Kenn Foubert

Impaired Fibrillin-1 Function Promotes Features of Plaque Instability in Apolipoprotein E–Deficient Mice

Background— Arterial stiffness has been associated with an increased cardiovascular risk. The aim of this study was to investigate the interaction between arterial stiffness and atherosclerosis. Methods and Results— Mice with a mutation (C1039G+/−) in the fibrillin-1 gene leading to fragmentation of the elastic fibers were crossbred with apolipoprotein E–deficient (ApoE−/−) mice. Subsequently, ApoE−/− and ApoE−/−C1039G+/− mice were fed a Western-type diet for 10 or 20 weeks. Our results show that the interaction between arterial stiffness and atherosclerosis is bidirectional. On the one hand, arterial stiffness in ApoE−/−C1039G+/− mice increased more rapidly in the presence of atherosclerotic plaques. On the other hand, arterial stiffness promoted the development of larger and more unstable plaques in ApoE−/−C1039G+/− mice. The plaque area at the aortic root was increased 1.5- and 2.1-fold in ApoE−/−C1039G+/− mice after 10 and 20 weeks of Western-type diet, respectively. After 10 weeks of Western-type diet, plaques of ApoE−/−C1039G+/− mice showed increased apoptosis of smooth muscle cells, which was associated with a decrease in collagen content, an enlargement of the necrotic core, and an increase in macrophages. After 20 weeks of Western-type diet, the number of buried fibrous caps was increased in atherosclerotic lesions of ApoE−/−C1039G+/− mice, not only at the level of the aortic valves but also in the brachiocephalic artery and in the upper, middle, and lower thoracic aorta. Furthermore, acute plaque rupture was observed. Conclusion— These results indicate that fragmentation of the elastic fibers leads to increased vascular stiffness, which promotes features of multifocal plaque instability.

Selective antileishmania activity of 13,28-epoxy-oleanane and related triterpene saponins from the plant families Myrsinaceae, Primulaceae, Aceraceae and Icacinaceae

Maesa saponins with the 13,28‐epoxy‐oleanane triterpene core skeleton were described recently to possess strong and selective in vitro and in vivo antileishmania activity. In the absence of direct chemical derivatization possibilities, a structure‐based literature search was carried out to explore a structure‐activity relationship. Crude alcohol extracts from several plant species of Myrsinaceae, Primulaceae, Aceraceae and Icacinaceae were evaluated for in vitro activity against Leishmania infantum intracellular amastigotes and cytotoxicity on MRC‐5SV2 cells, while the saponin content was evaluated qualitatively by TLC. A clear correlation was found between the presence of close analogue 13,28‐epoxy‐oleanane triterpene saponins and potent and selective antileishmania activity. This was most striking in Maesa species, except for M. macrosepala. Interesting activities were also found in extracts that did not exactly match the TLC characteristics of the Maesa saponin references, as was the case for Ardisia angusta, A. amherstiana, A. caudata, A. gigantifolia, A. roseiflora, Myrsine affinis, Acer brevipes and A. laurinum var. petelotii. This study indicates that the 13,28‐epoxy‐oleanane triterpene moiety is essential for selective antileishmania potential and that several other plant species could still be explored for antileishmania drug discovery. Copyright

Herbal Medicines and Infectious Diseases: Characterization by LC‑SPE‑NMR of Some Medicinal Plant Extracts Used against Malaria

The extracts of two medicinal plants used in traditionalmedicine against malariawere characterized by means of an LC‑SPE‑NMR and LC‑MS platform. The structure of a series of major constituents from Bafodeya benna, as well as minor constituents from Ormocarpum kirkii, was determined. Bafodeya benna was found to contain (2R,3R)-taxifolin-3-O-α-L-rhamnoside or astilbin, and its isomers neoastilbin, neoisoastilbin, and isoastilbin, as well as quercetin-3-O-α-L-rhamnoside. From Ormocarpum kirkii, a series of known flavonoids and biflavonoids was obtained, as well as three new compounds, i.e., 7,7′′-di-O-β-D-glucosyl-(−)-chamaejasmin, 7-O-β-D-glucosyl-(I-3,II-3)-biliquiritigenin, and isovitexin-(I-3,II-3)-naringenin. The isolated constituents may explain, at least in part, the traditional use against malaria. LC‑SPE‑NMR, in combination with LC‑MS, is a powerful tool for the fast characterization of plant extracts, in order to define priorities at an early stage of a fractionation procedure. In addition, herbal medicinal products can completely be characterized, both with regard to their major as well as their minor constituents.

Rapid isolation and identification of minor natural products by LC–MS, LC–SPE–NMR and ECD: Isoflavanones, biflavanones and bisdihydrocoumarins from Ormocarpum kirkii

The combination of the hyphenated techniques LC-MS and LC-SPE-NMR constitutes a powerful platform for the rapid isolation and identification of minor components from natural sources. Electronic circular dichroism (ECD) is a useful tool to determine the absolute configuration of small quantities of chiral molecules. In order to search for minor constituents present in an Ormocarpum kirkii extract, these techniques were applied for the separation and structure elucidation of a series of isoflavanones, biflavanones and biscoumarins. After optimization of chromatographic conditions and subsequent isolation, MS and 1D and 2D NMR data were collected. Experimental and calculated ECD spectra were used in conjunction with NMR data to confirm the absolute configuration of these compounds. Eight compounds were identified for the first time and six have been previously reported. The present approach offers a strategy for accelerating research on natural products.

Antiprotozoal and antiangiogenic saponins from Apodytes dimidiata.

Bioassay-guided isolation was performed on the leaves of Apodytes dimidiata E. Mey. Ex Arn. (Icacinaceae), based on previously demonstrated activity against Leishmania. Six saponins never isolated from nature before were elucidated with LC-MS/MS, GC-MS and 1D and 2D NMR. The compounds apodytine A-F are responsible at least in part for the antiprotozoal activity, but also possess haemolytic activity and display antiangiogenic activity in the rat aorta ring assay, an effect which may be due to a non-selective toxicity.

LC-MS analysis of 13,28-epoxy-oleanane saponins in Maesa spp. extracts with antileishmanial activity.

INTRODUCTION Saponins are natural products that are well known for a wide range of biological activities. For saponins of Maesa balansae, selective antileishmanial activity has been described. OBJECTIVE In view of their pharmacological interest, several Maesa species from the National Botanical Garden of Meise (Belgium) and wild-grown plants from Vietnam were screened for their antileishmanial potential and saponin content. METHODOLOGY Different parts of the plants (mainly leaves and twigs) were collected, dried and extracted. Plant extracts were evaluated by liquid chromatography/mass spectrometry (LC-MS) using electrospray ionisation in the negative ion mode and their saponin content was compared with those of Maesa balansae (maesabalides) and Maesa lanceolata (maesasaponins). RESULTS Several Maesa species (M. ambigua, M. argentea, M. brevipaniculata, M. japonica and M. perlarius) showed potent antileishmanial activity (<0.1 microg/mL) and indeed contained known maesasaponins and maesabalides. However the leaves of M. argentea also revealed two new compounds. Two saponins with [M - H]- ions at m/z 1465 and 1477 were characterised. Their mass spectrometric fragmentation pattern revealed a structure that was the same or closely related to maesasaponin V.3 and VI.2, respectively, but had a glycan part with one additional hexose residue. CONCLUSION Several known as well as new saponins from Maesa species active against leishmaniasis were characterised using LC-MS.

Bridging the gap between comprehensive extraction protocols in plant metabolomics studies and method validation

It is vital to pay much attention to the design of extraction methods developed for plant metabolomics, as any non-extracted or converted metabolites will greatly affect the overall quality of the metabolomics study. Method validation is however often omitted in plant metabolome studies, as the well-established methodologies for classical targeted analyses such as recovery optimization cannot be strictly applied. The aim of the present study is to thoroughly evaluate state-of-the-art comprehensive extraction protocols for plant metabolomics with liquid chromatography-photodiode array-accurate mass mass spectrometry (LC-PDA-amMS) by bridging the gap with method validation. Validation of an extraction protocol in untargeted plant metabolomics should ideally be accomplished by validating the protocol for all possible outcomes, i.e. for all secondary metabolites potentially present in the plant. In an effort to approach this ideal validation scenario, two plant matrices were selected based on their wide versatility of phytochemicals: meadowsweet (Filipendula ulmaria) for its polyphenols content, and spicy paprika powder (from the genus Capsicum) for its apolar phytochemicals content (carotenoids, phytosterols, capsaicinoids). These matrices were extracted with comprehensive extraction protocols adapted from literature and analysed with a generic LC-PDA-amMS characterization platform that was previously validated for broad range phytochemical analysis. The performance of the comprehensive sample preparation protocols was assessed based on extraction efficiency, repeatability and intermediate precision and on ionization suppression/enhancement evaluation. The manuscript elaborates on the finding that none of the extraction methods allowed to exhaustively extract the metabolites. Furthermore, it is shown that depending on the extraction conditions enzymatic degradation mechanisms can occur. Investigation of the fractions obtained with the different extraction methods revealed a low resolving power for phytochemicals for all methods. Nevertheless, an overall good repeatability was observed for all extraction methods, which is essential to allow direct comparison between samples. In summary, no single procedure outperforms the others and compromises will have to be made during method selection.

Interplay between Lactobacillus rhamnosus GG and Candida and the involvement of exopolysaccharides

A number of clinical studies have shown protective effects of lactobacilli against Candida species in the gastrointestinal tract, the urogenital tract and the oral cavity, while others did not show clear effects. Evidence on the mode of action of lactobacilli against Candida is also still lacking. In this study, the anti‐Candida activity of the model probiotic strain Lactobacillus rhamnosus GG was explored in different assays to determine molecular interactions. We found that L. rhamnosus GG was able to interfere with Candida growth, morphogenesis and adhesion. These three aspects of Candidas physiology are all crucial to its opportunistic pathogenesis. In follow‐up assays, we compared the activity of L. rhamnosus GG wild‐type with its exopolysaccharide (EPS)‐deficient mutant and purified EPS to evaluate the involvement of this outer carbohydrate layer. Our data demonstrate that purified EPS can both interfere with hyphal formation and adhesion to epithelial cells, which indicates that EPS is part of a combined molecular mechanism underlying the antihyphal and anti‐adhesion mechanisms of L. rhamnosus GG.

Antioxidant and Antiglycating Constituents from Leaves of Ziziphus oxyphylla and Cedrela serrata

Ziziphus oxyphylla and Cedrela Serrata plants have a folkloric use in Pakistan for treatments of different ailments, i.e., Jaundice, Hepatitis, Diabetes, and antimicrobial. Until now, none of the research studies have reported any phytochemical work on leaves of these two plants. This study aimed to isolate and perform phytochemical analysis in order to search for the constituent having the active role in treatment of the aforementioned ailments. A bioassay-guided fractionation and isolation procedure was used to isolate the concerned phytochemicals present in leaf extracts of Z. oxyphylla and C. serrata. The process involved the hyphenated techniques, i.e., Flash Chromatography, Semi-Preparative HPLC/DAD, UPLC/MS, and NMR in order to isolate and elucidate the structure of the phytochemicals. Seven compounds (1–7) were isolated and identified as flavonoids, more in particular glycosides of quercetin and kaempferol. They showed DPPH scavenging activity, compound 3 (isoquercitrin) being the most active one with an IC50 of 10.8 µg/mL (positive control quercetin; IC50 3.6 µg/mL). The superoxide-radical scavenging and total antioxidant (ABTS) assays indicated IC50 values ranging from 200 to 910 µg/mL and 170 to 320 µg/mL, respectively (positive control quercetin: 374 and 180 µg/mL, respectively). Furthermore, these compounds had low IC50 values for inhibition of protein glycation (AGEs inhibition), ranging from 530 to 818 µg/mL, comparable to aminoguanidine (510 µg/mL) used as a positive control. This study resulted in the identification of seven flavonoid glycosides for the first time from the leaves of Z. oxyphylla and C. serrata with antioxidative and antiglycating activities.

Methylated flavonoids as anti-seizure agents: Naringenin 4′,7-dimethyl ether attenuates epileptic seizures in zebrafish and mouse models

&NA; Epilepsy is a neurological disease that affects more than 70 million people worldwide and is characterized by the presence of spontaneous unprovoked recurrent seizures. Existing anti‐seizure drugs (ASDs) have side effects and fail to control seizures in 30% of patients due to drug resistance. Hence, safer and more efficacious drugs are sorely needed. Flavonoids are polyphenolic structures naturally present in most plants and consumed daily with no adverse effects reported. These structures have shown activity in several seizure and epilepsy animal models through allosteric modulation of GABAA receptors, but also via potent anti‐inflammatory action in the brain. As such, dietary flavonoids offer an interesting source for ASD and anti‐epileptogenic drug (AED) discovery, but their pharmaceutical potential is often hampered by metabolic instability and low oral bioavailability. It has been argued that their drug‐likeness can be improved via methylation of the free hydroxyl groups, thereby dramatically enhancing metabolic stability and membrane transport, facilitating absorption and highly increasing bioavailability. Since no scientific data is available regarding the use of methylated flavonoids in the fight against epilepsy, we studied naringenin (NRG), kaempferol (KFL), and three methylated derivatives, i.e., naringenin 7‐O‐methyl ether (NRG‐M), naringenin 4′,7‐dimethyl ether (NRG‐DM), and kaempferide (4′‐O‐methyl kaempferol) (KFD) in the zebrafish pentylenetetrazole (PTZ) seizure model. We demonstrate that the methylated flavanones NRG‐DM and NRG‐M are highly effective against PTZ‐induced seizures in larval zebrafish, whereas NRG and the flavonols KFL and KFD possess only a limited activity. Moreover, we show that NRG‐DM is active in two standard acute mouse seizure models, i.e., the timed i.v. PTZ seizure model and the 6‐Hz psychomotor seizure model. Based on these results, NRG‐DM is proposed as a lead compound that is worth further investigation for the treatment of generalized seizures and drug‐resistant focal seizures. Our data therefore highlights the potential of methylated flavonoids in the search for new and improved ASDs. Graphical abstract Figure. No caption available. HighlightsMethylated flavonoids are proposed as a source for anti‐seizure drug discovery.Methylated flavanones are highly active against seizures in zebrafish larvae.Naringenin 4′,7‐dimethyl ether is highly active against seizures in zebrafish larvae.Naringenin 4′,7‐dimethyl ether is active against drug‐resistant seizures in mice.Naringenin 4′,7‐dimethyl ether is proposed as a lead anti‐seizure compound.