1240 Original Papers

Effect of Hautriwaic Acid Isolated from

Dodonaea viscosa in a Model of Kaolin/
Carrageenan-Induced Monoarthritis

Authors David Osvaldo Salinas-Sánchez 1, 3, Alejandro Zamilpa 1, Salud Pérez 2, Maribel Herrera-Ruiz 1, Jaime Tortoriello 1,
Manasés González-Cortazar 1, Enrique Jiménez-Ferrer 1

1
Affiliations Centro de Investigación Biomédica del Sur (CIBIS), Instituto Mexicano del Seguro Social (IMSS), Xochitepec, Morelos, México
2
Departamento de Sistemas Biológicos, UAM-Xochimilco, México, D. F., México
3
Centro de Investigación en Biodiversidad y Conservación (CIByC), Universidad Autónoma del Estado de Morelos,
Cuernavaca, Morelos, México

Key words Abstract negative control group (animals with damage

l
" Dodonaea viscosa
! without any treatment). The negative control

Downloaded by: University of Illinois. Copyrighted material.

l
" Sapindaceae
In the present work, the antiarthritic activity of group presented a decrease in the concentration
l
" hautriwaic acid
hautriwaic acid is reported. This ent-clerodane di- of interleukin-10, while the groups that received
l
" clerodane

terpene isolated from Dodonaea viscosa was eval- hautriwaic acid at different dose exhibited an in-
l
" arthritis

l
" kaolin/carrageenan uated in mice using a kaolin/carrageenan-in-
duced monoarthritis model. The inflammation
observed in the joint (knee) on days 1–8 ranged
from 50–70 %. After 10 days of treatment with dif-
ferent doses of hautriwaic acid (5, 10, 20 mg/kg), a
decrease in knee inflammation was detected. This
recovery was observed with both reference drugs,
methotrexate (1 mg/kg) and diclofenac (0.75 mg/
kg). In these groups of mice, the concentration of
proinflammatory cytokines interleukin-1 beta,
interleukin-6, and tumor necrosis factor alpha in
the joint was significantly lower than that of the
crease in this interleukin. This anti-inflammatory
cytokine was not modified in the joint of mice
with diclofenac, but in mice that received metho-
trexate, a significant decrease was observed. Hau-
triwaic acid isolated from D. viscosa diminished
the joint edema induced by this mixture of poly-
saccharides (carrageenan), possibly by acting as
immunomodulator of the inflammatory re-
sponse.
Supporting information available online at
http://www.thieme-connect.de/products

Introduction toxicity and bone marrow toxicity, among others,

received Sept. 11, 2014
revised March 26, 2015
accepted May 21, 2015
Bibliography
DOI http://dx.doi.org/
10.1055/s-0035-1546197
Published online July 10, 2015
Planta Med 2015; 81:
1240–1247 © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Dr. Enrique Jiménez-Ferrer
Centro de Investigación
Biomédica del Sur (CIBIS)
Instituto Mexicano del Seguro
Social (IMSS)
Argentina #1, Col. Centro
62790 Xochitepec, Morelos
México
Phone: + 52 77 73 61 21 55
Fax: + 52 77 73 61 21 55
enriqueferrer_mx@yahoo.com
! generating an increase in the deterioration of the
Rheumatoid arthritis (RA) is a chronic, multisys-
temic immune disease whose main clinical sign
is a polyarticular inflammation with predomi-
nance in the small joints of hands and feet. It is
characterized by an uncontrolled proliferation of
synovial tissue. Prevalence is estimated at 1 %
worldwide, with a greater tendency for women
to have the disease [1]. For treating RA, different
types of drugs have been utilized, such as the fol-
lowing: Disease-modifying antirheumatic drugs
(DMARD), among which immune system sup-
pressors are prominent (Leflunomide and Rituxi-
mab); tumor necrosis factor alpha (TNF-α) inhib-
itors, including Infliximab, Etanercept, and Adali-
mumab; interleukin-1 beta (IL-1β) inhibitors
(Anakinra), and interleukin-6 (IL-6) inhibitors,
such as Tocilizumab [2]. In addition, some non-
steroidal anti-inflammatory drugs (NSAID) and
corticoids continue to be employed [3]. However,
many of these treatments cause side effects,
which range from gastric discomfort to hepato-
health of patients [4]. Medicinal plants comprise a
potential source of effective therapeutics for the
treatment of several illnesses. These species gen-
erally contain active principles that not only work
as anti-inflammatories in the case of RA, but also
possess immunomodulator effects and even “dis-
ease improvers”. At the Sierra de Huautla Bio-
sphere Reserve (REBIOSH) in Morelos State, Mex-
ico, we find Dodonaea viscosa (L.) Jacq., a plant be-
longing to the Sapindaceae Family and that is
widely employed for the treatment of illnesses
whose pathophysiological background is inflam-
mation and pain, such as RA [5, 6]. Previous phar-
macological studies of D. viscosa have shown its
antidiarrheic, antibacterial, analgesic, antiviral,
antiulcer, antioxidant, gastroprotective, and anti-
inflammatory activit [7–13]. Chemical studies of
this plant demonstrate the presence of terpenoids
and flavonoids [14–17]. On the other hand, in a
study carried out by our work group, the anti-in-
flammatory activity of hautriwaic acid (HA,

Salinas-Sánchez DO et al. Effect of Hautriwaic … Planta Med 2015; 81: 1240–1247


Fig. 1 Chemical struc-
ture of HA.
l" Fig. 1) isolated from a dichloromethane extract (DCME) of D.
viscosa was observed. This diterpene exhibited an anti-inflam-
matory effect in a model of edema in the mouse ear by 12-O-tet-
radecanoylforbol-13-acetate (TPA) administered topically and in-
traperitoneally (i. p.) [18]. Additionally, recent investigations sug-
gested that the HA present in the methanolic extract of D. viscosa
is one of the hepatoprotective constituents of this plant species
[19].
The objective of the present work was to measure the immuno-
modulatory effect of different doses of HA (5, 10, and 20 mg/kg)
in an experimental model of RA induced by kaolin/carrageenan
(K/C) injection in the right knee of mice, quantifying the levels
of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and the
Fig. 2 Effect of the oral administration of MTX (1.0 mg/kg), HA (5, 10,
20 mg/kg), and Diclo (0.75 mg/kg) on the size of the right knee joint in CD-1
mice that had induced monoarthritis with kaolin/carrageenan. Each point
Salinas-Sánchez DO et al. Effect of Hautriwaic …
Original Papers 1241
anti-inflammatory cytokine (IL-10) in the synovial capsule of
the joints of healthy and experimental, sick animals.
The differences between the TPA mouse model [18] and this
model of monoarthritis included that the inflammation is in-
duced by administering carrageenan (C), based on the fact that
the former generates an inflammation stimulus through the
arachidonic acid cascade and the latter causes the system to es-
tablish inflammatory disease modeling [20, 21].
In the research, diclofenac (Diclo) and methotrexate (MTX) were
used as positive controls; Diclo possesses an NSAID mechanism
in which cyclooxygenase is inhibited, and MTX modifies or alters
the activity of immune-stimulated cells, decreasing their activity
by inhibiting folic acid synthesis.
Results and Discussion
!
Previous pharmacological and chemical studies allowed the iso-
lation of the clerodane-type diterpene HA from D. viscosa leaves
[17, 22, 23] and demonstrated its anti-inflammatory effect [18].
Although such an effect of this compound has been demonstrat-
ed [18], to our knowledge, this is the first time that its antiar-
Downloaded by: University of Illinois. Copyrighted material.
thritic activity has been reported.
In l" Fig. 2, the results of the septic monoarthritis induced for a
15-day period in the knee of CD-1 mice by means of kaolin (4%)
and carrageenan (2%) injection are shown, resulting in cartilage
damage with prolonged inflammation of the synovial tissue,
which was evaluated as a percentage of inflammation with re-
represents the average ± S. E., n = 12, ANOVA, Bonferroni post-test, * p < 0.05
compared with the negative control.
Planta Med 2015; 81: 1240–1247
1242 Original Papers
spect to the baseline level (% of change). K/C-induced arthritis de-
velops rapidly, in a matter of hours, and persists for weeks [24].
The damage was visually observed due to its producing pro-
longed edema in comparison with baseline values, which were
established prior to the induction of the arthritis on day 1. The
inflammation could also be compared with the group of mice
not receiving K/C and that was monitored during the 15-day bio-
assay. Treatments were administered orally from 24 h after K/C
injection and daily up to day 15. In all groups, K/C administration
caused a pronounced level of edema from day 1 with peak in-
flammation at days 7 and 9, depending on the treatment. From
that moment, the inflammation began to cede significantly with
respect to the negative control group mice, which only received
the vehicle with K/C, but had no treatment (*p < 0.05).
Group 1 negative control mice presented a progressive inflam-
matory process. Day 1 initiated with a 40 % edema increase, with
the edema gradually increasing and being maintained until day 9,
when it reached maximal inflammation at 69%. On the following
days (10–15), the inflammation remained at between 68 and
53 %; data were not significantly different from those of day 9
(l" Fig. 2).
In group 2, which corresponds to mice treated with MTX (1.0 mg/
kg, orally – p. o.), a growing inflammation process was also ob-
served, beginning on day 1, when it reached 43 % of edema com-
pared with the baseline group; edema development was parallel
to that observed in the group with damage, although the inflam-
mation was less intense. The gradual increase of edema reached
its maximal level on day 7, recording 55 % edema in the baseline
control group. From day 8 of treatment with MTX, a drop in in-
flammation was observed to baseline levels (l " Fig. 2). The ani-
mals did not exhibit significant changes in behavior or in their
general state.
Group 3, treated with HA at 5 mg/kg p. o., presented 41 % edema
on day 1, increasing to 61 % on day 7. The inflammation de-
creased beginning on day 8 (with 59 %) and fell slowly until day
15, when the mice reached 27 %; on this same day, the animals
were sacrificed (l " Fig. 2).
Group 4, which included HA-treated mice at 10.0 mg/kg p. o., ex-
hibited 40 % edema at initiation on day 1, which increased to 58 %
at day 8. Later, the inflammation diminished slowly until reach-
ing 20 % on the last day of the bioassay (l " Fig. 2).
In group 5, with HA at a dose of 20 mg/kg p. o., the animals
showed an initial edema of 54% on day 1. On the following days,
an inflammation decrease was recorded of up to 13% on the day
the animals were sacrificed (l " Fig. 2). Animals in groups treated
with HA at different doses did not present significant behavioral
changes, nor evidence of toxicity, such as spiky hair or weight
loss.
Finally, in group 6, in which the animals were treated with Diclo
(0.75 mg/kg p. o.), edema developed gradually, with 36% at initia-
tion on day 1; maximal inflammation was observed at 45 % on
day 12, which slowly diminished from day 15 on (17%). On the
final treatment days, this group achieved baseline levels
(l" Fig. 2). On the other hand, during this treatment, the mice pre-
sented important alterations in their general condition; they ex-
hibited notable changes, such as aggressiveness, spiky hair, and
weight loss, and four deaths were registered.
These results showed that oral administration of MTX, HA at dif-
ferent doses, and Diclo substantially reduced edema in the dam-
aged right joint (*p < 0.05). Data represent the average ± standard
error of the mean (SEM) of the percentage of change in right knee
size and were significantly different from the results of negative
Salinas-Sánchez DO et al. Effect of Hautriwaic … Planta Med 2015; 81: 1240–1247
control group edema. This demonstrates that HA is capable of di-
minishing the inflammation generated by diverse irritating
agents, such as TPA [18] or carrageenan. This suggests that com-
pounds from D. viscosa can exert anti-inflammatory activity by
means of several mechanisms of action, because HA was proven
effective in the acute TPA-associated inflammation that was
caused and was effective in decreasing K/C-related chronic joint
inflammation. TPA is known to cause an increase in calcium in-
flux, resulting in the activation of phospholipase D, which stimu-
lates the conversion of arachidonic acid into prostaglandins and
leukotrienes [25] and regulates the release of cytokines [26]. Fur-
thermore, the chronic inflammation model by means of K/C ad-
ministration implies that the inflammatory stimulus is main-
tained at least for 2 weeks, due to poor kaolin absorption; thus,
tissue damage, characteristic of chronic inflammation, is estab-
lished [27], involving tissue infiltration mainly by macrophages,
lymphocytes, and plasma cell [28]. All of this causes tissue de-
struction and scarring processes involving angiogenesis, but es-
pecially fibrosis [29].
As shown in l " Figs. 3 and 4, the results correspond to the level of
proinflammatory cytokines (IL-1β and IL-6) and TNF-α in l " Fig. 5,
taken from the right knee of animals exposed to K/C administra-
Downloaded by: University of Illinois. Copyrighted material.
tion. The first column of each graph corresponds to the baseline
concentration of these cytokines. We observed that daily admin-
istration of Diclo, MTX, and HA (5, 10, and 20 mg/kg) avoided the
increase of proinflammatory cytokine IL-1β and IL-6 concentra-
tions induced by K/C, whose value was significantly less than that
of the negative control group (*p < 0.05) (l " Figs. 3 and 4).
In l" Fig. 5, we can observe that the administration of Diclo, MTX,
and HA at different doses avoids the increase in TNF-α concentra-
tion induced by K/C, whose value was significantly less than that
of the negative control group (*p < 0.05); however, the effect of
MTX on this cytokine was significantly different from that of the
group with damage, and less than that exerted on the proinflam-
matory interleukins.
Finally, l" Fig. 6 shows that the group of animals with damage
presents a decrease in joint concentration of cytokine IL-10.
Treatment with MTX does not cause significant changes in the
concentration of this protein in comparison with that of the neg-
ative control group (*p > 0.05). Diclo causes a significant decrease
of this cytokine. It is noteworthy that the difference between Di-
clo, MTX, and HA (5, 10, and 20 mg/kg) causes a significant in-
crease in IL-10 levels, while the synthetic drugs did not (l " Fig. 6).
Rheumatoid arthritis is a chronic, inflammatory autoimmune
disease and its preferential target is synovial tissue, cartilage,
and bone. Multiple cytokines produced by the innate and adap-
tive immune cells are implicated in the pathogenesis of RA. The
imbalance between anti-inflammatory and proinflammatory cy-
tokines leads to autoimmunity, chronic inflammation, and carti-
lage destruction. When the immune response is not suppressed
in a timely fashion, it can trigger immunological alterations with
collateral damage to the initial process, that is, to autoimmunity,
the developmental hub of RA. Due to the importance that cyto-
kines present in RA, they constitute important targets of thera-
peutic interest [30].
Experimental arthritis caused by K/C injection in mice is an ex-
perimental model utilized for the evaluation of potential antiar-
thritic drugs [31]. The joint inflammation produced by this toxic
mixture is based on its formulation, in that it contains a polysac-
charide isolated from some species of red algae, with an alumi-
num silicate (kaolin) as adjuvant, which presents a very low or
null absorption level. Thus, the proinflammatory stimulus is
 Original Papers 1243

Fig. 3 Effect of Diclo (0.75 mg/kg), MTX (1.0 mg/

kg), and HA (5, 10, 20 mg/kg) from D. viscosa on
increasing concentrations of 1 L‑1β in joint swelling
mice with K/C. Each bar represents the average ±
S. D., n = 12, ANOVA, Bonferroni post-test, *p < 0.05
compared with the negative control group. (Color
figure available online only.)

Fig. 4 Effect of Diclo (0.75 mg/kg), MTX (1.0 mg/

Downloaded by: University of Illinois. Copyrighted material.

kg), and HA (5, 10, 20 mg/kg) from D. viscosa on
increasing concentrations of 1 L‑6 in joint swelling
mice with K/C. Each bar represents the average ±
S. D., n = 12, ANOVA, Bonferroni post-test, *p < 0.05
compared with the negative control group. (Color
figure available online only.)

Fig. 5 Effect of Diclo (0.75 mg/kg), MTX (1.0 mg/

kg), and HA (5, 10, 20 mg/kg) from D. viscosa on
increasing concentrations of TNF-α in joint swelling
mice with K/C. Each bar represents the average ±
S. D., n = 12, ANOVA, Bonferroni post-test, *p < 0.05
compared with the negative control group. (Color
figure available online only.)

maintained for a prolonged time period, rendering the stimulus In the present work, it was possible to carry out the quantifica-
chronic, even though it has a one-application-only scheme. tion of cytokines in the damaged knee of animals with K/C and

Salinas-Sánchez DO et al. Effect of Hautriwaic … Planta Med 2015; 81: 1240–1247

1244 Original Papers
to compare this with the levels of these proteins in the knee of
the group of healthy animals. We observed that HA at different
doses not only possesses important anti-inflammatory activity,
but it is also capable of modulating cytokine concentrations. The
latter leads us to propose this diterpenic acid as an anti-inflam-
matory and also as an immunomodulator of response to chronic
damage, as is the case of pathophysiological mechanisms during
RA.
It has been demonstrated that in carrageenan-induced pain and
inflammation models, carrageenan promotes the increase of
TNF-α and IL-1β [32] concentration, and even IL-17, a cytokine
that has been proposed as an immune response marker in RA
[33]. This evidence indicates that carrageenan is an agent that ac-
tivates the immune response against the damage it generates
and, in the case of the present work, we observed that the joint
responded directly to this damage with a high production of
proinflammatory cytokines such as IL-1β and TNF-α.
TNF-α is produced by monocytes and macrophages. It is a pri-
mary immune response activator that stimulates chondrocytes
to release metalloproteinases, which in turn degrade the main
constituents of cartilage; TNF-α induces the release of IL-1β, con-
solidating the proinflammatory response [34]. The experimental
data indicate that treatments with clerodane isolated from D. vis-
cosa were capable of causing a decrease in the concentration of
this cytokine in the joint, a similar effect to that observed with
Diclo as well as with MTX.
On the other hand, IL-1β is a primary immune response activator
and it directs the inflammatory response and cellular prolifera-
tion of the synovial membrane; it also regulates the IL-6 expres-
sion of synoviocytes [35]. IL-6 is fundamental for stimulating B
lymphocytes and for generating antibodies that are inducers of
the aggravated inflammatory response triggering the course of
chronic inflammation in the joints [36, 37]. These results demon-
strated that daily oral administration of Diclo, MTX, and HA at
different doses avoided the increase of the proinflammatory cy-
tokine IL-1β and IL-6 concentrations caused by K/C injection, the
value achieved below that of the control group (−). Therefore, it is
noteworthy that the main compound isolated from D. viscosa at
different doses produces a diminution of these proinflammatory
cytokine levels. It is transcendental to highlight the importance
of the existence of this diminution and, concretely, a blockage of
these cytokines; thus, it has been manifested as a target for scien-
Salinas-Sánchez DO et al. Effect of Hautriwaic … Planta Med 2015; 81: 1240–1247
Fig. 6 Effect of Diclo (0.75 mg/kg), MTX (1.0 mg/
kg) and HA (5, 10, 20 mg/kg) from D. viscosa on
concentrations of IL-10 in joint swelling mice with
K/C. Each bar represents the average ± S. D., n = 12,
ANOVA, Bonferroni post-test, *p < 0.05 compared
with the negative control group. (Color figure avail-
able online only.)
tific research on RA and, in the future, for taking these drugs into
the clinic.
Downloaded by: University of Illinois. Copyrighted material.
The results demonstrated that HA at different doses increased the
concentration of IL-10, which is produced by different cell types
during the immune response. One of its main functions is to limit
the advance of the inflammatory process in order to protect the
host from the excessive immune overstimulation that generates
tissue destruction, which is characteristic of RA [38]. IL-10 activ-
ities represent a key homeostatic mechanism for controlling the
extent and duration of the immune response. It acts as a potent
anti-inflammatory, selectively blocking the expression of proin-
flammatory genes encoding for cytokines and chemokines, and
improving the production and expression of anti-inflammatory
molecules [39]. Although IL-10 is produced in substantial
amounts in the joints during RA, these levels are insufficient for
controlling inflammation. Thus, the interpretation can be that an
increase in the local concentration of this cytokine can in turn
give rise to the decrease in the levels and proinflammatory ac-
tions of other proteins, as in the case of TNF-α, IL-6, and IL-1β.
Therefore, the drugs making this action possible can be proposed
as modulators of the response to the damage, as in the case of HA
at different doses. These treatments were capable of increasing
IL-10 production and, in parallel fashion, were capable of causing
the decrease of proinflammatory cytokines. While the anti-in-
flammatory effect of D. viscosa and other plants of the genus has
been well described, to our knowledge, this is the first report that
indicates the pharmacological potential of this plant species for
the treatment of RA. Isolation of the HA anti-inflammatory cler-
odane from this plant has been achieved, which demonstrated
that it is capable of modifying local cytokine concentrations,
leading us to propose it as a substance with immunomodulator
activity. The results of this work comprise the previously re-
ported extension of HA pharmacological characterization, which
was reported previously [18]. That report included only the es-
tablished anti-inflammatory capacity of HA in an acute inflam-
mation model. This activity can probably be explained as a de-
crease in the production of eicosanoids. At present, there is evi-
dence of an immunomodulatory effect possessing dose-depen-
dent behavior. In this, the damage is decreased, evidenced by
the low concentration of proinflammatory interleukins, particu-
larly IL-6, already reported to be associated with the generation
of fibrosis [29]. In such a way, it can be concluded that HA exerts

its immunodulatory effect in the model of RA, meaning through
increasing the concentration of IL-10 in the joint.
Materials and Methods
! were maintained at a temperature of 22 ± 3 °C, at 70 ± 5% humid-
General information
Carrageenan was acquired from Sigma Chemical Co. The alumi-
num silicate (kaolin) was obtained from Merck. Indomethacin
(99%) and Methotrexate (98%) were purchased from Sigma
Chemical Co. The origin of the IL-1β, TNF-α, IL-6, and IL-10 cyto-
kine kits was BD Biosciences, Inc. Acetone, dichloromethane
(DCM), ethyl acetate, and methanol solvents were purchased
from Mallinckrodt Baker, Inc. The organic whole extracts, as well
as the subfractions, were separated and analyzed by application
of traditional chromatographic methods such as column chroma-
tography and thin-layer chromatography (TLC). Silica gel (70–230
mesh), reverse-phase silica gel C18 (40–63 µm, Merck), and chro-
matographic plates were acquired from Merck KGaA. The re-
agents utilized were ceric ammonium sulfate (CAS) at 1% in
H2SO4 2 N and Komarowsky reagent (terpene detection). HA
was characterized by NMR spectra analysis [18].
Plant material
D. viscosa was collected at the REBIOSH and a sample was depos-
ited at the Autonomous University of the State of Morelos Her-
barium (HUMO) with registry no. HUMO-26620. Identification
was conducted by Botanist Juan Carlos Juárez, B.Sc., of the Cuer-
navaca, Morelos-based Center for Research in Biodiversity and
Conservation (CIByC).
Hautriwaic acid isolation
HA isolation and purification was carried out following the previ-
ously reported method [18]. Plant material (dried and milled
leaves, 1000 g) was extracted with dichloromethane (DCM; reac-
tive-grade solvents; Baker) (5 l) by exhaustive maceration for 3
days and 3 times. The solvent was eliminated by reduced-pres-
sure distillation with a Heidolph Laborota 4000 rotary evaporator
[40]. The dry extracts were compared by TLC (Merck). Due to
their chemical similarity, the extracts were mixed with a total
yield of 2.03 %. The extract (DCME, 15 g) was fractionated in an
open column chromatograph (4 × 50 cm) previously packed with
80 g of silica gel (70–230 mesh; Merck). An n-hexane-ethyl ace-
tate gradient system (reactive-grade solvents; Baker) was em-
ployed for the mobile phase, starting with 100 % of the low polar-
ity solvent with 5 % polarity increments and finalizing with 100 %
ethyl acetate. Aliquots of 150 ml were collected. Advance of the
separation was monitored in TLC and samples with a similar RF
profile were integrated into four fractions until the identification
of clerodanes (F1). This fraction (8 g) was dissolved in a mixture
of acetone/methanol; then, 1.5 g of HA were obtained, whose
chemical structure was described previously. This clerodane pro-
duces a blue stain when revealed with Komarowsky reagent [41].
The purity of HA was checked by NMR and was > 90 %.
Animals and experimental design
We used CD-1 female mice weighing 25–30 g each, provided by
the Production and Experimentation Unit of Laboratory Animals
(UPEAL) of the Metropolitan Autonomous University Xochimilco
Campus (UAM Xochimilco) in Mexico City. The experiments were
performed according to Official Mexican Norm 062-ZOO-1999
(Technical Specifications for the Production, Care and Use of Lab-
Salinas-Sánchez DO et al. Effect of Hautriwaic …
Original Papers 1245
oratory Animals) and the international ethical guidelines for the
care and use of laboratory animals. The experimental protocol
was authorized by the local Health Research Committee (Mexi-
can Institute of Social Security [IMSS] with registration no. R-
2010-1701-33; see Fig. 3S, Supporting Information). The animals
ity, and with 12 h-12 h light-dark cycles, with water and food ad
libitum. In the bioassays, a control group was utilized that re-
ceived a K/C injection in the knee joint and oral administration
of the vehicle without any drugs (the negative control); two pos-
itive control groups, one administered with Diclo, and the other
group with MTX and an intra-articular injection of K/C. A base-
line group of healthy animals was also used; these animals were
injected with physiological saline solution [PSS] into the knee.
Arthritis model induced by kaolin/carrageenan
This experimental design lasted 15 days. There were seven
groups of 12 mice each as follows: group 1, negative control, mice
without any treatment; group 2, mice treated with MTX; group 3,
mice treated with HA (5 mg/kg); group 4, mice treated with HA
(10 mg/kg); group 5, mice treated with HA (20 mg/kg); group 6,
mice treated with Diclo, and group 7, baseline, healthy mice
Downloaded by: University of Illinois. Copyrighted material.
(PSS). Initially, measurements were taken from the baseline size
of the right and left knees with a Mitutoyo-brand digital micro-
meter. Then, the animals of groups 1–5 were anesthetized with
pentobarbital sodium administered i. p. at a dose of 55 mg/kg.
Later, these mice were slowly injected with a kaolin (4%, 40 µl)
solution in the joint cavity of the right knee. Consecutively, flex-
ions and extensions were performed during 15 min in the joint
administered with the drug. Immediately after the 15 min of flex-
ions, the mice were injected in the same right heel joint cavity
with 40 µL of carrageenan at 2 %. Similarly, flexions were carried
out for additional 5 min [42, 43]. Administration of the treat-
ments was conducted by the oral route and was initiated 24 h
after damage induction as follows: Diclo (0.75 mg/kg), MTX
(1.0 mg/kg), and HA (5, 10, 20 mg/kg). Afterwards, joint edema
was measured every 24 h, and on day 15 the mice were sacrificed
and suspended from a surgical table to dissect the right knee of
each animal. The extracted knees were frozen at − 70 °C for later
homogenization of the bone marrow and determination of proin-
flammatory cytokines (IL-1β, IL-6, TNF-α) and anti-inflammatory
IL-10 by the enzyme-linked immunosorbent assay (ELISA) meth-
od.
Homogenization of joint tissue
The right knee was thawed to a temperature of 4 °C, at which the
samples were manipulated for their analysis. The bone tissue was
placed in a mortar and covered with dry ice. Then, the tissue was
pulverized and completely disintegrated until the dry ice was
sublimated. The disintegrated tissue was placed in a vial with
2000 µL of phosphate buffered saline (PBS, pH 7.4) with phenyl-
methylsulfonyl fluoride (PMSF) at 0.01 % dissolved in isopropyl
alcohol (Merck). To homogenize the joint completely, we used
an Ultra Turrax homogenizer T-10 during 15 sec for the disinte-
gration. It was allowed to rest for 30 sec and the procedure was
repeated five additional times. Afterward, the samples were
placed in a centrifuge at 12 000 rpm during 5 min. We obtained
300-µL aliquots in centrifuge microtubes; each tube served to
quantify a different cytokine. These were immediately stored at
− 70 °C for their later use in the ELISA method.
Planta Med 2015; 81: 1240–1247
1246 Original Papers
Quantification of cytokines interleukin-1β,
interleukin-6, tumor necrosis factor-α, and
interleukin-10 by enzyme-linked immunosorbent
assay
The quantification of each of these cytokines was carried out by
the ELISA method utilizing a kit (OptEIA™ ELISA sets; BD Bio-
sciences) and following the manufacturerʼs instructions. Briefly,
we added 100 µl/well of the antibody uptake to 96-well plates.
The plates were incubated for 12 h at 4 °C. Once this time had
elapsed, the plate was washed with PBS buffer (0.05% of Tween-
20, 300 µl/well × three times). We added 100 µl of PBS with fetal
bovine serum (FBS) at 10%, pH 7.0, during 1 h at room tempera-
ture. The contents were discarded and the plate was washed with
PBS buffer (0.05% of Tween-20, 300 µl/well × three times). We
added 100 µl of the standard, the target (PBS with FBS), and the
test samples to the corresponding wells. The plate was incubated
for 2 h at room temperature. The contents were discarded and
the plate was washed with PBS buffer (0.05 % of Tween-20,
300 µl/well × five times). For TNF-α, IL-6, and IL-10, we added
100 µl/well of antibody detection plus a streptavidine-horserad-
ish peroxidase (HRP) enzyme, which was incubated for 1 h and
washed with PBS 0.05 % of Tween-20, 300 µl/well × seven times).
For IL-1β, we added 100 µl/well of antibody detection; this was
incubated for 1 h and washed with PBS 0.05 % of Tween-20,
300 µl/well × five times), followed by the streptavidine-HRP en-
zyme (100 µl/well), which was incubated at room temperature
and washed with PBS 0.05 % of Tween-20, 300 µl/well × seven
times. To each well, we added 100 µl of O-phenylendiamine
(OPD) substrate that had been previously prepared (one tablet of
OPD and one of urea dissolved in 20 ml of distilled water). This
was incubated for 30 min at room temperature under conditions
of total darkness, and we then added a stop solution (2 N H2SO4).
Reading of the plate was carried out on an Awareness Technolo-
gies Stat Fax 2100 spectrophotometer at a 450-nm wavelength at
37 °C.
Statistical analysis
The results are expressed as average ± SEM, and the data were an-
alyzed utilizing analysis of variance (ANOVA) and the post hoc
Bonferroni test (*p < 0.05). We employed the SPSS ver. 17 statisti-
cal software package.
Supporting information
1
H and 13C NMR spectra of compound HA as well as results of an
Irwin test to determine the toxic effects of HA are available as
Supporting Information.
Acknowledgments
! A. Anti-inflammatory activity of hautriwaic acid isolated from Dodo-
This work was financially supported by FIS-IMSS/PROT/G11/1930
and PROMEP/103.5/10/5444/UAEMOR‑113. We are grateful to
Ernesto Sánchez Mendoza (UAM-Xochimilco) for his technical as-
sistance. The authors thank Juan Carlos Juarez Delgado from the
HUMO Herbarium (University of Morelos) for his support in iden-
tifying the species. This paper is taken in part from the Ph.D. the-
sis of David Osvaldo Salinas Sánchez.
Conflict of Interest
!
The authors declare no conflict of interest.
Salinas-Sánchez DO et al. Effect of Hautriwaic … Planta Med 2015; 81: 1240–1247
References
1 Murphy G, Caplice N, Molloy M. Fractalkine in rheumatoid arthritis: a
review to date. Rheumatology 2008; 47: 1446–1451
2 Aguillón JC, Cruzat A, Contreras J, Dotte A, Pesce B, Aravena O, Salazar L,
Catalán D, Abello P, Aguirre A, Llanos C, Cuchacovich M. Artículo de Re-
visión: Terapias emergentes en artritis reumatoide. Rev Med Chile
2005; 133: 969–976
3 Olsen NJ, Stein CM. New drugs for rheumatoid arthritis. N Engl J Med
2004; 350: 2167–2179
4 Saag KG, Teng GG, Patkar NM, Anuntiyo J, Finney C, Curtis JR, Paulus HE,
Mudano A, Pisu M, Elkins-Melton M, Outman R, Allison JJ, Almazor MS,
Bridges SL, Chatham WW, Hochberg M, Maclean C, Mikuls T, Moreland
LW, OʼDell J, Turkiewicz A, Furst DE. American college of rheumatology
recommendations for the use of nonbiologic and biologic disease-
modifying antirheumatic drugs in rheumatoid arthritis. Arthritis Care
Res 2008; 59: 762–784
5 Maldonado A, Ortiz S, Dorado O. Preparados galénicos e imágenes de
plantas medicinales, una alternativa para promotoras de la salud en la
reserva de la biosfera sierra de Huautla. Cuernavaca, Morelos: Univer-
sidad Autónoma del Estado de Morelos, Centro de Educación Ambien-
tal e Investigación Sierra de Huautla; 2004: 7–11
6 Dorado O, Maldonado B, Arias D, Sorani V, Ramírez R, Leyva E, Valenzue-
la D. Programa de conservación y manejo reserva de la biosfera sierra
de Huautla. Morelos, México: Comisión Nacional de Áreas Naturales
Protegidas; 2005: 3–4, 33–34, 54, 139–170
7 Rajamanickam V, Rajasekaran A, Anandarajagopal K, Sridharan D, Sel-
Downloaded by: University of Illinois. Copyrighted material.
vakumar K, Rathinaraj BS. Anti-diarrheal activity of Dodonaea viscosa
root extracts. Int J Pharm Bio Sci 2010; 1: 182–185
8 Anilreddy B. Preparation, characterization and biological evaluation of
some overview of Dodonea viscosa Linn. J Pharm Sci Technol 2009; 1:
1–9
9 Khurranm M, Khan MA, Hameed A, Abbas N, Qayum A, Inayat H. Anti-
bacterial activities of Dodonaea viscosa using contact bioautography
technique. Molecules 2009; 14: 1332–1341
10 Pengelly A. Medicinal activity of Dodonaea viscosa. A preliminary study.
Newcastle, Australia: RIRDC; 2008. Available at http://www.rirde.gov.
au. Accessed June 15, 2011.
11 Khalil NM, Sperotto JS, Manfron MP. Antiinflammatory activity and
acute toxicity of Dodonaea viscosa. Fitoterapia 2006; 77: 478–480
12 Arun M, Asha VV. Gastroprotective effects of Dodonaea viscosa on vari-
ous experimental ulcer models. J Ethnopharmacol 2008; 118: 460–465
13 Alagarsamy V, Venket-Narayanan R, Thangathirupathy A, Amuthalaksh-
mi S, Slvakamisundari P, Jubie S, Syed-Ali AKS, Suresh M. Antiinflamma-
tory activity of Dodonaea viscosa Linn leaf extracts. Indian Drugs 2007;
44: 559–560
14 Sachdev K, Kulshreshtha DK. Flavonoids from Dodonaea viscosa. Phyto-
chemistry 1983; 22: 1253–1256
15 Mata R, Contreras JL, Crisanto D, Pereda-Miranda R, Castañeda P, Del Rio
F. Chemical studies on Mexican plants used in traditional medicine,
XVIII. New secondary metabolites from Dodonaea viscosa. J Nat Prod
1991; 54: 913–917
16 Ortega A, García PE, Cárdenas J, Mancera C, Marquina S, Garduño ML,
Maldonado E. Methyl dodonates, a new type of diterpenes with a modi-
fied clerodane skeleton from Dodonaea viscosa. Tetrahedron 2001; 57:
2981–2989
17 Niu HM, Zeng DQ, Long CL, Peng YH, Wang YH, Luo JF, Wang HS, Shi YN,
Tang GH, Zhao FW. Clerodane diterpenoids and prenylated flavonoids
from Dodonaea viscosa. J Asian Nat Prod Res 2010; 12: 7–14
18 Salinas-Sánchez DO, Herrera-Ruiz M, Pérez S, Jiménez-Ferrer E, Zamilpa
naea viscosa leaves. Molecules 2012; 17: 4292–4299
19 Hamid A, Nurul K, Akhtar M, Mohammad RS, Syed GM, Naveed I, Said N.
Hautriwaic acid as one the hepatoprotective constituent of Dodonaea
viscosa. Phytomedicine 2014; 21: 131–140
20 Berin C, Buell MG. Phorbol myristate acetate ex vivo model of enhanced
colonic epithelial permeability. Digest Dis and Sci 1995; 40: 2268–
2279
21 Uzkeser H, Cadirci E, Halici Z, Odabasoglu F, Polat B, Yuksel TN, Atalay F.
Anti-inflammatory and antinociceptive effects of salbutamol on acute
and chronic models of inflammation in rats: involvement of an antiox-
idant mechanism. Mediators Inflamm 2012; 2012: 438912
22 Hsü HY, Chen YP. Structure of hautriwaic acid. Phytochemistry 1971;
10: 2813–2814

23 Arriaga FJ, Wollenweber E, Schober I, Dostal P, Braun S. 2β-hydroxyhau-
triwaic acid, a clerodane type ditepenoid and other terpenoids from
three Baccharis species. Phytochemistry 1986; 25: 719–721
24 Neugebauer V, Han JS, Adwanikar H, Fu Y, Ji G. Techniques for assessing
knee joint pain in arthritis. Mol Pain 2007; 3: 1–13
25 Daniel LW, Sciorra VA, Ghosh S. Phospholipase D, tumor promoters,
proliferation and prostaglandins. Biochim Biophys Acta 1999; 1439:
265–276
26 Newton K, Dixit VM. Signaling in innate immunity and inflammation.
Cold Spring Harb Perspect Biol 2012; 4: a006049
27 Chung HJ, Lee HS, Shin JS, Lee SH, Park BM, Youn YS, Lee SK. Modulation
of acute and chronic inflammatory processes by a traditional medicine
preparation GCSB‑5 both in vitro and in vivo animal models. J Ethno-
pharmacol 2010; 130: 450–459
28 Liew FY. TH1 and TH2 cells: a historical perspective. Nat Rev Immunol
2002; 2: 55–60
29 Fielding CA, Jones GW, McLoughlin RM, McLeod L, Hammond VJ, Uceda J,
Williams AS, Lambie M, Foster TL, Liao CT, Rice CM, Greenhill CJ, Colmont
CS, Hams E, Coles B, Kift-Morgan A, Newton Z, Craig KJ, Williams JD, Wil-
liams GT, Davies SJ, Humphreys IR, OʼDonnell VB, Taylor PR, Jenkins BJ,
Topley N, Jones SA. Interleukin-6 signaling drives fibrosis in unresolved
inflammation. Immunity 2014; 40: 40–50
30 Yarilina A, Xu K, Chan C, Ivashkiv LB. Regulation of inflammatory re-
sponses in tumor necrosis factor-activated and rheumatoid arthritis
synovial macrophages by JAK inhibitors. Arthritis Rheum 2012; 64:
3856–3866
31 Yun YC, Koo ST, Kim CH, Lyu Y, Grady JJ, Chung JM. Two variables that can
be used as pain indices in experimental animal models of arthritis.
J Neurosci Meth 2002; 115: 107–113
32 De Lima FO, Alves V, Filho JM, Da Silva Almeida JR, Rodrigues LC, Soares
MB, Villarreal CF. Antinociceptive effect of lupeol: evidence for a role of
cytokines inhibition. Phytother Res 2013; 27: 1557–1563
Salinas-Sánchez DO et al. Effect of Hautriwaic …
Original Papers 1247
33 Ritter AM, Dominicano TP, Verri Jr. WA, Zarpelon AC, Da Silva LG, Barbosa
CP, Natali MR, Cuman RK, Bersani-Amado CA. Antihypernociceptive ac-
tivity of anethole in experimental inflammatory pain. Inflammophar-
macology 2012; 21: 187–197
34 Balkwill F, Joffroy C. TNF: a tumor suppressing factor or a tumor-pro-
moting factor? Future Oncol 2010; 6: 1833–1836
35 Iwakura Y. Roles of IL‑1 in the development of rheumatoid arthritis:
consideration from mouse models. Cytokine Growth Factor Rev 2002;
13: 341-355
36 Kishimoto T. Interleukin-6: from basic science to medicine-40 years in
immunology. Annu Rev Immunol 2005; 23: 1–21
37 Jazayeri JA, Carroll GJ, Vernallis AB. Interleukin-6 subfamily cytokines
and rheumatoid arthritis: role of antagonists. Int Immunopharmacol
2010; 10: 1–8
38 Sabat R, Grütz G, Warszawska K, Warszawska K, Kirsch S, Witte EK, Wolk
K, Geginat J. Biology of interleukin-10. Cytokine Growth Factor Rev
2010; 21: 331–344
39 Moore KW, De Waal MR, Coffman RL, OʼGarra A. Interleukin-10 and in-
terleukin 10 receptor. Annu Rev Immunol 2001; 19: 683–765
40 Kuklinski C. Farmacognosia/estudio de las drogas y sustancias medica-
mentosas de origen natural. Barcelona: Ediciones Omega S. A.; 2000:
33
41 Wagner H, Bladt S. Plant drug analysis. A thin layer chromatography at-
las, 2nd edition. Munich: Springer; 2009: 362
42 Hartmann P, Szabó A, Erős G, Gurabi D, Horváth G, Németh I, Ghyczy M,
Boros M. Anti-inflammatory effects of phosphatidylcholine in neutro-
Downloaded by: University of Illinois. Copyrighted material.
phil leukocyte-dependent acute arthritis in rats. Eur J of Pharmacol
2009; 622: 58–64
43 Sluka KA, Bailey K, Bogush J, Olson R, Ricketts A. Treatment with either
high or low frequency TENS reduces the secondary hyperalgesia ob-
served after injection of kaolin and carrageenan into the knee joint.
Pain 1998; 77: 97–102
Planta Med 2015; 81: 1240–1247