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Cancer remains a significant global health concern, with mortality rates steadily rising and prompting an urgent search for effective treatments. This study focuses on the medicinal properties of plants from the Phyllanthus genus, specifically Phyllanthus amarus and Phyllanthus niruri, which have shown promise in traditional medicine. Through bioguided fractionation using preparative high-performance liquid chromatography (HPLC), bioactive compounds were isolated and identified using ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC-QTOF-MS E ) and nuclear magnetic resonance (NMR) spectroscopy. Chemometric analyses such as principal component analysis (PCA) aided in understanding metabolite distribution. Biological assays demonstrated cytotoxic activities of specific fractions against cancer cell lines, notably the PhyN 4n fraction from P. niruri, which induced S-phase cell cycle arrest and apoptosis in HL60 cells. These findings underscore the anticancer potential of Phyllanthus species and lay the groundwork for future drug development efforts. The study's integration of advanced analytical techniques, chemometrics, and biological assays provides valuable insights for harnessing natural products in the fight against cancer., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

The good phytotoxicity and selectivity against weeds versus tomato or cress make saponin-rich fractions from Agave macroacantha , A. colorata , A. parryi , and A. parrasana attractive candidates as bioherbicides. The saponin contents have only previously been reported for A. macroacantha , and as a consequence, simultaneous dereplication has been performed on saponin-rich fractions from the other plants by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. This strategy enables the identification of a total of 26 saponins, 14 of which have been described previously and 12 of which are proposed as new saponins. They include isomers and a new sugar chain with a β-d-apiofuranose unit. The method is corroborated by the isolation of eight dereplicated saponins from A. colorata .

ABSTRACT Metagenome-assembled genomes (MAGs) expand our understanding of microbial diversity, evolution, and ecology. Concerns have been raised on how sequencing, assembly, binning, and quality assessment tools may result in MAGs that do not reflect single populations in nature. Here, we reflect on another issue, i.e., how to handle highly similar MAGs assembled from independent data sets. Obtaining multiple genomic representatives for a species is highly valuable, as it allows for population genomic analyses; however, when retaining genomes of closely related populations, it complicates MAG quality assessment and abundance inferences. We show that (i) published data sets contain a large fraction of MAGs sharing >99% average nucleotide identity, (ii) different software packages and parameters used to resolve this redundancy remove very different numbers of MAGs, and (iii) the removal of closely related genomes leads to losses of population-specific auxiliary genes. Finally, we highlight some approaches that can infer strain-specific dynamics across a sample series without dereplication.

Species of the genus Pleiocarpa are used in traditional medicine against fever and malaria. The present study focuses on the isolation and identification of bioactive compounds from P. bicarpellata extracts, and the evaluation of their antiprotozoal activity. Fractionation and isolation combined to LC-HRMS/MS-based dereplication provided 16 compounds: seven indole alkaloids, four indoline alkaloids, two secoiridoid glycosides, two iridoid glycosides, and one phenolic glucoside. One of the quaternary indole alkaloids (7) and one indoline alkaloid (15) have never been reported before. Their structures were elucidated by analysis of spectroscopic data, including 1D and 2D NMR experiments, UV, IR, and HRESIMS data. The absolute configurations were determined by comparison of the experimental and calculated ECD data. The extracts and isolated compounds were evaluated for their antiprotozoal activity towards Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum, as well as for their cytotoxicity against rat skeletal myoblast L6 cells. The dichloromethane/methanol (1:1) root extract showed strong activity against P. falciparum (IC50 value of 3.5 µg/mL). Among the compounds isolated, tubotaiwine (13) displayed the most significant antiplasmodial activity with an IC50 value of 8.5 µM and a selectivity index of 23.4. Therefore, P. bicarpallata extract can be considered as a source of indole alkaloids with antiplasmodial activity.

Restingas are extreme environments present in the Atlantic Rainforest biome. These ecosystems show peculiar characteristics, like sandy soil, high salinity, and high solar exposure, which brings scientific interest to their native species. Stachytarpheta schottiana is an endemic Brazilian species found in Jurubatiba Restinga, and just like other species of the genus Stachytarpheta, is used in folk medicine. In this paper, we describe, for the first time, 28 secondary metabolites from S. schottiana polar extract, among them iridoids, flavonoids, lignans and phenylethanoids, with the use of LC-HRMS/MS dereplication and molecular networking methodologies. Many of these compounds have not been described yet for the genus Stachytarpheta, like astragalin, taxifolin, lamiide and the lignans secondary metabolites class. Also, in this paper, High Speed Counter-Current Chromatography (HSCCC) isolation and Nuclear Magnetic Resonance (NMR) of two compounds were used to correct a misidentification in the dereplication procedure and to create seeds for molecular networking. Some of the suggested known compounds found in this work have had their biological activities described in the literature, such as the hepatoprotective activity of verbascoside, that matches those already related for the genus Stachytarpheta and for the folk use of Stachytarpheta schottiana itself. [ABSTRACT FROM AUTHOR]

Introduction: Metabolomics is the approach of choice to guide the understanding of biological systems and its molecular intricacies, but compound identification is yet a bottleneck to be overcome. Objective: To assay the use of NMRfilter for confidence compound identification based on chemical shift predictions for different datasets. Results: We found comparable results using the lead tool COLMAR and NMRfilter. Then, we successfully assayed the use of HMBC to add confidence to the identified compounds. Conclusions: NMRfilter is currently under development to become a stand-alone interactive software for high-confidence NMR compound identification and this communication gathers part of its application capabilities. [ABSTRACT FROM AUTHOR]

Alkaloids represent a major group of natural products (NPs), derived from highly diverse organisms. These structurally varied specialized metabolites are widely used for medicinal purposes and also known as toxic contaminants in agriculture and dietary supplements. While the detection of alkaloids is generally facilitated by GC- or LC-MS, these techniques do require considerable efforts in sample preparation and method optimization. Bypassing these limitations and also reducing experimental time, matrix-free laser desorption ionization (LDI) and related methods may provide an interesting alternative. As many alkaloids show close structural similarities to matrices used in matrix-assisted laser desorption ionization (MALDI), they should ionize upon simple laser irradiation without matrix support. With this in mind, the current work presents a systematic evaluation of LDI properties of a wide range of structurally diverse alkaloids. Facilitating a direct comparison between LDI and ESI-MS fragmentation, all tested compounds were further studied by electrospray ionization (ESI). Moreover, crude plant extracts of Atropa belladonna, Cinchona succirubra, and Colchicum autumnale were analyzed by LDI in order to evaluate direct alkaloid detection and dereplication from complex mixtures. Finally, dose-dependent evaluation of MALDI and LDI detection using an extract of Rosmarinus officinalis spiked with atropine, colchicine, or quinine was conducted. Overall, present results suggest that LDI provides a versatile analytical tool for analyzing structurally diverse alkaloids as single compounds and from complex mixtures. It may further serve various potential applications ranging from quality control to the screening for toxic compounds as well as the build up of MS databases. [ABSTRACT FROM AUTHOR]

Mistletoes are extremely important plants and their extracts have been widely used as a complementary medicine towards cancer therapy. In this context, we have studied the leaves of a South Indian Mistletoe, Macrosolen capitellatus (Wight & Arn.) Danser, a parasitic plant belonging to family Loranthaceae. A bioassay-guided isolation and LC-MS dereplication strategies have led to the isolation of pinocembrin-7-O-[4", 6"-(S)-hexahydroxy diphenoyl (HHDP)]-β-D-glucose (1). This is the first report showing the presence of 1 not only in this plant but also in the genus Macrosolen. In this study, the aforementioned parasitic plant was collected from two different host plants, Mangifera indica and Phyllanthus emblica, which were extensively studied chemically by various research groups. We have observed that 1 is present in both samples of M. capitellatus, while a thorough literature search showed that this compound 1 has not been reported from either Mangifera indica or Phyllanthus emblica. These observations indicated that the producer of 1 is nothing but parasitic plant M. capitellatus. This compound has shown moderate anti-proliferative activity against MCF-7 cell line (IC50 value-15 μM). A computational protein binding study has been performed on 1 against 25 crystallographic proteins of the BRCA-1 gene of breast cancer. The results showed interactions having -9.33 and -8.99 kcal/mol binding energy with the proteins IDs 1T15 and 1T29 respectively; which suggests that this scaffold has the potential to develop further. [ABSTRACT FROM AUTHOR]

• Thirty-two glycosides including flavonoids, phenolic acids and coronillins were identified. • 13C NMR-based dereplication was used to identify major compounds of the extracts. • A bioactivity-guided fractionation lead to the isolation of three new triglycosylated flavonoids. • The structures of compounds were determined by 1D and 2D NMR spectra and MS spectra. • Three quercetin glycosides exhibited a significant radical-scavenging activity. This study presents the bio-guided chemical investigation of a 80% methanol extract of Hippocrepis emerus flowers, a perennial non-climbing shrub. Liquid-liquid partitioning in solvents of increasing polarity combined to biological screening enabled to determine the EtOAc and n -BuOH soluble fractions as the most active parts of the extract. These fractions were chemically profiled by using a 13C NMR-based dereplication method, resulting in the identification of twenty-six compounds. The dereplication process was completed by purification of some unknown or minor compounds of the n -BuOH fraction. Three new glycosylated flavonoids, namely kaempferol-3- O -β- d -glucopyranosyl-7- O -β- d -glucopyranosyl-(1→3)-α- l -rhamnopyranoside (1), isorhamnetin-3- O -β- d -glucopyranosyl-7- O -β- d -glucopyranosyl-(1→3)-α- l -rhamnopyranoside (2) and quercetin-3- O -β- d -glucopyranosyl-7,4′- O -α- l -dirhamnopyranoside (3), together with twelve known compounds (4 – 15) were isolated. Their structures were elucidated by spectroscopic methods including NMR, HR-ESI-MS and UV. The antioxidant activity of fractions and isolated compounds were evaluated using DPPH and hydroxyl radical scavenging and CUPRAC assays. In parallel, their inhibitory properties against mushroom tyrosinase and human neutrophil elastase enzymes were assessed. Three quercetin glycosides exhibited a significant radical-scavenging activity and two flavonoids showed a moderate elastase inhibitory activity. [ABSTRACT FROM AUTHOR]

Gymnopilins are long chain oligoisoprenoids produced through the condensation of isoprene units from MEV and MEP biosynthetic pathways. In Gymnopilus , these carotenoid-like molecules are recognized as major compounds in some species. In the present study, oligoisoprenoids derived from gymnopilins were dereplicated from Gymnopilus imperialis, a mushroom-forming basidiomycete, using liquid chromatographic coupled with high-resolution mass spectrometry (tandem LC-HRMS/MS) and GNPS. From the dichloromethane extract (Gym-DCM) of G. imperialis we annotated 3 oligoisoprenoids from the GNPS molecular library spectra and 15 analogs from the curation of the molecular networking. Data from NMR spectroscopic of the extract confirmed the annotation of the metabolites. Based on the literature data suggesting the neurotoxic effect of gymnopilins, we investigated the effects of the administering different doses of gymnopilin extracts (1, 4 or 10 mg/kg) and diazepam (4 mg/kg) on the acquisition of object recognition memory (ORM) in mice. By studying novel object recognition memory (ORM), a type of non-aversive memory. ORM was assessed based on the total time of spontaneous exploration of both objects, the discrimination index (DI), and the frequency of contact with both objects. Our present findings reveal, for the first time, that gymnopilins treatment before training modulates ORM in a dose-dependent manner. It is also suggested that differential effects on memory might be related to differential effects on GABA A receptors but do not exclude its effects in other neurotransmitter systems. Another class of secondary metabolites, alkaloids, might modulate AChR, which is essential for maintaining object recognition memory over time. [Display omitted] • Dereplication of G. imperialis by LC-HRMS/MS allowed the annotation of eighteen oligoisoprenoids derived from gymnopilins. • Treatment of mice with gymnopilins at a higher dose did not prevent the acquisition and retrieval of memory for objects. • Gymnopilins in all doses did not modulate anxiety-like behavior in mice, as well as did not result in sedative effects. • Gymnopilins treatment in mice before training modulates ORM in a dose-dependent manner. [ABSTRACT FROM AUTHOR]

Three endophytic fungi isolated from Moquiniastrum polymorphum (Less.) G. Sancho (Asteraceae) were cultivated using the one strain many compounds (OSMAC) strategy to evaluate the production of griseofulvin derivatives. Extracts obtained were analyzed by HPLC–MS/MS and the chromatographic and spectrometric data used to elaborate a feature-based molecular network (FBMN) through the GNPS platform. This approach allowed the observation of differences such as medium-specific and strain-specific production of griseofulvin derivatives and variations of cytotoxic activity in most extracts. To evaluate the efficiency of the OSMAC approach allied with FBMN analysis in the prospection of compounds of biotechnological interest, griseofulvin and 7-dechlorogriseofulvin were isolated, and the relative concentrations were estimated in all culture media using HPLC–UV, allowing for the inference of the best strain–medium combinations to maximize its production. Malt extract-peptone broth and Wickerham broth media produced the highest concentrations of both secondary metabolites.

Since the golden age of antibiotics in the 1950s and 1960s actinomycetes have been the most prolific source for bioactive natural products. However, the number of discoveries of new bioactive compounds decreases since decades. New procedures (e.g., activating strategies or innovative fermentation techniques) were developed to enhance the productivity of actinomycetes. Nevertheless, compound identification remains challenging among others due to high rediscovery rates. Rapid and cheap genome sequencing as well as the advent of bioinformatical analysis tools for biosynthetic gene cluster identification in combination with mass spectrometry-based molecular networking facilitated the tedious process of dereplication. In recent years several studies have been dedicated to accessing the biosynthetic potential of Actinomyces species, especially streptomycetes, by using integrated genomic and metabolomic screening in order to boost the discovery rate of new antibiotics. This review aims to present the various possible applications of this approach as well as the newly discovered molecules, covering studies between 2014 and 2021. Finally, the effectiveness of this approach with regard to find new bioactive agents from actinomycetes will be evaluated.

The aim of this study was to assess the possibility to use Baikal endemic amphipods as a source for screening of novel natural products. Analysis of amphipod’s E. verrucosus hemolymph by HPLC-MS was performed for the first time. A number of natural products with new molecular mass for Baikal endemic macroinvertebrates were found. We found a compound as chrysin by comparison of MS1, MS2 profiles and molecular mass of natural products registered in Dictionary of Natural Products and MZ-cloud databases. Also, we found new compound with mass 493.80277 Da, circulating in the amphipod’s hemolymph.

The aim of the study was to conduct a dereplication analysis of crude extract of deepwater Baikal’s endemic amphipod species, Ommatogammarus albinus. Detected masses provide some evidence that amphipod’s extract might contain both host and microbiota secondary metabolites. Some of the masses correspond with known structures isolated from eubacteria. Also, two compounds did not match with any registered natural products from the database Dictionary of Natural Products. Those findings allow suggesting that Baikal’s endemics and their microbiota are promising sources of novel natural products.

ABSTRACT The isolation of microorganisms from microbial community samples often yields a large number of conspecific isolates. Increasing the diversity covered by an isolate collection entails the implementation of methods and protocols to minimize the number of redundant isolates. Matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) mass spectrometry methods are ideally suited to this dereplication problem because of their low cost and high throughput. However, the available software tools are cumbersome and rely either on the prior development of reference databases or on global similarity analyses, which are inconvenient and offer low taxonomic resolution. We introduce SPeDE, a user-friendly spectral data analysis tool for the dereplication of MALDI-TOF mass spectra. Rather than relying on global similarity approaches to classify spectra, SPeDE determines the number of unique spectral features by a mix of global and local peak comparisons. This approach allows the identification of a set of nonredundant spectra linked to operational isolation units. We evaluated SPeDE on a data set of 5,228 spectra representing 167 bacterial strains belonging to 132 genera across six phyla and on a data set of 312 spectra of 78 strains measured before and after lyophilization and subculturing. SPeDE was able to dereplicate with high efficiency by identifying redundant spectra while retrieving reference spectra for all strains in a sample. SPeDE can identify distinguishing features between spectra, and its performance exceeds that of established methods in speed and precision. SPeDE is open source under the MIT license and is available from https://github.com/LM-UGent/SPeDE. IMPORTANCE Estimation of the operational isolation units present in a MALDI-TOF mass spectral data set involves an essential dereplication step to identify redundant spectra in a rapid manner and without sacrificing biological resolution. We describe SPeDE, a new algorithm which facilitates culture-dependent clinical or environmental studies. SPeDE enables the rapid analysis and dereplication of isolates, a critical feature when long-term storage of cultures is limited or not feasible. We show that SPeDE can efficiently identify sets of similar spectra at the level of the species or strain, exceeding the taxonomic resolution of other methods. The high-throughput capacity, speed, and low cost of MALDI-TOF mass spectrometry and SPeDE dereplication over traditional gene marker-based sequencing approaches should facilitate adoption of the culturomics approach to bacterial isolation campaigns.

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 13 C NMR dereplication analyses for the quick identification of a number of triterpenes (friedelin, lupeol, betulinic acid), sterols (euphol, β -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 1 H and 13 C NMR spectroscopic data for paralycolin A is reported for the first time and the antioxidant and antiAGEs activity of both metabolites is described.

Women have been found to be at a higher risk of morbidity and mortality from type 2 diabetes mellitus (T2DM) and asthma. α-Glucosidase inhibitors have been used to treat T2DM, and arachidonic acid 15-lipoxygenase (ALOX15) inhibitors have been suggested to be used as treatments for asthma and T2DM. Compounds that inhibit both enzymes may be studied as potential treatments for people with both T2DM and asthma. This study aimed to determine potential anti-diabetic and anti-inflammatory bioactive hits from Coriaria intermedia Matsum. stem and Dracontomelon dao (Blanco) Merr. & Rolfe bark. A bioassay-guided fractionation framework was used to generate bioactive fractions from C. intermedia stem and D. dao bark. Subsequently, dereplication through ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and database searching was performed to putatively identify the components of one bioactive fraction from each plant. Seven compounds were putatively identified from the C. intermedia stem active fraction, and six of these compounds were putatively identified from this plant for the first time. Nine compounds were putatively identified from the D. dao bark active fraction, and seven of these compounds were putatively identified from this plant for the first time. One putative compound from the C. intermedia stem active fraction (corilagin) has been previously reported to have inhibitory activity against both α-glucosidase and 15-lipoxygenase-1. It is suggested that further studies on the potential of corilagin as an anti-diabetic and anti-inflammatory treatment should be pursued based on its several beneficial pharmacological activities and its low reported toxicity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Fabian, Astorga, Atis, Pilapil and Hernandez.)

Introduction: Natural deep eutectic solvents (NADES) have emerged as interesting extractants to develop botanical ingredients. They are nontoxic and biodegradable, nonflammable, easy to prepare, and able to solubilize a wide range of molecules. However, NADES extracts remain difficult to analyze because the metabolites of interest stay highly diluted in the nonvolatile viscous NADES matrix., Objective: This study presents a robust analytical workflow for the chemical profiling of NADES extracts. It is applied to Hypericum perforatum aerial parts extracted with the neutral mixture fructose/glycerol/water (3/1/1, w/w/w), and compared to the chemical profiling of a classical dry methanol extract., Methods: Exploiting polarity differences between metabolites, the H. perforatum NADES extract was partitioned in a liquid-liquid solvent system to trap the hydrophilic NADES constituents in the lower phase. The upper phase, containing a diversity of secondary metabolites from H. perforatum, was fractionated by centrifugal partition chromatography. All fractions were chemically investigated using a 13 C NMR dereplication method which involves hierarchical clustering analysis of the whole NMR dataset, a natural metabolite database for metabolite identification, and 2D NMR analyses for validation. Liquid chromatography-mass spectrometry (LC-MS) analyses were also performed to complete the identification process., Results: A range of 21 metabolites were unambiguously identified, including glycosylated flavonols, lactones, catechins, phenolic acids, lipids, and simple sugars, and 15 additional minor extract constituents were annotated by LC-MS based on exact mass measurements., Conclusion: The proposed identification process is rapid and nondestructive and provides good prospects to deeply characterize botanical extracts obtained in nonvolatile and viscous NADES systems., (© 2023 John Wiley & Sons Ltd.)

Microorganisms associated with termites are an original resource for identifying new chemical scaffolds or active metabolites. A molecular network was generated from a collection of strain extracts analyzed by liquid chromatography coupled to tandem high-resolution mass spectrometry, a molecular network was generated, and activities against the human pathogens methicillin-resistant Staphylococcus aureus, Candida albicans and Trichophyton rubrum were mapped, leading to the selection of a single active extract of Penicillium sclerotiorum SNB-CN111. This fungal species is known to produce azaphilones, a colorful family of polyketides with a wide range of biological activities and economic interests in the food industry. By exploring the molecular network data, it was shown that the chemical diversity related to the P. sclerotiorum metabolome largely exceeded the data already reported in the literature. According to the described fragmentation pathways of protonated azaphilones, the annotation of 74 azaphilones was proposed, including 49 never isolated or synthesized thus far. Our hypothesis was validated by the isolation and characterization of eight azaphilones, among which three new azaphilones were chlorogeumasnol (63), peniazaphilone E (74) and 7-deacetylisochromophilone VI (80).

The discovery and identification of novel natural products of medicinal importance in the herbal medicine industry becomes a challenge. The complexity of this process can be reduced by dereplication strategies. The current study includes a method based on high-performance liquid chromatography (HPLC), using the evaporative light scattering detector (ELSD) to identify the 12 most common secondary metabolites in plant extracts. Twelve compounds including rutin, taxifolin, quercetin, apigenin, kaempferol, betulinic acid, oleanolic acid, betulin, lupeol, stigmasterol, and β-sitosterol were analyzed simultaneously. The polarity of the compounds varied greatly from highly polar (flavonoids) to non-polar (triterpenes and sterols). This method was also tested for HPLC-DAD and HPLC-ESI-MS/MS analysis. Oleanolic acid and ursolic acid could not be separated in HPLC-ELSD analysis but were differentiated using LC-ESI-MS/MS analysis due to different fragment ions. The regression values (R2 > 0.996) showed good linearity in the range of 50–1000 µg/mL for all compounds. The range of LOD and LOQ values were 7.76–38.30 µg/mL and 23.52–116.06 µg/mL, respectively. %RSD and % trueness values of inter and intraday studies were mostly

Solid-phase extraction embedded dialysis (SPEED technology) is an innovative procedure developed to physically separate in-situ, during the cultivation, the mycelium of filament forming microorganisms, such as actinomycetes and fungi, and the XAD-16 resin used to trap the secreted specialized metabolites. SPEED consists of an external nylon cloth and an internal dialysis tube containing the XAD resin. The dialysis barrier selects the molecular weight of the trapped compounds, and prevents the aggregation of biomass or macromolecules on the XAD beads. The external nylon promotes the formation of a microbial biofilm, making SPEED a biofilm supported cultivation process. SPEED technology was applied to the marine Streptomyces albidoflavus 19-S21, isolated from a core of a submerged Kopara sampled at 20 m from the border of a saltwater pond. The chemical space of this strain was investigated effectively using a dereplication strategy based on molecular networking and in-depth chemical analysis. The results highlight the impact of culture support on the molecular profile of Streptomyces albidoflavus 19-S21 secondary metabolites.

Chamaecrista diphylla (L.) Greene (Fabaceae/Caesalpiniaceae) is a herbaceous plant that is widely distributed throughout the Americas. Plants from this genus have been used in traditional medicine as a laxative, to heal wounds, and to treat ulcers, snake and scorpion bites. In the present study, we investigated the chemical composition of Chamaecrista diphylla leaves through a mass spectrometry molecular network approach. The oxygen radical absorbance capacity (ORAC) for the ethanolic extract, enriched fractions and isolated compounds was assessed. Overall, thirty-five compounds were annotated for the first time in C. diphylla. Thirty-two of them were reported for the first time in the genus. The isolated compounds 9, 12, 24 and 33 showed an excellent antioxidant capacity, superior to the extract and enriched fractions. Bond dissociation energy calculations were performed to explain and sustain the antioxidant capacity found. According to our results, the leaves of C. diphylla represent a promising source of potent antioxidant compounds.

Natural products isolated by microorganisms are interesting in the search for new compounds with several biological activities. However, low concentration and structural diversity make the isolation a time-consuming step. Tandem mass spectrometry is a well-established technology for the identification and characterization of target microbial natural products due to high sensitivity and selectivity of these experiments. We developed a method employing neutral loss experiments (LC-ESI-MS/MS) to identify luminacins in microbial crude extracts. The luminacins class exhibited conserved fragmentation pattern with loss at 172 Da relative to glycosides fragment and this loss was used in searching for compounds belonging to this class. Therefore, the crude extract produced by Streptomyces sp. 39 PL was analysed and five luminacins were isolated - one is a novel luminacin I at 466 Da. [ABSTRACT FROM AUTHOR]

Keywords: high-throughput (HT) approaches; culturomics; microbial biological resource centers; antimicrobial resistance (AMR); strain typing; dereplication Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), based identification of microorganisms, appears the most suitable technique for this role. Though, the limited database and bias of commercial databases toward microbes of clinical or food safety relevance restrict the application of MALDI-TOF MS. [Extracted from the article]

Introduction: Hyptis verticillata Jacq. (Lamiaceae) is a Mexican medicinal plant for the treatment of skin infections and illness affecting the respiratory and gastrointestinal systems. Objective: To associate the efficient resolution provided by ultra‐high‐performance liquid chromatography combined to the accuracy of a hybrid Fourier‐transform (FT) mass spectrometer in order to dereplicate podophyllotoxin‐type lignans in a plant extract. Methods: An ultra‐high‐performance liquid chromatography‐photodiode array‐high resolution electrospray ionisation tandem mass spectrometry (UHPLC‐PDA‐HRESI‐MS/MS) method was applied in an Orbitrap hybrid FT spectrometer for dereplication of podophyllotoxin and related cytotoxic lignans in wild bushmint. This procedure included high‐resolution mass values for positively charged ions [M + H]+ and [M + NH4]+, MS/MS data, and comparison of UV maxima and retention times with pure compounds. Results: Podophyllotoxin in addition to seven aryltetralins, four arylnaphthalenes, and one dibenzylbutyrolactone were dereplicated from the methanol extract in a short‐time analysis (5 min). 4′‐O‐Demethyl‐dehydro‐deoxypodophyllotoxin was identified as a new natural product. Conclusion: The applied UHPLC‐MS/MS dereplication method is suitable for a rapid analysis of podophyllotoxin‐type lignans and the resulting chemical fingerprinting could be valuable in quality control of herbal drugs and their phytopharmaceuticals. Hyptis verticillata Jacq. is used for the treatment of skin infections and illness affecting the respiratory and gastrointestinal systems. An ultra‐high‐performance liquid chromatography‐photodiode array‐high resolution electrospray ionisation tandem mass spectrometry method was applied in an Orbitrap hybrid Fourier‐transform spectrometer for dereplication of podophyllotoxin and related cytotoxic lignans. Podophyllotoxin in addition to seven aryltetralins, four arylnaphthalenes, and one dibenzylbutyrolactone were dereplicated from the methanol extract. 4'‐O‐Demethyl‐dehydro‐deoxypodophyllotoxin was identified as a new natural product. [ABSTRACT FROM AUTHOR]

• 2D LC/UHPSFC-Q-TOF-MS system was established for characterization. • GNPS was applied for dereplication with high efficiency and accuracy. • A mass of isomers were separated and exposed. • Two subclasses compounds were found for the first time. • Two new bufadienolides were identified to verify the strategy. Comprehensive analysis and identification of chemical components are very important to evaluate the efficacy and safety of traditional Chinese medicine (TCM). Meanwhile, the discovery of new natural compounds is of great significance for drug exploitation and development. Although two-dimensional liquid chromatography (2D LC) systems expand the peak capacity and improve selectivity and resolution, interpreting the post-processing data is tedious and time-consuming. In this study, an off-line two-dimensional liquid chromatography/ultra-high performance supercritical fluid chromatography tandem quadrupole time-of-flight mass spectrometry (2D LC/UHPSFC-Q-TOF/MS) system was established for systematic chromatographic separation and identification of bufadienolides. Subsequently, the Global Natural Product Social Molecular Networking (GNPS) was applied for dereplication of chemical components of adjacent fractions with high efficiency and accuracy. The key parameters which affected separation and detection with respect to chromatographic conditions and mass spectrometry conditions were optimized. The extract of Venenum Bufonis was fractionated into forty fractions by first-dimensional reversed phase high-performance liquid chromatography (HPLC), which were further analyzed by the second-dimensional UHPSFC-Q-TOF/MS in positive ion mode. The data of forty fractions was imported into GNPS and processed automatically within about five hours. Furthermore, the chemical components with similar featured fragments were classified into the same cluster, which was helpful for components identification. A total of 229 bufadienolides were characterized and two subclasses of compounds (bufogenins conjugated with carboxylic acid and N -heterocyclic bufogenins) were found in Venenum Bufonis for the first time. In addition, UHPSFC exhibited powerful separation ability of isomers in Venenum Bufonis. In this analysis, two new compounds were isolated and fully characterized by NMR verifying the feasibility of this combined analytical strategy. This integrated strategy can improve the efficiency in the detection of new compounds and offer greater observation of isomers from medicinal herbs and other natural sources. [ABSTRACT FROM AUTHOR]

• MALDI-TOF MS is applicable as high-resolution and high-throughput tool. • The classification and characterization of cultivable microorganisms is targeted. • Advantageous are its simple sample preparation and short measurement time. • It accelerates the dereplication of isolates from large-scale screening campaigns. • Applications for studying microbial diversity and future trends are discussed. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) has become one of the most popular methods for the rapid, cost-effective and accurate classification and characterization of cultivable microorganisms. Due to its simple sample preparation and short measurement time, MALDI-TOF MS is an excellent choice for the high-throughput study of microbial isolates from rhizospheres or plants grown under diverse environmental conditions. While clinical isolates have a higher identification rate than environmental isolates due to the focus of commercial mass spectral libraries on the former, no identification is necessary in the dereplication step of large environmental studies. The grouping of large sets of isolates according to their intact protein profiles can be performed without knowledge of their taxonomy. Thus, this method is easily applicable to environmental samples containing microorganisms from yet undescribed phylogenetic origins. The main strategies applied to achieve effective dereplication are, first, expanding existing mass spectral libraries and, second, using an additional statistical analysis step to group measured mass spectra and identify unique isolates. In this review, these aspects are addressed. It closes with a prospective view on how MALDI-TOF MS-based microbial characterisation can accelerate the exploitation of plant-associated microbiota. [ABSTRACT FROM AUTHOR]

The classical approach to drug discovery from natural products (NP's) requires strenuous and complex purification steps for the isolation and structural elucidation. Modern strategies as dereplication aim to accelerate the identification of known compounds present in a crude or partially purified extract. In this work, we investigated the influence of the solid-phase extraction (Oasis, Plexa, and Agilent C18 cartridges with and without organic modifiers) chemical profile obtained by UPLC-QTOF-MS and NMR and cytotoxicities of aqueous extracts from Phyllanthus niruri and P. amarus. Our results showed differences between the SPE cartridges and the mass recovered. P. niruri showed higher mass recovery than P. amarus indicating a higher amount of secondary metabolites. The UPLC-QTOF-MS analysis revealed that P. niruri crude extract presents higher contents of phenolic compounds than P. amarus. According to NMR analysis, P. niruri contained more tyrosine, corilagin, and glycosidic residues while P. amarus , presented higher content of ellagic acid. The different stationary phases, as well as mobile phases for exploratory SPE, enabled the exploitation of the different chemical functionalities within the Phyllanthus species. The SPE (MeOH:H 2 O 70:30 with C18 cartridges) samples showed greater in vitro cytotoxicity than the crude extracts, with IC 50 ranging from 8.01 to 94.92 μg mL−1 against the tumor lines tested. The solid phase extraction allowed the concentration of molecules with desirable physicochemical characteristics, which might increase the hit of therapeutically useful substances. • UPLC-HRMS combined with multivariate analyzes were applied to investigate the metabolomic profiles of the Phyllanthus spp. • The SPE methodology was attested by 1H NMR analysis which shows the reductions of free carbohydrates. • It was possible to identify 33 possible biomarkers. Some of these were supposedly correlated with cytotoxic activity. • The SPE samples showed greater in vitro cytotoxicity than the crude extracts, against the tumor cell lines tested. [ABSTRACT FROM AUTHOR]

Abstract Lycopodium alkaloids (LAs) are the characteristic metabolites of club mosses. Chemical differences often exist in different specimens of a single plant species collected from different geographic origins. In this study, a preliminary LC-MS detection and dereplication analyses of alkaloidal constituents of Lycopodium cernuum var. sikkimense (Müll. Hal.) C.B. Clarke (LCVS2) collected from Fujian province led to the isolation and characterization of three undescribed LAs, lycocernuskines A–C, and six known cernuane-type LAs. The known compounds were previously isolated from the same plant species (LCVS1) collected from Chongqing, and so their dereplication in LCVS2 was accomplished based on their retention times (t R) and the quasi-molecular ion peaks in the LC-MS fingerprint. Chemical structures were identified by spectroscopic methods, single-crystal X-ray diffraction analysis, and electronic circular dichroism calculations. Lycocernuskines A and B are the first two examples of C-12 hydroxylated phlegmarane-type LAs bearing a nitrone residue at the quinoline ring. The isolates were evaluated for their anti-AChE and neuroprotective effects. Graphical abstract LC-MS method was applied to the isolation and dereplication of Lycopodium alkaloids (LAs) from samples of the same Lycopodiaceae species (Lycopodium cernuum var. sikkimense) collected in two different locations. Three undescribed LAs were thereafter obtained and characterized. Lycocernuskines A (1) and B represent the first two examples of C-12 hydroxylated phlegmarane-type LAs bearing a nitrone residue at the quinoline ring. Image 1 Highlights • LC-MS guided isolation and dereplication of Lycopodium alkaloids (LAs) was applied. • Three undescribed LAs were obtained from Lycopodium cernuum var. sikkimense. • Lycocernuskines A and B are unusual nitrone-containing 12-hydroxy-phlegmarane LAs. • The absolute configurations of lycocernuskines A and B were determined. [ABSTRACT FROM AUTHOR]

Introduction: Thin layer chromatography (TLC) is frequently used to obtain the fingerprint of a plant extract. Although the retardation factor and the response to visualisation give primary information about compound identification, the direct TLC‐mass spectrometry (MS) coupling allows a more detailed characterisation of samples. Objectives: To demonstrate the potential for the flavonoid dereplication using an inorganic matrix‐assisted laser desorption ionisation time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) method with and without TLC separation. Material and Methods: Samples derived from wine, apple or rose were deposited on an aluminium‐backed silica gel TLC sheet compatible with the MS adapter. Unlike the wine sample, for apple and rose samples compound derivatisation was necessary. These two samples were deposited twice and the plate was cut in two parts. One half was oversprayed with Neu‐Peg reagent to visualise flavonoids while the inorganic matrix was deposited on each flavonoid zone on the second half for MS ionisation. Results: Mass spectra obtained for samples without plate development showed numerous ions corresponding to glycosylated flavonoids. The lower m/z observed could be due either to aglycone flavonoids or to in‐source fragment ions. After plate development, a separation of many spots was observed and each spot was analysed separately leading to a deeper identification of the present flavonoids. Moreover, isobaric flavonoids with different hRf values could be differentiated. Conclusion: TLC‐MALDI‐TOF‐MS using an inorganic matrix enabled the analysis of anthocyanins in positive mode and of flavonols, flavanols, dihydrochalcones and phenolic acids in negative mode, reducing adduct, aggregate forms giving thus simple and reliable spectra for the dereplication approach of flavonoids in complex samples. Flavonoid compounds contained in various plant samples were analyzed using TLC/MALDI‐TOF MS with and without TLC plate development. Ionization of flavonoid was assisted by an inorganic matrix deposited on each spot zone of interest. The use of tis inorganic matrix enables anthocyanin ionization in positive mode whereas the other flavonoid derivatives were analysed in negative mode and reduce formation of adduct, aggregate forms during ionization giving thus simple and reliable MS spectra suitable for compound identification. [ABSTRACT FROM AUTHOR]

Introduction: Molecular networks are now established as the method of choice for tandem mass spectrometry dereplication and similarity‐based structure elucidation. Node identification can be used to start the propagation of the structure elucidation of unknown compounds progressively. Objective: To demonstrate the capabilities of using the LipidXplorer data results along with molecular networking to identify nodes and aid sequential structure elucidation of unknown compounds. Material and Methods: Molecular fragmentation query language (MFQL) files were written to identify glycoalkaloids based on known structures described for Solanum species. A dataset generated from liquid chromatography‐high resolution mass spectrometry (LC‐HRMS) analysis of Solanum pseudoquina sample were submitted to dereplication on both LipidXplorer software and Global Natural Products Social Molecular Network (GNPS) online system. The resulting attribute table from GNPS calculations was merged with the LipidXplorer results and this merged file was used for network visualisation in Cytoscape. Nodes in the molecular network were labelled using the LipidXplorer identifiers, thus assisting the structure elucidation of unidentified compounds. Results: The combination of the LipidXplorer glycoalkaloids list and GNPS analysis was used in Cytoscape to label nodes in the molecular network. The analysis of the network using these labelled starting points triggered the structure elucidation of closely related nodes leading to the identification of 30 compounds using the LipidXplorer output and four purified and structure elucidated compounds, including a new glycoalkaloids identified as 3‐O‐(β‐d‐xylopyranosyl)‐(20R,25S)‐22,26‐epimino‐16‐acetyl‐cholesta‐5,22(N)‐diene. Conclusion: A significant compound identification completely based on molecular formula and fragmentation queries was achieved. This new and effective approach could help researches to expand the identification rate of compounds in dereplication studies using molecular networks. To extend the compound identification rate for dereplication studies using molecular networking, we implemented the direct use of the data generated from the database independent method developed on LipidXplorer in the Global Natural Products Social Molecular Network (GNPS) table results. Features detected using the molecular formula restrictions translated into molecular fragmentation query language (MFQL) files were merged with the attribute table from GNPS to enable Cytoscape to plot the LipidXplorer identifiers as node labels. Thus, due to the flexibility of the MFQL files to annotate and classify compounds based on their molecular formula and fragmentation patterns together with the structure elucidation capability of molecular network we were able to significantly increase the putative compound identification. This application was employed to identify several glycoalkaloids from Solanum pseudoquina as well as a new glycoalkaloid identified as 3‐O‐(β‐d‐xylopyranosyl)‐(20R,25S)‐22,26‐epimino‐16‐acetyl‐cholesta‐5,22(N)‐diene. [ABSTRACT FROM AUTHOR]

Marine micro-organisms in the deep-sea hydrothermal vent systems are considered as potential sources of bioactive natural products. Sixteen bacterial strains were isolated from a deep-sea hydrothermal field and screened for bioactive metabolism studies. After the strains were subjected to bioactive testing at different culture media, chemical dereplication by HPLC coupled to high-resolution mass spectrometer was performed to analyse or determine the main secondary metabolisms in those strains. Strain 06204 was large-scale fermented with relative optimal media, for isolating the desired sulphur compound. Butyrolactone I 3-sulphate was isolated and structurally identified from the extract, guided by dereplication and showed moderate antivirus activities against H3N2 and EV71 viruses. Our study suggests that deep-sea hydrothermal bacteria are good sources of sulphur natural products. Meanwhile, the described approach, mainly bioactive screening, dereplication and targeted isolation, is effective and efficient to discover interesting bioactive compounds in hydrothermal bacteria. [ABSTRACT FROM AUTHOR]

We investigated the antidiarrhoeic effect of the aqueous extract of Annona crassiflora leaves (AEAC). The AEAC decreased the diarrhoeic stools and enteropooling induced by castor oil, without altering total faecal output; moreover, the distance travelled by charcoal meal in the intestine was increased. Twenty-eight compounds were identified by LC-DAD-MS in the AEAC, including flavonoids, alkaloids and proanthocyanidins. In addition, two oligomeric series of condensed tannins of up to nine flavan-3-ol units were characterised by MALDI-MS. These data suggest that the antidiarrhoeic effect of the AEAC is related to its ability to inhibit intestinal secretion and/or to increase intestinal absorption. Moreover, the prokinetic effect of AEAC, together with its inhibitory effect on enteropooling induced by castor oil, explains why this extract decreased diarrhoeic faeces without altering the total faecal output. All these effects are in agreement with the pharmacological activity reported in the literature for many of the secondary metabolites identified. [ABSTRACT FROM AUTHOR]

Stryphnodendron pulcherrimum is a species known to have a high content of tannins. Accordingly, its preparations are used in southern Pará, Brazil, for their anti-inflammatory and antimicrobial activities, but so far, its chemical profile composition remains essentially unknown. We herein describe the compounds present in a hydro-acetonic extract from S. pulcherrimum leaves as revealed by dereplication via ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry. The data were combined with spectral organization, spectral matching through the Global Natural Products Social platform, in silico annotation and taxonomical ponderation. Several types of phenolic compounds were identified such as gallic acids, flavan-3-ols and flavone-like compounds. From these, 5 have been recently reported by our group, whereas 44 are reported here for the first time in this tree species, and 41 (out of 49) for this genus. The results highlight the possible role of Stryphnodendron pulcherrimum as a renewable source for natural bioactive products with potential pharmaceutical applications.

Three species of the lichen Usnea (U. baileyi (Stirt.) Zahlbr., U. bismolliuscula Zahlbr. and U. pectinata Stirt.) and nine associated endolichenic fungi (ELF) were evaluated using a metabolomics approach. All investigated lichen crude extracts afforded antibacterial activity against Staphylococcus aureus (minimum inhibitory concentration (MIC): 0.0625 mg/mL), but none was observed against Escherichia coli, while the ELF extract Xylaria venustula was found to be the most active against S. aureus (MIC: 2.5 mg/mL) and E. coli (MIC: 5 mg/mL). X. venustula was fractionated and tested for to determine its antibacterial activity. Fractions XvFr1 to 5 displayed bioactivities against both test bacteria. Selected crude extracts and fractions were subjected to metabolomics analyses using high-resolution LC–MS. Multivariate analyses showed the presence of five secondary metabolites unique to bioactive fractions XvFr1 to 3, which were identified as responsible for the antibacterial activity of X. venustula. The p-values of these metabolites were at the margin of significance level, with methyl xylariate C (P_60) being the most significant. However, their high variable importance of projection (VIP) scores (>5) suggest these metabolites are potential diagnostic metabolites for X. venustula for “dual” bioactivity against S. aureus and E. coli. The statistical models also showed the distinctiveness of metabolites produced by lichens and ELF, thus supporting our hypotheses of ELF functionality similar to plant endophytes.

Introduction: The conventional process of natural drug development involves the purification and isolation of bioactive compounds from natural extracts, followed by manual structure elucidation using spectral data (e.g., NMR, MS). Two major challenges in this process are: (1) isolating and identifying new bioactive compounds in a reasonable time and cost, and (2) producing compounds with desired activity and toxicity profiles. Dereplication, which utilizes existing data on previously isolated bioactive compounds to identify the presence/absence of new compounds with minimal human intervention, offers a solution. With advancements in computer technology and machine learning, there is an opportunity to develop a computational platform integrating spectral data and experimental data of natural substances. Methods: A new database was curated consisting of various existing databases with the option of integrating future in-house isolated compound data, specifically for dereplication. Chemical compounds in the database were represented as tokens/vectors generated by calculating various chemical descriptors. Machine learning models, including naive Bayes classifier, support vector machines, random forest, and convolutional neural networks, were developed for structure elucidation in the dereplication process. The performance of these models was evaluated using precision, recall, F1-score, and AUC of the ROC curve. Results: The developed machine learning models were able to predict compound structures from preliminary spectral measurements with a precision of 78%, recall of 67%, an F1-score of 0.72, and an AUC of 0.79. These results are promising and the machine learning models can still be optimized further with additional data. Conclusion: The integration of machine learning algorithms with a curated database offers a promising approach for automating the dereplication process and discovery of active compounds from natural materials, potentially accelerating natural drug development. Future potential directions include external validation of the developed machine learning models using manual structure elucidation of natural product isolates. [ABSTRACT FROM AUTHOR]

[Display omitted] • 13C-NMR dereplication identified sterols, triterpenes and coumarins from Mesua lepidota. • A new and active anti-butyrylcholinesterase coumarin, lepidotin C was isolated. • Lepidotin B was the most potent butyrylcholinesterase inhibitor. 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), 3β-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 K i 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. [ABSTRACT FROM AUTHOR]

The isolation and identification of bioactive metabolites from complex extracts obtained from microbial growth media is a time consuming, costly, and labor-intensive task. A strategy to rapidly identify secondary metabolites isolated from extracts obtained from the culture media of marine-derived and endophytic fungal strains is described. Identification was achieved by HPLC-UV-MS and 1H NMR analyses in combination with data obtained from the Dictionary of Natural Products. Among the compounds identified, (-)-naphthoquinoneimine, citreorosein, emodin, pyrenocine A and harzianopyridone displayed moderate to potent antiviral activity. (-)-Naphthoquinoneimine was isolated as the enantiomer of its previously reported dextrorotatory congener, while 6,7-dihydroxy-2,2-dimethyl-4-chromanone is herein reported for the first time as a natural product.

The marine-derived fungus Stilbella fimetaria is a chemically talented fungus producing several classes of bioactive metabolites, including meroterpenoids of the ascochlorin family. The targeted dereplication of fungal extracts by UHPLC-DAD-QTOF-MS revealed the presence of several new along with multiple known ascochlorin analogues (19–22). Their structures and relative configuration were characterized by 1D and 2D NMR. Further targeted dereplication based on a novel 1,4-benzoquinone sesquiterpene derivative, fimetarin A (22), resulted in the identification of three additional fimetarin analogues, fimetarins B–D (23–25), with their tentative structures proposed from detailed MS/HRMS analysis. In total, four new and eight known ascochlorin/fimetarin analogues were tested for their antimicrobial activity, identifying the analogues with a 5-chloroorcylaldehyde moiety to be more active than the benzoquinone analogue. Additionally, the presence of two conjugated double bonds at C-2′/C-3′ and C-4′/C-5′ were found to be essential for the observed antifungal activity, whereas the single, untailored bonds at C-4′/C-5′ and C-8′/C-9′ were suggested to be necessary for the observed antibacterial activity.

Many natural extracts used as cosmetic ingredients are available as solutions prepared in high-boiling-point solvents, called carrier solvents, such as propylene glycol (1,2-propanediol), propanediol (1,3-propanediol) and glycerin. The upstream chemical profiling of these extracts represents a major asset for the cosmetic industry, because it accelerates product development. A new workflow for the rapid characterization of the main metabolites present in natural extracts diluted in propylene glycol and 1,3-propanediol is presented here as an extension of previous works on glycerin-containing extracts. This method is an optimized version of a well-established dereplication procedure and consists of a fractionation by centrifugal partition chromatography followed by 13C nuclear magnetic resonance analysis and dedicated data processing. The concentration by evaporation under reduced pressure was considered as a pertinent preliminary step, particularly adapted to the analysis of highly diluted extracts. A dried hydro-ethanolic extract of Leontopodium alpinum Cass. was prepared at laboratory scale and used for method validation. Three solutions at 5% wt. of dry extract were prepared with propylene glycol/water (1:1), 1,3-propanediol/water (1:1) and glycerin/water (1:1) as carrier solvents. The dereplication workflow was applied to the three resulting L. alpinum extracts. Each study led to the quick identification of 26 metabolites including five flavonoids (luteolin and its derivatives), five hydroxycinnamic acids (among which are leontopodic acids), sugars and organic acids.

The role and importance of the identification of natural products are discussed in the perspective of the study of secondary metabolites. The rapid identification of already reported compounds, or structural dereplication, is recognized as a key element in natural product chemistry. The biological taxonomy of metabolite producing organisms, the knowledge of metabolite molecular structures, and the availability of metabolite spectroscopic signatures are considered as the three pillars of structural dereplication. The role and the construction of databases is illustrated by references to the KNApSAcK, UNPD, CSEARCH, and COCONUT databases, and by the importance of calculated taxonomic and spectroscopic data as substitutes for missing or lost original ones. Two NMR-based tools, the PNMRNP database that derives from UNPD, and KnapsackSearch, a database generator that provides taxonomically focused libraries of compounds, are proposed to the community of natural product chemists. The study of the alkaloids from Urceolina peruviana, a plant from the Andes used in traditional medicine for antibacterial and anticancer actions, has given the opportunity to test different approaches to dereplication, favoring the use of publicly available data sources.