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A study on the basis of the Dietary Reference Intakes of the U.S. Department of Agriculture surveys the technological effects of juice processing on the mineral element content in sea buckthorn juice to assess the nutritional value. A beneficial source of chromium, copper, manganese, molybdenum, iron and potassium are obtained on drinking seabuckthorn juice.

The various processing effects and storage stability of phylloquinone in sea buckthorn berries (Hippophae rhamnoides L. ssp. rhamnoides) and related products are explained. The content of vitamin [K.sub.1] in these berries is found to be completely temperature dependent.

The primary vitamin in sea buckthorn berries is vitamin C containing values of approximately 400 mg/100 g. Processing effects were investigated during juice and concentrate production from sea buckthorn berries ( Hippophaë rhamnoides) and storage stability of juices was determined for up to 7 d using berries and juices from 2 different growing areas. During industrial juice production the technological processing of the berries caused a loss of about 5% to 11% total ascorbic acid (TAA) in the generated juice. The production of the concentrated juice resulted in 50% depletion of TAA. Sea buckthorn berries and juice were stored at 6, 25, and 40 °C for up to 7 d to investigate the temperature effects on TAA during storage. Analysis of kinetic data suggested that the degradation follows a 1st-order model. The results of the experiments showed that storage of sea buckthorn juices for 7 d at cold temperature (6 °C) already resulted in a degradation of TAA of about 11% to 12%. [ABSTRACT FROM AUTHOR]

Di-2-ethylhexyl phthalate (DEHP) is a commonly used plasticizer that is harmful to human health. magnetic resonance spectroscopy from the edible fruit flesh of Benincasa hispida (wax gourd) of the plant family Curcurbitaceae. The DEHP content of seven wax gourd samples collected from southern and northern provinces in China was determined as (mean  ±  SD): 18.3  ±  0.43, 2.64  ±  0.44, 44.0  ±  0.34, 62.5  ±  0.48, 52.0  ±  0.42, 58.3  ±  0.55 and 75.5  ±  0.63  mg  kg -1 fresh weight, respectively, indicating that most wax gourds were severely contaminated with DEHP. [ABSTRACT FROM AUTHOR]

C21H34O3, orthorhombic, P212121 (no. 19), a = 7.5420(7) Å, b = 11.6929(11) Å, c = 22.657(2) Å, V = 1998.1(3) Å3, Z = 4, Rgt(F) = 0.0383, wRref(F2) = 0.113, T = 293(2) K.

Phylloquinone (vitamin K1) is the primary dietary source of vitamin K. Processing effects and stability of phylloquinone were investigated during juice and concentrate production from sea buckthorn ( Hippophaë rhamnoides) using berries from 2 different growing areas. During industrial juice production the technological processing of the berries caused a loss of about 36% to 54% phylloquinone in the generated juice. The following processing steps leading to the concentrated juice resulted in a complete depletion of phylloquinone. Sea buckthorn berries and juice were stored at 6, 25, and 40 °C for up to 7 d to determine the temperature effects on phylloquinone during storage. Content of vitamin K1 in sea buckthorn berries was affected by storage time and storage temperature. Storage of freshly harvested berries resulted in a significant increase ( P < 0.01) of phylloquinone ranging from 21% up to 186% (wet weight). The juices showed almost identical significant degradation ( P < 0.01) of phylloquinone of about 18% to 32% at 6, 25, and 40 °C indicating that intensity of decomposition is independent of temperature (6 to 40 °C) and storage time in the range of consumer storage conditions. [ABSTRACT FROM AUTHOR]

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% H 2 O, 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 EC 50 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 EC 50 values of 24.9, 12.8, and 2.7 μM, respectively. Nonetheless, tested compounds 1 - 8 displayed no cytotoxicity against mammalian cells (CC 50 > 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 K D -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).

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.

Betacyanins and their decarboxylated derivatives from fresh and dried edible leaves of Atriplex hortensis L. var. "Rubra" were fractionated for the first time by high-speed countercurrent chromatography. Pigments present in fresh leaf extract were separated in systems: ethanol - acetonitrile - n-propanol - ammonium sulphate - water (0.5:0.5:0.5:1.2:1.0, v/v/v/v/v) (tail-to-head mode) and tert-butyl methyl ether - n-butanol - acetonitrile - water with 0.7% heptafluorobutyric acid (2:2:1:5, v/v/v/v) (head-to-tail mode). The mobile phase flow rate was 2 mL/min and the retention of the stationary phase was 79.8 and 75.2%, respectively. Pigments from dried leaves were separated in a similar ion-pair system with heptafluorobutyric acid in different volume proportions 1:3:1:5 (head-to-tail mode) and the flow rate of the mobile phase 3 mL/min. The stationary phase retention was 64.0%. The application of the countercurrent chromatography for the fractionation of betacyanins from leaves of Atriplex hortensis enabled to isolate and pre-concentrate the pigments for further low- and high-resolution liquid chromatographic-tandem mass spectrometric detection. This study revealed the presence of 10 betacyanins in fresh and 16 in dried leaves of Atriplex hortensis. Two compounds were not previously identified in the whole Amaranthaceae family. Additionally, instead of (iso)amaranthin, celosianin and its epimer were dominant betacyanins in the Atriplex hortensis., (© 2021 Wiley-VCH GmbH.)

N -Ethyl-2-pyrrolidinone-substituted flavanols (EPSF) are marker compounds for long-term stored white teas. However, due to their low contents and diasteromeric configuration, EPSF compounds are challenging to isolate. In this study, two representative epimeric EPSF compounds, 5''' R - and 5''' S -epigallocatechin gallate-8-C N -ethyl-2-pyrrolidinone ( R -EGCG-cThea and S -EGCG-cThea), were isolated from white tea using centrifugal partition chromatography (CPC). Two different biphasic solvent systems composed of 1. N -hexane-ethyl acetate-methanol-water (1:5:1:5, v / v / v / v ) and 2. N -hexane-ethyl acetate-acetonitrile-water (0.7:3.0:1.3:5.0, v / v / v / v ) were used for independent pre-fractionation experiments; 500 mg in each separation of white tea ethyl acetate partition were fractionated. The suitability of the two solvent systems was pre-evaluated by electrospray mass-spectrometry (ESI-MS/MS) analysis for metabolite distribution and compared to the results of the CPC experimental data using specific metabolite partition ratio K D values, selectivity factors α, and resolution factors &nbsp; R S . After size-exclusion and semi-preparative reversed-phase liquid chromatography, 6.4 mg of R -EGCG-cThea and 2.9 mg of S -EGCG-cThea were recovered with purities over 95%. Further bioactivity evaluation showed that R - and S -EGCG-cThea possessed in vitro inhibition effects on α -glucosidase with IC 50 of 70.3 and 161.7 μM, respectively.

Medicinal plants grown under stress conditions reveal higher concentrations of relevant specialized metabolites than well-watered plants, putatively due to an enhanced biosynthesis. Yet, stress also reduced the biomass gain. Accordingly, the concentration increase in comparison to control plants could also be due to lesser biomass employed as the reference value, whereas the rate of biosynthesis may remain unchanged. For an unequivocal proof that stress indeed enhances the biosynthesis, the total amount of the substances per plant has to be determined. In this study, we investigated the stress-induced impact on the alkaloids accumulated in Catharanthus roseus and quantified both, the changes in concentration and in the entire amount of alkaloids. At any time, all Catharanthus roseus plants grown under drought stress exhibited a markedly higher alkaloid concentration compared to the well-watered controls. However, by calculating the entire alkaloid content per plant, a corresponding increment occurred only within the first two weeks of drought stress. Thereafter, no significant differences among drought treatments and control were detected. Finally, within the last week, the alkaloid content per plant decreased markedly, although there was a meaningfully higher concentration of alkaloids in the drought-stressed plants. In contrast, when plants had been exposed to high salt concentrations, the alkaloid concentrations were quite the same in stressed and control plants. The related total contents were significantly lower in plants exposed to salt stress. These results display that both phenomena, an increased rate of biosynthesis and lesser reference values, i.e., the biomass, contribute to the stress-related increase in the concentration of natural product. Moreover, it has to be considered that the enhancement of biosynthesis could be due to either an "active" up-regulation of biosynthetic capacity or a "passive" shift caused by the over-reduced status as a result of the stress-induced stomatal closure., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The detailed metabolite profiling of Laguncularia racemosa was accomplished by high-performance countercurrent chromatography (HPCCC) using the three-phase system n -hexane- tert -butyl methyl ether-acetonitrile-water 2:3:3:2 ( v / v / v / v ) in step-gradient elution mode. The gradient elution was adjusted to the chemical complexity of the L. racemosa ethyl acetate partition and strongly improved the polarity range of chromatography. The three-phase solvent system was chosen for the gradient to avoid equilibrium problems when changing mobile phase compositions encountered between the gradient steps. The tentative recognition of metabolites including the identification of novel ones was possible due to the off-line injection of fractions to electrospray ionization mass spectrometry (ESI-MS/MS) in the sequence of recovery. The off-line hyphenation profiling experiment of HPCCC and ESI-MS projected the preparative elution by selected single ion traces in the negative ionization mode. Co-elution effects were monitored and MS/MS fragmentation data of more than 100 substances were used for structural characterization and identification. The metabolite profile in the L. racemosa extract comprised flavonoids, hydrolysable tannins, condensed tannins and low molecular weight polyphenols.

Extracts of Opuntia stricta var. dillenii fruits were fractionated by semi-preparative high-performance countercurrent chromatography (HPCCC) to study the secondary metabolite formation, whereby HPCCC showed a superior separation capacity to fractionate minor metabolites compared to HPLC. A family of new peptides was detected in semi-polar fractions when monitoring the HPCCC separation by off-line injections of fractions to ESI-MS/MS. Planar structures of the major compounds, two 14-ring-membered cyclopeptide alkaloids, which were named opuntisines A and B, were elucidated by 1D- and 2D-NMR spectroscopy and HR-ESI-MS/MS spectrometry, while a combination of chemical derivatisation and degradation revealed the stereo-configurations. Specifically, the methods of Marfey and Mosher indicated l-Glu, l-Ile, l-Phe and 1S-configurations, respectively; ROESY correlations revealed 8S, 9S. The novel opuntisine A showed moderate activity against the Gram-negative bacterium Escherichia coli, but no further antibacterial, antifungal nor cytotoxic effects. This bioactive natural product class is reported for the first time in the plant family Cactaceae., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Transfection is the process to deliver nucleic acid into eukaryotic cells. Different transfection techniques already exist. However, they can be expensive and toxic toward subjected cells. Previous research shed light on natural occurring molecules called triterpene saponins that have great potential for the non-viral gene delivery. Using a combination of different chromatographic techniques and in vitro transfection bioassays, a new triterpenoid saponin (agrostemmoside E) from the plant Agrostemma githago L. was isolated. Agrostemmoside E was characterized by mass spectrometry, intense NMR spectroscopy and was identified as 3-{O-ß-D-Galactopyranosyl-(1→2)]-[ß-D-xylopyranosyl-(1→3)]-ß-D-glucuronopyranosyl} quillaic acid 28-O-{[ß-D-4,6-di-(O-acetyl)-glucopyranosyl-(1→3)]-[ß-D-xylopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→2)}-[3,4-di-(O-acetyl)-ß-D-quinovopyranosyl-(1→4)]-ß-D-fucopyranoside ester. Agrostemmoside E has a great potential for delivery of gene loaded nanoplexes and increased the transfection efficiency by 70% compared to 2% without agrostemmoside E. By comparative toxicity studies, we show that agrostemmoside E can be applied at high concentrations without toxicity, justifying its use as a new tool for gene transfections., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Food authenticity in the field of food dyes can be interpreted as the correctness of the coloring ingredients indicated. The Rapid UV/vis Spectroscopic Dye Authentication Assay (RaSDAY) presented in this work was used to verify the authenticity of water-soluble reddish colorings for food use. RaSDAY includes the processing of samples under different experimental conditions with pH variations and heat exposure. The absorbances measured are analyzed by principal component analysis and a k -nearest neighbors algorithm. As a result, classification of anthocyanins, betalains, and carmine and the detection of Monascus pigments, undeclared artificial food dyes, and reactive textile azo dyes can be performed by utilizing a rapid screening method. In 17 out of 20 samples of coloring food additives that were included in this work, reactive dyes, unpermitted Monascus pigments, and artificial food dyes were detected using the developed method. "Reactive Red 120", "Reactive Red 195", and "Reactive Red 198" were identified by subsequent 1 H NMR spectroscopy in eight of those samples.

Nectandra leucantha (Lauraceae) is a tree indigenous to the tropical Atlantic forests of Brazil, one of the most biodiverse flora hotspots worldwide. This plant species contains high concentrations of neolignan and dehydrodieugenol derivatives that express significant in-vitro activities against various parasite strains. These activities are however responsible for severe tropical human infections, such as Leishmaniasis (Leishmania spp.) and Chagas disease (Trypanosoma cruzi), which have been classified by the World Health Organization (WHO) as Neglected Tropical Diseases (NTDs). In order to optimize the isolation process for these target metabolites, n-hexane extract of the leaves was separated by means of semi-preparative high performance countercurrent chromatography (HPCCC) and scale-up spiral-coil countercurrent chromatography (sp-CCC) systems. Several biphasic solvent mixtures were evaluated for their partitioning effects on neolignans, resulting in the selection of an optimized system n-hexane - ethylacetate - methanol - water (7:3:7:3, v/v/v/v). The chromatographic experiments on the HPCCC and sp-CCC were run in the head-to-tail mode with 500 mg and 16 g injections, respectively. For specific and multiple metabolite detection, the recovered CCC-fractions were off-line injected, in the sequence of recovery, to an electrospray mass-spectrometry (ESI-MS/MS) device. A projection of the single ion traces of the target compounds, in the positive ionization mode at a scan range of m/z 100-1500, located chromatographic areas where the co-elution effects occurred and pure target metabolites were present. Five major target neolignans were specifically detected, which enabled the accurate pooling of CCC-fractions for an optimum recovery of the metabolites. The direct comparison of the performance characteristics of the two CCC-devices, with very different mechanical designs was achieved by the conversion of the time axis into a partition ratio (K D ) separation scale. As a result, the compound specific K D -elution values of the target neolignan were determined in high precision, while the comparison of the calculated separation factor (α) and resolution factor (R S ) values revealed a superior separation performance for the HPCCC system. Also, the reproducibility of detected metabolites in the two CCC experiments was confirmed by small variations (ΔK D  ±0.1). Neolignan target compounds with anti-parasite activities were successfully isolated in the 100 mg to 4 g range in a single lab-scale countercurrent chromatographic process step., (Copyright © 2019. Published by Elsevier B.V.)

Electrospray mass spectrometry-profiling guided the metabolome investigation of a C18 reversed phase adsorbate of Opuntia stricta var. dillenii fruits following analytical, and semi-preparative high-performance countercurrent chromatography (HPCCC) fractionation, and visualization of molecular weight elution profiles based on selected single ion-traces. Experimental partition-ratio values K D , and peak widths for detected metabolites were determined. Structural characterization of metabolites, and co-elution effects were monitored in the scan range m/z 150-2200. The polar ion-pair activated solvent system tert.-butylmethylether - n-butanol - acetonitrile - water (0.7% trifluoroacetic acid) [2:2:1:5]) was used for partition-ratio K D improvement of ionic betalains. HPCCC operations were in the head-to-tail mode using the elution-extrusion approach. Selected ESI-MS ion traces visualized the elution of fourty-three metabolites, whereas twenty-one were identified as known betalain pigments, and their classic degradation products, and chlorinated betacyanin artefacts. Potentially, novel fruit metabolites of Opuntia were recognized by unknown molecular weights, and MS/MS fragmentations. Off-line ESI-MS fraction monitoring determined peak elution windows, and resulted in K D -based chromatographic scales. Detectable metabolites were compared by separation- α, and resolution-factors R S revealing a better performance of the analytical HPCCC experiment. Experimental metabolite K D -values from analytical and semi-preparative HPCCC runs were widely consistent, and confirmed the reproducibility of the technique based on the used sample material., (Copyright © 2019. Published by Elsevier B.V.)

The ability of certain triterpenoid saponins to modulate the endosomal release during the process of endocytosis and to ensure a nontoxic and efficient transfection recently led to an exceptional interest in the field of nonviral gene delivery. In vitro and in vivo studies demonstrated promising results in terms of tumor growth inhibition after the delivery of a suicide gene such as saporin and dianthin. With that, the question arises which structural features are necessary or advantageous to achieve an effective endosomal escape. Former studies described certain important characteristics a potent saponin should have. Particularly SA1641 ( Gypsophila paniculata ) and SO1861 ( Saponaria officinalis ) played an utmost important role to get a first insight into the structure-activity relationship. However, a number of issues such as the purpose of functional groups on the aglycon and the substitution of sugars and their modification remain unsolved and their value needs to be specified. By conducting a screening of several diverse saponins in terms of their transfection improving ability, we aimed to examine these questions in more detail and get a better understanding of the relevant features. The transfection of Neuro-2A-cells with GFP-DNA containing peptide-based nanoplexes provided a reliable method in order to compare the activity of the saponins. With that, we were able to provide new and essential insights regarding the structure-activity relationship of transfection-modulating saponins and give an idea of how a highly potent saponin for future gene therapies may look like., Competing Interests: The authors declare no conflicts of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Salicornia species have just been introduced to the European market as a vegetable named 'samphire', 'green asparagus', or 'sea asparagus'. Due to its increasing attention, and associated value, minor compounds of Salicornia gaudichaudiana Moq were investigated. The use of countercurrent chromatography and mass spectrometry enabled the search for known, as well as potentially novel natural products. Their identification was achieved based on molecular weights and mass-spectrometric fragmentation data. Low detection limits enabled the visualization of all compounds with their identification in almost real time close to the preparative countercurrent chromatography experiment. A list of known natural products from Salicornia genus guided the identification process of compounds occurring in Salicornia gaudichaudiana Moq by tandem mass spectrometry fragment comparison. The natural product classes were divided into four groups: chlorogenic acid derivatives; flavonoid derivatives; pentacyclic triterpenoid saponins; and other compounds., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To this date, a number of different Gypsophila species from the family of Caryophyllaceae were phytochemically characterized and tested for diverse pharmacological effects. With Gypsophila elegans M. Bieb., we investigated a scarcely explored Gypsophila species, providing a number of potential transfection enhancing triterpene saponins, and so-called sapofection agents. So far triterpene saponins have not been isolated in Gypsophila elegans M.Bieb. Crude extracts from roots and seeds, as well as each purification step were tested for delivery modulation of gene-loaded nanoplexes into neuroblastoma cells. The application of the bioassay guided isolation strategy enabled the assessment of the most active Gypsophila compound, the bisdesmosidic triterpene saponin gypsophilosid A. Gypsophilosid A was isolated by chromatographic techniques, and characterized by electrospray mass spectrometry and intense NMR-spectroscopy, using a variety of 1D and 2D-NMR experiments such as HSQC, HMBC, HQQC, TOCSY and NOESY. In neuroblastoma cells, gypsophilosid A increased the transfection efficiency of gene-nanoplexes up to 80% compared to 2% in the control group without saponin. Our results proved the successful applicability of the implemented methods to detect, isolate and identify saponins, which are biochemically active in terms of transfection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Porcelia macrocarpa (Warm.) R. E. Fries (Annonaceae) is an endemic plant in Brazil where its tasty pulp has been eaten fresh. The hexane extract from its flowers was subjected to chromatographic procedures to afford four acetylene derivatives identified as octadec-9-ynoic (stearolic acid - 1), (11E)-octadec-11-en-9-ynoic (santalbic acid - 2), 8-hydroxyoctadec-9,11-diynoic (3) and 8-hydroxyoctadec-17-en-9,11-diynoic (isanolic acid - 4) acids by NMR and HRESIMS. Among tested compounds against trypomastigote forms of T. cruzi, octadec-9-ynoic acid (1) displayed higher potential with IC 50  = 27.6 µM and a selectivity index (SI) higher than 7. Compounds 2 and 3 showed IC 50 of approximately 60 µM while compound 4 was inactive. The lethal action of the compound 1 was investigated using spectrofluorometric techniques to detect ROS content, plasma membrane permeability and plasma membrane potential by flow cytometry. Compound 1 showed no alteration in the production of ROS of treated trypomastigotes and no alteration of the plasma membrane permeability was observed as detected by the fluorescent probe SYTOX-green after 120 min of incubation. However, by using the potential-sensitive fluorescent probe DiSBAC 2 (3), compound 1 caused depolarization of the plasma membrane potential when compared to untreated parasites. Our results demonstrated the anti-T. cruzi effects of compounds 1-3 isolated from flowers of P. macrocarpa and indicated that the lethal effect of compound 1 in T. cruzi could be associated to the plasma membrane disturbance of the parasite., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Neuroblastoma represents the third most common malign neoplasm occurring in children and the most common in newborn. Although mortality in childhood cancer declined in the last decade, high-risk patients have poor prospects, due to the aggressiveness of the cancer. In the recent past, we underlined the potential of sapofectosid as novel and efficient transfection enhancer, demonstrating non-toxic gene delivery, but its value in tumor therapies has yet to be elucidated. A suicide gene, coding for saporin, a ribosome-inactivating protein type I, was incorporated into targeted, peptide-based nanoplexes. The nanoplexes were characterized for their size, zeta potential and appearance by electron microscopy. Gene delivery was observed via confocal imaging. In vitro transfections were conducted to monitor the real-time cell viability. After initial tolerability studies, NMRI nu/nu-mice bearing tumors from Neuro-2A-Luc-cells (murine neuroblastoma cells, transduced with a luciferase gene), were treated with targeted nanoplexes (30 μg saporin-DNA i.v./treatment) and sapofectosid (30 μg s.c. treatment). The treatment was compared to a vehicle (PBS) control and treatment without sapofectosid in terms of body weight, tumor growth and integrated density of tumor luminescence. The study revealed an anti-tumoral effect of the sapofectosid mediated gene therapy in the Neuro-2A-tumor model. The treatments were well tolerated by the animals indicating the applicability of this approach. With these results, we were able to proof the efficacy of a therapy, consisting of targeted suicide gene nanoplexes and sapofectosid, a novel and potent transfection enhancer. This study points out the enormous value for future targeted cancer and gene therapies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the title compound, C 21 H 24 O 4 (systematic name: 4,5'-diallyl-2,2',3'-tri-meth-oxy-diphenyl ether), the aromatic rings lie almost perpendicular to each other [dihedral angle = 85.96 (2)°]. The allyl side chains show similar configurations, with C ar -C-C=C (ar = aromatic) torsion angles of -123.62 (12) and -115.54 (12)°. A possible weak intra-molecular C-H⋯O inter-action is observed. In the crystal, mol-ecules are connected by two C-H⋯O hydrogen bonds, forming undulating layers lying parallel to the bc plane. Weak C-H⋯π and π-π stacking inter-actions also occur.

Different methods are being deployed for non-viral DNA/RNA delivery. However non-viral formulations for DNA/RNA-delivery are often accompanied by severe toxicity and thus low efficiency. Particular costly cell culture media are required as well. Here we introduce sapofection as a valuable enhancing method for non-viral DNA/RNA delivery. Sapofection is based on the application of DNA/RNA nanoplexes and sapofectosid, a plant derived natural transfection reagent. Sapofectosid was produced from plant raw material by chromatographic methods and characterized by tandem mass spectrometry and intensive one and two dimensional NMR-spectroscopy. Sapofectosid did enhance the transfection efficiency of different DNA- and RNA-nanoplexes formulated with liposomes, polyethylenimine (PEI) or targeted and non-targeted oligo-lysine peptides. All nanoplexes were characterized physicochemically and the influence of sapofectosid on the nanoplex integrity was determined by DNA complexation assays. The nanoplexes and sapofectosid were administered to a variety of cancer cell lines and the transfection efficiency was investigated by flow cytometry and confocal microscopy. Dependent on the cell line the transfection efficiencies varied from 6 to 76%. The saponin- and receptor-mediated endocytosis of nanoplexes was investigated by flow cytometry. As demonstrated by impedance based live cell imaging sapofection was non-toxic. The findings show the great potential of sapofection to be used as an effective and non-toxic transfection enhancing method., (Copyright © 2017 Elsevier B.V. All rights reserved.)

There is a vast amount of information about the nutritional and medicinal properties of honey as a result of its numerous benefits. However, honeys have been found to be contaminated with hepatotoxic and carcinogenic pyrrolizidine alkaloids (PAs) on account of bees foraging on PA-containing plants. This study deals with the analysis of PAs in tropical honeys emanating from different agro-ecological zones of Ghana in order to assess its potential health risk. PAs of 48 honey samples were analysed using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results show that a total of 85% of the honeys from various agro-ecological zones were PA positive including all honeys from supermarkets. The highest concentration of PAs was 2639 μg kg -1 , while the average PA concentration of the samples was 283 μg kg -1 . The study also found Chromolaena odorata pollens in majority of the honeys, thus indicating the plant as major source of PA contamination of honeys in the tropical regions.

The occurrence of potentially toxic pyrrolizidine alkaloids (PAs) in herbal medicines (HMs) is currently intensely being discussed in Europe. Pyrrolizidine alkaloids, particularly the 1,2-unsaturated PAs, are undesired compounds in HMs due to their potential hepatotoxic and carcinogenic properties. In this study, 98 widely patronized HMs from six popular German retail supermarkets/drugstores, as well as from pharmacies, were analyzed by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the presence of PAs. The results showed that about 63% of the HMs were PA positive, whereas the average PA concentration of the samples was 201 μg/kg, the highest concentration of PAs (3270 μg/kg) was attributed to a product that was purchased from the pharmacy and contained Hypericum perforatum L. (St. John's Wort) as an active ingredient. In addition, H. perforatum-containing products were frequently contaminated with PAs from Echium spp., while both Cynara cardunculus L. products and fixed-combination products of Gentiana lutea L., Rumex acetosa L., Verbena officinalis L., Sambucus nigra L., and Primula veris L. products were commonly contaminated with PAs of Senecio spp. The study showed that H. perforatum, C. cardunculus, Urtica dioica L., and fixed-combination products were frequently contaminated with PA levels above the recommended values of both the German and European Medicines Agencies. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Chagas disease is a neglected tropical disease, caused by the protozoan parasite Trypanosoma cruzi, which affects more than eight million people in Tropical and Subtropical countries especially in Latin America. Current treatment is limited to nifurtimox and benznidazole, both with reduced effectiveness and high toxicity. In this work, the n-hexane extract from leaves of Nectandra leucantha (Lauraceae) displayed in vitro antitrypanosomal activity against T. cruzi. Using several chromatographic steps, four related neolignans were isolated and chemically characterized as dehydrodieugenol B (1), 1-(8-propenyl)-3-[3'-methoxy-1'-(8-propenyl)-phenoxy]-4,5-dimethoxybenzene (2), 1-[(7S)-hydroxy-8-propenyl]-3-[3'-methoxy-1'-(8'-propenyl)-phenoxy]-4-hydroxy-5-methoxybenzene (3), and 1-[(7S)-hydroxy-8-propenyl]-3-[3'-methoxy-1'-(8'-propenyl)-phenoxy]-4,5-dimethoxybenzene (4). These compounds were tested against intracellular amastigotes and extracellular trypomastigotes of T. cruzi and for mammalian cytotoxicity. Neolignan 4 showed the higher selectivity index (SI) against trypomastigotes (>5) and amastigotes (>13) of T. cruzi. The investigation of the mechanism of action demonstrated that neolignan 4 caused substantial alteration of the plasma membrane permeability, together with mitochondrial dysfunctions in trypomastigote forms. In silico studies of pharmacokinetics and toxicity (ADMET) properties predicted that all compounds were non-mutagenic, non-carcinogenic, non-genotoxic, weak hERG blockers, with acceptable volume of distribution (1.66-3.32 L/kg), and low rodent oral toxicity (LD 50 810-2200 mg/kg). Considering some clinical events of cerebral Chagas disease, the compounds also demonstrated favorable properties, such as blood-brain barrier penetration. Unfavorable properties were also predicted as high promiscuity for P450 isoforms, high plasma protein binding affinity (>91%), and moderate-to-low oral bioavailability. Finally, none of the isolated neolignans was predicted as interference compounds (PAINS). Considering the promising chemical and biological properties of the isolated neolignans, these compounds could be used as starting points to develop new lead compounds for Chagas disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The title compound, C 20 H 22 N 2 O 2 , an alkaloid isolated from the Madagascar periwinkle, crystallizes in P 1 with two independent but closely similar mol-ecules in the unit cell. The mol-ecules are linked into pairs by two N-H⋯O=C hydrogen bonds. The absolute configuration was confirmed by anomalous dispersion effects as S at the 3 and 15 positions, and R at the 7 position.

Ageing is often accompanied by chronic inflammation. A fat- and sugar-rich Western-type diet (WTD) may accelerate the ageing phenotype. Cell culture studies have indicated that artepillin C-containing Brazilian green propolis exhibits anti-inflammatory properties. However, little is known regarding its anti-inflammatory potential in mouse liver in vivo. In this study, female C57BL/6NRj wild-type mice were fed a WTD, a WTD supplemented with Brazilian green propolis supercritical extract (GPSE) encapsulated in γ-cyclodextrin (γCD) or a WTD plus γCD for 10 weeks. GPSE-γCD did not affect the food intake, body weight or body composition of the mice. However, mRNA levels of the tumour necrosis factor α were significantly downregulated ( p < 0.05) in these mice compared to those in the WTD-fed controls. Furthermore, the gene expression levels of other pro-inflammatory markers, including serum amyloid P, were significantly ( p < 0.001) decreased following GPSE-γCD treatment. GPSE-γCD significantly induced hepatic ferritin gene expression ( p < 0.01), which may contribute to its anti-inflammatory properties. Conversely, GPSE-γCD did not affect the biomarkers of endogenous antioxidant defence, including catalase, glutathione peroxidase-4, paraoxonase-1, glutamate cysteine ligase and nuclear factor erythroid 2-related factor-2 (Nrf2). Overall, the present data suggest that dietary GPSE-γCD exhibits anti-inflammatory, but not antioxidant activity in mouse liver in vivo. Thus, GPSE-γCD has the potential to serve as a natural hepatoprotective bioactive compound for dietary-mediated strategies against chronic inflammation., Competing Interests: Keiji Terao is the president of CycloChem Bio Co., Ltd.

Ethnopharmacological Relevance: Herbal medicines have been used for centuries for the management and treatment of various ailments due to the belief that they pose only little or no health risk and side effects, and also, in part, due to their availability, affordability and/or self-supply. However, the increasing information over the recent years on the occurrence of pyrrolizidine alkaloids (PAs) in honey, herbal food and tea products has raised concerns about the safety of herbal medicines with respect to contamination. To this day, little is known on the occurrence of toxic PAs in herbal medicines, especially in tropical West Africa. The aim of this study was therefore to determine the PA content of 70 well-known and widely patronized plant-derived medicinal preparations, which are commercialized in Ghana and some West African countries, in order to ascertain their potential health risk., Materials and Methods: PAs of the herbal medicinal products, sourced from specialized drugstores and mostly regulatory approved, were analyzed for their PA content by a HPLC-ESI-MS/MS sum parameter method., Results: The results show that a total of 60% of the analyzed herbal products were PA positive, indicating an average PA-concentration of 25.0μg/kg. The maximum PA level (1290.0μg/kg) was attributed to a regulatory-approved herbal medicine not known, according to the list of declared ingredients, to contain PA-plant parts. Interestingly, higher PA content (average, 30.2μg/kg) was detected in regulatory-approved herbal medicines, in contrast to lower amount (average, 8.0μg/kg) detected in non-regulatory-approved products., Conclusion: The findings of this study clearly demonstrate that herbal medicines containing PA plants as ingredients, as well as some of those containing plant species not known to produce PAs, are likely to contain hepatotoxic PA at levels higher than the daily dose in food and herbal medicinal products proposed by the European Medicines Agency (i.e. 0.35μg PA per day for 50kg adult and 0.14μg PA per day for 20kg children). Hence, regulatory authorities are advised to carry out more rigorous quality control tests with respect to PAs so as to minimize the exposure of the consumers to these toxic compounds., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

Protein-based targeted toxins play an increasingly important role in targeted tumor therapies. In spite of their high intrinsic toxicity, their efficacy in animal models is low. A major reason for this is the limited entry of the toxin into the cytosol of the target cell, which is required to mediate the fatal effect. Target receptor bound and internalized toxins are mostly either recycled back to the cell surface or lysosomally degraded. This might explain why no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date although more than 500 targeted toxins have been developed within the last decades. To overcome the problem of insufficient endosomal escape, a number of strategies that make use of diverse chemicals, cell-penetrating or fusogenic peptides, and light-induced techniques were designed to weaken the membrane integrity of endosomes. This review focuses on glycosylated triterpenoids as endosomal escape enhancers and throws light on their structure, the mechanism of action, and on their efficacy in cell culture and animal models. Obstacles, challenges, opportunities, and future prospects are discussed.

Background: From a previous screening of Brazilian biodiversity for antiprotozoal activity, the hexane extract from leaves of Nectandra leucantha (Nees & Mart.) (Lauraceae) demonstrated activity against Trypanosoma cruzi. Chromatographic separation of this extract afforded bioactive dehydrodieugenol (1). Furthermore, methylated derivative 2 (dehydrodieugenol dimethyl ether) was prepared and also tested against T. cruzi., Purpose: To examine the therapeutical potential of compounds 1 and 2 against T. cruzi as well as to elucidate the mechanism of action of bioactive compound 1 against T. cruzi., Methods/study Design: Crude hexane extract from leaves was subjected to chromatographic steps to afford bioactive compound 1. In order to analyze the effect of additional methyl group in the antiparasitic activity of 1, derivative 2 was prepared (both are no pan-assay interference compounds - PAINS). These compounds were evaluated in vitro against T. cruzi (trypomastigote and amastigote forms) and analyzed for the potential effect in host cells through the production of nitric oxide and reactive oxygen species. Finally, the plasma membrane effect of the most potent compound 1 was investigated in T. cruzi trypomastigotes., Results: Compounds 1 and 2 displayed activity against amastigotes of T. cruzi. Although both compounds promoted activity against intracellular amastigotes, the production of nitric oxide and reactive oxygen species of host cells were unaltered, suggesting an antiparasitic activity other than host cell activation. Considering 1 the most effective compound against T. cruzi, the interference in the plasma membrane of the trypomastigotes was investigated using the fluorescent probe SYTOX ® Green. After a short-term incubation, the fluidity and integrity of the plasma membrane was completely altered, suggesting it as a primary target for compound 1 in T. cruzi., Conclusion: Compounds 1 and 2 selectively eliminated the intracellular parasites without host cell activation and could be important scaffolds for the search of new hit compounds., (Copyright © 2016 Elsevier GmbH. All rights reserved.)