Antibacterial activity of 3,3′-dihydroxycurcumin (DHC) is associated with membrane perturbation

Article abstract of DOI:10.1016/j.bioorg.2019.103031

Curcumin is a plant diphenylheptanoid and has been investigated for its antibacterial activity. However, the therapeutic uses of this compound are limited due to its chemical instability. In this work, we evaluated the antimicrobial activity of diphenylhe

Full text of DOI:10.1016/j.bioorg.2019.103031

Accepted Manuscript
Antibacterial activity of 3,3’-dihydroxycurcumin (DHC) is associated with
membrane perturbation
Carlos R. Polaquini, Luana G. Morão, Ana C. Nazaré, Guilherme S. Torrezan,
Guilherme Dilarri, Lúcia B. Cavalca, Débora L. Campos, Isabel C. Silva,
Jessé A. Pereira, Dirk-Jan Scheffers, Cristiane Duque, Fernando R. Pavan,
Henrique Ferreira, Luis O. Regasini
PII:
S0045-2068(19)30486-9
https://doi.org/10.1016/j.bioorg.2019.103031
103031
DOI:
Article Number:
Reference:
YBIOO 103031
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 March 2019
24 May 2019
3 June 2019
Please cite this article as: C.R. Polaquini, L.G. Morão, A.C. Nazaré, G.S. Torrezan, G. Dilarri, L.B. Cavalca, D.L.
Campos, I.C. Silva, J.A. Pereira, D-J. Scheffers, C. Duque, F.R. Pavan, H. Ferreira, L.O. Regasini, Antibacterial
activity of 3,3’-dihydroxycurcumin (DHC) is associated with membrane perturbation, Bioorganic Chemistry (2019),
doi: https://doi.org/10.1016/j.bioorg.2019.103031
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Bio-Organic Chemistry
Antibacterial activity of 3,3’-dihydroxycurcumin (DHC) is
associated with membrane perturbation
a,1
a
a
Carlos R. Polaquini , Luana G. Morão b,1, Ana C. Nazaré , Guilherme S. Torrezan ,
Guilherme Dilarri b, Lúcia B. Cavalca b,c, Débora L. Campos d, Isabel C. Silva b,d, Jessé
e
c
e
d
A. Pereira , Dirk-Jan Scheffers , Cristiane Duque , Fernando R. Pavan , Henrique
Ferreira b,*, Luis O. Regasini a,*
a
Department of Chemistry and Environmental Sciences, Institute of Biosciences,
Humanities and Exact Sciences, São Paulo State University (Unesp), São José do Rio
Preto 15054-000, Brazil
b
Department of Biochemistry and Microbiology, Institute of Biosciences, São Paulo
State University (Unesp), Rio Claro 13506-900, Brazil
c
Department of Molecular Microbiology, Groningen Biomolecular Sciences and
Biotechnology Institute, University of Groningen, Groningen 9747, The Netherlands
d
Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo
State University (Unesp), Araraquara 14800-903, Brazil
e Department of Pediatric Dentistry and Public Health, School of Dentistry, São Paulo
State University (Unesp), Araçatuba 16015-050, Brazil
1 C.R. Polaquini and L.G. Morão contributed equally to this work.
* Corresponding authors.
E-mail addresses:
luis.regasini@unesp.br (L.O. Regasini)
henrique.ferreira@linacre.oxon.org (H. Ferreira)
1

ABSTRACT
Curcumin is a plant diphenylheptanoid and has been investigated for its antibacterial
activity. However, the therapeutic uses of this compound are limited due to its chemical
instability. In this work, we evaluated the antimicrobial activity of diphenylheptanoids
derived from curcumin against Gram-positive and Gram-negative bacteria, and also
against Mycobacterium tuberculosis in terms of MIC (Minimum Inhibitory
Concentration) and MBC (Minimum Bactericidal Concentration) values. 3,3’-
Dihydroxycurcumin (DHC) displayed activity against Enterococcus faecalis,
Staphylococcus aureus and M. tuberculosis, demonstrating MIC values of 78 and 156
µg/mL. In addition, DHC was more stable than curcumin in acetate buffer (pH 5.0) and
o
phosphate buffer (pH 7.4) for 24 h at 37 C. We proposed that membrane and the cell
division protein FtsZ could be the targets for DHC due to that fact that curcumin
exhibits this mode of antibacterial action. Fluorescence microscopy of Bacillus subtilis
stained with SYTO9 and propidium iodide fluorophores indicated that DHC has the
ability to perturb the bacterial membrane. On the other hand, DHC showed a weak
inhibition of the GTPase activity of B. subtilis FtsZ. Toxicity assay using human cells
indicated that DHC has moderate capacity to reduce viability of liver cells (HepG2 line)
and lung cells (MRC-5 and A549 lines) when compared with doxorubicin. Alkaline
comet assay indicated that DHC was not able to induce DNA damage in A549 cell line.
These results indicated that DHC is promising compound with antibacterial and
antitubercular activities.
Keywords:
Curcumin
Curcuminoid
Antibacterial
Antitubercular
Membrane
FtsZ
Bacillus subtilis
2

1. Introduction
Curcumin is the main diphenylheptanoid from rhizomes of Curcuma longa,
commonly named as turmeric. It has been considered a privileged natural product due to
its therapeutic potential [1]. Its abilities to interact with multiple targets has recognized
role in polypharmacology of complex diseases such as cancer and Alzheimer [2].
Additionally, several clinical studies have supported its safety and tolerability for
humans [3].
Curcumin has demonstrated a broad spectrum of in vitro antibacterial activity
against Gram-positive, Gram-negative and Mycobacteria species [4], [5]. In vivo
investigations showed that curcumin was able to eliminate Helicobacter pylori from
mice stomach [6] and to decrease the mortality of mice infected with methicillin-
sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus
aureus (MRSA) [7]. The mode of antibacterial action of curcumin has been reported as
a combination of membrane disruption [8] and cell division blockage [9]. Activity of
curcumin on the membrane was attributed to its amphiphilicity, which allows its
insertion into phospholipid bilayers, increasing the permeability of membranes [8], [10].
The ability of curcumin to perturb bacterial cell division has been attributed to a direct
action on FtsZ, a prokaryotic cytoskinesis protein. Curcumin inhibited FtsZ
protofilament assembly and enhanced the GTPase activity of this protein by altering its
secondary structure [9], [11]. The FtsZ polymerization into protofilaments require GTP
as a stabilizer and an energy source, leading to the formation of the Z-ring, a pivotal
structure in the assembly of the divisome [12].
Although curcumin has shown to be a promising antibacterial agent, its poor
chemical stability has not supported advances of its use in clinical and pharmaceutical
studies [13], [14]. In addition, curcumin exhibits low oral bioavailability due to its poor
absorption and extensive intestinal and first-pass metabolism, resulting in rapid
excretion [15]. Wang and collaborators described 90% curcumin decomposed rapidly in
buffer systems at neutral-basic pH conditions similar to physiological matrices [16].
However, the addition of glutathione, ascorbic acid or phosphate buffer in culture media
increased its stability [16]. Interestingly, the presence of human blood also decreased its
degradation [16] which may be correlated with effect of plasma proteins, mainly serum
albumin and fibrinogen [17]. Thereby, efforts have been made in order to search for
more stable analogs [18], [19].
3

In our previous work, curcumin was used as framework to design antibacterial
simplified monoketones, which were more active and stable than curcumin [20]. In the
current work, we explored the role of substituents on the aromatic rings of curcumin
framework, including electron-donating groups (OH, OMe, Me, N(Me)2 and OAc) and
halogens (F, Cl, Br and CF3) toward antimicrobial activity. We synthesized a series of
diphenylhepatanoids based on curcumin framework (curcuminoids) and evaluated their
antibacterial and antitubercular activities. The chemical stability of most active
curcuminoid (DHC) was compared with curcumin in acetate buffer (pH 5.0) and
phosphate buffer (pH 7.4) for 24 h at 37 oC. DHC was investigated regarding its effects
on the membrane and FtsZ of Bacillus subtilis. In order to understand toxicity of DHC
toward human cells, cytotoxicity and alkaline comet assays were carried out.
2. Results and Discussion
2.1. Chemistry
One-pot synthesis of curcuminoids 1–12 was achieved by Pabon protocol [21],
with minor modifications [22]. Reactions between respective benzaldehydes and
acetylacetone were performed through successive boron complexes, resulting in yields
of 57–95% (Scheme 1). In general, a clear and unambiguous relationship between the
stereoelectronic effect of substituents on aromatic ring of benzaldehyde derivatives and
reaction yields cannot be established. Ester 13 was synthesized by acetylation of
compound 5 using acetic anhydride and pyridine, with yield of 87%.
Scheme 1.
1
13
Structure of curcuminoids was confirmed by their melting point and H and C
Nuclear Magnetic Resonance (NMR) spectral data analyses. Spectra were presented in
Supplementary Material. For all curcuminoids, NMR data, including chemical shifts,
integrations multiplicities and coupling constants, corresponded to the proposed
structures. In 1H NMR spectrum, aromatic hydrogens were observed as signals in range
of δH 6.50–7.67 ppm. Hydrogens α and β resonated as doublets and clearly indicated the
formation of two olefines. Coupling constants values of olefin hydrogens signals
ranging from 15 to 16 Hz, corroborating their trans configuration. In addition, singlet
4

with resonance at δH ~ 5.80 ppm and integration to one hydrogen was attributed to
methyne hydrogen of enol moiety [23].
2.2. Antibacterial and Antitubercular Activities
The antibacterial and antitubercular activities of curcumin and curcuminoids
1−13 were evaluated against Enterococcus faecalis, Staphylococcus aureus, Bacillus
subtilis, Pseudomonas aeruginosa, Escherichia coli and Mycobacterium tuberculosis.
Antimicrobial potency was expressed as Minimum Inhibitory Concentration (MIC) and
Minimum Bactericidal Concentration (MBC) values in µg/mL. Ciprofloxacin and
isoniazid were used as reference antibacterial and antitubercular drugs, respectively
(Table 1).
Table 1.
Curcumin displayed activity against E. faecalis, S. aureus and B. subtilis,
with MIC values of 78 and 156 μg/mL. However, curcumin demonstrated MIC > 156
μg/mL against M. tuberculosis, E. coli and P. aeruginosa. Although curcumin has
been recognized for its broad antibacterial spectrum, some studies reported that its
activity against Gram-positive bacteria has been higher than against Gram-negative
species [24], [25], corroborating our results. Activity of curcumin against S. aureus
has been extensively reported. A review about curcumin activity against MSSA
strains indicated MIC values ranged from 125 to 256 μg/mL by using broth
microdilution method. These values varied according to strain types and vehicles
(DMSO, EtOH or DMF) [26]. In addition, there are studies demonstrating ability of
curcumin to reverse methicillin resistance in MRSA [27] and its efficacy against
osteomyelitis in rats induced by MRSA, suppressing bacterial growth [28].
First, in order to investigate preliminary relationships between
antibacterial/antitubercular activities and substituents on aromatic rings, we
compared the activity of curcumin with the unsubstituted curcuminoid (1).
Compound 1 was inactive (MIC and MBC values > 156 μg/mL), indicating that
substituents OMe and OH at meta and para positions, respectively, are relevant for
antibacterial activity of curcumin against Gram-positive species.
5

Second, we evaluated the effect of m-OMe and p-OH separately, testing
simplified analogs 3,3’-dimethoxycurcumin (2) and 4,4’-dihydroxycurcumin (3),
respectively. Compounds 2 and 3 had MIC and MBC values > 156 µg/mL,
suggesting that antibacterial activity of curcumin can be correlated to simultaneous
presence of OMe and OH at meta and para positions, respectively. Also, compound 3
exhibits a natural occurrence in turmeric (also named as bisdesmethoxycurcumin or
curcumin III) [14]. Its antibacterial activity was previously studied by Péret-Almeida
and co-authors, who reported their inactivity against S. aureus, E. coli and
Salmonella choleraesuis [29], corroborating our current findings.
Third, we evaluated two regioisomers of compounds 2 and 3, which were
designed by replacement of OMe and OH positions, furnishing 4,4’-
dimethoxycurcumin (4) and 3,3’-dihydroxycurcumin (5 or DHC), respectively.
Compound 4 was inactive (MIC and MBC values > 156 μg/mL). On the other hand,
DHC demonstrated antibacterial activity similar to curcumin against S. aureus and B.
subtilis with MIC values of 156 μg/mL. Furthermore, DHC was more potent than
curcumin against E. faecalis and M. tuberculosis with MIC values of 78 and 156
μg/mL, respectively.
Fourth, we evaluated the effect of other electron-donating groups on
aromatic rings, including Me (curcuminoid 6) and N(Me)2 (curcuminoid 7), which
release electrons by inductive and mesomeric effect, respectively. Curcuminoids 6
and 7 were inactive (MIC and MBC values > 156 µg/mL). The comparison among
the curcuminoids DHC, 6 and 7 indicated that the electron donation on aromatic
rings is not relevant to antibacterial and antitubercular activities. However, the
bioactivity seems to be related to acidity of phenol group in DHC, because neutral
(Me, curcuminoid 6) and basic (N(Me)2, curcuminoid 7) groups did not improve
antimicrobial potency.
Fifth, we assayed a set of curcuminoids substituted by p-F, p-Cl, p-Br, p-CF3
and m,p-diCl (compounds 8 to 12, respectively). All halogenated curcuminoids were
not able to inhibit bacterial growth (MIC and MBC values > 156 µg/mL), suggesting
electron-withdrawing substituents on aromatic rings did not increase antibacterial and
antitubercular activities.
In our previous work, we described that diacetylated curcumin (DAC) was
more active than curcumin against Streptococcus mutans, MSSA and MRSA
planktonic and biofilm forms [30, 31]. Thus, we hypothesized that the acetylation of
6

DHC could lead to a more potent analog. Unfortunately, ester 13 was inactive (MIC
and MBC values > 156 µg/mL), corroborating the relevance of acidic OH at meta
position (phenol moiety) to antibacterial and antitubercular activities. In general,
phenolic compounds are recognized for their antibacterial activity [32], [33]. Recent
studies have elucidated the importance of the hydroxyl group position in phenolic
natural product derivatives concerning antibacterial potency, including
cinnamylideneacetophenones [34], gallates, protocatechuates, gentisates and β-
resorcylates [35].
Curcuminoids 1–13 were not able to inhibit growth of Gram-negative
species, including P. aeruginosa and E. coli. Gram-negative bacteria demonstrate a
complex barrier, including two membranes, which makes them less susceptible to
antibacterial agents. As part of this barrier, the cell wall exerts central role in the
permeation of compounds due to their complex composition (lipopolysaccharides,
phospholipids and proteins) [36], [37].
We selected DHC for further assays, aiming to evaluate its chemical
stability and the elucidation of its molecular targets, which could be related to the
mode of antibacterial action exhibited by curcumin. In previous studies by our group,
curcumin disrupted the membrane of B. subtilis, which was confirmed by microscopy
assays. Also, we described that curcumin blocked B. subtilis cell division, stimuling
the GTPase activity of FtsZ [20].
2.3. Evaluation of DHC chemical stability
Although curcumin is a promising antibacterial agent, its clinical application is
limited due to its poor stability under physiological conditions [14]. Thus, chemical
stability of DHC was evaluated at pH 5.0 and 7.4 by monitoring the decrease in its UV-
Vis absorption at 37 °C and compared with curcumin. As expected, both compounds
were more stable under acidic than basic conditions (Figure 1). Curcumin was rapidly
degraded at pH 5.0 and 7.4, reducing 52% and 63% after 24 h, respectively. However,
DHC was more stable than curcumin at pH 5.0 and 7.4, reducing only 16% and 24% of
its initial concentration after 24 h, respectively. Liu and co-authors also reported a
higher stability of DHC when compared with curcumin in RPMI media at 25 °C [38],
corroborating our current findings.
7

Wang and collaborators reported feruloylmethane, vanillin and ferulic acid as
o
degradation compounds of curcumin under phosphate buffer at pH 7.2 (at 37 C) [16].
These compounds are produced by cleavage of the β-diketone moiety. Thus, stabilizing
effects on transition state of these reactions can improve the degradation rate. In this
context, curcumin exhibits a hydroxyl group at para-position, which resonates to β-
diketone moiety, dispersing π-electron density. On the other hand, DHC presents a
hydroxyl group at meta-position, which did not allow that alone electron pairs of
hydroxyl group resonate to β-diketone moiety. Thereby, extended conjugation between
4-OH and β-diketone in curcumin structure can be correlated to its degradation.
Furthermore, in basic conditions hydroxyl group at para-position is deprotonated,
facilitating conjugation and degradation of curcumin.
Figure 1.
2.4. Effect of DHC on the membrane of B. subtilis
In order to evaluate the effects of DHC on the bacterial membrane, we used B.
subtilis strain 168. Bacterial cells were exposed to DHC at the MIC (78 µg/mL) for 15
and 30 min. Two fluorescent dyes propidium iodide (PI) and SYTO9 were added, which
stain cells with damaged membrane (in red) and intact membrane (in blue) [8], [39].
Nisin was used as a reference for membrane disruption due to produce membrane pores
in B. subtilis. Representative fluorescence microscopy images of the negative control
(cells treated with 1% DMSO), the positive control (nisin at 5 µg/mL) and DHC are
shown in Figure 2. Cells exposed to DMSO and nisin appear labeled in blue and red,
respectively. Exposure to DHC, in both periods of 15 and 30 min, led to a prevalence of
pink-red stained cells, indicating membrane permeability.
Figure 2.
Percentages of cells with damaged membrane were quantified from the
microscope images and are shown in Figure 3. Cultures treated with 1% DMSO had
approximately 2% of the cells with membrane permeabilized. On the other hand,
treatment with nisin demonstrated 99% of the cells stained with PI due to its ability to
make pores in the bacterial membrane [40]. Treatment with DHC (at the MIC) for 15
8

min and 30 min displayed damage percentages of 72 and 85%, respectively, evidencing
the membrane disruption of B. subtilis by DHC is time-dependent effect.
Membranes seems to be a target of phenolic compounds [41], [42]. They
induce changes in the membrane structure and function, including increased
permeability [33], [43]. Some antibacterial drugs exhibited the membrane as a primary
target such as daptomycin [44] and polymyxin B [45] or a secondary one such as
telavancin [46]. Furthermore, the combined use of compounds that increase membrane
permeability with other antibacterial agents may be a good alternative against bacterial
resistance [47], [48].
Figure 3.
2.5. Effect of DHC on the GTPase activity of B. subtilis FtsZ
In order to investigate bacterial division as target of DHC, we evaluated its
ability to affect the GTPase activity of Bacillus subtilis FtsZ (BsFtsZ). Compounds that
affect the GTP hydrolysis of FtsZ are promising inhibitors of bacterial division, since
GTP and GDP levels regulate the Z-ring dynamics [49]. We assessed the protein GTP
hydrolysis rates during treatment with DHC at the MIC (78 µg/mL) and 2×MIC (156
µg/mL). BsFtsZ was incubated with 1% DMSO and used as negative control. BsFtsZ
hydrolysis activity was slightly reduced by DHC when compared to the negative
control. At the MIC and 2×MIC values, the percentage of GTPase activity reduction
was of 10% and 26%, respectively. Thus, the weak inhibition of bacterial division was
less relevant to antibacterial activity than membrane disruption.
2.6. Assessment of DHC cyto-genotoxicity and determination of selectivity index
The investigation of new antibacterial agents against eukaryotic cells is an
important step for evaluation of their selectivity and safety. Toxicity of DHC was
evaluated against human cells from liver (tumorigenic HepG2 cell line) and lung
(tumorigenic A549 cell and normal MCR-5 cell lines). Hepatocytes are widely used to
evaluate the toxicity of bioactive compounds due to biotransformation abilities [50] and
lung cells were chosen based on their importance as host site for infections of E.
faecalis, S. aureus and M. tuberculosis [51], [52]. Toxic effect of curcumin and DHC
9

was expressed as the concentration required to reduce the viability of cells at 50%
(IC50) (Table 2).
Table 2.
DHC displayed IC50 values ranging from 9.6 to 10.6 µg/mL and it was
slightly more toxic than curcumin (15.5 µg/mL ≤ IC50 ≤ 32.3 µg/mL).
Biotransformation capacity of hepatocytes did not affect cytotoxicity of DHC,
because its IC50 value was similar to those of lung cell lines. In addition, no
significant difference was observed in the toxic effects of DHC against lung lines.
Normal human fibroblasts (MCR-5) and adenocarcinoma cells (A549) showed
similar sensitivity to DHC, indicating altered genes and proteins expression of the
tumorigenic lines did not alter the toxicity effect. When compared to doxorubicin
(0.03 µg/mL ≤ IC50 ≤ 0.9 µg/mL), a compound known for its cytotoxic properties
[53], DHC had moderade toxicity against human cells. In order to compare the effect
towards prokaryotic cells versus eukaryotic cells, the selectivity indexes (SI) of DHC
were determined as the ratio between IC50 and MIC values. We used MIC value of 78
µg/mL and IC50 for each human cell line. SI values were of 0.12, 0.13 and 0.12 for
HepG2, MRC-5 and A459, respectively. SI values < 1 indicated DHC was not selective,
displaying higher toxicity against human cells than bacteria.
Several studies have correlated toxicity of curcumin toward human cells to
DNA damage, due to its oxidizing properties [54], [55]. These effects toward DNA are
due to phenolic nature of curcumin and its quinone products, as well as chelatogenic
diketone. In order to study genotoxicity of DHC, we performed single cell gel
electrophoresis assay (alkaline comet assay) with A549 cells, which was the most
sensitive line. Comet assay is capable of evaluating damage induced by alkylating,
intercalating or oxidizing agents in the DNA of individual cells [56], [57]. The DNA of
an undamaged nucleus maintains its spherical organization, whereas fragmented DNA
migrates across gel (tail) [58]. Hydrogen peroxide (1 mM) was used as reference
genotoxic compound due to its strong oxidizing properties and high capacity to induce
long comet tail. Lower concentrations than IC50 were used to guarantee high
percentagens of metabolically viable cells. We selected the concentrations referred to ½
IC50 (4.8 µg/mL) and ¼ IC50 (2.4 µg/mL) for DHC. Also, curcuminin was evaluated at
½ IC50 (16.2 µg/mL) and ¼ IC50 (8.1 µg/mL).
10

Tail moment and percentage of DNA in the tail is directly proportional to DNA
break [59]. DHC demonstrated no significant genotoxic effects compared with the
negative control (cells treated with 1% DMSO). Furthermore, the genotoxic parameters
of DHC were closely similar to curcumin (Figure 4). Both compounds were three times
less able to induce DNA breaks than H2O2, indicating weak genotoxic effect toward
A549 cell.
Figure 4.
3. Conclusion
In conclusion, we synthesized a series of curcuminoids as part of our ongoing
search for antibacterial agents based on curcumin structure. 3,3’-Dihydroxycurcumin
(DHC) was found to be an important hit with antibacterial activity against Gram-
positive species and M. tuberculosis. The mode of action of DHC involved membrane
disruption, which is an important target for the development of novel antibacterial
agents. Furthermore, preliminary in vitro investigations of toxicity and stability
indicated that DHC is potentially safe and stable at pH 5.0 and 7.4. Altogether, our
results corroborate the potential of curcumin as a privileged framework for the
discovery of compounds for the treatment of bacterial infections and tuberculosis.
4. Experimental
4.1. Chemistry
4.1.1. Material and Instruments
All starting materials and reagents were purchased from Merck. Melting points
were determined on a Tecnopon PFM-II® apparatus and were uncorrected. NMR
spectra were recorded on two spectrometers: Bruker® Avance III 600 MHz (14.1 T) and
Bruker® Avance III 400 MHz (9.4 T). Compounds were dissolved in CDCl3 or DMSO-
d6 and its non-deuterated residue was used as internal standard for establishment of
1
13
chemical shift values (δH and δC). H and C NMR parameters, including δH and δC,
integrations, multiplicities and coupling constants (J in Hz) were measured. NMR
parameter values were compared to previous descriptions, confirming proposed
1
structures. The H and 13C NMR spectra of curcuminoids 1–13 and their detailed
analyses are in Supplementary Material.
11

4.1.2 Synthesis of curcuminoids 1–12
Compounds 1−12 were synthesized by Pabon method [21], according to
Venkateswarlu and co-authors [22], with minor modifications. Acetylacetone (5.0
mmol, 0.5 mL) was added to suspension of B2O3 (5.0 mmol) in DMF (2.0 mL) and
stirred for 15 min at 60 °C. Tributyl borate (10 mmol) was added and stirred for 15 min
at 60 °C. Respective benzaldehyde derivatives (10 mmol) was added and stirred for 5
min at 60 °C. Mixture of amylamine (0.1 mL) and acetic acid (0.3 mL) in DMF (1 mL)
was added to the reaction and heated to 90 °C for 4 h. After cooling, aqueous solution of
acetic acid (20%, 50 mL) was added and stirred for 1 h at 70 °C. Reaction medium was
poured onto crushed ice from deionized water. Precipitated crude products were filtered,
washed with cold water and dried at room temperature. Compounds 1, 6 and 8−12 were
recrystallized in cold ethanol. Compounds 2−5 and 7 were purified by gel filtration
column chromatography (LH-20, Sephadex) using ethanol as mobile phase.
4.1.3. Synthesis of curcuminoid 13
Compound 13 was synthesized by acetylation reaction of DHC according to
Sardi and co-authors [30], with minor modifications [60]. Acetic anhydride (10 mL)
was added to a solution of DHC (1 mmol) in pyridine (10 mL) and the reaction mixture
was kept under magnetic stirring at 100 °C. After 48 h, the residue was partitioned with
ethyl acetate (3×25 mL) and dried at room temperature. The crude product was purified
by column chromatography over silica gel using hexane and ethyl acetate (4:1).
4.2. Antibacterial assay
Bacterial species used in this work were Enterecoccus faecalis (ATCC
51299), Staphylococcus aureus (ATCC 14458), Bacillus subtilis strain 168,
Pseudomonas aeruginosa (ATCC 15442) and Escherichia coli (ATCC 10536). MIC
and MBC values were determined by the broth microdilution method in 96-well
microtiter plates, following protocol of National Committee for Clinical Laboratory
Standands [61]. Minor modifications in this protocol and their detailed procedures
were reported in previous work by Polaquini and co-authors [34]. Stock solutions (at
10 mg/mL) of curcumin and curcuminoids 1–13 were prepared in DMSO and tested
12

in concentrations ranging from 156 to 2.4 μg/mL. Ciprofloxacin was used as a
reference antibacterial drug. Three independent assays were performed.
4.3. Antitubercular assay
Antitubercular activity was performed toward Mycobacterium tuberculosis
H37Rv strain (ATCC 27294). MIC values were determined by Resazurin Microtiter
Assay (REMA), following protocol described by Palomino and co-authors [62].
Minor modifications in this protocol and their detailed procedures were reported in
previous work by Polaquini and co-authors [34]. Stock solutions (at 10 mg/mL) of
curcumin and curcuminoids 1–13 were prepared in DMSO and tested in
concentrations ranging from 156 to 2.4 μg/mL. Isoniazid was used as reference
antitubercular drug. Three independent assays were performed.
4.4. Chemical stability assay
Stability of DHC (at λmax 398 nm) and curcumin (at λmax 426 nm) was assessed
by monitoring their UV-visible absorptions in acetate buffer (pH 5.0) and phosphate
buffer (pH 7.4) at 37 °C [19], [38]. Compounds were solubilized in DMSO and
evaluated at 13.5 µg/mL. The spectra were run against blanks containing buffer
solution and DMSO.
4.5. Membrane perturbation assay
The membrane disruption assay was performed as described by Morão and co-
authors [20]. The analysis was carried out with the Live/Dead Baclight Bacterial
Viability kit (Thermo-Scientific L7012). All analyses were performed at 15 and 30 min.
All pictures were taken using an Olympus BX-61 microscope equipped with an
OrcaFlash-2.8 monochromatic camera (Hamamatsu) and guided by the CellSens
software version 11. Experiments were performed in triplicates, and the quantification
analyses considered at least 200 individuals (n=200) per treatment.
4.6. Anti-FtsZ assay
FtsZ of Bacillus subtilis (BsFtsZ) was expressed and purified using the
ammonium sulfate precipitation method as described previously [63]. The BsFtsZ GTP
hydrolysis rate was determined using the malachite green phosphate assay described by
13

Król and co-authors [63] with minor modifications. Stocks of DHC at MIC and 2×MIC
with FtsZ (24 µM), MgCl2 (20 mM) and Triton X-100 (0.02 %) prepared in
polymerization buffer (Hepes 50 mM, KCl 300 mM, pH 7.5) were stabilized at 30 ºC
for 5 min. Then, GTP (2 mM) dissolved in the polymerization buffer was added in
different time points (the final concentrations were half of the stock solutions). The
reactions were kept at 30 ºC and developed in accordance with MAK307 (Sigma-
Aldrich®) kit. 1% DMSO was used as negative control.
4.7. Cytotoxicity assay
The toxicity against human cells was evaluated according to Silva and co-
authors [64]. The cell lines used were HepG2 (human liver carcinoma cell line ATCC
HB-8065), MRC-5 (normal human lung fibroblast cell line ATCC CCL-171) and A549
(human lung adenocarcinoma epithelial cell line ATCC CCL-185). For the cytotoxicity
assay, 2.5 × 104 cells/mL were seeded into a 96-well cell culture plate with a total
volume of 100 µL for 24 h. Cells were treated with curcumin and DHC in
concentrations ranging from 0.8 to 100 µg/mL. After 24 h incubation, the medium was
removed, and 50 µL of resazurin (0.01% w/v) was added to each well, and the plates
were incubated at 37 °C for 3 h. The absorbance was measured with a microplate reader
in which an excitation (530 nm) and an emission (590 nm) filters were used. Three
independents assays were performed. IC50 value that represents the concentration
required to reduce the viability of cells at 50% was calculated from an analytical curve
through a regression analysis. Doxorubicin was used as reference cytotoxic drug.
4.8. Genotoxicity assay
The genotoxicity of curcumin and DHC was evaluated using the alkaline
version of the comet assay (single cell gell electrophoresis) by Tice and co-authors
protocol [56], with minor modifications [64]. For the comet assay, 2.5 × 104 A549
cells/well were seeded and treated with DHC and curcumin in concentrations at ½ IC50
and ¼ IC50 values for 24 h. After resuspending the cells, the following steps of the assay
were carried out: (i) the microscope slides were coated with normal-melting-point
agarose; (ii) the treated cell suspension was transferred to a microcentrifuge tube; (iii)
20 μL cells were suspended in 100 μL of a 0.5 % low-melting agarose solution at 37 °C;
(iv) 100 μL of this suspension were placed on the slides, which had been kept in freezer
14

for 5 min. In summary, 100 μL cells were taken, homogenized with low-melting point
agarose, spread on microscope slides, which were pre-coated with normal-melting-point
agarose and covered with a coverslip. After 5 min at 4 °C, the coverslip was removed
from the slides and they were immersed in cold lysing solution (2.4 M NaCl; 100 mM
EDTA; 10 mM Tris, 10% DMSO and 1% Triton-X, pH 10) for 24 h. After the lysis, the
slides were placed in electrophoresis chamber, covered with electrophoresis buffer
(300 mM NaOH plus 1 mM EDTA, pH > 13) and left for 20 min for DNA to unwind.
The electrophoresis ran for 20 min (25 V and 300 mA), after which slides were
submerged for 15 min in neutralization buffer (0.4 M Tris–HCl, pH 7.5) and fixed in
ethanol. Duplicate slides were stained with SYBR Green and 50 cells per sample were
screened in fluorescent microscope equipped with excitation filter of 515–560 nm,
barrier filter of 590 nm and objective (magnification × 40). The level of DNA damage
was assessed by image analysis system (TriTek CometScore™ 1.5, 2006) and the
percentage of DNA in the tail and tail moment were calculated. The results were
expressed as the mean ± standard error of three independent experiments performed in
duplicate.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgments
The authors gratefully acknowledge financial support from the Coordination
for the Improvement of Higher Education Personnel (CAPES, finance code 001),
Brazilian Council for Scientific and Technological Development (CNPq) (Grants
471129/2013-5, 306251/2016-7 and 429322/2018-6), the São Paulo Research
Foundation (FAPESP) (Grants 2013/07296-2, 2013/50367-8, 2014/18330-0,
2015/08089-6, 2015/50162-2, 2017/14936-9, 2017/50216-0 and 2018/15083-2), the
Netherlands Organization for Scientific Research (NWO, 729.004.005) and Multiuser
Centre for Biomolecular Innovation (FAPESP 2009/53989-4). CRP thanks National
Institute of Science and Genomics Technology for Citrus Improvement (INCT Citrus)
and CAPES (finance code 001) for his schoolarships (Grants FAPESP 2014/50880-0
and CNPq 465440/2014-2).
15

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1. B2O3, DMF, 60 °C
2. (n-BuO)2B, 60 °C
3. ArCOH, DMF, 60 °C
4. CH3(CH2)4NH2, HOAc, 90 °C
5. HOAc, H2O, 70 °C
O
OH
O
O
Ar
Ar
57-95%
Scheme 1. One-pot synthesis of curcuminoids 1–12. Ar = phenyl ring substituted by
electron-donating groups or electron-withdrawing groups.
25

Table 1. Antibacterial and antitubercular activities of curcumin and curcuminoids 1–13
expressed as MIC and MBC values in µg/mL
O
OH
R
R
Ef
MIC MBC MIC MBC MIC MBC MIC
156 >156 156 >156 78 156 >156
> 156 >156 >156 >156 > 156 >156 >156
> 156 >156 >156 >156 > 156 >156 >156
> 156 >156 >156 >156 > 156 >156 >156
> 156 > 156 >156 > 156 > 156 > 156 >156
Sa
Bs
Mt
Compound
R
curcumin
m-OMe,p-OH
H
1
2
3
4
m-OMe
p-OH
p-OMe
m-OH
p-Me
DHC or 5
78
156
156
>156
78
156
156
6
7
8
9
10
11
12
> 156 > 156 >156 > 156 > 156 > 156 >156
> 156 > 156 >156 > 156 > 156 > 156 >156
> 156 > 156 >156 > 156 > 156 > 156 >156
> 156 > 156 >156 > 156 > 156 > 156 >156
> 156 > 156 >156 > 156 > 156 > 156 >156
> 156 > 156 >156 > 156 > 156 > 156 >156
> 156 > 156 >156 > 156 > 156 > 156 >156
> 156 > 156 >156 > 156 > 156 > 156 >156
p-N(Me)2
p-F
p-Cl
p-Br
p-CF3
m,p-diCl
m-OAc
─
13
ciprofloxacin
isoniazid
0.25
0.25
0.015
0.03
0.015 0.015
─
─
─
─
─
─
─
0.04
─
MIC = minimal inhibitory concentration; MBC = minimum bactericidal concentration;
Ef = Enterococcus faecalis; Sa = Staphylococcus aureus; Bs = Bacillus subtilis and Mt
= Mycobacterium tuberculosis; ─ not determined.
26

Fig. 1. Chemical stability of DHC and curcumin at 13.5µg/mL (pH 5.0 and 7.4) by
monitoring the decrease in their maximum absorbance at 37 °C for 24 h. Data are
represented as means ± SEMs and expressed as percentage.
27

A
C
B
D
Fig 2. Fluorescence microscopy of B. subtilis strain 168 stained with Live/Dead
Baclight kit. A) negative control (cells treated with 1% DMSO); B) positive control
(cells treated nisin at 5 µg/mL, after 30 min) C) DHC (cells treated at MIC, after 15
min); D) DHC (cells treated at MIC, after 30 min). Magnification 100 ×; bar 5 μm.
28

120
100
80
60
40
20
0
***
***
***
NC
PC
DHC - 15 min DHC - 30 min
Fig 3. Percentage of permeabilized cells of B. subtilis. NC: negative control (cells
treated with 1% DMSO); PC: positive control (cells treated with nisin at 5 µg/mL, after
30 min); DHC (cells treated at MIC, after 15 and 30 min). *** p < 0.05, ANOVA with
Dunnett’s post-test.
29