Dietary supplementation with green tea extract improves the antioxidant status and oocyte developmental competence in Italian Mediterranean buffaloes.

The aim of this work was to assess the antioxidant status and the developmental competence of oocytes recovered by ovum pick-up (OPU) in Italian Mediterranean buffaloes supplemented with green tea extracts (GTE) for 90 days. Buffalo cows (n = 16) were randomly assigned to a control group receiving no supplement and a treatment group, receiving GTE starting 90 days before OPU, carried out for five consecutive sessions. Blood samples were collected before the start of supplementation with GTE (T0) and at day 45 (T1) and day 90 (T2) of supplementation, to measure ferric reducing activity (FRAP), total antioxidant capacity (TAC), superoxide dismutase (SOD) and catalase (CAT). The antioxidant status of follicles was measured as TAC on the follicular fluid collected from the dominant follicle just prior OPU, coinciding with T2, and at the end of five repeated OPU sessions (T3). Another objective was to assess in vitro the protective effects of green tea extracts on hepatic cells exposed to methanol insult. Different concentrations of GTE (0.5 µM and 1 µM) were tested on cultured hepatic cells and viability, morphology and SOD activity were assessed at 24, 48 and 72 h. Supplementation with GTE increased (P < 0.05) the number of total follicles (8.7 ± 0.5 vs 6.9 ± 0.5), the number and the percentage of Grade A + B cumulus-oocyte complexes (COCs) compared with the control (3.7 ± 0.4 vs 2.3 ± 0.3 and 57.5 ± 4.2 vs 40.4 ± 4.9 %, respectively). Oocyte developmental competence was improved in the GTE group as indicated by the higher (P < 0.05) percentages of Grade 1,2 blastocysts (44.8 vs 29.1 %). In the GTE group, plasma TAC was higher both at T1 and T2, while FRAP increased only at T2, with no differences in SOD and CAT. The TAC of follicular fluid was higher (P < 0.05) in the GTE compared to the control both at T2 and at T3 The in vitro experiment showed that co-treatment with methanol and 1 µM GTE increased (p < 0.01) cell viability at 24 h (P < 0.01), 48 h (P < 0.05) and 72 h (P < 0.01) compared with the methanol treatment co-treatment with 1 µM GTE prevented the decrease in SOD activity observed with methanol at 24 and 48 h of culture. In conclusion, the results of in vivo and in vitro experiments suggest that supplementation with GTE increases buffalo oocyte developmental competence, by improving oxidative status and liver function.

Impact of obesity and nitric oxide synthase gene G894T polymorphism on essential hypertension.

Hypertension is a multifactorial disease caused by environmental, metabolic and genetic factors, but little is currently known on the complex interplay between these factors and blood pressure. The aim of the present study was to assess the potential impact of obesity, and angiotensin-converting enzyme (ACE) I/D polymorphism and endothelial nitric oxide synthase gene (NOS3) 4a/4b, G894T and -T786C variants on the essential hypertension. The study group consisted of 1,027 Caucasian adults of Polish nationality (45.5 ± 13.6 years old), of which 401 met the criteria for hypertension. Body weight, height and blood pressure were measured and data on self-reported smoking status were collected. Fasting blood glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides were determined by standard procedures. The ACE I/D polymorphism and three polymorphisms in NOS3 gene (4a/4b, G894T, -T786C) were detected by the PCR method. Multivariable logistic regression demonstrated that age above 45 years, diabetes, dyslipidemia, smoking and male sex are important risk factors for hypertension and no significant influence of variants in ACE and NOS3 genes on this risk was recognized. Obese subjects had a 3.27-times higher risk (OR = 3.27, 95% CI: 2.37 - 4.52) of hypertension than non-obese, and in obese the NOS3 894T allele was associated with 1.37 fold higher risk of hypertension (P = 0.031). The distribution of NOS3 G894T genotypes supported the co-dominant (OR = 1.35, P = 0.034, Pfit = 0.435) or recessive (OR = 2.00, P = 0.046, Pfit = 0.286), but not dominant model of inheritance (P = 0.100). The study indicates that in obese NOS3 G894T polymorphism may enhance hypertension risk. However, in the presence of such strong risk factors as age, diabetes and smoking, the impact of this genetic variant seems to be attenuated. Further studies are needed to reveal the usefulness of G894T polymorphism in hypertension risk assessment in obese.

Voltammetric determination of betulinic acid at lead film electrode after chromatographic separation in plant material.

The construction and application of a novel electrochemical detection system with an in situ plated lead film electrode for high-performance liquid chromatography (HPLC) is described. The working electrode of interest was tested as a potential sensor for the adsorptive stripping voltammetric determination of betulinic acid (BA). The high sensitivity of the proposed electrochemical detection results from the accumulation by adsorption of BA on the lead film surface before the proper electrode process. In a solution of sodium hydroxide, used as a supporting electrolyte for the proposed voltammetric method, the oxidation signal for BA was found to be proportional to the BA concentration in the range from 0.02 to 0.5 µg L(-1) with a limit of detection equal to 0.009 µg L(-1) (with preconcentration for 15 s). The voltammetric detection was successfully applied to the determination of BA in the birch bark (Betula verrucosa) extracts after HPLC separation. The content of BA obtained by the proposed method was in close agreement with that obtained by HPLC coupled with photodiode array detector (HPLC-PAD). This appears to be the first application of electrochemical detection to the determination of pentacyclic triterpene.

Nociceptin and its natural and specifically-modified fragments: Structural studies.

The vibrational structures of Nociceptin (FQ), its short bioactive fragments, and specifically-modified [Tyr¹]FQ (1-6), [His¹]FQ (1-6), and [His(1,4)]FQ (1-6) fragments were characterized. We showed that in the solid state, all of the aforementioned peptides except FQ adopt mainly turn and disordered secondary structures with a small contribution from an antiparallel ß-sheet conformation. FQ (1-11), FQ (7-17) [His¹]FQ (1-6), and [His(1,4)]FQ (1-6) have an α-helical backbone arrangement that could also slightly influence their secondary structure. The adsorption behavior of these peptides on a colloidal silver surface in an aqueous solution (pH = ∼8.3) was investigated by means of surface-enhanced Raman scattering (SERS). All of the peptides, excluding FQ (7-17), chemisorbed on the colloidal silver surfaces through a Phe4 residue, which for FQ, FQ (1-11), FQ (1-6), [Tyr¹]FQ (1-6), and [His¹]FQ (1-6) lies almost flat on this surface, while for FQ (1-13) and FQ (1-13)NH2 adopts a slightly tilted orientation with respect to the surface. The Tyr¹ residue in [Tyr¹]FQ (1-6) does not interact with the colloidal silver surface, suggesting that the Tyr¹ and Phe4 side chains are located on the opposite sides of the peptide backbone, which can be also true for His¹ and Phe4 in [His¹]FQ (1-6). The lone pair of electrons on the oxygen atom of the ionized carbonyl group of FQ (1-13) and FQ (7-17) appears to be coordinated to the colloidal silver nanoparticles, whereas in the case of the remaining peptides, it only assists in the adsorption process, similar to the --NH4 group. We also showed that upon adsorption, the secondary structure of these peptides is altered.

The influence of Calendulae officinalis flos extracts on cell cultures, and the chromatographic analysis of extracts.

Three extracts of Calendulae officinalis flos (Asteraceae): heptane, ethyl acetate and methanol were introduced to a human skin fibroblast (HSF) cells culture and a culture of human breast cancer cells (T47D), cell culture collection ECACC number 85102201. The ethyl acetate but not the heptane and methanol extracts in concentrations above 25 microg/mL, can stimulate cell proliferation and cellular metabolism by increase of mitochondrial dehydrogenase activity. However, concentrations exceeding 75 microg/mL are toxic for cells. The second part of the study concerned elaborating of optimal chromatographic systems for quantitative analysis of these extracts by the use of HPTLC with densitometry. Oleanolic acid, beta-amyrin, beta-amyrin acetate, rutin, narcissin, 3-glucoside of isorhamnetin, quercetin, isoquercitrin, vanillic acid, caffeic acid, chlorogenic acid, protokatechuic acid, p-coumaric acid and syringic acid were all identified.

Asymmetric induction in the 1,3-dipolar cycloaddition of chiral nitrile oxide derived from (2R)-bornane-10,2-sultam.

The efficient preparation of the chiral nitrile oxide derived from N-glyoxyloyl-(2R)-bornane-10,2-sultam is presented. The nitrile oxide was trapped in situ with substituted olefins as dipolarophiles to furnish optically active 2-isoxazolines.