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Grapevine roots, as a side-stream of a vineyard, are a sustainable resource for the recovery of oligomeric stilbenoids, such as the bioactive r-viniferin. The aim of this study is to evaluate an in silico-supported method, based on the Conductor-like Screening Model for Real Solvents (COSMO-RS), for selection of environmentally friendly natural deep eutectic solvents (NADES) with regard to the extraction of grapevine roots. The most suitable NADES system for ultrasonic-assisted extraction of r-viniferin was choline chloride/1,2-propanediol. The optimal extraction parameters for r-viniferin were determined using single-factor experiments as follows: choline chloride/1,2-propanediol 1/2 mol/mol, 10 wt% H2O, biomass/NADES ratio 1/10 g/g, and 10 min extraction time. Under optimized conditions, the extraction yield of r-viniferin from grapevine roots reached 76% of the total r-viniferin content. Regarding stability, stilbenoids in choline chloride/1,2-propanediol remained stable during 128 days of storage at ambient temperature. However, fructose/lactic acid-based NADES were observed to degrade stilbenoids; therefore, the removal of the NADES will be of interest, with a suitable method implemented using Amberlite® XAD-16N resin. As green solvents, the NADES have been used as effective and environmentally friendly extractants of stilbenoid-containing extracts from grapevine roots for potential applications in the cosmetic and pharmaceutical industry or as nutraceuticals in the food industry.

Endemic in 21 countries, Chagas disease, also known as American Trypanosomiasis, is a neglected tropical disease (NTD) caused by the protozoan parasite Trypanosoma cruzi. The available drugs for the treatment of this disease, benznidazole and nifurtimox, are outdated and display severe side effects. Thus, the discovery of new drugs is crucial. Based on our continuous studies aiming towards the discovery of natural products with anti-T. cruzi potential, the MeOH extract from aerial parts of Baccharis sphenophylla Dusén ex. Malme (Asteraceae) displayed activity against this parasite and was subjected to high-performance countercurrent chromatography (HPCCC), to obtain one unreported syn-labdane diterpene — sphenophyllol (1) — as well as the known compounds gaudichaudol C (2), ent-kaurenoic acid (3), hispidulin (4), eupafolin (5), and one mixture of di-O-caffeoylquinic acids (6–8). Compounds 1–8 were characterized by analysis of nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. When tested against trypomastigote forms, isolated labdane diterpenes 1 and 2 displayed potent activity, with EC50 values of 20.1 μM and 2.9 μM, respectively. The mixture of chlorogenic acids 6–8, as well as the isolated flavones 4 and 5, showed significant activity against the clinically relevant amastigotes, with EC50 values of 24.9, 12.8, and 2.7 μM, respectively. Nonetheless, tested compounds 1–8 displayed no cytotoxicity against mammalian cells (CC50 > 200 μM). These results demonstrate the application of HPCCC as an important tool to isolate bioactive compounds from natural sources, including the antitrypanosomal extract from B. sphenophylla, allowing for the development of novel strategic molecular prototypes against tropical neglected diseases.

Grapevine canes are an important source of bioactive compounds, such as stilbenoids. This study aimed to evaluate an in silico method, based on the Conductor-like Screening Model for Real Solvents (COSMO-RS) to isolate stilbenoids from a grapevine cane extract by offline heart-cut high-performance countercurrent chromatography (HPCCC). For the following extraction of resveratrol and ε-viniferin from grapevine canes, natural deep eutectic solvents (NADES) were used as an environmentally friendly alternative to the traditionally used organic solvents. In order to evaluate a variety of combinations of hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) for the targeted extraction of stilbenoids, COSMO-RS was applied. In particular, ultrasonic-assisted extraction using a solvent mixture of choline chloride/1,2-propanediol leads to higher extraction yields of resveratrol and ε-viniferin. COSMO-RS calculations for NADES extraction combined with HPCCC biphasic solvent system calculations are a powerful combination for the sustainable extraction, recovery, and isolation of natural products. This in silico-supported workflow enables the reduction of preliminary experimental tests required for the extraction and isolation of natural compounds.

Peanut hulls (Arachis hypogaea, Leguminosae), which are a side stream of global peanut processing, are rich in bioactive flavonoids such as luteolin, eriodictyol, and 5,7-dihydroxychromone. This study aimed to isolate these flavonoid derivatives by liquid-liquid chromatography with as few steps as possible. To this end, luteolin, eriodictyol and 5,7-dihydroxychromone were isolated from peanut hulls using two different techniques, high-performance countercurrent chromatography (HPCCC) and fast-centrifugal partition chromatography (FCPC). The suitability of the biphasic solvent system composed of n-hexane/ethyl acetate/methanol/water (1.0/1.0/1.0/1.5; v/v/v/v) was determined by the Conductor like Screening Model for Real Solvents (COSMO-RS), which allowed the partition ratio KD-values of the three main flavonoids to be calculated. After a one-step HPCCC separation of ~1000 mg of an ethanolic peanut hull extract, 15 mg of luteolin and 8 mg of eriodictyol were isolated with purities over 96%. Furthermore, 3 mg of 5,7-dihydroxychromone could be isolated after purification by semi-preparative reversed-phase liquid chromatography (semi-prep. HPLC) in purity of over 99%. The compounds were identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy (NMR).

Opuntia stricta var. dillenii and Atriplex hortensis var. rubra, two plant species with abundant betalain and flavonoid content, were studied by high performance countercurrent chromatography (HPCCC) coupled with ESI-MS/ MS in an off-line detection process. Both CCC runs used tert.-butyl-methyl-ether/n-butanol/acetonitrile/water 1% trifluoroacetic acid (v/v/v/v, 2:2:1:5) as a biphasic solvent system within an elution-extrusion mode. The resulting fractions were scanned by an off-line sequential injection to ESI-MS/MS approach. The separation of Opuntia stricta var. dillenii extract showed a partial isolation of principal betalains (betanin) from their derivatives (betanidin, phyllocactin, feruloyl-betanin, betaxanthin), degraded products (hydroxyl-neobetanin, mono decarboxy-betanins), and flavonoid glycosides (isorhamnetin-rutinoside, kaempferol-rutinoside). In case of Atriplex hortensis var. rubra, this study confirmed the coexistence of several flavonoids (quercetin-Omalonyl glucoside, kaempferol-O-malonyl glucoside) and generated few pure pigment fractions (celosianin II) for structural elucidation.

Previously, different Hydrangea macrophylla ssp. serrata cultivars were investigated by untargeted LC-MS analysis. From this, a list of tentatively identified and unknown compounds that differ significantly between these cultivars was obtained. Due to the lack of reference compounds, especially for dihydro-isocoumarins, we aimed to isolate and structurally characterise these compounds from the cultivar ‘Yae-no-amacha’ using NMR and LC-MS methods. For purification and isolation, counter-current chromatography was used in combination with reversed-phase preparative HPLC as an orthogonal and enhanced purification workflow. Thirteen dihydro-isocoumarins in combination with other metabolites could be isolated and structurally identified. Particularly interesting was the clarification of dihydrostilbenoid glycosides, which were described for the first time in H. macrophylla ssp. serrata. These results will help us in further studies on the biological interpretation of our data.