Consumption of Brussels sprouts protects peripheral human lymphocytes against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and oxidative DNA-damage: results of a controlled human intervention trial

To find out if the cancer protective effects of Brussels sprouts seen in epidemiological studies are due to protection against DNA-damage, an intervention trial was conducted in which the impact of vegetable consumption on DNA-stability was monitored in lymphocytes with the comet assay. After consumption of the sprouts (300 g/p/d, n = 8), a reduction of DNA-migration (97%) induced by the heterocyclic aromatic amine 2-amino-1-methyl-6-phenyl-imidazo-[4,5-b]pyridine (PhIP) was observed whereas no effect was seen with 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2). This effect protection may be due to inhibition of sulfotransferase 1A1, which plays a key role in the activation of PhIP. In addition, a decrease of the endogenous formation of oxidized bases was observed and DNA-damage caused by hydrogen peroxide was significantly (39%) lower after the intervention. These effects could not be explained by induction of antioxidant enzymes glutathione peroxidase and superoxide dismutase, but in vitro experiments indicate that sprouts contain compounds, which act as direct scavengers of reactive oxygen species. Serum vitamin C levels were increased by 37% after sprout consumption but no correlations were seen between prevention of DNA-damage and individual alterations of the vitamin levels. Our study shows for the first time that sprout consumption leads to inhibition of sulfotransferases in humans and to protection against PhIP and oxidative DNA-damage.     

Determining the critical micelle concentration of a novel lipid-lowering agent, disodium ascorbyl phytostanyl phosphate (FM-VP4), using a fluorescence depolarization procedure

The objective of this study was to determine the critical micelle concentration (CMC) of a novel water-soluble plant sterol derivative (FM-VP4) using a fluorescence depolarization method. The CMC was determined by 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence depolarization. Test solutions of various concentrations of sodium dodecylsulphate (SDS) as a positive control or FM-VP4 in water were spiked with 2 µL of 4 mM DPH in tetrahydrofuran (THF) and left overnight to equilibrate in a dark chamber. Fluorescence of each solution was measured at room temperature using a Perseptive Biosystems Cytofluor Series 4000 multi-well plate reader. Fluorescence intensity increases as DPH is incorporated into the hydrophobic core of micelles. Thus, the CMC is the value at which an abrupt increase in intensity is observed. These points were observed at 8 mM and 0.014 mM for SDS and FM-VP4, respectively. Sodium dodecylsulphate was used as a positive control and supports the validity of our results, as the literature values of SDS are reported to be between 8-8.3 mM. The CMC of FM-VP4 is reported to be 0.014 mM.     

Development and characterization of liposomal disodium ascorbyl phytostanyl phosphates (FM-VP4)

The specific objectives of this project were (1) to develop liposomal disodium ascorbyl phytostanyl phosphate (FM-VP4) formulations, (2) to develop a liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) assay for quantification of FM-VP4 in liposomal formulations and plasma sample, and (3) to characterize liposomal FM-VP4 formulations by finding optimal drug-to-lipid ratios and determining the degradation of FM-VP4 in liposomes. Section 2 describes an LC/MS/MS assay developed for the identification and quantification of FM-VP4 in liposomal formulations to provide estimates of drug concentrations and encapsulation efficiency. The extra step of removing plasma proteins prior to LC/MS/MS assay yields an analysis of FM-VP4 in plasma samples. Section 3 describes experiments designed to find the optimal drug-to-lipid ratio for liposomal FM-VP4 formulations by comparing encapsulation efficiencies and varying the lipid compositions. Additionally, this section details our degradation studies to determine if liposomes have any protective effects on FM-VP4; these studies tested various lipid compositions at 37°C in rabbit plasma. The mechanism of how FM-VP4 lowers low-density lipoprotein (LDL) cholesterol and total cholesterol levels in various animal models is presently unknown. However, before the mechanism of action could be studied, FM-VP4 first had to be delivered efficiently into plasma or cultured cell. The low systemic bioavailability and cellular uptake of FM-VP4 further suggested the importance of finding an efficient delivery vehicle for this drug. This project proposed a framework for such delivery and paves the way for further investigation into how FM-VP4 works in vivo and in vitro.     

Activation of Akt (protein kinase B) stimulates metaphase I to metaphase II transition in bovine oocytes

In somatic cells, the serine/threonine kinase Akt (or protein kinase B) was shown to contribute to processes linked to cellular growth, cell survival and cell cycle regulation. In contrast to these findings, the function of Akt during the meiosis of mammalian oocytes remains to be investigated. We analysed the phosphorylation pattern and the activity of Akt during meiotic maturation (transition from prophase I to metaphase II) of bovine oocytes. The oocytes were matured in vitro (IVM) for 0, 10 and 24 h to reach the germinal vesicle (GV), metaphase I (M I) and metaphase II (M II) stages respectively. The abundance and phosphorylation pattern of Akt was revealed by Western blotting using total Akt or phosphoso-Akt-specific antibodies. The activity of this particular kinase was determined by an in vitro kinase assay. Furthermore, functional properties were analysed by cultivating oocytes in the presence of the Akt inhibitor SH6. The results showed that the overall abundance of Akt did not change significantly during IVM. On the other hand, Akt became phosphorylated at Thr 308 and Ser 473, reaching its maximum at the M I phase. In the GV and M II stages, only low basal phosphorylation levels were observed on both sides. This phosphorylation profile corresponded strictly to the activity of the kinase. The cultivation of oocytes in the presence of the phosphatidylinositol analogue SH6 for 24 h showed that, with higher concentrations, up to 65% of the oocytes were arrested in the M I stage. This result indicated that Akt is involved in the M I/M II transition during the meiotic maturation of bovine oocytes. The physiological aspects of the Akt function will be discussed.     

Thermosensitive hydrogels for modified release of ellagic acid obtained from Alchemilla vulgaris L. extract

Stimulus-sensitive hydrogels are used as carriers for modified release of pharmaceuticals. The synthesis of thermosensitive hydrogels poly(N-isopropylacrylamide), p(NIPAM), and poly(N-isopropylacrylamide-co-2-hydroxypropyl methacrylate), p(NIPAM-HPMet), is performed. The synthesized hydrogels are characterized using FTIR and scanning electron microscope methods and swelling properties, and applied for modified release of ellagic acid (EA). This work presents the selective extraction of EA, as a natural antioxidant, from the aerial parts of Alchemilla vulgaris L. EA and A. vulgaris extract are incorporated into p(NIPAM) and p(NIPAM-HPMet) hydrogels and characterized by FTIR method. The EA content in the extract by the UHPLC–DAD–HESI–MS/MS method is determined (0.64?mg?cm^?3). The total flavonoids content in the A. vulgaris extract was determined by the spectrophotometric method. Antioxidant activity of the A. vulgaris extract and EA is examined using the 2,2-diphenyl-1-picrylhydrazyl assay. The p(NIPAM-HPMet) shows a better incorporation and release at 37°C of EA standard and A. vulgaris extract (98.87 and 96.45% respectively), compared with p(NIPAM).     

Improved rapid authentication of vanillin using δ13C and δ2H values

Vanilla still remains one of the most important and widely used flavours in the food industry and is also extensively employed by fragrance and pharmaceutical manufacturing companies. Natural vanilla flavour, extracted from the pods of the tropic orchid vanilla, is considerably more expensive than synthetic vanillin. The disparity of prices between natural vanillin and that derived from other sources has given rise to many cases of fraudulent adulteration, and for more than 30 years, strenuous efforts have been made to authenticate sources of vanillin. Stable isotope analysis is one of the most powerful analytical tools to distinguish between natural vanillin and that originating from other sources. Recently, a rapid and precise method for analysis of both δ¹³C and δ²H values of plant methoxyl groups has been published. Here, we report an application of the method for the control of authenticity of vanillin. Carbon and hydrogen stable isotope values of the vanillin molecule and vanillin methoxyl groups of vanillin samples of different origins including authentic and synthetic samples were measured. The results clearly show that use of this approach provides a rapid and reliable authenticity assessment of vanillin. The technique used for these studies is robust and rapid, involves minimum sample preparation and requires only a small amount of vanillin sample, usually 1 mg for stable carbon and 4 mg for stable hydrogen analysis.     

Isotope-Guided Metabolomics Reveals Divergent Incorporation of Valine into Different Flavor Precursor Classes in Chives

Plants of the genus Allium such as chives, onions or garlic produce S-alk(en)yl cysteine sulfoxides as flavor precursors. Two major representatives are S-propenyl cysteine sulfoxide (isoalliin) and S-propyl cysteine sulfoxide (propiin), which only differ by a double bond in the C₃ side chain. The propenyl group of isoalliin is derived from the amino acid valine, but the source of the propyl group of propiin remains unclear. Here, we present an untargeted metabolomics approach in seedlings of chives (Allium schoenoprasum) to track mass features containing sulfur and/or ¹³C from labeling experiments with valine-¹³C₅ guided by their isotope signatures. Our data show that propiin and related propyl-bearing metabolites incorporate carbon derived from valine-¹³C₅, but to a much lesser extent than isoalliin and related propenyl compounds. Our findings provide new insights into the biosynthetic pathways of flavor precursors in Allium species and open new avenues for future untargeted labeling experiments.