Effects of Otostegia persica (Burm.) Boiss on morphine withdrawal syndrome in mice

Authors

Abstract

Effect of Otostegia persica on naloxone-induced morphine withdrawal syndrome was studied in male mice. Dependence was induced using daily subcutaneous injections of morphine for three days. Morphine was injected 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, morphine (90mg/kg) was injected 1h before oral administration and 1.5h before intraperitoneal (i.p.) injection of hydroalcoholic and hexane extracts of the plant. Naloxone was injected (5mg/kg, i.p.) 2h after the final dose of morphine and the withdrawal signs including jumping, rearing, diarrhoea, piloerection, tremor and ptosis were recorded during a period of 30 minutes. While oral and i.p. administration of hydroalcoholic extract reduced the number of jumping and rearing, the hexane extract could not exert any significant change. Also the hydroalcoholic extract (1500mg/kg) significantly (p<0.05) reduced diarrhoea, piloerection, tremor and ptosis. The hexane extract only significantly (p<0.05 ) inhibited diarrhoea. Results of this study indicated that the extract of Otostegia persica contained component(s) that alleviate morphine withdrawal syndrome and the responsible constituent(s) is(are) found in polar fraction since the hexane extract had only a negligible effect.

Keywords


Effects of Otostegia persica (Burm.) Boiss on morphine withdrawal syndrome in mice

Iranian Journal of Pharmaceutical Research (2004) 3: 171-175
Received: December 2003
Accepted: April 2004

Copyright ? 2004 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services

Original Article

Effects of Otostegia persica (Burm.) Boiss on morphine withdrawal syndrome in mice

Valiollah Hajhashemi*a, Mohammad Rabbania, Gholam-Reza Asgharib and Zaman Karami-Saravia 

aDepartment of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran. bDepartment of Pharmacognosy, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

* Corresponding author: E-mail: vhajhashemi@hotmail.com

 

Abstract


Effect of Otostegia persica on naloxone-induced morphine withdrawal syndrome was studied in male mice. Dependence was induced using daily subcutaneous injections of morphine for three days. Morphine was injected 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, morphine (90mg/kg) was injected 1h before oral administration and 1.5h before intraperitoneal (i.p.) injection of hydroalcoholic and hexane extracts of the plant. Naloxone was injected (5mg/kg, i.p.) 2h after the final dose of morphine and the withdrawal signs including jumping, rearing, diarrhoea, piloerection, tremor and ptosis were recorded during a period of 30 minutes. While oral and i.p. administration of hydroalcoholic extract reduced the number of jumping and rearing, the hexane extract could not exert any significant change. Also the hydroalcoholic extract (1500mg/kg) significantly (p<0.05) reduced diarrhoea, piloerection, tremor and ptosis. The hexane extract only significantly (p<0.05 ) inhibited diarrhoea. Results of this study indicated that the extract of Otostegia persica contained component(s) that alleviate morphine withdrawal syndrome and the responsible constituent(s) is(are) found in polar fraction since the hexane extract had only a negligible effect.

Keywords: Otostegia persica; Morphine dependence; Naloxone withdrawal syndrome

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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