β-glycyrrhetinic acid exerts an exfoliating effect on marginal gingivitis-inducing plaque biofilms

β-glycyrrhetinic acid exerts an exfoliating effect on marginal gingivitis-inducing plaque biofilms | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article β-glycyrrhetinic acid exerts an exfoliating effect on marginal gingivitis-inducing plaque biofilms Shinya Kato, Xiangtao Ma, Kayo Sato, Aya Okumura, Nobuo Yoshinari, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6615678/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract β-glycyrrhetinic acid (BGA) possesses antibacterial effects against human supragingival plaque bacteria and inhibit biofilm formation. This study analyzed and compared the effects of BGA on preformed supragingival plaque biofilms with those of cetylpyridinium chloride (CPC). All the experiments were performed using biofilms formed by incubating supragingival plaque bacteria for 24 h. First, we analyzed the number of viable and dead bacteria in the biofilms following BGA and CPC application. The number of viable bacteria was significantly reduced by BGA treatment than by the control. However, the viable/dead bacterial ratios did not significantly vary. Conversely, the turbidity in the supernatants (optical density at 600 nm [OD 600 ]) was 0.237 ± 0.003, 0.136 ± 0.002, and 0.096 ± 0.002 for the BGA, CPC, and control groups, respectively, indicating superior ability of BGA in biofilm exfoliation. Finally, we evaluated the strength of the biofilms based on the physical impact of BGA or CPC treatment. The biofilm amount ratio before and after sonication was significantly reduced by BGA and CPC than by the control. Furthermore, CPC demonstrated significantly greater reduction in biofilm formation than BGA following sonication. BGA demonstrated excellent bactericidal and exfoliating effects on human supragingival biofilms. These effects are presumably attributed to a mechanism different from that of CPC. Biological sciences/Microbiology Biological sciences/Microbiology/Antimicrobials Biological sciences/Microbiology/Biofilms Health sciences/Health care/Disease prevention Health sciences/Health care/Disease prevention/Preventive medicine Health sciences/Diseases/Oral diseases/Gingivitis Health sciences/Diseases Health sciences/Diseases/Infectious diseases Health sciences/Diseases/Oral diseases Health sciences/Health care/Dentistry/Oral microbiology Health sciences/Health care/Dentistry/Oral microbiology/Dental biofilms Health sciences/Health care Health sciences/Health care/Dentistry β-glycyrrhetinic acid (BGA) biofilm supragingival plaque gingivitis exfoliation effect Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Stability of the host-microbial interface across mucosal surfaces in the human body is essential for the maintenance of oral health. [ 1 ] This is especially relevant concerning the mucosal surfaces, which present a constant microbial challenge in the host epithelial barriers. [ 2 ] Under oral conditions, commensal bacteria actively interact with the gingival tissue to maintain healthy neutrophil surveillance and normal tissue and bone turnover processes. [ 3 – 5 ] The disruption of this homeostatic host-bacteria relationship occurs during gingivitis, marking the initiation of periodontitis. [ 6 , 7 ] Gingivitis is caused by substances derived from microbial plaque accumulation at or near the marginal gingiva; thus, an increase in the bacterial burden increases gingival inflammation. [ 8 – 12 ] Therefore, controlling gingivitis-causing biofilms is important for periodontal disease control. Biofilm control warrants the use of antimicrobial substances that do not rely on traditional antimicrobials owing to their low sensitivity to bacteria in biofilms and their potential to increase the number of resistant bacteria. [ 13 , 14 ] Glycyrrhiza glabra , also known as licorice, is a herbaceous perennial plant that has been used as a therapeutic agent for thousands of years. β-glycyrrhetinic acid (BGA) is obtained by hydrolyzing glycyrrhizic acid extracted from licorice [ 15 , 16 ] and has been reported to have strong anti-inflammatory, [ 17 – 19 ] antioxidative, [ 20 ] and antibacterial activities. [ 21 – 24 ] Previous investigations confirmed that BGA reduced the biofilm formation and virulence expression of Psudomonas aeruginosa , a representative multidrug-resistant species. [ 21 , 25 ] Another study reported that BGA promoted cell survival and reduced pro-inflammatory cytokines production, during carbapenem-resistant Klebsiella pneumoniae -induced human pulmonary epithelial cell. [ 26 ] Thus, BGA is effective against drug-resistant bacteria, including multidrug-resistant bacteria, which are increasingly emerging owing to the excessive inappropriate use of antimicrobial agents. [ 23 , 27 ] Although antimicrobial agents may be used to treat gingivitis and periodontitis, they should not be used extensively owing to their effects on the flora of other organs, such as the intestinal tract, and emergence of drug-resistant bacteria. [ 28 ] New anti-infection strategies are warranted to control the spread of resistant bacteria. Thus, we previously elucidated the inhibitory effect of BGA on supragingival plaque formation. [ 29 ] The present study aimed to analyze the effect of BGA on previously formed supragingival plaque and clarify its effect on supragingival biofilms. MATERIALS AND METHODS Ethical considerations All procedures were conducted in accordance with the guidelines of the Ethics Committee of the Faculty of Dentistry, Matsumoto Dental University (No. 0295) and the Declaration of Helsinki (64th WMA General Assembly, Fortaleza, October 2013). [ 30 ] For assays using human supragingival plaques, plaque samples were obtained from healthy volunteers after obtaining written informed consent. Bacterial strains and culture The bacterial strains used in this study are listed in Table 1 . All the bacteria were cultured as described previously. [ 29 ] Briefly, Streptococcus and Actinomyces species were inoculated in Bacto™ Brain Heart Infusion (BD Biosciences, Franklin Lakes, NJ, USA) broth at 37°C under anaerobic conditions. [ 31 ] Aggraegatibacter actinomycetemcomitans was inoculated in trypticase soy broth (TSB; Becton Dickinson, Sparks, MD, USA) supplemented with 0.6% yeast extract (Becton Biosciences) and 0.04% sodium bicarbonate at 37°C in a 5% CO 2 atmosphere. Prevotella spp., Fusobacterium nucleatum , and Porphyromonas gingivalis were grown in Gifu Anaerobic Medium (GAM; Nissui Medical Co., Tokyo, Japan) at 37°C under anaerobic conditions. P. gingivalis was inoculated in GAM broth supplemented with 5 µg of hemin per mL, 1.0 µg of menadion per mL, and 1.0% l -cysteine at 37°C under anaerobic conditions. [ 32 ] Table 1 Bacterial strains and MICs and MBCs of BGA Bacterial species strains MIC (µg/ml) MBC (µg/ml) MBC/MIC Streptococcus mutans MT 8148 128 1024 8 Ingbrid 32 1024 32 XC 32 1024 32 OMZ175 32 1024 32 10449 32 1024 32 Streptococcus sobrinus GTC278 16 1024 64 6715 32 512 16 Streptococcus anginosus NTCT10713 64 1024 16 Streptococcus mitis 9811 64 1024 16 Streptococcus sanguinis ATCC10556 64 1024 16 Streptococcus salivarius JCM5707 512 1024 2 HHT 64 512 8 HT9A 128 1024 8 Streptococcus gordonii DL1 64 512 8 Streptococcus oralis 557 64 512 8 Actinomyces naeslundii ATCC12104 64 512 8 Actinomyces viscosus ATCC15987 64 1024 16 Aggregatibacter actinomycetemcomitans JP2 128 512 4 Y4 64 1024 16 Prevotella denticola JCM8528 128 512 4 Prevotella nigrescens ATCC33563 64 2048 32 Fusobacterium nucleatum JCM6328 128 1024 8 Porphyromonas gingivalis W83 32 2048 64 ATCC33277 64 512 8 Prevotella intermedia ATCC25611 256 1024 4 Supragingival plaque collection Supragingival plaque samples were collected using a sterile curette from the mandibular left first molars of 12 healthy participants. The age of the participants ranged from 26 to 58 years, with an average age of 36.0 ± 9.8 years. Plaque samples were collected immediately above the gingival margin in 1.0 mL of sterile phosphate-buffered saline (PBS, FUJIFILM Wako Pure Chemical Co. Osaka, Japan). Plaque samples collected from the 12 volunteers were combined in equal proportions to form single biofilms that were used for the supragingival plaque assays. This mixture of supragingival plaque was stored at -20°C until use and incubated in BHI medium to a cell density of 1.0 optical density at 600 nm (OD 600 ), and then used as a supragingival plaque solution for experiments. BGA and cetylpyridinium chloride BGA was obtained commercially (Alps Pharmaceutical, Inc., Co., Ltd., Gifu, Japan), and dissolved in 100% dimethyl sulfoxide (DMSO) (FUJIFILM Wako Pure Chemical Co.). Stock solutions of the reagents were prepared at a concentration of 128 mg/mL and diluted in the medium to the appropriate concentrations for each experiment. Cetylpyridinium chloride (CPC) (FUJIFILM Wako Pure Chemical Co.) was used as a control in the study. Minimum inhibitory concentrations and minimum bactericidal concentration determination of BGA The antibacterial activity of BGA was evaluated by determining the MICs and MBCs using the microdilution method, as previously described. [ 33 ] Briefly, BGA was adjusted to 1024 µg/mL in BHI and two-fold serial dilutions were prepared in 96-well microplates (0, 8, 16, 32, 64, 128, 256, 512, and 1024 µg/mL; 96-well culture plate U-shape bottom, WATSON, Tokyo, Japan). Overnight bacterial cultures were adjusted to an OD 600 of 1.0 (10 8 cells/mL) and diluted to 1:100 with 100 µL of BHI (10 6 cells/mL). To each well, 10 µL of bacterial cultures was added, resulting in 100-µL cultures. The MICs of BGA were determined after 24 h of anaerobic incubation at 37℃. From the wells where bacteria did not grow, the medium was incubated on GAM agar medium (Nissui Medical Co.) supplemented with 5 µg of hemin per mL, 1.0 µg of menadion per mL, and 1.0% l -cysteine without antimicrobials, and the BGA concentration with a bacterial count of 10 or less was used as the MBC. Analysis of the number of bacteria in the biofilms Flat-bottomed polystyrene microtiter plates (96-well Easy Wash; Corning Inc., Corning, N.Y.) containing 100 µL of BHI per well were inoculated with 1 µL supragingival plaque solution obtained from 12 volunteers for 24 h at 37°C to form biofilms. The formed biofilms were washed with PBS and incubated in test mediums (i.e. the BHI medium with 128 µg/mL BGA and 40 µg/mL CPC dissolved in DMSO, and BHI medium with only DMSO without antibiotic (Supplementary Table S1 ) for an additional 6 hours. The colony-forming units (CFU) in the biofilm remaining at the bottom of the plate were measured using the 10-fold dilution method. LIVE/DEAD staining To each well of an 8-well plate (Nunc Lab-Tek II Chamber Slide System, Thermo Fisher Scientific, Waltham, MA), 495 µL of BHI and 5 µL of supragingival plaque solution were added and incubated for 24 hours to form biofilms. After biofilm formation, test media (Supplementary Table S1 ) were added to each well and allowed to incubate for 6 h. The LIVE/DEAD BacLight Bacterial Viability Kit (L7012, Invitrogen, Mount Waverley, Australia) was used for 15 min, and LIVE/DEAD staining was performed according to the manufacturer’s instructions. Confocal laser scanning microscope analysis of biofilms LIVE/DEAD-stained biofilms were imaged using confocal laser scanning microscopy (CLSM, Axiovert 200M Inverted Microscope, Carl Zeiss, Jena, Germany) and rendered in the x–y–z planes using ZEN 3.6 software (Carl Zeiss) for analyzing the bactericidal effect. In accordance with previous reports, the degree of unviable, based on the green (viable cells) and red (dead/damaged cells) pixel intensities for every pixel in the x–y–z planes, was evaluated using ImageJ software (National Institutes of Health (NIH)). [ 34 ] Analysis of the biofilm amount Flat-bottomed polystyrene microtiter plates (96-well Easy Wash; Corning Inc., Corning, N.Y., USA) containing 100 µL of BHI per well were inoculated with supragingival plaque solution, and incubated 24 h at 37°C to form biofilms. After biofilm formation, test media (Supplementary Table S1 ) were added to each well and allowed to stand for 6 h. After incubation, 25 µL of 1% (wt/vol) crystal violet (CV) solution was added to each well. After 15 min, the wells were rinsed three times with 200 µL of distilled water and air dried. The CV on the abiotic surfaces was solubilized in 95% ethanol and the OD 600 was measured. [ 35 ] Analysis of the exfoliating action of BGA on biofilms Biofilms were formed on 6-well polystyrene plates (Corning Inc.) using a supragingival plaque solution and incubated for 6 h following addition of the test media (Supplementary Table S1 ). The amount of suspended solids and number of viable bacteria in the supernatant were subsequently analyzed. The amount of suspended solids was analyzed by measuring the absorbance at OD 600 using a microplate reader (iMark microplate reader; Bio-Rad Laboratories Inc., Hercules, CA, USA). The CFU in the supernatant were measured using the 10-fold dilution method. Analysis of the embrittlement effect of BGA on biofilms Biofilms were formed in 6-well plates, incubated for 6 h following the addition of the test media (Supplementary Table S1 ), and allowed to stand for 6 h. The biofilm was then subjected to physical vibration (Amplitude: 20%, 1 pulse) using an ultrasonic sonication machine (UP-200S; Hielscher Ultrasonics GmbH, Teltow, Germany), and the amount of exfoliated biofilm was quantified to analyze the effect of antimicrobial agents on deterioration of the biofilm. The amount of biofilm formed before and after physical vibration was determined using a CV assay. [ 35 ] Statistical analysis Statistical analyses were performed using a statistical software (IBM SPSS Statistics version 28.0.0.0, Armonk, NY, USA). Comparisons between the three groups (DMSO, CPC, and BGA) were conducted using the 1-way analysis of variance for a priori comparisons and the Scheffé test for a post-hoc test. Comparisons before and after ultrasound were performed using a paired t-test. RESULTS Bactericidal effects of BGA on oral bacteria We previously analyzed the MIC of BGA against various oral bacteria. [ 29 ] In this study, we analyzed the MBC to determine the bactericidal effect of BGA (Table 1 ). The MBCs of BGA against Streptococcus mutans strains were 1024 µg/mL for all five strains (Table 1 ). The MBCs of BGA against Streptococcus sobrinus strains 6715 and GTC 278 were 512 and 1024 µg/mL, respectively. The MBCs of BGA against other Streptococcus strains was 1024 µg/mL, except for Streptococcus salivarius HHT (512 µg/mL), Streptococcus gordonii DL1 (512 µg/mL), and Streptococcus oralis 557 (512 µg/mL). The MBCs of BGA against both Actinomyces naeslundii ATCC 12104 and Actinomyces viscosus ATCC 15987 were 512 and 1024 µg/mL, respectively. The MBC/MIC ratios for various Streptococcus species varied within the range of 2–64. Of the 15 Streptococcus species, four had an MBC/MIC ratio of 32, five had an MBC/MIC ratio of 16, and five had an MBC/MIC ratio of 8. The lowest MBC/MIC ratio was 2 for Streptococcus salivarius JCM5703 and the highest was 64 for Streptococcus sobrinus GTC278. The MBC/MIC ratios for Actinomyces species were 8 and 16 for A. naeslundii ATCC 12104 and A. viscosus ATCC 15987, respectively. The MBCs of BGA against the Gram-negative rods listed in Table 1 varied from 512 to 2048 µg/mL. The lowest MBC was 512 µg/mL for Prevotella denticola JCM8528, while the highest was 2048 µg/mL for Prevotella nigrescens ATCC 33563. Based on the Clinical and Laboratory Standards Institute, a drug is considered to exhibit bactericidal activity when the MBC/MIC ratio is ≤ 4, whereas the drug is considered bacteriostatic when the corresponding MBC/MIC ratio is ≥ 8. [ 36 , 37 ] BGA appeared to be bacteriostatic against majority of the oral bacteria but exhibited bactericidal activity against some bacteria: Prevotella denticola JP2, Prevotella denticola JCM8528, Prevotella denticola ATCC25611. BGA reduces the number of viable bacteria in biofilms Previous studies have demonstrated that the MIC of BGA for supragingival plaques was 128 µg/mL. [ 29 ] The same measurements were performed to confirm that the MIC of CPC or supragingival plaque was 40 µ/ml (date not shown). We established the concentration of BGA at 128 µg/mL and CPC at 40 µ/ml and analyzed its effect on the bacteria in the biofilm. Supragingival plaque bacteria were cultured in 6-well polystyrene plates in BHI medium for 24 h to form a biofilm at the bottom of the plate. The biofilm formed was cultured in BHI medium containing BGA, CPC, and DMSO alone as controls (Supplementary Table S1 ) for another 6 h. The number of viable cells in the biofilms exposed to DMSO alone implied a CFU of 1.8 x 10 7 , while the number of viable cells in biofilms exposed to CPC was 2.0 × 10 0 , which was significantly reduced by 99.9% following exposure to CPC than DMSO (P < 0.001, Fig. 1 ). In contrast, the viable count of biofilms exposed to BGA implied a CFU of 4.0 x 10 5 , which was significantly (97.8%) lower (P < 0.001, Fig. 1 ) than that of BGA exposed to DMSO alone. These results indicate that BGA acts on the bacteria in biofilms and reduces the number of viable bacterial; however, it is not as effective as CPC. Fluorescence microscopy using LIVE/DEAD staining revealed that BGA treatment reduces the number of viable bacterial in biofilms We analyzed the number of viable and dead bacteria in the biofilms formed by human supragingival plaque bacteria following BGA and CPC application by fluorescence microscopy using LIVE/DEAD staining (Fig. 2 ). The percentage of dead bacteria in the biofilm on the polystyrene plate treated with CPC for 6 h was 85.7% (P < 0.001; Fig. 3 ), whereas the percentage of dead bacteria in the biofilm treated with BGA was 60%. However, BGA demonstrated no significant increase in the percentage of dead bacteria compared to that in the DMSO group (P = 0.145, Fig. 3 ). BGA decreases the amount of biofilm The results shown in Fig. 1 indicate that BGA reduced the number of viable bacteria by affecting the biofilms formed by supragingival plaque bacteria. Next, we analyzed the effects of BGA on biofilm formation. The amount of biofilm formed was reduced by 27.6% following BGA application to the BHI medium when compared to that of the control group (P < 0.001; Fig. 4 ). Comparing the amount of reduction in biofilms treated with CPC and BGA, the percentage reduction caused by CPC (31.4%) was nearly the same as that caused by BGA (27.6%); however, it was slightly higher for CPC (Fig. 4 ). BGA eliminates biofilms including dead and viable bacteria The experiment in Fig. 4 shows that although both CPC and BGA reduced biofilm formation to the same degree, the differences in the biofilm reduction mechanisms between BGA and CPC are unknown. Therefore, we analyzed the turbidity and number of viable bacteria in the supernatants removed from the biofilms. Culture supernatants of the BHI medium containing BGA and CPC were collected and their turbidity was measured; the turbidity of the culture supernatant of BHI medium containing BGA was the highest (OD 600 = 0.237 ± 0.003). This was significantly higher than the turbidity following CPC (OD 600 = 0.136 ± 0.002) and only DMSO (OD 600 = 0.096 ± 0.002) application (P < 0.001, Fig. 5 (A)). The turbidity of the culture supernatant following CPC application was also higher than that following only DMSO application (P < 0.001, Fig. 5 ). Next, the biofilms were treated with BGA, CPC, or DMSO alone and analyzed for the number of viable bacteria in their culture supernatants. Nearly no viable bacteria were found in the culture supernatant following CPC treatment (CFU unmeasurable), whereas the culture supernatant following BGA treatment contained viable bacteria with a CFU of 2.5 × 10 3 . Viable bacterial counts were significantly higher in the culture supernatant following BGA treatment than following CPC treatment (P < 0.001, Fig. 5 (B)). Although the number of viable bacteria in the BGA group was significantly lower than that in the DMSO group, which was not treated with antimicrobial substances (P < 0.001, Fig. 5 (B)), the biofilm eliminated by BGA treatment contained sufficient number of viable bacteria. These results indicate that the mechanisms underlying the effects of BGA and CPC on biofilm reduction are completely different. Furthermore, BGA treatment resulted in a bacterial mass in the culture supernatant, which was not observed in the presence of CPC or without antimicrobial treatment. Therefore, we performed time-lapse imaging of the formation of bacterial aggregates in BGAs over time (Supplementary Figure S1 ); a bacterial mass formed over time with the addition of BGA. These results indicated that biofilms activated and exfoliated by BGA contained a high proportion of viable bacteria, whereas those exfoliated by CPC treatment were mostly dead. BGA induces physical fragilization of the biofilm Thus far, these results indicate that BGA treatment causes biofilm exfoliation via a mechanism different from that of CPC, thus raising the question that whether the physical strength of the biofilm was altered by the BGA or CPC treatment. Therefore, we evaluated the effect of BGA and CPC treatment on the biofilm strength. The biofilms exposed to BGA, CPC, and only DMSO underwent sonication, and the amount of biofilm remaining at the bottom of the polystyrene plates was measured and analyzed and compared with the group that did not undergo sonication. After sonication, the amount of biofilm in the BGA, CPC, and DMSO groups was significantly lower than that before sonication (P < 0.001, Fig. 6 (A)). Next, the percentage of residual biofilm before and after sonication was compared among the BGA, CPC, and DMSO groups; compared to the DMSO group, the BGA and CPC groups demonstrated significantly lesser residual biofilm due to sonication (P < 0.01, P < 0.001, respectively, Fig. 6 (B)). Furthermore, between the BGA and CPC groups, CPC significantly reduced the amount of residual biofilm upon sonication (P < 0.01, respectively, Fig. 6 (B)). These results indicate that both BGA and CPC treatments provide some degree of fragility to physical impact when compared to DMSO as control. However, the extent to which fragility to physical impact is involved in the biofilm-exfoliating effect of BGA and CPC treatments is unclear. DISCUSSION In this study, we analyzed the effects of BGA on human supragingival plaque biofilms. BGA exhibited bactericidal activity against biofilms; however, it was not as strong as that of CPC. After BGA exposure, the number of CFU in the remaining biofilm decreased (Fig. 1 ); however, the ratio of viable to dead bacteria remained unchanged compared to that in the control (Fig. 3 ), which was attributed to the decrease in the amount of biofilm formed (Fig. 4 ). Furthermore, when the number of viable bacteria in the supernatant was compared to that in the control, BGA reduced the number of viable bacteria (Fig. 5 (B)). These results demonstrate the bactericidal effect of BGA. In these experiments, CPC demonstrated higher bactericidal efficacy than BGA (Fig. 1 , 3 , and 4 ). However, the use of CPC reportedly results in the growth of resistant bacteria. Although there are no studies have demonstrated the long-term effects of CPC exposure on the oral flora, concerns about the emergence of CPC-resistant oral bacteria exist. [ 38 ] In recent years, quorum sensing has received extensive attention as a drug discovery target to find new anti-infection strategies for controlling the spread of resistant bacteria. [ 39 , 40 ] BGA reportedly affects biofilm composition and inhibits biofilm formation by acting on quorum sensing. [ 25 , 41 ] BGA penetrates the biofilm matrix in Pseudomonas aeruginosa , [ 21 ] and exfoliates biofilms. [ 26 ] In this study, BGA was demonstrated greater effectiveness than CPC in exfoliating biofilms (Fig. 5 (A, B), Supplementary Figure S1 ). We believe that these anti-biofilm effects are probably related to the effect of BGA on quorum sensing; we aim to investigate the relationship between BGA quorum-sensing inhibition and exfoliation of supragingival plaque biofilms. This is the first report on the bactericidal and exfoliative effects of BGA on dental plaque. Thus, the use of BGA in oral care products not only as an anti-inflammatory agent but also as an antibacterial agent may be considered. We intend to accumulate further clinical data such as plaque and gingivitis scores and verify the safety of BGA in the body. In addition, we plan to evaluate the biofilm exfoliation effect of BGA at the molecular level and elucidate the detailed characteristics of this antimicrobial substance. In conclusion, BGA demonstrated a bactericidal action against supragingival plaque biofilms and superior biofilm-exfoliating effect by a mechanism different from that of CPC in this study (Supplementary Figure S2 ). Declarations ACKNOWLEDGMENTS We would like to thank Editage (www.editage.jp) for English language editing. AUTHOR CONTRIBUTIONS SK contributed to the conception, design, data collection and interpretation, and critical revision of the manuscript. XM conceived and designed the study, collected, and interpreted the data, and critically revised the study and manuscript. KS contributed to the conception, design, interpretation, and critical revision of the study and the manuscript. She contributed to the drafting and revision of the manuscript. AO contributed to the conception, design, interpretation, and critical revision of the study and the manuscript. She contributed to the drafting and revision of the manuscript. NY contributed to the conception, design, interpretation, and critical revision of the manuscript. AY contributed to the conception, design, interpretation, and critical revision of the study and the manuscript. He contributed to drafting and revising the manuscript. All the authors have approved the final version of the manuscript for publication. DATA AVAILABILITY STATEMENT Data supporting the findings of this study are available upon reasonable request from the corresponding author. The data are not publicly available owing to privacy and ethical restrictions. ADDITIONAL INFORMATION Funding This research was funded by the Kao Corporation. Competing interests Kayo Sato and Aya Okumura are paid employees of Kao Corporation, Tokyo, Japan. This research was funded by Kao Corporation. The authors declare no conflicts of interest. ETHICS APPROVAL STATEMENT : This study was approved by the Ethics Committee of the Faculty of Dentistry at Matsumoto Dental University (No. 0295). All the volunteers participating in this study have consented to the publication of this study. References Medzhitov, R. Origin and physiological roles of inflammation. Nature 454 , 428–435 (2008). Ptasiewicz, M. et al. Armed to the teeth-the oral mucosa immunity system and microbiota. Int. J. Mol. Sci. 23 , 882 (2022). Fine, N. et al. Distinct oral neutrophil subsets define health and periodontal disease states. J. Dent. Res. 95 , 931–938 (2016). Tonetti, M. S., Imboden, M. A. & Lang, N. P. Neutrophil migration into the gingival sulcus is associated with transepithelial gradients of interleukin-8 and ICAM-1. J. Periodontol. 69 , 1139–1147 (1998). Cortés-Vieyra, R., Rosales, C. & Uribe-Querol, E. Neutrophil functions in periodontal homeostasis. J. Immunol. Res. 2016 , 1396106 (2016). Darveau, R. P. Periodontitis: A polymicrobial disruption of host homeostasis. Nat. Rev. Microbiol. 8 , 481–490 (2010). Hajishengallis, G. & Lamont, R. J. Polymicrobial communities in periodontal disease: their quasi-organismal nature and dialogue with the host. Periodontol. 2000 86 , 210-230 (2021). Bamashmous, S. et al. Human variation in gingival inflammation. Proc. Natl Acad. Sci. U. S. A. 118 , e2012578118 (2021). Theilade, E., Wright, W. H., Jensen, S. B. & Löe, H. Experimental gingivitis in man. II. A longitudinal clinical and bacteriological investigation. J. Periodont. Res. 1 , 1-13 (1966). Page, R. C. Gingivitis. J. Clin. Periodontol. 13 , 345-359 (1986). Zemouri, C. et al . Resistance and resilience to experimental gingivitis: a systematic scoping review. BMC Oral Health 19 , 212 (2019). Trombelli, L. et al. Experimental gingivitis: reproducibility of plaque accumulation and gingival inflammation parameters in selected populations during a repeat trial. J. Clin. Periodontol. 35 , 955-960 (2008). Costerton, J. W., Stewart, P. S. & Greenberg, E. P. Bacterial biofilms: a common cause of persistent infections. Science 284 , 1318-1322 (1999). Theuretzbacher, U., Outterson, K., Engel, A. & Karlén, A. The global preclinical antibacterial pipeline. Nat. Rev. Microbiol. 18 , 275-285 (2020). Kwon, Y. J., Son, D. H., Chung, T. H. & Lee, Y. J. A review of the pharmacological efficacy and safety of licorice root from corroborative clinical trial findings. J. Med. Food 23 , 12-20 (2020). Baltina, L. A. et al. Hydrolysis of β-glycyrrhizic acid. Pharm. Chem. J. 30 , 263-266 (1996). Feng, Y., Mei, L., Wang, M., Huang, Q. & Huang, R. Anti-inflammatory and pro-apoptotic effects of 18beta-Glycyrrhetinic acid in vitro and in vivo models of rheumatoid arthritis. Front. Pharmacol. 12 , 681525 (2021). eCollection 2021. Chen, B. et al. 18beta-Glycyrrhetinic acid inhibits IL-1beta-induced inflammatory response in mouse chondrocytes and prevents osteoarthritic progression by activating Nrf2. Food Funct. 12 , 8399-8410 (2021). Kao, T. C., Shyu, M. H. & Yen, G. C. Glycyrrhizic acid and 18β-glycyrrhetinic acid inhibit inflammation via PI3K/Akt/GSK3beta signaling and glucocorticoid receptor activation. J. Agric. Food Chem. 58 , 8623-8629 (2010). Bayav, I., Darendelioğlu, E. & Caglayan, C. 18β-Glycyrrhetinic acid exerts cardioprotective effects against BPA-induced cardiotoxicity through antiapoptotic and antioxidant mechanisms. J. Biochem. Mol. Toxicol. 38 , e23655 (2024). Kannan, S., Sathasivam, G. & Marudhamuthu, M. Decrease of growth, biofilm and secreted virulence in opportunistic nosocomial Pseudomonas aeruginosa ATCC 25619 by glycyrrhetinic acid. Microb. Pathog. 126 , 332-342 (2019). Kowalska, A. & Kalinowska-Lis, U. 18β-glycyrrhetinic acid: its core biological properties and dermatological applications. Int. J. Cosmet. Sci. 41 , 325-331 (2019). Long, D. R., Mead, J., Hendricks, J. M., Hardy, M. E. & Voyich, J. M. 18β-glycyrrhetinic acid inhibits methicillin-resistant Staphylococcus aureus survival and attenuates virulence gene expression. Antimicrob. Agents Chemother. 57 , 241-247 (2013). Zhao, Y. & Su, X. Antibacterial activity of 18β-glycyrrhetinic acid against Neisseria gonorrhoeae in vitro. Biochem. Biophys. Rep. 33 , 101427 (2023). . Paul Bhattacharya, S. P., Mitra, A., Bhattacharya, A. & Sen, A. Quorum quenching activity of pentacyclic triterpenoids leads to inhibition of biofilm formation by Acinetobacter baumannii . Biofouling 36 , 922-937 (2020). Guan, X. et al. Glycyrrhetinic acid prevents carbapenem-resistant Klebsiella pneumoniae -induced cell injury by inhibiting mitochondrial dysfunction via Nrf-2 pathway. Microb. Pathog. 177 , 105825 (2023). Oyama, K. et al. Antibacterial effects of glycyrrhetinic acid and its derivatives on Staphylococcus aureus . PLOS One 11 , e0165831 (2016). Fernandes, Â. R., Rodrigues, A. G. & Cobrado, L. Effect of prolonged exposure to disinfectants in the antimicrobial resistance profile of relevant micro-organisms: a systematic review. J. Hosp. Infect. 151 , 45-59 (2024). Dewake, N. et al. β-glycyrrhetinic acid inhibits the bacterial growth and biofilm formation by supragingival plaque commensals. Microbiol. Immunol. 65 , 343-351 (2021). World Medical Association. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA 310 , 2191-2194 (2013). Yoshida, A., Ansai, T., Takehara, T. & Kuramitsu, H. K. LuxS-based signaling affects Streptococcus mutans biofilm formation. Appl. Environ. Microbiol. 71 , 2372-2380 (2005). Nakamura, S. et al. Porphyromonas gingivalis hydrogen sulfide enhances methyl mercaptan-induced pathogenicity in mouse abscess formation. Microbiology (Reading) 164 , 529-539 (2018). . So Yeon, L. & Si Young, L. Susceptibility of oral streptococci to chlorhexidine and cetylpyridinium chloride. Biocontrol Sci. 24 , 13-21 (2019). Nance, W. C. et al. A high-throughput microfluidic dental plaque biofilm system to visualize and quantify the effect of antimicrobials. J. Antimicrob. Chemother. 68 , 2550-2560 (2013). Yoshida, A. & Kuramitsu, H. K. Multiple Streptococcus mutans Genes Are involved in biofilm formation. Appl. Environ. Microbiol. 68 , 6283-6291 (2002). Huang, X. J. et al. The antibacterial properties of 4, 8, 4’, 8’-tetramethoxy (1,1’-biphenanthrene) -2,7,2’,7’-Tetrol from Fibrous Roots of Bletilla striata. Indian J. Microbiol. 61 , 195-202 (2021). French, G. L. Bactericidal agents in the treatment of MRSA infections—the potential role of daptomycin. J. Antimicrob. Chemother. 58 , 1107-1117 (2006). Mao, X. et al. Cetylpyridinium chloride: mechanism of action, antimicrobial efficacy in biofilms, and potential risks of resistance. Antimicrob. Agents Chemother. 64 , e00576-20 (2020). Rumbaugh, K. P. & Sauer, K. Biofilm dispersion. Nat. Rev. Microbiol. 18 , 571–586 (2020). . Peng, B. et al. A bibliometric analysis on discovering anti-quorum sensing agents against clinically relevant pathogens: current status, development, and future directions. Front. Microbiol. 14 , 1297843 (2023). Bhattacharya, S. P., Bhattacharya, A. & Sen, A. A comprehensive and comparative study on the action of pentacyclic triterpenoids on Vibrio cholerae biofilms. Microb. Pathog. 149 , 104493 (2020). Additional Declarations No competing interests reported. Supplementary Files supportingdata1.mp4 supportingdata2.tif supportingTable1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6615678","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":465155880,"identity":"1e3b9d67-c60c-4e45-a6cf-856667f72fb3","order_by":0,"name":"Shinya Kato","email":"","orcid":"","institution":"Matsumoto Dental University","correspondingAuthor":false,"prefix":"","firstName":"Shinya","middleName":"","lastName":"Kato","suffix":""},{"id":465155881,"identity":"77edf1b3-b4df-4778-968d-91113e6ecf8c","order_by":1,"name":"Xiangtao Ma","email":"","orcid":"","institution":"Matsumoto Dental University","correspondingAuthor":false,"prefix":"","firstName":"Xiangtao","middleName":"","lastName":"Ma","suffix":""},{"id":465155882,"identity":"3de82357-978b-4f4c-b508-e3098091cfb3","order_by":2,"name":"Kayo Sato","email":"","orcid":"","institution":"Kao Corporation (Japan)","correspondingAuthor":false,"prefix":"","firstName":"Kayo","middleName":"","lastName":"Sato","suffix":""},{"id":465155883,"identity":"80853b31-81f7-4626-8c3d-136a79b89cf8","order_by":3,"name":"Aya Okumura","email":"","orcid":"","institution":"Kao Corporation (Japan)","correspondingAuthor":false,"prefix":"","firstName":"Aya","middleName":"","lastName":"Okumura","suffix":""},{"id":465155884,"identity":"b347cece-51f4-40ac-b30b-9ca806c02dba","order_by":4,"name":"Nobuo Yoshinari","email":"","orcid":"","institution":"Matsumoto Dental University","correspondingAuthor":false,"prefix":"","firstName":"Nobuo","middleName":"","lastName":"Yoshinari","suffix":""},{"id":465155885,"identity":"ca16b69a-8fcf-403f-9e25-bf7c7e23525f","order_by":5,"name":"Akihiro Yoshida","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYDACHsY2hgQIk40hgY1BjgHKZSZaizERWoAqGWBagCixAWYCLsDfc7jtwcM9NgwGx9ufPXhQZpe+tj3HgOFHDQO7OQ4tEmcb2w0SnqUxGJw5Y26QcC45d9uZNwaMPccYmC0bcOg5z9gmkXDgMIPBjRw2icQ25txtN4C28DYwMBscwK5DHqEl/RlQS326GVAL4188WgzONsK0JJgBtRxOAGlhxmeL4ZmDIC1pPJJnzphJJJw7brjtzLOCwzLHJHD6Re5M+jPJHwds5PiAISb5o6xa3ux48saHb2psknGFGAzwKCA7A8iWSDYgoIVBHt0ZdgS1jIJRMApGwUgBANEgXohUYf9pAAAAAElFTkSuQmCC","orcid":"","institution":"Matsumoto Dental University","correspondingAuthor":true,"prefix":"","firstName":"Akihiro","middleName":"","lastName":"Yoshida","suffix":""}],"badges":[],"createdAt":"2025-05-08 01:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6615678/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6615678/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83857943,"identity":"d1ed816f-35f4-40b4-80fd-0c95558dde3b","added_by":"auto","created_at":"2025-06-03 18:09:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":231546,"visible":true,"origin":"","legend":"\u003cp\u003eColony forming units (CFUs) of bacteria in the biofilms at minimum inhibitory concentration (MIC) of β-glycyrrhetinic acid (BGA) and cetylpyridinium chloride (CPC). These experiments were carried out in triplicate. Error bars denote standard deviation. *P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6615678/v1/f53cec8be9384e66a120ce31.png"},{"id":83857950,"identity":"c086a378-6035-4c6e-944f-1c39329f917c","added_by":"auto","created_at":"2025-06-03 18:09:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":19051860,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative biofilm renderings (x–y plane) following each treatment. Green signal indicates viable live cells (Syto 9) and red signal indicates damaged/dead cells (propidium iodide). All images were taken at 200× magnification. minimum inhibitory concentration (MIC) of β-glycyrrhetinic acid (BGA) (A), MIC of cetylpyridinium chloride (CPC) (B), dimethyl sulfoxide (DMSO) as negative control (C).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6615678/v1/3a679cf46e9a754961c22275.png"},{"id":83857945,"identity":"c8eea97e-f1fc-41bd-bdfc-cbbf44a8bb54","added_by":"auto","created_at":"2025-06-03 18:09:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":809010,"visible":true,"origin":"","legend":"\u003cp\u003eAverage percentage signal from biofilms accounted for by \"dead\"/damaged (grey bars) and :live\"/viable (white bars) signals in relation to the total signal captured for both. These experiments were carried out in quadruple. Error bars denote standard deviation. *P\u0026lt;0.05 **P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6615678/v1/c3fecfcbf3011d6f342e4920.png"},{"id":83857947,"identity":"0f02d44e-2d80-4d29-be50-2a0c2743d6af","added_by":"auto","created_at":"2025-06-03 18:09:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":496756,"visible":true,"origin":"","legend":"\u003cp\u003eAmount of biofilm following each treatment. Biofilm biomass determined by crystal violet assays. Crystal violet assays were carried out in triplicate. Error bars denote standard deviation. *P\u0026lt;0.01, ** P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6615678/v1/8c53a265b8fd8b2926806f34.png"},{"id":83858084,"identity":"d724e38c-8d3c-49a2-b636-9ab7036cdf98","added_by":"auto","created_at":"2025-06-03 18:17:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":428449,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorbance (A) and colony forming unit (CFU) (B) of the biofilm supernatant following each treatment. All the experiments were carried out in triplicate. Error bars denote standard deviation. *P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6615678/v1/9a4c3108f37e0687a0dd20b9.png"},{"id":83857948,"identity":"f0134678-92a7-45da-a85c-84a4100082e6","added_by":"auto","created_at":"2025-06-03 18:09:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":680650,"visible":true,"origin":"","legend":"\u003cp\u003eAmount of biofilm following each treatment before and after sonication (A) and ratio of biofilm amount before and after sonication (B). Biofilm biomass determined by crystal violet assays. All the experiments were carried out in triplicate. Error bars denote standard deviation. *P\u0026lt;0.01, ** P\u0026lt;0.001 (Ultrasound -) †P\u0026lt;0.001 (Ultrasound +) \u003csup\u003e§\u003c/sup\u003e P\u0026lt;0.05, \u003csup\u003e§§\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e§§§\u003c/sup\u003eP\u0026lt;0.001. Comparisons before and after ultrasound were performed using t-test. \u003csup\u003e‡\u003c/sup\u003e P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6615678/v1/83d83f7241dcd1278cf804c1.png"},{"id":86913161,"identity":"279d19a4-4eb9-47a3-828e-a0e1b772795a","added_by":"auto","created_at":"2025-07-17 05:43:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21957799,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6615678/v1/7ce3f24d-09b2-4532-b9fd-5e928edb2b7c.pdf"},{"id":83857951,"identity":"a081d5ab-118c-44fa-ba6f-e16b7029e72c","added_by":"auto","created_at":"2025-06-03 18:09:15","extension":"mp4","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4165271,"visible":true,"origin":"","legend":"","description":"","filename":"supportingdata1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6615678/v1/9af5bd4dd5de2553169aa3d2.mp4"},{"id":83857952,"identity":"a40be371-219d-4fdd-b209-8ad48117a171","added_by":"auto","created_at":"2025-06-03 18:09:15","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2247832,"visible":true,"origin":"","legend":"","description":"","filename":"supportingdata2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6615678/v1/92f0f27140e34a73528064db.tif"},{"id":83858566,"identity":"6b58cdab-659d-4d37-8adb-271f53fb8af3","added_by":"auto","created_at":"2025-06-03 18:25:15","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13946,"visible":true,"origin":"","legend":"","description":"","filename":"supportingTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6615678/v1/6c7a8c1eaac03b126bde7fe4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eβ\u003cstrong\u003e-glycyrrhetinic acid exerts an exfoliating effect on marginal gingivitis-inducing plaque biofilms\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eStability of the host-microbial interface across mucosal surfaces in the human body is essential for the maintenance of oral health.\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e This is especially relevant concerning the mucosal surfaces, which present a constant microbial challenge in the host epithelial barriers.\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e Under oral conditions, commensal bacteria actively interact with the gingival tissue to maintain healthy neutrophil surveillance and normal tissue and bone turnover processes.\u003csup\u003e[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e The disruption of this homeostatic host-bacteria relationship occurs during gingivitis, marking the initiation of periodontitis.\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e Gingivitis is caused by substances derived from microbial plaque accumulation at or near the marginal gingiva; thus, an increase in the bacterial burden increases gingival inflammation.\u003csup\u003e[\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e Therefore, controlling gingivitis-causing biofilms is important for periodontal disease control. Biofilm control warrants the use of antimicrobial substances that do not rely on traditional antimicrobials owing to their low sensitivity to bacteria in biofilms and their potential to increase the number of resistant bacteria.\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eGlycyrrhiza glabra\u003c/em\u003e, also known as licorice, is a herbaceous perennial plant that has been used as a therapeutic agent for thousands of years. β-glycyrrhetinic acid (BGA) is obtained by hydrolyzing glycyrrhizic acid extracted from licorice\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e and has been reported to have strong anti-inflammatory,\u003csup\u003e[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e antioxidative,\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e and antibacterial activities.\u003csup\u003e[\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e Previous investigations confirmed that BGA reduced the biofilm formation and virulence expression of \u003cem\u003ePsudomonas aeruginosa\u003c/em\u003e, a representative multidrug-resistant species.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e Another study reported that BGA promoted cell survival and reduced pro-inflammatory cytokines production, during \u003cem\u003ecarbapenem-resistant Klebsiella pneumoniae\u003c/em\u003e-induced human pulmonary epithelial cell.\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e Thus, BGA is effective against drug-resistant bacteria, including multidrug-resistant bacteria, which are increasingly emerging owing to the excessive inappropriate use of antimicrobial agents.\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAlthough antimicrobial agents may be used to treat gingivitis and periodontitis, they should not be used extensively owing to their effects on the flora of other organs, such as the intestinal tract, and emergence of drug-resistant bacteria.\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e New anti-infection strategies are warranted to control the spread of resistant bacteria. Thus, we previously elucidated the inhibitory effect of BGA on supragingival plaque formation.\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e The present study aimed to analyze the effect of BGA on previously formed supragingival plaque and clarify its effect on supragingival biofilms.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthical considerations\u003c/h2\u003e \u003cp\u003e All procedures were conducted in accordance with the guidelines of the Ethics Committee of the Faculty of Dentistry, Matsumoto Dental University (No. 0295) and the Declaration of Helsinki (64th WMA General Assembly, Fortaleza, October 2013).\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e For assays using human supragingival plaques, plaque samples were obtained from healthy volunteers after obtaining written informed consent.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBacterial strains and culture\u003c/h3\u003e\n\u003cp\u003eThe bacterial strains used in this study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All the bacteria were cultured as described previously.\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e Briefly, \u003cem\u003eStreptococcus\u003c/em\u003e and \u003cem\u003eActinomyces\u003c/em\u003e species were inoculated in Bacto\u0026trade; Brain Heart Infusion (BD Biosciences, Franklin Lakes, NJ, USA) broth at 37\u0026deg;C under anaerobic conditions.\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e \u003cem\u003eAggraegatibacter actinomycetemcomitans\u003c/em\u003e was inoculated in trypticase soy broth (TSB; Becton Dickinson, Sparks, MD, USA) supplemented with 0.6% yeast extract (Becton Biosciences) and 0.04% sodium bicarbonate at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. \u003cem\u003ePrevotella\u003c/em\u003e spp., \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e, and \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e were grown in Gifu Anaerobic Medium (GAM; Nissui Medical Co., Tokyo, Japan) at 37\u0026deg;C under anaerobic conditions. \u003cem\u003eP. gingivalis\u003c/em\u003e was inoculated in GAM broth supplemented with 5 \u0026micro;g of hemin per mL, 1.0 \u0026micro;g of menadion per mL, and 1.0% \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-cysteine at 37\u0026deg;C under anaerobic conditions.\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBacterial strains and MICs and MBCs of BGA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBacterial species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003estrains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMIC (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMBC (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMBC/MIC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus mutans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMT 8148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIngbrid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOMZ175\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus sobrinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTC278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6715\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus anginosus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNTCT10713\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus mitis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9811\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus sanguinis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCC10556\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus salivarius\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJCM5707\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHHT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHT9A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus gordonii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus oralis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e557\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eActinomyces naeslundii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCC12104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eActinomyces viscosus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCC15987\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eAggregatibacter actinomycetemcomitans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eY4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotella denticola\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJCM8528\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotella nigrescens\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCC33563\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusobacterium nucleatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJCM6328\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eW83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCC33277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotella intermedia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCC25611\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eSupragingival plaque collection\u003c/h3\u003e\n\u003cp\u003eSupragingival plaque samples were collected using a sterile curette from the mandibular left first molars of 12 healthy participants. The age of the participants ranged from 26 to 58 years, with an average age of 36.0\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8 years. Plaque samples were collected immediately above the gingival margin in 1.0 mL of sterile phosphate-buffered saline (PBS, FUJIFILM Wako Pure Chemical Co. Osaka, Japan). Plaque samples collected from the 12 volunteers were combined in equal proportions to form single biofilms that were used for the supragingival plaque assays. This mixture of supragingival plaque was stored at -20\u0026deg;C until use and incubated in BHI medium to a cell density of 1.0 optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e), and then used as a supragingival plaque solution for experiments.\u003c/p\u003e\n\u003ch3\u003eBGA and cetylpyridinium chloride\u003c/h3\u003e\n\u003cp\u003eBGA was obtained commercially (Alps Pharmaceutical, Inc., Co., Ltd., Gifu, Japan), and dissolved in 100% dimethyl sulfoxide (DMSO) (FUJIFILM Wako Pure Chemical Co.). Stock solutions of the reagents were prepared at a concentration of 128 mg/mL and diluted in the medium to the appropriate concentrations for each experiment.\u003c/p\u003e \u003cp\u003eCetylpyridinium chloride (CPC) (FUJIFILM Wako Pure Chemical Co.) was used as a control in the study.\u003c/p\u003e\n\u003ch3\u003eMinimum inhibitory concentrations and minimum bactericidal concentration determination of BGA\u003c/h3\u003e\n\u003cp\u003eThe antibacterial activity of BGA was evaluated by determining the MICs and MBCs using the microdilution method, as previously described.\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e Briefly, BGA was adjusted to 1024 \u0026micro;g/mL in BHI and two-fold serial dilutions were prepared in 96-well microplates (0, 8, 16, 32, 64, 128, 256, 512, and 1024 \u0026micro;g/mL; 96-well culture plate U-shape bottom, WATSON, Tokyo, Japan). Overnight bacterial cultures were adjusted to an OD\u003csub\u003e600\u003c/sub\u003e of 1.0 (10\u003csup\u003e8\u003c/sup\u003e cells/mL) and diluted to 1:100 with 100 \u0026micro;L of BHI (10\u003csup\u003e6\u003c/sup\u003e cells/mL). To each well, 10 \u0026micro;L of bacterial cultures was added, resulting in 100-\u0026micro;L cultures. The MICs of BGA were determined after 24 h of anaerobic incubation at 37℃. From the wells where bacteria did not grow, the medium was incubated on GAM agar medium (Nissui Medical Co.) supplemented with 5 \u0026micro;g of hemin per mL, 1.0 \u0026micro;g of menadion per mL, and 1.0% \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-cysteine without antimicrobials, and the BGA concentration with a bacterial count of 10 or less was used as the MBC.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the number of bacteria in the biofilms\u003c/h2\u003e \u003cp\u003eFlat-bottomed polystyrene microtiter plates (96-well Easy Wash; Corning Inc., Corning, N.Y.) containing 100 \u0026micro;L of BHI per well were inoculated with 1 \u0026micro;L supragingival plaque solution obtained from 12 volunteers for 24 h at 37\u0026deg;C to form biofilms. The formed biofilms were washed with PBS and incubated in test mediums (i.e. the BHI medium with 128 \u0026micro;g/mL BGA and 40 \u0026micro;g/mL CPC dissolved in DMSO, and BHI medium with only DMSO without antibiotic (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) for an additional 6 hours. The colony-forming units (CFU) in the biofilm remaining at the bottom of the plate were measured using the 10-fold dilution method.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLIVE/DEAD staining\u003c/h3\u003e\n\u003cp\u003eTo each well of an 8-well plate (Nunc Lab-Tek II Chamber Slide System, Thermo Fisher Scientific, Waltham, MA), 495 \u0026micro;L of BHI and 5 \u0026micro;L of supragingival plaque solution were added and incubated for 24 hours to form biofilms. After biofilm formation, test media (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were added to each well and allowed to incubate for 6 h. The LIVE/DEAD BacLight Bacterial Viability Kit (L7012, Invitrogen, Mount Waverley, Australia) was used for 15 min, and LIVE/DEAD staining was performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eConfocal laser scanning microscope analysis of biofilms\u003c/h3\u003e\n\u003cp\u003eLIVE/DEAD-stained biofilms were imaged using confocal laser scanning microscopy (CLSM, Axiovert 200M Inverted Microscope, Carl Zeiss, Jena, Germany) and rendered in the x\u0026ndash;y\u0026ndash;z planes using ZEN 3.6 software (Carl Zeiss) for analyzing the bactericidal effect. In accordance with previous reports, the degree of unviable, based on the green (viable cells) and red (dead/damaged cells) pixel intensities for every pixel in the x\u0026ndash;y\u0026ndash;z planes, was evaluated using ImageJ software (National Institutes of Health (NIH)).\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the biofilm amount\u003c/h2\u003e \u003cp\u003eFlat-bottomed polystyrene microtiter plates (96-well Easy Wash; Corning Inc., Corning, N.Y., USA) containing 100 \u0026micro;L of BHI per well were inoculated with supragingival plaque solution, and incubated 24 h at 37\u0026deg;C to form biofilms. After biofilm formation, test media (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were added to each well and allowed to stand for 6 h. After incubation, 25 \u0026micro;L of 1% (wt/vol) crystal violet (CV) solution was added to each well. After 15 min, the wells were rinsed three times with 200 \u0026micro;L of distilled water and air dried. The CV on the abiotic surfaces was solubilized in 95% ethanol and the OD\u003csub\u003e600\u003c/sub\u003e was measured.\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the exfoliating action of BGA on biofilms\u003c/h2\u003e \u003cp\u003eBiofilms were formed on 6-well polystyrene plates (Corning Inc.) using a supragingival plaque solution and incubated for 6 h following addition of the test media (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The amount of suspended solids and number of viable bacteria in the supernatant were subsequently analyzed. The amount of suspended solids was analyzed by measuring the absorbance at OD\u003csub\u003e600\u003c/sub\u003e using a microplate reader (iMark microplate reader; Bio-Rad Laboratories Inc., Hercules, CA, USA). The CFU in the supernatant were measured using the 10-fold dilution method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the embrittlement effect of BGA on biofilms\u003c/h2\u003e \u003cp\u003eBiofilms were formed in 6-well plates, incubated for 6 h following the addition of the test media (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and allowed to stand for 6 h. The biofilm was then subjected to physical vibration (Amplitude: 20%, 1 pulse) using an ultrasonic sonication machine (UP-200S; Hielscher Ultrasonics GmbH, Teltow, Germany), and the amount of exfoliated biofilm was quantified to analyze the effect of antimicrobial agents on deterioration of the biofilm. The amount of biofilm formed before and after physical vibration was determined using a CV assay.\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using a statistical software (IBM SPSS Statistics version 28.0.0.0, Armonk, NY, USA). Comparisons between the three groups (DMSO, CPC, and BGA) were conducted using the 1-way analysis of variance for a priori comparisons and the Scheff\u0026eacute; test for a post-hoc test. Comparisons before and after ultrasound were performed using a paired t-test.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBactericidal effects of BGA on oral bacteria\u003c/h2\u003e \u003cp\u003eWe previously analyzed the MIC of BGA against various oral bacteria.\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e In this study, we analyzed the MBC to determine the bactericidal effect of BGA (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The MBCs of BGA against \u003cem\u003eStreptococcus mutans\u003c/em\u003e strains were 1024 \u0026micro;g/mL for all five strains (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The MBCs of BGA against \u003cem\u003eStreptococcus sobrinus\u003c/em\u003e strains 6715 and GTC 278 were 512 and 1024 \u0026micro;g/mL, respectively. The MBCs of BGA against other \u003cem\u003eStreptococcus\u003c/em\u003e strains was 1024 \u0026micro;g/mL, except for \u003cem\u003eStreptococcus salivarius\u003c/em\u003e HHT (512 \u0026micro;g/mL), \u003cem\u003eStreptococcus gordonii\u003c/em\u003e DL1 (512 \u0026micro;g/mL), and \u003cem\u003eStreptococcus oralis\u003c/em\u003e 557 (512 \u0026micro;g/mL). The MBCs of BGA against both \u003cem\u003eActinomyces naeslundii\u003c/em\u003e ATCC 12104 and \u003cem\u003eActinomyces viscosus\u003c/em\u003e ATCC 15987 were 512 and 1024 \u0026micro;g/mL, respectively. The MBC/MIC ratios for various \u003cem\u003eStreptococcus\u003c/em\u003e species varied within the range of 2\u0026ndash;64. Of the 15 \u003cem\u003eStreptococcus\u003c/em\u003e species, four had an MBC/MIC ratio of 32, five had an MBC/MIC ratio of 16, and five had an MBC/MIC ratio of 8. The lowest MBC/MIC ratio was 2 for \u003cem\u003eStreptococcus salivarius\u003c/em\u003e JCM5703 and the highest was 64 for \u003cem\u003eStreptococcus sobrinus\u003c/em\u003e GTC278. The MBC/MIC ratios for \u003cem\u003eActinomyces\u003c/em\u003e species were 8 and 16 for \u003cem\u003eA. naeslundii\u003c/em\u003e ATCC 12104 and \u003cem\u003eA. viscosus\u003c/em\u003e ATCC 15987, respectively. The MBCs of BGA against the Gram-negative rods listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e varied from 512 to 2048 \u0026micro;g/mL. The lowest MBC was 512 \u0026micro;g/mL for \u003cem\u003ePrevotella denticola\u003c/em\u003e JCM8528, while the highest was 2048 \u0026micro;g/mL for \u003cem\u003ePrevotella nigrescens\u003c/em\u003e ATCC 33563. Based on the Clinical and Laboratory Standards Institute, a drug is considered to exhibit bactericidal activity when the MBC/MIC ratio is \u0026le;\u0026thinsp;4, whereas the drug is considered bacteriostatic when the corresponding MBC/MIC ratio is \u0026ge;\u0026thinsp;8.\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e BGA appeared to be bacteriostatic against majority of the oral bacteria but exhibited bactericidal activity against some bacteria: \u003cem\u003ePrevotella denticola\u003c/em\u003e JP2, \u003cem\u003ePrevotella denticola\u003c/em\u003e JCM8528, \u003cem\u003ePrevotella denticola\u003c/em\u003e ATCC25611.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBGA reduces the number of viable bacteria in biofilms\u003c/h2\u003e \u003cp\u003ePrevious studies have demonstrated that the MIC of BGA for supragingival plaques was 128 \u0026micro;g/mL.\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e The same measurements were performed to confirm that the MIC of CPC or supragingival plaque was 40 \u0026micro;/ml (date not shown). We established the concentration of BGA at 128 \u0026micro;g/mL and CPC at 40 \u0026micro;/ml and analyzed its effect on the bacteria in the biofilm. Supragingival plaque bacteria were cultured in 6-well polystyrene plates in BHI medium for 24 h to form a biofilm at the bottom of the plate. The biofilm formed was cultured in BHI medium containing BGA, CPC, and DMSO alone as controls (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) for another 6 h.\u003c/p\u003e \u003cp\u003eThe number of viable cells in the biofilms exposed to DMSO alone implied a CFU of 1.8 x 10\u003csup\u003e7\u003c/sup\u003e, while the number of viable cells in biofilms exposed to CPC was 2.0 \u0026times; 10\u003csup\u003e0\u003c/sup\u003e, which was significantly reduced by 99.9% following exposure to CPC than DMSO (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, the viable count of biofilms exposed to BGA implied a CFU of 4.0 x 10\u003csup\u003e5\u003c/sup\u003e, which was significantly (97.8%) lower (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) than that of BGA exposed to DMSO alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results indicate that BGA acts on the bacteria in biofilms and reduces the number of viable bacterial; however, it is not as effective as CPC.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescence microscopy using LIVE/DEAD staining revealed that BGA treatment reduces the number of viable bacterial in biofilms\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe analyzed the number of viable and dead bacteria in the biofilms formed by human supragingival plaque bacteria following BGA and CPC application by fluorescence microscopy using LIVE/DEAD staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The percentage of dead bacteria in the biofilm on the polystyrene plate treated with CPC for 6 h was 85.7% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), whereas the percentage of dead bacteria in the biofilm treated with BGA was 60%. However, BGA demonstrated no significant increase in the percentage of dead bacteria compared to that in the DMSO group (P\u0026thinsp;=\u0026thinsp;0.145, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBGA decreases the amount of biofilm\u003c/h2\u003e \u003cp\u003eThe results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicate that BGA reduced the number of viable bacteria by affecting the biofilms formed by supragingival plaque bacteria. Next, we analyzed the effects of BGA on biofilm formation. The amount of biofilm formed was reduced by 27.6% following BGA application to the BHI medium when compared to that of the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Comparing the amount of reduction in biofilms treated with CPC and BGA, the percentage reduction caused by CPC (31.4%) was nearly the same as that caused by BGA (27.6%); however, it was slightly higher for CPC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBGA eliminates biofilms including dead and viable bacteria\u003c/h2\u003e \u003cp\u003eThe experiment in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows that although both CPC and BGA reduced biofilm formation to the same degree, the differences in the biofilm reduction mechanisms between BGA and CPC are unknown. Therefore, we analyzed the turbidity and number of viable bacteria in the supernatants removed from the biofilms. Culture supernatants of the BHI medium containing BGA and CPC were collected and their turbidity was measured; the turbidity of the culture supernatant of BHI medium containing BGA was the highest (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.237\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003). This was significantly higher than the turbidity following CPC (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.136\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002) and only DMSO (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.096\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002) application (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(A)). The turbidity of the culture supernatant following CPC application was also higher than that following only DMSO application (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, the biofilms were treated with BGA, CPC, or DMSO alone and analyzed for the number of viable bacteria in their culture supernatants. Nearly no viable bacteria were found in the culture supernatant following CPC treatment (CFU unmeasurable), whereas the culture supernatant following BGA treatment contained viable bacteria with a CFU of 2.5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e. Viable bacterial counts were significantly higher in the culture supernatant following BGA treatment than following CPC treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(B)). Although the number of viable bacteria in the BGA group was significantly lower than that in the DMSO group, which was not treated with antimicrobial substances (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(B)), the biofilm eliminated by BGA treatment contained sufficient number of viable bacteria.\u003c/p\u003e \u003cp\u003eThese results indicate that the mechanisms underlying the effects of BGA and CPC on biofilm reduction are completely different. Furthermore, BGA treatment resulted in a bacterial mass in the culture supernatant, which was not observed in the presence of CPC or without antimicrobial treatment. Therefore, we performed time-lapse imaging of the formation of bacterial aggregates in BGAs over time (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e); a bacterial mass formed over time with the addition of BGA. These results indicated that biofilms activated and exfoliated by BGA contained a high proportion of viable bacteria, whereas those exfoliated by CPC treatment were mostly dead.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eBGA induces physical fragilization of the biofilm\u003c/h2\u003e \u003cp\u003eThus far, these results indicate that BGA treatment causes biofilm exfoliation via a mechanism different from that of CPC, thus raising the question that whether the physical strength of the biofilm was altered by the BGA or CPC treatment. Therefore, we evaluated the effect of BGA and CPC treatment on the biofilm strength.\u003c/p\u003e \u003cp\u003eThe biofilms exposed to BGA, CPC, and only DMSO underwent sonication, and the amount of biofilm remaining at the bottom of the polystyrene plates was measured and analyzed and compared with the group that did not undergo sonication. After sonication, the amount of biofilm in the BGA, CPC, and DMSO groups was significantly lower than that before sonication (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(A)). Next, the percentage of residual biofilm before and after sonication was compared among the BGA, CPC, and DMSO groups; compared to the DMSO group, the BGA and CPC groups demonstrated significantly lesser residual biofilm due to sonication (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(B)). Furthermore, between the BGA and CPC groups, CPC significantly reduced the amount of residual biofilm upon sonication (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(B)).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results indicate that both BGA and CPC treatments provide some degree of fragility to physical impact when compared to DMSO as control. However, the extent to which fragility to physical impact is involved in the biofilm-exfoliating effect of BGA and CPC treatments is unclear.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we analyzed the effects of BGA on human supragingival plaque biofilms. BGA exhibited bactericidal activity against biofilms; however, it was not as strong as that of CPC. After BGA exposure, the number of CFU in the remaining biofilm decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); however, the ratio of viable to dead bacteria remained unchanged compared to that in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which was attributed to the decrease in the amount of biofilm formed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, when the number of viable bacteria in the supernatant was compared to that in the control, BGA reduced the number of viable bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(B)). These results demonstrate the bactericidal effect of BGA. In these experiments, CPC demonstrated higher bactericidal efficacy than BGA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, the use of CPC reportedly results in the growth of resistant bacteria. Although there are no studies have demonstrated the long-term effects of CPC exposure on the oral flora, concerns about the emergence of CPC-resistant oral bacteria exist.\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e In recent years, quorum sensing has received extensive attention as a drug discovery target to find new anti-infection strategies for controlling the spread of resistant bacteria.\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e BGA reportedly affects biofilm composition and inhibits biofilm formation by acting on quorum sensing.\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e BGA penetrates the biofilm matrix in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e,\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e and exfoliates biofilms.\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e In this study, BGA was demonstrated greater effectiveness than CPC in exfoliating biofilms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(A, B), Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We believe that these anti-biofilm effects are probably related to the effect of BGA on quorum sensing; we aim to investigate the relationship between BGA quorum-sensing inhibition and exfoliation of supragingival plaque biofilms.\u003c/p\u003e \u003cp\u003eThis is the first report on the bactericidal and exfoliative effects of BGA on dental plaque. Thus, the use of BGA in oral care products not only as an anti-inflammatory agent but also as an antibacterial agent may be considered. We intend to accumulate further clinical data such as plaque and gingivitis scores and verify the safety of BGA in the body. In addition, we plan to evaluate the biofilm exfoliation effect of BGA at the molecular level and elucidate the detailed characteristics of this antimicrobial substance.\u003c/p\u003e \u003cp\u003eIn conclusion, BGA demonstrated a bactericidal action against supragingival plaque biofilms and superior biofilm-exfoliating effect by a mechanism different from that of CPC in this study (Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe would like to thank Editage (www.editage.jp) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSK contributed to the conception, design, data collection and interpretation, and critical revision of the manuscript. XM conceived and designed the study, collected, and interpreted the data, and critically revised the study and manuscript. KS contributed to the conception, design, interpretation, and critical revision of the study and the manuscript. She contributed to the drafting and revision of the manuscript. AO contributed to the conception, design, interpretation, and critical revision of the study and the manuscript. She contributed to the drafting and revision of the manuscript. NY contributed to the conception, design, interpretation, and critical revision of the manuscript. AY contributed to the conception, design, interpretation, and critical revision of the study and the manuscript. He contributed to drafting and revising the manuscript. All the authors have approved the final version of the manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are available upon reasonable request from the corresponding author. The data are not publicly available owing to privacy and ethical restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADDITIONAL INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Kao Corporation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKayo Sato and Aya Okumura are paid employees of Kao Corporation, Tokyo, Japan. This research was funded by Kao Corporation. The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL STATEMENT\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the Faculty of Dentistry at Matsumoto Dental University (No. 0295). All the volunteers participating in this study have consented to the publication of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMedzhitov, R. Origin and physiological roles of inflammation. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e454\u003c/strong\u003e, 428\u0026ndash;435 (2008).\u003c/li\u003e\n\u003cli\u003ePtasiewicz, M. \u003cem\u003eet al.\u003c/em\u003e Armed to the teeth-the oral mucosa immunity system and microbiota. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 882\u003cem\u003e \u003c/em\u003e(2022).\u003c/li\u003e\n\u003cli\u003eFine, N. \u003cem\u003eet al.\u003c/em\u003e Distinct oral neutrophil subsets define health and periodontal disease states. \u003cem\u003eJ. Dent. Res.\u003c/em\u003e \u003cstrong\u003e95\u003c/strong\u003e, 931\u0026ndash;938 (2016).\u003c/li\u003e\n\u003cli\u003eTonetti, M. S., Imboden, M. A. \u0026amp; Lang, N. P. Neutrophil migration into the gingival sulcus is associated with transepithelial gradients of interleukin-8 and ICAM-1. \u003cem\u003eJ. Periodontol.\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 1139\u0026ndash;1147 (1998).\u003c/li\u003e\n\u003cli\u003eCort\u0026eacute;s-Vieyra, R., Rosales, C. \u0026amp; Uribe-Querol, E. Neutrophil functions in periodontal homeostasis. \u003cem\u003eJ. Immunol. Res.\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, 1396106 (2016).\u003c/li\u003e\n\u003cli\u003eDarveau, R. P. Periodontitis: A polymicrobial disruption of host homeostasis. \u003cem\u003eNat. Rev. Microbiol.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 481\u0026ndash;490 (2010).\u003c/li\u003e\n\u003cli\u003eHajishengallis, G. \u0026amp; Lamont, R. J. Polymicrobial communities in periodontal disease: their quasi-organismal nature and dialogue with the host. \u003cem\u003ePeriodontol. 2000\u003c/em\u003e \u003cstrong\u003e86\u003c/strong\u003e, 210-230 (2021).\u003c/li\u003e\n\u003cli\u003eBamashmous, S. \u003cem\u003eet al.\u003c/em\u003e Human variation in gingival inflammation. \u003cem\u003eProc. Natl Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, e2012578118 (2021).\u003c/li\u003e\n\u003cli\u003eTheilade, E., Wright, W. H., Jensen, S. B. \u0026amp; L\u0026ouml;e, H. Experimental gingivitis in man. II. A longitudinal clinical and bacteriological investigation. \u003cem\u003eJ. Periodont. Res.\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 1-13 (1966).\u003c/li\u003e\n\u003cli\u003ePage, R. C. Gingivitis. \u003cem\u003eJ. Clin. Periodontol.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 345-359 (1986).\u003c/li\u003e\n\u003cli\u003eZemouri, C. \u003cem\u003eet al\u003c/em\u003e. Resistance and resilience to experimental gingivitis: a systematic scoping review. \u003cem\u003eBMC Oral Health\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 212 (2019).\u003c/li\u003e\n\u003cli\u003eTrombelli, L. \u003cem\u003eet al.\u003c/em\u003e Experimental gingivitis: reproducibility of plaque accumulation and gingival inflammation parameters in selected populations during a repeat trial. \u003cem\u003eJ. Clin. Periodontol.\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 955-960 (2008).\u003c/li\u003e\n\u003cli\u003eCosterton, J. W., Stewart, P. S. \u0026amp; Greenberg, E. P. Bacterial biofilms: a common cause of persistent infections. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e284\u003c/strong\u003e, 1318-1322\u003cem\u003e \u003c/em\u003e(1999).\u003c/li\u003e\n\u003cli\u003eTheuretzbacher, U., Outterson, K., Engel, A. \u0026amp; Karl\u0026eacute;n, A. The global preclinical antibacterial pipeline. \u003cem\u003eNat. Rev. Microbiol.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 275-285\u003cem\u003e \u003c/em\u003e(2020).\u003c/li\u003e\n\u003cli\u003eKwon, Y. J., Son, D. H., Chung, T. H. \u0026amp; Lee, Y. J. A review of the pharmacological efficacy and safety of licorice root from corroborative clinical trial findings. \u003cem\u003eJ. Med. Food\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 12-20 (2020).\u003c/li\u003e\n\u003cli\u003eBaltina, L. A. \u003cem\u003eet al.\u003c/em\u003e Hydrolysis of \u0026beta;-glycyrrhizic acid. \u003cem\u003ePharm. Chem. J.\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 263-266\u003cem\u003e \u003c/em\u003e(1996).\u003c/li\u003e\n\u003cli\u003eFeng, Y., Mei, L., Wang, M., Huang, Q. \u0026amp; Huang, R. Anti-inflammatory and pro-apoptotic effects of 18beta-Glycyrrhetinic acid in vitro and in vivo models of rheumatoid arthritis. \u003cem\u003eFront. Pharmacol.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 681525 (2021). eCollection 2021.\u003c/li\u003e\n\u003cli\u003eChen, B. \u003cem\u003eet al.\u003c/em\u003e 18beta-Glycyrrhetinic acid inhibits IL-1beta-induced inflammatory response in mouse chondrocytes and prevents osteoarthritic progression by activating Nrf2. \u003cem\u003eFood Funct.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 8399-8410\u003cem\u003e \u003c/em\u003e(2021).\u003c/li\u003e\n\u003cli\u003eKao, T. C., Shyu, M. H. \u0026amp; Yen, G. C. Glycyrrhizic acid and 18\u0026beta;-glycyrrhetinic acid inhibit inflammation via PI3K/Akt/GSK3beta signaling and glucocorticoid receptor activation. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 8623-8629 (2010).\u003c/li\u003e\n\u003cli\u003eBayav, I., Darendelioğlu, E. \u0026amp; Caglayan, C. 18\u0026beta;-Glycyrrhetinic acid exerts cardioprotective effects against BPA-induced cardiotoxicity through antiapoptotic and antioxidant mechanisms. \u003cem\u003eJ. Biochem. Mol. Toxicol.\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, e23655 (2024).\u003c/li\u003e\n\u003cli\u003eKannan, S., Sathasivam, G. \u0026amp; Marudhamuthu, M. Decrease of growth, biofilm and secreted virulence in opportunistic nosocomial \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 25619 by glycyrrhetinic acid. \u003cem\u003eMicrob. Pathog.\u003c/em\u003e \u003cstrong\u003e126\u003c/strong\u003e, 332-342 (2019).\u003c/li\u003e\n\u003cli\u003eKowalska, A. \u0026amp; Kalinowska-Lis, U. 18\u0026beta;-glycyrrhetinic acid: its core biological properties and dermatological applications. \u003cem\u003eInt. J. Cosmet. Sci.\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 325-331 (2019).\u003c/li\u003e\n\u003cli\u003eLong, D. R., Mead, J., Hendricks, J. M., Hardy, M. E. \u0026amp; Voyich, J. M. 18\u0026beta;-glycyrrhetinic acid inhibits methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e survival and attenuates virulence gene expression. \u003cem\u003eAntimicrob. Agents Chemother.\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 241-247 (2013).\u003c/li\u003e\n\u003cli\u003eZhao, Y. \u0026amp; Su, X. Antibacterial activity of 18\u0026beta;-glycyrrhetinic acid against Neisseria gonorrhoeae in vitro. \u003cem\u003eBiochem. Biophys. Rep.\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 101427 (2023).\u003c/li\u003e\n\u003cli\u003e\u003cem\u003e. \u003c/em\u003ePaul Bhattacharya, S. P., Mitra, A., Bhattacharya, A. \u0026amp; Sen, A. Quorum quenching activity of pentacyclic triterpenoids leads to inhibition of biofilm formation by \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e. \u003cem\u003eBiofouling\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 922-937\u003cem\u003e \u003c/em\u003e(2020).\u003c/li\u003e\n\u003cli\u003eGuan, X. \u003cem\u003eet al.\u003c/em\u003e Glycyrrhetinic acid prevents carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e-induced cell injury by inhibiting mitochondrial dysfunction via Nrf-2 pathway. \u003cem\u003eMicrob. Pathog.\u003c/em\u003e \u003cstrong\u003e177\u003c/strong\u003e, 105825 (2023).\u003c/li\u003e\n\u003cli\u003eOyama, K. \u003cem\u003eet al.\u003c/em\u003e Antibacterial effects of glycyrrhetinic acid and its derivatives on \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. \u003cem\u003ePLOS One\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e0165831 (2016).\u003c/li\u003e\n\u003cli\u003eFernandes, \u0026Acirc;. R., Rodrigues, A. G. \u0026amp; Cobrado, L. Effect of prolonged exposure to disinfectants in the antimicrobial resistance profile of relevant micro-organisms: a systematic review. \u003cem\u003eJ. Hosp. Infect.\u003c/em\u003e \u003cstrong\u003e151\u003c/strong\u003e, 45-59 (2024).\u003c/li\u003e\n\u003cli\u003eDewake, N. \u003cem\u003eet al.\u003c/em\u003e \u0026beta;-glycyrrhetinic acid inhibits the bacterial growth and biofilm formation by supragingival plaque commensals.\u003cem\u003e Microbiol. Immunol.\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 343-351\u003cem\u003e \u003c/em\u003e(2021).\u003c/li\u003e\n\u003cli\u003eWorld Medical Association. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. \u003cem\u003eJAMA\u003c/em\u003e \u003cstrong\u003e310\u003c/strong\u003e, 2191-2194 (2013). \u003c/li\u003e\n\u003cli\u003eYoshida, A., Ansai, T., Takehara, T. \u0026amp; Kuramitsu, H. K. LuxS-based signaling affects \u003cem\u003eStreptococcus mutans\u003c/em\u003e biofilm formation. \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e \u003cstrong\u003e71\u003c/strong\u003e, 2372-2380 (2005). \u003c/li\u003e\n\u003cli\u003eNakamura, S. \u003cem\u003eet al.\u003c/em\u003e \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e hydrogen sulfide enhances methyl mercaptan-induced pathogenicity in mouse abscess formation. \u003cem\u003eMicrobiology (Reading)\u003c/em\u003e \u003cstrong\u003e164\u003c/strong\u003e, 529-539\u003cem\u003e \u003c/em\u003e(2018). \u003c/li\u003e\n\u003cli\u003e\u003cem\u003e. \u003c/em\u003eSo Yeon, L. \u0026amp; Si Young, L. Susceptibility of oral streptococci to chlorhexidine and cetylpyridinium chloride. \u003cem\u003eBiocontrol Sci.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 13-21\u003cem\u003e \u003c/em\u003e(2019).\u003c/li\u003e\n\u003cli\u003eNance, W. C. \u003cem\u003eet al.\u003c/em\u003e A high-throughput microfluidic dental plaque biofilm system to visualize and quantify the effect of antimicrobials. \u003cem\u003eJ. Antimicrob. Chemother.\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 2550-2560\u003cem\u003e \u003c/em\u003e(2013). \u003c/li\u003e\n\u003cli\u003eYoshida, A. \u0026amp; Kuramitsu, H. K. Multiple \u003cem\u003eStreptococcus mutans\u003c/em\u003e Genes Are involved in biofilm formation. \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 6283-6291 (2002).\u003c/li\u003e\n\u003cli\u003eHuang, X. J. \u003cem\u003eet al.\u003c/em\u003e The antibacterial properties of 4, 8, 4\u0026rsquo;, 8\u0026rsquo;-tetramethoxy (1,1\u0026rsquo;-biphenanthrene) -2,7,2\u0026rsquo;,7\u0026rsquo;-Tetrol from Fibrous Roots of Bletilla striata. \u003cem\u003eIndian J. Microbiol.\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 195-202 (2021).\u003c/li\u003e\n\u003cli\u003eFrench, G. L. Bactericidal agents in the treatment of MRSA infections\u0026mdash;the potential role of daptomycin. \u003cem\u003eJ. Antimicrob. Chemother.\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 1107-1117\u003cem\u003e \u003c/em\u003e(2006).\u003c/li\u003e\n\u003cli\u003eMao, X. \u003cem\u003eet al.\u003c/em\u003e Cetylpyridinium chloride: mechanism of action, antimicrobial efficacy in biofilms, and potential risks of resistance.\u003cem\u003e Antimicrob. Agents Chemother.\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, e00576-20 (2020).\u003c/li\u003e\n\u003cli\u003eRumbaugh, K. P. \u0026amp; Sauer, K. Biofilm dispersion. \u003cem\u003eNat. Rev. Microbiol.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 571\u0026ndash;586 (2020).\u003c/li\u003e\n\u003cli\u003e\u003cem\u003e. \u003c/em\u003ePeng, B. \u003cem\u003eet al.\u003c/em\u003e A bibliometric analysis on discovering anti-quorum sensing agents against clinically relevant pathogens: current status, development, and future directions. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1297843\u003cem\u003e \u003c/em\u003e(2023).\u003c/li\u003e\n\u003cli\u003eBhattacharya, S. P., Bhattacharya, A. \u0026amp; Sen, A. A comprehensive and comparative study on the action of pentacyclic triterpenoids on Vibrio cholerae biofilms. \u003cem\u003eMicrob. Pathog.\u003c/em\u003e \u003cstrong\u003e149\u003c/strong\u003e, 104493 (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"β-glycyrrhetinic acid (BGA), biofilm, supragingival plaque, gingivitis, exfoliation effect","lastPublishedDoi":"10.21203/rs.3.rs-6615678/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6615678/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eβ-glycyrrhetinic acid (BGA) possesses antibacterial effects against human supragingival plaque bacteria and inhibit biofilm formation. This study analyzed and compared the effects of BGA on preformed supragingival plaque biofilms with those of cetylpyridinium chloride (CPC). All the experiments were performed using biofilms formed by incubating supragingival plaque bacteria for 24 h. First, we analyzed the number of viable and dead bacteria in the biofilms following BGA and CPC application. The number of viable bacteria was significantly reduced by BGA treatment than by the control. However, the viable/dead bacterial ratios did not significantly vary. Conversely, the turbidity in the supernatants (optical density at 600 nm [OD\u003csub\u003e600\u003c/sub\u003e]) was 0.237\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003, 0.136\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002, and 0.096\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 for the BGA, CPC, and control groups, respectively, indicating superior ability of BGA in biofilm exfoliation. Finally, we evaluated the strength of the biofilms based on the physical impact of BGA or CPC treatment. The biofilm amount ratio before and after sonication was significantly reduced by BGA and CPC than by the control. Furthermore, CPC demonstrated significantly greater reduction in biofilm formation than BGA following sonication. BGA demonstrated excellent bactericidal and exfoliating effects on human supragingival biofilms. These effects are presumably attributed to a mechanism different from that of CPC.\u003c/p\u003e","manuscriptTitle":"β-glycyrrhetinic acid exerts an exfoliating effect on marginal gingivitis-inducing plaque biofilms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 18:09:10","doi":"10.21203/rs.3.rs-6615678/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e290be9b-ca61-4222-acd4-f56e15afdac3","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49476588,"name":"Biological sciences/Microbiology"},{"id":49476589,"name":"Biological sciences/Microbiology/Antimicrobials"},{"id":49476590,"name":"Biological sciences/Microbiology/Biofilms"},{"id":49476591,"name":"Health sciences/Health care/Disease prevention"},{"id":49476592,"name":"Health sciences/Health care/Disease prevention/Preventive medicine"},{"id":49476593,"name":"Health sciences/Diseases/Oral diseases/Gingivitis"},{"id":49476594,"name":"Health sciences/Diseases"},{"id":49476595,"name":"Health sciences/Diseases/Infectious diseases"},{"id":49476596,"name":"Health sciences/Diseases/Oral diseases"},{"id":49476597,"name":"Health sciences/Health care/Dentistry/Oral microbiology"},{"id":49476598,"name":"Health sciences/Health care/Dentistry/Oral microbiology/Dental biofilms"},{"id":49476599,"name":"Health sciences/Health care"},{"id":49476600,"name":"Health sciences/Health care/Dentistry"}],"tags":[],"updatedAt":"2025-07-17T05:26:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-03 18:09:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6615678","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6615678","identity":"rs-6615678","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}