ARTICLE IN PRESS

Phytomedicine 12 (2005) 338–344

www.elsevier.de/phymed

Pharmacological assay of Cordia verbenacea V: oral and topical

anti-inflammatory activity, analgesic effect and fetus toxicity of
a crude leaf extract
J.A.A. Sertié, R.G. Woisky, G. Wiezel, M. Rodrigues
Laboratório de Farmacologia e Toxicologia de Produtos Naturais, Departamento de Farmacologia, Instituto de Ciências
Biomédicas, Universidade de São Paulo, SP, Brazil

Received 11 August 2003; accepted 22 September 2003

Abstract
Cordia verbenacea D.C. (Borraginaceae) is a perennial bush plant that grows widely along the southeastern coast of
Brazil. Its leaves have been used in folk medicine for their anti-ulcer, anti-inflammatory and cicatrizing activities. We
have already described the anti-inflammatory properties of C. verbenacea and its low toxicity in different acute animal
models. In the present study, we investigated the anti-inflammatory activity in sub-chronic animal models of a crude
leaf lyophilized extract when administered by oral route or topically applied, and concomitantly, its analgesic potency
and toxicity to the fetus. Topical administration of the extract inhibited nystatin-induced edema proportionally to the
doses used, and this effect at a dose of 4.56 mg/kg body wt. was similar to that observed with 6.0 mg/kg body wt. of
naproxen. In miconazole-induced edema, the leaf extract at a dose of 1.24 mg/kg body wt., orally administered, has a
very similar effect as compared to nimezulide (2.5 mg/kg body wt.) and dexamethasone (0.2 mg/kg body wt.). At an
oral dose of 2.48 mg/kg body wt. the extract showed a very low analgesic effect, and total absence of fetus toxicity at
doses of less than 7.44 mg/kg body wt.
r 2004 Elsevier GmbH. All rights reserved.

Keywords: Cordia verbenacea; Alcoholic extract; Inflammation; Analgesia; Fetus toxicity

Introduction tannins, cordialins and essential oils (1.5/100 ml leaves)

(Jorge et al., 1998; Sertié et al., 1990, 1991).
Cordia verbenacea DC (Borraginaceae) a perennial We reported previously that the hydroalcoholic
bush plant, distributed widely along the southeastern lyophilized extract of C. verbenacea leaves exhibited an
Brazilian coast, is reputed in folk medicine as an anti- anti-inflammatory activity, allied to an important
ulcer, anti-inflammatory and cicatrizing drug (Smith protective effect on the gastric mucosa, and a very low
Lyman, 1970). Phytochemical screening of an ethanolic toxicity in acute models of experimentation in rats, when
extract of C. verbenacea leaves showed flavonoids administered by oral route (Sertié et al., 1988, 1991;
(quercetin, artemetin and hydroxyartemetin), mucilages, Oliveira et al., 1998). Based on these data, the objectives
of the present work were to evaluate whether the same
Corresponding author. Tel.: +55 022 3091 7315; hydroalcoholic extract shows anti-inflammatory activity
fax: +55 022 3091 7322. when administered by oral route or topical application
E-mail address: [email protected] (J.A.A. Sertié). in the subchronic animal model and, concomitantly, to

0944-7113/$ - see front matter r 2004 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2003.09.013
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verify both its analgesic potency and its possible toxic Miconazole-induced edema
side-effects after chronic oral administration on the
offspring of treated rats. The estrous cycle and fetal The inhibitory activity of the crude drug administered
osseous formations were also determined. daily by the oral route for 10 days, on miconazole-
induced paw edema, was compared with that of
nimizulide and dexamethasone, according to the method
Material and method of Hanada et al. (1994). One hour prior to the
administration of agar 1% (control), extract (1.24 mg/
Experimental kg body wt. — oral ED50 value determined by Sertié et
al., 1988), nimizulide (2.5 mg/kg body wt., Swingle and
C. verbenacea leaves were collected at Praia Grande, Moore, 1984) and dexamethasone (0.2 mg/kg body wt.,
State of São Paulo, in March, 1990 and identified by Basile et al., 1988), 0.1 ml of miconazole nitrate (2.0 mg/
Prof. Dr. S. Panizza of the Departamento de Botânica, paw) in CMC 0.5% was injected into the sub-plantar
Instituto de Biocências, Universidade de São Paulo, area of the left hind-paw of unanesthetized rats. An
Brazil, where a voucher specimen has been deposited. equal volume of CMC 0.5% was injected into the right
The fresh material (200 g) was extracted by maceration hind-paw. At various intervals over a 10-day period, the
with 70% EtOH (1 l) at room temperature. After volume of each paw up tibio-tarsal articulation was
filtration, the dark green decoction was concentrated determined according to the plethysmographic method
in vacuo at 50 1C to yield an aqueous viscous mass, of Winder et al. (1957). The results were expressed as
which was then lyophilized (1 mg equivalent to 14.28 mg percentage of the initial paw volume.
of fresh leaves).

Animals Analgesic effect

Virgin male and female Wistar Hanover rats, weigh- Rats were fasted 12 h before each experiment. They
ing 160710 g each, were maintained under standard were treated with 1.24 or 2.48 mg/kg body wt. of
conditions of temperature (22–25 1C), relative humidity lyophilized extract and 60, 120, 180 min later the pain
(40–60%) and light/dark cycle. They had free access to threshold in each paw was determined according to
Nuvilab CR-1s (Nuvital) chow and water. Swingle et al. (1971), using an Ugo-Basile analgesimeter.
Agar 1% was administered to the control group,
Nystatin-induced edema acetylsalicylic acid (100 mg/kg body wt.) was used as
reference drug. The pain threshold to force exerted on
Edema was induced according to Schiatti et al. (1970), the animal’s paw was represented as analgesic coefficient
i.e., 0.1 ml (47,600 units) of 8.5% nystatin (Squibb) (i), which was calculated as the following expression:
suspension were injected into the plantar side of the left
ðA60 þ A120 þ A180Þ
hind paw of unanesthetized rats. An equal volume of i¼ ;
saline was injected into the right hind paw. Six and 10 h ðB60 þ B120 þ B180Þ
later, the animals, housed individually, were divided into where A is the force measured in the treated animals,
four experimental groups: one group (control) received and B is the force measured in control, at different times
only acetone/water (7:3) topically; another two groups indicated in numbers.
were treated topically with either 2.28 or 4.56 mg/kg
body wt. (direct in the left hind paw) of extract (ED50
value and 2
ED50 value, respectively, as determined by
Basile et al., 1989) and the last one was treated with Estrous cycle in virgin rats
6.0 mg/kg body wt. of naproxen (Sigma) (Croker, 1967).
Both drugs were dissolved in 45 ml of acetone/water The animals were gavaged daily for 30 consecutive
(7:3). An equal volume of solvent was applied to the days with 1% agar (4.0 ml/kg body wt.) and the extract
right hind paw of all animals. The paw volume was at doses 1.24 and 7.44 mg/kg body wt. Every day, a
determined using the plethysmographic method at 4, 6, small volume of saline was introduced into the vagina of
8, 10, 12 and 24 h after nystatin injection. The the rats using a dropper and rapidly aspirated and added
displacement of the mercury column was measured with to a microscope slide, fixed, colored in the usual
a Gold Pressure Transducer (Model P23ID) and histological manner, and examined in a Olympus
registered on a Gould Polygraph (Model R.S. 3400). binocular microscope. The phases of the cycle (proes-
The results were expressed as percentage of the initial trous, estrous, metaestrous and diestrous) were identi-
paw volume. fied by the observation of the desquamation cells form.
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340 J.A.A. Sertié et al. / Phytomedicine 12 (2005) 338–344

Fetus toxicity in the offspring of females treated Statistical analysis

prior to the mating
All values are expressed as mean7SEM. One-way
Sexually mature virgin females were treated daily by analysis of variance was performed. Sequential differ-
gavage for 45 days with 1% agar (4.0 ml/kg body wt.) ences among means (po0.05; 0.001) were calculated,
and the extract (1.24 and 7.44 mg/kg body wt.). After using Tukey contrast analysis (Sokal and Rohlf, 1969).
this period, the females were caged with virgin and
untreated males (2:1) for 10 days, and then the females
were transferred to individual cages until delivery, when Results
we observed the numbers, weight, length and eventual
gross malformation of the litters. After that, about one-
Nystatin-induced edema
fourth of the litters were sacrificed with ether, and their
liver, spleen and kidney macroscopically examined The edema induced by nystatin suspension increased
under a stereomicroscope for the presence of gross
progressively for 12 h, when the control group reached
lesions. Both the weight and relative weight (organ/
an increase in the paw volume of about 55% (Fig. 1).
body) were noted. The behavior and fertility rate of the
The extract (2.28 and 4.56 mg/kg body wt.) applied
remaining litters were subsequently observed until
topically, produced a significant inhibitory effect as
sexual maturity.
compared to the control group, starting 8 h after
nystatin induction, and both doses reached a significant
(po0.001) inhibitory effect on the edema 12 h after the
injection of the flogystic agent. However, the extract at
Fetus toxicity in the offspring of males treated prior
dose of 2.28 mg/kg body wt. was less active than
to mating
naproxen (6.0 mg/kg body wt.).
After 45 consecutive days of treatment by oral route
with 1% agar (4.0 ml/kg body wt.) and extract (1.24 mg/ Miconazole-induced edema
kg body wt. and 7.44 mg/kg body wt.), virgin male rats
were caged with untreated virgin females (1:2). The fetus The edema induced by miconazole increased progres-
toxicity in the offspring was evaluated as described sively for 1 day (Fig. 2). The oral administration of the
above. extract (1.24 mg/kg body wt.) produced a significant
inhibitory effect when compared to the control and
nimezulide groups 9 h and on the 1st and 2nd days after
miconazole injection, and this effect persisted for 10
Fetus toxicity in the offspring of rats treated during
pregnancy

Virgin and untreated females were caged with virgin

and untreated males (2:1). Copulation was established
by checking the presence of sperm in the vaginal smears
(pregnancy day ‘‘0’’), then the females were treated daily
by oral route with 1% agar (4.0 ml/kg body wt.) or
extract (1.24 and 7.44 mg/kg body wt.). On the 20th day
of gestation, half of the pregnant rats were sacrificed
with ether and the abdominal wall and uterine horns
were opened, the fetuses were removed and their weights
and lengths noted. Subsequently, about two-thirds of
the fetuses were dissected out and their liver, spleen and
kidney macroscopically examined under a stereomicro-
scope for the presence of gross lesions. Their weights
Fig. 1. Effect of the topical application of C. verbenacea leaf
and relative weights (organ/body) were noted. The other
extract on nystatin-induced edema. Each point represents the
one-third was cleared in 1% of KOH for staining with mean7 S.E.M. of eight rats. aSignificantly different from the
alizarin red dye, for examination of osseous defects. The control (po0.05, Tukey); bsignificantly different from the
remaining half of the females had a normal pregnancy. control (po0.0001); csignificantly different from the control
After birth, the littermates were counted and their (po0.001, Tukey); dsignificantly different from a dose of
weights and lengths noted. The behaviors and fertility 2.28 mg/kg body wt. (po0.01, Tukey); esignificantly different
rate were observed until sexual maturity. from doses of 2.28 and 4.56 mg/kg body wt. (po0.01, Tukey).
 ARTICLE IN PRESS
J.A.A. Sertié et al. / Phytomedicine 12 (2005) 338–344 341

days. It is important to note that 1 day after miconazole- verbenacea extract was significant only in the group
induced edema, the extract presented an inhibitory effect treated with 2.48 mg/kg body wt. at only at 60 min after
similar to that of the nimizulide and dexamethasone. its oral administration. At the ED50 value anti-
inflammatory dose (1.24 mg/kg body wt.) the extract
Analgesic effect did not produce any increase in the analgesic effect. The
analgesic coefficients (i) obtained were 1.00 (control),
Fig. 3 shows force exerted on paws, respectively, at 1.00 (extract at dose 1.24 mg/kg body wt.), 1.17 (extract
60, 120 and 180 min. The analgesic effect of the C. at dose of 2.48 mg/kg body wt. and 1.48 (acetylsalicylic
acid)).

Estrous cycle and fetotoxicity

The effect of extract on the estrous cycle, and

offspring of males and females treated before mating
and females treated during pregnancy are shown in
Tables 1–4, respectively. In all types of treatment used,
the crude drug did not interfere with the osseous
formation (Fig. 4), development, sexual maturity or
fertility of the offspring.

Discussion
Fig. 2. Effect of oral administration of C. verbenacea leaf
extract on miconazol-induced edema. Each point represents Previous studies conducted in different acute models
the mean7S.E.M. of eight rats. aSignificantly different from of inflammation and toxicity revealed a potent anti-
the control (po0.01, Tukey); bsignificantly different from inflammatory activity of C. verbenacea leaf extract with
nimezulide (po0.05, Tukey); csignificantly different from the a very low toxicity (Sertié et al., 1988, 1991; Basile et al.,
control (po0.001, Tukey); dsignificantly different from nimi- 1989; Oliveira et al., 1998); The results of the present
zulide (po0.05, Tukey). study, conducted in two sub-chronic models of inflam-
mation, revealed an anti-inflammatory activity, asso-
ciated with a very low analgesic effect.
Niemegeers et al. (1975) reported that nystatin alters
the lysosomal membrane, causing the release of proteo-
lytic enzymes. Its local inflammation has a long
duration, reaching the maximal response 12 h after
injection of the phlogistic agent. After 6 h (just before
the topical administration of the drugs), all the groups
presented the same degree of edema, showing that any
further variation in the paw volume would have to be
due to different treatment administered.
In our assay, the topical administration of extract of
C. verbenacea extract inhibited nystatin-induced edema

Table 1. Effects of oral administration of C. verbenacea leaf

extract during the estrous cycle

Treatment Dose/kg No. of estrous Intervals

body wt. in 30 days between the
estrous (days)

Fig. 3. The analgesic effect of an orally administered leaf 1% agar 4.0 ml 6.470.30 4.170.23
extract from C. verbenacea on the mean weight (g) supported (control)
by rat hind paws. The results are expressed as the Extract 1.24 mg 6.670.37 3.970.17
mea7S.M.E. of 10 rats. aSignificantly different from the 7.44 mg 6.270.35 4.070.26
control and a dose of 2.48 mg/kg body wt. (po0.001, Tukey);
b
significantly different from the control (po0.05, Tukey). Each value represents the mean 7 S.E.M. of 10 rats.
 342
Table 2. Body, liver, spleen and kidney weights with respective relative weights in offspring from females treated orally for 45 days with C. verbenacea leaf extract, prior to
mating

Treatment Dose/kg Mated Litters/~ Litters Weights (g
102) and relative weights K/B
body wt. ~(No.) (No.) evaluated
Body (B) Liver (L) L/B Spleen (S) S/B Kidney (K)

Agar 1% 4.0 ml 11 8.8270.3 20 562717 35.871.2 0.06470.003 1.370.001 0.00270.0001 5.570.3 0.00970.0004
Extract 1.24 mg 11 10.5970.3 30 588712 38.3701.3 0.06670.004 1.770.01 0.00270.0001 6.170.3 0.01070.0003
7.44 mg 11 10.0870.3 30 616720 40.572.1 0.06570.002 1.870.02 0.00270.0002 5.970.3 0.00970.0002

Each value represents the mean 7 S.E.M.

J.A.A. Sertié et al. / Phytomedicine 12 (2005) 338–344

Table 3. Body, liver, spleen and kidney weights with respective relative weights in offspring from males treated orally for 45 days with C. verbenacea leaf extract, prior to
mating

ARTICLE IN PRESS
Treatment Dose/kg Mated # Litters/~ Litters Weights (g
102) and relative weights
body wt. (No.) (No.) evaluated
Body (B) Liver (L) L/B Spleen (S) S/B Kidney (K) K/B

Agar 1% 4.0 ml 8 9.8871.4 18 577710 34.876.1 0.06270.002 1.370.001 0.00270.0001 5.670.9 0.00970.0004
Extract 1.24 mg 8 9.0670.9 18 57274.6 43.375.5 0.07570.006 1.270.002 0.00270.0003 5.370.9 0.00970.0003
7.44 mg 8 9.5370.3 18 58775.3 32.877.0 0.06670.002 1.870.05 0.00270.0007 5.970.9 0.00970.0002

Each value represents the mean 7 S.E.M.

Table 4. Body, liver, spleen and kidney weights with respective relative weights in fetuses from females treated orally with C. verbenacea leaf extract during pregnancy

Treatment Dose/kg Pregnants Fetuses Weights (g
102) and relative weights

body wt. (No.) evaluated
Body (B) Liver (L) L/B Spleen (S) S/B Kidney (K) K/B

Agar 1% 4.0 ml 8 20 381711 35.872.0 0.0970.003 0.4170.003 0.00170.0003 3.370.08 0.00870.0001

Extract 1.24 mg 8 20 39778 35.271.9 0.0970.003 0.4870.003 0.00170.0007 2.970.10 0.00770.0001
7.44 mg 8 20 38075 31.571.0 0.0970.001 0.3870.02 0.00170.0005 2.670.1 0.00770.0001

Each value represents the mean7S.E.M.

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local inflammatory process. The extract probably pro-

duces its analgesic effect through a central mechanism.
In earlier toxicological studies the LD50 value of the
extract when administered orally to rats was found to be
more than 20 times the ED50 value, and significant
alterations in the biochemical, hematological and
pattern of E.C.G were observed (Sertié et al., 1988;
Oliveira et al., 1998). This low acute toxicity, associated
with the absence of fetus toxicity, comportment and
reproducibility of neonates observed in the present sub-
chronic study confirm the low toxicity of the extract.
In previous studies, two flavonoids were isolated and
identified in the C. verbenacea extract (Sertié, 1990).
According to Alcaraz and Jimenez (1988) some flavo-
noids can affect the inflammatory response in different
models of experimental inflammation, associated with a
Fig. 4. Osseous formation in fetuses from females treated
high margin of safety and weak analgesic effects.
during pregnancy with daily oral administration of C.
verbenacea leaf extract at a dose of 7.44 mg/kg body wt: (a)
Although the exact mechanism by which this extract
control; (b) extract. produces its activity is not entirely clear, several studies
have suggested that the anti-inflammatory effect of
flavonoids is probably related to anti-histamine, anti-
dose dependently. The inhibitory effect observed with bradykinin, or anti-serotonin properties or to inhibition
the topical administration of 4.56 mg/kg body wt. was of enzymes such as 12-lipoxygenase and 5-lipoxygenase
not significantly different from that observed with (Prabhakar et al., 1981; Agarwal, 1982; Kimura et al.,
6.0 mg/kg body wt. of naproxen 8 h after nystatin 1985).
injection, suggesting that the highest dose of C. Independent of the exact mechanism of action
verbenacea has an inhibitory effect similar to that of involved in the anti-inflammatory and analgesic affects
an NSAID. of this extract, the results presented here give experi-
According to Hanada et al. (1994), edema induced by mental support for the usage of the leaf extract of C.
miconazole, like carrageenin, results in a biphasic and verbenacea as an anti-inflammatory drug in folk
circumscribed inflammation, with the advantage of a medicine.
long-lasting effect. Miconazole treatment provoked an
increase in circulation leukocyte counts, which is
attributed to an increase in polymorphonuclear and References
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