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

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

Coumarinolignoid and Indole Alkaloids from the Roots of the Hybrid Plant Citrus × paradisi Macfad (Rutaceae).

A phytochemical investigation of the roots of Citrus × paradisi Macfad. (Rutaceae) led to the isolation of two new compounds, namely 1-formyl-5-hydroxy-N-methylindolin-1-ium (1) and decyloxycleomiscosin D (2), along with ten known compounds: 1,1-dimethylpyrrolidin-1-ium-2-carboxylate (3), furan-2,3-diol (4), 5-methoxyseselin (5), umbelliferone (6), scopoletin (7), citracridone I (8), citracridone II (9), citracridone III (10), limonin (11) and lupeol (12). The structures were determined through the comprehensive spectroscopic analysis of 1D and 2D NMR and EI- and ESI-MS, as well as a comparison with the published data. Notably, compounds 3 and 4 from the genus Citrus are reported here for the first time. In addition, the MeOH extract of the roots and compounds 1-7 were screened against the human adenocarcinoma alveolar basal epithelial cell line A549 and the Caucasian prostate adenocarcinoma cell line PC3 using the MTT assay. While the extract showed significant activity, with IC50 values of 35.2 and 38.1 µg/mL, respectively, compounds 1-7 showed weak activity, with IC50 values of 99.2 to 250.2 µM and 99.5 to 192.7 µM, respectively.

Untargeted LC-MS/MS-Based Multi-Informative Molecular Networking for Targeting the Antiproliferative Ingredients in Tetradium ruticarpum Fruit.

The fruit of Tetradium ruticarpum (TR) is commonly used in Chinese herbal medicine and it has known antiproliferative and antitumor activities, which can serve as a good source of functional ingredients. Although some antiproliferative compounds are reported to be present in TR fruit, most studies only focused on a limited range of metabolites. Therefore, in this study, the antiproliferative activity of different extracts of TR fruit was examined, and the potentially antiproliferative compounds were highlighted by applying an untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based multi-informative molecular networking strategy. The results showed that among different extracts of TR fruit, the EtOAc fraction F2-3 possessed the most potent antiproliferative activity against HL-60, T24, and LX-2 human cell lines. Through computational tool-aided structure prediction and integrating various data (sample taxonomy, antiproliferative activity, and compound identity) into a molecular network, a total of 11 indole alkaloids and 47 types of quinolone alkaloids were successfully annotated and visualized into three targeted bioactive molecular families. Within these families, up to 25 types of quinolone alkaloids were found that were previously unreported in TR fruit. Four indole alkaloids and five types of quinolone alkaloids were targeted as potentially antiproliferative compounds in the EtOAc fraction F2-3, and three (evodiamine, dehydroevodiamine, and schinifoline) of these targeted alkaloids can serve as marker compounds of F2-3. Evodiamine was verified to be one of the major antiproliferative compounds, and its structural analogues discovered in the molecular network were found to be promising antitumor agents. These results exemplify the application of an LC-MS/MS-based multi-informative molecular networking strategy in the discovery and annotation of bioactive compounds from complex mixtures of potential functional food ingredients.

Characterization of alkaloids in bark extracts of Geissospermum vellosii by HPLC-UV-diode array-multistage high-resolution mass spectrometry.

A number of analytical studies, started in the sixties of the last century, concerning the stem bark of Geissospermum vellosii, have documented the presence of a number of indole alkaloids whose molecular identity was defined by NMR technique. The potential bioactivity of these compounds has inspired more recent analogous studies either devoted to structural elucidation of new alkaloid molecules or to the investigation of the role of some of them in cancer therapy. Anyway, a complete fingerprinting of the bark content is still lacking. In this paper, after a suitable extraction step, we obtain a chromatographic separation showing a number of components higher than the number of alkaloids so far described. Considering the great number of substances present in the stem bark, their identification is practically impossible to reveal by NMR techniques. As we presume that there are other stem bark unidentified alkaloids with important bioactivity, we propose to characterize their molecular structures by UV-Vis Diode Array spectrophotometry and High-Resolution Multistage Mass Spectrometry. The two adopted detection techniques were first tested on the already known Geissospermum vellosii molecules, and, after an inspection of their efficacy, were applied to the substances that have not yet been described. Herewith we propose the molecular structures of 10 substances that were never previously described, and in addition we provide experimental evidence of the presence of 6 already known substances which were never reported in the Geissospermum genus. A far more detailed description of the bark constituents is therefore provided.

Kratom (Mitragyna speciosa) Validation: Quantitative Analysis of Indole and Oxindole Alkaloids Reveals Chemotypes of Plants and Products.

Many consumers are turning to kratom (Mitragyna speciosa) to self-manage pain and opioid addiction. In the United States, an array of capsules, powders, and loose-leaf kratom products are readily available. Additionally, several online sites supply live kratom plants. A prerequisite to establishing quality control and quality assurance standards for the kratom industry, or understanding how alkaloid levels effect clinical outcomes, is the identification and quantitation of major and minor alkaloid constituents within available products and preparations. To this end, an ultra-high performance liquid chromatography-high resolution mass spectrometry method was developed for the analysis of 8 indole alkaloids (7-hydroxymitragynine, ajmalicine, paynantheine, mitragynine, speciogynine, isopaynantheine, speciociliatine, and mitraciliatine) and 6 oxindole alkaloids (isomitraphylline, isospeciofoleine, speciofoline, corynoxine A, corynoxeine, and rhynchophylline) in US-grown kratom plants and commercial products. These commercial products shared a qualitatively similar alkaloid profile, with 12 - 13 detected alkaloids and high levels of the indole alkaloid mitragynine (13.9 ± 1.1 - 270 ± 24 mg/g). The levels of the other major alkaloids (paynantheine, speciociliatine, speciogynine, mitraciliatine, and isopaynantheine) and the minor alkaloids varied in concentration from product to product. The alkaloid profile of US-grown M. speciosa "Rifat" showed high levels of the indole alkaloid speciogynine (7.94 ± 0.83 - 11.55 ± 0.18 mg/g) and quantifiable levels of isomitraphylline (0.943 ± 0.033 - 1.47 ± 0.18 mg/g). Notably, the alkaloid profile of a US-grown M. speciosa seedling was comparable to the commercial products with a high level of mitragynine (15.01 ± 0.20 mg/g). This work suggests that there are several M. speciosa chemotypes.

A Fast and Reliable UHPLC-MS/MS-Based Method for Screening Selected Pharmacologically Significant Natural Plant Indole Alkaloids.

Many substances of secondary plant metabolism have often attracted the attention of scientists and the public because they have certain beneficial effects on human health, although the reason for their biosynthesis in the plant remains unclear. This is also the case for alkaloids. More than 200 years have passed since the discovery of the first alkaloid (morphine), and several thousand substances of this character have been isolated since then. Most often, alkaloid-rich plants are part of folk medicine with centuries-old traditions. What is particularly important to monitor for these herbal products is the spectrum and concentrations of the present active substances, which decide whether the product has a beneficial or toxic effect on human health. In this work, we present a fast, reliable, and robust method for the extraction, preconcentration, and determination of four selected alkaloids with an indole skeleton, i.e., harmine, harmaline, yohimbine, and ajmalicine, by ultra-high performance liquid chromatography coupled with tandem mass spectrometry. The applicability of the method was demonstrated for tobacco and Tribulus terrestris plant tissue, the seeds of Peganum harmala, and extract from the bark of the African tree Pausinystalia johimbe.

Metabolomics with 15N Labeling for Characterizing Missing Monoterpene Indole Alkaloids in Plants.

Monoterpene indole alkaloids (MIAs) in medicinal plants remain uncharacterized owing to their complicated structure by metabolomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) despite their pharmaceutical importance. We demonstrate an untargeted metabolome analysis with 15nitrogen (N) labeling to characterize MIAs having an indolic skeleton in the flowers, leaves, petioles, stems, and roots of Catharanthus roseus. Principal component analysis using 15N- and nonlabeled metabolome data showed that N-containing metabolites (N-metabolites) are labeled with 15N. Paring of the 15N- and nonlabeled precursor ions were performed using the criteria of retention time, difference of m/z value, and a nonlabeled product ion at m/z 144.08 that indicates an indolic skeleton. The mass shift of the m/z value of the product and precursor ions to their 15N-labeled ions identified the number of N of their ions. Finally, molecular formula of 45 MIAs was unambiguously identified using the identified N number. The alkaloid network analysis using the MS/MS similarity showed the structural commonness and uniqueness among the MIAs. Of them, antirhine was identified using an authentic standard compound. Multimetabolomics using LC-MS/MS and imaging mass spectrometry showed that antirhine accumulates considerably in the epidermis and vascular cylinder of the roots. The developed approach showed the existence of the missing MIAs. The modification of this approach will identify other MIAs that contain a hydroxylated or methoxylated indolic skeleton.

Analysis of dihydroindole-type alkaloids in Strychnos nux-vomica unprocessed and processed seeds by high-performance liquid chromatography coupled with diode array detection and mass spectrometry.

The ripened seeds of Strychnos nux-vomica L. have been extensively used as herbal medicines in Asian countries. Dihydroindole-type alkaloids are not only the active constituents but also the toxicants in Strychnos. However, the simultaneous determination of these alkaloids in both crude and processed Semen Strychni is still lacking. The present study represents the first quantitation and relative quantitation assay of 12 dihydroindole-type alkaloids in Strychnos nux-vomica unprocessed and sand-processed seeds using high-performance liquid chromatography coupled with diode array detection and mass spectrometry. The relative concentration of ten alkaloids was calculated by semi-quantification using the internal standard and their amounts in unprocessed and detoxified Semen Strychni were compared. We report here for the first time the significant increase of the two alkaloids, 19-N-methyl-strychnine, and 2,3-dimethoxy-19-N-methyl-strychnine, during the processing of Semen Strychni. Our study provides new insight into the true complexity of seed processing procedure and valuable information for assessing the efficacy and safety for clinical applications of Semen Strychni-containing drugs.

The genuine localization of indole alkaloids in Vinca minor and Catharanthus roseus.

Based on the occurrence of indole alkaloids in so-called "chloroform leaf surface extracts", it was previously deduced that these alkaloids are present in the cuticle at the leaf surface of Catharanthus roseus and Vinca minor. As no symplastic markers were found in these extracts this deduction seemed to be sound. However, since chloroform is known to destroy biomembranes very rapidly, these data have to be judged with scepticism. We reanalyzed the alleged apoplastic localization of indole alkaloids by employing slightly acidic aqueous surface extracts and comparing the corresponding alkaloid patterns with those of aqueous total leaf extracts. Whereas in the "chloroform leaf surface extracts" all alkaloids are present in the same manner as in the total leaf extracts, no alkaloids occur in the aqueous leaf surface extracts. These results clearly show that chloroform had rapidly destroyed cell integrity, and the related extracts also contain the alkaloids genuinely accumulated within the protoplasm. The related decompartmentation was verified by the massively enhanced concentration of amino acids in aqueous surface extracts of chloroform treated leaves. Furthermore, the chloroform-induced cell disintegration was vividly visualized by confocal laser scanning microscopical analyses, which clearly displayed a strong decrease in the chlorophyll fluorescence in chloroform treated leaves. These findings unequivocally display that the indole alkaloids are not located in the apoplastic space, but exclusively are present symplastically within the cells of V. minor and C. roseus leaves. Accordingly, we have to presume that also other leaf surface extracts employing organic solvents have to be re-investigated.

In vitro antiplasmodial activity and identification, using tandem LC-MS, of alkaloids from Aspidosperma excelsum, a plant used to treat malaria in Amazonia.

ETHNOPHARMACOLOGICAL RELEVANCE: Aspidosperma excelsum Benth. (Apocynaceae), a native tree in the Brazilian Amazonia, is traditionally used to treat various diseases, including malaria. AIM OF STUDY: To investigate the chemical constitution, antiplasmodial activity and cytotoxicity of samples obtained from A. excelsum trunk bark by different procedures aiming to evaluate their potential as an antimalarial phytomedicine. MATERIALS AND METHODS: A hydroethanolic extract and alkaloid extracts were prepared and assayed for antiplasmodial activity and cytotoxicity against chloroquine-resistant Plasmodium falciparum (W2 strain) and HepG2 cells, respectively. Taking into account the known occurrence and antimalarial activity of Aspidosperma monoterpene indole alkaloids (MIA), acid-base extractions were carried out and the fractions were assayed for antiplasmodial activity and cytotoxicity. All the samples were analysed by hyphenated chromatographic techniques, such as UPLC-DAD-ESI-MS/MS and HRMS (HPLC-MS MicroTOF), comparing their chemical composition to the literature data. RESULTS: The hydroethanolic extract disclosed a moderate in vitro activity against chloroquine-resistant Plasmodium falciparum (W2 strain) with IC50 23.68 ± 3.08 µg/mL), low cytotoxicity to HepG2 cells (> 250 µg/mL) and good SI (> 10.56). A total of 20 known monoterpene indole alkaloids were identified, seven of which are here firstly described for A. excelsum. Known highly active alkaloids, namely demethylaspidospermine, aspidocarpine, and ochrolifuanine are present in active alkaloid fractions and might contribute to their observed antiplasmodial effect. An alkaloid fraction (Ae-Alk2), obtained directly from trunk bark by extraction with dil. aqueous HCl, pointed out for its activity (IC50 8.75±2.26 µg/mL, CC50 185.14±1.97 µg/mL, SI 21.16) and should be highlighted as the most promising out of the assayed samples. CONCLUSION: The present results represent a preliminary support to the alleged antimalarial use of A. excelsum trunk bark and allowed to highlight alkaloid fractions as promising phytomedicines.

Diagnostic fragmentation filtering for the discovery of new chaetoglobosins and cytochalasins.

RATIONALE: Microbial natural products are often biosynthesized as classes of structurally related compounds that have similar tandem mass spectrometry (MS/MS) fragmentation patterns. Mining MS/MS datasets for precursor ions that share diagnostic or common features enables entire chemical classes to be identified, including novel derivatives that have previously been unreported. Analytical data analysis tools that can facilitate a class-targeted approach to rapidly dereplicate known compounds and identify structural variants within complex matrices would be useful for the discovery of new natural products. METHODS: A diagnostic fragmentation filtering (DFF) module was developed for MZmine to enable the efficient screening of MS/MS datasets for class-specific product ions(s) and/or neutral loss(es). This approach was applied to series of the structurally related chaetoglobosin and cytochalasin classes of compounds. These were identified from the culture filtrates of three fungal genera: Chaetomium globosum, a putative new species of Penicillium (called here P. cf. discolor: closely related to P. discolor), and Xylaria sp. Extracts were subjected to LC/MS/MS analysis under positive electrospray ionization and operating in a data-dependent acquisition mode, performed using a Thermo Q-Exactive mass spectrometer. All MS/MS datasets were processed using the DFF module and screened for diagnostic product ions at m/z 130.0648 and 185.0704 for chaetoglobosins, and m/z 120.0808 and 146.0598 for cytochalasins. RESULTS: Extracts of C. globosum and P. cf. discolor strains revealed different mixtures of chaetoglobosins, whereas the Xylaria sp. produced only cytochalasins; none of the strains studied produced both classes of compounds. The dominant chaetoglobosins produced by both C. globosum and P. cf. discolor were chaetoglobosins A, C, and F. Tetrahydrochaetoglobosin A was identified from P. cf. discolor extracts and is reported here for the first time as a natural product. The major cytochalasins produced by the Xylaria sp. were cytochalasin D and epoxy cytochalasin D. A larger unknown "cytochalasin-like" molecule with the molecular formula C38 H47 NO10 was detected from Xylaria sp. culture filtrate extracts and is a current target for isolation and structural characterization. CONCLUSIONS: DFF is an effective LC/MS data analysis approach for rapidly identifying entire classes of compounds from complex mixtures. DFF has proved useful in the identification of new natural products and allowing for their partial characterization without the need for isolation.

Genetic structure and chemical diversity in natural populations of Uncaria guianensis (Aubl.) J.F.Gmel. (Rubiaceae).

Uncaria guianensis is native to the Amazon and is used traditionally as an anti-inflammatory. Natural populations of the species have declined markedly in recent times because of strong anthropic pressure brought about by deforestation and indiscriminate collection. The aim of the present study was to assess the genetic and chemical diversity among eight natural populations of U. guianensis located in the Brazilian states of Acre, Amapá and Amazonas. A set of four primer combinations was employed in sequence-related amplified polymorphism (SRAP) amplifications of leaf DNA, and the fragments were analyzed in an LI-COR model 4300 DNA Analyzer. Genetic variability within the populations (81%) was substantially greater than that detected between them (19%). The highest percentage of polymorphic loci (90.21%) and the largest genetic variability were observed in the population located in Mazagão, Amapá. Genetic differentiation between populations was high (Fst = 0.188) and the studied populations formed three distinct genetic groups (K = 3). The population located in Assis Brasil, Acre, presented the highest average content of the mitraphylline (0.60 mg/g dry weight,). However, mitraphylline and isomitraphylline not detected in most individuals in the studied populations, and it is questionable whether they should be considered as chemical markers of the species. The genetic data confirm the urgent need for conservation programs for U. guianensis, and for further studies aimed at ascertaining the genetic basis and heritability of alkaloid accumulation.

An enhanced strategy integrating offline two-dimensional separation and step-wise precursor ion list-based raster-mass defect filter: Characterization of indole alkaloids in five botanical origins of Uncariae Ramulus Cum Unicis as an exemplary application.

Comprehensive chemical profiling is of great significance for understanding the therapeutic material basis and quality control of herbal medicines, which is challenging due to its inherent chemical diversity and complexity, as well as wide concentration range. In this study, we introduced an enhanced strategy integrating offline two-dimensional (2D) separation and the step-wise precursor ion list-based raster-mass defect filter (step-wise PIL-based raster-MDF) scan by tandem LTQ-Orbitrap mass spectrometer. A comprehensive analysis of indole alkaloids in five botanical origins of Uncariae Ramulus Cum Unicis (Gou-Teng) was used as an exemplary application. A positively charged reversed phase (PR) × conventional RP LC system in different pH conditions was constructed with the orthogonality of 74%. A theoretical step-wise PIL among 310-950 Da with the step-size of 2 Da was developed to selectively trigger fragmentations and extend the coverage of potential indole alkaloids. Simultaneously, by defining parent mass width (PMW) of the step-wise PIL to ±55 mDa, a raster-MDF screening was achieved in the acquisition process. Additionally, subtype classification and structural elucidation were facilitated by a four-step interpretation strategy. As a result, a total of 1227 indole alkaloids were efficiently exposed and characterized from five botanical origins of Gou-Teng, which showed high chemical diversity. A systematic comparison among five species was first performed and only 66 indole alkaloids were common. For method validation, three new alkaloid N-oxides were isolated and unambiguously identified by NMR. The present study provides a novel data-dependent acquisition method with improved target coverage and high selectivity. The integrated strategy is practical to efficiently expose and comprehensively characterize complex components in herbal medicines.

Quality Assessment of Kumu Injection, a Traditional Chinese Medicine Preparation, Using HPLC Combined with Chemometric Methods and Qualitative and Quantitative Analysis of Multiple Alkaloids by Single Marker.

Kumu injection (KMI) is a common-used traditional Chinese medicine (TCM) preparation made from Picrasma quassioides (D. Don) Benn. rich in alkaloids. An innovative technique for quality assessment of KMI was developed using high performance liquid chromatography (HPLC) combined with chemometric methods and qualitative and quantitative analysis of multi-components by single marker (QAMS). Nigakinone (PQ-6, 5-hydroxy-4-methoxycanthin-6-one), one of the most abundant alkaloids responsible for the major pharmacological activities of Kumu, was used as a reference substance. Six alkaloids in KMI were quantified, including 6-hydroxy-ß-carboline-1-carboxylic acid (PQ-1), 4,5-dimethoxycanthin-6-one (PQ-2), ß-carboline-1-carboxylic acid (PQ-3), ß-carboline-1-propanoic acid (PQ-4), 3-methylcanthin-5,6-dione (PQ-5), and PQ-6. Based on the outcomes of twenty batches of KMI samples, the contents of six alkaloids were used for further chemometric analysis. By hierarchical cluster analysis (HCA), radar plots, and principal component analysis (PCA), all the KMI samples could be categorized into three groups, which were closely related to production date and indicated the crucial influence of herbal raw material on end products of KMI. QAMS combined with chemometric analysis could accurately measure and clearly distinguish the different quality samples of KMI. Hence, QAMS is a feasible and promising method for the quality control of KMI.

Sceletium tortuosum may delay chronic disease progression via alkaloid-dependent antioxidant or anti-inflammatory action.

The link between obesity-induced systemic inflammation and decreased insulin signalling is well-known. It is also known that peripherally produced inflammatory cytokines can cross the blood-brain barrier, resulting in the release of neurotoxins that can ultimately lead to the demise of central nervous system integrity. A high-mesembrine Sceletium tortuosum extract was recently shown to possess cytoprotective and mild anti-inflammatory properties in monocytes and to target specific p450 enzymes to reduce adrenal glucocorticoid synthesis. This is significant since the aetiology of both obesity and diabetes is linked to inflammation and excess glucocorticoid production. Given the interlinked nature of glucocorticoid action and inflammation, central immunomodulatory effects of two Sceletium tortuosum extracts prepared by different extraction methods were investigated. Human astrocytes were pre-treated for 30 min, before exposure to Escherichia coli lipopolysaccharide for 23.5 h (in the presence of treatment). Cytotoxicity, mitotoxicity and cytokine responses (basally and in response to inflammatory stimulus) were assessed. In addition, total polyphenol content, antioxidant capacity and selected neural enzyme inhibition capacity were assessed for both extracts. The high-mesembrine Sceletium extract exerted cytoprotective and anti-inflammatory effects. In contrast, the high delta7-mesembrenone extract, rich in polyphenols, exhibited potent antioxidant effect, although with relatively higher risk of adverse effects with overdose. We conclude that both Sceletium tortuosum extracts may be employed as either a preventative supplement or complimentary treatment in the context of obesity and diabetes; however, current data also highlights the impact that extraction methods can have on plant product mechanism of action.

A UPLC-ESI-Q-TOF method for rapid and reliable identification and quantification of major indole alkaloids in Catharanthus roseus.

We developed a novel ultra performance liquid chromatography-quadrupole time-of-flight (UPLC-Q-TOF) mass spectrometry method that allows sensitive, rapid, and reliable detection and identification of six representative indole alkaloids (vincristine, vinblastine, ajmalicine, catharanthine, serpentine, and vindoline) that exhibit physiological activity in Catharanthus roseus (C. roseus). The alkaloids were eluted on a C18 column with acetonitrile and water containing 0.1% formic acid and 10 mM ammonium acetate, and separated with good resolution within 13 min. Electrospray ionization-Q-TOF (ESI-Q-TOF) analysis was performed to characterize the molecules and their fragment ions, and the characteristic ions and fragmentation patterns were used as to identify the alkaloids. The proposed analytical method was verified in reference to the ICH guidelines and the results showed excellent linearity (R2 > 0.9988), limit of detection (1 ng/mL to 10 ng/mL), limit of quantification (3 ng/mL to 30 ng/mL), intra-day and inter-day precisions, and extraction recovery rates (92.8% to 104.1%) for all components. The validated UPLC-Q-TOF method was applied to the analysis of extracts from the root, stem, and leaves of C. roseus, allowing the identification of six alkaloids by comparison of retention times, molecular ions, and fragmentation patterns with those of reference compounds. Sixteen additional indole alkaloids were tentatively identified by comparison of chromatograms to chemical databases and literature reports. The contents of bis-indole alkaloids (vincristine and vinblastine) were high in the aerial parts, while the contents of mono-indole alkaloids (ajmalicine, catharanthine, serpentine, and vindoline) were high in the roots. The present results demonstrate that the proposed UPLC-Q-TOF method can be useful for the investigation of phytochemical constituents of medicinal plants.

Aqueous extracts from Uncaria tomentosa (Willd. ex Schult.) DC. reduce bronchial hyperresponsiveness and inflammation in a murine model of asthma.

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.

Simultaneous quantitative determination of bioactive terpene indole alkaloids in ethanolic extracts of Catharanthus roseus (L.) G. Don by ultra high performance liquid chromatography-tandem mass spectrometry.

A rapid, sensitive and reproducible method using ultra-high-performance liquid chromatography coupled with electrospray ionization hybrid triple quadrupole-linear ion trap mass spectrometry (UHPLC-ESI-QqQLIT-MS/MS) in multiple reaction monitoring (MRM) mode was developed and validated for simultaneous quantitation of anticancer (vincristine, vinblastine, vindesine), antihypertensive (ajmaline, ajmalicine, reserpine), aphrodisiac (yohimbine), sedative (serpentine) agents, dietary supplement (vinpocetine, yohimbine) and precursor of vinblastine (vindoline) from crude extracts of Catharanthus roseus. The precursor to product ion transitions for these compounds were observed at m/z 327 → 144, 355 → 144, 754 → 355, 353 → 144, 349 → 317, 825 → 225, 811 → 224, 458 → 188, 351 → 280 and 609 → 195, respectively in positive ionization mode. Chromatographic separation of all targeted TIAs was performed on ACQUITY UPLC BEH™ C18 column (1.7 µm, 2.1 mm × 50 mm). The calibration curves were linear within the concentration range 0.5-1000 ng/mL and correlation coefficients (R2) were closer to 1. Limit of detection (LOD) and limit of quantitation (LOQ) ranged from 0.039-0.583 ng/mL and 0.118-1.767 ng/mL, respectively. The intra-day (0.23-2.71% RSD) and inter-day (0.40-2.90% RSD) precision, stability (0.69-3.45% RSD) and recovery (99.63-104.30% ±â€¯%RSD ≤ 3.03%) were acceptable indicating good accuracy of the developed method. The method was successfully applied in ethanolic extracts of 39 samples of C. roseus parts (leaf, stem and root) collected from five different locations in India. Serpentine was detected as one of the most abundant TIA. Principal component analysis (PCA) was able to successfully discriminate among C. roseus samples on the basis of content of targeted TIAs.

Characteristic Chromatogram: A Method of Discriminate and Quantitative Analysis for Quality Evaluation of Uncaria Stem with Hooks.

It remains a challenge to establish new monographs for herbal drugs derived from multiple botanical sources. Specifically, the difficulty involves discriminating and quantifying these herbs with components whose levels vary markedly among different samples. Using Uncaria stem with hooks as an example, a characteristic chromatogram was proposed to discriminate its five botanical origins and to quantify its characteristic components in the chromatogram. The characteristic chromatogram with respect to the components of Uncaria stem with hooks with the five botanical origins was established using 0.02% diethylamine and acetonitrile as the mobile phase. The total analysis time was 50 min and the detection wavelength was 245 nm. Using the same chromatogram parameters, the single standard to determine multicomponents method was validated to simultaneously quantify nine indole alkaloids, including vincosamide, 3α-dihydrocadambine, isocorynoxeine, corynoxeine, isorhynchophylline, rhynchophylline, hirsuteine, hirsutine, and geissoschizine methyl ether. The results showed that only the Uncaria stem with hooks from Uncaria rhynchophylla, the most widely used in the herbal market, showed the presence of these nine alkaloids. The conversion factors were 1.27, 2.32, 0.98, 1.04, 1.00, 1.02, 1.26, 1.33, and 1.25, respectively. The limits of quantitation were lower than 700 ng/mL. The total contents of 31 batches of Uncaria stem with hooks were in the range of 0.1 - 0.6%, except for Uncaria hirsuta Havil and Uncaria sinensis (Oliv.) Havil. The results also showed that the total content of indole alkaloids tended to decrease with an increase in the hook diameter. This showed that the characteristic chromatogram is practical for controlling the quality of traditional Chinese medicines with multiple botanical origins.

Mass defect filtering-oriented classification and precursor ions list-triggered high-resolution mass spectrometry analysis for the discovery of indole alkaloids from Uncaria sinensis.

Discovery of new natural compounds is becoming increasingly challenging because of the interference from those known and abundant components. The aim of this study is to report a dereplication strategy, by integrating mass defect filtering (MDF)-oriented novelty classification and precursor ions list (PIL)-triggered high-resolution mass spectrometry analysis, and to validate it by discovering new indole alkaloids from the medicinal herb Uncaria sinensis. Rapid chromatographic separation was achieved on a Kinetex® EVO C18 column (<16min). An in-house MDF algorithm, developed based on the informed phytochemistry information and molecular design, could more exactly screen the target alkaloids and divide them into three novelty levels: Known (KN), Unknown-but-Predicted (UP), and Unexpected (UN). A hybrid data acquisition method, namely PIL-triggered collision-induced dissociation-MS2 and high-energy C-trap dissociation-MS3 with dynamic exclusion on a linear ion trap/Orbitrap mass spectrometer, facilitated the acquisition of diverse product ions sufficient for the structural elucidation of both indole alkaloids and the N-oxides. Ultimately, 158 potentially new alkaloids, including 10 UP and 108 UN, were rapidly characterized from the stem, leaf, and flower of U. sinensis. Two new alkaloid compounds thereof were successfully isolated and identified by 1D and 2D NMR analyses. The varied ring E and novel alkaloid-acylquinic acid conjugates were first reported from the whole Uncaria genus. Conclusively, it is a practical chemical dereplication strategy that can enhance the efficiency and has the potential to be a routine approach for the discovery of new natural compounds.