Jwmwl of ~~~~~h&~o~g~, 31 W91) 239- 247 239

Elsevier Scientific Publishers LrelandLtd.

P~~ACOLOG~CAL ASSAY OF CoRDlA V~~~~AC~ III:

ORAL AND TOPICAL ~T~I~F~~ATORY ACTIVITY AND
GASTROTOXICITY OF A CRUDE LEAF EXTRACT

J.A.A. SERTI&, A.C. BASIL@, S. PANfZZAb, T.T. OSHIRO’, C.P. AZZOLrN~’ and
S.C. PENNk

Summary

The a~tiin~ammatory effects and gastrotoxieity of a lyoph~ized 70%

ethanol extract of the leaves of COTTA ~erbe~c~u were investigated
through experimental models in rats and mice. The oral administration of
1.24 mg/kg of the extract SignificantIy inhibited nystatin-induced oedema.
Topical application of the extract at a dose of 0.09 mgjear in mice was clearly
more effective than 1.0 mglear of naproxen in the reduction of the ear oed-
ema induced by croton oil. At antiinflammatory doses, the extract showed an
important protective effect on the gastric mucosa, reducing significantly the
number of gastric lesions.

Cordia ~eT~enuce~ IX (Boraginaceae) is reasonably well defined and

occurs generally near the Brazilian coast from Ceari to Sao Paulo. This spe-
cies is a spieated-powered shrub about 40-80 inches high. Its leaves are
elongate and lanceolate, 2-4 inches long by OX- 1.2 inches in breadth and
with abundant minute tubereles or murieations that are morpholo~ca~y the
bases of undeveloped trichomes. The spikes are usually slender and elon-
gate and tend to be interrupted by age (Johnston, 1930).
In Brazil, the leaves of C. ~e~&e~~ceu have a widespread use in popular
medicine as an antiinflammatory when administered orally or by topical
application.
The goal of this paper is to investigate the antiinflammatory activity of a
1yophiIized extract of the leaves when administered by the oral route or by
topical application in various animal models. concomitantly, its gastrotoxicol-
ogieal effect will be studied.

0378~7~1~03.~ 0 1991 Elsevier Scientific Pubiishers Ireiand Ltd.

Published and Printed in Ireland
240

Materials and Methods

Animub
Fasted male Wistar stock rats weighing 170- 190 g and male Swiss-
Webster mice weighing 20-30 g, having free access to water, were used in
these experiments. Their normal diet was Nuvilab CR-l (Nuvitall.

Plant ~xt~t~on
The leaves of C. verbenaceo were collected at Praia Grande, State of Sao
Paulo, and identified by the staff of the herbarium of the Department of
Botany, Institu~ de Bioci&mias, Uuive~idade de S&o Paulo, Brazil. The fresh
material (200 gl was extracted with 70% ethanol at roam temperature. After
filtration, the dark green deco&ion was eon~entrated under reduced pres-
sure at 50°C to give an aqueous, viscous mass which was then lyophilized (1
mg equivalent to 14.28 of fresh leaf). The pharmacological assays were car-
ried out with l”k, aqueous agar (Difeol or a~etone~ater (7:31solution~s~spen-
sions of the dried material.

Oedema was produced according to the model described by Schiatti et al.

(19701, i.e. 0.1 ml (47,600 units) of 8.5% nystatin (Squibb) suspension was
injected into the plantar area of the left hind paw of unanaesthetized rats.
An equal volume of saline was injected into the right hind paw. Six hours
later, the animals were treated orally with 1.24 mgtkg of extract (Sertie et
al., 19881, 5.0 mg&g of indomethacin (Sigma) (Arrigoni-Marteili et al., 19711.
The paw volume was determined by the plethysmo~aphie method described
by Winder et al. (19571 4, 6, 8, 10, 12, 24 and 48 h after nystatin injection.
The displacement of the mercury column was measured with a Gould pres-
sure transducter (Model P23IDl and registered on a Gould polygraph (Model
RS 3.4001.

Croton 04 dermatitis
The inflammation was induced in mice according to the procedure
described by Tubaro et al. (1987~ modified. Two hundred micrograms of
croton oil (Sigma) dissolved in 20 C;tof acetone/water (7:3f was applied to the
inner surface of the right ear. An equal volume of the solvents was applied
in the same manner to the left ear. Thirty minutes later the animals were
treated topically with 20 ~1 of 0.09 mglear of extract (Basile et al., 19891, 1.0
mgjear of naproxen (Sigma) or 0.015 mglear of dexamethasone (Merck, Sharp
and Dohmel dissolved in acetone/water (7:31.Mice were killed 6 h later and a
sample of 8 mm diameter was punched from both treated and untreated
ears. The antiphlogistic response was monitored by measuring the differen-
tial weights of two samples and then calculating percentage of inhibition.

Restraint gas trdc hidOnS

destruct-induct lesions were induced according to the methods of
 241

Takagi et al. (19641 and Takagi and Okabe (19681 modified. Twenty-hour
fasted rats with free access to water were treated orally with agar 1% (con-
trol), 1.24 mg/kg of extract (Sertie et al., 19881, 5.0 mg/kg of piroxicam
(Pfizer) (Brogden et al., 19811or 50.0 mglkg of calcium phenylbutazone fBoeb-
ringer-Ingelheiml (Basile et al., 19881.Thirty minutes later the animals were
placed in the stress box and immersed in circulating water at 25OC to the
level of the xiphoid process for a period of 17 h under fluorescent lighting.
After this period, the animals were killed and the stomach removed and
opened along the greater curvature. The lesions were examined with a bino-
cular stereomicroscope (Nikon SMZ-101 at 10 x and the lesions were scored
as follows: ( -t- 11petechial, ( + 21 moderate and ( + 31extensive.

Lesions and in~ammation were induced according to the method of Rains-

ford and Whitehouse (19771 modified. Twenty-hour fasted rats with free
access to water were treated orally with 1% agar ~control~,extract or acetyl-
salieylic acid (Aspirin, Bayer) at doses yielding similar antiphlogistic effects.
Immediately the animals were transfered for 45 min to a cold room ( - lS°C)
individually in special cages designed to prevent cold injury to the paws.
After this time, 0.1 ml of 1% carrageenin (Marine Colloids, RE-9~0) in saline
was injected into the subplantar area of the left hind-paw of these unan-
aesthetized rats. An equal volume of saline was injected into the right kind-
paw. Ninety minutes later, the volume of each paw up to the tibio-tarsal
articulation was determined. The displacement of the mercury column was
measured with a Gould pressure transducer (Model P 23 ID1 and registered
on a Gould polygraph (Model RS 3.4001. After determination of the paw vol-
umes, each animal was killed and the stomach removed and opened along the
greater curvature. The lesions were examined with a binocular stereomicro-
scope (Nikon SM~-10) 10 x and quantified as to severity, number and index.
In order to avoid variations in the lesion and antipholo~stic response due to
circadian fluctuations in the levels of corticosteroids, the experiments were
started l&O0 for the mice (Tubaro et al., 19851and 1500 for the rats (unpub-
lished observations).

A one-way analysis of variance was performed. Sequential differences

among means Cp < 0.051 were calculated, using Tukey contrast analysis 1So-
kal and Rohlf, 19691.

Results

The oedema induced by nystatin suspension increased progressively for 12

h when the control group experienced an approximate 75% increase in pedal
volume (Fig. 11. The extract (1.24 mgntgl produced a significant inhibitory
effect when compared to the control group (1% agarf 8 h after nystatin and
Fig, 1. Effect d oral ~~~~~~tr&t~~~of Cw& +xf&mzeea Leaf extract an ays~tin-~nd~eed oed-
ema in rats- Agar 1% ~~n~o~~4.0 m&g 4----I, aids verbslaacen extract 1.24 m&kg 6 - -1 and
~ndurn~tka~~6.# mgjkg g. - e.8.Each paint represents the mean f S.E.M. oMained frum I rats.
(a): Significantly different from the control 1p < 0.05, Tukeyk Eb):si~~~~a~t~ydifferent from the
control and extract Ur < 0.05, Tukeyl

this effect persisted for more than 40 h; however, this effect was less dra-
matic than the one obtained from ~~dorn@tba~in15rn~~g~.

lose-respo~so curves for crotorr oil versus ~aproxo~ and dexameth~so~e

were run initially to establish the concentrations giving maximal responses.
A dose of 200 fig/ear of the irritant was chosen which gave a response very
close to the maximal inflammatory response seen with 300 pglear (Fig. 2).
Doses of 1.0 mglear of naproxen and 0.015 mglear of dexamethasone were
also chosen for this experiment.
Table 1 shows that the extract at, 0.09 mgjear produced a signifi~~t inhi-
bitory effect P < 0.05) when compared to the control with the effect inter-
mediate to that seen with naproxen and dexamethaso~e*

Restraint gastric ksions

Table 2 shows the severity of lesions in stressed animals submitted to oral
 243

50 100 200 300

CROTON OIL CONCENTRAltON (rrg /ear)
Fig. 2. Development of ear dermatitis in mice with different doses of crotoa oif. Each point rep
resents the mean f S.E.M. obtained from 5 animals.

antiphlogistic doses of non-steroidal antiinflammatory drugs and the extract.

The incidence of lesions was significantly lowered P < 0.05) in the group
treated with the crude extract for all levels of lesion severity. When com-
pared to the control group, the extract reduced the number of lesions on
average about 34%.

TABLE 1

EFFECT OF TOPICAL APPLICATION OF CORDIA VERBENACEA EXTRACT ON THE

OEDEMATOUS RESPONSE TO CROTON OIL

Each value represents the mean -t S.E.M. obtained from 6 mice.

Treatment Dose Ear differential Inkibition (If
4mglear1 weights lrngf
Control 17.8 i 0.9
Extract 0.09 a.5 * 1.1
Naproxen 1.0 12.2 & 1.5
Dexamethasone 0.015 4.2 f 0.8
Y3ignificantIydifferent from control f_P< 0.05: Tukey).
bSignifieantlydifferent from naproxen (P < 0.05; Tukeyi.
*S~~iB~an~y different from extract (P < 0.05; Tukey)..
TABLE 2
L
EFFECT OF ORAL ADMINISTRATION OF CORDIA KERBENACEA EXTRACT ON INCIDENCE OF GASTRIC LESIONS INDUCED BY
RESTRAINT AND WATER IMMERSION AT 25OCFOR 17 H

Each value represents the mean f S.E.M. obtained from 7 rats.

Treatment Doselirg Grade +l Grade +2 Grade +3
Lesion Change Lesion Change Lesion Change
number (96) number (%I number (%I
Agar 1% 4.0 ml 13.7 f 1.0 - 5.0 * 0.3 - 4.5 * 1.0
(control)
Extract 1.24 mg 9.7 + 0.4’ - 29.2 3.3 t 0.8’ - 34.0 2.8 f: 0.4= - 37.8
Piroxicam 6.0 mg 24.7 zk l.wb +803 11.2 f O.&b + 124.0 5.0 * 1.2b + 11.1
Phenyibutasone @Jmg 22.3 f 0.9” + 82.8 12.8 f 0.7”b + 188.0 5.5 f 0.8b + 22.2
~Significantlydifferent from control 0, < 0.05, Tukey).
bSigni&antly different from extract u3 < 5.55, Tukeyl.

TABLE 3

ULCEROGENIC AND ANTI~FLAMMATORY ACTIVITIES OF CCRDZA VRRBENACEA EXTRACT ORALLY

Each value represents the mean f S.E.M. obtained from 7 rats.

Treatment Do=& Paw volume Lesion severity Lesion number Index
(96)
+1 i-2 +3
Agar 1% (control) 4.0 ml 41.7 f 1.1 94 2 0 13.7 + 0.4 14.9
Extract 2.98 ml 12.4 f 0.4’ 88 2 0 12.6 f 0.5 13.7
Aspirin 70.0 mg 13.2 k 0.5” 119 51 5 26.0 + 0.9b 80.8
‘significantly different from control U, < 0.55, Tukey).
bSignificantlydifferent from extract (P < 5.08, Tukey).
 The lyophilized extract and aeetylsalicylic acid administered in doses of
2.98 and 70.0 mglkg, respectively, showed equipotent antiinflammatory
effects, with reductions of paw volume significantly different P < 0.051from
the control. Under these experimental conditions, the lesion index values
changed about 4 and 0.9 times, respectively, with acetylsa~cyli~ acid and the
extract (Table 31.

The results of the present study indicate significant antiinflammatory

activity for the 6. verbenacea leaf extract in two different models of experi-
mental inflammation. The extract, at antiin~ammatory doses, does not
appear to facilitate the formation of gastric lesions.
It has been reported by ~iemergeers et al. (19751that nystati~ alters the
lysosomal membrane causing a release of proteolytic enzymes. Its local
in~ammatory response has a long duration, reaching maximal effects 8 h
after injection of the phlogogenic agent. After 6 h (just before the treatment
with drugs), all the groups presented the same degree of oedema showing
that any further variation of the paw volume would have to be due to the
different treatments administered.
In our assay, the extract of C. verbemecs signifi~ntly inhibited nystatin-
induced inflammation at a dose of 1.24 mglkg, but 5.0 mglkg of indomethacin
was clearly more effective.
Among the tests available to detect the activity of topical antiinflamma-
tory drugs, the croton oil ear test in mice is one of the most commonly used.
In this test, the in~ammatory response is usually quantified by measuring
the change in ear plug weight at a single time interval after the croton oil
effect is maximal.
According to Swingle et al. (19811, croton oil acts as a vascular ~de~al~
irritant with secondary polymorphonuclear leukocyte infiltration and inter-
cellular oedema, Our experience with eroton oil is consistent with this view
(unpublished observations).
In the croton oil model, 1.0 mglear of naproxen strongly inhibited the
inflammatory response, but 0.09 mg/ear of the leaf extract was clearly more
effective.
The mechanism by which this extract produees its antiin~ammatory activ-
ity is not entirely clear, but the pharmacology of croton oil dermatitis indi-
cates that it is generally sensitive to eyclooxygenase inhibitors (Berkenkopf
et al, 19851. In our experiment C. verbenuceu showed greater activity than
naproxen, a classical cyclooxygenase inhibitor.
It is well known that all common antii~ammatory drugs now avail-
able have the potential for gastrointestinal erosion, The combination of
antiin~ammatory and anti-ulcer actions as seen here is, therefore, both
unusual and therape~ti~aily desirable. C. ~e~be~e~ appears to exert an
246

important protective effect on the gastric mucosa. In comparisons with

piroxicam, calcium phenylbutazone and aeetylsali~ylic acid, only the extract
si~~ieantly reduced the number of gastric lesions.
The present results confirm our recent work (Sertii! et al., 1988; Basile et
al., 19891 about the potent antiinflammatory activity of the leaf extract and
demonstrate its low gastrotoxicity. This paper gives experimental support
for the use of a decoction of C. verbenacea leaves in folk medicine.
Recently one flavonoid isolated from a leaf extract of this species showed
marked antiinflammatory activity using certain experimental models in rats
(Sertie et al., 1990) indicating that this flavonoid may be one of the sub-
stances responsible for this effect. Pharmacological investigations on the
potential antiinflammatory activity of this compound are now under way.

Acknowledgements

The authors are grateful to Covenant Universidad de Slo Paulo Grupo

Ache for financial assistance and to Dra. Luiziania Ferreira da Silva for teeh-
nical assistance.

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