Judit Remenyik

Chemoenzymatic synthesis of 2-chloro-4-nitrophenyl β-maltoheptaoside acceptor-products using glycogen phosphorylase b

In the present work, we aimed at developing a chemoenzymatic procedure for the synthesis of beta-maltooligosaccharide glycosides. The primer in the enzymatic reaction was 2-chloro-4-nitrophenyl beta-maltoheptaoside (G(7)-CNP), synthesised from beta-cyclodextrin using a convenient chemical method. CNP-maltooligosaccharides of longer chain length, in the range of DP 8-11, were obtained by a transglycosylation reaction using alpha-D-glucopyranosyl-phosphate (G-1-P) as a donor. Detailed enzymological studies revealed that the conversion of G(7)-CNP catalysed by rabbit skeletal muscle glycogen phosphorylase b (EC 2.4.1.1) could be controlled by acarbose and was highly dependent on the conditions of transglycosylation. More than 90% conversion of G(7)-CNP was achieved through a 10:1 donor-acceptor ratio. Tranglycosylation at 37 degrees C for 30 min with 10 U enzyme resulted in G(8-->12)-CNP oligomers in the ratio of 22.8, 26.6, 23.2, 16.5, and 6.8%, respectively. The reaction pattern was investigated using an HPLC system. The preparative scale isolation of G(8-->11)-CNP glycosides was achieved on a semipreparative HPLC column. The productivity of the synthesis was improved by yields up to 70-75%. The structures of the oligomers were confirmed by their chromatographic behaviours and MALDI-TOF MS data.

Study of the defensive mechanism against drought in French bean (Phaseolus vulgaris L.) varieties

The response to drought of six green- and yellow-podded varieties of green bean was investigated in a phytotron. These varieties were also grown without irrigation in field experiments and analysis of leaf samples was carried out three times; before and under flowering and pod ripening, respectively. At first, the carotene and raffinose contents of leaves increase during mild drought (30/15°C). High temperature (35/25°C) combined with water deficiency resulted in a considerable decrease in leaf weight, chlorophyll b, maltose content, as well as the level of water-soluble antioxidants of the leaves, while the content of lipid-soluble antioxidants and raffinose content increased when compared with the control. The yellow-podded varieties of green beans responded to drought much more sensitively than the green-podded ones. The results suggested that a selection method based on the activity of antioxidants in the leaves can be used for testing the adaptability of numerous bean genotypes to drought.

Fenugreek (Trigonella Foenum-Graecum) Seed Flour and Diosgenin Preserve Endothelium-Dependent Arterial Relaxation in a Rat Model of Early-Stage Metabolic Syndrome

Fenugreek is a common herb possessing several bioactive components including diosgenin. Here, dietary fenugreek seed flour and diosgenin were evaluated on a model of endothelium-dependent vasorelaxation by abdominal aortas isolated from rats receiving high-fat, high-sugar diet (HFHSD). 60 male Wistar rats were randomized into six groups: (i) negative control getting conventional rat feed regimen; (ii) positive control receiving HFHSD; (iii) a test group fed 2 g/kg bw/day fenugreek seed flour (containing 10 mg/kg bw/day diosgenin) + HFHSD; (iv) three test groups fed 1, 10 and 50 mg/kg bw/day diosgenin + HFHSD. Alimentary treatments were carried out for six weeks. The abdominal aortas were isolated, and 2 mm wide rings were sectioned off and mounted at a resting tension of 10 mN in organ baths containing Krebs solution (36 °C) exposed to 95% O2 and 5% CO2. After 60-min incubation, a norepinephrine concentration-response (E/c) curve was generated to determine their half-maximal effective concentration (EC50) value. After 60-min wash-out, a pre-contraction with norepinephrine EC50 was made, followed by an acetylcholine E/c curve. Plasma glutathione levels, glutathione-handling enzyme activities and blood antioxidant capacities were also determined. HFHSD significantly decreased the dilatory response to acetylcholine and increased plasma glutathione levels and these effects were significantly reversed by fenugreek seed flour, 10 and 50 mg/kg bw/day diosgenin. Both fenugreek and diosgenin treatments prevent HFHSD-induced endothelial dysfunction and redox changes. As fenugreek treatment was more effective at lower acetylcholine concentrations than diosgenin treatments, components of fenugreek other than diosgenin may contribute to the beneficial effects of dietary fenugreek seed flour.

Protective Effect of Prunus Cerasus (Sour Cherry) Seed Extract on the Recovery of Ischemia/Reperfusion-Induced Retinal Damage in Zucker Diabetic Fatty Rat

Among diabetes patients, ophthalmological complications are very frequent. High blood glucose and (consequential) ischemia-reperfusion (I/R) injury contribute significantly to the severity of retinopathies. Diabetic retinopathy is among the leading causes of blindness. Our study demonstrates the effect of sour cherry seed extract (SCSE) on blood glucose and function of the retina with electroretinography (ERG) in a diabetic setting with or without ischemia-reperfusion (I/R) injury in Zucker Diabetic Fatty (ZDF) rats. Our results prove that the SCSE has a retinoprotective effect in diabetic rats: according to ERG measurements, SCSE treatment mitigated the retinal function-damaging effect of diabetes, and proved to be protective in the diabetic eye against ischemia-reperfusion injuries of the retina. Outcomes suggest that the protective effects of SCSE may occur through several pathways, including HO-1 dependent mechanisms. The observation that SCSE treatment decreases blood glucose is also novel. These findings offer the possibility for development of novel therapeutic strategies utilizing this emerging functional food, in particular in the prevention of conditions resulting from high blood glucose or I/R injury, such as deterioration of retinal microcirculation.

Erratum: Action pattern and subsite mapping of Bacillus licheniformis α-amylase (BLA) with modified maltooligosaccharide substrates (FEBS LETTERS (2002) 518 (79-82) PII: S0014579302026492)

0014-5793 / 02 /

Protective Effect of Pure Sour Cherry Anthocyanin Extract on Cytokine-Induced Inflammatory Caco-2 Monolayers

22.00 = 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 1 4 5 7 9 3 ( 0 2 ) 0 2 7 6 8 0 Fig. 2. Action pattern of BLA with modi¢ed maltooligosaccharides at 50‡C. G, glucosyl residues; NP, 4-nitrophenyl groups; CNP, 2chloro-4-nitrophenyl groups (NP and CNP are connected to the reducing end in L-form); Bnl groups (connected to the non-reducing end as Bnl acetals); DP, degree of polymerisation. Numbers above the arrows represent percentages of cleavage frequencies of glycosidic bonds. Bold numbers are experimental data, the bigger ones indicate the most favourable positions of cleavages. Normal numbers are related to the calculated data based on the optimised subsite map.

Anthocyanin composition, antioxidant efficiency, and α-amylase inhibitor activity of different Hungarian sour cherry varieties (Prunus cerasus L.)

Anthocyanins have several beneficial effects, especially on inflammatory and oxidative conditions. The pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), induce damage in the intestinal barrier and participate in the pathogenesis of chronic bowel diseases. A number of fruits have high anthocyanin contents with strong biological activity which can support protective actions. Sour cherry (Prunus cerassus) is one of the richest fruits in anthocyanins; especially it has high content of cyanidins. The aim of this study was to test the biological effects of a pure sour cherry anthocyanin extract under inflammatory conditions on the intestinal barrier. Caco-2 monolayers were stimulated with 50 ng/mL TNF-α and 25 ng/mL IL-1β, and the protective effects of the anthocyanin extract were examined. We demonstrated the safety of 500, 50, 5 and 0.5 µM anthocyanin extracts through cell impedance measurements. The 50 µM anthocyanin extract inhibited the cytokine-induced Caco-2 permeability and the nuclear translocation of NF-κB p65 subunits. The extract significantly reduced the release of IL-6 and IL-8 production in intestinal cells and glutathione peroxidase activity stimulated by cytokines. We demonstrated, for the first time, the beneficial effects of pure sour cherry anthocyanin extract on inflammatory Caco-2 monolayers, indicating that this substance could be protective in inflammatory bowel diseases and is an excellent raw material for further applications and formulations.