Publications by authors named "Laurence Marcourt"

84 Publications

Hypoglycemic active principles from the leaves of Bauhinia holophylla: Comprehensive phytochemical characterization and in vivo activity profile.

PLoS One 2021 24;16(9):e0258016. Epub 2021 Sep 24.

Faculty of Sciences, São Paulo State University (UNESP), Bauru, São Paulo, Brazil.

Bauhinia holophylla leaves, also known as "pata-de-vaca", are traditionally used in Brazil to treat diabetes. Although the hypoglycemic activity of this medicinal plant has already been described, the active compounds responsible for the hypoglycemic activity have not yet been identified. To rapidly obtain two fractions in large amounts compatible with further in vivo assay, the hydroalcoholic extract of B. holophylla leaves was fractionated by Vacuum Liquid Chromatography and then purified by medium pressure liquid chromatography combined with an in vivo Glucose Tolerance Test in diabetic mice. This approach resulted in the identification of eleven compounds (1-11), including an original non-cyanogenic cyanoglucoside derivative. The structures of the isolated compounds were elucidated by nuclear magnetic resonance and high-resolution mass spectrometry. One of the major compounds of the leaves, lithospermoside (3), exhibited strong hypoglycemic activity in diabetic mice at the doses of 10 and 20 mg/kg b.w. and prevents body weight loss. The proton nuclear magnetic resonance (1H NMR) quantification revealed that the hydroalcoholic leaves extract contained 1.7% of lithospermoside (3) and 3.1% of flavonoids. The NMR analysis also revealed the presence of a high amount of pinitol (4) (9.5%), a known compound possessing in vivo hypoglycemic activity. The hypoglycemic properties of the hydroalcoholic leaves extract and the traditional water infusion extracts of the leaves of B. holophylla seem thus to be the result of the activity of three unrelated classes of compounds. Such results support to some extent the traditional use of Bauhinia holophylla to treat diabetes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0258016PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8462688PMC
September 2021

Kaempferol-3-O-α-(3,4-di-E-p-coumaroyl)-rhamnopyranoside from Nectandra oppositifolia releases Ca from intracellular pools of Trypanosoma cruzi affecting the bioenergetics system.

Chem Biol Interact 2021 Sep 16;349:109661. Epub 2021 Sep 16.

Center of Natural Sciences and Humanities, Federal University of ABC, Santo Andre, SP, 09210-180, Brazil. Electronic address:

Phytochemical analysis of EtOH extract from leaves of Nectandra oppositifolia afforded three flavonoids: kaempferol (1), kaempferol-3-O-α-rhamnopyranoside (2) and kaempferol-3-O-α-(3,4-di-E-p-coumaroyl)-rhamnopyranoside (3), which were characterized by NMR and ESI-HRMS. When tested against the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, flavonoids 1 and 3 were effective to kill the trypomastigotes with IC values of 32.0 and 6.7 μM, respectively, while flavonoid 2 was inactive. Isolated flavonoids 1-3 were also tested in mammalian fibroblasts and showed CC values of 24.8, 48.7 and 153.1 μM, respectively. Chemically, these results suggested that the free aglycone plays an important role in the bioactivity while the presence of p-coumaroyl unities linked in the rhamnoside unity is important to enhance the antitrypanosomal activity and reduce the mammalian cytotoxicity. The mechanism of cellular death was investigated for the most potent flavonoid 3 in the trypomastigotes using fluorescent and luminescent-based assays. It indicated that this compound induced neither permeabilization of the plasma membrane nor depolarization of the membrane electric potential. However, early time incubation (20 min) with flavonoid 3 resulted in a constant elevation of the Ca levels inside the parasite. This effect was followed by a mitochondrial imbalance, leading to a hyperpolarization and depolarization of the mitochondrial membrane potential, with reduction of the ATP levels. During this time, the levels of reactive oxygen species levels (ROS) were unaltered. The leakage of Ca from the intracellular pools can affect the bioenergetics system of T. cruzi, leading to the parasite death. Therefore, flavonoid 3 can be a useful tool for future studies against T. cruzi parasites.
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http://dx.doi.org/10.1016/j.cbi.2021.109661DOI Listing
September 2021

Isolation and Identification of Isocoumarin Derivatives With Specific Inhibitory Activity Against Wnt Pathway and Metabolome Characterization of .

Front Chem 2021 12;9:664489. Epub 2021 Aug 12.

School of Pharmaceutical Sciences, University of Geneva, CMU, Geneva, Switzerland.

The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancers are dependent on Wnt pathway overactivation mostly through dysregulation of pathway component protein expression, which necessitates the search for therapeutically relevant compounds targeting them. Highly diverse microorganisms as endophytes represent an underexplored field in the therapeutic natural products research. In the present work, the objective was to explore the chemical diversity and presence of selective Wnt inhibitors within a unique collection of fungi isolated as foliar endophytes from the long-lived tropical palm . The fungi were cultured, extracted with ethyl acetate, and screened for their effects on the Wnt pathway and cell proliferation. The endophytic strain was prioritized for scaled-up fractionation based on its selective activity. Application of geometric transfer from analytical HPLC conditions to semi-preparative scale and use of dry load sample introduction enabled the isolation of 15 pure compounds in a single step. Among the molecules identified, five are original natural products described for the first time, and six are new to this species. An active fraction obtained by semi-preparative HPLC was re-purified by UHPLC-PDA using a 1.7 µm phenyl column. 75 injections of 8 µg were necessary to obtain sufficient amounts of each compound for structure elucidation and bioassays. Using this original approach, in addition to the two major compounds, a third minor compound identified as ()-(-)-5-hydroxymellein (18) was obtained, which was found to be responsible for the significant Wnt inhibition activity recorded. Further studies of this compound and its structural analogs showed that only 18 acts in a highly specific manner, with no acute cytotoxicity. This compound is notably selective for upstream components of the Wnt pathway and is able to inhibit the proliferation of three triple negative breast cancer cell lines. In addition to the discovery of Wnt inhibitors of interest, this study contributes to better characterize the biosynthetic potential of .
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http://dx.doi.org/10.3389/fchem.2021.664489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8397479PMC
August 2021

Metabolomics reveals biomarkers in human urine and plasma to predict cytochrome P450 2D6 (CYP2D6) activity.

Br J Pharmacol 2021 Aug 7. Epub 2021 Aug 7.

Division of Clinical Pharmacology and Toxicology, Geneva University Hospitals, Geneva, Switzerland.

Background And Purpose: Individualized assessment of cytochrome P450 2D6 (CYP2D6) activity is usually performed through phenotyping following administration of a probe drug to measure the enzyme's activity. To avoid any iatrogenic harm (allergic drug reaction, dosing error) related to the probe drug, the development of non-burdensome tools for real-time phenotyping of CYP2D6 could significantly contribute to precision medicine. This study focuses on the identification of markers of the CYP2D6 enzyme in human biofluids using an LC-high-resolution mass spectrometry-based metabolomic approach.

Experimental Approach: Plasma and urine samples from healthy volunteers were analysed before and after intake of a daily dose of paroxetine 20 mg over 7 days. CYP2D6 genotyping and phenotyping, using single oral dose of dextromethorphan 5 mg, were also performed in all participants.

Key Results: We report four metabolites of solanidine and two unknown compounds as possible novel CYP2D6 markers. Mean relative intensities of these features were significantly reduced during the inhibition session compared with the control session (n = 37). Semi-quantitative analysis showed that the largest decrease (-85%) was observed for the ion m/z 432.3108 normalized to solanidine (m/z 398.3417). Mean relative intensities of these ions were significantly higher in the CYP2D6 normal-ultrarapid metabolizer group (n = 37) compared with the poor metabolizer group (n = 6). Solanidine intensity was more than 15 times higher in CYP2D6-deficient individuals compared with other volunteers.

Conclusion And Implications: The applied untargeted metabolomic strategy identified potential novel markers capable of semi-quantitatively predicting CYP2D6 activity, a promising discovery for personalized medicine.
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http://dx.doi.org/10.1111/bph.15651DOI Listing
August 2021

, a Treatment for Uncontrolled Hypertension. Pilot Comparative Intervention.

Plants (Basel) 2021 May 19;10(5). Epub 2021 May 19.

Antenna Foundation, Avenue de la Grenade 24, 1207 Geneva, Switzerland.

In Iraq, in 2019, there were about 1.4 million Internally Displaced Persons (IDP); medical treatments were often interrupted. The feasibility of using () decoction to curb hypertension was evaluated. A multicentric comparative pilot intervention for 121 participants with high blood pressure (BP) (≥140/90 mmHg) was conducted. Participants of the intervention group (with or without conventional medication) received decoction on a dose regimen starting from 10 grams per day. BP was measured five times over six weeks. The major active substances were chemically quantified. Results: After 6 weeks, 61.8% of participants from the intervention group ( = 76) reached the target BP < 140/90 mmHg, compared to 6.7% in the control group ( = 45). In the intervention group, a mean (±SD) reduction of 23.1 (±11.8) mmHg and 12.0 (±11.2) for systolic and diastolic BP, respectively, was observed, while in the control group the reduction was 4.4 (±10.2)/3.6 (±8.7). The chemical analysis of the starting dose indicated a content of 36 mg of total anthocyanins and 2.13 g of hibiscus acid. The study shows the feasibility of using HS decoction in IDP's problematic framework, as hibiscus is a safe, local, affordable, and culturally accepted food product.
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http://dx.doi.org/10.3390/plants10051018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8160910PMC
May 2021

Identification of Potential Antiseizure Agents in using Zebrafish and Mouse Epilepsy Models.

ACS Chem Neurosci 2021 05 29;12(10):1791-1801. Epub 2021 Apr 29.

School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland.

The resin of the tree Flueck. (synonym: ; Burseraceae), also known as "frankincense", is a traditional remedy used for central nervous system disorders in East Africa. Here we report the evaluation of its antiseizure activity in zebrafish and mouse epilepsy models to identify novel antiseizure compounds. The resin was extracted by solvents of increasing polarity. The hexane extract demonstrated the strongest antiseizure activity and was therefore subjected to bioactivity-guided isolation, which leaded to the isolation of eight terpene derivatives. A new prenylbicyclogermacrene derivative () was isolated along with seven other compounds (, -). Among them, the triterpene β-boswellic acid () showed the strongest activity and reduced 90% of pentylenetetrazole (PTZ)-induced seizures at 100 μg/mL. In parallel to , a commercial extract of was also evaluated and showed moderate bioactivity (45% reduction at 30 μg/mL). The extract of was subjected to targeted isolation of other boswellic acid derivatives (-), which were evaluated for antiseizure activity in comparison with . In the whole series, β-boswellic acid () was the most active (60% reduction at 200 μM), and its potency was also confirmed with its purchased standard (). Pure nanoparticles of and a commercially formulated extract of were tested in a PTZ-kindling mouse seizure model. This notably revealed that the administration reduced seizures by 50% in this mouse model, which was consistent with its detection and quantification in plasma and brain samples. This study and the preclinical evaluation performed indicate that β-boswellic acid, common to various species of , has some potential as an antiseizure agent.
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http://dx.doi.org/10.1021/acschemneuro.1c00044DOI Listing
May 2021

Coumarins from Simonk.: Isolation by Liquid-Liquid Chromatography and Potential Anxiolytic Activity Using an In Vivo Zebrafish Larvae Model.

Int J Mol Sci 2021 Feb 12;22(4). Epub 2021 Feb 12.

Independent Laboratory of Natural Products Chemistry, Medical University of Lublin, 20-093 Lublin, Poland.

Different types of anxiety disorders have become the number one mental health issue in developed countries. The search for new, safer and effective drug-like molecules among naturally derived substances faces two difficulties: an efficient method of isolation compounds with a high-purity and high-throughput animal model for activity assay. Thus, the aim of the present study was to isolate by liquid-liquid chromatography high-purity rare coumarins from the fruits of Simonk. and evaluate their anxiolytic effect (defined as reversed thimotaxis) using a 5-days post-fertilization (dpf) larvae model. Liquid-liquid chromatography enabled the isolation of one simple hydroxycoumarin (devenyol) and four pyranocoumarins (cis-khellactone, d-laserpitin, isolaserpitin and octanoyllomatin). The anxiolytic effect was defined as a decrease in the time spent in the boundaries of the living space (also described as reversed thigmotaxis). Our results show that all isolated courmarins exerted a significant influence on the anxiety behavior (anxiolytic activity) in the zebrafish larvae model. According to our knowledge, this is the first report of anxiolytic activity of pyranocoumarins and devenyol.
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http://dx.doi.org/10.3390/ijms22041829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918798PMC
February 2021

Metabolite profile of Nectandra oppositifolia Nees & Mart. and assessment of antitrypanosomal activity of bioactive compounds through efficiency analyses.

PLoS One 2021 25;16(2):e0247334. Epub 2021 Feb 25.

Center of Natural Sciences and Humanities, Federal University of ABC, Santo Andre, Sao Paulo, Brazil.

EtOH extracts from the leaves and twigs of Nectandra oppositifolia Nees & Mart. shown activity against amastigote forms of Trypanosoma cruzi. These extracts were subjected to successive liquid-liquid partitioning to afford bioactive CH2Cl2 fractions. UHPLC-TOF-HRMS/MS and molecular networking were used to obtain an overview of the phytochemical composition of these active fractions. Aiming to isolate the active compounds, both CH2Cl2 fractions were subjected to fractionation using medium pressure chromatography combined with semi-preparative HPLC-UV. Using this approach, twelve compounds (1-12) were isolated and identified by NMR and HRMS analysis. Several isolated compounds displayed activity against the amastigote forms of T. cruzi, especially ethyl protocatechuate (7) with EC50 value of 18.1 μM, similar to positive control benznidazole (18.7 μM). Considering the potential of compound 7, protocatechuic acid and its respective methyl (7a), n-propyl (7b), n-butyl (7c), n-pentyl (7d), and n-hexyl (7e) esters were tested. Regarding antitrypanosomal activity, protocatechuic acid and compound 7a were inactive, while 7b-7e exhibited EC50 values from 20.4 to 11.7 μM, without cytotoxicity to mammalian cells. These results suggest that lipophilicity and molecular complexity play an important role in the activity while efficiency analysis indicates that the natural compound 7 is a promising prototype for further modifications to obtain compounds effective against the intracellular forms of T. cruzi.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247334PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906415PMC
August 2021

Saponins from Saffron Corms Inhibit the Gene Expression and Secretion of Pro-Inflammatory Cytokines.

J Nat Prod 2021 03 18;84(3):630-645. Epub 2021 Feb 18.

Pharmaceutical Biology, Pharmacenter, University of Basel, 4056 Basel, Switzerland.

Corms are obtained as a byproduct during the cultivation of saffron (). In a project aimed at the valorization of this waste product, we observed that a 70% EtOH extract of the corms and a sugar-depleted MeOH fraction of the extract inhibited the TNF-α/IFN-γ-induced secretion and gene expression of the chemokines IL-8, MCP-1, and RANTES in human HaCaT cells. The effects were in part stronger than those of the positive control hydrocortisone. For preparative isolation, the 70% EtOH extract was partitioned between -BuOH and water. Separation of the -BuOH-soluble fraction by centrifugal partition chromatography, followed by preparative and semipreparative HPLC, afforded a series of bidesmosidic glycosides of echinocystic acid bearing a 3,16-dihydroxy-10-oxo-hexadecanoic acid residue attached to the glycosidic moiety at C-28. They include azafrines 1 and 2, previously reported in saffron, and eight new congeners named azafrines 3-10. Saffron saponins significantly inhibited TNF-α/IFN-γ-induced secretion of RANTES in human HaCaT cells at 1 μM ( < 0.001). Some of them further lowered TNF-α/IFN-γ-induced gene expression.
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http://dx.doi.org/10.1021/acs.jnatprod.0c01220DOI Listing
March 2021

A Cytotoxic Porphyrin from North Pacific Brittle Star .

Mar Drugs 2020 Dec 29;19(1). Epub 2020 Dec 29.

Translational Research Center in Oncohaematology, Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland.

Triple-negative breast cancer (TNBC) represents the deadliest form of gynecological tumors currently lacking targeted therapies. The ethanol extract of the North Pacific brittle star presented promising anti-TNBC activities. After elimination of the inert material, the active extract was submitted to a bioguided isolation approach using high-resolution semipreparative HPLC-UV, resulting in one-step isolation of an unusual porphyrin derivative possessing strong cytotoxic activity. HRMS and 2D NMR resulted in the structure elucidation of the compound as (3,4)-14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid. Never identified before in Ophiuroidea, porphyrins have found broad applications as photosensitizers in the anticancer photodynamic therapy. The simple isolation of a cytotoxic porphyrin from an abundant brittle star species we describe here may pave the way for novel natural-based developments of targeted anti-cancer therapies.
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http://dx.doi.org/10.3390/md19010011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824513PMC
December 2020

A Mass Spectrometry Based Metabolite Profiling Workflow for Selecting Abundant Specific Markers and Their Structurally Related Multi-Component Signatures in Traditional Chinese Medicine Multi-Herb Formulae.

Front Pharmacol 2020 3;11:578346. Epub 2020 Dec 3.

School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland.

In Traditional Chinese Medicine (TCM), herbal preparations often consist of a mixture of herbs. Their quality control is challenging because every single herb contains hundreds of components (secondary metabolites). A typical 10 herb TCM formula was selected to develop an innovative strategy for its comprehensive chemical characterization and to study the specific contribution of each herb to the formula in an exploratory manner. Metabolite profiling of the TCM formula and the extract of each single herb were acquired with liquid chromatography coupled to high-resolution mass spectrometry for qualitative analyses, and to evaporative light scattering detection (ELSD) for semi-quantitative evaluation. The acquired data were organized as a feature-based molecular network (FBMN) which provided a comprehensive view of all types of secondary metabolites and their occurrence in the formula and all single herbs. These features were annotated by combining MS/MS-based spectral match, manual evaluation of the structural consistency in the FBMN clusters, and taxonomy information. ELSD detection was used as a filter to select the most abundant features. At least one marker per herb was highlighted based on its specificity and abundance. A single large-scale fractionation from the enriched formula enabled the isolation and formal identification of most of them. The obtained markers allowed an improved annotation of associated features by manually propagating this information through the FBMN. These data were incorporated in the high-resolution metabolite profiling of the formula, which highlighted specific series of related components to each individual herb markers. These series of components, named multi-component signatures, may serve to improve the traceability of each herb in the formula. Altogether, the strategy provided highly informative compositional data of the TCM formula and detailed visualizations of the contribution of each herb by FBMN, filtered feature maps, and reconstituted chromatogram traces of all components linked to each specific marker. This comprehensive MS-based analytical workflow allowed a generic and unbiased selection of specific and abundant markers and the identification of multiple related sub-markers. This exploratory approach could serve as a starting point to develop more simple and targeted quality control methods with adapted marker specificity selection criteria to given TCM formula.
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http://dx.doi.org/10.3389/fphar.2020.578346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756971PMC
December 2020

In Vitro Anti-Inflammatory Activity in Arthritic Synoviocytes of Root Extracts and Its Unusual Dimeric Flavonoids.

Molecules 2020 Nov 9;25(21). Epub 2020 Nov 9.

School of Pharmaceutical Sciences, University of Geneva, 1211 Geneva-4, Switzerland.

is a plant commonly used for the treatment of kidney stones, arthritis and pain in traditional Brazilian medicine. Different in vitro and in vivo activities, ranging from antinociceptive to anti-, have been reported for the dichloromethane root extract of (DCMAB) and isolated compounds. This work aimed to assess the in vitro anti-inflammatory activity in arthritic synoviocytes of the DCMAB, the hydroethanolic extract (HEAB) and three dimeric flavonoids isolated from the DCMAB. These compounds, brachydin A (), B () and C (), were isolated both by medium pressure liquid and high-speed counter current chromatography. Their quantification was performed by mass spectrometry on both DCMAB and HEAB. IL-1β activated human fibroblast-like synoviocytes were incubated with both extracts and isolated compounds to determine the levels of pro-inflammatory cytokine IL-6 by enzyme-linked immunosorbent assay (ELISA). DCMAB inhibited 30% of IL-6 release at 25 µg/mL, when compared with controls while HEAB was inactive. IC values determined for and were 3-fold higher than . The DCMAB activity seems to be linked to higher proportions of compounds and in this extract. These observations could thus explain the traditional use of roots in the treatment of osteoarthritis.
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http://dx.doi.org/10.3390/molecules25215219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665123PMC
November 2020

Phosphatidylcholines from eggs activate an immune response in Arabidopsis.

Elife 2020 09 28;9. Epub 2020 Sep 28.

Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland.

Recognition of conserved microbial molecules activates immune responses in plants, a process termed pattern-triggered immunity (PTI). Similarly, insect eggs trigger defenses that impede egg development or attract predators, but information on the nature of egg-associated elicitors is scarce. We performed an unbiased bioactivity-guided fractionation of eggs of the butterfly . Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry of active fractions led to the identification of phosphatidylcholines (PCs). PCs are released from insect eggs, and they induce salicylic acid and HO accumulation, defense gene expression and cell death in , all of which constitute a hallmark of PTI. Active PCs contain primarily C16 to C18-fatty acyl chains with various levels of desaturation, suggesting a relatively broad ligand specificity of cell-surface receptor(s). The finding of PCs as egg-associated molecular patterns (EAMPs) illustrates the acute ability of plants to detect conserved immunogenic patterns from their enemies, even from seemingly passive structures such as eggs.
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http://dx.doi.org/10.7554/eLife.60293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521926PMC
September 2020

Production of Highly Active Antiparasitic Compounds from the Controlled Halogenation of the Crude Plant Extract.

J Nat Prod 2020 09 9;83(9):2631-2640. Epub 2020 Sep 9.

School of Pharmaceutical Sciences and Institute of Pharmaceutical Sciences of Western Switzerland (ISPSW), University of Geneva, CMU, Rue Michel Servet 1, 1211 Geneva 4, Switzerland.

Direct halogenation of phenolic compounds present in the CHCl extract of the roots of was investigated to enhance chemodiversity. The approach is based on eco-friendly reactions using NaBr, NaI, and NaCl in aqueous media to generate multiple "unnatural" halogenated natural products from crude extracts. The halogenation reactions, monitored by UHPLC-PDA-ELSD-MS, were optimized to generate mono-, di-, or trihalogenated derivatives. To isolate these compounds, the reactions were scaled up and the halogenated analogues were isolated by semipreparative HPLC-UV and fully characterized by NMR and HR-MS data. All of the original 16 halogenated derivatives were evaluated for their antiparasitic activities against the parasites and . Compounds presenting selective antiparasitic activities against one or both parasites with IC values comparable to the reference were identified.
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http://dx.doi.org/10.1021/acs.jnatprod.0c00433DOI Listing
September 2020

Symphytum officinale L.: Liquid-liquid chromatography isolation of caffeic acid oligomers and evaluation of their influence on pro-inflammatory cytokine release in LPS-stimulated neutrophils.

J Ethnopharmacol 2020 Nov 31;262:113169. Epub 2020 Jul 31.

Department of Pharmacognosy, Grigore T. Popa University of Medicine and Pharmacy Iasi, 700115, Iasi, Romania; Biothermodynamics, TUM School of Life and Food Sciences Weihenstephan, Technical University of Munich, 85354, Freising, Germany. Electronic address:

Ethnopharmacological Relevance: Symphytum officinale L. (comfrey, Boraginaceae) has been traditionally used for millennia in joint distortions, myalgia, bone fractures and hematomas. However, key activity-determining constituents and molecular mechanisms underlying its use have not been completely elucidated.

Aim Of The Study: The objective of this study was to isolate and identify the major compounds from a hydroethanolic root extract of S. officinale and evaluate their antioxidant potential, alongside their effect on the cytokine production of ex vivo stimulated neutrophils, thus providing scientific support for the traditional use of comfrey root.

Material And Methods: Four caffeic acid oligomers were isolated from comfrey roots by liquid-liquid chromatography, their structures being established by MS and NMR analyses. In vitro antioxidant evaluation was performed by DPPH and ABTS assays. The cytotoxicity of isolated compounds was established by flow cytometry. The effect on cytokine release, such as interleukin (IL)-1β, IL-8 and tumor necrosis factor alpha (TNF-α), in lipopolysaccharide (LPS)-stimulated neutrophils was determined by enzyme-linked immunosorbent assay (ELISA).

Results: The main constituents found in comfrey root were represented by four caffeic acid oligomers, namely globoidnan B (1), rabdosiin (2), rosmarinic acid (3) and globoidnan A (4). Rabdosiin, globoidnans A and B were isolated for the first time from S. officinale. In the in vitro antioxidant tests, compound 2 was the most active, with EC values in DPPH and ABTS assays of 29.14 ± 0.43 and 11.13 ± 0.39, respectively. Neutrophils' viability over the tested concentration domain of 12.5-50 μM was not altered. At 50 μM, all compounds significantly inhibited IL-1β release, with compound 3 (45.60% release vs. LPS stimulated neutrophils) being the most active, followed by compounds 1 (53.85%), 2 (69.89%) and 4 (60.68%).

Conclusions: The four caffeic acid oligomers reported in S. officinale root may contribute to the overall anti-inflammatory activity for which comfrey preparations are used in traditional medicine.
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http://dx.doi.org/10.1016/j.jep.2020.113169DOI Listing
November 2020

Generation of Stilbene Antimicrobials against Multiresistant Strains of through Biotransformation by the Enzymatic Secretome of .

J Nat Prod 2020 08 24;83(8):2347-2356. Epub 2020 Jul 24.

School of Pharmaceutical Sciences, University of Geneva, CMU, Rue Michel Servet 1, 1211 Geneva 4, Switzerland.

The biotransformation of a mixture of resveratrol and pterostilbene was performed by the protein secretome of Several reaction conditions were tested to overcome solubility issues and to improve enzymatic activity. Using MeOH as cosolvent, a series of unusual methoxylated compounds was generated. The reaction was scaled-up, and the resulting mixture purified by semipreparative HPLC-PDA-ELSD-MS. Using this approach, 15 analogues were isolated in one step. Upon full characterization by NMR and HRMS analyses, eight of the compounds were new. The antibacterial activities of the isolated compounds were evaluated in vitro against the opportunistic pathogens and . The selectivity index was calculated based on cytotoxic assays performed against human liver carcinoma cells (HepG2) and the human breast epithelial cell line (MCF10A). Some compounds revealed remarkable antibacterial activity against multidrug-resistant strains of with moderate human cell line cytotoxicity.
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http://dx.doi.org/10.1021/acs.jnatprod.0c00071DOI Listing
August 2020

Phytochemical and Biological Investigation of Helianthemum nummularium, a High-Altitude Growing Alpine Plant Overrepresented in Ungulates Diets.

Planta Med 2020 Nov 9;86(16):1185-1190. Epub 2020 Jul 9.

Univ. Grenoble Alpes, CNRS, DPM, Grenoble, France.

is a European shrub growing at high altitude where it copes with a high level of stress. It was found to be overexpressed in ungulates diets compared to more abundant surrounding plants. These elements combined with the fact that from the Alps has never been investigated prompted us to study the phytochemical composition of its aerial parts. The analysis of the polar extract allowed for the isolation of eight compounds: -hydroxybenzoic acid, tiliroside, kaempferol, astragalin, quercetin, plantainoside B, quercetin-3--glucoside, and quercetin-3--glucuronide. We investigated the effect of the polar extract and isolated compounds on nuclear factor erythroid 2-related factor 2 transcription factor, which regulates the expression of a wide variety of cytoprotective genes. We found that the ethanolic extract activates the expression of nuclear factor erythroid 2-related factor 2 in a dose-dependent manner, whereas the pure compounds were much less active. The activation of the nuclear factor erythroid 2-related factor 2 pathway by the plant extract could pave the way for studies to promote healthy aging through protection of cells against oxidative stress. Moreover, the isolated compounds could be investigated alone or in combination in the perspective of making the link between the ungulate's preference for this plant and possible use of it for self-medication.
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http://dx.doi.org/10.1055/a-1197-2898DOI Listing
November 2020

Metabolite Profiling of Javanese Ginger and Identification of Antiseizure Metabolites via a Low-Cost Open-Source Zebrafish Bioassay-Guided Isolation.

J Agric Food Chem 2020 Jul 19;68(30):7904-7915. Epub 2020 Jul 19.

School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland.

The rhizomes of "Bangle", were investigated for its antiseizure properties using a streamlined and cost-effective zebrafish screening strategy and a mouse epilepsy assay. Its hexane extract demonstrated strong antiseizure activity in zebrafish epilepsy assay and was, therefore, selected for bioactivity-guided fractionation. Twelve compounds (-) were isolated, and two bioactive phenylbutenoids, - () and -banglene (), reduced up to 70% of pentylenetetrazole (PTZ)-induced seizures. These compounds showed moderate activity against PTZ-induced seizures in a mouse epilepsy assay. To understand the specificity of active compounds, its chemical profile was compared to that of . Their composition was assessed by differential metabolite profiling visualized by a molecular network, which revealed only vanillin derivatives and terpenoids as common metabolites and gave a comprehensive view of composition. This study demonstrates the efficacy of a streamlined zebrafish epilepsy assay, which is therefore suitable for routine screening in phytochemistry laboratories.
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http://dx.doi.org/10.1021/acs.jafc.0c02641DOI Listing
July 2020

Metabolomics of Populations in Brazilian Savanna Reveals Strong Influence of Environmental Factors on Its Specialized Metabolism.

Molecules 2020 Jun 26;25(12). Epub 2020 Jun 26.

School of Pharmaceutical Sciences, Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva (IPSWS), CH-1211 Geneva 4, Switzerland.

Environmental conditions influence specialized plant metabolism. However, many studies aiming to understand these modulations have been conducted with model plants and/or under controlled conditions, thus not reflecting the complex interaction between plants and environment. To fully grasp these interactions, we investigated the specialized metabolism and genetic diversity of a native plant in its natural environment. We chose due to its medicinal interest and occurrence in Brazilian savanna regions with diverse climate and soil conditions. An LC-HRMS-based metabolomics approach was applied to analyze 271 samples harvested across seven regions during the dry and rainy season. Genetic diversity was assessed in a subset of 40 samples using amplified fragment length polymorphism. Meteorological factors including rainfall, temperature, radiation, humidity, and soil nutrient and mineral composition were recorded in each region and correlated with chemical variation through multivariate analysis (MVDA). Marker compounds were selected using a statistically informed molecular network and annotated by dereplication against an in silico database of natural products. The integrated results evidenced different chemotypes, with variation in flavonoid and tannin content mainly linked to soil conditions. Different levels of genetic diversity and distance of populations were found to be correlated with the identified chemotypes. These observations and the proposed analytical workflow contribute to the global understanding of the impact of abiotic factors and genotype on the accumulation of given metabolites and, therefore, could be valuable to guide further medicinal exploration of native species.
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http://dx.doi.org/10.3390/molecules25122954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356273PMC
June 2020

Rutamarin: Efficient Liquid-Liquid Chromatographic Isolation from L. and Evaluation of Its In Vitro and In Silico MAO-B Inhibitory Activity.

Molecules 2020 Jun 9;25(11). Epub 2020 Jun 9.

Independent Laboratory of Natural Products Chemistry, Department of Pharmacognosy, Medical University of Lublin, 20-093 Lublin, Poland.

Naturally occurring coumarins are a group of compounds with many documented central nervous system (CNS) activities. However, dihydrofuranocoumarins have been infrequently investigated for their bioactivities at CNS level. Within the frame of this study, an efficient liquid-liquid chromatography method was developed to rapidly isolate rutamarin from L. (Rutaceae) dichloromethane extract (DCM). The crude DCM (9.78 mg/mL) and rutamarin (6.17 M) were found to be effective inhibitors of human monoamine oxidase B (MAO-B) with inhibition percentages of 89.98% and 95.26%, respectively. The inhibitory activity against human monoamine oxidase A (MAO-A) for the DCM extract was almost the same (88.22%). However, for rutamarin, it significantly dropped to 25.15%. To examine the molecular interaction of rutamarin with MAO- B, an evaluation was implemented. A docking study was performed for the two enantiomers ()-rutamarin and ()-rutamarin. The ()-rutamarin was found to bind stronger to the MAO-B binging cavity.
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http://dx.doi.org/10.3390/molecules25112678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321355PMC
June 2020

Antiseizure potential of the ancient Greek medicinal plant Helleborus odorus subsp. cyclophyllus and identification of its main active principles.

J Ethnopharmacol 2020 Sep 21;259:112954. Epub 2020 May 21.

School of Pharmaceutical Sciences, Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel Servet 1, CH-1211, Geneva 4, Switzerland. Electronic address:

Ethnopharmacological Relevance: Ethnopharmacological data and ancient texts support the use of black hellebore (Helleborus odorus subsp. cyclophyllus, Ranunculaceae) for the management and treatment of epilepsy in ancient Greece.

Aim Of The Study: A pharmacological investigation of the root methanolic extract (RME) was conducted using the zebrafish epilepsy model to isolate and identify the compounds responsible for a potential antiseizure activity and to provide evidence of its historical use. In addition, a comprehensive metabolite profiling of this studied species was proposed.

Materials And Methods: The roots were extracted by solvents of increasing polarity and root decoction (RDE) was also prepared. The extracts were evaluated for antiseizure activity using a larval zebrafish epilepsy model with pentylenetetrazole (PTZ)-induced seizures. The RME exhibited the highest antiseizure activity and was therefore selected for bioactivity-guided fractionation. Isolated compounds were fully characterized by NMR and high-resolution tandem mass spectrometry (HRMS/MS). The UHPLC-HRMS/MS analyses of the RME and RDE were used for dereplication and metabolite profiling.

Results: The RME showed 80% inhibition of PTZ-induced locomotor activity (300 μg/ml). This extract was fractionated and resulted in the isolation of a new glucopyranosyl-deoxyribonolactone (1) and a new furostanol saponin derivative (2), as well as of 20-hydroxyecdysone (3), hellebrin (4), a spirostanol glycoside derivative (5) and deglucohellebrin (6). The antiseizure activity of RME was found to be mainly due to the new furostanol saponin (2) and hellebrin (4), which reduced 45% and 60% of PTZ-induced seizures (135 μM, respectively). Besides, the aglycone of hellebrin, hellebrigenin (S34), was also active (45% at 7 μM). To further characterize the chemical composition of both RME and RDE, 30 compounds (A7-33, A35-37) were annotated based on UHPLC-HRMS/MS metabolite profiling. This revealed the presence of additional bufadienolides, furostanols, and evidenced alkaloids.

Conclusions: This study is the first to identify the molecular basis of the ethnopharmacological use of black hellebore for the treatment of epilepsy. This was achieved using a microscale zebrafish epilepsy model to rapidly quantify in vivo antiseizure activity. The UHPLC-HRMS/MS profiling revealed the chemical diversity of the extracts and the presence of numerous bufadienolides, furostanols and ecdysteroids, also present in the decoction.
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http://dx.doi.org/10.1016/j.jep.2020.112954DOI Listing
September 2020

Zebrafish bioassay-guided isolation of antiseizure compounds from the Cameroonian medicinal plant Cyperus articulatus L.

Phytomedicine 2020 Jan 23;70:153175. Epub 2020 Jan 23.

Institute of Pharmaceutical Sciences of Western Switzerland, CMU - Rue Michel-Servet 1, Geneva 4 CH-1211, Switzerland. Electronic address:

Background: Epilepsy is a chronic neurological disorder affecting more than 50 million people worldwide, of whom 80% live in low- and middle-income countries. Due to the limited availability of antiseizure drugs (ASDs) in these countries, medicinal plants are the first-line treatment for most epilepsy patients. In Cameroon, a decoction of Cyperus articulatus L. rhizomes is traditionally used to treat epilepsy.

Purpose: The aim of this study was to identify and isolate the active compounds responsible for the antiseizure activity of C. articulatus in order to confirm both its traditional medicinal usage and previous in vivo studies on extracts of this plant in mouse epilepsy models.

Methods: The dried rhizomes of C. articulatus were extracted with solvents of increasing polaritie (hexane, dichloromethane, methanol and water). A traditional decoction and an essential oil were also prepared. These extracts were evaluated for antiseizure activity using a larval zebrafish seizure model with seizures induced by the GABA antagonist pentylenetetrazole (PTZ). The hexane extract demonstrated the highest antiseizure activity and was therefore selected for bioassay-guided fractionation. The isolated bioactive compounds were characterized by classical spectroscopic methods. Since they were found to be volatile, they were quantified by GC-FID. In addition, the absorption of the active compounds through the gastrointestinal tract and the blood-brain barrier was evaluated using a hexadecane and a blood-brain barrier parallel artificial membrane permeability assays (HDM-PAMPA and PAMPA-BBB).

Results: The hexane extract of C. articulatus exhibited the highest antiseizure activity with a reduction of 93% of PTZ-induced seizures, and was therefore subjected to bioassay-guided fractionation in order to isolate the active principles. Four sesquiterpenoids were identified as cyperotundone (1), mustakone (2), 1,2-dehydro-α-cyperone (3) and sesquichamaenol (4) and exhibited significant antiseizure activity. These volatile compounds were quantified by GC in the hexane extract, the essential oil and the simulated traditional decoction. In addition, the constituents of the hexane extract including compounds 1 and 2 were found to cross the gastrointestinal barrier and the major compound 2 crossed the blood-brain barrier as well.

Conclusion: These results highlight the antiseizure activity of various sesquiterpene compounds from a hexane extract of C. articulatus dried rhizomes and support its use as a traditional treatment for epilepsy.
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http://dx.doi.org/10.1016/j.phymed.2020.153175DOI Listing
January 2020

Puerariae lobatae root extracts and the regulation of brown fat activity.

Phytomedicine 2019 Nov 23;64:153075. Epub 2019 Aug 23.

HanseMerkur Center for Traditional Chinese Medicine at the University Medical Center Hamburg-Eppendorf, Breitenfelder Straße 15, D-20251 Hamburg, Germany. Electronic address:

Background: Obesity is one of the major health problems worldwide. The induction of brown adipocyte formation and activity represents a promising therapeutic option by increasing energy expenditure. Asian herbs have the potential to treat obesity, however, pharmacological effects should be well documented at the molecular level first.

Hypothesis: A novel hypothesis-driven screening approach identified the root of Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep (PLR) to have potential effects on obesity by stimulating brown adipocytes.

Study Design: This study explored the metabolic effects of PLR water extract (PLRE) in a high-fat diet-induced obesity mouse model and characterized its secondary metabolite composition.

Methods: Animals were orally treated daily for two weeks and the bioactivity of PLRE evaluated by measuring various parameters including body weight, circulating metabolites, energy expenditure and insulin sensitivity. The chemical composition of the mains components was obtained by HPLC-MS-ELSD-PDA. Based on the dereplication results and semi-quantitative estimation, pure molecules were selected for tests on adipocytes in vitro.

Results: PLRE induces brown adipocyte activity and triggers the formation of brown-like cells in inguinal fat tissue, weight loss, and improved glucose metabolism. These effects are primarily caused by cell-autonomous activation of brown adipocytes and not by autonomic nervous system regulation. Even though the analysis of PLRE revealed puerarin as the most abundant secondary metabolite, it showed no effect on brown adipocyte formation and function. Brown adipocyte activity was induced dose-dependently by two other isoflavones, daidzein, and genistein. Daidzein is present in a very small amount in PLRE, but various glycosidic isoflavones, including puerarin, may release daidzein after metabolism.

Conclusion: This approach demonstrated the positive effects of PLRE on a diet-induced obesity mouse model and provided clues on the mode of action of PLRE at the molecular level.
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http://dx.doi.org/10.1016/j.phymed.2019.153075DOI Listing
November 2019

Discovery of Lipid Peroxidation Inhibitors from Species Prioritized through Multivariate Data Analysis and Multi-Informative Molecular Networking.

Molecules 2019 Aug 17;24(16). Epub 2019 Aug 17.

Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok 65000, Thailand.

A major goal in the discovery of bioactive natural products is to rapidly identify active compound(s) and dereplicate known molecules from complex biological extracts. The conventional bioassay-guided fractionation process can be time consuming and often requires multi-step procedures. Herein, we apply a metabolomic strategy merging multivariate data analysis and multi-informative molecular maps to rapidly prioritize bioactive molecules directly from crude plant extracts. The strategy was applied to 59 extracts of three species (, and ), which were profiled by UHPLC-HRMS and screened for anti-lipid peroxidation activity. Using this approach, six lipid peroxidation inhibitors ‒ of three spp. were discovered, three of them being new compounds: monnieraside IV (), monnieraside V () and monnieraside VI (). The results demonstrate that this combined approach could efficiently guide the discovery of new bioactive natural products. Furthermore, the approach allowed to evidence that main semi-quantitative changes in composition linked to the anti-lipid peroxidation activity were also correlated to seasonal effects notably for .
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http://dx.doi.org/10.3390/molecules24162989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719142PMC
August 2019

Chemical Constituents of as Inhibitors of Sirtuins.

Molecules 2019 Apr 3;24(7). Epub 2019 Apr 3.

Instituto Gonçalo Moniz, FIOCRUZ, Salvador, BA 40296-710, Brazil.

Benznidazole and nifurtimox, the only drugs available for the treatment of Chagas disease, have limited efficacy and have been associated with severe adverse side effects. Thus, there is an urgent need to find new biotargets for the identification of novel bioactive compounds against the parasite and with low toxicity. Silent information regulator 2 (Sir2) enzymes, or sirtuins, have emerged as attractive targets for the development of novel antitrypanosomatid agents. In the present work, we evaluated the inhibitory effect of natural compounds isolated from cashew nut (, L. Anacardiaceae) against the target enzymes TcSir2rp1 and TcSir2rp3 as well as the parasite. Two derivates of cardol (, ), cardanol (, ), and anacardic acid (, ) were investigated. The two anacardic acids (, ) inhibited both TcSir2rp1 and TcSir2rp3, while the cardol compound () inhibited only TcSir2rp1. The most potent sirtuin inhibitor active against the parasite was the cardol compound (), with an EC value of 12.25 µM, similar to that of benznidazole. Additionally, compounds (, ), which were inactive against the sirtuin targets, presented anti- effects. In conclusion, our results showed the potential of compounds for the development of potential sirtuin inhibitors and anti- agents.
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http://dx.doi.org/10.3390/molecules24071299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479711PMC
April 2019

Utility of dry load injection for an efficient natural products isolation at the semi-preparative chromatographic scale.

J Chromatogr A 2019 Aug 21;1598:85-91. Epub 2019 Mar 21.

School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU, 1, Rue Michel Servet, 1211 Geneva 4, Switzerland.

Semi-preparative HPLC is one of the main techniques used for the purification of natural products (NPs). Generally, the sample has to be solubilized in organic solvent and injected on column through a loop valve. Since the solubility of crude natural extracts is often limited, a high solvent volume is needed for injection. This significantly compromises the resolution and increases the risk of overpressure in the system. To overcome this problem, a dry load injection procedure was developed to ensure optimum resolution even at high sample loading. The approach was first validated with a representative mixture of NPs standards, and successfully applied to two representative crude plant extracts: the dichloromethane extract of Annacardium occidentale and the methanolic extract of Hypericum perforatum. In all cases, the dry loading injection setup enabled an efficient introduction of the samples in the semi-preparative HPLC system. Different overload conditions of the columns were tested and the results demonstrated the robustness of the method and the possibility of applying it with a limited loss of resolution compared to liquid injection and without increasing pressure. The chromatographic resolutions were close to those obtained at the analytical level and separation were of much better quality when compared to liquid injection. This approach is especially relevant when purifying compounds isolated with high resolution from extracts that are poorly soluble in low volume of injection solvent due to the presence of lipophilic compounds and are thus not compatible for loop injection in typical reversed phase conditions. In addition, the dry load setup was also found to be useful when relatively polar components have to be separated in reversed phase conditions. In this case, loop injection with methanol generates strong peak distortion and broadening, while the dry load injection affords symmetrical peaks.
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http://dx.doi.org/10.1016/j.chroma.2019.03.042DOI Listing
August 2019

Antimycobacterial activity in a single-cell infection assay of ellagitannins from Combretum aculeatum and their bioavailable metabolites.

J Ethnopharmacol 2019 Jun 23;238:111832. Epub 2019 Mar 23.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211, Geneva 11, Switzerland. Electronic address:

Ethnopharmacological Relevance: The water decoction of Combretum aculeatum aerial parts is traditionally used in Senegal to treat tuberculosis (TB). The extract shows significant antimycobacterial activity in a validated single-cell infection assay.

Aim Of The Study: The main aim of this study was to identify the antimycobacterial compounds in the water decoction of Combretum aculeatum. Since the traditional preparations are used orally, a bioactivity assessment of the possible bioavailable human metabolites was also performed.

Materials And Methods: The Combretum aculeatum water decoction extract was first fractionated by flash chromatography. The fractions were submitted to an antibiotic assay against Mycobacterium marinum and to a single-cell infection assay involving Acanthamoeba castellanii as a host. Using these approaches, it was possible to correlate the antimycobacterial activity with two zones of the chromatogram. In parallel with this liquid chromatography (LC)-based activity profiling, high-resolution mass spectrometry (UHPLC-HRMS/MS) revealed the presence of ellagitannin (Et) derivatives in the active zones of the chromatogram. Isolation of the active compounds was performed by preparative chromatography. The structures of the isolated compounds were elucidated by nuclear magnetic resonance (NMR). Additionally, the main human metabolites of commercially available Ets were biologically evaluated in a similar manner.

Results: The in vitro bioassay-guided isolation of the Combretum aculeatum water extract led to the identification of three Ets (1-3) and ellagic acid (4). The major compounds 2 and 3 (α- and β-punicalagin, respectively), exhibited anti-infective activity with an IC of 51.48 μM. In view of the documented intestinal metabolism of these compounds, some metabolites, namely, urolithin A (5), urolithin B (6) and urolithin D (7), were investigated for their antimycobacterial activity in the two assays. Urolithin D (7) exhibited the strongest anti-infective activity, with an IC of 345.50 μM, but this was moderate compared to the positive control rifampin (IC of 6.99 μM). The compounds assayed had no observable cytotoxicity towards the amoeba host cells at concentrations lower than 200 μg/mL.

Conclusion: The observed antimycobacterial properties of the traditional water decoction of Combretum aculeatum might be related to the activity of Ets derivatives (1-3) and their metabolites, such as ellagic acid (4) and urolithin D (7). Despite the relatively weak activity of these metabolites, the high consumption of tannins achieved by taking the usual traditional decoction doses should lead to an important increase in the plasmatic concentrations of these active and bioavailable metabolites. These results support to some extent the traditional use of Combretum aculeatum to treat tuberculosis.
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http://dx.doi.org/10.1016/j.jep.2019.111832DOI Listing
June 2019

High-performance countercurrent chromatographic isolation of acylated iridoid diglycosides from Verbascum ovalifolium Donn ex Sims and evaluation of their inhibitory potential on IL-8 and TNF-α production.

J Pharm Biomed Anal 2019 Mar 18;166:295-303. Epub 2019 Jan 18.

Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, Chodzki 1, 20-093 Lublin, Poland.

In traditional folk medicine, Verbascum species have been used since ancient times to treat respiratory disorders, hemorrhoids, diarrhea, wounds, eczema and other types of inflammatory skin conditions. Despite the fact that more than 200 bioactive constituents (phenylethanoids, flavonoids, neolignan glycosides, phenolic acids, iridoids, saponins and polysaccharides) have been previously isolated from various Verbascum species, to date preparative high-performance countercurrent chromatography (HPCCC) has never been employed for this purpose. Therefore, in this study, simple HPCCC methods were successfully developed with the aim to primarily isolate acylated iridoid diglycosides from Verbascum ovalifolium Donn ex Sims (oval-leaved mullein). By the use of several biphasic solvent systems containing n-hexane, ethyl acetate, n-butanol/methanol and water, premnacorymboside B (3, 4 mg, 95.4% purity), saccatoside (4, 6 mg, 95.7% purity), premnacorymboside A (7, 6 mg, 98.3%), scorodioside (8, 11 mg, 96.0%) and 6-O-(3'',4''-di-O-trans-cinnamoyl)-α-L-rhamnopyranosylcatalpol (9, 8 mg, 95.3%) were afforded; compounds 7, 8 and 9 have not been previously reported in Verbascum genus. Additionally, two phenolic acids (1, 2), two flavonoids (6, 10) and verbascoside (5) were secondarily isolated. Evaluation of interleukin 8 (IL-8) and tumor necrosis factor α (TNF-α) inhibitory properties of the acylated iridoid diglycosides proved that these compounds down-regulated TNF-α release more efficiently than IL-8 secretion. The activity might be dependent on the degree of esterification, as diacyl derivatives showed more potent effects than monoesters. The HPCCC methods herein developed could serve to large scale isolation of constituents from Verbascum genus for extensive biological investigations.
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http://dx.doi.org/10.1016/j.jpba.2019.01.031DOI Listing
March 2019

High-performance counter-current chromatography isolation and initial neuroactivity characterization of furanocoumarin derivatives from Peucedanum alsaticum L (Apiaceae).

Phytomedicine 2019 Feb 26;54:259-264. Epub 2018 Oct 26.

Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, 1 Chodźki Str., 20-093 Lublin, Poland. Electronic address:

Background: Medicinal plants are a proven source of drug-like small molecules with activity towards targets relevant for diseases of the central nervous system (CNS). Plant species of the Apiaceae family have to date yielded a number of neuroactive metabolites, such as coumarin derivatives with acetylcholinesterase inhibitory activity or anti-seizure activity.

Purpose: To accelerate the discovery of neuroactive phytochemicals with potential as CNS drug leads, we sought to rapidly isolate furanocoumarins, primary constituents of the dichloromethane (DCM) extract of the fruits of Peucedanum alsaticum L. (Apiaceae), using high-performance counter-current chromatography (HPCCC) and to evaluate their neuroactivity using both in vitro and in vivo microscale bioassays based on cholinesterase ELISAs and zebrafish epilepsy models.

Research Methods And Procedure: In this study the DCM extract was subjected to HPCCC for the efficient separation (60 min) and isolation of furanocoumarins. Isolated compounds were identified with TOF-ESI-MS and NMR techniques and examined as inhibitors of AChE and BChE using ELISA microtiter assays. Anti-seizure properties of the extract and of the isolated compounds were evaluated using a zebrafish epilepsy model based on the GABA antagonist pentylenetetrazol (PTZ), which induces increased locomotor activity and seizure-like behavior.

Results: The solvent system, composed of n-heptane, ethyl acetate, methanol and water (3:1:3:1, v/v/v/v), enabled the isolation of 2.63 mg lucidafuranocoumarin A (purity 98%) and 8.82 mg bergamottin (purity 96%) from 1.6 g crude DCM extract. The crude extract, at a concentration of 100 µg/ml, exhibited a weak inhibitory activity against acetylcholinesterase (AChE) (9.63 ± 1.59%) and a moderate inhibitory activity against butyrylcholinestrase (BChE) (49.41 ± 2.19%). Lucidafuranocoumarin A (100 µg/ml) was inactive against AChE but showed moderate inhibition towards BChE (40.66 ± 1.25%). The DCM extract of P. alsaticum fruits (0.62-1.75 µg/ml) and bergamottin (2-10 µm) exhibited weak anti-seizure activity, while lucidafuranocoumarin A (10-16 µm) was found to significantly inhibit PTZ-induced seizures. The percentage of seizure inhibition for the isolated compounds, at their most bioactive concentration, was 26% for bergamottin and 69% for lucidafuranocoumarin A.

Conclusion: Our findings underscore the utility of HPCCC for the rapid isolation of rare coumarin derivatives, and the potential of microscale in vivo bioassays based on zebrafish disease models for the rapid assessment of neuroactivity of these drug-like natural products.
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http://dx.doi.org/10.1016/j.phymed.2018.10.030DOI Listing
February 2019

Chemo-Diversification of Plant Extracts Using a Generic Bromination Reaction and Monitoring by Metabolite Profiling.

ACS Comb Sci 2019 03 17;21(3):171-182. Epub 2019 Jan 17.

Microbiology Unit, Department of Botany and Plant Biology , University of Geneva , 30 quai Ernest-Ansermet , 1211 Geneva 4, Switzerland.

A generic procedure for direct bromination of polyphenol in crude plant extracts was developed to generate multiple "unnatural" halogenated natural products for further bioassay evaluation. To better control the halogenation procedure, the bromination was optimized with a flavonoid standard, and the reactions were monitored by high-performance liquid chromatography photometric diode array coupled to the evaporative light scattering detection (ELSD). ELSD detection was successfully used for a relative yield estimation of the compounds obtained. From the halogenation of hesperitin (11), five brominated compounds were obtained. After optimization, the reaction was successfully applied to the methanolic extract of Citrus sinensis peels, a typical waste biomass and also to the methanolic extract of the medicinal plant Curcuma longa. In both cases, the methanolic extracts were profiled by NMR for a rapid estimation of the polyphenol versus primary metabolite content. An enriched secondary metabolites extract was obtained using vacuum liquid chromatography and submitted to bromination. Metabolite profiling performed by ultrahigh purity liquid chromatography time-of-flight high-resolution mass spectrometry revealed the presence of various halogenated products. To isolate these compounds, the reactions were scaled up, and six halogenated analogues were isolated and fully characterized by NMR and high-resolution electrospray ionization mass spectrometry analyses. The antibacterial properties of these compounds were evaluated using in vitro bioassays against multiresistant strains of Staphylococcus aureus and Pseudomonas aeruginosa. Some of the halogenated derivatives obtained presented moderate antibacterial properties.
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http://dx.doi.org/10.1021/acscombsci.8b00132DOI Listing
March 2019
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