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Development of Antimicrobial Dental Varnish Against Oral Pathogens Using Isolated Cocoa Polyphenols: An in-vitro Study.

Introduction

Oral biofilm is a unique form of biofilm comprising mixed species of microorganisms. Oral biofilms harbouring planktonic bacteria serves as a major virulent factor in developing oral diseases mainly dental caries [1]. The dental plaque biofilm is active site of bacterial proliferation and growth in addition to acid production spot [2,3]. The major cariogenic bacteria associated with dental plaque biofilm are streptococci group, lactobacillus and some actinomyces species [4,5]. These cariogenic bacteria contact the tooth surface and produce bacterial organic acids which aid in demineralization and cavitation of tooth structure [6,7]. The manipulation of dental bio film formation can act as one of the stage in prevention of dental caries.

Investigators have tried various therapeutic measures to prevent, controll and treat tooth decay process. Since tooth decay is a bacterial linked disease, antibacterial therapy is most favourable approach to reduce caries formation. Synthetic antibacterial agents used mainly are povidone iodine, chlorhexidine, cetylpyridinium chloride, triclosan and zinc citrate [8,9].

The vehicles used to deliver antimicrobial agents to the teeth are generally gels, pastes, tablets, solutions and mouth washes [10,11]. The major drawback of these therapeutic drug delivery systems is their reduced substantivity in the oral cavity and lack of prolonged therapy. Therefore intra oral sustained-release device with antibacterial agents seated have been developed. The main advantages of sustained-release devices are that they stretch the availability of the drug, promote increased concentration of drug on the dental biofilm and better inmost ingression of drug into the dental biofilm [12].

Varnishes are the largest approved form of sustained release delivery system enforced directly on the tooth surface. Varnish preparation varies in polymer matrix, pharmaceutical additivies and therapeutical active agents like fluoride derivatives, chlorhexidene and cetylpyridinium chloride [13]. The main drawbacks of these synthetic therapeutic compounds are dental fluorosis, vomiting, diarrhea and tooth discoloration [14]. Hence in recent years, much concentration has been focused for substitute compounds for prevention and treatment of dental caries that is natural, safe, economical, stable and more effective than synthetic therapeutic agents. Hence, the search led to identification of natural products with disease preventing and health promoting benefits. The natural products derived from medicinal plants are rich sources of antimicrobial phytochemicals capable of benefiting oral health with promising results and fewer side effects. The natural products with polyphenolic compounds is studied effective in altering the oral microbiota, suppress the development of dental biofilm and reduce the progression of dental caries. One such natural product with abundant sources of phytochemicals along with other compounds is cocoa. Since recent years, in-vitro studies have shown that cocoa has anti-cariogenic, anti-atherogenic, anti-microbial, anti-inflammatory, anti-oxidant and remineralizing properties.

The effect of cocoa in controlling dental caries is receiving interest due to the presence of cariostatic substances. The cariostatic substances present in cocoa shows anti-glucosyltransferase activity, anti-oxidant, antibacterial and remineralization activity. This brings change in cariogenic flora into non-cariogenic flora by reducing growth, metabolism and bioflim formation by cariogenic bacteria. The beneficial property exhibited from cocoa is because of compounds such as polyphenols, methylxanthines and fatty acids. The main polyphenolic composition of cocoa are catechins, proanthocyanidins, quercetin and phenolic acids such as gallic acid. Several studies have demonstrated the antimicrobial activity of these cocoa polyphenols against cariogenic microgranisms. However, these cocoa polyphenolic compounds coupled with varnish components for the use of sustained release systems for the delivery of antimicrobial agents as not reported. Hence, the aim of the present study was to develop antimicrobial dental varnish that might be effective for eliminating Streptococcus mutans and Lactobacillus acidophilus from the oral cavity.

Materials and Methods

Plant material

Cocoa beans collected from Puttur, Managalore, India was used for the study. The cocoa extract was prepared using soxhlet extraction method. The extract was subjected to column chromatography for isolation and fractions analysed by HPLC to confirm catechin, epicatechin, gallic acid and quercetin. These isolated compounds were used to assess antimicrobial properties along with other components added in varnish.

Chemicals

The polyphenolic standards such as catechin, epicatechins, gallic acid and quercetin was purchased from sigma Aldrich (USA). The other chemicals like Benzoin, agar, ethanol and purified water was purchased from Himedia laboratories Ltd. 0.12% Chlorhexidine varnish was procured from sigma Aldrich (USA and used as standard.

Bacterial growth

The bacterial strain used in the present study was Streptococcus mutans and Lactobacillus acidophilus obtained from Himedia laboratories Ltd. Cell suspension was grown on tryptone broth and activity was checked on Soya bean-casein digest agar medium at 37[degrees]C under aerobic environment supplemented with 5% C[O.sub.2]. Lactobacillus acidophilus was also grown on tryptone broth and activity checked using Soya bean-casein digest agar medium at 37[degrees]C under anaerobic environment.

Antibacterial varnish preparation

Different concentrations of individual components were assessed using standards to determine the concentration in the varnish formulation. The final concentration of varnish comprised of 0.3% wt/v agarose, catechin (0.05% wt/v) epicatechin (0.05% wt/v), Gallic acid (2% wt/v), quercetin (2% wt/v), Benzoin (2% wt/v) and 20% ethanol.

Physicochemical characterization of the developed formulation

The formulation was characterized for smoothness, homogeneity, gelation temperation, gel time, viscosity, casting time, spreadability, syringebilty, water resistance, pH, consistency, surface morphology, mucoadhesive strength and mucoadhesive force.

Smoothness and Homogeneity

The homogeneity of the sample is assessed by visual method after 48 hrs of preparation of the varnish. After 48 hrs, the sample is checked by rubbing between fingers for their appearance and for the presence of any aggregates, grittiness and smoothness.

Surface morphology

The sample was prepared by applying 50 pL of formulations on the glass slide using a small brush and allowed to dry to form a film. This dried glass slides were coated with gold before studying morphology of the surface under scanning electron microscope. The SEM photographs were taken at magnifications of 500 x.

Determination of pH

The pH of the formulation was determined using digital pH meter. The measurement of the pH was recorded in triplicate and average value was considered for record purpose.

Varnish casting time

A casting time or drying time of the varnish was determined by applying relatively 50 [micro]L of varnish onto the clean buccal surface of prepared enamel block measuring about 0.5 [cm.sup.2]. The casting time of varnish on tooth surface was determined using compressed air and without compressed air. This procedure is repeated in triplicate and average is considered for record. The casting film formed on the tooth surface was also assessed for integrity of the film with and without brushing. The integrity was determined by brushing method and immersing the enamel block with casted film in water.

Spreadability

The spreadability of the formulation was measured by spreading about 0.5g of formulation on the glass plate with premarked circle of 2cm diameter. The second glass plate was placed on the premarked glass plate. A weight measuring about 125gm was lodged on the upper glass plate for 5min. Later, the diameter of the spread formulation was measured.

Extrudability

A closed collapsible tube is loaded with 20 mg of the formulation and pressed firmly near the crimped part and a clamp is placed to prevent any roll back of the formulation. The cap was removed and the tube was compressed. The extrudability was determined by measuring the amount of formulation extruded in 10 seconds. The total amount of formulation extruded was collected and weighed. The percentage of the extruded formulation from the tube is calculated.

Water resistance

Repeated layer of varnish is applied till the thickness of varnish is upto 1mm (A). After complete drying immerse the glass slide inside a beaker containing water and leave it for 2mins. After 2mins remove the glass slide from the beaker and dry completely. After thorough drying again check the thickness of varnish using vernier calliper (B). Repeat this again for 4 min (C) and 6 min (D). Then, the water resistance is calculated by using formula A-B, A-C, A-D.

Viscosity

Viscosity of varnish was analyzed using on Brookfield viscometer, spindle number 3 at 60 revolutions per minute at constant temperature. The measurement was made over different range of speed settings from 10 to 100 rpm.

Gel time

Gelation time of the formulation are determined using test-tube inverting method. 2 ml of formulation is taken in a 15 ml borosilicate glass test tube. This glass test tube is placed in water bath maintained at 37[degrees]C. The gelation time is noted when the formulation stops flowing when the test tube is inverted.

Gel temperature

The temperature of the formulation was again determined using test tube inversion method. A 2 ml of formulation was taken in a test tube and intial formulation was checked using thermometer. The test tube was immersed in water bath at 15[degrees]C. The temperature of the water bath was gradually increased every 2 min. The gelation temperature was recorded when the formulation stops flowing upon test tube inversion. The readings are recorded.

Syringeability study

For the drug delivery on the tooth surface the injectable systems is useful. The use of injectable is easy and rapid. Syringeability of gel formulations was evaluated through 21 G needle. The syringeability of the formulations was considered as either pass or fail

Mucoadhesive strength determination

A glass slide was fixed to the apparatus with an instant adhesive.

The formulation was placed on the fixed glass slide using small brush. The diameter of the exposed glass surface was 1.5 cm. The other surface of formulation was attached to a pan through a pulley. A constant force for 2 min is applied to obtain close contact with the formulation and glass surface. The pan was used to keep the weights. Initially a weight of 5 g was placed and checked whether formulation was detaching or not from the other surface of the slide. Weight was added to the pan until the formulation detached from the glass surface. The detachment force gives the mucoadhesive strength of the formulation in grams. The mucoadhesive strength is calculated using the formula: T = mxg/bxc kg/[mm.sup.2].

Biological characterization of developed varnish: In vitro antimicrobial susceptibility test

The in-vitro antibacterial susceptibility was assessed using Agar diffusion method as per NCCLS guide lines 2005. Streptococcus mutans and Lactobacillus acidophilus was used as test microorganisms in the experiment. To assess the antibacterial activity the samples were grouped into 3 groups i.e Group 1 is chlorhexidine varnish group (Standard), Group 2 is water (Control) and Group 3 is developed Varnish.

Results and Discussion

Sustained release antimicrobial varnish was prepared from isolated cocoa polyphenolic compounds mainly catechins, epicatechins, gallic, quercetin, Benzoin and agar. The varnish was evaluated for various physicochemical parameters. The varnish was yellow in color and opaque in appearance and showed smooth and homogenous feeling on application with the absence of lumps and grittiness. The SEM analysis of surface morphology showed uniform, homogenous and with intimate contact to the surface of slide (Fig 1). There was no evidence of separation between the film and the glass slide noticed. The pH of the formulation throughtout the study was found between 6.8 [+ or -] 7 which is suitable for oral condition.

The casting time of the varnish without compressed air on the tooth surface was 20 min. The casting time employing after of the varnish using compressed air was about 2 min. The integrity of the varnish was maintained even after 24 hrs of immersion in water. The formulation was easily removed by brushing technique. The value of spreadabilty (4.6 [+ or -] 4.9) showed that the formulation have small amount of shear. The formulation showed good extrudability with value of between 88 [+ or -] 1% extrusion. This shows that good consistency for clinical application. The results for water resistance for the formulation showed that the formulation was resistance in presence of water. The thickness of the formulation film was maintained same to 1 mm from 2 min till 6 min placement in water. The viscosity result of the formulation shows the formulation is neither too thick nor too thin. The values of viscosity increases with decrease in rpm suggesting the formulation has shear thickening system (Table 2). The gelation time for the formulation was between 9mins to 11mins and initial gelation temperature of 31[degrees]C reaching to final temperature of 29[degrees]C. The syringeability test for the formulation suggests that the formulation was syrineable through 21 guaze needle. The formulation showed sufficient mucoadhesive force and mucoadhesive strength which helps in stronger binding of the formulation to the mucosal surface. This factor increases the retention time and decreasing the leakage by mucosal secretions (Table 1).

Antimicrobial activity was executed against Streptococcus mutans and Lactobacillus acidophilus. Dental varnish showed significant zone of inhibition of about 10 mm against Streptococcus mutans. The standard antibacterial chlorhexidine varnish showed slight more zone of inhibition of about 12 mm (Fig 2). The varnish antibacterial activity against Lactobacillus acidophilus showed zone of inhibition of about 7 mm, whereas chlorhexidine varnish showed 11mm zone of inhibition (Fig 3). The control group i.e water showed no zone of inhibition for Streptococcus mutans and Lactobacillus acidophilus. By comparing the zone of inhibition of varnish with standard chlorhexidine varnish, we could interpret that the zone of inhibition was near to standard chlorhexidine varnish. A Similar study showed 20.9% reduction of streptococcus mutans count when cocoa bean husk was added to mouthrinse for children [15]. Another study when incorporated into hamster diets showed reduction in caries score [16]. Thus the present study result suggest that antimicrobial dental varnish will prevent the formation of cariogenic biofilm.

Conclusion

The antibacterial dental varnish with cocoa polyphenols were successfully developed and characterized in this study. The antibacterial activity of the formulated varnish was similar to chlorhexidine varnish especially against Streptococcus mutans and Lactobacillus acidophilus which are the prime microorganisms involved in dental caries formation.

Received 1 October 2017

Accepted 25 October 2017

Published online 30 April 2018

References

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[8.] Jeon JG, Rosalen PL, Falsetta ML, Koo H: Nat Prod Caries Res 2009, 45:243-263.

[9.] Sajjan PG, Nagesh L, Sajjanar M, Reddy SK, Venktesh UG: Comparative evaluation of chlorhexidine varnish and fluoride varnish on plaque Streptococcus mutans count-an in vivo study. Int J Dent Hyg 2013, 11:191-197.

[10.] L. G. Petersson, K. Magnusson, H. Andersson, B. Almquist, and S. Twetman, Effect of quarterly treatments with a chlorexidine and a fluoride varnish on approximal caries in cariessusceptible teenagers: a 3-year clinical study, Caries Research, 2000, vol. 34, no. 2, pp. 140-143.

[11.] Q. Zhang, W. H. Van Palenstein Helderman, M. A. Van't Hof, and G.-J. Truin, Chlorhexidine varnish for preventing dental caries in children, adolescents and young adults: a systematic review, European Journal of Oral Sciences, vol. 114, no. 6, pp. 449-455, 2006.

[12.] Doron steinberg and Michael Friedman, Drug Dev.Res. 2000,50, 555-565.

[13.] Steinberg R, Rozen E.A, Klausner B, Zachs M and Friedman, Formulation, development and characterization of sustained release varnishes containing amine and stannous fluorides, Caries Res, 2002,36,411-416.

[14.] Franca, J.R., De Luca, M.P., Ribeiro, T.G., Castilho, R.O., Moreira, A.N., Santos, V.R. and Faraco, A.A., 2014. Propolis-based chitosan varnish: drug delivery, controlled release and antimicrobial activity against oral pathogen bacteria. BMC complementary and alternative medicine, 14(1), p.478.

[15.] Srikanth R.K., Shashikiran N.D., Subba Reddy V.V., J. Indian Soc. Pedod. Prev. Dent. 2008:67

[16.] Stralfors A.Arch Oral Biol 1967;12: 959

C. Pushpalatha (1), Swaroop Hegde (2), R. Deveswaran (3), Latha Anandakrishna (1)

(1) Department of Pedodontics and Preventive Dentistry, (2) Department of Conservative Dentistry & Endodontics, Faculty of Dental Sciences, (3) Department of Pharmaceutics, Faculty of Pharmacy, M.S. Ramaiah University of Applied Sciences, Begaluru, India

* Coresponding author.

E-mail address: drpushpalaiha29@ggnail.com (Dr. C. Pushpalatha)

Caption: Figure 1: SEM image for the formulation

Caption: Figure 2: Zone of inhibition for streptococcus mutans (1Developed varnish, S--Antibacterial standard varnish, Ccontrol (water))

Caption: Figure 3: Zone of inhibition for Lactobacillus acidophilus (1Developed varnish, S--Antibacterial standard varnish, C control (water))
Table 1: Physicochemical properties value of the
varnish

Parameters                               Values

pH                                       6.9 [+ or -] 7.0
Casting time
  Natural casting time                   20 mins
  Compressed air assisted casting time   3 mins
Spreadability                            4.6 [+ or -] 4.8
Extrudability                            98 [+ or -] 89%
Water resistance                         1mm
Gel time                                 9 [+ or -] 10 mins
Gelation temperature
  Initial gelation temperature           31[degrees]C
  Initial gelation temperature           28[degrees]C
Syringeability                           pass
Mucoadhesive strength                    2450 kg/[mm.sup.2]
Mocoadhesive Force                       0.245 N

Table 2: Viscosity of the formulation

Speed (RPM)   Viscosity (CPS)

50                 32.5
40                 42.1
30                 51.5
20                 65.5
10                 77.0

Table 3: Antibacterial activity of developed varnish, standard
antibacterial varnish and control against Streptococcus. mutants
and Lactobacillus. acidophilua

SI          Sample                       zone of inhibition
no
                                I. mutant *         L.acidophilus

1    Chlorhexidine varnish   12.0 [+ or -] 0.00   11.0 [+ or -] 0.00
2       Water(Control)              --                   --
3      Developed varnish     10.0 [+ or -] 0.00    7.0 [+ or -] 0.00
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Title Annotation:Original Article
Author:Pushpalatha, C.; Hegde, Swaroop; Deveswaran, R.; Anandakrishna, Latha
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Jan 1, 2018
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