848 Notes Biol. Pharm. Bull. 24(7) 848—851 (2001) Vol. 24, No.

Antidepressant Effects of Apocynum venetum Leaves in a Forced

Swimming Test
Veronika BUTTERWECK,*,a Sansei NISHIBE,b Tsutomu SASAKI,c and Masaru UCHIDAc
Institute of Pharmacology and Toxicology, WWU-Muenster,a Domagkstrasse 12, 48149 Muenster, Germany, Faculty of
Pharmaceutical Sciences, Health Sciences University of Hokkaido,b Ishikari-Tobetsu, Hokkaido 061–0293, Japan, and
Tokiwa Phytochemical Co., Ltd.,c 158 Kinoko Sakura, Chiba 285–0801, Japan.
Received February 2, 2001; accepted March 21, 2001

An extract of the leaves of Apocynum venetum L. (Apocynaceae) markedly shortened the immobility time of
male rats in a forced swimming test (FST) in a dose range of 30—125 mg/kg, indicating a possible antidepressant
activity. This effect was comparable to that of the tricyclic antidepressant imipramine (20 mg/kg). Neither
imipramine (20 mg/kg) nor the Apocynum extract in various doses (30, 60, 125 mg/kg) produced any overt behav-
ioural change or motor dysfunction in the open field test. This result confirms the assumption that the antide-
pressant effect of an Apocynum extract in the FST is specific. Further, it can be speculated that this effect might
be related to hyperoside and isoquercitrin which are major flavonoids in the extract.
Key words Apocynum venetum; leave extract; antidepressant effect; flavonoid

Apocynum venetum L. (Luobuma in Chinese, Apocy-
naceae) is a wild shrub widely distributed in mid and north-
western China. Since ancient times the leaves of A. venetum
have been used in traditional Chinese medicine for the treat-
ment of hypertension, nephritis and neurasthenia.1) Previous
papers describe the diuretic,2) antihyperlipemic3) and sedative
effects4) of the plant. The leaves of A. venetum are also used
in China as an anti-aging agent.1) This effect might be related
to the relatively high amount of flavonoids. A recent paper
demonstrated that hyperoside in Apocynum leaves had a
strong inhibitory effect in a lipid-peroxidation assay.5)
Flavonoids are polyphenolic compounds that are ubiqui-
tously present in foods of plant origin. Flavonoids are capa-
ble of modulating the activity of enzymes and affect the be-
haviour of many cell systems, suggesting that the compounds
may possess significant antihepatotoxic, antiallergic, anti-in-
flammatory, antiosteoporotic and even antitumour activi-
ties.6,7) Interestingly, the list of plant flavonoids did not in-
clude effects on the central nervous system (CNS) up to 1990
when Paladini et al. described the anxiolytic potency of
chrysin (5,7-dihydroxyflavone) and apigenin (5,7,49-trihy-
droxyflavone).8) Since then, a great deal of research has been
done in this field. Recently, it was shown that a flavonoid
fraction obtained from a crude extract of Hypericum perfora-
tum (St. John’s wort) was remarkably active in a forced
swimming test (FST).9) Isolates obtained from this frac-
tion including hyperoside, isoquercitrin, miquelianin and
quercitrin were also active in the FST. These data gave the
first evidence that some natural flavonoids possess antide-
pressant activity.
In the present study it was therefore of interest to investi-
gate the effects of an extract of A. venetum leaves (Apocynum
extract), containing an amount of 2.1% hyperoside and 2.7%
isoquercitrin, in the FST. The FST was chosen as this test is a
valid animal model to detect antidepressant compounds.10)
MATERIALS AND METHODS
Animals Male CD rats (230—250 g, Charles River
WIGA, Sulzfeld, Germany) were used. The animals were
∗ To whom correspondence should be addressed. e-mail: butterv@uni-muenster.de
kept in a 12 h light/dark cycle at a constant temperature of
2561 °C with free access to food (altromin 1324) and tap
water. Animals were divided randomly into control and ex-
perimental groups. Animal experiments were officially ap-
proved by the Regierungspräsident, Münster (A 38/93).
Preparation of Apocynum Extract Leaves of Apo-
cynum venetum (100 g) was refluxed for 1 h in aqueous
ethanol (70% v/v, 60 ml) twice and the combined alcoholic
extract was evaporated to dryness (28 g). The extract (13.5 g)
was dissolved in hot water (200 ml), and adjusted to pH 3.0
with sulfuric acid, then filtered. The filtrate was chro-
matographed on DIAION HP-20 (3.6 cm i.d.318 cm) and
eluted with water (200 ml) and then aquous ethanol (70% v/v,
200 ml). The aqueous ethanol fraction was collected and
evaporated to dryness to obtain Apocynum extract (4.2 g).
The extract contained 2.1% hyperoside and 2.7% iso-
quercitrin, respectively. HPLC analytical conditions were as
follows: Column: SHISEIDO CAPCELL PAKC18 (UG) 4.6
mm i.d.3150 mm, detector at 330 nm, mobile phase: 0.1%
TFA in water/0.1%TFA in acetonitrile 85 : 15, flow rate: 1.0
ml/min. Equipment: Waters 600 with a Waters 2487 UV de-
tector. Hyperoside appeared at 13.5 min and isoquercitrin at
14.7 min. Authentic hyperoside and isoquercitrin were pur-
chased from Funakoshi (Tokyo, Japan).
Drug Treatment Imipramine-HCL was purchased from
Sigma (Deisenhofen) and dissolved in deionized water. For
all experiments, powdered Apocynum extract (drug/extract
ratio: 25/2 prepared by Tokiwa Phytochemical Co., Ltd.,
Chiba, Japan) was used. Apocynum extract was dissolved ho-
mogeneously in water by sonication. Control animals re-
ceived deionized water only. All substances were adminis-
tered orally by gavage.
FST after Acute Pre-treatment The purpose of this ex-
periment was to investigate whether an Apocynum extract
possesses antidepressant activity in the FST. The adminis-
tered dose range of the extract was chosen in the same man-
ner as that of St. John’s wort extract9) since Apocynum extract
contains an almost equal amount of flavonoids to those of St.
John’s wort extract. Subsequently, a group of 40 animals was
subdivided as follows: 8 animals were treated with imipramine
© 2001 Pharmaceutical Society of Japan
July 2001 849

(30 mg/kg, p.o.), 24 with Apocynum extract (125, 250, 500 between 8—9 a.m.
mg/kg, p.o., 8 animals/group), and 8 animals received vehi- Statistics All data were expressed as the mean6S.E.M.
cle (deionized water). Test solutions were administered 24, 5 Group mean differences were ascertained with analysis of
and 1 h before the test. This procedure has been found to pro- variance (ANOVA). Multiple comparisons among treatment
duce more marked pharmacological effects than a single ap- means were checked with the Fisher-PLSD. The results were
plication 1h before the test.11) considered significant if the probability of error was ,5%.
FST after Repeated Treatment This experiment was
designed to validate the results of the acute experiment. The RESULTS
administered dose range of Apocynum extract was chosen in
the same manner as that of St. John’s wort extract9) since As shown in Fig. 1, immobility was significantly reduced
Apocynum extract contains an almost equal amount of after acute pre-treatment by imipramine (30 mg/kg) and
flavonoids to those of St. John’s wort extract. Consequently, a Apocynum extract (125 mg/kg). No effects after acute admin-
group of 40 animals was subdivided as follows: 8 animals istration were observed in the FST for the higher doses of the
were treated with imipramine (20 mg/kg, p.o.), 24 with Apo- extract (250, 500 mg/kg). However, 14 d after daily pre-treat-
cynum extract (30, 60, 125 mg/kg, p.o., 8 animals/group), and ment, the extract markedly reduced immobility at a dosage of
8 animals received vehicle (deionized water). Test solutions 30 and 125 mg/kg (Fig. 2). This effect was comparable to that
were administered once daily over a period of 14 d. Adminis- of the tricyclic antidepressant imipramine (20 mg/kg), al-
tration was performed between 3—4 p.m. every day. though the effect of the synthetic substance was more pro-
A dosage of 30 mg/kg imipramine was chosen for the nounced. The dosage of 60 mg/kg of the extract slightly re-
acute study because former experiments indicated that duced immobility, but the effect was not significant. Neither
dosages below 30 mg were only partially active; for chronic imipramine (20 mg/kg) nor the Apocynum extract in various
treatment 20 mg/kg proved to be an active dosage without doses (30, 60, 125 mg/kg) produced any overt behavioural
any toxic effects, whereas higher doses of imipramine change or motor dysfunction in the open field test (Table 1).
showed toxic effects in the chronic treatment (Butterweck, In fact, none of the drugs increased open field activity.
unpublished results).
Measurement of Immobility This experiment was per-
formed using the FST according to the method published by
Porsolt et al.12) Male rats were placed in a plexiglass cylinder
(40318 cm i.d.) containing a column of 17 cm of water at
2561 °C. According to Porsolt the rats learned in a pretest
session of 15 min that they could not escape from the cylin-
der. In the test period, 24 h later, the animals were exposed to
the experimental conditions for 5 min. A rat was judged to be
immobile whenever it remained floating in the water, in an
upright position, making only small movements to keep its
head above the water.
The FST for both treatment paradigms was performed be-
tween 1—3 p.m. and recorded by a video camera. The tapes
were evaluated by an observer who was not informed about
the kind of treatment each animal had received.
Fig. 1. Activity of Apocynum Extract in the FST after Acute Treatment
Measurement of Locomotor Activity In order to see
Test solutions were administered orally 24, 5 and 1 h before the test. n58; ∗ p,0.05
whether changes in immobility were associated with changes vs. control.
in motor activity as described by Porsolt et al.11) for ampheta-
mine and caffeine, animals treated with the various sub-
stances were tested for activity in an open field. For measure-
ment of locomotor activity naive rats were placed in an open
field apparatus. The open field apparatus was an arena 70 cm
in diameter divided into 18 approximately equal areas. Hand-
operated counters were employed to score locomotion (num-
ber of line crossings within 5 min) and rearing frequencies
(number of times an animal stood on its hind legs). In this
experiment the animals received the same drugs and doses as
used for the measurement of immobility after repeated treat-
ment. For open field observations, each rat was individually
placed in the center of the arena 15 h after the last treatment
and its behavioural parameters were recorded for 5 min. The
open field apparatus was washed with a detergent solution
before each behavioural test to eliminate possible odor clues
left by previous subjects. To minimize circadian changes in Fig. 2. Activity of Apocynum Extract in the FST after Repeated Treatment
the rat open-field behaviour, control and experimental ani- Test solutions were administered orally once daily over a period of 14 d. n58;
mals were intermixed. Open field observations were made ∗ p,0.05 vs. control.
850
Table 1. Locomotor Activity in the Open Field Test
Treatment Locomotion Rearing
Control 78615 1162
Imipramine 20 mg/kg 7469 1462
Apocynum ext. 30 mg/kg 71611 1062
Apocynum ext. 60 mg/kg 67614 863
Apocynum ext. 125 mg/kg 8267 1262
The behavioural parameters were recorded for 5 min. Locomotion; number of line
crossings within 5 min. Rearing; number of times an animal stood on its hind legs.
Values are expressed as mean6S.E.
DISCUSSION
Our present experiments show for the first time that an ex-
tract of the leaves of A. venetum L. possesses antidepressant
activity in the FST, a valid animal model for screening anti-
depressant drugs.10) A major problem in the search for new
antidepressant drugs is the question which animal model is
most appropriate to identify diverse antidepressant treat-
ments correctly, without making errors of omission or com-
mission and whether antidepressant effects are only present
on, or still exist after chronic administration.10) One model
which attempts to meet those requirements is the FST devel-
oped by Porsolt et al.12) The authors described procedures for
producing a state resembling depression in rats. The test is
based on the observation that rats when forced to swim in a
restricted space from which they cannot escape will cease ap-
parent attempts to escape and become immobile exhibiting
only small movements necessary to keep their heads above
the water.11) The authors suggested that this characteristic im-
mobile posture reflects a state of despair in the rat; it is as-
sumed that the animals have “given up hope of escaping.”
Immobility is reduced by a variety of agents which are thera-
peutically active in depression11) including tricyclics, mono-
amine-oxidase-inhibitors (MAO’s) and newer antidepressants
but not clearly 5-HT uptake inhibitors.13) There is, in fact, a
significant correlation between clinical potency and potency
of antidepressants in the FST14); this was not found in any
other model. The specificity of this test for antidepressants
has been questioned as a large number of non-antidepres-
sants also reduce immobility. False positive results have been
reported for stimulants, anticholinergics, antihistaminics,
pentobarbital and opiates.14—18) It has been found that
whereas response to antidepressants continues by chronic
treatment, the response to antihistaminics disappeared on
chronic administration.19) The effects of psychostimulants
such as caffeine or amphetamine can be excluded by addi-
tional open field tests which would markedly increase loco-
motor activity.11)
However, in our present study an Apocynum extract signif-
icantly reduced immobility time in the FST after acute ad-
ministration at a dosage of 125 mg/kg, an effect which was
comparable to that of the synthetic antidepressant imipramine
at a dosage of 30 mg/kg. Higher doses of the Apocynum ex-
tract (250, 500 mg/kg) remained inactive after acute pretreat-
ment. To exclude false positive results which might be due to
effects of antihistaminics, an additional FST was performed
after 14 d of daily treatment. After repeated treatment, Apo-
cynum extract markedly shortened the immobility time in the
FST at a dose range of 30—125 mg/kg, although the effect
Vol. 24, No. 7
may not be in a dose-dependent fashion, similar to that of St.
John’s wort extract.9) These could be preliminary data which
Defecation
gave the first hints of possible antidepressant activity of
3.060.8 Apocynum extract and further dose range tests might be
2.060.5 needed. The effect of the extract in the FST after a pre-treat-
1.360.7 ment period of 14 d was comparable to that of the tricyclic
1.760.6 antidepressant imipramine (20 mg/kg), although the effect of
1.060.4
the synthetic substance was more pronounced after repeated
treatment. Neither imipramine (20 mg/kg) nor the Apocynum
extract at various doses (30, 60, 125 mg/kg) produced any
overt behavioural change or motor dysfunction in the open
field test, so that the observed effects in the FST were not
due to a stimulation of locomotor activity. The results of the
FST after repeated treatment and after additional open field
tests confirms the assumption that the antidepressant effect of
an Apocynum extract in the FST is specific. Further, it can be
speculated that this effect might be related to hyperoside and
isoquercitrin, as both flavonoids recently showed antidepres-
sant actvity in the FST.9) A further reason for this assumption
is that the effective extract doses used in the present study
correlate well with the effective hyperoside and isoquercitrin
doses applied in the previous study by Butterweck et al.9) In
detail, Butterweck et al. showed that pure hyperoside signifi-
cantly shortened immobility time in the FST in a dose range
of 0.6—1.3 mg/kg, whereas the effects of doses below 0.6
mg/kg and above 1.3 mg/kg were not significantly different
from control in St. John’s wort extract.9) Pure isoquercitrin
was also active in the FST at a dosage of 0.6 mg/kg.9) This
amount of hyperoside as well as of isoquercitrin is approxi-
mately reached with 30 mg/kg of the Apocynum extract,
which was active in the FST after repeated treatment. The
fact that 125 mg/kg of the Apocynum extract was also active
in the FST after acute and repeated treatment leads to the as-
sumption that this extract probably contains other substances
with possible antidepressant potential. Further investigations
in this direction are necessary to confirm this hypothesis.
Acknowledgments These study results were followed by
application for a Japanese Patent (Application No. 2000-
334122). We thank Dr. S. Seo of Tokiwa Phytochemical Co.,
Ltd., for supporting our Patent application.
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