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.

Mitragynine and Morphine Produce Dose-dependent Bimodal Action on Food but not Water Intake in Rats.

Novel compounds such as mitragynine, the primary alkaloid in kratom (Mitragyna Speciosa), have emerged as alternative self-treatments for opioid use disorder. Mitragynine binds numerous receptor types, including opioid receptors, which are known to modulate food consumption. However, the ability of acute mitragynine to modulate food consumption remains unknown. The current study assessed the effects of acute mitragynine or morphine administration on unconditioned food and water intake in 16 Sprague Dawley rats. Food and water intake changes were monitored in response to morphine, mitragynine (1.78-56 mg/kg, intraperitoneal), saline, or vehicle controls for 12 hours starting at the onset of the dark cycle. Naltrexone pretreatment was utilized to examine pharmacological specificity. Both morphine and mitragynine demonstrated a biphasic food intake dose-effect, with low doses (5.6 mg/kg) increasing and high doses (56 mg/kg) decreasing food intake. All morphine doses reduced water intake; however, only the highest dose of mitragynine (56 mg/kg) reduced water intake. Naltrexone attenuated both stimulatory and inhibitory effects of morphine on food intake, but only the stimulatory effect of mitragynine. In conclusion, low doses of mitragynine stimulate food intake via opioid-related pathways, while high doses likely recruit other targets.

Using rDNA ITS2 barcoding to identify kratom (Mitragyna speciosa) from the genus Mitragyna and Neolamarckia cadamba.

This study collected 80 samples of suspected kratom plant powder. A polymerase chain reaction sequence analysis was conducted using two sets of DNA barcode primers for plant ribosomal (r)DNA internal transcribed spacers (ITSs), namely, ITS3/ITS4 and ITS-p3/ITS-u4. Among the 80 samples, 40 were analyzed using the ITS3/ITS4 primer pair, and then DNA sequences were subjected to a National Center for Biotechnology Information-Basic Local Alignment Search Tool (NCBI-BLAST) comparison. Results showed that 29 samples had a 100% match (364/364) with Mitragyna speciosa (kratom), and 6 samples had a 99.73% match (363/364) with M. speciosa, whereas 5 samples had disordered and unreadable sequences. The 5 unreadable samples and an additional 40 suspected kratom samples were then analyzed using the ITS-p3/ITS-u4 primer pair, followed by an NCBI-BLAST comparison. Among these, 32 samples had a 100% match (404/404) with M. speciosa, and 11 samples had a 99.75% match (403/404) with M. speciosa. Among the samples with sequences matching M. speciosa, three distinct types were observed (no variance/404, 287M/404, and 287A/404). One sample had a 99.51% match (404/406) with Neolamarckia cadamba, and another sample had a sequencing length of 305 bp, with 25 positions showing mixed base pairs, indicating a mixture of different species. Analysis of the mixed base pair pattern suggested a possible mixture of M. speciosa and N. cadamba. Actually, M. speciosa and N. cadamba have very similar external morphologies. This indicates that the ITS-p3/ITS-u4 primer pair is effective in distinguishing mixtures of M. speciosa and N. cadamba and is thus more suitable than ITS3/ITS4 for identifying and analyzing samples of suspected kratom plant powder.

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.

Controversies in Assessment, Diagnosis, and Treatment of Kratom Use Disorder.

PURPOSE OF REVIEW: We apply the Diagnostic and Statistical Manual of Mental Disorders Fifth Edition (DSM-5) criteria for substance use disorders (SUDs) to the herbal product kratom. Similarities and differences between kratom use disorder (KUD) and other SUDs are explored, along with assessment, diagnostic, and therapeutic recommendations for KUD. RECENT FINDINGS: Literature reports of "kratom addiction" or KUD rarely specify the criteria by which patients were diagnosed. Individuals meeting DSM-5 KUD criteria typically do so via tolerance and withdrawal, using more than intended, and craving, not functional or ​psychosocial disruption, which occur rarely. Most clinicians who use medication to treat patients with isolated KUD select buprenorphine formulations, although there are no controlled studies showing that buprenorphine is safe or efficacious in this patient population. Diagnosis and treatment decisions for KUD should be systematic. We propose an algorithm that takes into consideration whether KUD occurs with comorbid opioid use disorder.

Severe jaundice with life-threatening liver failure after Kratom use: Reversed by plasma exchange.

Kratom is an herbal supplement which is used for its stimulating properties and pain reduction due to interaction with opioid receptors. Kratom overdose may cause fatality. A 56-year-old man was admitted to the emergency department with severe jaundice and liver failure. His total bilirubin reached at 70.6 mg/dL, but extensive workup did not show any liver mass. Family informed that the patient was taking Kratom. Plasma exchange was suggested as an unconventional therapy and consent from the patient was obtained because this procedure has never been performed to treat Kratom toxicity before. After four procedures, his total bilirubin was reduced to 23.9 mg/dL and his clinical condition improved significantly. Finally on day 5 he was discharged at stable condition with a total bilirubin value of 21.3 mg/dL. There is no antidote for Kratom, and treatment is supportive. To our knowledge this is the first report of reversing Kratom poisoning using plasma exchange.

Kratom (Mitragyna speciosa) Use and Mental Health: A Systematic Review and Multilevel Meta-Analysis.

INTRODUCTION: Kratom (Mitragyna speciosa) is a medicinal tree native to Southeast Asia. The present multilevel meta-analysis describes the association between kratom use and the positive and negative indicators of mental health. METHODS: A total of thirty-six articles were included in the meta-analysis to examine the associations, using a random-effects model. RESULTS: The pooled effect size showed a very small positive association between kratom use and negative indicators of mental health {r = 0.092, 95% confidence interval (CI) = [0.020, 0.164], p < 0.05}, while no significant association was found with positive indicators of mental health (r = -0.031, 95% CI = [-0.149, 0.087], p > 0.05). Pooled effect sizes of specific mental health outcomes indicated that kratom use showed only a small positive correlation with externalizing disorders (r = 0.201, 95% CI = [0.107, 0.300], p < 0.001). No significant association was found between kratom use and quality of life (r = 0.069, 95% CI = [-0.104, 0.242], p > 0.05) and internalizing disorders (r = -0.001, 95% CI = [-0.115, 0.095], p > 0.05). Multilevel moderator analysis showed that the pooled effect size of the association between kratom use and substance use disorder was stronger in Malaysia (r = 0.347, 95% CI = [0.209, 0.516], p < 0.001), and with the mean age (ß1 = -0.035, 95% CI = [-0.055, -0.014], p = 0.003), and the drug profile of those who were not co-using other drugs (r = 0.347, 95% CI = [0.209, 0.516], p < 0.001). CONCLUSION: The meta-analysis supports the kratom instrumentalization concept, in that a positive gain from kratom consumption can be achieved without any significant adverse associations with mental health.

Botanical-induced toxicity: Liver injury and botanical-drug interactions. A report on a society of Toxicology Annual Meeting symposium.

Botanical supplements and herbal products are widely used by consumers for various purported health benefits, and their popularity is increasing. Some of these natural products can have adverse effects on liver function and/or interact with prescription and over-the-counter (OTC) medications. Ensuring the safety of these readily available products is a crucial public health concern; however, not all regulatory authorities require premarket safety review and/or testing. To address and discuss these and other emerging needs related to botanical safety, a symposium was held at the Society of Toxicology Annual Meeting in Salt Lake City (UT) on March 11, 2024. The symposium addressed the latest research on botanical-induced liver toxicity and botanical-drug interactions, including new approach methods to screen for toxicity, challenges in assessing the safety of botanicals, and relating human adverse events to specific products. The presentations and robust panel discussion between the speakers and audience highlighted the need for further research and collaboration to improve the safety of botanical supplements and herbal products, with the ultimate goal of protecting consumer health. Although utility of many of the modern tools presented in the symposium requires further study, the synergistic efforts of diverse experts hold promise for effective prediction and evaluation of botanical-induced hepatotoxicity and botanical-drug interaction potential.

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.

Characterization of kratom use and knowledge at a rural, Oregon community health center.

BACKGROUND: Kratom is an herbal supplement that has drawn attention for its use in the self-treatment of opioid withdrawal, and its widespread availability with minimal restrictions. Past web-based research has attempted to determine patterns and trends of use, but generalizability to underserved populations is unclear. OBJECTIVE: The purpose of this study was to characterize behavior related to kratom, attitudes toward kratom, and knowledge of kratom in a rural, underserved population. METHODS: We developed, refined, and administered a cross-sectional, 36-item survey to examine use, attitudes, and knowledge of kratom. We recruited participants and administered the survey alongside medical office appointments between January and April 2023. Data were summarized using descriptive statistics. RESULTS: A convenient sample of 186 patients (of the 907-patient clinic panel) were invited to participate and 150 returned the survey. A majority of patients were female (52.0%) and White (86.6%), and about half had an income below the federal poverty level (48.5%). Seventeen participants reported prior experience with kratom use, with one actively using kratom. The most commonly reported reasons for use were pain (47.1%) and mental health (41.2%). Kratom knowledge was low regardless of kratom use history, with the majority of respondents correctly answering between 1 and 3 questions (n = 71 of 86; 82.3%) of the 5 knowledge-focused items. CONCLUSION: Results suggest that while active kratom use is uncommon in this Oregon population, one in ten surveyed had used kratom. Regardless of past use, respondents had limited knowledge of kratom. Future research should focus on understanding trends in kratom use behaviors in underserved populations, addressing patient knowledge gaps, and evaluating patient safety and health equity implications.

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.

Red kratom and red corvette: A case of mania induced by kratom withdrawal.

Background: Kratom, derived from the Mitragyna speciose, a  plant native to Southeast Asia, is a substance that is gaining popularity in the United States as a self-medication tool for opiate withdrawal and pain management. Its active compounds, mitragynine, and 7-hydroxymitragynine, interact with various receptors in the body, resulting in a spectrum of clinical effects.Case: A 56-year-old male with a history of depression and cannabis use disorder arrived at the Emergency Department in a state of severe agitation with erratic behavior and aggression. Further evaluation revealed a recent abrupt discontinuation of Kratom use. A psychiatric consultation was requested and done by our consultation-liaison team, diagnosing Kratom withdrawal as the most likely cause of his acute mania. Neuropsychiatric symptoms arising from Kratom toxicity is a well-documented phenomenon; however, to our knowledge, this may be the first documented case of acute mania induced by Kratom withdrawal.

Prevalence, Characteristics, and Reasons for Kratom Use among Psychiatrically Ill Inpatients Who Use Substances.

OBJECTIVE: Despite kratom impacting neurobiological systems involved in psychiatric disorders, little is known about the prevalence of use among patients with severe psychopathologies. Here, we investigated the prevalence of kratom use, motives for use, and the clinical associations among inpatients with severe psychiatric disorders. METHODS: A total of 578 patients, aged 18 to 65, were evaluated by New Hampshire Hospital's Addiction Services from January 1, 2020, to February 28, 2022. The study collected demographic information and used chi-square tests, multivariable logistic regression, and subgroup analyses with 95% confidence intervals to examine trends among kratom users. A receiver operating characteristic curve analysis was also conducted. All statistical tests were performed using IBM SPSS Version 28.0.1. RESULTS: Of the patients assessed, 2.2% (n = 13) reported using kratom. The reasons for kratom use were managing withdrawal symptoms (15.4%), maintaining sobriety and reducing cravings for opioids (53.8%), improving focus and concentration (30.8%), alleviating low moods (38.5%), and managing pain (15.4%). Compared to non-kratom users, the only factor with a fair to good association with kratom use is postsecondary education (Area Under Curve, AUC = 0.77). CONCLUSIONS: Prevalence of kratom use among patients with serious mental illness at our site aligns with that reported in the general population. Users often cite self-management of cravings and sobriety from opioids, as well as treatment of low mood states, as motivations for consumption. While observations suggest a possible association between kratom use and individuals with post-secondary education, multiple substance use, and experience of substance-induced psychosis or mood disorders, it is essential to interpret these links cautiously until further rigorous studies are carried out to substantiate these findings.

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.

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.

The effects of Mitragyna speciosa extracts on intestinal microbiota and their metabolites in vitro fecal fermentation.

BACKGROUND: Kratom (Mitragyna speciosa) has a long history of traditional use. It contains various alkaloids and polyphenols. The properties of kratom's alkaloids have been well-documented. However, the property of kratom's polyphenols in water-soluble phase have been less frequently reported. This study assessed the effects of water-soluble Mitragyna speciosa (kratom) extract (MSE) on gut microbiota and their metabolite production in fecal batch culture. RESULTS: The water-soluble kratom extract (MSE0) and the water-soluble kratom extract after partial sugar removal (MSE50) both contained polyphenols, with total phenolic levels of 2037.91 ± 51.13 and 3997.95 ± 27.90 mg GAE/g extract, respectively and total flavonoids of 81.10 ± 1.00 and 84.60 ± 1.43 mg CEQ/g extract. The gut microbiota in fecal batch culture was identified by 16S rRNA gene sequencing at 0 and 24 h of fermentation. After fermentation, MSE50 stimulated the growth of Bifidobacterium more than MSE0. MSE0 gave the highest total fatty acids level among the treatments. The phenolic metabolites produced by some intestinal microbiota during fecal fermentation at 24 h were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The major metabolite of biotransformation of both water-soluble MSEs by intestinal microbiota was pyrocatechol (9.85-11.53%). CONCLUSION: The water-soluble MSEs and their produced metabolites could potentially be used as ingredients for functional and medicinal food production that supports specific gut microbiota. © 2024 Society of Chemical Industry.

The prevalence of kratom use and association with co-occurring substance use among adolescents: a 2022 Bangkok behavioral surveillance survey, Thailand.

Thailand removed kratom from the list of prohibited substances in 2021, possession and consumption of Kratom is now legal. It is prohibited from selling Kratom to anyone under the age of 18 and/or who is pregnant or breastfeeding. While there are benefits from kratom use with few reported adverse effects, escalating dose and increased use frequency raise the risk for toxic events in the setting of polysubstance use or development of a use disorder. We utilized data from the Behavior Surveillance Survey in Bangkok (n = 5,740) to examine the use of kratom with other substances use in the 12 months before the survey. The prevalence of past-year kratom use among students was 9.3% (95%CI = 8.7-9.9), with higher proportions of males (12.4 versus 6.1%, p < 0.001). The factors associated with past 12-month kratom use were academic performance (Medium GPA; AOR = 2.41, 95% CI = 1.76-3.29; Low GPA; AOR = 4.15, 95% CI = 2.94-5.87), close friend use substance (AOR = 1.94, 95% CI = 1.44-2.59), cannabis use (AOR = 6.84, 95% CI = 4.61-10.15), consumed alcohol (AOR = 2.32, 95% CI = 1.77-3.02), smoked conventional cigarettes (AOR = 4.20, 95% CI = 3.16-5.58), used e-cigarettes (AOR = 4.37, 95% CI = 3.30-5.79) used illicit opioids (AOR = 8.13, 95% CI = 4.35-15.18), and other illicit drug use (AOR = 9.15, 95% CI = 3.78-22.14). These findings may be useful for the initial targeting of efforts to reduce adolescent consumption of kratom. Future studies should examine the effect of regulatory policies or other Thai FDA-related policies use of illicit drugs and e-cigarettes on kratom use.

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.

Development and validation of ELISA for screening of Kratom (Mitragyna speciosa) habitual users using urinary AZ122 biomarker.

Kratom (Mitragyna speciosa Korth) has been used traditionally in Southeast Asia for its therapeutic properties. The major alkaloid of kratom, mitragynine, binds to opioid receptors to give opioid-like effects that causes addiction. In our previous study, we have identified AZ122 as a unique biomarker in habitual or regular kratom users through analysis of their urinary protein profiles. We aimed to develop and validate a screening method by means of enzyme-linked immunosorbent assay (ELISA) for detection of kratom habitual users. An ELISA approach was applied for the development of a screening method using urinary AZ122 as biomarker. Method validation was carried out using three quality control materials at different concentration of AZ122. The data was analyzed statistically using SPSS (Version 25). The ELISA was presented with Pearson correlation coefficient of 0.9993. The repeatability and reproducibility were presented at CV <7%, while the accuracy ranged from 78 to 96% at various AZ112 concentrations. Upon testing on 176 male respondents (n = 88 regular kratom users and n = 88 healthy controls), the specificity and sensitivity of the assay were both 100%. The ELISA has been validated and can be potentially used as a reliable screening test for detection of kratom habitual users.

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.