13C NMR-based dereplication using MixONat software to decipher potent anti-cholinesterase compounds in Mesua lepidota bark.

A bio-assay guided fractionation strategy based on cholinesterase assay combined with 13C NMR-based dereplication was used to identify active metabolites from the bark of Mesua lepidota. Eight compounds were identified with the aid of the 13C NMR-based dereplication software, MixONat, i.e., sitosterol (1), stigmasterol (2), α-amyrin (3), friedelin (6), 3ß-friedelinol (7), betulinic acid (9), lepidotol A (10) and lepidotol B (11). Further bio-assay guided isolation of active compounds afforded one xanthone, pyranojacareubin (12) and six coumarins; lepidotol A (10), lepidotol B (11), lepidotol E (13), lepidotin A (14), and lepidotin B (15), including a new Mammea coumarin, lepidotin C (16). All the metabolites showed strong to moderate butyrylcholinesterase (BChE) inhibition. Lepidotin B (15) exhibited the most potent inhibition towards BChE with a mix-mode inhibition profile and a Ki value of 1.03 µM. Molecular docking and molecular dynamics simulations have revealed that lepidotin B (15) forms stable interactions with key residues within five critical regions of BChE. These regions encompass residues Asp70 and Tyr332, the acyl hydrophobic pocket marked by Leu286, the catalytic triad represented by Ser198 and His438, the oxyanion hole (OH) constituted by Gly116 and Gly117, and the choline binding site featuring Trp82. To gauge the binding strength of lepidotin B (15) and to pinpoint pivotal residues at the binding interface, free energy calculations were conducted using the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) approach. This analysis not only predicted a favourable binding affinity for lepidotin B (15) but also facilitated the identification of significant residues crucial for the binding interaction.

Chemodiversity of propolis samples collected in various areas of Benin and Congo: Chromatographic profiling and chemical characterization guided by 13 C NMR dereplication.

INTRODUCTION: Propolis is a resinous natural substance collected by honeybees from buds and exudates of various trees and plants; it is widely accepted that the composition of propolis depends on the phytogeographic characteristics of the site of collection. OBJECTIVES: The aim of this study was to determine the phytochemical composition of ethanolic extracts from eight propolis batches collected in different regions of Benin (north, center, and south) and Congo, Africa. MATERIAL AND METHODS: Characterization of propolis samples was performed by using different hyphenated chromatographic methods combined with carbon-13 nuclear magnetic resonance (13 C NMR) dereplication with MixONat software. Their antioxidant or anti-advanced glycation end-product (anti-AGE) activity was then evaluated by using diphenylpicrylhydrazyl and bovine serum albumin assays, respectively. RESULTS: Chromatographic analyses combined with 13 C NMR dereplication showed that two samples from the center of Benin exhibited, in addition to a huge amount of pentacyclic triterpenes, methoxylated stilbenoids or phenanthrenoids, responsible for the antioxidant activity of the extract for the first one. Among them, combretastatins might be cytotoxic. For the second one, the prenylated flavanones known in Macaranga-type propolis were responsible for its significant anti-AGE activity. The sample from Congo was composed of many triterpene derivatives belonging to Mangifera indica species. CONCLUSION: Therefore, propolis from the center of Benin seems to be of particular interest, due to its antioxidant and anti-AGE properties. Nevertheless, as standardization of propolis is difficult in tropical zones due to its great chemodiversity, a systematic phytochemical analysis is required before promoting the use of propolis in food and health products in Africa.

13C NMR Dereplication Using MixONat Software: A Practical Guide to Decipher Natural Products Mixtures.

The growing use of herbal medicines worldwide requires ensuring their quality, safety, and efficiency to consumers and patients. Quality controls of vegetal extracts are usually undertaken according to pharmacopeial monographs. Analyses may range from simple chemical experiments to more sophisticated but more accurate methods. Nowadays, metabolomic analyses allow a fast characterization of complex mixtures. In the field, besides mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR) has gained importance in the direct identification of natural products in complex herbal extracts. For a decade, automated dereplication processes based on 13C-NMR have been emerging to efficiently identify known major compounds in mixtures. Though less sensitive than MS, 13C-NMR has the advantage of being appropriate to discriminate stereoisomers. Since NMR spectrometers nowadays provide useful datasets in a reasonable time frame, we have recently made available MixONat, a software that processes 13C as well as distortionless enhancement by polarization transfer (DEPT)-135 and -90 data, allowing carbon multiplicity (i.e., CH3, CH2, CH, and C) filtering as a critical step. MixONat requires experimental or predicted chemical shifts (δ C) databases and displays interactive results that can be refined based on the user's phytochemical knowledge. The present article provides step-by-step instructions to use MixONat starting from database creation with freely available and/or marketed δ C datasets. Then, for training purposes, the reader is led through a 30 - 60 min procedure consisting of the 13C-NMR based dereplication of a peppermint essential oil.

Using 13 C-NMR dereplication to aid in the identification of xanthones present in the stem bark extract of Calophyllum brasiliense.

INTRODUCTION: Xanthones are metabolites with a variety of biological properties. The Clusiaceae family, which until recently included the genus Calophyllum, is recognised for its production of monohydroxylated and polyhydroxylated xanthones. Presently, C. brasiliense is the only Calophyllum spp. known to occur in the Yucatan peninsula. OBJECTIVE: To use a combination of traditional phytochemical methods and carbon-13 nuclear magnetic resonance (13 C-NMR) dereplication analysis to identify xanthones in the stem bark of C. brasiliense. MATERIAL AND METHODS: Initial fractionation and purification of the stem bark extract of C. brasiliense produced macluraxanthone (1). Additional xanthones, together with chromanones and terpenoids, were identified using 13 C-NMR dereplication analysis in different semipurified fractions obtained from the low and medium polarity fractions of the stem bark extract of C. brasiliense. RESULTS: Initial identification of macluraxanthone (1) was confirmed by 13 C-NMR dereplication analysis; additionally, 13 C-NMR dereplication analysis allowed the identification of a number of monohydroxylated and polyhydroxylated xanthones, together with chromanones and terpenoids. CONCLUSION: This study confirms C. brasiliense as a rich source of xanthones and the 13 C-NMR dereplication analysis as a suitable method to quickly identify the presence of different families of secondary metabolites in semipurified fractions.

MixONat, a Software for the Dereplication of Mixtures Based on 13C NMR Spectroscopy.

Whether chemists or biologists, researchers dealing with metabolomics require tools to decipher complex mixtures. As a part of metabolomics and initially dedicated to identifying bioactive natural products, dereplication aims at reducing the usual time-consuming process of known compounds isolation. Mass spectrometry and nuclear magnetic resonance are the most commonly reported analytical tools during dereplication analysis. Though it has low sensitivity, 13C NMR has many advantages for such a study. Notably, it is nonspecific allowing simultaneous high-resolution analysis of any organic compounds including stereoisomers. Since NMR spectrometers nowadays provide useful data sets in a reasonable time frame, we have embarked upon writing software dedicated to 13C NMR dereplication. The present study describes the development of a freely distributed algorithm, namely MixONat and its ability to help researchers decipher complex mixtures. Based on Python 3.5, MixONat analyses a {1H}-13C NMR spectrum optionally combined with DEPT-135 and 90 data-to distinguish carbon types (i.e., CH3, CH2, CH, and C)-as well as a MW filtering. The software requires predicted or experimental carbon chemical shifts (δc) databases and displays results that can be refined based on user interactions. As a proof of concept, this 13C NMR dereplication strategy was evaluated on mixtures of increasing complexity and exhibiting pharmaceutical (poppy alkaloids), nutritional (rosemary extracts) or cosmetics (mangosteen peel extract) applications. Associated results were compared with other methods commonly used for dereplication. MixONat gave coherent results that rapidly oriented the user toward the correct structural types of secondary metabolites, allowing the user to distinguish between structurally close natural products, including stereoisomers.

Absolute Configuration of Mycosporine-Like Amino Acids, Their Wound Healing Properties and In Vitro Anti-Aging Effects.

Mycosporine-like amino acids (MAAs) are water-soluble metabolites, reported to exhibit strong UV-absorbing properties. They have been found in a wide range of marine organisms, especially those that are exposed to extreme levels of sunlight, to protect them against solar radiation. In the present study, the absolute configuration of 14 mycosporine-like-amino acids was determined by combining the results of electronic circular dichroism (ECD) experiments and that of advanced Marfey's method using LC-MS. The crystal structure of a shinorine hydrate was determined from single crystal X-ray diffraction data and its absolute configuration was established from anomalous-dispersion effects. Furthermore, the anti-aging and wound-healing properties of these metabolites were evaluated in three different assays namely the inhibition of collagenase, inhibition of advanced glycation end products (AGEs) and wound healing assay (scratch assay).

Additional Insights into Hypericum perforatum Content: Isolation, Total Synthesis, and Absolute Configuration of Hyperbiphenyls A and B from Immunomodulatory Root Extracts.

Phytochemical investigation of the root extracts of Hypericum perforatum led to the isolation of two biphenyl derivatives named hyperbiphenyls A and B (1 and 2) and four known xanthones (3-6). These structures were elucidated by spectroscopic and spectrometric methods including UV, NMR, and HRMS. The absolute configuration of the biphenyl derivatives was defined by two different approaches: biomimetic total synthesis of racemic hyperbiphenyl A followed by 1H and 19F NMR Mosher's esters analysis and stereoselective total synthesis of hyperbiphenyl B, permitting assignment of the S absolute configuration for both compounds. The bioactivity of compounds 1-6 toward a set of biomolecules, including major histocompatibility complex (MHC) molecules expressed on vascular endothelial cells, was measured. The results showed that the major xanthone, i.e., 5- O-methyl-2-deprenylrheediaxanthone B (3), is a potent inhibitor of MHC that efficiently reduces HLA-E, MHC-II, and MICA biomolecules on cell surfaces.

Lepidotol A from Mesua lepidota Inhibits Inflammatory and Immune Mediators in Human Endothelial Cells.

Phytochemical investigation on the fruits of Mesua lepidota (Calophyllaceae) led to the isolation of seven new phenylcoumarin derivatives named lepidotols A-E (1-5) and lepidotins A and B (6, 7). These structures were elucidated by spectroscopic and spectrometric methods including UV, NMR, and HRMS. Lepidotol A (1), the major compound, was evaluated for its inhibitory effect on inflammation and immunity using endothelial cell-based cellular assays. At 10 µM, 1 exhibited an anti-inflammatory activity, with a significant inhibition of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 expression induced by tumor necrosis factor-α. Lepidotol A also showed a mild immunosuppressive effect, with inhibition of the major histocompatibility complex molecules, namely, human leukocyte antigen (HLA)-DR and HLA-E.

Identification of Minor Benzoylated 4-Phenylcoumarins from a Mammea neurophylla Bark Extract.

Through dereplication analysis, seven known Mammea coumarins were identified in a fraction obtained from Mammea neurophylla dichloromethane bark extract selected for its ability to prevent advanced glycation end-product (AGE) formation. Among them, a careful examination of the NMR dataset of pedilanthocoumarin B led to a structural revision. Inspection of LC-DAD-MS(n) chromatograms allowed us to predict the presence of four new compounds, which were further isolated. Using spectroscopic methods (¹H-, (13)C- and 2D-NMR, HRMS, UV), these compounds were identified as new benzoyl substituted 4-phenylcoumarins (iso-pedilanthocoumarin B and neurophyllol C) and 4-(1-acetoxypropyl)coumarins cyclo F (ochrocarpins H and I).

Combination of Machine Learning and Empirical Computation for the Structural Validation of Trirosaline, a Natural Trimeric Monoterpene Indole Alkaloid from Catharanthus roseus.

Chemical investigation of the emblematic Catharanthus roseus led to the discovery of trirosaline (1), the first example of a tris-ajmalicine-type monoterpene indole alkaloid and the first natural trimeric MIA ever reported from this deeply dug plant species. Its structure was primarily elucidated based on NMR and HRESIMS analyses, and the nature of its unique intermonomeric linkages was firmly confirmed based on a combination of empirical computation and ML-J-DP4 study. Its absolute configuration was mitigated by comparison of experimental and TDDFT-simulated electronic circular dichroism (ECD) spectra. A possible biosynthetic pathway for trirosaline (1) was postulated.

Fluorescent self-assembled monolayers of umbelliferone: a relationship between contact angle and fluorescence.

Self-assembled monolayers (SAMs) that contain fluorophore units are nowadays widely used to tune surface properties and design new chemical sensor chips. It is well-known that the nature of the substrate may strongly interfere with the emission properties of the grafted molecules, but the organization of the monolayer may also have an important role. To study the influence of the SAM organization on the luminescence properties, we prepared different coumarin-based derivatives endowed with tethered chains of different lengths and elaborated the corresponding SAMs on glass slides. Besides SAM structural characterizations by atomic force microscopy and X-ray reflectivity, we carried out contact angle measurements and applied the Van Oss-Chaudhury-Good theory, which was rarely used previously for self-assembled monolayers. As expected, by increasing the tethered chain length, a higher surface coverage, a higher degree of organization, and a stronger molecular packing were observed. However, it appears to facilitate the self-quenching process, and thus, this strongly affects the fluorescent properties of the SAMs.

Tuning a 96-well microtiter plate fluorescence-based assay to identify AGE inhibitors in crude plant extracts.

Advanced glycation end-products (AGEs) are involved in the pathogenesis of numerous diseases. Among them, cellular accumulation of AGEs contributes to vascular complications in diabetes. Besides using drugs to lower blood sugar, a balanced diet and the intake of herbal products potentially limiting AGE formation could be considered beneficial for patients' health. The current paper presents a simple and cheap high-throughput screening (HTS) assay based on AGE fluorescence and suitable for plant extract screening. We have already implemented an HTS assay based on vesperlysines-like fluorescing AGEs quickly (24 h) formed from BSA and ribose under physiological conditions. However, interference was noted when fluorescent compounds and/or complex mixtures were tested. To overcome these problems and apply this HTS assay to plant extracts, we developed a technique for systematic quantification of both vesperlysines (λ(exc) 370 nm; λ(em) 440 nm) and pentosidine-like (λ(exc) 335 nm; λ(em) 385 nm) AGEs. In a batch of medicinal and food plant extracts, hits were selected as soon as fluorescence decreased under a fixed threshold for at least one wavelength. Hits revealed during this study appeared to contain well-known and powerful anti-AGE substances, thus demonstrating the suitability of this assay for screening crude extracts (0.1 mg/mL). Finally, quercetin was found to be a more powerful reference compound than aminoguanidine in such assay.

Synthesis and evaluation of naphthoic acid derivatives as fluorescent probes to screen advanced glycation end-products breakers.

Advanced glycation end-products, namely AGEs, are involved in the pathogenesis of numerous diseases. If AGEs inhibitors are well-known, only few products are described as compounds able to destroy those deleterious products. In this work, we describe naphthoic acid derivatives, particularly 1-(naphthalen-1-yl)propane-1,2-dione 9, allowing the simple and rapid detection of AGEs breakers using a 96-well microplate fluorescence assay. Since the inaugurate publication about AGEs breakers whose activity was demonstrated using HPLC analysis, this work proposes the first assay suitable for automated and high throughput screening of AGEs breakers.

Preparative isolation, fast centrifugal partition chromatography purification and biological activity of cajaflavanone from Derris ferruginea stems.

INTRODUCTION: The Derris genus is known to contain flavonoid derivatives, including prenylated flavanones and isoflavonoids such as rotenoids, which are generally associated with significant biological activity. OBJECTIVE: To develop an efficient preparative isolation procedure for bioactive cajaflavanone. METHODOLOGY: Fast centrifugal partition chromatography (FCPC) was optimised to purify cajaflavanone from Derris ferruginea stems in a single step as compared to fractionation from the cyclohexane extract by successive conventional solid-liquid chromatography procedures. The purification yield, purity, time and solvent consumption per procedure are described. The anti-fungal, anti-bacterial, anti-leishmanial, anti-plasmodial, anti-oxidant activities and the inhibition of advanced glycation end-products (AGEs) by cajaflavanone accumulation are described. RESULTS: FCPC enabled cajaflavanone purification in a single separation step, yielding sufficient quantities to perform in vitro biological screening. Interestingly, cajaflavanone had an inhibitory effect on the formation of AGEs, without displaying any in vitro anti-oxidant activity. CONCLUSION: A simple and efficient procedure, in comparison with other preparative methods, for bioactive cajaflavone purification has been developed using FCPC.

Phytoestrogens and Breast Cancer: Should French Recommendations Evolve?

Breast cancer (BC) occurs less frequently in Asia, where there is high soy consumption. It has been hypothesized that soy isoflavones could be protective against BC recurrence and mortality. At the same time, health organizations in several countries have differing recommendations for soy consumption (soy foods or dietary supplements) in BC survivors. The objective of this review is to analyze the literature and to determine whether it is justified to advise avoiding soy in dietary supplements and/or food in women with a history of BC. We conducted a systematic literature search with the Medline/Pubmed and Web of Science databases. Only prospective cohort studies published since 2009 were retained. The endpoint of studies was BC recurrence and/or mortality, and the association with soy isoflavone intake was specifically targeted. Seven studies were included. None of these studies found statistically significant adverse effects of soy consumption on BC recurrence or mortality (specific or all-cause). Overall, only one study was not able to find beneficial effects of soy intake on BC patients. The other studies concluded that there were positive associations but in very variable ways. Two studies found a decrease in BC recurrence associated with a higher isoflavone intake only for post-menopausal women. The other four studies concluded that there were positive associations regardless of menopausal status. Four studies showed better results on women with hormonal-sensitive cancer and/or patients receiving hormonal treatment. Only one found a stronger association for patients with ER-negative BC. No adverse effects of soy isoflavones on BC mortality/recurrence were found. Soy isoflavones may exert beneficial effects. These results coincide with other recent works and suggest that soy isoflavone intake is safe for BC survivors. Thus, these data no longer seem to coincide with the French recommendations, which could then be brought to evolve. However, in order to confirm the current results, larger studies are needed.

13C NMR dereplication-assisted isolation of bioactive polyphenolic metabolites from Clusia flava Jacq.

Presently it is estimated that many of the approximately 4000 new natural products isolated every year following complicated, long, and expensive isolation processes are already known; because of this, developing new strategies for locating secondary metabolites of interest in complex extracts or fractions is important. Currently, chromatographic and spectroscopic techniques are being used to optimize the isolation and identification of natural products. In this investigation we have used 13C NMR dereplication analyses for the quick identification of a number of triterpenes (friedelin, lupeol, betulinic acid), sterols (euphol, ß-sitosterol) and fatty acids (palmitic acid) present in semipurified fractions obtained from the stem bark extract of Clusia flava and to assist in the isolation of the bioactive metabolites trapezifolixanthone and paralycolin A. The complete and correct assignment of the 1H and 13C NMR spectroscopic data for paralycolin A is reported for the first time and the antioxidant and antiAGEs activity of both metabolites is described.

Chemical constituents of Antidesma bunius aerial parts and the anti-AGEs activity of selected compounds.

Thirty-three natural products were isolated from the aerial parts of Antidesma bunius, Euphorbiaceae, a plant used in Vietnamese traditional medicine against rheumatoid arthritis. All compounds were reported the first time for this species, and nine constituents resembled undescribed natural products, noticeably three coumarinolignans with 2,2-dimethyl-1,3-dioxolane moiety, two cyclopeptides, and two furofuran-type lignans connected with a phenylpropanoid moiety. The individual structures were elucidated by combining NMR and MS data, and their configuration was established by NOESY and ECD experiments and NMR calculations. Compounds with sufficient amount were analyzed for their inhibition of advanced glycation endproducts (AGEs) formation, metabolites involved in many diseases like Alzheimer, joint diseases or diabetes. With IC50 values below 0.2 mM rutin and p-hydroxyphenethyl trans-ferulate showed to be moderately active, both still being 10-times more active than the positive control aminoguanidine.

Automating a 96-well microtiter plate assay for identification of AGEs inhibitors or inducers: application to the screening of a small natural compounds library.

Advanced glycation end-products (AGEs) are involved in the pathogenesis of numerous affections such as diabetes and neurological diseases. AGEs are also implied in various changes in tissues and organs. Therefore, compounds able to break them or inhibit their formation may be considered as potential drugs, dietary supplements, or bioactive additives. In this study, we have developed a rapid and reliable (Z' factor calculation) anti-AGEs activity screening based on the overall fluorescence of AGEs. This method was successfully evaluated on known AGEs inhibitors and on a small library of natural compounds, yielding coherent results when compared with literature data.

Isoquinolines from the roots of Thalictrum flavum L. and their evaluation as antiparasitic compounds.

Alkaloids from Thalictrum flavum L. (Ranuculaceae) growing in the Loire valley (France) were isolated and evaluated for their antiplasmodial and leishmanicidal activities. Berberine was identified as a major component but its analogue, pseudoberberine, was isolated for the first time from this plant. As far as bisbenzylisoquinolines are concerned, thalfoetidine was also isolated and, besides, its nor- derivative, northalfoetidine, was identified as a new compound. Previously isolated alkaloids from Thalictrum species such as northalidasine, northalrugosidine, thaligosidine, thalicberine, thaliglucinone, preocoteine, O-methylcassythine and armepavine were newly described in the roots of T. flavum. Tertiary isoquinolines, and particularly bisbenzylisoquinolines, were found to be leishmanicidal against L. major. Thalfoetidine appeared as the most potent but its new nor- derivative northalfoetidine, as well as northalidasine, were of particular interest due to the fact that their potential leishmanicidal activity was not associated to a strong cytotoxicity.

Targeting MHC Regulation Using Polycyclic Polyprenylated Acylphloroglucinols Isolated from Garcinia bancana.

Modulation of major histocompatibility complex (MHC) expression using drugs has been proposed to control immunity. Phytochemical investigations on Garcinia species have allowed the isolation of bioactive compounds such as polycyclic polyprenylated acylphloroglucinols (PPAPs). PPAPs such as guttiferone J (1), display anti-inflammatory and immunoregulatory activities while garcinol (4) is a histone acetyltransferases (HAT) p300 inhibitor. This study reports on the isolation, identification and biological characterization of two other PPAPs, i.e., xanthochymol (2) and guttiferone F (3) from Garcinia bancana, sharing structural analogy with guttiferone J (1) and garcinol (4). We show that PPAPs 1-4 efficiently downregulated the expression of several MHC molecules (HLA-class I, -class II, MICA/B and HLA-E) at the surface of human primary endothelial cells upon inflammation. Mechanistically, PPAPs 1-4 reduce MHC proteins by decreasing the expression and phosphorylation of the transcription factor STAT1 involved in MHC upregulation mediated by IFN-γ. Loss of STAT1 activity results from inhibition of HAT CBP/p300 activity reflected by a hypoacetylation state. The binding interactions to p300 were confirmed through molecular docking. Loss of STAT1 impairs the expression of CIITA and GATA2 but also TAP1 and Tapasin required for peptide loading and transport of MHC. Overall, we identified new PPAPs issued from Garcinia bancana with potential immunoregulatory properties.