Effect of Nepeta glomerulosa Boiss. Aerial Parts Aqueous Extract on Morphine Withdrawal Syndrome in Mice

Document Type: Research Paper


Department of Pharmacodynamy and Toxicology, Pharmaceutical Research Center, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran


      The effect of aqueous extract of Nepata glomerulosa Boiss. aerial parts on morphine withdrawal syndrome was investigated in mice. Dependence was induced by subcutaneous injections of morphine for 3 consecutive days. On day 4, morphine was injected 2 h prior to intraperitoneal injection of naloxone. The number of jumps during a 20 min. period after naloxone injection was considered as a measure of the withdrawal syndrome. Open field and rotarod tests were also performed. The results indicated that the aqueous extract (0.1 to 2.8 g/kg, s.c.) and diazepam (5 mg/kg) reduced the number of jumps (58±2.3 to 10±0.82, pvs normal saline, 87±3.4). The extracts also reduced motion balance and locomotion activity. It is concluded that the aqueous extract of N. glomerulosa aerial parts could diminish morphine withdrawal syndrome.


1. Introduction


     The withdrawal syndrome is typically observed following abrupt termination of morphine intake or by the administration of a narcotic antagonist such as naloxone that has been shown to cause a specific set of behavioral effects in morphine dependent rodents. Stereotyped jumping behavior is the most dominant syndrome among them [1] and is widely considered the most sensitive and reliable index of withdrawal intensity [2-4]. It is suggested that jumping is the most suitable sign of measuring abstinence quantitatively because jumps are easily counted [5]. This behavioral phenomenon has a central origin and a direct relation to the intensity of the abstinence syndrome due to morphine withdrawal [6].

     Nepeta glomerulosa (Lamiaceae) is a medicinal plant found in Asia (widely distributed in Iran, especially areas of Khorasan and Isfahan provinces), America and Europe. The aerial parts of N. glomerulosa are empirically used in folk medicine as antitussive. Some Nepeta species have been reported to have antinociceptive (N. italica; N. cataria), antimicrobial (N. crispa.) and antiviral (N. cataria) effects [7-10]. The ingredients of N. glomerulosa extract are alkaloid, saponin and oils [11]. It’s oil contains over 35 components. The major components were α-pinene (18.3%), 1,8-cineole (13.9%), limonene (9.7%), gernyl acetate (9.3%), caryophyllene oxide (8.0%), linalool (4.8%), trans-β-ocimene (4.7%), humulene epoxide (4.2%), trans-α-bergamotene (3.5%), α-humulene (3.2%) and camphene (3.1%) [12, 13].

     Because of some the Lamiaceae plants such as Rosmarinus officinalis [14] and Salvia leriifolia [15] have been reported to decrease morphine withdrawal syndrome symptoms, therefore, this study was conducted to investigate the effect of N. glomerulosa on morphine dependence.


Figure 1. Effects of aqueous extracts of Nepeta glomerulosa Boiss. on withdrawal syndrome in male mice (10 mg/kg morphine s.c. injected, 3 times a day, during 3 days and one dose injected on day 4). Withdrawal syndrome was induced by the i.p. injection of naloxone (5 mg/kg) 90 min. after the i.p. treatments of extracts, diazepam or normal saline. Data are reported as Mean±SEM. ***p < 0.001 vs NS, Tukey-Kramer test.

2. Materials and methods


2.1. Plant material


     The aerial parts of N. glomerulosa Boiss. were collected from Torbat Jam Bazd mountains (26.5.2005, at an altitude of 1850 m). The plant was identified by Mr. Ahi, and voucher samples (152-1407-2) are preserved for reference at the herbarium of the department of Pharmacognosy, Faculty of Pharmacy, Mashhad University of Medical Sciences.


2.2. Animals


    Adult male albino mice weighing 25-30 g were obtained from the animal house of Mashhad University of Medical Sciences. Animals were housed in plastic cages in an animal colony room 12/12 h light/dark cycle at 21±2 °C with access to food and water ad libitum. Groups of animals of 8 mice were chosen randomly for the studies.


2.3. Preparation of extract


     The powder of aerial parts was extracted using the aqueous decoction. In the decoction method, 100 g of powder was added to 1 L of boiling water for 15 min. and then filtered through a cloth. The extract was then concentrated under reduced pressure (in a rotary evaporator at 40 °C) to the desired volume. The yield of the extract was 10% (w/w).


2.4. Morphine dependence


     The mice were treated with subcutaneous injection of morphine three times a day (08.00, 11.00 and 14.00 h) for three consecutive days. The doses of morphine were 50, 50 and 75 mg/kg, respectively. The higher dose at the daily 14 h injection was aimed at minimizing any overnight withdrawal. On day 4, they received a last dose of morphine (50 mg/kg) [16].


Figure 2. Effects of aqueous decoction extracts of Nepeta glomerulosa Boiss. on motor function in Rotarod system in 8 male mice, 30 and 60 min. after injection of extract, diazepam or normal saline, Data was reported as Mean±SEM. ***p < 0.001 vs NS, Tukey-Kramer test.

2.5. Morphine withdrawal


     Physical dependence was inferred if a withdrawal syndrome could be precipitated with the opioid receptor antagonist, naloxone. Groups of mice were tested for the occurrence of jumping after 10 injections of morphine and two hours after the last administration of morphine, animals were given naloxone (5 mg/kg) ip. and placed individually in glass cube boxes (28 cm diameter, 50 cm height). The number of jumps was recorded over a 20 min. period beginning 1 min. after the injection of naloxone (2 h after the final administration of morphine) [16].


2.6. Drug and extracts treatment


     The extracts (0.3 ml) were injected inter-aperitoneally 0.5 h after the final dose of morphine. Diazepam (5 mg/kg) and normal saline (10 ml/kg) containing Tween 80 (1%, v/v) were injected i.p. as positive and negative controls, respectively.


2.7. Rotarod test


     Motor coordination and balance were tested using accelerating rotarod (TSE Rotarod System). Mice were placed on a horizontal metal coated rod with rubber (3 cm diameter) rotating at an initial speed of 10 rpm/min. Terminal speed of the rod was 20 rpm in accelerated studies and rotational velocity of the rod was linearly increased from 10 to 20 rpm within 20 s. The time each animal was able to maintain its balance walking on the top of the rod was recorded. Mice were given two trials with a maximum time of 300 s and a 30 to 60 min. inter-trial rest interval [18]. Before the beginning of all experiments, the riding ability of the animals on the rotarod was checked. Thus, the mice were initially put on a rotating rod, and mice that immediately dropped off (within 30 s) were removed from the experiment [18].


2.8. Open field test


      The apparatus, made of white wood, had a floor of 100×100 cm divided by red lines into 25 squares of 20×20 cm. The walls (50 cm high) were also painted in white. The test room was illuminated at the same intensity at the colony room. Each mouse was placed in the center of the open field, and its behavior was observed for 5 min. The parameters evaluated were the total number of squares crossed, the number of outer squares (those adjacent to the walls) crossed and the number of inner squares crossed; the three measures were referred to as total, peripheral, and central locomotion, respectively.

      The numbers leanings (one or two paws in contact with the wall) rearing, grooming (face cleaning, paw licking, fur liking, head scarping and rubbing), and defecations were also recorded. At the end of each test, the whole area was cleaned with a wet sponge and a dry paper towel [19].


Figure 3(a and b). Effects of Nepeta glomerulosa Boiss. aqueous decoction extracts on open field test factors in 8 male mice, 60 min. after injection of extracts, diazepam or normal saline. Data were reported as Mean ± SEM, * p < 0.05 , ** P < 0.01 ,   *** P < 0.001 vs NS, Tukey-Kramer test.

2.9. Statistical analysis


    Statistical analysis was performed using analysis of variance (ANOVA) followed by Tukey-Kramer test for multiple comparison. Differences with a p < 0.05  were considered significant.


3. Results


     The administration of N. glomerulosa aqueous extracts (0.1 to 2.8 g/kg) 90 min. after the last dose of morphine on the 4th day, significantly reduced the jumping episodes. The maximum effect was observed at a dose of 2.8 g/kg (Figure 1). Furthermore, in confirmation to withdrawal syndrome declining, extracts (0.1 to 2.8 g/kg) decreased motion balance and function in rotarod system at the 30th min. after injections (Figure 2). The extract also decreased open field test factors (Figure 3a and 3b). Diazepam also decreased withdrawal signs, open field factors and motion balance (Figures 1, 2, 3a and b).



4. Discussion


     The present results indicate that the boiling water extract of N. glomerulosa aerial parts reduced the withdrawal signs of morphine, dose-dependently. Benzodiazepines have been shown to have an inhibitory effect on the dependence to morphine which has been suggested to be via GABAA receptors [20, 21]. It has been reported that morphine caused an increase in the whole brain GABA concentration in mice. Increase of GABA indiscrete parts of the thalamus and the spinal cord of the rats have been also reported [22,23].

     N. glomerulosa has some saponins which have depression effects on CNS [11, 24]. There is also a possibility that N. glomerulosa acts through complex ways to affect morphine dependence. It is well recognized that open field behavior is under the influence of several processes [19]. Our results showed that the extracts decrease locomotion activity factors like total, peripheral and central entrances with all doses. The extracts reduced the number of leaning, rearing, grooming and defecation by all doses except a dose of 0.1 g/kg. In our study, the reduction in locomotion activity with the extract is similar to diazepam effects. Drugs that depress spinal neurons can cause relaxation and decrease muscle spasmic reflexes and motion balance [25, 26]. Diazepam and possibly the extracts showed this effect via GABAA receptors inspinal neurons [26, 27].

     The results of this study showed that N. glomerulosa aqueous extracts decreased the morphine withdrawal syndrome. The mechanism of action of this effect is not clear. Besides the induction of muscle relaxations and decrease of motor functions, other mechanisms are possibly involved in the mechanism of action of the extract in decreasing morphine withdrawal syndromes.




     The authors are thankful to the Vice Chancellor of Research, Mashhad University of Medical Sciences for financial support.





[1]      Berthold H, Borel JF, Fluckiger E. Enigmatic action of cyclosporine A on the naloxone-precipitated morphine withdrawal syndrome in mice. Neuroscience 1989; 31: 97-103.

[2]      El Kadi AOS, Sharif SI. The influence of various experimental conditions on the expression of naloxone-induced withdrawal symptoms in mice. Gen Pharmacol 1994; 25: 1505-10.


[3]      Kest B, Hopkins E, Palmese CA, Chen ZP, Mogil JS, Pintar JE. Morphine tolerance and dependence in nociceptin/orphanin FQ transgenic knock-out mice. Neuroscience 2001; 104: 217-22.


[4]      Miyamoto Y, Takemori AE. Relative involvement of supraspinal and spinal mu opioid receptors in morphine dependence in mice. Life Sci 1993a; 52: 1039-44.


[5]      Zafar S, Ahmad MA, Siddiqui TA. Protective role of Delphinium denudatum (yadwar) against morphine induced tolerance and dependence in mice. J Ethnopharmacol 2001; 78: 95-8.


[6]      Bourin M, Malinge M, Colombel MC, Vasar E. Cholecystokinin receptor agonists block the jumping behavior precipitated in morphine-dependent mice by naloxone. Eur Neuropsychopharmacol 1999; 9: 37-43.


[7]      Aydin S, Demir T, Oztark Y, Baayer, KH. Analgesic activity of Nepeta italica L. Phytother Res 1999; 13: 20-3.


[8]      Sonboli A, Salehi P, Yousefzadei M. Antimicrobial activity and chemical composition of the essential oil of Nepeta crispa Wild. from Iran. Z Naturforsh C 2004; 59: 653-6.


[9]      Aydin S, Beis R, Oztark Y, Baser KH, Baser C. Nepetalacton: A new opioid analgesic from

 Nepeta  caesarea  Boiss.  J  Pharm  Pharmacol  1998; 50: 813-7.


[10]   Ganzera M, Crockett S, Tellez MR, Khan IA. Determination of nepetalactose in Nepeta cataria by reversed phase high performance liquid chromatography. Pharmazi 2001; 56: 896-7.


[11]   Fazly Bazzaz BS, Haririzadeh G, Imami SA, Rashed MH. Survey of Iranian plants for alkaloids, flavonoids, saponins, and tannins (Khorasan province). Int J Pharmacy 1997; 35: 17-30.


[12]   Ebrahim Sajjadi S, Ghassemi N. Volatile constituents of Nepeta glomerulosa Boiss. subsp. carmanica. Flav Frag J 1999; 14: 265-7.


[13]   Sefidkon F. Essential oil of Nepeta glomerulosa Bioss. from Iran. J Essent Oil Res 2001; 13:422-3.


[14]   Hosseinzadeh H, Nourbakhsh M. Effect of Rosmarinus officinalis L. aerial parts extract on morphine withdrawal syndrome in mice. Phytother Res 2003; 17: 938-41.


[15]    Hosseinzadeh H, Lary P. Effect of Salvia leriifolia leaf extract on morphine dependence in mice.

Phytother Res 2000; 14:1-4.


[16]   Rezayat M, Azizi N, Zarrindast MR. On the mechanism(s) of cholecystokinin (CCK): Receptor stimulation attenuates morphine dependence in mice. Pharmacol Toxicol 1997; 81: 124-9.


[17]   McIlwain KL, Merriweather MY, Yuva-Paylor LA, Paylor R. The use of behavioral test batteries: Effects of training history. Physiol Behav 2001, 73:705-17.


[18]    Sugarman MC, Yamasaki TR, Oddo S, Echegoyen JC, Murphy MP, Golde TE, Jannatipour M, Leissring MA, Laferal FM. Inclusion body

myositis-like phenotype induced by transgenic overexpression of βApp in skeletal muscle. Pro Natl Acad Sci USA. 2002; 99: 6334-9.


[19]   Pardon MC, Perez Diaz F, Joubert C, Cohen Salmon C. Age dependent effects of a chronic ultramild stress procedure on open field behavior in B6D2F1 female mice. Physiology Behavio 2000; 70: 7-13.


[20]   Suzuki T, Tsuda M, Narita M, Funada M, Mizoguchi H, Misawa M. Diazepam pretreatment suppresses morphine withdrawal signs in the mouse. Life Sci 1996; 58: 349-57.


[21]   Puntillo K, Casella V, Reid M. Opioid and benzodiazepine tolerance and dependence: Application of theory to critical care practice. Heart Lung 1997; 26: 317-24.


[22]   Zarrindast MR, Mousa Ahmadi E. Effects of GABAergic system on naloxone-induced jumping in morphine-dependent mice. Eur J Pharmacol 1999; 381: 129-33.


[23]   Dunham NM, Miya TS. A note on a simple apparatus for detecting neurological deficit in rats and mice. J Am Pharm Assoc 1957; 46: 208-9.


[24]   Hussain, MM, Sokomba EN, Shok M. Pharma-cological effects of Gardenia erubescens in mice, rats and cats. Int J Pharmacogn 1991, 29: 94-100.


[25]   Hosseinzadeh H, Ramezani M, Namjo N. Muscle relaxant activity of Elaeagnus angostifolia L. fruit seeds in mice. J Ethnopharmacol 2003; 84: 275-8.


[26]  Houghton PI, Zarka R, Delas Heras B, Hoult RS. Fixed oil of Nigella sativa and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation. Planta Med 1995; 61: 31-6.


[27]  Jion ZH, Chen CC, Lu HY, Chu CP, Ng JJ, Yu S. Effects of Jawey siwu on rat activity. Am J Chin Med 2001; 29: 509-16.