Seasonal and Geographic Variation in Alkaloid Content of Kratom (Mitragyna speciosa (Korth.) Havil.) from Thailand.

The objective of this study was to obtain data on the distribution of alkaloids in kratom plants grown in Thailand. Two collections were performed, covering the southern, central, and northern regions of Thailand and different seasons. The contents of alkaloids, including mitragynine (MG), paynantheine (PAY), and speciogynine (SG), were determined using the validated HPLC method. The 134 samples in the first collection were collected from Nam Phu subdistrict, Ban Na San, Surat Thani, Thailand, during June and October 2019 and January 2020. The maximum mitragynine content was 4.94% w/w in June (late summer), and the minimum content was 0.74% w/w in October (rainy season). To expand the study area after kratom decriminalization, 611 samples were collected in June-August 2021, October-December 2021, and January-April 2022. The accumulation of MG ranged from 0.35 to 3.46% w/w, 0.31 to 2.54% w/w, and 0.48 to 2.81% w/w, respectively. The meteorological data supported the climate's effect on alkaloid production. Soil analysis revealed the importance of Ca and Mg in promoting alkaloid production. Geographical locations played a role in the variation of MG in kratom leaves, but did not affect the color of leaf veins. In conclusion, the present study suggested that the alkaloid content in kratom diverges based on seasonal and geographical origin.

Effects of alkaloid-rich extract from Mitragyna speciosa (Korth.) Havil. on naloxone-precipitated morphine withdrawal symptoms and local field potential in the nucleus accumbens of mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Mitragyna speciosa (Korth.) Havil. (M. speciosa) is among the most well-known plants used in ethnic practice of Southeast Asia. It has gained increasing attention as a plant with potential to substitute morphine in addiction treatment program. However, its action on the central nervous system is controversial. AIM OF THE STUDY: This study investigated the effects of M. speciosa alkaloid extract on naloxone-precipitated morphine withdrawal and neural signaling in the nucleus accumbens (NAc, brain reward center) of mice. MATERIALS AND METHODS: The effects of M. speciosa alkaloid extract and mitragynine, a pure major constituent, on naloxone-precipitated morphine withdrawal were examined. Male Swiss Albino (ICR) mice were rendered dependent on morphine before injection with naloxone, a nonspecific opioid antagonist, to induce morphine withdrawal symptoms. The intensity of naloxone-precipitated morphine withdrawal was assessed from jumping behavior and diarrhea induced during a period of morphine withdrawal. To test possible addictive effect of M. speciosa alkaloid extract, mice were implanted with intracranial electrode into the NAc for local field potential (LFP) recording. Following M. speciosa alkaloid extract (80mg/kg) and morphine (15mg/kg) treatment, LFP power spectra and spontaneous motor activity were analyzed in comparison to control levels. RESULTS: One-way ANOVA and multiple comparisons revealed that M. speciosa alkaloid extract (80 and 100mg/kg) significantly decreased the number of jumping behavior induced by morphine withdrawal whereas mitragynine did not. Additionally, M. speciosa alkaloid extract significantly decreased dry and wet fecal excretions induced by morphine withdrawal. LFP analysis revealed that morphine significantly decreased alpha (9.7-12Hz) and increased low gamma (30.3-44.9Hz) and high gamma (60.5-95.7Hz) powers in the NAc whereas M. speciosa alkaloid extract did not. Spontaneous motor activity was significantly increased by morphine but not M. speciosa alkaloid extract. CONCLUSIONS: Taken together, M. speciosa alkaloid extract, but not mitragynine, attenuated the severity of naloxone-precipitated morphine withdrawal symptoms. Neural signaling in the NAc and spontaneous motor activity were sensitive to morphine but not M. speciosa alkaloid extract. Therefore, treatment with the M. speciosa alkaloid extract may be useful for opiate addiction treatment program.

Quantitative analysis of mitragynine, codeine, caffeine, chlorpheniramine and phenylephrine in a kratom (Mitragyna speciosa Korth.) cocktail using high-performance liquid chromatography.

A simple HPLC technique for determining mitragynine, codeine, caffeine, chlorpheniramine and phenylephrine in 'kratom cocktail' was developed. The analytical method for mitragynine, codeine and caffeine used an Eclipse XDB-C8 column. A Lichrospher CN column was using for analysing chlorpheniramine and phenylephrine. The correlation coefficient of each standard was between 0.9957 and 0.9993. The precision of the methods were between 0.700 and 7.108% RSD. The concentration of mitragynine, codeine, caffeine, chlorpheniramine and phenylephrine in 'kratom cocktail' was 90.021, 234.174, 73.986, 7.053 and 1.486 mg/L, respectively.

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.

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.

Pharmacokinetics of ivermectin in cats receiving a single subcutaneous dose.

Ivermectin is effective against ecto- and endoparasites. It is included in a plan of the Filariasis Division, Thailand for filariasis control and prevention by interrupting transmission of Brugia malayi-microfilariae from cat reservoirs to humans via mosquitoes. The pharmacokinetics of ivermectin in eight healthy cats receiving a single subcutaneous dose of 0.2mg/kg was investigated. Jugular blood samples were collected periodically for up to 30days after dosing. The serum ivermectin concentrations were measured by high performance liquid chromatography with fluorescence detection. The pharmacokinetic parameters (mean+/-S.D.) derived from one-compartment model analysis were as follows: T(max) 1.22+/-0.49day, C(max) 16.75+/-4.04ng/mL, k(ab) 2.62+/-1.86day(-1), t(1/2)(ab) 0.27+/-0.25day, k(el) 0.27+/-0.14day(-1), t(1/2)(el) 2.53+/-2.24day, V(d)/F 9.81+/-5.41L/kg, Cl/F 2.21+/-0.69L/kg/day and AUC(0-->infinity) 98.31+/-30.52ngday/mL. In conclusion, the pharmacokinetics of ivermectin in cats receiving a single dose of 0.2mg/kg by subcutaneous injection revealed a rapid absorption, high distribution, slow elimination and high possibility for the elimination of B. malayi-microfilariae from currently endemic regions.

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.

A high-performance liquid chromatographic method for determination of mitragynine in serum and its application to a pharmacokinetic study in rats.

A simple HPLC technique for determining mitragynine levels in serum was developed. The separation system consisted of a C18 column heated to 35 degrees C, a methanol-water (80:20, v/v) mobile phase, a flow rate of 0.8 mL/min and detection in the ultraviolet at 225 nm. Mitragynine, with a retention time of 10.09 min, was well resolved from any interferences in human serum and the internal standard peak. The calibration curve was linear from 0.1 to 10 microg/mL (r = 0.9995). Extraction of mitragy-nine from alkalinized serum using diethyl ether gave a high recovery (>or=85%). The intra- and inter-day precisions of the method were 4.29-5.88%RSD and 7.06-8.45%RSD, respectively. The accuracy ranged from -9.54 to +0.67%DEV. The limit of detection was 0.03 microg/mL and the lower limit of quantification was 0.1 microg/mL. Mitragynine in the stock solution was stable during 30 days of storage at 4 degrees C. This method was successfully applied to determine the pharmacokinetic characteristics of mitragynine levels in the serum of rats after it was administered orally.