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The dose-dependent consumptive effect of kratom and its potential application as an alternative source of medicine to mitigate opioid withdrawal symptoms has brought considerable attention to this plant. Increased interest in the application and use of kratom has emerged globally, including North America. Although the chemistry and pharmacology of major kratom alkaloids, mitragynine and 7-hydroxymitragynine, are well documented, foundational information on the impact of plant production environment on growth and kratom alkaloids synthesis is unavailable. To directly address this need, kratom plant growth, leaf chlorophyll content, and alkaloid concentration were evaluated under three lighting conditions: outdoor full sun, greenhouse unshaded, and greenhouse shaded. Nine kratom alkaloids were quantified using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Contents of six alkaloids to include: mitragynine, speciogynine, speciociliatine, mitraphylline, coynantheidine, and isocorynantheidine were not significantly impacted by lighting conditions, whereas 7-hydroxymitragynine was below the lower limit of quantification across all treatments. However, paynantheine concentration per leaf dry mass was increased by 40% and corynoxine was increased by 111% when grown under shade conditions in a greenhouse compared to outdoor full sun. Additionally, total alkaloid yield per plant was maximized when plants were under such conditions. Greenhouse cultivation generally promoted height and width extension, leaf number, leaf area, average leaf size, and total leaf dry mass, compared to outdoor full sun condition. Rapid, non-destructive chlorophyll evaluation correlated well (r2 = 0.68) with extracted chlorophyll concentrations. Given these findings, production efforts where low-light conditions can be implemented are likely to maximize plant biomass and total leaf alkaloid production.

Kratom (Mitragyna speciosa) is a psychoactive plant popular in the United States for the self-treatment of pain and opioid addiction. For standardization and quality control of raw and commercial kratom products, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of ten key alkaloids, namely: corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine, mitragynine, mitraphylline, paynantheine, speciociliatine, and speciogynine. Chromatographic separation of diastereomers, or alkaloids sharing same ion transitions, was achieved on an Acquity BEH C18 column with a gradient elution using a mobile phase containing acetonitrile and aqueous ammonium acetate buffer (10mM, pH 3.5). The developed method was linear over a concentration range of 1-200 ng/mL for each alkaloid. The total analysis time per sample was 22.5 minutes. The analytical method was validated for accuracy, precision, robustness, and stability. After successful validation, the method was applied for the quantification of kratom alkaloids in alkaloid-rich fractions, ethanolic extracts, lyophilized teas, and commercial products. Mitragynine (0.7%-38.7% w/w), paynantheine (0.3%-12.8% w/w), speciociliatine (0.4%-12.3% w/w), and speciogynine (0.1%-5.3% w/w) were the major alkaloids in the analyzed kratom products/extracts. Minor kratom alkaloids (corynantheidine, corynoxine, corynoxine B, 7-hydroxymitragynine, isocorynantheidine) were also quantified (0.01%-2.8% w/w) in the analyzed products; however mitraphylline was below the lower limit of quantification in all analyses.

Kratom, Mitragyna speciosa Korth., is being widely consumed in the United States for pain management and the reduction of opioid withdrawal symptoms. The central nervous system (CNS) active alkaloids of kratom, including mitragynine, 7-hydroxymitragynine, and numerous additional compounds, are believed to derive their effects through opioid receptor activity. There is no literature describing the systemic exposure of many of these alkaloids after the consumption of kratom. Therefore, we have developed and validated a bioanalytical method for the simultaneous quantitation of 11 kratom alkaloids (mitragynine, 7-hydroxymitragynine, corynantheidine, speciogynine, speciociliatine, paynantheine, corynoxine, corynoxine-B, mitraphylline, ajmalicine, and isospeciofoline) in rat plasma. The validated method was used to analyze oral pharmacokinetic study samples of lyophilized kratom tea (LKT) and a marketed product, OPMS liquid shot, in rats. Among the 11 alkaloids, only mitragynine, 7-hydroxymitragynine, speciociliatine, and corynantheidine showed systemic exposure 8 h postdose, and the dose-normalized systemic exposure of these four alkaloids was higher (1.6-2.4-fold) following the administration of the commercial OPMS liquid. Paynantheine and speciogynine levels were quantifiable up to 1 h postdose, whereas none of the other alkaloids were detected. In summary, the method was successfully applied to quantify the exposure of individual kratom alkaloids after an oral dose of traditional or commercial products. This information will contribute to understanding the role of each alkaloid in the overall pharmacology of kratom and elucidating the pharmacokinetic differences between traditional and commercial kratom products.

Leaves harvested from the Southeast Asian tree Mitragyna speciosa (kratom) have a history of use as a traditional ethnobotanical source of medicine to combat fatigue, improve work productivity, and to reduce opioid-related withdrawal symptoms. Kratom leaves contain an array of alkaloids thought to be responsible for the bioactivity reported by users. Interest in the consumptive effects of kratom has led to its recent popularity and use in North America, Western Europe, and Australia. Although the chemistry and pharmacology of select kratom alkaloids are understood, studies have not examined the influence of production environment on growth and alkaloidal content. To directly address this need, 68 kratom trees were vegetatively propagated from a single mother stock to reduce genetic variability and subjected to four varying fertilizer application rates. Leaves were analyzed for chlorophyll concentration, biomass, and alkaloidal content to understand the physiological response of the plant. While increasing rates of fertilizer promoted greater plant growth, relationships with alkaloidal content within leaves were highly variable. Fertility rate had little influence on the concentration of mitragynine, paynantheine, speciociliatine, mitraphylline, and corynoxine per leaf dry mass. 7-Hydroxymitragynine was below the lower limit of quantification in all the analyzed leaf samples. Low to medium rates of fertilizer, however, maximized concentrations of speciogynine, corynantheidine, and isocorynantheidine per leaf dry mass, suggesting a promotion of nitrogen allocation for secondary metabolism occurred for these select alkaloids. Strong correlations (r2 = 0.86) between extracted leaf chlorophyll and rapid, non-destructive chlorophyll evaluation (SPAD) response allowed for development of a reliable linear model that can be used to diagnose nutrient deficiencies and allow for timely adjustment of fertilization programs to more accurately manage kratom cultivation efforts. Results from this study provide a greater understanding of the concentration and synthesis of nine bioactive alkaloids in fresh kratom leaves and provide foundational information for kratom cultivation and production.

The purified oxindole alkaloids, isomitraphylline and mitraphylline from Uncaria perrottetii, revealed their ability to break amyloid aggregates in vitro suggesting their therapeutic potentials in Alzheimer’s disease (AD). Thioflavin-T assay for assessing amyloid-beta (Aβ) aggregation of these alkaloids exhibited inhibitions at 60.321% ± 2.61 (50 μM) for isomitraphylline and 43.17% ± 3.48 (50 μM) for mitraphylline. Neuroprotective effects were elaborated against Aβ-induced SH-SY5Y cells at 20 μM and 10 μM for isomitraphylline, and 20 μM for mitraphylline. In addition, both alkaloids attenuated and protected the H(2)O(2)-induced SH-SY5Y cell cytotoxicity at 20 μM. The intracellular ROS levels of SH-SY5Y cells from H(2)O(2)-induced oxidative stress were reduced at 20 μM and 10 μM, and the mitochondrial membrane potentials of Aβ-induced SH-SY5Y cells were protected at 20 μM. The overall results suggested the potentials of both alkaloids to target certain pathological biomarkers of AD and could be further investigated as therapeutic or preventive drug leads against AD. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02535-4) contains supplementary material, which is available to authorized users.

Phytochemical investigation on the aerial parts of Uncaria perrottetii led to the isolation of three pentacyclic oxindole alkaloids uncarine A or isoformosanine (1), uncarine E or isopteropodine (2), and rauniticine-allo-oxindole A (3). Five oxindole alkaloids, isomitraphylline (4), mitraphylline (5), uncarine B or formosanine (6), uncarine F (7), corynoxine (8), and uncarine D or speciophylline (9), were isolated from the leaves of Uncaria lanosa f. philippinensis. Their structures were determined by spectroscopic techniques and in comparison with the literature data. These compounds proved to be important chemotaxonomic markers in the genus Uncaria. Keywords: Rubiaceae, Uncaria, Oxindole alkaloids, Chemotaxonomy, Uncarine

Spiro-oxindole scaffolds have been studied due to their promising therapeutic potential. In the Amazon rainforest there are two important Uncaria species known as “cat’s claw”, which biosynthesize spirocyclic oxindole alkaloids; Uncaria tomentosa (Willd. ex Schult.) DC. and Uncaria guianensis (Aublet) Gmell. We carried out a precursor-directed biosynthesis approach with U. guianensis and successfully obtained oxindole alkaloid analogues with molecular mass corresponding to the addition of a methyl or fluorine group on the oxindole ring using tryptamine analogue precursors. Two of these novel oxindole alkaloid analogues (3b-7-methyl-isomitraphylline and 3c-6-fluoro-isomitraphylline) were isolated and characterized by NMR spectroscopy and ESI-QTOF-MS/MS. Having established a substrate feeding protocol for these plantlets, the biosynthetic route for mitraphylline (1), rhynchophylline (2), isomitraphylline (3) and isorhynchophylline (4) was also investigated using 13C-precursors (1-13C-D-glucose, 2-13C-tryptophan, 1-13C-DL-glyceraldehyde, and methyl-13C-D-methionine).

The usual quality control for Uncaria tomentosa (Willd. ex Schult.) DC. barks requires highly specific analytical standards and methods based on high-performance liquid chromatography (HPLC), which impacts the costs of the analytical process and the final products.To obtain an analytical reference standard of mitraphylline by isolation from U. tomentosa barks and develop a spectrophotometric method for determination of total alkaloids in samples of U. tomentosa.An alkaloid-enriched extract was obtained by acid-base partition and mitraphylline was selectively precipitated using an 80:20 v/v toluene/hexane solution. The compound was characterised by HPLC-UV/DAD (diode-array detector), mass spectrometry, UV-visible, infrared (IR) andMitraphylline with a purity of 98% was isolated in 0.05% m/m yield. All characterisation results were in agreement with previous published data. The spectrophotometric method showed linear range between 0.40 and 20 μg/mL; limits of detection and quantification of 0.15 and 0.49 μg/mg, respectively; dispersion of results lower than 5% for repeatability and intermediate precision; statistically proven accuracy by comparison with reference values obtained by Soxhlet and an HPLC-UV/DAD method; and robustness in relation to sample mass extracted and extraction time.The methods developed to obtain mitraphylline analytical standard from U. tomentosa barks and to determine total alkaloids by spectrophotometry provided a cheaper and faster quality control alternative for U. tomentosa samples.

International audience; Ethnopharmacological relevance Uncaria tomentosa (Willd. Ex Schult) DC is used by indigenous tribes in the Amazonian region of Central and South America to treat inflammation, allergies and asthma. The therapeutic properties of U. tomentosa have been attributed to the presence of tetracyclic and pentacyclic oxindole alkaloids and to phenolic acids. Aims of the study To characterize aqueous bark extracts (ABE) and aqueous leaf extracts (ALE) of U. tomentosa and to compare their anti-inflammatory effects. Materials and methods Constituents of the extracts were identified by ultra performance liquid chromatography-mass spectrometry. Anti-inflammatory activities were assessed in vitro by exposing lipopolysaccharide-stimulated macrophage cells (RAW264.7-Luc) to ABE, ALE and standard mitraphylline. In vivo assays were performed using a murine model of ovalbumin (OVA)-induced asthma. OVA-sensitized animals were treated with ABE or ALE while controls received dexamethasone or saline solution. Bronchial hyperresponsiveness, production of Th1 and Th2 cytokines, total and differential counts of inflammatory cells in the bronchoalveolar lavage (BAL) and lung tissue were determined. Results Mitraphylline, isomitraphylline, chlorogenic acid and quinic acid were detected in both extracts, while isorhyncophylline and rutin were detected only in ALE. ABE, ALE and mitraphylline inhibited the transcription of nuclear factor kappa-B in cell cultures, ALE and mitraphylline reduced the production of interleukin (IL)−6, and mitraphylline reduced production of tumor necrosis factor-alpha. Treatment with ABE and ALE at 50 and 200 mg kg−1, respectively, reduced respiratory elastance and tissue damping and elastance. ABE and ALE reduced the number of eosinophils in BAL, while ALE at 200 mg kg−1 reduced the levels of IL-4 and IL-5 in the lung homogenate. Peribronchial inflammation was significantly reduced by treatment with ABE and ALE at 50 and 100 mg kg−1 respectively. Conclusion The results clarify for the first time the anti-inflammatory activity of U. tomentosa in a murine model of asthma. Although ABE and ALE exhibited distinct chemical compositions, both extracts inhibited the production of pro-inflammatory cytokines in vitro. In vivo assays revealed that ABE was more effective in treating asthmatic inflammation while ALE was more successful in controlling respiratory mechanics. Both extracts may have promising applications in the phytotherapy of allergic asthma.

Persistent inhalation of mitragynine (MG), a major alkaloid in the leaves of Mitragyna speciosa, causes various systemic adverse effects such as seizure, diarrhea and arthralgias, but its toxicity to endothelial cells and effects on barrier function of the cells are poorly understood. In this study, we compared toxicities of MG and mitraphylline, another constituent of the leaves, against human aortic endothelial (HAE), bronchial BEAS-2B, neuronal SK-N-SH, hepatic HepG2, kidney HEK293, gastric MKN45, colon DLD1, lung A549, breast MCF7 and prostate LNCaP cells, and found that MG, but not mitraphylline, shows higher toxicity to HAE cells compared to the other cells. Forty-eight-hours incubation of HAE cells with a high concentration of MG (60 µM) provoked apoptotic cell death, which was probably due to signaling through enhanced reactive oxygen species (ROS) generation and resultant caspase activation. Treatment of the cells with MG at sublethal concentrations less than 20 µM significantly lowered transendothelial electrical resistance and elevated paracellular permeability, without affecting the cell viability. In addition, the MG-elicited lowering of the resistance was abolished by a ROS inhibitor N-acetyl-L-cysteine and augmented by H2O2 and 9,10-phenanthrenequinone, which generates ROS through its redox cycle. These results suggest the contribution of ROS generation to the increase in endothelial barrier permeability.

The leaves of the South Asian plant kratom are described as having stimulating effects at low doses, and opiate-like analgesic and euphoric effects at high doses. A long history of use and abuse has led to the classification of kratom as a controlled substance in its native Thailand and other South Asian countries. However, kratom is not controlled in the United States, and the ready availability of kratom has led to its emergence as an herbal drug of abuse. With the growing popularity of kratom, efficient procedures are needed to detect kratom use. In the current study, both ultra-high-performance liquid chromatography and high-performance liquid chromatography-tandem mass spectrometry methods have been developed and validated for monitoring the major alkaloids and metabolites found in urine following kratom use. The primary unique alkaloid mitragynine is quantified in human urine from 1.00-500.00 ng/mL using mitraphylline as an internal standard. In addition, two metabolites (5-desmethylmitragynine and 17-desmethyldihydromitragynine) and the related active, alkaloid 7-hydroxy-mitragynine, are simultaneously qualitatively monitored. The presence of analytes are confirmed by an information-dependent acquisition-enhanced product ion procedure generating full fragmentation data used to positively identify detected analytes. The validated method has been utilized for clinical and forensic analyses of urine for the detection of kratom use.

Introduction Uncaria tomentosa (“cat's claw”) is widely used for the treatment of some infectious and inflammatory diseases. Oxindole alkaloids are regarded as the most important components responsible for the biological activities attributed to the plant. Their analysis require efficient sample preparation and suitable reference standards but few are commercially available. Objective To develop and validate a HPLC analytical method for oxindole alkaloids in Uncaria tomentosa with emphasis on sample preparation. Method Factorial experimental designs were used for the optimisation of both sample preparation and chromatographic separation. The optimised sample preparation involved extraction with aqueous ethanol, and the granulometry of the powdered plant material significantly influenced extraction yields. Mitraphylline was used as a calibration reference for the determination of total alkaloids. Results The method was fully validated and showed good selectivity, linearity (r2 ≥ 0.9996), accuracy (≥ 96%) and precision (RSD

Preparations from Uncaria tomentosa, a South American Rubiaceae, have been used in the Peru- vian traditional medicine for the treatment of in- fective, inflammatory and tumoral processes. In this study, the pentacyclic oxindole alkaloid mi- traphylline was isolated from the dried inner bark of this plant species, and its structure elucidated by analysis of NMR spectroscopic data. Mitraphyl- line was differentially identified from its stereoisomeric pair isomitraphylline by 15N‑NMR. Its antiproliferative and cytotoxic effects have been tested on human Ewingʼs sarcoma MHH‑ES‑1 and breast cancer MT-3 cell lines, using cyclo- phosphamide and vincristine as reference con- trols. A Coulter counter was used to determine viable cell numbers, followed by the application of the tetrazolium compound MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)- 2-(4-sulfophenyl)-2H-tetrazolium] an inner salt. A colorimetric method was employed to evaluate cell viability in this cytotoxic assay. Micromolar concentrations of mitraphylline (5 µM to 40 µM) inhibited the growth of both cell lines in a dose- dependent manner. The IC50 ± SE values were 17.15 ± 0.82 µM for MHH‑ES‑1 and 11.80 ± 1.03 µM for MT-3 for 30 hours, smaller than those obtained for the reference compounds. This ac- tion suggests that the pentacyclic oxindole alka- loid mitraphylline might be a new promising agent in the treatment of both human sarcoma and breast cancer.

Oxindole alkaloids, isolated from the bark of Uncaria tomentosa [Willd. ex Schult.] Rubiaceae, are considered to be responsible for the biological activity of this herb. Five pentacyclic and two tetracyclic alkaloids were studied by solid-state NMR and theoretical GIAO DFT methods. The (13)C and (15)N CPMAS NMR spectra were recorded for mitraphylline, isomitraphylline, pteropodine (uncarine C), isopteropodine (uncarine E), speciophylline (uncarine D), rhynchophylline and isorhynchophylline. Theoretical GIAO DFT calculations of shielding constants provide arguments for identification of asymmetric centers and proper assignment of NMR spectra. These alkaloids are 7R/7S and 20R/20S stereoisomeric pairs. Based on the (13)C CP MAS chemical shifts the 7S alkaloids (delta C3 70-71ppm) can be easily and conveniently distinguished from 7R (deltaC3 74.5-74.9ppm), also 20R (deltaC20 41.3-41.7ppm) from the 20S (deltaC20 36.3-38.3ppm). The epiallo-type isomer (3R, 20S) of speciophylline is characterized by a larger (15)N MAS chemical shift of N4 (64.6ppm) than the allo-type (3S, 20S) of isopteropodine (deltaN4 53.3ppm). (15)N MAS chemical shifts of N1-H in pentacyclic alkaloids are within 131.9-140.4ppm.

Uncaria tomentosa inner bark extract is a popular plant remedy used in folk medicine to treat tumor and inflammatory processes. In this study, the anti-tumoral effects of its pentacyclic alkaloid mitraphylline were investigated. Furthermore, its growth-inhibitory and cytotoxic effects on glioma GAMG and neuroblastoma SKN-BE(2) cell lines were studied using cyclophosphamide and vincristine as controls. A colter counter was used to determine viable cell numbers, followed by application of the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium], inner salt, colorimetric method to evaluate cell viability in this cytotoxicity assay. Micromolar concentrations of mitraphylline (from 5 to 40 microM) inhibited the growth of both cell lines. It inhibited the growth of the two cell lines studied in a dose-dependent manner. The IC(50) values were 12.3 microM (30h) for SKN-BE(2) and 20 microM (48 h) for GAMG, respectively. This action suggests that mitraphylline is a new and promising agent in the treatment of human neuroblastoma and glioma.

In metabolomics, the analysis of product ions in tandem mass spectrometry (MS/MS) is noteworthy to chemically assign structural information. However, the development of relevant analytical methods are less advanced. Here, we developed a method to boost sensitivity in liquid chromatography-Fourier transform ion cyclotron resonance-tandem mass spectrometry analysis (MS/MS boost analysis). To verify the MS/MS boost analysis, both quercetin and uniformly labeled (13)C quercetin were analyzed, revealing that the origin of the product ions is not the instrument, but the analyzed compounds resulting in sensitive product ions. Next, we applied this method to the analysis of monoterpene indole alkaloids (MIAs). The comparative analyses of MIAs having indole basic skeleton (ajmalicine, catharanthine, hirsuteine, and hirsutine) and oxindole skeleton (formosanine, isoformosanine, pteropodine, isopteropodine, rhynchophylline, isorhynchophylline, and mitraphylline) identified 86 and 73 common monoisotopic ions, respectively. The comparative analyses of the three pairs of stereoisomers showed more than 170 common monoisotopic ions in each pair. This method was also applied to the targeted analysis of MIAs in Catharanthus roseus and Uncaria rhynchophylla to profile indole and oxindole compounds using the product ions. This analysis is suitable for chemically assigning features of the metabolite groups, which contributes to targeted metabolome analysis.

© 2015 Elsevier Ireland Ltd. All rights reserved. Ethnopharmacological relevance Uncaria tomentosa (Willdenow ex Roemer & Schultes) DC. (Rubiaceae) is a Peruvian thorny liana, commonly known as >cat's claw>, and traditionally used in folk medicine to deal with several inflammatory diseases. Mitraphylline (MTP) is the most abundant pentacyclic oxindolic alkaloid (POA) from U. Tomentosa and has been reported to modify the inflammatory response. Herein, we have sought to identify the mechanisms underlying this modulatory effect of MTP on primary human monocytes and its ability to regulate differentiation processes on human primary monocyte and monocyte-derived macrophages. Material and methods In vitro studies with human primary monocytes and monocyte-derived macrophages were performed. Monocytes and M0 macrophages were exposed to MTP (25 μM) and LPS (100 ng/mL). M0 macrophages were polarized to M1 and M2 phenotypes in the absence or presence of MTP. The activation state of monocytes/macrophages was assessed by flow cytometry, gene expression and protein analysis of different specific markers. Results In human primary monocytes, the incubation of MTP for 24 h reduced the number of classical (CD14++CD16-) and intermediate (CD14++CD16+) subsets when compared to untreated or LPS-treated cells. MTP also reduced the chemotactic capacity of human primary monocytes. In addition, MTP promoted the polarization of M0 macrophages toward an anti-inflammatory M2 phenotype, the abrogation of the release of pro-inflammatory cytokines such as TNFα, IL-6 or IL-1β, as well as the restoration of markers for M2 macrophages in LPS-treated M1 macrophages. Conclusions Our results suggest that MTP may be a key modulator for regulating the plasticity of monocytes/macrophages and the attenuation of the inflammatory response., This work was supported by the University of Seville, “V Own Research Plan” contract to BB and QA. MS has the benefit of a FPI fellowship (BES-2012–056104) of MICINN.

Natural products are still an untapped source of promising lead compounds for the generation of antineoplastic drugs. Here, we investigated for the first time the antiproliferative and apoptotic effects of highly purified oxindole alkaloids, namely isopteropodine (A1), pteropodine (A2), isomitraphylline (A3), uncarine F (A4) and mitraphylline (A5) obtained from Uncaria tomentosa, a South American Rubiaceae, on human lymphoblastic leukaemia T cells (CCRF-CEM-C7H2). Four of the five tested alkaloids inhibited proliferation of acute lymphoblastic leukaemia cells. Furthermore, the antiproliferative effect of the most potent alkaloids pteropodine (A2) and uncarine F (A4) correlated with induction of apoptosis. After 48 h, 100 micromol/l A2 or A4 increased apoptotic cells by 57%. CEM-C7H2 sublines with tetracycline-regulated expression of bcl-2, p16ink4A or constitutively expressing the cowpox virus protein crm-A were used for further studies of the apoptosis-inducing properties of these alkaloids. Neither overexpression of bcl-2 or crm-A nor cell-cycle arrest in G0/G1 phase by tetracycline-regulated expression of p16INK4A could prevent alkaloid-induced apoptosis. Our results show the strong apoptotic effects of pteropodine and uncarine F on acute leukaemic lymphoblasts and recommend the alkaloids for further studies in xenograft models.

The Uncaria genus is an important source of medicinal natural products, particularly alkaloids and triterpenes. The collected information is an attempt to cover the more recent developments in the ethnobotany, pharmacology and phytochemistry of this genus. During the past 20 years, alkaloids, terpenes, quinovic acid glycosides, flavonoids and coumarins have been isolated from Uncaria. Fifty-three novel structures are reported in this review. The species in which the largest number of compounds has been identified is the Peruvian Uncaria tomentosa or 'cat's claw.' Pharmacological studies are described according to cytotoxicity, anti-inflammatory, antiviral, immunostimulation, antioxidant, CNS-related response, vascular, hypotensive, mutagenicity and antibacterial properties. The potential for development of leads from Uncaria continues to grow, particularly in the area of immunomodulatory, anti-inflammatory and vascular-related conditions. The information summarized here is intended to serve as a reference tool to practitioners in the fields of ethnopharmacology and natural products chemistry.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:48:33Z No. of bitstreams: 1 S0100-40422004000600007.pdf: 221665 bytes, checksum: c447ca6529b7c3940429d633e2f49c75 (MD5) Made available in DSpace on 2013-08-22T18:48:33Z (GMT). No. of bitstreams: 1 S0100-40422004000600007.pdf: 221665 bytes, checksum: c447ca6529b7c3940429d633e2f49c75 (MD5) Previous issue date: 2004-12-01 Made available in DSpace on 2013-09-30T19:38:31Z (GMT). No. of bitstreams: 2 S0100-40422004000600007.pdf: 221665 bytes, checksum: c447ca6529b7c3940429d633e2f49c75 (MD5) S0100-40422004000600007.pdf.txt: 21180 bytes, checksum: 716ba7827e843aeb26f1337177d0921f (MD5) Previous issue date: 2004-12-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:21:04Z No. of bitstreams: 2 S0100-40422004000600007.pdf: 221665 bytes, checksum: c447ca6529b7c3940429d633e2f49c75 (MD5) S0100-40422004000600007.pdf.txt: 21180 bytes, checksum: 716ba7827e843aeb26f1337177d0921f (MD5) Made available in DSpace on 2014-05-20T14:21:04Z (GMT). No. of bitstreams: 2 S0100-40422004000600007.pdf: 221665 bytes, checksum: c447ca6529b7c3940429d633e2f49c75 (MD5) S0100-40422004000600007.pdf.txt: 21180 bytes, checksum: 716ba7827e843aeb26f1337177d0921f (MD5) Previous issue date: 2004-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Phytochemical studies with leaves of Uncaria guianensis resulted in the isolation of the oxindole alkaloids isomitraphylline (1), 3-isoajmalicine (2) mitraphylline (3), and isomitraphylinic acid (4). Structural assignments of these alkaloids, including relative configurations and conformations, were performed through spectral data and physical properties. 1D and 2D homonuclear and heteronuclear NMR spectroscopy was a valuable tool for the establishment of the relative stereochemistry of those compounds. Universidade Estadual Paulista Instituto de Química Universidade Estadual Paulista Instituto de Botânica Seção de Fisiologia e Bioquímica de Plantas Universidade Estadual Paulista Instituto de Química Universidade Estadual Paulista Instituto de Botânica Seção de Fisiologia e Bioquímica de Plantas

The single-crystal X-ray structures of speciophylline, mitraphylline, and rhynchophylline, oxindole alkaloids from the Peruvian climbing vine Uncaria tomentosa (Rubiaceae), were determined. The three compounds show N⋅⋅⋅HN hydrogen bonding, which has not been observed in the crystal structures of the related alkaloids pteropodine and isopteropodine. In the tetracyclic alkaloid rhynchophylline, the side chain is rotated out of the ring plane into a position perpendicular to it. This is in contrast to the situation of the pentacyclic analogue mitraphylline, which possesses a conformationally rigid tricyclic core. This conformational difference possibly causes the competitive antagonism of these two types of alkaloids.

Pteropodine and isopteropodine are heteroyohimbine-type oxindole alkaloid components of Uncaria tomentosa (Willd.) DC, a Peruvian medicinal plant known as cat's claw. In this study, the effects of these alkaloids on the function of Ca(2+)-activated Cl(-) currents evoked by stimulation of G protein-coupled muscarinic M(1) acetylcholine and 5-HT(2) receptors were studied in Xenopus oocytes in which rat cortex total RNA was translated. Pteropodine and isopteropodine (1-30 microM) failed to induce membrane current by themselves. However, these alkaloids markedly enhanced the current responses evoked by both acetylcholine and 5-hydroxyhyptamine (5-HT) in a concentration-dependent and reversible manner with the maximal effects at 30 microM. Pteropodine and isopteropodine produced 2.7- and 3.3-fold increases in the acetylcholine response with EC(50) values of 9.52 and 9.92 microM, respectively, and 2.4- and 2.5-fold increases in the 5-HT response with EC(50) values of 13.5 and 14.5 microM, respectively. In contrast, in oocytes injected with total RNA from the rat cerebellum or spinal cord, neither alkaloid had an effect on the metabotropic current responses mediated by glutamate receptor(1 and 5) (mGlu(1/5)) receptors or ionotropic responses mediated by N-methyl-D-aspartate, kainic acid or glycine. Pteropodine and isopteropodine (10 microM) significantly reduced the EC(50) values of acetylcholine and 5-HT that elicited current responses, but had no effect on the maximal current responses elicited by acetylcholine and 5-HT. On the other hand, mitraphylline, a stereoisomer of pteropodine, failed to modulate acetylcholine- and 5-HT-induced responses. These results suggest that pteropodine and isopteropodine act as positive modulators of muscarinic M(1) and 5-HT(2) receptors.

Mitragyna speciosa (kratom) is a popular herb in Southeast Asia, which is traditionally used to treat withdrawal symptoms associated with opiate addiction. Mitragynine, 7-hydroxymitragynine, and mitraphylline are reported to be the central nervous system active alkaloids which bind to the opiate receptors. Mitraphylline is also present in the bark of Uncaria tomentosa (catʼs claw). Several therapeutic properties have been reported for these compounds but limited information is available on the absorption and distribution properties. This study focuses on evaluating the absorption, distribution, metabolism, and excretion (ADME) properties of these compounds and their effect on major efflux transporter P-glycoprotein, using in vitro methods. Quantitative analysis was performed by the Q-TOF LC-MS system. Mitragynine was unstable in simulated gastric fluid with 26 % degradation but stable in simulated intestinal fluid. 7-Hydroxymitragynine degraded up to 27 % in simulated gastric fluid, which could account for its conversion to mitragynine (23 %), while only 6 % degradation was seen in simulated intestinal fluid. Mitraphylline was stable in simulated gastric fluid but unstable in simulated intestinal fluid (13.6 % degradation). Mitragynine and 7-hydroxymitragynine showed moderate permeability across Caco-2 and MDR-MDCK monolayers with no significant efflux. However, mitraphylline was subjected to efflux mediated by P-glycoprotein in both Caco-2 and MDR-MDCK monolayers. Mitragynine was found to be metabolically stable in both human liver microsomes and S9 fractions. In contrast, both 7-hydroxymitragynine and mitraphylline were metabolized by human liver microsomes with half-lives of 24 and 50 min, respectively. All three compounds exhibited high plasma protein binding (> 90 %) determined by equilibrium dialysis. Mitragynine and 7-hydroxymitragynine inhibited P-glycoprotein with EC50 values of 18.2 ± 3.6 µM and 32.4 ± 1.9 µM, respectively, determined by the calcein-AM fluorescent assay, while no inhibition was seen with mitraphylline. These data indicate the possibility of a drug interaction if mitragynine and 7-hydroxymitragynine are coadministered with drugs that are P-glycoprotein substrates.

The antitumor activity of Uncaria tomentosa, a native vine from the Amazonian rainforest, has been ascribed to pentacyclic oxindole alkaloids occurring in its bark. Former studies have shown that this activity, as well as its intensity, depends on whether catʼs claw alkaloids occur as original compounds or isomerized derivatives. This work addresses this aspect, using T24 and RT4 human bladder cancer cell lines for that purpose. Bark samples were extracted by dynamic maceration, prepurified with cross-linked polyvinylpyrrolidone and properly fractioned by an ion exchange process to obtain an oxindole alkaloid purified fraction. Alkaloid isomerization was induced by heating it under reflux at 85 °C. Samples collected after 5, 15, and 45 min of heating were analyzed by HPLC-PDA, freeze-dried at once, and separately assayed using the non-isomerized purified fraction for comparison purposes. The latter showed significant and dose-dependent cytotoxic activity against both T24 and RT4 cancer cell lines (IC50: 164.13 and 137.23 µg/mL, respectively). However, results for both cell lines were equivalent to those observed for isomerized samples (p > 0.05). The alkaloid isomerization induced by the incubation conditions (buffered medium pH 7.4 and temperature 37 °C) helps to explain the similar results obtained from non-isomerized and isomerized samples. Mitraphylline, speciophylline, uncarine F, and, to a lesser degree, pteropodine were more susceptible to isomerization under the incubation conditions. Thus, the alkaloid profile of all fractions and their cytotoxic activities against T24 and RT4 human bladder cancer cell lines are determined to a large extent by the incubation conditions.

Dissertação de mestrado em Técnicas de Caracterização e Análise Química, Uncaria tomentosa (Willd) DC., conhecida como unha de gato, é uma liana tropical nativa da Amazónia brasileira e peruana. A espécie é reconhecida pelas suas propriedades antiinflamatória, imunoestimulante, antioxidante e antiviral, geralmente atribuídas aos polifenóis e, principalmente aos alcaloides oxindólicos pentacíclicos. Essa espécie enfrenta ultimamente uma diminuição alarmante no seu habitat nativo, devido principalmente à sua colheita indiscriminada, dado o alto grau de comercialização das cascas para uso medicinal. Nesse âmbito, o possível aproveitamento das folhas para fins terapêuticos ganha relevância para a sobrevivência da espécie e a sustentabilidade do mercado. Contudo, as folhas ainda não são suficientemente conhecidas do ponto de vista do conteúdo alcaloídico, e até o momento, não existem estudos sobre seu conteúdo fenólico. Este trabalho teve como objetivo analisar o perfil químico e determinar o teor de alcaloides (quantificados pelo marcador mitrafilina) e polifenóis (quantificados pelo ácido clorogênico) por HPLC em fase reversa, complementado com informações obtidas por TLC, em cinco amostras de folhas de Uncaria tomentosa colhidas em diferentes localidades do Brasil. Foram comparados os produtos da extração das folhas secas com metanol puro e com etanol/água 1:1; obtendo-se para cada um deles, frações enriquecidas em alcaloides e fenóis, mediante procedimentos de partição. A quantificação da mitrafilina e do ácido clorogênico nas amostras foi realizada através do método HPLC-DAD, utilizando retas de calibração externa com seus respetivos padrões. Em ambos os casos, foram avaliados os parâmetros de linearidade, limite de deteção e quantificação e foram efetuados ensaios de recuperação. Três amostras da fração sob partição básica apresentaram conteúdos de mitrafilina entre 0,88-2,15 mg/g de folhas secas. As cinco amostras indicaram, na partição ácida, um conteúdo de ácido clorogênico de 0,14-1,71 mg/g de folhas secas. Uma das amostras apresentou o flavonoide campferitrina como um sinal proeminente no cromatograma, sugerindo que provavelmente esta amostra não provém da U. tomentosa, ainda que as folhas sejam morfologicamente similares. Estes resultados destacam a adequabilidade da técnica HPLC aplicada ao controle de qualidade de matéria-prima vegetal com fins medicinais. A análise por UPLC-DAD-MS corroborou a presença dos marcadores químicos (mitrafilina, ácido clorogênico e campferitrina) nos extratos de U. tomentosa analisados., Uncaria tomentosa (Willd) DC., known as cat´s claw is a vine native to tropical Brazilian and Peruvian Amazon. The species is known for its anti-inflammatory, immunostimulant, antioxidant and antiviral properties, generally attributed to polyphenols, and especially to the pentacyclic oxindole alkaloids. This species is lately facing alarming decrease in its native habitat, mainly due to the indiscriminate harvesting, given the high degree of commercialization of the bark for medicinal use. In this context, the possible use of the leaves for therapeutic use gains relevance for the survival of the species and the sustainability of the market. However, the leaves are not yet sufficiently known from the point of view of alkaloid content, and so far, there are no studies on its phenolic content. This study aimed to analyze the chemical profile and determine the content of alkaloids (quantified by mitraphylline marker) and polyphenols (quantified by chlorogenic acid) by reversed phase HPLC, supplemented with information obtained by TLC in five samples of leaves of Uncaria tomentosa harvested in different Brazilian locations. The products of the extraction of dried leaves with pure methanol and with ethanol / water 1:1 were compared, for each one of them, fractions enriched in alkaloids and phenols by partition procedures were obtained. The quantification of mitraphylline and chlorogenic acid in the samples was performed though the HPLC-DAD method using external calibration curves with their respective standards. In both cases the parameters evaluated were: linearity, limit of detection and quantification and recovery experiments were also performed. Three samples of the fraction under basic partition showed mitraphylline content between 0.88 and 2.15 mg / g of dried leaves. The five samples indicated in the acidic partition a chlorogenic acid content from 0.14 to 1.71 mg / g of dried leaves. One of the samples showed the kaempferitrin flavonoid as a prominent sign on the chromatogram, suggesting that this sample is probably not from U. tomentosa, even if the thorns are morphologically similar. These results highlight the suitability of the HPLC technique applied to quality control of plant material for medicinal purposes. The analysis by UPLC-DAD-MS confirmed the presence of chemical markers (mitraphylline, chlorogenic acid and kaempferitrin) in the extracts of U. tomentosa analyzed.

Uncaria tomentosa has been widely used in several countries as an anticancer remedy, but due to its pharmacological effects this plant has been extensively harvested and faces extinction. Regardless of its traditional anti-tumoral use, root bark water extract was the lesser active extract. Hydroalcoholic preparation of the root bark showed LD50 ranging from 150 to 500 μg/mL and leaf extracts (water and hydroalcoholic) showed LD50 as low as 180.1μg/mL. Selectivity index showed that except for astrocytoma and leukemia, all tumor cells were 2-8 times more sensitive to the extracts than to non tumor cells. Oxindole pentacyclic alkaloids (mitraphylline, isomitraphylline, uncarine C and uncarine E) are present in the extracts and they probably play a role in the cytotoxic effect on tumor cell lines. Our study suggets that Uncaria tomentosa roots could be replaced by the leaves in order to obtain good pharmacological results, protecting the plant from extinction.

The isomerization of the spiro oxindole alkaloids mitraphylline, isomitraphylline, pteropodine, isopteropodine, speciophylline and uncarine F in water has been studied at several temperatures and the rate coefficients have been determined. The effect of pH on the rate of reaction and the equilibrium composition has been investigated. The rate coefficients in water and in organic solvents correlate satisfactorily with the Dimroth–Reichardt solvent polarity parameter. The present results support the existence of a zwitterionic intermediate stabilized by polar solvents and show that protonation of the alkaloids inhibits the isomerization. The crystal structure of pteropodine has been determined.

The alkaloids of Uncaria tomentosa micropropagated plantlets and root cultures were isolated and identified by NMR and mass spectrometry. Plantlets yielded pteropodine (1), isopteropodine (2), mitraphylline (3), isomitraphylline (4), uncarine F (5), speciophylline (6), rhynchophylline (7) and isorhynchophylline (8). In plantlets growing under continuous light, tetracyclic alkaloids 7 and 8 decreased from 20 ± 1.8 at 2 months to 2.2 ± 0.33 mg/g dry wt at 6 months, while the pentacyclic alkaloids 1–4 increased from 7.7 ± 1.4 to 15 ± 0.05 mg/g dry wt, supporting their biogenetic conversion. Micropropagated plantlets produced four times more alkaloids (27.6 ± 3.1 mg/g dry wt) than greenhouse plants. Plantlet roots yielded 3, 4, 8 and the glucoindole alkaloids 3α-dihydrocadambine (9) and dolichantoside (10), the last one not previously found in Uncaria.

Ethnopharmacological relevance Uncaria tomentosa (Willd. ex Roem. & Schult.) DC. (Rubiaceae) is widely used by populations living in South America to treat many ailments associated with inflammatory disorders. Mitraphylline was shown to be the major pentacyclic oxindolic alkaloid present in the bark chloroformic extract of this plant. Its activity against cytokines involved in inflammation process was tested in a murine model in vivo . Materials and methods Mice received mitraphylline once a day for 3 days at 30 mg/kg/day by oral route. Then, they were subjected to bacterial lipopolysaccharide (LPS) endotoxin (15 mg/kg) and the LPS-induced production of 16 different cytokines was determined by Elisa multiplex. Control group received dexamethasone orally at 2 mg/kg/day. Toxicity on K565 cells and murine peritoneal macrophages, in vitro , at doses up to 100 μM was monitored by XTT-colorimetric assay. Results and conclusions For the first time mitraphylline was tested in vivo against a large range of cytokines that play a crucial role in inflammation. Mitraphylline inhibited around 50% of the release of interleukins 1α, 1β, 17, and TNF-α. This activity was similar to dexamethasone. It also reduced almost 40% of the production of interleukin 4 (IL-4) while the corticoid did not. Lastly it did not show any toxicity on K565 cells nor murine macrophages at doses up to 100 μM.

Complete assignments of the proton and carbon signals as well as conformational analysis of eleven basic heteroyohimbine-type oxindole alkaloids were performed by using modern NMR techniques.

The complete analysis of the 1H and 13C NMR has been performed for four pentacyclic oxindole alkaloids: mitraphylline, isomitraphylline, speciophylline and pteropodine. The total assignment of the 1H NMR parameters was achieved from combined evaluation of homonuclear shift correlation and J-resolved diagrams, while DEPT spectra and selective decoupling experiments provided all carbon chemical shifts.

The primary aim of this work was to determine the interactions of an oxindole alkaloid (mitraphylline) isolated from Uncaria tomentosa with β-amyloid 1-40 (Aβ1-40 protein) apply­ing the capillary electrophoresis (CE) method. Specifically the Hummel-Dreyer method and Scatchard analysis were performed to study the binding of oxindole alkaloids with Aβ1-40 protein. Prior to these studies extraction of the alkaloid of interest was carried out. Identifica­tion of the isolated alkaloid was performed by the use of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) combined with electrospray ionization mass spectrometry (ESI-MS). The proposed approach was proved to be an efficient and accurate method for specific compound isolation and identification purposes. Moreover, ana­lytical information from the CE approach can be considered as the valuable tool for binding constant determination. The binding constant of mitraphylline with Aβ1-40 protein deter­mined by the Hummel-Dreyer method and Scatchard analysis equals K = 9.95 x 105 m-1. The results obtained showed the significant binding of the tested compound with Aβ1-40 protein. These results are discussed and interpreted in the view of developing a strategy for identifica­tion of novel compounds of great importance in Alzheimer disease therapy.

The leaves of the plant Mitragyna parvifolia have afforded two alkaloids, 16,17-dihydro-17beta-hydroxy isomitraphylline (1) and 16, 17-dihydro-17beta-hydroxy mitraphylline (2), together with two known alkaloids, isomitraphylline (3) and mitraphylline (4). The structures of 1 and 2 were elucidated using 1D and 2D NMR spectral methods, including (1)H-(1)HCOSY, DEPT, HSQC, and HMBC. Mitraphylline was the main alkaloid constituent.

The effects of Uncaria tomentosa total alkaloid and its oxindole alkaloid components, uncarine E, uncarine C, mitraphylline, rhynchophylline and isorhynchophylline, on the impairment of retention performance caused by amnesic drugs were investigated using a step-down-type passive avoidance test in mice. In this test, the retention performance of animals treated with the amnesic and test drugs before training was assessed 24 h after training. Uncaria tomentosa total alkaloid (10–20 mgkg−1, i.p.) and the alkaloid components (10–40 mg kg−1, i.p.), as well as the muscarinic receptor agonist oxotremorine (0.01 mgkg−1, i.p.), significantly attenuated the deficit in retention performance induced by the muscarinic receptor antagonist scopolamine (3mgkg−1, i.p.). The effective doses of uncarine C and mitraphylline were larger than those of other alkaloid components. Uncarine E (20mgkg−1, i.p.) also blocked the impairment of passive avoidance performance caused by the nicotinic receptor antagonist mecamylamine (15 mgkg−1, i.p.) and the N-methyl-D-aspartate (NMDA) receptor antagonist (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 7.5 mgkg−1, i.p.), but it failed to affect the deficit caused by the benzodiazepine receptor agonist diazepam (2 mgkg−1, i.p.). Rhynchophylline significantly reduced the mecamylamine-induced deficit in passive avoidance behaviour, but it failed to attenuate the effects of CPP and diazepam. These results suggest that Uncaria tomentosa total alkaloids exert a beneficial effect on memory impairment induced by the dysfunction of cholinergic systems in the brain and that the effect of the total alkaloids is partly attributed to the oxindole alkaloids tested. Moreover, these findings raised the possibility that the glutamatergic systems are implicated in the anti-amnesic effect of uncarine E.

Mitragyna speciosa (Rubiaceae) has traditionally been used in the tropical regions of Asia, Africa and Indonesia as a substitute for opium. Indole alkaloids are the most common compounds that have been isolated. We investigated the constituents of the leaves of M. speciosa that was grown at the University of Mississippi. Several alkaloids were isolated, including ajmalicine, corynantheidine, isomitraphylline, mitraphylline, paynantheine, isocorynantheidine, 7-hydroxymitragynine and mitragynine, but their percentages were lower than those in a commercial Thai sample of “kratom”. In addition, we isolated the flavonoid epicatechin, a saponin daucosterol, the triterpenoid saponins quinovic acid 3- O-β-D-quinovopyranoside, quinovic acid 3- O-β-D-glucopyranoside, as well as several glycoside derivatives including 1- O-feruloyl-β-D-glucopyranoside, benzyl-β-D-glucopyranoside, 3-oxo-α-ionyl- O-β-D-glucopyranoside, roseoside, vogeloside, and epivogeloside. This is the first report of the last group of compounds having been isolated from a Mitragyna species. Biological studies are currently underway to test these compounds for opioid activity.

Ajmalicine, formosanine, isomitraphylline and mitraphylline have been isolated from the bark of Uncaria elliptica . Differences in alkaloid content within the species have been observed. © 1997 Elsevier Science Ltd. All rights reserved

The isolation of the indole alkaloids uncarine E (17), C (16), D (21), glabratine (29) and deoxycordifoline (30) from the fresh bark of Uncaria glabrata DC. ( Rubiaceae ) is reported. The high-field 1H and 13C n.m.r. spectra of these uncarines as well as uncarine B (14) and mitraphylline (12) are recorded and interpreted.