Tm

SSa

b

a

ARRAA

KMKMNMC

1

itIhIo2imcnkr

a

0d

Journal of Ethnopharmacology 129 (2010) 344–349

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

he neuromuscular blockade produced by pure alkaloid, mitragynine andethanol extract of kratom leaves (Mitragyna speciosa Korth.)

omsmorn Chittrakarna,∗, Niwat Keawpradubb, Kitja Sawangjaroena,upaporn Kansenalaka, Benjamas Janchaweea

Department of Pharmacology, Faculty of Science, Prince of Songkla University, 15 Kanchanavanich, Hat-Yai, Songkhla 90112, ThailandDepartment of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand

r t i c l e i n f o

rticle history:eceived 29 December 2009eceived in revised form 24 February 2010ccepted 29 March 2010vailable online 3 April 2010

eywords:itragyna speciosaratom

a b s t r a c t

Aim of the study: The effects of pure alkaloid, mitragynine and a methanolic extract of kratom leaves wereinvestigated on neuromuscular junction and compound nerve action potential.Materials and methods: Wistar rats were killed by cervical dislocation and decapitated. The phrenicnerve–hemidiaphragms, hemidiaphragms and sciatic nerve were isolated.Results: Kratom methanolic extract present at 0.1–1 mg/mL and mitragynine (0.0156 mg/mL) decreasedthe muscle twitch on the isolated phrenic nerve–hemidiaphragm and hemidiaphragm preparation.Muscle relaxation caused by kratom extract (1 mg/mL) was greater than the effect of mitragynine. Pan-curonium and succinylcholine potentiated the effect of kratom extract. It also had a direct relaxation

itragynineeuromuscular junctionuscle relaxation

ompound action potential

effect on the hemidiaphragm muscle. The muscle relaxation caused by kratom extract was not antago-nized by neostigmine, tetraethylammonium and calcium chloride. High concentrations of kratom extract(10–40 mg/mL) and mitragynine (2 mg/mL) blocked the nerve conduction, amplitude and duration ofcompound nerve action potential.Conclusions: The mechanism of action of kratom extract might not act as a competitive antagonist ofacetylcholine yet its dominant effect was at the neuromuscular junction and not at the skeletal muscle

or somatic nerve.

. Introduction

Kratom (Mitragyna speciosa Korth.) is a tall leafy tree, in the fam-ly Rubiaceae, and is native to Southeast Asia. It grows in hot, wetropical areas such as Thailand, where it is generally called kratom.t grows mostly in the southern regions of this country. The leavesave long been for “medicinal purposes” and as a narcotic drug.

t was also classified in Category V of a five category classificationf narcotics by the Thai government enacted the Narcotics Act B.E.522, placing kratom along with marijuana. This means that it is

llegal to buy, sell, import, or growing and harvesting. This lawakes planting the tree illegal and requires existing trees to be

ut down. However it is not fully effective, since the tree is indige-

ous to the country and native people prefer to use them. Hence,ratom remains a popular drug in Thailand, especially in southernegions.

Kratom has been traditionally used in Thailand, and there arelso reports of some use in Malaysia. There are two kinds of kratom,

∗ Corresponding author. Tel.: +66 74 288178; fax: +66 74 446678.E-mail address: somsmorn.c@psu.ac.th (S. Chittrakarn).

378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2010.03.035

© 2010 Elsevier Ireland Ltd. All rights reserved.

distinguished by the color of veins in the leaf, red or green. Localpeople preferred to use both of them. In addition to being used,in its own right, as a narcotic drug, it is often used as a substi-tute for opium when opium is unavailable, or to moderate opiumaddiction. Kratom has been reported to be a central nervous systemstimulant, and also depressant. It helps to increase work efficiencyand tolerance to hard work under a scorching sun (Suwanlert,1975). It also uses to treat muscle ache and fatigue (Chucheun,2005).

Over 25 alkaloids have been isolated from kratom leaveswith mitragynine being the most dominant (Chittrakarn etal., 2005). Other alkaloids are mitraphylline, speciogynine, 7-hydroxymitragynine, etc. Mitragynine has an antinociceptiveaction through the supraspinal opioid receptors and descendingnoradrenergic and serotonergic systems (Matsumoto et al., 1996).Mitragynine inhibited the vas deferens contraction elicited bynerve stimulation, probably through its blockage of neuronal Ca2+

channels (Matsumoto et al., 2005). Mitragynine inhibits guinea-pig

ileum contraction in vitro via the opioid receptor (Watanabe et al.,1997). 7-Hydroxymitragynine has a more potent analgesic activitythan that of morphine (Matsumoto et al., 2004). In folk medicine,it has been used to treat diarrhea. It was found that methanolicextract of kratom had the antidiarrhreal activity and decreased

hnoph

bast

slt

2

2

pFtPn

2

ettVr7

2

waeewetaAmmapNndy1

2

fU2dlf

2

c

2.7. Isolated sciatic nerve preparation

The rat was anesthetized by intraperitoneal injection of sodiumpentobarbital, 50 mg/kg body weight. Both the left and right sci-

S. Chittrakarn et al. / Journal of Et

ody weight (Chittrakarn et al., 2008). The fresh leaves are usu-lly chewed, often continuously, by workers or manual laborerseeking a numbing, stimulating effect that helps to improve theirolerance to work and relieves muscle strains.

From the traditional medicine use for relief muscle ache andtrain, the effects of mitragynine and methanol extract of kratomeaves on neuromuscular junction and somatic nerves were inves-igated in this study.

. Materials and methods

.1. Plant material

Kratom leaves (red vein type) were collected from Satoonrovince, in the southern part of Thailand during the months ofebruary–May 2005. Specimens of this plant have been deposited athe PSU Herbarium of the Department of Biology, Faculty of Science,rince of Songkla University, Thailand with the specimen voucherumber PSU 012821.

.2. Preparation of the methanolic extract

Air-dried leaves were pulverized by grinding and then mac-rated, at room temperature, with absolute methanol for 7 days,wice, while stirring 2–3 times/day. The extracts were mixed, fil-ered and concentrated using a rotary evaporator (BUCHI, B 169acumn-System, Switzerland). Then they were freeze-dried (Cor-osion Resistant Freezer Drier, FTS System, Inc., USA). The yield was.92% (w/w).

.3. Isolation of mitragynine

The dried product from the methanolic extract of kratom leavesas dissolved in 10% acetic acid solution. This solution was shaken

nd left overnight. The acidic filtrate was washed with petroleumther, adjusted to pH 9 with 25% ammonia solution, and thenxtracted with chloroform. The chloroform extract was washedith distilled water, dried over anhydrous sodium sulfate and

vaporated to yield a dry crude alkaloid extract. According tohe isolation procedure, the yield of crude alkaloid extract waspproximately 0.25% based on fresh weight of Mitragyna speciosa.n aliquot (2.5 g) was then subjected to silica gel column chro-atography, eluted with 5% methanol in chloroform to obtain aajor alkaloid (1.25 g), which appeared as a single spot on TLC

nalysis (four different solvent systems). It was found to be aure compound upon spectroscopic analysis (including 1H and 13CMR, IR, and mass spectrometry), and identified to be mitragy-ine by comparing the obtained spectra data with the publishedata (Shellard et al., 1978; Houghton et al., 1991). Over all, theield of mitragynine in the methanolic extract was approximately.56%.

.4. Experimental animals

Wistar rats of either sex, weighing 200–250 g, were obtainedrom the Southern Laboratory Animal Facility, Prince of Songklaniversity. They were housed in a temperature controlled room at5 ± 2 ◦C with a relative humidity at 50 ± 5% and a 12 h light/12 hark cycle. They were fed with animal food pellets and water ad

ibitum. The study protocol was approved by the Ethics Committeeor Experimental Animals, Prince of Songkla University.

.5. Isolated phrenic nerve–hemidiaphragm preparation

Rats were killed by cervical dislocation and decapitated. Thehest was opened and the diaphragm was divided into right

armacology 129 (2010) 344–349 345

and left parts. Each part of the diaphragm with its attachedphrenic nerve was removed (Bulbring, 1946). Isolated phrenicnerve–hemidiaphragm preparations were mounted vertically intoan 80 mL organ bath containing Krebs solution (mM: NaCl 118.07,NaHCO3 25, KCl 4.69, MgSO4·7H2O 1.18, KH2PO4 1.18, CaCl2 2.52,glucose 10.09) and continuously aerated with 95% O2 and 5% CO2at a temperature of 30 ◦C. The muscle tension was maintainedat 2 g and attached to an FT-03 force transducer (Grass Instru-ment Co., Quincy, MA, USA), connected to a Grass 79D polygraph(Grass Instrument Co., Quincy, MA, USA) for recording isotonic con-traction. The phrenic nerve was gently drawn through the loopsof a bipolar platinum stimulating electrode connected to a GrassS88 stimulator via a SIU5 stimulus isolation unit (Grass Instru-ment Co., Quincy, MA, USA). The nerve–muscle preparation wasleft in the organ bath for 30 min to reach equilibrium before car-rying out experiments. The neurally evoked twitch was recordedby stimulation with electrical pulses of supramaximal voltage at afrequency of 0.4 Hz and duration of 0.6 ms throughout all experi-ments.

2.6. Isolated hemidiaphragm preparation

The preparation was established as previously mentioned forthe isolated phrenic nerve–hemidiaphragm. The phrenic nerve wasremoved from the hemidiaphragm. The fan shaped hemidiaphragmwas then transferred into the organ bath containing 80 mL Krebssolution, aerated with 95% O2 and 5% CO2 at a temperature of 30 ◦C.The muscle tension was set at 2 g. The needle platinum electrodewas passed through the basement of hemidiaphragm. The directmuscle twitch was recorded by electrical stimulation of supramax-imal voltage at a frequency of 0.4 Hz and duration of 0.6 ms. Thepreparation was completely curarized by adding 0.005 mM pan-curonium (Chongrak, 1985). Thus, the contractile response was dueonly to the skeletal muscle response.

Fig. 1. The percentage relaxation (mean ± SEM, n = 8) of the twitch amplitudeproduced by the kratom extract present at 0.1, 0.25, 0.5 and 1 mg/mL and mitragy-nine (MG) present at 0.0156 mg/mL on the isolated phrenic nerve–hemidiaphragmpreparation. *Statistical significance p < 0.05 vs. kratom extract present at1 mg/mL.

3 hnoph

acpc(Ntts0(pUUd

muscle relaxation. The percent change of nerve conduction, ampli-

Fn

46 S. Chittrakarn et al. / Journal of Et

tic nerves were dissected out. The nerve was placed in a threeompartment chamber which bipolar stimulating and recordinglatinum electrodes were placed under the nerve. The centralompartment had a volume of 1 mL containing Tyrode’s solutionmM: NaCl 140.31, NaHCO3 11.90, KCl 2.68, MgCl2·6H2O 1.08,aH2PO4·2H2O 0.38, CaCl2 1.8, glucose 5.05) or test drugs. The

wo sides of the chamber contained liquid paraffin to moistenhe nerve (Chongrak, 1985). The nerve was stimulated with aupramaximal voltage at a frequency of 0.6 Hz and duration of.04 ms by a Grass S88 stimulator via a SIU5 stimulus isolation unitGrass Instrument Co., Quincy, MA, USA). The compound action

otentials were displayed on an oscilloscope (Tektronix TDS310,SA) via an AM502 Differential Amplifier (Tektronix TM502A,SA) for recording nerve conduction (ms), amplitude (mV) anduration (ms).

ig. 2. Potentiative muscle relaxation of pancuronium (Panc 0.6 �M), succinylcholinerve–hemidiaphragm preparation.

armacology 129 (2010) 344–349

2.8. Standard drugs

Pancuronium, d-tubocurarine, succinylcholine, neostigmine,tetraethylammonium and sodium pentobarbital were purchasedfrom Sigma–Aldrich (St. Louis, USA). Xylocaine was purchased fromOLIC (Thailand) Limited.

2.9. Data analysis

Results were expressed as a mean ± SEM, n = 8 of the percentage

tude and duration of the compound nerve action potential werealso expressed as a mean ± SEM, n = 6. Significant differences wereanalyzed by SPSS 11 for windows followed by a Tukey test for com-parison of more than two groups and independent-samples t test

e (Suc 3.1 �M) and kratom extract (MS 0.1 mg/mL) on the isolated phrenic

hnopharmacology 129 (2010) 344–349 347

fw

3

3

tmerpawt(oocNdtdcttae

3

aae1mk

Fo0s

S. Chittrakarn et al. / Journal of Et

or comparison of two groups. A probability level of less than 5%as considered significant.

. Results

.1. Isolated phrenic nerve–hemidiaphragm preparation

Kratom extract produced a decrease of the twitch contraction ofhe rat phrenic nerve–hemidiaphragm preparation. The degree of

uscle relaxation depended on the concentration and time. Kratomxtract present at 0.25, 0.5 and 1 mg/mL produced a completeelaxation in 50, 25 and 15 min, respectively (Fig. 1). Mitragynineresent at 0.0156 mg/mL also produced a complete muscle relax-tion in 45 min. The percent relaxation produced by mitragynineas significantly difference from kratom extract in the concentra-

ion of 1 mg/mL at p < 0.05. A low concentration of pancuronium0.6 �M) and also succinylcholine (3.1 �M) increased the effectf the kratom extract (Fig. 2). The concentration–response curvef kratom extract and kratom extract in the presence of pan-uronium or succinylcholine was also shifted to the left (Fig. 3).eostigimine (20 �M) antagonized the muscle relaxation caused by-tubocurarine (5 �M). However this standard drug did not reversehe effect of the kratom extract (Fig. 4). Muscle contraction wasecreased in Krebs solution which lack off calcium chloride and cal-ium chloride (1.25 mM) could be increased muscle contraction inhis condition (Fig. 5a). Tetraethylammonium (1 mM) antagonizedhe effect of pancuronium (0.005 mM) (Fig. 5b). Calcium chloridend tetraethylammonium could not antagonize the effect of kratomxtract (Fig. 5).

.2. Isolated hemidiaphragm preparation

The kratom extract and mitragynine were also investigated forny direct effect on the diaphragm skeletal muscle. Kratom extract

nd mitragynine decreased the muscle twitch response. Kratomxtract produced complete muscle relaxation at a concentration ofmg/mL in 55 min time (Fig. 6). Although, mitragynine produceduscle relaxation significantly difference less than the effect of

ratom extract.

ig. 3. The synergistic effect of pancuronium (0.6 �M) and succinylcholine (3.1 �M)n the muscle relaxation effect (mean ± SEM, n = 8) of kratom extract (MS) present at.1 mg/mL on the isolated phrenic nerve–hemidiaphragm preparation. *Statisticalignificance p < 0.05 vs. MS.

Fig. 4. Effect of neostigmine (Neo 20 �M) ond-tubocurarine (d-Tc 5 �M) and kratomextract (MS 0.5 mg/mL) on the isolated phrenic nerve–hemidiaphragm preparation.

3.3. Compound action potential on the isolated sciatic nerve

The kratom extract (10, 20 and 40 mg/mL) was tested on theisolated rat sciatic nerve. The nerve conduction, amplitude andduration of the compound nerve action potential were recorded.Kratom extract decreased nerve conduction, amplitude and dura-tion. Kratom extract present at 10 and 20 mg/mL prolonged theduration of the compound action potential at the first period of timebut at 40 mg/mL it blocked the compound action potential com-pletely at the 55 min time (Table 1). Mitragynine present at 2 mg/mLcaused a gradual decrease of the nerve conduction, amplitude andduration of the compound action potential. However, xylocainepresent at 0.1% could block the compound action potential com-pletely within 25 min.

4. Discussion

Kratom extract decreased muscle contraction on the isolatedphrenic nerve–hemidiaphragm preparation. It caused completemuscle relaxation at a concentration of more than 0.1 mg/mL.Kratom extract at the highest concentration used (1 mg/mL)completely relaxed muscle contraction within 15 min. Electricalstimulation via the phrenic nerve causes acetylcholine liberationfrom the nerve ending. Acetylcholine binds to the nicotinic recep-tors of the motor endplates and causes diaphragm contraction(Silverthorn, 2007). Kratom extract may interfere with the neuro-muscular junction so that the skeletal muscle could not contract.Low concentrations of pancuronium (0.6 �M) and succinylcholine(3.1 �M) potentiated the effect of 0.1 mg/mL kratom extract. Pan-curonium is a typical non-depolarizing curare-mimetic musclerelaxant. It acts as a competitive antagonist to acetylcholine atnicotinic receptors. Succinylcholine acts as a depolarizing neuro-muscular blocker. It imitates the action of acetylcholine at theneuromuscular junction and causes spontaneous depolarizationupon association with the nicotinic receptors at the neuromuscularjunction. Then it causes an endplate potential that is less than theaction potential. The muscle undergoes flaccid paralysis (Taylor,2006a). Kratom extract may act at the neuromuscular junction asdoes pancuronium and succinylcholine.d-Tubocurarine is a classical competitive antagonist acting at

the neuromuscular junctions. This causes muscle paralysis, as

does pancuronium. Neostigmine is a reversible cholinesteraseinhibitor that prevents the breakdown of acetylcholine (Taylor,2006b). Acetylcholine competes with d-Tubocurarine at the nico-tinic receptors and skeletal muscle increases its twitch contraction.Neostigmine reverses the effect of d-tubocurarine, pancuronium

348 S. Chittrakarn et al. / Journal of Ethnopharmacology 129 (2010) 344–349

F (Ca2+

1 the i

adfnrnnc

doefm

saito

Fohp

ig. 5. Effect of calcium chloride (Ca2+ 1.25 mM) on calcium free in Krebs solutionmM) on pancuronium (Panc 0.005 mM) and (b) kratom extract (MS 0.5 mg/mL) on

nd competes with other competitive neuromuscular blockingrugs. Tetraethylammonium increases acetylcholine liberationrom motor nerve endings (Stovner, 1958). Then it can antago-ize the neuromuscular block caused by pancuronium. The muscleelaxation produced by kratom extract was not antagonized byeostigmine and tetraethylammonium. Thus kratom extract mightot therefore act as a competitive or non-depolarized neuromus-ular blocking drug.

The neuromuscular transmission can be interrupted by calciumeficiency. It would appear to be caused by a reduced transmitterutput from presynaptic site. Calcium chloride did not reverse theffect of kratom extract. In this way the block may differ sharplyrom that produced by pancuronium or d-tubocurarine. Kratom

ight not affect acetylcholine release.Kratom extract was also tested directly on the hemidiaphragm

keletal muscle. It caused muscle relaxation. The percentage relax-tion of the kratom extract on the skeletal muscle was less thants effect on the isolated phrenic nerve–hemidiaphragm prepara-ion. Complete relaxation of the muscle by kratom extract was onlybserved at a concentration of 1 mg/mL. The skeletal muscle con-

ig. 6. Percentage relaxation (mean ± SEM, n = 8) produced by increasing amountsf kratom extract (0.1–1 mg/mL) and mitragynine (0.0156 mg/mL) on the isolatedemidiaphragm preparation. *Statistical significance p < 0.05 vs. kratom extractresent at 1 mg/mL.

free) and kratom extract (MS 0.5 mg/mL); (a) effect of tetraethylammonium (TEAsolated phrenic nerve–hemidiaphragm preparation.

tracts by the electrical impulse pass to the sarcoplasmic reticulum.The calcium gates in the membrane of the sarcoplasmic reticulumopen. As a result, calcium diffuses out of the sarcoplasmic reticulumand among the myofilaments. Then the muscle fibers get contrac-tion. The kratom extract may interfere these processes of musclecontraction.

The sciatic nerve was isolated to test the effect of kratomextract. Kratom extract, present at the high concentrations of 10,20 and 40 mg/mL blocked the compound nerve action potential.It decreased nerve conduction, amplitude and duration of thecompound action potential. The compound action potential wascompletely blocked at 40 mg/mL of kratom extract. The nerveaction potential occurs when sodium ions enter the cell throughsodium channels to induce depolarization. Then, the potassiumchannels open, causing potassium ions rush out to produce repo-larization. Kratom extract may interrupt the influx or efflux of theseions. Kratom extract therefore had its dominant effect on the neu-romuscular junction rather than on the skeletal muscle and thesomatic motor nerve.

Mitragynine, a major alkaloid from Mitragyna speciosa Korth.,was also tested on the isolated phrenic nerve–hemidiaphragm,hemidiaphragm and sciatic nerve preparation. Muscle relaxationcaused by mitragynine present at 0.0156 mg/mL was significantdifference less than the effect of kratom extract (1 mg/mL) bothin the isolated phrenic nerve–hemidiaphragm and in the directedhemidiaphragm preparation. According to the isolation of mitragy-nine, the methanolic kratom extract contained 1.56% mitragynine.Hence, kratom extract in the concentration of 1 mg/mL containedmitragynine 0.0156 mg/mL. Muscle relaxation produced by kratommay be effected by mitragynine and other alkaloids in kratomleaves. High concentrations of mitragynine (2 mg/mL) produced ablockage of the compound nerve action potential but it did not pro-duce a complete blockage. Xylocaine solutions containing lidocainehydrochloride is used as a common local anesthetic and antiar-rhythmic drug. Lidocaine blocks the fast voltage gated sodium(Na+) channels in the cell membrane. With sufficient blockage,the membrane of the presynaptic neuron will not depolarize and

so fails to transmit an action potential, leading to its anestheticeffects (Catterall and Mackie, 2006). Xylocaine (0.1%) completelyblocked the compound action potential within 15 min. A methanolextract of kratom leaf contained 1.6% of the major alkaloid mitrag-ynine. Hence the kratom extract added at 40 mg/mL contained only

S. Chittrakarn et al. / Journal of Ethnoph

Tab

le1

Effe

ctof

0.1%

xylo

cain

e,m

itra

gyn

ine

(MG

)p

rese

nt

at2

mg/

mL

and

krat

omex

trac

t(M

S)p

rese

nt

at10

,20

and

40m

g/m

Lon

the

per

cen

tage

chan

geof

ner

veco

nd

uct

ion

,am

pli

tud

ean

dd

ura

tion

ofth

eco

mp

oun

dac

tion

pot

enti

al(m

ean

±SE

M,n

=6)

onth

eis

olat

edra

tsc

iati

cn

erve

.

Tim

e(m

in)

%ch

ange

inn

erve

con

du

ctio

n%

chan

gein

amp

litu

de

%ch

ange

ind

ura

tion

0.1%

xylo

cain

eM

G2

mg/

mL

MS

10m

g/m

LM

S20

mg/

mL

MS

40m

g/m

L0.

1%xy

loca

ine

MG

2m

g/m

LM

S10

mg/

mL

MS

20m

g/m

LM

S40

mg/

mL

0.1%

xylo

cain

eM

G2

mg/

mL

MS

10m

g/m

LM

S20

mg/

mL

MS

40m

g/m

L

00

±0

0±

00

±0

0±

00

±0

0±

00

±0

0±

00

±0

0±

00

±0

0±

00

±0

0±

00

±0

55.

9±

10.5

1.8

±5.

2−1

.5±

1.2

−5.4

±3.

6−1

0.0

±5.

0−6

5.5

±6.

32.

4±

10.7

−5.0

±3.

2−1

1.2

±3.

6−3

2.0

±16

.117

.8±

5.8

3.5

±5.

04.

3±

4.2

6.8

±2.

3−1

3.0

±17

.710

22.5

±31

.51.

8±

5.2

−3.2

±1.

6−7

.1±

3.4

−15.

8±

4.4

−87.

4±

4.0

3.4

±15

.7−6

.4±

5.6

−13.

0±

4.2

−33.

0±

17.0

2.6

±21

.34.

8±

5.0

8.1

±4.

68.

2±

1.9

−15.

0±

17.5

15−4

5.8

±35

.61.

8±

5.2

−7.4

±3.

1−1

1.3

±4.

2−3

8.3

±14

.7−9

6.0

±3.

0−0

.3±

19.5

−10.

6±

5.5

−19.

4±

5.6

−41.

9±

18.7

−58.

3±

27.1

9.7

±6.

511

.9±

5.4

10.6

±2.

4−2

8.5

±22

.720

−79.

2±

20.8

1.8

±5.

2−9

.0±

3.5

−12.

9±

3.6

−52.

5±

18.0

−99.

4±

0.6

− 16.

4±

18.6

−14.

9±

7.9

−27.

5±

7.8

−47.

5±

17.4

−77.

8±

22.2

22.3

±9.

614

.8±

6.3

11.8

±4.

5−3

0.0

±22

.325

−100

.0±

01.

8±

5.2

−9.0

±2.

9−1

2.9

±3.

6−5

5.8

±16

.5−1

00.0

±0

−34.

0±

17.8

−22.

3±

12.3

−39.

1±

11.8

−56.

6±

14.2

−100

.0±

024

.6±

14.8

16.9

±7.

916

.3±

6.0

−31.

3±

21.8

30−3

.7±

24.1

−11.

7±

3.6

−14.

6±

5.2

−60.

0±

17.2

−56.

2±

14.8

−30.

6±

12.2

−44.

8±

12.8

−69.

4±

9.8

16.9

±27

.010

.2±

10.3

−0.6

±12

.9−3

5.8

±20

.635

−12.

4±

19.0

−17.

4±

5.7

−24.

2±

8.4

−65.

0±

16.3

−69.

1±

12.0

−31.

5±

20.4

−65.

1±

13.3

−82.

6±

7.3

1.1

±24

.87.

2±

10.8

−31.

5±

18.9

−36.

7±

23.8

40−1

2.9

±21

.3−3

7.4

±13

.3−5

6.7

±13

.4−7

8.3

±14

.2−8

3.1

±9.

4−4

9.2

±18

.2−7

2.7

±10

.9− 9

2.4

±5.

4−3

0.0

±31

.9−1

8.7

±17

.6−4

1.8

±19

.8−6

4.4

±22

.645

−20.

4±

18.6

−57.

4±

14.9

−69.

6±

16.5

−86.

7±

13.3

−92.

4±

5.7

-63.

0±

17.1

−81.

0±

9.5

−98.

3±

1.7

−40.

0±

28.0

−35.

3±

24.1

−57.

2±

18.0

−86.

1±

13.9

50−8

2.9

±15

.6−6

0.4

±13

.9−7

0.8

±18

.7−8

6.7

±13

.3−9

7.1

±2.

9−7

5.5

±14

.1−8

7.6

±7.

7−9

9.6

±0.

4−7

5.6

±24

.4−4

9.4

±24

.8−6

7.5

±16

.9−8

4.7

±15

.355

−86.

6±

13.3

−72.

5±

13.6

−75.

0±

17.1

−100

.0±

0−9

8.5

±1.

5−8

2.8

±11

.0−9

1.5

±6.

5−1

00.0

±0

−75.

6±

24.4

−53.

8±

22.3

−76.

6±

15.7

−100

.0±

060

−86.

6±

13.3

−74.

5±

12.8

−87.

5±

12.5

−99.

5±

0.5

−88.

8±

9.4

−95.

0±

3.3

−79.

5±

20.5

−73.

1±

19.3

−77.

3±

15.3

armacology 129 (2010) 344–349 349

0.61 mg/mL of mitragynine. Thus the inhibition of the compoundaction potential may be due to mitragynine and also other alkaloidspresent in the kratom leaf extract.

5. Conclusion

A methanol extract of kratom leaf and a major alkaloid, mitragy-nine produced skeletal muscle relaxation. Its mechanism of actionwas not by a competitive antagonism of acetylcholine binding. Ithad a synergistic effect with pancuronium and succinylcholine.Kratom extract and mitragynine also had a direct effect on skele-tal muscle by decreasing the muscle twitch. The compound actionpotential was blocked by high concentrations of kratom extract andmitragynine. Thus, kratom extract had a greater effect at the neuro-muscular junction than on the skeletal muscle or somatic nerve. It ispossible that alkaloid components of the kratom extract other thanmitragynine may have an effect on the compound action poten-tial.

Acknowledgement

This research project was supported by a grant from Prince ofSongkla University through Contract No. SCI50173.

References

Bulbring, E., 1946. Observations on the isolated phrenic nerve diaphragm prepa-ration of the rat. British Journal of Pharmacology and Chemotherapy 1,38–61.

Catterall, W.A., Mackie, K., 2006. Local anesthetics. In: Brunton, L.L., Lazo, J.S., Parker,K.L. (Eds.), Goodman & Gilman’s The Pharmacological Basis of Therapeutics.McGraw-Hill, New York, pp. 369–388.

Chittrakarn, S., Keawwongsri, P., Kumarnsit, E., Sawangjaroen, K., Keawpradub, N.,2005. Botanical description and pharmacological activity. In: Assanangkorn-chai, S., Siriwong Na Ayutthaya, A. (Eds.), Kratom in Thai Social. Bangkokblock,Bangkok, pp. 97–112.

Chittrakarn, S., Sawangjaroen, K., Prasettho, S., Janchawee, B., Keawpradub, N., 2008.Inhibitory effects of kratom leaf extract (Mitragyna speciosa Korth.) on the ratgastrointestinal tract. Journal of Ethnopharmacology 116, 173–178.

Chongrak, S., 1985. The Pharmacological Study of Cimetidine on MyoneuralJunction and Cardiovascular System in Rat. Graduate School, Chiang MaiUniversity.

Chucheun, C., 2005. Social, culture and human life in kratom community. In:Assanangkornchai, S., Siriwong Na Ayutthaya, A. (Eds.), Kratom in Thai Social.Bangkokblock, Bangkok, pp. 17–96.

Houghton, P.J., Latiff, A., Said, I.M., 1991. Alkaloids from Mitragyna speciosa. Phyto-chemistry 30, 347–350.

Matsumoto, K., Horie, S., Ishikawa, H., Takayama, H., Aimi, N., Ponglux, D., Watanabe,K., 2004. Antinoceptive effect of 7-hydroxymitragynine in mice: discovery of anactive opioid analgesic from the Thai medicinal herb Mitragyna speciosa. LifeSciences 74, 2143–2155.

Matsumoto, K., Mizowaki, M., Takayama, H., Sakai, S., Aimi, N., Watanabe, H., 1996.Suppressive effect of mitragynine on the 5-methoxy-N,N-dimethyltryptamine-induced head-twitch response in mice. Pharmacology Biochemistry andBehavior 57, 319–323.

Matsumoto, K., Yamamoto, L.T., Watanabe, K., Yano, S., Shan, J., Pang, P.K.T., Ponglux,D., Takayama, H., Horie, S., 2005. Inhibitory effect of mitragynine, an analgesicalkaloid from Thai herbal medicine, on neurogenic contraction of the vas defer-ens. Life Sciences 78, 187–194.

Shellard, E.J., Houghton, P.J., Resha, M., 1978. The Mitragyna species of Asia. PartXXXI. The alkaloids of Mitragyna speciosa Korth from Thailand. Planta Medica34, 26–36.

Silverthorn, D.U., 2007. Human Physiology. Pearson Education, San Francisco.Stovner, J., 1958. The anticurare activity of tetraethylammonium (TEA). Acta Phar-

macology and Toxicology 14, 317–332.Suwanlert, S., 1975. A study of kratom eaters in Thailand. ODCCP-Bulletin on Nar-

cotics 27, 21–27.Taylor, P., 2006a. Agents acting at the neuromuscular junction and autonomic

ganglia. In: Brunton, L.L., Lazo, J.S., Parker, K.L. (Eds.), Goodman & Gilman’sThe Pharmacological Basis of Therapeutics. McGraw-Hill, New York, pp. 217–236.

Taylor, P., 2006b. Anticholinesterase agents. In: Brunton, L.L., Lazo, J.S., Parker,

K.L. (Eds.), Goodman & Gilman’s The Pharmacological Basis of Therapeutics.McGraw-Hill, New York, pp. 201–216.

Watanabe, K., Yano, S., Horie, S., Yamamoto, L.T., 1997. Inhibitory effect of mitrag-ynine, an alkaloid with analgesic effect from Thai medicinal plant Mitragynaspeciosa, on electrically stimulated contraction of isolated guinea-pig ileumthrough the opioid receptor. Life Sciences 60, 933–942.