Antimicrobial Activity of Monoketone Curcuminoids Against Cariogenic Bacteria

Article abstract of DOI:10.1002/cbdv.201800216

We evaluated the antimicrobial activity of 25 monoketone curcuminoids (MKCs) against a representative panel of cariogenic bacteria in terms of their minimum inhibitory concentration (MIC) values. Curcumin A (10) displayed promising activity against Streptococcus mutans (MIC?=?50?μg/ml) and Streptococcus mitis (MIC?=?50?μg/ml) as well as moderate activity against S.?sanguinis (MIC?=?100?μg/ml), Lactobacillus casei (MIC?=?100?μg/ml), and Streptococcus salivarius (MIC?=?200?μg/ml). Results indicated higher activity of compound 10 than that of its bis-β-diketone analog. Additionally, compounds 3a (1,5-bis(4-methylphenyl)pentan-3-one) and 7b (1,5-bis(4-bromophenyl)pentan-3-ol) were moderately active against S.?mitis (MIC?=?100?μg/ml) and S.?salivarus (MIC?=?200?μg/ml).

Full text of DOI:10.1002/cbdv.201800216

Article Type: Full Paper
Antimicrobial activity of monoketone curcuminoids against
cariogenic bacteria
Tatiana M. Vieiraa, Isabella A. dos Santosa, Thayná S. Silvab, Carlos H. G. Martinsb, Antônio E.
M. Crottia
aDepartamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto,
SP, Brazil
b Laboratório de Pesquisa em Microbiologia Aplicada, Universidade de Franca, Franca, SP, Brazil
*millercrotti@ffclrp.usp.br
We evaluated the antimicrobial activity of twenty-five monoketone curcuminoids (MKCs) against a representative panel of
cariogenic bacteria in terms of their minimum inhibitory concentration (MIC) values. Curcumin A (10) displayed promising
activity against Streptococcus mutans (MIC = 50 μg/ml) and Streptococcus mitis (MIC = 50 μg/ml) as well as moderate activity
against S. sanguinis (MIC = 100 μg/ml), Lactobacillus casei (MIC = 100 μg/ml), and Streptococcus salivarius (MIC = 200 μg/ml).
Results indicated higher activity of compound 10 as compared to its bis-β-diketone analog. Additionally, compounds 3a (1,5-
bis-p-tolylpentan-3-one) and 7b (1,5-bis-(4-bromophenyl)pentan-3-ol) were moderately active against S. mitis (MIC = 100
μg/ml) and S. salivarus (MIC = 200 μg/ml).
Keywords: cariogenic bacteria; curcumin A; monoketone curcuminoids; oral pathogens; Streptococcus mutans
Introduction
Dental caries is an infectious and transmissible disease that affects large part of the population of many countries worldwide.[1]
In Brazil, 40% of the children aged 0-5 years and 53.6% of the children aged 12 years have caries despite water fluoridation.[2]
This pathology is associated with acidogenic and aciduric bacteria, which form a biofilm on the tooth surface.[3] Among the
colony-forming bacteria present in the oral microbiota, Streptococcus mutans plays a key role in caries emergence: this
bacterium produces compounds that favor adhesion and accumulation of other microorganisms, which then form a resistant
extracellular matrix, a mechanism known as co-aggregation.[4] The most effective way to prevent caries and other periodontal
diseases is to remove biofilm by brushing and flossing the teeth. However, most people fail to avoid biofilm accumulation
through mechanical removal, which makes the use of chemicals necessary.[5] Antimicrobial agents like chlorhexidine, fluorides,
ampicillin, penicillin, and vancomycin, among others, have been used to inhibit bacterial growth; chlorhexidine is the most
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widely employed anti-cariogenic agent. However, regular use of these agents often causes several adverse effects. For example,
such agents may disturb the oral and intestinal flora.[6]
The term ‘curcuminoid’ is used to describe curcumin and other α,β-unsaturated bis-β-diketone analogs. Curcumin, also known
as diferuloylmethane, is a major hydrophobic phenolic compound that occurs in Curcuma longa L. (Zingiberaceae) rhizomes.
The scientific literature contains reports about the various biological activities of curcumin, including its insecticide,[7]
antioxidant,[8] anti-inflammatory,[9] trypanocidal,[10] and schistosomicidal[11] actions. In addition, curcumin has been recently
described to suppress S. mutans adhesion to the tooth surface and to reduce biofilm formation through inhibition of the
enzyme sortase.[12-14] Notwithstanding its efficacy and safety, curcumin has not yet been approved as therapeutic agent because
it is relatively little bioavailable, a result of its low absorption and rapid metabolism.[15] Moreover, there is in vitro evidence that
curcumin exhibits keto-enolic tautomerism under physiological pH conditions, and enol hydroxyl is responsible for the
instability of this compound.[16] In this scenario, curcumin has been used as model to synthesize more effective analogs, such as
monoketone curcuminoids (MKCs). These compounds are very stable at different pH, including alkaline pH and presence of
reducing agents,[17] but their antiproliferative activity is conserved.[18] In vitro studies have reported promising MKC trypanocidal
activity against Trypanosoma brucei.[19]
As part of our ongoing project on the antimicrobial activity of synthetic and natural products,[20-22] in this manuscript we
investigate the antimicrobial activity of twenty-five synthetic MKCs against a representative panel of cariogenic bacteria.
Results and Discussion
The monoketone curcuminoids (MKCs) investigated in this study were obtained by Claisen-Schmidt condensation between
acetone and different aromatic aldehydes, followed by catalytic hydrogenation, as shown in Scheme 1. Table 1 summarizes the
results of the antimicrobial activities assays with the twenty-five synthetic MKCs against the selected cariogenic bacteria. The
antimicrobial activity was based on minimum inhibitory concentration (MIC) values as compared to chlorhexidine
dihydrochloride, which we used as positive control. The broth dilution method in 96-well microplates was employed.
Table 1. Minimum inhibitory concentration (MIC) values [μg/ml] of the monoketone curcuminoids 1-9, 1a-9a, 1b-9b, and 10 against selected
cariogenic bacteria.*
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Enterococcus
faecalis
Streptococcus
salivarius
(ATCC
Lactobacillu
s casei
Streptococcu
s mitis
Streptococc
us sanguinis
(ATCC
Streptococc
us sobrinus
(ATCC
Streptococc
us mutans
(ATCC
Microorgani
sm
(ATCC 4082)
(ATCC
(ATCC
25975)
11578)
49456)
10556)
33478)
25175)
>400 (1.70)
>400
(1.70)a
>400 (1.70)a
400 (1.70)a
>400 (1.70)a
400 (1.70)a
>400 (1.70)a
1
a
>400 (1.68)
>400
(1.68)a
>400
>400 (1.68)a
200 (0.83)a
>400 (1.36)a
400 (1.68)a
400 (1.66)a
400 (1.36)a
400 (1.68)a
400 (1.66)a
400 (1.36)a
>400 (1.68)a
>400 (1.66)a
>400 (1.36)a
>400 (1.68)a
>400 (1.66)a
>400 (1.36)a
1a
1b
2
a
400 (1.66) a
(1.66)a
>400
>400 (1.36)
a
(1.36)a
>400 (1.34)
>400 (1.34)a
400 (1.33)a
>400 (1.34)a
400 (1.33)a
>400 (1.34)a
>400 (1.33)a
400 (1.34)a
>400 (1.34)a
400 (1.33)a
>400 (1.34)a
>400 (1.33)a
2a
a
400 (1.33)a
400 (1.33)a
2b
3a
7
200 (0.75)a
>400 (1.02)a
100 (0.37) a
400 (1.02)a
200 (0.75)a
400 (1.02)a
200 (0.75)a
>400 (1.02)a
400 (1.50)a
400 (1.02)a
>400 (1.50)a
>400 (1.02)a
>400 (1.50)a
>400 (1.02)a
>400 (1.01)a
400 (0.96)a
50 (0.15)a
100 (0.27)a
400 (0.96)a
50 (0.15)a
200 (0.50)a
400 (0.96)a
200 (0.61)a
400 (1.01)a
>400 (0.96)a
100 (0.30)a
>400
(1.01)a
>400 (1.01)a
400 (0.96)a
>400 (1.22)a
>400 (1.01)a
>400 (0.96)a
100 (0.30)a
7b
>400
8
(0.96)a
400 (1.22)a
10
Positive
control
0,92 b
(0.0015)a
1.84 b
(0.00318)a
1.84 b
(0.00318)a
0.92b
(0.0015)a
0.92b
(0.0015)a
0.92 b
(0.0015)a
7.37b (0.012)a
a Values in mM. bChlorhexidine dihydrochloride. *Compounds 3, 4, 4a, 4b, 5, 5a, 5b, 6, 6a, 6b, 7a, 9, 9a, and 9b were not active in the range of
tested concentrations (MIC > 400 μg/ml), so they were not included.
Scheme 1. Reaction conditions for the synthesis of compounds 1-9, 1a-9a, 1b-9b, and 10 from aromatic aldehydes and acetone, r.t., and catalytic
hydrogenation with H2, Pd/C (10%), 100 psi, r.t.
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According to the literature, MIC values lower than 100 μg/ml, 100 ≤ MIC < 500 μg/ml, and 500 ≤ MIC < 1000 μg/ml correspond
to promising, moderate, and weak activities, respectively, whereas MIC values greater than 1000 μg/ml denote inactivity.[23-26]
On the basis of these criteria, all the compounds displayed moderate antimicrobial activity (MIC ≤ 400 μg/ml) against at least
one of the selected microorganisms, except for 3, 4, 4a, 4b, 5, 5a, 5b, 6, 6a, 6b, 7a, 9, 9a, and 9b (data not shown). The lowest
MIC values were obtained for compound 10 (Table 1). This compound displayed promising activity against Streptococcus
mutans (MIC = 50 μg/ml; 0.15 mM) and S. mitis (MIC = 50 μg/ml; 0.15 mM) as well as moderate activity against S. sanguinis
(MIC = 100 μg/ml; 0.30 mM), Lactobacillus casei (MIC = 100 μg/ml; 0.30 mM), and S. salivarus (MIC = 200 μg/ml; 0.61 mM). The
activity of compound 10 against S. mutans was remarkable because very few natural compounds can inhibit this microorganism,
which is one of the primary causative agents of dental caries.[4] Compounds 3a and 7b also displayed moderate activity against
Streptococcus mitis (MIC = 100 μg/ml, 0.37 mM and 0.27 mM, respectively). However, these compounds were weakly active or
inactive against all the other tested cariogenic bacteria (MIC values higher than 400 μg/ml).
Comparison between the MIC values of the tested MKCs indicated that most of the compounds bearing a double bond
between C2 and C3, and C2’ and C3’ were inactive against the tested microorganisms in the range of tested concentrations,
except for compounds 1, 2, 8, and 10. Considering that compounds 2, 8, and 10 have a methoxyl group at the aromatic ring,
the presence of this substituent could be related to the antimicrobial activity of these MKCs. These findings agreed with
previous reports on the antibacterial activity of chalcones (α,β-unsaturated ketones), which is potentialized by electron-releasing
groups at the aromatic ring, such as methoxy and hydroxyl groups.[27] However, compounds 4 and 6, which also have methoxyl
groups attached to the aromatic rings, were inactive in the range of tested concentrations. These results indicated that the
presence of two methoxyl groups in adjacent carbons of the aromatic ring could decrease the antimicrobial activity against the
tested cariogenic bacteria.
For compounds that bear a double bond between C2 and C3, and C2’ and C3’, the presence of a methyl or a halogen (Cl or Br)
at the aromatic ring did not affect the antimicrobial activity. Compounds 1a and 2a displayed similar activity to the activity of
compounds 1 and 2, from which they were obtained by catalytic hydrogenation. On the other hand, compounds 3a and 7a had
increased activity as compared to their precursors 3 and 7. Although hydrogenation of the double bond between C2 and C3 (or
C2’ and C3’) tended to increase the antimicrobial activity against the tested cariogenic bacteria slightly, the role played by the
double (or single) bond between these two carbons was not clear, as the bond also seemed to be associated with the nature of
the substituent attached to the aromatic ring. In addition, reduction of the ketone group to produce a secondary alcohol had
little or no effect on the antimicrobial activity of most MKCs, except for compounds 1b and 7b. The higher activity of
compounds 1b and 7b as compared to their precursors 1a and 7a could be associated with increased hydrophilicity of these
compounds due to the hydroxyl group at C1.
We also compared the antimicrobial activity of curcumin A (10) with that of curcumin, which is its β-diketone analog.[28] As
previously mentioned in this paper, the ability of curcumin to suppress S. mutans adhesion to the tooth surface by inhibiting the
activity of the enzyme sortase has been previously described.[14, 29, 30] Despite the anti-HIV activity of curcumin A,[28] its activity
against cariogenic bacteria has not been previously reported. According to the literature, curcumin is effective against S. mutans
(MIC = 250 μg/ml; 0.68 mM) and S. mitis (MIC = 62.5 μg/ml; 0.17 mM) [31, 32]. In this study, curcumin A at 50 μg/ml (0.15 mM)
was active against S. mutans and S. mitis. Therefore, together, our data indicated that curcumin A (10) is more effective against
these two cariogenic bacteria than curcumin.
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Conclusions
Among the twenty-five MKCs tested herein, curcumin A (10) is the most active. The MIC values obtained for curcumin A against
S. mutans are even lower as compared to the MIC values previously reported for its β–diketone analog, curcumin. The promising
activity of curcumin A against S. mutans is a remarkable result and deserves further investigation. Studies to explore the
structure-activity relationships of MKCs more deeply are underway.
Experimental Section
Synthesis of monoketone curcuminoids 1-9, 1a-9a, 1b-9b, and curcumin A (10)
The monoketone curcuminoids 1-9 were synthesized by Claisen-Schmidt condensation between acetone and different aromatic
aldehydes, according to methodologies previously described in the literature, with some modifications.[33] Reactions were
conducted by using basic catalysis at room temperature for the time indicated in the experimental protocol.[33] Derivatives 1a-
9a and 1b-9b were obtained by further catalytic hydrogenation. In this procedure, the monoketone curcuminoids were
completely dissolved in HPLC grade ethyl acetate together with the catalyst and transferred to a high-pressure reactor under
stirring, H2 atmosphere, and 100 psi at room temperature for 18 h.[15] Compound 10 was obtained by Claisen-Schmidt
condensation in acidic conditions according to a previously published methodology.[28] Acetic acid (50 ml) was saturated with
hydrogen chloride at 0 ºC. A mixture of vanillin (3.04 g, 20 mmol) and acetone (580 mg, 10 mmol) was slowly added to acetic
acid. The reaction mixture was brought to room temperature and stirred for 24 h. The crude reaction mixture was poured into
ice-cold water (200 ml) and extracted with ethyl acetate (3 x 30 ml). The organic portion was dried over MgSO4 and filtered.
Then, the solvent was removed by evaporation under reduced pressure in a rotary evaporator. The resulting solid was purified
by silica gel flash column chromatography with isocratic elution with hexane/ethyl acetate 1:1 (v/v) (Scheme 1). NMR, EI-MS,
and IR analyses were used to confirm the structures y (see Support Information).
Antibacterial Assays
The in vitro antimicrobial action of monoketone curcuminoids 1-9, 1a-9a, and 1b-9b and curcumina A (10) was evaluated in
terms of their minimum inhibitory concentration (MIC) values.[34] To this end, Streptococcus mutans (ATCC 25175), Streptococcus
mitis (ATCC 49456), Streptococcus salivarius (ATCC 25975), Streptococcus sanguinis (ATCC 10556), Streptococcus sobrinus (ATCC
33478), Enterococcus faecalis (ATCC 4082), and Lactobacillus casei (ATCC 11578) were assayed by the broth microdilution
method, in 96-well microplates. The bacterial colonies were cultured at 37 ºC for 24 h in blood agar (Difco Labs, Detroit, MI,
USA). Further standardization of the inoculum quantity was accomplished on a spectrophotometer Femto (São Paulo, Brazil)
operating at a wavelength of 625 nm, to match 0.5 in the McFarland scale (1.5 x 108 CFU/ml). The microorganism suspensions
were diluted to a final concentration of 5 x 105 CFU/ml. Samples of compounds 1-9, 1a-9a, 1b-9b, and 10 were dissolved in
DMSO (Merck, Darmstadt, Germany) and tryptic soy broth (TSB, Difco), to obtain final concentrations varying from 1.9 to 400
μg/ml. Inoculated microplate wells containing DMSO (1%) and TSB (1:5 v/v and 100%) were employed as negative control. A
non-inoculated well was also added, to ensure medium sterility. Chlorhexidine dihydrochloride (Sigma-Aldrich, St. Louis) at
concentrations ranging from 0.0115 to 5.9 μg/ml in TSB (Difco) was the positive control. The microplates were sealed with
plastic film and incubated at 37 ºC for 24 h. Next, 30 μl of revealing 0.02% resazurin (Sigma-Aldrich, St. Louis) was added to
each microplate well, to indicate microbial viability.[34] Experiments were conducted in three replicates for each microorganism.
MIC values were assessed by analysis of the DNB capacity to prevent the color of the resazurin solution from changing.[35]
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Supplementary Material
Supporting information for this article is available on the WWW at http://dx.doi.org/10.1002/MS-number.
Acknowledgements
The authors thank the Brazilian foundations FAPESP (Proc. 2016/19272-9 and 2013/20094-0) and CNPq for fellowships and
grants.
Author Contribution Statement
T. M. V. and I.A.S. synthesized the MKCs; T.S.S. performed the antimicrobial assays; C.H.G.M. supervised the antimicrobial assays;
A.E.M.C. designed and drafted the manuscript. All the authors have read the final manuscript and approved its submission.
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