ORIGINAL ARTICLEPhDPharmacology Assoc Prof Department of Clinical Pharmacy PhDPharmacognosy Asst ProfDepartment of Pharmacognosy and Pharmaceutical Botany PhDPharmacology Asst ID: 945697
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Effects of the extracts from Korth.Wantana Reanmongkoland Kitja SawangjaroenReanmongkol, W., Keawpradub, N. and Sawangjaroen, K.Effects of the extracts from Songklanakarin J. Sci. Technol., March 2007, 29(Suppl. 1) : 39-48The leaves of Mitragyna speciosa Korth. () were extracted with methanol to give methanolextract. The methanol extract was made in acid and then in alkaline and extracted with chloroform to givealkaloid extract. The effects of the methanol and alkaloid extracts on analgesic activities in hot plate test inin mice, were examined. In acute toxicity test, the LD values of oral administration of the methanol and leaves in mice were 4.90 g/kg and 173.20 mg/kg, respectively. Oral admin-istration (50, 100 and 200 mg/kg) of the methanol extract of M. speciosa leaves significantly prolonged thelatency of nociceptive response on hot plate test in mice. The alkaloid extract of also increasedthe pain response latency at the dose of 20 mg/kg but less potent than those of the methanol extract (100 mg/kg) in mice (comparing 5-10 mg/kg alkaloid extract with corresponding to approximately 200 mg/kg of ORIGINAL ARTICLEPh.D.(Pharmacology), Assoc. Prof., Department of Clinical Pharmacy, Ph.D.(Pharmacognosy), Asst. Prof.,Department of Pharmacognosy and Pharmaceutical Botany, Ph.D.(Pharmacology), Asst. Prof., Departmentof Pharmacology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, 90112 Thailand.Corresponding e-mail: wantana.r@psu.ac.th ORIGINAL ARTICLEPh.D.(Pharmacology), Assoc. Prof., Department of Clinical Pharmacy, Ph.D.(Pharmacognosy), Asst. Prof.,Department of Pharmacognosy and Pharmaceutical Botany, Ph.D.(Pharmacology), Asst. Prof., Department Effects of the extracts from methanol extract). The antinociceptive action of either methanol extract (100 mg/kg, p.o.)
or alkaloid extract leaves was blocked by naloxone (2 mg/kg, i.p.) in mice. Neither the methanolextract nor the alkaloid extract significantly prolonged latency of nociceptive response on tail flick test insleep in mice, respectively. These results suggest that the methanol and alkaloid extracts of leavespossess the analgesic activity which partly acted at opioid receptors in the supraspinal opioid system.Key words:6 # 7 6 6# 8 6# 8 9:(7 ( 6 !" ($%& &#x=;&#x= 6;&#x#;&#x ;&#x ;&#x ;&#x ;&#x;&#x#00;; -@AB CDEBF BFGB1 &#x=;&#x= 6;&#x#;&#x ;&#x ;&#x ;&#x ;&#x;&#x#00; " -BEHD IDHJK BFGB1 !'$9 L= 7 = 6# ' 9 ( &#x=;&#x= 6;&#x#;&#x ;&#x ;&#x ;&#x ;&#x;&#x#00;# ? # / ' *+ 8 4),+ M) N ) + )M) 6 O � ;&#x ;'0;&#x.;&#x' ;&#x-107;&#x.600; ("# # ? O -*+P ++ ++ )M)1 "# !L Q ; &#x=;&#x ;&#x 0;&#x=;&#x ;&#x 0; = 6 = 6# ? (!L Q R =" + )M) S" -++ )M)1 &#x=;&#x ;&#x 0;# ? - (" *3+ )M) " S ++)M)1 $%& " -++ )M)1 ( - + )M)1 "#&#x=;&#x ;&#x 0; ? Q9; - )M) 9 . " 1 &#x=
;&#x ;&#x 0;# ? T (7! 6 L Q = S &#x=;&#x ;&#x 0;;= = 6 6 (7! 6 L= 7 .40;L = 6# ' 9 ( # ? ! T ("# $%& 9$%& = 9U(.40;# 9U(= LV R V R V R V R V R'$0/ ( S V R/ ( V R V R S / (Mitragyna speciosaM. speciosa) Korth.(Rubiaceae) is traditionally used in Thailand andfor opium when opium is unavailable, or tooften used to treat diarrhea. A small minority ofonly need a few leaves to obtain the desired effects. Effects of the extracts from M. speciosa have been reported. Several 9- leaves(Takayama The pharmacological activities of the leaves have beenstudied. Mitragynine, a major indole-alkaloid, leaves, exerts inhibitoryeffect on electrically stimulated contraction ofisolated guinea-pig ileum (Watanabe on forskolin-stimulated cAMP formation inNG108-15 cells (Tohda and has morphine-like action on gastric acidstimulation (Tsuchiya leading to a potent inhibition of electricallydelta-receptors (Yamamoto , 1999; Takayama, 2002). 7-hydroxymitragynine, a minorpotent opioid effect on the electrically-stimulatedanalgesic effect based on activation of mu-opioid, 2005; Takayama, 2004;Matsumoto, 2004; Matsumoto et al., 2005b).9-hyhroxycorynantheidine, synthesized frommitragynine, has partial agonist properties onmu-opioid receptors in the guinea-pig ileum leaves have been reported toshow antinociceptive activity, no evaluation of theanalgesic ac
tivity of the methanol and alkaloidextracts of M. speciosa leaves has been clearlyreported. In the present study, we investigated the leavesrats. The general behaviors using locomotor activityThe fresh leaves of Korth.2005. Authentication of plant material was carriedout at the Department of Pharmacognosy andPharmaceutical Botany, Faculty of PharmaceuticalSciences, Prince of Songkla University, Songkhla,Thailand, where the herbarium vouchers (No.Preparation of the methanol and alkaloidextracts from the leaves of (5 kg) weremacerated with methanol for 72 hours. Then filter-C to obtain a syrupy mass. The marc wasremacerated with methanol twice, filtered andevaporated. All syrupy masses were combined togive crude methanol extract 396 g. An aliquot (300was washed with petroleum ether, made alkalinewith portions of chloroform. The combined chlo-roform extract was washed with distilled water,dried over anhydrous sodium sulfate and evapor-used as the test extract. All doses were expressedfrom the Animal House, Faculty of Science, Princeof Songkla University, Hat Yai, Songkhla, Thailand.Male Swiss mice and Wistar rats with the weightranging from 30-39 g and 150-230 g, respectively, Effects of the extracts from were used. The rats were handled for 5-10 mindaily for several days before experiments. Thewater were given ad libitum unless otherwisespecified. All procedures described were reviewedEthical Use of Animals, Prince of Songkla Uni-versity, Thailand.alkaloid extracts of M. speciosa leaves were1.Hot plate testThe hot plate test was carried outaccording to the method described by Woolfe &(15 min after administration), the nociceptivecutaneously. The cut-off time was 45 sec. Only the2.Tail flick test& Smith, 1941). The tail-flick r
eflex latency (sec)extract (50, 100, 200 mg/kg), the alkaloid extract leaves or co-solvent except morphine was subcutaneouslyadministered 15 min (10 mg/kg). The rats whosecarded and a cut-off time of 10 sec was maintainedthe extracts by pre-treatment with naloxonedose of 2 mg/kg ( i.p.). After 10 min the test agentswere given. The assessments were conducted byLocomotor activity was recorded in an1996). The mice were placed in the cage foradministration of drugs. Temperature, sound andthe course of the experiments. Measurementscounts were recorded for a 1 h period. Experi-mg/kg) intraperitoneally to induce sleep. Theduration of sleep was measured as the periodreflex. The methanol extract (50, 100, 200 mg/kg), (5, 10 and 20 mg/St. Louis, U.S.A.); methanol, petroleum ether,chloroform (AR grade, Merck, Germany); sodiumCarlo Erba, Germanny). The methanol and alkaloid leaves were dissolved in Effects of the extracts from and administered subcutaneously. All drug solutionswere prepared immediately before starting thewere analyzed statistically by one-way ANOVAprocedures, followed by Dunnett's test. A differ-included lethargy, tremor, fatigue, paralysis, lossand death. The LD values of orally administra- leaves in mice were 4.90 g/kg and 173.20mg/kg, respectively. leaves and morphine on nociceptiveresponse induced by heat in hot plate testAs shown in Table 1, oral administration (50, leaves significantly prolonged the latency leaves also leaves and morphine in leaves was blockedas shown in Table 2. leaves and morphine on nociceptiveresponse in tail-flick testkg, p.o.) of leaves significantlyTable 1.Effects of the methanol and alkaloid extracts of morphine on nociceptive response in hot plate test. Latency of nociceptive response (sec)15304560 m
inCosolvent-9.7±0.611.4±1.010.4±1.311.7±1.5Morphine1024.0±3.8*28.4±3.9*23.1±4.2*21.9±4.0*5013.2±2.211.5±0.717.2±3.4*14.3±1.2(methanol)10013.6±1.217.4±3.2*18.5±3.4*16.3±3.3*20014.0±1.012.9±1.116.4±2.8*13.9±1.2Cosolvent-10.0±0.710.0±1.29.3±0.99.2±1.4Morphine1017.5±2.3*21.6±3.0*19.7±3.2*17.8±3.3*510.1±0.69.3±0.68.5±0.68.7±0.6(alkaloid)1011.5±0.69.7±0.89.6±1.09.4±0.92013.5±1.1*13.1±1.4*12.3±1.110.3±0.9the nociceptive response was measured every 15 min over a 60-min period. Each datum represents thelatency of nociceptive responses (sec) ± S.E.M. (n=10). * ped with the control group Effects of the extracts from generated by the tail flick apparatus whilethe latency of nociceptive response (Table 3). leaves and methamphetamine onkg, p.o.) of leaves significantlyincreased the motor activity, compared with thecosolvent group (Table 4). leaves on pentobarbital-induced leaves had a significanteffect on pentobarbital-induced sleep in mice(Table 5).The results demonstrate that the methanol exerted the antinociceptive response leavesheat-induced pain in hot plate test in mice. Thealkaloid extract also increased the pain latencyalkaloid extract significantly affected the painaction in hot plate and tail flick tests involvessupraspinal (Yaksh and Rubi, 1976) and spinal1974). The antinociceptive activity of the methanoland alkaloid extracts is due to action at theantagonist (Gutstein and Akil, 2001). These resultssuggest that the antinociceptive activity of theextracts partly acts at opioid receptors in the leaves, exhibited antinociceptiveTable 2.Effects of the naloxone on methanol and alkaloid extracts of morphine in nociceptive response in hot plate test. Latency of nociceptive response (sec)15304560 minNaloxone + Cosolvent-9.4±0.710.9±0.911.9±
0.810.8±0.8Naloxone + Morphine1010.8±1.211.5±1.212.8±1.317.3±1.7*10012.3±1.215.4±1.213.5±1.115.6±1.9Naloxone + Cosolvent-9.2±0.79.2±0.68.4±0.67.7±0.7Naloxone + Morphine1010.0±0.59.0±0.710.7±1.210.9±1.3209.9±0.79.7±0.88.8±0.78.4±0.6Naloxone (2 mg/kg) was intraperitoneally injected 10 min before test agents administration in mice.The nociceptive response was measured every 15 min over a 60-min period. Each datum represents thelatency of nociceptive responses (sec) ± S.E.M. (n=10). *ped with the control group Dose(mg/kg, p.o.) Effects of the extracts from actions by involving the descending noradrenergicand serotonergic systems of the supraspinal opioidsystem on the mechanical noxious stimulationIn this study, the animals were orally10-30 leaves per day (Anon, 2006). The effectiveTable 3.Effects of the methanol and alkaloid extracts of M. speciosa leaves and morphineon nociceptive response in the tail-flick test. Latency of nociceptive response (sec)15304560 minCosolvent-2.8±0.62.1±0.31.9±0.12.2±0.3Morphine109.5±0.4*9.6±0.4*9.8±0.2*8.1±0.8*503.4±0.62.5±0.32.2±0.22.4±0.3(methanol)1004.2±1.02.9±0.32.5±0.53.5±0.52002.6±0.43.0±0.32.2±0.32.9±0.6Cosolvent-3.8±1.24.0±1.23.4±1.52.6±0.3Morphine109.5±0.3*9.8±0.2*10.0±0.0*9.7±0.3*53.7±1.32.9±0.83.9±0.54.8±1.3(alkaloid)103.2±1.14.1±1.02.8±0.72.1±0.2204.1±1.54.2±1.52.8±0.33.1±0.6the nociceptive response was measured every 15 min over a 60-min period. Each datum representsthe latency of nociceptive responses (sec) ± S.E.M. (n=6) * ped with the control group Dose(mg/kg, p.o.) Table 4.Effects of the methanol and alkaloid extracts of DrugDoseLocomotor activity(mg/kg, p.o.)(counts/30 min)Cosolvent-343.6±48.5Methamphetamine (i.p.)1 1315.9±135.7*50233.3±93.7(methanol)100163.5±63.8200159.2±34.4Cosolvent- 507.5±179.9Methamphetamine (i.p.)1 1
343.5±279.5*5252.9±87.2(alkaloid)10 269.4±101.220258.9±93.9injected intraperitoneally in mice, changes in spontaneous motor activity were measuredover a 30-min period. Each datum represents the mean ± S.E.M. from 10 mice. *p0.05, compared with the control group (Dunnett's test). Effects of the extracts from Table 5.Effects of the methanol and alkaloid extracts of DrugDoseDuration of pentobarbital-(mg/kg, p.o.)induced sleep (min)Cosolvent-62.2±4.15070.1±3.3(methanol)10073.0±3.620075.5±4.9Cosolvent-91.6±7.8586.8±6.6(alkaloid)1090.0±5.32086.1±5.8 leaves were orally administered.measured. Each datum represents the mean ± S.E.M. (n = 10). ciceptive activity. Although the alkaloid extracthowever, it had weak analgesic activity. Takinginto account the yield of 5-10 mg/kg alkaloid leaves,showed no significant antinociceptive effect. values of orally admin-173.20 mg/kg and 4.90 g/kg, respectively, in mice.in the methanol extract have synergistic effects onthe analgesic action. Thus the preparation of leaves or kratom in the methanol extractform has more analgesic efficacy and is less toxicIn general behavioral study, the methanol leaves had nosignificant effect on pentobarbital-induced sleep inmice, so a sedative effect could be excluded fromthis study. Neither of the extracts of activity.The authors are very grateful to the ThailandReferencesAnon. 2006. Mitragyna speciosa - Kratom. Availablefrom http://www.entheology.org/edoto/anmviewer.asp?a=63&z=5.Bruce, R.D. 1985. An up- and down procedure for acutetoxicity testing. Fundam. Appl. Toxicol., 5: 151-Capasso, A., Di Giannuario, A., Loizzo, A., Pieretti,S. and Sorrentino, L. 1996. DexamethasoneD'Amour, F.E. and Smith, D.L. 1941. A method forExp. Ther., 72: 74-79.Ferrini, R., Miragoli, G. and Taccardi, B. 1974. Neuro-pharmacologica
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