Synergistic effect between cryptotanshinone and antibiotics in oral pathogenic bacteria

Cryptotanshinone (CT), a major tanshinone of medicinal plant Salvia miltiorrhiza Bunge, demonstrated effective in vitro antibacterial activity against all oral bacteria tested in this experiment. The antibacterial activities of CT against oral bacteria were assessed using the checkerboard and time-kill methods to evaluate the synergistic effects of treatment with ampicillin or gentamicin. The CT was determined against oral pathogenic bacteria with MIC and MBC values ranging from 0.5 to 16 and 1 to 64 μg/mL; for ampicillin from 0.0313 to 16 and 0.125 to 32 μg/mL; for gentamicin from 2 to 256 and 4 to 512 μg/mL respectively. The range of MIC50 and MIC90 were 0.0625 8 μg/mL and 1 64 μg/mL, respectively. The combination effects of CT with antibiotics were synergistic (FIC index < 0.5) against tested oral bacteria except additive, Streptococcus sobrinus, S. criceti, and Actinobacillus actinomycetemcomitans (FIC index < 0.75 1.0). The MBCs were shown reducing ≥4 8-fold, indicating a synergistic effect as defined by a FBCI of ≤0.5. Furthermore, a time-kill study showed that the growth of the tested bacteria was completely attenuated after 3 6 h of treatment with the 1/2 MIC of CT, regardless of whether it was administered alone or with ampicillin or gentamicin. The results suggest that CT could be employed as a natural antibacterial agent against cariogenic and periodontopathogenic bacteria.


INTRODUCTION
Dental caries and periodontal disease are prevalent worldwide.Bacteria existing in the dental plaque or biofilm play an important role in the development of both dental caries and periodontal disease [1,2].Of the more than 750 species of bacteria that inhabit the oral cavity, a number are implicated in oral diseases [3].The development of dental caries involves acidogenic and aciduric gram-positive bacteria (mutans streptococci, lactobacilli and actinomycetes) [4].Periodontal diseases have been linked to anaerobic gram-negative bacteria (Porphyromonas gingivalis, Actinobacillus, Prevotella, and Fusobacterium) [5].Antibiotics such as ampicillin, chlorhexidine, erythromycin, penicillin, tetracycline and vancomycin have been very effective in preventing dental caries [6].Of the selected putative periodontal species, strains of Prevotella intermedia, Fusobacterium nucleatum and to the first time, Tannerella forsythia, were β-lactamase positive, with P. intermedia being the most frequently detected enzyme positive species [7].Subgingival isolates of P. gingivalis, P. intermedia and F. nucleatum in a group of subjects increase in the MIC values of tetracycline [8].This clinical observation led to studies that established metronidazole as an important antibiotic for anaerobic infection.Since then, this compound has also played an important role in treating anaerobe related infection in the oral cavity, abdomen, and female genital tract, among others [9].Oral bacteria have been reported to show increased resistance towards common antibiotics such as penicillin, cephalosporin, erythromycin, tetracycline, and metronidazole which have been used therapeutically for the treatment of oral infection [10,11].The increase in resistance and adverse effects has lead researchers to explore novel anti-infective herbal compounds which could be used for effective treatment of oral diseases [12,13].
Salvia miltiorrhiza Bunge (Danshen) is an herb commonly used in traditional oriental medicine the treatment of several pathologies including cardiovascular diseases, hepatitis, menstrual disorders, diabetes, and chronic renal failure [14][15][16][17].Cryptotanshinone (CT) is one of the principal active constituents in Danshen extract and has several pharmacological effects, such as anti-inflamma-tory, anti-oxidative, anti-bacterial, anti-angiogenic, antimutagenic, anti-platelet aggregation, anti-human hepatocellular carcinoma effects, and anti-cyclooxygenase-2 (COX-2) functions [18][19][20][21][22][23].CT exhibits antimicrobial activity against a broad range of Gram-positive, including S. aureus, and Gram-negative bacteria as well as other microorganisms [24].Although CT exhibited fairly high levels of activity against S. aureus, there have been no reports related to the inhibitory mechanisms of CT against S. aureus.For over 100 years, chemical compounds isolated from medicinal plants have served as the models for many clinically proven drugs and are now being reassessed as antimicrobial agents [25].Plant-derived antibacterials are always a source of novel therapeutics.
We have investigated the antibacterial activity of cryptotanshinone, a major bioactive constituent isolated from Salvia miltiorrhiza Bunge (Danshen) against oral pathogens when used alone and in combination with antibiotics.

Bacterial Strains
The cariogenic bacterial strains used in this study were:

Minimum Inhibitory Concentrations/ Minimum Bactericidal Concentrations Assay
The minimum inhibitory concentrations (MICs) were determined for CT by the broth dilution method [26], 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.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 and MIC 90 s, defined as MICs at which, 50 and 90%, respectively 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 CT that kills 99.9% of the test bacteria by plating out onto each appropriate agar plate.Ampicillin (Sigma) and gentamicin (Sigma) were used as standard antibiotics in order to compare the sensitivity of CT against test bacteria.

Checker-Board Dilution Assay
The antibacterial effects of a combination of CT, which exhibited the highest antimicrobial activity, and antibiotics were assessed by the checkerboard test as previously described [26,27].The antimicrobial combinations assayed included CT with ampicillin or gentamicin.Serial dilutions of two different antimicrobial agents were mixed in cation-supplemented Mueller-Hinton broth.After 24 h of incubation at 37˚C, the MIC was determined to be the minimal concentration at which there was no visible growth.The fractional inhibitory concentration index (FICI) is the sum of the FICs of each of the drugs, which in turn is defined as the MIC of each drug when it is used in combination divided by the MIC of the drug when it is used alone.The interaction was defined as synergistic if the FIC index was less than or equal to 0.5, additive if the FIC index was greater than 0.5 and less than or equal 1.0, indifferent if the FIC index was greater than 1.0 and less than or equal to 2.0, and antagonistic if the FIC index was greater than 2.0 [26,27].

Statistical Analysis
All the data are expressed as a mean ± standard error (SE) of triplicate experiments.

Synergistic Effect of CT with Antibiotics
Activity of antibiotics plus the plant extract was determined using the checkerboard technique [26,28].The synergistic effect of CT with ampicillin or gentamicin in oral bacteria was presented in Tables 3 and 4, respectively.In combination of CT with ampicillin, the MIC ranges were observed in cariogenic bacteria at 0.125 μg/mL to 1 μg/mL and reduced ≥4-fold, producing a synergistic effect as defined by FICI ≤ 0.5, except additive effect in S. sobrinus and S. criceti by FICI ≤ 0.75 -1.0.The MBC ranges (0.5 μg/mL to 4 μg/mL) of CT with ampicillin were reduced ≥4-fold in S. ratti, S. anginosus, and S. gordonii (Table 3).In periodontopathogenic bacteria, the MIC values of CT with ampicillin was also observed by ≥4-fold, producing a synergistic effect as defined by FICI ≤ 0.5, except additive effect in A. actinomycetemcomitans by FICI ≤ 0.75 and the MBC values (4 μg/mL to 16 μg/mL) of CT with ampicillin were reduced ≥4-fold in F. nucleatum.The combination of CT with gentamicin was observed resulted in the decrease ≥4-fold in MIC for most of tested bacteria, except S. sanguinis, S. ratti, A. actinomycetemcomitans, and P. intermedia by FICI ≤ 0.5 and in MBC for S. mutans, S. criceti, S. anginosus, and P. gingivalis by FBCI ≤ 0.5 but additive for S. sanguinis, S. ratti, A. actinomycetemcomitans, and P. intermedia by FIBI ≤ 0.75 (Table 4).

Time-Kill Curves
The synergistic effect of CT with ampicillin or gentamicin against oral bacteria was confirmed by time-kill curve experiments.The cultures of all bacteria, with a cell density of 10 5 CFU/ml, were exposed to MIC or 1/2 MIC of CT alone and with 1/2 MIC of ampicillin or gentamicin.We observed that CT with antibiotics resulted rate of killing increasing in CFU/ml at time-dependent manner (Figures 1 and 2).In order to assess the effects of combinations of CT and antibiotics, the MIC 50 values of the antibiotics were determined as these provide the reference point for defining the interactions.

DISCUSSION
With the increase in the incidence of resistance to antibiotics, alternative natural products of plants could be of interest.Some plant extracts and phytochemicals are known to have antimicrobial properties, which could be of great importance in the therapeutic treatments [25][26][27]29].Many plants have been evaluated not only for direct antimicrobial activity but also as resistance-modifying agents [30,31].In this study, the antibacterial activities and synergistic effects of CT or with antibiotics were exhibited in oral bacteria.The results of the antimicrobial activity showed that CT exhibited antimicrobial activities gainst cariogenic bacteria at 0.5 to 4 µg/mL of MICs a   and MIC 90 were 0.125 μg/mL and 0.5 μg/mL.These diterpene quinines are found exclusively in the genus Salvia and show antibacterial, antifungal, antioxidant, anti-inflammatory, and anti-platelet aggregation effects [32][33][34].The cryptotanshinone and dihydrotanshinone I exhibit strong antimicrobial activity and MIC values of these on the spore germination of M. oryzae were 6.25 μg/mL and 3.13 μg/mL, respectively and on A. tumefaciens, E. coli, P. lachrymans, R. solanacearum, X. vesicatoria, B. subtilis, S. aureus, and S. haemolyticus ranged from 6.25 μg/mL to 100 μg/mL, and the median inhibitory concentration (IC50) values from 3.66 μg/mL to 57.38 μg/mL.Combinations of some herbal materials and different antibiotics might affect the inhibitory effect of these antibiotics [26,27,29].In combination of CT with ampicillin, the MIC ranges were observed in cariogenic bacteria at 0.125 μg/mL to 1 μg/mL and reduced ≥4-fold, producing a synergistic effect as defined by FICI ≤ 0.5 and in periodontopathogenic bacteria.The MIC values of CT with ampicillin was also observed by ≥ 4-fold, producing a synergistic effect as defined by FICI ≤ 0.5, except additive effect in A. actinomycetemcomitans by FICI ≤ 0.75 and the MBC values, 4 μg/mL to 16 μg/mL of CT with ampicillin were reduced ≥4-fold in F. nucleatum.The combination of CT with gentamicin was observed resulted in the decrease ≥4-fold in MIC and MBC for most of tested bacteria by FICI ≤ 0.5 but additive for S. sanguinis, S. ratti, A. actinomycetemcomitans, and P. intermedia by FIBI ≤ 0.75.Such combinations would be synergistic if there is a decrease in the MIC of each agent of four-fold; partially synergistic if there is a MIC decrease for one drug of four-fold and a decrease of two-fold of the other agent; additive if there is a two-fold reduction in the MIC of both agents; indifference is all interactions not meeting the criteria listed above and not being antagonistic  [27,35].Antagonistic response refers to where a MIC increase of four-fold for each drug would be observed in combination [36].The synergistic effect of CT with ampicillin or gentamicin against oral bacteria was confirmed.1 -3 hours of treatment with 1/2 MIC of CT 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.Phenolic acids with a variety of bioactivities, including antimicrobial, antioxidant, anti-thrombosis, anti-hypertension, antivirus and antitumor properties, are widely distributed in the plant kingdom [37][38][39].CT demonstrates effective in vitro antibac-terial activity against all 21 S. aureus strains [24].Affymetrix GeneChips are utilized to determine the global transcriptional response of S. aureus ATCC 25,923 to treatment with subinhibitory concentrations of CT [24].Those results were found similar to our results which were evaluated as strong antibacterial activity against gram-positive bacteria, cariogenic bacteria.
In conclusion, the results suggest that combinations of CT with antibiotics should be investigated further for possible use in antibacterial products.Particularly, these may be useful in the future for the treatment of cariogenic bacteria.

Table 1 .
List of strains and culture media used for antimicrobial experiment.

Table 2 .
Antibacterial activity of cryptotanshinone and antibiotics in oral bacteria.

Table 3 .
Synergistic effects of cryptotanshinone with ampicillin against oral bacteria.