Physiological effects of chicory root preparations with various levels of fructan and polyphenolic fractions in diets for rats

The experiment was aimed at studying the effects of easily fermentable oligosaccharides and phenolic compounds from chicory root meal (CRM) on the fermentative processes in the caecum, the antioxidative status and the lipoprotein profile of rats. Five different diets were fed ad libitum to 40 Wistar rats (eight animals per group, individually housed): a control group (C); group PCM (10% processed CRM, deprived of polyphenolic fraction); group PCMO (8% processed CRM and 1.6% oligofructose); group UCM (10% unprocessed CRM); and group FP (8.3% fructan-polyphenol concentrate from CRM). Diets PCM, PCMO, UCM and FP induced favourable metabolic changes in the caecum, blood lipid profile and the antioxidative status of the body. In the caecum, the experimental diets increased the production of volatile fatty acids (VFA) and acidification of digesta as well as a decrease in the ammonia concentration and bacterial β-glucuronidase activity. In blood serum, the total cholesterol concentration was reduced and, simultaneously, the proportion of HDL in the total cholesterol concentration was increased. The presence of the polyphenolic fraction in the unprocessed meal (diets UCM and FP) evoked a significant increase in the total antioxidative status in blood serum. Dietary fibre and the polyphenolic fraction present in diet UCM and the FOS-polyphenol concentrate in diet FP did not exhibit an antagonistic activity regarding the physiological parameters analysed, except for in the intensity of caecal fermentation. The results of the experiment point to the benefits of dietary supplementation with chicory preparations containing both prebiotic saccharides and polyphenolic compounds, which enable us to take advantage of the physiological traits of both components.     

In vitro synthesis of fractofuranosyl-only oligosaccharides from inulin and fructose by purified chicory root fructan:fructan fructosyl transferase

Besides the non-reducing inulin series of gluco-frueto oligosaccharides which are common in Asteraceae, two series of reducing fructofuranosyl-only oligosaccharides occur in overwintering, forced, and sprouting chicory roots. Such inulo-n-oses could be produced by the action of an endo-inulinase on gluco-fructo oligosaccharides. We show here that similar series of inulo-n-oses as found in vivo can be produced by incubating fructose and inulin with a purified fructamfructan tructosyl transferase (FFT; EC 2.4.1.100) from chicory roots ( Cichorium intybus L.). Therefore fructose (but not glucose) can act as an acceptor for FFT. Since the inulo-n-oses only appear in vivo once the endogenous fructose concentration increases, we conclude that they are not the result of a putative endo-inulinase but of FFT activity.     

Effect of nitrogen concentration on fructan and fructan metabolizing enzymes in young chicory plants (Cichorium intybus)

Witloof chicory seeds ( Cichorium intybus L. var. foliosum cv. Flash) were sown in acid-washed vermiculite in a controlled environment growth chamber. Plants received a nitrogen poor ("N-poor": 0.2 m M NH₄NO₃) but otherwise complete medium, or a nitrogen rich ("N-rich": 2 m M NH₄NO₃) medium. After 1 month of growth the fructan concentration in the "N-poor" plants was about five times higher and also the activity of sucrose:sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) was twice as high as in "N-rich" plants. The activities of the catabolic enzymes fructan 1-exohydrolase (1-FEH; EC 3.2.1.80) and acid invertase (EC 3.2.1.26) were higher in the "N-rich" plants where significant energy was invested in root and leaf growth. After one month of growth, part of the "N-poor" plants were switched to the "N-rich" medium. One day after this switch, a sharp decrease in sucrose and glucose concentration was observed in the roots. During the following days, both the activities of 1-SST and fructan:fructan 1-fructosyl transferase (1-FFT; EC 2.4.1.100) decreased and the 1-FEH and invertase activities increased. These changes were correlated with a decrease in fructan concentration. Ten days after the switch, glucose and sucrose concentrations increased again and fructan synthesis resumed. During this period 1-SST activity increased and 1-FEH activity decreased. Apparently 1-SST, 1-FFT and 1-FEH simultaneously control fructan in young chicory roots. The rather unexpected finding that 1-FEH activity, which was believed to occur only in older material, can be induced in very young roots indicates that this enzyme can be induced at any physiological stage.     

Effect of defoliation on fructan pattern and fructan metabolizing enzymes in young chicory plants (Cichorium intybus)

Witloof chicory ( Cichorium intybus L. var. foliosum cv. Flash) was sown in acid-washed vermiculite in a controlled growth chamber. After 1 month of growth, one half of the chicory plants were defoliated whereas the intact chicory plants remained as a control. Twenty-four hours after defoliation, a very sharp decrease in hexose, sucrose, and total fructan concentration was observed in the roots. This coincided with a strong decrease in sucrose:sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) activity and a strong increase in fructan 1-exohydrolase (1-FEH; EC 3.2.1.80) activity. After day 5, 1-SST activity increased and 1-FEH activity decreased. However, from day 5 to 15, both the activities of 1-SST and acid invertase (EC 3.2.1.26) remained significantly lower than in the control plants. From 10 days after defoliation, fructan synthesis resumed and hexose and sucrose concentrations increased. Up to now, 1-FEH activity was believed to occur only in mature tissues (end of the growing season, storage, forcing, or sprouting). Therefore, the rather unexpected finding that 1-FEH can also be induced in very young chicory roots after defoliation suggests that 1-FEH can be considered a 'survival' enzyme that can be induced at any physiological stage when energy demands increase.     

Influencing the binding configuration of sucrose in the active sites of chicory fructan 1-exohydrolase and sugar beet fructan 6-exohydrolase

The hydrolytic plant enzymes of family 32 of glycoside hydrolases (GH32), including acid cell wall type invertases (EC 3.2.1.26), fructan 1-exohydrolases (1-FEH; EC 3.2.1.153) and fructan 6-exohydrolases (6-FEH; EC 3.2.1.154), are very similar at the molecular and structural levels, but are clearly functionally different. The work presented here aims at understanding the evolution of enzyme specificity and functional diversity in this family by means of site-directed mutagenesis. It is demonstrated for the first time that invertase activity can be introduced in an S101L mutant of chicory (Cichorium intybus) 1-FEH IIa by influencing the orientation of Trp 82. At high sucrose and enzyme concentrations, a shift is proposed from a stable inhibitor configuration to an unstable substrate configuration. In the same way, invertase activity was introduced in Beta vulgaris 6-FEH by introducing an acidic amino acid in the vicinity of the acid-base catalyst (F233D mutant), creating a beta-fructofuranosidase type of enzyme with dual activity against sucrose and levan. As single amino acid substitutions can influence the donor substrate specificity of FEHs, it is predicted that plant invertases and FEHs may have diversified by introduction of a very limited number of mutations in the common ancestor.     

Variation in the in vitro generated fructan pattern from sucrose as a function of the purified chicory root 1-SST and 1-FFT concentrations

At low concentrations of purified chicory root 1-SST, only 1-kestosewas produced from a physiologically relevant sucrose concentration.As the 1-SST concentration increased, some higher oligofructanswere also detected, showing that 1-SST has some 1-FFT activityafter sucrose exhaustion in the reaction mixtures. The consequencesfor the interpretation of fructan synthesizing activities invitro are discussed. With a mixture of both purified 1-SST and1-FFT it was found that the higher the enzyme concentration,the higher the maximal DP of the fructans that could be synthesized.The higher the enzyme concentration, the higher the relativeabundance of the larger DP fructans and fructose in the reactionmixtures. Taken together with previous results, there is confidencethat the final fructan pattern obtained in vitro is a functionof the (1-SST+1-FFT)/sucrose ratio and suggest that the latterratio in situ could affect the highly variable tissue- or species-specificpattern of fructans produced in vivo. Key words: 1-FFT, 1-SST, chicory, enzyme concentration, sucrose     

Structural changes in liver parenchyma of rats kept on diets with various protein contents

The influence of diets with different protein content on the liver structural organization has been studied. Adaptation processes such as hepatocyte proliferation and an increase in the number of secondary lysosomes with the following atrophy of hepatocytes were observed in rats on low protein diet. High protein diet caused hypertrophy of hepatocytes and hyperplasia of subcellular structures.     

Liver and plasma copper concentrations in rats fed diets containing various proteins

The question was addressed whether the type of dietary protein influences copper (Cu) concentrations in liver and plasma of rats. For this purpose, weanling female rats were fed diets containing as the sole source of protein either soybean protein, casein, amino acid mixtures simulating soybean protein or casein, lactalbumin, ovalbumin, or herring meal. The diets were balanced for residual Cu in the protein preparations. The type of protein and the composition of the amino acid mixture did not differentially influence liver Cu concentrations. Liver Cu was significantly lowered after feeding the amino acid mixtures when compared with the intact proteins. Plasma Cu concentrations were not affected by the type of protein in the diet.     

Hypolipidemic and antioxidant effects of Morus alba L. (Egyptian mulberry) root bark fractions supplementation in cholesterol-fed rats

The 70% alcohol extract of the Egyptian Morus alba L. root bark was fractionated over cellulose CC eluted with water, 50% methanol and finally with 100% methanol to yield 3 fractions (MRBF-1, MRBF-2 and MRBF-3), respectively. In continuation of chromatographic purification of 70% alcohol extract fractions of the Egyptian M. alba L. root bark, 4 compounds namely: mulberroside A, 5,7,2'-trihydroxyflavanone-4'-O-beta-D-glucoside and albanols A and B were isolated from MRBF-2 for the first time from the Egyptian plant. Experimentally induced atherosclerosis was produced by feeding rats a diet enriched in coconut oil (25% by weight) and cholesterol (2% by weight) for 21 days. Then, hypercholesterolemic rats were orally administered (MRBF-1, MRBF-2 and MRBF-3 fractions) in a dose of 500 mg kg(-1) day(-1) for 15 successive days, in order to evaluate their expected hypocholesterolemic activity. Lipid profile parameters such as plasma total cholesterol, LDL-C, VLDL-C, LDL:HDL ratio and triglycerides, as well as plasma and liver lipid peroxides and glutathione-S-transferase enzyme levels, serum paraoxonase enzyme level, LDL oxidation, LDL aggregation and LDL retention, were measured. Plasma and liver glutathione-S-transferase enzyme levels were unaffected in all studied groups. The results revealed that the administration of (MRBF-2 and/or MRBF-3) fractions resulted in alleviation of atherosclerotic state. Administration of MRBF-3 significantly retained plasma and liver peroxides towards their normal levels, and also, produced significant increase in resistance towards major atherogenic modifications; namely LDL oxidation, LDL aggregation and LDL retention by 44%, 30%, and 33%, respectively. Thus, it can be concluded that the consumption of MRBF-2 and (MRBF-3, in some extent) fractions of M. alba L. root bark 70% alcohol extract may act as a potent hypocholesterolemic nutrient and powerful antioxidant via the inhibition of LDL atherogenic modifications and lipid peroxides formation in hypercholesterolemic rats.     

Diet supplemented either with dried chicory root or chicory inulin significantly influence kidney and liver mineral content and antioxidative capacity in growing pigs

According to the Regulation No. 1831/2003 of the European Parliament and European Union Council, the use of antibiotics as a dietary supplements has been prohibited. It seems that the administration of prebiotics, instead of antibiotics, into the pig's diet, may regulate the intestinal microbiota and has a long-term health-related impact on the host. Inulin-type fructans can stimulate mineral absorption from the gut. Additionally, it may regulate energy metabolism and activate enzymatic mechanisms preventing oxidative stress. The goal of the present study was to estimate the influence of dietary supplementation with dried chicory root or native chicory inulin on 1) liver histology; 2) liver and kidney lipid metabolism indices, activity of selected enzymes, concentration of macro- and micronutrients and heavy metals; 3) blood plasma, liver and kidney oxidative stress biomarkers and 4) blood plasma water–electrolyte homeostasis indices in growing pigs. The nutritional study was conducted on 24 piglets assigned to 3 dietary groups (n = 8): control (C) fed a basal diet and two experimental groups receiving basal diet supplemented with 2% of inulin (IN) either 4% of dried chicory root (CR). The animals were fed with a group-specific diets for 40 days and then subjected to euthanasia. Subsequently, blood, liver and kidney samples were harvested for further processing. In the control and experimental groups, no apparent morphological abnormalities in the liver tissues were seen. The percent of periodic acid Schiff positive glycogen liver cells was significantly lower in the CR group as compared to C and IN groups (P < 0.001). Chicory root supplementation improved blood plasma prooxidative-antioxidative balance – PAB (P < 0.001) and liver PAB (P < 0.01) and thiobarbituric acid reactive substances – thiobarbituric acid-reactive substances (P < 0.05). Feeding the CR diet increased calcium (P < 0.001) and potassium (P < 0.05) and decreased cadmium (P ≥ 0.05) content in the liver when compared to the C group. Administration of the CR and IN diets increased selenium (Se) and sodium concentrations, whereas decreased zinc content both in the liver (P < 0.01; P < 0.05 and P < 0.05, respectively) and in the kidney (P < 0.01; P < 0.001 and P < 0.001, respectively) of pigs. Additionally, a higher concentration of lead (P < 0.05) was observed in the kidney of pigs fed the CR diet. In conclusion, both dietary supplements had a potential to significantly improve the Se status and oxidoreductive homeostasis in growing pigs.     

Brain processing of hemorphin-7 peptides in various subcellular fractions from rats

Hemorphins are multifunctional peptides derived from hemoglobin or blood processing. They have been found at high levels within the central nervous system where they have a direct effect on neuronal cells via peptidergic receptors. As relatively few studies have examined their metabolic stability in the brain, such investigation was performed to locate the cellular distribution of enzymatic activity against these peptides. High-performance liquid chromatography (HPLC) combined with electrospray ionisation mass spectrometry (ESI-MS) allows identification of degradation products resulting from incubation of hemorphin-7 peptides (LVV-hemorphin-7, VV-hemorphin-7 and hemorphin-7) with subcellular fractions isolated from rat brain tissue. Metabolic activities were found against the three peptides in brain homogenate and subcellular fractions with the highest metabolic activity (<3% peptide remaining after 10 min) observed in the microsomal fraction which processed hemorphin-7 peptides mainly into N-terminal fragments (giving LVVH5) suggesting action of brain-membrane enzymes with C-terminal specificity. Incubation of the ACE inhibitor captopril (0.2 μM) with microsomal fraction, together with LVVH7, decreased the processing of LVVH7 to form LVVH5 by 85%.     

Soil carbon fractions in grasslands respond differently to various levels of nitrogen enrichments

Background and Aims

Soil contains many different C fractions which have diverse physical and chemical compositions. Examining these differential soil C fractions in response to N enrichment is helpful for better understanding soil C changes under the predominantly increasing N deposition. In this study, we used a field N addition experiment in a grassland to explore the effects of various N enrichment levels on soil C fractions.

Methods

We conducted a field manipulative experiment which used a Latin square design with six N addition levels of 0, 2, 4, 8, 16 and 32 g N m^?2 year^?1 since 2003 in a semiarid grassland in northern China. Soil samples were collected in August (when plants have the greatest biomass), 2011. We measured C and N concentrations in soil light fraction, microbial biomass, extractable organic matter, heavy fraction, and total soil C and N.

Results

The results showed that total soil C and N, and heavy fraction C and N were not significantly affected by N addition after 9 years of treatments. In contrast, different N enrichment levels changed soil light fraction C and N, ranging from 4.3 to 27.7 % and 3.3–30.0 %, respectively. Moreover, both light fraction C and N had a nonlinear relationship with N addition rates, and the threshold for N-induced change in light fraction C and N was near 16 g N m^?2 year^?1 in this semiarid grassland. Increases of soil light fraction C and N primarily resulted from changes in biotic (N-stimulated aboveground biomass) and abiotic (soil temperature, moisture and pH) factors under N enrichment. Soil microbial biomass exponentially declined with increasing N, but extractable organic C showed a positive linear response to N enrichment rates. Changes in microbial biomass C and extractable organic C were primarily due to the reduced soil pH under N addition.

Conclusions

Our findings suggest that various soil C fractions differentially respond to elevated N, because different sets of biotic and abiotic factors regulate those fractions under N enrichment.     

Time-varying effects in mice and rats of several synthetic ACTH preparations

Circadian stage-dependent effects characterize synthetic ACTH 1-17 preparation (HOE 433 = Synchrodyn 1-17), tested in mice and rats, with reference notably to corticosterone and aldosterone production in vitro and to the behavior of rhythms in these two corticoids as an aspect of the adrenal cortical pacemaker of the circadian system. The possibility to advance or delay the rhythm in serum corticosterone by ACTH 1-17 also is demonstrated, as is a differential behavior of the circadian rhythm in serum aldosterone. Differences in timing of circadian corticosterone and aldosterone responses also are described and await further scrutiny for ultradian and infradian (notably circannual) modulation.