Introduction
Drug dependence is a serious problem in
most regions of the world. Many investigators have worked on
systems or drugs which alleviate morphine withdrawal syndrome.
These include dopaminergic(1, 2), adrenergic(3), excitatory
amino acids(4-6), purinergic(7,8), NMDA (9,10), nitric oxide
(10) and serotoninergic systems (11). Also some medicinal
plants including ginseng (12), passion flower (13), Salvia
leriifolia (14) and Ferula gummosa (15) have been studied in
this regard. Otostegia persica (Labiatae) grows in different parts
of Iran. In Sistan-Baluchestan province the plant is
traditionally used to alleviate opium withdrawal syndrome. This
study was aimed to find pharmacological support for this usage.
Experimental
Plant material
Aerial parts of O. persica were collected
in summer 2003 from a region 25 km north of Zabul in
Sistan-Baluchestan province (Iran). Voucher samples were
preserved for further reference at the Herbarium of Department
of Pharmacognosy, Faculty of Pharmacy, Isfahan, Iran.
Preparation of hydroalcoholic and
hexane extracts of O. persica
For preparation of hydroalcoholic extract,
air-dried and powdered aerial parts of the plant (100g) were
macerated with 400ml of ethanol-water (7:3) for 48 hours. After
shaking and filtering, the solution was concentrated under
reduced pressure (16). Hexane extract was prepared with the
same procedure but the solvent was n-hexane. Evaporation and
solvent removal of hydroalcoholic and hexane extracts gave
semi-solid masses (yield 29% and 14% respectively).
Phytochemical screening The
hydroalcoholic extract of O.
persica was screened for alkaloids,
anthraquinones, cardiac glycosides, saponins, steroids, tannins
and triterpenoids (17).
Animals
Male albino mice weighing 25-35g were
obtained from Pasteur institute (Tehran, Iran) and maintained
in animal house of Isfahan university of medical sciences in
12/12h light/dark cycle at 21±2 °C. They had free
access to food and water.
Grouping of animals and administration
of extracts
Ninety male mice were randomly divided in
groups of six in each. All animals were rendered dependent on
morphine. One series of animals orally received either saline
(10 ml/kg), different doses of hydroalcoholic extract of O. Persica (500,
1000 and 1500 mg/kg) or clonidine (0.2 mg/kg) as a reference
drug one hour prior to naloxone injection. The second series
were given i.p. injections of either saline (10ml/kg), the same
doses of hydroalcoholic extract of O.
Persica (500, 1000 and 1500 mg/kg)
or clonidine (0.2 mg/kg) thirty minutes before naloxone
injection. The third series orally received either saline
(10ml/kg), hexane extract of O.
Persica (500, 1000 and 1500 mg/kg)
or clonidine (0.2 mg/kg) one hour prior to naloxone injection.
Morphine dependence
Morphine was injected subcutaneously to
mice at doses of 30 and 45mg/kg on day 1 and 60 and 90mg/kg on
day 2 (8:00 am and 6:00 pm). On day 3, a single dose of
morphine (90mg/kg) was injected at 8:00 am (18)
Naloxone- precipitated withdrawal
syndrome
Withdrawal signs were elicited by i.p.
injection of naloxone hydrochloride (5mg/kg) 2h after the last
injection of morphine. Counted and checked signs were evaluated
during a 30min period starting just after naloxone injection.
Jumping and rearing were counted and checked signs including
diarrhea, ptosis, tremor and piloerection were evaluated over
3x10 min periods with one point given for the presence of each
sign during each period ( maximum score : 3) (18).
Statistical analysis
The data were expressed as mean ±
S.E.M. One-way ANOVA followed by Duncan test was used for
comparison of data and P values less than 0.05 were considered
significant. The Mann-Whitney U test was used for comparison of
checked signs data. All statistical calculations were done with
SPSS for windows (SPSS 10) software.
Results and discussion
Phytochemical screening
Preliminary phytochemical screening of
hydroalcoholic extract of O.
persica showed the presence of
flavonoids, steroids, tannins and triterpenoids.
Pharmacological study
Both oral and i.p. administration of the
hydroalcoholic extract reduced the number of jumping episodes
in a dose dependent manner (fig. 1 & 2 ). Oral doses of
500, 1000 and 1500mg/kg of hydroalcoholic extract produced 18,
45 and 89% reduction of jumps, respectively. Intraperitoneal
injection of the same doses decreased the number of jumps by
58, 82 and 91% respectively. Oral and i.p. administration of
hydroalcoholic extract also significantly (P<0.05) reduced
the number of rearing (fig. 1 & 2). Clonidine as a
reference drug significantly (p<0.01) reduced the number of
jumping and rearing. The effects of the high dose of
hydroalcoholic extract on these signs were comparable to those
of clonidine. Hexane extract at doses of 500, 1000 and
1500mg/kg could not exert any significant effect on number of
jumping and rearing episodes (figure 3). Diarrhea,
piloerection, ptosis and tremor were also suppressed by
clonidine and the hydroalcoholic extract. The hexane extract at
applied doses only inhibited diarrhea (Table 1 and 2).
The results of the present study indicate
that hydroalcoholic extract of O.
persica has component(s) that
could alleviate the morphine withdrawal syndrome. Hexane
extract of this plant, which contains non-polar substances,
could not produce any significant decrease in number of jumping
and rearing episodes and therefore it seems that
hexane-extractable constituents are not involved in alleviation
of morphine withdrawal syndrome. The results of
phytochemical study showed that flavonoids are found in
the plant and probably these compounds may have some role in
observed pharmacological effects. There is also a report
that flavonoids could suppress opioid withdrawal syndrome
(19). In comparison with intraperitoneal injection, oral
route of the hydroalcoholic extract at doses of 500 and
1000mg/kg had less effect on jumping. In general this could be
due to incomplete oral absorption (19) or high first pass
effect of the active compounds (20).
In addition to jumping which is a very
specific behavior of the morphine withdrawal syndrome (18) the
other withdrawal signs including ptosis, piloerection, diarrhea
and tremor were also significantly reduced by the
hydroalcoholic extract of O.
persica and according to our
results it seems that this extract contains active
constituent(s) which merit further works. As mentioned in
introduction section, various systems including dopaminergic
(1,2), adrenergic (3), excitatory amino acids (4-6), purinergic
(7,8), NMDA (9,10), nitric oxide (10) and serotoninergic (11)
are involved in suppression of opioid withdrawal syndrome.
However, the mechanism of action of this plant is not known and
further investigations are needed to clarify it.
Acknowledgement
This work has been supported by research
council of Isfahan University of Medical Sciences.
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