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Kratom – Biological Effects and Chemical Composition

Kratom - Biological Effects and Chemical Composition

The safety and efficacy of kratom (Mitragyna speciosa) for treatment of pain is highly controversial. Kratom produces more than 40 structurally related alkaloids, but most studies have focused on just two of these, mitragynine and 7-hydroxymitragynine. Here, we profiled 53 commercial kratom products using untargeted LC–MS metabolomics, revealing two distinct chemotypes that contain different levels of the alkaloid speciofoline. Both chemotypes were confirmed with DNA barcoding to be M. speciosa.

To evaluate the biological relevance of variable speciofoline levels in kratom, we compared the opioid receptor binding activity of speciofoline, mitragynine, and 7-hydroxymitragynine. Mitragynine and 7-hydroxymitragynine function as partial agonists of the human µ-opioid receptor, while speciofoline does not exhibit measurable binding affinity at the µ-, δ- or ƙ-opioid receptors. Importantly, mitragynine and 7-hydroxymitragynine demonstrate functional selectivity for G-protein signaling, with no measurable recruitment of β-arrestin.

Overall, the study demonstrates the unique binding and functional profiles of the kratom alkaloids, suggesting potential utility for managing pain, but further studies are needed to follow up on these in vitro findings. All three kratom alkaloids tested inhibited select cytochrome P450 enzymes, suggesting a potential risk for adverse interactions when kratom is co-consumed with drugs metabolized by these enzymes.


According to the US Department of Health and Human Services, opioids were responsible for more than 42,000 deaths in the US in 2016, the highest in recorded history1. More than 40% of these deaths involved a prescription opioid. Some individuals who suffer from chronic pain are turning to other options, one of which is the plant known as kratom [Mitragyna speciosa (Korth.) Havil. (Rubiaceae)], a tropical tree native to peninsular Thailand, Myanmar, Malaysia and other countries in Southeast Asia. Kratom has skyrocketed in popularity in western countries in the past decade; current estimates are that as many as 5 million individuals in the US use kratom on a regular basis. There has been considerable controversy over the safety and efficacy of kratom use by US consumers. Citing safety concerns (one study reports that kratom has been implicated as at least partially involved in 91 deaths), the United States Department of Agriculture (USDA) has made it their practice to confiscate shipments of kratom into the US. The US Drug Enforcement Administration (DEA) threatened to assign kratom as a schedule 1 controlled substance, which would make possession of kratom illegal. The DEA then suspended that decision in response to a backlash from some US consumers, who claim that it is a safer alternative to opioids for treatment of pain and/or opioid addiction. The effectiveness of kratom for these purposes continues to be a hotly debated and politically charged topic, and one that requires rigorous scientific investigation.

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Widespread kratom use is a relatively new phenomenon in the US. However, the medicinal use of kratom in southeast Asia has a long history. Kratom was first documented in the scientific literature in 1836, in a paper that described use of kratom leaves by the Malays as a substitute for opium. The isolation of mitragynine was reported in 1921 by Ellen Field, who ended the introduction of her paper with the following statements, “According to Redley………..Mitragyne [sic] speciosa is used in Perak against the opium habit, whilst, according to Dr. P.P. Laidlaw, mitragynine is a local anaesthetic [sic]…” Thus, controversy over the use and effectiveness of kratom was documented nearly 100 years ago. It is a controversy that continues today.

Consistent with the claim that kratom can be effective in the treatment of pain, extracts from this plant demonstrated opioid-receptor mediated analgesic effects in mouse model studies. This activity has generally been attributed to alkaloids that the plant produces, of which mitragynine (1) and 7-hydroxymitragynine (2) (Fig. 1) have been the focus of multiple pharmacological investigations. Mitragynine and 7-hydroxymitragynine both bind to the human µ-opioid and ƙ-opioid receptors (hMOR, hKOR) with nanomolar affinity, and function as partial agonists at the µ-opioid receptor and weak antagonists at ƙ-opioid and δ-opioid receptors. 7-Hydroxymitragynine exhibits approximately fivefold greater affinity at the μ-opioid receptor compared to mitragynine. Upon receptor activation, mitragynine and 7-hydroxymitragynine exhibit functional selectivity for G-protein signaling, with no measurable recruitment of β-arrestin14. In antinociception assays, 7-hydroxymitragynine exhibits 40-fold greater potency than mitragynine and tenfold greater potency than morphine, whereas mitragynine is less potent than morphine in antinociceptive assays. Combined administration of mitragynine with morphine increases antinociception compared with morphine alone and prevents the development of morphine tolerance. In contrast to mitragynine, repeated administration of 7-hydroxymitragynine produces antinociceptive tolerance as well as cross-tolerance to morphine’s antinociceptive action and induces physical dependence. Furthermore, mitragynine does not exhibit abuse liability and decreases the reinforcing effects of morphine whereas 7-hyroxymitragynine demonstrates abuse liability and increased morphine self-administration in rats . Interestingly, a recent study by Kruegel et al. shows that mitragynine can be converted to 7-hydroxymitragynine both in vitro and in a mouse model, therefore, some of the in vitro activity attributed to mitragynine may in fact be due to the action of it metabolite 7-hydroxymitragynine.


kratom scientific report figure 1

Structures of select alkaloids present in kratom (Mitragyna speciosa), mitragynine (1), 7-hydroxymitragynine (2) and speciofoline (3).

Collectively, the scientific data on mitragynine and 7-hydroxymitragynine suggest that kratom tea might indeed be an effective alternative to opioids against pain. However, kratom products are sold to consumers under a variety of trade names with no data regarding the chemical composition of plant material being consumed. Kratom has been documented to produce variable levels of its more than 40 alkaloids, depending on genetic variation and differences in growth and processing conditions. What are the potential health implications of the variability in chemical content of kratom preparations? As of yet, this question has been difficult to answer because mechanistic studies of kratom alkaloids have focused largely on isolated alkaloids without consideration of how these alkaloids are represented in kratom materials used medicinally. Herein, we sought to connect the dots between investigations of pure alkaloids and their relevance to medicinally used kratom. Towards this goal, we employed untargeted metabolomics to determine which alkaloids vary in content across commercial kratom products. We then isolated these relevant alkaloids and compared their biological effects in vitro, including activity at the µ-opioid receptors and inhibitory effects on CYP isoforms involved in the metabolism of opioids and other drugs. Our ultimate objective with this study was to capture the variability of commercial kratom products (kratom powderkratom capsuleskratom extracts, etc) being employed by US consumers and to evaluate (based on vitro studies) the potential implications of this variability in terms of safety and efficacy.

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Consistent with previous literature, our results raise concern about potential drug interactions that may occur when kratom is consumed concurrently with opiates or other drugs metabolized by the cytochrome P450s. Following up on these findings, future clinical studies to evaluate the safety and efficacy of kratom and its alkaloids would be of great benefit to the public that may employ this botanical for the treatment of pain, currently or in the future. In such studies, it would be useful to consider the potential differences in activity that would be observed by complex M. speciosa preparations as compared to their isolated alkaloids, and to account for potential differences in alkaloid profile in different kratom materials. Chemical profiling of samples prior to evaluating their biological activity is clearly crucial, and it is important that the biological studies be informed by the results of the chemical analyses. This study demonstrates how untargeted mass spectrometry metabolomics followed by targeted mass spectrometric quantitation can be effectively employed to generate chemical data valuable to inform biological evaluation. As such, our approach can serve as a model for the design of future studies.



DAMGO, quinidine, ketoconazole, morphine sulfate, and alprazolam were purchased from MilliporeSigma (St Louis, MO). Mitragynine, 7-hydroxymitragynine, midazolam, 1′-hydroxymidazolam, sulfaphenazole, and NADPH were obtained from Cayman Chemical Company (Ann Arbor, MI). Diclofenac, 4′-hydroxydiclofenac, dextromethorphan, and dextrorphan were obtained from Toronto Research Chemicals, Inc. (Toronto, Canada). Potassium phosphate buffer salts were obtained from Fisher Scientific (Fair Lawn, NJ). Human liver microsomes were obtained from XenoTech, LLC (H0604, mixed gender, pool from 15 donors, lot no. 1010191; Kansas City, KS). All other chemicals and reagents used were analytical grade.

Acquisition of Kratom plant material

Fifty commercial products labeled as kratom were obtained from vendors within the US. A table with details about these products is available in the supporting information (Table S1). The commercial kratom products were obtained as dried powders and were various shades of grey-green, with the exception of product K49, which was obtained as a cut leaf. All plant material was stored dry at room temperature until time of analysis.

A cultivated plant of kratom (Mitragyna speciosa) (coded as K55) was generously donated to the project by Shon Lenzo. The plant was grown from the cutting of a 7-year-old tree from a strain labeled as “rifat.” Prior to shipping to the University of North Carolina Greensboro, the cutting was grown indoors in a greenhouse under a 1000 W high pressure sodium (HPS) LED light for 6 months. Upon receipt, the plant was repotted and transferred to an outdoor location near the University of North Carolina Greensboro, where it was grown for several summer months. Leaves were cut from this plant, dried at room temperature and powdered for extraction using a Wiley Mill Standard Model No. 3 (Arthur Thomas Company). A sample of leaves from this plant, harvested at the coordinates 36°4′53.616″ N 79°47′6.18″ W, was pressed and submitted to the University of North Carolina Herbarium, with accession #670043 (sernecportal.org catalog number NCU00433756).

Isolation of speciofoline

Kratom plant material (K52, 1 kg) was extracted exhaustively with 2 L of CHCl3/CH3OH (1:1) and 50 mL of KOH (10%) by maceration over 24 h at room temperature. The mixture was filtered, and the solvent was evaporated under reduced pressure. The dried extract was reconstituted in 1 N HCl solution and hexanes (1:1), transferred to a separatory funnel, and shaken vigorously. After removal of the hexanes phase, the pH of the aqueous phase was adjusted to 9.0 with concentrated NH4OH solution. The basic phase was exhaustively extracted with CHCl3, and the organic phase was evaporated to dryness to yield 1.0 g of the dried extract. Fractionation of the extract was conducted with normal phase flash chromatography using a silica column (24 g) and a gradient solvent system of hexane–acetone-CH3OH over 52 min at a flow rate of 35 mL/min. In total, thirteen pooled fractions were obtained. Fraction 4 was subjected to preparative reversed phase HPLC over a Luna PFP(2) (pentafluorophenyl) column (Phenomenex, 250 mm × 21.2 mm, 5 µm) using a gradient of 70:30 to 100:0 CH3OH–H2O (10 mM of ammonium acetate in both phases) over 20 min at a flow rate of 20 mL/min. This process yielded 25.0 mg of speciofoline (95% purity based on UPLC-UV), the structure of which was solved by comparison of the calculated ([M + H]+ C22H29N2O5, 401.2076) and measured (401.2067) m/z values and by comparison of key 1H-NMR signals (Figure S1 and Table S2) and the ECD spectra (Figure S2) with those reported in the literature.

Extraction for metabolomics profiling

For each extraction of powdered kratom sample, 50 mg of dried kratom plant material was extracted with 5.0 mL of methanol in a 25 mL scintillation vial. Extracts were shaken at 150 rpm for 12 h and the powder was allowed to settle. The extracts were decanted into clean scintillation vials and dried under nitrogen. Extracts were resuspended in methanol containing 0.125 µg/mL of mitragynine-D3 (MilliporeSigma, St. Louis, MO, USA) for analysis. All diluted extracts contained mitragynine-D3 at a final concentration of 0.125 µg/mL as an internal standard to monitor the consistency of the instrument performance.

Read more about: Kratom – Biological Effects and Chemical Composition

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