Glaucolides D and E, sesquiterpene lactones isolated from Vernonia liatroides, were found to relax smooth muscle contractions in isolated rat aorta and uterus in a dose-dependent manner. Glaucolide E was more potent than glaucolide D at relaxing contractions induced by high potassium levels or agonists like noradrenaline and oxytocin. In contrast, a hirsutinolide-type sesquiterpene lactone from Vernonia paniculata did not have any effect on smooth muscle relaxation. The relaxant effects of glaucolides D and E, which lack an alpha-methylene gamma-lactone moiety present in the related compound

1. 112 Notes Biol. Pharm. Bull. 26(1) 112—115 (2003) Vol. 26, No. 1
Relaxation of Uterine and Aortic Smooth Muscle by Glaucolides D and E
from Vernonia liatroides
María CAMPOS,*,a Martha OROPEZA,a Héctor PONCE,a Jaquelina FERNÁNDEZ,a
Manuel JIMENEZ-ESTRADA,b Héctor TORRES,b and Ricardo REYES-CHILPAb
a Unit of Medical Research in Pharmacology, National Medical Center S. XXI, Mexican Institute of Social Security; San
Francisco 350–502, Col. Del Valle, Mexico City, 03100, Mexico: and b Institute of Chemistry, National University of
Mexico; Mexico City, 04510, Mexico. Received July 29, 2002; accepted September 30, 2002
Vernonia spp. (Asteraceae) are used in herbolaria in Latin America in menstrual and stomach disorders,
suggesting smooth muscle relaxing properties of some of their chemical constituents. For pharmacological sup-port
for this belief, sesquiterpene lactones glaucolides D and E were assayed on isolated rat smooth muscle.
Glaucolide E proved more potent than glaucolide D to relax high KCl- or noradrenaline-induced contractions in
aorta and to relax the high KCl-contraction in uterus. Hirsutinolide-type sesquiterpene lactone also was tested
but displayed no effect. Relaxation of smooth muscle by structurally related sesquiterpene lactone parthenolide
has been attributed mainly to the a-methylene g-lactone moiety; because glaucolides D and E lack this func-tional
group, their relaxant properties may rely on other alkylating sites such as C10 of the germacra-1(10),4-
diene-4-epoxide skeleton.
Key words Vernonia; sesquiterpene lactone; glaucolide; hirsutinolide; smooth muscle; parthenolide
Vernonia spp. (Asteraceae) are reported in Mexican herbo-laria
for used in menstrual disorders, hair regeneration,
dysentery, and as a coagulant.1) Brazilian folk medicine has
shown that the crude extract prepared from leaves of V. con-densata
prevents stomach and liver disturbances (sic).2) Sev-eral
of these applications suggest presence of compounds ac-tive
on smooth muscle. Sesquiterpene lactones, mainly glau-colide-
and hirsutinolide-type, have been isolated from Ver-nonia
spp. –sensu latu–.3—6) However, several hirsutinolides
might be artifacts formed from glaucolides on exposure to
acidic adsorbents during chromatography.7—9) Glaucolides
have been reported to elicit molluscicidal, antitumoral, and
antimicrobial properties5,10,11); but their effects on smooth
muscle have not been investigated previously. Parthenolide, a
structurally related sesquiterpene lactone (Fig. 1D) bearing
also a germacra-1(10),4-diene-4-epoxide skeleton is known
to inhibit smooth muscle contractility likely due to the a -
methylene g -lactone moiety.12) Therefore, the effects of glau-colides
D (Fig. 1A) and E (Fig. 1B) on vascular and uterine
smooth muscle contraction were studied. The effect of a hir-sutinolide
type sesquiterpene lactone (Fig. 1C) lacking both
germacrane skeleton and a -methylene g -lactone was also in-vestigated.
We have assayed the action of these compounds
on smooth muscle pre-contracted with either depolarizing
high-KCl solution or an agonist (noradrenaline for aorta and
oxytocin for uterus). This model was selected as mean to in-dicate
whether glaucolides D and E are able to relax the con-traction
dependent on different pathways of calcium influx.
MATERIALS AND METHODS
Compounds Glaucolides D (Fig. 1A) and E (Fig. 1B)
were isolated from Vernonia liatroides by chromatography
and identified by spectroscopic methods as previously de-scribed.
3) The hirsutinolide [1R,4S,5R,6S,8S,10R]-8,10,13-
triacetoxy-1(4)-epoxy-1,5-dihydroxygermacr-7(11)-en-6(12)-
olide (Fig. 1C) was obtained from Vernonia paniculata as
transformation product of glaucolide B treated with bentonite
earth.7) Vernonia liatroides was collected in Ixtapan de la Sal,
State of Mexico, while V. paniculata was collected in
Matatlán, State of Oaxaca, in Mexico. Herbarium specimens
are deposited in IMMS and MEXU herbaria, respectively.
Animals Sprague-Dawley rats, female for uterine exper-iments
and male for aorta experiments, body weight approxi-mately
300 g, were used. Virgin female rats received a subcu-taneous
injection of estradiol benzoate (40m g/kg) 24 h prior
to the experiment. Animal experiments were performed fol-lowing
the recommendations of the Policies on Animal Ex-perimentation
of the Scientific Committee of the Instituto
Mexicano del Seguro Social; animals were decapitated, and
uterus or descending thoracic aorta were isolated, fat and
connective tissue were removed, and tissues were placed in
Krebs–Ringer bicarbonate solution.
Uterus Experiments Uterine rings (5—7 mm long)
were placed in a 5ml organ bath containing Krebs–Ringer
bicarbonate (KRb) solution with the following composition
(mM): NaCl, 120; KCl, 4.6; CaCl2, 1.5; NaHCO3, 20;
KH2PO4, 1.2; MgSO4, 1.2, and glucose 11, maintained at
37 °C and bubbled with a mixture of 95% O2–5% CO2
(pH7.4). Contractions were recorded isometrically by a
force–displacement transducer (Grass, FT03) connected to a
Grass polygraph (model 7B). Tissues were allowed to equili-brate
for 60 min during which KRb was changed every 20
min and maintained under optimal tension of 1 g prior initia-tion
of the experimental protocol. After equilibration, uterine
rings were bathed in a depolarizing solution (60mM KCl)
prepared by equimolar substitution of NaCl for KCl to ex-pose
tissues to a single sub-maximal concentration of KCl
(60mM), and to elicit contractile response. Control contrac-tile
responses were considered as two successive similar re-sponses.
To study the effect of the compounds on a depolar-ization-
induced contraction, a third maximal contraction to
KCl 60mM was recorded during 20 min to obtain steady re-sponse.
After reaching plateau response, each compound (A,
B, or C) was added in progressively increasing cumulative
concentrations (3, 10, 30, 100m M) to the bath solution and
∗ To whom correspondence should be addressed. e-mail: mariagcampos@yahoo.com © 2003 Pharmaceutical Society of Japan

2. January 2003 113
Fig. 1. Structures of Glaucolides-D (A), E (B), [1R,4S,5R,6S,8S,10R]-8,10,13-Triacetoxy-1(4)-epoxy-1,5-dihydroxygermacr-7(11)-en-6(12)-olide (C), and
Parthenolide (D)
the effect of each concentration on the tonic contraction was
observed for 5 min. In every set of experiments, a control tis-sue
was run concurrently; this control was treated in the
same manner as test tissues but only vehicle was added. To
study the effects of compounds on agonist-induced contrac-tile
response, contraction was induced by 10 mU/ml oxy-tocin.
After 10 min, the compound was added in progres-sively
increasing cumulative concentrations (3, 10, 30, 100
m M) on contractile response.
Aorta Experiments Aortic rings (approximately 5mm
in length) were mounted in organ baths containing 5 ml KRb
solution, maintained at 37 °C and bubbled with a mixture of
95% O2–5% CO2 (pH7.4). The equilibration period was
similar to that of uterine rings, but optimal tension was 2 g.
Subsequently, a protocol similar to that of uterine rings was
performed to elicit KCl (60mM)-induced contractile re-sponses
and to assay glaucolide effect. To study the effects of
glaucolides on agonist-induced contractile response, the
tonic contraction was induced by 106
M noradrenaline con-centration,
producing sub-maximal contraction. To avoid no-radrenaline
oxidation in the bath solution, 50m M ascorbic
acid was added to noradrenaline solution freshly prepared for
each experiment. When contractile response to noradrenaline
was stable, the compound was added in progressively in-creasing
cumulative concentrations (3, 10, 30, 100mM) at 5
min-intervals.
Drugs L-Noradrenaline bitartrate, 17b -estradiol-3-ben-zoate,
and ascorbic acid were obtained from Sigma Chemical
Co. (St. Louis MO, U.S.A.). Dimethyl sulfoxide and ethanol
were purchased from Mallinckrodt Baker (Mexico), while
oxytocin was obtained from Sandoz (Mexico). Glaucolides
were dissolved in dimethyl sulfoxide/ethanol (2 : 5, v/v).
Final concentration of DMSO/ethanol in the bathing solution
did not exceed 0.2%, innocuous to tissue contractile activity.
Estradiol benzoate was dissolved in corn oil (100m g/ml).
Data Analysis Maximal tissue contractile response was
determined as the response elicited by 60mM KCl or by ago-nist
(106
M noradrenaline or 10 mU/ml oxytocin). Relax-ation
induced by compounds A, B, and C was assessed as
percentage of relaxation of maximal contractile response.
EC50 is the effective concentration 50 (concentration of com-pounds
causing 50% relaxation of smooth muscle contractile
response). Results are expressed as meanS.D. of 4—10 or
more preparations each obtained from different animals.
One-way analysis of variance and Bonferroni test for multi-ple
comparisons were used to evaluate statistical differences,
and p0.05 was considered significant.
RESULTS
Effect of Glaucolides on High KCl-Induced Contrac-tions
Smooth muscle contractions induced by 60mM potas-sium
in both aorta and uterus were relaxed by glaucolides D
and E in a dose-dependent manner (Fig. 2). No effect was ob-served
when hirsutinolide was assayed. EC50 and Emax values
indicate that glaucolide E was more potent, and more effec-tive
than glaucolide D to relax high KCl-induced contraction
in both tissues (Table 1). When the effect of glaucolide D
was compared between high KCl- and agonist-induced con-tractions,
glaucolide D showed to be more potent acting on
high KCl-induced contractions in aorta (p0.001) and on
oxytocin-induced contractions in uterus (p0.05). Regarding
glaucolide E, this showed to be more potent and effective act-ing
on high KCl- than on noradrenaline-induced contractions
in aorta (p0.01). The effect of glaucolide E on high KCl-and
oxytocin-induced contractions in uterus was not statisti-cally
different (Tables 1, 2).
Effect of Glaucolides on Agonist-Induced Contractions
Glaucolides D and E relaxed in a dose-dependent manner
contractions induced by noradrenaline (106
M) and oxytocin
(10 mU/ml) in aorta and uterus, respectively (Fig. 3). The
hirsutinolide lacked effect on both tissues. Glaucolide E
showed to be more potent and effective than glaucolide D on
noradrenaline-induced contractions in aorta (p0.05). No
significant difference was observed between glaucolides D
and E on oxytocin-induced contractions in uterus (Table 2).
DISCUSSION
To our knowledge, this is the first report concerning relax-ant
effects of glaucolides on smooth muscle. We have tested
the action of these compounds on smooth muscle contraction
induced by either high KCl or agonist (noradrenaline for
aorta, oxytocin for uterus).
Glaucolides D and E relaxed contractions induced by high
KCl or agonist in aorta and uterus; the hirsutinolide was de-void
of activity. These results suggest that sesquiterpenoids
lacking a -methylene g -lactone moiety could also relax
smooth muscle contraction as long as they bear alternative
alkylating sites such as C-10 provided by germacra-1(10),4-
diene-4-epoxide skeleton. The importance of alternative
alkylating- reactive sites in addition to a -methylene g -lac-

3. 114 Vol. 26, No. 1
Fig. 2. Glaucolides D and E Relaxed in a Dose-Dependent Manner the High KCl-Induced Contraction in Rat Smooth Muscle
Bars represent the mean of 4—8 observations, vertical lines represent the standard deviation.
tone was first addressed by Fischer et al.,13) who examined
the antimicobacterial activity of a variety of sesquiterpene
lactones including parthenolide. In fact, it was proposed that
facile transannular cyclization of parthenolide may explain
the high activity of this compound. A similar mechanism
may be operating on smooth muscle contractility in the case
of glaucolides D and E. It is noteworthy that hirsutinolide,
transformed by a 1—4 transannular cyclization, lacked activ-ity,
whereas glaucolide E, which bears a reactive a -methyl-ene
in the acyl substituent, was more potent than glaucolide
D, exhibiting an epoxide.
Spasmogenic responses of smooth muscle to high potas-sium
solutions can be explained in terms of calcium influx
from extracellular milieu via voltage-operated calcium chan-nels;
14,15) this mechanical response to KCl can be completely
inhibited by calcium entry blockers.16,17) The relaxing effect
of several products isolated from medicinal plants has been
related to blockade of calcium influx to smooth muscle
cell.18,19) Additionally, agonists such as noradrenaline or oxy-tocin
induce calcium influx by activating receptor-operated
Table 1. Relaxant Effect of Glaucolides D and E on High KCl-Induced
Contraction in Rat Smooth Muscle
Glaucolide D Glaucolide E
n
EC50 (m M) Emax (%) EC50 (m M) Emax (%)
Aorta 4 10.422.18a,b 59.368.00a 7.001.74 94.2511.66
Uterus 8 25.573.06c,d 83.2812.96d 13.201.45 98.063.71
Values represent meanS.D. a) p0.05 vs. glaucolide E/high KCl/aorta.
b) p0.001 vs. glaucolide D/agonist/aorta. c) p0.05 vs. glaucolide D/agonist/
uterus. d) p0.01 vs. glaucolide E/high KCl/uterus.
Table 2. Relaxant Effect of Glaucolides D and E on Agonist-Induced Con-traction
in Rat Smooth Muscle
Glaucolide D Glaucolide E
n
EC50 (m M) Emax (%) EC50 (m M) Emax (%)
Aorta 6 26.114.69 50.126.02 20.873.54a 73.119.71a
Uterus 10 18.503.33 93.256.23 16.463.12 94.286.26
Values represent meanS.D. a) p0.05 vs. glaucolide D/agonist/aorta and glau-colide
E/high KCl/aorta.
Fig. 3. Glaucolides D and E Relaxed in a Dose-Dependent Manner the Agonist-Induced Contraction in Rat Smooth Muscle
Bars represent the mean of 6—10 observations, vertical lines represent the standard deviation.

4. January 2003 115
channels.20)
In the present study, glaucolides D and E relaxed the con-traction
induced by either high potassium or agonist (nora-drenaline
on vascular smooth muscle, oxytocin on uterus).
Based on previous reports,14—20) we suggest that this finding
might be explained by the inhibition or blockade of calcium
influx via voltage- and receptor-operated channels in vascu-lar
and uterine smooth muscles. However, at the tested doses,
glaucolide E was more potent and effective than glaucolide D
to relax KCl-induced contraction in both tissues and in nora-drenaline-
induced contraction in aorta, whereas in the oxy-tocin-
induced contraction of uterine smooth muscle both
glaucolides displayed a similar effect. A likely explanation
for this finding might be that glaucolide E has higher affinity
than glaucolide D for calcium channels. However, this would
not explain the similar effect on oxytocin-induced contrac-tion
in uterus. In this case, oxytocin-induced contraction in-volves
calcium influx through receptor operated-channels
and calcium release from internal stores.21) Uterine intracel-lular
calcium stores may be very efficient, i.e., oxytocin-in-duced
contractile response in myometrium is extremely resis-tant
to extracellular calcium removal.22) This might be the
reason we observed no difference between the relaxing effect
of glaucolides E and D on oxytocin-induced contractile re-sponse
in uterine smooth muscle. On the other hand, these
compounds might have other sites of action, such as protein
kinase C, phospholipase C, etc. Therefore, more studies are
needed to investigate the precise mechanism of action of
glaucolides on cellular pathways of calcium entry and/or on
the mechanism of calcium release from internal stores.
REFERENCES
1) Aguilar A., Camacho J. R., Chino S., Jacquez P., Lopez M. E.,
“Herbario Medicinal del Instituto Mexicano del Seguro Social. Infor-macion
Etnobotanica,” IMSS, Mexico D. F., 1994, p. 64.
2) Frutuoso V. S., Gurjao M. R., Cordeiro R. S., Martins M. A., Planta
Med., 60, 21—25 (1994).
3) Maldonado J. E., Martínez R., Martínez V. M., Rev. Latinoamer.
Quím., 11, 58—59 (1980).
4) Mabry T. J., Bedel-Baset Z. H., Padolina W. G., Jones S. B., Biochem.
Sys. Ecol., 2, 185—192 (1975).
5) Alarcon M. C., Callegari J. L., Herz W., Planta Med., 56, 271—273
(1990).
6) Bazon J. N., Callegari Lopes L., Vichnewski W., Dias D. A., Nagamiti
K., Cunha W. R., Herz W., Phytochemistry, 44, 1535—1536 (1997).
7) Jiménez M., Ortega A., Navarro A., Maldonado E., Van Calesteren M.
R., Jankowski C. K., J. Natural Products, 58, 424—427 (1995).
8) Borkosky S., Bardón A., Catalán C. A. N., Díaz J. G., Herz W., Phyto-chemistry,
44, 465—470 (1997).
9) Kotowickz C., Bardón A., Catalán C. A. N., Cerda-García-Rojas C.
M., Joseph-Nathan P., Phytochemistry, 47, 425—428 (1998).
10) Jisaka M., Ohigashi H., Takegawa K., Huffman M. A., Koshimizu K.,
Biosci. Biotechnol. Biochem., 57, 833—834 (1993).
11) Oketch-Rabah H. A., Christensen S. B., Frydenvang K., Dossaji S. F.,
Theander T. G., Cornett C., Watkins W. M., Kharazmi A., Lemmich
E., Planta Med., 64, 559—562 (1998).
12) Hay A. J., Hamburger M., Hostettmann K., Hoult J. R., Br. J. Pharma-col.,
112, 9—12 (1994).
13) Fischer H. H., Lu T., Cantrell C. L., Castañeda-Acosta J., Quijano L.,
Franzblaus S. G., Phytochemistry, 49, 559—564 (1998).
14) Amedée T., Mironneau C., Mironneau J., Br. J. Pharmacol., 88, 873—
880 (1986).
15) Edwards D., Good D., Granger S., Hollingsworth M., Robson A.,
Small R. C., Weston A. H., Br. J. Pharmacol., 88, 899—908 (1986).
16) Calixto J. B., Loch S., Br. J. Pharmacol., 85, 189—195 (1985).
17) Granger S. E., Hollingsworth M., Weston A. H., Br. J. Pharmacol., 85,
255—262 (1985).
18) Rojas A., Cruz S., Rauch V., Bye R., Linares E., Mata R., Phytomedi-cine,
2, 51—55 (1995).
19) Campos M., Valencia A., Ponce-Monter H., Uribe C., Osuna L.,
Calderon J., Phytotherapy Res., 11, 11—16 (1997).
20) Karaki H., Weiss G. B., Gastroenterol. 87, 960—970 (1984).
21) Anwer K., Sanborn B. M., Endocrinology, 124, 17—23 (1989).
22) Anselmi E., D’Ocon M. P., Villar A., Prostaglandins, 34, 351—358
(1987).