Evaluation of preliminary anti-microbial efficacy of curcumin and chitosan nanoparticles with and without photoactivation against candida albicans – An invitro study

Introduction

The success of root canal treatment in contemporary endodontic therapy mainly depends upon three important aspects like debridement, cleaning and shaping followed by three-dimensional obturation.1 The first step in root canal treatment is to eliminate the microorganisms which are considered to be the primary source of endodontic infections. Endodontic microflora is diverse containing different species of bacteria, fungi and viruses.

The most commonly found fungi in root canal infections are Candida albicans with a prevalence ranging from 0.5%–55%.2 The Candida albicans biofilms possess virulence factors that may play a role in the onset of endodontic pathologies.

As the root canal anatomy is complicated complete removal of these microorganisms is not possible from all areas which are difficult to reach by instruments. Root canal irrigants are important adjuvants in root canal debridement and disinfection which can reach all parts of the root canal. 3 Sodium hypochlorite is considered as an ideal root canal irrigant, however, this popular irrigant has various side effects such as unpleasant taste and odour, tissue toxicity, inability to remove the smear layer, inability to fully eradicate microbes from the infected canals, allergic potential, risk of emphysema on overfilling, and discolourations of clothes.4 To overcome these disadvantages various newer irrigants having antimicrobial activity have been tried in endodontics.

Most herbal alternatives researched are Neem, Tulsi, Green tea, Propolis, Triphala and Curcumin. Curcuma longa (commonly called as turmeric) belonging to the Zingiberaeceae family has been used as a traditional medicine from ancient times. Curcumin (diferuloylmethane) is the main yellow bioactive component of turmeric and has been shown to have a wide spectrum of actions like anti-inflammatory, antioxidant, antibacterial, antifungal, antiprotozoal and antiviral activities.5

Antimicrobial efficacy can also be enhanced by using Chitosan Nanoparticles, which are particles sized less than 100 nm, have demonstrated broad-spectrum antimicrobial activity. Nanoparticles have the ability to exert their biological activity without causing adverse reactions neither local nor systemic in patients receiving the therapy.6

Because of the increasing incidence of fungal infections, coupled with the growing resistance against antifungal agents, an alternative technique of root canal disinfection involves photodynamic therapy (PDT). Photodynamic inactivation of micro-organisms is based on the combination of a drug, known as photosensitizer, and the delivery of visible light of the appropriate wavelength to excite the photosensitizer molecule.

Hence the aim of this study was to evaluate the preliminary anti-microbial efficacy of Curcumin and Chitosan nanoparticles with and without photoactivation against Candida albicans.

Materials and Methods

Pure strain of Candida albicans ATCC strain 10231 was obtained from the Department of Microbiology, MMNGH Institute of Dental Sciences and Research Centre, Belagavi.

Ethanolic extract of turmeric was prepared by adding 200mg of Curcumin in 20ml ethanol for preparation of irrigant.

Chitosan nanoparticle solution was prepared by dissolving commercially procured Chitosan in dilute acetic acid (2 % v/v), the pH was adjusted to 5.0 using 4M NaOH. This solution was stirred at 600 rpm on a magnetic stirrer. Afterwards, sodium tripolyphosphate (HiMedia Laboratories Pvt. Ltd, Mumbai, India) (TPP, 0.1 % w/v) solution was added to the above mixture. The suspension obtained was stirred further for 30 min at room temperature and centrifuged at 12857 × g at 20 °C for 20 min to pelletize the prepared nanoparticles. The prepared pellet was subsequently redispersed in deionized water and stored at 4^oC.

Other irrigants used were commercially available 3%Sodium hypochlorite, Normal saline

Results

And MBC values in triplicates

Table 1

MIC values in triplicates and mean value

Groups	1	2	3	Mean
Nonphotoactivated Curcumin	0.4 μg/ml	0.8 μg/ml	0.8 μg/ml	0.66
Photoactivated Curcumin	0.2 μg/ml	0.2 μg/ml	0.4 μg/ml	0.26
Nonphotoactivated Chitosan nanoparticles	0.8 μg/ml	0.8 μg/ml	0.8 μg/ml	0.80
Photoactivated Chitosan nanoparticles	1.6 μg/ml	0.4 μg/ml	0.8 μg/ml	0.66
Sodium hypochlorite 3% (Positive control)	3.12 μg/ml	3.12 μg/ml	12.5 μg/ml	4.24
Sterile saline (Negative control)	12.5 μg/ml	100 μg/ml	100 μg/ml	70.83

Figure 1

Comparison of six groups with MIC values against Candida albicans.

Table 2

MBC values in triplicates andmean

Groups	1	2	3	Mean
Nonphotoactivated Curcumin	1.6μg/ml	0.8 μg/ml	0.8 μg/ml	1.0
Photoactivated Curcumin	0.8 μg/ml	0.4 μg/ml	0.4 μg/ml	0.53
Nonphotoactivated Chitosan nanoparticles	1.6 μg/ml	0.4 μg/ml	0.8 μg/ml	0.66
Photoactivated Chitosan nanoparticles	1.6 μg/ml	0.4 μg/ml	0.8 μg/ml	0.93
Sodium hypochlorite 3% (Positive control)	6.25 μg/ml	1.6 μg/ml	12.5 μg/ml	4.7
Sterile saline (Negative control)	25μg/ml	100 μg/ml	100 μg/ml	75

MBC

Figure 2

Comparison of six groups with MBC values against Candida albicans.

Mean MIC values of all 6 groups were compared. Photoactivated Curcumin with mean value of (0.26 μg/ml) showed better antimicrobial efficacy followed by Curcumin without photoactivation( 0.66 μg/ml), Chitosan nanoparticles with photoactivation(0.66 μg/ml), Chitosan nanoparticles without photoactivation (0.80 μg/ml), NaOCl(4.24 μg/ml) and Sterile saline (70.83 μg/ml).

Mean MBC values of all 6 groups were compared. Photoactivated Curcumin with mean value of (0.53μg/ml) showed better antimicrobial efficacy followed by Chitosan nanoparticles without photoactivation(0.66μg/ml), Chitosan nanoparticles with photoactivation(0.93 μg/ml)Curcumin without photoactivation(1μg/ml), NaOCl(4.7 μg/ml) and Sterile saline (75 μg/ml).

Discussion

Endodontic treatment success, to great extent, depends on the depletion or elimination of microorganisms from the root canal system. The use of irrigating solutions with proper anti-microbial and anti-fungal properties during canal debridement and preparation are considered enormously important.

Candida albicans was chosen for this study as it is the most common fungus seen in the root canals with a prevalence of 21% in primary infections and 18% in cases of retreatments. The portal of entry of yeasts into an infected endodontic eco-niche is likely to be through cracks and leakage around faulty restorations. They may also enter through dentinal tubules of deep carious lesions and invade frank pulpal exposures. They survive harsh environmental conditions, thereby making the penetration of antimicrobial agents and irrigants difficult and thus adding to the virulence of microorganisms.7

Nara et al reported Sodium hypochlorite (NaOCl) as a gold standard irrigant. As it has high antimicrobial action and the ability to dissolve organic material. But it has deleterious effects like tissue toxicity, unpleasant taste and odour, and inability to remove the smear layer. In the present study to overcome these disadvantages, we have used Curcumin and Chitosan nanoparticles with and without photoactivation to check for antimicrobial efficacy.

Curcuma longa a member of the ginger family possesses anti-inflammatory, antioxidant, antimicrobial and anticancer activity. In an in vitro study conducted by Neelakantan P et al. (2011), it has been shown that curcumin has significant antibacterial activity against E.faecalis and can be used as an alternative to sodium hypochlorite for root canal irrigation.8

Mean MIC values of all 6 groups were compared. Photoactivated Curcumin with mean value of (0.26 μg/ml) showed better antimicrobial efficacy followed by Curcumin without photoactivation (0.66 μg/ml), Chitosan nanoparticles with photoactivation (0.66 μg/ml), Chitosan nanoparticles without photoactivation(0.80 μg/ml), NaOCl (4.24 μg/ml) and Sterile saline(70.83 μg/ml).

Mean MBC values of all 6 groups were compared. Photoactivated Curcumin with mean value of (0.53μg/ml) showed better antimicrobial efficacy followed by Chitosan nanoparticles without photoactivation (0.66μg/ml), Chitosan nanoparticles with photoactivation(0.93 μg/ml)Curcumin without photoactivation (1μg/ml), NaOCl (4.7 μg/ml) and Sterile saline (75 μg/ml).

There are various modes to activate or increase the efficacy of irrigants. One among them is photodynamic therapy. Photodynamic therapy (PDT) is a novel therapeutic strategy based on the interaction between a nontoxic photosensitizer and a safe source of visible light at a low intensity; the combination of these two leads to the development of reactive oxygen species (ROS) which are toxic and cause oxidative damage to microorganisms and tumour cells. Cells treated with photo-sensitizers in PDT are susceptible to eradication by exposure to light.

This study clearly demonstrates the efficacy of curcumin and chitosan nanoparticles with and without light activation using a blue light source on the Candida albicans. In this study, special attention was given to the protocol of light exposure by the blue light device during photoactivation. The PDT was able to promote anti-fungal effects against C. Albicans and was able to produce significant reductions in viable Candida counts when compared to other groups. Although the statistical analysis did not find significant differences between photoactivated curcumin and nonphotoactivated curcumin groups.

In the present study, Curcumin without photoactivation also showed good results. It may be because of the main yellow bioactive component of turmeric as it has shown to have a wide spectrum of antifungal action. Which causes leakage of potassium ions from the fungal cytosol, increase in membrane permeability and disruption of membrane.

In a study Jing Ma et al used one standard strain ATCC 90028 and two clinical isolates from HIV (CCA1) and oral lichen planus (CCA2) patients' oral cavities. Biofilms were photosensitized with 60 μM Curcumin and irradiated by a light emitting diode (LED) under the wavelength of 455 nm and energy densities of 2.64, 5.28, 7.92, 10.56, 13.2 J/cm². Then the antifungal effects of Curcumin-photodynamic therapy were evaluated by XTT reduction assay and confocal light scanning microscopy (CLSM) observations. The study concluded that the combination of curcumin with blue LED light was effective against Candida albicans.9

In dentistry, Chitosan has been used due to its antibacterial characteristics for the prevention of dental caries. Chitosan nanoparticles have superior activities, including antimicrobial effects, drug, gene and/or vaccine delivery systems, and antitumor effects. 10 Louise et al studied the effect of Chitosan nanoparticles (ChNPs) on the inhibition of Candida spp. biofilm on the denture base surface. Results showed the ChNPs inhibited C. Albicans biofilm and reduced Candida biofilm on resin.10

The results of our study were in accordance with Fabio et al who showed that combining photodynamic therapy (PDT) and chitosan reduced the endodontic infection caused by E. faecalis compared to Chitosan without photoactivation.11

Therefore, within the limitation of this study, it can be concluded that photoactivated Curcumin has shown the best antimicrobial results as compared to Curcumin, Chitosan nanoparticles (photoactivated and nonphotoactivated) and 3% Sodium hypochlorite. Further studies should concentrate on the evaluation of the antimicrobial activity of photoactivated Chitosan nanoparticles on different species of microorganisms and also evaluate the effect of Curcumin with photoactivation in invivo situations using animal models and on humans.

Source of Funding

None.

Conflict of Interest

None.