Photodiagnosis and Photodynamic Therapy (2013) 10, 313—319

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/pdpdt

Photodynamic potential of curcumin and

blue LED against Streptococcus mutans in a
planktonic culture
Marco Aurelio Paschoal DDS, MS a,∗, Caroline C. Tonon b,
Denise M.P. Spolidório c, Vanderley S. Bagnato d,
Juçaíra S.M. Giusti d, Lourdes Santos-Pinto a

a
Department of Pediatric Dentistry, Araraquara Dental School, UNESP — Univ Estadual Paulista,
Araraquara, SP 14801-903, Brazil
b
Araraquara Dental School, UNESP — Univ Estadual Paulista, Araraquara, SP 14801-903, Brazil
c
Department of Pathology and Physiology, Araraquara Dental School, UNESP — Univ Estadual Paulista,
Araraquara, São Paulo 14801-903, Brazil
d
São Carlos Institute of Physics, University of São Paulo, USP, São Carlos, São Paulo, Brazil
Available online 6 April 2013

KEYWORDS Summary
S. mutans; Background: The photodynamic therapy (PDT) involves the use of light of specific wavelength to
Photochemotherapy activate a nontoxic photosensitizing agent or dye in the presence of oxygen for eradication of
target cells. In dentistry, this therapy is used to suppress the growth of microorganisms involved
directly with dental decay and periodontitis process. There are evidences that curcumin dye is
able to control microbial activity when illuminated with specific wavelength. The purpose of
this study was to evaluate the in vitro efficacy of PDT using curcumin dye (Cur-C) in combination
with a blue LED (L) device on a planktonic model of Streptococcus mutans (S. mutans).
Methods: Suspensions (0.5 mL) containing S. mutans at 1 × 107 CFU mL−1 were prepared and
divided into 4 groups: Group C − L− (control: no treatment and 1 experimental condition),
Group C + L− (curcumin at 3 different concentrations: 2000; 4000 and 8000 ␮M and 3 exper-
imental conditions), Group C − L+ (LED at 3 different dosages: 24, 48 and 72 J cm−2 and 3
experimental conditions), and Group C + L+ (PDT group: curcumin at respective concentrations
combined to LED dosages and 9 experimental conditions). Samples of each experimental con-
dition were cultured in Petri dishes of BHI agar. Incubation in micro-aerophilia at 37 ◦ C for 48 h
was performed for subsequent visual counting of CFU/mL. Data were transformed into log10
and analyzed by two-way ANOVA and Tukey’s test at p < 0.05.

∗
Corresponding author at: Araraquara Dental School, Rua Humaitá, 1680 Araraquara, São Paulo 14801-903, Brazil. Tel.: +55 16 33016330.
E-mail addresses: marcobpaschoal@hotmail.com (M.A. Paschoal), carolinectonon@hotmail.com (C.C. Tonon), dmps@foar.unesp.br
(D.M.P. Spolidório), vander@ifsc.usp.br (V.S. Bagnato).

1572-1000/$ — see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.pdpdt.2013.02.002
314
Results: Group C + L+, in specific experimental conditions, demonstrated a log bacterial reduction
70% higher than Group C − L−. Both groups C − L+ and C + L− presented a slight decrease in log
bacterial counting.
Conclusion: This in vitro method was able to reduce the number of S. mutans in a planktonic
suspension.
© 2013 Elsevier B.V. All rights reserved.
Introduction
The control of dental caries activity is still one of the
major challenges to dental professionals [1]. The relation-
ship between bacterial growth present in dental plaque and
the progression of tooth decay is well established [2—4]. The
dental calcified tissue is affected when biofilm disorganiza-
tion becomes inefficient and if untreated, it progresses to
destruction of the organic portion [5—7].
An option for decreasing the level of microorganisms
involved in the etiology of dental caries is the association of
chemical agents to mechanical debridement [8,9]. An ideal
chemical solution has to be safe to oral cavity as well as
effective in eliminating the pathogens without causing side
effects to the host organism [10,11]. In this field, chlorhexi-
dine solutions are the main choice for this approach [12,13].
Many studies have highlighted chlorhexidine as an anti-
septic with known outcomes, such as decreasing the number
of salivary bacteria in oral cavity (due to its interactions with
external cell components, thus causing a high rate of intra-
cellular damage) and releasing its components over time
[14,15]. On the other hand, alteration in taste, staining
of both teeth and restorations, and burning sensation are
the main side effects related to the use of this substance
[16,17].
In this context, alternative antimicrobial products that
cause bacterial reduction with no adverse side effects are
needed. To overcome this problem, photodynamic ther-
apy (PDT) has been appointed as a promising method of
treatment that offers the possibility of efficient intra-oral
disinfection [18—22]. PDT involves the use of dyes or pho-
tosensitizing compound (PS) associated with proper source
of lights [23,24]. This therapy consists of PS activation
with appropriate wavelength, consequently resulting in the
production of reactive oxygen species (ROS), such as free
radicals and singlet oxygen, which exert a bactericidal
effect that are not toxic to host cells. Light and PS are non-
toxic by themselves; hence only cells containing dyes and
receiving light are affected by this intervention [25].
PDT has been extensively studied for the control growth
of several microorganisms in both in vitro and clinical assays
[18,26]. Rapidness and non-invasiveness are the main char-
acteristics of this approach, besides offering good result at
low costs. Thus, PDT emerges as a suitable process to reduce
bacterial contamination in oral cavity, increasing the thera-
peutic success [27].
PS should be biologically stable, photochemically active,
and minimally toxic to tissues of the organism [10,11].
The most used PS in PDT includes hematoporphyrin deriva-
tives (Photogem® and Photosan® ) [28,29] phenotiazinium
(toluidine blue O and methylene blue) [22,28,30,31] and
phthalocyanines [32,33]. These PS have effective photo-
killing effects on bacteria and fungi [34,35]. However, the
M.A. Paschoal et al.
process of purification presents high costs and possi-
bility of tooth staining makes its clinical applicability a hard
task [28,32].
Curcumin is the major constituent of turmeric powder,
extracted from rhizomes of the plant Curcuma longa and
used as a traditional Indian medicine [36,37]. Turmeric con-
tains 2—5% (w/w) of curcumin and exhibits a wide range of
pharmacological effects, including anti-inflammatory, anti-
carcinogenic, and anti-infection activities [36]. As a potent
anti-oxidant, curcumin has also shown anti-proliferative and
anti-carcinogenic properties in a wide variety of cell lines
and animals [38,39]. Some studies have also proposed that
these effects may possibly be enhanced by light applica-
tion [40,41]. Curcumin absorbs light in the blue wavelength
range (300—500 nm) of the visible spectrum. Blue light has a
lower penetration depth into the tissue compared to red
light due to scattering and absorption by biomolecules;
therefore, the use of curcumin in PDT is restricted to
topical use for superficial wounds (e.g. skin and mouth)
[42].
The applicability of curcumin focusing on microbial
decontamination is a recent approach. Hegge et al. [42]
showed the phototoxic effects against Eschericia coli and
Enterococcus faecalis when using curcumin associated
with methylated ␤-cyclodextrin at 0.01%. This combi-
nation resulted in a high photo-toxicity in the studied
species when irradiated with proper blue light intensity
(29 J cm−2 ).
Fungal species like Candida albicans has shown high
photo-killing rate to some curcumin preparations in both
planktonic and biofilm form. In addition, parameters related
to pre-irradiation time (PIT) and the best intensities of blue
light were investigated [43].
Although photo-toxicity on carcinogenic microorganisms
have been proved, further studies on the use of curcumin
and blue light for eliminating Streptococcus mutans should
be encouraged in order to define the optimal parameters
of these combination against one of the most important
bacteria involved in the dental caries process. Hence, the
purpose of this study was to evaluate in vitro the photody-
namic effect on the viability of S. mutans in a planktonic
suspension by mediating curcumin in association with a blue
LED device.
Materials and methods
Photosensitizer
A solution of curcumin [1,7-bis-(4-hydroxy-3-
methoxyphenyl)-1,6-heptadiene-3,5-dione] (PDT Pharma,
Cravinhos — SP, Brazil) was prepared and dissolved in sterile
Photodynamic potential of curcumin and blue LED
Figure 1 Absorption spectrum of curcumin dye.
distilled water to obtain a stock solution, which was then
stored in the dark until use.
Curcumin was excited with a LED in the blue region of
the spectrum, where PS absorbs efficiently the light deliv-
ered. The characteristics of ultraviolet—visible absorption
spectrum of the dye are illustrated in Fig. 1.
Light source
A light emitting-diode (LED) in the blue wavelength was used
to excite the curcumin [43]. The device (Prototype, Project
Finep/Gnatus LED Edixeon, Edison Opto Corporation, New
Taipei City, Taiwan) provided a light emission ranging from
400 to 500 nm with predominant central wavelength of
450 nm ± 30 at intensity (power density) of 240.1 mW cm−2 .
The irradiances (energy fluency) tested in the study were
24, 48 and 72 J cm−2 and the irradiation was performed
in a non-contact mode with a focused beam at 5 mm of
working distance (distance between the light source and
cell line surface). To achieve such energy fluencies, light
irradiation time was calculated according to the following
equation:
Fluency (J cm−2 ) = power density (W cm−2 )
× irradiation time (s)
Preparation of the bacterial inoculum
A standard suspension of S. mutans (strain ATCC 25175) con-
taining 1 × 107 viable cells mL−1 was prepared as follows:
brain heart infusion broth (BHI, Becton, Dickinson Com-
pany, Sparks, MD USA) was inoculated with S. mutans and
incubated for 18 h at 37◦ C under micro-aerophilic condition
(10% of CO2 ). The bacterial culture was then centrifuged at
3000 rpm for 5 min and the supernatant was discarded. Then,
the cell pellet was re-suspended in 5 mL of sterile solution
of 0.9% sodium chloride (NaCl).
The number of viable cells in the suspension was
determined with a spectrophotometer (Eppendorf AG,
315
Hamburg, Germany) operating at a wavelength of 600 nm
and optical density of 0.15—0.20 (corresponding to
1 × 107 cells mL−1 ).
Photodynamic treatment
Aliquots of 500 ␮L of S. mutans standard suspension were
individually transferred to separate wells of a 24-well cul-
ture cell plate. On the day of the experiment, the stock
solution was diluted in sterile saline to concentrations of
4000 ␮M, 8000 ␮M and 16,000 ␮M. An equal volume (500 ␮L)
of each prepared concentration was added to the wells
to give final tested concentrations of 2000 ␮M, 4000 ␮M
and 8000 ␮M. After dark incubation for 60 s (pre-irradiation
time), the wells were illuminated by a blue LED device for
100, 200 and 300 s (corresponding to 24, 48 and 72 J cm−2 ,
respectively) (treated with curcumin and LED: Group C + L+).
To determine whether curcumin alone induced any toxic
effects on bacterial viability, additional wells containing the
bacteria suspension were exposed to curcumin under iden-
tical conditions to those described above, but not exposed
to LED (treated only with curcumin: Group C + L−). Exposing
cells to irradiation determined the isolated effect of LED
with no previous exposure to curcumin (treated only with
LED: Group C − L+). The control wells consisted of S. mutans
suspension exposed to neither curcumin nor LED (no treat-
ment: C − L−; control group). Ten fold-serial dilutions of the
contents from each well were obtained and aliquots were
transferred in triplicate to Petri dishes containing BHI agar.
After incubation (37◦ C for 48 h), the total number of colony
forming units (CFU) was determined by using a digital colony
counter (CP 600 Plus, Phoenix Ind. Com. Equipamentos Cien-
tificos Ltda, Araraquara, SP, Brazil). The number of CFUs per
millimeter (CFU mL−1 ) was obtained and transformed into
logarithm (log10 ).
Statistical analysis
In order to verify the differences between groups, the vari-
able reduction in viable bacterial colony counts promoted
by each group was analyzed by two-way ANOVA and Tukey’s
test. The statistical significance cutoff level was set as
p < 0.05. The BioEstat 5.0 software for Windows (Sociedade
Civil Mamiraua, Manuas, AM, Brazil) was used for data anal-
ysis.
Results
The results of reduction in viable bacterial colony counts
(CFU log10 mL−1 ) are summarized in Table 1. All the asso-
ciations of curcumin and LED were able to decrease the
number of viable bacteria compared to control group. A
significant reduction of S. mutans viability was achieved
with the association of 4000 ␮M curcumin and exposure
to 48 and 72 J cm−2 , promoting reductions of 60.66% and
71.07%, respectively. In spite of these results, the increase
of curcumin concentration (8000 ␮M) showed that bacterial
photo-killing rate did not demonstrate a cumulative effect,
indicating a non-direct dose dependent relationship. Groups
C − L+ and C + L− showed similar results with slight bacterial
reduction.
316 M.A. Paschoal et al.

Table 1 Mean values of log10 (CFU mL−1 ) for all experimental conditions and log10 reduction (%) in the number of viable cells
for experimental groups compared to control group. C − L+: blue LED alone; C + L−: Cur alone; C + L+: Cur and blue LED; C − L−:
control group.

Groups Experimental situations CFU log10 mL−1 p value Percentage of log

reduction (%)

C − L+ 24 J cm−2 7.23 <0.05 3.44a*

48 J cm−2 7.28 <0.05 2.73a*
72 J cm−2 7.29 >0.05 2.56b*
C + L− 2000 ␮M 7.20 >0.05 3.79b*
4000 ␮M 7.24 >0.05 3.31b*
8000 ␮M 7.05 <0.05 5.80a*
C + L+ 2000 ␮M 24 J cm−2 5.17 <0.05 30.91a*
48 J cm−2 4.62 <0.05 38.28a*
72 J cm−2 3.29 <0.05 55.98a*
4000 ␮M 24 Jcm−2 4.95 <0.05 33.83a*
48 J cm−2 2.94 <0.05 60.66a*
72 J cm−2 2.16 <0.05 71.07a*
8000 ␮M 24 J cm−2 5.98 <0.05 20.09a*
48 J cm−2 5.81 <0.05 22.41a*
72 J cm−2 5.50 <0.05 26.47a*
C − L− 7.48 0.0b*

* Control group is represented by b character. The presence of a different character (a) at any experimental condition indicates

statistical difference (p < 0.05) compared to control group.

Discussion investigations related to this dye. To verify the susceptibility

The results showed the effective potential of curcumin in
PDT procedure when irradiated with proper source of light.
Additionally, S. mutans standard strain is a major pathogen
involved in the dental caries process, presenting a high sen-
sitivity to the studied parameters. The synergism between
curcumin and light (C + L+) was confirmed with a log reduc-
tion varying from 1.5 log10 to 5.3 log10 (p < 0.05) whereas the
reduction observed in Groups C − L+ and C + L− were less
than 1 log10 .
The present investigation used a blue LED with average
wavelength of 450 nm, since the studied dye (curcumin) has
an absorption peak ranging between 300 and 500 nm. The
curcumin concentrations tested (2000, 4000 and 8000 ␮M)
are too low and do not cause damage to oral tissues [35]. The
concentration of 4000 ␮M associated with 48 and 72 J cm−2
of blue LED resulted in a bacterial reduction over than 60%.
This result is in agreement with a previous in vivo study [44].
On the other hand, when the curcumin concentration was
increased there was not a higher photokilling rate. This fact
could be probably due to optical quenching by excess dye
in solution preventing the light from reaching the bacteria.
The role of curcumin in the field of health presents beneficial
outcomes in the treatment of tumors, infections and healing
process. Recently, microbial reduction has been investigated
as another important characteristic of this PS when irradi-
ated with a light in the blue wavelength [43,45]. However,
no in vitro study using S. mutans has been reported.
Since there is no guidance in demonstrating the opti-
mal concentration of curcumin combined with a dosage
of irradiation, it is mandatory proceed with preliminary
of the studied microorganism to the novel treatment, grown
in suspension is the first step on trying performing a proto-
col. A great number of studies have shown that oral bacteria
are susceptible to the action of PDT when suspended in
planktonic cultures [8,46]. It is known that dental caries
result from biofilms on teeth. However, the effectiveness of
PDT tends to be minimized by the presence of this biofilm,
since the mechanisms responsible for the reduced suscepti-
bility are the existence of biofilm bacteria in slow-growing
or starved state, and distinct and protected phenotypes
expressed by biofilm species [47]. Thus, perform previous
tests in planktonic model aiming biofilm application is nec-
essary to provide some idea in how to proceed with the PDT
components.
Bevilacqua et al. [48] were the first investigators who
demonstrated a high sensitivity of planktonic S. mutans to
toluidine blue O (TBO) associated with a red LED. In their
study, the authors achieved 100% of bacterial reduction.
Similar results were obtained in the studies by Williams et al.
[49] and Giusti et al. [28], who found a significant bacterial
reduction in a group submitted to both PS and light. These
results highlighted the need for dye-light conjugation to
ensure the effectiveness of PDT. However, in all cited stud-
ies, a slight bacterial reduction was found when either PS or
light was tested alone. Our results showed that the curcumin
concentration of 8000 ␮M achieved a log bacterial reduction
(%) of 5.80 in Group C + L−. It can be hypothesized that the
more concentrated the PS, the more it diffuses through the
membrane, causing a greater degree of bacterial reduction
[50] when compared to another curcumin concentrations
in the present investigation. According to this statement,
Photodynamic potential of curcumin and blue LED
Wilson and Mia [51] demonstrated a microbial reduction of
20% when PS was tested alone. A small reduction in bacterial
counts was also observed in the study by Myiabe et al. [52],
who used a group submitted to methylene blue (MB) at a con-
centration of 3 ␮M for photosensitization of Staphylococcus
strains. Furthermore, our study showed that fluencies of 24
and 48 J cm−2 achieved a log bacterial reduction (%) of 3.44
and 2.73, respectively. Gois et al. [53] also reported a reduc-
tion in the number of Staphylococcus aureus cells of 0.97
and 1.05 log10 when irradiated with red LED (20 J cm−2 ) and
diode laser (20 and 40 J cm−2 ), respectively, in the absence
of PS. Other studies on fungal species also confirm these out-
comes [11,45,54]. From a clinical point of view, although
it can cause a slight reduction in the amount of bacteria
present in dental plaque, there would remain many viable
microorganisms that could form new biofilms.
An ideal PS should present no toxicity to host cells and
possess biological stability and can be activated photo-
chemically. Phenotiazinium dyes (e.g. toluidine blue O and
methylene blue) are common substances used in PDT and
have been shown to be effective in reduction of bacteria
[8,28,46,50] and fungi [21,55]. However, when used for an
in vivo study, the color of teeth and restorations can be
damaged [28]. Curcumin was used by Araujo et al. [44],
who aimed to decrease the number of S. mutans present
in the saliva of 13 volunteers when irradiated with a blue
LED device. The sample demonstrated no burning sensa-
tion, oral soreness, or ulcers. Overall, decontamination of
oral cavity was achieved by a simple approach and can be
used in the dental practice. Moreover, blue LED is a device
easily found in the dental office, also promoting effec-
tive activation of curcumin [19]. Affordable price, simple
manipulation and great effectiveness of curcumin associ-
ated with light source can make PDT procedure a clinical
reality [43,44].
Curcumin displays a variety of biological properties such
as proliferative activity against cancer cells and antioxi-
dant activity. Moreover, it exhibits significant antimicrobial
properties in vitro against a number of gram-negative and
gram-positive bacteria [56,57]. The penetration of PS into
the cell is not a passive process. From the point of view
of bacteria and PS interaction, the effectiveness of PDT
is mostly related to three main aspects: PS capability of
interacting with the bacterial membrane, PS ability of pen-
etration and action inside the cell and reactive singlet
oxygen (ROS) formation around the bacterial cell by illu-
mination of PS [58]. S. mutans is a gram-positive bacteria
that present a thick membrane which is relatively permeable
that blocks the passage of hydrophobic components [59]. It
is possible that this relatively porous layer of peptidoglycan
and lipoteichoic acid outside the cytoplasmic membrane of
gram-positive specie allows the PS to diffuse into sensitive
sites [32]. Since the cell membrane acts a selective bar-
rier to free diffusion, penetration of PS molecules depends
on their size, charge and solubility [60]. Results of recent
studies have indicated that curcumin binds preferentially
to lipid membrane and some cell proteins [61]. On the
other hand, it can be hypothesized that the probable way
of curcumin action is beyond bacterial DNA intercalation
more than membrane acting; generating strand breaks in the
organism’s nucleic acid, with resultant genetic mutation and
photodamage [46]. The possible reasons are that this PS is
317
relatively insoluble in water and presents a high molecular
weight (368.38 g mol−1 ), not allowing it to diffuse more eas-
ily as well. Another point that should be emphasized is the
correlation between ROS production and its interaction with
bacteria and consequently, the efficacy of the treatment
regimen [24]. The relative production of ROS by curcumin
was not determined in the present study, although previ-
ous studies have demonstrated that PS irradiated by blue
light sources presented lower amount of ROS than the other
PS irradiated by red lights [46]. In addition, a phototoxic
effect can be reached by production of hydrogen peroxide
in the presence of light and oxygen and curcumin-mediated
growth inhibition of Helicobacter pylori and Bacillus sub-
tilis is exerted by perturbing activities of enzymes that are
crucial for bacterial growth as well [62,63]. In the present
study, curcumin exhibited some toxicity to the bacteria,
even without radiation. However, irradiation increased the
antimicrobial activity of curcumin, suggesting that the ROS
formed after irradiation is responsible for this increase.
Thus, an antimicrobial effect can be reached by other fac-
tors related to the pharmacodynamic of PS such as cellular
penetration, localization and influx and efflux parameters;
the intracellular stability and activity of the PS; and suffi-
cient targeting of the PS toward the pathogen [60].
The development of this investigation field has as main
goal its applicability as an adjunct tool with a traditional
mechanical dental plaque debridement. Due to proximity of
the bacteria present on the plaque and the cells present
in the periodontal region, it is important to investigate the
potential toxic effects of PDT to the host cells, which also
are susceptible to ROS species, illumination and PS presence
[64]. Paulino et al. [8] investigated the effects of PDT medi-
ated by rose bengal acting on S. mutans and fibroblasts.
The results demonstrated that under light exposure con-
centrations of the PS above 5 ␮M all bacteria were killed
with no cytotoxic effects to fibroblasts. Soukos et al. [65]
showed that toluidine blue O is cytotoxic to Streptococ-
cus sobrinus in concentrations of 2.5 g mL−1 (after He—Ne
irradiation) while similar treatment did not affect the oral
keratinocytes viability. Similar results were obtained in a
study using phorphyrin-mediated PDT that reduced the via-
bility of human dermal fibroblasts by 66.5% and generated
inactivation of 99% of methicillin-resistant S. aureus [66].
However, an investigation using curcumin concentration at
20 ␮M reduced the metabolism of cultured fibroblast cells by
80% after blue LED light illumination, but it was not sufficient
to cause complete inhibition of the SDH enzyme, suggesting
that the microorganisms studied were more susceptible to
PDT than the fibroblasts [64].
The success of PDT is based on some factors such as
type, concentration, incubation time of the PS, the stud-
ied microorganism, light source and doses. Considering the
current results, this in vitro planktonic model investigation
is a mandatory step, since this approach is able to guide
future in vivo protocols/parameters of both illumination
issues (related to light source) and concentration (related
to dyes) and could demonstrate the efficiency in controlling
S. mutans growth in a planktonic suspension.
The present investigation has indicated that curcumin-
mediated PDT was able to reduce the number of viable
cells of S. mutans standard strain according to the studied
parameters.
318
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