Life Sciences 79 (2006) 1062 – 1068

www.elsevier.com/locate/lifescie

Ursolic acid mediates the vasorelaxant activity of Lepechinia caulescens via

NO release in isolated rat thoracic aorta☆
Francisco Aguirre-Crespo a , Jorge Vergara-Galicia a , Rafael Villalobos-Molina b,c ,
Juan Javier López-Guerrero b , Gabriel Navarrete-Vázquez a , Samuel Estrada-Soto a,⁎
a
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Colonia Chamilpa, 62210, Cuernavaca, Morelos, México
b
Departamentode Farmacobiología, Centro de Investigación y de Estudios Avanzados Sede Sur, Calz. Tenorios 235, Col. Granjas Coapa,
México, D.F., 14330, México
c
Unidad de Biomedicina, Facultad de Estudios Superiores-Iztacala, Universidad Nacional Autónoma de México, Avenida de los Barrios 1,
Los Reyes Iztacala, Tlalnepantla, México 54090, México
Received 3 November 2005; accepted 8 March 2006

Abstract

We have determined that the methanolic extract of L. caulescens (MELc) produced a significant vasodilator effect in a concentration-dependent
and endothelium-dependent manner. This relaxation was blocked by Nω-nitro-L-arginine methylester (L-NAME), indicating that MELc vasodilator
properties are endothelium mediated due to liberation of nitric oxide (NO). In this paper we aimed to corroborate its mode of action. MELc effects
on noradrenaline (NA)-induced contraction in isolated rat aortic thoracic rings with endothelium (+ E), in the presence of atropine (0.1 μM) and 1-
H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, 1 μM) were conducted. MELc relaxation curve was significantly shifted to the right in the
presence of ODQ and atropine, thus confirming that its mode of action is related with activation of nitric oxide synthase (NOS) and the consequent
increment in NO formation. Bio-guided study of MELc allowed the isolation of ursolic acid (UA, 50 mg) and ursolic–oleanolic acids mixture
[UA/OA (7:3), 450 mg]. The relaxant effect of UA (0.038–110 μM) was evaluated in functional experiments. UA induced a significant relaxation
in a concentration- and endothelium-dependent manner (IC50 = 44.15 μM) and did not produce a vasorelaxant effect on contraction evoked by KCl
(80 mM). In addition, NA-induced contraction was significantly displaced to the right by UA (30 μM). In order to determine its mode of action,
UA-induced relaxant effect was evaluated in the presence of atropine (0.1 μM), indomethacin (10 μM), L-NAME (100 μM) and ODQ (1 μM).
Relaxation was blocked by L-NAME and ODQ. On the other hand, UA (3 μM) provoked a significant displacement to the left in the relaxation
curve induced by sodium nitroprusside (SNP, 0.32 nM to 0.1 μM), but it was not significant in the presence of Carbamoyl choline (carbachol,
1 nM to 10 μM). These results indicate that UA-mediated relaxation is endothelium dependent, probably due to NO release, and the consequent
activation of vascular smooth muscle soluble guanylate cyclase (sGC), a signal transduction enzyme that forms the second messenger cGMP.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Lepechinia caulescens; Ursolic acid; Oleanolic acid; Relaxation; Aortic smooth muscle; Nitric oxide; cGMP

Introduction public health problems (INEGI/SSA/CONAPO, 2002). Anti-

The systemic arterial hypertension (SAH) is characterized by
high blood pressure (> 140/90 mm Hg) and is a major risk factor
to develop cardiac, renal and endothelial insufficiency, diabetes
mellitus, metabolic syndrome and heart attack (Sardana and
Madan, 2003). In México, SAH is one of the most important
☆
Taken in part from the Ph.D. Thesis of F. Aguirre-Crespo.
⁎ Corresponding author. Tel./fax: +52 777 3 29 70 89.
E-mail address: enoch@uaem.mx (S. Estrada-Soto).
0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2006.03.006
hypertensive drugs influence arterial blood pressure at four
effector sites: the resistance vessels, the capacitance vessels, the
heart, and the kidney. Important classes of antihypertensive
drugs are diuretics, sympatholytic drugs, vasodilators, calcium
channel blockers, angiotensin-II blockers and angiotensin
converting enzyme inhibitors (Hoffman, 2005). In recent
years, nitric oxide (NO) has been demonstrated to be an
important regulator of vascular functions by controlling blood
vessel tone as well as blood cell interactions with the vascular
wall (Yi-Ching et al., 2005). The NO/cGMP signaling cascade
 F. Aguirre-Crespo et al. / Life Sciences 79 (2006) 1062–1068 1063

plays an essential role in vascular smooth muscle relaxation,
and clinical studies indicate that endothelium-derived NO is
involved in normal and pathological blood pressure regulation
in humans. Targeted inactivation of endothelial NO synthase
causes hypertension (Yi-Ching et al., 2005). The action of NO
as a vasodilator is mediated by the activation of vascular smooth
muscle soluble Guanylate Cyclase (sGC), a signal transduction
enzyme that forms the second messenger molecular cGMP
(Schlossmann and Hofmann, 2005).
Although there is availability of low-cost therapy, the
Mexican folk medicine policies promote the use of medicinal
plants for the treatment of different diseases (SSA, 2004), and
some herbal medicines are real choices for treatment of
hypertension (Aguilar et al., 1994; Monroy-Ortíz and Castillo,
2000). Lepechinia caulescens is one of these plants used as
antihypertensive agent. In previous investigations, a phyto-
chemical analysis of L. caulescens had led to the isolation of
terpenoids and sterols (Delgado et al., 1992, 1994), and
pharmacological evaluations showed that L. caulescens have
antidiabetic (Román-Ramos et al., 2001) and spasmolytic
(Rodríguez-López et al., 2003) activities. Recently we also
showed that this plant has vasorelaxant properties and this effect
is related with NO release by activation of eNOS (Aguirre-
Crespo et al., 2005), then it is possible that the vasodilatory
action could be related to the presence of oleanolic (OA) and
produce eleven pooled fractions (F-I to F-XI) based on their
TLC profiles. According to the initial vasorelaxant test, only
fractions F-I, F-II, F-V and F-VI were active (50% of
inhibition), inhibiting NA-precontracted rat aorta when tested
at the IC50 of the MeOH extract (4.1 μg/mL). From the active
fraction F-II, UA and UA/OA were spontaneously obtained by
precipitation and the structural elucidation was carried out by
comparison of experimental values from IR, NMR1H, 13C and
GC–MS with those previously reported (Seo et al., 1975).
Animals
Male Wistar rats were used and maintained under standard
laboratory conditions with free access to food and water. All
animal procedures were conducted in accordance with our
Federal Regulations for Animal Experimentation and Care
(SAGARPA, NOM-062-ZOO-1999, México), and approved by
the Institutional Animal Care and Use Committee.
a
-20
0
20 *
% of Relaxation

ursolic acids (UA), both triterpenoids have been isolated from

*
this species (Delgado et al., 1994). These compounds were 40
isolated from other plants and were described as antihyperten-
sive, antioxidant, antiatherosclerotic, antihyperlipidemic (Liu, *
60
2005), cardiotonic and antidysrhythmic agents (Somova et al., *
2004). However, the underlying mechanisms involved in the 80 *
antihypertensive effect of UA are not clear. In this context, the
100 MELc
present study was undertaken to determine the vasorelaxant MELc + Atropine (0.1 μM)
effect of UA and to elucidate its mode of action by functional
100 101 102
experiments.
Log μg ml-1

Materials and methods b

Chemicals and drugs -20

0
Carbamoyl choline (carbachol), noradrenaline HCl (NA), *
ω
N -Nitro-L-arginine methyl ester HCl (L-NAME), indometha- *
% of Relaxation

20
cin, atropine, sodium nitroprusside (SNP) and 1-H-[1,2,4]- *
oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ) were purchased 40
from Sigma-Aldrich Co. (St. Louis, MO, USA). Ursolic acid *
(UA) was purchased from MP Biomedicals, Inc. (Aurora, OH, 60
USA). All other reagents were analytical grade from local *
sources. 80

MELc
Isolation of UA and UA/OA 100 MELc + ODQ 1 μM

MELc was obtained previously (Aguirre-Crespo et al., 100 101 102

2005). MELc (89.52 g) was subjected to column chromatog- Log μg ml-1

raphy. The column was eluted using the following solvent
systems: Hex-CH2Cl2 (1:0 → 0:1), CH2Cl2–AcOEt (1:0 → 0:1),
followed by AcOEt–MeOH (1:0 → 0:1). Two hundred and sixty
eight fractions (550 mL each) were collected and combined to
Fig. 1. Relaxant effect of MELc on isolated rat aortic rings pre-contracted with
NA (0.3 μM) in the presence of atropine (0.1 μM; a) and ODQ (1 μM; b).
Results are expressed as the mean ± S.E.M. of six experiments (⁎p < 0.05 vs.
control).
1064 F. Aguirre-Crespo et al. / Life Sciences 79 (2006) 1062–1068
Functional studies
Rats (250–350 g body weight) were killed by exposure to
ether. The thoracic aorta was cleaned of adhering connective
tissue and was cut into 3–5 mm length rings. In some rings, the
endothelium was removed. Then, tissue segments were
mounted in stainless steel hooks, under an optimal tension of
3 g, in 10 mL organ baths containing warmed (37 °C) and
oxygenated (O2/CO2, 19:1) Krebs solution (composition, mM:
NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2 mM; KH2PO4,
1.2; NaHCO3, 25.0; EDTA, 0.026 and glucose, 11.1, pH 7.4).
Changes in tension were recorded by Grass-FT03 force
transducers (Astromed, West Warwick, RI, USA) connected
to a MP100 analyzer (Biopac Instruments, Santa Barbara, CA,
USA), as previously described (Aguirre-Crespo et al., 2005).
After equilibration, rings were contracted by NA (0.3 μM) and
washed every 30 min for 2 h. The absence of endothelium was
confirmed by the lack of a relaxing response to carbachol
(1 μM). After precontraction with NA or KCl (80 mM), the test
Aerial parts from Lepechinia
Plant material
Plant material
Plant material
F-I** F-II** F-III F-IV
R1
R2
COOH
HO
R1= H; R2= CH3 (Ursolic acid)
R1= CH3; R2= H (Oleanolic acid)
Fig. 2. Diagram for extraction and the bio-guided fractionation of MELc. Activity of the extract and fractions are included.
samples (UA, vehicle and positive control) were added to the
bath in a volume of 100 μL; then cumulative concentration–
response curves were obtained for each ring (0.86–50 μg/mL
for MELc and 0.038–110 μM for UA). In order to avoid fatigue
of the arterial preparation, a 60 min recovery period was
allowed between curves. The relaxant effect of extracts and
positive controls were determined by comparing the muscular
tone of the contraction before and after addition of the test
materials. Muscular tone was calculated from the tracings, using
Acknowledge software (Biopac®).
In order to confirm the mode of action of MELc and to
establish the mechanism of UA, three sets of experiments were
performed:
1) When required by the experimental protocol, indomethacin
(10 μM), L-NAME (100 μM), atropine (0.1 μM) and ODQ
(1 μM) were added to the organ bath, and incubated during
15 min before contracting the arterial rings (+ E) with NA
(0.3 M) then, MELc and/or UA were added at different
caulescens 89.52 g
Extraction by maceration (3 times x
72 hrs.)
Filtered and evaporated in vacuum.
Hexanic extract
N.T*
CH2Cl2 extract
N.T*
MeOH extract
(90.52 g, IC50 +E= 10 ± 0.52 μg/mL, Emax = 90.17 ± 4.68%)
Fractionation by CC (Si Gel), using
a gradient of polarity
F-V** F-VI** F-VII F-VIII F-IX F-X
*N. T. Not tested
** IC50 ≥ 50% of inhibition
 F. Aguirre-Crespo et al. / Life Sciences 79 (2006) 1062–1068 1065

a curve fitting program (ORIGIN 6.0). The statistical significance

(p < 0.05) of differences between means was assessed by an
0
analysis of Student's t test (Daniel, 2002; Bailey, 1995).

20 Results
% of Relaxation

*
40 Pharmacological evaluation of MELc and isolation of UA and
UA/OA
60 *
To continue the functional pharmacological characteriza-
tion of MELc (Aguirre-Crespo et al., 2005), we evaluated its
80 Carbachol relaxant effect in the presence of atropine (0.1 μM, antagonist
*
UA E+
UA E-
of M2-acetylcholine receptor) and ODQ (1 μM, inhibitor of
100 sGC) in rat aortic rings pre-contracted with NA (0.3 μM).
10-2 10-1 100 101 102
Atropine and ODQ induced a significant displacement to the
Log μM
right of the relaxant curve of L. caulescens and the Emax was
markedly reduced (Fig. 1) [(IC50: 4.1 ± 0.52 μg/mL and Emax
b
90.17 ± 4.68% vs. 12.13 ± 1.82 μg/mL and 84.94 ± 5.56% in
0 the presence of atropine) (IC50: 4.10 ± 0.52 μg/mL and Emax
90.17 ± 4.68% vs. 25.58 ± 6.1 μg/mL and 56.5 ± 5.5% in the
20
presence of ODQ)].
The bio-guided fractionation of MELc allowed the isolation
% of Relaxation

of UA and UA/OA. The structural elucidation of UA and UA/

40 OA was carried out by comparison of experimental values from
IR, NMR1H, 13C and GC–MS with values previously reported
60 (Seo et al., 1975). Fig. 2 summarizes the procedure followed to
obtain UA and UA/OA.
80
Nitrendipine Pharmacological evaluation of UA
UA E+
UA E-
100 UA showed a significant relaxant effect after NA action
10-2 10-1 100 101 102
on endothelium-intact aortic rings (IC50 = 44.15 ± 6.1 μM,
Log μM
Emax = 97.2 ± 2.1%); however, in endothelium-denuded rings,
Fig. 3. Relaxant effect of UA on isolated rat aortic rings pre-contracted with NA no effect was observed (Fig. 3a). Moreover, UA did not produce
(0.3 μM; a) and KCl (80 mM; b) Results are expressed as the mean ± S.E.M. of a significant vasorelaxation on contraction evoked by KCl (Fig.
six experiments [⁎p < 0.05 compare the relaxant effect on the endothelium (+E) 3b). Carbachol and nitrendipine were used as positive controls
vs. (−E)]. (aortic rings + E and − E, respectively). On the other hand, UA

concentrations and cumulative concentration–response 4.0

Control
curves were obtained. 3.5 UA 3 μM
2) Aortic rings (+ E) were incubated with UA (3 μM) during UA 30 μM
15 min before contracting the arterial rings (+ E) with NA 3.0

(0.3 μM) then, carbachol and SNP were added at different

Contraction (g)

2.5
concentrations and cumulative concentration–response
2.0 *
curves were obtained. *
3) Aortic rings (+ E) were incubated with UA (3 and 30 μM) 1.5 *
during 15 min, and then NA was added at different *
1.0
concentrations (1 nM to 10 μM).
0.5
The effect of MELc and UA was determined and calculated 0.0
as described before.
-0.5
10-3 10-2 10-1 100 101
Data analysis
NA (log μM)

Results are expressed as the mean of six experiments ± S.E.M. Fig. 4. Effect of 3 and 30 μM of UA on NA cumulative concentration–effect
Concentration responses curves (CRC) were plotted and the curve (1 nM to 30 μM) on rat aorta rings (+ E). Each point represents the
experimental data from the CRC were adjusted by the nonlinear, mean ± S.E.M. of six experiments (⁎p < 0.05 vs. control).
1066 F. Aguirre-Crespo et al. / Life Sciences 79 (2006) 1062–1068

(30 μM) induced a marked depression on the cumulative a

concentration–response curve for NA (1 nM to 1 μM) on aortic 0
rings (+ E) (Fig. 4). The vasorelaxant effect of AU on
20
a

% of Relaxation
*
0 40

60
*
20
*
% of Relaxation

80 *
40 *
Control
100
UA 3 μM
60
10-4 10-3 10-2 10-1
SNP (log μM)
80
Control b
Atropine 0.1 μM
100 0
100 101 102
UA (log μM)
20

b % of Relaxation

0 40
*
* * 60
20
% of Relaxation

*
80
40 Control
* UA 3 μM
100
60 * 10-3 10-2 10-1 100 101
Carbachol (log μM)

80 Control Fig. 6. Relaxant effect of SNP and carbachol on aortic rings (+E) pre-contracted
L - NAME 100 μM
with NA (0.3 μM) in the presence of AU (3 μM). Results are expressed as the
Indomethacin 10 μMI
100 mean ± S.E.M. of six experiments (⁎p < 0.05 vs. control).
10-1 100 101 102
UA (log μM)
endothelium-intact aortic rings, precontracted with NA, was
c also investigated in the presence of atropine, L-NAME,
*
indomethacin and ODQ. Pretreatment with L -NAME
0
* (100 μM) and ODQ (1 μM) produced a significant change of
the response and vasorelaxation was markedly inhibited (Fig.
20 * 5b and c). With indomethacin (10 μM) the relaxant curve of UA
% of Relaxation

was significant shifted to the left (Fig. 5b); in contrast, with

atropine (0.1 μM) did not show a significant influence in UA
40
effect (Fig. 5a). Finally, preincubation with 3 μM of UA,
provoked a significant shift to the left of the relaxant curve of
60 SNP (0.32 nM to 0.1 μM) and in the presence of carbachol
(1 nM to 10 μM) (Fig. 6a and b) the effect was not significant
(p < 0.05).
80 Control
ODQ 1 μM
Discussion
10-3 10-2 10-1 100 101 102
UA (log μM) In a previous investigation, we showed that MELc induced a
significant relaxation in a concentration- and endothelium-
Fig. 5. Relaxant effect of UA on isolated rat aortic rings pre-contracted with
NA (0.3 μM) in the presence of atropine (0.1 μM; a), L-NAME (100 μM; b), dependent manner (Aguirre-Crespo et al., 2005). At the same
indomethacin (10 μM; b) and ODQ (1 μM; c). Results are expressed as the time, we also found that MELc mode of action to induce
mean ± S.E.M. of six experiments (⁎p < 0.05 vs. control). vasorelaxation is related to NO release (Aguirre-Crespo et al.,
 F. Aguirre-Crespo et al. / Life Sciences 79 (2006) 1062–1068
2005). In order to continue this investigation and to support this
proposal, MELc relaxation effect was evaluated on aortic rings
preincubated with atropine and ODQ, the concentration–
response curve to MELc was significantly shifted to the right,
reflecting the involvement of NO in the endothelium-dependent
relaxation (Aktan, 2004). Bio-guided fractionation of MELc
allowed the isolation of UA and UA/OA. Pharmacological
evaluation of UA showed that this pentacyclic triterpenoid
caused both concentration- and endothelium-dependent relax-
ation of rat thoracic aortic rings pre-contracted with NA and the
effect was less potent than carbachol (positive control). This
effect suggested that cyclooxygenase or nitric oxide synthase
pathways were involved in the response (Vanhoutte, 2001).
However, since vasorelaxation was blocked by L-NAME and
ODQ, while indomethacin and atropine did not inhibit the
effect, endothelium-derived NO seems to be involved in UA
action (Moncada et al., 1997), and allowed us to discard the
possible role of cyclooxygenases or a direct action on
cholinergic muscarinic receptor in the endothelium-dependent
relaxation (Schlossmann and Hofmann, 2005). However,
preincubation with indomethacin provoked a significant
displacement to the left, indicating that UA relaxant activity is
synergized. Experiments are in progress to understand that
behavior. Relaxation due to SNP and carbachol was shifted to
the left in the presence of UA, but only in the presence of SNP
was significant, indicating a synergic effect on the vasorelaxant
activity. Finally, UA (30 μM) caused a marked depression of the
Emax in the cumulative concentration–response curve for NE
contraction in aortic rings (+ E), suggesting that the possible
mode of action of this compound is related with a direct
stimulation of eNOS and NO release (Aktan, 2004). The fact
that vasorelaxation was blocked by the NOS inhibitor L-NAME
in endothelium-intact artery, indicates that the enzyme is
involved in UA action; in support of this contention, inhibiting
another step in the pathway, i.e., inhibition of Guanylyl Cyclase
by ODQ produces the same result: decrease in UA-induced
relaxation. In addition, the presence of SNP, a NO-donor,
increases the relaxing effect of UA, which suggests that the NO-
cGMP pathway is stimulated by UA, most probably via NOS
activity. We consider that the vasodilator effect showed by L.
caulescens could be related to the presence of UA and OA.
Recently, Rodríguez-Rodríguez et al. (2004) reported that
oleanolic acid (OA) isolated from “Orujo” olive oil was able to
relax, in a concentration-dependent fashion, the contractions
induced by phenylephrine in rat aortic rings with functional
endothelium. OA mechanism of action seems to be mainly
mediated by endothelial NO production. These results are in
concordance with the relaxant effect observed for UA in this
investigation, and support the hypothesis that UA and OA or the
UA/OA mixture, are responsible of the vasorelaxant activity of
L. caulescens.
Since UA and OA have a broad spectrum of pharmaco-
logical activities (Liu, 2005), we suggest that these
compounds could be leads for the development of new
therapeutic agents, including antihypertensive drugs. In this
context, experiments are in progress in order to provide new
data to clarify the precise mechanism by which MELc and
1067
UA produce their vasorelaxant effect. These experiments will
determine the levels of cGMP, NOS activity and NOS
mRNA in the aorta to demonstrate the direct involvement of
NO in the endothelium-dependent vasorelaxation. In addition,
we are generating some semi-synthetic derivatives to
establish new structure–activity relationships for these
molecules and contribute to the characterization of their
biological activity.
Finally, it is important to mention that a drug therapy for
cardiovascular and pulmonary diseases involves the modulation
of NO-cGMP signalling pathway. Classic compounds are the
NO-releasing organic nitrates: nitroglycerine, isosorbide-mono-
nitrate and dinitrate, which are used mainly to alleviate the
symptoms of various diseases related with different physiolog-
ical functions including smooth muscle tone, platelet aggrega-
tion and intestinal secretion (Schlossmann and Hofmann, 2005).
However, continuous administration eventually leads to nitrate
tolerance. In these circumstances, the development of drugs
with NO-release properties via stimulation of eNOS could be
important for the treatment of hypertension and as we
mentioned before, triterpenoids isolated from L. caulescens
could be the chemical structure leads to obtain these new drugs.
In conclusion, to our knowledge this is the first report
showing that the vascular activity of UA is dependent upon the
presence of a functional endothelium and possible related to NO
release. These results confirm that vasorelaxation showed by
MELc is produced by the presence of UA and OA and gives an
additional support of the medicinal uses of L. caulescens for the
treatment of hypertension.
Acknowledgements
This study was financed by grants from SEP-CONACyT
(43440) and PROMEP-SEP. Authors are grateful to Itzell
Gallardo-Ortiz for technical assistance. F. Aguirre-Crespo is
grateful to CONACyT for the scholarship grant (181772) to
carry out graduate studies.
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