JKAOH Journal of Korean Academy of Oral Health J Korean Acad Oral Health 1225-388X 2093-7784 Journal of Korean Academy of Oral Health 10.11149/jkaoh.2020.44.1.13 jkaoh-44-13 Original Article Antibacterial effect of different concentrations of Galla Chinensis extract on cariogenic bacteria in a biofilm model KimEun-Jeong1 JinBo-Hyoung2 Dental Research Institution, Korea Department of Preventive & Social Dentistry, School of Dentistry, Seoul National University, Seoul, Korea Corresponding Author: Bo-Hyoung Jin Department of Preventive & Social Dentistry, School of Dentistry, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Korea Tel: +82-2-740-8783 Fax: +82-2-766-8781 E-mail: jjbh@snu.ac.kr https://orcid.org/0000-0003-3526-6805 032020 15032020 441 1319 19022020 07032020 07032020 Copyright © 2020 by Journal of Korean Academy of Oral Health 2020 This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Objectives

Galla chinensis inhibited the adherence of planktonic oral bacteria and acid production by cariogenic bacteria. However, little is known about the relevant conditions of Galla Chinensis extract (GCE) exposure time and concentration and the effect of GCE on the structural and functional activity of cariogenic bacteria. The antibacterial effects of natural G. Chinensis extract on S. mutans, S. sanguinis, and S. oralis biofilms were evaluated in vitro.

 Methods

Biofilms formed on glass surfaces were treated with different concentrations of GCE at different exposure times. The effects were assessed by examining the bactericidal activity, acido-genesis, minimum inhibitory concentration, and morphology.

 Results

There was a statistically significant difference in the bacterial growth inhibition depending on the concentration of the GCE, with bacterial growth being inhibited as the concentration of GCE increased. A concentration of 1.0 mg/ml GCE had similar bactericidal effects against S. mutans and S. oralis biofilms to those produced by 2.0 mg/ml CHX. In the 1.0 mg/ml GCE group, incomplete septa were also observed in the outline of the cell wall, together with disruption of the cell membrane. In addition, there was also a slight exudation of the intracellular content from the bacteria in the 1.0 mg/ml GCE and 2 mg/ml CHX groups.

 Conclusions

These results indicate that GCE inhibits the growth of S. mutans, S. sanguinis, and S. oralis with increasing concentrations. It alters the microstructure of S. mutans biofilms. These results suggest that GCE might be a useful anti-bacterial agent for preventing dental caries.

 Antibacterial effects Biofilms Galla Chinensis Streptococcus mutans References HamadaSKogaTOoshimaTVirulence factors of Streptococcus mutans and dental caries preventionJ Dent Res198463407411 LoescheWJRole of Streptococcus mutans in human dental decayMicrobiol Rev198650353380 MonchoisVWillemotRMMonsanPGlucansucrases: mechanism of action and structure-function relationshipsFEMS Microbiol Rev199923131151 ScheieAAModes of action currently known chemical antiplaque agents other than chlorhexidineJ Dent Res19896816091616 ZhangTTGuoHJLiuXJChuJPZhouXDGalla chinensis compounds remineralize enamel caries lesions in a rat modelCaries Res201650159165 CheynierVPolyphenols in foods are more complex than often thoughtAm J Clin Nutr200581223S229S TagashiraMUchiyamaKYoshimuraTShirotaMUemitsuNIni-hibition by hop bract polyphenols of cellular adherence and water-insoluble glucan synthesis of mutans streptococciBiosci Biotechnol Biochem199761332335 FurigaALonvaud-FunelADorignacGBadetCIn vitro anti-bacterial and anti-adherence effects of natural polyphenolic compunds on oral bacteriaJ Appl Microbiol200810514701476 HuangSGaoSChengLYuHCombined effects of nano-hydroxyapatite and Galla Chinensis on remineralization of initial enamel lesion in vitroJ Dent201038811819 CostertonJWIntroduction to biofilmInt J Antimicrob Agents199911217221 HaffajeeADSocranskySSIntroduction to microbial aspects of periodontal biofilm communities, development and treatmentPeri-odontol 2000200642712 NascimentoGGLocatelliJFreitasPCSilvaGLAntibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteriaBraz J Microbiol200031247256 XieQLiJZhouXAnticaries effect of compounds extracted from Galla Chinensis in a multispecies biofilm modelOral Microbiol Im-munol200823459465 National Committee for Clinical Laboratory StandardsMethods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: Approved Standard M7-A5NCCLSWayne, PA, USA Clinical and Laboratory Standards InstituteMethods for antimicro-bial susceptibility testing of anaerobic bacteria; approved standard M11-A88th ed.PA, USACLSI2012 GamboaFEstupinanMGalindoAPresence of streptococcus mutans in saliva and its relationship with dental caries: antimicrobial susceptibility of the isolatesUniversitas Scientiarum200492327 HuangXDengMLiuMChengLExterkateRAMLiJComparison of composition and anticaries effect of Galla Chinensis extracts with different isolation methodsOpen Dent J201711447459 AndrewsJMDetermination of minimum inhibitory concentrationsJ Antimicrob Chemother200148516 MieleWHEfficacy of grapefruit seed extract against Salmonella typhi, Escherichia coli, and Staphylococcus aureus, Microbiological food analysis report reviewed and approved by Southern Testing and Research LaboratoriesNCWilson Inc198815 KimJEKimHEHwangJKLeeHJKwonHKKimBIAntibacterial characteristics of Curcuma xanthorrhiza extract on Streptococcus mutans biofilmJ Microbiol200846228232 DuarteSGregoireSSinghAPVorsaNSchaichKBowenWHInhibitory effects of cranberry polyphenols on formation and ac-idogenicity of Streptococcus mutans biofilmsFEMS Microbiol Lett20062575056 HaslamENatural polypheonols (vegetable tannins) as drugs: possible modes of actionJ Nat Prod199659205215 BurtSEssential oils: Their antibacterial properties and potential applications in foods - a reviewInt J Food Microbiol200494223253 Figures and Tables

The bacterial growth curve of S. mutans, S. sanguinis and S. oralis biofilms by GCE concentration. After the biofilms had been exposed to the test solutions for 3, 6, 9, 12, and 24 hours, the number of colonies was counted to determine the CFU. There was a significant difference over time at all concentrations.

TEM images of the S. mutans biofilm after 1 hour of treatment. The black arrows indicate the wall of S. mutans, and the white arrows indicate the intracellular contents. The scale bar is 100 nm. (A) 1% DMSO, (B) 2 mg/ml CHX, and (C) 1.0 mg/ml GCE. After the S. mutans biofilms had been exposed to 1% DMSO for 1 h as the negative control, the TEM showed a clear outline of the S. mutans cell wall and a peptidoglycan layer. However, most of the peptidoglycan layers of S. mutans in the CHX group had disappeared. In the 1.0 mg/ml GCE group, incomplete septa were also observed in the outline of the cell wall. In addition, there was a slight exudation of the intracellular contents in the 1.0 mg/ml GCE and 2 mg/ml CHX groups.

MIC induced by GCE at different concentrations

Treatment group S. mutans* S. sanguis* S. oralis*
GCE 0.1 mg/ml M±SD 2.43±0.04a 2.58±0.03a 2.60±0.02a
Proportion (%) 84.04 80.51 87.18
GCE 0.2 mg/ml M±SD 2.32±0.04a 2.14±0.12a,b 2.16±0.04b
Proportion (%) 75.75 52.05 56.5
GCE 0.4 mg/ml M±SD 1.95±0.14b 2.10±0.07a,b 2.41±0.06b
Proportion (%) 47.83 50.21 48.68
GCE 0.8 mg/ml M±SD 1.69±0.08b 2.08±0.08a,b 1.95±0.05c
Proportion (%) 36.96 41.51 46.18
GCE 1.0 mg/ml M±SD 1.48±0.10c 1.54±0.12c 1.35±0.08d
Proportion (%) 30.13 28.92 25.68
CHX 2.0 mg/ml M±SD 1.40±0.30c 1.35±0.06c 1.25±0.05d
Proportion (%) 28.81 24.1 23.23
1% DMSO M±SD 2.69±0.05a 2.69±0.03a,b 2.74±0.01a
Proportion (%) 102.35 89.72 107.59

Data are presented as log10 colony-forming units (CFU)/disc and percentages. M, Ln (Log10 CFU); SD, standard deviation; Proportion, If the OD value is less than 90-95% of the control 100%, the bacteria is affected and has not grown.

The different superscripts in the same column indicate a statistically significant difference from each group (P<0.05). Post hoc Tukey’s HSD, *P<0.05.

General linear model including time and treatment groups

Source df Mean square F Sig. Partial eta square
Treatment groups 6 15.12 5694.59 <.001 0.995
Time 4 10.11 3806.47 <.001 0.989
Treatment groups×Time 24 0.89 337.88 <.001 0.979

R Squared=.997 (Adjusted R Squared=.996).