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.
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).
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
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 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
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).
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
Preliminary phytochemical screening of
hydroalcoholic extract of O.
persica showed the presence of
flavonoids, steroids, tannins and triterpenoids.
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.
This work has been supported by research
council of Isfahan University of Medical Sciences.
(1) Puri S and Lal H. Effect of
dopaminergic stimulation or blockade on morphine withdrawal
Psychopharmacologia (1973) 32: 113-120
(2) Lal H and Numan R. Blockade of
morphine-withdrawal body shakes by haloperidol. Life Sci. (1976) 18: 163-167
(3) Ambrosio E, Igalesias V,
Garcia-Lecumberri C, Orensanz L and Algauacil L F. Effect of
yohimbine on the development of morphine dependence in the rat:
lack of involvement of cortical beta-adrenoceptor
(4) Wange L, Milne B and Jhamandas K.
Involvement of excitatory amino acid pathways in the expression
of precipitated opioid withdrawal in the rostral ventrolateral
medulla: an in vivo voltametric study. Brain Res. (1995) 697:
(5) Tokuyama S, Wakabayashi H and Ho I K.
Direct evidence for a role of glutamate in the expression of
the opioid withdrawal syndrome. Eur.
J. Pharmacol. (1996) 295:
(6) Gonzalez P, Cabello P, Germany A,
Norris B and Contreas E. Decrease of tolerance to, and physical
dependence on morphine by glutamate receptor antagonists. Eur. J. Pharmacol. (1997) 332: 257-262
(7) Michalska E and Malec D. Agonists and
antagonists of adenosine receptors and morphine withdrawal
syndrome in rats. Pol. J.
Pharmacol. (1993) 45: 1-9
(8) Capasso A and Loizzo A. Purinoreceptors
are involved in the control of morphine withdrawal. Life Sci. (2001)
(9) Tokuyama S, Zhu H, Oh S, Ho I K and
Yamamoto T. Further evidence for a role of NMDA receptors in
the locus coeruleus in the expression of withdrawal syndrome
from opioids. Neurochem. Int. (2001) 39: 103-109
(10) Herman B H, Vocci F and Bridge P. The
effects of NMDA receptor antagonists and nitric oxide synthase
inhibitors on opioid tolerance and withdrawal: Medication
development issues for opiate addiction. Neuropsychopharmacol. (1995)
(11) El-Kadi A O S and Sharif S I. The role
of 5-HT in the expression of morphine withdrawal in mice. Life Sci. (1995)
(12) Bhargava H N. Diversity of agents that
modify opioid tolerance, physical dependence, abstinence
syndrome and self administrative behavior. Pharmacol. Rev. (1994)
(13) Akhondzadeh S, Kashani L, Mobasheri M,
Hosseini S H and Khani M. Passion flower in the treatment of
opiate withdrawal. J. Clin.
Pharmacol. Ther. (2001) 26:
(14) Hosseinzadeh H and Lary P. Effect of Salvia leriifolia leaf extract on morphine dependent in mice. Phytother. Res. (2000)
(15) Ramezani M, Hosseinzadeh H and
Mojtahehdi K. Effects of Ferula
gummosa Boiss. Fractions on
morphine dependence in mice. J.
Ethnopharmacol. (2001) 77: 71-75
(16) Sajjadi S E, Movahedian-Atar A M and
Yektaian A. Antihyperlipidemic effect of hydroalcoholic extract
and polyphenolic fraction from
Dracocephalum Kotschyi Boiss.
(17) Ghasemi N and Moatar F. Laboratory methods in pharmacognosy. Medical University of Isfahan
Publication, Isfahan (1996) 11-32
(18) Broseta I, Rodriguez-Arias M, Stinus L
and Minarro J. Ethological analysis of morphine withdrawal with
different dependence programs in male mice. Neuropsychopharmacol. Biol. Psych. (2002) 26: 335-347
(19) Capasso A, Piacente S, Pizza C and
Sorrention L. Flavonoids reduce morphine withrawal in-vitro.
J. Pharm. Pharmacol. (1998) 50: 561-564
(20)Hollman P C and Katan M B. Absorption,
metabolism and health effects of dietary flavonoids in man . Biomed. Pharmacother. (1997) 51: 305-310