Effects of Hibiscus sabdariffa L. extract on multispecies Porphyromonas gingivalis and Treponema denticola biofilms in vitro

Angela Winson; Trijani Suwandi; Olivia Nauli Komala

doi:10.4103/SDJ.SDJ_4_23

ORIGINAL ARTICLES

Year : 2023 | Volume : 7 | Issue : 1 | Page : 26-32

Effects of Hibiscus sabdariffa L. extract on multispecies Porphyromonas gingivalis and Treponema denticola biofilms in vitro

Angela Winson¹, Trijani Suwandi², Olivia Nauli Komala²
¹ Faculty of Dentistry, Universitas Trisakti, Jakarta, Indonesia
² Department of Periodontology, Faculty of Dentistry, Universitas Trisakti, Jakarta, Indonesia

Date of Submission	06-Dec-2022
Date of Decision	11-Jan-2023
Date of Acceptance	13-Feb-2023
Date of Web Publication	17-May-2023

Correspondence Address:
Trijani Suwandi
Department of Periodontology, Faculty of Dentistry, Universitas Trisakti, Kyai Tapa No. 260, West Jakarta
Indonesia

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/SDJ.SDJ_4_23

Abstract

Background: Porphyromonas gingivalis and Treponema denticola are bacteria that play a crucial role in periodontitis. Hibiscus sabdariffa L. contains flavonoids, alkaloids, saponins, and tannins; compounds which are expected to inhibit biofilm formation with minimal side effects. Objectives: To determine the secondary metabolite compounds present in H. sabdariffa L. and its effect on multispecies biofilms formation consisted of P. gingivalis and T. denticola. Methods: This study used H. sabdariffa L. calyx extract with concentrations of 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, and 1.5625%. The positive control used in this study was 0.2% chlorhexidine, and the negative control was brain heart infusion broth. The bacterial biofilm was grown for 1, 6, and 24 hours. Results: Hibiscus sabdariffa L. 1.5625% extracted in a 1-h incubation period, 6.25% extracted in a 6-h incubation period, and 3.25% extracted in a 24-h incubation period are more effective than chlorhexidine 0.2% in inhibiting multispecies P. gingivalis and T. denticola biofilms (P < 0.05). Conclusion: Hibiscus sabdariffa L. calyx extract significantly inhibits multispecies P. gingivalis and T. denticola biofilm with the most effective concentration at 100% at 1-h, 6-h, and 24-h incubation periods. Future studies are needed to test the toxicity of H. sabdariffa L. on cells in the oral cavity.

Keywords: Biofilm, Hibiscus sabdariffa L., Porphyromonas gingivalis, Treponema denticola

How to cite this article:
Winson A, Suwandi T, Komala ON. Effects of Hibiscus sabdariffa L. extract on multispecies Porphyromonas gingivalis and Treponema denticola biofilms in vitro. Sci Dent J 2023;7:26-32

How to cite this URL:
Winson A, Suwandi T, Komala ON. Effects of Hibiscus sabdariffa L. extract on multispecies Porphyromonas gingivalis and Treponema denticola biofilms in vitro. Sci Dent J [serial online] 2023 [cited 2023 Jun 2];7:26-32. Available from: https://www.scidentj.com/text.asp?2023/7/1/26/377189

Background

Periodontitis is a disease that affects the periodontal tissue and is characterized by chronic inflammation that is destructive, progressive, and can affect the attachment apparatus.^[1] The main cause of periodontitis is the accumulation of biofilms on the tooth surface. Biofilms are communities of microorganisms attached to each other and embedded in the extracellular matrix (EPS).^[2]Porphyromonas gingivalis (P. gingivalis) and Treponema denticola (T. denticola) are two bacteria species that show a synergistic relationship in the formation of polymicrobial biofilm. These two bacteria play a crucial role in the pathogenesis of periodontitis.^[1]

The risk of periodontal disease can be minimized by reducing biofilm formation.^[3] In addition to the main mechanical methods, mouth rinses can be utilized to lessen the production of biofilm. The gold standard commonly used as an anti-plaque agent mouthwash is chlorhexidine.^[3] However, chlorhexidine has side effects such as tooth discoloration, burning sensation, and mucosal irritation.^[4] According to the World Health Organization (WHO), in recent years, many people have started using traditional medicinal plants, which are believed to have minimal side effects compared to chemical anti-plaque agents. One of which is Hibiscus sabdariffa L. (Roselle).^[5]

Hibiscus sabdariffa L., a member of the Malvaceae family of plants, is well known as a plant that has many beneficial uses, such as being used to make drinks, food flavorings, and herbal medicines^.[3]Hibiscus sabdariffa L. is also known to have antibacterial, antipyretic, anti-cholesterol, and antioxidant properties.^[6] The antibacterial properties of H. sabdariffa L. calyx are shown by the presence of secondary metabolites such as flavonoids, alkaloids, saponins, and tannins.^[7] Previous studies have shown that H. sabdariffa L. calyx extract has antibacterial properties against Streptococcus pyogenes^[7] and can inhibit the biofilm of Fusobacterium nucleatum,^[8]P. gingivalis,^[8] and Streptococcus sanguinis in vitro.^[9] However, the effect of H. sabdariffa L. calyx extract on the formation multispecies P. gingivalis and T. denticola has not yet been studied.

Materials and Methods

This research was carried out in vitro at the Microbiology Centre of Research and Education (MiCORE) Laboratory, Faculty of Dentistry, Trisakti University as an experimental laboratory study with a post-test only control group design. Hibiscus sabdariffa L. used in this study was obtained and tested for its constituents at the Balai Penelitian Tanaman Rempah dan Obat (BALITTRO), Bogor, Indonesia.

Hibiscus sabdariffa L. calyx extract

Hibiscus sabdariffa L. calyx was collected from Balai Penelitian Tanaman Rempah dan Obat (BALITTRO), Bogor, Indonesia and identified by the Lembaga Ilmu Pengetahuan Indonesia. Hibiscus sabdariffa L. calyxes were dried in the open air at room temperature for 14–21 days, weighed at 1000 g and then ground into powder using a grinder. The obtained simplisa powder was immersed in 96% ethanol with a ratio of 1:10 under sun-shielded conditions and covered with aluminum foil for 3 days, with periodic shaking of the solution.^[10] The solution was then filtered and evaporated using an evaporator at a maximum temperature of 50ºC. Hibiscus sabdariffa L. calyx extract was diluted with aquadest (Schurchard, Hohenburnn, Germany) into six concentrations: 50%, 25%, 12.5%, 6.25%, 3.125%, and 1.5625%. The extracts were then placed in a closed container and further stored in a refrigerator with a temperature of 4ºC before use in the biofilm experiment.

Phytochemical assay

The ethanol extract of H. sabdariffa L. calyx extracts was examined for phytochemical assay. This assay was conducted qualitatively to evaluate the presence of alkaloids, saponins, tannins, phenolics, flavonoids, triterpenoids, steroids, and glycosides. Thus, the qualitative results are shown by (+) for the presence and (–) for the absent of these substances.

Bacterial culture and biofilm assay

Porphyromonas gingivalis (ATCC 33277) and T. denticola (ATCC 35405) from laboratorium stock (–80ºC) was first grown on Brain Heart Infusion (BHI) agar medium, and incubated for 24 h at 37ºC in an anaerobic jar containing gaspack. The bacterial colonies were further inoculated in BHI broth (Sigma Aldrich, St Louis, Missouri, United States) for 2 × 24 h. The cultured bacterial was each diluted and standardized to achieve an optical density (OD) of 0.25–0.3 or 1 × 10⁷ colony-forming units (CFU)/mL.

This experiment was performed in triplicate and was conducted once without repetition. For the positive control, 0.2% chlorhexidine was used, and BHI was used as the negative control. The samples of 200 µL, each containing 1 × 10⁷ CFU/mL of mixture P. gingivalis and T. denticola bacterial suspension was first distributed into a 96-well-flat-bottom microplate (Nest Biotech, Wuxi, Jiangsu, China) and incubated for 48 h at the temperature of 37ºC and under microaerophilic condition as stated above. After the 48-h incubation period, the supernatant in each well of the plate was removed and rinsed by using the phosphate buffer saline (PBS) (Oxoid, Hampshire, United Kingdom). Hibiscus sabdariffa L. calyx extract with concentrations of 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, and 1.5625% were then added to each well-plate and incubated for 1 h, 6 h, and 24 h before once more being rinsed with PBS. Biofilm staining was performed using crystal violet (0.05%, w/v) (Merck, Darmstadt, Germany) for a 15-min incubation period and rinsed again with PBS. The quantitative analysis of biofilm production was performed by adding 200 μL of 96% ethanol to destain the wells and measured using a microplate reader (Safas, Monaco, Europe) at 490 nm.

Statistical analysis

The statistical tests were performed using Statistical Package for the Social Sciences (SPSS) version 26.0 (IBM, Armonk, New York, United States). Saphiro-Wilk and Levene’s tests were used to test for finding normality and homogeneity, respectively. If data are normally distributed (P > 0.05), then the statistical test is continued with one-way analysis of variance (ANOVA) with a significance level of P < 0.05. This was followed by Least Significant Difference post-hoc test (P < 0.05) if the data are homogenous, and Tamhane’s T2 test (P < 0.05) if the data are heterogenous.^[11]

Results

Phytochemical test showed that the extract of Hibiscus sabdariffa L. calyx with 96% ethanol solvent contains alkaloids, saponins, tannins, phenolics, flavonoids, triterpenoids, and glycosides [Table 1].

Table 1: Phytochemical test results of the ethanol extract of Hibiscus sabdariffa L.

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The results of this study showed that H. sabdariffa L. calyx extract was able to inhibit multispecies biofilm formation consisted of P. gingivalis and T. denticola (OD ± mean) at all incubation periods (P < 0.05) set in the current study.

At the 1-h incubation period, all concentrations of H. sabdariffa L. calyx extract show significantly lower OD values (P < 0.05) compared to the positive control (OD 1.462 ± 0.041). The smallest concentration that was more effective in inhibiting P. gingivalis and T. denticola multispecies biofilm, compared to the positive control, at the 1-h incubation period was 1.5625% (OD 0.359 ± 0.064) [Table 2], [Figure 1].

Table 2: The average of OD ± SD H. sabdariffa L. ethanol extract against multispecies P. gingivalis and T. denticola at 1-hour incubation period

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Figure 1: Graph showing the effectiveness of the H. sabdariffa L. calyx extract against multispecies P. gingivalis and T. denticola biofilms after a treatment period of 1 h. Each graph shows the average of triplicate experiments (*P<0.05)

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At the 6-h incubation period, H. sabdariffa L. calyx extract with a concentration of 100%, 50%, 25%, 12.5%, and 6.25% showed significantly lower OD values, compared to the positive control (OD 1.130 ± 0.027). The smallest concentration that was more effective in inhibiting P. gingivalis and T. denticola multispecies biofilm compared to the positive control at the 6-h incubation period was 6.25% (OD 0.287 ± 0.059). The difference was statistically significant (P < 0.05). The result was seen in [Table 3] and [Figure 2].

Table 3: The average of OD ± SD H. sabdariffa L. ethanol extract against multispecies P. gingivalis and T. denticola at 6-hour incubation period

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Figure 2: Graph showing the effectiveness of the H. sabdariffa L. calyx extract against multispecies P. gingivalis and T. denticola biofilms after a treatment period of 6 h. Each graph shows the average of triplicate experiments (*P < 0.05)

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At the 24-h incubation period, H. sabdariffa L. calyx extract with a concentration of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125% showed significantly lower OD values compared to the positive control (OD 1.042 ± 0.023). The smallest concentration that was more effective in inhibiting P. gingivalis and T. denticola multispecies biofilm compared to the positive control at 24 h incubation period was 3.125% (OD of 0.171 ± 0.070). The difference was statistically significant (P < 0.05). The result was seen in [Table 4] and [Figure 3].

Table 4: The average of OD ± SD H. sabdariffa L. ethanol extract against multispecies P. gingivalis and T. denticola at 24-h incubation period

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Figure 3: The effectiveness of the H. sabdariffa L. calyx extract against multispecies P. gingivalis and T. denticola biofilms after a treatment period of 24 h. Each graph shows the average of triplicate experiments (*P < 0.05)

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Discussion

In this study, our data showed that based on the group of concentration H. sabdariffa L. calyx extract, concentration of 100% is the most effective in inhibiting multispecies of P. gingivalis and T. denticola biofilm formation. The smallest concentration that was more effective in inhibiting P. gingivalis and T. denticola multispecies biofilm formation, compared to the 0.2% chlorhexidine at 1-h, 6-h, and 24-h incubation periods was 1.5625%, 6.25%, and 3.125%, respectively.

The incubation time of this study was chosen to evaluate the effect of H. sabdariffa L. calyx extract on each different phase of the formation of biofilms. An incubation period of 1 hour indicates the initial attachment phase (0–4 h), an incubation period of 6 h indicates the active accumulation phase (4–20 h), and an incubation period of 24 h indicates the biofilm maturation phase (after 24 h).^[9]

This study showed that the H. sabdariffa L. calyx extract displayed the best outcomes at the 1-h incubation period between the incubation periods. This is because the bacteria’s attachment is still reversible and easily removed during the initial adhesion phase. Contrary to the 1-h incubation period, the 24-h incubation period exhibits a higher OD. This may occur because in the maturation phase it is more difficult for antibacterial agents to enter the biofilm due to the presence of a glycocalyx matrix that strengthens and protects bacterial immunity to antibacterial agent.^[12]

This study showed that higher concentrations were more effective at inhibiting the formation of multispecies biofilms. This means that the H. sabdariffa L. calyx effects on the formation of the biofilm are concentration-dependent. This is supported by research conducted by Ji et al.,^[7] regarding the antibacterial effect of H. sabdariffa L. calyx extract on Streptococcus pyogenes using the Kirby-Bauer agar diffusion method, which showed that H. sabdariffa L. calyx extract with the concentration of 5%, 10%, 20%, and 30% resulted in the inhibition zone of 7.13, 8.4, 10.2, and 13.3 mm, respectively. This suggests that the extract’s antibacterial activity increases with extract concentration.

Phytochemical component of H. sabdariffa L. calyx extract is the key for the extract’s ability to inhibit the formation of biofilms. The results of the phytochemical tests showed that the ethanol extract of H. sabdariffa L. calyx used in this study contains secondary metabolites such as alkaloids, saponins, tannins, phenolics, flavonoids, triterpenoids, and glycosides. Our finding was supported by a phytochemical screening conducted by Suniarti et al.,^[13] which found that the flower calyx extract of H. sabdariffa L. contains saponins, tannins, phenols, flavonoids, triterpenoids, and glycosides.

The result obtained showed that H. sabdariffa L. calyx extract could inhibit P. gingivalis and T. denticola biofilm formation because OD of bacterial biofilm treated in the form of H. sabdariffa L. calyx extract was lower than OD bacterial biofilm that was not given the extract. The mechanisms underlying the inhibitory effects of H. sabdariffa L. calyx extract on biofilm formation is yet unknown; however, it is believed to be due to the flavonoid and tannin present in the H. sabdariffa L.^[14] According to a prior Lee et al.^[15] study, flavonoids and tannins have the potential to inhibit biofilm growth because they can inhibit intercellular adhesion genes icaA and icaD. This gene can produce polysaccharide Intercellular Adhesion (PIA), which is crucial for cell aggregation and formation of EPS during the formation of biofilm. Flavonoids themselves can interfere with the quorum sensing mechanism that functions as a communication media between bacteria, affecting the formation of biofilms.^[16] This result is supported by research conducted by Federika et al.,^[16] that stated that flavonoid has antibiofilm against P. gingivalis.

In addition, flavonoids contain hydroxyl groups that can disrupt bacterial nutrient transport, and thus can cause changes in organic components in bacteria that can cause toxic effects on the bacteria.^[17] Flavonoids also have the ability to inhibit bacterial attachment by interfering with the glucosyltransferase enzyme in the production of glucan.^[16] Tannins have the ability to pass through the bacterial cell wall so that it can interfere with the metabolism of the bacterial cell wall and cause damage to the bacterial cell.^[18] Alkaloid can affect the peptidoglycan component of bacterial cells so that the bacterial cell layer is not formed completely, resulting bacterial cell death.^[7] Saponins also have the ability to interfere with the permeability of the outer membrane of bacterial cells so that they can cause bacterial cell leakage and also result in bacterial cell death.^[19] Phenol is a compound that can cause changes in bacterial cell proteins and can cause damage to bacterial cell membranes.^[17] Triterpenoids have the ability to react with transmembrane proteins found in the outer membrane of the bacterial cell wall by forming strong polymer bonds and causing damage to transmembrane proteins, which causes bacterial death.^[20] Glycosides have the ability to penetrate membrane in the bacterial cell wall, potentially causing damage to the bacterial cell wall.^[21]

Therefore, in this study, H. sabdariffa L. calyx extract may inhibit biofilm formation due to its antibacterial properties and its capacity to prevent the formation of EPS by preventing the expression of the PIA gene. This is also supported by the statistical test result value of P < 0.05 which indicates that there are significant differences between the groups that treated with the H. sabdariffa L. calyx extract and the group that was not treated with the extract. Thus, it is clear that H. sabdariffa L. calyx extract has the ability to inhibit the multispecies of P. gingivalis and T. denticola biofilm formation.

The sequential interactions of many species facilitate the complex process of multispecies biofilm development. The structural and functional dynamics of the entire biofilm community are frequently changed by these highly specific interactions within the bacterial community, enhancing both virulence and protective properties.^[22] Hence, this research is conducted to determine the effects of H. sabdariffa L. calyx extract on the multispecies P. gingivalis and T. denticola biofilms. The research was conducted with the crystal violet assay method, which is the “gold standard” for measuring biofilms in a microtiter dish. It is a simple and affordable assay that may be routinely performed.^[23]

Previous research conducted by Indriani and Dharmautama^[24] showed that H. sabdariffa L. extract has also had antimicrobial effects on monospecies of P. gingivalis bacteria using the agar method. It showed that H. sabdariffa L. flower petal extract at concentrations of 2.5%, 5%, 7.5%, and 10% with average inhibition zones were 6.676, 8.043, 13.586, and 11.013 mm. In addition, there was also research on the effect of H. sabdariffa calyx extract on the activity of Rgp and Kgp which are the major proteases of P. gingivalis bacteria. This study demonstrated that H. sabdariffa L. calyx extract reduced the activity of Rgp and Kgp activity about 90% and 70%, respectively, at 2.2 mg/mL.^[25] This indicates that H. sabdariffa L. petal and calyx extract not only have antimicrobial properties, but also inhibit the virulence factor of monospecies P. gingivalis.

This study is also supported by research conducted by Sebastian and Widyarman, who investigated the effect of H. sabdariffa L. calyx extract using the biofilm assay method. The results of this study indicated that at an incubation period of 1 h, 6 h, and 24 h, H. sabdariffa L. calyx extract had the ability to inhibit the formation of monospecies P. gingivalis in vitro. This study showed that extract concentration of 25% at 1-h incubation period and extract concentration of 12.5% at 1 h and 24 h incubation period could lower OD value of P. gingivalis monospecies biofilms compared to 0.2% chlorhexidine.^[8] Study conducted by Sebastian et al. also showed that at an incubation period of 1 hour, 6 hours, and 24 hours, H. sabdariffa L. have ability to inhibit the multispecies Fusobacterium nucleatum and P. gingivalis biofilm formation with the best concentration for inhibiting multispecies biofilm was 100%.^[8]

Research by Suwandi et al. also showed that H. sabdariffa L. calyx extract could reduce Streptococcus sanguinis biofilm viability. An H. sabdariffa L. extract concentration of 10% at 20 and 24 h of incubation is equal to 0.2% chlorhexidine in reducing the growth potential of S. sanguinis biofilms.^[9] The research conducted by Sulistyani et al.^[14] also states that H. sabdariffa L. calyx extract showed antibacterial activity against Prevotella intermedia, F. nucleatum, and P. gingivalis, with the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 7.2–28.8 and 14.4–57.6 respectively. There is also research that demonstrates the antibacterial activity against Streptococcus mutans, S. sanguinis, Capnocytophaga gingivalis, and Staphylococcus aureus with the MIC and MBC values of 5–20 and 25–33 mg/mL respectively.^[26]

In accordance with the finding described above and previously existing research, H. sabdariffa L. calyx extract is proved to have an antibacterial effect and also has the potential to inhibit biofilm formation not only on multispecies, but also on monospecies Gram-negative bacteria and Gram-positive bacteria. This research was only conducted one time with no repetition, so the data will be less accurate than if it is done several times. Hence, further study is needed to examine this research more than once so that more accurate results can be obtained.

Conclusion

Hibiscus sabdariffa L. calyx extract is able to inhibit the formation of multispecies P. gingivalis and T. denticola biofilms. The most effective concentration at 1-h, 6-h, and 24-h incubation period is 100% with significance difference (P < 0.05). However, further research is still needed to repeat this experiment and evaluate the H. sabdariffa L. calyx extract toxicity effects to cells in the oral cavity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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