In vitro and in vivo anti-inflammatory properties of imine resveratrol analogues

Article abstract of DOI:10.1016/j.bmc.2018.08.029

Resveratrol is a natural polyphenol found mainly on red grapes and in red wine, pointed as an important anti-inflammatory/immunomodulatory molecule. However, its bioavailability problems have limited its use encouraging the search for new alternatives agents. Thus, in this study, we synthetize 12 resveratrol analogues (6 imines, 1 thioimine and 5 hydrazones) and investigated its cytotoxicity, antioxidant activity and in vitro anti-inflammatory/immunomodulatory properties. The most promising compounds were also evaluated in vivo. The results showed that imines presented less cytotoxicity, were more effective than resveratrol on DPPH scavenger and exhibited an anti-inflammatory profile. Among them, the imines with a radical in the para position, on the ring B, not engaged in an intramolecular hydrogen-interaction, showed more prominent anti-inflammatory activity modulating, in vivo, the edema formation, the inflammatory infiltration and cytokine levels. An immunomodulatory activity also was observed in these molecules. Thus, our results suggest that imines with these characteristics presents potential to control inflammatory disorders.

Full text of DOI:10.1016/j.bmc.2018.08.029

Accepted Manuscript
In vitro and in vivo anti-inflammatory properties of imine resveratrol analogues
Danielle Cristina Zimmermann-Franco, Bruna Esteves, Leticia Moroni Lacerda,
Isabela de Oliveira Souza, Juliana Alves dos Santos, Nicolas de Castro Campos
Pinto, Elita Scio, Adilson David da Silva, Gilson Costa Macedo
PII:
S0968-0896(18)30884-8
https://doi.org/10.1016/j.bmc.2018.08.029
BMC 14516
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
6 June 2018
14 August 2018
23 August 2018
Please cite this article as: Cristina Zimmermann-Franco, D., Esteves, B., Moroni Lacerda, L., de Oliveira Souza, I.,
Alves dos Santos, J., de Castro Campos Pinto, N., Scio, E., David da Silva, A., Costa Macedo, G., In vitro and in
vivo anti-inflammatory properties of imine resveratrol analogues, Bioorganic & Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.bmc.2018.08.029
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In vitro and in vivo anti-inflammatory properties of imine resveratrol analogues
Danielle Cristina Zimmermann-Franco1, Bruna Esteves1, Leticia Moroni Lacerda1,
Isabela de Oliveira Souza2, Juliana Alves dos Santos2, Nicolas de Castro Campos Pinto3,
Elita Scio3, Adilson David da Silva2, Gilson Costa Macedo1,*
1- Departamento de Parasitologia, Microbiologia e Imunologia, Instituto de Ciências
Biológicas, Universidade Federal de Juiz de Fora, Campus Universitário, Bairro
Martelos, Juiz de Fora, Minas Gerais 36036-900, Brazil.
2- Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Juiz
de Fora, Campus Universitário, Bairro Martelos, Juiz de Fora, Minas Gerais 36036-
900, Brazil.
3- Departamento de Bioquímica, Instituto de Ciências Biológicas, Universidade Federal
de Juiz de Fora, Campus Universitário, Bairro Martelos, Juiz de Fora, Minas Gerais
36036-900, Brazil.
* Corresponding author: Gilson Costa Macedo, gcmbio@gmail.com
1

ABSTRACT
Resveratrol is a natural polyphenol found mainly on red grapes and in red wine, pointed
as an important anti-inflammatory/immunomodulatory molecule. However, its
bioavailability problems have limited its use encouraging the search for new alternatives
agents. Thus, in this study, we synthetize 12 resveratrol analogues (6 imines, 1 thioimine
and 5 hydrazones) and investigated its cytotoxicity, antioxidant activity and in vitro anti-
inflammatory/immunomodulatory properties. The most promising compounds were also
evaluated in vivo. The results showed that imines presented less cytotoxicity, were more
effective than resveratrol on DPPH scavenger and exhibited an anti-inflammatory profile.
Among them, the imines with a radical in the para position, on the ring B, not engaged
in an intramolecular hydrogen-interaction, showed more prominent anti-inflammatory
activity modulating, in vivo, the edema formation, the inflammatory infiltration and
cytokine levels. An immunomodulatory activity also was observed in these molecules.
Thus, our results suggest that imines with these characteristics presents potential to
control inflammatory disorders.
KEYWORDS: Resveratrol; analogues; imine, anti-inflammatory; immomodulatory
2

ABBREVIATIONS USED:
1
13C NMR, carbon-13 nuclear magnetic resonance; H NMR, proton nuclear magnetic
resonance; 5-LOX, 5-lipoxygenase; AP-1, activator protein 1; AR, resveratrol analogs;
BSA, Bovine serum albumin; CCL-2, C-C motif chemokine ligand 2; CD, cluster of
differentiation; COX, cyclooxigenase; DMSO, dimethyl sulfoxide; DPPH, 2,2-diphenyl-
1-picrylhydrazyl; EDTA, Ethylenediamine tetraacetic acid; ELISA, Enzyme-Linked
Immunosorbent Assay; HE, hematoxylin and eosin; HTAB, hexadecyl trimethyl
ammonium bromide; IC, inhibitory concentration; IFN-γ, Interferon-γ; IL, interleukin;
iNOS, Nitric oxide synthases; LPS, Lipopolysaccharide; MCP-1, monocyte
chemoattractant protein 1; MHC, major histocompatibility complex; MPO,
myeloperoxidase;
MTT,
(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide); NF-κB, nuclear factor-κB; NO, nitric oxide; OD, optical density; OPD,
Ophenylenediamine; PMSF, phenylmethylsulfonyl fluoride; SD, standard desviation; TH,
T helper cell; TMS, tetramethylsilane; TNF-α, tumor necrosis factor- α; v/v, volume (of
solute) per volume (of solvent).
3

1 INTRODUCTION
Resveratrol (3,5,4′-trihydroxystilbene) is a natural polyphenolic phytoalexin
stilbene that naturally occurs in cis and trans isomeric forms. This compound may be
isolated from several food sources as peanuts, gramberries and blueberries. However,
higher concentrations may be found on skins and seeds of red grapes and in red wine.1,2
Several studies have shown that resveratrol presents a vast list of beneficial
effects, including antioxidant3, protection against heart disease,4-6 antitumor,7-10 anti-
inflammatory,11-16 antiaging17 and immunomodulatory activities.13 Resveratrol is also
endowed with the ability to enhance antitumor activities of commercial medicine as
melphalan,18 herceptin19 and tamoxifen.20
Regarding to inflammation, previous studies reports that resveratrol is able to
inhibit the synthesis and release of several pro-inflammatory mediators, such as nitric
oxide (NO)5,16 and a wide range of cytokines (as IL-1β, IL-6, IL-12 and TNF-α,).11, 12, 21-
23
Moreover, resveratrol induces modifications of eicosanoid production and suppress
important ezymes involved in synthesis of
pro-inflamatory mediators, as
cyclooxygenase-1 (COX-1) or cyclooxygenase-2 (COX-2).24 The mechanisms
responsible for its biological activities are not completely elucidated, however they are
probably related to modulation of transcription factors as nuclear factor – κB (NF-κB)
and activator protein-1 (AP-1).11,25,26 Resveratrol also presents immunosuppressive
activities through down-regulation of costimulatory molecules (CD80 and CD86) and
CD28, inhibition of B and T lymphocyte proliferation and function of T helper -1 (TH1)
and TH2 cells.15
4

Despite of several benefits presented by resveratrol, this compound shows, in
mammalian, a very low bioavailability, being quickly eliminated. Its metabolic instability
has restrict its use in vivo. 27 To solve this problem, several studies have been performed
in an attempt to obtain synthetic analogues of resveratrol with improved bioavailability,
maintaining its desirable characteristics.28, 29
Thus, in this study, 12 compounds (imines, thioimine and hydrazones) analogues
of resveratrol (AR) were produced (Fig. 1) and evaluated for its cytotoxicity. The less
cytotoxic were evaluated for anti-oxidant, anti-inflammatory and immunomodulatory
activities in vitro. The most promising analogues also were tested in vivo.
R1
R2
A
R1
HN N
OH
OH
B
R3
HO
R5
R2
ring A
XH
R1
ring B
N
A
B
R3
Resveratrol
R5
R4
12 resveratrol analogues
Figure 1: Chemical structure of the resveratrol and resveratrol analogues
2 RESULTS
2.1 Cytotoxicity of resveratrol analogues
In order to determine the cytotoxicity of resveratrol analogues, RAW 264.7 cells
were treated with the analogues at 25 or 50 µM, and the viability was evaluated after 48
hours. As showed in Fig. 2, at 50 µM, the analogues AR16, AR32, AR35, AR38, AR39
and AR76 induced significant cell viability reduction when compared with not-treated
cells or with resveratrol and thus, were excluded. As expected, resveratrol did not show
5

significant cytotoxicity on evaluated concentrations. Decrease of cell viability by
resveratrol was only observed in concentrations of 100 µM or higher (data not shown).
No difference was found between non-treated cells and cells treated with ethanol (1%)
used as solvent.
30
As the cell lines may differ from the primary cells in diverse aspects,
the
cytotoxicity of the resveratrol analogues (50 µM) also was evaluated in peritoneal
macrophages from C57BL/6 mice. The results were similar to those of RAW264.7 cells
(data not show).
Figure 2. Effects of resveratrol and analogues on cell viability. RAW 264.7 cells (2 x 105
cells/well) were treated with resveratrol (RVT) or analogues (AR) (25 µM or 50 µM) for 48 hours.
Cell viability was evaluated by MTT assay and expressed as mean SD relative to untreated
control (NT) (*p<0.05 vs NT, #p<0,05 vs RVT, ANOVA/Dunnet).
2.2 Antioxidant Activity of resveratrol analogues
As several studies suggest a relation between the radical scavenging activity
presented by resveratrol and its beneficial properties,31 the antioxidant potential of
resveratrol analogues was assessed by DPPH assay. The table 1 report that all resveratrol
analogues were more effective DPPH scavengers than resveratrol (IC50: 65.6 µM). The
most prominent antioxidant compounds were AR08, AR13, AR25 and AR26. These
6

analogues exhibited a very similar activity, presenting an IC50 ranging between 34.1 µM
to 36.6 µM.
Table 1. Effects of resveratrol and analogues on ion capture DPPH (IC50).
COMPOUND
AR07
IC50 (µM)a
45.8 3.3*
35.9 3.2#
34.5 1.6#
49.1 5.3*
36.6 2.9#
34.1 1.9#
65.6 3.2&
AR08
AR13
AR22
AR25
AR26
RVT
aValues were expressed as inhibitory concentration of 50% DPPH· ions (IC50 µM). Different
symbols (*, # or &) indicate significant differences among them. Results are representative of
three independent experiments (p<0,05; one-way ANOVA/Tukey). RVT- resveratrol
2.3 Effect of resveratrol analogues on inhibition of pro-inflammatory mediators
Has been evidenced that in vitro treatment with resveratrol can reduce NO
16, 32
levels.11,
Thus, the effect of the analogues on NO production by LPS+IFN-γ
stimulated RAW 264.7 cells was evaluated. As evidenced on Table 2, treatment with
resveratrol (50 µM) was able to induce a significantly decrease on NO production (around
25%), compared to stimulated cells. However, in a very interesting way, all imines
exhibited stronger inhibitory activity than resveratrol. The compounds AR07, AR08,
AR13, AR22 and AR25 were the most effective, reducing the NO levels in approximately
90% (p<0,05, Tukey test).
7

In order to investigate whether imines also affect cytokine/chemokine production,
the levels of IL-1β, IL-6, IL-12, TNF-α and CCL-2 (MCP-1) produced by LPS stimulated
cell lines (RAW 264.7 or J774A.1) were examined. In this assay, J774A.1 cells were used
to evaluate IL-1β production since RAW264.7 is a poor producer of this cytokine.33
The results confirm that resveratrol presents a prominent anti-inflammatory
profile, decreasing the levels of all inflammatory mediators evaluated (Table 2).
Consistently, the analogues also reduced the production of IL-1β, IL-6, IL-12 and CCL-
2. However, the influence on IL-12 and TNF-α production was noticeable. All analogues
were powerful to reduce IL-12 production (better than resveratrol) suggesting an ability
to influence the TH1 profile. Despite of this, only the analogues AR07, AR22 and AR25
were effectives to reduce TNF-α levels, suggesting that these compounds can be the more
effective to modulate the inflammatory response.
Table 2: Influence of resveratrol and analogues on pro-inflammatory mediators production
NO
CCL-2
IL-6
IL-12
TNF-α
IL-1β
(pg/mL)b
(µM)
(ng/mL)
(ng/mL)
(pg/mL)
(ng/mL)
0.4 0.09*
#
2,90 0,0*
0,4 0,1*
2,9 0,3
2,1 0,2*
38,7 49,5*
15,4 3,6*
58,6 7,4
44,2 4,7*
58,4 6,7
#
#
#
Medium
ND
55.0 2.6
Stimulusa
AR07
12,73 0,10
7,90 0,04*
7,20 0,10a
382,3 50,0
4839,2 2,2
4.8 0.5*
5.9 0.8*
8.1 0.3*
#
#
#
88,2 11,4*
62,3 27,1*
4,7 0,3*
#
#
2,5 0,2*
2,4 0,1*
2,3 0,1*
2,2 0,2*
2,3 0,2*
1,6 0,1*
5,5 2,1*
#
#
AR08
AR13
AR22
AR25
AR26
RVT
9,70 0,05*
5,50 0,08*
120,3 28,1*
188,3 11,5*
7,7 0,6*
#
#
60,5 0,8
14.6 0.9*
#
9,3 1,5*
44,8 4,7*
#
9.5 1.0*
#
3,70 0,04*
6,10 0,05*
6,60 0,01*
53,4 16,7*
12,7 1,3*
30,6 3,9*
57,4 3,1
#
#
#
22.6 0.8*
44.5 2.6*
#
76,7 16,3*
34,0 7,5*
#
#
159,7 33,5*
1036.0 99,6*
31,0 0,8*
8

aRAW 264.7 cells were pre-stimulated with LPS or LPS + IFN-γ to determine cytokines or NO
production, respectively. One hour later, the cells were treated with resveratrol or analogues (50
µM). The levels of IL-6, IL-12, TNF-α, CCL-2 or NO were determined on supernatant after 48
hours. Data are expressed as mean SD of quadruplicates. ND – Not determined.
bJ774A.1 cells were used to evaluate IL-1β production after 24h of treatment, following the same
protocol. Data are expressed as mean SD of quadruplicates.
* p<0,05 vs stimulus; # p<0,05 vs resveratrol (RVT); one-way ANOVA/Tukey;
2.4 Anti-proliferative activity of resveratrol analogues
An important characteristic of resveratrol is its ability to inhibit proliferation of T
and B cell.12, 15 Thus, some experiments were performed to determine whether the
analogues also are able to influence on in vitro lymphocytes proliferation. Therefore,
splenocytes from C57BL/6 mice were stimulated with LPS (a B-cell mitogen) or
Concanavalin A (a T-cell mitogen) and treated with the analogues. The percentage of
proliferation was determined after 72h by MTT assay.
As expected, resveratrol was effective to inhibit both LPS or Con A-induced
proliferation (Figure 3). A similar behavior was observed after treatment with the
analogues AR07, AR08 and AR25 that reduced the splenocyte proliferation similar to
resveratrol. The analogue AR26 also influenced the proliferation but only after LPS
stimulation. These results suggest that, as resveratrol, the analogues can modulate the B
and T lymphocyte proliferation.
9

Figure 3: Anti-proliferative activity of resveratrol analogues: Splenocytes from C57BL/6
were stimulated with LPS (A) or Concanavalin A - ConA (B) and treated with resveratrol or
analogues (50µM). After 72 hours, the proliferation was evaluated by MTT assay. Data are
presented as the mean SD of percentage of proliferation relative to untreated stimulated cells
(ConA or LPS). *p<0,05 vs untreated stimulated cells, one-wayANOVA/Dunnet.
2.5 Down-regulation of MHC II and costimulatory molecules
As resveratrol and analogues were able to reduce the lymphocyte proliferation and
cytokines production by macrophages, their influence on antigen presentation was
determined through the modulation of costimulatory molecules (CD80/CD86) or MHC
II.
As expected, LPS-activated macrophages significantly up-regulate the expression
of CD80, CD86 and MHCII compared to non-stimulated cells. According with previous
reports, the treatment with resveratrol reduced the expression of CD80 (Fig. 4.A) and
CD86 (Fig. 4.B) but did not affect MHCII (Fig. 4.C) levels.15 Regarding analogues,
10

except for AR08 and AR26, all the others were able to reduce CD80 expression compared
to stimulated cells. Nevertheless, only the analogues AR07 and AR25 were able to down-
modulate CD86 expression. This indicates that these compounds can influence T cell
activation by disturbing the optimal antigen presentation. As resveratrol, none analogue
affected the MHCII expression.
Figure 4: Modulation of MHC and costimulatory molecules. Flow cytometry analysis of LPS
(1ug/mL) stimulated Raw264.7 cells after treatment with resveratrol or analogues (50uM). Data
are expressed as MFI (mean fluorescence intensity SD) of the expression of CD80 (A), CD86
(B) or MHC II (C). Results are representative of two independent experiments. *p<0,05 vs
untreated cells (LPS).
2.6 Effect of resveratrol analogues on in vivo acute inflammation
11

Due the encouraging results presented by of AR07 and AR25, their anti-
inflammatory activity were also evaluated in vivo by croton oil-induced ear edema model
in mice. As shown in Fig. 5, the topical treatment with resveratrol or analogues inhibited
the edema by approximately 40%. Dexamethasone (Dexa), used as a reference drug, was
more effective, reducing the edema by90%. This edema reduction induced byresveratrol,
analogues or Dexa was followed by a considerably decrease of leucocyte infiltration,
when compared to non-treated mice (Fig. 6A-E). These results demonstrate that the
topical application of AR07 and AR25 analogues exhibited similar anti-inflammatory
effects than resveratrol.
The analysis of pro-inflammatory mediators on ear tissue showed that, in vivo,
resveratrol and analogues were not able to influence the production of IL-1β. However,
they were very effective to decrease myeloperoxidase activity. Nevertheless, despite of
similar effect on ear edema, AR07 and AR25 were also able to reduce the production of
IL-6 and CCL-2, while resveratrol not affect its cytokines (Fig. 7). These results
suggested that these analogues can be more effective than resveratrol to modulate chronic
inflammation due to their action in reducing the production of important inflammatory
cytokines in vivo.
12

Figure 5: Effect of resveratrol and analogues in mice ear edema. Mice ear edema was induced
by topical application of croton oil on the inner surface of right ear, followed by topically
treatment with 0,1 mg/ear of resveratrol, analogues or Dexamethasone. Left ears were used as
control. Values in each column represent the mean S.D. of the weight difference between right
and lefts ears. One-way ANOVA followed by Newman–Keuls test was used for statistical
analysis. (* p<0,05; **p<0,01; *** p<0,001 vs vehicle).
13

Figure 6: Representative photomicrographs of sections of mice ear plugs from
croton oil induced ear edema test. Mice ear edema was induced by topical application
of croton oil followed by treatment with (A) vehicle; (B) Dexamethasone; (C) AR07; (D)
AR25 or (E) resveratrol for 4 h. The arrows indicate infiltration of inflammatory cells.
(400x magnification, H&E staining)
Figure 7: Effect of resveratrol and analogues on pro-inflammatory mediators levels in ear
tissue of croton oil induced ear edema test. Levels of (A) IL-1β; (B) IL-6; (C) CCL-2 and (D)
Myeloperoxidase activity was represented by the mean S.D. a p<0,05 vs vehicle, One-way
ANOVA followed by Newman–Keuls.
14

3 DISCUSSION
Resveratrol is a very promising compound responsible to a wide range of
beneficial actions mainly on inflammatory process. However, its poor bioavailability
have refrained a possible therapeutic use. Thus, the present study evaluated a series of
resveratrol analogues on immune effector cell function in order to find new alternative
compounds. An important advantage of the synthetics analogues is to offer a
supplementary source of resveratrol-like anti-inflammatory compounds in a satisfactory
amount. Furthermore, analogues can maintain or improve the qualities of the precursor
molecule and correct structural problems as well as the bioavailability.34-36
On this context, 12 synthetic polyphenols - hydrazones, imines and thioimines-
analogues from resveratrol were produced. The CH group on C=C of resveratrol was
replaced by a nitrogen atom and hydrazine group, obtained the Aza-stilbene C=N31 and
hydrazone skeleton. As previously reported,36 this replacement facilitates the generation
of resveratrol analogues and increases the possibility to develop compounds with better
bioavailability keeping the fundamental properties of precursor compound.31,36 Among
the analogues, only the imines showed satisfactory cytotoxicity and were selected to
further investigations. These results suggest that the present of hydrazone (AR16, AR32,
AR35, AR38 and AR39) and/or nitro radical (AR32, AR38, AR39 and AR76) in the
structure can increase the toxicity of the compound for in vitro cultured cells.
Although the exact mechanism by which resveratrol exerts beneficial effects is
not completely understood, its prominent antioxidant activity seems to be essential,
influencing important pathways related with immune response as well as production of
reactive oxygen species (ROS) and activation of NF-κB.37-40 As showed on Table 1, all
the imine have a prominent antioxidant activity compared to resveratrol. Thus, since this
15

ability seems to be related to suppression of inflammation,38 it may to explain many
properties of resveratrol analogues associated to preventing several oxidative stress-
related diseases.
Inflammation is a common risk in the pathogenesis of several conditions such as
infections, arthritis, type 2 diabetes mellitus, obesity and cancer.37 Therefore,
pharmacological modulation of this process is desirable. Thus, the anti-inflammatory
activity of the imine resveratrol analogues was evaluated. The focus was primarily on
macrophages due to it central role on inflammation process through the production of
several inflammatory mediators (as NO, cytokines and chemokines), largely involved on
many pathophysiological events associated with inflammatory diseases.25,26,41,42 In this
context, modulation of macrophage-mediated inflammatory responses is very important
to create a new therapeutic approach against inflammatory diseases.43
An overall analysis of the pro-inflammatory mediators produced by macrophages
reveals that, as resveratrol, the imine exhibited a potential to modulate the inflammatory
response since are able to reduce the production of NO, IL-1β, IL-6, TNF-α and CCL-2
that are crucial molecules to establish an inflammatory process. In this context, previous
studies showed that resveratrol might inhibit diverse pathways related to the
inflammatory response as AP-1, NF-kB and proteasome pathways.44 These inhibition
results on decrease of pro-inflammatory cytokines production (mainly IL-1β, IL-6 and
TNF-α) as well as on suppression of inducible nitric oxide synthase gene (iNOS).25,45
Thus, as the analogues present very similar structures to resveratrol and showed similar
behavior on inflammatory mediators, is plausive to suppose that the new compounds also
act on AP-1, NF-kB and proteassome. However, more studies are necessary to confirm
this hypothesis.
16

Although NO is an important mediator to combat infectious organisms and to
regulate the functional activity, growth and death of many immune and inflammatory cell
types,46 its action also increased the inflammatory response and it is associated with
several diseases, including autoimmune pathologies.47 Thus, decreasing NO levels has a
direct impact in the reduction of inflammation and its damage. As demonstrated (Table
2), the analogues are more effective on NO reduction than resveratrol. These finds suggest
that analogues are more potent to inhibit NF-kB and iNOS enzyme (as previously
described for resveratrol)45 or that they can act by a mechanism other than that proposed
for resveratrol. Despite of this, the analogues demonstrate more potential to prevent
damages caused by exacerbation of NO production.
Regarding IL-12 production, in a very interesting way, the analogues were notably
more powerful than resveratrol, reducing its production to basal levels. IL-12 is a
heterodimeric pro-inflammatory cytokine critical for the differentiation of TH1 cells and
consequent production of IFN-γ.23 Several studies demonstrate that IL-12 is important in
the pathogenesis of diverse autoimmune disease such as multiple sclerosis48, rheumatoid
arthritis,49 inflammatory bowel disease50 and others51,52. Thus, the substantial reduction
of IL-12 induced by resveratrol analogues suggest a beneficial effect on control of these
pathologies. Furthermore, considering that IL-12p40 subunit (evaluated in the present
study) is also part of IL-2323 which in turn is important to maintenance of TH17 cells,22
our results suggest that the analogues can be able to modulate the TH1 and TH17 profiles.
In accordance, Imler and Petro, 200953 demonstrate that resveratrol decrease the severity
of animal model of Multiple Sclerosis and this effect was associated to reduction of IL-6
and IL-12/IL-23p40 subunit.
Some studies have shown that resveratrol inhibits the proliferation of spleen cells
induced by LPS,15 Concanavalin-A, IL-2, or alloantigens.12 In agreement, we also find a
17

significant reduction on splenocytes proliferation after treatment with resveratrol or
analogues. However, only compounds AR07, AR08 and AR25 presented behavior similar
to resveratrol reducing both T and B cell proliferation. These results suggest that, as
resveratrol, the analogues also are able to modulate cellular and humoral immune
response. This characteristic of the analogues can be associated to inhibition of NF-kB
pathway and reduction of cytokines production (mainly IL-2) as already demonstrated for
resveratrol12. The antioxidant properties can also contribute to this result.54,55
Costimulatory molecules, as CD80 and CD86, play a key role on regulation of T-
cell activation and its absence can prevent the immune cell function.56 In this study, all
analogues were able to decrease the expression of CD80 but only AR07 and AR25
reduced the CD86 expression. Thus, AR07 and AR25 could suppress the activation of T
cells by down-regulating crucial costimulatory molecules present on antigen-presentating
cells (as macrophages). This immunomodulatory effect is important on treatment of
autoimmune disease or hypersensitivity reactions.
Summarizing, the in vitro results indicated that, among the imine, AR07 and AR25
are the most promising once decrease all inflammatory mediators (NO, cytokines and
chemokines) as well as cellular proliferation and costimulatory molecules
(immunomodulation). Thus, these compounds present potential to modulate
inflammatory and/or autoimmune disease and were selected for in vivo tests.
Structurally, the analogue AR07 presents a methoxy group in the para position on
the ring B, not compromised with an intramolecular hydrogen interaction. A similar
structure can be observed in AR25 that shown a carboxyl group on the ring B, also in the
para position and not involved in an intramolecular hydrogen bond. Thus, this
characteristic seems to favor the anti-inflammatory activity. In comparison, AR13 also
presents a methoxy group in the para position on the ring B, however the presence of the
18

OH in the meta position compromise the methoxy group with a hydrogen bond, reducing
the activity. Regard the AR26, it also present a radical carboxyl in the structure (on the
ring B) however, in the ortho position. Again, this position favor the intramolecular
hydrogen interaction that seems to compromise the radical. Consistent with this
hypothesis, the compound AR22 present a radical (hydroxyl) in the para position not
engaged in intramolecular hydrogen bound. This analogue also demonstrate a good anti-
inflammatory property reducing the production of all mediators evaluates.
Croton oil is a potent activator of diverse pathways related to inflammatory
response as NF-κB and AP-1, which are important to the production of inflammatory
cytokines as TNF-α, IL-6 and IL-1β. Furthermore, this irritant agent also activate
phospholipase A2 which induce aracdonic acid formation and consequently, the
production of prostaglandin and leukotrienes by cyclooxygenase (COX) and 5-
lipoxygenase (5-LOX) enzymes respectively57. These inflammatory mediators induce a
rapid and intense inflammatory response evidence by edema and leukocyte migration.58,59
The treatment of croton oil-induced inflammation with AR07, AR25 or resveratrol
showed very similarities. All of them reduced the edema and the accumulation of
inflammatory cells in a similar way. These results can be associated with the suppression
of eicosanoids production since resveratrol is a potent COX inhibitor.60 In addition, the
analogues also reduced the activity of MPO, an enzyme considered as a marker of
polymorphonuclear leukocyte influx into inflamed tissues, contributing to decreasing of
inflammatory infiltrate.61
As previously reported,62-64 resveratrol can modulate important pathways related to
inflammatory process resulting on reduction of inflammatory mediators. This
characteristic was confirmed in our in vitro studies. However, resveratrol not show the
same behavior in vivo, not affecting the levels of cytokines/chemokines. These
19

discrepancies can be related to the resveratrol concentration used, administration route
(topical) or the time between treatment and analyses (only 4 hours). In contrast, AR07
and AR25 were effective to reduce the production of IL-6 and CCL-2 also in vivo. These
results suggest that analogues can be more effective than resveratrol to down-modulate
the cytokine production pathways (as NF-κB and AP-1) or can influence another
pathways not described for resveratrol. Although more experiment are necessary to clear
this question, these finds reinforce the hypothesis that AR07 and AR25 can be useful to
control inflammatory problems.
Ultimately, despite of these encouraging results, is important to emphasize that, in
ear edema model, the compounds were topically administered, so that they were not
submitted to previous metabolism. Thus, as resveratrol presents bioavailability
problems65 and discrepant in vitro and in vivo immunological effects13, it is relevant to
perform a new parallel between resveratrol and analogues using others administration
routs.
4 CONCLUSION
This study showed that the presence of a radical in the para position on the ring
B, not engaged with intramolecular hydrogen bond could facilitate the anti-inflammatory
properties of the imines resveratrol analogues. Furthermore, although the mechanisms of
action are not fully elucidated, our results point the analogues AR07 and AR25 as
potential alternatives to control inflammatory diseases since their modulate parameters of
innate and acquired immunity.
20

5 EXPERIMENTAL SECTION
General procedures: Drug synthesis was performed as previously reported by Da Silva
et al. 2016.66 In brief, the synthesis of the resveratrol imine analogues (series AR07,
AR08, AR13, AR22, AR25, AR26, AR76) and resveratrol hydrazine analogues (series
AR16, AR32, AR35, AR38 and AR39) were obtained by condensation of aromatic
aldehydes with the equimolar amount of 2-hydroxyaniline, phenylhydrazine, 2,4-
dinitrophenylhydrazine or 2-aminothiophenol in ethanol, at room temperature (Scheme
1), as solids after being filtered, washed with ethanol and dried in an oven (yield 33-90%).
OH
NH2
R1
R2
R4
OH
AR07 R3 = OMe, R1 = R2 = R4 = R5= H
AR08 R5 = OH, R1 = R2 = R3 = R4 = H
AR13 R2 = OH, R3 = OMe, R1 = R4= R5 = H
AR22 R5 = R3 = OH, R1 = R2= R4 = H
AR25 R3 = CO2H, R1 = R2 = R4 = R5= H
AR26 R1 = CO2H, R2 = R3 = R4 = R5= H
R1
R2
R4
EtOH
r.t.
O
N
R3
R3
R5
R5
2-hydroxyaniline
R1
aromatic aldehydes
R1
R2
AR16 R1 = R2 = H; R5 = OH; R3 = OMe
AR35 R1 = R2 = R5 = H; R3 = CO2H
AR32 R1 = NO2; R2 = OH; R3 = OMe
AR38 R1 = NO2; R2 = R5 = H; R3 = CO2H
AR39 R1 = NO2; R2 = Br; R3 = H; R5 = OH
O
EtOH
r.t.
R1
HN
N
R1
HN NH2
R3
R3
R2
R5
phenylhydrazine or
2,4-dinitrophenylhydrazine
aromatic aldehydes
SH
SH
O
EtOH
r.t.
NH2
N
R3
AR76
NO2
2-aminothiophenol
aromatic aldehydes
Scheme 1. Synthetic pathway for resveratrol analogues.
All the compounds were characterized via one-dimensional nuclear magnetic
resonance (1D-NMR), infrared (IR) spectroscopy, and melting point (MP) assays, and the
results were in accordance with data in the literature: AR07 and AR08,66,67 AR13 and
21

AR22; AR16;69 AR35;70 AR76.40 The compounds AR25,71 AR2671 and AR32,72,
68
AR3872 and AR3972 were send it to Patent Deposit Services.
1
MP were determined using a MQAPF-301-Microquimica digital apparatus. H
NMR spectra were recorded on a Bruker AC-300 and 13C NMR spectra were recorded on
a Bruker AC-300 at 75 MHz. The chemical shifts (δ) are given in parts per million relative
to tetramethylsilane (TMS). Spectra were acquired in dimethyl sulfoxide (DMSO-d6).
Reagents and materials were obtained from commercial suppliers and were used without
purification. All reagents used were analytical reagent grade. The resveratrol was
obtained from Fragon BR, (99% purity).
To perform the biological assays, resveratrol and analogues were solubilized in
ethanol, and dilute in RPMI medium. The maximum concentration of ethanol reached
was 1%.
Cells: The murine macrophage cell lines RAW264.7 and J774.A1 was kindly provided by Dr.
Ana Paula Ferreira and Dr. Henrique Couto Teixeira (Federal University of Juiz de Fora, Brazil)
respectively. Cells were routinely maintained in RPMI-1640 medium (Gibco) supplemented
with 10% bovine fetal serum (Gibco), 1% streptomycin and 100 U/ml penicillin solution
(Gibco), at 37ºC in humidified CO2 (5%) atmosphere.
Cytotoxicity: RAW264.7 or J774A.1 were seeded in a 96-well dish (2 x 105 cells/well) and
cultured for 24 hours before exposure to resveratrol or analogues (25µM or 50µM). After 48
hours of the treatment, MTT solution (0,5mg/mL) was added to each well and the plate were
incubated for additional 2h at 37ºC, 5% CO2. Then, the supernatant was removed and the
farmazan crystals was solubilized with DMSO and optical density (OD) was measured at 595
nm with spectrophotometer.
22

Determination of antioxidant activity: The antioxidant activity was evaluated using the
2,2diphenyl-1-picrylhydrazyl (DPPH; Sigma-Aldrich, USA) assay, described by Sreejayan and
Rao73, with minor modifications. A 150 µL of a DPPH ethanolic solution of 300 µM (120
µg/mL) were added to 50 µL of ethanol solution of analogues at concentrations from 3,12 to
400 µM in 96-well plates. Resveratrol was used as a standard at the same concentrations.
Reactions elapsed at room temperature for 30 minutes in the dark and then the absorbance was
read in a spectrophotometer (λ= 510 nm). The concentration that provided 50% inhibition
(IC50) was calculated graphically using a calibration curve in the linear range by plotting the
extract concentration vs the corresponding scavenging activity. This procedure was done
twice and assays were performed in triplicate.
Nitric Oxide production: RAW 264.7 cells (2 x 105 cells/well) were stimulated with
Escherichia coli LPS (1µg/mL - Sigma-Adrich) + IFN-γ (20U/mL - Peprotech) for 1 h and
treated with resveratrol or analogues (50 µM). After 48 horas, the NO production was
determined by measuring the nitrite content in culture supernatant thought Griess
method. Briefly, culture supernatants (50 µl) were mixed with 50 µl of Griess reagent (1%
sulfanilamide, 0.1% naphthylethyline diamine dihydrochloride, and 2.5% phosphoric acid) in
triplicate. After 5 minutes at room temperature, the OD was determined at 540 nm. Sodium
nitrite was used as a standard.
Cytokines/chemokine production: RAW 264.7 or J774A.1 cells (2 x 105 cells/well in
triplicate) were stimulated with Escherichia coli lipopolysaccharide (LPS -1ug/mL) for 1 h and
treated with resveratrol or analogues (50 µM). After 24 (IL-1β in J774A.1 cells) or 48 hours
(IL-6, IL-12, TNF-α and CCL-2), the cytokine production was evaluated by ELISA using
23

commercial specific kits (R&D system or BD biosciences), according to manufacturer's
protocol. The levels of cytokine were determined by a spectrophotometer at 450 nm.
Analysis of the expression of CD80, CD86 and MHC II: Raw 264.7 cells (4 x 105 cells/well
in a 24-well dish in triplicate) were stimulated with Escherichia coli LPS (1µg/mL - Sigma-
Adrich) for 1 h and treated with resveratrol or analogues (50 µM). After 24 hours, cells were
removed, blocking with anti-mouse CD16/32 (Clone 2.4G2) and stained with FITC-conjugated
antibodies against CD80 (Clone 16-10A1) or CD86 (clone GL1) or MHC II (I-Ab Clone AF6-
120) from BD Bioscience. Samples were acquired (minimum of 30.000 events) on a
FACScanto II flow cytometer (BD Biosciences). Acquired data were analyzed by using FlowJo
vX software (Tree Star).
Animals: Female C57/BL6 and Balb/c mice 8–10 weeks old were obtained from the
Reproductive Biology Center, Federal University of Juiz de Fora. All mice were maintained in
a temperature-controlled facility with a 12 h light/dark cycle, with chow and water ad libitum.
All animal experimentation was in accordance with the principles of Brazilian Code for the Use
of Laboratory Animals and was approved by UFJF Ethics Committee for the Use of Laboratory
Animals (CEEA- UFJF 027/2013 and 031/2016).
Cell proliferation assay: Mitogen-induced proliferation assay was performed using whole
spleen cells according Sharma et al.15 Splenocytes from C57BL/6 were cultured (1x105
cell/well) in 96-well round-bottomed microplates in RPMI 1640 medium (Gibco, Carlsbad, CA,
USA) supplemented with 10% FBS, 100 U/mL of sodium penicillin, 100 µg/mL of
streptomycin sulphate. Concanavalin A (Con A - 5µg/mL) or Escherichia coli LPS (1µg/mL)
(Sigma) were used as nonspecific T and B-lymphocyte mitogen, respectively. After an hour of
24

stimulation (37°C in 5% CO2), the splenocytes were treated with resveratrol or analogues
(50µM) and cultured for additional 72 hours. Proliferation was evaluated by MTT assay (as
previously reported) after 72 hours of incubation (37°C in 5% CO2). Results were presented as
the mean SD of percentage of proliferation relative to untreated stimulated cells.
Ear edema test: This test was carried out in accordance to Pinto et al.74 Balb/c mice (n =
6/group) was immobilized and received 20 µL of a croton oil solution (2.5% v/v) on the inner
surface of the right ear. The same volume of acetone (vehicle) were applied on the inner surface
of the left ear. Immediately, the animals were topically treated with 20 µL of analogues
(0,1mg/ear), dexamethasone (0.1 mg/ear), or acetone (negative control). Four hours after
inflammation induction, the mice were sacrificed and a plug (6 mm diameter) was removed
from both the treated and untreated ears. Edema was evaluated as the weight difference between
the plugs. The data were expressed as the mean SD of ear measurements.
Subsequently, part of the ear plugs were fixed to histopathological analysis and part
were stored at -80ºC in order to evaluate cytokines (IL-1β and IL-6) and chemokine (CCL-2)
levels and MPO activity.
Histological analyses: Ear plugs were fixed for 24 h in ethanol 70% and then preserved in
10% formalin. Subsequently, the tissues were dehydrated, blocked in paraffin, and sectioned
with a microtome (4 µm). Than, the sections were stained with hematoxylin and eosin (HE).
Representative areas were selected for qualitative light microscopic analysis. Images were
captured using the Image-Pro Plus® 6.0 software.
Cytokines/chemokine levels on inflamed ears: Half of the ears plugs were homogenized in
800µL of cytokines extraction solution (BSA 0.5%, PMSF 0.1% benzethonium chloride 0.1M,
25

EDTA 10mM, and aprotinin 20kIU/ml ) and centrifuged at 440g for 15 min at 4ºC. The
supernatant was used to evaluate the levels of IL-1β, IL-6 or CCL-2 by ELISA as previously
reported. The pellet was used to evaluate MPO activity.
Myeloperoxidase Activity (MPO). Ear tissue was homogenized in phosphate-buffered saline
(PBS) and centrifuged (12000 x g, 10 min, 4ºC). The resulting pellet was suspended in 0.5%
hexadecyl trimethyl ammonium Bromide (HTAB 2mL/100mg tissue - Sigma) in PBS,
homogenized and submitted to 3 cycles of freeze/thawing in liquid nitrogen. After new
centrifugation (10000 x g, 15 min 4ºC), MPO activity was determined by mixing equal volumes
(100 µL) of the supernatnat and OPD (6 mM Ophenylenediamine) solution in 10 mM citrate
(pH = 4.5), followed by the addition of 20% H2O2. After 15 min, the reaction was stopped with
50 µL of 1 M H2SO4. The absorbance was determined at 492 nm on a spectrophotometer
(SpectraMax 190).
Statistical analysis: Data were analyzed using Graph Pad Prism version 6.0 (Graph Pad
Software, San Diego, CA, USA). The data of in vitro experiment were statistically analyzed
using one-way variance analysis followed by Tukey’s or Dunnet test. The in vivo analysis were
analysed using one-way variance analysis followed by Newman–Keuls test. Differences were
considered significant at p < 0.05.
SUPPORTING INFORMATION:
Supplementary data (experimental procedure and general information about the synthesis of
AR32, AR38, and AR39 compounds) associated with this article can be found in the online
file.
26

DECLARATIONS OF INTEREST:
None
ACKNOWLEDGEMENTS:
The authors are grateful to Centro de Tecnologia Celular e Imunologia Aplicada (Dr. Henrique
Couto Teixeira and Dr. Ana Paula Ferreira) for laboratory and equipments support.
FUNDING:
This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais –
FAPEMIG (APQ-02140-14 and Rede Mineira de Imunobiologicos, RED-00140-16 ) and
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The fellowships
were supplied by CAPES, PROBIC/FAPEMIG and BIC/UFJF.
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