Journal of Ethnopharmacology,

Elsevier Scientific Publishers

26 (1989) 197 - 203

Ireland Ltd.

197

Short Communication

PHARMACOLOGICAL
SCREENING AND ANTIMICROBIAL
ACTIVITY
OF THE ESSENTIAL
OIL OF ARTEMISIA
CAERULESCENS
SUBSP. GALLICA

A. MORAN.

M.J. MONTERO,

M.L. MARTIN

and L. SAN ROMAN

Laboratory
of Phamacognosy
and Phamnacodynamics,
Department
of Physiology
and
Pharmacology,
Faculty of Pharmacy,
University
of Salamanca, 37007 Salamanca (Spain)
(Accepted

December

6, 1988)

Introduction
Artemisia caerulescens subsp. gallica (Willd.) K. Persson is a plant of the
Compositae
(Asteraceael
family in which certain species with important
pharmacological
activities are found. Pharmacological
properties have been
ascribed to as many as 100 species and, in particular, to the genus Artemisti
e.g. stimulant, tonic, antispasmodic, anthelminthic activity, etc. (Boldina and
Nazarenko, 1967; Gala1 et al., 1968; Jamwal et al., 1972; Ross et al., 19801.
Regarding
A. caerulescens
subsp. gall&,
according to a review of the
literature carried out prior to this study, few chemical data were found
relating to its essential oil (Villar et al., 19831 and to that found in related
species (Paris and Moyse, 19711 and no relevant information about possible
pharmacological properties.
This plant species was selected as the focus of a Research and Study
Project
of Castilian-Leonese
medicinal flora carried out jointly by our
laboratory and the Department of Organic-Pharmaceutical
Chemistry.
The object
of the present
research
was to carry out a general
pharmacological
study of the different
fractions
of this plant species,
concentrating
on the essential oil upon which its antimicrobial
action
depends.

Materials and methods

Plant material
Artemisia

Correspondence

subsp. gallica was collected in Aldeamayor de San

Spain) during flowering. Its identification was confirmed

caerulescens

Martin (Valladolid.

to: M.L. Martin.

0 1989 Elsevier
0378.8741/89/.$02.80
Published and Printed in Ireland

Scientific Publishers Ireland Ltd.

198

by the Dept. of Botany of the Faculty of Pharmacy. A voucher specimen is

deposited in the Herbarium of that Faculty with the number SALAF 4854.
The aerial parts of the plant were dried at room temperature and stored in
hermetically sealed containers, protected from light and humidity.
A portion of the plant material was successively
fractionated into four
extracts
according
to the procedures
of Farnsworth
et al. (19661 and
following the exact process described by Martin et al. (19871. These Soxhletderived extracts were hexane, chloroform, butanol and water, which were
refrigerated at 4OC until subsequently assayed in rats.
Another portion of the plant material was distilled in a Clevenger apparatus for 4 h. The essential oil obtained was dried with anhydrous sodium
sulphate and preserved at 4OC.
Pharmacological

screening

The hippocratic screening method of Malone and Robichaud (19621, was

chosen as the preliminary screening protocol. This was performed with doses
below 1000 mglkg for the extracts and not above 1 ml/kg for the essential oil
in albino Wistar rats (weight range 150-200
gl from the Animalarium of the
Faculty of Pharmacy of Salamanca University. The experimental conditions
were those described
by Martin et al. (19871. The suspension vehicles
employed were 0.25% agar for the aqueous, butanol and essential oil fractions, 0.25% agar plus three drops of Tween80 for the chloroform extract,
and peanut oil for the hexane extract. Documentation of observed symptoms
was done according to the indications of Malone and Robichaud (19621 and
Malone (1973, 19771 using a numerical scale with scores ranging between 0
and 4.
The LD,, of the extracts was determined in male Swiss mice supplied by
the Animalarium of the Faculty of Pharmacy and quantitated using the
double-integration
method of Reed and Muench (19381 as modified by Pizzi
(19501 following the experimental conditions described by Martin et al. (19871.
Antimicrobial
In vitro
conducted
Carron et
wells. The
de Cultivos

activity

studies on the antimicrobial activity of the essential oil were

according to the agar plate diffusion method as proposed by
al. (1987). The essential oil was placed in 4- and 6-mm diameter
microorganism cultures were supplied by the Coleccion Espanola
Tipo Nalencial.

Results
Hexane extract (3.4% yield). The i.p. LD,, in mice was above 3000 mglkg.
Intraperitoneal
doses of 707, 840 and 1000 mg/kg suspended in peanut oil
were screened in rats. Lower doses of this extract were inactive. The most

199

significant dose-related symptoms observed were a decrease in motor activity and tone, respiratory depression, hypothermia and hyperglycemia.
Chloroform extract (2.1%1 yield). The i.p. LD, in mice was above 3000
mg/kg. Intraperitoneal doses of 316, 749 and 1000 mglkg in rats showed a
decrease in motor activity and tone, respiratory depression, hyperemia,
hypothermia, catalepsy and hyperglycemia; maximum effects were observed
between 30 min and 3 h postinjection.
Butanol extract (8.3% yield). The i.p. LD, of this extract in mice was 982
mg/kg (95% CL = 897 - 10741.Intraperitoneal doses of 316, 562 and 749 mgl
kg of this extract in rats produced a positive Robichaud test and catalepsy,
together with a certain degree of CNS depression and a decrease in motor
activity and tone. Hypothermia and hyperemia were also seen.
Aqueous extract (9.6% yield). The i.p. LD, in mice was above 3000 mglkg.
Dose-related symptoms with doses of 464, 681 and 1000 mg/kg i.p. were
observed in rats with the most prominent being hyperemia of ears and paws,
together with a decrease in motor activity and tone. Hypothermia and catalepsy were also seen.
Essential oil (1% v/w). The i.p. LD, in mice was 1.35 ml/kg (95% CL =
1.14-1.591. The screening doses administered to rats were 0.1, 0.3, 0.6 and 1
ml/kg i.p. and the dose-related symptoms observed corresponded to a
decrease in motor activity and tone, ataxia, loss of reflexes (normal, corneal,
pineal and ipsalateral), analgesia, palpebral ptosis, a decrease in respiratory
rate and an increase in deep respiration, hypothermia, catalepsy and hyperglycemia. Body weight increased with the lowest doses assayed (0.1 and 0.3
ml/kg) but decreased with doses of 0.6 and 1 ml/kg.
Tables 1 and 2 summarize the results of the pharmacological screening.
The inhibition halos obtained against the different microorganisms
assayed with the essential oil are shown in Table 3 and confirm both
antibacterial and antifungal activity. These results are similar to those
obtained with extracts of this plant (Carron et al., 19871.
Discussion

According to the results obtained in the hippocratic screening, the most

active fraction of A. caerulescens
subsp. gallica seems to be the essential oil.
Its components could be at least partly responsible for the activity observed
with the less polar extracts (hexane and chloroform). All the dose-related
symptoms observed after the administration of these two extracts also
appear in rats given the essential oil, with the exception of the hyperemia
produced by the chloroform extract. This hyperemia also appears with the
aqueous and butanol fractions, apparently indicating the existence in this
species of some more polar compounds with a possible vasodilatory action.
In general, all the actions are dose-dependent with the exception of the
changes observed in body weight with the essential oil. In this case, body
weight increased with low doses but decreased with high ones. In view of

SCREENING

OF A. CAERULESCENS

subsp. GALLICA

EXTRACTS IN CONSCIOUS RATS

Motor activity
Reflexes
Respiratory rate
Palpebrai ptosis
Hyperemia
Pilomotor erection
Urine production (ml)
Rectal temperature (OC)
Weight change fg)
Body tone
Body touch passive
Catalepsy
Bfood glucose lo!)

Parameters

N
N
-1
0
0
0
10
- 2.7
-6
0
0
0
+47

-1
N
-2
0
0
0
9
- 2.9
- 13
-1
+1
0
i42

-2
N
-2
0
0
0
7
- 3.3
-18
-1
+l
0
+ 73

-1
N
-1
0
0
0
12
- 2.0
i-8
-I
0
0
N

-2
N
-2
0
+1
0
10
- 3.2
-4
-2
+1
+2
+22

749

316

lQ60

707

840

Chloroform (mglkg)

observed

Hexane (mg/hgI

Mean maximum response

-3
N
-2
0
+2
0
8
- 3.5
- 15
-3
+2
+2
-I-100

1000
-1
N
-1
+l
0
0
18
-1.7
-5
0
0
0
N

464
-2
N
-1
$1
+1
0
9
-2.1
il
-2
0
+2
N

681

Aqueous (mg/hg)

-3
-1
-3
+2
+2
0
15
- 3.5
-1
-2
+l
+3
N

1000

-1
N
N
0
0
+1
15
- 1.4
-10
-1
+1
0
N

316

-2
-1
-2
il
il
+1
21
- 1.8
-4
-2
+2
+1
N

562

Butanol (mg/kg)

-3
-2
-1
+2
+2
+2
17
- 2.4
+1
-3
+3
+2
N

749

With the exception of rectal temperature, body weight urine production and blood glucose, symptoms were evaluated on a O-4 scale. Minus values indicate a decrease of function, while positive values indicate the presence of increased function of the 63 parameters evaluated. Measured
parameters with little to no change in function are not listed here. N = normal control activity: 0 = absence of effect.

SUMMARY OF PHARMACOLOGICAL

TABLE 1

201
TABLE

SUMMARY
OF
CAERULESCENS

PHARMACOLOGICAL
subsp. GALLICA

SCREENING

OF

With the exception of rectal temperature, body weight,

symptoms were evaluated on a O-4 scale. Minus values
positive values indicate the presence of increased function
ured parameters with little to no change in function are
activity; 0 = absence of effect.

Motor activity
Ataxia
Loss righting reflex
Analgesia
Reflexes
Respiratory rate
Palpebral ptosis
Urine production (ml)
Robichaud test
Rectal temperature (C)
Weight change (g)
Body tone
Body touch passive
Catalepsy
Blood glucose (%)

5
10
15
25
50

OIL

OF A.

urine production and blood glucose,

indicate a decrease of function, while
of the 63 parameters evaluated. Measnot listed here. N = normal control

0.1

0.3

0.6

1.0 ml/kg

-1

-2

0
N
0
N
N
0
24
0
- 3.7
+ 12
0
0
0
+51

+/+1
+2
-1
-1
+1
10
0
- 4.5

-3
+2
+3
+3
-2
-2
+2
30
+I
- 7.5
-6
-3
-3
+2
+ 98

-4
+3
+4
+4
-3
-3
+3
46
+1
- 7.8
-17
-4
-4
+3
+ 158

+ 11
-2
-2
+1
+ 76

INHIBITION
ARTEMISIA
Essential
oil per
well ($1

ESSENTIAL

Mean maximum response observed

Parameters

TABLE

THE

OF GROWTH
CAERULESCENS

OF MICROORGANISMS
subsp. GALLICA

BY

THE

ESSENTIAL

OIL

OF

Mean + S.E.M. !JV = 3) diameter of zone inhibition (cm)

Bacillus
subtilis
1.00
1.20
1.50
2.00
2.90

f
f
+
f
f

0.05
0.11
0.11
0.07
0.07

Escherichia
coli

Candida
albicans

0.80
1.00
1.20
2.60

0.90
1.20
1.40
1.80
3.80

f
f
f
+

0.06
0.05
0.11
0.08

f
rt
-c
-c
+

Saccharom
cerevisiae
0.03
0.12
0.00
0.05
0.07

1.00
1.10
1.30
1.40
1.50

k
f
f
f
f

yces

0.00
0.08
0.00
0.05
0.05

Aspergillus
niger
1.20
1.90
2.20
2.50
3.30

f
f
f
2
f

0.10
0.00
0.05
0.06
0.15

202

the considerable
degree
of CNS depression
exerted
by the essential
oil in
rats, where at a dose of 1 ml/kg the animals
remain immobile
for long periods of time, the weight loss is not surprising.
It is probably
due to a low food
intake, although certain metabolic alterations may have passed unnoticed in
this test. An increased production of urine may also contribute to the weight
loss.
A marked CNS depressive action can thus be inferred from the symptoms
of neurolepsy, ataxia, analgesia and even a certain degree of anaesthesia. In
the light of the components that other authors have found in the essential oil
of A. caerulescens
(Villar et al., 19831 such as a-thujone
and camphor,
together with other terpene compounds, such activities can be justified.
Camphor and a-thujone, which are convulsants
at high doses, have a
sedative action at low doses (Duke, 1986; Litter, 19861. Certain authors
propose
the use of plant species with essential oils rich in terpene
compounds for their sedative properties (Paris and Moyse, 1971; Vigneau,
19851.
Thus we can propose for this plant species a sedative action with a
possible depressive action on respiration, as well as antipyretic and analgesic
activities. It also exhibits both antibacterial and antifungal capacities.
At the present moment we are carrying out a series of studies aimed at
quantifying
the
analgesic
activity
and establishing
the
compounds
responsible
for this action. Since the species seems to exert a dual
whether an antiantipyretic-analgesic
action, we shall also investigate
inflammatory capacity is present.
Acknowledgements
The authors thank Dr. M. Ladero for authenticating the plant species used
in the present work and thank the Autonomous Government of Castilla-Leon
for funding.

Boldina, LG. and Nazarenko, M.U. (1967) Pharmacological properties

of chamazulene from

Artemisia
sieversiana.
Rastitelnye
Resursy 3 (11, 67-71.
Carron, R., Moran, A., Montero, M.J., Fernandez-Lago,
L. and Dominguez,
A. (1987) Propietcs
antimicrobiennes
des differents
extraits
obtenus
de quelques
plantes
mediterraneennes
dint&et
medicinal. Plantes Medicinales
et Phytotherapie
21 (4), 195-202.
Duke, J.A. (19861 Handbook of Medicinal Herbs, CRC Press Inc., Boca Raton, Florida, pp. 69-70.
Farnsworth,
N.R., Henry, L.K., Svoboda, G.H., Blomster, R.N., Yates, M.J. and Euler, K.L. (19661
Biological and phytochemical
evaluation
of plants. I: Biological test procedures
and results
from 200 accesions. Lloydiu 29, lOl- 122.
Galal, E.E., Madkour, M.K. and Nasr, A. (19681 Pharmacological
and toxicological
studies of
judaicin, a crystalline
bitter principle isolated from Artemisiu
judaica
Drug Research
1111,
129- 162.
Jamwal, KS., Sharma, M.L., Chandhoke,
N. and Ghatak, B.J. (19721 Pharmacological
fraction of
6,7-dimethoxy
coumarin (scoparone) isolated from Artemisia
scopariu Indian Journal Medical
Research
60(5), 763-771.

203

Litter, M. (1986) Farmacologia experimental y clink, Ed. Ateneo, Buenos Aires, pp. 421-424.
Malone, M.H. and Robichaud, R.C. (19621 A hippocratic screen for pure or crude drug materials.
Lloydia 25,320-332.
Malone, M.H. (1973)Observational (hippocraticl screening. In: M.H. Malone and J.L. McLaughlin
(Eds.), Experiments
in the Pharmaceutical
Biological Sciences,
Biological Sciences Section of
the Conference of Teachers of the American Association of Colleges of Pharmacy, Bethesda
MD, pp. 120- 123.
Malone, M.H. (1977) Pharmacological approaches to natural product screening and evaluation. In:
H. Wagner and P. Wolff (Eds.), New Natural Products and Plant Drugs with Pharmacological, Biological or Therapeutical
Activity,
Springer-Verlag, Berlin, pp, 23-53.
Martin, M.L., Moran, A. and San Roman, L. (1987) Pharmacological screening of Pulsatilla alpina
subsp. apiifolia Journal of Ethnopharmacology
21,201206.
Paris, R.R. and Moyse, H. (1967) Traits de Matiere Medicale, Vol. 3, Ed. Masson, Paris, pp. 411420.
Pizzi. M. (19501 Sampling variation of the fifty percent end-point determined
by the ReedMuench (Behrensl method. Human Biology 22, 151- 180.
Reed, L.J. and Muench, H. (19381 A simple method of estimating fifty percent end-points. American Journal

of Hygiene

27, 493 - 497.

Ross, S.A., Megalla, S.E., Bishay, D.W. and Awad, A.M. (1980) Studies for determining antibiotic
sustances in some Egyptian plants. Part I: Screening for antimicrobial activity. Fitoterapia
6(51), 303 - 308.

Vigneau, C. (19851Pluntes Medicinules, Ed. Masson, Paris, pp. 249-256.

Villar, A., Calduch, M.L. and Zafra-Polo, M.C. (1983) Aceites esenciales
Artemisias. Archive Pharmaceutics
2, 149- 159.

de diversas

especies

de