Research Article

Kaurenic acid: An in vivo experimental study of its antiinflammatory and antipyretic effects
Miriam C. Sosa-Sequera, Omar Surez, Nelson L. Dal

ABSTRACT
Research Unit in Experimental
Pharmacology, School of Medicine,
Universidad Centroccidental
Lisandro Alvarado, Barquisimeto,
Venezuela.
Received: 24-06-2010
Revised: 01-07-2010
Accepted: 13-07-2010
DOI: 10.4103/0253-7613.70205
Correspondence to:
Dr. Miriam C. Sosa-Sequera,
E-mail: msosas2001@yahoo.es

Objective: This study was designed to investigate the anti-inflammatory and antipyretic
effects of kaurenic acid (KA), a tetracyclic diterpenoid carboxylic acid, using in vivo
experimental animal models.
Material and Methods: The anti-inflammatory activity of KA was evaluated in rats, using
egg albumin-induced paw edema (acute test) and Freunds complete adjuvant-induced paw
edema (subacute test), whereas the antipyretic effect was studied in rabbits by peptoneinduced pyresis. Acute and subacute toxicity of KA were analyzed in NMRI mice.
Results: KA showed anti-inflammatory and antipyretic properties, and the effect caused
was significantly dose-related (P < 0.001) in both cases. The mean lethal doses of KA were
439.2 and 344.6 mg/kg for acute and subacute toxicity, respectively.
Conclusion: On the basis of these findings, it may be inferred that KA has an antiinflammatory and antipyretic potential.
KEY WORDS: Antipyretic, anti-inflammatory, kaurenic acid, tetracyclic diterpenoid

Introduction
Kaurenic acid [()-kaur-16-en-19-oic acid] (KA) is a
diterpenoid acid derived from an ent-kaurene tetracyclique
hydrocarbure isolated from the aerial parts of the species
Espeletia semiglobulata.[1] It is described that Kaur-16-en-19oic acid derivatives may be promising compounds for leading
new chemopreventive strategies as well as having contributed
to development of antibiotic and antimicrobial agents.[2,3]
Ent-kaurenes and many natural derivatives of these
diterpenes have significant anti-inflammatory, anti-hypertensive,
and diuretic biological effects in vivo, as well as antimicrobial,
smooth muscle relaxant, and cytotoxic actions in vitro.[4]
Numerous findings indicate that ent-kauranes are potential
anti-inflammatory agents, with a specific mechanism in which
both the inhibition of nuclear factor kappa B (NF-kappa B)
translocation and the consequent decrease of proinflammatory
mediators are implicated. The antipyretic effects also have been
analyzed in different diterpene-types.[5]
KA is a bioactive compound with demonstrated
anticonvulsant, sedative,[6] and hypotensive effects.[7] This study
has been carried out in order to evaluate the anti-inflammatory
and antipyretic properties of KA using in vivo experimental
animal models for inflammation and pyrexia.
Materials and Methods
Drugs and reagents
KA was isolated from E. semiglobulata, and purified as

previously described.[1] KA, as sodium kaurenate [C20H29NaO2],

a gift from Dr. Alfredo Usubillaga (Research Institute, ULA,
Mrida, Venezuela) was diluted in distilleddeionised water for
assays.[8,9] Only high purity reagents were used in the different
assays. Acetylsalicylic acid (ASA) and Freunds complete
adjuvant (FCA) were purchased from Sigma (St. Louis, MO). Egg
albumin was obtained from Merck AG (Darmstadt, Germany).
Dexamethasone (DX, Decobel) was from Productos Ronava
(Caracas, Venezuela). Acetaminophen (ACTP) (Tempra) was
from Mead Johnson (Venezuela), and Peptone (PT) was from
Himedia Laboratories (Mumbai, India).
Animals
Adult male and female animals were used in this study.
Sprague-Dawley rats (body weight, 240300 g), NMRI mice
(body weight, 2535 g), and New Zealand male rabbits (body
weight, 24 kg) were supplied by the Veterinary School of
Universidad Centroccidental Lisandro Alvarado, Venezuela.
All animals had free access to food and water and were kept
on 12/12 h lightdark cycle. Before each study, animals were
submitted to fasting for at least 12 h. Animals were managed
according to the Guide to the Care and Use of Experimental
Animals, Canadian Council on Animal Care (1984), and the
Guide to the Use of Laboratory Animal, National Institute of
Health, Bethesda, USA. (Publication 8623, 1985).
Evaluation of Anti-inflammatory activity
Acute anti-inflammatory activity
This study was performed using the egg albumin
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Sosa-Sequera, et al.: Kaurenic acid: Anti-inflammatory and antipyretic effects

model. [10] Sprague-Dawley rats (n = 10) were randomly

allocated in three groups: an experimental group received KA
(20, 40, 80, and 160 mg/kg i.p.), a standard treatment group
was treated with acetylsalicylic acid (ASA: 50, 100, 200, and
250 mg/kg), and a control group received 0.9% saline solution (1
mL). In order to induce inflammation, all animals were injected
subcutaneously (s.c.) in the left hind paw with 0.1 mL of an
albumin solution diluted as 0.5% w/v in 0.9% saline solution,
30-min post-treatment. Acute inflammation was measured as
the linear diameter using callipers. Measurements were carried
out every 30 min for 5 h after administration of experimental
and control treatments.[11]
The anti-inflammatory effect of KA and ASA was calculated
and compared with the inflammation in the control group. Acute
anti-inflammatory activity was also confirmed by the volume
displacement method [12] using a Digital Plethysmometer LE
7500 (Letica, Barcelona, Spain). The mean effective dose (ED50)
was calculated for KA and ASA, and the therapeutic index was
determined in relation to acute LD50.
Subacute anti-inflammatory activity
These experiments were performed using the Freunds
adjuvant arthritis model. The administration of the Freunds
complete adjuvant (FCA, 0.1 mL, s.c.) was done according to
what was previously described.[13] KA (20, 40, 80, and 160 mg/
kg/day) was administered during 3 days. Dexamethasone (DX:
0.2, 2.0, and 3.8 mg/kg/day, i.p.) and saline 0.9% (1 ml/day, i.p.)
were given by the same route at the same periods of time and
were used as control groups. The subacute inflammation level
was measured by the volumetric method using plethysmometer
and by determining the linear diameter in a parallel
assay.[11,12] Paw swelling was determined at 0, 1, 6, 24 h, and
during the next 6 days. The average volume and thickness of
the paw in the experimental group (KA) was calculated and
compared with values obtained from the standard and control
groups (DX and saline) according to what was described
before.[13] The ED50 was calculated, and the therapeutic index
was estimated in relation to the values of subacute LD50.
Acute antipyretic activity
One hour prior to the assay, the animals were acclimatized
at room temperature (21C). New Zealand rabbits (n = 6) were
randomly divided into four groups. Three of them were treated
with peptone (PT), a pyrexia inductor, in 1% solution at doses of
0.1 mL/kg, i.v. and control group received only 0.9% saline (5
mL, i.v.). Experimental group treated with PT, also received KA
in a doses of 10, 20, 40, 80, and 160 mg/kg i.m. The standard
group received acetaminophen (ACTP: 25, 50, and 100 mg/
doses, i.m.). PT was given to all groups after recording basal
body temperature for 1 h poststabilization in the cuff. Core
body temperature (Tb) was measured in the rectum every 15
min during 4 h as described elsewhere.[14] Pyretic or antipyretic
effects were considered when the differences between maximal
and minimal temperatures were 0.6C or larger. The ED50
was calculated for experimental and control groups, and the
therapeutic index was determined with respect to acute LD50.
Acute and subacute toxicity testing
NMRI mice (n = 10) received KA intraperitoneally. In acute
assays, the animals were treated with a single-dose ranging
between 10 and 600 mg/kg body weight and mice were observed
294 Indian J Pharmacol | October 2010 | Vol 42 | Issue 5 | 293-296

for gross behavioural effects and mortality for a period of 24 h.

The control group received saline solution (0.5 mL). Accordingly,
in subacute studies, the selected doses of KA were 40, 80, 160,
320, and 460 mg/kg, i.p. administered during 5 days. The LD50
was estimated.
Statistical analysis
The percentage of lethality was determined using nonlinear
curve regression analysis. Variance analysis (two-way ANOVA)
followed by a Bonferroni post-test were used for significance
analysis of differences of inflammation and pyresis data,
respectively. The data were expressed as mean SD obtained
from at least three independent experiments performed by
triplicate. Values P < 0.05 were reported as statistically
significant. All statistical analysis were done using Prism
Software version 4.0 (GraphPad Software Inc., San Diego, CA).
Results
Acute anti-inflammatory activity
KA showed dose-related anti-inflammatory effects at 2 h
with an ED50 of 83.37 0.29 mg/kg (95% CI: 3.51197.8) which
was comparable to ASA = 97.62 0.10 mg/kg at 2 h) (95% CI:
36.50261.1). The anti-inflammatory effect was 60.26% and
81% for doses of 80 and 160 mg/kg, respectively. These results
were statistically significant when compared to those of control
group receiving albumin plus saline (P < 0.01; P < 0.001). By
comparison, ASA 200 mg/kg inhibited the edema significantly
(P < 0.01) which represented 58.93% of anti-inflammatory
activity at 3.5 h. Similarly, plethysmometric measurements
showed 77.55% inhibition for KA (160 mg/kg i.p.) that was
comparable to ASA (60.45%, P < 0.001). Anti-inflammatory
activity of KA compared to control groups is shown in Figure
1. The therapeutic index value corresponded to 5.26.
Subacute anti-inflammatory activity
Evaluation of the subacute anti-inflammatory effect using
the two methods indicated that KA caused inhibition from 50%
and 89.57% at doses of 80 and 160 mg/kg, respectively, after
2 days of treatment (P <0.001). The anti-inflammatory effect

Figure 1: Effect of KA on albumin-induced acute edema. Data were

calculated as percent of control and expressed as mean SD (n =
10) of triplicate measurements of three separated experiments. ***P
< 0.001 vs. edema-inductor.

Sosa-Sequera, et al.: Kaurenic acid: Anti-inflammatory and antipyretic effects

was dose-related. The ED50 was found to be 74.85 0.50 mg/

kg (95% CI: 62.4489.74) in relation to DX = 1.41 0.51 mg/
kg/day (95% CI: 0.0446.79) at 3 days post-treatment, being
the therapeutic index for KA of 4.60 in this subacute model.
The percent inhibition for DX (2 mg/kg) was 66%, and it was
significantly different from FCA plus saline control animals
(P < 0.001) [Figure 2]

The results of the study suggest that KA has antiinflammatory and antipyretic activities in the experimental
models studied. This indicates that KA is a bioactive diterpenoid;
which corresponds with the reports that describe the properties
of diterpenes kaurenoic.[8,15-18] KA was also as potent as ASA and
ACTP in the acute models, but with minor potency in relation to
DX. The ED50 value for the anti-inflammatory effect represented

1/6 of LD50, whereas the ED50 value for the antipyretic effect
was 1/20 of LD50. Although the therapeutic index for the antiinflammatory activity was low, no deaths were observed in
these animals at the doses used in the assays.
The development of acute edema in the paw of the rat has
been described as a three-phase event. It is mainly attributed
to the release of histamine, serotonin in the initial phase (02
h); cytokines, in the second phase (3 h); and prostaglandin, in
the third phase (>4 h).[19] The anti-inflammatory effects of KA
observed in the acute model lasted for up to 5 h. This shows that
KA acted on all inflammatory phases, whereas ASA acted much
slowly. In addition, KA in the subacute model maintained its antiinflammatory effect for 6 days, which was more accentuated
in relation to DX; nevertheless, higher doses were required.
These findings suggest that KA exhibited a potent capacity for
inhibiting acute and subacute edema.
Although elucidating the mechanism of action of KA was
not the main aim of this study; the anti-inflammatory effect of
most terpenes has been explained in terms of the inhibition of
NF-kappa B. In this regard, it has been reported that ent-kaurene
diterpenes inhibit the expression of inducible nitric oxide
synthase (iNOS) and the release of tumor necrosis factor-alpha
(TNF-).[20] It is also known that several diterpenoids isolated
from the Chinese herb Isodon rubescens are potent inhibitors of
NF-kappa B transcriptional activity which subsequently target
iNOS and cycloxygenase-2 (COX-2), with the latter being less
sensitive to diterpenoids.[21] Other diterpenoids isolated from
Chinese herbal remedy (Tripterygium wilfordii Hook.f.) inhibit
the interleukin-1-induced production of PGE2 by selectively
suppressing the gene expression and production of COX-2, but
not those of COX-1.[22] Furthermore, recently the ent-kaurene
isolated from the roots of Siegesbeckia pubescens was shown
to inhibit iNOS, COX-2, and TNF- protein via down-regulation
of NF-kappa B binding activity.[23] In relation to this mechanism,
we have previously reported evidence that KA, administered in
mice (1 mg/kg), inhibits the expression of genes for the proteins
iNOS, eNOS, neuronal NOS (nNOS), and Bcl-xL in murine
melanoma cells.[24] In addition, it appears that the effect of

Figure 2: Effect of KA on adjuvant-induced subacute edema. Data

were calculated as percent of control and expressed as mean SD
(n = 10) of triplicate measurements of three separated experiments.
***P < 0.001 vs. edema-inductor.

Figure 3: Effect of KA on body temperature in conditions of peptoneinduced fever. Results are expressed as mean SD (n = 6) of triplicate
measurements of three separated experiments. **P < 0.01, ***P < 0.001
vs. increment in PT-induced fever.

Acute antipyretic activity

The measurements of body temperature (Tb) are represented
in Figure 3. The saline group showed a constant basal Tb of
37.6 0.15C over a 4-h period. An increase in Tb to 38.56
0.18C was observed at 1 h post-PT. However, when animals
were treated with KA (10 and 20 mg/kg) at 2.30 h after PT
injection a decrease of 0.6 0.2C and 1.2 0.4C, respectively,
was found. This finding was significantly different in animals
treated with PT only (P < 0.01 and P < 0.001, respectively)
and mimicked with ACTP, which showed a decrease of 0.81
0.43C (P < 0.01). The effects were dose-dependent. The ED50
for the antipyretic effect of KA was 23.56 0.9 mg/kg (95%
CI: 21.3625.99) in relation to 44.38 0.41 mg/dose (95%
CI: 1.37143) for ACTP. The therapeutic index of KA for acute
model was 18.64.
Acute and subacute toxicity
The estimated LD50 for KA in the acute assay was 439.2 mg/
kg, i.p. (95% CI: 413.2466.9). In contrast, LD50 in the subacute
study performed during 5 days was found to be 344.6 mg/kg,
i.p. (95% CI: 243.1488.4). During treatment, mice showed
some degree of somnolence, weakness of the hind limbs, and
accelerated breathing at dose of 10 mg/kg and higher.
Discussion

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Sosa-Sequera, et al.: Kaurenic acid: Anti-inflammatory and antipyretic effects

terpenes on the gene expression of COX-2 is less sensitive than

that of the iNOS gene.[23] Therefore, kaurenes could be acting
as anti-inflammatory agents through a novel mechanism of
action totally different from that of common nonsteroidal antiinflammatory drugs (NSAID) that inhibit COX-1 with subsequent
side effects on the gastric mucosa and platelets aggregation. The
results obtained in our study are coincident with these reports
in relation to the potent anti-inflammatory effect of diterpenes.
The antipyretic effect of diterpenes could also be explained
by its potent inhibition of iNOS, because NO has been shown to
be an important mediator of febrile responses. In this regard,
when fever is induced, activation of iNOS is responsible for
triggering and maintaining lipopolysaccharide-induced fever in
rats.[25,26] In agreement with these data, KA-induced antipyretic
effect with a latency of 1 h, during a period of 4 h and with a
good therapeutic index. As an antipyretic, KA could be working
via a nitrergic mechanism due to its inhibition of NOS;[27] but a
prostanoid pathway has not been investigated yet.
In this work, evidence is presented that KA has antiinflammatory and antipyretic effects; nonetheless, further
studies are needed for evaluating levels of NO or interleukins
to explain underlying mechanisms involved in these events. The
discovery of diterpenes as specific inhibitors capable of blocking
NF-kappa B activation could be an excellent approach for the
treatment of inflammatory diseases. Our results justify further
investigations in order to evaluate diterpenes as potentially
useful agents for the treatment of inflammation and fever.
Acknowledgments
This work was supported by Research Council (CDCHT) of the
Universidad Centroccidental Lisandro Alvarado (Grant # 016-ME-2001).
The authors thank to Dr. Alfredo Usubillaga, The Andes University, for his
gently donation of the assayed drug. We wish to express our gratitude to
Dr. Juan C. Pia-Crespo and Dr. Liduska Sosa-Burgos for his invaluable
help in the scientific reviewing of the manuscript and to Francisco Mujica,
Griseth Linrez, Addelina Escobar, and Maria Eugenia Camargo for their
technical assistance.

9.
10.
11.
12.

13.
14.
15.

16.
17.
18.
19.
20.
21.
22.
23.

References
1. Usubillaga A, Capra M. Chemical constituents of Espeletia semiglubulata.
Fitoterapia 1987;59:383-4.
2. Ruiz Y, Rodrgues J, Arvelo F, Usubillaga A, Monsalve M, Diez N et al. Cytotoxic
and apoptosis-inducing effect of ent-15-oxo-kaur-16-en-19-oic acid, a derivative
of grandiflorolic acid from Espeletia schultzii. Phytochemistry 2008; 69:432-8.
3. De Albuquerque M, Lyra F, De Mllo J, De Lima O, Monache F, Diu e Laurinete
Costa M. Antimicrobial substances of superior plants. 44. Isolation of diterpene
acid from Croton aff. argyrophylloides Muell arg. (Euphorbiaceae). Rev Inst
Antibiot 1974; 14:83-9.
4. Cavalcanti BC, Costa-Lotufo LV, Morales MO, Burbano RR, Silveira ER, Cunha
KM, et al. Genotoxicity evaluation of kaurenoic acid, a bioactive diterpenoid
present in Copaiba oil. Food Chem Toxicol 2006;44:388-92.
5. Vit P, Melendez P, Schwartzenberg J. Espeletia Schutzii Wedd. Botanical
description of apicola interest in Venezuela, N 5 frailejn. Rev Fac Farm
2003;45:180-2.
6. Suebsasana S, Pongnaratorn P, Sattayasai J, Arkaravichien T, Tiamkao
S, Aromdee C. Analgesic, antipyretic, anti-inflammatory and toxic effects
of andrographolide derivatives in experimental animals. Arch Pharm Res
2009;32:1191-200.
7. Dal NL, Sosa-Sequera MC, Usubillaga A. On the anticonvulsant activity of
kaurenic acid. Invest Clin 2007;48:349-58.
8. Sosa-Sequera MC, Linarez G, Testa M, Escobar A, Neira N, Mujica F, et

296 Indian J Pharmacol | October 2010 | Vol 42 | Issue 5 | 293-296

24.

25.
26.
27.

al. Comparation of hypotensive effect from sodium kaurenate with sodium

nitroprusiate in hypertensive rats. Acta Cient Venez 2003;54:93.
Usubillaga A, Romero M, Aparicio R. Kaurenic acid in Espeletiinae. Acta Hortic
2003;597:129-30.
Viloria E, Rojas L, Usubillaga A. Analysis of kaurenic acids methyl esters by gas
chromatography. High Res Chromatogr 1997;20:50-1.
Saidu K, Onah T, Orisadipe A, Olusola A, Wambebe C, Gamaniel K.
Antiplasmodial, analgesic and anti-inflammatory activities of the aqueous extract
of the stem bark of Erythrina senegalensis. J Ethnopharmacol 2000;6:275-80.
Sordelli DO, Fontan PA, Meiss RP, Ruggiero RA, Bustuoabad OD. Antiinflammation induced by counter-irritation or by treatment with non-steroidal
agents inhibits the growth of a tumour of non-detected immunogenicity. Brit J
Cancer 1989;60:734-8.
Winter CA, Risley EA, Nuss GW. Carrageenan-induced edema in hind paw of rat
as an assay for antiinflammatory drugs. Proc Soc Exp Biol Med 1962;111:544-7.
Yang Y, Hutchinson P, Morand EF. Inhibitory effect of annexin I on synovial
inflammation in rat adjuvant arthritis. Arthritis Rheum 1999;42:1538-44.
Josa M, Urizar JP, Rapado J, Dios-Viitez C, Castaeda-Hernndez G, FloresMurrieta F, et al. Pharmacokinetic / pharmacodynamic modeling of antypiretic
and anti-inflammatory effects of naproxen in the rat. J Pharmacol Exp Ther
2001;297:198-205.
Davino SC, Giesbrecht AM, Roque NF. Antimicrobial activity of kaurenoic acid
derivatives substituted on carbon-15. Braz J Med Biol Res 1989;22:1127-9.
Miles DH, Chittawong V, Payne A. Cotton boll weevil antifeedant activity and
antifungical activity (Rhizoctonia solani and Pythium ultimun) of extracts of the
stems of Wedelia biflora. J Agric Food Chem 1990;38:1591-4.
Paiva LA, Gurgel LA, Silva RM, Tome AR, Gramosa NV, Silveira ER, et al. Antiinflammatory effect of kaurenoic acid, a diterpene from Copaifera lansdorffi on
acetic acid-induced colitis in rats. Vascul Pharmacol 2002;39:303-7.
Vieira HS, Takahashi JA, Oliveira AB, Chiari E, Boaventura MA. Novel derivatives
of kaurenoic acid. J Braz Chem Soc 2002;13:151-7.
Dimo T, Fotio AL, Nguelefack TB, Asongalem EA, Kamtchouing P. Antiinflammatory
activity of leaf extracts of Kalanchoe crenata Andr. Indian J Pharmacol
2006;38:115-9.
Castrillo A, de Las Heras B, Hortelano S, Rodrguez B, Villar A, Bosca L. Inhibition
of the nuclear factor kappa B (NF-kappa B) pathway by tetracyclic kaurene
diterpenes in macrophages. J Biol Chem 2001;276:15854-60.
Leung CH, Grill SP, Lam W, Han QB, Sun HD, Cheng YC. Novel mechanism of
inhibition of nuclear factor- kappa B DNA-binding activity by diterpenoids isolated
from Isodom rebescens. Mol Pharmacol 2005;68:286-97.
Lin N, Sato T, Ito A. Tritolide, a novel diterpenoids triepoxide from Tripterygium
wilfordii Hook. f., suppresses the production and gene expression of promatrix metalloproteinases 1 and 3 and augments those of tissue inhibitors
of metalloproteinases 1 and 2 in human synovial fibroblasts. Arthritis Rheum
2001;44:2193-200.
Park HJ, Kim IT, Won JH, Jeong SH, Park EY, Nam JH, et al. Anti-inflammatory
activities of ent-16alphaH, 17-hydroxy-kauran-19-oic acid isolated from the
roots of Siegesbeckia pubescens are due to the inhibition of iNOS and COX-2
expression in RAW 264.7 macrophages via NF-kappa B inactivation. Eur J
Pharmacol 2007;558:185-93.
Rivero MB, Mendoza HJ, Chiurillo MA, Usubillaga A, Sosa-Sequera MC.
Apoptotic gene expression in melanoma cells treated with kaurenic acid. Basic
Clin Pharmacol Toxicol 2007;101:76.
Soszynski D, Chelminiak M. Intracerebroventricular injection of neuronal and
inducible nitric oxide synthase inhibitors attenuate fever due to LPS in rats. J
Physiol Pharmacol 2007;58:551-61.
Aquila S, Weng ZY, Zeng YQ, Sun HD, Ros JL. Inhibition of NF-kappa B activation
and iNOS induction by ent-kaurane diterpenoids in LPS-stimulated RAW264.7
murine macrophages. J Nat Prod 2009;72:1269-72.

Source of Support: Research Council (CDCHT) of the

Universidad Centroccidental Lisandro Alvarado (Grant # 016-ME2001). Conflict of Interest: None declared.