Healthberry 865® and a Subset of Its Single Anthocyanins Attenuate Oxidative Stress in Human Endothelial In Vitro Models.

Oxidative stress and inflammation play a pivotal role in the development of cardiovascular diseases, an ever-growing worldwide problem. As a non-pharmacological approach, diet, especially a flavonoid-rich diet, showed promising results in the reduction of cardiovascular diseases and alleviation of their symptoms. In this study, in vitro systems based on human microvascular endothelial cells (hmvEC) and human umbilical cord endothelial cells (HUVEC) were established to determine the effect of Healthberry 865® (HB) and ten of its relating single anthocyanins on oxidative stress. Furthermore, five metabolites were used in order to examine the effect of anthocyanin's most common breakdown molecules. The results showed an effect of HB in both models after 24 h, as well as most of its single anthocyanins. Cyanidin-rutinoside, peonidin-galactoside, and petunidin-glucoside had a model-specific effect. For the metabolites, phloroglucinaldeyhde (PGA) showed an effect in both models, while vanillic acid (VA) only had an effect in HUVEC. When combined, a combination of several anthocyanins did not have a cumulative effect, except for combining glucosides in hmvEC. The combination of PGA and VA even revealed an inhibitive behavior. Overall, the study demonstrates the antioxidative effect of HB and several of its single anthocyanins and metabolites, which are partially model specific, and coincides with animal studies.

A Novel Combination of High-Load Omega-3 Lysine Complex (AvailOm®) and Anthocyanins Exerts Beneficial Cardiovascular Effects.

Omega-3 fatty acids have been shown to exert several beneficial effects in the prevention of cardiovascular and cerebrovascular diseases. The objective of the present study was to analyze the effects of a novel high-load omega-3 lysine complex, AvailOm®, its related constituents and a novel mixture of AvailOm® with specific vasoactive anthocyanins on vascular function in mice resistance artery. Pressure myograph was used to perform vascular reactivity studies. Nitric oxide and oxidative stress were assessed by difluorofluorescein diacetate and dihydroethidium, respectively. Increasing doses of AvailOm® exerted a dose-response vasorelaxation via AMPK-eNOS-mediated signaling. Omega-3 Ethyl Ester was identified as the main bioactive derivative of AvailOm®, being capable of inducing vasorelaxant action to the same extent of entire product. The combination of AvailOm® with a mix of potent vasoactive anthocyanins (C3-glu + DP3-glu + Mal3-glu + Mal3-gal + PEO3-gal), strongly protected mesenteric arteries from vascular dysfunction and oxidative stress evoked by oxidized-LDL. These data demonstrate for the first time the direct effects of AvailOm® on resistance arteries. The evidence that the combination of specific vasoactive anthocyanins and AvailOm® further enhanced the vasculoprotective properties of these compounds, may offer new promising perspectives for preventing the onset of cardiovascular and cerebrovascular events.

Healthberry 865® and Its Related, Specific, Single Anthocyanins Exert a Direct Vascular Action, Modulating Both Endothelial Function and Oxidative Stress.

In recent years, epidemiological studies have identified a relationship between diet and cerebro-cardiovascular disease (CVD). In this regard, there is a promising dietary group for cardiovascular protection are polyphenols, especially anthocyanins. Vascular reactivity studies were performed using Healthberry 865® and constituent single anthocyanins to characterize vasomotor responses; immunofluorescence analysis with dichlorofluorescein diacetate and dihydroethidium were used to evaluate nitric oxide and oxidative stress; lucigenin assay was used to measure NADPH oxidase activity; and gel electrophoresis and immunoblotting were used to dissect the molecular mechanisms involved. We demonstrated that Healthberry 865® exerts an important vasorelaxant effect of resistance artery functions in mice. Its action is mediated by nitric oxide release through the intracellular signaling PI3K/Akt. Moreover, behind its capability of modulating vascular tone, it also exerts an important antioxidant effect though the modulation of the NADPH oxidase enzyme. Interestingly, its cardiovascular properties are mediated by the selective action of different anthocyanins. Finally, the exposure of human dysfunctional vessels to Healthberry 865® significantly reduces oxidative stress and improves NO bioavailability. Although further investigations are needed, our data demonstrate the direct role of Healthberry 865® on the modulation of vasculature, both on the vasorelaxation and on oxidative stress; thus, supporting the concept that a pure mixture of anthocyanins could be helpful in preventing the onset of vascular dysfunction associated with the development of CVD.

A Review of the Properties of Anthocyanins and Their Influence on Factors Affecting Cardiometabolic and Cognitive Health.

The incidence of cardiovascular and metabolic diseases has increased over the last decades and is an important cause of death worldwide. An upcoming ingredient on the nutraceutical market are anthocyanins, a flavonoid subgroup, abundant mostly in berries and fruits. Epidemiological studies have suggested an association between anthocyanin intake and improved cardiovascular risk, type 2 diabetes and myocardial infarct. Clinical studies using anthocyanins have shown a significant decrease in inflammation markers and oxidative stress, a beneficial effect on vascular function and hyperlipidemia by decreasing low-density lipoprotein and increasing high-density lipoprotein. They have also shown a potential effect on glucose homeostasis and cognitive decline. This review summarizes the effects of anthocyanins in in-vitro, animal and human studies to give an overview of their application in medical prevention or as a dietary supplement.

Calcium phosphate increases the encapsulation efficiency of hydrophilic drugs (proteins, nucleic acids) into poly(d,l-lactide-co-glycolide acid) nanoparticles for intracellular delivery.

Calcium phosphate/poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles with a diameter below 200 nm, loaded with either nucleic acids or proteins, were synthesized by a water-in-oil-in-water (W1/O/W2) emulsion solvent evaporation technique. The particles were stabilized by polyvinyl alcohol (PVA) and had a negative charge (zeta potential -26 mV). By the addition of calcium phosphate into the inner aqueous phase of the W1/O/W2-emulsion, the encapsulation efficiency of siRNA was increased to 37%, of DNA to 52%, and of bovine serum albumin to 78%, i.e. by a factor of 3 to 10 compared to PLGA nanoparticles without calcium phosphate. Total loadings of 8 µg siRNA, 5 µg DNA and 280 µg fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA) per mg of PLGA nanoparticles were achieved by this method. The addition of an outer layer of either chitosan or polyethyleneimine (PEI) reversed the charge of the particles (zeta potential > +30 mV) and improved the cellular uptake as well as the endosomal escape of these particles as demonstrated by confocal laser scanning microscopy. Calcium phosphate-PLGA nanoparticles loaded with DNA encoding for enhanced green fluorescent protein (eGFP-DNA) showed a good transfection efficiency for epithelial cells (HeLa). Gene silencing with HeLa cells expressing eGFP gave knockdown efficiencies of 53% for anionic nanoparticles, of 68% for chitosan-coated cationic nanoparticles, and of 89% for polyethyleneimine-coated cationic nanoparticles.

A novel concept in enteric coating: a double-coating system providing rapid drug release in the proximal small intestine.

A novel double-coating enteric system was developed to accelerate drug release in conditions resembling the upper small intestine. The system comprises an inner coat (partially neutralised EUDRAGIT L 30 D-55 and organic acid) and an outer coat (standard EUDRAGIT L 30 D-55). Prednisolone tablets were coated with double layer formulations with inner coats neutralised to pH 5.6 in the presence of 10% citric acid or adipic acid. A conventional single coating was also applied for comparison purposes. There was no drug release from the single coated or double-coated tablets in 0.1M HCl for 2 h using USP II apparatus. The lag times of drug release in subsequent pH 5.6 phosphate buffer (to resemble the pH condition of the proximal small intestine) were 102, 42 and 28 min for the single coated, adipic acid and citric acid double-coated tablets respectively. The lag time for release from the double-coated tablets was further reduced to 5 min when the inner coat was neutralised to pH 6.0 in the presence of 10% citric acid. The rapid drug release from the double-coating system was associated with faster polymer dissolution rates compared to the single coating. The novel double-coated system has the potential to provide rapid drug release in the proximal small intestine, overcoming the limitations of conventional enteric coatings.

SEM/EDX and confocal microscopy analysis of novel and conventional enteric-coated systems.

A novel double coating enteric system (comprising an inner layer of neutralised EUDRAGIT) L 30 D-55 and organic acid, and an outer layer of standard EUDRAGIT) L 30 D-55) was developed to provide fast dissolution in proximal small intestinal conditions. The mechanisms involved in the dissolution of the double coating were investigated and compared with a conventional single layer enteric coating and an hypromellose (HPMC) sub-coated enteric system. Rates of drug release from coated prednisolone pellets were established using USP II dissolution methods (0.1M HCl for 2h and subsequently pH 5.5 phosphate buffer) and the coating dissolution process was illustrated using confocal laser scanning microscopy (CLSM). The distribution of sodium, as a representative ion, in the double-coating system during dissolution was determined using scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX). The double-coating system showed faster dissolution compared to the single coating and the HPMC sub-coated system in pH 5.5 buffer. The dissolution process of the double-coating was unusual; the inner coat dissolved before the outer coat and this accelerated the dissolution of the outer coat. During dissolution, sodium ions diffused from the inner coat to the outer coat. This migration of ions and the increased ionic strength and buffer capacity of the inner coat contribute to the rapid dissolution of the double-coating system.

Modulated release metoprolol succinate formulation based on ionic interactions: in vivo proof of concept.

A modulated release, multiunit oral drug delivery technology using a system based on ionic interactions of anions of salts with quaternary ammonium ions of the ammoniomethacrylate polymer is described. The system consisted of a drug layered, EUDRAGIT NE-coated salt core which was further coated with EUDRAGIT RS. The relative effects of different anions on the polymer permeability have been investigated by studying their influence on the in vitro drug release. A prototype formulation of metoprolol succinate using this technology was developed and the drug release from the formulation was adjusted to have a release profile which would match the circadian rhythm i.e. a higher amount of drug would be available after an initial lower release (accelerated type of release). The formulation was tested in vivo in 12 healthy human volunteers in an open label, randomized, two-treatment, two-period, single dose crossover bio-study with reference formulation Beloc-zok. The in vivo release demonstrated that compared to the reference, a higher amount of drug was available in the plasma from the 7th hour onwards. A higher AUC of the drug was also observed compared to the reference formulation. An in vitro-in vivo correlation was attempted to identify a bio-relevant in vitro dissolution medium for the formulation.