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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.45 No.4 pp.143-151
DOI : https://doi.org/10.11620/IJOB.2020.45.4.143

Inhibitory effects of Coptis chinensis extract on the growth and biofilm formation of Streptococcus mutans and Streptococcus sobrinus

Si Yeong Kim1, Yuri Song1, Hyun Ah Lee1, Hee Sam Na1, Chul Jong Jung2, Gyung Yun Bek2, Jin Chung1*
1Department of Oral Microbiology, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea
2Department of Okchundang Research Institute, Daegu 41059, Republic of Korea
*Correspondence to:Jin Chung, E-mail: jchung@pusan.ac.kr
February 18, 2020 November 30, 2020 December 3, 2020

Abstract


Streptococcus mutans and Streptococcus sobrinus play important roles in dental caries. Coptis chinensis is a natural product with antimicrobial activity against enterobacteria; however, its effects on oral streptococci are still unknown. Therefore, the effects of C. chinensis on the growth and biofilm formation of the representative cariogenic bacteria S. mutans and S. sobrinus were investigated for the possible use of C. chinensis as an anticaries agent. The C. chinensis extract was diluted with sterile distilled water, and 0.1–2.5% of the extract was used in the experiment. The effects of the C. chinensis extract on the growth and glucan formation of S. mutans and S. sobrinus were measured by viable cell counting and spectrophotometry at 650 nm absorbance, respectively. Crystal violet staining was also carried out to confirm the C. chinensis extract’s inhibitory effect on biofilm formation. The C. chinensis extract significantly inhibited the growth of S. mutans and S. sobrinus at concentrations of ≥ 0.3% as compared with the control group. The viable cell count of colonies decreased by 1.7-fold and 1.2-fold at 2.5% and 1.25%, respectively, compared with the control group. The biofilm formation of S. mutans and S. sobrinus was inhibited by > 20-fold at C. chinensis extract concentrations of ≥ 1.25% as compared with the control group. In summary, the C. chinensis extract inhibited the growth and biofilm and glucan formation of S. mutans and S. sobrinus . Therefore, C. chinensis might be a potential candidate for controlling dental caries.


Coptis , Growth , Biofilm , Streptococcus mutans , Streptococcus sobrinus

초록

    Pusan National University(PNU)
    © The Korean Academy of Oral Biology. All rights reserved.

    This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Introduction

    According to a survey of Korean children’s oral health status in 2018, more than half of 12-year-olds experienced permanent teeth caries. And the average number of caries experienced by 12-year-olds was 1.84 which is higher than the average of 1.2 Organization for Economic Cooperation and Development (OECD) member countries [1]. When bacteria that cause early dental caries are isolated, two strains of Streptococcus mutans and Streptococcus sobrinus are separated into 80% and 20%, respectively [2]. They produce glucan and fructan using sucrose as an efficient energy source by glucosyltransferase (GTFase) and fructosyltransferase, respectively. Among them, S. mutans has a close correlation with early dental caries in humans, and extracellular insoluble glucan which is synthesized from sucrose in food by the action of GTFase of S. mutans, is the cause of promoting dental caries [3]. Especially, insoluble glucan makes oral bacteria to adhere onto teeth sur- face and support to form microbial biofilm. Cariogenic bacteria such as S. mutans and S. sobrinus produce organic acids from glucan as well as dietary sugars. The organic acid, especially, lactic acid decomposes hydroxyapatite, a chemical component of tooth enamel. In addition, glucan makes biofilm dense and insoluble, thus, it acts as a barrier to prevent saliva penetration into biofilm resulting in reduced saliva buffering capacity and then, causes acid stagnates locally, which finally leads to dental caries [4]. Therefore, dental caries can be greatly decreased by inhibiting the growth of S. mutans and S. sobrinus . Until now, many studies have been performed to inhibit the growth of S. mutans and S. sobrinus for the control of dental caries. Several agents including antibiotics were developed but they showed adverse effects such as disturbance of intestinal flora, digestive disorders, and hypersensitivity reactions [5]. There are also several natural extracts such as Camellia sinensis [6-8], propolis [9], Akebia quinata [10,11], Magnolia officinalis [12,13], and Xylitol [14] which have inhibitory effects on dental caries. They show excellent antibacterial activities against cariogenic bacteria, however they still have several adverse effects [15- 18]. Thus, we need to find out the effective agent to control dental caries.

    Coptis chinensis (CC) which belongs to Ranunculaceae family has long been used as a herbal medicine, proving its stability [19]. CC is a perennial herb that grows in the mountainous region. It is an uneven columnar shape, 2–4 cm long, slightly curved, tastes bitter, and can remain yellowish on saliva. The roots of CC have various alkaloids such as berberine, worenine, coptisine, palmatine, and sanguinarine [20]. Among them, berberine, a major component of CC, inhibits bacterial growth by inhibiting bacterial carbohydrate metabolism and protein synthesis [19]. Traditionally, it has been used for detoxification, disinfection, treatment of eczema and burn, and hemostasis as well as removal of fever. It was also used to treat various diseases including conjunctivitis, otitis, hypertension and diabetes [21]. CC extract has significant antimicrobial activity against a variety of microorganisms including bacteria, viruses, fungi, protozoans, helminths, and Chlamydia [22-24]. However, the effect of CC on oral streptococci including S. mutans and S. sobrinus are not still investigated.

    In this study at first, we tested the antibacterial activities of several extracts which have been reported to be effective for oral diseases in oriental medicine for the development of preventive agents of dental caries. Among them, CC extract showed the most excellent antibacterial effect. Thus the aim of this study was to study the anticariogenic activity of CC including the inhibitory effects on bacterial growth, glucan formation and biofilm formation.

    Materials and Methods

    1. Bacterial culture

    S. mutans and S. sobrinus were cultured aerobically in brain heart infusion (BHI) at 37℃ in a 5% CO2 atmosphere up to the late log phase of growth.

    2. Preparation and treatment of CC extract and other natural extracts

    CC extract and other natural extracts used in the study were taken from the Okcheondang located in Yeongcheon. All extracts were extracted with hydrothermal water using a reflux cooling extractor with 10 times (w/v) distilled water per 100 g. The extract was filtered with a filter paper (Whatman No. 2; Sigma-Aldrich, St. Louis, MO, USA) and then concentrated with the rotary vacuum evaporator (Rotavapor R-100; Buchi, Essen, Germany) before freeze drying it (TFD5505; ilShin Bio- Base, Dongducheon, Korea). The concentration of the extracts was adjusted by dissolving in phosphate-buffered saline (PBS) from 0.1% (1 mg/mL) to 10% (100 mg/mL).

    3. Screening experiments of herbal extracts against S. mutans and S. sobrinus

    Mentha piperascens, Ulmus macrocarpa Hance, Syzygium aromaticum, Polygonum tinctorium and CC extract suspended in PBS at the concentration of 0 to 5% were added into a 96 well plate and the bacteria were inoculated to 1 × 105 CFU/ well. After incubating at 37℃ for 24 hours, the absorbance was measured at 650 nm using a spectrophotometer (Tecan, Männedorf, Switzerland). The control group was conducted without any herbal extracts.

    4. Measurement of growth of S. mutans and S. sobrinus

    The equal amount of BHI broth and CC extract were added to the 96 well plate and each bacterial species was inoculated to 1 × 105 CFU/well. After incubating at 37℃ for 24 hours, absorbance was measured at 650 nm using spectrophotometer. The control group was conducted without CC extract.

    5. Viable cell count

    CC extract (5 mL) was added into each tube at different concentrations 0 to 2.5%. S. mutans and S. sobrinus (1 × 104 CFU/mL) grown and diluted in BHI broth were inoculated into CC extract containing tube. After 18 hours of incubation at 5% CO2 incubator, diluted with PBS and inoculated into BHI agar to measure the number of viable cells after 48 hours of incubation.

    6. Measurement of biofilm formation

    Each BHI broth with 5% sucrose in a 24 well plate was inoculated with S. mutans and S. sobrinus , respectively and mixed with CC extract to the concentration of 0 to 10%. After incubation at 37℃ for 72 hours to induced biofilm production, the biofilm was washed three times with PBS and then stained with 0.1% crystal violet solution for 10 minutes. After washing three times with PBS and drying in the hood for 15 minutes, the biofilm was dissolved by adding 100% ethanol and the absorbance was measured at 570 nm using a spectrophotometer.

    7. Glucan synthesis test

    S. mutans and S. sobrinus were inoculated to 1 × 105 CFU/ mL into glass test tubes containing BHI broth with or without 5% sucrose and then CC extract were added to the concentration of 0 to 2.5%. After incubation at 37℃ for 24 hours, 1 mL of the culture supernatant was centrifuged and 200 μL of supernatant was transferred into 96 well plate, and then the absorbance was measured at 650 nm by a spectrophotometer. The control group was performed without CC extract.

    Results

    1. Screening of natural extract with antibacterial activity

    The extracts from Mentha piperascens, Ulmus macrocarpa Hance, Syzygium aromaticum, Polygonum tinctorium, and CC were tested for the inhibitory effect on bacterial growth. As shown in Fig. 1, CC extract most inhibited the growth of S. mutans and S. sobrinus compared to other natural extracts. Thus, CC extract was selected for further experiments.

    2. Effect of CC extract on the growth of S. mutans and S. sobrinus

    In order to examine the antibacterial activity of CC extract, S. mutans, S. sobrinus, Streptococcus oralis, Streptococcus mitis, and Streptococcus salivarius were treated with CC extract at 37℃ for 24 hours and the optical density was measured. All concentration of CC extract inhibited the growth of all Streptococcal species used in the experiment, and growth of all test strains was inhibited at concentrations above 0.6% (Fig. 2A). According to viable cell count, 2.5% CC extract significantly decreased the number of viable cells of S. mutans and S. sobrinus compared to control group (Fig. 2B).

    3. Effect of CC extract on biofilm formation

    We examined whether CC extract inhibited the biofilm formation because it is critical for causing dental caries. As shown in Fig. 3, the biofilm formation by S. mutans and S. sobrinus was decreased 20-fold more at the concentrations of 1.25 and 2.5% of CC, compared to the control.

    4. Effect of CC extract on glucan synthesis

    We examined the effect on glucan synthesis which is an essential component for biofilm development. CC extract inhibited glucan synthesis (Fig. 4). Especially, 1.25% and 2.5% of CC extract significantly inhibited glucan synthesis by both S. mutans and S. sobrinus compared to the control.

    Discussion

    CC is a traditional Chinese herbal medicine that has been used to treat diabetes for more than 1,000 years [25]. It has also been used to treat gastroenteritis and diarrhea [26]. In particular, Illustrated Book of Korean Medical Herbs describes the various applications of the efficacy of the CC in relation to infectious diseases [27]. CC has been reported to possess the bactericidal activity in skin infection [27]. Although the anticariogenic effect of CC extract on S. mutans has been previously reported [19], the effects on biofilm formation or glucan synthesis associated with S. sobrinus have not been fully investigated.

    CC is composed of several active ingredients such as coptisine, epiberberine, berberine, and palmatine [28]. Among these, coptisine, epiberberine, and plamatine were reported to inhibit Helicobacter pylori urease and to exhibit antimicrobial effect against H. pylori [29-31]. In this study, CC extract showed strong inhibitory effect on the growth of S. mutans and S. sobrinus in a dose-dependent manner. In particular, the number of viable bacteria was significantly reduced when S. mutans and S. sobrinus were exposed to CC extract with concentration of 2.5% for 18 hours. Therefore, these results suggested that the antibacterial effect of CC extract can also be used to inhibit dental caries by S. mutans and S. sobrinus.

    Next, we examined the effects of CC extract on biofilm formation produced by S. mutans and S. sobrinus. S. mutans and S. sobrinus play an important role in oral biofilm formation [32]. Bacterial populations form biofilms adhering to teeth surfaces and to synthesizing bacterial membranes composed of polysaccharides. Biofilm significantly increases resistance to various environmental stresses such as nutrient depletion, acidity changes, and osmotic pressure compared to floating bacteria, and affect oral health [33-36]. Therefore, it is necessary to control the biofilm formation by S. mutans and S. sobrinus to effectively prevent dental caries. In this experiments, as shown in Fig. 3, we examined the inhibitory effect of CC extract on the biofilm formation by S. mutans and S. sobrinus.

    Finally, we investigated the effects of CC extract on glucan synthesis by S. mutans and S. sobrinus . Glucan promotes bacterial attachment to teeth and interrupts their separation owing to physical forces such as masticatory movements. S. mutans attaches to the tooth surface via sucrose-dependent and sucrose-independent pathways [30-32]. Sucrosedependent adhesion is mediated by glucan binding proteins and glucans produced by GTFase enzymes [37]. The present results showed that CC extract reduced glucan synthesis, suggesting that CC extract could contribute to inhibiting GTFase activty. This result is similar to previous reports that other oriental medicines such as Aconitum koreanum extract and Radix pulsatillae extract, inhibited glucan synthesis and further showed anti-caries effects [4,38]. Taken together, CC extract suppressed the synthesis of biofilm and glucan which is critical for dental plaque formation and dental caries development. This might be caused by the inhibitory effect of CC extract on the growth of S. mutans and S. sobrinus.

    In summary, our study showed that CC extract inhibited the growth of S. mutans and S. sobrinus, the biofilm formation and glucan synthesis by S. mutans and S. sobrinus. Therefore, CC extract might be suggested as a potential candidate for the control of dental caries through the antibacterial effects.

    Acknowledgements

    This work was supported by a 2-Year Research Grant of Pusan National University.

    Figure

    IJOB-45-4-143_F1.gif

    Effect of natural extracts on bacterial growth. Natural extracts were treated to (A) Streptococcus mutans and (B) Streptococcus sobrinus, respectively and the growth inhibitory effects were measured by detecting absorbance at 650 nm after incubation of 37℃ for 24 hours.

    OD, optical density.

    IJOB-45-4-143_F2.gif

    Effect of Coptis chinensis (CC) extract on oral streptococci. (A) Oral streptococcal species were treated with CC extract solution of 0.1% to 2.5% and optical densities (OD) were measured at 650 nm after incubation of 37℃ for 24 hours. (B) Streptococcus mutans and Streptococcus sobrinus were treated with or without 1.25%, 2.5% CC extract at 37℃ overnight and viable cells were counted on brain heart infusion agar plates.

    Con, control.

    *p < 0.05, **p < 0.01.

    IJOB-45-4-143_F3.gif

    Effect of Coptis chinensis extract on biofilm formation. The biofilm was produced by Streptococcus mutans and Streptococcus sobrinus at 37℃ for 72 hours, washed with phosphate-buffered saline, stained with crystal violet and measured by detecting optical densities (OD) at 540 nm using spectrophotometer.

    *p < 0.05, **p < 0.01, ***p < 0.001.

    IJOB-45-4-143_F4.gif

    Effect of Coptis chinensis (CC) extract on glucan synthesis. Streptococcus mutans and Streptococcus sobrinus were grown in BHIS broth with or without CC extract at 37℃ overnight. The culture solution was centrifuged and the optical densities (OD) of the culture supernatants were measured at 650 nm to determine glucan synthesis.

    **p < 0.01, ***p < 0.001.

    Table

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