Understanding the physicochemical properties of mitragynine, a principal alkaloid of Mitragyna speciosa, for preclinical evaluation.

Varied pharmacological responses have been reported for mitragynine in the literature, but no supportive scientific explanations have been given for this. These studies have been undertaken without a sufficient understanding of the physicochemical properties of mitragynine. In this work a UV spectrophotometer approach and HPLC-UV method were employed to ascertain the physicochemical properties of mitragynine. The pKa of mitragynine measured by conventional UV (8.11 ± 0.11) was in agreement with the microplate reader determination (8.08 ± 0.04). Mitragynine is a lipophilic alkaloid, as indicated by a logP value of 1.73. Mitragynine had poor solubility in water and basic media, and conversely in acidic environments, but it is acid labile. In an in vitro dissolution the total drug release was higher for the simulated gastric fluid but was prolonged and incomplete for the simulated intestinal fluid. The hydrophobicity, poor water solubility, high variability of drug release in simulated biological fluids and acid degradable characteristics of mitragynine probably explain the large variability of its pharmacological responses reported in the literature. The determined physicochemical properties of mitragynine will provide a basis for developing a suitable formulation to further improve its solubility, stability and oral absorption for better assessment of this compound in preclinical studies.

Anxiolytic-like effects of mitragynine in the open-field and elevated plus-maze tests in rats.

The effects of mitragynine on anxiety-related behaviours in the open-field and elevated plus-maze tests were evaluated. Male Sprague-Dawley rats were orally treated with mitragynine (10, 20 and 40 mg/kg) or diazepam (10 mg/kg) 60 min before behavioural testing. Mitragynine doses used in this study were selected on the basis of approximately human equivalent doses with reference to our previous literature reports. Acute administration of mitragynine (10, 20 and 40 mg/kg) or diazepam (10 mg/kg) increased central zone and open arms exploration in the open-field and elevated plus-maze tests respectively. These anxiolytic-like effects of mitragynine were effectively antagonized by intraperitoneal administration of naloxone (2 mg/kg), flumazenil (10 mg/kg), sulpiride (0.5 mg/kg) or SCH 23390 (0.02 mg/kg) 15 min before mitragynine treatments. These findings reveal that the acute administration of mitragynine produces anxiolytic-like effects and this could be possibly attributed to the interactions among opioidergic, GABAergic and dopaminergic systems in brain regions involved in anxiety.

A simple HPLC-DAD method for the detection and quantification of psychotropic mitragynine in Mitragyna speciosa (ketum) and its products for the application in forensic investigation.

Mitragyna speciosa, a native plant of Thailand and Malaysia known as 'ketum', is a plant of considerable interest. It exhibits strong antinociceptive effect and yet, acts like a psychostimulant. Due to the affordability and its ease of availability, the abuse of this plant as a substitute for other banned narcotics has become a major concern in many societies. In countries such as Thailand, Myanmar, Australia and Malaysia, the use of ketum is illegal. However, for a person to be charged for possessing or selling ketum, a reliable analytical method is needed in order to detect and identify the plant and its products. Mitragynine is the major alkaloid of ketum. This compound manifests its antinociceptive effects by acting on the opioid receptors. Since M. speciosa contain large quantity of mitragynine and it is exclusive to the species, the present analytical method is developed and validated for the purpose of screening ketum products based on this unique compound as the analytical marker. The method uses a HPLC-DAD system with Inertsil C8 (4.6 mm × 150 mm, 5 µm) as the column and a mixture of acetonitrile and formic acid, 50:50 (v/v), as the mobile phase. This method not only detects mitragynine, it can also be used to quantify the amount of mitragynine in the sample. The limit of detection is 0.25 µg/ml, while the limit of quantification is 0.50 µg/ml. The method is quick, simple and reliable with an accuracy of 97.27-101.74% and coefficient of variations of between 0.91 and 3.96%. The method has been tested and found suitable for the identification and quantification of mitragynine in dried plants, a variety of ketum extracts, as well as ketum drink obtained from the market.

Determination of mitragynine in plasma with solid-phase extraction and rapid HPLC-UV analysis, and its application to a pharmacokinetic study in rat.

A new solid phase extraction method for rapid high performance liquid chromatography-UV determination of mitragynine in plasma has been developed. Optimal separation was achieved with an isocratic mobile phase consisting of acetonitrile-ammonium acetate buffer, 50 mM at pH 5.0 (50:50, v/v). The method had limits of detection and quantification of 0.025 and 0.050 microg/mL, respectively. The method was accurate and precise for the quantitative analysis of mitragynine in human and rat plasma with within-day and between-day accuracies between 84.0 and 109.6%, and their precision values were between 1.7 and 16.8%. Additional advantages over known methods are related to the solid phase extraction technique for sample preparation which yields a clean chromatogram, a short total analysis time, requires a smaller amount of plasma samples and has good assay sensitivity for bioanalytical application. The method was successfully applied in pharmacokinetic and stability studies of mitragynine. In the present study, mitragynine was found to be fairly stable during storage and sample preparation. The present study showed for the first time the detailed pharmacokinetic profiles of mitragynine. Following intravenous administration, mitragynine demonstrated a biphasic elimination from plasma. Oral absorption of the drug was slow, prolonged and was incomplete, with a calculated absolute oral bioavailability value of 3.03%. The variations observed in previous pharmacokinetic studies after oral administration of mitragynine could be attributed to its poor bioavailability rather than to the differences in assay method, metabolic saturation or mitragynine dose.

Evaluation of antioxidant and antibacterial activities of aqueous, methanolic and alkaloid extracts from Mitragyna speciosa (Rubiaceae family) leaves.

Studies on the antioxidant and antimicrobial activities of Mitragyna speciosa leaf extracts are lacking. In this study the antioxidant properties of water, methanolic and alkaloid M. speciosa leaf extracts were evaluated using the DPPH (2,2-diphenyl-1- picrylhydrazyl) radical scavenging method. The amount of total phenolics and flavanoid contents were also estimated. The DPPH IC(50) values of the aqueous, alkaloid and methanolic extracts were 213.4, 104.81 and 37.08 microg/mL, respectively. The total phenolic content of the aqueous, alkaloid and methanolic extracts were 66.0 mg, 88.4, 105.6 mg GAE/g, respectively, while the total flavanoid were 28.2, 20.0 and 91.1 mg CAE/g respectively. The antioxidant activities were correlated with the total phenolic content. This result suggests that the relatively high antioxidant activity of the methanolic extract compared to aqueous and alkaloid extract could be possibly be due to its high phenolic content. The aqueous, alkaloid and methanolic extracts were screened for antimicrobial activity. The extracts showed antimicrobial activity against Salmonella typhi and Bacillus subtilis. The minimum inhibitory concentrations (MICs) of extracts determined by the broth dilution method ranged from 3.12 to 6.25 mg/mL. The alkaloid extract was found to be most effective against all of the tested organisms.