Effect of olive leaves feeding on phenolic composition and lipolytic volatile profile in goat milk.

The aim of this study was to evaluate phenolic composition, antioxidant potential, and lipolytic events in raw milk obtained from goat fed a dietary supplementation with olive leaves (OL), a by-product of the olive oil production chain. For this purpose, 30 Saanen goats were randomly allocated into 2 groups of 15 goats each: the control group received a standard diet that was prepared by taking into account the nutritional needs of lactating goats, whereas the experimental group (EG) was fed with an OL-supplemented diet (10% on a dry matter basis). At the end of the 30 d of the trial, the individual milk samples were collected and immediately analyzed for total phenolic content and antioxidant activity (AOA). Subsequently, the individual phenolic compounds have been identified and quantified through an ultra-high-performance liquid chromatography system and a characterization of free fatty acids released in milk has been performed. The results showed a positive effect of dietary OL supplementation in improving total phenolic content and AOA; furthermore, 19 phenolic compounds, including phenolic acids, flavonoids, simple phenols, and secoiridoids, have been identified in EG milk. In addition to this, a reduced accumulation of free fatty acids has been found in EG milk, and this finding leads us to hypothesize an inhibitory action of the identified phenolic compounds toward the enzymes responsible for lipolytic events. The use of the molecular docking approach verified the interactions, defining a fairly interesting framework for cinnamic acid, which should be able to noncovalently bind these enzymes, interfering with the recruitment of the substrate and therefore, slowing down their hydrolytic activity. In any case, this information will be subjected to in vitro evaluations for an accurate characterization of the biochemical mechanisms that can be established in milk naturally enriched with bioactive compounds.

The binding of human serum transferrin to its specific receptor reconstituted into liposomes.

Human placental transferrin receptor (HPTR), purified following a procedure based on affinity chromatography step, was reconstituted by the detergent dialysis method into various kinds of phosphatidylcholine vesicles and the receptor ability to bind 125I-labelled human serum transferrin (HST) was then evaluated. In our experimental conditions, the binding of the labelled protein to its specific receptor showed several features, in particular: (1) in cholesterol/1-alpha-dipalmitoylphosphatidyl choline (CHO/DPPC) liposomes, a positive cooperatively of the transferrin binding resulted at the lowest cholesterol/phospholipids (C/P) ratio; 1-alpha-dioleylphosphatidyl choline (DOPC) and phosphatidic acid (PA) containing liposomes showed an opposite binding curve trend; (2) the apparent dissociation constant (K'd) did not change significantly as a function of the lipid composition, being always around 1.00 x 10(-6) M; (3) the encapsulation capacity of liposomes decreased from 27% to about 13% with increasing amounts of cholesterol and was around 20% in the presence of DOPC or PA; about 8-13% of this receptor was found to be functional; (4) receptor-loaded liposomes treated with polyclonal anti-HPTR rabbit antibodies showed a remarkable binding decrease for transferin. All these results seem to point out the crucial role played by the environment in the binding behaviour of the transferrin receptor.

Structural analysis of seminal and serum human transferrin by second derivative spectrometry and fluorescence measurements.

Denaturation of human seminal transferrin (HSmT) compared with human serum transferrin (HSrT) was followed to check structural differences between these two proteins. Second derivative UV spectroscopy indicated that treatment with 6 M guanidine hydrochloride (Gnd.HCl) induced greater structural changes in HSrT than in HSmT and, in particular; (i) the exposure value of tyrosinyl residues was almost 2.5-fold higher in native HSmT than in native HSrT; and (ii) a much more pronounced movement of tryptophanyl residues toward a higher polar environment could be noticed in HSrT after incubation with denaturing agent. Fluorescence measurements showed that: (i) a shift of the maximum emission wavelength of HSmT occurred (maximum emission was centered at 333 nm instead of 323 nm as for HSrT; excitation = 280 nm); (ii) the intrinsic tryptophan fluorescence intensity of HSmT increased after 36 hr in the range of 1.5-4.0 M of denaturant, whereas an opposite behavior was found for HSrT in the range 0.0-2.0 M; and (iii) the wavelength maximum of fluorescence emission changed in a biphasic manner for HSrT and, conversely, under the same experimental conditions, HSmT gave a linear and parallel increase of fluorescence emission after 1 and 36 hr. We can conclude that this different behavior of HSmT with respect to HSrT might be due mainly to the fact that both the number and the exposure of tyrosinyl and tryptophanyl residues are different. Lately, these effects are discussed in relationship with the fact that HSmT contains less than half disulphide bridges than HSrT.

PPIX induced photohemolysis of erythrocytes partially-depleted of cholesterol.

The protoporphyrin IX (PPIX)-sensitized hemolysis of erythrocytes depleted of cholesterol was investigated. From 20% to 30% of the total membrane cholesterol was removed from cells by incubation with old autologous plasma or by means of interaction with L-alpha-phosphatidylcholine dipalmitoyl (DPPC) liposomes. As expected, after this treatment, the cells show an overall increase in membrane fluidity revealed by means of specific fluorescent probes. The same cells are more susceptible to the photohemolysis induced by PPIX excited by visible light, but gave no lysis in the absence of the sensitizer. As a consequence, the primary oxidative damage which is produced during irradiation can be possibly assigned to the phospholipidic and/or proteic moiety instead of the steroidal moiety.

The ultraviolet derivative spectrophotometric determination of neutral liposome-entrapped beta-lactam antibiotics.

A simple, non-invasive spectrophotometric assay to measure the concentration of some beta-lactam antibiotics in turbid solutions containing liposomes was carried out. Since zero-order spectra gave strong interference because of sample turbidity, derivative spectrophotometry was used to enhance the spectral details. Derivative spectra showed bands in the ultraviolet region due to the presence of the cephalosporin and penicillin beta-lactams. A linear relationship between derivative amplitudes and antibiotic concentration was found when antibiotic-containing liposome solutions were measured. A saturative trend in the liposome-encapsulation was observed. The antibiotic entrapment was lowered by increasing the cholesterol-phospholipid ratio in the mixture used for liposome preparation. After treatment of antibiotic-loaded liposomes with beta-lactamase, a hydrolytic enzyme specific for beta-lactams, the remaining antibiotic concentration decreased significantly, showing that some of the antibiotic was retained on the outer surface of the vesicles.