Evolution and diversification of carboxylesterase-like [4+2] cyclases in aspidosperma and iboga alkaloid biosynthesis.

Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, and their biosynthetic construction has been a subject of intensive study for many years. The enzymatic basis for the production of aspidosperma and iboga alkaloids, which are produced exclusively by members of the Apocynaceae plant family, has recently been discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus and Tabernanthe iboga catalyze regio- and enantiodivergent [4+2] cycloaddition reactions to generate the aspidosperma (tabersonine synthase, TS) and iboga (coronaridine synthase, CorS; catharanthine synthase, CS) scaffolds from a common biosynthetic intermediate. Here, we use a combined phylogenetic and biochemical approach to investigate the evolution and functional diversification of these cyclase enzymes. Through ancestral sequence reconstruction, we provide evidence for initial evolution of TS from an ancestral CXE followed by emergence of CorS in two separate lineages, leading in turn to CS exclusively in the Catharanthus genus. This progression from aspidosperma to iboga alkaloid biosynthesis is consistent with the chemotaxonomic distribution of these MIAs. We subsequently generate and test a panel of chimeras based on the ancestral cyclases to probe the molecular basis for differential cyclization activity. Finally, we show through partial heterologous reconstitution of tabersonine biosynthesis using non-pathway enzymes how aspidosperma alkaloids could have first appeared as "underground metabolites" via recruitment of promiscuous enzymes from common protein families. Our results provide insight into the evolution of biosynthetic enzymes and how new secondary metabolic pathways can emerge through small but important sequence changes following co-option of preexisting enzymatic functions.

Genome-wide identification of GATA transcription factor family and the effect of different light quality on the accumulation of terpenoid indole alkaloids in Uncaria rhynchophylla.

Uncaria rhynchophylla is an evergreen vine plant, belonging to the Rubiaceae family, that is rich in terpenoid indole alkaloids (TIAs) that have therapeutic effects on hypertension and Alzheimer's disease. GATA transcription factors (TF) are a class of transcription regulators that participate in the light response regulation, chlorophyll synthesis, and metabolism, with the capability to bind to GATA cis-acting elements in the promoter region of target genes. Currently the charactertics of GATA TFs in U. rhynchophylla and how different light qualities affect the expression of GATA and key enzyme genes, thereby affecting the changes in U. rhynchophylla alkaloids have not been investigated. In this study, 25 UrGATA genes belonging to four subgroups were identified based on genome-wide analysis. Intraspecific collinearity analysis revealed that only segmental duplications were identified among the UrGATA gene family. Collinearity analysis of GATA genes between U. rhynchophylla and four representative plant species, Arabidopsis thaliana, Oryza sativa, Coffea Canephora, and Catharanthus roseus was also performed. U. rhynchophylla seedlings grown in either red lights or under reduced light intensity had altered TIAs content after 21 days. Gene expression analysis reveal a complex pattern of expression from the 25 UrGATA genes as well as a number of key TIA enzyme genes. UrGATA7 and UrGATA8 were found to have similar expression profiles to key enzyme TIA genes in response to altered light treatments, implying that they may be involved in the regulation TIA content. In this research, we comprehensively analyzed the UrGATA TFs, and offered insight into the involvement of UrGATA TFs from U. rhynchophylla in TIAs biosynthesis.

A Platform for the Synthesis of Corynantheine-Type Corynanthe Alkaloids.

Corynantheine-type alkaloids are major components of the Mitragyna speciosa, also known as kratom, that exhibit unique pharmacological activity. However, no universal method to prepare these alkaloids has been reported. Disclosed herein is a catalytic, asymmetric platform that enables rapid access to corynantheine alkaloids. The first enantioselective total synthesis of (-)-corynantheidine pseudoindoxyl is described. The first asymmetric syntheses of (+)-corynoxine and (-)-corynoxine B were also achieved, along with enantioselective syntheses of (-)-corynantheidol and (-)-corynantheidine. Through this work, all series of corynantheine alkaloids including indole, spirooxindole, and pseudoindoxyl can now be accessed in the laboratory, enabling comprehensive biological investigation of kratom alkaloids to be undertaken.

Integration of cell differentiation and initiation of monoterpenoid indole alkaloid metabolism in seed germination of Catharanthus roseus.

In Catharanthus roseus, monoterpenoid indole alkaloids (MIAs) are produced through the cooperation of four cell types, with final products accumulating in specialized cells known as idioblasts and laticifers. To explore the relationship between cellular differentiation and cell type-specific MIA metabolism, we analyzed the expression of MIA biosynthesis in germinating seeds. Embryos from immature and mature seeds were observed via stereomicroscopy, fluorescence microscopy, and electron microscopy. Time-series MIA and iridoid quantification, along with transcriptome analysis, were conducted to determine the initiation of MIA biosynthesis. In addition, the localization of MIAs was examined using alkaloid staining and imaging mass spectrometry (IMS). Laticifers were present in embryos before seed maturation. MIA biosynthesis commenced 12 h after germination. MIAs accumulated in laticifers of embryos following seed germination, and MIA metabolism is induced after germination in a tissue-specific manner. These findings suggest that cellular morphological differentiation precedes metabolic differentiation. Considering the well-known toxicity and defense role of MIAs in matured plants, MIAs may be an important defense strategy already in the delicate developmental phase of seed germination, and biosynthesis and accumulation of MIAs may require the tissue and cellular differentiation.

The leaf idioblastome of the medicinal plant Catharanthus roseus is associated with stress resistance and alkaloid metabolism.

Catharanthus roseus leaves produce a range of monoterpenoid indole alkaloids (MIAs) that include low levels of the anticancer drugs vinblastine and vincristine. The MIA pathway displays a complex architecture spanning different subcellular and cell type localizations, and is under complex regulation. As a result, the development of strategies to increase the levels of the anticancer MIAs has remained elusive. The pathway involves mesophyll specialized idioblasts where the late unsolved biosynthetic steps are thought to occur. Here, protoplasts of C. roseus leaf idioblasts were isolated by fluorescence-activated cell sorting, and their differential alkaloid and transcriptomic profiles were characterized. This involved the assembly of an improved C. roseus transcriptome from short- and long-read data, IDIO+. It was observed that C. roseus mesophyll idioblasts possess a distinctive transcriptomic profile associated with protection against biotic and abiotic stresses, and indicative that this cell type is a carbon sink, in contrast to surrounding mesophyll cells. Moreover, it is shown that idioblasts are a hotspot of alkaloid accumulation, suggesting that their transcriptome may hold the key to the in-depth understanding of the MIA pathway and the success of strategies leading to higher levels of the anticancer drugs.

Microbial allies: exploring fungal endophytes for biosynthesis of terpenoid indole alkaloids.

Terpenoid indole alkaloids (TIAs) are natural compounds found in medicinal plants that exhibit various therapeutic activities, such as antimicrobial, anti-inflammatory, antioxidant, anti-diabetic, anti-helminthic, and anti-tumor properties. However, the production of these alkaloids in plants is limited, and there is a high demand for them due to the increasing incidence of cancer cases. To address this research gap, researchers have focused on optimizing culture media, eliciting metabolic pathways, overexpressing genes, and searching for potential sources of TIAs in organisms other than plants. The insufficient number of essential genes and enzymes in the biosynthesis pathway is the reason behind the limited production of TIAs. As the field of natural product discovery from biological species continues to grow, endophytes are being investigated more and more as potential sources of bioactive metabolites with a variety of chemical structures. Endophytes are microorganisms (fungi, bacteria, archaea, and actinomycetes), that exert a significant influence on the metabolic pathways of both the host plants and the endophytic cells. Bio-prospection of fungal endophytes has shown the discovery of novel, high-value bioactive compounds of commercial significance. The discovery of therapeutically significant secondary metabolites has been made easier by endophytic entities' abundant but understudied diversity. It has been observed that fungal endophytes have better intermediate processing ability due to cellular compartmentation. This paper focuses on fungal endophytes and their metabolic ability to produce complex TIAs, recent advancements in this area, and addressing the limitations and future perspectives related to TIA production.

Catalytic selectivity and evolution of cytochrome P450 enzymes involved in monoterpene indole alkaloids biosynthesis.

Cytochrome P450 enzyme (CYP)-catalyzed functional group transformations are pivotal in the biosynthesis of metabolic intermediates and products, as exemplified by the CYP-catalyzed C7-hydroxylation and the subsequent C7-C8 bond cleavage reaction responsible for the biosynthesis of the well-known antitumor monoterpene indole alkaloid (MIA) camptothecin. To determine the key amino acid residues responsible for the catalytic selectivity of the CYPs involved in MIA biosynthesis, we characterized the enzymes CYP72A728 and CYP72A729 as stereoselective 7-deoxyloganic acid 7-hydroxylases (7DLHs). We then conducted a comparative analysis of the amino acid sequences and the predicted structures of the CYP72A homologs involved in camptothecin biosynthesis, as well as those of the CYP72A homologs implicated in the pharmaceutically significant MIAs biosynthesis in Catharanthus roseus. The crucial amino acid residues for the catalytic selectivity of the CYP72A-catalyzed reactions were identified through fragmental and individual residue replacement, catalytic activity assays, molecular docking, and molecular dynamic simulations analysis. The fragments 1 and 3 of CYP72A565 were crucial for its C7-hydroxylation and C7-C8 bond cleavage activities. Mutating fragments 1 and 2 of CYP72A565 transformed the bifunctional CYP72A565 into a monofunctional 7DLH. Evolutionary analysis of the CYP72A homologs suggested that the bifunctional CYP72A in MIA-producing plants may have evolved into a monofunctional CYP72A. The gene pairs CYP72A728-CYP72A610 and CYP72A729-CYP72A565 may have originated from a whole genome duplication event. This study provides a molecular basis for the CYP72A-catalyzed hydroxylation and C-C bond cleavage activities of CYP72A565, as well as evolutionary insights of CYP72A homologs involved in MIAs biosynthesis.

Polycyclic pyrroloindoline-containing natural products with a unique 3-heptyl-2a,4a-diazapentaleno[1,6-ab]indene core isolated from Alstonia scholaris.

Alscholarine C (1), featuring an unprecedented pyrroloindoline-containing natural product (PiNP) with a 6/5/5/5 tetracyclic carbon skeleton, and four known PiNPs (2-5), namely demethylalstoscholarinine E (2), Nb-demethylechitamine (3), winphylline A (4), and echitamine (5), were isolated from Alstonia scholaris. Compound 1 was characterized by a hexahydropyrrolo[2,3-b] indole (HPI) core fused to a unique 4-heptylimidazolidine motif, forming an unparalleled 3-heptyl-2a,4a-diazapentaleno[1,6-ab]indene ring system. Their structures were established by spectroscopic analysis, quantum-chemical calculated 13C NMR data with DP4+ probability analyses, and ECD calculations and comparison. A plausible biosynthetic pathway of 1 was proposed. Compound 1 exhibited potential anti-inflammatory activity against LPS-stimulated NO production in RAW264.7 cells.

Ajmalicine induces the pyroptosis of hepatoma cells to exert the antitumor effect.

Ajmalicine (AJM) is an alkaloid extracted from the root of Yunan Rauvolfia verticillata. At present, little research has reported the antitumor pharmacological action and mechanism of AJM. Therefore, this work aimed to conduct relevant research. The mouse hepatoma cell line H22 was intervened with a gradient concentration of AJM. Subsequently, the pyroptosis level was detected by flow cytometry. The expression of inflammatory factors and lactate dehydrogenase was measured by enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) expression was detected by dichlorodihydrofluorescein diacetate probe. In addition, the tumor-bearing model mice were also treated with AJM to analyze tumor growth as well as the expression levels of tissue inflammatory factors and proteins. According to our results, AJM promoted the pyroptosis of H22 cells, increased the pyroptosis rate, and upregulated the expression of inflammatory factors tumor necrosis factor α, interleukin-1ß, and interleukin-6. At the same time, it enhanced the openness of membrane pores and increased the expression of ROS. Moreover, AJM promoted the expression of Caspase-3 and N-terminal gasdermin E (GSDME). The AJM-induced pyroptosis was suppressed after N-acetylcysteine treatment to inhibit ROS, while Caspase-3 knockdown also inhibited the AJM-induced pyroptosis. In animals, AJM suppressed tumor growth. AJM can activate ROS to induce pyroptosis and exert the antitumor effect via the noncanonical Caspase-3-GSDME pyroptosis pathway.

Quinoline, Indole, and Isogranatanine Alkaloids from Malayan Leuconotis eugeniifolia.

Nine new alkaloids, eugeniinalines A-H (1-8) and (+)-eburnamenine N-oxide (9), comprising one quinoline, six indole, and two isogranatanine alkaloids, were isolated from the stem-bark extract of the Malayan Leuconotis eugeniifolia. The structures and absolute configurations of these alkaloids were established based on the analysis of the spectroscopic data, GIAO NMR calculations, DP4+ probability analysis, TDDFT-ECD method, and X-ray diffraction analysis. Eugeniinaline A (1) represents a new pentacyclic quinoline alkaloid with a 6/6/5/6/7 ring system. Eugeniinaline G (7) and its seco-derivative, eugeniinaline H (8), were the first isogranatanine alkaloids isolated as natural products. The known alkaloids leucolusine (10) and melokhanine A (11) were found to be the same compound, based on comparison of the spectroscopic data of both compounds, with the absolute configuration of (7R, 20R, 21S). Eugeniinalines A and G (1 and 7) showed cytotoxic activity against the HT-29 cancer cell line with IC50 values of 7.1 and 7.2 µM, respectively.

Alstomaphylines A-K, monoterpenoid bisindole alkaloids from Alstonia macrophylla with AChE inhibitory activity and cytotoxicity.

Eleven undescribed monoterpenoid bisindole alkaloids, alstomaphyines A-K (1-11), along with three known analogues were isolated from the leaves and stem bark of the Alstonia macrophylla. Compounds 1-3 were unprecedented dimerization alkaloids incorporating a macroline-type motif with an ajmaline-type motif via a C-C linkage. Their structures and absolute configurations were elucidated by extensive spectroscopic analysis, electronic circular dichroism (ECD) calculation, and CD exciton chirality method. Compounds 1-3 displayed potential inhibitory bioactivity against AChE with IC50 values of 4.44 ± 0.35, 3.59 ± 0.18, and 3.71 ± 0.23 µM, respectively. Enzyme kinetic study revealed compounds 1-3 as mixed competitive AChE inhibitors. Besides, compounds 8 and 12-14 exhibited better cytotoxicity against human cancer cell line HT-29 than cisplatin. Flow cytometry data revealed that compounds 8, 13, and 14 significantly induced the HT-29 cells arrest in G0/G1 phase in a concentration-dependent manner.

The role of the Golden2-like (GLK) transcription factor in regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

KEY MESSAGE: A GLK homologue was identified and functionally characterized in Catharanthus roseus. Silencing CrGLK with VIGS or the chloroplast retrograde signaling inducer lincomycin increased terpenoid indole alkaloid biosynthesis. Catharanthus roseus is the sole source of the chemotherapeutic terpenoid indole alkaloids (TIAs) vinblastine and vincristine. TIA pathway genes, particularly genes in the vindoline pathway, are expressed at higher levels in immature versus mature leaves, but the molecular mechanisms responsible for this developmental regulation are unknown. We investigated the role of GOLDEN2-LIKE (GLK) transcription factors in contributing to this ontogenetic regulation since GLKs are active in seedlings upon light exposure and in the leaf's early development, but their activity is repressed as leaves age and senesce. We identified a GLK homologue in C. roseus and functionally characterized its role in regulating TIA biosynthesis, with a focus on the vindoline pathway, by transiently reducing its expression through two separate methods: virus-induced gene silencing (VIGS) and application of chloroplast retrograde signaling inducers, norflurazon and lincomycin. Reducing CrGLK levels with each method reduced chlorophyll accumulation and the expression of the light harvesting complex subunit (LHCB2.2), confirming its functional homology with GLKs in other plant species. In contrast, reducing CrGLK via VIGS or lincomycin increased TIA accumulation and TIA pathway gene expression, suggesting that CrGLK may repress TIA biosynthesis. However, norflurazon had no effect on TIA gene expression, indicating that reducing CrGLK alone is not sufficient to induce TIA biosynthesis. Future work is needed to clarify the specific molecular mechanisms leading to increased TIA biosynthesis with CrGLK silencing. This is the first identification and characterization of GLK in C. roseus and the first investigation of how chloroplast retrograde signaling might regulate TIA biosynthesis.

Characterization of the ZCTs, a subgroup of Cys2-His2 zinc finger transcription factors regulating alkaloid biosynthesis in Catharanthus roseus.

KEY MESSAGE: The C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the terpenoid indole alkaloid pathway when highly expressed. Catharanthus roseus is the sole known producer of the anti-cancer terpenoid indole alkaloids (TIAs), vinblastine and vincristine. While the enzymatic steps of the pathway have been elucidated, an understanding of its regulation is still emerging. The present study characterizes an important subgroup of Cys2-His2 zinc finger transcription factors known as Zinc finger Catharanthus Transcription factors (ZCTs). We identified three new ZCT members (named ZCT4, ZCT5, and ZCT6) that clustered with the putative repressors of the TIA pathway, ZCT1, ZCT2, and ZCT3. We characterized the role of these six ZCTs as potential redundant regulators of the TIA pathway, and their tissue-specific and jasmonate-responsive expression. These ZCTs share high sequence conservation in their two Cys2-His2 zinc finger domains but differ in the spacer length and sequence between these zinc fingers. The transient overexpression of ZCTs in seedlings significantly repressed the promoters of the terpenoid (pLAMT) and condensation branch (pSTR1) of the TIA pathway, consistent with that previously reported for ZCT1, ZCT2, and ZCT3. In addition, ZCTs significantly repressed and indirectly activated several promoters of the vindoline pathway (not previously studied). The ZCTs differed in their tissue-specific expression but similarly increased with jasmonate in a dosage-dependent manner (except for ZCT5). We showed significant activation of the pZCT1 and pZCT3 promoters by the de-repressed CrMYC2a, suggesting that the jasmonate-responsive expression of the ZCTs can be mediated by CrMYC2a. In summary, the C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the TIA pathway when highly expressed.

Genome-wide identification and characterization of wall-associated kinases, molecular docking and polysaccharide elicitation of monoterpenoid indole alkaloids in micro-propagated Catharanthus roseus.

Wall-associated kinases (WAKs) are a unique family of proteins that are predominantly localized on the plasma membrane and simultaneously bound to the cell wall. WAKs play a pivotal role in signal transduction to regulate growth, defense, and response to environmental stimuli in plants. These kinases have been identified and characterized in various plant species, however, similar information for Catharanthus roseus is scarce. C. roseus is an evergreen ornamental plant that produces a repertoire of biologically active compounds. The plant is best characterized for the production of antineoplastic monoterpenoid indole alkaloids (MIAs) namely vinblastine and vincristine. Owing to the diverse composition of phytochemicals, C. roseus is known as a "model non-model" plant for secondary metabolite research. Genome analyses showed 37 putative CrWAK genes present in C. roseus, largely localized on the plasma membrane. Phylogenetic analysis revealed six clusters of CrWAKs. Diverse cis-acting elements, including those involved in defense responses, were identified on the promotor regions of CrWAK genes. The highest binding affinity (- 12.6 kcal/mol) was noted for CrWAK-22 against tri-galacturonic acid. Tri-galacturonic acid stimulated 2.5-fold higher production of vinblastine, sixfold upregulation of the expression of ORCA3 transcription factor, and 6.14-fold upregulation of CrWAK-22 expression. Based on these results it was concluded that the expression of CrWAK genes induced by biotic elicitors may have an important role in the production of MIAs. The current findings may serve as a basis for functional characterization and mechanistic explanation of the role of CrWAK genes in the biosynthesis of MIAs upon elicitation.

Engineered biosynthesis of plant heteroyohimbine and corynantheine alkaloids in Saccharomyces cerevisiae.

Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30-40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation. ONE SENTENCE SUMMARY: An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.

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.

The basic chemical substances of total alkaloids of Uncaria rhynchophylla and their anti-neuroinflammatory activities.

To clarify the chemical basis of the total alkaloids of Uncaria rhynchophylla, HPLC-VWD chromatogram of total alkaloids was established. Under its guidance, modern chromatographic and spectroscopic techniques were used to track, isolate and identify the representative principal components. As a result, one new monoterpenoid indole alkaloid, 3S,15S-N4-methoxymethyl-geissoschizine methyl ether (1), together with 20 known alkaloids (2-21), and 5 other known compounds (22-26) were obtained. Meanwhile, sixteen characteristic peaks were identified from the total alkaloids using HPLC analysis. Then, the anti-neuroinflammatory effect of compounds 1-21 was assessed through inhibiting nitric ---oxide (NO) production in lipopolysaccharide (LPS)-induced BV-2 microglial cells. Among them, compounds 1, 3, 7, 8, 11, 12, 19 and 21 showed potent inhibitory activities with IC50 values of 5.87-76.78 µM.

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.

[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.