Xanthone Inhibitors of Unfolded Protein Response Isolated from Calophyllum caledonicum.

The unfolded protein response (UPR) is a key component of fungal virulence. The prenylated xanthone γ-mangostin isolated from Garcinia mangostana (Clusiaceae) fruit pericarp, has recently been described to inhibit this fungal adaptative pathway. Considering that Calophyllum caledonicum (Calophyllaceae) is known for its high prenylated xanthone content, its stem bark extract was fractionated using a bioassay-guided procedure based on the cell-based anti-UPR assay. Four previously undescribed xanthone derivatives were isolated, caledonixanthones N-Q (3, 4, 8, and 12), among which compounds 3 and 8 showed promising anti-UPR activities with IC50 values of 11.7 ± 0.9 and 7.9 ± 0.3 µM, respectively.

Natural Products Targeting the Fungal Unfolded Protein Response as an Alternative Crop Protection Strategy.

Discovering new solutions for crop protection is a major challenge for the next decades as a result of the ecotoxicological impact of classical fungicides, the emergence of fungicide resistances, and the consequence of climate change on pathogen distribution. Previous work on fungal mutants deficient in the unfolded protein response (UPR) supported that targeting this pathway is a promising plant disease control strategy. In particular, we showed that the UPR is involved in fungal virulence by altering cell protection against host defense compounds, such as phytoalexins and phytoanticipins. In this study, we evaluated natural products targeting fungal IRE1 protein (UPR effector) and consequently increasing fungal susceptibility to plant defenses. Developing an in vitro cell-based screening assay allowed for the identification of seven potential IRE1 inhibitors with a focus on polyhydroxylated prenylated xanthones. Inhibition of hac1 mRNA splicing, which is mediated by IRE1, was then validated for the most active compound, namely, γ-mangostin 3. To study the mode of interaction between the binding site of IRE1 and active xanthones, molecular docking was also undertaken, revealing similar and novel interactions between the known inhibitor and the binding site. Eventually, active xanthones applied at subtoxic doses induced a significant reduction in necrosis size for leaves of Brassica oleracea inoculated with Alternaria brassicicola and Botrytis cinerea.

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

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

Screening an In-House Isoquinoline Alkaloids Library for New Blockers of Voltage-Gated Na+ Channels Using Voltage Sensor Fluorescent Probes: Hits and Biases.

Voltage-gated Na+ (NaV) channels are significant therapeutic targets for the treatment of cardiac and neurological disorders, thus promoting the search for novel NaV channel ligands. With the objective of discovering new blockers of NaV channel ligands, we screened an In-House vegetal alkaloid library using fluorescence cell-based assays. We screened 62 isoquinoline alkaloids (IA) for their ability to decrease the FRET signal of voltage sensor probes (VSP), which were induced by the activation of NaV channels with batrachotoxin (BTX) in GH3b6 cells. This led to the selection of five IA: liriodenine, oxostephanine, thalmiculine, protopine, and bebeerine, inhibiting the BTX-induced VSP signal with micromolar IC50. These five alkaloids were then assayed using the Na+ fluorescent probe ANG-2 and the patch-clamp technique. Only oxostephanine and liriodenine were able to inhibit the BTX-induced ANG-2 signal in HEK293-hNaV1.3 cells. Indeed, liriodenine and oxostephanine decreased the effects of BTX on Na+ currents elicited by the hNaV1.3 channel, suggesting that conformation change induced by BTX binding could induce a bias in fluorescent assays. However, among the five IA selected in the VSP assay, only bebeerine exhibited strong inhibitory effects against Na+ currents elicited by the hNav1.2 and hNav1.6 channels, with IC50 values below 10 µM. So far, bebeerine is the first BBIQ to have been reported to block NaV channels, with promising therapeutical applications.

A Descriptive Chemical Composition of Concentrated Bud Macerates through an Optimized SPE-HPLC-UV-MS2 Method-Application to Alnus glutinosa, Ribes nigrum, Rosa canina, Rosmarinus officinalis and Tilia tomentosa.

Concentrated bud macerates (CBMs) are obtained from meristematic tissues such as buds and young shoots by maceration in a solvent composed of glycerin, water and ethanol (1/1/1/, v/v). Their traditional utilization in gemmotherapy has gained interest in the past years, and the knowledge of their chemical characterization can provide commercial arguments, particularly to secure their quality control. Therefore, an optimized method for phytochemical analysis including glycerol removal by a preliminary solid phase extraction (SPE) followed by compound identification using high performance liquid chromatography coupled with ultra-violet and tandem mass detectors (HPLC-UV-MS2) was developed. This method was applied on 5 CBMs obtained from Alnus glutinosa, Ribesnigrum, Rosmarinus officinalis, Rosa canina and Tilia tomentosa in order to determinate their chemical composition. Their antioxidant effects were also investigated by radical scavenging activity assays (DPPH and ORAC). Glycerol removal improved the resolution of HPLC chemical profiles and allowed us to perform TLC antioxidant screening. Our approach permitted the identification of 57 compounds distributed in eight major classes, three of them being common to all macerates including nucleosides, phenolic acids and glycosylated flavonoids. Quantification of the later class as a rutin equivalent (RE) showed a great disparity between Rosa canina macerate (809 mg RE/L), and the other ones (from 175 to 470 mg RE/L). DPPH and ORAC assays confirmed the great activity of Rosa canina (4857 and 6479 µmol TE/g of dry matter, respectively). Finally, phytochemical and antioxidant analysis of CBMs strengthened their phytomedicinal interest in the gemmotherapy field.

Toxicity and mode of action of the aporphine plant alkaloid liriodenine on the insect GABA receptor.

Liriodenine is a biologically active plant alkaloid with multiple effects on mammals, fungi, and bacteria, but has never been evaluated for insecticidal activity. Accordingly, liriodenine was applied topically in ethanolic solutions to adult female Anopheles gambiae, and found to be mildly toxic. Its lethality was synergized in mixtures with dimethyl sulfoxide and piperonyl butoxide. Recordings from the ventral nerve cord of larval Drosophila melanogaster showed that liriodenine was neuroexcitatory and reversed the inhibitory effect of 1 mM GABA at effective concentrations of 20-30 µM. GABA antagonism on the larval nervous system was equally expressed on both susceptible and cyclodiene-resistant rdl preparations. Acutely isolated neurons from Periplaneta americana were studied under patch clamp and inhibition of GABA-induced currents with an IC50 value of about 1 µM were observed. In contrast, bicuculline did not reverse the effects of GABA on cockroach neurons, as expected. In silico molecular models suggested reasonable structural concordance of liriodenine and bicuculline and isosteric hydrogen bond acceptor sites. This study is the first assessing of the toxicology of liriodenine on insects and implicates the GABA receptor as one likely neuronal target, where liriodenine might be considered an active chemical analog of bicuculline.

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

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

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

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

Collected mass spectrometry data on monoterpene indole alkaloids from natural product chemistry research.

This Data Descriptor announces the submission to public repositories of the monoterpene indole alkaloid database (MIADB), a cumulative collection of 172 tandem mass spectrometry (MS/MS) spectra from multiple research projects conducted in eight natural product chemistry laboratories since the 1960s. All data have been annotated and organized to promote reuse by the community. Being a unique collection of these complex natural products, these data can be used to guide the dereplication and targeting of new related monoterpene indole alkaloids within complex mixtures when applying computer-based approaches, such as molecular networking. Each spectrum has its own accession number from CCMSLIB00004679916 to CCMSLIB00004680087 on the GNPS. The MIADB is available for download from MetaboLights under the identifier: MTBLS142 ( https://www.ebi.ac.uk/metabolights/MTBLS142 ).

Prenylated Polyphenols from Clusiaceae and Calophyllaceae with Immunomodulatory Activity on Endothelial Cells.

Endothelial cells (ECs) are key players in inflammation and immune responses involved in numerous pathologies. Although attempts were experimentally undertaken to prevent and control EC activation, drug leads and probes still remain necessary. Natural products (NPs) from Clusiaceous and Calophyllaceous plants were previously reported as potential candidates to prevent endothelial dysfunction. The present study aimed to identify more precisely the molecular scaffolds that could limit EC activation. Here, 13 polyphenols belonging to 5 different chemical types of secondary metabolites (i.e., mammea coumarins, a biflavonoid, a pyranochromanone acid, a polyprenylated polycyclic acylphloroglucinol (PPAP) and two xanthones) were tested on resting and cytokine-activated EC cultures. Quantitative and qualitative changes in the expression of both adhesion molecules (VCAM-1, ICAM-1, E-selectin) and major histocompatibility complex (MHC) molecules have been used to measure their pharmaceutical potential. As a result, we identified 3 mammea coumarins that efficiently reduce (up to >90% at 10 µM) both basal and cytokine-regulated levels of MHC class I, class II, MICA and HLA-E on EC surface. They also prevented VCAM-1 induction upon inflammation. From a structural point of view, our results associate the loss of the free prenyl group substituting mammea coumarins with a reduced cellular cytotoxicity but also an abrogation of their anti-inflammatory potential and a reduction of their immunosuppressive effects. A PPAP, guttiferone J, also triggers a strong immunomodulation but restricted to HLA-E and MHC class II molecules. In conclusion, mammea coumarins with a free prenyl group and the PPAP guttiferone J emerge as NPs able to drastically decrease both VCAM-1 and a set of MHC molecules and to potentially reduce the immunogenicity of the endothelium.

Antifungal and antibacterial metabolites from a French poplar type propolis.

During this study, the in vitro antifungal and antibacterial activities of different extracts (aqueous and organic) obtained from a French propolis batch were evaluated. Antifungal activity was evaluated by broth microdilution on three pathogenic strains: Candida albicans, C. glabrata, and Aspergillus fumigatus. Antibacterial activity was assayed using agar dilution method on 36 Gram-negative and Gram-positive strains including Staphylococcus aureus. Organic extracts showed a significant antifungal activity against C. albicans and C. glabrata (MIC80 between 16 and 31 µg/mL) but only a weak activity towards A. fumigatus (MIC80 = 250 µg/mL). DCM based extracts exhibited a selective Gram-positive antibacterial activity, especially against S. aureus (SA) and several of its methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) strains (MIC100 30-97 µg/mL). A new and active derivative of catechin was also identified whereas a synergistic antimicrobial effect was noticed during this study.

Chemical composition, antioxidant and anti-AGEs activities of a French poplar type propolis.

Accumulation in tissues and serum of advanced glycation end-products (AGEs) plays an important role in pathologies such as Alzheimer's disease or, in the event of complications of diabetes, atherosclerosis or renal failure. Therefore, there is a potential therapeutic interest in compounds able to lower intra and extracellular levels of AGEs. Among them, natural antioxidants (AO) with true anti-AGEs capabilities would represent good candidates for development. The purpose of this study was to evaluate the AO and anti-AGEs potential of a propolis batch and then to identify the main compounds responsible for these effects. In vivo, protein glycation and oxidative stress are closely related. Thus, AO and antiglycation activities were evaluated using both DPPH and ORAC assays, respectively, as well as a newly developed automated anti-AGEs test. Several propolis extracts exhibited very good AO and anti-AGEs activities, and a bioguided fractionation allowed us to identify pinobanksin-3-acetate as the most active component.

Bioguided fractionation and isolation of natural inhibitors of advanced glycation end-products (AGEs) from Calophyllum flavoramulum.

Advanced glycation end-products (AGEs) are associated with many pathogenic disorders such as Alzheimer's disease, pathogenesis of diabetes, atherosclerosis or endothelial dysfunction leading to cardiovascular events. Clusiaceae and Calophyllaceae families are rich in compounds like polyphenols which are able to inhibit their formation and are therefore of great interest. Calophyllum flavoramulum Hend. & Wyatt-Sm., a native Malaysian plant, was selected after an anti-AGEs screening conducted on DCM and MeOH extracts from plants belonging to these aforementioned families. In a first study, bioguided fractionation of the MeOH leaf extract of C. flavoramulum afforded amentoflavone, 3-methoxy-2-hydroxyxanthone, 3,4-dihydroxy-tetrahydrofuran-3-carboxylic acid, quercitrin, 3,4-dihydroxybenzoic acid, canophyllol and apetalactone. Amentoflavone and 3-methoxy-2-hydroxyxanthone were found to be very potent (IC(50)=0.05 and 0.06 mM respectively), while anti-AGEs activities of quercitrin and 3,4-dihydroxybenzoic acid appeared as moderately strong (IC(50)=0.5 mM). In a second study, a systematic phytochemical study of the cyclohexane, DCM and EtOAc extracts obtained from the same plant was conducted to isolate the following products: flavoramulone, 6-deoxyjacareubin, rheediachromenoxanthone, 2,3-dihydroamentoflavone and benzoic acid. 3,4-Dihydroxy-tetrahydrofuran-3-carboxylic acid and flavoramulone were isolated for the first time and their structures were identified by means of IR, MS and NMR spectrometries.

In vitro and in vivo bactericidal activities of vancomycin dispersed in porous biodegradable poly(epsilon-caprolactone) microparticles.

Treatment of methicillin-resistant Staphylococcus aureus osteomyelitis requires a prolonged antibiotic therapy with vancomycin. Because of its weak diffusion, the in situ implantation of vancomycin could be interesting. The activity of vancomycin encapsulated in microparticles was evaluated in vitro and in vivo on rabbit osteomyelitis and showed a good activity compared to intravenous administration.

Prenylated xanthones and tocotrienols from Garcinia virgata.

Two xanthones, namely virgataxanthone A and B, have been isolated from the stem bark of Garcinia virgata, together with two formylated tocotrienols and the known delta-tocotrienol, griffipavixanthone and 2,6-dihydroxy-4-methoxybenzophenone (cotoin). Their structures were mainly established using one and two-dimensional NMR and mass spectroscopies. When sufficient material was available, the antioxidant activities of the crude extracts as well as the isolated compounds were evaluated.