Metabolic engineering of yeast for de novo production of kratom monoterpene indole alkaloids.

Monoterpene indole alkaloids (MIAs) from Mitragyna speciosa ("kratom"), such as mitragynine and speciogynine, are promising novel scaffolds for opioid receptor ligands for treatment of pain, addiction, and depression. While kratom leaves have been used for centuries in South-East Asia as stimulant and pain management substance, the biosynthetic pathway of these psychoactives have only recently been partially elucidated. Here, we demonstrate the de novo production of mitragynine and speciogynine in Saccharomyces cerevisiae through the reconstruction of a five-step synthetic pathway from common MIA precursor strictosidine comprising fungal tryptamine 4-monooxygenase to bypass an unknown kratom hydroxylase. Upon optimizing cultivation conditions, a titer of ∼290 µg/L kratom MIAs from glucose was achieved. Untargeted metabolomics analysis of lead production strains led to the identification of numerous shunt products derived from the activity of strictosidine synthase (STR) and dihydrocorynantheine synthase (DCS), highlighting them as candidates for enzyme engineering to further improve kratom MIAs production in yeast. Finally, by feeding fluorinated tryptamine and expressing a human tailoring enzyme, we further demonstrate production of fluorinated and hydroxylated mitragynine derivatives with potential applications in drug discovery campaigns. Altogether, this study introduces a yeast cell factory platform for the biomanufacturing of complex natural and new-to-nature kratom MIAs derivatives with therapeutic potential.

Antioxidant and antibacterial efficiency of the ethanolic leaf extract of Kratom (Mitragyna speciosa (Korth.) Havil) and its effects on growth, health, and disease resistance against Edwardsiella tarda infection in Nile tilapia (Oreochromis niloticus).

The research examined the impact of an ethanolic extract from the leaves of Kratom (Mitragyna speciosa (Korth.) Havil.) on the growth, antioxidant capacity, immune-related gene expression, and resistance to disease caused by Edwardsiella tarda in Nile tilapia (Oreochromis niloticus). The findings revealed that the extract had the important phytochemical content in the extract included total phenolics content, total flavonoids content, vitamin C, and total antioxidant capacity and 5.42 % of the crude extract was mitragynine. The extract demonstrated antioxidant activity, as evidenced by its IC50 values against ABTS and DPPH radicals and its ferric reducing power in vitro. Moreover, the MIC-IC50 value of 0.625 mg/mL indicated that the growth of the bacteria was reduced by approximately 50 %, and the MBC was 2.50 mg/mL against E. tarda. Furthermore, the orally administered Kratom leaf extract to fingerling tilapia for 8 weeks exhibited a noticeable increase in oxidative stress, as demonstrated by the increase in MDA production in the 10 and 25 g/kg groups. It also exhibited an increase in acetylcholinesterase (AChE) activity in muscle tissue at the 50 g/kg group. However, when administered at a feeding rate of 5-10 g/kg feed, the extract showed an increase in the expression of immune-related genes (IL1, IL6, IL8, NF-kB, IFNγ, TNFα, Mx, CC-chemokine, CD4, TCRß, MHC-IIß, IgM, IgT, IgD) and enhanced resistance to E. tarda infection in fish. Conversely, administering the extract at 25-50 g/kg feed resulted in contrasting effects, suppressing and reducing the observed parameters. Nevertheless, feeding the extract at all concentrations for 8 weeks did not produce any changes in the histology or systemic functioning of the liver and intestines, as indicated by blood biochemistry. These findings suggest that the ethanolic leaf extract from Kratom has the potential to be used as a substitute for antibiotics in the management of bacterial infections in Nile tilapia culture, with a recommended dosage of 5-10 g/kg feed/day for a maximum of 8 weeks.

Quantitative analysis of selected alkaloids of Mitragyna speciosa using 1H quantitative nuclear magnetic resonance spectroscopy.

Mitragyna speciosa is a perennial plant native to Asia, well known for its psychoactive properties. Its major alkaloid mitragynine is known to have sedative and euphoric effects. Hence, the plant has been a subject of abuse, leading to addiction, necessitating efficient analytical methods to detect its psychoactive constituents. However, current chromatography-based methods for detecting the alkaloids are time consuming and costly. Quantitative nuclear magnetic resonance (qNMR) spectroscopy emerges as a promising alternative due to its nondestructive nature, structural insights, and short analysis time. Hence, a rapid and precise qNMR method was developed to quantify selected major psychoactive alkaloids in various parts of M. speciosa. Mitragynine, specioliatine, and speciogynine were quantified in relation to the integral value of the -OCH3 groups of the alkaloids and the internal standard 1,4-dinitrobenzene. The precision and reproducibility of the method gave a relative standard deviation (RSD) of 2%, demonstrating the reliability of the method. In addition, the method showed excellent specificity, sensitivity, high linearity range (R2 = 0.999), and limits of detection (LOD) and quantification (LOQ) values. The analysis revealed that the red-veined M. speciosa leaves contained higher levels of mitragynine (32.34 mg/g), specioliatine (16.84 mg/g) and speciogynine (7.69 mg/g) compared to the green-veined leaves, stem bark, or fruits.

Rapid mitragynine quantification and fingerprinting of products from Mitragyna speciosa Korth. leaf (Kratom) using high-performance thin-layer chromatography.

INTRODUCTION: Kratom (leaves from Mitragyna speciosa Korth.; Rubiaceae) is a herbal medicine known for its analgesic properties and psychoactive effects. Kratom in Thailand is currently legal; however, it is prohibited in some countries and considered a narcotic plant. OBJECTIVE: Our aim was to establish a reliable, simple, and rapid method for quantifying mitragynine in Kratom leaves and related products through a combination of high-performance thin-layer chromatography (HPTLC) and densitometry. METHODOLOGY: A densitometric HPTLC method was developed and validated in terms of specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, and robustness. The fingerprints of kratom leaves, Mitragyna spp., and related products were constructed. RESULTS: For HPTLC, samples were applied to silica gel 60 F254 plates, and the mobile phase comprised n-hexane, ethyl acetate, and triethylamine (1:1:0.15, v/v/v). Densitometric detection was carried out under ultraviolet light at a wavelength of 226 nm. The validated method exhibited a range of 14.31-143.10 µg/mL, yielding a correlation coefficient of 0.9993. Spiked recovery rates were within a range of 98.3%-100.9%, and the LOD and LOQ were 3.80 and 11.53 µg/mL, respectively. Kratom samples were analyzed with the developed method, and the correlation coefficient was 0.9641, compared to the high-performance liquid chromatography-diode-array detection (HPLC-DAD) method. The HPTLC fingerprints displayed a distinctive pattern, facilitating discrimination among different plant parts and Mitragyna spp. CONCLUSION: The established method offers the advantages of simplicity, ease of use, and speed of analysis, serving as a practical alternative for mitragynine quantification in kratom leaf and its related products.

An In Vitro Examination of Whether Kratom Extracts Enhance the Cytotoxicity of Low-Dose Doxorubicin against A549 Human Lung Cancer Cells.

Doxorubicin is an effective chemotherapeutic agent in the treatment of solid hematological and non-hematological carcinoma. However, its long-term usage could result in side effects, such as cardiomyopathy, chronic heart failure, neurotoxicity and cancer cell resistance. In this study, we reported the sensitivity enhancement of A549 human lung cancer cells on doxorubicin at a low dose (0.1 ppm) in combination with 10-60 ppm of crude and alkaloid extracts derived from the leaves of Kratom (Mitragyna speciosa (Korth.) Havil. Rubiaceae). A549 cancer cell lines were insensitive to the crude extract containing low mitragynine (MG) (4-5%), while these cells were moderately inhibited by the alkaloid extract containing 40-45% MG (IC50 of 48-55 ppm). The alkaloid extract was found to inhibit A549 cancer cells via apoptosis as suggested by the higher relative fluorescence intensity with Annexin compared to that in propidium iodide (PI), i.e., a positive Annexin and a negative PI. The combination of crude extract and doxorubicin sensitized A549 cancer cells to doxorubicin by 1.3 to 2.4 times, while the combination with the alkaloid induced a 2.6- to 3.4-fold increase in sensitivity. The calculated combination index (CI) for doxorubicin with the crude and alkaloid extracts was 0.6 and 0.3, respectively, showing potential synergistic combinations to reduce the level of dosage of doxorubicin used in chemotherapy. In addition, the synergistic enhancement effect of crude extract on the cytotoxic activity of doxorubicin provides insights into the plausibility of non-alkaloids to influence the biological activities of Kratom.

Human Mitragynine and 7-Hydroxymitragynine Pharmacokinetics after Single and Multiple Daily Doses of Oral Encapsulated Dried Kratom Leaf Powder.

Kratom leaves, consumed by millions worldwide as tea or ground leaf powder, contain multiple alkaloids, with mitragynine being the most abundant and responsible for most effects. Mitragynine is a partial µ-opioid receptor agonist and competitive antagonist at κ- and δ-opioid receptors; however, unlike morphine, it does not activate the ß-arrestin-2 respiratory depression pathway. Due to few human mitragynine data, the largest randomized, between-subject, double-blind, placebo-controlled, dose-escalation study of 500-4000 mg dried kratom leaf powder (6.65-53.2 mg mitragynine) was conducted. LC-MS/MS mitragynine and 7-hydroxymitragynine plasma concentrations were obtained after single and 15 daily doses. Mitragynine and 7-hydroxymitragynine Cmax increased dose proportionally, and AUC was slightly more than dose proportional. The median mitragynine Tmax was 1.0-1.3 h after single and 1.0-1.7 h after multiple doses; for 7-hydroxymitragynine Tmax, it was 1.2-1.8 h and 1.3-2.0 h. Steady-state mitragynine concentrations were reached in 8-9 days and 7-hydroxymitragynine within 7 days. The highest mean mitragynine T1/2 was 43.4 h after one and 67.9 h after multiple doses, and, for 7-hydroxymitragynine, it was 4.7 and 24.7 h. The mean 7-hydroxy-mitragynine/mitragynine concentration ratios were 0.20-0.31 after a single dose and decreased (0.15-0.21) after multiple doses. These mitragynine and 7-hydroxymitragynine data provide guidance for future clinical kratom dosing studies and an interpretation of clinical and forensic mitragynine and 7-hydroxymitragynine concentrations.

[Kratom-From natural remedy to addictive drug and back]. / Kratom ­ vom natürlichen Heilmittel zur Suchtdroge und zurück.

BACKGROUND: Kratom/ketum is a psychoactive herbal preparation that has been used for a long time as a remedy and performance-enhancing substance in Southeast Asia. The advancement of globalization is making kratom increasingly more available in the western world, where it is becoming increasingly more used. OBJECTIVE: The current research on kratom and its ingredients is presented. MATERIAL AND METHODS: An overview of the use and effects of kratom is exemplary given on the basis of reports. The instrumentalization of the drug and its consequences up to the development of addiction are discussed. RESULTS: Consumption is accompanied by several instrumentalizeable effects so that kratom is used as a therapeutic substance in the self-management of pain, anxiety and depression as well as other substance addictions. Another benefit comes from the performance-enhancing effects on physical work and in a social context. Consumption is usually well controlled, rarely escalates and has few and mostly mild aversive side effects. The danger arises from consumption particularly when there is an escalation of the dose and from mixed consumption with other psychoactive substances. The main alkaloid mitragynine and the more potent 7­hydroxy-mitragynine are considered mainly responsible for the effect. Both have a complex pharmacology that involves partial µ­opioid receptor agonism. DISCUSSION: Epidemiological, clinical and neurochemical studies have shown that kratom only has a limited addictive drug profile, which might suggest a medical use as a remedy or substitute in addiction treatment.

Biosynthesis of kratom opioids.

Mitragynine, an analgesic alkaloid from the plant Mitragyna speciosa (kratom), offers a safer alternative to clinical opioids such as morphine, owing to its more favorable side effect profile. Although kratom has been traditionally used for stimulation and pain management in Southeast Asia, the mitragynine biosynthesis pathway has remained elusive. We embarked on a search for mitragynine biosynthetic genes from the transcriptomes of kratom and other members of the Rubiaceae family. We studied their functions in vitro and in vivo. Our investigations led to the identification of several reductases and an enol methyltransferase that forms a new clade within the SABATH methyltransferase family. Furthermore, we discovered a methyltransferase from Hamelia patens (firebush), which catalyzes the final step. With the tryptamine 4-hydroxylase from the psychedelic mushroom Psilocybe cubensis, we accomplished the four-step biosynthesis for mitragynine and its stereoisomer, speciogynine in both yeast and Escherichia coli when supplied with tryptamine and secologanin. Although we have yet to pinpoint the authentic hydroxylase and methyltransferase in kratom, our discovery completes the mitragynine biosynthesis. Through these breakthroughs, we achieved the microbial biosynthesis of kratom opioids for the first time. The remarkable enzyme promiscuity suggests the possibility of generating derivatives and analogs of kratom opioids in heterologous systems.

Upregulation of Brain's Calcium Binding Proteins in Mitragynine Dependence: A Potential Cellular Mechanism to Addiction.

Background: Mitragyna speciosa Korth or Kratom is widely used traditionally for its medicinal values. The major alkaloid content of kratom leaves is mitragynine, which binds to opioid receptors to give opioid-like effects. This study aimed to analyse the brain proteome of animals that displayed addictive behaviors. Design and Methods: Six groups (n=6-8) of rats made up of negative control, positive control using morphine (10 mg/kg), and treatment groups at low (1mg/kg) and high doses of mitragynine (30 mg/kg) for 1 and 4 days. The rats' behaviors were evaluated and subsequently the rats' brains were harvested for proteomic analysis that was performed by using 2D gel electrophoresis and LC/MS/MS. Results: The rats developed physical dependence only on day 4 following morphine and mitragynine (1 and 30mg/kg) treatments. Among the proteins that were up-regulated in treatment groups were four calcium-binding proteins, namely calretinin, F-actin, annexin A3 and beta-centractin. Conclusions: Upregulation of calretinin acted as low Ca2+ buffering upon the blockage of Ca2+ ion channel by mitragynine in the brain, which subsequently caused a reduction of GABA released and inversely increased the dopamine secretions that contributed to dependence indicators.

Evaluation of toxicity profile of kratom (Mitragyna speciosa Korth) decoction in rats.

Mitragyna speciosa Korth also known as kratom, is an herbal drug preparation for its therapeutic properties and opioid-replacement therapy. Kratom is consumed in a brewed decoction form in Malaysia and to date, no studies have characterized its chemical and toxicity profile. Thus, this study aims to evaluate kratom decoction's safety and toxicity profile after 28 days of treatment. Mitragynine content was quantified in kratom decoction and used as a marker to determine the concentration. Male and female Sprague Dawley rats were orally treated with vehicle or kratom decoction (10, 50 or 150 mg/kg) and two satellite groups were treated with vehicle and kratom decoction (150 mg/kg). Blood and organs were collected for hematology, biochemical and histopathology analysis at the end of treatment. No mortality was found after 28 days of treatment and no significant changes in body weight and hematology profile, except for low platelet count. High amounts of uric acid, AST, ALT and alkaline phosphatase were found in the biochemical analysis. Histological investigation of the heart and lungs detected no alterations except for the kidney, liver and brain tissues. In conclusion, repeated administration of kratom decoction provided some evidence of toxicity in the kidney and liver with no occurrence of mortality.

A Critical Review of the Neuropharmacological Effects of Kratom: An Insight from the Functional Array of Identified Natural Compounds.

Kratom (Mitragyna speciosa Korth. Havil) has been considered a narcotic drug for years, barred by the law in many parts of the world, while extensive research over the past few decades proves its several beneficial effects, some of which are still in ambiguity. In many countries, including Thailand, the indiscriminate use and abuse of kratom have led to the loss of life. Nonetheless, researchers have isolated almost fifty pure compounds from kratom, most of which are alkaloids. The most prevalent compounds, mitragynine and 7-hydroxy mitragynine, are reported to display agonist morphine-like effects on human µ-opioid receptors and antagonists at κ- and δ-opioid receptors with multimodal effects at other central receptors. Mitragynine is also credited to be one of the modulatory molecules for the Keap1-Nrf2 pathway and SOD, CAT, GST, and associated genes' upregulatory cascades, leading it to play a pivotal role in neuroprotective actions while evidently causing neuronal disorders at high doses. Additionally, its anti-inflammatory, antioxidative, antibacterial, and gastroprotective effects are well-cited. In this context, this review focuses on the research gap to resolve ambiguities about the neuronal effects of kratom and demonstrate its prospects as a therapeutic target for neurological disorders associated with other pharmacological effects.

A systematic review of (pre)clinical studies on the therapeutic potential and safety profile of kratom in humans.

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.

Asymmetric Total Syntheses of Mitragynine, Speciogynine, and 7-Hydroxymitragynine.

A number of alkaloids found in Mitragyna species belonging to the Rubiaceae family have been shown to have potent biological activity such as analgesic properties. Here, we report the asymmetric total syntheses of mitragynine, speciogynine, and 7-hydroxymitragynine, which are classified as corynantheine-type monoterpenoid indole alkaloids, isolated from Mitragyna speciosa. These syntheses were accomplished within 12 steps and in >11% total yield from commercial 3-(trimethylsilyl)propanal using an organocatalytic anti-selective Michael reaction and bioinspired transformations.

Sulfobutylation of Beta-Cyclodextrin Enhances the Complex Formation with Mitragynine: An NMR and Chiroptical Study.

Mitragynine (MTR), the main indole alkaloid of the well-known plant kratom (Mitragyna speciosa), is one of the most studied natural products nowadays, due to its remarkable biological effects. It is a partial agonist on the opioid receptors, and as such relieves pain without the well-known side-effects of the opioids applied in the clinical practice. MTR and its derivatives therefore became novel candidates for drug development. The poor aqueous solubility and low bioavailability of drugs are often improved by cyclodextrins (CyDs) as excipients through host-guest type complex formation. Among the wide variety of CyDs, sulfobutylether-beta-cyclodextrin (SBEßCyD) is frequently used and official in the European and U.S. Pharmacopoeia. Herein, the host-guest complexation of MTR with ßCyD and SBEßCyD was studied using chiroptical and NMR spectroscopy. It was found by NMR measurements that MTR forms a rather weak (logß11 = 0.8) 1:1 host-guest complex with ßCyD, while the co-existence of the 2MTR∙SBEßCyD and MTR∙SBEßCyD species was deducted from 1H NMR titrations in the millimolar MTR concentration range. Sulfobutylation of ßCyD significantly enhanced the affinity towards MTR. The structure of the formed inclusion complex was extensively studied by circular dichroism spectroscopy and 2D ROESY NMR. The insertion of the indole moiety was confirmed by both techniques.

Kratom Alkaloids, Natural and Semi-Synthetic, Show Less Physical Dependence and Ameliorate Opioid Withdrawal.

Chronic administration of opioids produces physical dependence and opioid-induced hyperalgesia. Users claim the Thai traditional tea "kratom" and component alkaloid mitragynine ameliorate opioid withdrawal without increased sensitivity to pain. Testing these claims, we assessed the combined kratom alkaloid extract (KAE) and two individual alkaloids, mitragynine (MG) and the analog mitragynine pseudoindoxyl (MP), evaluating their ability to produce physical dependence and induce hyperalgesia after chronic administration, and as treatments for withdrawal in morphine-dependent subjects. C57BL/6J mice (n = 10/drug) were administered repeated saline, or graded, escalating doses of morphine (intraperitoneal; i.p.), kratom alkaloid extract (orally, p.o.), mitragynine (p.o.), or MP (subcutaneously, s.c.) for 5 days. Mice treated chronically with morphine, KAE, or mitragynine demonstrated significant drug-induced hyperalgesia by day 5 in a 48 °C warm-water tail-withdrawal test. Mice were then administered naloxone (10 mg/kg, s.c.) and tested for opioid withdrawal signs. Kratom alkaloid extract and the two individual alkaloids demonstrated significantly fewer naloxone-precipitated withdrawal signs than morphine-treated mice. Additional C57BL/6J mice made physically dependent on morphine were then used to test the therapeutic potential of combined KAE, mitragynine, or MP given twice daily over the next 3 days at either a fixed dose or in graded, tapering descending doses. When administered naloxone, mice treated with KAE, mitragynine, or MP under either regimen demonstrated significantly fewer signs of precipitated withdrawal than control mice that continued to receive morphine. In conclusion, while retaining some liabilities, kratom, mitragynine, and mitragynine pseudoindoxyl produced significantly less physical dependence and ameliorated precipitated withdrawal in morphine-dependent animals, suggesting some clinical value.

In silico and in vitro anti-AChE activity investigations of constituents from Mytragyna speciosa for Alzheimer's disease treatment.

Acetylcholinesterase (AChE), one of the major therapeutic strategies for the treatment of Alzheimer's disease (AD) is to increase the acetylcholine (ACh) level in the brain by inhibiting the biological activity of AChE. In this present work, a set of alkaloids and flavonoids against AChE enzyme were screened by computational chemistry techniques. The docking results showed that among alkaloid compounds the oxindole alkaloid namely mitragynine oxidole B (MITOB) and the indole alkaloids namely mitragynine (MIT) exhibited a good binding affinity towards AChE. These two compounds were then studied by molecular dynamics (MD) simulations. The binding free energy calculation and ligand-protein binding pattern suggested that both alkaloids could interact with AChE very well. Since MIT is the main alkaloid constituent of Mytragyna speciose leaves, this compound was isolated from M. speciose leaves and tested for anti-AChE activity. As a result, the isolated MIT had an inhibitory activity with pIC50 value of 3.57. This finding provided that the mitragynine compound has the potential to be as a therapeutic agent for further anti-AChE drug development in treatment of Alzheimer's disease.

Seizures among young adults consuming kratom beverages in Malaysia: A case series.

We report a case series of young adults who were admitted to hospital with seizures after regular kratom beverage consumption. This study aimed to determine kratom consumption habits and seizure characteristics and to explore whether chronic kratom ingestion without concomitant drug abuse leads to recurrent seizure or epilepsy. All patients underwent blood investigations, a brain computed tomography (CT) scan, electroencephalography, and urine testing for mitragynine and drug toxicology. Eleven participants who had a positive urine mitragynine test were included in the study. The longest duration of kratom consumption was 84 months: - most drank more than eight times per month (>200 mL/drink). Seizure developed within 10 minutes or up to 72 hours post-ingestion. Seizure occurred one to three times per year in most cases. Four patients had a focal to bilateral tonic-clonic seizure whereas the remaining participants had a generalized tonic-clonic seizure. Four patients mixed kratom with diphenhydramine syrup, and one patient took methamphetamine. Two patients had positive urine results for recreational drugs (opioid and amphetamine). This study provided indirect evidence that chronic kratom use with or without concomitant drug abuse can cause recurrent seizures in susceptible individuals, which may progress to epilepsy or require antiepileptic medication.

A comparative analysis of kratom exposure cases in Thailand and the United States from 2010-2017.

Background: Interest in the Southeast Asian natural remedy kratom has increased in Western countries recently, along with increasing concern over its potential toxic effects.Objective: To describe and compare demographics, common co-exposure substances, clinical effects, treatments, and medical outcomes of kratom "abuse" exposures in the United States (US) and Thailand.Methods: This is a retrospective analysis of kratom "abuse" exposures, defined as use when attempting to gain a psychotropic effect, reported to the National Poison Data System (NPDS) in the US and the Ramathibodi Poison Center (RPC) in Thailand from 2010 to 2017. Multivariate analysis identified risk factors for severe medical outcomes, defined as both ICU admissions and death.Results: Nine-hundred-twenty-eight cases were included (760 from NPDS and 168 from RPC). A greater proportion of cases involved co-exposures in Thailand (64.8% versus 37.4%; odds ratio [OR] = 3.10, 95% confidence interval [CI] = 2.15-4.47, p < .01). Both countries had a similar prevalence of opioid and benzodiazepine co-ingestions, but the US had more co-ingestions with other sedatives (4.6% versus 0%, OR = 0, 95% CI = 0-0.47, p < .01). Common clinical effects included tachycardia (30.4%), agitation/irritability (26.2%), and drowsiness/lethargy (21.1%). Six deaths occurred, including one single-substance exposure in the US, three multiple-substance exposures in the US, and two multiple-substance exposures in Thailand. Severe medical outcomes were reported more frequently in the US (OR = 18.82, 95% CI = 5.85-60.56, p < .01).Conclusions: Despite lower frequencies of co-ingestants overall, US kratom abuse exposures yielded greater clinical severity. This disparity may be attributable to differences in the products labeled "kratom," greater sedative co-exposures in the US, and/or differences in population genetics or use patterns.

Accelerated Solvent Extractions (ASE) of Mitragyna speciosa Korth. (Kratom) Leaves: Evaluation of Its Cytotoxicity and Antinociceptive Activity.

Mitragyna speciosa Korth (kratom) is known for its psychoactive and analgesic properties. Mitragynine is the primary constituent present in kratom leaves. This study highlights the utilisation of the green accelerated solvent extraction technique to produce a better, non-toxic and antinociceptive active botanical extract of kratom. ASE M. speciosa extract had a dry yield (0.53-2.91 g) and showed a constant mitragynine content (6.53-7.19%) when extracted with organic solvents of different polarities. It only requires a shorter extraction time (5 min) and a reduced amount of solvents (less than 100 mL). A substantial amount of total phenolic (407.83 ± 2.50 GAE mg/g and flavonoids (194.00 ± 5.00 QE mg/g) were found in ASE kratom ethanol extract. The MTT test indicated that the ASE kratom ethanolic leaf extract is non-cytotoxic towards HEK-293 and HeLa Chang liver cells. In mice, ASE kratom ethanolic extract (200 mg/kg) demonstrated a better antinociceptive effect compared to methanol and ethyl acetate leaf extracts. The presence of bioactive indole alkaloids and flavonols such as mitragynine, paynantheine, quercetin, and rutin in ASE kratom ethanolic leaf extract was detected using UHPLC-ESI-QTOF-MS/MS analysis supports its antinociceptive properties. ASE ethanolic leaf extract offers a better, safe, and cost-effective choice of test botanical extract for further preclinical studies.

Development of an ELISA for detection of mitragynine and its metabolites in human urine.

An enzyme-linked immunosorbent assay for detection of mitragynine, other closely related Kratom alkaloids and metabolites was developed using polyclonal antibodies. Mitragynine was conjugated to a carrier protein, cationized-bovine serum albumin using Mannich reaction. The synthesized antigen was injected into rabbits to elicit specific polyclonal antibodies against mitragynine. An enzyme conjugate was synthesized for evaluating its performance with the antibodies produced. The assay had an IC50 of 7.3 ng/mL with a limit of detection of 15 ng/mL for mitragynine. Antibody produced have high affinity for mitragynine (100%), other closely related Kratom alkaloids such as paynantheine (54%), speciociliatine (63%), 7α-hydroxy-7H-mitragynine (83%) and cross-reacted with metabolites 9-O-demethyl mitragynine (79%), 16-carboxy mitragynine (103%), 9-O-demethyl mitragynine sulfate (263%), 9-O-demethyl mitragynine glucuronide (60%), 16-carboxy mitragynine glucuronide (60%), 9-O-demethyl-16-carboxy mitragynine sulfate (270%) and 17-O-demethyl-16,17-dihydro mitragynine glucuronide (34%). It showed cross-reactivity less than 0.01% to reserpine, codeine, morphine, caffeine, methadone, amphetamine, and cocaine. Ten-fold dilution urine was used in the assay to reduce the matrix effects. The recovery ranged from 83% to 112% with variation coefficients in intraday and interday less than 8% and 6%, respectively. The ELISA turned out to be a convenient tool to diagnose mitragynine, other closely related Kratom alkaloids and metabolites in human urine samples.