In vitro Antimicrobial and Antibiofilm Activity of Artocarpus Lakoocha (Moraceae) Extract against Some Oral Pathogens

Purpose: To evaluate the antimicrobial and antibiofilm activity of A. lakoocha extract against oral pathogens by an in vitro method. Methods: The dried powder of the aqueous extract of A. lakoocha was purchased from a Thai traditional drug store. Representative strains of oral pathogens (Streptococcus mutans ATCC 25175, Streptococcus sobrinus ATCC 33478, Enterococcus faecalis ATCC 19433, Lactobacillus fermentum ATCC 14931, Lactobacillus salivarius ATCC 11741, Aggregatibacter actinomycetemcomitans ATCC 33384, Porphyromonas gingivalis ATCC 33277, Prevotella intermedia ATCC 25611, Prevotella nigrescens ATCC 25261, Fusobacterium nucleatum ATCC 25586 and Tanerella forsythia ATCC 43037) were tested for minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using a microdilution technique, as well as by a time kill assay. Antibiofilm activity was investigated by a 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium-bromide (MTT) assay. Results: All tested strains were susceptible to A. lakoocha extract with variable degrees of antimicrobial inhibition. The extract was effective against both Gram-negative bacteria (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis) and Gram-positive bacteria (Streptococcus mutans, Streptococcus sobrinus), with MIC ranging from 0.10 – 0.39 mg/ml and MBC from 0.10 – 3.12 mg/ml. Killing activity depended on time and concentrations of the extract. The extract acted as a potent antibiofilm agent with dual actions, preventing biofilm formation and also eradicating the existing biofilm. Conclusion: A. lakoocha extract possesses compounds with good antimicrobial properties that may be used for oral infectious diseases caused by certain oral pathogens associated with dental caries and/or periodontal diseases. For the application, A. lakoocha extract may be incorporated in mouthwash or toothpaste.


INTRODUCTION
Antimicrobial properties have been derived from a wide range of plant extracts. Natural products are sources of chemical compounds that can be used as antimicrobial agents. Artocarpus lakoocha Roxb (Moraceae) is a traditional herbal medicine, which is commonly found in tropical areas such as India and Thailand. The oxyresveratrol, 2, 4, 3´, 5´-tetrahydroxystilbene, is the major constituent of aqueous extract of Artocarpos lakoocha which has been revealed as a natural anthelmintic [1] and anti-herpes simplex virus (HSV) both in vitro and in vivo studies [2,3]. It has also been reported to show in vitro antivaricella zoster virus activity [4,5]. Another study has suggested that oxyresveratrol was neuroprotective and inhibited apoptotic cell death in transient ischemia in a rat model [6]. A. lakoocha extract has a potential application as a novel skin whitening agent in cosmetic preparations [7], due to its potent tyrosinase inhibitory [8] and antioxidant activities [9].
Oral diseases, including dental caries and periodontal diseases, are commonly caused by a wide range of microorganisms associated with oral biofilm or dental plaque [10,11]. Recently study reported that oxyresveratrol exhibited antibacterial activities against periodontal pathogens, P. gingivalis and A. actinomycetemcomitans [12]. Our previous study indicated that A. lakoocha extract may be a useful antimicrobial medication for endodontic treatment, due to its ability to inhibit growth of Enterococcus faecalis [13]. However, information of the antibacterial property of A. lakoocha extract against oral pathogens is still limited.
This study is aimed at investigating the antimicrobial and antibiofilm activities of A. lakoocha extract against oral pathogens as well as the potential health benefits of the extract.

EXPERIMENTAL Preparation of A. lakoocha extract
The dried powder of the aqueous extract of A. lakoocha was purchased from a Thai traditional drug store at Chiang Mai province, Thailand Organisms were maintained on either brain heart infusion agar (BHA) with 5 % (v/v) blood for facultative bacteria and supplemented with a 0.5 % (w/v) yeast extract, haemin and vitamin K for anaerobic bacteria. The strains were grown under aerobic or anaerobic (10 % H2, 10 % CO 2 and 80 % N 2 ) conditions as appropriate.

Agar diffusion test
The broth culture of each tested strain (approximately 10 7 cfu/ml) was mixed thoroughly with the sterile BHA (20 ml) and then poured into a plate with 6-mm diameter metal cups. The metal cups were removed after the medium has set, and then in the wells were added 100 µl of 10 % A. lakoocha extract, while 10 % DMSO was used as the control. The plate was incubated in appropriate conditions according to microorganisms tested at 37 °C for 24 h. Antimicrobial activity was evaluated by measuring inhibition zone diameters; the experiments were conducted in triplicate.

Minimal inhibitory and minimal bactericidal concentration
The minimal inhibitory concentration (MIC) of A. lakoocha extract against each tested strain was determined using the Clinical and Laboratory Standards Institute-(CLSI) recommended broth microdilution assay as modified Briefly, two fold serial dilutions of A. lakoocha extract were prepared with brain heart infusion broth (BHI) at a total volume of 100 µl per well in the 96-well plates. The final concentrations of A. lakoocha extract ranged from 25-0.01 mg/ml. The microtiter plate wells were inoculated with 100 µl of 0.5 McFarland for each tested strain per well. After overnight incubation at 37 °C appropriate conditions, absorbance was measured at 600 nm using a microtiter plate reader (Expert Plus UV; ASYS Hitech GmbH Eugendorf, Austria) to assess the cell growth. The negative control consisted of BHI broth and bacterial cell suspension without the agent, and the blank control contained only the medium. The MIC end-point was defined as the lowest concentration of the test agent that completely inhibited growth or produced at least 90 % reduction of absorbance in comparison with the negative control. All experiments were performed in triplicate and the average values were reported as MIC.
The minimal bactericidal concentration (MBC) was defined as the lowest concentration of wells that did not allow visible growth when 10 µl of the well contents was plated on agar and grown 24 -48 h at 37 °C in appropriate conditions.

Time kill assay
Bactericidal activity of A. lakoocha extract was examined using a time kill assay. Growing cultures (10 6 cfu/ml) of each representative strain, Gram positive (S. mutans ATCC 25175) and Gram negative bacteria (A. actinomycetemcomitans ATCC 33384), were added to appropriate medium and exposed to 1×, 2× and 4× the MIC of A. lakoocha extract. Samples were taken for colony counts at 0, 30 min, 2, 4, 6, 8, 10, 12 and 24 h. The viable counts were determined after appropriate incubation and each experiment was performed in triplicate. Chlorhexidine diacetate monohydrate (CHX, 0.1 %) and extract free medium were used as the positive and negative controls, respectively.

Inhibition of biofilm formation
The effect of A. lakoocha extract on biofilm formation of each representative strain, S. mutans ATCC 25175 and A. actinomycetemcomitans ATCC 33384, was examined using the modified microdilution method of Tang et al [14]. Briefly, two-fold serial diluted concentrations from 25 to 0.01 mg/ml of A. lakoocha extract were made in a flat-bottom 96-well microtiter plate. The CHX (0.1 %), phosphate buffered saline and the medium alone were used as positive, non-treated and blank controls, respectively. An equal volume of the tested strains (1 × 10 6 cfu/ml) was added and mixed with the agents, except in the well with medium alone (the blank control). Following incubation at 37 °C for 24 h, supernatants were discarded and washed three times with phosphate buffered saline Biofilm formation was quantified using a 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium-bromide (MTT) assay. The numbers of surviving bacteria were determined by measuring their ability to reduce the yellow tetrazolium salt to a purple formazan product at 570 nm. The OD values indicated a number of surviving microorganisms in the biofilm. Percentage inhibition was calculated using an equation [1 -(A570 of the test/A570 of non-treated control)] × 100. The biofilm inhibition concentration (MBIC 50 and MBIC 90 ) was defined as the concentrations that showed 50 % and 90 % inhibition of biofilm formation.

Eradication of biofilm formation
The antimicrobial activity of A. lakoocha extract in the biofilm was also examined using the minimum biofilm eradication concentration (MBEC) assay. Briefly, 200 µl (10 6 cfu/ml) of each representative strain, S. mutans ATCC 25175 and A. actinomycetemcomitans ATCC 33384, was inoculated into each well of the flatbottom 96-well microtiter plate and incubated for 24 h in appropriate conditions at 37 °C. After biofilm formation, the medium was then blotted out and the well carefully washed three times with sterile phosphate buffered saline in order to remove non-adherent cells. A. lakoocha extract was then added to the biofilms in two-fold dilutions serially from 25 to 0.01 mg/ml and incubated for 24 h in appropriate conditions at 37 °C. At the end-point of the treatment of the biofilms with A. lakoocha extract, the adherent bacteria were washed three times with sterile phosphate buffered saline. The numbers of surviving bacteria were determined by a MTT assay. The MBEC value was defined as the concentrations that showed 50 % and 90 % inhibition of biofilm formation on the biofilm. And 0.1 % CHX, phosphate buffered saline and the medium alone were used as the positive, nontreated and blank controls, respectively. The percentage eradication was calculated using an equation [1 -(A570 of the test/A570 of nontreated control)] × 100.

Scanning electron microscopy
A scanning electron microscopy (SEM) was performed to examine the morphology changes of each representative strain in S. mutans ATCC 25175 and A. actinomycetemcomitans ATCC 33384 after treatment with 10 % DMSO or 0.1 mg/ml A. lakoocha extract as the control and treated sample, respectively. After 8 h incubation at 37 °C in an appropriate condition, the bacterial pellet was collected and washed twice with phosphate buffered saline with pH 7 by centrifugation at 1000 rpm at 4 °C for 5 min. The bacterial pellet was fixed overnight in 2.5 % glutaraldehyde and 0.1 M cacodylate buffer at cool temperature, and then dehydrated in a graded series of ethanol solutions for 30 min. The samples were subsequently dried by a critical point drying method and coated with gold. The microbial morphology was observed with a field emission SEM.

Statistical analysis
Data were expressed as mean and standard deviation (S.D.) by computational analysis from triplicate independent experiments.

RESULTS
A. lakoocha extract was evaluated for antimicrobial potential against oral pathogens by an agar diffusion assay, the results of which are shown in Table 1. All tested strains were susceptible to A. lakoocha extract with variable degrees of inhibition zones. The extract was found effective against both Gram positive bacteria and Gram negative bacteria. The solvent control, 10 % DMSO, did not affect microorganism growth. The MIC and MBC of A. lakoocha extract as evaluated by a microdilution assay are shown in Table 1 The concentrations of A. lakoocha extract required to inhibit of the ≥ 50 % biofilm formation (MBIC 50 ) of S. mutans and A. actinomycetemcomitans were 0.39 ± 0.11 and 0.10 ± 0.09 mg/ml, and for ≥ 90 % inhibition of biofilm growth (MBIC 90 ) were 3.12 ± 0.02 and 0.39 ± 0.10 mg/ml, respectively (Figure 2A). The eradication of the biofilm formation of S. mutans and A. actinomycetemcomitans by A. lakoocha extract at various concentrations and with 0.1 % CHX is demonstrated in Figure 2B. The concentrations of A. lakoocha extract to eradicate of the ≥ 50 % biofilm formation (MBEC 50 ) of S. mutans and A. actinomycetemcomitans were 1.56 ± 0.13 and 0.78 ± 0.12 mg/ml, and for ≥ 90 % of the eradication of biofilm growth (MBEC 90 ) were 6.25 ± 0.34 and 3.12 ± 0.18 mg/ml, respectively.
In Figure 3, the change in morphology of S. mutans and A. actinomycetemcomitans was observed using SEM after treating 0.1 mg/ml of A. lakoocha extract in comparison with the control (10 % DMSO). Bacterial cells of S. mutans and A. actinomycetemcomitans in control groups showed a regular, smooth surface as shown in Figure 3A and C, respectively. It revealed that the bacterial cells after treatment with A. lakoocha extract lost their original shape showing a distorted, irregular cell wall structure, which was clearly observed in A. actinomycetemcomitans ( Figure 3D).

DISCUSSION
The search for novel antimicrobial agents from plants has been of great interest in the last few decades. Throughout human history, infectious diseases are known to have been treated with compounds derived from plants and herbal remedies.

Results
of the present study clearly demonstrated that A. lakoocha extract revealed good antibacterial agent activity against both cariogenic bacteria (e.g. S. mutans, S. sobrinus) and periodontopathogens including P. gingivalis, F. nucleatum, T. forsythia and A. actinomycetemcomitans. Gram negative bacteria were generally found to be more susceptible than Gram positive bacteria. Our results are in agreement with Phoolcharoen et al [12] who demonstrated the antimicrobial activity of A. lakoocha extract against periodontopathogens Gram negative bacteria. However, cariogenic bacteria were not susceptible to A. lakoocha extract in that study [12]. This may be due to the different strains used in that study; however, no details of the strains were given. Besides, the in vitro antimicrobial activity of A. lakoocha extract against oral pathogen planktonic cells, the antibiofilm activity of A. lakoocha extract was also assessed in this study. Experiments comparing biofilms of oral strains with broth cultures have demonstrated that higher concentrations of A. lakoocha extract are required to significantly inhibit existing biofilm cells. This is an expected result since bacteria in the biofilm are strongly protected and less susceptible to antimicrobial agents than in planktonic form. It was shown that A. lakoocha extract was able to eradicate oral biofilm in a dose and time dependent manner. Our results indicate that A. lakoocha extract acts as a potent antibiofilm agent that has dual actions preventing biofilm formation and eradication of existing biofilm.
Oral infections are commonly caused by imbalance of microorganisms in dental plaque or biofilm. Pathologies such as dental caries or periodontitis arise when an imbalance of microorganisms occurs in the biofilm resulting in a decrease of the indigenous bacteria and in favour of pathogenic strains. An effective approach for controlling dental plaque is to prevent formation of biofilm or to remove biofilm by using antimicrobial or chemical agents [15]. The use of CHX solution, a commercial antimicrobial agent, for clinical application is limited due to its bitter taste and staining of the tooth surface [16]. Thus, it is reasonable to develop alternative antiplaque agents from natural sources that exhibit few or no sideeffects. It was found that A. lakoocha extract has an efficacy for the prevention of plaque-related diseases such as dental caries and periodontitis. Although the MIC and MBC of A. lakoocha extract obtained were higher than for CHX, the extract exhibited a better antimicrobial activity than other herb extract such as garlic [17], Polygonum cuspidatum root [18], Coco cravo [19] and Rhei Rhizoma [20] with MICs against S. mutans of 71.4, 1 -2, 4 and > 5 mg/ml, respectively.
The exact mechanism of action exerted by oxyresveratrol, the major constituent of A. lakoocha extract, has not been previously reported. SEM images in this study indicated that oxyresveratrol might have effect on the cell wall integrity of bacterial cells. Exposure of S. mutans and A. actinomycetemcomitans to A. lakoocha extract revealed an irregular cell wall structure. All the results above indicated that A. actinomycetemcomitans is more sensitive to A. lakoocha extract than S. mutans. This may be due to Gram positive bacteria contained in the thicker peptidoglycan layer than Gram negative bacteria.

CONCLUSION
This study suggests that A. lakoocha extract possesses compounds with potential antimicrobial properties that may be useful for treatment of oral infectious diseases caused by certain oral pathogens. However, since this study was conducted on artificial biofilm in vitro, the actual effects of A. lakoocha extract on dental plaque would need to be evaluated clinically.