The Effects of Anthocyanin-Rich Bilberry Extract on Transintestinal Cholesterol Excretion

Hypercholesterolemia is one of the modifiable and primary risk factors for cardiovascular diseases (CVD). Emerging evidence suggests the stimulation of transintestinal cholesterol excretion (TICE), the nonbiliary cholesterol excretion, using natural products can be an effective way to reduce CVD. Bilberry (Vaccinium myrtillus L.) has been reported to have cardioprotective effects by ameliorating oxidative stress, inflammation, and dyslipidemia. However, the role of bilberry in intestinal cholesterol metabolism is not well understood. To examine the effects of bilberry in intestinal cholesterol metabolism, we measured the genes for cholesterol flux and de novo synthesis in anthocyanin-rich bilberry extract (BE)-treated Caco-2 cells. BE significantly decreased the genes for cholesterol absorption, i.e., Niemann-Pick C1 Like 1 and ATP-binding cassette transporter A1 (ABCA1). In contrast, BE significantly upregulated ABCG8, the apical transporter for cholesterol. There was a significant induction of low-density lipoprotein receptors, with a concomitant increase in cellular uptake of cholesterol in BE-treated cells. The expression of genes for lipogenesis and sirtuins was altered by BE treatment. In the present study, BE altered the genes for cholesterol flux from basolateral to the apical membrane of enterocytes, potentially stimulating TICE. These results support the potential of BE in the prevention of hypercholesterolemia.

Old Plant, New Possibilities: Wild Bilberry (Vaccinium myrtillus L., Ericaceae) in Topical Skin Preparation

Bilberry represents a valuable source of antioxidant substances responsible for its application for the treatment of different conditions (such as inflammation, cardiovascular disease, cancer, diabetes, and different age-related diseases) associated with increased oxidative stress. As oxidative stress might cause skin impairments, we aim to evaluate a topical preparation containing bilberry leaves extract and bilberry seeds oil, obtained as a byproduct of the food industry. To obtain the extracts, the conventional maceration technique for leaves, and supercritical carbon dioxide extraction for seeds were employed. The chemical profile of both actives was achieved by HPLC and GC methods, revealing the presence of phenolic acids (chlorogenic being the most abundant), flavonoids (isoquercetin in the highest amount), and resveratrol in leaves extract, while in seeds oil the essential ω-3 and ω-6 fatty acids were determined in favorable ratio, almost being 1. Antioxidant potential of the wild bilberry extract and seed oil was evaluated using in vitro DPPH and FRAP assays. Finally, effects of the oil-in-water creams with mentioned wild bilberry isolates on the skin were investigated in an in vivo study conducted on healthy human volunteers, revealing the significant beneficial effects when topically applied.

Differential protection by anthocyanin-rich bilberry extract and resveratrol against lipid micelle-induced oxidative stress and monolayer permeability in Caco-2 intestinal epithelial cells

Excess dietary fat, and associated bile acids, can impair intestinal barrier integrity, produce intestinal or systemic inflammation and promote tumorigenesis.

Bilberry (Vaccinium myrtillus L.) Extracts Comparative Analysis Regarding Their Phytonutrient Profiles, Antioxidant Capacity along with the In Vivo Rescue Effects Tested on a Drosophila melanogaster High-Sugar Diet Model

Bilberries (Vaccinium myrtillus L.) have been reported to hold a plentitude of health-promoting properties beyond basic nutrition, mainly attributed to their anthocyanin content and antioxidant activity. In this article, we built the phytochemical profile of three wild bilberry fruit extract formulations (aqueous, methanolic, and hydro-methanolic) using UHPLC-ESI-MS/MS putative analysis, identifying 88 individual phytochemicals, mainly flavonoids (total content 8.41 ± 0.11 mg QE/g dw), free amino acids, polyphenols (total content 21.68 ± 0.19 mg GAE/g dw), carboxylic acids, and vitamins. Furthermore, the antioxidant activity of the extract was assessed, reaching 78.03 ± 0.16% DPPH free radical scavenging activity, comparable to literature values determined for bilberry extracts of other origin. Due to the increased prevalence of metabolic syndrome and based on the reviewed benefits of bilberries, we tested the most potent formulation of our bilberry extracts in this biological context. The in vivo rescue effect of a bilberry extract supplemented diet on Drosophila melanogaster was assessed by monitoring biochemical and genomic markers. Hemolymph trehalose levels were halved upon addition of 3% hydro-methanolic bilberry extract to a high-sugar (1.5 M sucrose) diet, as compared to the non-supplemented high-sugar diet. Noteworthy, the rescue seen for flies kept on the bilberry extract supplemented high-sugar diet appeared to parallel the trehalose levels observed in the case of the control diet (50 mM sucrose) flies. Moreover, next to the trehalose-lowering type of in vivo effects, other gene expression related rescues were also detected for genes such as InR, Akh, AstA, AstC, Irk, Npc2g, and CCHa2 upon supplementation of the high-sugar diet with our hydro-methanolic bilberry fruit extract. Our findings suggest that such a bilberry fruit extract could generate physiological and genomic type of compensatory mechanisms so that further translational approaches would advance the understanding of some human specific pathological conditions.

Assessment of the In Vivo Antioxidant Activity of an Anthocyanin-Rich Bilberry Extract Using the Caenorhabditis elegans Model

Anthocyanins have been associated with several health benefits, although the responsible mechanisms are not well established yet. In the present study, an anthocyanin-rich extract from bilberry (Vaccinium myrtillus L.) was tested in order to evaluate its capacity to modulate reactive oxygen species (ROS) production and resistance to thermally induced oxidative stress, using the nematode Caenorhabditis elegans as an in vivo model. The assays were carried out with the wild-type N2 strain and the mutant strains daf-16(mu86) I and hsf-1(sy441), which were grown in the presence of two anthocyanin extract concentrations (5 and 10 μg/mL in the culture medium) and further subjected to thermal stress. The treatment with the anthocyanin extract at 5 μg/mL showed protective effects on the accumulation of ROS and increased thermal resistance in C. elegans, both in stressed and non-stressed young and aged worms. However, detrimental effects were observed in nematodes treated with 10 μg/mL, leading to a higher worm mortality rate compared to controls, which was interpreted as a hormetic response. These findings suggested that the effects of the bilberry extract on C. elegans might not rely on its direct antioxidant capacity, but other mechanisms could also be involved. Additional assays were performed in two mutant strains with loss-of-function for DAF-16 (abnormal DAuer Formation factor 16) and HSF-1 (Heat Shock Factor 1) transcription factors, which act downstream of the insulin/insulin like growth factor-1 (IGF-1) signaling pathway. The results indicated that the modulation of these factors could be behind the improvement in the resistance against thermal stress produced by bilberry anthocyanins in young individuals, whereas they do not totally explain the effects produced in worms in the post-reproductive development stage. Further experiments are needed to continue uncovering the mechanisms behind the biological effects of anthocyanins in living organisms, as well as to establish whether they fall within the hormesis concept.

Authentication of the Bilberry Extracts by an HPLC Fingerprint Method Combining Reference Standard Extracts

A simple and fast high-performance liquid chromatography (HPLC) fingerprint method combining reference standard extract for the identification of bilberry extract was developed and validated. Six batches of bilberry extract collected from different manufactures were used to establish the HPLC fingerprint. Other berry extracts—such as blueberry extracts, mulberry extracts, cranberry extracts, and black rice extracts—were also analyzed for their HPLC chromatograms. The fingerprints of five batches of bilberry extract showed high similarities, while one batch was distinguished from others. Additionally, the content of anthocyanin Cyanidin-3-O-glucoside (Cy-3-glc) in each berry extract was analyzed and compared. The results indicate that this HPLC fingerprint method, combining reference standard extracts, could be used for the authentication and quality control of bilberry extracts.