Comparative analysis of whole plant, flower and root extracts of Chamomilla recutita L. and characteristic pure compounds reveals differential anti-inflammatory effects on human T cells.

Introduction: Chronic inflammation is a hallmark of chronic wounds and inflammatory skin diseases. Due to a hyperactive and prolonged inflammation triggered by proinflammatory immune cells, transitioning to the repair and healing phase is halted. T cells may exacerbate the proinflammatory milieu by secreting proinflammatory cytokines. Chamomilla recutita L. (chamomile) has been suggested for use in several inflammatory diseases, implying a capability to modulate T cells. Here, we have characterized and compared the effects of differently prepared chamomile extracts and characteristic pure compounds on the T cell redox milieu as well as on the migration, activation, proliferation, and cytokine production of primary human T cells. Methods: Phytochemical analysis of the extracts was carried out by LC-MS/MS. Primary human T cells from peripheral blood (PBTs) were pretreated with aqueous or hydroethanolic chamomile extracts or pure compounds. Subsequently, the effects on intracellular ROS levels, SDF-1α induced T cell migration, T cell activation, proliferation, and cytokine production after TCR/CD3 and CD28 costimulation were determined. Gene expression profiling was performed using nCounter analysis, followed by ingenuity pathway analysis, and validation at protein levels. Results: The tested chamomile extracts and pure compounds differentially affected intracellular ROS levels, migration, and activation of T cells. Three out of five differently prepared extracts and two out of three pure compounds diminished T cell proliferation. In line with these findings, LC-MS/MS analysis revealed high heterogeneity of phytochemicals among the different extracts. nCounter based gene expression profiling identified several genes related to T cell functions associated with activation and differentiation to be downregulated. Most prominently, apigenin significantly reduced granzyme B induction and cytotoxic T cell activity. Conclusion: Our results demonstrate an anti-inflammatory effect of chamomile- derived products on primary human T cells. These findings provide molecular explanations for the observed anti-inflammatory action of chamomile and imply a broader use of chamomile extracts in T cell driven chronic inflammatory diseases such as chronic wounds and inflammatory skin diseases. Importantly, the mode of extract preparation needs to be considered as the resulting different phytochemicals can result in differential effects on T cells.

Anti-Allergic, Anti-Inflammatory and Anti-Hyperglycemic Activity of Chasmanthe aethiopica Leaf Extract and Its Profiling Using LC/MS and GLC/MS.

This study aims to comprehensively explore the phytoconstituents as well as investigate the different biological activities of Chasmanthe aethiopica (Iridaceae) for the first time. Metabolic profiling of the leaf methanol extract of C. aethiopica (CAL) was carried out using HPLC-PDA-ESI-MS/MS. Twenty-nine compounds were annotated belonging to various phytochemical classes including organic acids, cinnamic acid derivatives, flavonoids, isoflavonoids, and fatty acids. Myricetin-3-O-rhamnoside was the major compound identified. GLC/MS analysis of the n-hexane fraction (CAL-A) resulted in the identification of 45 compounds with palmitic acid (16.08%) and methyl hexadecanoic acid ester (11.91%) representing the major constituents. CAL-A exhibited a potent anti-allergic activity as evidenced by its potent inhibition of ß-hexosaminidase release triggered by A23187 and IgE by 72.7% and 48.7%, respectively. Results were comparable to that of dexamethasone (10 nM) in the A23187 degranulation assay showing 80.7% inhibition for ß-hexosaminidase release. Both the n-hexane (CAL-A) and dichloromethane (CAL-B) fractions exhibited potent anti-inflammatory activity manifested by the significant inhibition of superoxide anion generation and prohibition of elastase release. CAL showed anti-hyperglycemic activity in vivo using streptozotocin-induced diabetic rat model by reducing fasting blood glucose levels (FBG) by 53.44% as compared with STZ-treated rats along with a substantial increase in serum insulin by 22.22%. Molecular modeling studies indicated that dicaffeoylquinic acid showed the highest fitting with free binding energies (∆G) of -47.24 and -60.50 Kcal/mol for human α-amylase and α-glucosidase, respectively confirming its anti-hyperglycemic activity. Thus, C. aethiopica leaf extract could serve as an effective antioxidant natural remedy combating inflammation, allergy, and hyperglycemia.

What do minerals in the feces of Bearded Vultures reveal about their dietary habits?

The diet of Bearded Vultures Gypaetus barbatus consists mainly of bones, which are completely digested in the gastrointestinal tract, unwanted bone minerals being discarded via the feces. Chemical analyses of feces therefore provide a noninvasive technique for studying the diet of this species. We analysed the inorganic and organic remains in feces collected from Bearded Vulture nests in the Spanish Pyrenees and discussed these results with the diet of individuals determined by video camera observations. Of the food items delivered to the nest, taxonomically 65% were bone fragments of Ovis/Capra spp. (range 56-75%) and anatomically 76% (74-81%) bones from the extremities, indicating a selective preference. At least 15% of the diet was meat based, mainly originating from small prey (e.g. small carnivores, birds). The fecal analyses show that calcium and phosphorus are the most abundant mineral constituents, accounting for 41.3-44.4% of the mineral part of the feces. Among the minor elements identified, the variation in the concentrations of iron, silicon and zinc suggest differences in food selection between territories, although this could be related to varying amounts of accidentally ingested soil particles present in the food. We found variation in the content of uric acid in the feces, ranging between 0.5 and 4.6%. Higher values of uric acid might be due to a more meat or marrow bone-based diet. However, no relationship was found between the amount of calcium and uric acid levels, suggesting that the metabolites of meat digestion (uric acid) and those of bone digestion (calcium) are not negatively correlated as expected. In conclusion, our chemical analyses of feces collected from the nests of Bearded Vultures confirm that their diet consists mainly of bone remains and that these bones are digested completely. However, the direct observations of the prey items delivered to the nest produced more detailed information than the chemical analyses.

Antioxidant, Cytotoxic, and Antimicrobial Activities of Glycyrrhiza glabra L., Paeonia lactiflora Pall., and Eriobotrya japonica (Thunb.) Lindl. Extracts.

Background: The phytochemical composition, antioxidant, cytotoxic, and antimicrobial activities of a methanol extract from Glycyrrhiza glabra L. (Ge), a 50% ethanol (in water) extract from Paeonia lactiflora Pall. (Pe), and a 96% ethanol extract from Eriobotrya japonica (Thunb.) Lindl. (Ue) were investigated. Methods: The phytochemical profiles of the extracts were analyzed by LC-MS/MS. Antioxidant activity was evaluated by scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals and reducing ferric complexes, and the total phenolic content was tested with the Folin⁻Ciocalteu method. Cytotoxicity was determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in murine macrophage RAW 264.7 cells. Antimicrobial activity of the three plant extracts was investigated against six bacterial strains with the broth microdilution method. Results: Only Pe showed high antioxidant activities compared to the positive controls ascorbic acid and (-)-epigallocatechin gallate (EGCG) in DPPH assay; and generally the antioxidant activity order was ascorbic acid or EGCG > Pe > Ue > Ge. The three plant extracts did not show strong cytotoxicity against RAW 264.7 cells after 24 h treatment with IC50 values above 60.53 ± 4.03 µg/mL. Ue was not toxic against the six tested bacterial strains, with minimal inhibitory concentration (MIC) values above 5 mg/mL. Ge showed medium antibacterial activity against Acinetobacter bohemicus, Kocuria kristinae, Micrococcus luteus, Staphylococcus auricularis, and Bacillus megaterium with MICs between 0.31 and 1.25 mg/mL. Pe inhibited the growth of Acinetobacter bohemicus, Micrococcus luteus, and Bacillus megaterium at a MIC of 0.08 mg/mL. Conclusions: The three extracts were low-cytotoxic, but Pe exhibited effective DPPH radical scavenging ability and good antibacterial activity; Ue did not show antioxidant or antibacterial activity; Ge had no antioxidant potential, but medium antibacterial ability against five bacteria strains. Pe and Ge could be further studied for their potential to be developed as antioxidant or antibacterial candidates.

In vitro metabolic activation of vitamin D3 by using a multi-compartment microfluidic liver-kidney organ on chip platform.

Organ-on-chip platforms provide models that allow the representation of human physiological processes in cell-based miniaturized systems. Potential pre-clinical applications include drug testing and toxicity studies. Here we describe the use of a multi-compartment micro-fluidic chip to recapitulate hepatic vitamin D metabolism (vitamin D to 25-hydroxyvitamin D) and renal bio-activation (25-hydroxyvitamin D to 1,25-dihydroxyvitamin D) in humans. In contrast to cultivation in conventional tissue culture settings, on-chip cultivation of HepG2 and RPTEC cells in interconnected chambers, used to mimic the liver and kidneys, respectively, resulted in the enhanced expression of vitamin D metabolizing enzymes (CYP2R1, CYP27B1 and CYP24A1). Pump-driven flow of vitamin D3-containing medium through the microfluidic chip produced eluate containing vitamin D3 metabolites. LC-MSMS showed a strong accumulation of 25-hydroxyvitamin D. The chip eluate induced the expression of differentiation markers in HL-60 (acute myeloid leukemia) cells, assessed by qPCR and FACS analysis, in a manner similar to treatment with reference standards indicating the presence of fully activated 1,25 dihydroxyvitamin D, although the latter was not detected in the eluate by LC-MSMS. Interestingly, 25-hydroxyvitamin D by itself led to weak activation of HL-60 cells suggesting that 25-hydroxyvitamin D is also an active metabolite. Our experiments demonstrate that complex metabolic interactions can be reconstructed outside the human body using dedicated organ-on-chip platforms. We therefore propose that such systems may be used to mimic the in vivo metabolism of various micronutrients and xenobiotics.

Extracts of Glycyrrhiza uralensis and isoliquiritigenin counteract amyloid-ß toxicity in Caenorhabditis elegans.

Alzheimer's disease is a rising threat for modern societies as more and more people reach old age. To date, there is no effective treatment for this condition. In this study, we investigated the potential of Glycyrrhiza uralensis to counteract amyloid-ß toxicity, one of the key features of Alzheimer's disease. An LC-MS/MS analysis revealed glycyrrhizic acid and glycosylated forms of isoliquiritigenin and liquiritigenin as major constituents of water and methanol extracts of G. uralensis. These extracts and the pure compounds were tested for their activity in two Caenorhabditis elegans models of amyloid-ß aggregation and amyloid-ß toxicity, respectively. The number of amyloid-ß aggregates decreased by 30% after treatment with isoliquiritigenin, the methanol extract could reduce the number by 14%, liquiritigenin and glycyrrhizic acid by 15%, and the aglycon of glycyrrhizic acid, glycyrrhetinic acid, by 20%. Both extracts and isoliquiritigenin also showed significant activity against acute amyloid-ß toxicity in transgenic C. elegans that express human amyloid-ß peptides, delaying the paralysis in this model by 1.8 h and 1.1 h, respectively. We conclude that secondary compounds of G. uralensis may become interesting drug candidates for the treatment of Alzheimer's disease, which, however, need further analysis in other model systems.

Toxicity, dioxin-like activities, and endocrine effects of DDT metabolites--DDA, DDMU, DDMS, and DDCN.

BACKGROUND, AIM, AND SCOPE: 2,2-bis(chlorophenyl)-1,1,1-trichloroethane (DDT) metabolites, other than those routinely measured [i.e., 2,2-bis(chlorophenyl)-1,1-dichloroethylene (DDE) and 2,2-bis(chlorophenyl)-1,1-dichloroethane (DDD)], have recently been detected in elevated concentrations not only in the surface water of Teltow Canal, Berlin, but also in sediment samples from Elbe tributaries (e.g., Mulde and Havel/Spree). This was paralleled by recent reports that multiple other metabolites could emerge from the degradation of parent DDT by naturally occurring organisms or by interaction with some heavy metals. Nevertheless, only very few data on the biological activities of these metabolites are available to date. The objective of this communication is to evaluate, for the first time, the cytotoxicity, dioxin-like activity, and estrogenicity of the least-studied DDT metabolites. METHODS: Four DDT metabolites, p,p'-2,2-bis(chlorophenyl)-1-chloroethylene (DDMU), p,p'-2,2-bis(chlorophenyl)-1-chloroethane (DDMS), p,p'-2,2-bis(4-ch1oropheny1)acetonitrile (DDCN), and p,p'-2,2-bis(chlorophenyl)acetic acid (DDA), were selected based on their presence in environmental samples in Germany such as in sediments from the Mulde River and Teltow Canal. O,p'-DDT was used as reference in all assays. Cytotoxicity was measured by neutral red retention with the permanent cell line RTG-2 of rainbow trout (Oncorhynchus mykiss). Dioxin-like activity was determined using the 7-ethoxyresorufin-O-deetylase assay. The estrogenic potential was tested in a dot blot/RNAse protection-assay with primary hepatocytes from male rainbow trout (O. mykiss) and in a yeast estrogen screen (YES) assay. RESULTS: All DDT metabolites tested revealed a clear dose-response relationship for cytotoxicity in RTG-2 cells, but no dioxin-like activities with RTL-W1 cells. The dot blot/RNAse protection-assay demonstrated that the highest non-toxic concentrations of these DDT metabolites (50 µM) had vitellogenin-induction potentials comparable to the positive control (1 nM 17ß-estradiol). The estrogenic activities could be ranked as o,p'-DDT > p,p'-DDMS > p,p'-DDMU > p,p'-DDCN. In contrast, p,p'-DDA showed a moderate anti-estrogenic effect. In the YES assay, besides the reference o,p'-DDT, p,p'-DDMS and p,p'-DDMU displayed dose-dependent estrogenic potentials, whereas p,p'-DDCN and p,p'-DDA did not show any estrogenic potential. DISCUSSION: The reference toxicant o,p'-DDT displayed a similar spectrum of estrogenic activities similar to 17ß-estradiol, however, with a lower potency. Both p,p'-DDMS and p,p'-DDMU were also shown to have dose-dependent estrogenic potentials, which were much lower than the reference o,p'-DDT, in both the vitellogenin and YES bioassays. Interestingly, p,p'-DDA did not show estrogenic activity but rather displayed a tendency towards anti-estrogenic activity by inhibiting the estrogenic effect of 17ß-estradiol. The results also showed that the p,p'-metabolites DDMU, DDMS, DDCN, and DDA do not show any dioxin-like activities in RTL-W1 cells, thus resembling the major DDT metabolites DDD and DDE. CONCLUSIONS: All the DDT metabolites tested did not exhibit dioxin-like activities in RTL-W1 cells, but show cytotoxic and estrogenic activities. Based on the results of the in vitro assays used in our study and on the reported concentrations of DDT metabolites in contaminated sediments, such substances could, in the future, pose interference with the normal reproductive and endocrine functions in various organisms exposed to these chemicals. Consequently, there is an urgent need to examine more comprehensively the risk of environmental concentrations of the investigated DDT metabolites using in vivo studies. However, this should be paralleled also by periodic evaluation and monitoring of the current levels of the DDT metabolites in environmental matrices. RECOMMENDATIONS AND PERSPECTIVES: Our results clearly point out the need to integrate the potential ecotoxicological risks associated with the "neglected" p,p'-DDT metabolites. For instance, these DDT metabolites should be integrated into sediment risk assessment initiatives in contaminated areas. One major challenge would be the identification of baseline data for such risk assessment. Further studies are also warranted to determine possible additive, synergistic, or antagonistic effects that may interfere with the fundamental cytotoxicity and endocrine activities of these metabolites. For a more conclusive assessment of the spectrum of DDT metabolites, additional bioassays are needed to identify potential anti-estrogenic, androgenic, and/or anti-androgenic effects.

Uptake of S-(3-amino-3-oxopropyl)-cysteine by Caco-2 cells.

Acrylamide is a reactive neurotoxin with a high intestinal bioavailability. Recently we have shown that under the pH regime of the gut acrylamide can react with proteins and that this reaction reduces the uptake of acrylamide in a gut model. On the other hand, using radioactive labeled acrylamide, Bjellaas et al. [Toxicol. Sci. 100, 374-380 (2007)] showed that in vivo the vast majority of orally administered acrylamide is absorbed and excreted as N-acetyl-S-(3-amino-3-oxopropyl)-cysteine with the urine. Therefore, we tested whether intestinal proteases can degrade a protein with acrylamide bound to cysteine residues. Furthermore we tested whether the product of this reaction, S-(3-amino-3-oxopropyl)-cysteine, can pass the intestinal barrier. Here we showed that S-(3-amino-3-oxopropyl)-cysteine is indeed a product of proteolytic degradation of acrylamide-treated proteins. Using Caco-2 cells as a gut model, we further showed that the non-protein amino acid S-(3-amino-3-oxopropyl)-cysteine is a substrate for the neutral and cationic amino acid transporter system. Hence we concluded that protein-bound acrylamide can be released in the intestine and that the resulting product S-(3-amino-3-oxopropyl)-cysteine is transported through the intestinal barrier and later excreted via the urine.