Essentials in the acquisition, interpretation, and reporting of plant metabolite profiles.

Plant metabolite profiling reveals the diversity of secondary or specialized metabolites in the plant kingdom with its hundreds of thousands of species. Specialized plant metabolites constitute a vast class of chemicals posing significant challenges in analytical chemistry. In order to be of maximum scientific relevance, reports dealing with these compounds and their source species must be transparent, make use of standards and reference materials, and be based on correctly and traceably identified plant material. Essential aspects in qualitative plant metabolite profiling include: (i) critical review of previous literature and a reasoned sampling strategy; (ii) transparent plant sampling with wild material documented by vouchers in public herbaria and, optimally, seed banks; (iii) if possible, inclusion of generally available reference plant material; (iv) transparent, documented state-of-the art chemical analysis, ideally including chemical reference standards; (v) testing for artefacts during preparative extraction and isolation, using gentle analytical methods; (vi) careful chemical data interpretation, avoiding over- and misinterpretation and taking into account phytochemical complexity when assigning identification confidence levels, and (vii) taking all previous scientific knowledge into account in reporting the scientific data. From the current stage of the phytochemical literature, selected comments and suggestions are given. In the past, proposed revisions of botanical taxonomy were sometimes based on metabolite profiles, but this approach ("chemosystematics" or "chemotaxonomy") is outdated due to the advent of DNA sequence-based phylogenies. In contrast, systematic comparisons of plant metabolite profiles in a known phylogenetic framework remain relevant. This approach, known as chemophenetics, allows characterizing species and clades based on their array of specialized metabolites, aids in deducing the evolution of biosynthetic pathways and coevolution, and can serve in identifying new sources of rare and economically interesting natural products.

Cytotoxic diterpenoids from Salvia glutinosa and comparison with the tanshinone profile of danshen (Salvia miltiorrhiza).

The roots of Salvia miltiorrhiza are the source of the traditional Chinese medicine danshen and the class of tanshinones, particular quinoid nor-diterpenoids of the abietane type. Of these compounds, cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA, have been extensively studied for their anticancer potential, not only but as well because of their high abundance in S. miltiorrhiza and their thus easy availability. However, also additional Salvia species are known to contain tanshinones, mainly such of the subgenus Glutinaria, of which S. glutinosa is the only species widely occurring in Europe. Using UHPLC-DAD-MS, the tanshinone profile of S. glutinosa roots collected from two different locations was compared to the profile in S. miltiorrhiza roots. In addition, tanshinone IIA and another six diterpenoids from S. glutinosa were investigated for their antiproliferative and cytotoxic potential against MDA-MB-231 and HL-60 cells. Apart from dihydrotanshinone I, which has been previously characterized due to its anticancer properties, we determined danshenol A as a highly antiproliferative and cytotoxic agent, significantly surpassing the effects of dihydrotanshinone I. With regard to the diterpenoid profile, S. miltiorrhiza showed a higher concentration for most of the tanshinones, except for (+)-danshexinkun A, which was present in comparable amounts in both species. Danshenol A, in contrast, was only present in S. glutinosa as were dehydroabietic acid and (+)-pisiferic acid. The results of our study underlines the long traditional use of danshen due to its high amount on tanshinones, but also demonstrates the potential value of investigating closely related species for the discovery of new biologically active lead compounds.

Structure-dependent activity of plant natural products against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes for nosocomial infections and has been classified as "high priority pathogen" by the World Health Organization. Its ability to develop resistances has been a challenge for the last decades and is still a threat to health care systems, as strains with resistances to the so-called drugs of last resort have been discovered. Therefore, new antibiotics are urgently needed. Natural products are an important source for the development of new drugs, thereby mostly serving as lead compounds for further modification. In this review, the data on plant natural products with reported anti-MRSA activity until the end of 2022 is discussed, highlighting the most effective drugs with respect to their inhibitory concentrations as well as with regard to eventual synergistic effects with existing antibiotics. In the latter sense, the class of alkaloids must be mentioned, exhibiting additive or synergistic effects by inhibiting bacterial efflux pumps. With regard to the antibiotic activity, phloroglucinol derivatives certainly belong to the most promising compounds, revealing several candidates with remarkable effects, e.g., lupulone, ivesinol, rhodomyrtone, aspidinol, or hyperforin. Also, the class of terpenoids yielded noteworthy compounds, such as the sesquiterpene lactones parthenolide and lactopicrin as well as acetophenone sesquiterpenes and sphaerodiene type diterpenoids, respectively. In addition, pronounced effects were observed for the macrolide neurymenolide A and three flavonol dicoumaroylrhamnosides.

Momordica charantia L.-Diabetes-Related Bioactivities, Quality Control, and Safety Considerations.

Momordica charantia L. (Cucurbitaceae), commonly known as bitter gourd or bitter melon, is widely cultivated in many tropical and subtropical regions of the world, where its unripe fruits are eaten as a vegetable. Apart from its culinary use, M. charantia has a long history in traditional medicine, serving as stomachic, laxative or anthelmintic, and, most notably, for the treatment of diabetes and its complications. Its antidiabetic properties and its beneficial effects on blood glucose and lipid concentrations have been reported in numerous in vitro and in vivo studies, but the compounds responsible for the observed effects have not yet been adequately described. Early reports were made for charantin, a mixture of two sterol glucosides, and the polypeptide p-insulin, but their low concentrations in the fruits or their limited bioavailability cannot explain the observed therapeutic effects. Still, for many decades the search for more reasonable active principles was omitted. However, in the last years, research more and more focused on the particular cucurbitane-type triterpenoids abundant in the fruits and other parts of the plant. This mini review deals with compounds isolated from the bitter gourd and discusses their bioactivities in conjunction with eventual antidiabetic or adverse effects. Furthermore, methods for the quality control of bitter gourd fruits and preparations will be evaluated for their meaningfulness and their potential use in the standardization of commercial preparations.

Antimicrobial Prenylated Isoflavones from the Leaves of the Amazonian Medicinal Plant Vatairea guianensis Aubl.

Vatairea guianenis Aubl. (Fabaceae) is an Amazonian medicinal plant species traditionally used for treating skin diseases. In an initial screening, a V. guianensis leaf extract and its subextracts showed antibacterial and antifungal activities. The EtOAc subextract was selected for chemical workup and afforded five known (1-4 and 8) and six undescribed isoflavones, vatairenones C-H (5-7 and 9-11). All isoflavones are prenylated in position C-8, displaying either chain-prenylated (1-7) or ring-closed forms (8-11). The most bioactive compound (3) exhibited in vitro activity against clinically relevant bacteria and fungi with IC50 values ranging from 6.8 to 26.9 µM. Due to its broad antimicrobial activity and low general toxicity, compound 3 is a potential lead compound for structural modifications. The results of the present study support the ethnomedicinal use of V. guianensis in the treatment of dermatological disorders. 1H NMR spectra of some of the isolated compounds showed intricate signal patterns, which might explain repeated errors in assigning the correct structure of the isoflavonoid B-ring in the literature and which we resolved by higher order spectra simulations.

Insights into polyamine metabolism: homospermidine is double-oxidized in two discrete steps by a single copper-containing amine oxidase in pyrrolizidine alkaloid biosynthesis.

Polyamines are important metabolites in plant development and abiotic and biotic stress responses. Copper-containing amine oxidases (CuAOs) are involved in the regulation of polyamine levels in the cell. CuAOs oxidize primary amines to their respective aldehydes and hydrogen peroxide. In plants, aldehydes are intermediates in various biosynthetic pathways of alkaloids. CuAOs are thought to oxidize polyamines at only one of the primary amino groups, a process frequently resulting in monocyclic structures. These oxidases have been postulated to be involved in pyrrolizidine alkaloid (PA) biosynthesis. Here, we describe the identification and characterization of homospermidine oxidase (HSO), a CuAO of Heliotropium indicum (Indian heliotrope), involved in PA biosynthesis. Virus-induced gene silencing of HSO in H. indicum leads to significantly reduced PA levels. By in vitro enzyme assays after transient in planta expression, we show that this enzyme prefers Hspd over other amines. Nuclear magnetic resonance spectroscopy and mass spectrometry analyses of the reaction products demonstrate that HSO oxidizes both primary amino groups of homospermidine (Hspd) to form a bicyclic structure, 1-formylpyrrolizidine. Using tracer feeding, we have further revealed that 1-formylpyrrolizidine is an intermediate in the biosynthesis of PAs. Our study therefore establishes that HSO, a canonical CuAO, catalyzes the second step of PA biosynthesis and provides evidence for an undescribed and unusual mechanism involving two discrete steps of oxidation that might also be involved in the biosynthesis of complex structures in other alkaloidal pathways.

Prediction of the sweetening effect of Siraitia grosvenorii (luo han guo) fruits by two-dimensional quantitative NMR.

During the last decade, dried fruits of Siraitia grosvenorii (luo han guo), also known as monk fruit, have become popular food ingredients. Luo han guo extracts, which are promoted as non-caloric natural sweeteners, are now incorporated into dietary supplements, soft drinks, and energy shakes. The compounds responsible for the sweetening effect are glycosylated cucurbitane-type triterpenoids, the so-called mogrosides. However, of the more than 40 known mogroside compounds, only 11-α-hydroxy-mogrosides exhibit a sweetening effect, whereas the other triterpenoids are non- or bitter-tasting. We have used two-dimensional quantitative NMR to determine selectively the content of 11-α-hydroxy-mogrosides in these dried fruits and thus to predict their sweetening potential. Homonuclear (H,H COSY) and heteronuclear (HSQC) methods were developed, validated, and compared. Both techniques were found suitable for the quality control of luo han guo fruits and extracts, the COSY experiment being advantageous with regard to accuracy, precision, and limit of quantification.

Development of an activity assay for characterizing deoxyhypusine synthase and its diverse reaction products.

Deoxyhypusine synthase transfers an aminobutyl moiety from spermidine to the eukaryotic translation initiation factor 5A (eIF5A) in the first step of eIF5A activation. This exclusive post-translational modification is conserved in all eukaryotes. Activated eIF5A has been shown to be essential for cell proliferation and viability. Recent reports have linked the activation of eIF5A to several human diseases. Deoxyhypusine synthase, which is encoded by a single gene copy in most eukaryotes, was duplicated in several plant lineages during evolution, the copies being repeatedly recruited to pyrrolizidine alkaloid biosynthesis. However, the function of many of these duplicates is unknown. Notably, deoxyhypusine synthase is highly promiscuous and can catalyze various reactions, often of unknown biological relevance. To facilitate in-depth biochemical studies of this enzyme, we report here the development of a simple and robust in vitro enzyme assay. It involves precolumn derivatization of the polyamines taking part in the reaction and avoids the need for the previously used radioactively labeled tracers. The derivatized polyamines are quantified after high-performance liquid chromatography coupled to diode array and fluorescence detectors. By performing kinetic analyses of deoxyhypusine synthase and its paralog from the pyrrolizidine alkaloid-producing plant Senecio vernalis, we demonstrate that the assay unequivocally differentiates the paralogous enzymes. Furthermore, it detects and quantifies, in a single assay, the side reactions that occur in parallel to the main reaction. The presented assay thus provides a detailed biochemical characterization of deoxyhypusine synthase and its paralogs.

Two-dimensional qNMR of anthraquinones in Frangula alnus (Rhamnus frangula) using surrogate standards and delay time adaption.

In the absence of adequate reference material, a problem often encountered in natural product chemistry, we investigated the use of surrogate standards in two-dimensional qNMR for the quantification of anthraquinones in the bark of alder buckthorn (Frangula alnus). Using the integrals of cross signals in the HSQC spectrum obtained from commercial standards rutin and duroquinone and adapting the delays for the 1JCH coupling, we quantified the total amount of anthraquinones and anthraquinone glucosides, as well as the content of the value-determining glucofrangulins and frangulins. Thereby, duroquinone was used as an external standard to establish the calibration curve for the methylated anthraquinone scaffold, whereas calibration curves for the glycosides were obtained using the anomeric proton signals of the rutinose disaccharide. The method was validated for accuracy, precision, specificity, linearity and limit of quantitation and shows clear advantages over the method of the European Pharmacopeia, especially in terms of specificity and meaningfulness of the results. Apart from being a useful alternative in the quality control of alder buckthorn, the presented approach demonstrates, moreover, the versatility of sophisticated 2D measurements in quantitative NMR.

Dihydroresveratrol Type Dihydrostilbenoids: Chemical Diversity, Chemosystematics, and Bioactivity.

BACKGROUND: Dihydrostilbenoids, a diverse class of natural products differing from stilbenoids by the missing double bond in the ethylene chain linking the aromatic moieties, have been reported from fungi, mosses, ferns, and flowering plants. OBJECTIVE: Occurrence, structure, and bioactivity of naturally occurring dihydroresveratrol type dihydrostilbenoids are discussed in this review. METHOD: A Reaxys database search for dihydroresveratrol derivatives with possible substitutions on all atoms, but excluding non-natural products and compounds featuring additional rings involving the ethyl connecting chain, was performed. RESULTS: Structures include simple dihydroresveratrol derivatives, compounds substituted with complex side chains composed of acyl moieties and sugars, and compounds containing polycyclic cores attached to dihydrostilbenoid units. Dihydrostilbenoids have a wide spectrum of bioactivities ranging from expectable antioxidant and anti-inflammatory activities to interesting neuroprotective and anticancer activity. The anticancer activity in particular is very pronounced for some plant-derived dihydrostilbenoids and makes them interesting lead compounds for drug development. Apart from some reports on dihydroresveratrol derivatives as phytoalexins against plant-pathogenic fungi, only very limited information is available on the ecological role of these compounds for the organisms producing them. CONCLUSION: Dihydrostilbenoids are a class of natural products possessing significant biological activities; their scattered but not ubiquitous occurrence throughout the kingdoms of plants and fungi is not easily explained. We are convinced that future studies will identify new sources of dihydrostilbenoids, and we hope that the present review will inspire such studies and will help in directing such efforts to suitable source organisms and towards promising bioactivities.

Metabolic fingerprinting of Leontopodium species (Asteraceae) by means of ¹H NMR and HPLC-ESI-MS.

The genus Leontopodium, mainly distributed in Central and Eastern Asia, consists of ca. 34-58 different species. The European Leontopodium alpinum, commonly known as Edelweiss, has a long tradition in folk medicine. Recent research has resulted in the identification of prior unknown secondary metabolites, some of them with interesting biological activities. Despite this, nearly nothing is known about the Asian species of the genus. In this study, we applied proton nuclear magnetic resonance (¹H NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) metabolic fingerprinting to reveal insights into the metabolic patterns of 11 different Leontopodium species, and to conclude on their taxonomic relationship. Principal component analysis (PCA) of ¹H NMR fingerprints revealed two species groups. Discriminators for these groups were identified as fatty acids and sucrose for group A, and ent-kaurenoic acid and derivatives thereof for group B. Five diterpenes together with one sesquiterpene were isolated from Leontopodium franchetii roots; the compounds were described for the first time for L. franchetii: ent-kaur-16-en-19-oic acid, methyl-15α-angeloyloxy-ent-kaur-16-en-19-oate, methyl-ent-kaur-16-en-19-oate, 8-acetoxymodhephene, 19-acetoxy-ent-kaur-16-ene, methyl-15ß-angeloyloxy-16,17-epoxy-ent-kauran-19-oate. In addition, differences in the metabolic profile between collected and cultivated species could be observed using a partial least squares-discriminant analysis (PLS-DA). PCA of the LC-MS fingerprints revealed three groups. Discriminating signals were compared to literature data and identified as two bisabolane derivatives responsible for discrimination of group A and C, and one ent-kaurenoic acid derivative, discriminating group B. A taxonomic relationship between a previously unidentified species and L. franchetii and Leontopodium sinense could be determined by comparing NMR fingerprints. This finding supports recent molecular data. Furthermore, Leontopodium dedekensii and L. sinense, two closely related species in terms of morphology and DNA-fingerprints, could be distinguished clearly using ¹H NMR and LC-MS metabolic fingerprinting.

Quantitative analysis of cycloartane glycosides in black cohosh rhizomes and dietary supplements by RRLC-ELSD and RRLC-qTOF-MS.

In this study, a fast and reproducible RRLC-ELSD method for the quantitative analysis of 17 cycloartane glycosides and the aglycone cimigenol in black cohosh rhizomes and dietary supplements has been developed. Separation of the 18 triterpenes was achieved within 16 min using reversed phase material and a gradient elution system consisting of water, acetonitrile and methanol. The method was validated for accuracy (recovery rates from 96.79% to 102.86%), precision (intra-day variation ≤5.98%, inter-day variation ≤3.74%), repeatability (R.S.D. ≤ 6.94%) and sensitivity, with detection limits below 4.0 µg/mL and quantification limits lower than 13.2 µg/mL. Calibration curves were established in the range from 5-1,000 µg/mL, with correlation coefficients higher than 0.998 for all constituents investigated. Peak purity and peak assignment were confirmed by means of RRLC-qTOF-MS and in comparison with reference compounds. Three different MS sources (ESI, APCI and APPI) were compared for their ionisation potential regarding cycloartane derivatives. One of the isolated black cohosh constituents, 24-O-acetylhydroshengmanol-3-O-α-L-arabinopyranoside, could be identified as new natural compound.

Bioactivity-guided isolation of GABA(A) receptor modulating constituents from the rhizomes of Actaea racemosa.

Black cohosh (Actaea racemosa) is a frequently used herbal remedy for the treatment of mild climacteric symptoms. In the present study, the modulation of γ-aminobutryic acid (GABA)-induced chloride currents (I(GABA)) through GABA type A (GABA(A)) receptors by black cohosh extracts and isolated compounds was investigated. GABA(A) receptors, consisting of α(1), ß(2), and γ(2S) subunits, were expressed in Xenopus laevis oocytes, and potentiation of I(GABA) was measured using the two-microelectrode voltage clamp technique. In a bioactivity-guided isolation procedure the positive modulation of I(GABA) could be restricted to the plant terpenoid fractions, resulting in the isolation of 11 cycloartane glycosides, of which four significantly (p < 0.05) enhanced I(GABA). The most efficient effect was observed for 23-O-acetylshengmanol 3-O-ß-d-xylopyranoside (4, 100 µM), enhancing I(GABA) by 1692 ± 201%, while actein (1), cimigenol 3-O-ß-d-xylopyranoside (6), and 25-O-acetylcimigenol 3-O-α-l-arabinopyranoside (8) were significantly less active. In the absence of GABA, only 4 induced small (not exceeding 1% of I(GABA-max)) chloride inward currents through GABA(A) receptors. It is hypothesized that the established positive allosteric modulation of GABA(A) receptors may contribute to beneficial effects of black cohosh extracts in the treatment of climacteric symptoms.