Antibacterial and antibiofilm activities of Prangos acaulis Bornm . extract against Streptococcus mutans : an in silico and in vitro study

Introduction Dental caries is a multifactorial complication and one of the most common infectious diseases in the world (1). Streptococcus mutans is one of the key factors in development of dental plaque and caries due to the ability for formation of biofilms and colonization on the dental surface in the presence of sucrose. S. mutans has different virulence factors such as mutacins, lactate dehydrogenase (LDH), antigen B, fructosyltransferase (Ftf), non-enzymatic glucan-binding proteins (GBPs) and glucosyltransferases (GTFs) which have vital roles in colonization and accumulation on dental surfaces (2,3). Among the mentioned virulence factors, glucosyltransferase is the key factor in demineralization of the tooth surface, the biofilm formation and development of dental caries. The sticky polymer of sucrose named glucan produced by glucosyltransferase of S. mutans is an initiating factor for dental caries formation due to trapping oral bacteria, food debris and salivary components. The secreted acids by trapped bacteria in http://www.herbmedpharmacol.com doi: 10.15171/jhp.2018.29


Introduction
Dental caries is a multifactorial complication and one of the most common infectious diseases in the world (1).Streptococcus mutans is one of the key factors in development of dental plaque and caries due to the ability for formation of biofilms and colonization on the dental surface in the presence of sucrose.S. mutans has different virulence factors such as mutacins, lactate dehydrogenase (LDH), antigen B, fructosyltransferase (Ftf), non-enzymatic glucan-binding proteins (GBPs) and glucosyltransferases (GTFs) which have vital roles in colonization and accumulation on dental surfaces (2,3).Among the mentioned virulence factors, glucosyltransferase is the key factor in demineralization of the tooth surface, the biofilm formation and development of dental caries.The sticky polymer of sucrose named glucan produced by glucosyltransferase of S. mutans is an initiating factor for dental caries formation due to trapping oral bacteria, food debris and salivary components.The secreted acids by trapped bacteria in the glucan polymer break down the enamel on the surface of teeth causing dental caries (4,5).Due to key role of the glucosyltransferase in formation and development of dental caries, many studies have been conducted to introduce novel glucosyltransferase inhibitors (6)(7)(8)(9).Glucosyltransferase is an enzyme (GTF-SI) encoded by gtf gene, composed of eight chains including A, B, C, D, E, F, G and H.This enzyme plays a key role in biofilm formation and colonization of S. mutans on dental surface.The enzyme is a member of glycoside hydrolase family 70 that catalyzes the formation of glucan with different types of glucosidic linkage such as α (1-3), α (1-4) and α (1-6) (10)(11)(12).Presently, there is a lot of interest in investigation of natural materials especially plant materials as a good source of new inhibitors for glucosyltransferase of S. mutans and prevention of dental caries.Medicinal plants have long been used for prevention and treatment of infectious diseases due to their availability, proper effects, low cost, variety and presence of a wide range of herbal compounds with therapeutic effects (13)(14)(15)(16)(17)(18)(19).The genus Prangos that is known Jashir in Iran belongs to Apiaceae family.The plants of this genus are widely used in folk medicine to treat external bleeding, scars of worms, digestive disorders, leukoplakia and infectious diseases as the antimicrobial agent (20).P. acaulis Bornm. is one of the important species of genus Prangos in Iran that is used in folk medicine as Sedative and anti-infective agent (21,22).This plant is used in traditional medicine in some areas of Iran as pain relief and tooth whitener.Therefore, the plant was selected for investigation of the anti-bacterial and anti-biofilm activities against S. mutans.
Despite numerous drug properties, plants can include mutagenic compounds (23)(24)(25).Therefore, investigation of the mutagenic potential of plant materials is essential.Consequently, along with anti-bacterial properties of the plant extracts probable mutagenicity of them was also evaluated for the first time using Ames test.Designing or screening new drug compounds using conventional methods are costly and time consuming along with possibility of human mistakes.Therefore, in recent years in silico methods for drug discovery and development are highly regarded.Bioinformatics is playing an increasingly key role in drug target identification, drug candidate screening, and refinement as well as characterization of side effects and prediction of drug resistance (26,27).In the present study, the antibacterial and anti-biofilm as well as mutagenicity activity of methanolic extracts from different parts of P. acaulis were investigated.Furthermore, in silico analysis was used for screening new inhibitor(s) for glucosyltransferase of S. mutans.

Materials and Methods
In silico analysis Retrieval of receptor and ligands from database Fifteen dominant compounds from P. acaulis, reported in previous studies (28,29), were selected for in silico analysis (Table 1).The three-dimensional structures of all the phytochemicals and Benzyl penicillin as positive control were retrieved from PubChem database (http://pubchem.ncbi.nlm.nih.gov).Moreover, a three-dimension structure of glucosyltransferase as a receptor was obtained from protein data bank (http://www.rcsb.org)with PDB entry 3AIE.

Receptor and ligands preparation and optimization
The raw structure of glucosyltransferase and the ligands were further prepared for in silico analysis.For this aim, the receptor was initially made ready by removing all water molecules and none polar hydrogens, followed by subsequent energy minimization to remove the bad steric clashes using the UCSF Chimera-1.11.2 software.Also, all ligand molecules were minimized in term of energy and structure using ArgusLab 4.01 software.

In vitro analysis Plant materials
The fresh plant samples were collected from Kurdistan province of Iran from May to June 2016.The plants were authenticated by a botanist at the herbarium unit of the University of Isfahan, Iran.The plant parts were washed using tap water and then were separated into flower, leaf, stem, seed, and root.Afterward, various parts were air dried in shadow for one week at room temperature, then powdered for extraction.

Preparation of plant extracts
The methanol extracts of different parts of P. acaulis were prepared using maceration method (30).Briefly, 100g of a powdered plant part was soaked in 300 mL of methanol for 72 hours, then, filtered using Whatman filter paper.The collected extracts were concentrated by rotary vacuum evaporator (Stero glass, Italy) at 45°C and then dried using freeze dryer (Zirbus, Germany).All extracts were dissolved in dimethyl sulphoxide (DMSO) and diluted with to give concentrations of 250, 500, 1000, 1500, 2000 and 3000 μg/mL.

Microorganisms and culture condition
The tested strains in the study for antibacterial and mutagenicity evaluation of the plant were S. mutans (PTCC 1683) and Salmonella typhimurium TA98 which were respectively purchased from Iranian Biological Resources Center (Tehran, Iran) and Bio Reliance Corporation (Rockville, MD, USA).S. mutans was grown on Nutrient Broth medium at 37˚C for 8 hours to subculture and prepare for antibacterial analysis.Furthermore, S. typhimurium TA98 was cultured on minimal glucose agar (1.5% agar, 2.0% glucose, and 2.0% Vogel-Bonner medium) containing histidine and biotin at 37˚C for 18 hours to measure the mutagenic properties of plant materials.

Antibacterial activity
Antibacterial activity of the P. acaulis extracts against the single form of S. mutans was investigated using disk diffusion method (31).For this, sterile Whatman filter papers No.1 were prepared and soaked separately in each of the extracts in different concentrations for 5 minutes, then placed on the plate for 24 hours at 37°C.In the next step, the zone of inhibition around the disc, which indicated antibacterial activity of tested extracts, was measured.In the study, standard discs of penicillin (10 mg/disk) and DMSO were used as positive and negative controls, respectively.Furthermore, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of tested extracts were determined by microdilution method using 96-well microtiter plate (32).For these, 100 μL of each extract in tested concentration was added to all wells, which contained 100 μL of BHI (Brain Heart Infusion) medium and 20 μL of inoculums (containing about 6×10 4 colony) and incubated in 37°C for 24 hours.After incubation optical density of each well was read at 620 nm.In the study, a serial dilution of penicillin (25,50,100,200, 400, 800, 1600 and 3200 μg/ mL) was considered as positive control.The MIC value was defined as the lowest concentration which inhibited the growth or fewer than three discrete colonies which were detected.Similarly, an MBC value was defined as the lowest concentration of the plant extracts to kill the microorganisms.Plates were read in triplicate, and the average of the MIC and MBC values were recorded.

Inhibition of biofilm formation
The effects of P. acaulis extracts on S. mutans were evaluated using microtiter plate assay (15).For this, the extracts were placed at different concentrations in the wells of a sterile polystyrene microtiter plates, previously treated with Saliva.After this, the microtiter plate wells containing 100 μl BHI medium with 2% sucrose were inoculated with 3 μL of the overnight grown culture of S. mutans (1.0 × 107 cells/mL in BHI medium), and the samples were incubated at 37°C for 18 hours.After incubation, 30 μL saline (0.85% NaCl) was added to each well for removing loosely attached and nonadherent cells.In the next step, the wells were washed three times with sterile distilled water, and the plates were dried for 45 minutes before carrying out biofilm quantification.Biofilm growth and quantification were evaluated using the crystal violet staining method and measuring absorbance at 575 nm.The percentage of inhibition was calculated using the following formula:

Mutagenicity assay
The mutagenic potential of the extracts was evaluated using Ames test based on mutagenicity in S. typhimurium TA98 with some modifications, as described previously (20).For this, 100 μL of an overnight grown culture (10 7 CFU/mL) of the strain was added in sterile screw-capped tubes.Then, 2 mL of top agar (0.6% agar and 0.5% NaCl) and 100 μL of each extract were added to the tubes and vortexed.After this, the solution was poured onto a minimal glucose agar plate and was incubated at 37°C for 48 hours.After incubation, numbers of His+ revertant colonies were counted.The positive and negative controls in this assay were sodium azide and sterile distilled water, respectively.The mutagenic effects of tested extracts were estimated using the twofold rule according to the following formula.The substance was considered mutagen if the QM value was calculated higher than two.QM =Number of His+ revertant colonies from tested extracts/colonies from negative control

Statistical analysis
The data were obtained in 30 replicates from three separate experiments and were expressed as mean ± standard deviation (SD) and then analyzed by SPSS V.21 using oneway analysis of variance (ANOVA) and Duncan post hoc tests.Additionally, P values <0.05 were considered to be statistically significant between different samples.

Determination of binding site
The predicted binding sites of glucosyltransferase are presented in Table 2.The results showed that the enzyme has ten binding sites with different amino acid numbers.The size evaluation of predicted binding sits showed that the largest and smallest sits were 161 and 42 Å.

Drug-likeness and toxicity prediction
The results of calculation of drug-likeness and toxicity of the ligands are given in Table 3.The results confirmed that all studied compounds had suitable permeation and distribution with low toxicity.Furthermore, the results showed that the most studied compounds had low gastrointestine absorption except terpineol and nerol that GI absorption was predicted in a high level for them.The results also revealed that among studied compounds only camphene showed genotoxicity potential.

Molecular docking
The results of molecular docking studies are shown in Table 4. Results revealed that three compounds including ar-curcumene, d-limonene and α-pinene had appropriate

Active site
Size (Å) Residues and robust interactions with glucosyltransferase in comparison to penicillin and other tested phytochemicals.
The results also confirmed that most of the interactions are van der Waals forces and c-terminal amino acid residues of the enzyme have key roles in enzyme-phytocompounds interactions (Figure 1).Moreover, investigation of amino acids involved in interactions showed that the interactions between penicillin and glucosyltransferase occurred in the predicted cavity number 5, whilst often interactions of ar-curcumene and α-pinene occurred in cavity numbers 2 and 9, respectively, while amino acids involved in the interactions between d-limonene and glucosyltransferase were not in any of the predicted cavities.

Antibacterial activity
The results of the antibacterial activity of P. acaulis extracts against S. mutans are shown in Table 5.All tested extracts had antibacterial effects against the tested strain in a dose-dependent manner.Consequently, most antibacterial effects were observed at the highest concentrations.Results also revealed that root extract had the most antibacterial activity followed by flower, leaf, stem and seed extracts.The MIC and MBC values of the tested extract are presented in Table 5.Similarly, to results of disc diffusion assay root and seed extract showed most and least antibacterial activity with MIC values of 500-1000, and 2000-3000 respectively, as well as maximum and minimum MBC values, were determined for root and seed extract respectively.

Biofilm inhibition activity
The results of potential inhibition of S. mutans biofilm formation by P. acaulis extracts are shown in Table 6.The results indicated that all the tested extracts particularly in 2000 to 3000 µg/mL concentrations could inhibit the biofilm formation.Inhibition of the biofilm formation, like the antibacterial effects, was in dose-dependent manner, so that inhibitory function increased with increasing concentration.Results also showed that root and seed extracts had the most and the least efficacies in inhibition of biofilm formation with 66.40% and 22±0.20%reduction, respectively.

Mutagenicity assay
The results of Ames test for determination of the mutagenic potential of P. acaulis extracts are presented in Table 7.
The results confirmed that none of the tested extracts had mutagenic effect in the studied concentrations.
Comparative study of tested extracts indicated that the number of His+ revertant colonies caused by the extracts increases with increasing concentrations such that the highest number of the revertant colonies were observed in the highest concentration.Results also showed that root and seed extracts with QM values of 1.67±0.31and 1.33±0.20 in highest tested concentration had the most and the least effects on the number of the revertant colonies, respectively

Discussion
Dental caries is one of the costly diseases, most common preventable and a global oral health problem in the world.
Based on the World Health Organization (WHO) report, poor oral health may have a dreadful effect on quality of life and general health.This chron ic disease is caused by the interaction of oral microorganisms, diet and some host factors, which among them oral bacteria have a major role in the formation and development of dental caries.Acidic by-products from the bac terial fermentation of dietary carbohydrates are the main cause of the destruction of dental hard tissues that is referred to dental caries (1-3).About 700 different bacteria species have been identified in the oral cavity.Due to some special abilities of S. mutans such as acid production, build up glycogen reserves; synthesize extracellular polysaccharides and adherence to enamel surfaces.It is believed that the bacteria are the key etiological agents in formation and development dental caries (4).
Because of important role of S. mutans in dental caries formation, recently, many studies have been conducted to introduce novel anti-microbial agents, especially   (42).Also, some studies have been done to evaluate the anti-biofilm activity of medicinal plants.Barnabe and colleagues confirmed antibiofilm activity of obtained extract from Dioscorea altissima and Annona hypoglauca against a single-bacteria biofilm of S. mutans (43).The methanol extracts of Camellia japonica and Thuja orientalis were investigated for determining ability to inhibit S. mutans biofilm formation.The results showed that the mentioned plants could inhibit the biofilm with an effectiveness of over 90% (44).In a similar research lee et al revealed that S. mutans biofilm had the highest level of sensitivity for the extract of Sophora flavescens (45).Furthermore, several studies aimed at investigating the anti-bacterial and anti-biofilm activities of pure phytochemicals and some compounds such as linoleic, linolenic, oleanolic, betulinic acids, betulin, beta-sitosterol glucoside and carvacrol introduced as effective compounds against dental caries development and S. mutans biofilm formation (16,46,47).Due to costly and time consuming of convention methods for evaluation, drug likeness and anti-bacterial activities of phytochemicals currently in silico methods have been regarded.Therefore, some studies were planned to in silico screen the effective compounds with anti-biofilm and antibacterial activities (48,49).The results of the present study confirmed that ar-curcumene, d-limonene, and alpha-pinene had strong and appropriate interactions to glucosyltransferase and could be good candidates for inhibiting S. mutans biofilm formation and dental caries development.In this regard, our previous studies showed that some phytochemicals such as carvacrol, α-pinen, limonene, gama-terpinen, betabisabolene, gosferol, psoralen and p-cymen were good candidates for investigating the antibacterial activities (20).Similarly, in a structure-based virtual screening study it was revealed that betulin and 3,12-oleandione could inhibit S. mutans glycosyltransferase, efficiently (50).In another structure-based virtual screening study, it was found that quinoxaline derivative could be a good candidate as a potential glucosyltransferase inhibitor (8).
In conclusion, based on the results of the present study P. acaulis can be a good candidate for more study and development of a natural anti-dental caries agent.

Conclusion
This study was planned to in silico and in vitro evaluate the anti-bacterial and anti-biofilm activities of methanol extracts of P. acaulis and investigate their potential mutagenicities.The results showed that the extracts especially root extract had significant antibacterial activities against a single form of S. mutans.Results also showed that all the extracts could inhibit the biofilm formation in a dose-dependent manner without any mutagenic effects.In silico analysis revealed that arcurcumene, d-Limonene and alpha-Pinene could be good candidates for inhibition of S. mutans biofilm formation and development.Therefore, the plant and its by-products might be good candidates for introduction as new drugs.revised the manuscript for important intellectual content and MN submitted it.All read and confirmed the article ready for publication.

Table 1 . Major constitutes of Prangos acaulis (selected for molecular docking study)
for AutoDock4 runs were as follows: 200 docking runs, population size of 200, random starting position and conformation, translation step ranges of 2A, mutation rate of 0.02, cross-over rate of 0.8, local search rate of 0.06 and 2.5 million energy evaluations.Docked conformations were clustered by a tolerance of two Å root mean square deviations (RMSD).

Table 2 .
The predicted binding sites of glucosyltransferase

Table 4 .
The Results of molecular docking studies on dominant compounds of Prangos acaulis extract and Streptococcus mutans glucosyltransferase

Table 5 .
Antibacterial activity of Prangos acaulis against single form of Streptococcus

Table 6 .
The percentage of inhibition of biofilm structure of Streptococcus mutans