Document Type : Original Article
Authors
1 Associate Professor of Oral and Maxillofacial Medicine, Faculty of Dentistry, Babol University of Medical Science, Babol, Iran
2 Assistant Professor of Pediatric Dentistry, Faculty of Dentistry, Golestan University of Medical Science, Gorgan, Iran
Abstract
Keywords
Main Subjects
Introduction
Candidiasis is a very important fungal infection of oral cavity. The most important risk factors are endocrine alterations such as diabetes mellitus, pregnancy and renal failure, immune depression, poor oral hygiene, smoking, alcoholism and long term administration of drugs1-3. Oral candidiasis occurs because of increase in Candida species, chiefly Candida Albicans. Some pathogenicity factors in C. albicans cause better adherence to cell membrane than other species (4).
Management of oral candidiasis is typically drug based but the extensive use of azoles has resulted in resistance in Candida species and subsequent treatment failures. Problems associate to common antifungal drugs have led to new therapeutic methods to be thought (5).
Curcumin is the active substance of the herbal remedy and the spice turmeric (curcuma longa) 6. It is the element of yellow colour in spices turmeric and curry that is isolated from the rhizomes of the plant Curcuma longa so that this material is natural, healthy and safe (7). Spice turmeric has a lot of benefits in traditional medicine such as treatment of Jaundice, rheumatoid arthritis, liver disease, eye infections and dental pain (8, 9). Also the remedial effect of turmeric in new medicine has been approved such as anti-inflammatory, hypoglycemic, antioxidant, wound-healing, and antimicrobial activities (10, 11). The mechanism of its antifungal activity is not known but it has been suggested that its fungicidal mechanism may involve chitin accumulation on the cell wall outer layer12. However, curcumin has been suggested as an irresistible antiviral, antibacterial and antifungal agent, but it is still obscure that is curcumin as effective as common antifungal drugs especially against C. Albicans or not 13.
This study wants to evaluate the effect of curcumin against C. Albicans species in vitro. Also, compare the fungal effect of curcumin with the famous antifungal drug, Nystatin.
Methods and Materials
This is a prospective lab trial study with no ethical concerns. Pure standard specimen of C.albicans (PTCC5027, Merck, Germany) prepared in a way that is described below:
Three laboratory methods were used to evaluate antifungal effect that in all methods the plates that have been infected by bacteria excluded from the study and the process repeated for those plates.
A) Cell count technique, was used to evaluate the amount of C. Albicans after time, in different concentrations of Dimethyl sulfoxide (DMSO) :
The main solution was prepared in the form of curcumin inside DMSO solvent, with a concentration of 5gram in 100cc. Therefore, a solution consists of 10g curcumin in 200ɥlit DMSO was obtained. Then the solution diluted by DMSO solvent in a ratio of 1/2 in 5 microplates, as in the last plate, curcumin concentration in solvent got to 0.156g in 100cc. The content of solution in each microplate was 200μlit.
In the other side, Nystatin was prepared into the manufacturer program and 50μlit of that was mixed with 50μlit of liquid BHI culture. Then 100μlit of fungal suspension that was made in BHI culture, added to each microplate. To ensure that the solvent has no effect on antifungal activity of curcumin, the second and third testimonial prepared (table.1).
Previously mentioned, microplates were placed in 37˚c autoclave in a same condition for 24 and 48 hours to study the growth. This experiment was repeated 19 times.
B) Cup bioassay technique, in which inhibitory zones of curcumin in DMSO was evaluated in sabauraud culture plates:
Three cups with a diameter of 6 mm were created by the beak of sterile pipet in the sabauraud culture plates. The main solution was prepared in the form of curcumin inside DMSO solvent with a concentration of 5%, then the main solution diluted in a ratio of 1/2 in 2 experimental tubes (concentrations:5%, 2.5%, 1.25%), and poured from prepared concentrations in the plate as following bellow:
Plate1: 3 cups (30ɥlit of 5%curcumin, 30ɥlit DMSO, 30ɥlit Nystatin)
Plate2: 3 cups (30ɥlit of 2.5%curcumin, 30ɥlit DMSO, 30ɥlit Nystatin)
Plate3: 3 cups (30ɥlit of 1.25%curcumin, 30ɥlit DMSO, 30ɥlit Nystatin)
Plate4: 3cups (30ɥlit of 5%curcumin, 30ɥlit of 2.5%curcumin, 30ɥlit of 1.25%curcumin)
Plate5: 3 cups (30ɥlit of 5%curcumin, 30ɥlit of 2.5%curcumin, 30ɥlit Nystatin), for elimination of probable effect of DMSO on curcumin.
Plate6: 3 cups (10ɥlit of 5%curcumin, 10ɥlit of 2.5%curcumin, 10ɥlit Nystatin), for observation of probable changes with changing in using volumes.
Then the plates were placed in 37˚c autoclave for 48 hours to evaluate the results. This procedure was repeated 3times for each plate.
C) Disk diffusion, that inhibitory zones of dried disks which contained curcumin in DMSO was evaluated:
The main solution was prepared as foresaid and diluted in a ratio of 1/2 in 5 microplates (5%, 2.5%, 1.25%, 0.625%, 0.313%, 0.156%). 20μlit of each solution was poured on one of the disks by a sampler. In lower concentrations in the pilot examination was not seen any effect. 20μlit Nystatin was poured on each of disks that constituted Nystatin disks as well. The disks were placed in autoclaved to dry. In addition, they placed under UV ray for 24 hours because of infection control. Some disks of DMSO were prepared the same and autoclaved, too. Three disks were placed in each plate including one of curcumin concentrations, one Nystatin disk and one DMSO disk. The disks were placed on the culture and were autoclaved in 37˚c for 48 hours. This procedure performed for 18 plates (3 times for each concentration).
Statistical analysis:
Shapiro-wilk test was used to determine the parameter setting for the data. As the data was non-parametric, Wilcoxon and Kruskal-wallis tests where used to evaluating the results.
Table1. Component of experimental microplates
|
Testimonia l3 volume |
Testimonia l2 volume |
Testimonia l1 volume |
Tube volume number 1-6 |
Tube No. |
|
0.1 - 0.05 |
0.1 - -
|
0.1 - -
|
0.1 0.1 - |
suspension curcumin-DMSO solution Nystatine |
|
- 0.05 |
0.1 - |
- 0.1
|
- - |
DMSO BHI culture |
|
0.2 |
0.2 |
0.2 |
0.2 |
Final volume |
*Curcumin concentration in tube number1 was 0.05gr/cc and in tube number6 was 156×10-5 gr/cc
*All the volumes are in millilitre (cc) measuring unit.
Results
A) Evaluation of microplates showed that in testimonial1 microplate there was apparent growth of fungi but fungal growth was significantly lower in testimonial2 microplate. In other microplates, fungal growth was lower, especially in higher concentrations of curcumin (chart.1).
As the curcumin concentration increases, the fungal growth decreases (chart1). Also from testimonial2 (DMSO) to 5% curcumin there is a decreasing process, that is a sign of curcumin antifungal effect alone without the effect of DMSO.
Wilcoxon nonparametric test was used to compare fungal computation based on 24 and 48 hours groups. The difference was statistically significant, so that after 48 hours more antifungal effect was seen in comparison with 24 hours in each group.
Also By the results of Kruskal-wallis test that used to compare different groups of 24hours with each other, as well as 48 hours groups, the difference was significant. That means as curcumin concentration increases, antifungal activity increases too (table.2).
B) Evaluation of plates showed that around DMSO cup, there was an inhibition halo. There was a halo around curcumin cup too. In higher concentrations, it was significantly more than DMSO cup. The halo around nystatin cup was the biggest one (table3).
C) There was inhibition halo around the disks approximately similar to those around the cups. In this experiment, bigger halos in more concentrations were seen as well.
Chart.1 Inhibitory effect of different concentrations of curcumin on C. Albicans growth, 24 and 48 hours after receiving curcumin
Table 2. Average ± standard deviation of Candida Albicans cell count in different treatment groups (control and curcumin in various concentrations), 24 and 48 hours after beginning the treatment
|
0.156% curcumin |
0.313% curcumin |
0.625% curcumin |
1.25% curcumin |
2.5% curcumin |
5% curcumin |
DMSO
|
control |
Treatment groups Time Of effect |
||
|
26.58 2.69± |
34.21 4.89± |
26.21 5.23± |
17.53 15.69 ± |
20.21 16.59± |
27.74 18.17± |
30.63 16.73± |
1160.79 46.95 |
After 24 hours |
||
|
20.26 17.81± |
12.36 10.44± |
12.36 14.90± |
4.78 6.94± |
1.36 1.25± |
0.63 1.16± |
33.15 15.77± |
|
After 48 hours |
||
|
0.034 |
0.008 |
0.008 |
0.007 |
0.007 |
0.003 |
0.037 |
0.071 |
Comparison of P values |
Table3. Diameters of inhibitory halo of C. Albicans growth in different treatment groups (control and various curcumin concentrations), 48 hours after placing different concentrations of curcumin by the cup bioassay technique
|
Nystatin |
DMSO |
0.156% curcumin |
1.25% curcumin |
2.5% curcumin |
5% curcumin |
Cup |
|
20-22mm |
mm ~2 |
mm ~2 |
mm ~3 |
mm ~6 |
mm ~6 |
Diameter of Inhibitory halo |
Discussion
Curcumin is the effective substance of curcuma longa that has anti-fungal activity based on Shyh Ming Tsoa, CVB Martins, and M Sharma studies (14, 15, 16). And its antifungal activity against C. Albicans has been confirmed by Livia N. Dovigo study.
In 2000 Shyh Ming Tsoa showed that confection of curcumin with Amphotericin B or fluconazole provides more antifungal effect for systemic fungal infections 14. M sharma study confirmed the synergic effect of curcumin confecting Poliens or Azoles as well 16. In our study, as a secondary finding, due to antifungal activity of DMSO, confection of curcumin and DMSO, was increased the solution's antifungal activity.
In a study by Garia Gomes AS et al in 2012 concluded that curcumin have a great capability to inhibit Fluconazole resistance of the isolate of C. Albicans (17).
In another study in 2008 by CVB Martins et al, it was concluded that curcumin was much more efficient than Fluconazole for restraining C. Albicans adherence to the epithelium of buccal mucosa 15. In the current study, C. Albicans growth restrained by nystatin drug much more than curcumin. However, curcumin showed a significant effect too. This difference may be attributed to the different types of drug used in the studies. Moreover, they studied C. Albicans' adherence inhibition in-vivo, but in our study, growth inhibition of C. Albicans was evaluated, in vitro.
In the current study, first we tried cell count method, and curcumin diluted initially by distinct water, that redounded many huge crystallizations and prevented cell counting by microscope. To solve the problem we used another solvent named DMSO, as was used in Livia N. Dovigo et al, as well as Rana Pratap Singh et all studies (18,19). However, during the experiments it was revealed that DMSO has antifungal activity, itself. Whereas it have not been noted in other studies. To solve the problem, we decreased solvent concentration to 50% (in less concentrations curcumin sedimented). But the results didn't significantly change.
Next step, we decided to use Alcohol as solvent. The steps of experiment performed with methanol but it was revealed that Methanol has antifungal activity too. So we decided to use 50% DMSO that provided more elucidate solution.
Due to the noted results, we decided to use cup bioassay method to eliminate antifungal effect of solvent with another method.
In our study, inhibition halo observed for curcumin extract, and halo diameter was 6mm for 5% curcumin and it was bigger than DMSO halo.
These results are in contradiction to Ranna Pratap Singh study. In their study, the diameter of inhibition halo of curcumin was 18mm and for DMSO there was no halo (19).
This may be because in their study the curcumin concentration was 20mg/ml in DMSO solvent, but in our study maximum concentration was 0.05mg/ml and as noted before, the amount of antifungal activity is proportionate to concentration of curcumin.
In the other method, we used disks on the plates. In this experiment a little inhibition halo was created around DMSO disks, and the halo around curcumin disks was bigger than DMSO disks, especially in more concentrations. Whereas in Monika Sharma et al study, the use of curcumin1 alone or an antifungal drug (Nystatin or Amphotericin B) alone, did not create any halo after 48 hours. However, when curcumin was used simultaneously with each of two antifungal drugs, there was a 1-1.5mm halo because of synergic effect of drugs (16).
This may be because they used 23μg/ml concentration of curcumin and also 0.078ɥg/ml Amphotericin B, so that because of very low concentrations may each one alone was not effective. In another study, curcumin was shown to enhance the activity of azole and polyene antifungals (20).
In the current study due to more concentrations of curcumin, the halo created around curcumin disks, alone. Additionally, P-value for different concentrations of curcumin was significant, as it was in Livia N. study (18).
Conclusion
ccording to this study, we can conclude that curcumin has antifungal activity against C. Albicans , and this effect generally increases by increasing the dosage.
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