This study was designed to evaluate the analgesic effect of Teucrium polium leaf extract in the diabetic rat formalin test. For this purpose, streptozotocin (STZ)-diabetic rats received intraperitoneal injection of this extract (100 and 200 mg/kg per day) for a period of 2 weeks. It was found out that Teucrium polium-treated diabetic rats exhibited a lower nociceptive score as compared to untreated diabetics. The results may suggest therapeutic potential of Teucrium polium extract for the treatment of diabetic hyperalgesia. Â© 2004 Elsevier Ireland Ltd. All rights reserved

Baluchnejadmojarad, T.

Roghani, M.

Roghani-Dehkordi, F.

eprints Iran University of Medical Sciences

Journal of Ethnopharmacology 97 (2005) 207–210Antinociceptive effect of Teucrium polium leafextract in the diabetic rat formalin testTourandokht Baluchnejadmojarada, Mehrdad Roghanib,∗, Farshad Roghani-Dehkordica Department of Physiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iranb Department of Physiology, School of Medicine, Shahed University, Tehran, Iranc Department of Cardiology and Internal Medicine, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, IranReceived 20 October 2003; received in revised form 28 September 2004; accepted 11 October 2004Available online 4 January 2005AbstractThis study was designed to evaluate the analgesic effect of Teucrium polium leaf extract in the diabetic rat formalin test. For this purpose,streptozotocin (STZ)-diabetic rats received intraperitoneal injection of this extract (100 and 200 mg/kg per day) for a period of 2 weeks. Itwm©K11irMsfi1(ha(c0das found out that Teucrium polium-treated diabetic rats exhibited a lower nociceptive score as compared to untreated diabetics. The resultsay suggest therapeutic potential of Teucrium polium extract for the treatment of diabetic hyperalgesia.2004 Elsevier Ireland Ltd. All rights reserved.eywords: Teucrium polium; Pain; Formalin test; Diabetic rat. Introduction.1. PlantTeucrium polium, a wild-growing flowering plant belong-ng to the family Labiatae, was obtained from mountainousegions of Hamedan (Iran) in mid-July and authenticated byr. Ghamkhar (Department of Botany, Shahrekord Univer-ity, Shahrekord). The voucher number 2003-46 was speci-ed by Shahrekord University Herbarium..2. Reported activitiesTeucrium polium is a medicinal plant with antinociceptiveAbdollahi et al., 2003), antioxidant (Couladis et al., 2003),ypolipidemic (Rasekh et al., 2001), anti-inflammatory,nti-rheumatoid (Tariq et al., 1989), and hypoglycemicGharaibeh et al., 1988) properties. Therefore, this study wasarried out to evaluate the antinociceptive effect of aqueousTeucriumpolium leaf extract in streptozotocin (STZ)-diabeticrats using formalin test.2. Materials and methods2.1. ExtractionFresh leaves of Teucrium polium were separated, cleaned,and dried at room temperature. Thereafter, 150 g of driedleaves was grounded and the obtained powder was mixed with1000 ml of distilled boiling water for a period of 15 min undercontinuous stirring. The obtained mixture was filtered twicethrough a mesh and then one time through a filtered funnel,and the obtained liquid was dried on a magnet stirrer untila concentrated residue (69%, w/w) was obtained. This stockextract was maintained at −20 ◦C until being used. Lowerconcentrations of the extract were prepared by dilution of thestock with cold and sterile 0.9% saline solution.2.2. Experimental procedure∗ Corresponding author. Tel.: +98 21 8964792; fax: +98 21 8966310.E-mail address: mehjour@yahoo.com (M. Roghani).Male albino Wistar rats (Pasteur’s institute, Tehran,Iran) weighing 200–250 g (7–9 weeks old) were housed378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2004.10.030208 T. Baluchnejadmojarad et al. / Journal of Ethnopharmacology 97 (2005) 207–210in an air-conditioned colony room on a light/dark cycle at21± 3 ◦C and supplied with standard pelleted diet and tapwater ad libitum. Procedures involving animals and theircare were conducted in conformity with the institutionalguidelines of Iran University of Medical Sciences (Tehran,Iran) and in accordance with the NIH guidelines for the careand use of laboratory animals.The animals were randomly divided into control (n= 15)receiving 0.9% saline, Teucrium polium-treated control(n= 12), sodium salicylate (SS; Sigma Chemical)-treatedcontrol (n= 9), vehicle-treated diabetic (n= 13), SS-treateddiabetic (n= 7), and Teucrium polium-treated diabetic (n= 24in two equal-sized parts) groups. Diabetes was induced by asingle intraperitoneal injection of STZ (60 mg/kg/l; Upjohn)dissolved in cold 0.9% saline immediately before use. After2 weeks, Teucrium polium extract was intraperitoneally ad-ministered to diabetic rats at doses of 100 and 200 mg/kg perday for 2 weeks. Control rats received the Teucrium poliumextract only at a dose of 200 mg/kg and SS (200 mg/kg, i.p.)was administered 1 h before conducting the formalin test.Serum glucose level and body weight were monitored at thestart and end of the experiment. Diabetes was verified by aserum glucose level higher than 250 mg/dl (glucose oxidasekit, Zistchimie, Tehran).For assessment of pain, formalin test was used accordingt1tcsPnscipepsttbap2.3. Statistical analysisAll values were given as mean±S.E.M. Statistical analy-sis was carried out using student’s paired t-test and one-wayanalysis of variance (ANOVA) followed by Tukey’s post hoctest. Statistical P-value less than 0.05 was considered signif-icant.3. Results and discussionRegarding body weight and serum glucose level (Table 1),there was no significant differences between the groups be-fore the experiment. At the end of 4 weeks, the body weight ofthe untreated (P< 0.01) andTeucriumpolium-treated diabetic(P< 0.05) rats was found to be significantly lower as com-pared to control rats. In addition, untreated (P< 0.001) andTeucrium polium-treated (P< 0.005) diabetic rats also had el-evated serum glucose level over those of control rats. In thisrespect, treatment of diabetic rats with Teucrium polium ex-tract caused a significant reduction in the latter parameter incomparison with untreated diabetics (P< 0.01).Hind limb formalin injection produced a marked biphasicresponse in the rats of all groups. Hyperalgesia was signifi-cantly (P< 0.05) greater in untreated diabetic than in controlrdatcl(cbtosbwhmaiTB ium extWC 2C 2D 1D 2D 2* ol grouo the previously described method (Dubuisson and Dennis,977). Briefly, each animal was acclimatized to the observa-ion box before any testing began. Then, it was given a sub-utaneous injection of 50l of 2.5% formalin into the plantarurface of one hind paw. It was then immediately placed in alexiglas box. Observations continued for the next 60 min. Aociceptive score was determined for 5 min blocks by mea-uring the amount of time spent in each of the four behavioralategories: 0, the position and posture of the injected hind paws indistinguishable from the contralateral paw; 1, the injectedaw has little or no weight placed on it; 2, the injected paw islevated and is not in contact with any surface; 3, the injectedaw is licked, bitten, or shaken. Then, a weighted nociceptivecore, ranging from 0 to 3 was calculated by multiplying theime spent in each category by the category weight, summinghese products and dividing by the total time for each 5 minlock of time. The first 10 min post-injection was considereds the early phase, and the time interval 15–60 as the latehase.able 1ody weight and serum glucose level of control, diabetic, and Teucrium polBody weight (g)Week + 0ontrol 211.1± 3.7ontrol + Teucrium polium (200 mg/kg) 225.4± 3.9iabetic 217.4± 4.5iabetic + Teucrium polium (100 mg/kg) 231.1± 4.2iabetic + Teucrium polium (200 mg/kg) 220.1± 5.4P< 0.05, **P< 0.01, ***P< 0.005, and ****P< 0.001 (as compared to contrats in both phases of the test (Fig. 1). Treatment of control andiabetic rats with sodium salicylate (200 mg/kg, i.p.) causedsignificant reduction (P< 0.05) in nociceptive score only inhe second phase of the formalin test. In addition, treatment ofontrol rats withTeucriumpolium extract (200 mg/kg) causedower nociceptive scores in both phases of the formalin testP< 0.05). On the other hand, diabetic animals receiving Teu-rium polium extract showed also a less intensive nociceptiveehavior, especially in the first phase of the test, as comparedo untreated diabetic rats.There are two main conclusions to be drawn from thebtained results as follows: first, the results clearly demon-trated that there is an intensified nociceptive response inoth phases of the formalin test in diabetic rats. It is aell-established fact that diabetic rats display exaggeratedyperalgesic behavior in response to noxious stimuli that mayodel aspects of painful diabetic neuropathy (Freshwater etl., 2002) and for this reason, STZ-diabetic rats have beenncreasingly used as a model of painful diabetic neuropathyract-treated ratsSerum glucose (mg/dl)eek + 4 Week + 0 Week + 434.5± 4.8 103.6 ± 4.3 107.6 ± 5.133.2± 4.9 98.9 ± 4.7 90.7 ± 6.2*96.4± 4.3** 105.1 ± 4.5 389.1 ± 14.9****09.4± 5.1*,$ 97.1 ± 5.6 280.7 ± 13.1***,$$13.5± 6.1*,$ 108.2 ± 4.8 256.3 ± 12.3***,$$p); $P< 0.05 and $$P< 0.01 (as compared to diabetic group).T. Baluchnejadmojarad et al. / Journal of Ethnopharmacology 97 (2005) 207–210 209Fig. 1. The effect of Teucrium polium extract (100 and 200 mg/kg, i.p.) and sodium salicylate (SS, 200 mg/kg) on nociceptive scores in the first (early) andsecond (late) phases of the formalin test. All data represent mean±S.E.M. *P< 0.05 and **P< 0.01 (as compared to control group). P< 0.05 and P< 0.01 (ascompared to diabetic group).to assess the efficacy of potential analgesic agents (Fox etal., 1999). Although evaluation of mechanisms causing thesesymptoms is complicated because of the overlap betweenthe systemic effects of hyperglycemia and its toxic effectswithin the peripheral nervous system, but direct functionaltoxicity of hyperglycemia in the peripheral nervous system(Dobretsov et al., 2001), an increased activity of primary af-ferent fibers leading to an increased excitatory tone within thespinal cord, increased release of glutamate and activation ofthe NMDA receptor, reduced activity of both opioidergic andGABAergic inhibitory systems (Malcangio and Tomlinson,1998), decreased activity of nNOS–cGMP system in neuronsof dorsal root ganglion (Sasaki et al., 1998), altered sensitivityof the dopaminergic receptors and altered responsiveness ofthe dopaminergic system, possibly through the enhancementand/or deactivation of the endogenous Met-enkephalinergicsystem (Takeshita and Yamaguchi, 1998; Rutledge et al.,2002), and alterations in L-type Ca2+ channels (Gullapalli etal., 2002) could be involved in the modulation of nociceptionin diabetic rats. Secondly, it was demonstrated that intraperi-toneal administration of aqueous Teucrium polium extract ata dose of 200 mg/kg for a period of 2 weeks could producea significant analgesic effect in both phases of the formalintest in control and diabetic rats. On the other hand, sodiumsalicylate significantly reduced the nociceptive score only intri1topOtm1988) and antioxidant (Couladis et al., 2003) effects. Sincehyperglycemia in diabetic state could induce some functionalalterations in the nervous system (Dobretsov et al., 2001),this extract may have attenuated the hyperalgesic condition.To conclude, administration of aqueous Teucrium poliumextract could attenuate the hyperalgesic state of diabetic ratsand this may be of potential benefit in painful diabetic con-ditions.ReferencesAbdollahi, M., Karimpour, H., Monsef-Esfehani, H.R., 2003. Antinoci-ceptive effects of Teucrium polium L. total extract and essen-tial oil in mouse writhing test. Pharmacological Research 48, 31–35.Couladis, M., Tzakou, O., Verykokidou, E., Harvala, C., 2003. Screeningof some Greek aromatic plants for antioxidant activity. PhytotherapyResearch 17, 194–195.Dobretsov, M., Hastings, S.L., Stimers, J.R., Zhang, J.M., 2001. Me-chanical hyperalgesia in rats with chronic perfusion of lumbar dorsalroot ganglion with hyperglycemic solution. Journal of NeuroscienceMethods 110, 9–15.Dubuisson, D., Dennis, S.G., 1977. The formalin test: a quantitative studyof the analgesic effects of morphine, meperidine and brain stem stim-ulation in rats and cats. Pain 4, 161–174.Fox, A., Eastwood, C., Gentry, C., Manning, D., Urban, L., 1999. Criticalevaluation of the streptozotocin model of painful diabetic neuropathyFGGhe second phase of the formalin test in control and diabeticats. It has been known that central acting drugs like narcoticsnhibit both phases of the formalin test equally (Shibata et al.,989), while peripheral acting drugs like aspirin only inhibithe late phase (Rosland et al., 1990). Therefore, the effectf Teucrium polium extract in this study could be mediatedossibly through a central and/or a peripheral mechanism.ne of the possible mechanisms that could partially explainhe beneficial analgesic property of Teucrium polium extractay be attributed to its hypoglycemic (Gharaibeh et al.,in the rat. Pain 81, 307–316.reshwater, J.D., Svensson, C.I., Malmberg, A.B., Calcutt, N.A., 2002.Elevated spinal cyclooxygenase and prostaglandin release during hy-peralgesia in diabetic rats. Diabetes 51, 2249–2255.haraibeh, M.N., Elayan, H.H., Salhab, A.S., 1988. Hypoglycemic ef-fects of Teucrium polium. Journal of Ethnopharmacology 24, 93–99.ullapalli, S., Gurumoorthy, K., Kaul, C.L., Ramarao, P., 2002. Roleof L-type Ca2+ channels in attenuated morphine antinociception instreptozotocin-diabetic rats. European Journal of Pharmacology 435,187–194.210 T. Baluchnejadmojarad et al. / Journal of Ethnopharmacology 97 (2005) 207–210Malcangio, M., Tomlinson, D.R., 1998. A pharmacologic analysis ofmechanical hyperalgesia in streptozotocin-diabetic rats. Pain 76,151–157.Rasekh, H.R., Khoshnood-Mansourkhani, M.J., Kamalinejad, M., 2001.Hypolipidemic effects of Teucrium polium in rats. Fitoterapia 72,937–939.Rosland, J.H., Tjolsen, A., Maehle, B., Hole, K., 1990. The formalin testin mice-effect of formalin concentration. Pain 42, 235–242.Rutledge, L.P., Ngong, J.M., Kuperberg, J.M., Samaan, S.S., Soliman,K.F., Kolta, M.G., 2002. Dopaminergic system modulation of nocicep-tive response in long-term diabetic rats. Pharmacology, Biochemistry,and Behavior 74, 1–9.Sasaki, T., Yasuda, H., Maeda, K., Kikkawa, R., 1998. Hyperalgesia anddecreased neuronal nitric oxide synthase in diabetic rats. Neuroreport9, 243–247.Shibata, M., Ohkubo, T., Takahashi, H., Inoki, R., 1989. Modifiedformalin test: characteristic biphasic pain response. Pain 38, 347–352.Takeshita, N., Yamaguchi, I., 1998. Antinociceptive effects of morphinewere different between experimental and genetic diabetes. Pharmacol-ogy, Biochemistry, and Behavior 60, 889–897.Tariq, M., Ageel, A.M., al-Yahya, M.A., Mossa, J.S., Al-Said, M.S., 1989.Anti-inflammatory activity of Teucrium polium. International Journalof Tissue Reactions 11, 185–188.

Antinociceptive effect of Teucrium polium leaf extract in the diabetic rat formalin test

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Antinociceptive effect of Teucrium polium leaf extract in the diabetic rat formalin test

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