Ajda Ota

Effect of flavonoid structure on the fluidity of model lipid membranes.

We investigated how the structural properties of (+)-catechin, (-)-epicatechin, (-)-epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG) and butylated hydroxytoluene (BHT) correlate with structural changes of phosphatidylcholine plus sphingomyelin (2.4:1) model lipid membranes. Changes were measured by fluorescence anisotropy, electron paramagnetic resonance, and differential scanning calorimetry. Two fluorophores and two spin probes were used to monitor membrane characteristics close to water-lipid interface and in the middle of the bilayer. The data obtained were correlated to the amount of bounded compounds, the number of H-bonds, and the topological polar surface area (TPSA) of the compounds. These correlations reflect the behaviours of (+)-catechin, (-)-epicatechin, EGC, EGCG and BHT. Our results confirm that phenolics studied here are bounded to a membrane surface predominantly via hydrogen bonds, while BTH is inserted into the lipid bilayer.

Stability of diether C(25,25) liposomes from the hyperthermophilic archaeon Aeropyrum pernix K1.

Temperature and pH effects were studied for stability, structural organization, fluidity and permeability of vesicles from a polar lipid methanol fraction isolated from the Aeropyrum pernix. We determined the permeability of C(25,25) liposomes using fluorescence intensity of released calcein. At pH 7.0 and 9.0, and from 85°C to 98°C, only 10% of entrapped calcein was released. After 10 h at 90°C, calcein release reached 27%, independent of pH. Fluorescence anisotropy measurements of hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene revealed gradual changes up to 60°C. At higher temperatures, the anisotropy did not change significantly. Fluorescence alone did not provide detailed and direct structural information about these C(25,25) liposomes, so we used electron paramagnetic resonance spectroscopy (EPR) and differential scanning calorimetry (DSC). From EPR spectra, mean membrane fluidity determined according to maximal hyperfine splitting and empirical correlation times showed continuous increases with temperature. Computer simulation of EPR spectra showed heterogeneous membranes of these C(25,25) liposomes: at low temperatures, they showed three types of membrane regions characterized by different motional modes. Above 65°C, the membrane becomes homogeneous with only one fluid-like region. DSC thermograms of C(25,25) liposomes reveal a very broad and endothermic transition in the temperature range from 0°C to 40°C.

An Overview of Herbal Products and Secondary Metabolites Used for Management of Type Two Diabetes

Diabetes mellitus is a common effect of uncontrolled high blood sugar and it is associated with long-term damage, dysfunction, and failure of various organs. In the adult population, the global prevalence of diabetes has nearly doubled since 1980. Without effective prevention and management programs, the continuing significant rise in diabetes will have grave consequences on the health and lifespan of the world population, and also on the world economy. Supplements can be used to correct nutritional deficiencies or to maintain an adequate intake of certain nutrients. These are often used as treatments for diabetes, sometimes because they have lower costs, or are more accessible or “natural” compared to prescribed medications. Several vitamins, minerals, botanicals, and secondary metabolites have been reported to elicit beneficial effects in hypoglycemic actions in vivo and in vitro; however, the data remain conflicting. Many pharmaceuticals commonly used today are structurally derived from natural compounds from traditional medicinal plants. Botanicals that are most frequently used to help manage blood glucose include: bitter melon (Momordica charantia), fenugreek (Trigonella foenum graecum), gurmar (Gymnema sylvestre), ivy gourd (Coccinia indica), nopal (Opuntia spp.), ginseng, Russian tarragon (Artemisia dracunculus), cinnamon (Cinnamomum cassia), psyllium (Plantago ovata), and garlic (Allium sativum). In majority of the herbal products and secondary metabolites used in treating diabetes, the mechanisms of action involve regulation of insulin signaling pathways, translocation of GLUT-4 receptor and/or activation the PPARγ. Several flavonoids inhibit glucose absorption by inhibiting intestinal α-amylase and α-glucosidase. In-depth studies to validate the efficacies and safeties of extracts of these traditional medicinal plants are needed, and large, well designed, clinical studies need to be carried out before the use of such preparations can be recommended for treatment and/or prevention of diabetes. The main focus of this review is to describe what we know to date of the active compounds in these, along with their glucose-lowering mechanisms, which are either through insulin-mimicking activity or enhanced glucose uptake.

Archaeosomes can efficiently deliver different types of cargo into epithelial cells grown in vitro

Archaeosomes are a type of liposomes prepared from the polar lipids of various Archaeobacteria. These have unique structural features that increase the lipid bilayers stability even under high temperatures, low or high pH, presence of phospholipases and bile salts. This makes them ideal as basis for the development of new drug, gene and vaccine delivery systems. In this study we prepared large unilamellar archaeosomes (400nm size) from Aeropyrum pernix K1 and demonstrated their potential as base for the development of an efficient and universal system for drug or therapy delivery to epithelial cells. Our archaeosomes may be used to deliver small fluorescent molecules (calcein), smaller proteins (60kDa listeriolysin), large protein aggregates (e.g. keratin 14) and plasmid DNA, into epithelial cells grown in culture. The delivery efficiency for small molecules is already quite high at this initial stage of development, around 40%. Our unilamellar archaeosomes are also not toxic to keratinocytes even at high doses (500μg/ml).

Morphology, biophysical properties and protein-mediated fusion of archaeosomes.

As variance from standard phospholipids of eubacteria and eukaryotes, archaebacterial diether phospholipids contain branched alcohol chains (phytanol) linked to glycerol exclusively with ether bonds. Giant vesicles (GVs) constituted of different species of archaebacterial diether phospholipids and glycolipids (archaeosomes) were prepared by electroformation and observed under a phase contrast and/or fluorescence microscope. Archaebacterial lipids and different mixtures of archaebacterial and standard lipids formed GVs which were analysed for size, yield and ability to adhere to each other due to the mediating effects of certain plasma proteins. GVs constituted of different proportions of archaeal or standard phosphatidylcholine were compared. In nonarchaebacterial GVs (in form of multilamellar lipid vesicles, MLVs) the main transition was detected at Tm = 34. 2°C with an enthalpy of ΔH = 0.68 kcal/mol, whereas in archaebacterial GVs (MLVs) we did not observe the main phase transition in the range between 10 and 70°C. GVs constituted of archaebacterial lipids were subject to attractive interaction mediated by beta 2 glycoprotein I and by heparin. The adhesion constant of beta 2 glycoprotein I – mediated adhesion determined from adhesion angle between adhered GVs was in the range of 10−8 J/m2. In the course of protein mediated adhesion, lateral segregation of the membrane components and presence of thin tubular membranous structures were observed. The ability of archaebacterial diether lipids to combine with standard lipids in bilayers and their compatibility with adhesion-mediating molecules offer further evidence that archaebacterial lipids are appropriate for the design of drug carriers.

Effect of Growth Medium pH of Aeropyrum pernix on Structural Properties and Fluidity of Archaeosomes

The influence of pH (6.0; 7.0; 8.0) of the growth medium of Aeropyrum pernix K1 on the structural organization and fluidity of archaeosomes prepared from a polar-lipid methanol fraction (PLMF) was investigated using fluorescence anisotropy and electron paramagnetic resonance (EPR) spectroscopy. Fluorescence anisotropy of the lipophilic fluorofore 1,6-diphenyl-1,3,5-hexatriene and empirical correlation time of the spin probe methylester of 5-doxylpalmitate revealed gradual changes with increasing temperature for the pH. A similar effect has been observed by using the trimethylammonium-6-diphenyl-1,3,5-hexatriene, although the temperature changes were much smaller. As the fluorescence steady-state anisotropy and the empirical correlation time obtained directly from the EPR spectra alone did not provide detailed structural information, the EPR spectra were analysed by computer simulation. This analysis showed that the archaeosome membranes are heterogeneous and composed of several regions with different modes of spin-probe motion at temperatures below 70°C. At higher temperatures, these membranes become more homogeneous and can be described by only one spectral component. Both methods indicate that the pH of the growth medium of A. pernix does not significantly influence its average membrane fluidity. These results are in accordance with TLC analysis of isolated lipids, which show no significant differences between PLMF isolated from A. pernix grown in medium with different pH.

Nutritional, antioxidative, and antimicrobial analysis of the Mediterranean hackberry (Celtis australis L.)

Abstract Celtis australis is a deciduous tree commonly known as Mediterranean hackberry or the European nettle tree. The fruit of hackberry are seldom used for nutritional purposes. The nutritional and physicochemical properties of ripe hackberry fruit from Istria (Marasi village near Vrsar, Croatia) were determined, including water, total fiber, protein, vitamin, mineral, and phenolic contents. This analysis demonstrates that the hackberry fruit is a valuable source of dietary fiber, protein, and vitamins, and of pigments such as lutein, β‐carotene, zeaxanthin, and tocopherols. The seasonal differences associated with the different growth stages for the element composition, total phenolic content, and phenolic profile were also determined for hackberry mesocarp and leaves. Water and ethanol extracts were prepared from mesocarp and leaves harvested at different growth stages and their phenolic profiles and antioxidant and antimicrobial activities were investigated. This study demonstrates that water and ethanol extracts of hackberry fruit and leaves collected at different growth stages contain epicatechin, gallic acid, vanillic acid, 3,4‐dihydroxybenzaldehyde, delphinidin‐3,5‐di‐O‐glucoside, cyanidin‐3,5‐di‐O‐glucoside, and pelargonidin‐3,5‐di‐O‐glucoside. They also show some antimicrobial and antifungal activities. Further studies are needed to identify and define the active ingredients of these hackberry leaf ethanol extracts.

Biological potential of nanomaterials strongly depends on the suspension media: experimental data on the effects of fullerene C60 on membranes

Fullerenes (C60) are some of the most promising carbon nanomaterials to be used for medical applications as drug delivery agents. Computational and experimental studies have proposed their ability to enter cells by penetrating lipid bilayers. The aim of our study was to provide experimental evidence on whether pristine C60 in physiological media could penetrate cell membranes. The effect was tested on phospholipid vesicles (liposomes) composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and validated on isolated human red blood cells (RBCs). We incubated the liposomes in an aqueous suspension of C60 and dissolved the lipids and C60 together in chloroform and subsequently formatted the liposomes. By differential scanning calorimetry measurements, we assessed the effect of C60 on the phospholipid thermal profile. The latter was not affected after the incubation of liposomes in the C60 suspension; also, a shape transformation of RBCs did not occur. Differently, by dispersing both C60 and the phospholipids in chloroform, we confirmed the possible interaction of C60 with the bilayer. We provide experimental data suggesting that the suspension medium is an important factor in determining the C60-membrane interaction, which is not always included in computational studies. Since the primary particle size is not the only crucial parameter in C60-membrane interactions, it is important to determine the most relevant characteristics of their effects on membranes.

Genetic, Biochemical, Nutritional and Antimicrobial Characteristics of Pomegranate (Punica granatum L.) Grown in Istria

This study characterises the genetic variability of local pomegranate (Punica granatum L.) germplasm from the Slovenian and Croatian areas of Istria. The bioactive components and antioxidant and antimicrobial properties of ethanol and water extracts of different parts of pomegranate fruit were also determined, along with their preliminary nutritional characterisation. Twenty-six different genotypes identified with microsatellite analysis indicate the great diversity of pomegranate in Istria. The pomegranate fruit ethanol extracts represent rich sources of phenolic compounds (mean value of the mass fraction in exocarp and mesocarp expressed as gallic acid is 23 and 16 mg/g, respectively). The ethanol extracts of pomegranate exocarp and mesocarp showed the greatest antimicrobial activity against Candida albicans, Candida parapsilosis, Rhodotorula mucilaginosa, Exophiala dermatitidis and Staphylococcus aureus, and the same water extracts against S. aureus and Escherichia coli. To the best of our knowledge, this study represents the first report of the characterisation of pomegranate genetic resources from Istria at different levels, including the molecular, chemical, antimicrobial and nutritional properties.

Impact of Carrier Systems on the Interactions of Coenzyme Q10 with Model Lipid Membranes

We investigated the influence of carrier systems for different commercially available water-soluble formulations for coenzyme Q10 on structural changes of model lipid membranes formed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and by a mixture of phosphatidylcholine and sphingomyelin (2.4:1). Structural changes in the membranes were measured using fluorescence anisotropy, electron paramagnetic resonance, and differential scanning calorimetry. Two fluorophores and two spin probes were used to monitor membrane characteristics close to the water-lipid interface and in the middle of the bilayer of the model lipid membranes. Different water-soluble carrier systems were tested. These data show that different systems can facilitate penetration of CoQ10 in the lipid membranes, where an increase in the lipid order parameter was observed. In addition, water soluble CoQ10 formulations better protect lipids from oxidation in liposome solution. With the exception of the carriers in an emulsified formulation of CoQ10, those in the other samples did not have any significant effects on membrane fluidity.