Anti-inflammatory activity of ortho-trifluoromethoxy-substituted 4-piperidione-containing mono-carbonyl curcumin derivatives in vitro and in vivo

Article abstract of DOI:10.1016/j.ejps.2021.105756

Curcumin was reported as an anti-inflammatory agent. However, curcumin's poor bioavailability limited its clinical utility. Here, thirty ortho-substituted mono-carbonyl curcumin derivatives, containing acetone, cyclopentanone, cyclohexanone or 4-piperidione (N—H, N-methyl or N-acrylyl) moieties replacing β-diketone moiety of curcumin, were investigated for anti-inflammatory activity. Two active ortho-trifluoromethoxy-substituted 4-piperidione-containing derivatives 22 and 24 owned good cell uptake ability, and displayed excellent anti-inflammatory activity in both lipopolysaccharide-induced Raw264.7 macrophages and a dextran sulfate sodium (DSS)-induced mouse model of colitis. They inhibited the production of nitric oxide, reactive oxygen species, malonic dialdehyde and cyclooxygenase-2; and the expression of pro-inflammatory cytokines interleukin-1β, tumor necrosis factor-α and myeloperoxidase; the phosphorylation of mitogen-activated protein kinases; and the nucleus translocation of p65. What's more, 22 or 24 oral administered reduced the severity of clinical symptoms of ulcerative colitis (body weight and disease activity index), and reduced obviously DSS-induced colonic pathological damage (the colon length and histopathology analysis). These results suggested that ortho-trifluoromethoxy-substituted 4-piperidione-containing mono-carbonyl curcumin derivatives 22 and 24 were potential anti-inflammatory agents; and offered the important information for design and discovery of more potent anti-inflammatory drug candidates.

Full text of DOI:10.1016/j.ejps.2021.105756

European Journal of Pharmaceutical Sciences 160 (2021) 105756
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Anti-inflammatory activity of ortho-trifluoromethoxy-substituted 4-piperi-
dione-containing mono-carbonyl curcumin derivatives in vitro and in vivo
Ziqing Wang, Wenwen Mu, Pengxiao Li, Guoyun Liu*, Jie Yang*
School of Pharmaceutical Sciences, Liaocheng University, 1 Hunan Street, Liaocheng, Shandong 252059, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Curcumin was reported as an anti-inflammatory agent. However, curcumin’s poor bioavailability limited its
clinical utility. Here, thirty ortho-substituted mono-carbonyl curcumin derivatives, containing acetone, cyclo-
Mono-carbonyl curcumin derivatives
Anti-inflammatory
–
pentanone, cyclohexanone or 4-piperidione (N H, N-methyl or N-acrylyl) moieties replacing β-diketone moiety
Raw264.7 macrophages
Colitis
of curcumin, were investigated for anti-inflammatory activity. Two active ortho-trifluoromethoxy-substituted 4-
piperidione-containing derivatives 22 and 24 owned good cell uptake ability, and displayed excellent anti-
inflammatory activity in both lipopolysaccharide-induced Raw264.7 macrophages and a dextran sulfate so-
dium (DSS)-induced mouse model of colitis. They inhibited the production of nitric oxide, reactive oxygen
species, malonic dialdehyde and cyclooxygenase-2; and the expression of pro-inflammatory cytokines inter-
Ortho-trifluoromethoxy-substituted
4-piperidione
leukin-1β, tumor necrosis factor-α and myeloperoxidase; the phosphorylation of mitogen-activated protein ki-
nases; and the nucleus translocation of p65. What’s more, 22 or 24 oral administered reduced the severity of
clinical symptoms of ulcerative colitis (body weight and disease activity index), and reduced obviously DSS-
induced colonic pathological damage (the colon length and histopathology analysis). These results suggested
that ortho-trifluoromethoxy-substituted 4-piperidione-containing mono-carbonyl curcumin derivatives 22 and 24
were potential anti-inflammatory agents; and offered the important information for design and discovery of more
potent anti-inflammatory drug candidates.
1. Introduction
(NF-κB), toll-like receptor 4 (TLR4) and mitogen-activated protein ki-
nase pathways (MAPKs), and suppression of the expression of
Inflammatory bowel disease (IBD), containing Crohn’s disease (CD)
and ulcerative colitis (UC), is a kind of chronic non-specific intestinal
inflammatory disease with unclear etiology. Currently, the treatment
options for patients with IBD include corticosteroids, sulfasalazine,
mesalamine, and immunosuppressors or surgical treatments. However,
due to a marked immune response suppression, conventional treatments
have numerous side effects, and negative impacts on the quality of life of
sufferers (Bryant et al., 2015; Harbord et al., 2017; Kawalec and Mali-
nowski, 2015). Therefore, effective and safe therapeutic agents are still
necessary for prevention and treatment of IBD.
cyclooxygenase-2 (COX-2), nitric oxide (NO) and pro-inflammatory
cytokines (interleukin (IL)ꢀ 1β, IL-6, IL-18 and tumor necrosis
factor-alpha (TNF-α)) (Karimian et al., 2017; Zhou et al., 2015). How-
ever curcumin’s poor bioavailability, unstability and rapid degradation
under physiological conditions limited its clinical utility (Begum et al.,
2008; Siviero et al., 2015).
Natural products are the inspiration of drug discovery, and structural
modification of natural products is an important strategy in pharma-
ceutical discovery. The β-diketone moiety of curcumin has been
considered to be responsible for its unstability, rapid degradation and
poor bioavailability (Singh et al., 2019; Wiggers et al., 2017). In recent
years, a lot of mono-carbonyl curcumin derivatives were developed with
improved biological activities in preventing and treating various dis-
eases (Dinkova-Kostova et al., 2007; He et al., 2018) (such as cancer,
inflammation, et al.), through regulating a variety of molecular targets
in cells, such as inhibiting NF-κB pathways, activating c-Jun N-terminal
Curcumin, a natural yellow pigment isolated from the rhizomes of
Curcuma longa, could be applied in food coloration, cosmetics utility,
and fabric dying and so on. In addition, curcumin has been shown
multiple pharmacological activities, including antioxidant, anti-cancer,
anti-inflammatory, antimicrobial, and cardiovascular protective effects
(Chen et al., 2020). Curcumin displayed anti-inflammatory effect via
inhibition of different signaling pathways such as nuclear factor kappa-B
kinase (JNK) pathway or inhibiting B-cell lymphoma
2 (Bcl-2)
* Corresponding author.
E-mail addresses: guoyunliu@126.com (G. Liu), yangjie1110@163.com (J. Yang).
https://doi.org/10.1016/j.ejps.2021.105756
Received 20 November 2020; Received in revised form 5 February 2021; Accepted 9 February 2021
Available online 12 February 2021
0928-0987/© 2021 Elsevier B.V. All rights reserved.

Z. Wang et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105756
expression, etc. (Chen et al., 2018; Zhu et al., 2016). The structure de-
signs of mono-carbonyl curcumin derivatives not only replace β-dike-
tone by acythesizedetone, but also combine with cyclopentanone,
cyclohexanone, 4-piperidone, or N-substituted-4-piperidone moieties
and so on (Dai et al., 2015; Li et al., 2019; Liu et al., 2016; Wu et al.,
2013; Yang et al., 2020). In addition, among the design strategies of
mono-carbonyl curcumin derivatives, “ortho effect”, that ortho-sub-
stituents on the phenyl ring can contribute to the improvement of bio-
logical activity, is emphasized and utilized. For example, the presence of
a ortho group was proven to strengthen significantly the cytotoxicity of
mono-carbonyl curcumin derivatives (Dai et al., 2015; Liu et al., 2016),
cinnamaldehyde derivatives (Chew et al., 2010) and chalcone de-
rivatives (Gan et al., 2013), the potency of curcumin derivatives in
inducing phase II enzymes (Dinkova-Kostova et al., 2001), and the
interaction with certain target proteins by a conformational change of a
Firstly, a series of mono-carbonyl curcumin derivatives were tested
for their inhibitory effect on the NO production in the LPS-induced
Raw264.7 macrophage cells. Results were expressed as IC50 (Table 1),
the half maximal inhibitory concentration. From an “β-diketone moiety
modification” perspective, cyclopentanone and cyclohexanone modifi-
cations showed little or no contribution to improve the anti-
inflammation activity. For acetone derivatives, 2‑hydroxy-substituted
derivative 1, showed the best activity (2.47
rivatives, 2‑methoxy- and 2-trifluoromethoxy-substituted derivatives 10
and 22, showed excellent activity (2.48 and 2.73 M); for N-methyl-4-
piperidone derivatives, 2‑methoxy-substituted derivatives 11, showed
good activity (3.66 M); for N-acrylyl-4-piperidone derivatives,
2‑methoxy- and 2-trifluoromethoxy-derivatives 12 and 24, showed
μM); for 4-piperidione de-
μ
μ
excellent activity (2.77 and 2.02 μM) (Fig. 1).
Unfortunately, there were no obvious the structure-activity rela-
tionship. However, over half derivatives were more potent than their
¨
ligand (Schonherr and Cernak, 2013). Besides, the introduction of
fluorine and fluorinated groups into potential drug candidates is a
commonly employed approach to improve their biological activities,
including metabolic stability, high lipophilicity, enhanced binding in-
teractions, and bioavailability (Hagmann, 2008; Yerien et al., 2016).
Therefore, in this study, we designed and chose thirty mono-carbonyl
curcumin derivatives with drawing/donoring groups (hydroxyl,
methoxy, fluoro, trifluoromethyl and trifluoromethoxy) attaching to
ortho-position on the aromatic ring, together with acetone, cyclo-
parent molecule curcumin (19.43 μM). Moreover, the IC50 values of
eight derivatives (1, 4, 10, 11, 12, 18, 22, and 24) were less than 5
μ
mol/L, and all of eight active derivatives exhibited low cytotoxicity.
(Fig. 2)
2.2.2. Effects of active derivatives on the protective properties against t-
BHP-induced cytotoxicity in the Raw264.7 macrophage cells
Oxidative stress plays a key role in the inflammation, and is related to
–
´
pentanone, cyclohexanone, 4-piperidione (N H, N-methyl, and N-
IBD (Dudzinska et al., 2018). Oxidative stress could cause the over-
acrylyl) moieties respectively. Then, we focused on their anti-
inflammatory effect in lipopolysaccharide (LPS)-stimulated Raw264.7
macrophages and a dextran sulfate sodium (DSS)-stimulated mouse
model of colitis, for the sake of seeking the potent anti-inflammatory
agents in vivo.
production of ROS, and further aggravate the inflammatory reaction
(Lei et al., 2015). In order to supplement to the results of initial
screening, we further chose these eight active derivatives to test the
effects on the protective properties against tert‑butyl hydroperoxide
(t-BHP)-induced cytotoxicity in Raw264.7 macrophage cells, an oxida-
tive stress model, using an MTT assay. As shown in Fig. 3, after treat-
ment with t-BHP, cell viability was decreased in
a
2. Result and discussion
concentration-dependent manner relative to the control group. All of
eight active derivatives protected remarkably the cells from t-BHP-in-
duced oxidative cytotoxicity along with 2 mM t-BHP. Moreover, when
2.1. Mono-carbonyl curcumin derivatives
treatment with 2.5 μM active derivatives, the cell viability was increased
Thirty mono-carbonyl curcumin derivatives with drawing/donoring
groups (hydroxyl, methoxy, fluoro, trifluoromethyl and tri-
fluoromethoxy) in the ortho position were designed and synthesized by
our laboratories (Liu et al., 2018; Liu et al., 2018; Yang et al., 2018;
Yang et al., 2018; Zhang et al., 2018). β-diketone moiety of curcumin
were replaced with acetone, cyclopentanone, cyclohexanone, and
to above 70%. However, the same concentration of curcumin didn’t
show any protective effect, and treatment with 20 μM curcumin could
increase the cell viability to about 70%. These results suggested that
these eight derivatives owned more protective properties against
t-BHP-induced oxidative cytotoxicity than curcumin in the Raw264.7
macrophage cells. In addition, 1, 10, 22 and 24 were more effective than
–
4-piperidione (N H, N-methyl, and N-acrylyl) moieties respectively.
4, 11, 12 and 18 by comparing the effect of 0.5 μM.
2.2. In vitro study
2.2.4. Effect of cellular uptake activity
Next, these active derivatives 1, 10, 22 and 24 were examined to
compare their cellular uptake ability according to the previously pub-
lished paper (Dai et al., 2015; Kunwar et al., 2008) with some modifi-
cations (Liu et al., 2021). Fig. 4A showed a comparison of four active
derivatives in the cellular uptake in Raw264.7 cells by high performance
liquid chromatography analysis of methanol extracted cell lysates, after
2.2.1. The inhibitory effect of derivatives on the LPS-induced no production
The macrophage plays a critical role in the inflammation as a
response to injury or infection (Chawla et al., 2011). LPS is widely used
as the inducer of inflammation of macrophages, and it could induce the
production of NO, ROS, cytokines and so on. LPS-induced Raw264.7
macrophage cells is a classical model to cause NO production. NO is a
key inflammatory intermediate (Nagy et al., 2007). Excessive NO pro-
duction can cause significant inflammatory damage or disease. In-
hibitors of NO production might be potential anti-inflammatory agents.
treatment with 50 μM derivatives for different time. As shown in Fig. 4A,
22, and 24 displayed good cellular uptake in cells, compared with 1 and
10. Notably, 22, and 24 contained trifluoromethoxy groups; 1 and 10
Table 1
The inhibitory effect of derivatives on the NO production.
Num
IC50
(
μM)
Num
IC50
(μ
M)
Num
IC50
(μM)
Num
IC50
(μM)
Num
IC50(μM)
2-OH
2–OCH3
2-CF3
13
2-OCF3
19
2-F
25
26
27
28
29
30
acetone
1
2.47 ± 0.02
24.46 ± 0.44
9.89 ± 0.76
3.70 ± 0.35
5.22 ± 0.83
17.50 ± 0.53
19.43 ± 1.39
7
10.42 ± 0.96
>100
19.79 ± 1.32
49.95 ± 2.75
72.81 ± 6.30
25.27 ± 1.33
8.36 ± 0.67
4.89 ± 0.45
>200
18.06 ± 0.96
>100
cyclopentanone
cyclohexanone
2
8
14
20
52.01 ± 4.72
18.98 ± 1.13
2.73 ± 0.54
19.26 ± 1.48
2.02 ± 0.08
3
9
13.51 ± 1.57
2.48 ± 0.57
3.66 ± 0.14
2.77 ± 0.70
15
21
27.26 ± 1.37
15.72 ± 0.77
6.07 ± 0.79
5.80 ± 0.34
4-piperidione
4
10
11
12
16
22
N-methyl-4-piperidione
N-acrylyl-4-piperidione
5
17
23
6
18
24
Cur
2

Z. Wang et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105756
Fig. 1. Design and synthesis of mono-carbonyl curcumin derivatives.
of 4-piperidione. The ortho-trifluoromethoxy group could contribute to
its improved anti-inflammatory activity, cell uptake ability and meta-
bolic stability. Moreover, trifluoromethoxy group adopted an orthog-
onal direction relative to the aromatic plane, which might be
contributed to the protein-ligand interactions (Müller et al., 2007).
2.2.5. Effect on the intracellular ROS generation
The excessive ROS production was related to the activation and
aggravation of the inflammation (Mittal et al., 2014). To investigated
the anti-inflammatory mechanism of 22 and 24, we tested the effect of
22 and 24 on the production of ROS in LPS-induced Raw264.7 macro-
phage cells. As shown in Fig. 5, the ROS levels in the LPS-induced cells
substantially increased compared to the control cells. When treatment
with 22 or 24 at the same time, intracellular ROS levels were signifi-
cantly decreased in a concentration-dependent manner relative to
treatment with LPS only. And, 22 showed little more activity than 24. It
was suggested that 22 and 24 maybe play anti-inflammatory role via
inhibiting the production of ROS.
Fig. 2. Effect of eight active derivatives on cell viability in the Raw264.7
macrophage cells. Data were expressed as means ± SD (n = 3) from indepen-
dent experiments.
2.2.6. Evaluation of ability of 22 and 24 to inhibit LPS-mediated IL-1β and
TNF-α production
Pro-inflammatory cytokines, secreted by immune cells, has been
reported to serve vital roles in host defense (Akira et al., 2006). Herein,
contained hydroxyl or methoxy groups. These results provided evi-
dences that the incorporation of fluorine-containing moieties into de-
rivatives significantly contributed to the increased cellular uptake.
Besides, it was observed that only 5.81% or 3.01% accumulated in the
membrane for 22 or 24 (Fig 4B), respectively, which reflected that 22
and 24 could effectively penetrate the cellular membrane to enter the
cells.
we selected IL-1β and TNF-α as the representatives to evaluated the
inhibitory effects of 22 and 24 on pro-inflammatory cytokines in the
LPS-induced Raw264.7 macrophage cells, and the expressions were
detected by ELISA. As shown in Fig. 6, the formations of IL-1β and TNF-
α
in the control cells were low, but they were significantly increased when
treatment with LPS only. When treatment with 22 or 24 at the same
According to the initial screening in the LPS-induced NO over-
expression cell model, the t-BHP-induced oxidative stress cell model,
and cell uptake ability, we then selected two ortho-trifluoromethoxy-
substituted derivatives 22 and 24 for the follow-up studies. The different
of structures between 22 and 24 was the easily hydrolyzed amide bond
time, the expressions of IL-1β and TNF-
α
was inhibited in a
concentration-dependent manner relative to the LPS cells. And, 22
showed little more activity than 24. So, this result further indicated that
22 and 24 were the inspiring anti-inflammatory compounds owning the
ability to inhibit LPS-stimulated cytokine expressions.
3

Z. Wang et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105756
Fig. 3. Effects of eight active derivatives on the t-BHP-induced RAW264.7 macrophage cell cytotoxicity. (A) RAW 264.7 macrophage cells were subjected to cur-
cumin for 24 h, subsequently induced with different concentrations of t-BHP for 3 h. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group. #p <
0.05, ##p < 0.01, ###p < 0.001, compared with the 0.5 mM t-BHP group. •p < 0.05, ••p < 0.01, •••p < 0.001, compared with the 1 mM t-BHP group. ★p < 0.05, ★★p <
0.01, ★★★p < 0.001, compared with the 2 mM t-BHP group. ▴p < 0.05, ▴▴p < 0.01, ▴▴▴p < 0.001, compared with the 3 mM t-BHP group. (B) RAW 264.7 macrophage
cells were subjected to derivatives for 24 h, subsequently induced with t-BHP (2 mM) for 3 h. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group.
#p < 0.05, ##p < 0.01, ###p < 0.001, compared with the 2 mM t-BHP group. Measurement of cytotoxicity using the MTT assay. Data were expressed as means ± SD
(n = 3) from independent experiments.
Fig. 4. Cellular uptake of four active derivatives in whole cells (A) and membrane (B).
2.2.7. Evaluation of the ability of 22 and 24 to suppress LPS-induced COX-
2 production
of MAPK proteins were analyzed using western blot. As shown in Fig. 8,
LPS caused the increase of the phosphorylation of p38, JNK, and ERK.
Treatment with 22 could obviously decreased the phosphorylation of
MAPK proteins. But, 24 showed less effect on the decrease of the
phosphorylation of p-38 and p-JNK than 22, and showed little effect on
the decrease of the phosphorylation of p-ERK. This suggested that 22
and 24 could exert an anti-inflammatory activity, which was related to
the prevention the activation of the LPS-induced MAPK signal pathway.
COX-2 is an important regulator of inflammatory mediator produc-
tion in response to LPS or other stimuli. And the upregulation of COX-2
can promote chronic inflammation. Therefore, we next evaluated the
ability of 22 and 24 to modulate LPS-induced the expression of COX-2,
and the levels were analyzed using western blot. As shown in Fig. 7, LPS
caused a significant increase in the protein levels of COX-2 compared
with the control cells. Treatment with 22 or 24 reduced the LPS-induced
COX-2 expression.
2.2.9. Assessment of the ability of 22 and 24 to inhibit LPS-induced NF-κB
activation
These results presented above suggested that 22 and 24 could pre-
vent LPS-induced expression of inflammatory mediators (NO, ROS, IL-
NF-κB is an key transcription factor, which regulates the pro-
inflammatory mediators in activated macrophages. Before macrophage
cells are induced with LPS, NF-κB is inactive, is bounded with the in-
hibitor kappa B (IκB) and retained in the cytoplasm. When macrophage
cells are stimulated, activated IκB is phosphorylated, releasing the NF-κB
heterodimers (p65/p50), which then translocate into the nucleus, and
further to bind to pro-inflammatory genes (Kim et al., 2016). So, the p65
nuclear translocation is an important marker for the inflammatory
reaction.
1β, TNF-α and COX-2), and they were worthy of further study.
2.2.8. Assessment of the ability of 22 and 24 to inhibit the phosphorylation
of MAPKs in the LPS-induced Raw264.7 macrophage cells
The MAPK signaling pathway is important in the expression of in-
flammatory factors. During the inflammatory response, the phosphory-
lation of MAPK proteins (p38, JNK, and ERK) is key for the activation of
activating protein 1 (AP-1) and NF-κB, and the release of NO, IL-1β, TNF-
α
and so on in the LPS-induced macrophages. The phosphorylation levels
To investigate if the activation of NF-κB was inhibited by 22 or 24 in
4

Z. Wang et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105756
the colonic mucosa (Cosnes et al., 2011). A DSS-induced mouse model of
colitis can emulate the disease characteristics of human UC, including
weight loss, diarrhea, rectal bleeding, intestinal mucosal damage, and
pro-inflammatory cytokine elevation. This classic model has been
widely used to screen potential therapeutic agents of UC.
2.3.1. Effect of 22 and 24 on the development of DSS-induced colitis in
mice
As shown in Fig. 10A and 10B, after 4 days of DSS treatment, the
ratio of body weight change was markedly decreased in the DSS-treated
group relative to the control group. The loss of body weight was
remarkly reduced in 200 mg/kg/day 22-treated group and 100 mg/kg/
day 24-treated group, in which the effects were significantly better than
the effect of treatment with 300 mg/kg/day sulfasalazine (SASP), which
was effective for treating UC (Singh et al., 2009), and was used as the
reference anti-inflammatory drug.
The scores of disease activity index (DAI) of mice was determined
according to the scoring system (Table 2) (Cooper et al., 1993; Mu et al.,
2016). As shown in Fig. 10C and 10D, the scores of DAI of mice in 200
mg/kg/day 22- or 100 mg/kg/day 24-treated group were remarkly
lower than that in the DSS- or 300 mg/kg/day SASP-treated group. The
results of DAI indicated clearly that oral administration of 22 and 24
effectively alleviated the severity of clinical symptoms of colitis in the
DSS-induced mouse model.
Fig. 5. Effects of the active derivatives on ROS production in the LPS-induced
Raw264.7 macrophage cells, using DCFH-DA staining and flow cytometric
analysis. The levels of ROS were expressed as fold change relative to the levels
in the control cells. Data were expressed as means ± SD (n = 3) from inde-
pendent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the
control group. #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the
LPS group.
2.3.2. Effect of 22 and 24 on the colon in the DSS-induced mouse model of
colitis
the LPS-induced Raw264.7 macrophage cells, the expressions of p65 in
nuclear and cytoplasmic extracts were analyzed using western blot. As
shown in Fig. 9, when treatment with LPS only, the expression of p65 in
the nuclei markedly increased. However, the nuclear levels of p65 were
reduced obviously by 22 or 24 treatment. Meanwhile, the cytoplasmic
levels of p65 were similar. Therefore, the LPS-induced increase of p65
nuclear translocation in Raw264.7 macrophage cells was suppressed
obviously in 22 or 24 treatment group accoring to the relative protein
expression (the nuclear levels of p65/the cytoplasmic levels of p65); that
was to say, 22 and 24 could inhibit LPS-induced NF-κB activation. And,
22 showed little more activity than 24.
DSS administration leads to ulceration, hyperemia and bowel wall
thickening, and then cause significantly decreasing of colon length,
which is an obvious symptom. As shown in Fig. 11A, the colon length in
the DSS group was obviously short relative to the control group. How-
ever, shortening of colon length was attenuated significantly with 22
and 24 treatments at 200 and 100 mg/kg/day respectively.
Additionally, histopathological analysis was used to test the protec-
tive effect of 22 and 24 on colitis using hematoxylin-eosin (H&E)
staining, and representative images of colons and the histological scores
were shown in Fig. 11B. The histological scoring system was shown in
Table 3 (Mu et al., 2016). Compared with the control group, histological
observation of the DSS-treated group showed a significant acute in-
flammatory response, which was characterized by severe lesions in the
mucosa, loss of crypts, increase in neutrophil population and so on
(Mazzucchelli et al., 1994). However, after treatment with 22 or 24, the
2.3. In vivo study
A DSS-induced mouse model of colitis was used to verify whether 22
and 24 showed effective anti-inflammatory effect in vivo. UC is one of
the common nonspecific IBD, which causes inflammation and ulcers in
above symptoms of mice were significantly reduced in
a
Fig. 6. Effects of the active derivatives on the expression of IL-1β and TNF-α in the LPS-induced Raw264.7 macrophage cells. Data were expressed as means ± SD (n
= 3) from independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group. #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the
LPS group.
5

Z. Wang et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105756
Fig. 7. 22 and 24 inhibited LPS-induced expression of COX-2 in the RAW264.7 macrophage cells. The representative images were shown here. Data were expressed
as means ± SD (n = 3) from independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group. #p < 0.05, ##p < 0.01, ###p < 0.001,
compared with the LPS group.
Fig. 8. Effect of 22 and 24 on the activation of MAPK signaling pathways. The representative images were shown here. Data were expressed as means ± SD (n = 3)
from independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the LPS group of p-p38. #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the
LPS group of p-JNK. •p < 0.05, ••p < 0.01, •••p < 0.001, compared with the LPS group of p-ERK.
concentration-dependent manner relative to the DSS group. Contrasted
with control group, 300 mg/kg/day SASP, 50 mg/kg/day 22, and 50
mg/kg/day 24 groups, were reduced partially colonic tissue damage;
whereas, 100 mg/kg/day 22, and 100 mg/kg/day 24 groups, were
reduced significantly colonic tissue damage. These results suggested that
100 mg/kg/day 22 or 100 mg/kg/day 24 could protect remarkably the
colonic tissue against the DSS-induced damage.
the inhibitory effects of 22 and 24 on pro-inflammatory cytokines in
colon tissues of DSS-induced mouse model of colitis. As shown in Fig. 12,
compared with the control group, the levels of IL-1β and TNF-
α
increased remarkably in the DSS-treated group. After treatment with
300 mg/kg/day SASP, or different concentration of 22 and 24, these
levels were significantly inhibited, but without in a concentration-
dependent manner. These results indicated that 22 and 24 could
possess potent anti-inflammatory activity through inhibiting the
expression of pro-inflammatory cytokines in the DSS-induced mice.
2.3.3. Evaluation of ability of 22 and 24 to inhibit DSS-mediated IL-1β and
TNF-α production in colon
In addition to histopathological analysis, we further investigated the
inflammatory cytokines in the colon, and chose IL-1β and TNF- to test
α
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Z. Wang et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105756
Fig. 9. Effect of 22 or 24 on the NF-κB p65 nuclear translocation. (A, B) The expression of P65 proteins of cytoplasmic and nuclear proteins were assessed by western
blot analysis. The representative images were shown here. (C) Relative protein levels of nuclear and cytoplasmic P65 protein. Data were expressed as means ± SD (n
= 3) from independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group. #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the
LPS group.
2.3.4. Evaluation of ability of 22 and 24 to inhibit DSS-mediated NO,
MDA, and MPO production in colon
two active ortho-trifluoromethoxy-substituted 4-piperidione-containing
mono-carbonyl curcumin derivatives 22 and 24, owning the good cell
uptake ability, inhibiting effectively the LPS-induced overexpression of
NO, and protecting remarkably the Raw264.7 macrophage cells from t-
BHP-induced oxidative cytotoxicity, were gained and chose to further
study. In the LPS-induced Raw264.7 macrophage cells model, 22 and 24
showed excellent anti-inflammatory effect, which inhibited effectively
the levels of ROS, the expression of pro-inflammatory cytokines (IL-1β
Oxidative and nitrosative stress, that the continuously excessive
ROS/RNS production due to an imbalance of cellular redox homeostasis,
both act as a significant role in the process of inflammation and
inflammation-associated diseases (Yu et al., 2015). Here, we chose
several typical indexes of oxidative and nitrosative stress to further
measure. NO is a key inflammatory intermediate. Malonic dialdehyde
(MDA) is an important end product of lipid peroxidation. Myeloperox-
idase (MPO) is an enzyme of peroxidases found in azurophilic granules
of neutrophils.
and TNF-α), the production of COX-2, the phosphorylation of MAPKs,
and the nucleus translocation of p65.
What’s more, 22 and 24 exerted excellent anti-inflammatory effect
in the DSS-induced mouse model of colitis. Oral administration of 22 or
24, could reduce effectively the severity of clinical symptoms of UC
(body weight change and the scores of DAI), and reduced effectively
colonic tissue damage (the colon length and histopathology analysis).
Furthermore, 22 and 24 displayed anti-inflammatory activity through
suppressing the expression of pro-inflammatory cytokine (IL-1β and
As shown in Fig. 13, the levels of NO, MDA, and MPO in the DSS-
treated group significantly increased relative to the control group.
When treatment with 300 mg/kg/day SASP, these levels significantly
decreased compared with DSS group. These levels in 100 or 200 mg/kg/
day 22 or 24 groups were lower than these levels in both DSS and 300
mg/kg/day SASP groups. These results indicated that administration of
100 or 200 mg/kg/day 22 or 24 groups could inhibit the production of
RNS and ROS, and exert their anti-nitrosative and anti-oxidative stress
effects, which were related to the anti-inflammatory effect. And, the
inhibitory activity of the production of RNS and ROS was consistent with
the inhibition of pro-inflammatory cytokines, and the reduction of the
severity of clinical symptoms and the pathological damage of ulcerative
colitis in the DSS-induced mice. Moreover, the anti-inflammatory effect
of 22 and 24 was consistent in the DSS-induced mouse model of colitis
and in the LPS-induced Raw264.7 macrophage cells.
TNF-α), and the production of RNS/ROS (NO, MDA, and MPO) of colon
in the DSS-induced mouse model of colitis.
This study has provided the scientific evidences that ortho-tri-
fluoromethoxy-substituted 4-piperidione-containing mono-carbonyl
curcumin derivatives 22 and 24 were potential anti-inflammatory
agents; and offered the important information for design and discov-
ery of more potent anti-inflammatory drug candidates. Further study
will focus on the further molecular mechanism of anti-inflammatory
activity, metabolism, bioavailability and toxicological effects in the
animal model.
3. Conclusion
4. Materials and methods
In this paper, thirty ortho-substituted mono-carbonyl curcumin de-
rivatives were designed and chose, containing acetone, cyclopentanone,
4.1. Chemistry
–
cyclohexanone, or 4-piperidione (N H, N-methyl or N-acrylyl) moieties
replacing β-diketone moiety of curcumin. Through the initial screening,
All reagents and chemicals were commercially available from Sigma-
7

Z. Wang et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105756
Fig. 10. Anti-inflammatory effects of 22 and 24 in DSS-induced colitis. (A and B) Body weight change. (C and D) DAI score. Data were expressed as means ± SD (n
= 6).
4.2. Cell lines and culture conditions
Table 2
The DAI scoring system.
The Raw264.7 cell line, which were obtained from the Shanghai
Score
Body weight loss (%)
Diarrhea
normal
Rectal bleeding
none
Institute of Biochemistry and Cell Biology, Chinese Academy of Sci-
ences, were cultured in DMEM (Hyclone) supplemented with 10% fetal
bovine serum (Gibco), and 100 U/mL penicillin/streptomycin (Beyo-
time) in a humidified 5% CO2 atmosphere at 37 ◦C (Thermo Scientific
HERAceu150i).
0
1
2
3
4
0
1–5
6–10
10–15
>15
loose stools
diarrhea
slight
fecal blood
4.3. Measurement of the LPS-induced no production (Lee et al., 2009)
Aldrich, Aladdin, Tan sole, Energy Chemical et al., without further
purification.
100
μ
L Raw264.7 cells were seeded at 1 × 106 cells/well in 96-well
plates (Costar) for 24 h and then incubated with LPS (Sigma) (1 μg/mL)
4.1.1. The synthesis of ortho-substituted mono-carbonyl curcumin
derivatives
and different concentrations of the test compounds for 24 h. Then 75 μL
cell supernatant were mixed with the equal volumes of the fresh Griess
reaction solutions (1% sulfanilamide, 0.1% N-(1-naphthyl)-ethylenedi-
amine dihydro-chloride in 2.5% phosphoric acid) in a 96-well plate.
After 5 min of incubation at RT, the absorbance at 540 nm was measured
on a multiwallplate reader (Biotek Synergy H1 multi format microplate
readers). Assays were performed in triplicate and repeated three times.
The ortho-substituted mono-carbonyl curcumin derivatives together
–
with acetone, cyclopentanone, cyclohexanone, or 4-piperidione (N
H
or N-methyl) moieties were synthesized from ortho-substitued benza-
dehyde (2 equiv) and ketone (1.0 equiv) in ethanol (3 mol/L); and ortho-
substituted N-acryloyl-4-piperidione-containing mono-carbonyl curcu-
min derivatives were synthesized from corresponding 4-piperidione-
containing mono-carbonyl curcumin derivatives (1.0 equiv) and
acryloyl chloride (1.5 equiv) in acetone (0.3 mol/L), according to our
previously published paper. Their 1H and 13C nuclear magnetic (NMR)
spectroscopy witnesses our previously published work (Liu et al., 2018;
Liu and Zhang et al., 2018; Yang et al., 2018; Yang et al., 2018; Zhang
et al., 2018).
4.4. Cell cytotoxic assay. (for 2.2.1)
100
μ
L Raw264.7 cells were seeded at 1 × 106 cells/well in 96-well
plates for 24 h and then treated with different concentrations of the test
compounds for 24 h. After added the fresh solution of MTT (Beyotime)
(10 μ
L, 5 mg/mL), the plate was incubated for an additional 4 h at 37 ◦C.
8

Z. Wang et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105756
Fig. 11. Anti-inflammatory effects of 22 and 24 in DSS-induced colitis. (A) The length of colons. (B) The representative DSS-induced colonic pathological damage
images (40x and 200x) and the histological scores. Data were expressed as means ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control
group; #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the DSS-treated group. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
Finally, 300 μL of DMSO was added, and then 100 μL solution were
Table 3
distributed in a 96-well plate. The absorbance was measured at 570 nm
by a multiwallplate reader (Biotek Synergy H1 multi format microplate
readers). Assays were performed in triplicate and repeated three times.
The histological scoring system.
Score
Severity of
Extent of
Crypt damage
inflammation
inflammation
0
1
2
none
none
none
4.5. t-BHP-induced oxidative cytotoxicity (for 2.2.2)
mild
mucosa
1/3damaged
2/3damaged
moderate
mucosa and
submucosa
transmural
RAW 264.7 cells were grown in 96-well plates (2 × 104 cells/well,
3
4
severe
crypt loss by surface
epithelium present
both crypt and surface
epithelium lost
100 μL/well) for 24 h, and then treated with different concentrations of
compounds for 24 h. Cells were subsequently stimulated with t-BHP (2
mM) for an additional 3 h, and then washed with PBS buffer (10 mM, pH
7.4). MTT (100 μL, 0.5 mg/mL) was added to the cells and incubated for
another 4 h. Then the supernatant was removed, and 150 μL DMSO was
Fig. 12. Effects of the active compounds 22 and 24 on the production of IL-1β and TNF-α in DSS-induced colitis mice. (A) IL-1β levels. (B) TNF-α levels. Data were
expressed as means ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group; #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the
DSS-treated group.
9

Z. Wang et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105756
Fig. 13. Effects of 22 and 24 on NO production, MDA levels, and MPO activity in DSS-induced colitis mice. (A) NO production. (B) MDA levels. (C) MPO activity.
Data were expressed as means ± SD (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group; #p < 0.05, ##p < 0.01, ###p < 0.001, compared
with the DSS-treated group.
added to each well. The absorbance of MTT was measured at 570 nm by
a microplate reader (Biotek Synergy H1 multi format microplate
readers).
4.423 min, 310 nm, S (mAU*S) = 0.45776 + 14.72851 C (
μM); 24,
4.088 min, 310 nm, S (mAU*S) = 0.55896 + 16.22655 C ( M))
μ
4.7. Determination of intracellular ROS levels (Yang et al., 2020)
4.6. Cellular uptake activity
Raw264.7 cells were seeded at 3 × 106 cells/well in six-well plates
Raw264.7 cells were seeded at 3 × 106 cells/well (2 mL) in six-well
and incubated for 24 h. After treatment with LPS (1 μg/mL) and the test
plates and incubated for 24 h. After 0.5, 1, 2, 3, or 4 h of treatment with
compounds for 24 h, the cells were collected, and washed twice with
phosphate buffer solution (PBS, 10 mM, pH 7.4). And then resuspended
the test compounds 1, 10, 22 and 24 (50 μM), the cells were rapidly
washed twice with 1 mL ice-cold PBS. They were then extracted with ice-
cold methanol (800
L/well) at 4 ◦C overnight. The suspension was
centrifuged for 5 min at 10,000 rpm and 4 ◦C, and the supernatant (200
L) was determined with high performance liquid chromatography
(HPLC, Agilent Technologies 1260 Infinity). (Fig. 14)
After the cells were treated with 22 or 24 (50
in PBS and incubated with 3 μ
M DCFH-DA for 30 min at 37 ◦C in the
μ
dark. Then the cells were washed with PBS and analyzed immediately
for DCFH-DA fluorescence intensity with a FACSCanto flow cytometer
(Millipore Guava easyCyte 8HT benchtop flow cytometer) with excita-
tion and emission settings of 488 and 530 nm, respectively. The 10,000
events were acquired per sample. Three independent experiments are
carried out.
μ
μ
M) for 3 h, the cells
(six wells) were rapidly washed twice with 1 mL ice-cold PBS, and then
harvested. The samples were treated with the solution A of the mem-
brane and cytosol protein extraction kit (Beyotime Biotechnology,
China) to isolate cell membrane. Cell membrane was extracted with
4.8. Cytokine assays
methanol (200
with HPLC.
μ
L), and then the supernatant (200
μ
L) were determined
Raw264.7 cells were seeded at 3 × 106 cells/well in six-well plates
The standard curves were determined using 10
μL of 30, 20, 10 and 5
for 24 h, and then treated with LPS (1 μg/mL) and different concen-
μ
M different compounds 1, 10, 22 and 24 in methanol solution. The
methanol-water (90: 10) was used as mobile phase at the flow rate 1 mL/
min. (1, 3.302 min, 373 nm, S (mAU*S) = 0.01093 + 12.3076 C ( M);
10, 4.868 min, 350 nm, S (mAU*S) = ꢀ 0.69604 + 10.07493 C ( M); 22,
trations of the test compounds for 24 h. After incubation, the levels of IL-
1β and TNF-α in the supernatant were measured using a mouse IL-1 beta
μ
ELISA Kit and a mouse TNF-alpha ELISA Kit (RayBiotech), following the
μ
manufacture’s instructions.
Fig. 14. The representative HPLC chromatogram of cellular uptake of 1 with different times.
10

Z. Wang et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105756
4.9. Western blot analysis
4.16. Histopathology analysis
Raw264.7 cells were seeded at 3 × 106 cells/well in six-well plates
The colons of mice were fixed in buffered formalin (Beyotime),
embedded in paraffin, and then sectioned. The sections were staining
with hematoxylin and eosin (H&E), and photographed using an
Olympus microscope (BX53 + DP80).
and incubated for 24 h. After treatment with LPS (1 μg/mL) and the test
compounds for 24 h, the cells were harvested, washed twice with PBS,
and lysed with radio immunoprecipitation Assay (RIPA) buffer (Beyo-
time) or nuclear extraction kits (Beyotime). And then western blot was
carried out as the general process.
4.17. Statistical analysis
4.10. Animal
The data were presented as the means ± standard deviation (SD) and
analyzed using one-way ANOVA to determine any significant differ-
ences. p < 0.05 was considered as statistically significant.
Balb/c mice (8-week-old, male, 18–20 g) were purchased from
Pengyue Experimental Animal Breeding Co. LTD (Jinan, Shandong,
China). Mice were housed on a 12 h dark/light cycle at constant tem-
perature of 23 ± 2 ◦C with free access to standard diet and water. All
animal studies were conducted under the National Institute Guide for
the Care and Use of Laboratory Animals. Experimental protocols were
approved by the Ethics Committee of Liaocheng University.
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgements
4.11. Induction of acute colitis and treatment
This work was supported by the National Natural Science Foundation
of China (Grant No. 81901420).
After 7 days of adaptation period, the animals were randomly
assigned into nine groups (n = 8). Mice were supplied with distilled
water ad libitum in the control group, whereas all other experimental
groups were given 3.0% (w/v) DSS for 10 days. Besides, the mice of the
SASP (300 mg/kg/day), 22 (50, 100, and 200 mg/kg/day), and 24 (50,
100, and 200 mg/kg/day) treated groups were orally given SASP, 22, or
24 suspension with water from day 6 to day 10, respectively. Following
the last administration, the mice were deprived food for 12 h, anes-
thetized, and sacrificed. Unfortunately, two mice of DSS-treated group
dead in the experiment.
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