Development of a CZE-MS/MS method with dynamic pH junction sample pretreatment for analysis of kratom psychoactive alkaloids in urine.

BACKGROUND: Kratom is a herbal substance belonging to the group of new psychoactive substances. It contains psychoactive indole alkaloids mitragynine and 7-hydroxymitragynine. At low doses, they act as psychostimulants and at higher doses they mediate an opioid-like effect. The increasing misuse of kratom requires the development of analytical methods that will accurately and reliably identify and quantify its psychoactive alkaloids in biological samples. Therefore, the development of effective, precise, and reliable green analytical methods that are easy to implement in practice is of great importance. On-line combination of capillary zone electrophoresis with tandem mass spectrometry (CZE-MS/MS) seems to be a promising solution. RESULTS: We present a novel green approach based on capillary zone electrophoresis - tandem mass spectrometry (CZE-MS/MS) method with on-line dynamic pH junction sample pretreatment to identify and determine mitragynine and 7-hydroxymitragynine in urine samples. The separation was performed in a background electrolyte composed of 100 mM formic acid (pH 2.39). The dynamic pH junction was ensured by injection of a short plug of 12.5 % NH4OH before the sample. Under optimal conditions, the developed method was validated and parameters such as linearity (r2 > 0.99), precision (2.2-8.7 %), accuracy (89.2-102.5 %) or stability of the sample (86.6-114.7 %) met the defined FDA guideline criteria (%RSD and %RE values where within ±15 %). Introduction of a simple in-capillary preconcentration strategy based on dynamic pH junction enabled significant improvement in analytical signal intensity and also the applicability of the method. Applying the presented approach, high sensitivity was achieved as indicated by limit of detection values, which were 0.5 ng mL-1 and 2 ng mL-1 for mitragynine and 7-hydroxymitragynine, respectively. Greenness of the proposed approach was confirmed by the AGREE metrics (score 0.63). The application potential of the developed method was successfully verified using blinded urine model samples. SIGNIFICANCE: For the first time a fully validated CZE-MS/MS method for kratom alkaloids determination was introduced. The presented novel method is a cheaper and more ecological alternative to conventionally used chromatographic techniques what was clearly confirmed by its greenness evaluation and comparison with previously published liquid chromatography (LC) approaches. In-capillary sample pretreatment (dynamic pH junction) has been demonstrated to be an effective and fast tool in bioanalysis, minimizing the number of pretreatment steps and the manipulation with the sample. Moreover, LOD values comparable to those obtained by LC methods were recorded. High potential for the implementation of this approach into the toxicology environment in the near future is expected.

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.

The Mitragyna speciosa (kratom) alkaloid mitragynine: Analysis of adrenergic α2 receptor activity in vitro and in vivo.

Mitragynine, an alkaloid present in the leaves of Mitragyna speciosa (kratom), has a complex pharmacology that includes low efficacy agonism at µ-opioid receptors (MORs). This study examined the activity of mitragynine at adrenergic α2 receptors (Aα2Rs) in vitro and in vivo. Mitragynine displaced a radiolabeled Aα2R antagonist ([3H]RX821002) from human Aα2ARs in vitro with lower affinity (Ki = 1260 nM) than the agonists (-)-epinephrine (Ki = 263 nM) or lofexidine (Ki = 7.42 nM). Mitragynine did not significantly stimulate [35S]GTPγS binding at Aα2ARs in vitro, but in rats trained to discriminate 32 mg/kg mitragynine from vehicle (intraperitoneally administered; i.p.), mitragynine exerted an Aα2R agonist-like effect. Both α2R antagonists (atipamezole and yohimbine) and MOR antagonists (naloxone and naltrexone) produced rightward shifts in mitragynine discrimination dose-effect function and Aα2R agonists lofexidine and clonidine produced leftward shifts. In the mitragynine trained rats, Aα2R agonists also produced leftward shifts in discrimination dose-effect functions for morphine and fentanyl. In a separate rat cohort trained to discriminate 3.2 mg/kg i.p. morphine from vehicle, naltrexone produced a rightward shift, but neither an Aα2R agonist or antagonist affected morphine discrimination. In a hypothermia assay, both lofexidine and clonidine produced marked effects antagonized by yohimbine. Mitragynine did not produce hypothermia. Together, these data demonstrate that mitragynine acts in vivo like an Aα2R agonist, although its failure to induce hypothermia or stimulate [35S]GTPγS binding in vitro, suggests that mitragynine maybe a low efficacy Aα2R agonist.

Discovery of procyanidin condensed tannins of (-)-epicatechin from Kratom, Mitragyna speciosa, as virucidal agents against SARS-CoV-2.

Kratom, Mitragyna speciosa, is one of the most popular herbs in the West and Southeast Asia. A number of previous works have focused on bioactive alkaloids in this plant; however, non-alkaloids have never been investigated for their biological activities. Antiviral and virucidal assays of a methanol leaf extract of Kratom, M. speciosa, revealed that a crude extract displayed virucidal activity against the SARS-CoV-2. Activity-guided isolation of a methanol leaf extract of Kratom led to the identification of B-type procyanidin condensed tannins of (-)-epicatechin as virucidal compounds against SARS-CoV-2. The fraction containing condensed tannins exhibited virucidal activity with an EC50 value of 8.38 µg/mL and a selectivity index (SI) value >23.86. LC-MS/MS analysis and MALDI-TOF MS identified the structure of the virucidal compounds in Kratom as B-type procyanidin condensed tannins, while gel permeation chromatograph (GPC) revealed weight average molecular weight of 238,946 Da for high molecular-weight condensed tannins. In addition to alkaloids, (-)-epicatechin was found as a major component in the leaves of M. speciosa, but it did not have virucidal activity. Macromolecules of (-)-epicatechin, i.e., procyanidin condensed tannins, showed potent virucidal activity against SARS-CoV-2, suggesting that the high molecular weights of these polyphenols are important for virucidal activity.

Kratom as a potential substance use disorder harm reduction agent.

Substance use disorders contribute to considerable U.S. morbidity and mortality. While effective pharmacotherapy options are available to treat opioid and alcohol use disorders, for a variety of reasons, many patients lack access to treatment or may be reluctant to seek care due to concerns such as perceived stigma or a current lack of desire to completely curtail their substance use. Furthermore, treatment options are limited for patients with stimulant or polysubstance use disorders. Thus, there is considerable need to expand the substance use disorder harm reduction armamentarium. Kratom (Mitragyna speciosa Korth.) is an herbal substance that can produce both opioid and stimulant-like effects, and its use in the US is growing. Though there are concerns regarding adverse effects, dependence risk, and limited regulation of its manufacturing and sale, the pharmacology of kratom and early preclinical studies suggest a potential role as a harm reduction agent for various substance use disorders, and it has historically been used in Southeast Asia for such purposes. The goal of this review is to describe kratom's history of use, pharmacology, and early pre-clinical and observational research regarding its therapeutic potential in opioid use disorder, as well as alcohol, stimulant, and polysubstance use disorders, while also highlighting current concerns around its use, existing gaps in the literature, and directions for future research.

LC-MS-MS method for mitragynine and 7-hydroxymitragynine in hair and its application in authentic hair samples of suspected kratom abusers.

Kratom is a natural psychoactive product known primarily in Southeast Asia, including Thailand, Malaysia, etc. It is also known as krathom, kakuam, ithang, thom (Thailand), biak-biak, ketum (Malaysia) and mambog (Philippines) and is sometimes used as an opium substitute. It is stimulant at doses of 1-5 g, analgesic at doses of 5-15 g and euphoric and sedative at doses of >15 g. Mitragynine is the most abundant indole compound in kratom (Mitragyna speciosa) and is metabolized in humans to 7-hydroxymitragynine, the more active metabolite. Adverse effects include seizures, nausea, vomiting, diarrhea, tachycardia, restlessness, tremors, hallucinations and death. There are few studies on the analytical method for the detection of mitragynine and 7-hydroxymitragynine in hair. Therefore, this study proposes a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the analysis of kratom in hair. Hair samples were first weighed to ∼10 mg and washed with methanol. Then the washed hair samples were cut into pieces and incubated in methanol with stirring and heating (16 h/38℃). Extracts were then analyzed by LC-MS-MS. This method was validated by determining the limit of detection (LOD), limit of quantification, linearity, intra- and inter-day accuracy and precision, recovery and matrix effects. The intra- and inter-day precision (CV%) and accuracy (bias%) were within ±20%, which was considered acceptable. Using this newly developed LC-MS-MS method, the simultaneous detection of mitragynine and 7-hydroxymitragynine in six authentic hair samples was achieved to provide the direct evidence of kratom use in the past. Mitragynine concentrations ranged from 16.0 to 2,067 pg/mg (mean 905.3 pg/mg), and 7-hydroxymitragynine concentrations ranged from 0.34 to 15 pg/mg (mean 7.4 pg/mg) in six authentic hair samples from kratom abusers. This may be due to the higher sensitivity of the LOD in this study, with values of 0.05 pg/mg for mitragynine and 0.2 pg/mg for 7-hydroxymitragynine in hair.

Sequence variation of commercially available kratom products at universal DNA barcode regions.

Mitragyna speciosa, commonly known as kratom, is a narcotic plant that is used for its unique mood-enhancing and pain-relieving effects. It is marketed throughout the United States as a 'legal high' and has gained popularity as an alternative to opioids. However, kratom's increasing involvement in accidental overdoses, especially among polydrug users, has prompted warnings from the Drug Enforcement Agency (DEA) and the Food and Drug Administration (FDA). Despite these warnings, kratom remains legal federally, although it is banned in six states. This legal disparity complicates monitoring and enforcement efforts in states where kratom is illegal. Common forensic techniques using morphology or chemical analysis are beneficial in some instances but are not useful in source attribution because most seized kratom is powdered and the alkaloid content of samples can vary within products, making sourcing unreliable. This study focused on developing a DNA barcoding method to access sequence variation in commercial kratom products. It evaluated the utility of one nuclear barcode region (ITS) and three chloroplast barcode regions (matK, rbcL, and trnH-psbA) in assessing sequence variation across commercially available kratom products. Novel polymorphisms were discovered, and the ITS region showed the greatest variation between samples. Among the 15 kratom products tested, only two haplotypes were identified across the four barcoding regions. The findings highlight the potential of DNA barcoding as a forensic tool in the traceability and enforcement against illegal kratom distribution. Nonetheless, the limited haplotypic diversity points to a need for further development and expansion of the M. speciosa DNA sequence database.

Kratom addiction per DSM-5 SUD criteria, and kratom physical dependence: Insights from dosing amount versus frequency.

BACKGROUND: Kratom products are widely used in the United States, with inadequate understanding of how dosing amounts/frequencies relate to outcomes. METHODS: Between July-November 2022, we enrolled 395 active US adult kratom consumers into a remote study with a baseline survey. We examined self-reported typical dose amounts and frequencies across people and product types, and their associations with outcomes: multiple regression was used to examine whether amounts and frequencies (doses/day) were associated with acute effects, withdrawal symptoms, scores on the Subjective Opioid Withdrawal Scale (SOWS), and addiction (operationalized as DSM-5-based symptoms of kratom-use disorder, KUD). RESULTS: Participants were 54.9% male, aged 38.1 on average, and 81.3% White. Mean length of kratom use was 5.7 years. Most (95.9%) reported regularly using whole-leaf kratom products; 16 (4.1%) reported regular extract use. SOWS scores were mild to moderate on average (13.5, SD 11.9). KUD symptom counts were mostly in the mild/moderate range (80.7%). Withdrawal and KUD symptoms were more closely associated with dose frequency than dose amount. Men reported more acute effects, withdrawal symptoms with cessation, and KUD symptoms than women. CONCLUSIONS: Greater dose amount and frequency were systematically related to the number of withdrawal symptoms upon cessation and to KUD symptoms; the relationship was stronger for dose frequency than amount. Men may have more acute effects and more withdrawal and KUD symptoms than women. Although kratom may be used nonproblematically by some consumers, physical dependence (tolerance, withdrawal, or use to avoid withdrawal) and KUD become more likely with increasing dose frequency.

Formation of multiple ion types during MALDI imaging mass spectrometry analysis of Mitragyna speciosa alkaloids in dosed rat brain tissue.

Mitragyna speciosa, more commonly known as kratom, has emerged as an alternative to treat chronic pain and addiction. However, the alkaloid components of kratom, which are the major contributors to kratom's pharmaceutical properties, have not yet been fully investigated. In this study, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry was used to map the biodistribution of three alkaloids (corynantheidine, mitragynine, and speciogynine) in rat brain tissues. The alkaloids produced three main ion types during MALDI analysis: [M + H]+, [M - H]+, and [M - 3H]+. Contrary to previous reports suggesting that the [M - H]+ and [M - 3H]+ ion types form during laser ablation, these ion types can also be produced during the MALDI matrix application process. Several strategies are proposed to accurately map the biodistribution of the alkaloids. Due to differences in the relative abundances of the ions in different biological regions of the tissue, differences in ionization efficiencies of the ions, and potential overlap of the [M - H]+ and [M - 3H]+ ion types with endogenous metabolites of the same empirical formula, a matrix that mainly produces the [M + H]+ ion type is optimal for accurate mapping of the alkaloids. Alternatively, the most abundant ion type can be mapped or the intensities of all ion types can be summed together to generate a composite image. The accuracy of each of these approaches is explored and validated.

Mitragynine (Kratom)-Withdrawal behaviour and cognitive impairments can be ameliorated by an epigenetic mechanism.

BACKGROUND AND PURPOSE: Kratom is a preparation from Mitragyna speciosa, which is used as a natural drug preparation for many purposes around the world. However, an overdose of Kratom may cause addiction-like problems including aversive withdrawal states resulting in cognitive impairments via unknown mechanisms. Its main psychoactive alkaloid is mitragynine, showing opioid-like properties. EXPERIMENTAL APPROACH: Here, we analysed the neuropharmacological effects of mitragynine compared with morphine withdrawal in rats and searched for a pharmacological treatment option that may reverse the occurring cognitive deficits that usually aggravate withdrawal. KEY RESULTS: We found that withdrawal from 14-day mitragynine (1-10 mg·kg-1·day-1) treatment caused dose-dependent behavioural withdrawal signs resembling those of morphine (5 mg·kg-1·day-1) withdrawal. However, mitragynine (5 and 10 mg·kg-1·day-1) withdrawal also induced impairments in a passive avoidance task. Mitragynine withdrawal not only reduced hippocampal field excitatory postsynaptic potential (fEPSP) amplitudes in basal synaptic transmission and long-term potentiation (LTP) but also reduced epigenetic markers, such as histone H3K9 and H4K12 expression. At the same time, it up-regulates HDAC2 expression. Targeting the epigenetic adaptations with the HDAC inhibitor, SAHA, reversed the effects of mitragynine withdrawal on epigenetic dysregulation, hippocampal input/output curves, paired-pulse facilitation, LTP and attenuated the cognitive deficit. However, SAHA amplified the effects of morphine withdrawal. CONCLUSION AND IMPLICATIONS: The data from this work show that changes in histone expression and downstream hippocampal plasticity may explain mitragynine, but not morphine, withdrawal behaviours and cognitive impairments. Thus, it may provide a new treatment approach for aversive Kratom/mitragynine withdrawal and addiction.

Testing for Kratom alkaloids in fingernail clippings - not only mitragynine.

Kratom (Mitragyna speciosa) is a species of large tree that grows in Southeast Asia and is part of the Rubiaceae family. Its fresh leaves are harvested for their medicinal properties and used for their psychoactive effects. Kratom contains many biologically active alkaloids, including mitragynine and 7-OH-mitragynine, which are considered the two most important psychoactive components and constitute approximately 66% and 2% of the total alkaloid content. Other alkaloids are present in the plant, such as speciogynine, speciociliatine and paynantheine, but have less psychoactive activity. Over the past decade, the sale of kratom powder has increased on the Internet. This led to a significant increase in forensic cases. Given the lack of data existing in the literature, and the total absence of data in nails, the authors report a study to determine the best target alkaloids for documenting kratom consumption in this matrix. Fingernail clippings from a supposed kratom powder user were analyzed after liquid-liquid extraction, chromatography separation using a HSS C18 column and performed on an ultra-high performance liquid chromatography coupled to a tandem mass spectrometer. In the specimen, mitragynine was quantified at 229 pg/mg, speciogynine and paynantheine were both quantified at 2 pg/mg, and speciociliatine was quantified at 19 pg/mg. 7-OH-mitragynine was not detected. The interpretation of these concentrations is complex, since there is currently no reference in the literature, as this is the first identification of mitragynine and other kratom alkaloids in nails. Nevertheless, in view of the high concentration of mitragynine, the subject seems to be a repetitive user of kratom. According to the measured concentrations, it seems that mitragynine remains the best target to document kratom consumption, but the identification of the other alkaloids would enhance the specificity of the test.

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.

Kratom: The analytical challenge of an emerging herbal drug.

Mitragyna speciosa or kratom is emerging worldwide as a "legal" herbal drug of abuse. An increasing number of papers is appearing in the scientific literature regarding its pharmacological profile and the analysis of its chemical constituents, mainly represented by alkaloids. However, its detection and identification are not straightforward as the plant material is not particularly distinctive. Hyphenated techniques are generally preferred for the identification and quantification of these compounds, especially the main purported psychoactive substances, mitragynine (MG) and 7-hydroxymitragynine (7-OH-MG), in raw and commercial products. Considering the vast popularity of this recreational drug and the growing concern about its safety, the analysis of alkaloids in biological specimens is also of great importance for forensic and toxicological laboratories. The review addresses the analytical aspects of kratom spanning the extraction techniques used to isolate the alkaloids, the qualitative and quantitative analytical methods and the strategies for the distinction of the naturally occurring isomers.

Total Synthesis and Structural Plasticity of Kratom Pseudoindoxyl Metabolites.

Mitragynine pseudoindoxyl, a kratom metabolite, has attracted increasing attention due to its favorable side effect profile as compared to conventional opioids. Herein, we describe the first enantioselective and scalable total synthesis of this natural product and its epimeric congener, speciogynine pseudoindoxyl. The characteristic spiro-5-5-6-tricyclic system of these alkaloids was formed through a protecting-group-free cascade relay process in which oxidized tryptamine and secologanin analogues were used. Furthermore, we discovered that mitragynine pseudoindoxyl acts not as a single molecular entity but as a dynamic ensemble of stereoisomers in protic environments; thus, it exhibits structural plasticity in biological systems. Accordingly, these synthetic, structural, and biological studies provide a basis for the planned design of mitragynine pseudoindoxyl analogues, which can guide the development of next-generation analgesics.

Comparative metabolomics analysis reveals alkaloid repertoires in young and mature Mitragyna speciosa (Korth.) Havil. Leaves.

The fresh leaves of Mitragyna speciosa (Korth.) Havil. have been traditionally consumed for centuries in Southeast Asia for its healing properties. Although the alkaloids of M. speciosa have been studied since the 1920s, comparative and systematic studies of metabolite composition based on different leaf maturity levels are still lacking. This study assessed the secondary metabolite composition in two different leaf stages (young and mature) of M. speciosa, using an untargeted liquid chromatography-electrospray ionisation-time-of-flight-mass spectrometry (LC-ESI-TOF-MS) metabolite profiling. The results revealed 86 putatively annotated metabolite features (RT:m/z value) comprising 63 alkaloids, 10 flavonoids, 6 terpenoids, 3 phenylpropanoids, and 1 of each carboxylic acid, glucoside, phenol, and phenolic aldehyde. The alkaloid features were further categorised into 14 subclasses, i.e., the most abundant class of secondary metabolites identified. As per previous reports, indole alkaloids are the most abundant alkaloid subclass in M. speciosa. The result of multivariate analysis (MVA) using principal component analysis (PCA) showed a clear separation of 92.8% between the young and mature leaf samples, indicating a high variance in metabolite levels between them. Akuammidine, alstonine, tryptamine, and yohimbine were tentatively identified among the many new alkaloids reported in this study, depicting the diverse biological activities of M. speciosa. Besides delving into the knowledge of metabolite distribution in different leaf stages, these findings have extended the current alkaloid repository of M. speciosa for a better understanding of its pharmaceutical potential.

Inhibition of Advanced Glycation End-Products by Tamarindus indica and Mitragyna inermis Extracts and Effects on Human Hepatocyte and Fibroblast Viability.

Tamarindus indica and Mitragyna inermis are widely used by herbalists to cure diabetes mellitus. The aim of this study is to investigate the inhibitory potential of aqueous and various organic solvent fractions from both plants and some isolated compounds against advanced glycation end-products (AGEs). For this purpose, an in vitro BSA-fructose glycation model was used to evaluate the inhibition of AGE formation. Furthermore, the effects of the fractions on mouse fibroblast (NIH-3T3) and human hepatocyte (HepG2) survival were evaluated. The leaf, stem, and root fractions of both plants exhibited significant inhibition of AGEs formation. The IC50 values appeared to be less than 250 µg/mL; however, all fractions presented no adverse effects on NIH-3T3 up to 500 µg/mL. Otherwise, our phytochemical investigation afforded the isolation of a secoiridoid from the Mitragyna genus named secoiridoid glucoside sweroside (1), along with three known quinovic acid glycosides: quinovic acid-3ß-O-ß-d-glucopyranoside (2), quinovic acid-3-O-ß-d-6-deoxy-glucopyranoside, 28-O-ß-d-glucopyranosyl ester (3), and quinovic acid 3-O-α-l-rhamnopyranosyl-(4→1)-ß-d-glucopyranoside (4). In particular, 1-3 are compounds which have not previously been described in Mitragyna inermis roots. However, the isolated compounds did not exhibit AGE inhibitory activity. Further investigation on these potent antiglycation fractions may allow for the isolation of new antidiabetic drug candidates.

Analysis of Mitragynine and Speciociliatine in Umbilical Cord by LC-MS-MS for Detecting Prenatal Exposure to Kratom.

Kratom is an herbal drug that is legal in the USA. While it is marketed as a safer alternative to opioids, it can cause opioid-like withdrawal symptoms when discontinued after regular use. Several case studies have shown that kratom exposure in utero can lead to symptoms in newborns consistent with neonatal abstinence syndrome. Here, we present a validated method for the detection of kratom in umbilical cord by liquid chromatography--tandem mass spectrometry. The umbilical cord is homogenized in solvent and kratom analytes are purified by solid phase extraction (strong cation exchange). Diastereomeric kratom alkaloids mitragynine (MG), speciociliatine (SC), speciogynine and mitraciliatine are separated by reverse phase chromatography on a phenyl-hexyl column. Applying this method to residual umbilical cords submitted to our laboratory for drug testing, 29 positive specimens exhibiting varied kratom analyte distributions were observed. MG and SC were the most abundant kratom analytes and were selected as biomarkers of kratom exposure. A cutoff concentration of 0.08 ng/g was established for both MG and SC.

Drug testing for mitragynine and kratom: Analytical challenges and medico-legal considerations.

Mitragyna speciosa, known as kratom, is a tropical tree native to Southeast Asia that has long been used to increase energy and in traditional medicine. Kratom leaves contain several indole alkaloids including mitragynine, mitraciliatine, speciogynine, and speciociliatine, which have the same molecular formula and connectivity, but different spatial arrangements (i.e., diastereomers). A routine liquid-chromatographic-high-resolution mass-spectrometric (LC-HRMS) multi-analyte method for addictive and herbal drugs in urine did not separate mitragynine from speciogynine and speciociliatine. Separation and individual measurement of the four diastereomers was possible with an improved LC method. All diastereomers were detected in 29 patient urine samples who tested positive for mitragynine with the routine method, albeit at variable absolute amounts and relative proportions. The presence of all diastereomers rather than individual substances indicated that they originated from the intake of kratom (i.e., plant material). Speciociliatine dominated in most samples (66%), whereas mitragynine and mitraciliatine were the highest in 17% each. A kratom product (powdered plant material) marketed in Sweden contained all diastereomers with mitragynine showing the highest level. In Sweden, there are signs of an increasing use of kratom in society, based on the results from drug testing, the number of poisons center consultations on intoxications, and customs seizure statistics. Because there may be health risks associated with kratom use, including dependence, serious adverse reactions, and death, analytical methods should be able to identify and quantify all diastereomers. In Sweden, this is important from a legal perspective, as only mitragynine is classified, whereas the other three diastereomers, and kratom (plant material), are not.

Respiratory effects of oral mitragynine and oxycodone in a rodent model.

RATIONALE: Kratom derives from Mitragyna speciosa (Korth.), a tropical tree in the genus Mitragyna (Rubiaceae) that also includes the coffee tree. Kratom leaf powders, tea-like decoctions, and commercial extracts are taken orally, primarily for health and well-being by millions of people globally. Others take kratom to eliminate opioid use for analgesia and manage opioid withdrawal and use disorder. There is debate over the possible respiratory depressant overdose risk of the primary active alkaloid, mitragynine, a partial µ-opioid receptor agonist, that does not signal through ß-arrestin, the primary opioid respiratory depressant pathway. OBJECTIVES: Compare the respiratory effects of oral mitragynine to oral oxycodone in rats with the study design previously published by US Food and Drug Administration (FDA) scientists for evaluating the respiratory effects of opioids (Xu et al., Toxicol Rep 7:188-197, 2020). METHODS: Blood gases, observable signs, and mitragynine pharmacokinetics were assessed for 12 h after 20, 40, 80, 240, and 400 mg/kg oral mitragynine isolate and 6.75, 60, and 150 mg/kg oral oxycodone hydrochloride. FINDINGS: Oxycodone administration produced significant dose-related respiratory depressant effects and pronounced sedation with one death each at 60 and 150 mg/kg. Mitragynine did not yield significant dose-related respiratory depressant or life-threatening effects. Sedative-like effects, milder than produced by oxycodone, were evident at the highest mitragynine dose. Maximum oxycodone and mitragynine plasma concentrations were dose related. CONCLUSIONS: Consistent with mitragynine's pharmacology that includes partial µ-opioid receptor agonism with little recruitment of the respiratory depressant activating ß-arrestin pathway, mitragynine produced no evidence of respiratory depression at doses many times higher than known to be taken by humans.

Inhibition of α-Glucosidase and Pancreatic Lipase Properties of Mitragyna speciosa (Korth.) Havil. (Kratom) Leaves.

Kratom (Mitragyna speciosa (Korth.) Havil.) has been used to reduce blood sugar and lipid profiles in traditional medicine, and mitragynine is a major constituent in kratom leaves. Previous data on the blood sugar and lipid-altering effects of kratom are limited. In this study, phytochemical analyses of mitragynine, 7-hydroxymitragynine, quercetin, and rutin were performed in kratom extracts. The effects on α-glucosidase and pancreatic lipase activities were investigated in kratom extracts and mitragynine. The LC-MS/MS analysis showed that the mitragynine, quercetin, and rutin contents from kratom extracts were different. The ethanol extract exhibited the highest total phenolic content (TPC), total flavonoid content (TFC), and total alkaloid content (TAC). Additionally, compared to methanol and aqueous extracts, the ethanol extract showed the strongest inhibition activity against α-glucosidase and pancreatic lipase. Compared with the anti-diabetic agent acarbose, mitragynine showed the most potent α-glucosidase inhibition, with less potent activity of pancreatic lipase inhibition. Analysis of α-glucosidase and pancreatic lipase kinetics revealed that mitragynine inhibited noncompetitive and competitive effects, respectively. Combining mitragynine with acarbose resulted in a synergistic interaction with α-glucosidase inhibition. These results have established the potential of mitragynine from kratom as a herbal supplement for the treatment and prevention of diabetes mellitus.