International Journal of

Environmental Research
and Public Health

Article
In Vitro Evaluation of the Inhibitory Activity of
Thymoquinone in Combatting Candida albicans in
Denture Stomatitis Prevention
Ahmad M. Al-Thobity 1, * , Khalifa S. Al-Khalifa 2 , Mohammed M. Gad 1 ,
Mohammed Al-Hariri 3 , Aiman A. Ali 4,† and Talal Alnassar 5,6
1 Department of Substitutive Dental Sciences, College of Dentistry, University of Dammam, Dammam 32214,
Saudi Arabia; mmjad@uod.edu.sa
2 Department of Preventive Dental Sciences, College of Dentistry, University of Dammam, Dammam 32214,
Saudi Arabia; kalkhalifa@uod.edu.sa
3 Department of Physiology, College of Medicine, University of Dammam, Dammam 32214, Saudi Arabia;
mtalhariri@uod.edu.sa
4 Department of Biomedical Dental Sciences, College of Dentistry, University of Dammam, Dammam 32214,
Saudi Arabia; Aiman.a.ali@gmail.com
5 Department of Prosthetic Dental Sciences, College of Dentistry, Kind Saud University, Riyadh 11692,
Saudi Arabia; dr_tmt@hotmail.com
6 Department of Oral Rehabilitation, Dental College of Georgia, Augusta University, Augusta 30912, GA, USA
* Correspondence: aalthobity@uod.edu.sa; Tel.: +966-530203530
† Current address: Oral Pathology and Oral Medicine, Faculty of Dentistry, University of Toronto, 124 Edward
St. Room 314, Toronto, ON M5G 1G6, Canada.

Academic Editor: Paul B. Tchounwou

Received: 11 June 2017; Accepted: 3 July 2017; Published: 8 July 2017

Abstract: Candida albicans adhesion and proliferation on denture bases may lead to denture stomatitis,
which is a common and recurrent problem in denture wearers. The goal of this study was to assess the
inhibitory effect of thymoquinone incorporated in the polymethyl methacrylate denture base material
against Candida albicans. Eighty acrylic resin specimens were fabricated and divided into eight groups
(n = 10) according to thymoquinone concentrations of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, and 5% of acrylic
powder. Two methods were used to evaluate the effect of thymoquinone on Candida albicans: the slide
count and the serial dilution test. A multivariate analysis of variance (MANOVA) and the post-hoc
Tukey’s Honestly Significant Difference (HSD) test were performed to compare the difference of
means between the observations taken at various intervals with baseline. The p value was statistically
significant at ≤0.05. According to the slide count and the serial dilution test, the mean number of
adhered Candida albicans in the control group was 5436.9 ± 266 and 4691.4 ± 176.8; however, this
number dramatically decreased to 0 ± 0 and 32.4 ± 1.7 in group 8 (concentration 5%). These results
suggest that the incorporation of thymoquinone into the acrylic resin denture base material might be
effective in preventing Candida albicans adhesion.

Keywords: Candida albicans; black seeds; denture base; denture stomatitis; thymoquinone

1. Introduction
Denture stomatitis (DS) is a highly prevalent disease in complete or partial denture wearers, which
mainly affects the palatal mucosa [1,2]. Clinically, it appears as localized red erythematous patches or
diffused patches beneath the denture [2–4]. Different factors may contribute to the development of
DS, such as poor denture and oral hygiene, low flow of saliva, oral mucous membrane trauma, and

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Int. J. Environ. Res. Public Health 2017, 14, 743 2 of 9

microbial infection (basically Candida infection) [5–9]. DS has been found in approximately 30–75% of
denture wearers and has a high rate of recurrence, even if treated with antifungal therapy [10–13].
The role of Candida albicans in DS has been investigated and strong evidence suggesting
Candida albicans as the main fungal source has been shown [14–16]. The ability of Candida albicans to
develop biofilms has been discerned as the key factor in the DS pathogenesis. However, the denture
base material is porous in nature, which allows the Candida albicans to colonize and adhere into the
denture surface. Furthermore, this biofilm formation and adhesion reduce the cleansing efficacy
against the biofilms and increase its resistance to antifungal therapy [17–20].
Different management procedures have been developed to inhibit fungal growth into the denture
base, such as denture cleansing modalities [21–23], the addition of antifungal medications into the
denture lining or tissue conditioning materials [24,25], and the incorporation of antimicrobials into the
denture base resin powder [26]. On one hand, the usage of denture cleansers may lead to deterioration
of the denture base and increase its surface roughness, which makes it more susceptible to the biofilms’
accumulation [27–30]. On the other hand, studies have reported that the application of antimicrobial
therapy into the denture materials could enhance the fungal resistance and reduce the medication
effectiveness [19,20,31].
Nevertheless, the prevention of Candida albicans adherence to the denture base has been considered
as an effective protocol in DS prevention [32–35]. Yodmongkol et al. [32] observed that when coating
the acrylic resin specimens with silane-SiO2 nanocomposite films the adhesion of Candida albicans to
specimens’ surfaces was reduced without affecting its physical properties.
Natural products have been investigated experimentally by mixing them with the denture base
material to evaluate their effectiveness in the inhibition of Candida albicans growth [36,37]. Recently,
Nawasrah et al. [36] reported that adding 1% of henna powder to the denture material resulted in a
significant reduction in the Candida albicans count.
Nigella sativa is an annually flowering medicinal plant native to South and Southwest Asia; its
seeds are commonly known as black seeds or black cumin [38]. N. sativa seed extract includes essential
oil, alkaloids, fixed oil, proteins, and saponins. Its extract has been explored in the medical field and
has been found to have antibacterial, anti-inflammatory, anti-oxidant, and antitumor properties [39–42].
Thymoquinone (TQ) is the major ingredient in N. sativa seed essential oil, which has been proved to
have broad medical benefits [43–45]. In addition, N. sativa seed extract has been tested recently in the
dental field for a potential therapeutic effect against dental caries [46], pulpal diseases [47], gingival
and periodontal diseases [48], and oral ulcerations [49].
The lowest therapeutic concentration of a medical agent that inhibits the development and
growth of any microorganism is known as the Minimum Inhibitory Concentration (MIC) [50].
Harzallah et al. [51] reported that 2.13 mg/mL (MIC) of TQ has a strong antibacterial action against
Streptococcus mitis and Streptococcus mutans as cariogenic strains. The therapeutic effect of TQ in the
MIC against Candida albicans for the prevention of DS has not been reported in the literature. The goal
of this study was to assess the MIC of TQ incorporated in the polymethyl methacrylate (PMMA)
denture base material against Candida albicans.

2. Materials and Methods

The sample size was calculated using the results of a previous study [36]. The sample size was
calculated to be 20 per group keeping a confidence interval of 95% and a power of at least 80%. A total
of 80 specimens of heat polymerized acrylic resin (Major base 20 resin; Prodotti Dentari SPA, Moncalieri,
Italy) using a negative metal mold with the dimensions of 10 × 10 × 3 mm was prepared and waxed.
Wax specimens were flasked in stone then wax burned-out to create mold space. Acrylic resin material
was prepared by the adding of TQ (thymoquinone ≥98%; Sigma-Aldrich, Taufkirchen, Germany)
in concentrations of 0.5, 1, 1.5, 2, 2.5, 3 and 5 wt % of acrylic powder and properly mixed to attain
a homogenous color. To fabricate acrylic resin specimens, polymer and monomer were measured
and mixed according to manufacturer instructions. Mixing was done in a porcelain jar, which was
Int. J. Environ. Res. Public Health 2017, 14, 743 3 of 9

kneaded by hand upon achieving a dough-like consistency to increase its homogeneity and integrity.
At the dough stage, the mixture was packed and then processed in a heat curing unit at 74 ◦ C for
2 h and 100 ◦ C for 1 h. After the curing of all the specimens, the flasks were brought down to room
temperature and deflasked. The excess resins of the deflasked specimens were removed, and then
the specimens were finished and then stored at 37 ◦ C for 24 h in sterile distilled water to remove any
residual monomers. According to the different TQ concentrations, the specimens were divided into
8 groups (n = 10) represented in Table 1.

Table 1. Tested groups and description according to thymoquinone (TQ) concentrations.

Groups Description
0% (Control) heat polymerized specimens
0.5% heat polymerized specimens incorporated with 0.5% TQ
1% heat polymerized specimens incorporated with 1% TQ
1.5% heat polymerized specimens incorporated with 1.5% TQ
2% heat polymerized specimens incorporated with 2% TQ
2.5% heat polymerized specimens incorporated with 2.5% TQ
3% heat polymerized specimens incorporated with 3% TQ
5% heat polymerized specimens incorporated with 5% TQ

2.1. Microbiology Test

2.1.1. Exposing Acrylic Specimens to Candida albicans

Before microbiologic valuation, the acrylic specimens were sterilized in an autoclave (Ritter M11
UltraClave; Midmark International, Versailles, OH, USA) for 15 min under 15 bar at 121 ◦ C. All acrylic
plates’ specimens were immersed in artificial saliva (A.S. Orthana, Biofac A/S, Kastrup, Denmark)
containing 2,000,000 cells of Candida albicans (ATCC 10231) for two weeks at a temperature of 37 ◦ C
(Table 2). The acrylic plates were washed three times with phosphate-buffered saline (PBS) to remove
non-adherent cells and then placed in sterile tubes with 1 mL of Sabouraud’s dextrose broth (SDB
Acumedica Co., Manufacturers, Inc., Lansing, MI, USA) for 2 days. The plates were then vibrated
using a vortex mixer for 10 min followed by centrifuging the tubes at 4500 rpm for 5 min to get the
concentrated bullet of Candida albicans. At this stage, two methods were used to count the number of
alive Candida albicans for each sample:

Table 2. Composition of artificial saliva.

Artificial Saliva Composition

Mucin, methyl-4-hydroxybenzoate, benzalconium chloride,
A.S. Orthana, Biofac A/S, Kastrup, Denmark ethylenediaminetetraacetic acid (EDTA), H2 O2 , xylitol,
peppermint oil, spearmint oil and mineral salts

2.1.2. Evaluation
After centrifuging, the acrylic resin plates were removed from their tubes, and the concentrated
pellet was collected from the tube. Two methods of evaluation were used to calculate the amount of
Candida albicans adhered to each acrylic resin specimen as follows:

Slide Count
Samples were placed on a special slide count (Neubauer Slide Counter; Chambers-Marienfeld,
Lauda-Konigshofen, Germany) after adding 2.5 µL of Trypan Blue 0.4% solution in phosphate
(MP-Biomedicals, Santa Ana, CA, USA) to 7.5 µL of each sample to be evaluated under light microscope.
Trypan Blue stain can differentiate between dead and alive Candida albicans cells; dead Candida albicans
Int. J. Environ. Res. Public Health 2017, 14, 743 4 of 9

usually appear blue while alive Candida albicans appear transparent with a blue peripheral line.
To count the number of Candida albicans, a light microscope with a magnification of 10× was used.
Candida albicans were counted in two squares out of the four main squares of the slide count and
multiplied
Int. J. Environ.by
Res.2Public
to find out2017,
Health the 14, 743number of Candida albicans in each slide.
total 4 of 9

Serial
Serial Dilution
Dilution Test
Test
A
A 10 µ L of
10 µL of each
each bullet
bullet was
was taken,
taken, and
and then
then it
it was
was diluted
diluted serially
serially and
and spread
spread on
on aa petri
petri dish
dish
containing
containing Sabouraud dextrose agar (SDA) (Acumedica Co., Manufacturers, Inc.) and incubated for
Sabouraud dextrose agar (SDA) (Acumedica Co., Manufacturers, Inc.) and incubated for
48 ◦
48 hhat
at37
37 C.°C.AAmarker
markerpen counter
pen counter(Colony
(ColonyCounter;
Counter;Bel-Art Scienceware,
Bel-Art Wayne,
Scienceware, NJ, USA)
Wayne, was used
NJ, USA) was
to count
used the number
to count of Candida
the number albicans
of Candida colonies
albicans in eachinquadrant
colonies wherewhere
each quadrant acceptable growthgrowth
acceptable was noted
was
and
noted and the final number was corrected for the dilution factor. If the number of colonies or
the final number was corrected for the dilution factor. If the number of colonies was 500 wasmore,
500
it
orwas
more,considered as an overgrowth
it was considered (Figure 1)(Figure
as an overgrowth [37]. 1) [37].

Figure 1. Cultures of Candida albicans colonies based on different concentrations of thymoquinone: (A)
Figure 1. Cultures of Candida albicans colonies based on different concentrations of thymoquinone:
Control group (0%); (B) 0.5%; (C) 1%; (D) 1.5%; (E) 2%; (F) 2.5%; (G) 3%; (H) 5%.
(A) Control group (0%); (B) 0.5%; (C) 1%; (D) 1.5%; (E) 2%; (F) 2.5%; (G) 3%; (H) 5%.

2.2. Statistical Analysis

2.2. Statistical Analysis
SPSS-20.0 (IBM Inc., Armonk, NY, USA) was used for the statistical data analysis. The results of
SPSS-20.0 (IBM Inc., Armonk, NY, USA) was used for the statistical data analysis. The results of
the Candida albicans count from the two different methods were formulated into arithmetic means
the Candida albicans count from the two different methods were formulated into arithmetic means and
and standard deviations. The multivariate analysis of variance (MANOVA) was applied to compare
standard deviations. The multivariate analysis of variance (MANOVA) was applied to compare the
the mean effect on each interval with the baseline. The post-hoc Tukey’s Honestly Significant
mean effect on each interval with the baseline. The post-hoc Tukey’s Honestly Significant Difference
Difference (HSD) test was performed to compare the difference of means between the observations
(HSD) test was performed to compare the difference of means between the observations taken at
taken at various intervals with baseline. If the p value was ≤0.05, then it was considered statistically
various intervals with baseline. If the p value was ≤0.05, then it was considered statistically significant.
significant.
3. Results
3. Results
The antifungal effect of TQ on Candida albicans was examined using different concentrations.
The antifungal
The mean effectdeviation
and standard of TQ on Candida
values albicans was examined
were obtained for eachusing different
group. It canconcentrations. The
be observed, that
mean
the useand standard
of TQ deviation values
at a concentration werefrom
starting obtained
0.5% for
waseach group. Itwith
associated can abesignificant
observed,reduction
that the use
of
of TQ at a concentration starting from 0.5% was associated with a significant reduction
Candida albicans in the slide count (Table 3). In addition, the inhibitory effect of TQ on Candida of Candida
albicans
albicans insignificantly
increased the slide count (Table
with the 3). In addition,
concentration of TQ. the inhibitoryateffect
Interestingly, of TQ on Candida
the concentration 3% andalbicans
above,
increased significantly with the concentration of TQ. Interestingly,
there were no signs of any fungal growth using the slide count. at the concentration 3% and above,
thereTowere no signs
ensure of any fungal
the antifungal growth
effect of TQ,using the slide
colonies count.albicans were counted after applying
of Candida
the same doses of TQ using cell culture counts (Figure 1). Tablealbicans
To ensure the antifungal effect of TQ, colonies of Candida 3 shows were counted after
the significant applying
effect of TQ
the same doses of TQ using cell culture counts (Figure 1). Table 3 shows the significant effect of TQ
has on live Candida albicans using concentrations of 0.5% and higher. There were no differences in the
antifungal effect of TQ in either methods, it can be seen that the significant effect of TQ, which
indicates the significant inhibitory effect of TQ on Candida albicans. However, the significant effect of
TQ at a concentration of 2.5% and higher on Candida albicans was greater compared to the other
Int. J. Environ. Res. Public Health 2017, 14, 743 5 of 9

has on live Candida albicans using concentrations of 0.5% and higher. There were no differences in the
antifungal effect of TQ in either methods, it can be seen that the significant effect of TQ, which indicates
the significant inhibitory effect of TQ on Candida albicans. However, the significant effect of TQ at a
concentration of 2.5% and higher on Candida albicans was greater compared to the other concentrations.

Table 3. Effect of different concentration of TQ on Candida albicans count.

Slide Count Serial Dilution Test

TQ Concentration
Mean ± SD Mean ± SD
0% Control 5436.9 ± 266 4691.4 ± 176.8
0.5% 3776.10 ± 98.8 3334.7 ± 121.2
1% 3037.4 *** ± 39.2 2619.4 *** ± 50.1
1.5% 980.2 ** ± 10.8 894.6 ** ± 32.3
2% 466 ** ± 6.5 310.3 ** ± 8.2
2.5% 166.5 * ± 6 91.9 * ± 4.5
3% 0*±0 53.9 * ± 2.0
5% 0*±0 32.4 * ± 1.7
a Significantly different from control group; *** Significantly different at 0.05; ** Significantly different at 1;

* Significantly different at 1.5; using one-way analysis of variance at p = 0.05. SD: Standard Deviation.

4. Discussion
The goal of the present study was to assess the inhibitory effect of TQ (the active ingredient of
N. sativa) as natural and safe compound on Candida albicans adherence to PMMA acrylic resins as an
alternative method for the prevention of DS, which frequently occurs in patients who wear complete
dentures [52]. Using the slide method and the serial dilution test, the results showed that the TQ at the
MIC significantly reduced the number of Candida albicans. Furthermore, these results found that the
adding of 0.5% of TQ to the PMMA led to a significant reduction of Candida albicans. By increasing
the percentage of TQ from 0.5% to 5%, the number of Candida albicans dramatically decreased to zero
using the slide count evaluation method.
Several studies have investigated the medicinal effect of the N. sativa extract in different dental
applications [46–49]. Omar et al. [47] evaluated N. sativa oil as pulp capping medicaments in pediatric
dentistry using the animal model. They found that there was less infiltration of the inflammatory
cells and fewer degenerative changes after the application of N. sativa oil, when compared to the
formocresol medicament. They concluded that the N. sativa had an anti-inflammatory effect and could
be used as a pulp capping agent. Another study evaluated the effect of the biodegradable periodontal
chip, including the use of TQ in the management of patients with chronic periodontitis. They found
a significant reduction in the periodontal pockets, bleeding on probing and the plaque index, and a
significant increase in the clinical attachment in subjects treated with TQ [48].
DS is a disease that is most commonly associated with the use of acrylic dentures. Many factors
may contribute to the development of the denture stomatitis etiology; however, all of these factors
are related to the ability of Candida albicans to adhere to and colonize on the dentures and oral
mucosal surfaces [7]. Many studies have reported that mechanical and chemical cleansing for the
removable prostheses are not adequate enough to eliminate contaminating microorganisms [27–30].
The increased antimicrobial resistance emphasizes the need for a study and evaluation of a new
antifungal agent [17–20,53]. Different mechanisms have been suggested for the development of
resistance to treatment regimes in Candida albicans [19,20,31,54]. A number of factors, such as nutritional
factors, radiotherapy, surgical procedures, poor oral hygiene, and others, have been associated with
the increasing frequency of Candida albicans intra-orally [55].
Several studies have addressed ways to decrease the formation and the development of adherent
biofilms through modifying the denture base, modifying the denture surface, or through chemical
modification [21–25]. Modifying the denture base materials by incorporating TQ was investigated
Int. J. Environ. Res. Public Health 2017, 14, 743 6 of 9

in this study in an attempt to control and prevent the adhesion of Candida albicans on the surface of
acrylic resin dentures.
The Nigella sativa extract (6.6 mL/kg daily for the 3 days) has shown a marked ability to inhibit
the growth of Candida albicans in infected mice [56]. Few studies have been conducted on the effect of
using TQ on Candida albicans. One study has assessed the in vitro inhibitory activity of the ethanol
extracts of Nigella sativa, along with those of five other plants, against the oral candidal isolates
collected from 175 patients. It has been recorded that the smallest inhibition zone was noticed with
a 100 µg/mL concentration of Nigella sativa [57]. Another study compared in vitro the antifungal
activity of nanoparticulate TQ versus microstructured TQ, ketoconazole and amphotericin B against
Candida albicans. The study found nanosized TQ to be 2 to 4 times more active against Candida yeasts
and Candida biofilm [58].
The present work was conducted to evaluate the effect of TQ, as natural product, against
Candida albicans to prevent or treat DS. Based on our results, TQ exhibited antifungal effect at a 0.5%
concentration in the PMMA denture base material. These results also show that TQ can significantly
inhibit the growth of Candida albicans in agreement with a previous study that found TQ exhibited
a potent growth-inhibitory effect against Candida albicans [55]. The results also suggest that the
concentrations of TQ (2.5%, 3%, and 5%) are effective for inhibiting Candida albicans.
The ability of TQ to inhibit the Candida albicans as observed by the present study will promote
further investigations in determining the usefulness of TQ in combatting Candida albicans. Additionally,
this study may promote the use of TQ as an effective alternative method to prevent and/or treat patients
with this pathogen. The limitations of this study were that the oral environment consisted of several
microorganisms, not only Candida albicans, and that no aging procedure was performed. In the future
studies, under better-simulated conditions, varied microorganisms, including biofilm formations,
should be evaluated. However, investigating the biocompatibility of PMMA/TQ composite, comparing
the effect of TQ with different antifungal effect on Candida adhesion and evaluating the effect of the
TQ addition on the physical properties of the acrylic resin denture base materials are necessary.

5. Conclusions
Within the limitations of this study, it could be concluded that the incorporation of TQ as a natural
compound into the acrylic resin denture base material could be effective in preventing Candida albicans
adhesion and proliferation on the denture surface. Further investigations on the physical properties of
the PMMA/TQ composite are required.

Acknowledgments: Authors would like to thank Kumar Muralidharan and Badar T. Alsaqer for their assistant in
sample preparation and conducting the lab testing procedures. Also, we would like to thank Intisar Siddiqui for
his assistant in the biostatistics analysis.
Author Contributions: Research hypothesis and experiment design: Ahmad M. Al-Thobity, Khalifa S. AlKhalifa.
Performing the experiment: Mohammed M. Gad, Mohammed Al-Hariri. Data analysis: Aiman A. Ali,
Talal Alnassar. Writing the paper: Ahmad M. Al-Thobity, Khalifa S. AlKhalifa, Mohammed M. Gad, Mohammed
Al-Hariri, Aiman A. Ali, Talal Alnassar.
Conflicts of Interest: The authors declare no conflict of interest.

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