[Toxicological assessment of nanostructured silica. I. Integral indices, adducts of DNA, tissue thiols and apoptosis in liver].

Nanostructured amorphous silica (SiO2) is widely used in food additives, pharmaceuticals and cosmetics. Available data on the oral toxicity of this nanomaterial (NM) in vivo, obtained in acute and subacute experiments are contradictory. The purpose of this study is evaluation of some parameters of toxicity of nanostructured SiO2 when orally administered to rats for 3 months. We used commercial SiO2 preparation, obtained by gas-phase hydrolysis of tetrachlorosilane with a size of the primary nanoparticles close to 5-30 nm, which was characterized as NM by several independent methods. SiO2 in the form of sonicated aqueous dispersion was administered to male rats with initial weight of 80 +/- 4 g for the first 30 days by intragastric gavage and then for 62 days with consumed diets in daily dose of 0,1; 1,0; 10 and 100 mg/kg body weight. The control animals received vehicle--deionized water. Weight gain, relative mass of internal organs, intestinal permeability to protein macromolecules (determination of ovalbumin level in blood serum by solid-phase bivalent immunoassay), urinary excretion of oxidative degradation product of DNA 8-oxo-2-deoxyguanosine (8-oxo-G) (by reversed phase HPLC), the level of thiol compounds in liver (spectrophotometrically), liver cell apoptosis (flow cytometer), fixing efficiency of passive avoidance (CRPA) have been measured. It has been shown that three-month administration of nanostructured SiO2 in all doses resulted in animal body weight decrease by 10-15%; a significant increase in adrenal weight was noticed under doses of 1 and 10 mg/kg and urinary 8-oxo-G excretion was significantly reduced at the dose 10 mg/kg. At the maximum dose of NM, 100 mg/kg, after 2 months of administration the number of animals decreased that entered the dark compartment of the experimental setup at initial testing of CRPA. The rest of the studied indices did not experience any significant changes depending on the dose of NM. It is concluded that no toxic effect were expressed in indices studied under the influence of nanostructured SiO2 in rats at daily doses up to 100 mg per kg body weight for 3 months.

[Phytoecdysteroids influence on the hormonal status and apoptosis in growing rats].

The impact of the 15-day consumption of Serratula coronata extract containing phytoecdysteroids on some indicators of hormonal status and activity of apoptosis in various organs of growing male Wistar rats (initial body weight 127.8 +/-2.5 sigma) has been studied. The extract from the leaves of Serratula coronata was added to the water of animals of experimental groups 2 and 3 (n = 8 in each group) daily at the dose of 5 and 15 mg phytoecdysteroids per kg of body weight respectively. Animals of the control group 1 (n = 8) received water alone throughout the experiment. Daily volume of drunk fluid was recorded. At the 15th day of the experiment animals were taken out using the decapitation under the light ether anesthesia. The content of corticosterone, prostaglandin E2 and beta-endorphin in rat blood plasma were determined by ELISA test. Plasma level of noradrenaline was determined by HPLC. DNA damage and percentage of apoptotic cells (apoptotic index) were measured in isolated cells of the thymus, heart and brain by single-cell gel electrophoresis (the comet assay). Significantly lower concentration of norepinephrine was detected in plasma of experimental animals from groups 2 and 3 (10.3 +/- 1.1 and 7.2 +/- 0.8 ng/ml, respectively) compared to the same index in the control group (20.4 +/- 3.4 ng/ml). Significant differences of other biochemical parameters for all groups of animals have not been identified. Statistical significant difference in the ratio of corticosterone/norepinephrine compared with control animals was detected for a group of rats consumed the highest dose of phytoecdysteroids. There was no statistically significant difference in DNA fragmentation and apoptosis index in animals consumed phytoecdysteroids in compare with the control group of animals. The absence of the activity of apoptosis in cells of the heart, brain and thymus of rats treated with phytoecdysteroid extract may indicate the safety of its use in the diet of the animals.

[Influence of enzymatic hydrolyzate of mussel meat on growth and some indicators of general adaptation syndrome in rats].

The impact of the 15-day consumption of enzymatic hydrolyzate of the mussels meat as a part of semi-synthetic diet on some stress biomarkers and apoptosis activity in various organs of growing male Wistar rats have been studied. Enzymatic hydrolyzate of the mussels meat (EMM) was obtained in pilot conditions using the enzyme preparation "Protozim". The animals of control group 1 (n = 8 with initial body weight of 179.4 ± 5.9 g) and experimental group 2 (n = 8, 176.3 ± 4.5 g) received a semi synthetic diet; the animals of the experimental group 3 (n = 8, 177.6 ± 4.0 g) received the same semi synthetic diet in which 50% of the casein was replaced by the peptides of EMM. On the penult day of the experiment animals of groups 2 and 3 were subjected to stress exposure by electric current on their paws (current 0.4 mA for 8 seconds) and were placed in metabolic cages for the collection of daily urine. At the 15th day of the study, all control and test animals were killed by decapitation under ether anesthesia and necropsied. The content of prostaglandin E2 and ß-endorphin in blood plasma was determined by ELISA test. The concentration of urine corticosterone was measured by HPLC. DNA damage and percentage of apoptotic cells (apoptotic index) were calculated in thymus by single-cell gel electrophoresis assay (Comet assay). The relative body weight increase of animals treated with EMM was significantly (p < 0.05) higher (68.2 ± 3.0%) than those in animals of groups 1 and 2 (57.2 ± 4.0 and 59.7 ± 2.8%, respectively). The apoptotic index in thymus cells of tested groups of animals (2 and 3) was significantly (p < 0.05) higher (1.13 ± 0.09 and 1.09 ± 0.01%) compared to intact animals of control group (1.04 ± 0.01%). Determination of ß-endorphin and prostaglandin E2 levels did not shown any significant differences between the groups. Significantly (p < 0.05) lower concentrations of corticosterone was found in the daily urine of stressed animals from group 3 (452 ± 78 ng/ml), treated with EMM, compared to stressed animals of group 2 that received a casein diet (834 ± 167 ng/ml). It has been shown that consumption of EMM with a high content of short and medium peptides has an impact on effectiveness of body weight gain of growing laboratory animals, and restrict the increase of corticosterone level in rats blood, which is typical for general adaptation syndrome.

[Effect of vitamin sufficiency on adaptation syndrome in growing rats].

The influence of vitamin supply of growing male -Wistar rats (n=21) with an initial body weight 53,5±0,9 g on their resistance to a single distress induced by the electric shock has been investigated. Control rats within 21 days received a complete semisynthetic diet,providingadequate amounts of vitamins. Combined vitamin deficiency in experimental rats was caused by 5-fold decrease of vitamin mixture amount in the feed and the total vitamin E exclusion from the mixture. On the 21st day, one day before the end of the experiment, both groups of rats were subjected to stress impact (electrocutaneous irritation on paws, 0,4 mA for 8 sec) and then animals were placed in metabolic cages to collect urine. By the end of the experiment, the animals with the combined vitamin deficiency lag behind in growth. Vitamin B2, A, B1 and E liver content decreased in experimental rats by 1,6, 2,3, 4,4 and 15 fold accordingly. Retinol plasma concentration was significantly reduced by 18%, α-tocopherol level - by 5 fold, urinary excretionof riboflavin and 4-pyridoxic acid (vitamin B6 metabolite) was significantly reduced by 6,5 and 2,46 times accordingly. MDA blood plasma concentration and the urinary ratio of oxidized and not oxidized form of 8-hydroxy-2'-deoxy-guanosine did not differ in both groups of rats. Urinary excretion of stress biomarker corticosterone in rats with combined vitamin deficit was 2,5-fold higher than in control rats. Thus, reducing of vitamins supply resulted in an increase of urine corticosterone in stressed rats, that characterized the intensity of general adaptation syndrome. This fact shows the importance of optimal sufficiency with vitamins in nonspecific (general) resistance to stress.

[Complex of zinc with enzymatic hydrolysate of pigspleen protein - in vivo investigation].

Experimental sample of complex zinc with enzymatic hydrolysate of pigspleen protein (Zn-EHPS) was produced in laboratory with ultra- and nanofiltration technologies; its composition was analyzed; male rats Wister (initial body weight 132±2,4 g) were under observation in vivo: rats weregiven zinc-deficient ration (ZDR) "Zinc Deficient Diet, Egg White Base", containing not more than 1,3 mg zinc/kg, and water ad libitum during 18 days. Animals were divided into 3 groups (10 in each): group I was given ZDR and distilled water; group 2 - ZDR and water zinc sulphate solution (ZnSO4) with zinc concentration of 16,3 mcg/ml; group 3 - ZDR and water Zn-EHPS solution with zinc concentration of 16,3 mcg/ml. All rats were daily observed with registration of water consumption. Unconditioned reflex (photophobia) or crossing time from light to darkness on special system before the beginning of experiment was tested out. An increase of body weight (ΔW) during the experiment, unconditioned reflex stability and training to conditioned passive avoidance reflex (CPAR) after paw painful electric stimulation were studied. On 18 day in 16 hours after paw painful electric stimulation alkaline phosphatase activity (AP) and corticosterone concentration in rat blood serum were examined. Insignificant changes in distribution of peptide fractions according its molecular mass and practical complete remove of free metal ions from final product were observed when Zn-EHPS-technology were used. During experiment in all groups correspondently ΔW was 23,87 and 82% (p<0,05); AP activity - 1,70±0,06; 3,10±0,25; 4,19± 0,26 mcmol/l/s (p<0,01); corticosterone concentration - 20,7±7,2; 21,5±6,3; 22,1±16,5 ng/ml (p>0,1) were observed. Significant (p<0,05) worse change of unconditioned reflex (photophobia) in group 1 (ZDR) in comparison with group 2 and group 3 (latent period - LP, s: 50,9±12,0; 18,2±16,0; 16,0±4,2) as well as to initial test (LP, s - 16,8±8,0) was found on the 14th day of the experiment. According to LP-test CPAR in all groups in 24 and in 96 hours after paw painful electric stimulation was found to be maintained. Data obtained let to suggest high bioavailability of new zinc organic form - Zn-EHPS and its usage possibility in fortification of special foods.

[Effect of intragastrically injection of phytoecdysteroids on some indicators of hormonal status in rats].

The experiment in vivo in growing male Wistar rats was carried out. The animals of the experimental 2-4 groups were daily intragastrically injected water solutions of the dried extract from the leaves of Seratulla coronata L. in volume of 1.0 ml, containing 2, 20 and 50 mg of phytoecdysteroids per kg of animal weight, accordingly. Animals of control group were daily injected 1.0 ml of water. The content of phytoecdysteroids in the dry extract was analyzed by high performance liquid chromatography (HPLC). The concentration of the sum of phytoecdysteroids in dry extract was 6.15%, 66% of which was 20-hydroxyecdysone and 23% was 25S-inokosteron. On the 15th day animals were taken out of the experiment by the decapitation. The content of corticosterone, prostaglandin E2 and beta-endorphin in rat blood plasma were determined by ELISA test. The pathological--anatomical analysis and weighing of the liver did not reveal any adverse changes of this organ in the animals of all groups. The average concentration of blood plasma corticosterone reduced with increasing of the dose of the extract injected to the animals, reaching significant differences relative to the control group (60.9 +/- 9.4 ng/ml) for 3 and 4 groups (22.7 +/- 6.6 and 17.6 +/- 7.3 ng/ml, accordingly). Beta-endorphin and prostaglandin E2 levels did not differ. The ratio of the mediator of stress (corticosterone) and inhibitors of stress (beta-endorphin and prostaglandin E2) has been calculated. A monotonic decrease of corticosterone/beta-endorphin and corticosterone/prostaglandin E2 ratio has been found with extract dose increasing. Taken together the results of determination of biochemical parameters of the general adaptation syndrome the dose-dependent stress-protective effect of Seratulla coronata L extract has been demonstrated.

[Comparative evaluation of the influence of homogenate and enzymatic hydrolyzate of mussels' meat in the growth rats and their protein assimilation].

The experiment in vivo by a comparative assessment of the biological value of peptide fractions in structure of enzymatic hydrolyzates of mussels' meat and protein in structure of homogenate of mussels' meat (raw material) was carried out. The experiment was made in growing male rats of Wistar line, who got semi-synthetic diet with a source of protein presented by homogenate of mussels meat and enzymatic hydrolyzate of mussels meat. Throughout the study were defined the individual parameters of palatability of feed and increase of body weight of each animal; during the so-called exchange period, besides the listed parameters, were taken into account the quantity of nitrogen removed with excrement. Biological value of protein in structure of homogenate of mussels meat and peptide fractions in structure of enzymatic hydrolyzate of mussels meat was evaluated by determining the coefficient of efficiency of the protein and true digestibility of the protein. The findings show a high biological value of new source of protein - enzymatic hydrolyzate of mussels meat, the prospectivity of the further researches by evaluating a possible antistress properties of enzymatic hydrolyzate of mussels meat, as well as the opportunity of its use as a component of preventive food product of a broad spectrum of action.

[20-Hydroxyecdysone--plant adaptogen: an anabolic effect, possible use in sports nutrition].

In the review the presentation about plant adaptogens--biologically active compounds is given. Its administration can help to achieve non-specific state of high resistance. The hypothetical mechanism of action: adaptogens are prostressors, reducing excessive increase of stress mediators in the following stress exposure. The features of adaptogenic effect of phytoecdysteroids, polyhydroxylated sterols, which are analogs of hormones of molting and metamorphosis of arthropodas, and are structurally similar to glucocorticoids on the example of the most widely studied phytoecdysteroid--20-hydroxyecdysone--are described. The results of studies of anabolic action of 20-hydroxyecdysone in experiments on laboratory animals and the possible explanation (existing in the modern scientific literature) of the mechanism of this phenomenon are discussed. Scientific publication testifying on the application of phytoecdysteroids to remove chronic fatigue syndrome, reducing nerve and muscle fatigue, improve memory and attention processes are presented. The prospects of using the 20-hydroxyecdysone in the composition of food supplements and specialized products for athletes are discussed.

[Optimization of amino-acid profile of protein-peptide products used in preparation of functional beverages].

The message is devoted to optimization of amino acid structure of protein/peptide composition for its use as a component (a key ingredient) of functional drinks of various purposes. A basis of a composition forms protein/peptide product (PPP) received by enzymatic hydrolysis of meat and bone residues of processing of a bird, representing rather unvaluable food raw material. Molecular-mass distribution of fractions in PPP is quantitatively determined by an exclusion chromatography method of average pressure with the subsequent integration of chromatograms with a weight method. It can be supported the essential decrease of initial antigenicity of processed food raw materials as almost 80% from all sum protein/peptides and peptide/amino acid fractions are submitted by amino acids and peptides which molecular weight < or = 6 kD. The analysis of amino acid structure of PPP shows that the increase of its biological value assumes the increase of its score on four irreplaceable amino acids: leucine, tryptophan, phenylalanine and tyrosine (totally). For getting of the protein/peptide compositions of high biological value, the "above enrichment" of PPP by total protein of the cow milk is carried out. The modeling of a process of getting a two-component mix is carried out by a graphic method. The least percentage of the added total protein of a cow milk to PPP, at which is reached the amino acid score of the mix equal to 1,0, makes 26% from weight of the mix. The subsequent publications will be devoted to an estimation of biological value of a key component of functional food in the form of instant drinks in experiments in vivo on laboratory animals.

[Assessment of biological value of protein-peptide ingredients of functional drink in growing rats].

The abstract is devoted to research in vivo on the growing rats of biological value of protein hydrolyzate of meat-and-bone residues of poultry, fortified by defatted cow milk. Cow milk casein was chosen as a template. PER was determined by growing method, based on lab animals' growth rate assessment. True protein digestibility was determined by balance method. The indices were fixed individually for each animal and then the group mean value was calculated. Accessibility of protein hydrolyzate of meat-and-bone residues of poultry fortified by defatted cow milk equals 100%. PER is 1.25 lower in comparison with casein, which can be explained by a lower retention.

[New sources of organic form zinc].

The Comparative analysis of the efficiency of the enzymatic hydrolysis of the mussels meat has been carried out, using enzymatic preparations with proteolytic action called "Protozim", "Flavoenzyme" and "Pancreatine". Complexes of fermentolyzates of the mussels meat with zink have been obtained by the reaction of chelating. Prepared fermentolyzates and their complexes with zink have been determined by physicochemical methods. Composition of amino acids has been determined in fermentolyzates and in their complexes with zink has been characterized the content of zink. The valuation of molecular-mass distribution of peptide fractions of fermentolyzates and their complexes with zink has been given.