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Attenuated total reflectance-infrared spectroscopy (ATR-IR) coupled with partial least squares regression (PLSR) was evaluated as a rapid, label free and cost-effective tool to quantify water content in extracts obtained from spirulina wet biomass using a glucose glycerol natural deep eutectic solvent (NADES). NADESs are green, renewable and biodegradable solvents with unique properties outcompeting existing organic solvents, for instance, for plant or biomass extraction. The properties of NADESs depend critically on their water concentration, and therefore, it is essential to develop methods to monitor it, to ensure optimal extraction efficiency and experimental repeatability to achieve a better standardization of extraction protocols. First, Karl Fischer titration was performed on a set of 20 NADES extracts in order to obtain reference water concentrations. Secondly, ATR-IR spectra were collected and subjected to datamining to construct PLSR predictive models. An

International audience; Natural deep eutectic solvents (NADES) are ionic solutions, of great interest for extraction from biomass, biocatalysis, and nanoparticle synthesis. They are easily synthesised and eco-friendly, have low volatility and high dissolution power, and are biodegradable. However, water content in NADES is a critical parameter, affecting their optimal use and extraction efficiency. Vibrational spectroscopic techniques are rapid, label-free, non-destructive, non-invasive, and cost-effective analytical tools that can probe the molecular composition of samples. A direct comparison between a previous study using attenuated total reflectance infrared (ATR-IR) spectroscopy for water quantification in NADES and the same investigation performed with Raman spectroscopy is presently reported. Three NADES systems, namely betaine-glycerol (BG), choline chloride-glycerol (CCG), and glucose-glycerol (GG), containing a range of water concentrations between 0% (w/w) and 40% (w/w), have been analysed with Raman spectroscopy coupled to partial least squares regression multivariate analysis. The values of root mean square error of cross-validation (RMSECV) obtained from analysis performed on the pre-processed spectra over the full spectral range (150–3750 cm−1) are respectively 0.2966% (w/w), 0.4703% (w/w), and 0.2351% (w/w) for BG, GG, and CCG. While the direct comparison to previous ATR-IR results shows essentially similar outcomes for BG, the RMSECV is 33.14% lower and 65.84% lower for CG and CCG. Furthermore, mean relative errors obtained with Raman spectroscopy, and calculated from a set of samples used as independent samples, were 1.452% (w/w), 1.175% (w/w), and 1.188% (w/w). Ultimately, Raman spectroscopy delivered performances for quantification of water in NADES with similar accuracy to ATR-IR. The present demonstration clearly highlights the potential of Raman spectroscopy to support the development of new analytical protocols in the field of green chemistry.

Natural Deep Eutectic solvents (NADES) are green, ionic solutions prepared by mixing vegetal cellular constituents. They are renewable and biodegradable solvents composed of hydrogen bonded donor and acceptor compounds. NADES have a wide range of applications, mainly as solvents for extraction of plants, biocatalysis, and nanoparticle synthesis. Water content is very critical, impacting significantly on NADES properties. Therefore, controlling H2O concentration appears essential for optimal use of NADES. In the present study, Attenuated Total Reflectance – infrared spectroscopy (ATR-IR) coupled to multivariate analysis, namely Partial Least Square s regression (PLSR), was investigated as a rapid, label free and cost-effective tool for measuring the water concentration of NADES. Betaine_Glycerol (BG), Choline Chloride_Glycerol (CCG) and Glucose_Glycerol (GG) were the selected model systems, each over a range of concentrations between 0% (w/w) and 40% (w/w). PLSR results demonstrate the robustness of the analysis, yielding R2 values of 0.99 and Root Mean Square Error of Cross Validation (RMSECV) of respectively 0.2602% w/w, 0.6883% w/w and 0.7034% w/w. Moreover, the % relative error achieved is below a 5% threshold, further highlighting the suitability of ATR-IR for water content monitoring. This preliminary work demonstrates the appropriateness of developing easily accessible analytical tools to support the development of green chemistry solvents, which currently represent a major focus of interest in both research and industrial environments.

Background/Objectives: Hibiscus sabdariffa L. (H. sabdariffa (HS)) extract has a vascular relaxant effect on isolated rat thoracic aorta, but data on small resistance arteries, which play an important role on the development of hypertension, are still missing. The purposes of this study were (1) to assess the effect on isolated mesenteric arteries (MA) from normotensive (Wistar and Wistar-Kyoto (WKY)) and spontaneous hypertensive rats (SHR), (2) to elucidate the mechanism(s) of action underling the relaxant effect in light of bioactive components. Methods: Vascular effects of HS aqueous fraction (AF) on isolated MA rings, as well as its mechanisms of action, were assessed using the contractility and intracellular microelectrode technique. The patch clamp technique was used to evaluate the effect of HS AF on the L-type calcium current. Extraction and enrichment of AF were carried out using liquid&ndash, liquid extraction, and the yield was analyzed using HPLC. Results: The HS AF induced a concentration-dependent relaxant effect on MA rings of SHR (EC50 = 0.83 &plusmn, 0.08 mg/mL), WKY (EC50 = 0.46 &plusmn, 0.04 mg/mL), and Wistar rats (EC50 = 0.44 &plusmn, 0.08 mg/mL) pre-contracted with phenylephrine (10 &micro, M). In Wistar rats, the HS AF maximum relaxant effect was not modified after endothelium removal or when a guanylate cyclase inhibitor (ODQ, 10 &micro, M) and a selective &beta, 2-adrenergic receptor antagonist (ICI-118551, 1 &micro, M) were incubated with the preparation. Otherwise, it was reduced by 34.57 &plusmn, 10.66% when vascular rings were pre-contracted with an 80 mM [K+] solution (p &lt, 0.001), which suggests an effect on ionic channels. HS AF 2 mg/mL significantly decreased the peak of the L-type calcium current observed in cardiac myocytes by 24.4%. Moreover, though the vasorelaxant effect of HS, AF was reduced by 27% when the nonselective potassium channels blocker (tetraethylammonium (TEA) 20 mM) was added to the bath (p &lt, 0.01). The extract did not induce a membrane hyperpolarization of smooth muscle cells, which might suggest an absence of a direct effect on background potassium current. Conclusion: These results highlight that the antihypertensive effect of HS probably involves a vasorelaxant effect on small resistance arteries, which is endothelium independent. L-type calcium current reduction contributes to this effect. The results could also provide a link between the vasorelaxant effect and the bioactive compounds, especially anthocyanins.

Biofilm-related infections are a matter of concern especially because of the poor susceptibility of microorganisms to conventional antimicrobial agents. Innovative approaches are needed. The antibiofilm activity of extracts of cyanobacteria Arthrospira platensis, rich in free fatty acids, as well as of extract-loaded copper alginate-based nanocarriers, were studied on single- and dual-species biofilms of Candida albicans and Cutibacterium acnes. Their ability to inhibit the biofilm formation and to eradicate 24 h old biofilms was investigated. Concentrations of each species were evaluated using flow cytometry. Extracts prevented the growth of C. acnes single-species biofilms (inhibition &gt, 75% at 0.2 mg/mL) but failed to inhibit preformed biofilms. Nanovectorised extracts reduced the growth of single-species C. albicans biofilms (inhibition &gt, 43% at 0.2 mg/mL) while free extracts were weakly or not active. Nanovectorised extracts also inhibited preformed C. albicans biofilms by 55% to 77%, whereas the corresponding free extracts were not active. In conclusion, even if the studied nanocarrier systems displayed promising activity, especially against C. albicans, their efficacy against dual-species biofilms was limited. This study highlighted that working in such polymicrobial conditions can give a more objective view of the relevance of antibiofilm strategies by taking into account interspecies interactions that can offer additional protection to microbes.

Microalgae are a sustainable source of bioactive compounds like carotenoids, free fatty acids and especially polyunsaturated fatty acids. The extraction of such nonpolar metabolites usually involved nonpolar or toxic solvent like methanol or hexane. We report the first screening of Natural Deep Eutectic Solvents for the extraction of pigments and free fatty acids from spirulina. Natural Deep Eutectic Solvent with a wide range of polarity was screened, and the resulting extracts were compared according to their pigment and free fatty acid contents. The extraction performance was increased, and various metabolite profiles were obtained. A Natural Deep Eutectic Solvent based on glycerol/glucose exhibited a wide range of metabolites, which ranged from polar phycobiliproteins to free fatty acids. Natural Deep Eutectic Solvent based on a fatty acid mixture demonstrated the highest selectivity towards free fatty acids. After intensification of the extraction process, 6 spirulina- Natural Deep Eutectic Solvent formulations and Natural Deep Eutectic Solvent alone were evaluated in terms of the effects on cutaneous inflammation induced by Staphylococcus aureus, as well on 4 bacterial species that are part of the skin microbiota. The glycerol/glucose formulation exhibited an anti-inflammatory effect on keratinocytes stimulated by S. aureus. Nonpolar Natural Deep Eutectic Solvent alone and formulations impacted bacterial viability, especially for S. aureus, thus providing a new approach for the regulation of skin microbiota or product preservation.

International audience; Aristolochic acids are biologically active and highly toxic secondary metabolites that recently became an important pharmacological tool for in vivo models of acute kidney disorders. Only aristolochic acid I and a mixture of aristolochic acids I and II are currently commercially available. A method based on the strong-ion exchange displacement mode in centrifugal partition chromatography was developed to extract and purify arictolochic acids, naturally present in Aristolochia bracteolata Lam. (Aristolochiaceae). The extraction and purification of aristolochic acids were performed at a flow rate of 2 mL min−1 in the descending mode by using the biphasic solvent system methyl-ter-butylether, acetone, methanol and water (3:1:1:3, v/v/v/v). Trioctylmethylammonium chloride (Aliquat 336®) was used as the anion exchanger in the organic stationary phase and sodium iodide as the displacer in the aqueous mobile phase. From 5 g of crude extract of A. bracteolata, 45.5 mg, 77.2 mg and 35.1 mg of aristolochic acids I, II and IIIa were obtained respectively with purities greater than 95%.

The growing expansion range of drug resistant parasitic nematode populations threatens the sustainability of ruminant farming worldwide. In this context, nutraceuticals, i.e. feed that would both fulfil dietary requirements while ensuring parasite control, would contribute to increase farming sustainability in developed and low resource settings. In this study, we characterized the anthelmintic potential of lupin seed extracts against major ruminant trichostrongylids, i.e. H. contortus and T. circumcincta. Our observations showed that total seed extracts from commercially available lupin varieties significantly inhibited larval migration. This anthelmintic effect was sustained on multidrug resistant field isolate and across parasite species and was mediated by the seed alkaloid content. Analytical chemistry revealed a set of four lupanine and sparteine-derivatives with anthelmintic activity and electrophysiology assays on recombinant nematode acetylcholine receptors suggested an antagonistic mode of action for lupin alkaloids. While commercial lupin seeds did not exert direct anthelmintic effect in H. contortus infected lupin-fed ewes and goats, it significantly dampened blood production losses suffered by goats. Lupin seed extracts hence provide a working basis for the development of novel anthelmintic compounds able to break drug resistance in the field, while they could be used to increase the resilience of infected dairy ruminants.

International audience; Cyanobacteria and especially Spirulina platensis are recognized as abundant resources of lipids and polyunsaturated fatty acids (PUFA). The main valorization of these lipids has been the biofuel production but another approach was developed here: the investigation of S. platensis fatty acids as anti-biofilm agent for skin diseases. In fact, chronic wounds represent an important burden for occidental health care systems, and are usually related to the presence of a biofilm, especially of Candida species.In order to develop a biomimetic approach against Candida biofilm formation, free fatty acids (FFA) isolated from microalgae S. platensis were explored. Four solvents, including three bio-sourced ones, and three extraction conditions were screened. Lipid and pigment amounts, combined with FFA profiles were analyzed to select the optimal conditions: 30 min of ultrasonic extraction using dimethylcarbonate or ethylacetate as extraction solvent. A vectorization using a macroalgal-alginate nanocarrier was successfully performed. Vectorized extracts were safe towards keratinocytes and thus compatible with a topical use. Spirulina lipid-enriched extracts showed a high anti-biofilm growth activity at low concentrations (about 80% inhibition after 24 h at 0.2 mg/mL). The combination of extracts in copper-alginate nanocarriers potentiated the anti-biofilm growth activity and exhibited a good anti-biofilm activity (about 50% inhibition after 24 h at 0.1 mg/mL). The combined approach of encapsulated Spirulina lipid extracts represented thus a relevant concept to develop all in one anti-biofilm weapons against Candida and represent a new perspective for algae biomass valorization.

International audience; A fast quality control protocol based on reference BL-DMAC assay and High-performance thin-layer chromatography (HPTLC)-Densitometry was optimized for routine quality control of cranberry (Vaccinium macrocarpon) products. The sample preparation appeared as a key step to ensure a relevant comparison of products quality and biological data. Sequential solid/liquid extractions were required to fully extract polyphenols, regardless the extraction solvent chosen. The HPTLC-densitometry herein protocol used epicatechin, PAC-A2 and PAC-B2 as quality markers and densitometric measurements at 200 nm. The PAC-A2/Epicatechin ratio, obtained using HPTLC, appeared as an appropriate indicator of the quality of cranberry ingredient. Only two products, among the tested panel, exhibited a high quality cranberry-based ingredient using this criteria. UPLC-MS analyses coupled to multivariate analyses confirmed HPTLC-densitometry conclusions. This work highlighted the plural PAC composition of commercial products and the lack of standardization for cranberry-based products on the market, explaining the non-reproducibility of clinical trials.

Ethnopharmacoligical relevance Leaves of Crateva adansonii DC (Capparidaceae), a small bush found in Togo, are widely used in traditional medicine to cure infectious abscesses. Traditional healers of Lome harvest only budding leaves early in the morning, in specific area in order to prepare their drugs. Aim of the study The main goal was to validate the ancestral picking practices, and to assess the activity of C. adansonii medicine towards infectious abscesses. Materials and methods A phytochemical screening of various C. adansonii leaf samples was performed using an original HPTLC-densitometry protocol and major flavonoids were identified and quantified. C. adansonii samples were collected in different neighborhoods of Lome, at different harvesting-times and at different ages. Radical scavenging capacity, using DPPH assay, was used to quickly screen all extracts. Extracts were tested for anti- Staphylococcus aureus activity and anti-inflammatory effect on human primary keratinocytes infected by S. aureus . IL6, IL8 and TNFα expression and production were assessed by RT-PCR and ELISA assays. Results Using antioxidant activity as selection criteria, optimal extracts were obtained with budding leaves, collected at 5:00 am in Djidjole neighborhood. This extract showed the strongest anti-inflammatory effect on S. aureus -infected keratinocytes by reducing IL6, IL8 and TNFα expression and production. None of the extracts inhibited the growth of S. aureus . Conclusions Those results validate the traditional practices and the potential of C. adansonii as anti-inflammatory drug. Our findings suggest that traditional healers should add to C. adansonii leaves an antibacterial plant of Togo Pharmacopeia, in order to improve abscess healing.

Cranberry polyphenols are intensively studied for their anti-infectious and antioxidant properties. Nevertheless, in biofilm and infected-wound healing, the identity of active polyphenols still remains unclear. In order to identify active polyphenols, an innovative fractionation scheme based on Centrifugal Partition Chromatography (CPC) was developed. CPC is a support free liquid/liquid chromatographic technology used here for the first time to fractionate cranberry juice. The fractionation protocol involved 3 mains steps: firstly polyphenol and sugars separation; then polyphenols fractionation based of molecular weight and polarity, and finally CPC or on LH-20 resin. Following the second step, the 6 main fractions obtained were screened on Candida biofilm and toward infected wound. Fractions containing low molecular weight proanthocyanidins (Fr Ill and IV) and containing high molecular weight PAC (Fr V ad VI) exhibited the most interesting activities. Concerning Candida albicans biofilm a prophylactic effect was observed by prevention of adhesion. On infected wound, a wound-healing potential related to anti-inflammatory effect but not to antibacterial activity against Staphylococcus aureus was highlighted. These four active fractions were further purified using either CPC or LH-20 resin, to lead to almost 40 sub-fractions. Against fractions containing PACs of medium and high molecular weight exhibited the highest activity against adhesion step of C. albicans strains and infected wound healing. This work is a step further to understand mechanisms of action of cranberry polyphenols on C. albicans biofilm prevention and in infected wound healing.

Theme 4: Natural products as preventive and curative medicine for humans and animals Theme 4: Natural products as preventive and curative medicine for humans and animalsTheme 4: Natural products as preventive and curative medicine for humans and animals; Digestive strongyles are a serious handicap for sheep farming, due to production losses or associated veterinary costs. Meanwhile, the molecules currently available to control these parasites are no longer appropriate due to the emergence of resistant parasite populations but also because of the environmental impact of these molecules. The use of nutraceuticals, plants simultaneously presenting nutritional and medicinal qualities, could be an alternative solution to sustainable pest management. Lupine seed, Lupinus spp., is a protein crop used in animal farming. This plant also contains alkaloids that target nicotinic receptors [1], targets for anthelmintic drugs. These alkaloids provide toxic properties to the bitter ecotypes. The varieties used in animal feed legally contain less than 1.5% bitter seeds. Our study aims at validating and quantifying these properties through in vitro testing of seed extracts from alkaloid-rich or –poor varieties. Five varieties of sweet lupine (3 L. albus L. angustifolius, 1 L. luteus) and six bitter ecotypes (3 L. albus, 1 L. angustifolius, 1 L. luteus, 1 L. mutabilis), richer in alkaloids, were tested against H. contortus. Two L albus varieties, a sweet-one ENERGY and a bitter-one E063, exhibited the higher activity on H. contortus mobility. Chemical analyses revealed a higher alkaloid content in the alkaloid-rich (E063) (3.3% vs 0.04% in the alkaloid-poor) but a lower diversity than in the alkaloid-poor variety (ENERGY) (n=3 vs 5 respectively). Aqueous extracts of both varieties could significantly control infective larval stages and inhibited egg development. Extracted alkaloid fractions exhibited a stronger inhibitory effect, corroborating the working hypothesis, and could also control a multi-resistant isolate. These results are in favor of nutraceutical properties of lupin seed. Alkaloids of ENERGY variety exhibited original mechanism of action against cholinergic receptors, with low EC50.

Poster Session Wednesday; Antioxidative and wound healing properties of [i]Crateva adansonii[/i]. 9. Joint Natural Products Conference 2016

Poster Session Wednesday : Theme 5: Production and regulatory aspects of herbal preparations; Solid/liquid extraction as key step for quality assessment of commercial cranberry products using HPTLC-densitometry. 9. Joint Natural Products Conference 2016

Two heterodimers comprising anthraquinone and methylbenzoisocoumarin moieties (1 and 2) were isolated, together with emodin and physcion from the tubers of Pyrenacantha kaurabassana. The structures of 1 and 2 were established by NMR spectroscopy, including the analysis of a 2D INADEQUATE spectrum. On the basis of the data obtained, the structures that were previously proposed in the literature for these compounds were revised. Compounds 1 and 2 showed antibacterial activity against three different strains of Staphylococcus aureus. Compound 2 also showed bactericidal activity against Helicobacter pylori.