Parthenolide in Danish biodynamic and organic milk: A new source of exposure to an allergenic sesquiterpene lactone.

BACKGROUND: The sunflower family of plants (Compositae = Asteraceae) is currently the most allergenic plant family worldwide, according to the number of sensitizing species. Secondary plant metabolites, including the allergenic sesquiterpene lactones present in Compositae plants, may occur in food items either through their presence in, or through contamination of, plant-based raw materials, or through their occurrence in products of non-plant origin. OBJECTIVE: To analyse biodynamic, organic and conventional milk for the presence of the sesquiterpene lactone parthenolide. METHODS: The content of parthenolide in the milk samples was investigated in dichloromethane extracts obtained by liquid-liquid extraction, followed by gas chromatography-mass spectrometry analyses. RESULTS: The concentration of parthenolide was 0.07 ±0.004 ppm in biodynamic milk, 0.05 ±0.002 ppm in organic milk, and not detectable (<0.002 ppm) in conventional milk. CONCLUSIONS: This is the first report of a potent contact allergen in milk. There seems to be an association between the time that the dairy cattle spend grazing and the amount of parthenolide detected. Although the concentration is low, it is estimated to be high enough to elicit dermatitis in the most sensitive persons by direct contact with the milk.

Development of an In Vitro Screening Platform for the Identification of Partial PPARγ Agonists as a Source for Antidiabetic Lead Compounds.

Type 2 diabetes (T2D) is a metabolic disorder where insulin-sensitive tissues show reduced sensitivity towards insulin and a decreased glucose uptake (GU), which leads to hyperglycaemia. Peroxisome proliferator-activated receptor (PPAR)γ plays an important role in lipid and glucose homeostasis and is one of the targets in the discovery of drugs against T2D. Activation of PPARγ by agonists leads to a conformational change in the ligand-binding domain, a process that alters the transcription of several target genes involved in glucose and lipid metabolism. Depending on the ligands, they can induce different sets of genes that depends of their recruitment of coactivators. The activation of PPARγ by full agonists such as the thiazolidinediones leads to improved insulin sensitivity but also to severe side effects probably due to their behavior as full agonists. Partial PPARγ agonists are compounds with diminished agonist efficacy compared to full agonist that may exhibit the same antidiabetic effect as full agonists without inducing the same magnitude of side effects. In this review, we describe a screening platform for the identification of partial PPARγ agonists from plant extracts that could be promising lead compounds for the development of antidiabetic drugs. The screening platform includes a series of in vitro bioassays, such as GU in adipocytes, PPARγ-mediated transactivation, adipocyte differentiation and gene expression as well as in silico docking for partial PPARγ agonism.

Effect of resveratrol on experimental non-alcoholic fatty liver disease depends on severity of pathology and timing of treatment.

BACKGROUND AND AIM: Non-alcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease with few therapeutic options. Resveratrol (RSV) prevents the development of steatosis in a number of experimental fatty liver (non-alcoholic fatty liver [NAFL]) models, but the preventive or therapeutic effects on experimental NASH are not yet clarified, and clinical results on non-alcoholic fatty liver disease are ambiguous. Thus, we aimed to compare the RSV-mediated preventive and therapeutic effects on experimental NAFL and NASH. METHODS: We used a high-fat (HF) diet to generate a rat NAFL model and a high-fat, high-cholesterol (HFC) diet to generate a rat NASH model. The preventive and therapeutic potential of RSV was tested by adding RSV to the HF and HFC diet from study start or after 1 week of the diets. Animals were sacrificed after 8 weeks with appropriate controls. Blood and liver were harvested for analysis, including measurement of RSV metabolites. RESULTS: Resveratrol reduced the development of histological steatosis (P = 0.03) and partly triglyceride accumulation (fold change reduced from 3.6 to 2.4, P = 0.08) in the male NAFL model, although effects were moderate. In NASH prevention, RSV reduced the accumulation of triglyceride in hepatic tissue (P < 0.01), while there was no effect on biochemical, histopathological, or transcriptional NASH changes. Further, RSV had no therapeutic effect on established NASH. We found RSV metabolites but no parent RSV in serum or liver tissue, confirming low bioavailability. CONCLUSIONS: These experimental findings suggest that a weak hepatic benefit of RSV treatment is seen in prevention of steatosis only.

Identification of PPARγ Agonists from Natural Sources Using Different In Silico Approaches.

Peroxisome proliferator-activated receptor γ plays an important role in lipid and glucose homeostasis and is the target of many drug discovery investigations because of its role in diseases such as type 2 diabetes. Activation of peroxisome proliferator-activated receptor γ by agonists leads to a conformational change in the ligand-binding domain altering the transcription of several target genes involved in glucose and lipid metabolism, resulting in, for example, facilitation of glucose and lipid uptake and amelioration of insulin resistance, and other effects that are important in the treatment of type 2 diabetes. Peroxisome proliferator-activated receptor γ partial agonists are compounds with diminished agonist efficacy compared to full agonists; however, they maintain the antidiabetic effect of full agonists but do not induce the same magnitude of side effects. This mini-review gives a short introduction to in silico screening methods and recent research advances using computational approaches to identify peroxisome proliferator-activated receptor γ agonists, especially partial agonists, from natural sources and how these ligands bind to the peroxisome proliferator-activated receptor γ in order to better understand their biological effects.

Sunflower seeds as eliciting agents of Compositae dermatitis.

BACKGROUND: Sunflowers may cause dermatitis because of allergenic sesquiterpene lactones (SLs). Contact sensitization to sunflower seeds has also been reported, but the allergens are unknown. OBJECTIVES: To analyse sunflower seeds for the presence of SLs and to assess the prevalence of sunflower sensitization in Compositae-allergic individuals. PATIENTS/MATERIALS/METHODS: Sunflower-sensitive patients were identified by aimed patch testing. A dichloromethane extract of whole sunflower seeds was analysed by liquid chromatography-mass spectrometry and high-performance liquid chromatography. RESULTS: The prevalence of sensitivity to sunflower in Compositae-allergic individuals was 56%. A solvent wash of whole sunflower seeds yielded an extract containing SLs, the principal component tentatively being identified as argophyllin A or B, other SLs being present in minute amounts. CONCLUSIONS: The concentration of SLs on the sunflower seeds is considered high enough to elicit dermatitis in sensitive persons, and it seems appropriate to warn Compositae-allergic subjects against handling sunflower seeds.

Isomeric C12-alkamides from the roots of Echinacea purpurea improve basal and insulin-dependent glucose uptake in 3T3-L1 adipocytes.

Echinacea purpurea has been used in traditional medicine as a remedy for the treatment and prevention of upper respiratory tract infections and the common cold. Recent investigations have indicated that E. purpurea also has an effect on insulin resistance. A dichloromethane extract of E. purpurea roots was found to enhance glucose uptake in adipocytes and to activate peroxisome proliferator-activated receptor γ. The purpose of the present study was to identify the bioactive compounds responsible for the potential antidiabetic effect of the dichloromethane extract using a bioassay-guided fractionation approach. Basal and insulin-dependent glucose uptake in 3T3-L1 adipocytes were used to assess the bioactivity of extract, fractions and isolated metabolites. A peroxisome proliferator-activated receptor γ transactivation assay was used to determine the peroxisome proliferator-activated receptor γ activating properties of the extract, active fractions and isolated metabolites. Two novel isomeric dodeca-2E,4E,8Z,10E/Z-tetraenoic acid 2-methylbutylamides together with two known C12-alkamides and α-linolenic acid were isolated from the active fractions. The isomeric C12-alkamides were found to activate peroxisome proliferator-activated receptor γ, to increase basal and insulin-dependent glucose uptake in adipocytes in a dose-dependent manner, and to exhibit characteristics of a peroxisome proliferator-activated receptor γ partial agonist.

Falcarindiol Purified From Carrots Leads to Elevated Levels of Lipid Droplets and Upregulation of Peroxisome Proliferator-Activated Receptor-γ Gene Expression in Cellular Models.

Falcarindiol (FaDOH) is a cytotoxic and anti-inflammatory polyacetylenic oxylipin found in food plants of the carrot family (Apiaceae). FaDOH has been shown to activate PPARγ and to increase the expression of the cholesterol transporter ABCA1 in cells, both of which play an important role in lipid metabolism. Thus, a common mechanism of action of the anticancer and antidiabetic properties of FaDOH may be due to a possible effect on lipid metabolism. In this study, the effect of sub-toxic concentration (5 µM) of FaDOH inside human mesenchymal stem cells (hMSCs) was studied using white light microscopy and Raman imaging. Our results show that FaDOH increases lipid content in the hMSCs cells as well as the number of lipid droplets (LDs) and that this can be explained by increased expression of PPARγ2 as shown in human colon adenocarcinoma cells. Activation of PPARγ can lead to increased expression of ABCA1. We demonstrate that ABCA1 is upregulated in colorectal neoplastic rat tissue, which indicates a possible role of this transporter in the redistribution of lipids and increased formation of LDs in cancer cells that may lead to endoplasmic reticulum stress and cancer cell death.

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 ± 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

Dietary Polyacetylenic Oxylipins Falcarinol and Falcarindiol Prevent Inflammation and Colorectal Neoplastic Transformation: A Mechanistic and Dose-Response Study in A Rat Model.

Falcarinol (FaOH) and falcarindiol (FaDOH) are cytotoxic and anti-inflammatory polyacetylenic oxylipins, which are commonly found in the carrot family (Apiaceae). FaOH and FaDOH have previously demonstrated a chemopreventive effect on precursor lesions of colorectal cancer (CRC) in azoxymethane (AOM)-induced rats. The purpose of the present study was to elucidate possible mechanisms of action for the preventive effect of FaOH and FaDOH on colorectal precancerous lesions and to determine how this effect was dependent on dose. Gene expression studies performed by RT-qPCR of selected cancer biomarkers in tissue from biopsies of neoplastic tissue revealed that FaOH and FaDOH downregulated NF-κß and its downstream inflammatory markers TNFα, IL-6, and COX-2. The dose-dependent anti-neoplastic effect of FaOH and FaDOH in AOM-induced rats was investigated in groups of 20 rats receiving a standard rat diet (SRD) supplemented with 0.16, 0.48, 1.4, 7 or 35 µg FaOH and FaDOH g-1 feed in the ratio 1:1 and 20 rats were controls receiving only SRD. Analysis of aberrant crypt foci (ACF) showed that the average number of small ACF (<7 crypts) and large ACF (>7 crypts) decreased with increasing dose of FaOH and FaDOH and that this inhibitory effect on early neoplastic formation of ACF was dose-dependent, which was also the case for the total number of macroscopic neoplasms. The CRC protective effects of apiaceous vegetables are mainly due to the inhibitory effect of FaOH and FaDOH on NF-κB and its downstream inflammatory markers, especially COX-2.

Fast cleavage of phycocyanobilin from phycocyanin for use in food colouring.

Phycocyanins from cyanobacteria are possible sources for new natural blue colourants. Their chromophore, phycocyanobilin (PCB), was cleaved from the apoprotein by solvolysis in alcohols and alcoholic aqueous solutions. In all cases two PCB isomers were obtained, while different solvent adducts were formed upon the use of different reagents. The reaction is believed to take place via two competing pathways, a concerted E2 elimination and a SN2 nucleophilic substitution. Three cleavage methods were compared in terms of yield and purity: conventional reflux, sealed vessel heated in an oil bath, and microwave assisted reaction. The sealed vessel method is a new approach for fast cleavage of PCB from phycocyanin and gave at 120°C the same yield within 30min compared to 16h by the conventional reflux method (P<0.05). In addition the sealed vessel method resulted in improved purity compared to the other methods. Microwave irradiation increased product degradation.

Dietary polyacetylenes, falcarinol and falcarindiol, isolated from carrots prevents the formation of neoplastic lesions in the colon of azoxymethane-induced rats.

Falcarinol (FaOH) and falcarindiol (FaDOH) are found in many food plants of the Apiaceae family. Carrots are a major dietary source of these polyacetylenes. Feeding azoxymethane (AOM)-induced rats with carrots and purified FaOH have previously been shown to inhibit neoplastic transformations in the colon. FaOH and FaDOH have also shown to have a synergistic effect in vitro, resulting in a significant increased cytotoxic activity. Based on these findings the antineoplastic effect of FaOH and FaDOH (purity > 99%) was investigated in the AOM-induced rat model. Twenty rats received rat diet containing 7 µg FaOH per g feed and 7 µg FaDOH per g feed and 20 rats were controls receiving only rat diet. Then carcinogenesis was induced in all 40 rats with the carcinogen AOM. All animals received the designated diet for 2 weeks before AOM induction and continued on the designated diet throughout the experiment. Rats were euthanized 18 weeks after the first AOM injection and macroscopic polyp/cancers were measured, harvested and stained for histology. The difference in sizes of aberrant crypt foci (ACF) were analysed in a Wilcoxon rank sum test, in which the median number of small ACF was 218 in controls and 145 in polyacetylene treated rats (P < 0.001). Fifteen control rats and 8 treated rats had macroscopic tumors (P = 0.027). The number of tumors larger than 3 mm were 6 and 1 in control and treated rats, respectively (P = 0.032). In conclusion dietary supplements with FaOH and FaDOH reduced the number of neoplastic lesions as well as the growth rate of the polyps suggesting a preventive effect of FaOH and FaDOH on the development of colorectal cancer.

Polyacetylenes from carrots (Daucus carota) improve glucose uptake in vitro in adipocytes and myotubes.

A dichloromethane (DCM) extract of carrot roots was found to stimulate insulin-dependent glucose uptake (GU) in adipocytes in a dose dependent manner. Bioassay-guided fractionation of the DCM extract resulted in the isolation of the polyacetylenes falcarinol and falcarindiol. Both polyacetylenes were able to significantly stimulate basal and/or insulin-dependent GU in 3T3-L1 adipocytes and porcine myotube cell cultures in a dose-dependent manner. Falcarindiol increased peroxisome proliferator-activated receptor (PPAR)γ-mediated transactivation significantly at concentrations of 3, 10 and 30 µM, while PPARγ-mediated transactivation by falcarinol was only observed at 10 µM. Docking studies accordingly indicated that falcarindiol binds to the ligand binding domain of PPARγ with higher affinity than falcarinol and that both polyacetylenes exhibit characteristics of PPARγ partial agonists. Falcarinol was shown to inhibit adipocyte differentiation as evident by gene expression studies and Oil Red O staining, whereas falcarindiol did not inhibit adipocyte differentiation, which indicates that these polyacetylenes have distinct modes of action. The results of the present study suggest that falcarinol and falcarindiol may represent scaffolds for novel partial PPARγ agonists with possible antidiabetic properties.

Screening for bioactive metabolites in plant extracts modulating glucose uptake and fat accumulation.

Dichloromethane and methanol extracts of seven different food and medicinal plants were tested in a screening platform for identification of extracts with potential bioactivity related to insulin-dependent glucose uptake and fat accumulation. The screening platform included a series of in vitro bioassays, peroxisome proliferator-activated receptor (PPAR) γ-mediated transactivation, adipocyte differentiation of 3T3-L1 cell cultures, and glucose uptake in both 3T3-L1 adipocytes and primary porcine myotubes, as well as one in vivo bioassay, fat accumulation in the nematode Caenorhabditis elegans. We found that dichloromethane extracts of aerial parts of golden root (Rhodiola rosea) and common elder (Sambucus nigra) as well as the dichloromethane extracts of thyme (Thymus vulgaris) and carrot (Daucus carota) were able to stimulate insulin-dependent glucose uptake in both adipocytes and myotubes while weekly activating PPARγ without promoting adipocyte differentiation. In addition, these extracts were able to decrease fat accumulation in C. elegans. Methanol extracts of summer savory (Satureja hortensis), common elder, and broccoli (Brassica oleracea) enhanced glucose uptake in myotubes but were not able to activate PPARγ, indicating a PPARγ-independent effect on glucose uptake.