PHYTOTHERAPY RESEARCH

Phytother. Res. 14, 107–111 (2000)

Effect of Psidium guajava Leaves on Some

Aspects of the Central Nervous System in Mice

Huda M. Shaheen,1 Badreldin H. Ali,2* Ali A. Alqarawi2 and Ahmed K. Bashir1

1
Biology Department, Faculty of Science; U. A. E. University, Al-Ain, United Arab Emirates
2
Department of Veterinary Science, College of Agriculture and Veterinary Science, Buraydah, Al-Gaseem, Saudi Arabia

The present work examines the effects of hexane, ethyl acetate and methanol extracts of Psidium guajava
leaves (20,100,500 and 1250 mg/kg) on the central nervous system in mice. The three extracts exhibited
mostly dose-dependent antinociceptive effects in chemical and thermal tests of analgesia. The extracts
also produced dose-dependent prolongation of pentobarbitone-induced sleeping time. However, they had
variable and mostly non-significant effects on locomotor coordination, locomotor activity or exploration.
In the pharmacological tests used, the ethyl acetate extract seemed to be the most active, followed by the
hexane and then the methanol extracts. Copyright # 2000 John Wiley & Sons, Ltd.
Keywords: Psidium guajava; motor activity; analgesia; exploration; sedation.

INTRODUCTION METHODOLOGY

The common guava tree, Psidium guajava Linn. (Family
Myrtaceae), is grown in tropical and subtropical parts of
the world. The fleshy pulp of the fruits is eaten and is used
in manufacturing juices and jams. In many parts of the
world, and for many centuries, various parts of the tree
have been used in the indigenous medical practices of the
people (Lutterodt and Abdul-Maleque, 1988). The
pharmacological actions and the medicinal uses of the
leaves include the treatment of various types of pain,
vomiting, diarrhoea, inhibition of the peristaltic reflex,
gastroenteritis, spasmolytic activity, convulsions, epi-
lepsy, and in some countries the leaf extract is used to
treat insomnia (Middleton et al., 1981; Lutterodt and
Abdul-Maleque, 1988; Lutterodt, 1988; Lutterodt, 1992;
Morales et al., 1994; Meckes and Calzada, 1996). In the
folk medicinal practices of the Arabian Peninsula a
sweetened P. guajava decoction of the leaves is usually
taken to alleviate sore throat, common colds and
influenza.
More than 20 identified compounds from the P. gujava
leaf have been reported (Seshadri and Vasishta, 1965;
Osman et al., 1974; Lutterodt, 1988). The major
constituents of the leaf were identified to be tannins, b-
sitosterol, maslinic acid, essential oils (mainly caryo-
phyllene, b-bisabolene, aromadendrene, b-selinene, ner-
olidiol, caryophyllene oxide and sel-11-en-4-ol) and
triterpenoids, including oleanolic, ursolic, crategolic
and guaijavolic acids (Osman et al., 1974).
The present work was carried out to investigate the
effect of various extracts from the leaves of P. guajava
grown in Al-Ain region, UAE on central nervous system
activity of mice.
* Correspondence to: Dr B. H. Ali, Department of Veterinary Science,
College of Agriculture and Veterinary Science, Buraydah, Al-Gaseem, Saudi
Arabia.
Animals. Male albino mice (OT strain) weighing about
25 g were obtained from the Animal Facility of the UAE
University. They were housed six to a cage under
standard laboratory conditions of temperature (22 ° 
2 °C), humidity (50%–60%), and artificial light from
6.00 am to 6.00 pm. Pelleted food (Abu-Dhabi Animal
Feed Factory) and water were available freely to all
mice. Each animal was used only once.
Plant materials. P. guajava leaves were collected in
October 1996 from trees grown in Al-Ain region, UAE.
The plant material was botanically authenticated and
herbarium specimens deposited at the National Herbar-
ium, Desert and Marine Environment Research Centre,
UAE University.
Plant extracts. The leaves were dried in the shade and
coarsely powdered. The powder (1 kg) was successively
extracted with hexane, ethyl acetate and methanol using a
soxhlet extraction apparatus to give a hexane extract
(45.0 g), an ethyl acetate extract (12.6 g) and a methanol
extract (58.8 g).
The hexane and ethyl acetate extracts were taken in
polyethylene glycol and the methanol extract was taken
in distilled water. The extracts were prepared freshly just
before use. For each plant extract four doses (20, 100, 500
and 1250 mg/kg) were given orally by gavage. Control
animals were treated with the appropriate vehicles (poly-
ethylene glycol or distilled water) at a oral doses of
10 mL/kg.
Analgesic effect. Two types of analgesic tests were used.
For the abdominal constriction test, acetic acid (0.6%)
v/v was injected intraperitoneally (i.p.) in a volume of
12 mL/kg to control animals or animals treated 45 min
earlier with different doses of P. guajava extracts.

Received 1 March 1999

Copyright # 2000 John Wiley & Sons, Ltd. Accepted 22 September 1999
108 H. M. SHAHEEN ET AL.
Abdominal constrictions were then counted for 15 min
after the acetic acid administration (Koster et al., 1959).
For the hot plate test, A commercially available hot-
plate apparatus (Letica S.A., Model-DS 37, Spain) was
used. The heated surface was maintained at 52.0 ° 
0.2 °C. The animals were gently placed on the plate,
and the time required for paw licking or escape attempt
was taken as the response (Eddy and Leim Bach, 1953).
To avoid tissue damage, the cut-off time for the latency
of response in the animals was taken as 30 s. The test
was performed before oral administration of the extracts
(0 min) and at 30 and 60 min thereafter.
Sleeping time. This was carried out using sodium
pentobarbitone (40 mg/kg i.p.). The sleeping time was
calculated as the interval elapsing between the loss and
recovery of the righting reflex (Fujimori, 1965). The
barbiturate was given to rats pretreated 45 min earlier
with various oral doses of the plant extract.
Locomotor coordination. Impairment of motor control
was measured in control and treated mice as the time they
stayed on a rotarod treadmill. The method used was
adopted from that of Kuribara et al. (1977) which
employed a rota-rod treadmill for mice (7600 Ugo Basile,
Italy). A plastic rod (diameter 30 mm, length 30 cm),
with a non-slippery surface, was used 15 cm over the base
of the cage. The rod was divided into five equal sections
by six discs, thus enabling five animals to walk on the rod
at the same time. The rod was rotated at a speed of
25 rev/min. The interval between the animal mounting
the rod and falling off was recorded by means of a built-in
timer; and this was considered as the performance time.
Exploration. This was studied using the hole-board test.
In the hole-board the behaviour of the mouse confronted
with a new environment is studied (Boissier and Simon,
1964; Boissier et al., 1964). The test enables the initial
exploratory activity of the animal and its variations to be
assessed. The dimensions of the hole-board used (UGO
Basile, Italy) were 40  2.2 cm, and it had 16 flush
mounted tubes (holes) 3 cm deep. Each tube had an
emitter and a diametrically opposed receiver. The mouse
was placed at the centre of the board. At every head
plunging (nose-poking) the activity was registered
digitally. The time taken in each nose poke in the hole
was recorded using a chronometer (triple timer).
Measurement of motor activity. Locomotor (ambula-
tory) and behavioural (total) activity (Kulkarni and
Sharma, 1994) was measured using a computerized
animal activity meter (Opto Varimex Columbus Instru-
ments, Ohio, USA). Total activity included the ambula-
tory (actual relocation) activity score, and the activity
confined to a small space, that is, repeated crossing of a
single photocell. An array of 15 infrared emitter/detector
pairs, spaced at 2–5 cm intervals, measured the animal
activity along a single axis of motion. The counts of
photo interruptions were displayed on the front panel
meters as ambulatory activity and total activity. Im-
mediately after acute treatment, each rat was placed in
the transparent plastic cage (43 cm  43 cm  22 cm) of
the activity meter. After a 5 min habituation period in the
cage, the activity meter was zeroed and counts were then
taken after 10, 20 and 30 min. The measurements were
always taken between 08:00 and 12:00 h.
Copyright # 2000 John Wiley & Sons, Ltd.
Drugs and chemicals. The drugs and chemicals used
were: acetic acid (BDH, Poole, Dorset, England), ethyl
acetate (Univar Ajax Chemicals, Australia), hexane
(Riedel Hehaen, Germany), methanol (Univar Ajax
Chemicals, Australia), pentobarbitone (Sigma, St Louis,
MO, USA) and polyethylene glycol (Merck-Schuchardt,
Hohenbrunn, Germany).
Statistical analysis. Values reported are mean  SEM
(number of mice). Differences between the means of
different groups were analysed by a one-way analysis of
variance (ANOVA) followed by a multiple comparison
(Dunnett’s) test. p < 0.05 was considered significant.
RESULTS
Analgesia
Abdominal constriction test. There was a dose-
dependent decrease in the number of abdominal con-
strictions in mice treated with the three extracts, which
was significant at all doses except at 20 mg/kg of the
hexane and methanol extracts (Fig 1). On a comparative
basis, the analgesic effect was strongest with the ethyl
acetate extract, followed by hexane then methanol
extracts.
Hot plate. The results of this experiment are shown in
Fig 2. Treatment of mice with the hexane and ethyl
acetate extracts (500 and 1250 mg/kg), and methanol
extract (1250 mg/kg) significantly increased the time
spent on the hot plate (p < 0.005). Unlike the methanol
extract, lower doses of hexane (20 and 100 mg/kg) and
ethyl acetate (100 mg/kg) were also effective in increas-
ing the time spent on the hot plate. Thirty and sixty min
after the hexane extract was given at a dose of
1250 mg/kg, the percentage increase in the time spent
on the hot plate was 29.2% and 72.3%, respectively. The
ethyl acetate and the methanol extracts given at the same
dose increased the time spent on the hot plate by 51.9%
and 83.0% (ethyl acetate) and 13.4% and 15.7%
(methanol), 30 and 60 min after the treatment, respec-
tively.
Sleeping time
There was a dose-dependent increase in the sleeping time
of mice treated with the three extracts, which was
significant at doses of 500 and 1250 mg/kg of the ethyl
acetate and hexane extracts. On a dose to dose basis, the
ethyl acetate and hexane extracts were more effective
than the methanol extract (Fig. 3).
Locomotor coordination
All doses up to 1250 mg/kg of the methanol, hexane and
ethyl acetate extracts did not any elicit significant effect
on the time spent on the rotarod treadmill, except for a
small but statistically significant reduction (22%)
observed when a dose of 1250 mg/kg of the ethyl acetate
extract was given.
Phytother. Res. 14, 107–111 (2000)
 PSIDIUM GUAJAVA ON CNS
Figure 1. The effect of hexane, ethyl acetate and methanol
extracts of Psidium guagava leaves on abdominal constric-
tion test of mice. Each column and vertical bar represent
mean  SEM of 10 animals. Each extract was administered
orally at doses of 20, 100, 500 and 1250 mg/kg. * Denotes
signi®cant difference from the corresponding values ob-
tained from control rats.
Exploration
A dose of 500 mg/kg of hexane, ethyl acetate and
methanol extracts did not show any significant effect on
the frequency and time spent in the hole-board. However,
a small and statistically insignificant increase in
frequency (10%) and time (28%) was seen with the ethyl
acetate extract.
Motor activity
In view of the high degree of variation in the results, no
clear effects due to the plant extracts were seen in treated
mice.
Copyright # 2000 John Wiley & Sons, Ltd.
109
Figure 2. The effect of three extracts of Psidium guagava
leaves on the hot plate test; for zero minute (®lled columns),
30 min (cross-hatched columns) and 60 min (open columns).
Each column and vertical bar represent mean  SEM of 10
animals. The different extracts (hexane, ethyl acetate and
methanol) were administered orally at doses (20, 100, 500
and 1250 mg/kg). * Denotes signi®cant difference from the
corresponding values obtained from control.
DISCUSSION
In this work we compared the CNS activity of three
different extracts of P. guajava in order to select the most
active extract for future pharmacological characteriza-
tion. Two established chemical and thermal methods to
test the analgesic (antinociceptive) actions were em-
ployed. The results indicated that the three extracts were
dose-dependently effective in producing an antinocicep-
tive effect. On a dose to dose basis the ethyl acetate
extract was the most effective, followed by the hexane
and then the methanol extract. This variation in the
analgesic response could be due to the variation in the
polarity and chemical nature of the compounds present in
Phytother. Res. 14, 107–111 (2000)
110 H. M. SHAHEEN ET AL.
Figure 3. The effect of treating mice with three extracts of
Psidium guagava leaves on pentobarbitone-induced sleeping
time. Each extract was administered orally at doses of 20,
100, 500 and 1250 mg/kg. * Denotes signi®cant difference
from the corresponding values obtained from control.
the extract used. The difference between the actions of
the extracts may be due to differential extraction or phase
partition of active components during the extraction
process.
The analgesic effect seen with P. guajava was
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