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Natural products (NPs) continue to inform the discovery and development of a diversity of drugs, both marketed and investigational. Pain, one of the most common of human experiences and profound challenges in medicine and biology, has emerged at the core of an urgent societal problem, in the United States and globally. The present study employs a retrospective analysis of an extensive set of published literature curated in the NAPRALERT database to identify NPs with experimental evidence of bioactivity supporting the selection and prioritization of NP leads with promise in pain management. The NAPRALERT pain data set currently documents >38,000 pain-relevant experiments reported in >1,750 distinct journals. The evidence presented here was annotated from >10,000 distinct scientific publications identifying NP extracts and isolates with experimental biological data indicating positive mitigation of pain, inflammation, and/or modulation of nociceptive signaling targets. Correlation of ethnomedical uses with experimental data represents a value-added approach to the selection and prioritization of leads. Dissemination of this unique NP/pain data set, with experimental data and information applicable to basic, translational, and clinical science stakeholders alike, furnishes practical evidence in support of a rational selection of NPs for directed pain research. A large portion of the NAPRALERT pain-relevant data set, along with a set of query tools designed to assist user-directed selection and prioritization of leads, are presented as Supporting Information in order to mitigate the limitations inherent in presenting such a large data set in (print) format. To support user efforts, this report involves explication of NAPRALERT data organization and the articulation of rational approaches to user-guided selection of evidence-based NP leads.

Phytochemical investigation of Marsypopetalum modestum (Annonaceae) led to the isolation of a new phenylpropanoid glycoside, lyciumphenylpropanoid B (10), along with nine known compounds (1-9) from an aqueous methanolic extract of the stem. Most compounds are reported from this genus for the first time. The structures of the isolated compounds were elucidated using spectroscopic methods including NMR spectroscopy, high-resolution mass spectrometry, and quantum chemical electronic circular dichroism (ECD) calculations. Cytotoxic and antitubercular activities of several isolated compounds were evaluated. Dipyrithione (1) displayed anti-mycobacterial (MIC = 0.23 µM) and cytotoxic (IC50 = 0.8 µM in Hep G2 cells; 4.1 µM in HCT 116 cells) activities. Kelampayoside A (8) showed moderate cytotoxic activity against cancer cells.

An evaluation of select medicinal species from Chapter 5 was performed by exploring the global scientific literature for evidence in support of the folkloric uses, through convergence in bioactivity profiles or corroboration of therapeutic uses. Five common affliction categories in the primary ethnopharmacological data were explored: diarrhea, fever, pain, rheumatism, and liver complaints. The LOTUS and NAPRALERT datasets were queried for chemical profiles of the species, folkloric use, and experimental bioactivity data, respectively. The IUCN Red List and CITES Checklists supplied data on conservation status of species. The Plants of the World Online dataset confirmed taxonomic nomenclature and provided species distributions for the 99 species presented in this chapter. Keyword searching of online text aggregators Google Scholar, PubMed, and EMBASE produced a set of recommended citations for each selected plant species and use category, which were then followed up by a review of citations for confirmation. Corroboration of use was found in the literature for 50% of all species, distributed among the top two-thirds of species, ranked by number of citations. The lower third of species, those of more limited geographic distribution, remain essentially un-referenced. Evaluation of the medicinal flora among the six medicinal plant preserves in this study reveals that only 6% of species were found to have shared use between provinces. This divergence reflects the rich geographic, cultural, and biological diversity of Laos, and points to the value of the MBPs-MPPs in biodiversity and cultural conservation and introduces prospects for further scientific investigation of the medicinal flora of Laos.

Contemporary bioinformatic and chemoinformatic capabilities hold promise to reshape knowledge management, analysis and interpretation of data in natural products research. Currently, reliance on a disparate set of non-standardized, insular, and specialized databases presents a series of challenges to data access, either within the discipline or to integration and interoperability between related domains. The fundamental elements of exchange are referenced structure-organism pairs that establish relationships between distinct molecular structures and the living organisms from which they were identified. Consolidating and sharing such information via an open platform has strong transformative potential for natural products research and beyond. This is the ultimate goal of the newly established LOTUS initiative, which has now completed the first steps toward the harmonization, curation, validation and open dissemination of 700,000+ referenced structure-organism pairs. LOTUS data is hosted on Wikidata and regularly mirrored on https://lotus.naturalproducts.net. Data sharing within the Wikidata framework broadens data access and interoperability, opening new possibilities for community curation and evolving publication models. Furthermore, embedding LOTUS data into the vast Wikidata knowledge graph will facilitate new biological and chemical insights. The LOTUS initiative represents an important advancement in the design and deployment of a comprehensive and collaborative natural products knowledge base.

Two new diprenylated coumaric acid isomers (1a and 1b) and two known congeners, capillartemisin A (2) and B (3), were isolated from Artemisia scoparia as bioactive markers using bioactivity-guided HPLC fractionation. Their structures were determined by spectroscopic means, including 1D and 2D NMR methods and LC-MS, with their purity assessed by 1D 1H pure shift qNMR spectroscopic analysis. The bioactivity of compounds was evaluated by enhanced accumulation of lipids, as measured using Oil Red O staining, and by increased expression of several adipocyte marker genes, including adiponectin in 3T3-L1 adipocytes relative to untreated negative controls. Compared to the plant’s 80% EtOH extract, these purified compounds showed significant but still weaker inhibition of TNFα-induced lipolysis in 3T3-L1 adipocytes. This suggests that additional bioactive substances are responsible for the multiple metabolically favorable effects on adipocytes observed with Artemisia scoparia extract.

Curcuma longa (turmeric) has an extensive history of ethnomedical use for common ailments, and “curcumin”-containing dietary supplements (CDS) are a highly visible portion of today’s self-medication market. Owing to raw material cost pressure, CDS products are affected by economically motivated, nefarious adulteration with synthetic curcumin (“syncumin”), possibly leading to unexpected toxicological issues due to “residual” impurities. Using a combination of targeted and untargeted (phyto)chemical analysis, this study investigated the botanical integrity of two commercial “turmeric” CDS with vitamin and other additives that were associated with reported clinical cases of hepatotoxicity. Analyzing multisolvent extracts of the CDS by 100% quantitative 1H NMR (qHNMR), alone and in combination with countercurrent separation (CCS), provided chemical fingerprints that allowed both the targeted identification and quantification of declared components and the untargeted recognition of adulteration. While confirming the presence of curcumin as a major constituent, the universal detection capability of NMR spectroscopy identification of significant residual impurities, including potentially toxic components. While the loss-free nature of CCS captured a wide polarity range of declared and unwanted chemical components, and also increased the dynamic range of the analysis, (q)HNMR determined their mass proportions and chemical constitutions. The results demonstrate that NMR spectroscopy can recognize undeclared constituents even if they represent only a fraction of the mass balance of a dietary supplement product. The chemical information associated with the missing 4.8% and 7.4% (m/m) in the two commercial samples, exhibiting an otherwise adequate curcumin content of 95.2% and 92.6%, respectively, pointed to a product integrity issue and adulteration with undeclared synthetic curcumin. Impurities from synthesis are most plausibly the cause of the observed adverse clinical effects. The study exemplifies how the simultaneously targeted and untargeted analytical principle of the 100% qHNMR method, performed with entry-level high-field instrumentation (400 MHz), can enhance the safety of dietary supplements by identifying adulterated, non-natural “natural” products.

This Perspective of the published essential medicinal chemistry of cannabidiol (CBD) provides evidence that the popularization of CBD-fortified or CBD-labeled health products and CBD-associated health claims lacks a rigorous scientific foundation. CBD’s reputation as a cure-all puts it in the same class as other “natural” panaceas, where valid ethnobotanicals are reduced to single, purportedly active ingredients. Such reductionist approaches oversimplify useful, chemically complex mixtures in an attempt to rationalize the commercial utility of natural compounds and exploit the “natural” label. Literature evidence associates CBD with certain semiubiquitous, broadly screened, primarily plant-based substances of undocumented purity that interfere with bioassays and have a low likelihood of becoming therapeutic agents. Widespread health challenges and pandemic crises such as SARS-CoV-2 create circumstances under which scientists must be particularly vigilant about healing claims that lack solid foundational data. Herein, we offer a critical review of the published medicinal chemistry properties of CBD, as well as precise definitions of CBD-containing substances and products, distilled to reveal the essential factors that impact its development as a therapeutic agent.

In a continuing search for neuroprotective stilbenoids, two uncommon diastereomeric stilbenoid glucosides, africanoside A (1) and B (2) as well as fifteen known stilbenes, E-resveratrol, E-gnetifolin E, E-piceid, E-resveratroloside, E-gnetifolin K, E-gnetol, E-isorhapontigenin, E-isorhapontin, E-isorhapontigenin-4′-O-glucopyranoside, E-gnetin C, E-bisisorhapontigenin B, E-gnemonoside A, E-gnemonoside C, E-gnemonoside D, and E-gnetin E, were isolated from rhizomes of Gnetum africanum, using a combination of centrifugal partition chromatography and preparative HPLC. The structure of these two stilbenoids was investigated by NMR, vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) experiments. The absolute configurations of 1, and 2 were established by VCD as (7aS,8aS) and (7aR,8aR), respectively. Compound 1 significantly chelated Fe(II) at 100, 250 and 500 μM. The ability of stilbenoids to chelate ferrous ions which are implicated in physiopathological hallmark of Alzheimer’s disease provides useful data for potential treatment.

Chlorophylls are present in all extracts from the aerial parts of green plant materials. Chlorophylls may act as in vitro bioassay nuisance compounds, possibly preventing the reproducibility and accurate measurement of readouts due to their UV/vis absorbance, fluorescence properties, and tendency to precipitate in aqueous media. Despite the diversity of methods used traditionally to remove chlorophylls, details about their mode of operation, specificity, and reproducibility are scarce. Herein, we report a selective and efficient 45 min liquid–liquid/countercurrent chlorophyll cleanup method using Centrifugal Partition Chromatography (CPC) with a solvent system composed of hexanes–EtOAc–MeOH–water (5:5:5:5, v/v) in elution-extrusion mode. The broader utility of the method was assessed with four different extracts prepared from three well-characterized plant materials: Epimedium sagittatum (leaves), Senna alexandrina (leaves), and Trifolium pratense (aerial parts). The reproducibility of the method, the selectivity of the chlorophyll removal, as well as the preservation of the phytochemical integrity of the resulting chlorophyll-free (“degreened”) extracts were evaluated using HPTLC, UHPLC-UV, 1H NMR spectroscopy, and LC-MS as orthogonal phytochemical methods. The cleanup process adequately preserves the metabolomic diversity as well as the integrity of the original extracts. This method was found to be sufficiently rapid for the “degreening” of botanical extracts in higher-throughput sample preparation for further biological screening.

Whereas generic, LC-based pharmaceutical control quality procedures depend largely on the detection mode and can be particularly ‘blind’ to certain impurities, NMR is a more versatile and, thus, often more judicious detector. While adulteration presents ever-evolving challenges for the analysis of active pharmaceutical ingredients (APIs) and finished products sold in the worldwide (online) marketplace, research chemicals are usually trusted rather than being considered flawed or even adulterated.

This report shows how NMR analysis uncovered the unanticipated presence of substantial amounts of mannitol (20 and 43% w/w) as undeclared constituent in two custom synthetic peptides, DR and DRVYI, that were sourced commercially. Quantitative 1H NMR (qHNMR) readily detected the contaminant, even on a 60 MHz benchtop instrument, and quantified the highly polar and UV-transparent adulterant. Quantum-mechanical 1H iterative Full Spin Analysis (HiFSA) not only achieved unambiguous identification of both the mannitol and the peptides, but also confirmed the quantitative results.

Mass spectrometry (MS) offers unrivalled sensitivity for the metabolite profiling of complex biological matrices encountered in natural products (NP) research. The massive and complex sets of spectral data generated by such platforms require computational approaches for their interpretation. Within such approaches, computational metabolite annotation automatically links spectral data to candidate structures via a score, which is usually established between the acquired data and experimental or theoretical spectral databases (DB). This process leads to various candidate structures for each MS features. However, at this stage, obtaining high annotation confidence level remains a challenge notably due to the extensive chemodiversity of specialized metabolomes. The design of a metascore is a way to capture complementary experimental attributes and improve the annotation process. Here, we show that integrating the taxonomic position of the biological source of the analyzed samples and candidate structures enhances confidence in metabolite annotation. A script is proposed to automatically input such information at various granularity levels (species, genus, and family) and complement the score obtained between experimental spectral data and output of available computational metabolite annotation tools (ISDB-DNP, MS-Finder, Sirius). In all cases, the consideration of the taxonomic distance allowed an efficient re-ranking of the candidate structures leading to a systematic enhancement of the recall and precision rates of the tools (1.5- to 7-fold increase in the F1 score). Our results clearly demonstrate the importance of considering taxonomic information in the process of specialized metabolites annotation. This requires to access structural data systematically documented with biological origin, both for new and previously reported NPs. In this respect, the establishment of an open structural DB of specialized metabolites and their associated metadata, particularly biological sources, is timely and critical for the NP research community.

Ensuring identity, purity, and reproducibility are equally essential during synthetic chemistry, drug discovery, and for pharmaceutical product safety. Many peptidic APIs are large molecules that require considerable effort for integrity assurance. This study builds on quantum mechanical 1H iterative Full Spin Analysis (HiFSA) to establish NMR peptide sequencing methodology that overcomes the intrinsic limitations of principal compendial methods in identifying small structural changes or minor impurities that affect effectiveness and safety. HiFSA sequencing yields definitive identity and purity information concurrently, allowing for API quality assurance and control (QA/QC). Achieving full peptide analysis via NMR building blocks, the process lends itself to both research and commercial applications as 1D 1H NMR (HNMR) is the most sensitive and basic NMR experiment. The generated HiFSA profiles are independent of instrument or software tools and work at any magnetic field strength. Pairing with absolute or 100% qHNMR enables quantification of mixtures and/or determination of peptide conformer populations. Demonstration of the methodology uses single amino acids (AAs) and peptides of increasing size, including the octapeptide, angiotensin II, and the nonapeptide, oxytocin. The feasibility of HiFSA coupled with automated NMR and qHNMR for use in QC/QA efforts is established through case-based examples and recommended procedures.

With contributions from the global natural product (NP) research community, and continuing the Raw Data Initiative, this review collects a comprehensive demonstration of the immense scientific value of disseminating raw nuclear magnetic resonance (NMR) data, independently of, and in parallel with, classical publishing outlets. A comprehensive compilation of historic to present-day cases as well as contemporary and future applications show that addressing the urgent need for a repository of publicly accessible raw NMR data has the potential to transform natural products (NPs) and associated fields of chemical and biomedical research. The call for advancing open sharing mechanisms for raw data is intended to enhance the transparency of experimental protocols, augment the reproducibility of reported outcomes, including biological studies, become a regular component of responsible research, and thereby enrich the integrity of NP research and related fields.

Humans have co-evolved alongside numerous other organisms, some having a profound effect on health and nutrition. As the earliest pharmaceutical subject, pharmacognosy has evolved into a meta-discipline devoted to natural biomedical agents and their functional properties. While the acquisition of expanding data volumes is ongoing, contextualization is lagging. Thus, we assert that the establishment of an integrated and open databases ecosystem will nurture the discipline. After proposing an epistemological framework of knowledge acquisition in pharmacognosy, this study focuses on recent computational and analytical approaches. It then elaborates on the flux of research data, where good practices could foster the implementation of more integrated systems, which will in turn help shaping the future of pharmacognosy and determine its constitutional societal relevance.

This is an answer to some reactions to our review article on Curcumin.

Curcumin is a constituent (up to ∼5%) of the traditional medicine known as turmeric. Interest in the therapeutic use of turmeric and the relative ease of isolation of curcuminoids has led to their extensive investigation. Curcumin has recently been classified as both a PAINS (pan-assay interference compounds) and an IMPS (invalid metabolic panaceas) candidate. The likely false activity of curcumin in vitro and in vivo has resulted in >120 clinical trials of curcuminoids against several diseases. No double-blinded, placebo controlled clinical trial of curcumin has been successful. This manuscript reviews the essential medicinal chemistry of curcumin and provides evidence that curcumin is an unstable, reactive, nonbioavailable compound and, therefore, a highly improbable lead. On the basis of this in-depth evaluation, potential new directions for research on curcuminoids are discussed.

Chemical standardization, along with morphological and DNA analysis ensures the authenticity and advances the integrity evaluation of botanical preparations. Achievement of a more comprehensive, metabolomic standardization requires simultaneous quantitation of multiple marker compounds. Employing quantitative 1H NMR (qHNMR), this study determined the total isoflavone content (TIfCo; 34.5–36.5% w/w) via multimarker standardization and assessed the stability of a 10-year-old isoflavone-enriched red clover extract (RCE). Eleven markers (nine isoflavones, two flavonols) were targeted simultaneously, and outcomes were compared with LC-based standardization. Two advanced quantitative measures in qHNMR were applied to derive quantities from complex and/or overlapping resonances: a quantum mechanical (QM) method (QM-qHNMR) that employs 1H iterative full spin analysis, and a non-QM method that uses linear peak fitting algorithms (PF-qHNMR). A 10 min UHPLC-UV method provided auxiliary orthogonal quantitation. This is the first systematic evaluation of QM and non-QM deconvolution as qHNMR quantitation measures. It demonstrates that QM-qHNMR can account successfully for the complexity of 1H NMR spectra of individual analytes and how QM-qHNMR can be built for mixtures such as botanical extracts. The contents of the main bioactive markers were in good agreement with earlier HPLC-UV results, demonstrating the chemical stability of the RCE. QM-qHNMR advances chemical standardization by its inherent QM accuracy and the use of universal calibrants, avoiding the impractical need for identical reference materials.

Centrifugal partition chromatography (CPC) and all countercurrent separation apparatus provide chemists with efficient ways to work with complex matrixes, especially in the domain of natural products. However, despite the great advances provided by these techniques, more efficient ways of analyzing the output flow would bring further enhancement. This study describe a hyphenated approach made by coupling NMR with CPC through a hybrid-indirect coupling made possible by using a solid phase extraction (SPE) apparatus intended for high-pressure liquid chromatography (HPLC)-NMR hyphenation. Some hardware changes were needed to adapt the incompatible flow-rates and a reverse-engineering approach that led to the specific software required to control the apparatus. 1D 1HNMR and 1H–1H correlation spectroscopy (COSY) spectra were acquired in reasonable time without the need for any solvent-suppression method thanks to the SPE nitrogen drying step. The reduced usage of expensive deuterated solvents from several hundreds of milliliters to the milliliter order is the major improvement of this approach compared to the previously published ones.

Abstract from conference

NAPRALERT is a database on natural products, including data on ethnobotany, chemistry, pharmacology, toxicology, and clinical trials from literature dating back to the 19th century. Established in 1975 by Norman R. Farnsworth, it became a web accessible resource in 2005 but soon became stagnant while literature grew exponentially. After a complete rewrite of the platform, the focus is now on connecting this resource to the rest of the existing databases and expanding its usability. The creation of a Pharmacognosy/Natural Product ontology will foster better understanding of this domain, its linking potential with other resources and the ability to automatize literature annotation and entry efficiently.

The notion of data transparency is gaining a strong awareness among the scientific community. The availability of raw data is actually regarded as a fundamental way to advance science by promoting both integrity and reproducibility of research outcomes. Particularly, in the field of natural product and chemical research, NMR spectroscopy is a fundamental tool for structural elucidation and quantification (qNMR). As such, the accessibility of original NMR data, i.e., Free Induction Decays (FIDs), fosters transparency in chemical research and optimizes both peer review and reproducibility of reports by offering the fundamental tools to perform efficient structural verification. Although original NMR data are known to contain a wealth of information, they are rarely accessible along with published data. This viewpoint discusses the relevance of the availability of original NMR data as part of good research practices not only to promote structural correctness, but also to enhance traceability and reproducibility of both chemical and biological results.

Composite-based, tooth colored dental restorations, in spite of their aesthetic appeal, are limited by a short life span. Every subsequent restoration results in the loss of healthy dental tissue. Thus, a bio-mimetic approach has been developed to enhance the mechanical strength of dentin using plant-derived proanthocyanidins (PACs). From a panel of eight active plants, grape seed extract showed the highest dentin biomodification potential, a 15-fold enhancement of dentin stiffness measured in MPa. Fractions with varying degrees of polymerization (DP) were obtained using solvent partitioning and centrifugal partition chromatography (CPC). Oligomeric PACs (OPACs, DP 2 – 7) surfaced as the most promising dentin biomodifiers compared to the constituent monomers and polymers (Dp ≥8). OPACs with DP 3 to 4 showed the most efficacious dentin-PAC interaction as evaluated by bio-mechanical tests. While one arm of the separation focused on the development of a highly active custom-made tri- and tetra-meric OPAC enriched mixture (GSE3+4), OPACs were also purified as single chemical entities. OPACs with predominantly 4β→8/6 B-type interflavan linkages (IFLs) along with one having a unique 2→8 IFL were isolated. Structural characterization employed 1D and 2D NMR at low temperature (255K) to overcome line-broadening due to atropisomerism. The presence of gallate ester moieties is a characteristic feature of grape seed PACs and the biological evaluation also highlighted the enhanced effect of galloylated OPACs on dentin biomodification. Grape seeds are thus, a viable source of novel restorative dental biomaterials and highlight a novel application of plant-based natural products in the biomedical field.

NAPRALERT is a database on natural products, including data on the ethnobotany, chemistry, pharmacology, toxicology, and clinical trials. It was established in 1975 by the late Norman R. Farnsworth, at a time when computerized databases were just starting. It became web-accessible in 2005. Due to resource constraints, few enhancements were made to the existing database structure. Now, 10 years later, NAPRALERT faces the challenge of catching-up with other well-established resources.

Dereplication represents a key step for rapidly identifying known secondary metabolites in complex biological matrices. In this context, liquid-chromatography coupled to high resolution mass spectrometry (LC-HRMS) is increasingly used and, via untargeted data-dependent MS/MS experiments, massive amounts of detailed information on the chemical composition of crude extracts can be generated. An efficient exploitation of such data sets requires automated data treatment and access to dedicated fragmentation databases. Various novel bioinformatics approaches such as molecular networking (MN) and in-silico fragmentation tools have emerged recently and provide new perspective for early metabolite identification in natural products (NPs) research. Here we propose an innovative dereplication strategy based on the combination of MN with an extensive in-silico MS/MS fragmentation database of NPs. Using two case studies, we demonstrate that this combined approach offers a powerful tool to navigate through the chemistry of complex NPs extracts, dereplicate metabolites, and annotate analogues of database entries.

The revision of the structure of the sesquiterpene aquatolide from a bicyclo[2.2.0]hexane to a bicyclo[2.1.1]hexane structure using compelling NMR data, X-ray crystallography, and the recent confirmation via full synthesis exemplify that the achievement of “structural correctness” depends on the completeness of the experimental evidence. Archived FIDs and newly acquired aquatolide spectra demonstrate that archiving and rigorous interpretation of 1D 1H NMR data may enhance the reproducibility of (bio)chemical research and curb the growing trend of structural misassignments. Despite being the most accessible NMR experiment, 1D 1H spectra encode a wealth of information about bonds and molecular geometry that may be fully mined by 1H iterative full spin analysis (HiFSA). Fully characterized 1D 1H spectra are unideterminant for a given structure. The corresponding FIDs may be readily submitted with publications and collected in databases. Proton NMR spectra are indispensable for structural characterization even in conjunction with 2D data. Quantum interaction and linkage tables (QuILTs) are introduced for a more intuitive visualization of 1D J-coupling relationships, NOESY correlations, and heteronuclear experiments. Overall, this study represents a significant contribution to best practices in NMR-based structural analysis and dereplication.

High-throughput biology has contributed a wealth of data on chemicals, including natural products (NPs). Recently, attention was drawn to certain, predominantly synthetic, compounds that are responsible for disproportionate percentages of hits but are false actives. Spurious bioassay interference led to their designation as pan-assay interference compounds (PAINS). NPs lack comparable scrutiny, which this study aims to rectify. Systematic mining of 80+ years of the phytochemistry and biology literature, using the NAPRALERT database, revealed that only 39 compounds represent the NPs most reported by occurrence, activity, and distinct activity. Over 50% are not explained by phenomena known for synthetic libraries, and all had manifold ascribed bioactivities, designating them as invalid metabolic panaceas (IMPs). Cumulative distributions of ∼200,000 NPs uncovered that NP research follows power-law characteristics typical for behavioral phenomena. Projection into occurrence–bioactivity–effort space produces the hyperbolic black hole of NPs, where IMPs populate the high-effort base.

Plant derived proanthocyanidins are well-established compounds exhibiting high dentin biomodification potency. Among the effects, enhanced tissue biomechanics and biostability are of relevance to restorative dentistry. This study evaluated the adhesive properties of an enriched proanthocyanidin primer on the bond strength of experimental resin-based adhesives containing variable concentrations of HEMA.

The ability of certain oligomeric proanthocyanidins (OPACs) to enhance the biomechanical properties of dentin involves collagen cross-linking of the 1.3–4.5 nm wide space via protein–polyphenol interactions. A systematic interdisciplinary search for the bioactive principles of pine bark has yielded the trimeric PAC, ent-epicatechin-(4β→8)-epicatechin-(2β→O→7,4β→8)-catechin (3), representing the hitherto most potent single chemical entity capable of enhancing dentin stiffness. Building the case from two congeneric PAC dimers, a detailed structural analysis decoded the stereochemistry, spatial arrangement, and chemical properties of three dentin biomodifiers. Quantum-mechanics-driven 1H iterative full spin analysis (QM-HiFSA) of NMR spectra distinguished previously unrecognized details such as higher order J coupling and provided valuable information about 3D structure. Detection and quantification of H/D-exchange effects by QM-HiFSA identified C-8 and C-6 as (re)active sites, explain preferences in biosynthetic linkage, and suggest their involvement in dentin cross-linking activity. Mapping of these molecular properties underscored the significance of high δ precision in both 1H and 13C NMR spectroscopy. Occurring at low- to subppb levels, these newly characterized chemical shift differences in ppb are small but diagnostic measures of dynamic processes inherent to the OPAC pharmacophores and can help augment our understanding of nanometer-scale intermolecular interactions in biomodified dentin macromolecules.

1D NMR spectra contain a wealth of vital structural information that can enhance the description of bioactive molecules. The present study demonstrates how quantum-mechanics driven 1H iterative Full Spin Analysis (QM-HiFSA) is capable of distinguishing spectral detail that cannot be interpreted manually or visually, but provides important information of the 3D structure and bonding (re-)activity of the molecules. This approach is established by analyzing 1D NMR spectra of oligomeric proanthocyanidins (OPACs), which exhibit high dentin bioactivity, and were isolated from the inner bark of pine. The higher order coupling and proton-deuterium exchange effects observed in these complex molecules were fully explained and quantified by QM-HiFSA. Dimeric and trimer OPACs provide evidence that high δ precision is applicable to 13C, in addition to 1H 1D NMR spectra, requiring reporting to the ppb level and below. Both the nano chemical shifts (ppb) and the associated nano substituent chemical shifts (s.c.s.) are significant properties of the 1H and 13C NMR spectra and enable recognition of structural properties that are relevant to better understanding of the intermolecular interactions between the OPAC pharmacophores and dentin micromolecules triggering enhanced tissue mechanics.

The acquisition of 1D 1H NMR (HNMR) spectra is one of earliest steps in characterizing natural products and other organic molecules. For publication, HNMR information usually is “converted” into a table format, and sometimes spectral plots are provided. However, this transformation is lossy and frequently insufficient for unambiguous dereplication. This ambiguity can even lead to structural revision, such as in the recent case of aquatolide (1), a sesquiterpene lactone from Asteriscus aquaticus. Our study demonstrates that public dissemination of original (digital) HNMR data (FIDs) can be a powerful means of enhancing the reproducibility of structural assignments and, thus, any downstream biological studies. Using the archived 800 MHz HNMR spectrum, and employing a semi-automated quantum mechanics-driven spectral analysis (HiFSA), we were able to rule out the initial assignment (1a), confirm the revision (1b), and achieve the full interpretation of the HNMR fingerprints. Using additional examples of constitutional and diastereomeric isomers which exhibit complex and near-identical HNMR spectra, we show that the public sharing of original HNMR data (FIDs) is not only essential for robust structural assignments, but can enhance the reproducibility of research with bioactive natural products and other organic molecules simply and productively.

C-glycosylated flavones, including orientin, isoorientin, vitexin, and isovitexin, are minor but biologically significant constituents of fruit extracts of the chaste-tree (Vitex agnus-castus L.), a botanical supplement used to treat PMS and postmenopausal symptoms. The partition coefficient, or K-value, is the ratio of the concentration of a compound in each phase of a biphasic solvent mixture and is a physicochemical property of a particular compound in a particular solvent system. This value can be used to predict retention volume (V ret) in a countercurrent separation procedure. The K-values of C-glycosylflavones present in complex botanical fractions have been determined in a number of solvent system families (HEMWat, EBWat, HterAcWat, terAcWat) using the shake-flask technique combined with relative LC-MS quantification. This K-value database has been used to develop targeted centrifugal partition (CPC) and high-speed countercurrent chromatography (HSCCC) methods. In each separation procedure the actual K value and V ret was reasonably predicted by the shake-flask partition experiment, confirming the utility of this approach in choosing a solvent system and targeting the fraction that contains the desired compound. This K-value database allowed for the efficient isolation of C-glycosylflavones from V. agnus-castus using orthogonal CCC and CPC methods.

A recent article by Baell(1) on the problems experienced by medicinal chemists with pan-assay interference compounds (PAINS) and Shoichet’s work(2) on the impact of aggregation occurring in high throughput screening libraries, prompts a consideration of how these and other similar problems are experienced by pharmacognosists with promiscuous invalid metabolites as panaceas (PIMPs). Contrary to the classical definition of secondary metabolites as being species specific (or near specific), several natural products, particularly in the more extensively investigated plant kingdom, are common across species, genera, and even families (e.g. β-sitosterol). In the course of bioactivity-guided fractionation, PIMPs have shown up as major components in active fractions of a wide variety of pharmacological assays, i.e., they have been designated as panaceas. As in the case of PAINS, these assay results are almost invariably invalid and lead enthusiastic young scientists down a garden path. Why does this happen and how can it be avoided? Interestingly, the advances in modern methods of structure determination have exacerbated this problem, because it is possible to determine the structure of a compound when it is quite impure, and residual complexity is characteristic of chromatographic fractionation. That these residuals are often the source of the bioactivity is also frequently overlooked. Classic examples where this has occurred and ways to avoid it will be outlined.

Proanthocyanidins (PACs) are secondary plant metabolites that mediate nonenzymatic collagen cross-linking and enhance the properties of collagen based tissue, such as dentin. The extent and nature of cross-linking is influenced by the composition and specific chemical structure of the bioactive compounds present in certain PAC-rich extracts. This study investigated the effect of the molecular weight and stereochemistry of polyphenol compounds on two important properties of dentin, biomechanics, and biostability. For that, purified phenols, a phenolic acid, and some of its derivatives were selected: PAC dimers (A1, A2, B1, and B2) and a trimer (C1), gallic acid (Ga), its esters methyl-gallate (MGa) and propyl-gallate (PGa), and a pentagalloyl ester of glucose (PGG). Synergism was assessed by combining the most active PAC and gallic acid derivative. Mechanical properties of dentin organic matrix were determined by the modulus of elasticity obtained in a flexural test. Biostability was evaluated by the resistance to collagenase degradation. PACs significantly enhanced dentin mechanical properties and decreased collagen digestion. Among the gallic acid derivatives, only PGG had a significant enhancing effect. The lack of observed C1:PGG synergy indicates that both compounds have similar mechanisms of interaction with the dentin matrix. These findings reveal that the molecular weight of polyphenols have a determinant effect on their interaction with type I collagen and modulates the mechanism of cross-linking at the molecular, intermolecular, and inter-microfibrillar levels.

The polyphenol source and molecular structure complexity affects interactions of proanthocyanidins (PACs) with dentin matrix. Therefore, this study evaluated the dentin bioactivity of compounds from a non-galloylated PACs-rich extract.

Stilbenoids have received increasing attention over the last two decades since the discovery of resveratrol in wine. With an ever-growing rhythm, a multitude of biological activities of naturally occurring stilbenes are being reported. In this work, we investigated minor stilbenoid compounds from Vitis vinifera stalks. The compounds were purified by means of centrifugal partition chromatography (CPC), a countercurrent-separation technique. Electrospray ionization–ion trap mass spectrometry (ESI–IT-MS) after optimization proved to be extremely efficient for the detection of these new molecules, providing both structural information for structure elucidation and a means to achieve identification even with minute amounts. Here a new stereoisomer of E-miyabenol C, E-cis-cis-miyabenol C (2), along with the already reported E-trans-cis-miyabenol C (1) and E-cis-trans-miyabenol C (3), was purified from a complex Vitis vinifera cane extract, using adapted solvent systems K and L from the ‘Arizona’ solvent system range, without the need for any solid support. Moreover, compounds 1–3 showed an inhibitory activity on α-synuclein filament formation.

Dimeric stilbene glucosides 1–3 [two diastereomers of (−)-gnemonoside A (1a and 1b), (−)-gnemonoside C (2), and (−)-gnemonoside D (3)] as well as a mixture of the two enantiomers of gnetin C (4) were isolated from the rhizomes of Gnetum africanum. The two enantiomers of gnetin C, (+)-4 and (−)-4, were obtained from the aglycones of 1a and 1b, respectively. The configurations of these stilbenoids were investigated by NMR and vibrational circular dichroism (VCD) experiments. The absolute configurations of (−)-1a, (−)-2, (−)-3, and (−)-4 were established as 7aS,8aS by VCD spectroscopy in combination with density functional theory calculations. The antiamyloidogenic activity of the isolated stilbenes was also evaluated versus beta-amyloid fibrils. The four glucosides of gnetin C (1a, 1b, 2, and 3) were found to be the most active compounds, with inhibition percentages of 56, 56, 58, and 54 at 10 μM, respectively.

The present study demonstrates the importance of adequate precision when reporting the δ and J parameters of frequency domain 1H NMR (HNMR) data. Using a variety of structural classes (terpenoids, phenolics, alkaloids) from different taxa (plants, cyanobacteria), this study develops rationales that explain the importance of enhanced precision in NMR spectroscopic analysis and rationalizes the need for reporting Δδ and ΔJ values at the 0.1–1 ppb and 10 mHz level, respectively. Spectral simulations paired with iteration are shown to be essential tools for complete spectral interpretation, adequate precision, and unambiguous HNMR-driven dereplication and metabolomic analysis. The broader applicability of the recommendation relates to the physicochemical properties of hydrogen (1H) and its ubiquity in organic molecules, making HNMR spectra an integral component of structure elucidation and verification. Regardless of origin or molecular weight, the HNMR spectrum of a compound can be very complex and encode a wealth of structural information that is often obscured by limited spectral dispersion and the occurrence of higher order effects. This altogether limits spectral interpretation, confines decoding of the underlying spin parameters, and explains the major challenge associated with the translation of HNMR spectra into tabulated information. On the other hand, the reproducibility of the spectral data set of any (new) chemical entity is essential for its structure elucidation and subsequent dereplication. Handling and documenting HNMR data with adequate precision is critical for establishing unequivocal links between chemical structure, analytical data, metabolomes, and biological activity. Using the full potential of HNMR spectra will facilitate the general reproducibility for future studies of bioactive chemicals, especially of compounds obtained from the diversity of terrestrial and marine organisms.

Grapevine canes are rich in resveratrol and its complex derivatives. These compounds have many biological activities and are needed mainly for health purposes. Canes, which are often wasted, can be used to produce these high-value compounds at low cost. We studied sixteen Vitis vinifera L. cultivars among the most widely cultivated ones worldwide. Polyphenols were extracted from their canes and identified by liquid chromatography–nuclear magnetic resonance spectroscopy. We accurately determined the content of E-ε-viniferin, E-resveratrol, E-piceatannol, and vitisin B and, for the first time, that of hopeaphenol and miyabenol C. The canes did not contain these major stilbene compounds in similar proportions, and their abundance and order of abundance varied according to the cultivar. For instance, Pinot noir has very high levels of E-resveratrol and E-ε-viniferin; Gewurztraminer has very high levels of vitisin B, and Carignan and Riesling have very high levels of hopeaphenol. These findings suggest that the right cultivar should be used to obtain the highest yield of a polyphenol of interest.

Microglia-driven inflammatory processes are thought to play an important role in ageing and several neurological disorders. Since consumption of a diet rich in polyphenols has been associated with anti-inflammatory and neuroprotective effects, we studied the effects of twenty-five stilbenoids isolated from Milicia excelsa, Morus alba, Gnetum africanum, and Vitis vinifera. These compounds were tested at 5 and 10 µM on BV-2 microglial cells stimulated with bacterial lipopolysaccharide. Ten stilbenoids reduced lipopolysaccharide-induced nitric oxide production at 5 and/or 10 µM. Two tetramers, E-vitisin A and E-vitisin B, were the most effective molecules. Moreover, they attenuated the expression of the inducible NO synthase protein and gene.

Flavours extracted from oak wood during barrel ageing contribute to the organoleptic character of wines and spirits. The aim of this work was to identify the glycosidic precursors of the key volatile compounds responsible for oak wood aroma. Oak extract is a very complex matrix and, furthermore, precursors are present in very small quantities. Preparative centrifugal partition chromatography (CPC) is a promising solution for purifying the oak extract. The solvent system was selected on the basis of the partition coefficient of glycosidase enzyme activity (Kca). Thanks to the efficacy of CPC separation, three glucoside gallates were subsequently isolated by HPLC chromatography. Vanillin-(6′-O-galloyl)-O-β-d-glucopyranoside, 3,4,5-trimethoxyphenyl-(6′-O-galloyl)-O-β-d-glucopyranoside, and (6R,9R)-3-oxo-α-ionol-9-O-(6′-O-galloyl)-β-glucopyranoside (macarangioside E) were isolated and identified. This was the first time that vanillin-(6′-O-galloyl)-O-β-d-glucopyranoside was identified and the first time that macarangioside E was isolated from oak wood. Heating macarangioside E resulted in the formation of megastigmatrienone, which has an aroma reminiscent of tobacco.

Wine is a major dietary source of numerous potentially health promoting stilbenoids that have been the subject of many qualitative and quantitative studies. However, our initial HPLC–MS analyses of crude wine samples demonstrated the presence of compounds with molecular weights matching characteristic stilbenoid dimers, trimers, and tetramers that were unaccounted for in the literature. Due to the likelihood that these are known compounds, a chemical dereplication method is highly desirable. We developed such a method using LC-DAD–MS monitored fractionation steps, using adsorption and centrifugal partition chromatography (CPC), to obtain fractions rich in stilbenoids for analysis in stopped-flow LC–NMR. 1H NMR spectra and MS data were cross-referenced with our laboratory database and the literature for identification. This method yielded highly useful structural information, allowing the characterization of previously unidentified stilbenoids in wine, ampelopsin C, isohopeaphenol, quadrangularin A, and E-ω-viniferin. These results demonstrate the usefulness of stop-flow LC–NMR in conjunction with LC–MS guided fractionation for the dereplication of compounds of interest in general, and specifically for expanding the current knowledge of wine chemistry.

We present stilbenoid profiles of canes from 16 grapevines. Fifteen stilbenoids were obtained through isolation and structure identification using MS, NMR, and [α]D or as commercial standards. An HPLC–UV method for the simultaneous quantification of nine of these stilbenoids was developed and applied to canes of Vitis amurensis, Vitis arizonica, Vitis berlandieri, Vitis betulifolia, Vitis cinerea, Vitis × champini, Vitis × doaniana, Vitis labrusca, Vitis candicans (syn. Vitis mustangensis), Vitis riparia, Vitis rupestris, Vitis vinifera, Muscadinia rotundifolia, and a V. vinifera × M. rotundifolia hybrid. In these species, E-ampelopsin E, E-amurensin B, E-piceid, E-piceatannol, E-resveratrol, E-resveratroloside, E-ε-viniferin, E-ω-viniferin, and E-vitisin B were quantified, when found in sufficient amounts. Total concentrations ranged from ∼2.2 to 19.5 g/kg of dry weight. Additional stilbenoids, E-3,5,4′-trihydroxystilbene 2-C-glucoside, Z-ampelopsin E, Z-trans-miyabenol C, E-trans-miyabenol C, scirpusin A, and Z-vitisin B, were identified but not quantified. Our results indicate that canes, particularly those of non-vinifera species, have substantial quantities of valuable, health-promoting stilbenoids.

The interaction between Vitis vinifera and trunk disease fungi requires better understanding. We studied the role of phenolics as possible plant defense compounds in this context. The impact of 24 grapevine phenolic compounds was determined on 6 major wood decay fungi by an in vitro agar plate assay. Hydroxystilbenoids, especially oligomers such as miyabenol C, isohopeaphenol, and vitisin A and B, greatly reduced the growth of the fungi, except that of Phaeoacremonium aleophilum. A detailed investigation in 10 Botryosphaeriaceae strains revealed that all of the studied members of this family display a common susceptibility to phenolics that is more or less significant. Then we undertook a quantitative analysis of stilbenoid content in grapevine plantlets inoculated with Botryosphaeriaceae to investigate whether in planta these fungi have to counteract the most active phenolics. On the basis of our results, the possible role of phenolics in grapevine defense against trunk disease agents is discussed.

Countercurrent chromatography techniques are increasingly used in the fractionation and purification steps of natural products due to their excellent versatility and efficiency. However, one of the major bottlenecks in liquid-liquid chromatography is the solvent system selection. In this work, a NMR-based analysis of complex solvent mixtures was developed for biphasic system selection and modification. This allowed us to develop more versatile separation conditions, such as gradients. Additionally, we hyphenated our countercurrent chromatography device with NMR and mass spectrometry. Our hybrid technique afforded a time-saving approach with a significant reduction in compound handling, an asset in working with minor and/or sensitive compounds. Current results and further developments will be presented and discussed.

The phytochemical study of the root extract of the stilbenoid-rich Vitis riparia × Vitis berlandieri grapevine was carried out by centrifugal partition chromatography (CPC). For this reason, we developed a new elution mode we named back-step, which allowed us to obtain cleaner fractions and a more efficient separation process when used in conjunction with a classical elution approach. Three hydroxystilbenes: (E)-resveratrol, (E)-ɛ-viniferin and (E)-vitisin C, with greater than 90% purity were thus obtained through such process, with minimal sample handling and purification steps. Online coupling of CPC to ESI mass spectrometry was used for optimization of the separation parameters and to facilitate the characterization of the stilbenoids. This study details the first phytochemical investigation of stilbenoids from the hybrid species together with a new elution mode able to widen the range of ARIZONA biphasic systems.