International Journal of Botany Studies

International Journal of Botany Studies


International Journal of Botany Studies
International Journal of Botany Studies
Vol. 7, Issue 3 (2022)

Interference of naringin, a bioactive compound characterized from the methanolic leaf extract of Citrus maxima, with the biofilm-forming property of cariogenic bacterium Streptococcus mutans


Apoorva Udayashankara, Manjunath Kiragandur, Hemavathy E


International Journal of Botany Studies

www.botanyjournals.com

ISSN: 2455-541X

Received: 29-01-2022, Accepted: 14-02-2022, Published: 01-03-2022

Volume 7, Issue 3, 2022, Page No. 7-13

  Interference of naringin, a bioactive compound characterized from the methanolic leaf extract of Citrus maxima, with the biofilm-forming property of cariogenic bacterium Streptococcus mutans

Apoorva Udayashankara, Manjunath Kiragandur*, Hemavathy E

Department of Microbiology and Biotechnology, Bangalore University, Jnana Bharathi Campus, Bangalore, Karnataka, India

 

 

 

 
   

 

Abstract

Microbial adhesion on the tooth surface is the initiating factor for plaque formation. The majority of the early plaque flora is constituted by different Streptococci sp. In this study, we have reported the antimicrobial and antibiofilm activity of naringin against the cariogenic bacterium Streptococcus mutans MTCC 497. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the identified bioactive compound naringin were determined using a micro-dilution assay. The antibiofilm activity was screened at sublethal concentrations of naringin (75%, 50%, and 25 % of MBC) using the microtiter plate method. Further, the cytotoxicity of naringin and chlorhexidine mouth wash was tested on L132, normal human epithelial cell lines using a phase-contrast inverted microscope and MTT assay. Our results revealed, bioactive compound naringin exhibited a significant antimicrobial activity with MIC 12.5mg/ml, MBC 6.25mg/ml, and IC50 value 4.563mg/ml. There was more than an 85% reduction in the biofilm formation at 75% sub-MBC level Concentration. Naringin-treated cells, showed no signs of deformity even at the highest tested concentration while there was evidence of cell toxicity in the cells treated with the lowest concentration of chlorhexidine mouth wash. LC50 value of naringin was found to be 174.884 mg/ml indicating it to be safe. The findings of this study endorse that naringin has the strong ability to interfere In vitro with the biofilm formation of Streptococcus mutans, the primary etiological agent of dental caries. It also showed the least toxicity in comparison to chlorhexidine mouth wash, the choice of drug used to control dental plaque.

 

Keywords: naringin, antimicrobial, antibiofilm, dental plaque, Streptococcus mutans

 

 
   

 


 

 


 

Introduction

The microbes exist in two forms namely planktonic and sessile. Planktonic cells enter the sessile phase by attaching to a biotic or abiotic surface to form a biofilm. A biofilm is a complex habitat composed of not just one, but a divergent range of microorganisms living enclosed in a hostile environment, composed of a matrix of polysaccharides, proteins, lipids, nucleic acids, and other chemical materials [1]. This association is a reversible one where the planktonic cells can get detached to form a new biofilm by attaching themselves to a new surface. Dental plaque is an example of a biofilm. Once the dental plaque is formed, the cells start metabolizing the food particles by producing acids that destruct the tooth enamel leading to dental caries and gingivitis [2]. It is very difficult to combat this army of cells with static and cidal agents due to various factors like the hindrance offered by the thick matrix for the permeability of the chemicals, efficient multidrug efflux pump, horizontal gene transfer, and secretion of antibiotic modifying enzymes, and the quorum sensing defense. Due to this extreme resistance, the dental biofilms are often treated with high doses of antibiotic agents or disrupted by a physical intervention that damages the tooth surface [3]. This concern has forced investigators to find an alternative antibiofilm agent with minimal side effects. The development of novel therapeutic measures to control the biofilm formation rather than killing the pathogen using plant-based products appears to be a promising approach. In our study, we have used naringin separated and identified from the methanolic leaf extract of Citrus maxima, [29] to screen its antibiofilm activity against Streptococcus mutans, which is the primary etiological agent of dental caries. Since flavonoids are natural agents, they are safer in comparison to chemical agents and also have an upper edge over antibiotics as they have multiple approaches to killing the cell, making it hard for the bacteria to develop resistance against them [4]. Naringin is a flavanone glycoside, found in citrus fruits, grapefruits, cherries, beans, cocoa, oregano, and tomatoes, responsible for their bitter taste in them. It is produced from the flavanone naringenin and disaccharide neohesperidose [5]. Naringin and its derivatives have reported antimicrobial activity against pathogenic bacteria. [6, 7] It also exhibits strong anti-inflammatory and antioxidant activity [8], naringin and naringenin in combination have exhibited anti-cancerous activity [9] and are also known to have anti-quorum and antibiofilm activity [10, 11, 12]. The objective of the present study was to study the antimicrobial activity of flavonoid naringin, to screen its inhibitory influence on biofilm formation of Streptococcus mutans MTCC 497, and to evaluate its toxicity in comparison to chlorhexidine mouthwash.

Materials and Methods

Bacterial culture

The bacterium used in this study was Streptococcus mutans MTCC 497, which was kindly provided by Maharani Lakshmi Ammanni college for women, Bengaluru, Karnataka, India. It was cultured in brain heart infusion broth (BHB) followed by Brain heart infusion agar (BHA) with 0.1% sucrose at 37 ℃ in a 5% CO2 enriched condition. All the media components used were purchased from HiMedia Laboratories Pvt Ltd.

 

Plant materials and preparation of the naringin

The bioactive compound naringin was isolated, purified, and characterized from the methanolic leaf extract of Citrus maxima from our previous study as described by Apoorva et al., (2020) [30], The compound was dissolved in 1% Dimethyl sulfoxide at a concentration of 25mg/ml.

 

Determination of minimum inhibitory concentration and minimum bactericidal concentration

The minimum inhibitory concentration and minimum bactericidal concentration of the natural compound naringin were evaluated against the test organism S. mutans MTCC 476 using broth microdilution assay, following the procedure described by Oliveira et al., (2006) and Lima et al., (2012). 25mg/ml of naringin was dissolved in 1% DMSO, which was serially diluted in twofold dilution. 20µl of BHIB inoculated with the test bacteria approximately containing 1×108 CFC/ml were added to the wells containing antimicrobial agent at a final volume of 100µl/ well ranging from 25- 0.04mg/ml. The microplate was incubated at 37 ℃ and 5% CO2 for 24h. The bacterial growth was measured by absorbance at OD570 nm using, Thermo scientific MULTISCAN GO Micro ELISA auto reader. Gentamycin was used as a positive control at 25mg/ml and 1% DMSO as the negative control. The lowest concentration of the naringin which restricted the visible growth of the test bacteria was considered as the MIC. The samples were assayed in triplicates and repeated twice The MBC of the naringin was determined by streaking a loopful of each concentration ≥ MIC on the BHA medium. The plate was incubated at 37℃ and 5% CO2 for 24 to 48 h. MBC was defined as the lowest concentration of the bioactive compound showing no bacterial growth.

 

Antibiofilm assay

Inhibitory activity of naringin on the biofilm-forming property of S. mutans MTCC 497 was screened at sublethal concentrations of 75%, 50%, and 25 % of MBC, using 96 - well microtiter plate method by following the methodology used by Antunes et al., (2010). 300µl of trypticase soya yeast broth (TSY) inoculated with the test organism at 106 CFU/ml concentration was aliquoted into each well of the microplate. Chlorohexidine mouthwash at 0.2% concentration was used as a positive control and inoculated medium without the bioactive compound was used as the negative control. The plate was incubated at 37℃ and 5% CO2 for 24 to 48 h. At the end of incubation, the supernatant was carefully removed and each well was washed thoroughly with sterile distilled water to remove free-floating cells. The plate was then air-dried for 30min, the adhering biofilm was stained using a 0.1% aqueous solution of crystal violet for 15 min at room temperature. Following incubation, the excess stain was removed by washing the plate gently with sterile distilled water. The bound dye to the cells was solubilized by the addition of 250µl of 95% ethanol. After 15min of incubation, absorbance was measured at 570nm using a Thermo scientific MULTISCAN GO Micro ELISA auto reader. The percentage of biofilm inhibition was calculated using the formula

 

 

Cytotoxicity ASSAY

Cell lines and culture condition

The Normal, Human epithelial cell line, L132 was initially procured from National Centre for Cell Sciences (NCCS), Pune, India, and maintained in Dulbecco’s modified Eagles medium, DMEM (Sigma Aldrich, USA). The cell line was cultured in a 25 cm2 tissue culture flask with DMEM supplemented with 10% FBS, L-glutamine, sodium bicarbonate (Merck, Germany), and an antibiotic solution containing: Penicillin (100µg/ml), Streptomycin (100µg/ml), and Amphotericin B (2.5µg/ml). Cultured cell lines were kept at 37ºC in a humidified 5% CO2 incubator.

 

Cytotoxicity evaluation

Two days old confluent monolayer of cells were trypsinized and the cells were suspended in a 10% growth medium, 100µl cell suspension (5x103 cells/well) was seeded in 96 well tissue culture plate and incubated at 37ºC in a humidified 5% CO2 incubator. After 24 hours the growth medium was removed, freshly prepared naringin and chlorhexidine mouthwash in DMEM were five times serially diluted by two-fold dilution (100µL, 50µL, 25µL, 12.5µL, 6.25µL in 500µl of DMEM) and each concentration of 100µl was added in triplicates to the respective wells and incubated at 37ºC in a humidified 5% CO2 incubator. Non-treated control cells were also maintained.

 

Cytotoxicity assay by direct microscopic observation

The entire plate was observed after 24 hours of treatment in an inverted phase-contrast tissue culture microscope (Olympus CKX41 with Optika Pro5 CCD camera) and microscopic observations were recorded as images. [13] Any detectable changes in the morphology of the cells, such as rounding or shrinking of cells, granulation, and vacuolization in the cytoplasm of the cells were considered as indicators of cytotoxicity.

 

Cytotoxicity assay by MTT method

15mg of MTT (Sigma, M-5655) was reconstituted in 3 ml PBS until completely dissolved and sterilized by filter sterilization. After 24 hours of the incubation period, the sample content in wells was removed and 30µl of reconstituted MTT solution was added to all test and control wells, the plate was gently shaken well, then incubated at 37ºC in a humidified 5% CO2 incubator for 4 hours. After incubation, the supernatant was removed and 100µl of MTT Solubilization Solution (Dimethyl sulphoxide: DMSO, Sigma Aldrich, USA) was added and the wells were mixed gently by pipetting up and down to solubilize the formazan crystals. The absorbance values were measured by using a microplate reader at a wavelength of 540 nm [14].

The percentage of growth inhibition was calculated using the formula:

 

                                                               

Statistical analysis

All the results were represented as mean ± standard deviation of at least two independent experiments performed in triplicates. Statistical analysis was performed by using student t-test, ANOVA models, differences were accepted as statistically significant at P < 0.05.

 

Results

The alarming rate of increased resistance against conventional antibiotic treatment, across the globe, has forced our researchers to find substitutes for antibiotics, to avoid the dissemination of deadly resistant microbes. Hence in this study, we are focusing on using phytochemicals as one of the alternative natural sources to control dental plaque formation. Naringin isolated and identified by us, from the methanolic leaf extract of Citrus maxima [29] was used to screen its antimicrobial and antibiofilm activity against S. mutans MTCC 497.

 

Minimum inhibitory concentration and minimum bactericidal concentration

The mean absorbance value recorded at 570nm is as tabulated in Table no. 1. Naringin was observed to inhibit the growth of the test organism from 6.25mg/ml hence, the minimum inhibitory concentration of naringin that restricted the visible growth of the tested organism was found to be 12mg/ml. From Fig.1, the lowest dilution that showed no visible growth when streaked onto BHA agar plates, post-incubation was 6.25mg/ml and is considered as the MBC of naringin against S. mutans MTCC 497. To calculate the IC50 value the percentage of cell viability was calculated and plotted against the concentration of naringin using an online IC50 calculator AAT Bioquest. The concentration of naringin that inhibits the growth of half of an inoculum (IC50) of the tested organism was calculated to be 4.563mg/ml.

 

Table 1: MIC of naringin against S. mutans MTCC 497.

 

Organism

tested

Mean OD at 570nm

25 mg/ml

12.5 mg/ml

6.25 mg/ml

3.12 mg/ml

1.56 mg/ml

0.78 mg/ml

0.39 mg/ml

0.19 mg/ml

0.09 mg/ml

0.04 mg/ml

+

control

-

control

S. mutans

MTCC497

0.0476 ±

0.006

0.0777±

0.004

0.1806±

0.014

0.3053±

0.004

0.315±

0.004

1.3545±

0.006

2.5024±

0.070

2.604

±

0.008

2.814±

0.067

2.9131±

0.087

0.025

±

0.004

2.9688

±

0.136

Mean ± Standard deviation

 

 

a                                                                b

 

Fig 1: a- BHA agar plate streaked with different dilutions of naringin. b- Graph depicting the IC50 value of naringin for S. mutans MTCC 497.

Antibiofilm assay

The effect of naringin on the plaque-forming ability of S. mutans MTCC 497 was evaluated at sub-MBC concentrations of 75%, 50%, and 25%, and its effect is shown in Fig 2. At 75 % of MBC value, naringin was efficiently inhibiting the adhesive property of the tested organism.

 

 

Fig 2: Percentage of biofilm inhibition by chlorohexidine mouthwash used as positive control and naringin at sub-lethal MBC concentrations of 75%, 50%, 25%.

 

Cytotoxicity assay by direct microscopic observation