Niwat Keawpradub

The neuromuscular blockade produced by pure alkaloid, mitragynine and methanol extract of kratom leaves (Mitragyna speciosa Korth.)

AIM OF THE STUDY The effects of pure alkaloid, mitragynine and a methanolic extract of kratom leaves were investigated on neuromuscular junction and compound nerve action potential. MATERIALS AND METHODS Wistar rats were killed by cervical dislocation and decapitated. The phrenic nerve-hemidiaphragms, hemidiaphragms and sciatic nerve were isolated. RESULTS Kratom methanolic extract present at 0.1-1 mg/mL and mitragynine (0.0156 mg/mL) decreased the 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. Pancuronium and succinylcholine potentiated the effect of kratom extract. It also had a direct relaxation effect on the hemidiaphragm muscle. The muscle relaxation caused by kratom extract was not antagonized 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 of compound nerve action potential. CONCLUSIONS The mechanism of action of kratom extract might not act as a competitive antagonist of acetylcholine yet its dominant effect was at the neuromuscular junction and not at the skeletal muscle or somatic nerve.

Inhibitory effects of kratom leaf extract (Mitragyna speciosa Korth.) on the rat gastrointestinal tract

Kratom (Mitragyna speciosa Korth.) is an indigenous plant of Thailand used traditionally in folk medicine although it is claimed to cause addiction. It is used to treat diarrhea, however, there is no scientific evidence to support the use. The aim of this study is to investigate the effect of methanolic extract of kratom leaves on the rat gastrointestinal tract. Kratom extract at 50, 100, 200 and 400 mg/kg (p.o.) caused a dose dependent protection against castor oil-induced diarrhea in rats and also inhibited intestinal transit. The antidiarrheal effect was not antagonized by naloxzone. The inhibition of intestinal transit by kratom extract was significantly different from the control when treated with a single dose for 1 day. For longer-term treatments of 15 and 30 days, kratom extract did not decrease the intestinal transit time indicating that adaptation had occurred. Kratom extract at a dose level of 200 and 400 mg/kg for 30 days and morphine at 3 mg/kg (i.p.) caused a decrease in the increment of body weight that was significantly different from the control and kratom extract at lower doses (50 and 100 mg/kg). However it had no effect on the level of plasma cholecystokinin. The results suggested that methanolic kratom extract exhibited its antidiarrheal effect on rat gastrointestinal tract. The effects may occur via pathways in addition to the action on opioid receptors. High does of kratom extract decreased the increment of body weight similar to the effect of morphine.

Indole alkaloids fromAlstonia glaucescens

Abstract Three new indole alkaloids, 17- O -acetyl- N b -demethylechitamine, echitamidine N -oxide and echitaminic acid, have been isolated from the stem bark of Alstonia glaucescens along with one iridoid compound, sweroside, and five known indole alkaloids, i.e. echitamidine, N b - demethylechitamine, 20-epi-19ξ-echitamidine, N b -demethylechitamine N -oxide and echitamine. Their structures were elucidated by spectroscopic methods, especially 2D NMR.

Fos-like immunoreactivity in rat dorsal raphe nuclei induced by alkaloid extract of Mitragyna speciosa

Mitragyna speciosa (MS) has been traditionally used for medicinal purposes especially in southern Thailand. Previously, an alkaloid extract of this plant was demonstrated to mediate antinociception, partly, through the descending serotonergic system. The present study investigated the stimulatory effect of the MS extract on the dorsal raphe nucleus and its antidepressant-like activity. The MS extract containing approximately 60% mitragynine as a major indole alkaloid was used to treat the animals. The stimulatory effect of the MS extract was determined by detecting the expression of the immediate early gene, cfos, in the dorsal raphe nucleus of male Wistar rats. The immunohistochemistry was used to detect Fos protein, the protein product of cfos gene. The present data show that a significant increase in Fos expression was observed following long-term administration of the MS extract (40 mg/kg) for 60 consecutive days. In addition, the antidepressant-like activity of the MS extract was determined by using the forced swimming test (FST) in male mice. The results show that a single injection (either 60 or 90 mg/kg doses) significantly decreased immobility time in the FST. These findings indicate that the MS extract has a stimulatory effect on the dorsal raphe nucleus and an antidepressant-like activity. Stimulation of this brain area has been known to cause antinociception. These findings suggest that the MS extract might produce antinociceptive and/or antidepressive actions partly through activation of the dorsal raphe nucleus. Moreover, the dorsal raphe nucleus may be one of site of MS action in the central nervous system.

Cytotoxic activity of acetogenins and styryl lactones isolated from Goniothalamus undulatus Ridl. root extracts against a lung cancer cell line (COR-L23)

An investigation of the chemical constituents in a dichloromethnae extract of Goniothalamus undulatus root led to the isolation of three known styryl lactones (5-acetoxyisogoniothalamin oxide, O-acetylaltholactone and altholactone), and four known annonaceous acetogenins (annonacin, cis-annonacin, goniothalamicin and cis-goniothalamicin). These compounds were subjected to a sulphorhodamine B (SRB) cytotoxicity assay against human large cell lung carcinoma (COR-L23), and normal human fetal fibroblast (MRC-5), cell lines. The isolated acetogenins showed higher cytotoxic activity against COR-L23 compared to the styryl lactones, with IC₅₀ values in the range of 0.5-1.7 μM and 7.4-15.4 μM, respectively. A similar pattern of cytotoxicity was also observed against the other cell line (MRC-5); acetogenins IC₅₀ values were in the range of 11.8-31.4 μM, and those for styryl lactones were in the range of 48.7-102.8 μM. This is the first report of a bioassay-guided isolation of chemical constituents from G. undulatus and on cytotoxic studies of the isolated compounds using these particular lung cancer cell lines.

Study on glucose transport in muscle cells by extracts from Mitragyna speciosa (Korth) and mitragynine

The leaves of Mitragyna speciosa Korth (Rubiaceae) have been used in folk medicine for its unique medicinal properties. This study examined the water, methanolic and crude alkaloidal extracts from M. speciosa leaves and its major constituent mitragynine for the enhancement of glucose transport. Cellular uptake of radioactive 2-deoxyglucose was determined in rat L8 myotubes. Involving signalling pathway was determined with the specific inhibitors. Cell cytotoxicity was monitored by lactate dehydrogenase assay. Protein levels of glucose transporters (GLUTs) were measured by Western blotting. The results show that test samples significantly increased the rate of glucose uptake. The uptake was associated with increase in GLUT1 protein content. Co-incubation with insulin had no additional effect, but the cellular uptake was decreased by wortmannin and SB 203580, specific inhibitors of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38 MAPK), respectively. It is concluded that the increased glucose transport activity of M. speciosa is associated with increases in activities of the key enzymes dependent to the insulin-stimulated glucose transport for its acute action, and increases in the GLUT1 content for its long-term effect. This study demonstrated the effect of M. speciosa in stimulating glucose transport in muscle cells, implicating the folkloric use of M. speciosa leaves for treating diabetes.

Mitragyna speciosa: Hairy Root Culture for Triterpenoid Production and High Yield of Mitragynine by Regenerated Plants

Hairy root cultures of Mitragyna speciosa were established by infection of Agrobacterium rhizogenes ATCC 15834 and maintained in McCown woody plant medium (WPM) supplemented with 0.5 mg/l naphthaleneacetic acid. The hairy roots were identified for the rooting genes loci of rolA and rolB by polymerase chain reaction. For studying the secondary metabolite production, the n-hexane extract of the hairy roots was prepared and the compounds were isolated by silica gel column chromatography, affording triterpenoids (ursolic acid and oleanolic acid) and phytosterols (β-sitosterol and stigmasterol). The shoots from the hairy root cultures were regenerated and differentiated to the plantlets. For micropropagation, shoot multiplication was successfully induced from the axillary buds of the regenerated plantlets in WPM supplemented with 0.1 mg/l thidiazuron. The mitragynine contents of 5-monthold regenerated plants and in vitro plantlets (germinated from seeds) were determined using the TLC-densitometric method. The regenerated plants contained (14.25 ± 0.25) mg/g dry wt mitragynine, whereas the in vitro plantlets contained (4.45 ± 0.09) mg/g dry wt.

Development of indirect competitive ELISA for quantification of mitragynine in Kratom (Mitragyna speciosa (Roxb.) Korth.)

Monoclonal antibody (MAb) against mitragynine (MG), an analgesic alkaloid from Kratom leaves (Mitragyna speciosa), was produced. MG was coupled to carrier proteins employing either 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), a zero-length cross linker or a 5-carbon length glutaraldehyde cross linker. To confirm the immunogenicity, the hapten numbers were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Preparation of the MAb was accomplished by the electrofusion method. Hybridoma 1A6 that was constructed from the fusion between splenocytes of EDC/NHS conjugate immunized mice and SP2/0-Ag14 myeloma cells was selected, cloned twice and expanded. The cross-reactivities (CRs) of this MAb 1A6 with a series of indole alkaloids were 30.54%, 24.83% and 8.63% for speciogynine, paynantheine and mitraciliatine, respectively. Using this MAb, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with a measurement range of 32.92-250 μg/mL. Quantitative analysis of the MG contents in plant samples by icELISA correlated well with the standard high performance liquid chromatography method (R(2)=0.994). The MAb against mitragynine provided a tool for detection of MG in Kratom preparations.

Effects of Mitragynine and a Crude Alkaloid Extract Derived from Mitragyna speciosa Korth. on Permethrin Elimination in Rats

Detoxification and elimination of permethrin (PM) are mediated by hydrolysis via carboxylesterase (CES). Mitragyna speciosa (kratom) contains mitragynine (MG) and other bioactive alkaloids. Since PM and MG have the same catalytic site and M. speciosa is usually abused by adding other ingredients such as pyrethroid insecticides, the effects of MG and an alkaloid extract (AE) on the elimination of PM were investigated in rats. Rats were subjected to single and multiple pretreatment with MG and AE prior to receiving a single oral dose (460 mg/kg) of PM. Plasma concentrations of trans-PM and its metabolite phenoxybenzylalcohol (PBAlc) were measured. The elimination rate constant (kel) and the elimination half-life (t1/2 el) of PM were determined, as well as the metabolic ratio (PMR).A single and multiple oral pretreatment with MG and AE altered the plasma concentration-time courses of both trans-PM and PBAlc during 8–22 h, decreased the PMRs, delayed elimination of PM, but enhanced elimination of PBAlc. Results indicated that PM–MG or AE toxicokinetic interactions might have resulted from the MG and AE interfering with PM hydrolysis. The results obtained in rats suggest that in humans using kratom cocktails containing PM, there might be an increased risk of PM toxicity due to inhibition of PM metabolism and elimination.

Effects of an alkaloid-rich extract from Mitragyna speciosa leaves and fluoxetine on sleep profiles, EEG spectral frequency and ethanol withdrawal symptoms in rats.

BACKGROUND Many antidepressants are effective in alleviating ethanol withdrawal symptoms. However, most of them suppress rapid eye movement (REM) sleep. Thus, development of antidepressants without undesirable side effects would be preferable. Previously, crude alkaloid extract from Mitragyna speciosa (MS) Korth was found to produce antidepressant activities. It was hypothesized that the alkaloid extract from MS may attenuate ethanol withdrawal without REM sleep disturbance. METHODS Adult male Wistar rats implanted with electrodes over the frontal and parietal cortices were used for two separated studies. For an acute study, 10 mg/kg fluoxetine or 60 mg/kg alkaloid extract from MS were administered intragastrically. Electroencephalographic (EEG) signals were recorded for 3 h to examine sleep profiles and EEG fingerprints. Another set of animal was used for an ethanol withdrawal study. They were rendered dependent on ethanol via a modified liquid diet (MLD) containing ethanol ad libitum for 28 days. On day 29, fluoxetine (10 mg/kg) or alkaloid extract from MS (60 mg/kg) were administered 15 min before the ethanol-containing MLD was replaced with an isocaloric ethanol-free MLD to induced ethanol withdrawal symptoms. RESULTS The sleep analysis revealed that alkaloid extract from MS did not change any REM parameters which included average duration of each REM episode, total REM time, number of REM episode and REM latency whereas fluoxetine significantly suppressed all REM parameters and delayed REM latency. However, power spectral analysis revealed similar fingerprints for fluoxetine and alkaloid extract from MS characterized by decreasing powers in the slow frequency range in frontal and parietal cortical EEG. Neither treatment affected spontaneous motor activity. Finally, both alkaloid extract from MS and fluoxetine were found to significantly attenuate ethanol withdrawal-induced hyperexcitability (increases gamma activity) in both cortices and to reduce locomotor activity. CONCLUSION The present study demonstrated that the alkaloid extract from MS alleviates ethanol withdrawal severity with no side effect on REM sleep. In addition, these data suggest that suppressive effects on slow frequency powers but not REM sleep may be hallmarks of effective antidepressants for ethanol withdrawal treatment.