruskal-Wallis Test RESULTS This study evaluated the antibacterial properties of Z. mauritiana Lam in inhibiting the growth and formation of S. mutans biofilms. Table 1 summarizes the growth inhibition of S. mutans by various concentrations of Z. mauritiana Lam extract over 24, 48, and 72 hours. At a 50% concentration, the extract showed 10% inhibition at 24 hours, 9% at 48 hours, and 13% at 72 hours, with a significant p-value of 0.004 at 72 hours. The 25% concentration consistently inhibited growth by 9% at both 24 and 48 hours, increasing to 13% at 72 hours. The 12.5% concentration showed 6% inhibition at 24 hours, 9% at 48 hours, and 7% at 72 hours. At 6.25%, inhibition was 5% at 24 hours, 10% at 48 hours, and 14% at 72 hours. The 3.125% concentration consistently inhibited growth by 6% at 24 hours, 9% at 48 hours, and 9% at 72 hours. The control group using CHX showed consistent inhibition of 8% at both 24 and 48 hours, increasing to 13% at 72 hours. In contrast, the untreated S. mutans group showed significantly higher growth, with 56% at 24 hours, 46% at 48 hours, and 31% at 72 hours. The p-values indicate significant inhibition at 24 hours (0.02), but not at 48 hours (0.189) and 72 hours (0.188). The degradation of S. mutans biofilm mass due to the influence of various concentrations of Z. mauritiana Lam extract is depicted in Figure 1. At a concentration of 25% (B), the biofilm mass showed a slight reduction, similar to the positive control group (F). However, at concentrations of 50% (A), 12.5% (C), and 6.25% (D), there was a significant loss of biofilm mass, indicating that Z. mauritiana Lam extract is effective in inhibiting the formation of S. mutans biofilm in vitro. Table 2 shows the effect of Z. mauritiana Lam extract on inhibiting S. mutans biofilm formation over 24, 48, and 72 hours. At a 50% concentration, biofilm inhibition was 14% at 24 hours 0.53±0.58), increasing to 18% at 48 hours (0.365±0.481) and 17% at 72 hours (0.457± 0.397), with a p-value of 0.017 indicating statistical significance at 24 hours. At a 25% concentration, biofilm inhibition was 13% at 24 hours, 11% at 48 hours, and 13% at 72 hours. The 12.5% concentration showed the highest inhibition of 23% at 24 Figure 1. Mass of S. mutans biofilm. Mass degradation of S. mutans biofilm after being influenced by various concentrations of Z. mauritiana Lam extract. (A) 50%, (B) 25%, (C) 12.5%, (D) 6.25%, (E) 3.125%, (F) Positive control (CHX). Yellow arrow (biofilm mass), green arrow (missing biofilm mass), and red arrow (inactive biofilm mass). One percent crystal violet stain with 200x magnification. Copyright © 2024 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v57.i3.p189–194 https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v57.i3.p189-194 192Syafriza et al. Dent. J. (Majalah Kedokteran Gigi) 2024 September; 57(3): 189–194 hours, which decreased to 13% at 48 hours and 17% at 72 hours. At a 6.25% concentration, inhibition was 18% at 24 hours, 15% at 48 hours, and 14% at 72 hours. At a 3.125% concentration, biofilm inhibition was 17% at 24 and 48 hours and 15% at 72 hours. The control group using CHX showed consistent inhibition, with 15% at 24 hours, 26% at 48 hours, and 23% at 72 hours. Overall, higher concentrations of Z. mauritiana Lam extract tended to show greater biofilm inhibition. The 50% concentration showed significant inhibition at 24 hours, while CHX, as the control, demonstrated consistent inhibition but was most effective at 48 hours. The p-values indicate statistical significance at 24 hours (0.02) but not at 48 hours (0.061) and 72 hours (0.172). The extract of Z. mauritiana Lam demonstrated efficacy in reducing the growth frequency of S. mutans, leading to a consequent decrease in biofilm formation, as illustrated in Figures 1 and 2. The analysis revealed that, after 24 hours, the 50% and 25% concentrations exhibited a notably close interaction compared to the other experimental groups. At the 48-hour mark, a marked similarity in the interaction was observed between the 25% and 12.5% concentrations. By the 72-hour mark, the interaction between the 25% and 6.25% concentrations was very close. This interaction was methodically analyzed based on the percentage of inhibition gain attributed to each concentration of the test substance. DISCUSSIONS This study evaluated the antibacterial properties of Z. mauritiana Lam on the growth and formation of S. mutans biofilms. The study focuses on these two virulence properties because they are critical in decreasing adhesion in dental caries’ pathogenesis. Z. mauritiana Lam is reported to Figure 2. The relationship between growth inhibition and biofilm formation of S. mutans after being affected by Z. mauritiana Lam extract. The most substantial interaction occurred at 72 hours of incubation. The black box lines on the bar lines indicate the interaction between the growth and formation of S. mutans biofilms. Copyright © 2024 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v57.i3.p189–194 https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v57.i3.p189-194 193 Syafriza et al. Dent. J. (Majalah Kedokteran Gigi) 2024 September; 57(3): 189–194 have several secondary metabolites that act as antioxidants, antifungals, and antibacterials. The antibacterial role tends to be facilitated by flavonoids, saponins, and tannins.15 Arma et al.16 reported that Z. mauritiana Lam contains antioxidant and antifungal compounds, such as Vitamin E, squalene, and phytol. Several of these compounds have been reported to reduce the virulence properties of S. mutans potentially. Table 1 shows that Z. mauritiana Lam could inhibit the growth of S. mutans. All concentrations demonstrated excellent inhibitory ability against S. mutans 0.04–0.09 (< 300 CFU/mL). The power of Z. mauritiana Lam to inhibit the growth of S. mutans is related to the role of several active antibacterial compounds found in Z. mauritiana Lam. Jubair et al.17 reported that almost all plants are rich in secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found to have anti-microbial properties in vitro. Cellularly, antibacterial compounds increase the action potential on the surface of the cell membrane so that the cell surface exerts pressure, which causes changes in the membrane’s permeability properties, which impacts membrane leakage.13 These impacts can cause the release of intracellular fluid and cells, causing lysis.18 According to Giovannoni et al.,19 releasing cellular contents from the cell wall causes the cell wall to degrade. This phenomenon suggests a possible antibacterial mechanism by which Z. mauritiana Lam inhibits the growth of S. mutans and the cellular response to dental caries treatment. The release of intracellular fluid can cause a decrease in the cellular response to cell division activity so that cells do not form new daughters.20 The fungistatic properties of Z. mauritiana Lam have increased the sensitivity of the cellular response to its growth, thus indirectly influencing the development and response of infection to the host. Miller and Zachary21 reported that DNA activity often influences mitotic failure, with intracellular fluid outside the cell membrane wall disrupting genetic communication and consequently affecting protein synthesis, which DNA facilitates. Table 2 reports that Z. mauritiana Lam can inhibit the formation of S. mutans biofilms. The incubation time of 24 hours tends to result in strong biofilm inhibition, while the incubation time of 48 and 72 hours tends to yield moderate-scale biofilm inhibition. The biofilm inhibitory properties of Z. mauritiana Lam correlate with the intensity of bacterial growth (Figure 1). Based on the findings from this study, Z. mauritiana Lam can interfere with and inhibit the formation of S. mutans biofilms (Figure 1). This phenomenon indicates that Z. mauritiana Lam can interfere with communication between the bacterial cells involved in biofilm formation.16 The increase in conductivity caused by the chemical compounds in Z. mauritiana Lam could be involved in the interruption of communication between bacterial cells during quorum sensing formation,1 which could interfere with the biofilm formation of S. mutans. Meanwhile, the antibacterial compounds contained in Z. mauritiana Lam can inhibit biofilm formation by reducing the morphology or degrading the structure of the biofilm12 so that the biofilm mass loses cohesion and cannot adhere to the pathogenesis of caries infection. This study reported that the extract of Z. mauritiana Lam degraded the biofilm’s mass and decreased the biofilm’s morphology. From the perspective of biofilm inhibition modeling, Ghosh et al.22 reported that the inhibition of biofilms by antibacterial compounds could be initiated by inhibiting bacterial surface adhesion, disrupting the quorum-sensing system, disrupting the signaling of genetic information, interfering with biofilm maturation, and degrading biofilm mass. The small organic compounds present in several anti- infective plants can prevent bacterial infection by inhibiting the adhesion of the bacterial surface to the host cell and interfering with the quorum-sensing system in the biofilm formation process.22 In conclusion, Z. mauritiana Lam can be bacteriostatic against the growth of S. mutans and can inhibit the formation of biofilms by degrading the structure and mass of S. mutans biofilms. The limitation of this study was that there was no attempt to examine how the antibacterial properties of Z. mauritiana Lam might interfere with the communication of S. mutans cells with other bacteria in the formation of quorum sensing and biofilms. This study gives hope that Z. mauritiana Lam may be used as an active preventative against dental caries. ACKNOWLEDGMENT Universitas Syiah Kuala funded this research, Ministry of Education, Culture, Research, and Technology following the Letter of Assignment of Research Assignment for the Implementation of “Riset Asisten Ahli for 2022”, Number: 148/UN11/SPK/PNBP/2022 dated February 11, 2022. REFERENCES 1. Rahayu YC, Setiawatie EM, Rahayu RP, Ramadan DE. Analysis of antioxidant and antibacterial activity of cocoa pod husk extract (Theobroma cacao L.). Dent J. 2023; 56(4): 220–5. 2. Soraya C, Mubarak Z, Gani BA. The growth and biofilm formation of Enterococcus faecalis in ethanol extract of Citrus aurantiifolia Indonesian species. 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