Legal use of herbal health products must comply with quality and safety standards required by health regulatory authorities in order to protect consumers from risks associated with misidentification, falsification and undesirable toxic effects.Some plant-derived compounds can act as CYP450 inhibitors, potentially leading to herb-drug interactions and, in some cases, direct hepatotoxicity, particularly when CYP450-mediated bioactivation is involved. There is a critical need to develop reliable and efficient screening assays to identify potential CYP450 inhibitors in such complex mixtures and hepatotoxicity risks associated to CYP450 metabolization. In this context, we propose associating a HepaRG cell-based test with suitable qualitative and quantitative phytochemical analyses. A case study was conducted using Tinospora crispa (T. crispa) stems harvested in Laos (“boraphet”). This herb is traditionally used for its antidiabetic effects and is known to cause hepatotoxicity, most likely due to the presence of furanoditerpenoids bioactivated by cytochromes. A Molecular Networking-based UHPLC-(HRMS)2 fingerprint of furanoditerpenoids was established by analysing methanolic extracts, enriched fractions of a representative sample and standards, by applying the bioinformatic Feature-Based Molecular Networking workflow. The fingerprinting model enabled identification and comparison between three samples harvested from nearby geographical locations (Hinboun, Yommalath and Nakaiy) through the detection of fourteen furanoditerpenoids. Four of them were identified as borapetosides A-D, eight had not yet been isolated, and were tentatively assigned. Borapetoside C content, determined by UHPLC-UV is proposed as an assay for the quality control of T. crispa stems. The CYP3A4 inhibitory activities of the methanol extracts and of borapetoside C were measured in a HepaRG cell model, which is a validated model for studying CYP450 inhibitory effects. All extracts and borapetoside C exhibited CYP3A4 inhibition.This work highlights the interest in including a HepaRG cell-based test in the T. crispa stem specification to assess the toxicity of herbal constituents associated with CYP450 inhibition.

To synthesize a natural 28-norlupane endoperoxide previously isolated from an herbal source and displaying antiplasmodial activity, a four-step semisynthetic pathway was designed from natural betulinic acid and performed via Isayama-Mukaiyama cobalt-catalyzed hydroperoxyl silylation. The target 28-norlupane endoperoxide (2) was obtained along with its endoperoxide analogs (compd. 7-11) and other products (compd. 12-16). All products were identified by 1D and 2D nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS), and some were characterized by X-ray diffraction. All compounds were tested for their antiplasmodial activity against both Plasmodium falciparum chloroquine-sensitive 3D7 and chloroquine-resistant W2 strains as well as for their cytotoxicity. Compound 2 presented broad-spectrum activity by inhibiting both chloroquine-sensitive (3D7) and -resistant (W2) P. falciparum strains. Compound 11, featuring a novel rearranged seven-membered ring scaffold, showed potent antiplasmodial activity with a favorable selectivity index, while compound 13 exhibited moderate activity. These findings highlight the potential of novel 28-norlupane endoperoxides as promising leads in antimalarial drug discovery.

A screening of 824 plant extracts from Rutaceae and Annonaceae species against a human alphacoronavirus (HCoV-229E-Luc) identified Uvaria siamensis (Scheff.) L.L.Zhou, Y·C·F.Su & R.M.K.Saunders (Annonaceae) as a promising source of bioactive metabolites. Subsequent bioassay-guided fractionation of its ethyl acetate and methanol leaf extracts led to the isolation of three previously undescribed amide derivatives, melodamides B-D (3-5), together with melodamide A (2), uvariadiamide (6), (+)- and (-)-toussaintine C (1), and five known polyphenols (7-11). The structures of compounds 1-11 were characterized by comprehensive spectroscopic analysis and comparison with literature data. In HCoV-229E-infected Huh-7 cells, (+)-toussaintine C (1) and melodamide C (4) were the most potent metabolites, with IC50 values of 1.4 and 1.8 μM and selectivity indices of 43 and 39, respectively, while melodamide A (2) also displayed notable activity, with an IC50 of 4.2 μM and an SI of 11. In contrast, polyphenols 7-11 displayed weaker antiviral effects restricted to the low- to mid-micromolar range. Extension of the profiling to a pandemic clinical isolate of SARS-CoV-2 in Vero cells, showed that both enantiomers of toussaintine C, as well as melodamides A and C, retained measurable inhibition in the mid-micromolar range, whereas most polyphenols lost activity. Taken together, these results identify amide derivatives from U. siamensis as antiviral scaffolds with preferential activity against an alphacoronavirus model, and highlight this species as a valuable source of small-molecule coronavirus inhibitors.

Plant-based food supplements (FS) of doubtful traceability have now emerged as a new threat to human health. Food supplements adulterated with pharmaceutical ingredients are considered “medicines in disguise” by regulatory authorities, which is a sub-category of falsified medicines. In the context of illegal manufacture and trade, as well as in the absence of an official phyto- and/or pharmacovigilance system, emergency departments and poison control centers constitute a early warning system for detecting ingested suspect FS. In the present investigation, we set up efficient workflows for the systematic characterization of adulterated plant-based FS in the context of an original local early warning alert system (i.e., FalsiMedTrack) involving an emergency department, a poison center, and academic analytical chemistry laboratories. Fit-for-purpose cross-analytical methods were employed, including sophisticated methods such as liquid chromatography coupled to high-resolution mass spectrometry, nuclear magnetic resonance, X-ray powder diffraction, as well as the most accessible and affordable HPLC method with UV/DAD detection. The strategy was applied successfully to typical cases of suspect plant-based health products, i.e., sample incriminated in patients experiencing side effects and herbal products currently commercialized for their “amazing health benefits”. The samples contained active pharmaceutical ingredients, including diclofenac, piroxicam, dexamethasone 21-acetate, and sibutramine. We provided evidence of “medicines in disguise” presented as food supplements, which raises concerns about their quality and safety.

Triterpenoids of natural origin are of increasing interest due to their broad spectrum of biological activities. Previous studies on dipterocarpaceous plants have reported that they were valuable source of new active natural compounds. As part of an ongoing search for naturally occurring bioactive triterpenoids from Dipterocarpaceae, phytochemical investigation of Dipterocarpus costatus Gaertn. f. heartwood collected in Thailand was conducted. Seventeen triterpenoids (1-17) belonging to both 28-norlupane and dammarane series were isolated from the corresponding bioactive n-hexane extract. Among them, norlupane 1 was identified as previously undescribed and norlupane 2 was described for the first time from a natural source. Compound structures were elucidated by 1D/2D-NMR spectroscopy and mass (HRMS) spectrometry. All isolated molecules were tested for their cytotoxicity on two human colorectal carcinoma cell lines (i.e. HT-29 and HCT116), as well as for their antiplasmodial activity against chloroquine-resistant strain FcB1 of Plasmodium falciparum. Norlupanes 2 and 4 were shown to exhibit a good cytotoxic activity against HCT 116 (IC50=4.2 mu M) and potent antiplasmodial activity (IC50=3.74 mu M), respectively.

Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present feature-based molecular networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. FBMN enables quantitative analysis and resolution of isomers, including from ion mobility spectrometry.

Latent HIV reservoirs are the main obstacle to eradicate HIV infection. One strategy proposes to eliminate these viral reservoirs by pharmacologically reactivating the latently infected T cells. We show here that a 4-deoxyphorbol ester derivative isolated from Euphorbia amygdaloides ssp. semiperfoliata, 4β-dPE A, reactivates HIV-1 from latency and could potentially contribute to decrease the viral reservoir. 4β-dPE A shows two effects in the HIV replication cycle, infection inhibition and HIV transactivation, similarly to other phorboids PKC agonists such PMA and prostratin and to other diterpene esters such SJ23B. Our data suggest 4β-dPE A is non-tumorigenic, unlike the related compound PMA. As the compounds are highly similar, the lack of tumorigenicity by 4β-dPE A could be due to the lack of a long side lipophilic chain that is present in PMA. 4β-dPE activates HIV transcription at nanomolar concentrations, lower than the concentration needed by other latency reversing agents (LRAs) such as prostratin and similar to bryostatin. PKCθ/MEK activation is required for the transcriptional activity, and thus, anti-latency activity of 4β-dPE A. However, CD4, CXCR4 and CCR5 receptors down-regulation effect seems to be independent of PCK/MEK, suggesting the existence of at least two different targets for 4β-dPE A. Furthermore, NF-κb transcription factor is involved in 4β-dPE HIV reactivation, as previously shown for other PKCs agonists. We also studied the effects of 4β-dPE A in combination with other LRAs. When 4β-dPE A was combined with another PKC agonists such as prostratin an antagonic effect was achieved, while, when combined with an HDAC inhibitor such as vorinostat, a strong synergistic effect was obtained. Interestingly, the latency reversing effect of the combination was synergistically diminishing the EC50 value but also increasing the efficacy showed by the drugs alone. In addition, combinations of 4β-dPE A with antiretroviral drugs as CCR5 antagonist, NRTIs, NNRTIs and PIs, showed a consistent synergistic effect, suggesting that the combination would not interefer with antiretroviral therapy (ART). Finally, 4β-dPE A induced latent HIV reactivation in CD4 + T cells of infected patients under ART at similar levels than the tumorigenic phorbol derivative PMA, showing a clear reactivation effect. In summary, we describe here the mechanism of action of a new potent deoxyphorbol derivative as a latency reversing agent candidate to decrease the size of HIV reservoirs.

"Chiniy-tref" (CT) is a traditional preparation used in folk medicine in Martinique Island (French West Indies) that is nowadays mainly taken orally to prevent or act against any "manifestation of evil". CT is easily prepared at home by macerating larvae of the endemic swallowtail Battus polydamas (ssp.) cebriones (Dalman, 1823), sometimes accompanied by a leaf of its host-plant Aristolochia trilobata L., in commercial rum. We have previously reported the detection of nephrotoxic and carcinogenic aristolochic acids (AAs) I and II in CT, leading the Regional Health Agency (ARS) of Martinique to issue an alert regarding the potential risks associated with its consumption in 2015. In order to complete the toxicity risk assessment for oral consumption of CT, a full qualitative analysis of AAs and their analogues (AAAs) was performed, as well as a quantitative determination of the major AAs, namely AAs I and II. The phytochemical profiling of AAAs present in CT, that also corresponds to that of B. polydamas cebriones larvae feeding on A. trilobata, has been established for the first time by ultra-high performance liquid chromatography/electrospray ionization quadrupole time of-flight tandem mass spectrometry. AAs I and II were quantified in a small panel of tinctures by using a validated UHPLC/UV method, allowing us to estimate the probable daily intakes of these toxins by CT consumers. The results proved the existence of a real risk of renal toxicity and carcinogenicity associated with the chronic oral consumption of CT in Martinique, and more generally of similar "snake bottles" throughout the Caribbean.

In this paper, the inhibiting properties of an aqueous solution containing Hylocereus undatus towards CaCO3 formation were studied by using fast controlled precipitation and chronoamperometric methods, in order to study the calcium carbonate formation in solution and on a metallic surface, respectively. As a purpose of comparison, the antiscaling properties of citric acid were studied in the same experimental conditions. The results obtained by fast controlled precipitation method showed that Hylocereus undatus solution was more efficient than citric acid to inhibit CaCO3 formation in solution (optimal concentrations of 20 and 24 mg L−1, respectively). By using the chronoamperometric method, a concentration of citric acid at 160 mg L−1 was required to totally prevent the precipitation of CaCO3 whereas it was 180 mg L−1 for Hylocereus undatus solution. The morphology analysis by scanning electronic microscopy and X-ray diffraction revealed that vaterite was favored in the presence of Hylocereus undatus solution, whereas a mixture of calcite and vaterite was obtained with citric acid.

In this retrospective series of 97 cases of manchineel fruit ingestion reported to French Poison Control Centers between 2009 and 2017, we investigated cases of poisoning due to manchineel fruit (from the Hippomane mancinella tree). This fruit is known to be responsible for oropharyngeal and gastrointestinal tract lesions and possibly hypotension and bradycardia (previously attributed to the presence of physostigmine). The most commonly observed clinical signs were oropharyngeal pain, abdominal pain, diarrhea and oropharyngeal irritation. No major gastrointestinal tract lesions were observed in the five cases in which upper gastrointestinal (GI) endoscopy was performed. One case of laryngeal edema and one case of bradycardia were observed, but analysis of the harvested fruits did not confirm the presence of physostigmine. Ingestion of manchineel fruit can cause mild abdominal pain and digestive irritation, requiring medical attention. Rarely, when several fruits have been ingested, severe oropharyngeal injury or hemodynamic disorders may require otorhinolaryngological consultation or cardiac monitoring for several hours, respectively.

Calophyllum inophyllum L. (Calophyllaceae) is a tropical tree traditionally used in folk medicine as anti-inflammatory, fungicidal, healing agent and antimicrobial agent. Its properties are explained by a high content in coumarins, xanthones and triterpenes [1]. Particularly, tamanu oil is a traditional Tahitian medicine. This oil is extracted from dried fruits (picture below) and contains a resinous fraction [2] possessing antimicrobial properties. The aim of our study was to investigate the molecular content-activity relationships of the resinous fraction from tamanu oil by aims of molecular networks. Molecular networking is a recent approach which allows to represent LC-MS2 data according to similarities between fragmentation pathways [3] [4]. First of all, five major known specific markers were isolated and identified from the resin: inophyllum E, inophyllum J, tamanolid, inophyllum P and (E)-5-methoxy-2,2-dimethyl-6-(2-methylbut-2-enoyl)-10-propyl-2H,8H-pyrano[2,3-f]chromen-8-one [5], allowing the further annotation of specific clusters in molecular networks. Then, the antimicrobial activity of the crude oil-resin and fractions thereof was evaluated against seventeen bacteria strains and the minimal inhibitory concentration (MIC) measured. Finally, the molecular content of the tested samples including resin and its fractions was explored by UHPLC-HRMS2 analysis and a molecular network using a “fraction layout” was built. The relationship between antimicrobial activity of fractions and the presence of coumarins and acid chromanones will be discussed.

•H. undatus and citric acid are green inhibitors.•The antiscalant properties were tested by using FCP and chronoamperometry.•The scale inhibition effects in solution and on a surface have been examined.

Tinospora crispa is a popular traditional herbal plant commonly used throughout the world for treatment of various diseases, in particular type 2 diabetes mellitus. We report here a new case of toxic hepatitis in a 57-year old male patient in the French West Indies following the consumption of two aqueous extracts of fresh Tinospora crispa stems. It thus differs from two previously reported cases that concerned the chronic intake of powdered dry stems delivered in solid oral dosage forms (i.e. pellets and tablets). Liquid Chromatography-Diode Array Detection-Mass Spectrometry (LC/DAD/MS) analyses were performed on an aqueous extract of the offending sample that mimics the swallowed preparation. They revealed the presence of species-specific molecular marker borapetoside C (1) and thus enabled an unambiguous phytochemical identification. The exploration of tandem MS/MS data obtained by ultra-high performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-HRMS) allowed the identification of 17 additional cis-clerodane-type furanoditerpenoid lactones, analogues of 1. These results support the hypothesis that the mechanisms underlying hepatotoxicity of Tinospora crispa are the same as those encountered with furanoditerpenoids-containing plants such as Teucrium chamaedrys or Dioscorea bulbifera. In the context of type 2 diabetes treatment, we recommend that Tinospora crispa intake should be more closely monitored for signs of hepatotoxicity.

A supercritical fluid chromatography-based targeted purification procedure using tandem mass spectrometry and molecular networking was developed to analyze, annotate, and isolate secondary metabolites from complex plant extract mixture. This approach was applied for the targeted isolation of new antiviral diterpene esters from Euphorbia semiperfoliata whole plant extract. The analysis of bioactive fractions revealed that unknown diterpene esters, including jatrophane esters and phorbol esters, were present in the samples. The purification procedure using semipreparative supercritical fluid chromatography led to the isolation and identification of two new jatrophane esters (13 and 14) and one known (15) and three new 4-deoxyphorbol esters (16-18). The structure and absolute configuration of compound 16 were confirmed by X-ray crystallography. This compound was found to display antiviral activity against Chikungunya virus (EC50 = 0.45 μM), while compound 15 proved to be a potent and selective inhibitor of HIV-1 replication in a recombinant virus assay (EC50 = 13 nM). This study showed that a supercritical fluid chromatography-based protocol and molecular networking can facilitate and accelerate the discovery of bioactive small molecules by targeting molecules of interest, while minimizing the use of toxic solvents.

ABSTRACT A supercritical fluid chromatography-based targeted purification workflow using tandem mass spectrometry and molecular networking was developed to analyze, annotate and isolate secondary metabolites from complex mixture. This approach was applied for targeted isolation of new antiviral diterpene esters from Euphorbia semiperfoliata whole plant extract. The analysis of bioactive fractions revealed that unknown diterpene esters, including jatrophane esters and phorboids esters, were present in the samples. The purification procedure using semi-preparative-supercritical fluid chromatography led to the isolation and identification of two jatrophane esters ( 13 and 14 ) and four 4-deoxyphorbol esters ( 15 - 18 ). Compound 16 was found to display antiviral activity against chikungunya virus (EC 50 = 0.45 µ M), while compound 15 was found to be a potent and selective inhibitor of HIV-1 replication in a recombinant virus assay (EC 50 = 13 nM). This study showed that supercritical fluid chromatography-based workflow and molecular networking can facilitate and accelerate the discovery of bioactive small molecules by targeted molecules of interest, while minimizing the use of toxic solvents.

Chemical antiscalants are usually used to control scale depositions, a major concern in industry. Some of these products may be harmful towards environment or toxic for humans, so environmentally friendly antiscalant are needed. In this respect, plant extracts can be used as new green antiscalant agents. In this work, stock aqueous solution of Herniaria glabra (H. glabra) was obtained by infusion. H. glabra is used in traditional medicine to treat or prevent urolithiasis; it could be an interesting candidate as a green antiscaling agent. The properties of H. glabra towards CaCO3 formation were tested by using chronoamperometry and Fast Controlled Precipitation methods. An n-butanol solution was used to partition the aqueous solution of H. glabra, yielding a polar compounds-free fraction. This fraction was also studied by chronoamperometry and Fast Controlled Precipitation methods. The results showed a better inhibition efficiency towards CaCO3 precipitation for the n-butanol fraction compared to the crude H. glabra solution.

Piperlongumine is a natural amide alkaloid isolated from several species of Piper and is described in the literature as selectively cytotoxic to several cancer cell lines.Inhibiting cell migration has gained considerable interest as an approach for discovering antimetastatic agents because this process is fundamental to metastasis.Piperlongumine, selected from cell-based assay screening of NuBBE Database, inhibited the migration of MDA-MB-231 breast cancer cells with an EC 50 of 3.0 ± 1.0 µM by the Boyden chamber assay.A series of five analogous compounds based on the structure of piperlongumine were designed, synthesized and evaluated in cell migration and cytotoxicity assays.The analogue designed by molecular simplification ((E)-N-acryloyl-3-(3,4,5-trimethoxyphenyl)acrylamide) was the most active of the series, with an EC 50 of 1.5 ± 1 µM.Additionally, this compound was selectively cytotoxic, with a selectivity index (SI) of 4.4.