Diacetylcurcumin: Its potential antiarthritic effect on a freund’s complete adjuvant-induced murine model

Article abstract of DOI:10.3390/molecules24142643

The present study aims to evaluate the antiarthritic activity of diacetylcurcumin (DAC), a synthetic derivative where the free phenolic groups of curcumin are derivatized by acetylation, thereby conferring greater lipophilicity to the parent molecule and

Full text of DOI:10.3390/molecules24142643

molecules
Article
Diacetylcurcumin: Its Potential Antiarthritic Effect on a
Freund’s Complete Adjuvant-Induced Murine Model
Carolina Escobedo-Martínez 1,*, Silvia Laura Guzmán-Gutiérrez 2, María Isabel Carrillo-López 1,
Martha Alicia Deveze-Álvarez 1, Alfonso Trujillo-Valdivia 1, William Meza-Morales 3 and
Raúl G. Enríquez 3,
1
*
Departamento de Farmacia, División de Ciencias Naturales y Exactas, Universidad de Guanajuato,
Campus Guanajuato, Guanajuato, Gto. CP 36050, Mexico
CONACyT-Instituto de Investigaciones Biomédicas, Departamento de Inmunología,
Universidad Nacional Autónoma de México, CDMX. CP 04510, Mexico
Instituto de Química, Universidad Nacional Autónoma de México, México, CDMX. CP 04510, Mexico
Correspondence: c.escobedo@ugto.mx (C.E.-M.); enriquezhabib@gmail.com (R.G.E.);
Tel.: +52-473-732-0006 (ext. 5415) (C.E.-M.); +52-555-622-4404 (R.G.E.)
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 25 June 2019; Accepted: 20 July 2019; Published: 21 July 2019
Abstract: The present study aims to evaluate the antiarthritic activity of diacetylcurcumin (DAC),
a synthetic derivative where the free phenolic groups of curcumin are derivatized by acetylation,
thereby conferring greater lipophilicity to the parent molecule and partially overcoming the limited
systemic bioavailability of curcumin. Antiarthritic activity was evaluated on a Freund’s complete
adjuvant (FCA)-induced murine model of arthritis. Oral administration of DAC (60 and 120 mg/kg)
resulted in a significant inhibition of inflammation in the acute and chronic phases, respectively,
demonstrating an improved and sustained anti-inflammatory effect, comparable to that of curcumin
(150 mg/kg) in the chronic stage at a lower dose. Phenylbutazone (80 mg/kg) was used as a reference
drug. The pharmacological consequence of DAC or curcumin treatment is the prevention of secondary
lesions commonly associated with this biological model.
Keywords: diacetylcurcumin; curcumin; Freund’s complete adjuvant; antiarthritic
1. Introduction
Inflammation is an essential and protective immune response against microbial invasion, the
entrance of antigens, and damage to cells and tissues. This complex process involves different types
of cells and pro- and anti-inflammatory mediators that regulate cell migration, chemotaxis, and
proliferation in a highly coordinated manner. If acute inflammation fails to control pro-inflammatory
stimulus, it leads to chronic inflammation, auto-immunity, and excessive tissue damage [1,2].
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation that
causes varying degrees of damage to articular cartilage and subchondral bone [
common cause of physical disability in developed countries, where prevalence ranges between 0.3%
and 1.5%, with a 3:1 female-male ratio [ ]. Although several medications (steroidal or non-steroidal
anti-inflammatory and immunosuppressive drugs) have been used in recent decades to treat RA, more
specific drugs with fewer side effects are urgently needed [ ]. In this regard, plant-derived products
3]. RA is the most
4
5,6
are gaining interest since they are generally less toxic, less expensive, and easily accessible sources of
new drugs.
Curcumin, a good example of these candidate compounds, has been assayed in experimental and
clinic studies to validate its use against several human diseases [7,8].
Curcumin (diferuloylmethane) is the active component of turmeric (Curcuma longa), a spice
native to India. It is often referred to as a highly pleiotropic molecule with cicatrizing, antimicrobial,
Molecules 2019, 24, 2643; doi:10.3390/molecules24142643
www.mdpi.com/journal/molecules

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immunomodulatory, and antiproliferative activities, being capable of interacting with numerous
inflammation-related molecular targets [ 10]. Several studies have shown the positive effect of
8–
curcumin in the treatment of experimental autoimmune arthritis and animal models of rheumatoid
arthritis [11–13], incluiding freund’s complete adjuvant model [14].
This botanic dietary component has a potent anti-inflammatory activity, inhibiting all types of
inflammation mediators: cytokines, chemokines, adhesion molecules, and growth factors, as well as
cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) [15]. These effects of curcumin
are due to its capacity to inhibit the pathway of the nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-κB) and other proinflammatory signaling pathways, including the Activator
protein 1 (AP-1), early growth response protein 1 (Egr-1), and Janus kinase signal transducer and
transcription activator (JAK-STAT) [9].
Although there is a remarkably extensive record on the use of curcumin in traditional medicine, it
was not considered to be a drug candidate in western medicine until relatively recently [16]. In 1973,
Srimal and Dhawan [17] proposed the chemical synthesis of curcumin as a reliable source of research
material, noting its lack of toxicity, multifactorial anti-inflammatory action, and very low ulcerogenic
index. These desirable traits were highlighted when a possible use against RA was suggested [7], with
some advantages over non-steroidal anti-inflammatory drugs (NSAID). However, in the last three
decades, studies on animals have demonstrated that curcumin is hydrolytically unstable at intestinal
pH levels, it is also rapidly metabolized, conjugated in the liver, and excreted in feces [8]. Thus, the
systemic bioavailability of curcumin is limited by intestinal and hepatic glucuronidation as a part of its
metabolism [16], and different derivatives and analogs have been prepared over time to overcome this
major drawback [18,19].
Diacetylcurcumin (DAC) is a synthetic derivative of curcumin where phenolic OH groups are
protected with acetyl groups. This results in increased lipophilicity, possibly leading to a higher
bio-membrane penetration rate. DAC has been reported to have important biological properties, e.g.,
high antibacterial activity and anti-biofilm activity against methicillin-resistant Staphylococcus aureus
strains [20] and antimalarial activity in vitro against chloroquine-resistant Plasmodium falciparum [21].
DAC has potential as an antiproliferative agent in anticancer therapies [22]. Thus, studies on molecular
human colon cancer cells (HCT116) have shown that DAC prevents the correct formation of the mitotic
spindle, hindering chromosome alignment and, therefore, blocking cells in mitosis [23]. In addition,
there are controversial studies on the effect of DAC observed on carrageenan-induced inflammation,
ranging from a total absence of activity [24] up to a maximum value of activity in inhibition of edema,
followed only by aspirin and curcumin [25], including a potent anti-inflammatory effect on par with
other curcumin analogues [26]. Thus, we report herein for the first time the antiarthritic activity of
DAC on a RA murine model.
2. Results
2.1. Acute Toxicity Studies
No CD1 mice mortality was observed in the groups orally receiving curcumin or DAC at doses up
to 5000 mg/kg. The body weight of treated CD1 mice increased gradually with time, with no significant
differences with respect to the control group since the first day of treatment (Data not shown).
2.2. Anti-Inflammatory Activity
2.2.1. Behavior of Controls
The antiarthritic activity of DAC and curcumin was assessed in independent experiments. The
Freund’s complete adjuvant (FCA) injection in the footpad of the right rear limb of rats in the control
group (vehicle) used in the curcumin experiment caused an edema that reached its maximum volume
within the acute phase, that is, the first 3–5 days [27] (Figure 1A). In the following days, the edema

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showed a sudden increase on day 2 and decreased slowly from day 3 to day 10. On day 11, the edema
showed a small increase, which became marked after day 15, increasing and reaching its maximum
value on days 20–25 (Figure 2A). During this last period, such values were superimposed with those of
the period of edema formation in the chronic phase, emulating an arthritic status.
In the DAC control group experiment (using a vehicle), the maximum edema was observed 8 h
after FCA injection (Figure 1B). The edema decreased slowly until day 3, exhibiting a sudden increase
on day 4 and a marked and continuous decrease until day 7. After day 8, a daily and significant
increase of the edema was observed. The edema reached its highest value on days 18–22, showing a
small decrease until the end of the experiment (Figure 2B). These results are similar to those obtained
in the curcumin experiment for the chronic phase. The positive controls using phenylbutazone in both
experiments showed anti-edema activity (80 mg/Kg) throughout the acute phase (Figure 1A,B) and
chronic phase. This substance has been previously used in other models as a suitable reference drug to
inhibit the formation of edema caused by CFA injection in the hind paws of rats [28,29].
A
B
Figure 1. Acute effect of the oral administration of phenylbutazone (80 mg/kg), (
) diacetylcurcumin (DAC) (60, 120, and 150 mg/kg) on the edema produced by intradermal injection
of Freund’s complete adjuvant into the footpad of right rear limbs of Wistar rats. Each bar represents the
mean SEM (n = 5). ANOVA followed by Tukey’s test. p < 0.05, 0.01 and 0.001 (*, ** and *** respectively).
A) curcumin, and
(
B
±

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2.2.2. Effect of the Oral Administration of the Diacetylcurcumin and Curcumin against
FCA-Induced Arthritis
Continuous measurements of the footpad of the right rear limb of the animals with a
plethysmometer demonstrated that treatment with phenylbutazone, curcumin, or DAC at 60, 120,
and 150 mg/kg doses inhibited the formation of the edema during the period under study. During
the acute phase of the edema formation (Figure 1), the treatment with curcumin showed a significant
inhibitory effect at 4 h (120 mg/kg) and 24 h (120 mg/kg and 150 mg/kg) after FCA injection. Similar
findings were observed in the group treated with DAC at all doses (60, 120, and 150 mg/kg), which
significantly inhibited the formation of the edema during the acute phase by 37.21%, 47.67%, and
51.55%, respectively (Figure 1, see Supplementary Material). The reference drug (phenylbutazone,
80 mg/kg) significantly inhibited the edema at 4, 8, and 24 h in both experiments.
A
B
Figure 2. Antiinflammatory effect during the chronic phase of the oral administration of phenylbutazone
(80 mg/kg), (A) curcumin, and (B) diacetylcurcumin (DAC) (60, 120, and 150 mg/kg) upon the edema
induced by intradermal injection of Freund’s complete adjuvant into the footpad of right rear limbs of
Wistar rats. Each point represents the mean
0.01 and 0.001 (*, ** and *** respectively).
±
SEM (n = 5). ANOVA followed by Tukey’s test. p < 0.05,
During the chronic phase, the oral administration of curcumin caused a significant inhibition of
the edema on days 17–25 (150 mg/kg) (Figure 2A). Compared with phenylbutazone, the curcumin

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showed a slight increase in the percentages of edema inhibition. Furthermore, the administration of
DAC caused a significant decrease in the formation of the edema at different doses on days 4, 5, 6, 7, 10,
11, and 12 (60 mg/kg); on days 3, 4, 5, 6, 7, 9, 11, 12, and 15–25 (120 mg/kg); and on days 4, 5, 6, and
9–25 (150 mg/kg) (Figure 2B). In the chronic phase (after day 16), administration of DAC at 120 mg/kg
led to inhibition percentages that were equal to, or surpassed, those caused by the administration
of phenylbutazone.
The oral administration of DAC at doses of 120 and 150 mg/kg led to earlier, more sustained, and
more efficacious anti-edema activity (on days 3–25 and 4–25, respectively) than curcumin at a dose
of 150 mg/kg, with a significant reduction of swelling after day 17 until the end of the experiment
(Figure 2B). Visually, the footpads of rats treated with DAC at doses of 120 mg/kg and 150 mg/kg
were clearly healthier than the footpads of rats receiving curcumin. During the whole experiment,
no secondary lesions were observed in the non-injected footpads of the left rear limbs in any of the
groups treated with curcumin, DAC, or phenylbutazone (Figures 3 and 4). Additionally, all treated
groups showed acceptable joint mobility. The mean body weight showed no significant difference
(on day 25) between that of the animals receiving curcumin or DAC orally at any dose, and that of the
group treated with the vehicle. However, a loss in body weight seems to be a trait of the inflammation
model itself [30].
E
A
B
C
D
Figure 3. Edema status after oral treatment (on day 25) with curcumin at different doses. (
A) Vehicle,
(
B
) Curcumin 60 mg/kg, ( ) Curcumin 120 mg/kg, (D) Curcumin 150 mg/kg, and Phenylbutazone
C
(80 mg/kg) (E).
E
A
B
C
D
Figure 4. Edema status after oral treatment (on day 25) with diacetylcurcumin (DAC) at different
doses. ( ) Vehicle, ( ) DAC 60 mg/kg, ( ) DAC 120 mg/kg, ( ) DAC 150 mg/kg and Phenylbutazone
(80 mg/kg) (E).
A
B
C
D
3. Discussion
To assess the anti-arthritic effect of DAC in this study, one of the most important pharmacological
models was used. The arthritic status was induced by an injection of a dispersion of heat-killed and
freeze-dried mycobacteria into the footpad of the right rear limb of rats.
Although the pathogenesis of adjuvant arthritis is not clearly understood, there is a good amount
of evidence indicating that this disease is the result of an aberrant immune response. The humoral
immune response also plays a very important role in the initial phase of the disease [31]. On the other
hand, arthritis due to FCA injection at the footpad of the right rear limb of rats caused the primary
lesion, characterized by inflammation at the application site, while secondary lesions resulted in the
swelling of the footpad of the left rear limb and other parts of the body, such as ears or tail. There is an
alternative method to induce arthritis in a murine model, based on a single FCA injection at the base of
the tail. However, this method is considered unsatisfactory to cause arthritis [32].

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Our results showed that groups administered with the vehicle showed the peak of the chronic
phase to occur between days 18 and 21 [32]. Thus, it is safe to assume that the arthritic status existed in
all FCA-administered groups. Therefore, our results, suggesting a significant inhibition of inflammation
at the doses tested, are trustworthy. On the other hand, the reference drug phenylbutazone, used
herein at a lower dose than the 100 mg/kg reported by Perper et al. [32], was efficacious, providing
significant protection against primary and secondary lesions (Figures 3 and 4).
In the acute phase, observed 4 h after FCA injection, DAC showed better results against
inflammation than curcumin. Even at the lowest dose administered (60 mg/kg), DAC caused a
significant edema inhibition percentage (37.21%). In contrast, curcumin required a higher dose
(120 mg/kg) to cause a significant decrease of 41.67% in the edema. This fact is probably due to
the acetylation of the phenolic groups of the precursor molecule curcumin, resulting in increased
lipophilicity of the molecule and probably increased penetration in the gastrointestinal membrane.
During the chronic phase (days 17–25), the administration of curcumin (150 mg/kg) led to
inhibition percentages slightly higher than the reference drug: 43.9–52.4% and 42.7–49.3%, respectively
(Figure 2, see Supplementary Material). On the other hand, DAC administration resulted in a significant
inhibition of edema after day 4 at any dose. The best results were obtained at day 17, when DAC
inhibited the edema by 43.33–47.57% and 46.39–51.27% at doses of 120 and 150 mg/kg, respectively.
These effects were better than those of phenylbutazone (41.39–47.31%) in the same period (Figure 2, see
Supplementary Material). These results demonstrate the improved and sustained anti-inflammatory
effect of DAC, similar to curcumin at a dose of 150 mg/kg, but the effect of DAC starts at lower
doses. This action is probably due to the acetylation of the phenolic hydroxyl groups of the precursor
molecule, curcumin, which have increased the lipophilicity of the molecule. Consequently, the greater
penetration of the gastrointestinal membrane, like that which happened in in vitro studies when
evaluating the antibacterial activity of DAC and curcumin, shows that DAC has action at much lower
concentrations [20].
While non-steroidal anti-inflammatory drugs (NSAIDs) like aspirin, indomethacin, and naproxen
have been widely used to treat arthritis symptoms, they have been found to cause severe irritation
in the gastric mucosa after extended use [33,34]. This is due to the common mechanism of action
of NSAIDs, the inhibition of the cyclooxygenase known as prostaglandin-endoperoxide synthase
(PGH synthase), a speed-limiting enzyme that converts arachidonic acid into prostaglandins (PG).
Based on this, curcumin has been proven to be a biologically safe and efficient source material for
research, since its lack of toxicity, anti-inflammatory action, and very low ulcerogenic index have
been established [17]. These traits have been highlighted to propose its use against RA [7], with
various advantages over NSAIDs. The results reported here indicate that the curcumin analogue,
DAC, is an even more efficacious anti-inflammatory drug than its parent compound and also has a
shorter response time. On the other hand, DAC is practically non-toxic, allowing an oral intake of
5 g/kg. Moreover, DAC accumulation after a daily administration of the maximum dose (150 mg/kg)
for 14 days did not result in any signs of toxicity during the period under study. Thus, our results
support the anti-inflammatory potential of this curcumin analogue. Furthermore, because curcumin
is a major metabolite that naturally occurs in the plant species Curcuma longa and DAC is prepared
by synthetic modification of curcumin, both compounds were obtained with a high degree of purity,
as shown by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance
(NMR) and demonstrated by their usefulness as pharmacological therapeutic agents in a model of
rheumatoid arthritis.
4. Materials and Methods
4.1. Drugs and Chemicals
Phenylbutazone and Freund’s complete adjuvant (FCA) were purchased from Sigma (St. Louis,
MO, USA). Standard curcumin was purchased from ChromaDex (Irvine, CA, USA). Diacetylcurcumin

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(DAC) was synthetized at the Instituto de Qu
ímica, UNAM (Mexico City, Mexico). Curcuma longa
(Turmeric) was purchased from a local market in downtown Mexico City.
4.2. Extraction and Purification of Curcumin
Fresh rhizomes were cleaned, washed and sliced. Dried and ground C. longa (700 g) was macerated
with various solvent consecutive times. The first extraction was performed with dichloromethane,
followed by ethyl acetate and methanol, and, later, the extracts were evaporated. The percentages and
weights of the curcuminoid extracts were: Dichloromethane 4.3% (30.1 g), ethyl acetate 4.5% (31.5 g),
and methanol 5.6% (39.2 g). The methanol extract was subjected to column chromatography in a
silica gel (70–150 mesh) glass column. Chloroform and methanol solvent mixtures were used as the
mobile phase. All the collected fractions were monitored by thin-layer chromatography. Curcumin was
identified by running standard curcumin along with the samples. Crude curcumin was recrystallized
in isopropanol three times in a concentration of 6 g/L.
4.3. Synthesis of Diacetylcurcumin
Curcumin (5 g, 13.57 mmol) was dissolved in 70 mL of dichloromethane (CH2Cl2) with 2.20 mL
(27.14 mmol) of pyridine. This solution was kept under agitation for 15 min, followed by the dropwise
addition of acetic anhydride (1.4 mL, 13.57 mmol). The reaction mixture was kept under agitation
for 3 h at room temperature. Then, CH2Cl2 was evaporated and the residue was extracted with ethyl
acetate (3
×
60 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated
under reduced pressure. The resultant solid was recrystallized in ethyl acetate to yield yellow crystals
(4.2861 g of DAC, mp. 170.2 ◦C, yield 69.79%).
4.4. HPLC and NMR Analysis
The purity of curcumin (Figure 5) and DAC (Figure 6) was determined by high-performance
liquid chromatography (HPLC) in a Waters 1525 Binary HPLC pump equipment, controlled with the
Breeze 2 HPLC software (Waters Corporation, Milford, MA, USA). A 100–5 C18 Kromasil column
15 cm
×
4.6 mm i.d. and a diode array detector series 2998 (Waters Corporation, Milford, MA, USA),
the maximum absorption for curcumin and DAC was set at 265 and 257 nm, respectively) were used.
An acetonitrile-methanol-water-acetic acid (2%) isocratic gradient 40:20:40 was applied for 20 min at a
flow speed of 1 mL/min.
O
OH
1
7
3
5
6'
1'
5'
4'
2
4
1
6
2'
HO
OH
3'
O
O
2''
Figure 5. Chromatogram of the curcumin (1) (tR = 4.873 min) sample, injected at a concentration of
0.25 mg/mL, with detection at a wavelength of 265 nm.
O
OH
1
7
3
5
6'
1'
5'
4'
O
O
2
4
2
6
2'
1'''
O
O
3'
2'''
O
O
2''
Figure 6. Chromatogram of diacetylcurcumin (2) (tR = 9.190 min) samples, injected at a concentration
of 0.25 mg/mL, with detection at a wavelength of 257 nm.
The nuclear magnetic resonance (NMR) spectra of DAC and curcumin were acquired in Bruker
Avance III HD 500 MHz NMR Spectrometer (Bruker Scientific LLC, Billerica, MA, USA) equipment for

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1H and 13C in CDCl3 solution, using tetramethylsilane (TMS) as an internal standard (Tables 1 and 2).
NMR spectra were processed with the software MestReNova version: 12.0.0 (Mertrelab Research, S.L.,
Santiago de Compostela, España) and can be seen in the Supplementary Material. The chemical shift
values for DAC were compared with those reported in the literature [35].
Table 1. 1H-NMR spectral data of curcumin and diacetylcurcumin (DAC) (500 MHz) a.
Curcumin
Diacetylcurcumin
1H a
Chemical
Shift
Coupling
Constant (Hz)
Chemical
Shift
Coupling
Constant (Hz)
Multiplicity
Integral
Multiplicity
Integral
CH3-2000
CH3-200
H-4
s
s
s
d
d
m
6
6
1
2
2
4
2.33
3.88
5.85
6.56
7.06
s
s
s
d
d
6
6
1
2
2
3.93
5.82
6.48
6.91
7.07
H-2/H-6
J = 15.72
J = 8.01
J = 15.86
J = 8.11
H-50
H-2’/H-60
H-20
7.12
7.15
7.61
d
dd
d
2
2
2
J = 1.81
J = 8.15, 1.82
J = 15.78
H-60
H-1/H-7
O-Haryl
O-H
7.57
8.04
16.11
d
2
2
1
J = 15.72
Wide signal
Wide signal
15.85
Wide signal
1
a Data recorded in CDCl3. Chemical shifts (
δ) are expressed in ppm with respect to tetramethylsilane (TMS). Assigned
coupling patterns: s = singlet, d = doublet, dd = doublet of doublets, m = multiplet. All assignments were derived
from experiments: 1H-1H, 13C-1H, HSQC (heteronuclear single quantum coherence; couplings 1H-13C to a bond) and
HMBC (heteronuclear multiple bond correlation; couplings 1H-13C to 2 and 3 bonds). See Supplementary Material.
Table 2. 13C-NMR spectral data of curcumin and diacetylcurcumin (DAC) (125.7 MHz) a.
Chemical Shift
Carbon a
Curcumin
Diacetylcurcumin
C-2000
C-200
20.87
56.11
55.42
100.62
109.66
114.99
120.75
122.40
126.55
140.17
147.12
148.31
C-4
102.01
111.63
123.48
124.45
121.27
134.15
140.14
141.49
151.59
169.00
183.28
C-20
C-50
C-2 and C-6
C-60
C-10
C-1 and C-7
C-40
C-30
C-1000
C-3 and C-5
182.76
a
Data recorded in CDCl3. Chemical shifts (δ) are expressed in ppm with respect to tetramethylsilane (TMS). All
assignments were derived from experiments: 1H-1H, 13C-1H, HSQC (heteronuclear single quantum coherence;
couplings 1H-13C to a bond) and HMBC (heteronuclear multiple bond correlation; couplings 1H-13C to 2 and 3
bonds) 2D NMR spectra. See Supplementary Material.
4.5. Pharmacological Effect
4.5.1. Animals
All experiments were performed in accordance with the applicable ethical guidelines for avoiding
pain on experimental research animals [36] and the Mexican Official Norm for animal care and handling
(NOM-062-ZOO-1999). The experimental protocol (CIBIUG-P15-2018) and the study were approved
and supervised by the Institutional Committee of Ethics for the Care and Use of Laboratory Animals,
Universidad de Guanajuato. Inbred Wistar male rats (250–300 g, 7–8 weeks of age) and CD1 male
mice (20–25 g), obtained from the vivarium of the Division of Natural and Exact Sciences, Universidad
de Guanajuato, were used. All Wistar rats were housed in groups of five (n = 5) under controlled

Molecules 2019, 24, 2643
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temperature (23
±
2 ◦C) and humidity (55
±
10%) conditions, with a 12 h light/darkness cycle, and were
familiarized to the environment one week before the experiments. The animals were allowed food and
water ad libitum. Immediately after the experiments were completed, all animals were euthanized in a
CO2 chamber.
4.5.2. Acute Toxicity Studies
The mean lethal dose (LD50) by oral route (p.o.) was determined on CD1 male mice, as reported
by Lorke [37]. The drug concentration (DAC or curcumin) was adjusted to 3000 and 5000 mg/kg,
and administered p.o. (cannula 18G
were observed daily for 14 days, recording mortality, toxic effects, and/or weight changes, as well as
×
1.5”) with a volume of administration of 0.1 mL/10 g. Mice
behavior patterns.
4.5.3. Anti-Inflammatory Activity
The evaluation the effect of curcumin and DAC was done in independent experiments, and 25 rats
were required for each experiment (Five groups per experiment: Vehicle, phenylbutazone, and DAC or
curcumin (at 60, 120 and 150 mg/kg). The arthritis syndrome was induced by the intradermal injection
of 0.05 mL of FCA into the footpad of the right rear limb [28] of Wistar rats using a 20-gauge needle.
The edema evolution was determined by the volume displacement method using a digital
plethysmometer (Panlab, Barcelona, Spain). Volume displacement was recorded 24 h before FCA
injection and 4, 8, and 24 h after FCA injection. To complete the evaluation of drug activity on arthritis
induction, any variation in volume displacement at the footpad of the right rear limb of the subject rats
was determined daily until 25 days after injection. The reference drug (phenylbutazone, 80 mg/kg),
curcumin, and DAC (at a dose of 60, 120, or 150 mg/kg) were administered p.o. (cannula 16G
1 h before FCA injection and for 14 days after. The carrier vehicle used was corn oil in a maximum
administration volume of 0.1 mL/100 g.
×
3”)
The percentage of edema inhibition was calculated as the average measurement for each group
(n = 5) treated, with respect to the average measurement of the group only administered with the
vehicle. Differences between the controls and treatments were analyzed with Tukey’s test. A p-value
less than 0.05 was considered statistically significant. The data were processed with the statistics
software GraphPad PRISM 7.0b (GraphPad Software, San Diego, CA, USA).
5. Conclusions
The oral administration of diacetylcurcumin (60 mg/kg and 120 mg/kg) caused a significant
inhibition of inflammation during the acute and chronic phases, respectively, demonstrating an
enhanced and sustained anti-inflammatory effect, surpassing that of curcumin (150 mg/kg) in the
chronic stage, and showing a high therapeutic potential as an antiarthritic agent with fewer side effects
(low ulcerogenic index). This study supports the traditional use of Curcuma longa to treat inflammatory
conditions and the positive consequences that chemical modifications may have in improving its
therapeutic potential.
Supplementary Materials: The following are available online. Table S1: The effect of curcumin and DAC on the
edema induced by Freund’s complete adjuvant on a murine model (acute phase); Table S2: The effect of curcumin
and DAC on the edema induced by Freund’s complete adjuvant on a murine model (chronic phase); Figure S1:
1H-NMR of curcumin (
HMBC of curcumin ( ); Figure S5: HMBC of curcumin (
of dicaetylcurcumin (
); Figure S8: 13C-NMR of diacetylcurcumin (
1
); Figure S2: 13C-NMR of curcumin (
); Figure S6: HMBC of curcumin (
); Figure S9: HSQC of diacetylcurcumin (
); Figure S11: HMBC of diacetylcurcumin ( ); Figure S12: HMBC of
1
); Figure S3: HSQC of curcumin (
); Figure S7: 1H-NMR
);
1); Figure S4:
1
2
1
1
2
2
Figure S10: HSQC of diacetylcurcumin (
2
2
diacetylcurcumin (2).
Author Contributions: Formal analysis, S.L.G.-G. and W.M.-M.; Funding acquisition, C.E.-M., M.A.D.-Á., and
R.G.E.; Investigation, C.E.-M. and R.G.E.; Methodology, M.I.C.-L., A.T.-V., and W.M.-M.; Supervision, C.E.-M.;
Writing—review and editing, C.E.-M. and R.G.E.

Molecules 2019, 24, 2643
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Funding: This research was funded by PROGRAMA DE APOYO A PROYECTOS DE INVESTIGACI
E INNOVACI N TECNOL GICA (PAPIIT-UNAM), grant number IN208516, CONSEJO NACIONAL
DE CIENCIA Y TECNOLOG A-INVESTIGACI N CIENT FICA B SICA 2015, grant number 252524,
CONVOCATORIA INSTITUCIONAL DE INVESTIGACI N CIENT FICA 2016-2017-UG, grant number 1088/2016
and CONVOCATORIA INSTITUCIONAL DE INVESTIGACI N CIENT FICA 2019-UG, grant number 178/2019.
ÓN
Ó
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Ó
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Á
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Acknowledgments: M.I.C.-L. thanks the scholarship granted through the project No. 1088/2016, Convocatoria
Institucional de Investigaci n Cient fica 2016-2017-UG. Special thanks to Juan Pedro Galv n Ch a, Claudia Karina
ó
í
á
í
Sánchez Sánchez, and Víctor Rangel Picón for their technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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