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Behavioral and neurochemical characterization of kratom (Mitragyna speciosa) extract

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Mitragyna speciosa and its extracts are named kratom (dried leaves, extract). It contains several alkaloids and is used in traditional medicine to alleviate musculoskeletal pain, hypertension, coughing, diarrhea, and as an opiate substitute for addicts. Abuse and addiction to kratom is described, and kratom has attracted increasing interest in Western countries. Individual effects of kratom on opioidergic, adrenergic, serotonergic, and dopaminergic receptors are known, but not all of the effects have been explained. Pharmacokinetic and pharmacodynamic data are needed. The effects of kratom extract on mice behavior were investigated following oral (po), intraperitoneal (ip), and intracerebroventricular (icv) application. Receptor-binding studies were performed. In μ opioid receptor knockout mice (-/-) and wild type (+/+) animals, the extract reduced locomotor activity after ip and low po doses in +/+ animals, but not after icv administration. The ip effect was counteracted by 0.3 mg/kg of apomorphine sc, suggesting dopaminergic presynaptic activity. An analgesic effect was only found in -/- mice after icv application. Norbinaltorphimine abolished the analgesic effect, but not the inhibitory effect, on locomotor activity, indicating that the analgesic effect is mediated via κ opioid receptors. Oral doses, which did not diminish locomotor activity, impaired the acquisition of shuttle box avoidance learning. There was no effect on consolidation. Binding studies showed affinity of kratom to μ, δ, and κ opioid receptors and to dopamine D1 receptors. The results obtained in drug-naïve mice demonstrate weak behavioral effects mediated via μ and κ opioid receptors.
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ORIGINAL INVESTIGATION
Behavioral and neurochemical characterization of kratom
(Mitragyna speciosa) extract
Anne-Christin Stolt &Helmut Schröder &Hartmud Neurath &Gisela Grecksch &
Volker Höllt &Markus R. Meyer &Hans H. Maurer &Nancy Ziebolz &
Ursula Havemann-Reinecke &Axel Becker
Received: 4 April 2013 /Accepted: 24 June 2013 / Published online: 12 July 2013
#Springer-Verlag Berlin Heidelberg 2013
Abstract
Objective Mitragyna speciosa and its extracts are named
kratom (dried leaves, extract). It contains several alkaloids
and is used in traditional medicine to alleviate musculoskel-
etal pain, hypertension, coughing, diarrhea, and as an opiate
substitute for addicts. Abuse and addiction to kratom is de-
scribed, and kratom has attracted increasing interest in Western
countries. Individual effects of kratom on opioidergic, adren-
ergic, serotonergic, and dopaminergic receptors are known, but
not all of the effects have been explained. Pharmacokinetic and
pharmacodynamic data are needed.
Methods The effects of kratom extract on mice behavior were
investigated following oral (po), intraperitoneal (ip), and in-
tracerebroventricular (icv) application. Receptor-binding stud-
ies were performed.
Results In μopioid receptor knockout mice (/) and wild
type (+/+) animals, the extract reduced locomotor activity
after ip and low po doses in +/+ animals, but not after icv
administration. The ip effect was counteracted by 0.3 mg/kg
of apomorphine sc, suggesting dopaminergic presynaptic
activity. An analgesic effect was only found in /mice after
icv application. Norbinaltorphimine abolished the analgesic
effect, but not the inhibitory effect, on locomotor activity,
indicating that the analgesic effect is mediated via κopioid
receptors. Oral doses, which did not diminish locomotor
activity, impaired the acquisition of shuttle box avoidance
learning. There was no effect on consolidation. Binding
studies showed affinity of kratom to μ,δ, and κopioid
receptors and to dopamine D1 receptors.
Conclusions The results obtained in drug-naïve mice dem-
onstrate weak behavioral effects mediated via μand κopioid
receptors.
Keywords Kratom .Locomotor activity .Pain threshold .
Anxiety .Shuttle box learning .Receptor binding .Mice
Introduction
Kratom, which is botanically known as Mitragyna speciosa
Korth (Rubiaceae), is a tropical tree indigenous to Thailand,
Malaysia, Indonesia, and Papua New Guinea and is cultivat-
ed mostly for its leaves (Ahmad and Aziz 2012). More than
40 alkaloids have been isolated from its leaves (Shellard 1974).
The alkaloids are used in traditional medicine to alleviate mus-
culoskeletal pain, hypertension, coughing, and diarrhea; to im-
prove physical stamina and sexual performance; and as a sub-
stitute for morphine in treating addicts (Ahmad and Aziz 2012;
Apryanietal.2010; Assanangkornchai et al. 2007;Wardetal.
2011). On the other hand, kratom seems to have an addictive
potential (McWhirter and Morris 2010; Sheleg and Collins
2011;Hassanetal.2013). Chronic exposure to kratom can led
to withdrawal symptoms (Suwanlert 1975). Kratom has been
described as exerting both stimulant and depressive effects
(Ahmad Akhibi 2008). The effects of the predominant alkaloids,
i.e., mitragynine, 7-hydroxymitragynine, and paynantheine,
A.<C. Stolt :H. Schröder :G. Grecksch :V. Höllt :A. Becker (*)
Institute of Pharmacology and Toxicology, Medical Faculty, O. v.
Guericke University, Leipziger Str. 44, 30124 Magdeburg, Germany
e-mail: axel.becker@med.ovgu.de
H. Neurath
Center of Pharmacology and Toxicology, Georg August University,
Robert-Koch-Str. 40, 37075 Göttingen, Germany
M. R. Meyer :H. H. Maurer
Department of Experimental and Clinical Toxicology, Institute of
Experimental and Clinical Pharmacology and Toxicology,
Saarland University, D-66421 Homburg (Saar), Germany
N. Ziebolz :U. Havemann-Reinecke
Department of Psychiatry and Psychotherapy, Georg August
University, von Siebold-Str. 5, 37075 Göttingen, Germany
U. Havemann-Reinecke
DFG Center for Nanoscale Microscopy and Molecular Physiology
of the Brain, 37075 Göttingen, Germany
Psychopharmacology (2014) 231:1325
DOI 10.1007/s00213-013-3201-y
have mainly been explained by interactions with opioidergic,
adrenergic, serotonergic, and dopaminergic receptors (Boyer
et al. 2008; Horie et al. 2005; Matsumoto et al. 1996a,b,
1997; Reanmongkol et al. 2007; Shamima et al. 2012;
Yamamoto et al. 1999).
A number of publications report the effects of single
constituents of the extract. Beside its traditional use in its
countries of origin, kratom, in the form of dried leaves or
extract, has also attracted increasing interest in Western
countries. Here, it appears to enjoy widespread use among
specific populations (Ward et al. 2011). However, there is a
paucity of systematic pharmacokinetic, pharmacodynamic,
and toxicological data. Therefore, the aim of the present
study was to investigate the effects of commercially avail-
able kratom extract on mice behavior. We focused on behav-
ioral patterns that are associated with the function of opioid
receptors such as locomotor activity, anxiety, and pain
threshold using the κantagonist norbinaltorphimine and
disruption of the μsystem by receptor deletion. Moreover,
kratom effects on learning were studied. From the literature,
it is well known that alkaloids contained in kratom extract
interact with opioid receptors. Since the concentration of
these alkaloids varies dependent on the geographical origin
of the plant, the method of extraction, and others, we also
performed receptor binding studies to correlate alkaloid con-
centrations and behavioral effects.
Material and methods
The work reported here was conducted in accordance with
EC regulations and those of the National Act on the Use of
Experimental Animals (Germany). The experimental proto-
col was approved by the Ethics Commission of the Federal
State of Saxony-Anhalt.
Animals
To investigate the effects of kratom on analgesia, anxiety,
and interaction with apomorphine and norbinaltorphimine,
male wild type mice (+/+) and μopioid receptor (MOR)
knockout mice (/) were used. Breeding of the /animals
(Loh et al. 1998), genotyping,
3
H-[D-Ala
2
,N-MePhe
4
,Gl-ol
5
]-
enkephalin (DAMGO) receptor autoradiography, and results
of binding studies have been published earlier (Becker et al.
2000). After genotyping, homozygote animals were bred
according to a standard breeding protocol. To study the effects
of kratom on the acquisition and consolidation of a condi-
tioned reaction in a shuttle box, animals showing a moderate
learning performance are needed in order to assess improve-
ment or worsening due to drug effects. Therefore, male
C57Bl6/N mice (Charles River, Sulzfeld, Germany) were
used. For the binding assay, male RjHan:WI rats (Janvier, Le
Genest Saint Isle, France) were used.
The animals were kept under controlled laboratory condi-
tions with lighting regime LD = 12:12 (light on at 6:00 a.m.),
temperature 20 ± 2°°C, and relative air humidity 5060 %.
The animals had free access to standard rat pellets (R/H
maintenance, ssniff, Soest, Germany) and tap water. After
weaning on day 21 postpartum, the animals were separated
according to sex and sheltered litterwise in groups of five in
Macrolon III cages. Only the minimum number of animals
necessary to produce reliable scientific data was used.
Surgery
Seven-week-old mice were deeply anesthetized with etomidate
(Radenarcon®, Arzneimittelwerk Dresden, Germany, 10 mg/
kg ip) and fixed in a stereotactic frame (lambda 1 mm below
bregma). A hole was drilled into the bone at the stereotactic
coordinates AP 0.2 mm and lateral 0.2 mm (relative to bregma).
A microcannula (outer diameter 0.8 mm, inner diameter 0.6 mm,
length 2.1 mm) was inserted in the right lateral ventricle and
fixed with tissue adhesive (Histoacryl®, B. Braun Melsungen,
Germany). For final fixation, a socket made of dental cement
(Paladur®, Heraeus Kulzer GmbH, Wehrheim, Germany) was
mounted. The postsurgery convalescence period was 1 week.
On completion of the experiments, the mice were sacrificed
using an overdose of chloral hydrate. After icv injection of
5μl toluidine blue, the brains were removed and placement of
the cannulas was verified.
Behavioral testing
The male mice were aged 89 weeks at the beginning of the
experiments. Their body weight was 2429 g. All experi-
ments were performed between 8:00 am and 3:00 pm. The
animals were randomly ordered for testing to avoid the bias
of circadian rhythms. The animals were used once only.
Locomotor activity
Locomotor activity after solvent or kratom application was
measured using a computerized system (Moti-Test, TSE
Systems, Bad Homburg, Germany). The system consisted
of four identical boxes (46×46× 50 cm). The horizontal and
vertical activities of the animals were measured by the inter-
ruption of infrared light beams from cells located in the frames
of the apparatus. The illumination level in the sound-reduced
testing room was 30 lx. The boxes were cleaned and wiped
prior to the first test and after each test. After application, the
animals were placed for 60 min in the test box. Activity was
measured in terms of total activity time, which represents time
spent in horizontal activity + time spent in vertical activity.
14 Psychopharmacology (2014) 231:1325
Elevated plus maze
Situational anxiety was measured in the elevated plus maze.
The maze was made of black polyvinyl chloride and had two
open and two closed arms (50×10×40 cm) mounted 50 cm
above the floor. The floor of the arms was smooth. The light
level was 30 lx.
A mouse was placed in the central platform of the
apparatus facing a closed arm. A camera on the ceiling
of the test room was used to score and tape the animals'
behavior from an adjacent room for a period of 7 min.
The number of entries into open and closed arms, time
spent on open arms, and time spent on closed arms
were measured. An entry is defined as placing both
forepaws into the given compartment of the maze. The
maze was cleaned after each trial. The pretreatment time was
60 min.
Thermal pain threshold
Pain threshold in response to thermal stimulus in the
hot plate test was measured 60 min after kratom appli-
cation using an incremental hot/cold plate analgesia
meter (Stoelting, Wood Dale, IL, USA). The surface of
the hot plate was heated to a constant temperature of
42 °C. Mice were placed on the hot plate, which was
surrounded by a clear acrylic cage, and the start/stop
button on the timer was activated. The temperature of
the plate was increased constantly by 10°/min. The
latency to respond with either a hind paw lick or hind
paw trembling was measured by deactivating the timer
when the response was observed. The mouse was im-
mediately removed from the hot plate and returned to
its home cage.
Effect of the κopioid receptor antagonist
norbinaltorphimine (BNT)
Effect on locomotor activity after kratom ip application
Both /and +/+ animals were subcutaneously injected
with 10.0 mg/kg BNT. Twenty-four hours later, the
animals received either saline or 2 mg/kg mitragynine
+ 0.1 mg/kg paynantheine ip. Afterwards, locomotor activity
was measured over a period of 60 min as described above.
Effect on thermal pain threshold after kratom icv application
The /mice were subcutaneously injected with 10.0 mg/kg
BNT. Twenty-four hours later, the animals received 5 μlicv
saline or kratom (10.0 μgmitragynine+0.5μg paynantheine,
20.0 μgmitragynine+1.0μg paynantheine). Thermal pain
response was measured 60 min after kratom application
according to the method described above.
Interaction between kratom and apomorphine
To evaluate the possible dopaminergic effects of kratom, /
mice and +/+ animals were simultaneously injected with saline
or 0.3 mg/kg apomorphine (APO) sc and kratom ip (2.0 mg/kg
mitragynine + 0.1 mg/kg paynantheine), respectively. The an-
imals were put in the Moti-Test box, and locomotor activity was
measured as described above at 3060 min postapplication.
The following groups were used:
Route ip sc
Substance Control Saline
Control APO
0.3 mg/kg
Kratom Saline
(2.0 mg/kg mitragynine +
0.1 mg/kg paynantheine)
Kratom APO
(2.0 mg/kg mitragynine + 0.3 mg/kg
0.1 mg/kg paynantheine).
Two-way active avoidance learning: shuttle box
The automatic shuttle box (TSE Systems, Bad Homburg,
Germany) was divided into two compartments (15 cm ×
14 cm) separated by a 4-cm hurdle. The conditioned stimuli
were light (12-W bulbs located on the central ceiling of each
compartment) and a sound produced by a buzzer. The un-
conditioned stimulus was an electric footshock (0.41.2 mA
depending on individual sensitivity; footshock intensity was
adjusted at about vocalization threshold) delivered through
stainless steel rods forming the floor. The conditioned stim-
uliunconditioned stimulus interval was 4 s. The stimuli
were switched off after changing the shuttle box compart-
ment. One trial was limited to 20 s if the animal failed to react
within this period. Intertrial intervals lasted for a randomized
period of 2030 s. Sessions each consisted of 60 trials. Prior to
the training session, the animals were allowed to explore the
box for 5 min. Twenty-four hours later, a relearning session
was performed and 1 min was provided for habituation.
The number of escapes (reaction time > 4 s), conditioned
reactions (reaction time < 4 s), and the number of hurdle
crossings during the habituation as well as learning/relearning
sessions were recorded for further evaluation. If there were no
significant differences in the number of conditioned reactions
in the training session, the retention index (RI) was calculated
according to the following formula:
RI ¼Escapes trainingEscapes relearning
Escapes training 100
Psychopharmacology (2014) 231:1325 15
Diluted ethanol for control (Co) or kratom were given orally
either 30 min pretraining (0.25 mg/kg mitragynine + 0.0125
mg/kg paynantheine, 0.5 mg/kg mitragynine + 0.025 mg/kg
paynantheine, 1.0 mg/kg mitragynine + 0.05 mg/kg
paynantheine, 2.0 mg/kg mitragynine + 0.1 mg/kg
paynantheine) or immediately posttraining (1.0 mg/kg
mitragynine + 0.05 mg/kg paynantheine, 2.0 mg/kg mitragynine
+ 0.1 mg/kg paynantheine) in order to investigate drug effect on
memory acquisition and consolidation, respectively.
Cell culture and transfections of μopioid receptor receptors
in human embryonic kidney 293 cells
Human embryonic kidney (HEK) 293 cells were obtained
from the American Type Culture Collection (ATCC,
Rockville, MD, USA) and grown in Dulbecco's modified
eagle medium (DMEM, BF12-604 F, Cambrex, Verviers,
Belgium) supplemented with 10 % fetal calf serum (FCS,
Bachem, Heidelberg, Germany) in a humidified atmosphere
containing 10 % CO
2
at 37 °C. Cells were first transfected
with pEAK10-HA-MOR and pEAK10-HA-DOR plasmid
expression vector carrying the puromycin resistance gene,
using the calcium phosphate precipitation method (Schröder
et al. 2009). After selecting individual resistant clones, re-
ceptor expression was monitored using receptor-ligand bind-
ing assays and immunofluorescent staining. Cells were
preincubated in UltraMEM (BE12-743 F, Cambrex) for about
30 min prior to treatment with 10 μMDAMGO,morphine,or
extract of kratom for a further 30 min.
Binding assays
For
3
H-DAMGO,
3
H-naltrindole, and
3
H-diprenorphine
binding assay, tissue (striatadiprenorphine binding) or
MOR and δopioid receptor (DOR)-transfected HEK 293
(for MOR and DOR binding) was homogenized in 50 mM
TrisHCl buffer, pH 7.8, containing 1 mM EGTA, 2 mM
EDTA, and 5 mM MgCl
2
, centrifuged (50,000g, 15 min) and
washed twice with buffer (Schröder et al. 2009,2011). The
resulting pellet was resuspended with buffer. Aliquots of the
crude membrane suspension (50 μg protein) were incubated
for 40 min at 25 °C with 2.5 nM
3
H-DAMGO or 0.25 nM
3
H-naltrindole, or
3
H-diprenorphine at 30 °C for 60 min.
Specific binding was calculated by subtracting nonspecific
bindingdefined as that seen in the presence of 1 μM
unlabeled DAMGO (MOR) or 1 μM naltrindole (DOR) or
1μM diprenorphine with the addition of 10 nM funaltrexamine
as well as naltrindole to mask μ-andδ-opiate receptors (to
detect the κ-opiate receptor part)from total binding obtained
with radioligand alone. In parallel use of kratom extract,
mitragynine and paynantheine were used in all binding assays.
The incubation was terminated by the addition of ice-cold
buffer and rapid filtration through glass fiber filters. The filters
were washed, dried, a scintillation cocktail was added, and
radioactivity was counted using a β-counter. The EC
50
was
determined by the addition of agonists over a wide concentra-
tion range (10
10
to 10
4
M).
For guanosine 5-(gamma-thio)triphosphate (
35
S-GTPγS)
binding assay, the cells were harvested into PBS and stored
at 80 °C. After thawing, the cells were centrifuged for 5 min
at 20,000gand then homogenized in lysis buffer containing
50 mM TrisHCl, 10 mM EDTA, pH 7.4; membranes were
prepared by centrifugation at 50 000 gfor 15 min, washed
with lysis buffer, and resuspended in incubation buffer
(20 mM HEPES, 100 mM NaCl, 10 mM, MgCl
2
, pH 7.4).
Aliquots containing 2025 μg protein were incubated with
3μM GDP and 0.05 nM
35
S-GTPγS (specific activity: 46.3
TBq/mmol, PerkinElmer, USA) in the presence or absence of
agonists of either 1 μM DAMGO and morphine in compari-
son with kratom extracts, mitragynine, and paynantheine.
Assays were carried out in a final volume of 1 ml at 30 °C
for 30 min under continuous agitation. Nonspecific binding
was determined in the presence of 10 μM of unlabeled
GTPγS. The incubation was terminated by the addition of
ice-cold buffer and rapid filtration through glass fiber filters.
The filters were washed and dried. A scintillation cocktail was
added and radioactivity was counted using a β-counter. The
maximal agonist response (E
max
) and the EC
50
were deter-
mined by the addition of agonists over a wide concentration
range (10
10
to 10
4
M).
Western blot analysis
Cells were plated onto poly-L-lysine-coated 150-mm dishes
and grown up to 80 % confluence. Cells were then washed
twice with PBS and harvested into ice-cold lysis buffer
(10 mM TrisHCl, pH 7.6, 5 mM EDTA, 3 mM EGTA,
250 mM sucrose, 10 mM iodoacetamide, and the following
proteinase inhibitors: 0.2 mM phenylmethylsulfonyl fluo-
ride, 10 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml
aprotinin, and 10 μg/ml bacitracin). Iodoacetamide was in-
cluded in each buffer used for protein preparation to prevent
nonspecific disulfide linkages. Cells were swollen for 15 min
on ice and homogenized. The homogenate was centrifuged at
500gfor 5 min at 4 °C to remove unbroken cells and nuclei.
Membranes were then centrifuged at 20,000 × gfor 30 min at
4 °C, and pelleted membranes were lysed in detergent buffer
(20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA,
3 mM EGTA, 4 mg/ml β-dodecylmaltoside, 10 mM
iodoacetamide, and proteinase inhibitors as above) for 1 h
on ice. The lysed membranes were centrifuged at 20,000 × g
for 30 min at 4 °C. In the resulting supernatant, the glyco-
proteins were purified using wheat germ lectin. For the
enrichment of glycoproteins, 1 ml of the supernatant was
incubated with 100 μl of wheat germ agglutinin (WGA)
agarose beads (Pharmacia, Freiburg, Germany) for 90 min
16 Psychopharmacology (2014) 231:1325
at 4 °C with continuous agitation. Beads were washed five
times with detergent buffer and the adsorbed glycoproteins
were either subject to enzymatic deglycosylation or di-
rectly eluted into 200 μl of SDS sample buffer (62.5 mM
trisHCl, pH 6.8, 2 % SDS, 20 % glycerol, 200 mM
DL-dithiothreitol, and 0.005 % bromophenol blue) at 60 °C
for 20 min.
For immunoprecipitation, the lysates were precleared with
50 μl of protein A-agarose beads (Calbiochem, Bad Soden,
Germany) for 2 h. Receptor proteins were then
immunoprecipitated with 1 ml of the supernatant using 10 μg
of one of the following antibodies: anti-HA wheat germ beads.
Immunoprecipitations were carried out for 12 h at 4 °C with
continuous agitation. Immunocomplexes were collected using
100 μl of protein A-agarose beads for 3 h. Beads were washed
five times with detergent buffer and immunoprecipitates were
eluted from the beads with a 200-μl SDS sample buffer at
60 °C for 20 min. Equal amounts of protein from each sample
were then loaded onto regular 6 % SDS-polyacrylamide gels.
After electroblotting, membranes were incubated with rat anti-
HA at a concentration of 1 μg/mlfor12hat4°C.Bound
primary antibodies were detected with peroxidase-labeled
secondary antibodies, and immunoreactive proteins were
visualized using an enhanced chemiluminescence detection
system. In a further series of experiments for the detection
of HA-tagged MOR as well as phospho-MOR after
electroblotting, the membranes were incubated with anti-
phospho-Ser
375
antibody (1:1,000, Cell Signaling
Technology, Beverly, MA, USA) or anti-HA antibody
followed by detection using an enhanced chemilumines-
cence detection system (Amersham, Braunschweig,
Germany) described elsewhere (Schulz et al. 2004).
Mitragynine blood plasma levels
Blood plasma levels in naïve +/+ and /mice 1 h after po or ip
application of 10 mg/kg of kratom containing 0.2 mg/ml of
mitragynine were measured using an LXQ linear ion trap
(Thermo Fisher Scientific, TF, Dreieich, Germany) mass spec-
trometer equipped with a heated electrospray ionization source
and coupled to a TF Accela ultra UHPLC system consisting of
a degasser, a quaternary pump, and an autosampler. Conditions
were similar to those described previously (Wissenbach et al.
2011) with the following modifications. The gradient was
programmed to be 02 min from 2 to 10 % mobile phase B,
24minto25%B,48.5 min hold at 25 % B, 8.59minto
75 % B, 911minto98%B,and1114 hold at 2 % B. To
monitor mitragynine and the internal standard (IS)
trimipramine-d3, product ion spectra of the protonated mole-
cules at m/z399 and 298, respectively, were recorded and the
resulting fragment ions at m/z238 and 103, respectively, were
used as a quantifier. 7-Hydroxy-mitragynine was not quantified
during the study, since the formation of this of the metabolite
was not expected. In respective metabolism studies, this sub-
stance could not be detected (Philipp et al. 2009).
Sample preparation for mitragynine determination An ali-
quot of 100 μl of plasma was diluted with 400 μl of human
plasma and processed as described by Remane et al. (2010).
Method validation for mitragynine determination A simpli-
fied method validation was performed as proposed by Peters
et al. (Peters et al. 2007) and modified according to
Wille et al. (2013). The validation protocol included
specificity, matrix effects, limit of quantification, and
accuracy/precision.
Specificity Six blank plasma samples were analyzed for peaks
interfering with mitragynine detection. Additionally, two zero
samples were analyzed to check for interference induced by
the used IS.
Calibration Calibration was performed in duplicate at three
concentrations as follows: 1, 10, and 20 μg/l. The regression
lines were calculated using a weighted (1/×2) linear regres-
sion model. The back-calculated concentrations of all cali-
bration samples lay within 20 % of the target concentrations.
Matrix effects Matrix effect (ME) studies were performed at
QC low (2 μg/l) and high (15 μg/l) concentrations using six
plasma sources according to Matuszewski et al. (2003).
Accuracy and precision Eight quality control (QC) samples
at low and high concentrations were analyzed. Accuracy and
precision were calculated as recommended (Wille et al. 2013).
Quantification limits The limit of quantification (LOQ) of
the method was defined as the lowest point of the calibration
curve (1 μg/l) still providing a signal-to-noise ratio >10:1.
Drugs
Kratom (Thai dragonfly liquid extract, ethanolic extract) was
purchased from Worldherbals (Maassluis, the Netherlands).
Analysis revealed a mitragynine content of 0.2 mg/ml and a
paynantheine content of 0.01 mg/ml. The stock solution
contained 6 % ethanol. Dilutions were made with isotonic
saline. In the experiments based on po or ip administration,
6 % ethanol was diluted with isotonic saline in the same
relationship for control, i.e., 1:2, 1:4, etc. (Co). To obtain
higher concentrations, the extract was dried in a vacuum
freezer and the dry extract was resolved in 6 % ethanol.
For icv injection, the extract was dried as described above
and resolved in sterile saline. For sterile venting, Minisart®
syringe filters with polytetrafluorethylene membranes and
Psychopharmacology (2014) 231:1325 17
0.2-μm pore size were used. In this experiment, sterile saline
(sal) was used as control.
To simulate clinically relevant conditions, Thai dragon fly
was administered in the behavioral experiments and for deter-
mination of substance concentrations in the blood. All doses
refer to the main alkaloids, i.e., mitragynine + paynantheine.
Apomorphine (Apomorphin®, Teclapharm, Lüneburg,
Germany) and norbinaltorphimine hydrochloride (Tocris,
Bristol, UK) were dissolved in physiological saline. The
application volume for po and ip injection was 10 ml/kg
of body weight and, for icv application, it was 5 μlper
animal.
For the binding assay, [D-Ala
2
,N-MePhe
4
,Gl-ol
5
]-enkepha-
lin (DAMGO) was purchased from BIOTREND Chemicals AG
(Cologne, Germany). Proteinase inhibitors phenylmethylsulfonyl
fluoride, leupeptin, pepstatin A, aprotinin, and bacitracin were
purchased from Sigma RBI (Munich, Germany).
3
H-DAMGO
(SA: 1.43 TBq/mmol),
3
H-naltrindole (specific activity: 0.49
TBq/mmol) and
3
H-diprenorphine (SA: 1.85 TBq/mmol) were
obtained from PerkinElmer (Boston, USA). Kratom for the bind-
ing assay was a patient's exhibit at the Center of Pharmacology
and Toxicology, Georg August University Göttingen (narcotic
law allowance no. 82.04-4517970-0069/13) in powder form, and
a 10 % ethanolic extract was prepared.
Statistics
The data obtained in experiments using transgenic mice were
evaluated with two-way ANOVA, with strain (+/+ vs. /)
and treatment (sal or Co vs. kratom) being the independent
variables, followed by a one-way ANOVA and Bonferroni
post hoc test. Learning experiments were analyzed on the
basis of a one-way ANOVA and Bonferroni post hoc test.
The significance threshold was set at p< 0.05. Data from
ligand binding and GTPγ-S as well as the E
max
and EC
50
were analyzed by curve fitting using GraphPad Prism 3.0
software (two-tailed MannWhitney test).
Results
Effect on locomotor activity
Per oral administered kratom altered total activity time in a
specific manner. There was a significant effect of strain
(F
1, 89
= 13.49, p<0.001) and treatment (F
3, 89
=3.09,
p<0.031) but no strain × treatment interaction (F
3, 89
=1.85,
p<0.145). One-way ANOVA revealed significant differences
between the groups (F
7, 82
=4.34,p<0.001). Both /and +/+
animals injected with the solvent differed significantly
(p=0.021). With +/+ mice, oral administration of 0.5 mg/kg
mitragynine + 0.025 mg paynantheine resulted in a significant-
ly reduced total activity time (p=0.43). In all the other +/+
groups and between +/+KO, there were no differences
(p>0.05) (Fig. 1a).
Ip injection of kratom extract resulted in reduced total activity
time dependent on strain (F
1, 55
=5.68,p=0.021) and treatment
(F
2, 55
=20.07,p<0.001) but no strain × treatment interaction
(F
1, 55
=13.49,p<0.001). One-way ANOVA revealed signifi-
cant differences between the groups (F
5, 50
= 13.49, p<0.001).
In +/+, total activity time was decreased independently of dose,
whereas in /, it was dose dependently decreased.
In the experiments based on icv application (20.0 μg
mitragynine + 1.0 μg paynantheine), there was an effect of strain
(F
1, 59
=9.88,p= 0.003), but no effect of treatment (F
2, 59
=2.27,
p=0.11) and no strain × treatment interaction. The /animals
were less active compared with +/+ mice. Eight to 15 animals
were used per group.
Effect on behavior in the elevated plus maze
A comparison of %time spent in open arms and the numbers
of total arm changes (=number of entries into open and
closed arms) revealed significant differences between the
two strains, but these parameters were not altered by treatment
with kratom extract after po administration (%time open arms:
strain F
1, 65
=14.33,p<0.001, treatment F
2, 65
=1.17,
p=0.32, strain × treatment F
2, 65
=1.1, p= 0.34; total arm
changes: strain F
1, 65
=12.96,p=0.001, treatment F
2, 65
=
1.34, p=0.27, strain × treatment F
2, 65
=0.49, p=0.61) or icv
(%time open arms: strain F
1, 59
=9.03,p= 0.04, treatment F
2,
59
=1.26,p=0.293, strain × treatment F
2, 59
=2.2,p=0.58;
total arm changes: strain F
1, 59
=13.39, p=0.001, treatmentF
2,
59
=1.39,p= 0.26, strain × treatment F
2, 59
=0.49,p=0.61).
Moreover, there were no strain × treatment interactions,
suggesting no effect on situational anxiety (Fig. 1b, c).
As shown in Fig. 1b, %time spent in open arms was very
low in /mice. Therefore, these dataare difficult to interpret.
After ip application, there was a significant reduction in
the parameter %time spent in open arms in both strains
(strain F
1, 55
= 2.97, p=0.09, treatment F
2, 55
= 6.44,
p=0.0003, strain × treatment F
2, 55
= 0.78, p=0.46), indicat-
ing anxiety. However, in a similar way as was found in the
Moti-Test, the total number of arm changes that expresses
locomotor activity was diminished (strain F
1, 55
= 3.81,
p=0.06, treatment F
2, 55
= 13.659, p<0.001, strain × treat-
ment F
2, 55
= 1.98, p=0.148). Thus, no conclusions could be
drawn concerning the effects of kratom after ip injection on
situational anxiety. Eight to 15 animals were used per group.
Effect on thermal pain threshold
After po (strain F
1, 65
=2.6,p= 0.11, treatment F
2, 65
= 1.2,
p=0.3, strain × treatment F
2, 65
=0.6,p=0.94, Fig. 2a)andip
(strain F
1, 55
=1.8,p= 0.19, treatment F
2, 55
=0.67,p=0.51,
strain × treatment F
2, 55
=0.43,p=0.65, Fig. 2b) application of
18 Psychopharmacology (2014) 231:1325
kratom, we did not find changes in the thermal pain threshold
of either strain of mice (Fig. 2). After icv (Fig. 2c) application,
there was a significant effect of strain (F
1, 59
= 6.0, p=0.018),
treatment (F
2, 59
=4.7,p=0.013) and a significant strain ×
treatment interaction (F
2, 59
= 4.63, p=0.014). One-way
ANOVA revealed significant differences between the six
groups (F
5, 54
=4.94,p=0.001). In the +/+ mice, icv kratom
had no analgesic effect. In /mice, thermal pain threshold
was significantly increased in the group treated with 20.0 μg
mitragynine + 1.0 μg paynantheine but not in the group treated
with 1.0 μg mitragynine + 0.5 μg paynantheine. This clearly
suggests that kratom analgesia after icv administration is not
mediated via MOR. Eleven to 15 animals were used per group.
Effects of norbinaltorphimine
Effect of norbinaltorphimine on locomotor activity
Pretreatment with BNT did not abolish the depressive effect
of ip kratom (Fig. 3a). As found in the first experiment, both
strains of mice reacted in a similar way to kratom (strain F
1,
45
= 1.0, p=0.322; treatment F
1, 45
= 39.14, p<0.001, strain ×
treatment F
1, 45
= 3.21, p=0.8). One-way ANOVA revealed
significant differences between the four groups (F
3, 42
=
16.86, p<0.001). The groups treated with kratom exhibited
significantly lower locomotor activity than control mice
(p<0.05) (Fig. 3). Nine to 14 animals were used per group.
Effect of norbinaltorphimine on thermal pain threshold
Pretreatment with BNT antagonized the analgesic effect of
icv kratom in /mice completely (Fig. 3b). There were no
differences between the groups (F
2, 29
= 1.05, p=0.36). This
clearly indicates that analgesia is mediated via κopioid
receptors. Ten to 11 animals were used per group.
Kratom interactions with apomorphine
In this experiment, kratom (ip) and 0.3 mg/kg of apomor-
phine (sc) were injected simultaneously. Afterwards, the
mice were placed in a Moti-Test box and total activity time
(i.e., horizontal activity + vertical activity) was measured
3060 min postinjection. There was a significant effect of
apomorphine treatment (F
1, 114
= 24.75, p<0.001), treatment
with kratom (F
1, 114
= 75.34, p<0.001), a significant strain ×
apomorphine interaction (F
1, 114
= 9.87, p0.002), and a
significant apomorphine × kratom interaction (F
1, 114
=
30.37, p<0.001). The effect of kratom was independent of
strain (F
1, 114
= 2.8, p=0.96). One-way ANOVA between +/+
mice (Fig. 4a) revealed significant differences between the four
Fig. 1 a Total activity in wild type (+/+) and μopioid receptor-deficient
mice (/)060 min after po, ip, and icv administration of kratom extract.
b%time spent in open arm in the elevated plus maze of wild type (+/+) and
μopioid receptor-deficient mice 60 min after po, ip, and icv administration
of kratom extract. cNumber of total arm changes in the elevated plus maze
of wild type (+/+) and μopioid receptor-deficient mice 60 min after po, ip,
and icv administration of kratom extract. Eight to 15 animals were used per
group. po per oral, ip intraperitoneal, icv intracerebroventricular. *p<0.05.
po and ip doses are in mg/kg (mitragynine + paynantheine), icv dose in μg
per animal (mitragynine + paynantheine). Co diluted ethanol for control
Psychopharmacology (2014) 231:1325 19
experimental groups (F
3, 53
= 5.31, p=0.03). Apomorphine did
not significantly change activity time in +/+ animals (F
1, 52
=
3.76, p=0.061). As found in the test battery, kratom signifi-
cantly reduced activity time (+/+ p=0.04). In combination with
apomorphine, the effect of kratom was normalized (p=1.0).
Similar results were obtained in /animals (Fig. 4b). The four
experimental groups were significantly different (F
3, 56
=20.1,
p<0.001). In these animals, apomorphine had a depressive
effect on total activity time (p=0.049). The depressive effect
of kratom (p<0.001) was counteracted by apomorphine
(sal/krat vs. apo/krat p=0.003), but it did not reach the value
measured in the control group (p=0.03) (Fig. 4). Twelve to 16
animals were used per group.
Effect of po kratom on shuttle box learning
Application prior to the learning session
The number of conditioned reactions in the training sessions
was significantly different (F
4, 63
= 3.49, p=0.012) between
Fig. 2 Thermal pain threshold in wild type (+/+) (a) and μopioid
receptor-deficient mice (/)(b) 60 min after po, ip, and icv adminis-
tration of kratom extract. Eight to 15 animals were used per group. po
per oral, ip intraperitoneal, icv intracerebroventricular. *p<0.05. po and
ip doses are in mg/kg (mitragynine + paynantheine), icv doses in μg per
animal (mitragynine + paynantheine). Co diluted ethanol for control
Fig. 3 Effect of pretreatment with 10 mg/kg norbinaltorphimine 24 h
prior to measuring locomotor activity after intraperitoneal (ip) treatment
(a). Thermal pain threshold was only measured in /mice after
intracerebroventricular (icv) treatment (b) with kratom extract. +/+ wild
type mice, /μopioid receptor knockout mice. Total activity time was
measured in 914animals pergroup, and pain threshold was measured in
1011 animals per group. ip doses are in mg/kg (mitragynine +
paynantheine), icv doses in μg per animal (mitragynine + paynantheine).
*p<0.05. Co diluted ethanol for control
Fig. 4 Total activity time measured 3060 min after simultaneous appli-
cation of 0.3 mg/kg apomorphine (APO) or saline (sal) subcutaneously
and kratom extract (2.0 mg/kgmitragynine+ 0.1 mg/kg paynantheine) or
6 % ethanol (Co) intraperitoneally. a+/+ wild type mice, b,/μopioid
receptor knockout mice. Twelve to 16 animals were used per group
20 Psychopharmacology (2014) 231:1325
the groups of C57Bl6/N mice. The doseresponse curve is U-
shaped. Animals that were treated with 0.25 mg/kg mitragynine +
0.0125 mg/kg paynantheine or 0.5 mg/kg mitragynine + 0.025-
mg/kg paynantheine had fewer conditioned reactions compared
with the control group or the groups with higher doses of both
alkaloids (p< 0.05). A similar result was obtained in the
relearning session (F
4, 63
=9.49,p<0.001). Here, the same two
groups had a significantly lower number of conditioned reactions
(p<0.05) (Fig. 5).
Intertrial activity was also different (training session F
4, 63
=
12.71, p<0.001, relearning session F
4, 63
= 5.23, p=0.001).
Intertrial activity was greatest in the groups treated with 0.25-
mg/kg mitragynine+ 0.0125 mg/kg paynantheine or 0.5 mg/kg
mitragynine + 0.025 mg/kg paynantheine (p<0.05). Thirteen
to 14 animals were used per group.
Application after the training session
Oral post-training application of kratom to C57Bl6/N mice
did not alter any parameter measured in the shuttle box learn-
ing sessions. Treatment groups were designed according to the
number of escape reactions in the training session, so that
there were no differences between the groups (F
2, 37
=0.89,
p=0.92). There were no differences in the number of condi-
tioned reactions in the relearning session (F
2, 37
=0.45,
p=0.64) and intertrial activity (training session F
2, 37
=0.14,
p=0.98, relearning session F
2, 37
=0.9,p=0.9) (Fig. 6).
Thirteen animals were used per group.
Binding studies
Using binding studies with tritium-labeled ligands, it was
shown that kratom has an affinity for all three opioid receptors.
It must be pointed out that the affinity was 150- to 300-fold
lower for MOR, about 2,000-fold lower for DOR, and 750-
fold lower for κopioid receptors (KOR) when compared with
specific agonists of these receptors (Fig. 7ac). The competi-
tion at the MOR and KOR for both the kratom extract and the
major kratom alkaloids mitragynine and paynantheine had
comparable affinity values (EC
50
), whereas mitragynine did
not interact with DOR. As shown in Fig. 7d, kratom demon-
strates a low affinity to D1 dopaminergic binding sites, where-
as D2 binding sites were not affected (not shown). ED
50
values
are presented in Table 1(Figs. 7and 8).
The stimulation of the
35
S-GTPγS(guanosine5-(gamma-
thio)triphosphate) binding reflecting the signal transduction
activity of a drug at GPCR was measured using specific opioid
agonists, kratom extract, and the kratom alkaloids. The kratom
extract, as well as the alkaloids mitragynine and paynantheine,
has a low, less specific effect compared with DAMGO or
morphine (Fig. 7). Furthermore, the receptor phosphorylation
seems to be an important parameter of the receptor internali-
zation process. Measuring the phosphorylation of MOR in-
duced by opioid agonists, as well as kratom, we found that
DAMGO and morphine evoked a high phosphorylation of
MOR, whereas kratom has only a minor effect on this process
(Fig. 8).Theeffectofmorphineisatanintermediatelevel.The
ED50 values are presented in Tables 1and 2.
Mitragynine blood plasma levels
Quantification as described in Mitragynine blood plasma
levelsrevealed the following mitragynine concentrations.
After po application, mitragynine blood plasma concentration
Fig. 5 Effect of orally administered kratom extract on the acquisition of a
conditioned avoidance reaction (upper panel) and intertrial activity (lower
panel) in C57BL6/N mice in the shuttle box. The extract was administered
30 min prior to the learning session. Doses indicate mitragynine +
paynantheine mg/kg. Twelve to 16 animals were used per group
Fig. 6 Effect of orally administered kratom extract on the consolidation of
a conditioned avoidance reaction (upper panel) and intertrial activity
(lower panel) in C57BL6/N mice in the shuttle box. The extract was
administered immediately after the learning session. Doses indicate
mitragynine + paynantheine mg/kg. Thirteen animals were used per group
Psychopharmacology (2014) 231:1325 21
in +/+ animals was 14.33± 6.85 μg/L and 10.84±4.05 μg/l
in /animals (F
1, 15
=0.55,p=0.47). After ip application, it
Fig. 7 DAMGO (a), naltrindole (b), diprenorphine (c), and SCH 23.390 (d) binding to HEK cells or membranes of rat striatal cells
Table 1 The EC
50
values for the displacement of
3
H-labeled DAMGO,
diprenorphine, SCH 23.390, and naltrindole by different μ-, δ-andκ-
opioid receptor-specific drugs in comparison to kratom, mitragynine, and
paynantheine (mean ± S.E.M., n=68, competition curves in Fig. 7)
Binding EC
50
3
H-DAMGO Morphine 17.5± 2.01 nM
Kratom 2.7± 0.75 μM
Mitragynine 3.8± 0.5 μM
Paynantheine 3.8± 0.75 μM
3
H-SCH23.390 cis-Flupenthixol 3.7± 0.19 nM
Kratom 3.38± 3.3 μM
3
H-Diprenorphine Diprenorphine 2.58± 0.5 nM
Kratom 1.95± 85 μM
Mitragynine 0.85± 73 μM
Paynantheine 1.85± 197 μM
3
H-Naltrindole Naltrindole 16.0± 4.0 nM
Kratom 35.5± 3.5 μM
Mitragynine
Paynantheine 38.9± 1.0 μM
Fig. 8 DAMGO, morphine, kratom, mitragynine, and paynantheine
stimulated
35
S-GTPγS binding to membranes of MOR-transfected
HEK 293 cells. (Mean ± S.E.M., n=68)
22 Psychopharmacology (2014) 231:1325
was 30.28±7.04 μg/l, and in /animals, a concentration of
35.66±17.18 μg/l was found (F
1, 15
=1.35,p=0.26). Eight
animals were used per group.
Discussion
The alkaloids extracted from the tree Mitragyna speciosa
interact with different receptors, e.g., opioidergic, serotoner-
gic, adrenergic, and dopaminergic receptors. Their interac-
tion with opioidergic receptors, in particular, might explain
their traditional use in the treatment of musculoskeletal pain,
diarrhea, and coughing. The leaves of this tree, known as
kratomin Thailand and biak-biakin Malaysia, were tra-
ditionally used by laborers as a stimulant or as a substitute for
opium, and this might explain why the use of this plant is
illegal in these countries. Experiments with mitragynine, one
of the main constituents of the extract, administered in a dose
of 35 mg/kg, exhibited a clear analgesic effect in the hot plate
assay, and the tail pinch test that was reversible by naloxone,
naltrexone, and cyprodime suggested that analgesia was
mediated via MOR (Shamima et al. 2012; Thongpradichote
et al. 1998). Mitragynine is characterized by a weak potency
that does not completely explain the analgesic effect found in
the hot plate assay and in the tail pinch test. Therefore, other
constituents were tested. Among these compounds, 7-
hydroxymitragynine exhibited a 30-fold higher potency than
mitragynine and a 17-fold higher potency than morphine in
the guinea pig ileum contraction assay. The κreceptor antag-
onist BNT antagonized the effect in the tail-pinch test but not
analgesia measured in the hot plate test (Thongpradichote
et al. 1998), whereas AM25, a CB1 receptor antagonist, was
ineffective (Shamima et al. 2012). This indicates an involve-
ment of μand κopioid receptors in the analgesic effects, but
not of cannabinoid receptors.
Effects induced by chemically defined components are
not completely identical with those found after the applica-
tion of plant extracts that contain a variety of compounds
differing in efficacy and potency. For a better understanding
of the clinically desired and potentially harmful effects of
kratom, we investigated the extract in different behavioral
tests in mice. The standard was Dragonfly® containing
0.2 mg/ml of mitragynine and 0.01 mg/ml of paynantheine.
In MOR-transfected HEC cells, EC
50
for morphine = 17.5±
2.01 nM and for kratom, EC
50
= 2.7±0.15 μM.
The effect of kratom on MOR is quantitatively different to
that of morphine, the affinity is less, and kratom was not able to
induce a comparable phosphorylation (Fig. 9) and signal induc-
tion of MOR (GTPγS stimulation, Fig. 8). The effect of kratom
is less pronounced, and in comparison to the specific μ-opioid
agonists, it is about two orders of magnitude less potent. In
addition to MOR binding, the extract also binds to DOR, KOR,
and dopamine D1 receptors (Figs. 7and 8,Tables1and 2).
As shown in Fig. 1, kratom icv did not affect total activity
time in +/+ and /mice. However, after ip application, total
activity time was significantly reduced in both strains, which
clearly indicates that this effect was not mediated via MOR.
Moreover, pharmacokinetic differences can be excluded,
since mitragynine blood plasma levels were not different
between +/+ and /animals. Interestingly, after po appli-
cation, the depressant effect was only found in +/+ animals
dosed with 0.5 mg/kg mitragynine + 0.025 mg/kg
paynantheine. It seems likely that this effect is based on a
dopaminergic mechanism. Binding studies confirmed that the
extract binds to D1 receptors (Fig. 7d). APO, which acts via
D1/D2 receptors, was reported to activate D2 autoreceptors at
a dose of 0.3 mg/kg as a functional D2 antagonist (Wang et al.
2012). In +/+ mice, this dose of APO did not change total
activity time, but it abolished the depressant effect of kratom
completely (Fig. 4a). In /mice, this dose of APO reduced
the total activity time significantly (Fig. 4b). This might be the
consequence of altered D2 binding in /animals (Matthies
et al. 2000). Nevertheless, low-dose APO also counteracted
the depressant effect in /animals, which substantiates our
hypothesis that the kratom effect on total activity is mainly
mediated via dopaminergic mechanisms. The locus of action
of APO, i.e., presynaptic or postsynaptic, is dependent on the
Table 2 The EC
50
values of the stimulation of
35
S-GTPγS binding to
membranes of MOR transfected HEK 293 cells by the μ-opioid ago-
nists DAMGO and morphine compared to kratom, mitragynine, and
paynantheine (mean ± S.E.M., n=68)
EC
50
(nM)
Morphine 387± 96
Kratom 1,304± 171
Mitragynine 1,502± 114
Paynantheine 1,807± 390
DAMGO 25± 7
Fig. 9 MOR phosphorylation experiments: Western blot analysis of MOR
and pMOR (phospho-MOR using phospho S 375 antibody) in MOR-
expressing HEK 293 cells in the presence of 1 μMmorphine(MO),
DAMGO as well as 1:10 diluted (KRA 1) or original kratom (KRA 2)
extracts. Note that the DAMGO-induced phosphorylation is most drastic,
morphine to a lesser extent, and kratom extracts exhibit only slight effects.
Co means HEK 293 control without MOR (3 independent experiments)
Psychopharmacology (2014) 231:1325 23
dose administered. Thus, an increase in the kratom dose
should result in a switch from presynaptic (functional antag-
onistic) to postsynaptic (agonistic) action. For that reason, the
extract dose for oral administration was increased up to 8.0-
mg/kg mitragynine + 0.4 mg/kg paynantheine. This increase
changed neither total activity time nor thermal pain threshold
(data not shown). More research is needed to shed light on this
somewhat unconventional mode of action and the doseeffect
relationships. Blood plasma concentrations after ip adminis-
tration were twofold compared with concentrations after po
administration. However, the behavioral effects showed no
clear correlation with the blood concentrations. This phenom-
enon cannot be explained yet. In subsequent experiments, we
will also measure kratom levels in specific brain areas and this
might possibly help to answer some of the open questions.
In the elevated plus maze, po and icv treatments with kratom
did not change %time spent in open arms or the total number of
arm changes. After ip application, %time in open arms was
significantly reduced in +/+ as well as /animals. This is not
considered an indicator of anxiogenic action, since the number
of total arm changes was also reduced (Fig. 4b and c). In
general, the extract seems not to modify situational anxiety.
However, these results should be interpreted with caution.
There were differences in the number of arm changes after ip
injection and /mice spent very low time in open arms.
In several tests, mitragynine and 7-hydroxymitragynine
were found to exert moderate or powerful analgesic effects that
could be reversed with naloxone (Matsumoto et al. 2004;
Matsumoto et al. 1996b). In the doses administered in our
experiments, we found a significant increase in thermal pain
threshold after icv application in a dose-dependent manner in
/mice (Fig. 2c). Obviously, the analgesic effect of the extract
is independent of MOR. Pretreatment with BNT abolished the
kratom-induced analgesia completely (Fig. 3b), which clearly
indicates that analgesia is mediated via κopioid receptors. We
also tested kratom effects on total activity time after pretreat-
ment with BNT. As shown in Fig. 3a, the depressant effect on
total activity time occurred regardless of BNT treatment. It
seems that the analgesic effects of kratom are mainly mediated
via κopioidergic mechanisms, whereas dopaminergic mecha-
nisms are responsible for effects on motor/locomotor activity.
Recently, it was reported that chronic administration of
mitragynine significantly reduced the discrimination ratio time
in an object placement task and affected locomotor activity in
the open field (Apryani et al. 2010). In the present experiments,
we investigated the effect of acute kratom on the acquisition and
consolidation of a conditioned avoidance reaction in C57Bl6/N
mice. For that purpose, the extract was given po prior to the
training session or immediately after the training session.
Kratom administered prior to the training session signifi-
cantly impaired the acquisition of the avoidance reaction. Even
more interesting is that the doseresponse curve is U-shaped.
The same effect was found 24 h later in the relearning session
(Fig. 5). Whereas the doseresponse curve for conditioned
reactions is U-shaped, intertribal activity shows a bell-shaped
curve. In a dose of 0.5 mg/kg mitragynine + 0.025 mg/kg,
paynantheine had a depressant effect on total activity time,
whereas intertribal activity in the shuttle box was significantly
increased. This implies that kratom might not act as a general
sedative drug. Under stressful conditions in the shuttle box
(electric footshock), the sedative effect was ameliorated and
switched to a status characterized by increased motor activity.
From the literature, it is known that dopamine antagonists
disrupt conditioned avoidance learning (Sanger 1985,1987).
A presynaptic (=functional antagonistic) dopaminergic mech-
anism of kratom extract might well explain the deterioration in
the acquisition of shuttle box avoidance learning. In contrast,
kratom did not affect consolidation of this learning task.
Previously, it was shown that orally administered methanolic
Mitragyna speciosa extract facilitated two-way active avoid-
ance in SpragueDawley rats, but there was no benefit on long-
term memory consolidation (Senik et al. 2013b). In hippocam-
pal slices, the extract produced an irreversible reduction of field
excitatory postsynaptic potentials, prevented the induction of
long-term potentiation, and induced only short-term potentia-
tion in hippocampal CA1 neurons (Senik et al. 2013a). This
action on the neurophysiological level is consistent with the
results of the shuttle box experiment in our study (Fig. 5).
Taken together, kratom extract seems to exert analgesic
effects predominantly via κopioid receptors and depressant
effects on locomotor activity via presynaptic dopamine ef-
fects. This would also explain the poor shuttle box results. It
was reported that chronic administration of kratom can lead
to addiction (Suwanlert 1975; (Sheleg and Collins 2011;
Hassan et al. 2013). In addition to their analgesic effects, κ
agonists may induce dysphoria. Moreover, presynaptic do-
paminergic agonistic effects would also be inconsistent with
the concept of addiction. Thus, further experiments focusing
on kratom self-administration, conditioned place preference,
and kratom effects in opiate and alcohol addicted mice are
needed to clarify the addictive potential of the extract as well
as its usefulness as a substitute for drugs of abuse.
Acknowledgments We gratefully acknowledge the expert assistance
of Ms. M. Böx, Ms. P. Dehmel, Ms. D. Heidemann, Ms. G. Borkhardt
and Ms. C. Knape.
Conflict of interest None.
References
Ahmad Akhibi MFB (2008) Isolation of major alkaloids from Mitragyna
speciosa (Kratom). Faculty of Applied Sciences. University Teknologi,
Mara, Malaysia
Ahmad K, Aziz Z (2012) Mitragyna speciosa use in the northern states of
Malaysia: a cross-sectional study. J Ethnopharmacol 141:446450
24 Psychopharmacology (2014) 231:1325
Apryani E, Hidayat MT, Moklas MA, Fakurazi S, Idayu NF (2010)
Effects of mitragynine from Mitragyna speciosa Korth leaves on
working memory. J Ethnopharmacol 129:357360
Assanangkornchai S, Muekthong A, Sam-Angsri N, Pattanasattayawong
U (2007) The use of Mitragynine speciosa (Krathom), an addic-
tive plant, in Thailand. Subst Use Misuse 42:21452157
Becker A, Grecksch G, Brödemann R, Kraus J, Peters B, Schroeder H,
Thiemann W, Loh HH, Höllt V (2000) Morphine self-administration
in mu-opioid receptor-deficient mice. Naunyn Schmiedebergs Arch
Pharmacol 361:584589
Boyer EW, Babu KM, Adkins JE, McCurdy CR, Halpern JH (2008)
Self-treatment of opioid withdrawal using kratom (Mitragynia
speciosa Korth). Addiction 103:10481050
Hassan Z, Muzaimi M, Navaratnam V, Yusoff NH, Suhaimi FW,
Vadivelu R, Vicknasingam BK, Amato D, von Horsten S, Ismail
NI, Jayabalan N, Hazim AI, Mansor SM, Muller CP (2013) From
kratom to mitragynine and its derivatives: physiological and be-
havioural effects related to use, abuse, and addiction. Neurosci
Biobehav Rev 37:138151
Horie S, Koyama F, Takayama H, Ishikawa H, Aimi N, Ponglux D,
Matsumoto K, Murayama T (2005) Indole alkaloids of a Thai
medicinal herb, Mitragyna speciosa, that has opioid agonistic
effect in guinea-pig ileum. Planta Med 71:231236
Loh HH, Liu HC, Cavalli A, Yang W, Chen YF, Wei LN (1998) mu
Opioid receptor knockout in mice: effects on ligand-induced anal-
gesia and morphine lethality. Brain Res Mol Brain Res 54:321326
Matsumoto K, Horie S, Ishikawa H, Takayama H, Aimi N, Ponglux D,
Watanabe K (2004) Antinociceptive effect of 7-hydroxymitragynine
in mice: discovery of an orally active opioid analgesic from the Thai
medicinal herb Mitragyna speciosa. Life Sci 74:21432155
Matsumoto K, Mizowaki M, Suchitra T, Murakami Y, Takayama H,
Sakai S, Aimi N, Watanabe H (1996a) Central antinociceptive
effects of mitragynine in mice: contribution of descending norad-
renergic and serotonergic systems. Eur J Pharmacol 317:7581
Matsumoto K, Mizowaki M, Suchitra T, Takayama H, Sakai S, Aimi N,
Watanabe H (1996b) Antinociceptive action of mitragynine in
mice: evidence for the involvement of supraspinal opioid recep-
tors. Life Sci 59:11491155
Matsumoto K, Mizowaki M, Takayama H, Sakai S, Aimi N, Watanabe
H (1997) Suppressive effect of mitragynine on the 5-methoxy-N,
N-dimethyltryptamine-induced head-twitch response in mice.
Pharmacol Biochem Behav 57:319323
Matthies H, Schroeder H, Becker A, Loh H, Höllt V, Krug M (2000)
Lack of expression of long-term potentiation in the dentate gyrus
but not in the CA1 region of the hippocampus of mu-opioid
receptor-deficient mice. Neuropharmacology 39:952960
Matuszewski BK, Constanzer ML, Chavez-Eng CM (2003) Strategies
for the assessment of matrix effect in quantitative bioanalytical
methods based on HPLC-MS/MS. Anal Chem 75:30193030
McWhirter L, Morris S (2010) A case report of inpatient detoxification after
kratom (Mitragyna speciosa) dependence. Eur Addict Res 16:229231
Peters FT, Drummer OH, Musshoff F (2007) Validation of new
methods. Forensic Sci Int 165:216224
Philipp AA, Wissenbach DK, Zoerntlein SW, Klein ON, Kanogsunthornrat
J, Maurer HH (2009) Studies on the metabolism of mitragynine, the
main alkaloid of the herbal drug kratom, in rat and human urine using
liquid chromatography-linear ion trap mass spectrometry. J Mass
Spectrom 44:12491261
Reanmongkol W, Keawpradub N, Sawangjaroen K (2007) Effects of
the extracts from Mitragyna speciosa Korth. leaves on analgesic
and behavioral activities in experimental animals. J Sci Technol
29:3948
Remane D, Meyer MR, Peters FT, Wissenbach DK, Maurer HH (2010)
Fast and simple procedure for liquidliquid extraction of 136
analytes from different drug classes for development of a liquid
chromatographic-tandem mass spectrometric quantification meth-
od in human blood plasma. Anal Bioanal Chem 397:23032314
Sanger DJ (1985) The effects of clozapine on shuttle-box avoidance
responding in rats: comparisons with haloperidol and chlordiaz-
epoxide. Pharmacol Biochem Behav 23:231236
Sanger DJ (1987) The actions of SCH 23390, a D1 receptor antagonist,
on operant and avoidance behavior in rats. Pharmacol Biochem
Behav 26:509513
Schröder H, Wu DF, Seifert A, Rankovic M, Schulz S, Höllt V, Koch T
(2009) Allosteric modulation of metabotropic glutamate receptor 5
affects phosphorylation, internalization, and desensitization of the
micro-opioid receptor. Neuropharmacology 56:768778
Schröder H, Höllt V, Becker A (2011) Parecoxib and its metabolite
valdecoxib directly interact with cannabinoid binding sites in
CB1-expressing HEK 293 cells and rat brain tissue. Neurochem
Int 58:913
Schulz S, Mayer D, Pfeiffer M, Stumm R, Koch T, Höllt V (2004)
Morphine induces terminal micro-opioid receptor desensitiza-
tion by sustained phosphorylation of serine-375. EMBO J
23:32823289
Senik MH, Mansor SM, Rammes G, Tharakan JHJ, Abdullah JHB
(2013a) Mitragyna speciosa Korth standardized methanol extract
induced short-term potentiation of CA1 subfield in rat hippocam-
pal slices. J Med Plants Res 6:12341243
Senik MH, Mansor SM, Tharakan JHJ, Abdullah JHB (2013b) Effect of
acute administration of Mitragyna speciosa Korth. standardized
methanol extract in animal model of learning and memory. J Med
Plants Res 6:10071014
Shamima AR, Fakurazi S, Hidayat MT, Hairuszah I, Moklas MA,
Arulselvan P (2012) Antinociceptive action of isolated
mitragynine from Mitragyna speciosa through activation of opioid
receptor system. Int J Mol Sci 13:1142711442
Sheleg SV, Collins GB (2011) A coincidence of addiction to
kratomand severe primary hypothyroidism. J Addict Med
5:300301
Shellard EJ (1974) The alkaloids of Mitragyna with special reference to
those of Mitragyna speciosa. Korth Bull Narc 26:4155
Suwanlert S (1975) A study of kratom eaters in Thailand. Bull Narc
27:2127
Thongpradichote S, Matsumoto K, Tohda M, Takayama H, Aimi N,
Sakai S, Watanabe H (1998) Identification of opioid receptor sub-
types in antinociceptive actions of supraspinally-administered
mitragynine in mice. Life Sci 62:13711378
Wang YC, He BH, Chen CC, Huang AC, Yeh YC (2012) Gender
differences in the effects of presynaptic and postsynaptic dopamine
agonists on latent inhibition in rats. Neurosci Lett 513:114118
Ward J, Rosenbaum C, Hernon C, McCurdy CR, Boyer EW (2011)
Herbal medicines for the management of opioid addiction: safe
and effective alternatives to conventional pharmacotherapy? CNS
Drugs 25:9991007
Wille SMR, Peters FB, Di Fazio V, Samyn N (2013) Practical aspects
concerning validation and quality control for forensic and clinical
bioanalytical quantitative methods. Accredit Qual Assur.2011
279292
Wissenbach DK, Meyer MR, Remane D, Weber AA, Maurer HH
(2011) Development of the first metabolite-based LC-MS(n) urine
drug screening procedure-exemplified for antidepressants. Anal
Bioanal Chem 400:7988
Yamamoto LT, Horie S, Takayama H, Aimi N, Sakai S, Yano S, Shan J,
Pang PK, Ponglux D, Watanabe K (1999) Opioid receptor agonis-
tic characteristics of mitragynine pseudoindoxyl in comparison
with mitragynine derived from Thai medicinal plant Mitragyna
speciosa. Gen Pharmacol 33:7381
Psychopharmacology (2014) 231:1325 25
... Several ligand-binding studies have demonstrated that mitragynine exerts its effects by interactions with opioid receptors (i.e. mu-, delta-, and kappa-opioid receptors) with various degrees of affinity towards its every subtype [16,41,64,65]. However, most effects of mitragynine were reported to be mediated by the mu-opioid receptor [66][67][68] while subsequent functional analyses and ligand binding studies by Kruegel et al. [36] suggested that mitragynine acts as a partial agonist at the human mu-opioid receptor and as competitive antagonists at kappa-and delta-opioid receptors. ...
... It is also important to highlight that the mechanisms underlying dependence and addiction are multifaceted, often involving multiple CNS pathways and receptors [102], and are not fully understood. The complexity of kratom's pharmacological profile may be attributed to kratom's multiple active ingredients with multiple mechanisms of action that are yet to be fully characterized, and the fact that mitragynine alone interacts with multiple targets in the CNS [65,66,75]. It has been suggested that 7-hydroxymitragynine is the other contributor to the development of the plant's addictive liability [35,84]. ...
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Opioid use disorder (OUD) has become a significant public health issue worldwide. Methadone and buprenorphine are the most common medications used for treating OUD. These drugs have the potential to assist many patients in managing their opioid dependence and withdrawal but they are currently misused and associated with certain compliance issues, side effects, and risk of relapse. As an opioid-like herbal supplement, Mitragyna speciosa Korth or kratom has received increased attention for managing chronic pain and opioid withdrawal symptoms. Nevertheless, the use of kratom as a self-treatment medication for opioid dependence continues to be controversial due to concerns raised about its effectiveness, safety, and abuse liability. The main active alkaloid constituent of the plant, mitragynine, has been shown to act as a partial mu-opioid agonist. Given this pharmacology, studies have been focusing on this psychoactive compound to examine its potential therapeutic values as medication-assisted therapy (MAT). This review aims to provide a current preclinical overview of mitragynine as a prospective novel option for MAT and summarise the recent developments in determining if the plant’s active alkaloid could provide an alternative to opioids in the treatment of OUD.
... Twenty-three in vivo (mice, rats, or dogs) studies provided evidence for kratom's potential therapeutic use in the treatment of acute pain (Carpenter et al., 2016;Criddle, 2015;Fakurazi et al., 2013;Hiranita et al., 2019;Idid et al., 1998;Macko et al., 1972;Matsumoto et al., 1996aMatsumoto et al., , 1996bMatsumoto et al., , 2004Matsumoto et al., , 2005Matsumoto et al., , 2006Matsumoto et al., , 2008Mossadeq et al., 2009;Reanmongkol et al., 2007;Sabetghadam et al., 2010Sabetghadam et al., , 2013Shamima et al., 2012;Stolt et al., 2014;Takayama et al., 2002;Thongpradichote et al., 1998;Wilson et al., 2020) and chronic pain PREVETE ET AL. ...
... The studied preparations were Mitragyna speciosa (MS) aqueous or methanol or alkaloid extracts (Carpenter et al., 2016;Criddle, 2015;Mossadeq et al., 2009;Reanmongkol et al., 2007;Sabetghadam et al., 2010Sabetghadam et al., , 2013, lyophilized kratom tea (LKT) , mitragynine alone (Carpenter et al., 2016;Criddle, 2015;Fakurazi et al., 2013;Foss et al., 2020;Hiranita et al., 2019;Idid et al., 1998;Macko et al., 1972;Matsumoto et al., 1996aMatsumoto et al., , 1996bShamima et al., 2012;Thongpradichote et al., 1998), or mitragynine + paynantheine (Stolt et al., 2014), and its synthetic derivatives MG Pseudoindoxyl and [(E)-methyl 2-(3ethyl-7a,12a-(epoxyethanoxy)-9-fluoro-1,2,3,4,6,7,12,12b-octahydr (Matsumoto et al., 2008), or 7HMG (Matsumoto et al., 2004(Matsumoto et al., , 2005(Matsumoto et al., , 2006, and its derivatives (E)-methyl 2-((2S,3S,7aS,12aR, 12bS)-3-ethyl-7a-hydroxy-8-methoxy-1,2,3,4,6,7,7a,12,12a,12b-de cahydroindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate and (E)-methyl 2-((2S,3S,7aS,12aR,12bS)-3-ethyl-9-fluoro-7a-hydroxy-8-methoxy-1,2,3,4,6,7,7a,12,12a,12b-decahydroindolo[2,3a]quinolizin-2-yl)-3-methoxyacrylate (MGM-16) (Matsumoto et al., 2014). ...
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Introduction Kratom (Mitragyna speciosa) is a tropical plant traditionally used as an ethnomedicinal remedy for several conditions in South East Asia. Despite the increased interest in its therapeutical benefits in Western countries, little scientific evidence is available to support such claims, and existing data remain limited to kratom's chronic consumption. Objective Our study aims to investigate (pre)clinical evidence on the efficacy of kratom as a therapeutic aid and its safety profile in humans. Methods A systematic literature search using PubMed and the Medline database was conducted between April and November 2020. Results Both preclinical (N = 57) and clinical (N = 18) studies emerged from our search. Preclinical data indicated a therapeutic value in terms of acute/chronic pain (N = 23), morphine/ethanol withdrawal, and dependence (N = 14), among other medical conditions (N = 26). Clinical data included interventional studies (N = 2) reporting reduced pain sensitivity, and observational studies (N = 9) describing the association between kratom's chronic (daily/frequent) use and safety issues, in terms of health consequences (e.g., learning impairment, high cholesterol level, dependence/withdrawal). Conclusions Although the initial (pre)clinical evidence on kratom's therapeutic potential and its safety profile in humans is encouraging, further validation in large, controlled clinical trials is required.
... [9] Receptor binding study showed that M. speciosa possesses a different degree of affinity to all opioid receptors (μ, κ, and δ) and D1 dopamine receptors. [41] At a cellular level, M. speciosa extract, mitragynine and paynantheine, showed a minor effect on phosphorylation and signal transduction of μ-opioid receptor in a guanosine 5'-(gamma-thio) triphosphate (35S-GTPγS) binding assay which was not comparable to the specific agonist morphine. [41] Another systematic study was carried out by Kruegel et al. on the in vitro characterization of opioid receptor pharmacology and signaling of the mitragynine, 7-hydroxymitragynine, and other Mitragyna alkaloids in human embryonic kidney cells expressing human μ-, κ-, and δ-opioid receptors. ...
... [41] At a cellular level, M. speciosa extract, mitragynine and paynantheine, showed a minor effect on phosphorylation and signal transduction of μ-opioid receptor in a guanosine 5'-(gamma-thio) triphosphate (35S-GTPγS) binding assay which was not comparable to the specific agonist morphine. [41] Another systematic study was carried out by Kruegel et al. on the in vitro characterization of opioid receptor pharmacology and signaling of the mitragynine, 7-hydroxymitragynine, and other Mitragyna alkaloids in human embryonic kidney cells expressing human μ-, κ-, and δ-opioid receptors. Both mitragynine and 7-hydroxymitragynine showed partial agonist activity at the μ-opioid receptors, with 7-hydroxymitragynine being the stronger agonist. ...
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The plant Mitragyna speciosa Korth. is receiving increased attention as a therapeutic substitution for opioid use disorder (OUD). The active alkaloids constituents of the plant, particularly mitragynine and 7-hydroxymitragynine, have been shown to modulate opioid receptors, acting as agonists at mu-opioid receptors. Given this pharmacology, several studies have examined the abuse and dependence potential of M. speciosa and its alkaloids in various animal models of dependence. In addition to action on opioid receptors, the Mitragyna alkaloids also appear to exert diverse activities at other receptors in the central nervous system which may explain the complex pharmacological profile of these alkaloids. Hence, this review aims to provide an overview of the preclinical studies used to study M. speciosa dependence potential and describe recent progress made in assessing whether the plant or its active alkaloids can offer alternatives to opioids in the management of OUD. In conclusion, M. speciosa Korth. or its compound mitragynine may offer alternatives as a replacement therapy to opioid.
... Mitragynine binds to alpha 1-and alpha 2 adrenergic receptors, serotonin-1A and -2A receptors, and the dopamine D1 receptor. 22,23 The functional significance of such binding is unclear. ...
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Kratom is the common term for Mitragyna speciosa and its products. Its major active compounds are mitragynine and 7-hydroxymitragynine. An estimated 2.1 million US residents used kratom in 2020, as a "legal high" and self-medication for pain, opioid withdrawal, and other conditions. Up to 20% of US kratom users report symptoms consistent with kratom use disorder. Kratom use is associated with medical toxicity and death. Causality is difficult to prove as almost all cases involve other psychoactive substances. Daily, high-dose use may result in kratom use disorder and opioid-like withdrawal on cessation of use. These are best treated with buprenorphine.
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Opioid overprescribing, with resultant overdose and death, has led to a national focus on alternative treatments for pain. With the decline in legal access to opioids, kratom has gained popularity as a legal, "natural," and easily accessible nonprescription analgesic for consumers wishing to self-medicate for pain, opioid use disorder, and other mental health conditions. While implications of kratom use in patients with chronic pain and/or opioid use disorder have been published, information on perianesthetic implications is lacking. Anesthesiologists should be informed about kratom, including the potential for unexpected physiologic derangements and adverse drug interactions resulting from complex pharmacologic activity, cytochrome P450 interactions, and common adulterations of the drug that may result in unpredictable clinical effects. This article explores the relevance of kratom to perioperative anesthetic care, including suggestions for anesthesiologists extrapolated from published information in nonoperative settings that may improve patient safety in individuals using kratom.
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Mitragyna speciosa, a species of plant that is native to Thailand, Malaysia and Southeast Asia, contains two major psychoactive alkaloids: mitragynine and 7-hydroxymitragynine. Pharmacologically, the alkaloids exhibit biphasic effects - at low dose, stimulant effects are realized, while high doses exhibit sedative effects. For years, the plant has been used recreationally and medicinally for these effects, but its use has been implicated in and associated with intoxications and deaths. In this case report we describe two cases whereby decedents presented with single substance fatal intoxications by mitragynine in the absence of other postmortem toxicological findings. The cases entail young male decedents in outdoor settings (e.g. driving a vehicle and bicycle). Postmortem blood concentrations were 2,325 ng/mL and 3,809 ng/mL. The medical examiner (ME) certified cause of death (COD) as acute mitragynine intoxication in both cases. The toxicology results presented become useful when considering mitragynine to be the offending agent in lethal single drug intoxications; further, the information included is pertinent to medical examiners, forensic pathologists, forensic toxicologists, and emergency department personnel in evaluating possible poisoning and lethality by mitragynine.
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Ukraine today does not regulate the sale of products made of Kratom (Mitragyna speciosa Korth. (from the family Rubiaceae) and does not take measures to control the quality and safety of this product, despite its rapid spread throughout the country. Аim of the Work is to summarize the results of scientific research on the toxicity of alternative opioids contained in Mitragyna speciosa and combined products based on them. Material and methods. Reports from the World Health Organization (WHO), the European Monitoring Center for Drugs and Drug Addiction (EMCDDA), the United Nations Office on Drugs and Crime, the results of scientific reviews and individual studies on biochemistry, toxicology, forensic identification of substances contained in products made from Mitragyna speciosa, over the past 10 years (Elsevier, PubMed, ToxNet). Results and Discussion. Recently, kratom has been cultivated on different continents and entered the market under the name "Kратом", in English-language sources - "Kratom". Kratom leaves are dried and sold in the form of green powder, tablets, capsules, extracts and gummies. In Ukraine, kratom is sold under hundreds of commercial names on the Internet as "Kratom", "Kratom product", "Kratom organic tea", "Kratom ethnic tea", "Kratom tea" and others. More than 40 structurally related alkaloids, as well as several flavonoids, terpenoid saponins, polyphenols and various glycosides were found in kratom leaves. The pharmacological and toxic effects of kratom for most of its components have not been studied enough. Like other dietary supplements, kratom products should be standardized for alkaloids, microbial contamination, pesticides, heavy metals, residual solvents, benzo(a)pyrene, aflatoxins, etc., with appropriate labeling requirements. Conclusions. Quality products should enter the Ukrainian market - standardized leaf extract of kratom, or other safe products made on its basis. However, recent studies show that mitraginine contained in kratom has great potential for medical science as a model for developing new approaches in very relevant areas of medicine: to treat pain and get rid of opioid dependence. Key Words: Mitragyna speciosa, alternative opioids, toxicity.
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Background and objective Learning and memory are necessary for survival. The hippocampus plays a significant role in learning process. GABAA receptors in the hippocampus are effective in learning and memory mechanism. The present study effect of tarragon hydroalcoholic extract and coumarin on memory, tissue index and GABAA receptor gene expression in the hippocampus of male rats. Methodology 56 Wistar rats were used and randomized in 7 groups (N = 8). These groups included the intact, receiving DMSO, receiving tarragon hydroalcoholic extract doses of 25, 50 and 100 mg/kg and receiving coumarin dose of 3, 5 mg/kg. They have undergone treatment intraperitoneally once a day for two weeks. The shuttle box was used for the memory retention test. The rats were killed according to the research ethical codes after the tests were done. When the brains of rats were removed, 4 brains in each group were chosen for the histological test using Nissl staining. In the other four brains, the hippocampus was removed immediately. The hippocampus was located in a microtube and was frozen by liquid nitrogen. Finally, a gene expression test was performed using real-time PCR. Results the findings of the present study reveal that there was no significant difference between of solvent recipients and the intact group in the memory retention test, the number of healthy hippocampal pyramidal neurons, and the expression of the GABAA gene. The treated groups with various doses of hydroalcoholic extract of tarragon and coumarin showed decreased in the memory retention test and the number of healthy pyramidals neurons as well as a significant increase in GABAA- α5 and GABAA- α2 genes expression compared to the group receiving solvent. Conclusion Tarragon hydroalcoholic extract and coumarin affects memory impairment through increasing the GABAA-α5 and GABAA-α2 genes expression and decreasing the number of healthy hippocampal neurons.
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Background and Objectives: Learning is essential for understanding mental disorders, normal behavior, and forgetfulness. In this regard, the hippocampus plays an important role in the learning process. It has been reported that gamma-aminobutyric acid (GABA) receptors in the hippocampus are involved in learning and memory mechanisms and some diseases, such as epilepsy and Alzheimerchr('39')s. This study aimed to investigate the effect of coumarin on retention, tissue index, and GABA type A receptor gene expression in the hippocampus of male gonadectomy rats. Methods: The population of this study consisted of 40 Wistar rats, which were randomized into 5 groups (n=8 each). These groups included healthy without treatment, gonadectomized without treatment, gonadectomized receiving solvent or Dimethyl sulfoxide, and gonadectomized receiving coumarin at a dose of 3.5 mg/kg. The treatment was administered intraperitoneally once daily in 2 weeks. A shuttle box was used to test the memory retention of the rats. At the end of the research process, the rats were exterminated in accordance with research ethics. After removing the brains of rats, in each group, in four brains histology test was implemented with Niels staining, and in four other brains, the hippocampus was removed quickly. The hippocampi were placed inside the micro type and frozen with liquid nitrogen. Finally, a gene expression test was taken from the hippocampus using a real-time polymerase chain reaction. Results: Based on the findings, in the memory retention test of initial latency to enter the dark room (step through latency), the gonadectomy group showed a reduction, compared to the healthy group. Moreover, a significant decrease was observed in the number of healthy hippocampal pyramidal neurons; however, GABAA gene expression showed no significant difference. In the gonadectomy groups receiving treatments with different doses of coumarin, the amount of STL (Step Through Latency) and number of healthy pyramidal neurons in the memory retention test showed a significant decrease, compared to the gonadectomy group receiving solvent; nonetheless, a significant increase was revealed in the GABAA-α2 gene expression. Conclusion: It can be concluded that Gonadectomy caused memory impairment and coumarin affects memory impairment by increasing the GABAA-α2 gene expression and decreasing the number of healthy hippocampal neurons.
Chapter
Mitragyna speciosa (kratom), a Southeast Asian plant belonging to the Mitragyna genus, has a long history of traditional uses. There are several therapeutic properties attributed to kratom such as energy booster, pain reliever, mood enhancer, remedy for various ailments, and management of opiate addiction. In recent years, kratom leaves and derivatized botanical products (e.g., extracts, solutions) are being sold as dietary supplements and marketed worldwide via the internet for the management of pain, anxiety, and depression. Indole and oxindole alkaloids are the major chemical constituents that are most likely implicated in the pharmacological effects of kratom products. In Western countries, other than in Southeast Asia, several issues have been alarming, such as adulteration, substitution, and spiking the plant material with neuropharmacological and illicit substances. This chapter provides a summary of the ethnobotany and alkaloid chemistry of kratom including plant biosynthesis and chemical synthesis of alkaloid molecules. Recent developments in the alkaloid detection methods for kratom profiling and authentication of kratom products are also discussed in this chapter. Additionally, this chapter discusses a compilation of the available information from the literature related to the CNS exposure and interaction of major kratom alkaloids.
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Mitragyna speciosa Korth. or ketum is classified under medicinal plants, used by locals in Thailand and Malaysia to treat various types of diseases. Due to lack of information present on the effect of M. speciosa in learning and memory function, therefore this study is conducted to understand its effect on cognitive functions (learning and long-term memory). The aim of this study is to evaluate cognitive functions of rats after acute exposure of M. speciosa standardized methanol extract using avoidance tasks. Sprague-Dawley rats were treated with three different concentrations of M. speciosa standardized methanol extract (100, 500 and 1000 mg/kg). Morphine was given to positive control group and carboxyl-methyl-cellulose (CMC) and piracetam were given to negative control group. Learning and memory were evaluated using one-way passive avoidance test and two-way active avoidance test. This study showed significant improvement in learning acquisition in the M. speciosa standardized methanol extract treated group compared to controls, however no benefit was observed on memory consolidation, in both passive and active avoidance tests. In conclusion, acute administration of M. speciosa standardized methanol extract facilitated learning, but there was no benefit on long-term memory consolidation. Key words: Mitragyna speciosa standardized methanol extract (MS), one-way passive avoidance test, two-way active avoidance test, memory impairment.
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Mitragyna speciosa Korth or ketum or kratom has long been used by local people in Thailand and Malaysia to treat various types of diseases and to boost energy. There is lack of information available on the effect of M. speciosa Korth in learning and memory function, therefore this study was conducted to understand its effect using a cellular model (hippocampus). The objective of this study was to delineate the effect of M. speciosa Korth standardized methanol extract (MS), we used extracellular recording in rat hippocampal slices in vitro. Acute hippocampal slices were prepared from 4 weeks-old male Sprague dawley rats. Field excitatory post-synaptic potentials (fEPSP) were investigated after the application of test materials in concentrations of 0.0001, 0.001, 0.005, 0.01, 0.05 and 0.1% dissolved in 0.1% DMSO. The 50% inhibitory concentration (IC50) of test material was then calculated. Superfusion of MS (all concentrations) produced irreversible fEPSP amplitude reduction with an IC 50 of 0.008%. The same concentration of MS (0.008%) prevented the induction of long-term potentiation (LTP) and induced only short-term potentiation (STP) in CA1 neurons. In the CA1 region of the hippocampus, reduced concentration-dependently glutamatergic transmission and blocked LTP at the IC50. Key words: Mitragyna speciosa Korth standardized methanol extraction (MS), extracellular recording, field excitatory post-synaptic potential (fEPSP), short-term potentiation (STP).
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Mitragyna speciosa Korth. or ketum is classified under medicinal plants, used by locals in Thailand and Malaysia to treat various types of diseases. Due to lack of information present on the effect of M. speciosa in learning and memory function, therefore this study is conducted to understand its effect on cognitive functions (learning and long-term memory). The aim of this study is to evaluate cognitive functions of rats after acute exposure of M. speciosa standardized methanol extract using avoidance tasks. Sprague-Dawley rats were treated with three different concentrations of M. speciosa standardized methanol extract (100, 500 and 1000 mg/kg). Morphine was given to positive control group and carboxyl-methyl-cellulose (CMC) and piracetam were given to negative control group. Learning and memory were evaluated using one-way passive avoidance test and two-way active avoidance test. This study showed significant improvement in learning acquisition in the M. speciosa standardized methanol extract treated group compared to controls, however no benefit was observed on memory consolidation, in both passive and active avoidance tests. In conclusion, acute administration of M. speciosa standardized methanol extract facilitated learning, but there was no benefit on long-term memory consolidation. Key words: Mitragyna speciosa standardized methanol extract (MS), one-way passive avoidance test, two-way active avoidance test, memory impairment.
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Kratom (or Ketum) is a psychoactive plant preparation used in Southeast Asia. It is derived from the plant Mitragyna speciosa Korth. Kratom as well as its main alkaloid, mitragynine, currently spreads around the world. Thus, addiction potential and adverse health consequences are becoming an important issue for health authorities. Here we reviewed the available evidence and identified future research needs. It was found that mitragynine and M. speciosa preparations are systematically consumed with rather well defined instrumentalization goals, e.g. to enhance tolerance for hard work or as a substitute in the self-treatment of opiate addiction. There is also evidence from experimental animal models supporting analgesic, muscle relaxant, anti-inflammatory as well as strong anorectic effects. In humans, regular consumption may escalate, lead to tolerance and may yield aversive withdrawal effects. Mitragynine and its derivatives actions in the central nervous system involve μ-opioid receptors, neuronal Ca2+ channels and descending monoaminergic projections. Altogether, available data currently suggest both, a therapeutic as well as an abuse potential.
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