Antimicrobial Activity of Sophora Japonica Extract Against Oral Bacteria

Background: The flower and flower buds of Sophora japonica L. have the same medicinal uses, with significant biological activity, in the treatment of bleeding hemorrhoids, hematuria, hematemesis, hemorrhinia, uterine or intestinal hemorrhage, metrorrhagia, leukorrhea, conjunctivitis, pyoderma, arteriosclerosis, hypertension, and dizziness. Methods: This study aimed to investigate the synergistic antibacterial activity with existing antimicrobial agents against oral pathogen. The synergistic effects and anti biofilm of 50% ethanol extract of (SJEE) were evaluated against oral bacteria, either alone or with antibiotics, via broth microdilution method, time-kill method, and crystal violet assay. Results: MIC/MBC values for SJEE, Ampicillin, Gentamicin, Erythromycin, and Vancomycin against all the tested bacteria ranged between 0.1254/0.5-16 mg/mL, 0.0313-16/0.125-32 μg/mL, 2-256/4-512 μg/mL, 0.00832/0.016-64 μg/mL, and 0.25-64/1-128 μg/mL, respectively. The synergistic effects were exhibited on SJEE with antibiotics against oral bacteria at Fractional Inhibitory Concentration Index (FICI)<0.5. Moreover, SJEE and antibiotics were found to synergistically reduce biofilm formation. 1-6 hours of treatment with 1/2 MIC of SJEE with 1/2 MIC of antibiotics resulted from an increase of the rate of killing in units of CFU/mL to a greater degree than was observed with alone. Conclusion: The SJEE was exerted a strong bactericidal effect in drug combinations against oral bacteria and biofilm formation. Abbreviations: SJEE: Sophora japonica Ethanol Extract; MICs: Minimum Inhibitory Concentrations; MBCs: Minimum Bactericidal Concentrations; CFU: Colony Forming Unit; FIC index: Fractional Inhibitory Concentration; FBC Index: Fractional Bactericidal Concentration Index Su-Mi Cha1, Jeong-Dan Cha2, Soon-Il Yun2,5*, EunJin Jang3 and Sung-Mi Choi4,5 1Department of Oral Microbiology and Institute of Oral Bioscience, Chonbuk National University, Jeonju, Republic of Korea 2Department of Food Science & Technology, College of Agriculture & Life Sciences, Chonbuk National University, Jeonju, Republic of Korea 3Department of Dental Technology, Daegu Health College, Daegu, South Korea 4Department of Dental Hygiene, Daegu Health College, Daegu, South Korea 5Department of Dental Hygiene, Daegu Health College, Daegu, South Korea *Address for Correspondence Soon-Il Yun, Department of Food Science & Technology, College of Agriculture & Life Sciences, Chonbuk National University, 664-14 Duckjin-Dong, Duckjin-Ku, Jeonju, Chonbuk, 561-756 Republic of Korea, Tel: +82-63-270-2566; Fax: +8263-270-4049; E-mail: siyun@jbnu.ac.kr Submission: 12 October, 2017 Accepted: 28 November, 2017 Published: 07 December, 2017 Copyright: © 2017 Cha SM, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Research Article Open Access


Introduction
More than 700 different bacterial species have been detected in the oral cavity of humans [1].Saliva contains 10 8 to 10 9 bacteria per milliliter, and some of these adhere to the teeth and initiate formation of a dental biofilm, previously called dental plaque [2,3].Dental caries are caused by demineralization of the enamel of the tooth by acid produced from dietary sugars by micro-organisms growing as a biofilm or plaque [4,5].Until a few decades ago, development of caries was ascribed to a few gram-positive bacterial species in the biofilm, i.e. the specificbiofilm/plaque hypothesis, and Streptococcus mutans, Streptococcus sobrinus together with some Lactobacillus species were regarded as key pathogens [6].While especially S. mutans, Actinomyces, and Lactobacillus species were previously regarded as responsible for caries, the list of caries-associated bacteria now includes species of the genera Actinomyces, Lactobacillus, Dialister, Eubacterium, Olsenella, Bifidobacterium, Atopobium, Propionibacterium, Scardovir, Abiotrophia, Selenomonas, and Veillonella in addition to carbohydrate fermenting oral streptococci [6,7].
Periodontitis is characterized by inflammation of the periodontal tissues.Generally, the etiological agents of periodontal diseases are Gram-negative rods including Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Prevotella, Fusobacterium, and Porphyromonas gingivalis [8,9].Recent reports have suggested a potential role for periodontal infections in more serious systemic diseases including cardiovascular disease, respiratory infections, and diabetes, which are pathologies that significantly affect the overall health of the infected individual [10][11][12].
Mechanical dental plaque removal is an efficient procedure to prevent periodontitis and caries.However, the use of chemical compounds as a complementary method is also necessary and has proven to be a valuable tool to decrease tooth biofilm formation.Natural products are a major source of new natural drugs and their use as an alternative medicine for treatment of various diseases has been increased in the last few decades [13].Sophora japonica L. is a tree native to Korea, China, and Japan.Both the flower and flower buds have the same medicinal uses, with significant biological activity, in the treatment of bleeding hemorrhoids, hematuria, hematemesis, hemorrhinia, uterine or intestinal hemorrhage, metrorrhagia, leukorrhea, conjunctivitis, pyoderma, arteriosclerosis, hypertension, and dizziness [14][15][16].Flavones from the buds and pericarp were discovered as haemostatic constituents [17,18].Triterpenes, phospholipids, alkaloids, amino acids and fatty acids have been reported as the main chemical constituents of the seeds of this plant [19,20].Among these compounds, kaempferol, quercetin, rutin, isorhamnetin, genistein, and sophoricoside are the major active constituents of S. japonica [15,[17][18][19][20]. Rutin, in particular, is the most important and abundant constituent of S. japonica [15].Crude extracts in vitro, the ethanol extract from flower buds of S. japonica exhibits a significant antibacterial activity against Staphylococcus aureus, Propionibacterium avidum, and Propionibacterium acnes under weak acidic conditions [21].The Ethyl acetate (EtOAc)soluble fraction is effective in inhibiting Escherichia coli, Klebsiella pneumoniae, and S. aureus [22].
In this study, the antimicrobial activities of 50% ethanol extract of Sophora japonica (SJEE) against oral bacteria were assessed using broth microdilution method, time kill method, and crystal violet assay for synergistic effect and biofilm formation of the combination with antibiotics.

Plant material and preparation of 50% ethanol extract of Sophora japonica (SJEE)
Dried flowers from S. japonica (2 kg) were macerated and extracted three times with 50% EtOH (10 L) for 4 h at 80 °C.The combined 50% EtOH extract (30 L) was clarified by filtration and evaporated to obtain a dark brown syrup (200 g).One hundred mg/ mL of extract was dissolved in 10% dimethyl sulfoxide (DMSO, Sigma Chemical Co., St. Louis, MO, USA) and then, diluted with bacteria culture medium for testing.All of the extract was kept at 4 °C in the dark until further use.

Bacterial strains
The oral bacterial strains used in this study were: used for all bacterial strains except P. intermedia and P. gingivalis.For P. intermedia and P. gingivalis, BHI broth containing hemin 1 μg/mL (Sigma) and menadione 1 μg/mL (Sigma) was used.

Minimum inhibitory concentrations/minimum bactericidal concentrations assay
The minimum Inhibitory Concentrations (MICs) were determined for 50% ethanol extract of Sophora japonica (SJEE) by the broth dilution method, and were carried out in triplicate.The antibacterial activities were examined after incubation at 37 °C for 18 h (facultative anaerobic bacteria), for 24 h (microaerophilic bacteria), and for 1-2 days (obligate anaerobic bacteria) under anaerobic conditions, used a mix of H 2 and nitrogen (N 2 ) (5/95%) or N 2 /carbon dioxide (CO 2 )/H 2 (85/10/5%) to remove oxygen.MICs were determined as the lowest concentration of test samples that resulted in a complete inhibition of visible growth in the broth.MIC 50 s, defined as MICs at which, 50% of MIC of oral bacteria were inhibited, were determined.Following anaerobic incubation of MICs plates, the minimum bactericidal concentrations (MBCs) were determined on the basis of the lowest concentration of SJEE that kills 99.9% of the test bacteria by plating out onto each appropriate agar plate.Ampicillin, gentamicin, erythromycin, and vancomycin (Sigma) were used as standard antibiotics in order to compare the sensitivity of SJEE against oral bacteria.

Checkerboard dilution test
The antibacterial effects of a combination of SJEE and antibiotics were assessed by the checkerboard test as previously described [23,24].The antimicrobial combinations assayed included SJEE with antibiotics, ampicillin, gentamicin, erythromycin, and vancomycin.Serial dilutions of two different antimicrobial agents were mixed in cation-supplemented Mueller-Hinton broth.After 24-48 h of incubation at 37 °C, the MICs were determined to be the minimal concentration at which there was no visible growth and MBCs were determined on the basis of the lowest concentration of SJEE that kills 99.9% of the test bacteria by plating out onto each appropriate agar plate.The Fractional Inhibitory Concentration (FIC)/ Fractional Bactericidal Concentration (FBC) index was calculated according to the equation: The FIC and FBC index are the sum of the FICs and FBCs of each of the drugs, which in turn is defined as the MIC and MBC of each drug when it is used in combination divided by the MIC and MBC of the drug when it is used alone.The interaction was defined as synergistic if the FIC and FBC index was less than or equal to 0.5, additive if the FIC and FBC index was greater than 0.5 and less than or equal 1.0, indifferent if the FIC and FBC index was greater than 1.0 and less than or equal to 2.0, and antagonistic if the FIC and FBC index was greater than 2.0 [23].

Biofilm formation assay
Evaluation of the effect of SJEE on biofilm formation of oral bacteria by crystal violet biofilm formation assay was performed according to previous studies [25,26].Briefly, 200 µL aliquots of treated oral bacteria (final concentration of 1.0×10 6 CFU/mL) at sub-lethal dose of SJEE (1/2 MIC) plus antibiotics (1/8 MIC) was transferred to a flat-bottomed sterile polystyrene micro plate, and incubated for 24-48 h at 37 °C under anaerobic conditions to form biofilm.Then, cells were washed with Phosphate-Buffered Saline (PBS), stained with 0.1% (wt/vol) crystal violet solution for 15 min, washed with PBS, and de-stained with 96% ethanol 10 min in order to fix the cells.Thereafter, the wells were rinsed and air-dried.33% (vol/ vol) acetic acid was then added to each well and biofilm formation was quantified by measuring the absorbance of the solution at 540 nm using a micro plate reader (BMG LABTECH, USA).

Time-kill and growth inhibition curves assay
Bactericidal activities of SJEE and antibiotics under study were also evaluated using time kill curves on oral bacteria.Tubes containing Mueller-Hinton supplemented to which antibiotics had been added at concentrations of the 1/2 MIC were inoculated with a suspension of the test strain, giving a final bacterial count between 5~7×10 6 CFU/mL.The tubes were thereafter incubated at 37 °C in an anaerobic chamber and viable counts were performed at 0, 0.5, 1, 2, 3, 4, 5, 6, 12 and 24 h after addition of antimicrobial agents, on agar plates incubated for up to 48 h in anaerobic chamber at 37 °C.Antibiotic carryover was minimized by washings by centrifugation and serial 10-fold dilution in sterile phosphate-buffered saline, pH 7.3.Colony counts were performed in duplicate, and means were taken.The solid media used for colony counts were BHI agar for streptococci and BHI agar containing hemin and menadione for P. intermedia and P. gingivalis.

Statistical analysis
Experiments were performed three times and statistical analyses were performed with parametric tests (two-way analysis of variance [ANOVA] and Tukey's test) using commercial software (SPSS 22.0).The results were expressed as mean values±standard deviations (mean±SD) and were considered significant at the level of p<0.05 or/ and <0.01.

Minimum inhibitory concentrations/minimum bactericidal concentrations of SJEE and antibiotics
The use of natural products and herbal medicines has been documented in the past.They have been reported to be effective in the management of many infections in general.Some of these have been assessed in the recent past for their antimicrobial potential against oral bacteria [27][28][29].SJEE was evaluated for their antimicrobial activities against thirteen oral bacterial species present in the oral cavity.The results of the antimicrobial activity showed that SJEE exhibited antimicrobial activities against cariogenic bacteria at MICs, 0.125 to 4 mg/mL; MBCs, 0.5 to 16 mg/mL, against periodontopathogenic bacteria at MICs, 0.5 to 4 mg/mL; MBCs, 1 to 16 mg/mL and for ampicillin, either MIC/MBCs 0.0625/1 or 1/4 μg/mL; for gentamicin, either MIC/MBCs 2/8 or 256/512 μg/mL; for erythromycin, either 0.016/0.031or 32/64 μg/mL; for vancomycin, either 0.25/1 or 64/128 μg/mL on tested all bacteria (Table 1).The MIC50 and MIC90 ranges of SJEE were from 0.313 to 1 mg/mL and 0.125 to 4 mg/mL, respectively.The SJFF showed stronger antimicrobial activity against S. sobrinus, S. gordonii, A. actinomycetemcomitans and F. nucleatum at MIC/MBC, 0.125/0.5-0.5/2mg/mL than another bacteria at MIC/ MBC, 1-4/1-16 mg/mL.

Synergistic effect of SJEE with antibiotics
Many antimicrobial preparations, such as conventional antibiotics, chlorhexidine (CHX), phenolic compounds and triclosan, can inhibit bacteria biofilm effectively [30,31].However, extensive use of these antimicrobial agents can lead to some sideeffects, such as tooth staining, calculus formation, drug resistance and gastrointestinal reactions [32,33].Therefore, searching for new antimicrobial molecules, which exhibit few or no side effects and long term retention in oral cavity, has been intensified in recent years [34].The synergistic effects of SJEE with antibiotics or with antibiotics were evaluated in oral bacteria (Tables 2-5).In combination with 1 American Type Culture Collection (ATCC) 2 Korean collection for Oral Microbiology (KCOM) 3 Korean collection for Type Cultures (KCTC) ampicillin, SJEE was reduced ≥4-fold in tested bacteria, except S. ratti, S. criceti, and S. gordnii, producing a synergistic effect as defined by FICI≤0.5.The MBC for ampicillin was shown synergistic effects in S. parasanguinis, S. downei, S. anginosus, S. gordonii, A. actinomycetemcomitans, F. nucleatum, and P. gingivalis by FBCI≤0.5 (Table 2).In combination with SJEE, the MIC for gentamicin was reduced ≥4-8-fold in all tested bacteria, expect S. ratti by FICI≤0.5 and MBC in S. mutans, S. sobrinus, S. ratti, S. criceti, S. gordonii, and A. actinomycetemcomitans by FBCI≤0.5 (Table 3).Moreover, the MIC for erythromycin with SJEE was reduced ≥4-fold in tested bacteria, except S. sanguinis, producing a synergistic effect as defined by FICI≤0.5 and the MBC for erythromycin was shown synergistic effects, except S. mutans, S. sanguinis, S. sobrinus, S. rattii, S. criceti, S. gordonii, and A. actinomycetemcomitans by FBCI≥0.75 (Table 4).
In combination with SJEE, the MIC for vancomycin was reduced ≥4 fold in tested all bacteria, except S. sanguinis, S. criceti, S. downei, P.

Anti-biofilm formation of SJEE with antibiotics
Oral diseases, such as dental caries, periodontal disease are directly linked with the ability of bacteria to form biofilm [3,4,7,9].Prenylated flavonoids are widely distributed in the plant world and prenylation elevates hydrophobicity on the basic structure of the molecule, enhancing flavonoid biological functions [35].Prenylation facilitates flavonoid interaction with biofilm and uptake into bacteria through the membrane, enhancing inhibitory effects of the flavonoids on P. gingivalis growth and biofilm formation [36,37].The isorhamnetin-3-O-β-D-ru-tinoside from the dried flowers of S. japonica, which significantly inhibited the action of sortase A (SrtA)

Time kill of SJEE with antibiotics
Previous studies had reported the antimicrobial activities and mechanisms of SJEE against several kinds of bacteria [21,22].However, the type of microorganisms and their cell membrane structure and composition could play an important role in the susceptibility to antimicrobials [39,40].The MIC value was similar to the results that MIC values of SJEE against Staphylococcus aureus and Aeromonas hydrophila were 20 μg/mL and 50 μg/mL, respectively [22,41,42].The bacterial effect of SJEE with antibiotics, ampicillin, gentamicin, erythromycin, and vancomycin against oral bacteria was confirmed by time kill curve experiments.The SJEE (MIC or 1/2 MIC) alone resulted rate of killing increasing or not changing in CFU/ml at time dependent manner, with a more rapid rate of killing by SJEE (1/2 MIC) with ampicillin, gentamicin, erythromycin, or/and vancomycin (1/2 MIC) (Figures 2-4).A strong bactericidal effect was exerted in drug combinations.

Conclusion
In conclusion, these findings suggest that crude extract of S. japonica exhibited a wide range of pharmacological effects establish the conditions required of a novel cariogenic bacteria and periodontal pathogens, particularly bacteroides species drug and may be useful in the future in the treatment of oral bacteria biofilm.

Figure 1 :
Figure 1: Anti-biofilm effect of different concentrations of SJEE alone or/ and antibiotics on biofilm formation of oral bacteria, SM, SS, SP, SSO, SR, and SC.Cells stained with 0.1% (wt/vol) crystal violet solution for 15 min, washed with PBS, and de-stained with 96% ethanol 10 min in order to fix the cells.Thereafter, acetic acid was then added to each well and biofilm formation was quantified by measuring the absorbance of the solution at 540 nm using a micro plate reader.Data points are the mean values±S.E.M. of six experiments.SM: Streptococcus mutans; SS: Streptococcus sanguinis; SP: Streptococcus parasanguinis; SSO: Streptococcus sobrinus; SR: Streptococcus ratti; SC: Streptococcus criceti.

Figure 2 :
Figure 2: Anti-biofilm effect of different concentrations of SJEE alone or/and antibiotics on biofilm formation of oral bacteria, SD, SA, SG, AA, FN, PI, and PG.Cells stained with 0.1% (wt/vol) crystal violet solution for 15 min, washed with PBS, and de-stained with 96% ethanol 10 min in order to fix the cells.Thereafter, acetic acid was then added to each well and biofilm formation was quantified by measuring the absorbance of the solution at 540 nm using a microplate reader.Data points are the mean values±S.E.M. **Significantly different from the control group (1/8 MIC SJEE or antibiotics) (P<0.01) of six experiments.SD: Sreptococcus downei; SA: Streptococcus anginosus; SG: Streptococcus gordonii; AA: Aggregatibacter actinomycetemcomitans; FN: Fusobacterium nucleatum; PI: Prevotella intermedia; PG: Porphylomonas gingivalis

Table 1 :
Antibacterial activity of 50% ethanol extract of Sophora japonica (SJEE) and antibiotics in oral bacteria.