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.

CrJAT1 Regulates Endogenous JA Signaling for Modulating Monoterpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus.

Many monoterpenoid indole alkaloids (MIAs) produced in Catharanthus roseus have demonstrated biological activities and clinical potential. However, their complex biosynthesis pathway in plants leads to low accumulation, limiting therapeutic applications. Efforts to elucidate the MIA biosynthetic regulatory mechanism have focused on improving accumulation levels. Previous studies revealed that jasmonic acid (JA), an important plant hormone, effectively promotes MIA accumulation by inducing the expression of MIA biosynthesis and transport genes. Nevertheless, excessive JA signaling can strongly inhibit plant growth, decreasing MIA productivity in C. roseus. Therefore, identifying key components balancing growth and MIA production in the JA signaling pathway is imperative for effective pharmaceutical production. Here, we identify a homolog of the jasmonate transporter 1, CrJAT1, through co-expression and phylogenetic analyses. Further investigation demonstrated that CrJAT1 can activate JA signaling to promote MIA accumulation without compromising growth. The potential role of CrJAT1 in redistributing intra/inter-cellular JA and JA-Ile may calibrate signaling to avoid inhibition, representing a promising molecular breeding target in C. roseus to optimize the balance between growth and specialized metabolism for improved MIA production.

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.

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 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.

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.

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.

Pharmacokinetic and Pharmacodynamic Consequences of Cytochrome P450 3A Inhibition on Mitragynine Metabolism in Rats.

Mitragynine, an opioidergic alkaloid present in Mitragyna speciosa (kratom), is metabolized by cytochrome P450 3A (CYP3A) to 7-hydroxymitragynine, a more potent opioid receptor agonist. The extent to which conversion to 7-hydroxymitragynine mediates the in vivo effects of mitragynine is unclear. The current study examined how CYP3A inhibition (ketoconazole) modifies the pharmacokinetics of mitragynine in rat liver microsomes in vitro. The study further examined how ketoconazole modifies the discriminative stimulus and antinociceptive effects of mitragynine in rats. Ketoconazole [30 mg/kg, oral gavage (o.g.)] increased systemic exposure to mitragynine (13.3 mg/kg, o.g.) by 120% and 7-hydroxymitragynine exposure by 130%. The unexpected increase in exposure to 7-hydroxymitragynine suggested that ketoconazole inhibits metabolism of both mitragynine and 7-hydroxymitragynine, a finding confirmed in rat liver microsomes. In rats discriminating 3.2 mg/kg morphine from vehicle under a fixed-ratio schedule of food delivery, ketoconazole pretreatment increased the potency of both mitragynine (4.7-fold) and 7-hydroxymitragynine (9.7-fold). Ketoconazole did not affect morphine's potency. Ketoconazole increased the antinociceptive potency of 7-hydroxymitragynine by 4.1-fold. Mitragynine (up to 56 mg/kg, i.p.) lacked antinociceptive effects both in the presence and absence of ketoconazole. These results suggest that both mitragynine and 7-hydroxymitragynine are cleared via CYP3A and that 7-hydroxymitragynine is formed as a metabolite of mitragynine by other routes. These results have implications for kratom use in combination with numerous medications and citrus juices that inhibit CYP3A. SIGNIFICANCE STATEMENT: Mitragynine is an abundant kratom alkaloid that exhibits low efficacy at the µ-opioid receptor (MOR). Its metabolite, 7-hydroxymitragynine, is also an MOR agonist but with higher affinity and efficacy than mitragynine. Our results in rats demonstrate that cytochrome P450 3A (CYP3A) inhibition can increase the systematic exposure of both mitragynine and 7-hydroxymitragynine and their potency to produce MOR-mediated behavioral effects. These data highlight potential interactions between kratom and CYP3A inhibitors, which include numerous medications and citrus juices.

Metabolomic responses to the mechanical wounding of Catharanthus roseus' upper leaves.

Purpose: Plant secondary metabolites are used to treat various human diseases. However, it is difficult to produce a large number of specific metabolites, which largely limits their medicinal applications. Many methods, such as drought and nutrient application, have been used to induce the biosynthetic production of secondary metabolites. Among these secondary metabolite-inducing methods, mechanical wounding maintains the composition of secondary metabolites with little potential risk. However, the effects of mechanical stress have not been fully investigated, and thus this method remains widely unused. Methods: In this study, we used metabolomics to investigate the metabolites produced in the upper and lower leaves of Catharanthus roseus in response to mechanical wounding. Results: In the upper leaves, 13 different secondary metabolites (three terpenoid indole alkaloids and 10 phenolic compounds) were screened using an orthogonal partial least squares discriminant analysis (OPLS-DA) score plot. The mechanical wounding of different plant parts affected the production of secondary metabolites. Specifically, when lower leaves were mechanically wounded, the upper leaves became a strong source of resources. Conversely, when upper leaves were injured, the upper leaves themselves became a resource sink. Changes in the source-sink relationship reflected a new balance between resource tradeoff and the upregulation or downregulation of certain metabolic pathways. Conclusion: Our findings suggest that mechanical wounding to specific plant parts is a novel approach to increase the biosynthetic production of specific secondary metabolites. These results indicate the need for a reevaluation of production practices for secondary metabolites from select commercial plants.

Do Fungicides Affect Alkaloid Production in Catharanthus roseus (L.) G. Don. Seedlings?

The presence of endophytes in plants is undeniable, but how significant their involvement is in the host plant biosynthetic pathways is still unclear. The results reported from fungicide treatments in plants varied. Fungicide treatment in Taxus was found to decrease the taxol content. In Ipomoea asarifolia, Pronto Plus and Folicur treatments coincided with the disappearance of ergot alkaloids from the plant. In Narcissus pseudonarcissus cv. Carlton, a mixture of fungicide applications decreased the alkaloids concentration and altered the carbohydrate metabolism. Jacobaea plants treated with Folicur reduced the pyrrolizidine alkaloids content. There have not been any studies into the involvement of endophytic fungi on alkaloids production of Catharanthus roseus until now. Though there is a report on the isolation of the endophytic fungi, Fusarium oxysporum from C. roseus, which was reported to produce vinblastine and vincristine in vitro. To detect possible collaborations between these two different organisms, fungicides were applied to suppress the endophytic fungi in seedlings and then measure the metabolomes by 1HNMR and HPLC analysis. The results indicate that endophytic fungi were not directly involved in alkaloids biosynthesis. Treatment with fungicides influenced both the primary and secondary metabolism of C. roseus. The systemic fungicides Pronto Plus and Folicur caused an increase in loganin and secologanin levels. In contrast, control samples had higher level of catharanthine and vindoline. This means that fungicide treatments cause changes in plant secondary metabolism.

Establishment of recombinant Catharanthus roseus stem cells stably overexpressing ORCA4 for terpenoid indole alkaloids biosynthesis.

Catharanthus roseus is a perennial herb of the Apocynaceae family, from which about 200 kinds of alkaloids have been characterized. Most alkaloids from C. roseus are terpenoid indole alkaloids (TIAs), such as vinblastine and vincristine, which are widely used in the clinic for their good antitumor activity. However, they were only biosynthesized in C. roseus, and their content in C. roseus is extremely low. The access to these valuable compounds is by plant extraction or chemical semisynthesis from their precursors catharanthine and vindoline. Since catharanthine and vindoline are also obtained from C. roseus, the supply of vinblastine and vincristine makes it difficult to meet market demands. Therefore, how to improve the yield of TIAs is an attractive issue. In this study, we compared the regulatory effect of two critical transcription factors, octadecanoid-derivative responsive Catharanthus AP2-domain protein 3 (ORCA3) and octadecanoid-derivative responsive Catharanthus AP2-domain protein 4 (ORCA4), on the biosynthesis of TIAs in C. roseus. The results showed that overexpressing both two transcription factors could increase the accumulation of TIAs. The effect was more significant when ORCA4 was overexpressed. To acquire C. roseus TIAs on a continuous and consistent basis, we then created and acquired C. roseus stem cells stably overexpressing ORCA4. This is the first time a recombinant C. roseus stem cell system with stable ORCA4 overexpression has been developed, which not only provides new ideas for future research in this area but also breaches new life into the industrial application of using plant cell culture to obtain natural products.

Cell type matters: competence for alkaloid metabolism differs in two seed-derived cell strains of Catharanthus roseus.

Since the discovery of the anticancer drugs vinblastine and vincristine, Catharanthus roseus has been intensively studied for biosynthesis of several terpene indole alkaloids (TIAs). Due to their low abundance in plant tissues at a simultaneously high demand, modes of production alternative to conventional extraction are mandatory. Plant cell fermentation might become one of these alternatives, yet decades of research have shown limited success to certain product classes, leading to the question: how to preserve the intrinsic ability to produce TIAs (metabolic competence) in cell culture? We used the strategy to use the developmental potency of mature embryos to generate such strains. Two cell strains (C1and C4) from seed embryos of Catharanthus roseus were found to differ not only morphologically, but also in their metabolic competence. This differential competence became manifest not only under phytohormone elicitation, but also upon feeding with alkaloid pathway precursors. The more active strain C4 formed larger cell aggregates and was endowed with longer mitochondria. These cellular features were accompanied by higher alkaloid accumulation in response to methyl jasmonate (MeJA) elicitation. The levels of catharanthine could be increased significantly, while the concurrent vindoline branch of the pathway was blocked, such that no bisindole alkaloids were detectable. By feeding vindoline to MeJA-elicited C4 cells, vincristine became detectable; however, only to marginal amounts. In conclusion, these results show that cultured cells are not "de-differentiated", but can differ in metabolic competence. In addition to elicitation and precursor feeding, the cellular properties of the "biomatter" are highly relevant for the success of plant cell fermentation.

Single-cell RNA sequencing provides a high-resolution roadmap for understanding the multicellular compartmentation of specialized metabolism.

Monoterpenoid indole alkaloids (MIAs) are among the most diverse specialized metabolites in plants and are of great pharmaceutical importance. We leveraged single-cell transcriptomics to explore the spatial organization of MIA metabolism in Catharanthus roseus leaves, and the transcripts of 20 MIA genes were first localized, updating the model of MIA biosynthesis. The MIA pathway was partitioned into three cell types, consistent with the results from RNA in situ hybridization experiments. Several candidate transporters were predicted to be essential players shuttling MIA intermediates between inter- and intracellular compartments, supplying potential targets to increase the overall yields of desirable MIAs in native plants or heterologous hosts through metabolic engineering and synthetic biology. This work provides not only a universal roadmap for elucidating the spatiotemporal distribution of biological processes at single-cell resolution, but also abundant cellular and genetic resources for further investigation of the higher-order organization of MIA biosynthesis, transport and storage.

De Novo transcriptome assembly and differential expression analysis of catharanthus roseus in response to salicylic acid.

The anti-cancer vinblastine and vincristine alkaloids can only be naturally found in periwinkle (Catharanthus roseus). Both of these alkaloids' accumulations are known to be influenced by salicylic acid (SA). The transcriptome data to reveal the induction effect (s) of SA, however, seem restricted at this time. In this study, the de novo approach of transcriptome assembly was performed on the RNA-Sequencing (RNA-Seq) data in C. roseus. The outcome demonstrated that SA treatment boosted the expression of all the genes in the Terpenoid Indole Alkaloids (TIAs) pathway that produces the vinblastine and vincristine alkaloids. These outcomes supported the time-course measurements of vincristine alkaloid, the end product of the TIAs pathway, and demonstrated that SA spray had a positive impact on transcription and alkaloid synthesis. Additionally, the abundance of transcription factor families including bHLH, C3H, C2H2, MYB, MYB-related, AP2/ ERF, NAC, bZIP, and WRKY suggests a role for a variety of transcription families in response to the SA stimuli. Di-nucleotide and tri-nucleotide SSRs were the most prevalent SSR markers in microsatellite analyses, making up 39% and 34% of all SSR markers, respectively, out of the 77,192 total SSRs discovered.

Effect of the plant probiotic bacteria on terpenoid indole alkaloid biosynthesis pathway gene expression profiling, vinblastine and vincristine content in the root of Catharanthus roseus.

BACKGROUND: Catharanthus roseus is the sole resource of vinblastine and vincristine, two TIAs of great interest for their powerful anticancer activities. Increasing the concentration of these alkaloids in various organs of the plant is one of the important goals in C. roseus breeding programs. Plant probiotic bacteria (PBB) act as biotic elicitors and can induce the synthesis of secondary products in plants. The purpose of this research is to study the effects of PBB on expression of the TIA biosynthetic pathway genes and the content of alkaloids in C. roseus. METHODS AND RESULTS: The individual and combined effects of P. fluorescens strains 169 and A. brasilense strains Ab-101 was studied for expression of the TIA biosynthetic pathway genes (G10H, DAT, T16H and CrPRX) using qRT-PCR and the content of vinblastine and vincristine using HPLC method in roots of C. roseus. P. fluorescens. This drastically increased the content of vinblastine and vincristine alkaloids, compared to the control in the roots, to 174 and 589 (µg/g), respectively. Molecular analysis showed bacterium significantly increased the expression of more genes in the TIA biosynthetic pathway compared to the control. P. fluorescens increased the expression of the final gene of the biosynthetic pathway (CrPRX) 47.9 times compared to the control. Our findings indicate the correlation between transcriptional and metabolic outcomes. The same was true for A. brasilense. CONCLUSIONS: It can be concluded that seed treatments and seedling root treatments composed of naturally occurring probiotic bacteria are likely to be widely applicable for inducing enhanced alkaloid contents in medicinal plants.

An epigenetic modifier enhances the generation of anti-phytopathogenic compounds from the endophytic fungus Chaetomium globosporum of Euphorbia humifusa.

Endophytic fungi are striking resources rich in bioactive structures with agrochemical significance. In order to maximize the opportunity of search for bioactive compounds, chemical epigenetic manipulation was introduced to enhance the structural diversity of the fungal products, and an UPLC-ESIMS and bioassay-guided separation was used to detect novel bioactive metabolites. Consequently, four previously undescribed compounds including two cyclopentenones (globosporins A and B) and two monoterpenoid indole alkaloids (globosporines C and D), as well as three known compounds, were isolated from the endophytic fungus Chaetomium globosporum of Euphorbia humifusa by exposure to a DNA methyltransferase inhibitor 5-azacytidine. Their structures including the absolute configurations were elucidated by the analysis of NMR spectroscopic data, HRESIMS, and TD-DFT-ECD calculations. The indole alkaloids (globosporines C and D) showed antimicrobial activities against three phytopathogenic microbes (Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, and Pseudomonas syringae pv. lachrymans) with MICs in the range of 14-72 µg/mL. Mostly, globosporine D was proved to be potently anti-phytopathogenic against X. oryzae pv. oryzae in vitro and in vivo, which suggested that it has the potential to be developed as a candidate for the prevention of rice bacterial leaf blight. This work provides an efficient and environmentally friendly approach for expanding fungal products with agricultural importance.

A lesion-mimic mutant of Catharanthus roseus accumulates the opioid agonist, akuammicine.

Catharanthus roseus is a medicinal plant that produces an abundance of monoterpenoid indole alkaloids (MIAs), notably including the anticancer compounds vinblastine and vincristine. While the canonical pathway leading to these drugs has been resolved, the regulatory and catalytic mechanisms controlling many lateral branches of MIA biosynthesis remain largely unknown. Here, we describe an ethyl methanesulfonate (EMS) C. roseus mutant (M2-117523) that accumulates high levels of MIAs. The mutant exhibited stunted growth, partially chlorotic leaves, with deficiencies in chlorophyll biosynthesis, and a lesion-mimic phenotype. The lesions were sporadic and spontaneous, appearing after the first true bifoliate and continuing throughout development. The lesions are also the site of high concentrations of akuammicine, a minor constituent of wild type C. roseus leaves. In addition to akuammicine, the lesions were enriched in 25 other MIAs, resulting, in part, from a higher metabolic flux through the pathway. The unique metabolic shift was associated with significant upregulation of biosynthetic and regulatory genes involved in the MIA pathway, including the transcription factors WRKY1, CrMYC2, and ORCA2, and the biosynthetic genes STR, GO, and Redox1. Following the lesion-mimic mutant (LMM) phenotype, the accumulation of akuammicine is jasmonate (JA)-inducible, suggesting a role in plant defence response. Akuammicine is medicinally significant, as a weak opioid agonist, with a preference for the κ-opioid receptor, and a potential anti-diabetic. Further study of akuammicine biosynthesis and regulation can guide plant and heterologous engineering for medicinal uses.

Metabolism of Speciociliatine, an Overlooked Kratom Alkaloid for its Potential Pharmacological Effects.

Speciociliatine, a diastereomer of mitragynine, is an indole-based alkaloid found in kratom (Mitragyna speciosa). Kratom has been widely used for the mitigation of pain and opioid dependence, as a mood enhancer, and/or as an energy booster. Speciociliatine is a partial µ-opioid agonist with a 3-fold higher binding affinity than mitragynine. Speciociliatine has been found to be a major circulating alkaloid in humans following oral administration of a kratom product. In this report, we have characterized the metabolism of speciociliatine in human and preclinical species (mouse, rat, dog, and cynomolgus monkey) liver microsomes and hepatocytes. Speciociliatine metabolized rapidly in monkey, rat, and mouse hepatocytes (in vitro half-life was 6.6 ± 0.2, 8.3 ± 1.1, 11.2 ± 0.7 min, respectively), while a slower metabolism was observed in human and dog hepatocytes (91.7 ± 12.8 and > 120 min, respectively). Speciociliatine underwent extensive metabolism, primarily through monooxidation and O-demethylation metabolic pathways in liver microsomes and hepatocytes across species. No human-specific or disproportionate metabolites of speciociliatine were found in human liver microsomes. The metabolism of speciociliatine was predominantly mediated by CYP3A4 with minor contributions by CYP2D6.

Biotechnological interventions and indole alkaloid production in Rauvolfia serpentina.

Rauvolfia serpentina (L). Benth. ex Kurz. (Apocynaceae), commonly known as Sarpagandha or Indian snakeroot, has long been used in the traditional treatment of snakebites, hypertension, and mental illness. The plant is known to produce an array of indole alkaloids such as reserpine, ajmaline, amalicine, etc. which show immense pharmacological and biomedical significance. However, owing to its poor seed viability, lesser germination rate and overexploitation for several decades for its commercially important bioactive constituents, the plant has become endangered in its natural habitat. The present review comprehensively encompasses the various biotechnological tools employed in this endangered Ayurvedic plant for its in vitro propagation, role of plant growth regulators and additives in direct and indirect regeneration, somatic embryogenesis and synthetic seed production, secondary metabolite production in vitro, and assessment of clonal fidelity using molecular markers and genetic transformation. In addition, elicitation and other methods of optimization of its indole-alkaloids are also described herewith. KEY POINTS: • Latest literature on in vitro propagation of Rauvolfia serpentina • Biotechnological production and optimization of indole alkaloids • Clonal fidelity and transgenic studies in R. serpentina.

Molecular and biochemical characterization of Catharanthus roseus perivine-Nß-methyltransferase.

The biosynthesis of monoterpenoid indole alkaloids in Catharanthus roseus has been most extensively studied, leading to the detailed characterization of the pathway for the formation of their well-known anticancer alkaloids. The present study describes the identification, molecular cloning, and functional expression of C. roseus perivine-Nß-methyltransferase (PeNMT) that converts perivine to Nß-methylperivine (vobasine). PeNMT is member of a recently discovered γ-tocopherol-like N-methyltransferase (γ-TLMT) gene family that displays high substrate specificity and that appears to have evolved in the Vinceae tribe of Apocynaceae family where most N-methylated MIAs have been identified in the phytochemical literature.