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

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

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.

Bee Species, Botanical Sources and the Chemical Composition of Propolis from Yucatan, Mexico.

Propolis is used by corbiculated bees to protect the bee hive; it is mostly used to seal cracks, to reduce or prevent microbial growth and to embalm invaders. Different factors have been reported to influence the chemical composition of propolis, including bee species and the flora surrounding the hive. Nevertheless, the majority of the studies are focused on propolis produced by Apis mellifera, while studies on the chemical composition of propolis produced by stingless bees are still limited. In this investigation, the chemical composition of 27 propolis samples collected in the Yucatan Peninsula from A. mellifera beehives, together with 18 propolis samples from six different species of stingless bees, were analyzed by GC-MS. Results showed that lupeol acetate and ß-amyrin were the characteristic triterpenes in propolis samples from A. mellifera, while grandiflorenic acid and its methyl ester were the main metabolites present in samples from stingless bees. Multivariate analyses were used to explore the relationship between bee species and botanical sources on the chemical composition of the propolis samples. Differences in body size and, therefore, foraging abilities, as well as preferences for specific botanical sources among bee species, could explain the observed variation in propolis chemical composition. This is the first report on the composition of propolis samples from the stingless bees Trigona nigra, Scaptotrigona pectoralis, Nannotrigona perilampoides, Plebeia frontalis and Partamona bilineata.

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

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

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

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

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

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

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.

Implementation of a MS/MS database for isoquinoline alkaloids and other annonaceous metabolites.

This data descriptor reports on the upload to a public repository (GNPS) of the IQAMDB, IsoQuinoline and Annonaceous Metabolites Data Base, comprising 320 tandem mass spectra. This project originated from our in-house collection of isoquinolines. The diversity of compounds included in this database was further extended through the contribution of two additional laboratories involved in isoquinoline alkaloids research: University of Angers and University of Manaus. The generated MS/MS data were processed and annotated on an individual basis to promote their straightforward reuse by natural product chemists interested in either the description of new isoquinoline alkaloids or the dereplication of isoquinoline-containing samples. The interest of the current repertoire for dereplication purposes has been validated based on the molecular networking of the well-investigated plant model Annona montana against the IQAMDB-implemented GNPS.

Analogues of Natural Chalcones as Efficient Inhibitors of AKR1C3.

Naturally occurring substances are valuable resources for drug development. In this respect, chalcones are known to be antiproliferative agents against prostate cancer cell lines through various mechanisms or targets. Based on the literature and preliminary results, we aimed to study and optimise the efficiency of a series of chalcones to inhibit androgen-converting AKR1C3, known to promote prostate cancer. A total of 12 chalcones with different substitution patterns were synthesised. Structure-activity relationships associated with these modifications on AKR1C3 inhibition were analysed by performing enzymatic assays and docking simulations. In addition, the selectivity and cytotoxicity of the compounds were assessed. In enzymatic assays, C-6' hydroxylated derivatives were more active than C-6' methoxylated derivatives. In contrast, C-4 methylation increased activity over C-4 hydroxylation. Docking results supported these findings with the most active compounds fitting nicely in the binding site and exhibiting strong interactions with key amino acid residues. The most effective inhibitors were not cytotoxic for HEK293T cells and selective for 17ß-hydroxysteroid dehydrogenases not primarily involved in steroid hormone metabolism. Nevertheless, they inhibited several enzymes of the steroid metabolism pathways. Favourable substitutions that enhanced AKR1C3 inhibition of chalcones were identified. This study paves the way to further develop compounds from this series or related flavonoids with improved inhibitory activity against AKR1C3.

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.

Natural chalcones elicit formation of specialized pro-resolving mediators and related 15-lipoxygenase products in human macrophages.

Specialized pro-resolving mediators (SPMs) comprise lipid mediators (LMs) produced from polyunsaturated fatty acids (PUFAs) via stereoselective oxygenation particularly involving 12/15-lipoxygenases (LOXs). In contrast to pro-inflammatory LMs such as leukotrienes formed by 5-LOX and prostaglandins formed by cyclooxygenases, the SPMs have anti-inflammatory and inflammation-resolving properties. Although glucocorticoids and non-steroidal anti-inflammatory drugs (NSAIDs) that block prostaglandin production are still prime therapeutics for inflammation-related diseases despite severe side effects, novel concepts focus on SPMs as immunoresolvents for anti-inflammatory pharmacotherapy. Here, we studied the natural chalcone MF-14 and the corresponding dihydrochalcone MF-15 from Melodorum fruticosum, for modulating the biosynthesis of LM including leukotrienes, prostaglandins, SPM and their 12/15-LOX-derived precursors in human monocyte-derived macrophage (MDM) M1- and M2-like phenotypes. In MDM challenged with Staphylococcus aureus-derived exotoxins both compounds (10 µM) significantly suppressed 5-LOX product formation but increased the biosynthesis of 12/15-LOX products, especially in M2-MDM. Intriguingly, in resting M2-MDM, MF-14 and MF-15 strikingly evoked generation of 12/15-LOX products and of SPMs from liberated PUFAs, along with translocation of 15-LOX-1 to membranous compartments. Enhanced 12/15-LOX product formation by the chalcones was evident also when exogenous PUFAs were supplied, excluding increased substrate supply as sole underlying mechanism. Rather, MF-14 and MF-15 stimulate the activity of 15-LOX-1, supported by experiments with HEK293 cells transfected with either 5-LOX, 15-LOX-1 or 15-LOX-2. Together, the natural chalcone MF-14 and the dihydrochalcone MF-15 favorably modulate LM biosynthesis in human macrophages by suppressing pro-inflammatory leukotrienes but stimulating formation of SPMs by differential interference with 5-LOX and 15-LOX-1.

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.

Behavioral and Antennal Responses of Tribolium confusum to Varronia globosa Essential Oil and Its Main Constituents: Perspective for Their Use as Repellent.

Essential oils of aromatic plants represent an alternative to classical pest control with synthetic chemicals. They are especially promising for the alternative control of stored product pest insects. Here, we tested behavioral and electrophysiological responses of the stored product pest Tribolium confusum, to the essential oil of a Brazilian indigenous plant, Varronia globosa, collected in the Caatinga ecosystem. We analyzed the essential oil by GC-MS, tested the effects of the entire oil and its major components on the behavior of individual beetles in a four-way olfactometer, and investigated responses to these stimuli in electroantennogram recordings (EAG). We could identify 25 constituents in the essential oil of V. globosa, with anethole, caryophyllene and spathulenole as main components. The oil and its main component anethole had repellent effects already at low doses, whereas caryophyllene had only a repellent effect at a high dose. In addition, the essential oil abolished the attractive effect of the T. confusum aggregation pheromone. EAG recordings revealed dose-dependent responses to the individual components and increasing responses to the blend and even more to the entire oil. Our study reveals the potential of anethole and the essential oil of V. globosa in the management of stored product pests.

Exploration of Long-Chain Vitamin E Metabolites for the Discovery of a Highly Potent, Orally Effective, and Metabolically Stable 5-LOX Inhibitor that Limits Inflammation.

Endogenous long-chain metabolites of vitamin E (LCMs) mediate immune functions by targeting 5-lipoxygenase (5-LOX) and increasing the systemic concentrations of resolvin E3, a specialized proresolving lipid mediator. SAR studies on semisynthesized analogues highlight α-amplexichromanol (27a), which allosterically inhibits 5-LOX, being considerably more potent than endogenous LCMs in human primary immune cells and blood. Other enzymes within lipid mediator biosynthesis were not substantially inhibited, except for microsomal prostaglandin E2 synthase-1. Compound 27a is metabolized by sulfation and ß-oxidation in human liver-on-chips and exhibits superior metabolic stability in mice over LCMs. Pharmacokinetic studies show distribution of 27a from plasma to the inflamed peritoneal cavity and lung. In parallel, 5-LOX-derived leukotriene levels decrease, and the inflammatory reaction is suppressed in reconstructed human epidermis, murine peritonitis, and experimental asthma in mice. Our study highlights 27a as an orally active, LCM-inspired drug candidate that limits inflammation with superior potency and metabolic stability to the endogenous lead.

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.

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

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

Semisynthetic Vitamin E Derivatives as Potent Antibacterial Agents against Resistant Gram-Positive Pathogens.

New 5-substituted vitamin E derivatives were semisynthesized, and their antibacterial activity against human Gram-positive and Gram-negative pathogens was evaluated. Several vitamin E analogues were active against methicillin-resistant Staphylococcus aureus (MRSA) and/or methicillin-resistant Staphylococcus epidermidis (MRSE); structure-activity relationships (SARs) are discussed. As a result, it is shown that the presence of a carboxylic acid function at the C-5 position and/or at the end of the side chain is crucial for the antibacterial activity. The bactericidal or bacteriostatic action of three compounds against MRSA and MRSE was confirmed in a time-kill kinetics study, and the cytotoxicity on human cells was evaluated. The preliminary mechanism study by confocal microscopy indicated that those vitamin E analogues led to bacterial cell death through membrane disruption.

Curcumin Treatment Identifies Therapeutic Targets within Biomarkers of Liver Colonization by Highly Invasive Mesothelioma Cells-Potential Links with Sarcomas.

Investigations of liver metastatic colonization suggest that the microenvironment is preordained to be intrinsically hospitable to the invasive cancer cells. To identify molecular determinants of that organotropism and potential therapeutic targets, we conducted proteomic analyses of the liver in an aggressive model of sarcomatoid peritoneal mesothelioma (M5-T1). The quantitative changes between SWATH-MS (sequential window acquisition of all theoretical fragmentation spectra) proteotype patterns of the liver from normal rats (G1), adjacent non-tumorous liver from untreated tumor-bearing rats (G2), and liver from curcumin-treated rats without hepatic metastases (G3) were compared. The results identified 12 biomarkers of raised immune response against M5-T1 cells in G3 and 179 liver biomarker changes in (G2 vs. G1) and (G3 vs. G2) but not in (G3 vs. G1). Cross-comparing these 179 candidates with proteins showing abundance changes related to increasing invasiveness in four different rat mesothelioma tumor models identified seven biomarkers specific to the M5-T1 tumor. Finally, analysis of correlations between these seven biomarkers, purine nucleoside phosphorylase being the main biomarker of immune response, and the 179 previously identified proteins revealed a network orchestrating liver colonization and treatment efficacy. These results highlight the links between potential targets, raising interesting prospects for optimizing therapies against highly invasive cancer cells exhibiting a sarcomatoid phenotype and sarcoma cells.

Identification and Quantification of a Phytotoxic Metabolite from Alternaria dauci.

Alternaria dauci is the causal agent of Alternaria leaf blight (ALB) in carrot (Daucus carota) crops around the world. However, to date, A. dauci has received limited attention in its production of phytotoxic metabolites. In this investigation, the bioassay-guided isolation of the extract from liquid cultures of A. dauci resulted in the isolation of two metabolites identified as α-acetylorcinol (1) and p-hydroxybenzoic acid (2), based on their spectroscopic data and results from chemical correlation reactions. Testing of both metabolites in different assays showed an important phytotoxic activity for p-hydroxybenzoic acid (2) when tested in the leaf-spot assay on parsley (Petroselinum crispum), in the leaf infiltration assay on tobacco (Nicotiana alata) and marigold (Tagetes erecta), and in the immersion assay on parsley and parsnip (Pastinaca sativa) leaves. Quantification of the two metabolites in the crude extract of A. dauci kept at different times showed that p-hydroxybenzoic acid (2) is one of the first metabolites to be synthesized by the pathogen, suggesting that this salicylic acid derivative could play an important role in the pathogenicity of the fungus.

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

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