Identification of coumarins in DCM bark, leaf and fruit extracts from Mammea neurophylla (Calophyllaceae) by LC-PDA-MSn

4-phenyl and 4-propylcoumarins display a wide variety of biological activities including anti-oxidant and anti-inflammatory effects, antiparasitical activities against Leishmania or Plasmodium as well as antibacterial, antiviral (HIV) and cytotoxic activities. Using LC-PDA-MSn we have developed a specific protocol allowing the simultaneous and qualitative detection of 4-phenyl and 4-propylcoumarins in DCM bark, fruit and leaf extracts obtained from Mammea neurophylla. By comparison of their retention times, MS and UV data with that of authentic samples, nine, seven and five 4-phenylcoumarins could be directly identified in bark, leaf and fruit extracts respectively. On the other hand, interlocking UV spectra and ESI-MSn data analysis allowed us to deduce plausible structures of five, eight and four other coumarins in bark, leaf and fruit respectively by comparison with their reported spectral data. During this study new Mammea A/AA 9-hydroxy-cyclo F and Mammea A/AB 9-hydroxy-cyclo F were identified. We believe that this protocol will be useful in case of dereplicative studies of Mammea and related species

Identifying Natural Products (NPs) as potential UPR inhibitors for crop protection

As far as the future of agriculture is concerned, one major challenge will be to face an expected increase in health risks due to pesticides together with a lower efficiency of crop treatments. Therefore it is today necessary to develop new strategies to enhance the effectiveness and sustainability of current control methods. The so-called “Alternaria Leaf Spot“ is a common disease of crucifers caused by the fungal pathogen Alternaria brassicicola which affects different crops including cabbage, kale, Brussels sprout, cauliflower and broccoli. Indole phytoalexins camalexin and brassinin play in planta a key role in crop protection against this necrotrophic agent. However it has been shown that mutants become phytoalexin-resistant by activating at least three signaling pathways named as Cell Wall Integrity (CWI), High Osmolarity Glycerol (HOG) and Unfolded Protein Response (UPR) [1,2]. The latter is particularly involved in the fungus protection against phytoalexins since UPR deficient avirulent mutants of A. brassicicola appear as hypersensitive to camalexin and brassinin [3]. Since very few UPR inhibitors such as the synthetic STF-083010 [4] are known we decided to develop an original screening assay, detecting the production of a HAC1 fluorescence-induced protein, i.e. a transcriptional activator involved in the UPR pathway, in Saccharomyces cerevisiae cultures (Figure 1). The preliminary screening of an in-house NPs library [c.a. 70 compounds (polyphenols, terpenoids and alkaloids)] clearly revealed aescin (Aesculus hippocastanum)] as a potential UPR inhibitor

Screening an in house alkaloids library using Voltage-Sensor Probes for new modulators of voltage-gated sodium channels

Voltage-gated sodium channels (Nav) are molecular targets of clinically used drugs for treatments of various diseases (epilepsy, chronic pain, cardiac arrhythmia…) and also of numerous animal and plant neurotoxins. The development of easy-to-use screening assays for searching new ligands from chemicals libraries, animal venoms or plant extracts represents a challenge of a great interest to generate therapeutic hits. Here, we used the mammalian GH3B6 pituitary cell line, which constitutively expresses three different neuronal Nav channel isoforms (Nav1.2, Nav1.3 and Nav1.6), to identify novel compounds of pharmacological interest from a library of in-house vegetal alkaloids. The screening is based on a method using Voltage-Sensor Probes (VSPs) that we adapted to detect both activators and blockers of Nav channels. Over the 84 pure alkaloids or plant extracts that were screened, 17 increased the VSP signal. They operated as gating modifier, showing an action mechanism similar to that of batrachotoxin (BTX), known to strongly inhibit Nav channel inactivation. The remaining 67 plant products were assessed for their potency to inhibit BTX-induced VSP signal. We further selected 11 alkaloids as efficient Nav channels inhibitors. We focused our attention on two structural analogs belonging to the aporphine family, liriodenine and oxostephanine, which differ only by a methoxy group. Whereas liriodenine has been already described as a Nav channels blocker, oxostephanine has not been yet documented as an ion channel modulator. In conclusion, the novel VSPs-based screening assay we developed is a suitable method to challenge the discovery and to assess the activity of novel ligands on Nav channels

Low-impact mating system

An androgynous mating system for mating two exoatmospheric space modules comprising a first mating assembly capable of mating with a second mating assembly; a second mating assembly structurally identical to said first mating assembly, said first mating assembly comprising; a load ring; a plurality of load cell subassemblies; a plurality of actuators; a base ring; a tunnel; a closed loop control system; one or more electromagnets; and one or more striker plates, wherein said one or more electomagnets on said second mating assembly are capable of mating with said one or more striker plates on said first mating assembly, and wherein said one or more striker plates is comprised of a plate of predetermined shape and a 5-DOF mechanism capable of maintaining predetermined contact requirements during said mating of said one or more electromagnets and said one or more striker plates

Voltage Sensor Probes (VSPs) as an Efficient Tool to Screen for Inhibitors of Voltage-Gated Sodium Channels

Voltage-gated sodium channels (Nav) represent a therapeutically validated group of targets for the development of antiepileptic drugs, analgesics and antiarrhythmics [1]. However most of the existing drugs acting as Nav blockers suffer from multiple side effects, but the existence of a multigene family of Nav [2] suggests that the identification of new compounds that selectively block Nav isoforms might have better therapeutic efficiency and reduced side effects. Due to their molecular interference with numerous ion channels, alkaloids represent a group of natural products of particular interest. This is the reason why we have evaluated the efficiency of an in-house method to screen a library of isoquinoline alkaloids formerly isolated in our laboratory. Mammalian GH3 cells constitutively expressing Nav where used in conjunction with Voltage Sensor Probes (VSPs), the signals being read on a fluorescence plate reader. Thanks to this technique, we were able to precisely detect Nav channels activators or blockers. Among 62 compounds tested, 5 isoquinolines appeared as potent Nav channels inhibitors. References: 1. Salat, K. et al. (2014) EOID 23:1093-1104 2. Yu, F.H. et al (2003) Genome Biol. 4

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 (IC50 = 0.05 and 0.06 mM respectively), while anti-AGEs activities of quercitrin and 3,4-dihydroxybenzoic acid appeared as moderately strong (IC50 = 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