Comparative evaluation of antimicrobial properties of piperidine and apple cider vinegar on Streptococcus mutansand in-silico demonstration of the mechanism of action

INTRODUCTION

Oral biofilm, a structured community which consists of a wide range of microbes embedded with self-organized matrix of extracellular polysaccharides (EPS), is clearly recognized as a virulence factor to many oral infectious diseases including dental caries, gingivitis, periodontitis, periapical periodontitis and peri-implantitis.²¹ The bulk of the microorganisms that form the biofilm are the Streptococcus mutans and other anaerobes.

Streptococcus mutans, a member of the viridans group of streptococci, is recognized as part of the normal oral flora, and is an aetiological agent in smooth-surface dental caries. The three key virulence attributes of S. mutans that enable this organism to cause dental caries is the ability to form biofilms on the tooth, production of organic acids via metabolism of dietary carbohydrates; and the ability to grow and to continue to produce acids in a low pH environment, known as aciduricity.²² By contrast, little is understood about the most economic and effective ways of controlling oral biofilm due to the enhanced resistance to antibiotics and other antimicrobial agents.

Chlorhexidine is the most commonly used antimicrobial agent in denstistry as they destroy wide spectrum of microrganisms. Constant increase in resistant strains and side effects associated with the use of chlorhexidine is brownish discoloration of teeth, restoration and tongue has led to the search for alternative herbal metabolite. Various herbal extracts, such as neem and tulsi, Aloe vera, curcum longa, and turmeric, having antimicrobial, anti-inflammatory are promising to be used.² But there is a lack of sufficient studies reporting various herbal metabolite and their antimicrobial actions against S. mutans. Black pepper (Piper nigrum L.) the “King of Spices” a common household substance,⁵ is used in traditional medicinal systems as an antimicrobial due to presence of volatile compounds, tannins, phenols and various unknown substances^13,14. Similarly the acetic acid in apple cider vinegar penetrates cell membranes of microorganisms leading to bacterial cell death. The bacterial strains, temperature, pH, acid concentration, and ionic strength influence the antimicrobial activity of organic acids effective against several types of bacteria and acts as an antimicrobial agent³ .

The Computational method such as molecular docking is very useful and reasonably reliable for prediction of putative binding modes and affinities of ligands for macromolecules. Such methods are gaining popularity because the experimental determination of complex structures is rather difficult and expensive. Molecular docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex⁸ . Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules. Molecular docking is one of the most frequently used methods in structure-based drug design, due to its ability to predict the binding-conformation of small molecule ligands to the appropriate target binding site. Characterization of the binding behavior plays an important role in rational design of drugs as well as to elucidate fundamental biochemical processes⁹ .

The current study focuses on the evaluation of pipperidine and apple cider vinegar as an antimicrobial agents against S.mutans and also the mechanism of action of pipperidine as a biofilm quenching agent through in-silico docking method.

MATERIALS AND METHODOLOGY:

Sample preparation: S. mutans pure culture maintained in the laboratory of Dextrose Technologies Pvt. Ltd., was used for the study after sub-culturing in Brain Heart infusion broth (BHI). The organism was incubated for 48 hours prior to use for the study.

Experimental design: The study was designed with two test samples and on a control sample as illustrated below:

Group1: Piperidine

Group2: Apple cider vinegar

Group3: Chlorhexidine

100 µl of sub-cultured S. mutans was inoculated in 10ml BHI in 20 test tubes each including their respective positive and negative controls. Three different concentrations of the test samples like 50µl, 100µl & 150µl were added to BHI broth culture in duplicate and incubated at 37ºC for 24hrs for further analysis.

Turbidity analysis and Minimal bacterial count (MBC): The samples were analyzed spectrophotometrically at 600nm with uninoculated media as control 19. Briefly, 100 µl of sample was diluted with 900 µl of distilled water. 100µl of sample was inoculated in the pre-prepared BHI agar plates and incubated at 37ºC for 24 hours and the number of colony forming units were counted after incubation.

Biofilm Assay: Tube assay was performed for assessing the biofilm formation and biofilm quenching activity of the test samples on S. mutans.19 numbers of test tubes were taken with 10 ml of BHI broth. 100 µl of S. mutans culture was inoculated into each test tube and incubated at 37ºC for 24hrs. After incubation the culture was aspirated, and the bacterial biofilm was fixed by adding 500μl of crystal violet solution (0.2%) to each test tube. After 10 minutes, the excess crystal violet was removed and test tubes were washed twice with phosphate buffer and air dried. Finally, the cell bound crystal violet was dissolved in 33% acetic acid and the intensity of the color was measured at 595 nm using a spectrophotometer. Intensity of the color is proportional to the number of cells in the biofilm and hence considered as the parameter for quantifying the biofilm.

EPS estimation: The culture sample was centrifuged at 5000rpm for 10mints before adding ice cold ethanol. The sample was incubated overnight at 4˚C. EPS was analyzed by phenolsulfuric acid method (DuBois method) to determine total polysaccharide content (DuBois et al. 1956). Glucose was used as a calibration standard. After letting the sample to cool down for 30 min, sample absorbance was measured at 490 nm⁸ .

In-silico analysis: The mechanism of action of herbal extract against S. mutans was carried out by Molecular Docking technique using Autodock vina 4.2 against dihydrofolate reductase enzyme. Standard docking procedures for a rigid protein and flexible ligands were used as per the user guide. Default settings were used for parameters. About 10 docking runs were performed for the ligands to find out the target site which gives binding affinity. At the end of docking, the best binding modes were analyzed for various interactions.¹⁰

Docking studies:

Statistical analysis: The statistical analysis t-test was performed between the results of test and control groups as well as within the test groups using SPSS version 17 in order to evaluate the results.

RESULTS:

Turbidity analysis: PP: Piperidine; ACV: Apple cider vinegar; CH: Chlorhexidine was analyzed as mentioned above. Samples were analyzed in various concentrations. The OD value shows the turbidity or the microbial populations.

DISCUSSION:

Bacterial biofilm is infectious in nature and can result in infections. The microbial biofilm of caries is diverse and contains many facultative and obligate anaerobic bacteria.⁷ Classical biofilm lifecycle includes bacterial attachment, biofilm growth/ maturation, and biofilm dispersal.1⁰ S. mutans, is the most common acid producer, primarily associated with caries. Furthermore, some S. mutans strains are naturally competent for genetic transformation and are able to take up DNA from their environment. Additionally, the competence pathway of S. mutans is linked to the production of bacteriocins, which kill susceptible closely related species, thus eliminating competitors while increasing the genetic m⁶ . Due to the complexity of the oral cavity and the rapid clearance of saliva, topically applied antibacterial agents are not retained at the proper concentrations for a long enough duration.²² Measures that can disrupt any stage of biofilm cycle are considered as potential approach to the control of biofilm. Therefore, finding solutions with long term effectivity against dental pathogens is crucial for the success of treatments. Current study has been carried out with this intention and comparative effectivity of two compound/products have been tested.

Chlorhexidine was developed in 1940’s as a result of search of antiviral agents. It was found that chlorhexidine does not possess antiviral property but antibacterial activity.²³ Currently it is one of the most accepted and extensively used anti-microbial agent in the field of medical and dentistry. The mature black pepper contains a specific substancePiperidine, recently which is employed in the anti-inflammatory and anti-malarial treatment.² And apple cider vinegar is an anti-microbial agent which has been recognized but not investigated.³ An investigation of the inhibitory effect of these metabolites against planktonic (floating) and biofilm forms of microorganisms (streptococci mutans) is required before they may be proposed as anti-microbial agent to control and to promote oral health status. Therefore, these compounds were examined for the in-vitro antimicrobial activity against the cariogenic bacteria, to provide informative guidance for further investigations and development.

Methodology of this study followed the standard established tests. Study design presented in this paper is more consistent with other studies testing ability of antimicrobial action.⁹ Docking is a process by which two molecules fit together in 3D space. To perform docking software packages of various kinds have been used. Molecular docking is like a “lock-and-key”, where one is interested in finding the correct relative orientation of the “key” which will open up the “lock”¹⁰. During docking high affinity hydrogen bonds were formed with Piperidine. When the receptor molecule forms more hydrogen bonds, higher the binding efficacy of the herbal extract to the enzyme.

In this study, Molecular docking of Piperidine (active component of black pepper extract) and with the target protein Dihydrofolate Reductase enzyme was done. The docking sites are given in figure^6,7 (a,b). Dihydrofolate Reductase (DHFR) is a target for cancer chemotherapy and bacterial infection. Dihydrofolate reductase is a smallenzyme that plays a supporting role, but an essential role, in the DNA synthesis and other processes, thereby inhibiting bacterial proliferation. Molecular docking demonstrated high binding efficacy for pepper extracts.

This inhibits the DNA synthesis, thereby increasing the antibacterial efficacy of the herbal extracts. Computational approach for structure based drug discovery offer a valuable alternative to the costly and time consuming process of random screening and wet lab analysis. The synthetic programs directed towards the design of new DHFR inhibitors have most often followed an empirical medicinal approach involving replacement of ring nitrogen's by carbon atoms. An important quantification made during docking simulations of small molecules to enzymes or other complex protein structures can also be performed.

CONCLUSION:

In conclusion our study has manifested comparative evaluation of the antibacterial efficacy of pipperdine, apple cider vinegar and chlorhexidine against Streptococcus mutans. The EPS analysis; the crystal violet test and the EPS estimation by Spectrophotometric method has given a clear cut estimation of biofilm produced and being quenched by test materials. From, the statistical analysis it was proved that 150 µL of piperidine shows better results compared to apple cider vinegar and almost equivalent effect to the commercial available chemical agent chlorhexidine. Hence, In future it can replace the use of chemical chlorhexidine. The in-silico study has also manifested the mechanism of action of piperidine showing -4.2 binding affinity against dihydrofolate reductase a DNA precursor enzyme which clearly proves that piperidine can damage the DNA molecule in the S.mutans and thereby terminates the replication of the pathogen.