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Abstract: Drynaria fortunei (D. fortunei), widely used in traditional Korean medicine, is reportedly effective in treating inflammation, hyperlipidemia, bone fractures, oxidative damage, arteriosclerosis, rheumatism, and gynecological diseases. The objective of this study was to evaluate the antibacterial effects of the chloroform fraction of D. fortunei (DFCF) and assess the synergistic effects of DFCF with antibiotics against bacterial pathogens. This was carried out by calculating the minimal inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) and performing checkerboard dilution test and time-kill assays. The MICs/MBCs for DFCF, ampicillin, and gentamicin against all oral strains were >39-2,500/5,000 μg/mL, 0.25-64/0.25-64 µg/mL, and 0.5-256/1-512 µg/mL, respectively. DFCF exhibited the highest activity against the periodontic pathogens Prevotella intermedia and Porphylomonas gingivalis. DFCF in combination with ampicillin showed a strong synergistic effect against oral bacteria (fractional inhibitory concentration (FIC) index ≤0.5), whereas on combining with gentamicin, it reduced the on half-eighth times than used alone (FICI ≤ 0.5). DFCF combined with ampicillin or gentamicin killed 100%

of most tested bacteria within 3-4 h. The results of this study demonstrate the antimicrobial and synergistic activity of DFCF and antibiotics against oral patho-gens.

Keywords: Drynaria fortunei; oral bacteria; synergistic effect; antimicrobial activity; minimum inhibitory concentrations; minimum bactericidal concentrations.

IntroductionThe human oral microbiome contains approximately 700 predominant bacterial species that protect the oral cavity (1,2). However, the oral microbiota are often involved in the pathogenesis of a variety of systemic diseases including respiratory infections, cancer, diabetes melli-tus, rheumatoid arthritis, cardiovascular diseases, brain abscess, and premature birth (3-5). Dental caries and periodontal diseases are the most common oral and dental diseases (6,7). The bacteria living in the oral cavity occur in the form of biofilms and bacterial plaque accumulated on solid surfaces, such as the tooth enamel, fillings, restorations, and orthodontic appliances, and play a role in the etiology and pathogenesis of periodontal disease and dental caries, often resulting in teeth loss if left untreated (4,8). The accumulation of plaque around the gingival and subgingival regions may lead to a change in the microbial composition from predominantly Mutans streptococci to a larger number of Actinomyces spp., and capnophilic and obligatory anaerobic bacteria such as

Journal of Oral Science, Vol. 59, No. 1, 31-38, 2017

Original

Antimicrobial activity of the chloroform fraction of Drynaria fortunei against oral pathogens

Jeong-Dan Cha1), Eun-Kyung Jung2), Sung-Mi Choi3), Kyung-Yeol Lee1), and Sung-Wook Kang4)

1)Department of Oral Microbiology and Institute of Oral Bioscience, Chonbuk National University, Jeonju, Republic of Korea

2)Department of Dental Hygiene, Ulsan College, Ulsan, Republic of Korea3)Department of Dental Hygiene, Daegu Health College, Daegu, Republic of Korea4)Department of Public Health, Daegu Haany University, Daegu, Republic of Korea

(Received March 7, 2016; Accepted June 24, 2016)

Correspondence to Dr. Sung-Wook Kang, Department of Public Health, Daegu Haany University, 1 Haanydaero, Gyeongsan-si, Gyeongsangbuk-do 712-715, Republic of KoreaE-mail: health@dhu.ac.krdoi.org/10.2334/josnusd.16-0150DN/JST.JSTAGE/josnusd/16-0150

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Porphyromonas gingivalis (P. gingivalis) (9-11). Several antiseptic agents including chlorhexidine, cetylpyridin-ium chloride, fluorides, phenol derivatives, ampicillin, erythromycin, penicillin, tetracycline, and vancomycin have been used widely in dentistry to reduce bacterial growth (12-14). However, these chemicals may disturb the oral and intestinal flora, and may cause side effects such as microbe susceptibility, vomiting, diarrhea, and tooth staining (15-17). In order to address these issues, natural antimicrobial agents with fewer side effects and specificity for oral pathogens should be studied further. Plants have a large number of biologically active compounds, many of which have been reported to have antimicrobial properties (18-20).

Drynaria fortunei (D. fortunei; Gol-Se-Bo in Korean and Gu-Sui-Bu in Chinese), a traditional Korean medicine, has been known to be effective in treating inflammation, hyperlipidemia, arteriosclerosis, rheumatism, gynecolog-ical diseases, osteoporosis, and bone resorption (21-25). It is also used for treatment and prevention of orthopedic diseases, and is known to promote bone healing (22,26). Previous studies have reported that it exhibited anti-oxidant effects on rat osteoblasts undergoing hydrogen peroxide-induced death, and promoted the restoration of bone under similar pathologic conditions (27). The water extract of D. fortunei has been exhibited to have a significant protective effect against ototoxicity caused by treptomycin, streptomycin, and kanamycin in humans, and also inhibit the progression of bone loss induced by ovariectomy in rats (28,29).

This study aimed to determine the antimicrobial effects of the chloroform fraction of D. fortunei (DFCF) on the growth of cariogenic and periodontic pathogens. Additionally, a combination of DFCF with currently used antibiotics was also investigated.

Materials and MethodsPlant material and preparation of crude plant extracts D. fortunei was identified by Dr. Young-Sung Ju at the College of Oriental Medicine, Woosuk University, and purchased from the herbal medicine cooperative associa-tion of Jeonbuk Province, Korea, in November 2004. A voucher specimen (No. JS12A) has been deposited at the Division of Medicinal Chemistry and Cosmetics, Mokwon University. The powdered roots (1.2 kg) of D. fortunei were extracted by repeated refluxing with methanol (MeOH, Merck, Darmstadt, Germany) (12 L) for 4 h at 80°C. The combined MeOH extract (12 L) was clarified by filtration and evaporated to obtain a dark brown syrup (210 g). This was then suspended in water

(H2O) and partitioned with chloroform (CHCl3, Merck), ethyl acetate (EtOAc, Merck), and n-butanol (n-BuOH, Merck) successively. The organic solvent extracts were dried in vacuum at 45°C to yield a CHCl3 soluble fraction (0.23 g), EtOAc soluble fraction (8.94 g), and n-BuOH soluble fraction (22.47 g).

Bacterial strains Streptococcus mutans (S. mutans ATCC 25175), Streptococcus sanguinis (S. sanguinis ATCC 10556), Streptococcus sobrinus (S. sobrinus ATCC 27607), Streptococcus ratti (S. ratti KCTC 3294), Streptococcus criceti (S. criceti KCTC 3292), Streptococcus anginosus (S. anginosus ATCC 31412), Streptococcus gordonii (S. gordonii ATCC 10558), Aggregatibacter actinomycetem-comitans (A. actinomycetemcomitans ATCC 43717), Fusobacterium nucleatum (F. nucleatum ATCC 51190), Prevotella intermedia (P. intermedia ATCC 49046), and Porphylomonas gingivalis (P. gingivalis ATCC 33277) were identified using the broth dilution method. Faculta-tive anaerobic bacteria were incubated with Brain-Heart Infusion broth (BHI; Difco Laboratories, Detroit, MI, USA) for 18 h at 37°C. BHI supplemented with 1% yeast extract (Difco) was used to incubate A. actinomy-cetemcomitans, F. nucleatum, and P. intermedia for 24 h at 37°C, and BHI containing hemin (Sigma Aldrich, St Louis, MO, USA) and menadione (Sigma Aldrich) was used for incubating the obligate anaerobic bacteria, P. gingivalis, for 48 h at 37°C.

Minimum inhibitory concentrations/minimum bactericidal concentrations assayThe minimum inhibitory concentration (MIC) of the CHCl3 DFCF was determined using the broth dilution method, and this was repeated three times. The antibacte-rial activities were recorded after incubation at 37°C for 18-48 h under anaerobic conditions. MIC was defined as the lowest concentration of tested DFCF that completely inhibited visible growth in the broth. To measure the minimum bactericidal concentration (MBC), the lowest concentration of tested DFCF was subcultured on appro-priate agar plates. The number of colonies were counted and compared with the number of CFU/mL in the original inoculum after 24-48 h. The lowest concentration of DFCF that could kill 99.9% of bacteria was defined as the MBC.

Checker board dilution test The combination of DFCF and antibiotics (ampicillin and gentamicin, Sigma) was determined using a checker-board test against oral pathogens, as previously described

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(30,31). Combination assays were examined in 24-well plates, where the first agent was diluted twofold, followed by addition of the second agent at various concentra-tions. Thereafter, the bacteria were added to each well and incubated at 37°C for 24-48 h. The interaction was identified as being synergistic if the fraction inhibitory concentration (FIC)/fraction bactericidal concentration (FBC) index was less than or equal to 0.5, additive if the FIC/FBC index was greater than 0.5 and less than or equal to 1.0, indifferent if the FIC/FBC index was greater than 1.0 and less than or equal to 2.0, and antagonistic if the FIC/FBC index was greater than 2.0 (31).

Time-kill curves The bactericidal activities of the DFCF were also esti-mated using time-kill curves on oral pathogens. Tubes containing 1/2 MIC of DFCF with antibiotics individu-ally or in combination were inoculated with a suspension having a final bacterial count of approximately 1×106-5×106 CFU/mL. After incubation, 100 µL of bacterial aliquots were spread onto BHI agar to allow counting of the colony-forming units (CFU).

Statistical analysis The data have been presented as mean and S.E. for each individual experiment. One-way analysis of variance (ANOVA) was used to analyze the data, and P values less than 0.05 were considered statistically significant.

ResultsAntibacterial activity: MICs/MBCsDFCF exhibited antibacterial effects against all tested oral bacteria in a dose-dependent manner (Fig. 1). It had the strongest effect against P. interdedia and P. gingivalis (MICs, 39 μg/mL: MBCs, 39-78 μg/mL), and a moderate effect against S. mutans, S. sobrinus, and

gordonii (MICs, 78 μg/mL; MBCs, 156 μg/mL) (Table 1, Fig. 1). The MICs were either 0.25/0.25 or 64/64 μg/mL for ampicillin, and 0.5/1 or 256/512 μg/mL (Table 1) for gentamicin. The range of MIC50 and MIC90 was 9-313 µg/mL and 39-2,500 µg/mL, respectively. The DFCF showed stronger antimicrobial activity against S. gordonii, P. intermedia, and P. gingivalis compared to other bacteria (MIC/MBC, 39-78/39-156 µg/mL), and the range of MIC50 and MIC90 was 9 µg/mL and 39-78 µg/mL, respectively.

Combined effects: bacteriostatic synergismThe FIC and FBC indices for DFCF combined with ampicillin or gentamicin against oral bacteria have been presented in Tables 2 and 3, respectively. The combination of DFCF with ampicillin exhibited ≥4-8-fold reduction of the MICs for all tested bacteria except S. criceti, indi-cating a synergistic effect (FICI values of ≤0.25-0.5), and MBCs were shown as synergistic effect by FBCI values of ≤0.5, except S. mutans and S. creceti (FBCI values of ≥0.5) (Table 2). The combination of DFCF and genta-micin resulted in a ≥4-8-fold reduction of MIC/MBCs for all tested bacteria, with the ranges of 9-313/9-1250 μg/mL for gentamicin becoming 0.125-32/0.25-64 μg/mL. The FICI/FBCI indicated synergistic interactions for the combination of DFCF and gentamicin (FICI/FBCI values ≤0.125-0.5/0.5) for S. sanguinis, S. sobrinus, S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis, except S. mutans, S. ratti, S. criceti, and S. anginosus with additive (FICI/FBCI values of ≥0.75/≥0.75-1.0) (Table 3).

Combined effects: time-kill curveThe time-kill curve assay was used to examine the syner-gistic effect of DFCF with ampicillin or/and gentamicin against oral bacteria. Bacteria cultured at a cell density of

Table 1 Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the extract and several fractions of Drynaria fortunei against oral bacteria

StrainsDrynaria fortunei CHCl3 fraction (µg/mL) Ampicillin Gentamicin

MIC50 MIC90 MIC/MBCS. mutans ATCC 25175 39 78 78/156 4/4 8/8S. sanguinis ATCC 10556 78 313 313/313 32/32 8/16S. sobrinus ATCC 27607 19 78 78/156 2/2 4/8S. retti KCTC 3294 78 313 313/313 4/4 4/8S. criceti KCTC 3292 39 313 313/625 4/4 8/8S .anginosus ATCC 31412 19 156 156/156 4/4 16/16S. gordonii ATCC 10558 9 78 78/156 1/2 2/4A. actinomycetemcomitans ATCC 43717 313 2,500 2,500/5,000 64/64 2/2F. nucleatum ATCC 51190 313 1,250 1,250/1,250 0.25/0.25 16/32P. intermedia ATCC 49046 9 39 39/78 32/32 0.5/1P. gingivalis ATCC 33277 9 39 39/39 0.5/1 256/512ATCC, American type culture collection; KCTC, Korean collection for type cultures.

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106 CFU/mL were exposed to MICs of the DFCF alone or in combination with ampicillin or/and gentamicin. The combination with antibiotics exhibited a time-dependent increase in rate of killing, measured in CFU/mL than DFCF alone. The combination of 1/2 MIC DFCF with

1/2 MIC ampicillin or 1/2 gentamicin killed 100% of the bacteria within 3 and 4 h (Fig. 2). The results confirmed the synergistic effect of DFCF with antibiotics against oral bacteria.

Fig. 1 Antibacterial activity of CHCl3 fraction of D. fortunei against S. mutans, S. sanguinis, S. sobrinus, S. ratti, S. criceti, S. anginosus, S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis. Bacteria were incubated with several concentrations of CHCl3 fraction for 24 h. Data points represent mean values ± S.E.M. of the four experiments.

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DiscussionThe use of medicinal plants may be considered a suitable alternative for the treatment of different pathological diseases. A large range of plant extracts have been shown

to have antimicrobial effects and this can contribute to the development of new drugs, thus generating a significant improvement in management of various health disorders (20,32,33).

In the present study, DFCF exhibited strong antibac-

Table 2 Checkerboard assay of CHCl3 fraction of Drynaria fortunei and ampicillin against oral bacteria

Strains Agent MIC/MBC (µg/mL) FIC/FBC FICI/FBCI OutcomeAlone CombinationS. mutans ATCC 25175 CHCl3 78/156 19/39 0.25/0.25 0.5/0.75 Synergistic/Additive

Ampicillin 4/4 1/2 0.25/0.5S. sanguinis ATCC 10556 CHCl3 313/313 39/78 0.125/0.25 0.375/0.5 Synergistic/Synergistic

Ampicillin 32/32 8/8 0.25/0.25S. sobrinus ATCC 27607 CHCl3 78/156 19/39 0.25/0.25 0.5/0.5 Synergistic/Synergistic

Ampicillin 2/2 0.5/0.5 0.25/0.25S. retti 4KCTC 3294 CHCl3 313/313 39/78 0.125/0.25 0.5/0.75 Synergistic/Additive

Ampicillin 4/4 1/2 0.25/0.5S. criceti KCTC 3292 CHCl3 313/625 78/156 0.25/0.25 0.75/0.75 Additive/Additive

Ampicillin 4/4 2/2 0.5/0.5S. anginosus ATCC 31412 CHCl3 156/156 19/39 0.125/0.25 0.375/0.5 Synergistic/Synergistic

Ampicillin 4/4 1/1 0.25/0.25S. gordonii ATCC 10558 CHCl3 78/156 9/39 0.125/0.25 0.375/0.5 Synergistic/Synergistic

Ampicillin 1/2 0.25/0.5 0.25/0.25A. actinomycetemcomitans ATCC 43717

CHCl3 2,500/5,000 313/1,250 0.125/0.25 0.25/0.5 Synergistic/SynergisticAmpicillin 64/64 8/16 0.125/0.25

F. nucleatum ATCC 51190 CHCl3 1,250/1,250 156/313 0.125/0.25 0.25/0.5 Synergistic/SynergisticAmpicillin 0.25/0.25 0.0313/0.0625 0.125/0.25

P. intermedia ATCC 49049 CHCl3 39/78 9/19 0.25/0.25 0.375/0.5 Synergistic/SynergisticAmpicillin 32/32 4/8 0.125/0.25

P. gingivalis ATCC 33277 CHCl3 39/39 9/9 0.25/0.25 0.5/0.5 Synergistic/SynergisticAmpicillin 0.5/1 0.125/0.25 0.25/0.25

The MIC and MBC of the CHCl3 fraction with ampicillin. FIC, the fractional inhibitory concentration; FBC, index/fractional bactericidal concentration index; ATCC, American type culture collection; KCTC, Korean collection for type cultures.

Table 3 Checkerboard assay of CHCl3 fraction of Drynaria fortunei and gentamicin against some oral bacteria

Strains Agent MIC/MBC (µg/mL)FIC/FBC FICI/FBCI2 OutcomeAlone Combination1

S. mutans ATCC 25175 CHCl3 78/156 19/39 0.25/0.25 0.5/0.75 Synergistic/AdditiveGentamicin 8/8 2/4 0.25/0.5

S. sanguineis ATCC 10556 CHCl3 313/313 39/78 0.125/0.25 0.25/0.5 Synergistic/SynergisticGentamicin 8/16 1/4 0.125/0.25

S. sobrinus ATCC 27607 CHCl3 78/156 19/39 0.25/0.25 0.375/0.5 Synergistic/SynergisticGentamicin 4/8 0.5/2 0.125/0.25

S. retti 4KCTC 3294 CHCl3 313/313 78/156 0.25/0.5 0.75/0.75 Additive/AdditiveGentamicin 4/8 2/2 0.5/0.25

S. criceti KCTC 3292 CHCl3 313/625 156/156 0.5/0.25 0.75/0.75 Additive/AdditiveGentamicin 8/8 2/4 0.25/0.5

S. anginosus ATCC 31412 CHCl3 156/156 78/78 0.5/0.5 0.75/1.0 Additive/AdditiveGentamicin 16/16 4/8 0.25/0.5

S. gordonii ATCC 10558 CHCl3 78/156 19/39 0.25/0.25 0.375/0.375 Synergistic/SynergisticGentamicin 2/4 0.25/0.5 0.125/0.125

A. actinomycetemcomitans ATCC 43717

CHCl3 2,500/5,000 313/1,250 0.125/0.25 0.375/0.75 Synergistic/AdditiveGentamicin 2/2 0.5/1 0.25/0.5

F. nucleatum ATCC 51190 CHCl3 1,250/1,250 156/313 0.125/0.25 0.25/0.5 Synergistic/SynergisticGentamicin 16/32 2/8 0.125/0.25

P. intermedia ATCC 49049 CHCl3 39/78 9/19 0.25/0.25 0.5/0.5 Synergistic/SynergisticGentamicin 0.5/1 0.125/0.25 0.25/0.25

P. gingivalis ATCC 33277 CHCl3 39/39 9/9 0.25/0.25 0.375/0.375 Synergistic/SynergisticGentamicin 256/512 32/64 0.125/0.125

1The MIC and MBC of the CHCl3 fraction with gentamicin; 2The fractional inhibitory concentration (FIC) index/fractional bactericidal concentration (FBC) index. ATCC, American type culture collection; KCTC, Korean collection for type cultures.

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terial activity against oral bacteria in a dose and time dependent manner. The MIC and MBC values of DFCF were shown on 39-313 µg/mL and 39-625 mg/mL in tested bacteria, respectively, except for A. actinomy-

cetemcomitans and F. nucleatum where the MICs/MBCs values were shown on 1,250-2,500/1,250-5,000 µg/mL. The most sensitive bacterial strains were the periodontal pathogens such as P. intermedia and P. gingivalis (MIC/

Fig. 2 Time-kill curves of MICs of CHCl3 fraction alone and in combination with MICs of ampicillin or gentamicin against S. mutans, S. sanguinis, S. sobrinus, S. ratti, S. criceti, S. anginosus, S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis. Bacteria were incubated with CHCl3 fraction along (■), CHCl3 frac-tion with ampicillin (∆), and CHCl3 fraction with gentamicin (●) over time. Data points represent mean values ± S.E.M. of the four experiments. CFU, colony-forming units.

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MBCs ranging from 39/39-78 µg/mL) and the cariogenic species including S. sobrinus and S. gordonii (MIC/MBC = 78/156 µg/mL). Antibacterial agents such as chlorhexi-dine, ampicillin, erythromycin, penicillin, tetracycline, vancomycin, cetylpyridinium chloride, triclosan, and chlorine dioxide have been shown to have side effects when tested alone or in combination (34-38). In recent years, several studies have reported that various plant extracts or their derivatives can reduce these side effects (39,40), although some combinations of natural materials and antibiotics may affect the inhibitory effect of these antibiotics (41,42).

The n-BuOH, MeOH, and H2O extracts of D. fortunei contain alkaloids, phenolics, glycosides, proteins, and flavonoids. The CHCl3 extract contains naringin, hesperidin, caffeic acid, and procyanidin, but flavonoids was detected only in the n-BuOH extract for presence of organic acid (21,22,25,28,43). Some of the polyphenols isolated from plants exhibit anti-carious effects such as growth inhibition and inhibition of glucosyltransfer-ases against mutans streptococci (44-46). Flavonoid complexes attach with extracellular soluble proteins and the bacterial cell wall, and exhibit antibacterial activity (47,48). As DFCF is composed of many flavonoids, it may exhibit strong antibacterial activity against oral bacteria.

The combination of DFCF with ampicillin or genta-micin showed a rapid killing rate for all tested bacteria, eradicating almost 100% of the bacteria within 3-4 h. Moreover, this differential effect of D. fortunei extract and fractions on bacteria associated with caries and oral periodontal pathogens was also reflected in the time-kill curves performed for S. mutans and P. gingivalis, where Streptococcus was killed much slower than Porphy-romonas. As D. fortunei is composed of many flavonoids, it may exhibit strong antibacterial activity against oral bacteria.

In conclusion, this study presents preliminary evidence of the antimicrobial activity of DFCF against oral bacte-rial species. It seems likely been the development of a new oral therapies.

Conflict of interestThe authors have no conflict of interest to declare.

References1. Takahashi N (2015) Oral Microbiome metabolism: from

“who are they?” to “what are they doing?”. J Dent Res 94, 1628-1637.

2. Ting M, Whitaker EJ, Albandar JM (2016) Systematic review of the in vitro effects of statins on oral and perioral microor-

ganisms. Eur J Oral Sci 124, 4-10. 3. Patil S, Rao RS, Sanketh DS, Amrutha N (2013) Microbial

flora in oral diseases. J Contemp Dent Pract 14, 1202-1208.4. Relvas M, Diz P, Seoane J, Tomás I (2013) Oral health scales:

design of an oral health scale of infectious potential. Med Oral Patol Oral Cir Bucal 18, e664-670.

5. Słotwińska SM, Słotwiński R (2015) Host response, obesity, and oral health. Cent Eur J Immunol 40, 201-205.

6. Costalonga M, Herzberg MC (2014) The oral microbiome and the immunobiology of periodontal disease and caries. Immunol Lett 162, 22-38.

7. Celakovsky P, Kalfert D, Smatanova K, Tucek L, Cermakova E, Mejzlik J et al. (2015) Bacteriology of deep neck infec-tions: analysis of 634 patients. Aust Dent J 60, 212-215.

8. Pasich E, Walczewska M, Pasich A, Marcinkiewicz J (2013) Mechanism and risk factors of oral biofilm formation. Postepy Hig Med Dosw (Online) 67, 736-741.

9. Seneviratne CJ, Zhang CF, Samaranayake LP (2011) Dental plaque biofilm in oral health and disease. Chin J Dent Res 14, 87-94.

10. Wade WG (2013) The oral microbiome in health and disease. Pharmacol Res 69, 137-143.

11. Struzycka I (2014) The oral microbiome in dental caries. Pol J Microbiol 63, 127-135.

12. van Winkelhoff AJ (2012) Antibiotics in the treatment of peri-implantitis. Eur J Oral Implantol 5, Suppl, S43-S50.

13. Latimer J, Munday JL, Buzza KM, Forbes S, Sreenivasan PK, McBain AJ (2015) Antibacterial and anti-biofilm activity of mouthrinses comtaining cetylpyridinium chloride and sodium fluoride. BMC Microbiol 15, 169.

14. Verardi G, Cenci MS, Maske TT, Webber B, Santos LR (2016) Antiseptics and microcosm biofilm formation on titanium surfaces. Braz Oral Res, doi: 10.1590/1807-3107BOR-2016.

15. Takeuchi N, Chuma T, Mano Y (2004) Phenol block for cervical dystonia: effects and side effects. Arch Phys Med Rehabil 85, 1117-1120.

16. Lin S, Levin L, Weiss EI, Peled M, Fuss Z (2006) In vitro antibacterial efficacy of a new chlorhexidine slow-release device. Quintessence Int 37, 391-394.

17. McCoy LC, Wehler CJ, Rich SE, Garcia RI, Miller DR, Jones JA (2008) Adverse events associated with chlorhexidine use: results from the Department of Veterans Affairs Dental Diabetes Study. J Am Dent Assoc 139,178-183.

18. Leszczyńska A, Buczko P, Buczko W, Pietruska M (2011) Periodontal pharmacotherapy--an updated review. Adv Med Sci 56, 123-131.

19. Chandra Shekar BR, Nagarajappa R, Suma S, Thakur R (2015) Herbal extracts in oral health care--a review of the current scenario and its future needs. Pharmacogn Rev 9, 87-92.

20. Kouidhi B, Al Qurashi YM, Chaieb K (2015) Drug resistance of bacterial dental biofilm and the potential use of natural compounds as alternative for prevention and treatment. Microb Pathog 80, 39-49.

21. Long M, Qiu D, Li F, Johnson F, Luft B (2005) Flavonoid of

38

Drynaria fortunei protects against acute renal failure. Phyto-ther Res 19, 422-427.

22. Jeong JC, Lee BT, Yoon CH, Kim HM, Kim CH (2005) Effects of Drynariae rhizoma on the proliferation of human bone cells and the immunomodulatory activity. Pharmacol Res 51, 125-136.

23. Lee YE, Liu HC, Lin YL, Liu SH, Yang RS, Chen RM (2014) Drynaria fortunei J. Sm. improves the bone mass of ovari-ectomized rats through osteocalcin-involved endochondral ossification. J Ethnopharmacol 158 Pt A, 94-101.

24. Chang HC, Chen JC, Yang JL, Tsay HS, Hsiang CY, Ho TY (2014) The suppressive activities of six sources of medicinal ferns known as gusuibu on heat-labile enterotoxin-induced diarrhea. Molecules 19, 2114-2120.

25. Yang RC, Chang CC, Sheen JM, Wu HT, Pang JH, Huang ST (2014) Davallia bilabiata inhibits TNF-α-induced adhesion molecules and chemokines by suppressing IKK/NF-kappa B pathway in vascular endothelial cells. Am J Chin Med 42, 1411-1429.

26. Wang X, Zhen L, Zhang G, Wong MS, Qin L, Yao X (2011) Osteogenic effects of flavonoid aglycones from an osteo-protective fraction of Drynaria fortunei--an in vitro efficacy study. Phytomedicine 18, 868-872.

27. Wong KC, Pang WY, Wang XL, Mok SK, Lai WP, Chow HK et al. (2013) Drynaria fortunei-derived total flavonoid frac-tion and isolated compounds exert oestrogen-like protective effects in bone. Br J Nutr 110, 475-485.

28. Long M, Smouha EE, Qiu D, Li F, Johnson F, Luft B (2004) Flavanoid of Drynaria fortunei protects against gentamicin ototoxicity. Phytother Res 18, 609-614.

29. Liu X, Zhang S, Lu X, Zheng S, Li F, Xiong Z (2012) Meta-bonomic study on the anti-osteoporosis effect of Rhizoma Drynariae and its action mechanism using ultra-performance liquid chromatography-tandem mass spectrometry. J Ethno-pharmacol 139, 311-317.

30. Lee YS, Jang KA, Cha JD (2012) Synergistic antibacterial effect between silibinin and antibiotics in oral bacteria. J Biomed Biotechnol 2012, 618081.

31. Farooqui A, Khan A, Borghetto I, Kazmi SU, Rubino S, Paglietti B (2015) Synergistic antimicrobial activity of Camellia sinensis and Juglans regia against multidrug-resistant bacteria. PLoS One 10, e0118431.

32. Ferrazzano GF, Roberto L, Catania MR, Chiaviello A, De Natale A, Roscetto E et al. (2013) Screening and scoring of antimicrobial and biological activities of Italian vulnerary plants against major oral pathogenic bacteria. Evid Based Complement Alternat Med 2013, 316280.

33. Samprasit W, Opanasopit P, Sukma M, Kaomongkolgit R (2014) Antibacterial activity of Garcinia mangostana extracts on oral pathogens. Minerva Stomatol 63, 249-257.

34. Moshrefi A (2002) Chlorhexidine. J West Soc Periodontol Periodontal Abstr 50, 5-9.

35. Quirynen M, Soers C, Desnyder M, Dekeyser C, Pauwels M,

van Steenberghe D (2005) A 0.05% cetyl pyridinium chlo-ride/0.05% chlorhexidine mouth rinse during maintenance phase after initial periodontal therapy. J Clin Periodontol 32, 390-400.

36. Mombelli A (2012) Antimicrobial advances in treating peri-odontal diseases. Front Oral Biol 15, 133-148.

37. Pinto CF, Berger SB, Cavalli V, Da Cruz SE, Gonçalves RB, Ambrosano GM et al. (2015) In situ antimicrobial activity and inhibition of secondary caries of self-etching adhesives containing an antibacterial agent and/or fluoride. Am J Dent 28, 167-173.

38. Pankhurst C, Rautemaa-Richardson R, Seoudi N, Smith A, Wilson M (2016) Antimicrobial resistance: antibiotics and consultant oral microbiologist posts. Br Dent J 220, 2-3.

39. Smullen J, Koutsou GA, Foster HA, Zumbé A, Storey DM (2007) The antibacterial activity of plant extracts containing polyphenols against Streptococcus mutans. Caries Res 41, 342-349.

40. Ma XH, Zheng CJ, Han LY, Xie B, Jia J, Cao ZW et al. (2009) Synergistic therapeutic actions of herbal ingredients and their mechanisms from molecular interaction and network perspectives. Drug Discov Today 14, 579-588.

41. Hemaiswarya S, Kruthiventi AK, Doble M (2008) Synergism between natural products and antibiotics against infectious diseases. Phytomedicine 15, 639-652.

42. Jang EJ, Cha SM, Choi SM, Cha JD (2014) Combination effects of baicalein with antibiotics against oral pathogens. Arch Oral Biol 59, 1233-1241.

43. Qiao X, Lin XH, Liang YH, Dong J, Guo DA, Ye M (2014) Comprehensive chemical analysis of the rizomes of Drynaria fortunei by orthogonal pre-separation and liquid chromatog-raphy mass spectrometry. Planta Med 80, 330-336.

44. Ferrazzano GF, Amato I, Ingenito A, Zarrelli A, Pinto G, Pollio A (2011) Plant polyphenols and their anti-cariogenic properties: a review. Molecules 16, 1486-1507.

45. Goyal D, Sharma S, Mahmood A (2013) Inhibition of dextransucrase activity in Streptococcus mutans by plant phenolics. Indian J Biochem Biophys 50, 48-53.

46. Wittpahl G, Kölling-Speer I, Basche S, Herrmann E, Hannig M, Speer K et al. (2015) The polyphenolic composition of cistus incanus herbal tea and its antibacterial and anti-adherent activity against Streptococcus mutans. Planta Med 81, 1727-1735.

47. Levinger O, Bikels-Goshen T, Landau E, Fichman M, Shapira R (2012) Epigallocatechin gallate induces upregulation of the two-components VraSR system by evoking a cell wall stress response in Staphylococcus aureus. Appl Environ Microbiol 78, 7954-7959.

48. Wallock-Richards DJ, Marles-Wright J, Clarke DJ, Maitra A, Dodds M, Hanley B et al. (2015) Molecular basis of Streptococcus mutans sortase A inhibition by the flavonoid natural product trans-chalcone. Chem Commun (Camb) 51, 10483-10485.