Gerhard Sontag

Changes in chemical composition of pumpkin seeds during the roasting process for production of pumpkin seed oil. (Part 1: non-volatile compounds)

Abstract Pumpkin seed oil is a common salad oil in Austria. It is not only of interest because of its typical taste but also because of its potential in curing prostate disease. Besides the fatty acids, the micronutrients, which comprise vitamin E, phytosterols and lignans, are of special interest. Since the seeds are roasted before pressing of the oil, changes occur in the composition of the fatty acids and micronutrients. The oxidation-sensitive linoleic acid decreases from 54.6 to 54.2% whereas the concentrations of the vitamin E isomers show a decrease during the first 40 min of about 30% followed by an increase during the last 20 min to about the same level as at the beginning of the roasting process. The concentrations of α-tocopherol and γ-tocopherol in the fresh dried seeds are 37.5 and 383 μg/g, respectively. The concentration of the tocotrienols is about one third of the corresponding tocopherols. The initial concentration of the total sterols (1710 μg/g) increases to 1930 μg/g. The increases of the sterols and vitamin E during the roasting process could be attributed to the changes of the seed meal, since at the end of the roasting the oil emerges from the seeds resulting in altered chemical behaviour of the extraction process. Secoisolariciresinol, which is only detectable at the beginning with a concentration of 3.8 μg/g, is destroyed after 20 min.

Genotoxic effects of crude juices from Brassica vegetables and juices and extracts from phytopharmaceutical preparations and spices of cruciferous plants origin in bacterial and mammalian cells

Crude juices of eight Brassica vegetables as well as juices and extracts of spices and phytopharmaceutical preparations from cruciferous vegetables were tested for induction of point mutations in Salmonella TA98 and TA100, repairable DNA damage in E.coli K-12 cells and clastogenic effects in mammalian cells. In bacterial assays, all juices caused genotoxic effects in the absence of metabolic activation, the ranking order being: Brussels sprouts > white cabbage > cauliflower > green cabbage > kohlrabi > broccoli > turnip > black radish. In experiments with mammalian cells, six juices induced structural chromosome aberrations. Brussels sprouts, white and green cabbage caused the strongest effects (800 microliters of juice induced a 5-fold increase over the background). In sister chromatid exchange assays, positive results were measured as well, but the effects were less pronounced. With all juices the genotoxic effects seen in mammalian cells were paralleled by a pronounced decrease in cell viability. Column fractionation experiments showed that 70-80% of the total genotoxic activity of the juices is found in the fraction which contains isothiocyanates and other breakdown products of glucosinolates, whereas phenolics and flavonoids contributed to a lesser extent to the overall effects. On the basis of these findings, and considering the negative results obtained with non-cruciferous vegetables (tomato, carrot and green pepper), it seems likely that the genotoxic effects of the juices are due to specific constituents of cruciferous plants such as glucosinolates and/or their breakdown products, in particular, isothiocyanates, which we found previously to be potent genotoxins in bacterial and mammalian cells. Finally, spices (mustards and horse radish paste) and phytopharmaceutical preparations were tested in bacterial assays. Mustards and horse radish caused very weak effects while most of the pharmaceutical preparations gave negative results, except cabbage tablets, which caused a strong and dose dependent induction of his revertants in Salmonella TA100. The present findings clearly indicate that cruciferous vegetables contain DNA damaging constituents. These observations are in contrast to earlier findings, which emphasized the antimutagenic effects of vegetable juices and also raise the question whether greatly increased consumption of Brassica vegetables or their concentrated constituents as a means for cancer prevention is indeed recommendable.

Analytical and compositional aspects of isoflavones in food and their biological effects

This paper provides an overview of analytical techniques used to determine isoflavones (IFs) in foods and biological fluids with main emphasis on sample preparation methods. Factors influencing the content of IFs in food including processing and natural variability are summarized and an insight into IF databases is given. Comparisons of dietary intake of IFs in Asian and Western populations, in special subgroups like vegetarians, vegans, and infants are made and our knowledge on their absorption, distribution, metabolism, and excretion by the human body is presented. The influences of the gut microflora, age, gender, background diet, food matrix, and the chemical nature of the IFs on the metabolism of IFs are described. Potential mechanisms by which IFs may exert their actions are reviewed, and genetic polymorphism as determinants of biological response to soy IFs is discussed. The effects of IFs on a range of health outcomes including atherosclerosis, breast, intestinal, and prostate cancers, menopausal symptoms, bone health, and cognition are reviewed on the basis of the available in vitro, in vivo animal and human data.

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.

Identification of phenolic components in dried spices and influence of irradiation.

LC-ESI-MS analysis was used for identification of phenolic compounds in the methanolic extracts of commercially available dried oregano, sage and thyme. Rosmarinic acid, apigenin-glucuronide, luteolin-glucuronide, as well as quinic acid were present in all three spices. Whereas in thyme and sage only derivatives from flavonoid compounds were identified, in oregano also the aglycons eriodictyol, naringenin, hispidulin, apigenin and luteolin could be found. Some constituents, especially glucosides and glucuronides, were observed for the first time in the methanol extracts of these spices. To ascertain an irradiation influence (dose: 10kGy) the MS peak area counts of twenty constituents, among them 10 glycosides/glucuronides, were compared. Although the majority of those compounds exhibited a slight decrease with irradiation, the changes were not significant.

Flow-injection analysis with electrochemical detection for determination of salicylic acid in pharmaceutical preparations

Abstract An enzyme-based flow-injection system with electrochemical detection suitable for the determination of salicylic acid in pharmaceuticals is described. The method makes use of the enzyme salicylate hydroxylase (EC 1.14.13.1), which is covalently bound to glass beads and put into the reactor of a flow system. The enzyme catalyses the stoichiometric conversion of salicylic acid to catechol, which can then be detected amperometrically at +0.45 V (vs. Ag/AgCl/KCl sat. ). The electrode response is linearly proportional to the concentration of salicylic acid between 5 and 150 μg ml −1 . The detection limit is 0.4 μg ml −1 . This method is simple, rapid and specific. The assayed samples yielded relative standard deviations between 0.5 and 2.0% and recoveries between 93 and 98%.

Determination of 1-nitropyrene in herbs after selective enrichment by a sol-gel-generated immunoaffinity column.

Using the determination of 1-nitropyrene as an example the paper demonstrates the advantages of including a highly selective sol-gel-generated immunoaffinity column in the sequence of clean-up steps necessary to determine haptens in complex matrices. The sol-gel method to immobilise antibodies enlarges the variety of immunoaffinity columns available and leads to mechanically stable columns with constant retention characteristics. The sample preparation scheme proposed combines acetonitrile extraction, size-exclusion and immunoaffinity chromatography. 1-Nitropyrene is then separated by reversed-phase HPLC from interfering compounds and determined after catalytic on-line reduction to the corresponding amine by spectrofluorimetry. Concentrations in the range from 0.1 to 1.4 microg/kg 1-nitropyrene were detected in herbs.

Multichannel coulometric detection coupled with liquid chromatography for determination of phenolic esters in honey

A method for the determination of phenolic esters in different varieties of honey was developed. The substances were separated by RP-HPLC. A coulometric electrode-array system with sixteen electrodes arranged in series and set at increasing potentials (300-900 mV) was used for electrochemical detection of the compounds. Chromatographic peaks for methyl 4-hydroxybenzoate, methyl vanillate, methyl syringate, trans-p-methyl coumarate and trans-methyl ferulate were identified. The content of the esters varied between 1.3 and 5044 micrograms per kg of honey with detection limits of 0.1-1.0 microgram per kg of honey (S/N = 3). The method described is a sensitive assay to differentiate between rape honey and other varieties.

Comparison of sample preparation methods for analysis of isoflavones in foodstuffs

Due to the lack of one universally applicable and commonly used reference method, sample preparation in isoflavone (IF) analysis has been performed by many different methods which renders comparison and quality assessment of published IF contents in foodstuffs difficult. In the present work, the impact of different experimental parameters on the IF concentrations determined in soybeans, tofu, soy drink and textured vegetable protein by different extraction and hydrolysis methods was assessed and IF contents obtained by optimized orthogonal methods were compared. Chromatographic analysis was performed by HPLC-UV-ESI-MS. If possible sources of error - which are also pointed out in this work - are avoided, IF contents obtained by extraction, acid-, base- and enzymatic hydrolysis are similar. However, these sample preparation methods differ in the amount of time, standard compounds and instruments required, ruggedness, and in their applicability to analysis of complex composite samples containing soy as minor ingredient. Enzymatic hydrolysis with Helix pomatia juice after extraction by sonication with first 50, then 80% aqueous acetonitrile in the presence of zinc sulfate heptahydrate and after adjustment to </=10% organic solvent turned out to be the method of choice if only aglucone equivalent contents are required. The advantages of this method are short chromatographic run times, smallest danger of coelution, lowest achievable limits of quantitation and therefore best suitability for work-up of complex composite samples and that only aglucone standards are needed for quantitation.

Binding of heterocyclic aromatic amines by lactic acid bacteria: results of a comprehensive screening trial.

Aim of the present study was a comprehensive investigation of the detoxification capacities of lactic acid bacteria (LAB) towards heterocyclic aromatic amines (HCA) formed during cooking of meat. It has been postulated that LAB prevent genotoxic and/or carcinogenic effects of HCA in laboratory rodents and humans via direct binding mechanisms. We measured the removal of the most abundant cooked food mutagens (AalphaC, PhIP, IQ, MeIQx, DiMeIQx) by eight LAB species. From each species, twelve strains were tested in liquid binding experiments with HPLC coupled with coulometric electrode array detection. The highest removal rates were observed with the representatives of the L. helveticus and S. thermophilus groups, which were seven to eight times more effective than L. kefir and L. plantarum. Strong and statistically significant differences were seen in the binding behaviour of the individual amines, the ranking order of detoxification being AalphaC > DiMeIQx > MeIQx > IQ > PhIP. Results of Salmonella/microsome assays with strain TA98 showed that the binding of AalphaC and PhIP to LAB correlates with the reduction of their mutagenic activities. This study may contribute to the development of strategies concerning the adverse health effects of HCA utilizing highly protective LAB for the production of fermented foods.