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Biol. Pharm. Bull. 35(10) 18181820 (2012)

Note

Vol. 35, No. 10

Anti-arthritic Effect of Eugenol on Collagen-Induced Arthritis

Experimental Model
Renata Grespan,*,a Marcia Paludo,a Henrique de Paula Lemos,b Carmem Patrcia Barbosa,a
Ciomar Aparecida Bersani-Amado,a Marcia Machado de Oliveira Dalalio,c and
Roberto Kenji Nakamura Cumana
a

Department of Pharmacology and Therapeutics, State University of Maring; c Department of Clinical Analysis,
State University of Maring; Maring, Pr 87020-900, Brazil: and b Immunotherapy Center, Georgia Health Sciences
University; Augusta, GA 30912, U.S.A. Received February 7, 2012; accepted July 12, 2012
This study was designed to test the efcacy of eugenol, a compound obtained from the essential oil of
cloves (Syzygium aromaticum) in collagen-induced arthritis (CIA), a well characterized murine model of
rheumatoid arthritis. Macroscopic clinical evidence of CIA manifests rst as periarticular erythema and
edema in the hind paws. Treatment with eugenol starting at the onset of arthritis (day 25) ameliorated these
clinical signs of CIA. Furthermore, eugenol inhibited mononuclear cell inltration into the knee joints of
arthritic mice and also lowered the levels of cytokines (tumor necrosis factor (TNF)-, interferon (IFN)-
and tumor growth factor (TGF)-) within the ankle joints. Eugenol treatment did not affect the in vitro cell
viability as assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Therefore, eugenol ameliorates experimental arthritis and could be useful as a benecial supplement in treating human arthritis.
Key words

eugenol; collagen-induced arthritis; inammation; cytokine; mononuclear cell

Eugenol (4-allyl-2-methoxyphenol) is the major component in the essential oil of many aromatic plants, including
clove (Zyzygium aromaticum) and it is known to possess
antioxidant, analgesic and neuroprotective properties among
others.1,2) In addition, eugenol and related compounds exhibit
anti-inammatory activities, e.g. inhibition of lipopolysaccharide (LPS)-stimulated nuclear factor-kappa B (NF-B)
activation, cytokine release and cyclooxygenase-2 (COX-2) expression by macrophages in vitro3) and inhibition of 5-lipoxygenase activity in polymorphonuclear cells.4) However, despite
the anti-inammatory effects of eugenol described previously,
there is only one report of it exhibiting antirheumatic effects
in a model of adjuvant arthritis.5)
This new therapeutic strategy involving natural products
has been devised because the anti-inammatory and immunosuppressive drugs currently available cause many side effects
and show limited efcacy in the treatment of rheumatoid
arthritis.
Collagen-induced arthritis (CIA) is used as an experimental model that resembles human rheumatoid arthritis. Both
T cell and B cell responses to the autoantigen as well as the
production of cytokines by systemic and tissue-specic cell
populations are critical for the development (and eventual
diminution) of the autoimmune response to CII and to the
pathology in CIA.6) Due to the paucity of information on the
effect of eugenol on experimental models of arthritis, this
study was undertaken to investigate the efcacy of eugenol on
CIA model.

MATERIALS AND METHODS

Animals This study was conducted on male DBA1/J
mice, 23 months old, 2025 g body weight, obtained from
the University of So Paulo, Department of Pharmacology,
The authors declare no conict of interest.
* To whom correspondence should be addressed.

Faculty of Medicine of Ribeiro Preto, SP, Brazil. All mice

had access to water and standard rodent diet ad libitum. All
experimental procedures were approved by the Ethics Committee of the State University of Maring, Pr, Brazil.
Induction and Assessment of CIA Mice received 100 g
of bovine collagen type II (CII) (kind gift of David D Brand,
Memphis, Tennessee, U.S.A.) dissolved in 25 L of 0.05 m M
acetic acid and emulsied with an equal volume of complete
Freunds adjuvant (CFA, 4 mg/mL) (Difco Laboratories, Detroit, Michigan, U.S.A.). Emulsion (50 L) was injected intradermically (i.d.) into the base of the tail (day 0). Mice were
monitored daily for signs of arthritis and graded using a scale
of 04, where 0=normal, 1=mild swelling with erythema, 2=
signicant joint swelling, 3=severe swelling and digit deformity, and 4=maximal swelling with ankylosis. Each joint was
scored, with a maximum possible score of 4 per mouse that
did have disease, since we sum the values of four paws and
divide by four to put in the results.6)
Treatment Protocol Mice received 100 g of eugenol
(Biodinmica Laboratory, Ibipor, Paran, Brazil) or vehicle
(saline containing 1% Tween 80, v/v) orally, daily, from the
disease onset (day 25) until the end of the experiment. The
normal mice were immunized with CII but without treatment
or vehicle.
Cell Migration To evaluate leukocyte migration, the
articular cavities of knee joints were washed twice with 5 L
phosphate buffered saline (PBS) containing 1 m M ethylenediaminetetraacetic acid (EDTA) and then diluted to a nal
volume of 100 L with PBS/EDTA. Total cell counts were
performed in a Neubauer chamber under optical microscopy
(Nikon Eclipse E-200), and differential cell counts (100 cells)
were performed on cytocentrifuge slides (Cytospin 3; Shandon, Pittsburgh, PA, U.S.A.) stained with Rosenfelds stain.
Differential cell counts were performed using a light microscope (Zeiss, Wetzlar, Germany), and results were expressed
as the number of mononuclear cells per cavity.

e-mail: r_grespan@yahoo.com.br

2012 The Pharmaceutical Society of Japan

October 2012

Fig. 1.

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Eugenol Reduced the Progression and Severity of CIA and Cellular Recruitment in Knee Joints from Arthritic Mice

(A) Eugenol (100 g/mouse) was administered orally to CII-immunized mice once daily from day 25 to day 40. The severity of arthritis was evaluated by clinical arthritis score. (B) Mononuclear cells migration from Normal, Vehicle or Eugenol treated arthritic mice were evaluated at the end of the experiment. Data are expressed as
meansS.E.M. of 5 mice per group. # p<0.05 versus vehicle-treated CIA mice.

Measurements of Cytokine Levels by Enzyme-Linked

Immunosorbent Assay (ELISA) To measure cytokine concentrations in the inammatory site, articular tissues were
harvested and triturated in 500 L of PBS containing EDTA
by tissue-trimmer. Articular homogenates were centrifuged
and supernatants collected and stored at 70C for subsequent
determination of interferon (IFN)-, interleukin (IL)-10, tumor
growth factor (TGF)-, and tumor necrosis factor (TNF)-
concentrations by ELISA (R & D system), according to the
manufacturers instructions.
Cell Viability Assay The MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay is based
on the mitochondrial enzyme reduction of a tetrazolium dye
to detect and determine cell viability. Briey, the cells were
plated at a density of 1105 cells/well into 96-wells plates.
After 4 h exposure to eugenol (1, 3, 10, 30 or 90 g/mL of
0.05% dimethyl sulfoxide (DMSO)), 20 L of MTT (5 mg/mL,
Sigma, St. Louis, U.S.A.) stock solution was added to each
well. After 2 h incubation at 37C, the medium was removed
and 200 L of DMSO were added to each well. Cells were incubated for a further 10 min and then the absorbance was read
on a Bio-Tek (ELX 808IU) ELISA reader at the wavelength of
540 nm. Blank values were subtracted from each treated and
control reading. Cell viabilities were expressed as percentage
of viable cells determined as follows:

% viable cells = (the absorbance of the treated cells)

(the absorbance of the blank) 100 /
(the absorbance of the control)
(the absorbance of the blank)

RESULTS AND DISCUSSION

Among the several constituents of plant essential oils,
studies have shown that eugenol has antioxidant, anti-inammatory, DNA-protective, analgesic and antimicrobial properties.1,2) Our previous ndings indicate that Syzygium aromaticum, whose major compound is eugenol, has an immunomodulatory effect.7) In the present study, we tested if the presence
of eugenol has any effect on an established arthritis in mice.
Although only a few advances in understanding the etiology
of human autoimmune diseases has been achieved with the
use of experimental models, they do offer an opportunity to
investigate new potential therapeutic applications to improve

Fig. 2.

Effect of Eugenol on Neutrophils Viability

Neutrophils were incubated with eugenol at indicated concentrations for 4 h. Cell

viability was measured as described in Materials and Methods. Each value as represented as meanS.E.M. from three independent experiments.

joint inammation. As shown in Fig. 1A, eugenol-treated

arthritic mice exhibited clinical scores lower than the control group which received vehicle only. Even though eugenol
started to produce benecial effects on the inammation observed in CIA at the beginning of the treatment, a statistically
signicant reduction in the clinical score of CIA by eugenol
became apparent after 3540 d after immunization. Besides,
it was not observed any difference between vehicle-arthritic
mice and normal mice (Fig. 1).
Given the fact that inltration of leukocytes plays an essential role in experimental arthritis and could contribute to
articular damage,8) we tested whether eugenol could modulate
leukocyte recruitment to the knee joints and also cell viability.
As shown in Fig. 1B, eugenol signicantly reduced mononuclear cell migration to the knee joint when compared with
the vehicle-treated arthritic mice. Cell viability was measured
using an MTT test and expressed in terms of relative absorbance of eugenol-treated cells versus control cells. Our results
show that cells treated with eugenol remain viable (>80%)
even when exposed to the highest concentration (90 g/mL)
(Fig. 2).
Taking into account that cytokines are involved in direct
cell-to-cell communication and in the tissue damage observed
in rheumatoid arthritis,9) we investigated the effect of eugenol
on the concentrations of TNF-, IFN-, TGF- and IL-10 in
the affected ankle joints. Paw samples from arthritic mice
treated with vehicle contained signicantly higher concentrations of all of the above-mentioned cytokines when compared

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Fig. 3.

Vol. 35, No. 10

Decreased Inammatory Cytokines in Articular Joints from Eugenol-Treated Arthritic Mice

Ankle joints from vehicle or eugenol (100 g/mouse) treated arthritic mice were homogenized and the levels of TNF- (A), IL-10 (A), IFN- (B) and TGF- (B) in the
supernatants determined by ELISA. Results are expressed as meanS.E.M. of 4 mice per group. # p<0.05 versus vehicle-treated CIA mice.

to those of nave mice.10) On the other hand, mice treated with

eugenol showed a signicant reduction in TNF- (Fig. 3A),
IFN- and TGF- (Fig. 3B) levels. It has been previously reported that IL-10 inhibit autoimmune inammation in CIA.11)
In our results, we found a slight tendency for eugenol to enhance levels of the anti-inammatory cytokine IL-10 (Fig. 3A)
when compared with vehicle-treated arthritic mice.
In conclusion, the results presented herein give additional
insight into the previously described antiinammatory benecial effects of eugenol, suggesting that this compound may
be an alternative and/or supplemental treatment to chronic
inammatory diseases such as rheumatoid arthritis.
Acknowledgements We would like to thank Jailson G.
Dantas and Clia Regina Miranda for technical assistance.
This research was supported by CAPES.

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