Bilberry Anthocyanin Research Articles

Transcriptome and proteome profile of jejunum in chickens challenged with Salmonella Typhimurium revealed the effects of dietary bilberry anthocyanin on immune function.

The present study investigated the effects of bilberry anthocyanin (BA) on immune function when alleviating Salmonella Typhimurium (S. Typhimurium) infection in chickens. A total of 180 newly hatched yellow-feathered male chicks were assigned to three groups (CON, SI, and SI + BA). Birds in CON and SI were fed a basal diet, and those in SI + BA were supplemented with 100 mg/kg BA for 18 days. Birds in SI and SI + BA received 0.5 ml suspension of S. Typhimurium (2 × 109 CFU/ml) by oral gavage at 14 and 16 days of age, and those in CON received equal volumes of sterile PBS. At day 18, (1) dietary BA alleviated weight loss of chickens caused by S. Typhimurium infection (P < 0.01). (2) Supplementation with BA reduced the relative weight of the bursa of Fabricius (P < 0.01) and jejunal villus height (P < 0.05) and increased the number of goblet cells (P < 0.01) and the expression of MUC2 (P < 0.05) in jejunal mucosa, compared with birds in SI. (3) Supplementation with BA decreased (P < 0.05) the concentration of immunoglobulins and cytokines in plasma (IgA, IL-1β, IL-8, and IFN-β) and jejunal mucosa (IgG, IgM, sIgA, IL-1β, IL-6, IL-8, TNF-α, IFN-β, and IFN-γ) of S. Typhimurium-infected chickens. (4) BA regulated a variety of biological processes, especially the defense response to bacteria and humoral immune response, and suppressed cytokine-cytokine receptor interaction and intestinal immune network for IgA production pathways by downregulating 6 immune-related proteins. In summary, the impaired growth performance and disruption of jejunal morphology caused by S. Typhimurium were alleviated by dietary BA by affecting the expression of immune-related genes and proteins, and signaling pathways are related to immune response associated with immune cytokine receptors and production in jejunum.

Dietary supplementation of bilberry anthocyanin on growth performance, intestinal mucosal barrier and cecal microbes of chickens challenged with Salmonella Typhimurium

BackgroundAnthocyanins (AC) showed positive effects on improving the intestinal health and alleviating intestinal pathogen infections, therefore, an experiment was conducted to explore the protective effects of supplemented AC on Salmonella-infected chickens.MethodsA total of 240 hatchling chickens were randomly allocated to 4 treatments, each with 6 replicates. Birds were fed a basal diet supplemented with 0 (CON, and ST), 100 (ACL) and 400 (ACH) mg/kg of AC for d 60, and orally challenged with PBS (CON) or 109 CFU/bird (ST, ACL, ACH) Salmonella Typhimurium at d 14 and 16.Results(1) Compared with birds in ST, AC supplementation increased the body weight (BW) at d 18 and the average daily gain (ADG) from d 1 to 18 of the Salmonella-infected chickens (P < 0.05); (2) AC decreased the number of Salmonella cells in the liver and spleen, the contents of NO in plasma and inflammatory cytokines in ileal mucosa of Salmonella-infected chickens (P < 0.05); (3) Salmonella infection decreased the ileal villi height, villi height to crypt depth (V/C), and the expression of zonulaoccludins-1 (ZO-1), claudin-1, occludin, and mucin 2 (MUC2) in ileal mucosa. AC supplementation relieved these adverse effects, and decreased ileal crypt depth (P < 0.05); (4) In cecal microbiota of Salmonella-infected chickens, AC increased (P < 0.05) the alpha-diversity (Chao1, Pd, Shannon and Sobs indexes) and the relative abundance of Firmicutes, and decreased (P < 0.05) the relative abundance of Proteobacteria and Bacteroidota and the enrichment of drug antimicrobial resistance, infectious bacterial disease, and immune disease pathways.ConclusionsDietary AC protected chicken against Salmonella infection via inhibiting the Salmonella colonization in liver and spleen, suppressing secretion of inflammatory cytokines, up-regulating the expression of ileal barrier-related genes, and ameliorating the composition and function of cecal microbes. Under conditions here used, 100 mg/kg bilberry anthocyanin was recommended.

Bilberry Anthocyanins (Vaccinium myrtillus L.) Induced Apoptosis of B16-F10 Cells and Diminished the Effect of Dacarbazine

Anthocyanins have been reported to have potential as dietary or pharmaceutical supplements in the application of cancer prevention and adjunctive treatment. However, there are few studies on the effect of anthocyanins on melanoma, which have only been performed in cell lines. The objective of this work was to investigate the anticancer effects and mechanisms of bilberry anthocyanin extract (BAE) on melanoma In Vitro and In Vivo. Moreover, a primary study was done to investigate how BAE influenced C57BL/6 mice bearing subcutaneous B16-F10 tumors treated with dacarbazine (DTIC). BAE-induced apoptosis in B16-F10 cells was associated with activation of the mitochondrial pathway induced by increased reactive oxygen species. More, In Vivo anticancer activity studies indicated that BAE attenuated melanoma growth, as identified by hematoxylin-eosin staining, Ki-67, and TUNEL assays. Further western blot results revealed higher phospho-Akt expression with the combination of BAE and DTIC, indicating no suppression of the PI3K/AKT signaling pathway. In summary, this study demonstrated the anti-melanoma activity of BAE and investigated its mechanism. Notably, it should be careful to use products enriching BAE for those melanoma patients treated with DTIC.

Ultrasound-Assisted Natural Deep Eutectic Solvents Extraction of Bilberry Anthocyanins: Optimization, Bioactivities, and Storage Stability.

Bilberry fruits (Vaccinium myrtillus L.) are one of the richest natural sources of anthocyanins and are widely used due to their pharmacological and nutritional properties. To ensure their maximum application potential, it is necessary to overcome the limitations of conventional extraction solvents and techniques. This study aimed to develop a green method for bilberry anthocyanin extraction using natural deep eutectic solvents (NaDES) integrated with ultrasound-assisted extraction (UAE) in order to define extraction conditions that will prevent decomposition of the anthocyanins or the loss of bioactivity. After a screening of ten different NaDES, choline chloride:sorbitol (1:1) was selected as the most effective. Furthermore, the influence analysis and optimization of the NaDES–UAE extraction conditions were carried out employing response surface methodology. The optimal conditions were found to be an extraction time of 37.63 min, a temperature of 48.38 °C, and 34.79% (w/w) water in NaDES. The extraction yields of target compounds under optimized extraction conditions were 0.27 mg/g DW of cyanidin-3-O-glucoside and 2.12 mg CGE/g DW of TAC. The obtained optimized extract showed promising radical scavenging and antimicrobial activity. A stability study with the optimized extract revealed that refrigerated storage at 4 °C in the dark provided the best anthocyanins preservation. Overall, the developed NaDES-UAE method showed promising application potential and can be considered as a high-efficiency green alternative to conventional anthocyanins extraction methods, enabling the preservation of active ingredients and the bioactivity of extracts.

Protective Effect of Bilberry Anthocyanin Extracts on Dextran Sulfate Sodium-Induced Intestinal Damage in Drosophila melanogaster.

Inflammatory bowel disease (IBD) is a chronic recurrent disease that can be controlled by various natural extracts. Anthocyanins (ANCs) from bilberry have significant antioxidant capacity and are widely used as food colors and antioxidants. In this study, we investigated the protective effects of bilberry anthocyanin extracts (BANCs) against dextran sulphate sodium (DSS)-induced intestinal inflammation in a Drosophila melanogaster (D. melanogaster) model, and the effects on the lifespan, antioxidant capacity, intestinal characteristics, and microbiome and gene expression profiles were analyzed to elucidate the underlying biological mechanisms. In DSS-induced normal and axenic D. melanogaster, BANCs significantly increased the survival rate, maintained the intestinal morphology and integrity, and reduced the number of dead intestinal epithelial cells and the ROS level of these cells. BANC supplementation had no significant effect on the intestinal microflora of DSS-induced D. melanogaster, as demonstrated by a 16S rDNA analysis, but improved the antioxidant capacity by activating the relative gene expression of NRF2 signaling pathways in the intestine of D. melanogaster with DSS-induced inflammation. Therefore, the results demonstrate that BANCs effectively alleviate intestinal inflammatory injury induced by DSS and improve the antioxidant capacity of D. melanogaster by modulating NRF2 signaling pathways, and could thus promote the application of BANCs as functional foods.

Correction: Bilberry anthocyanin improves the serum cholesterol in aging perimenopausal rats via the estrogen receptor signaling pathway.

Correction for 'Bilberry anthocyanin improves the serum cholesterol in aging perimenopausal rats via the estrogen receptor signaling pathway' by Na Li et al., Food Funct., 2019, 10, 3430-3438, DOI.

Preparation of Lipid-Soluble Bilberry Anthocyanins through Acylation with Cinnamic Acids and their Antioxidation Activities.

To enable the use of anthocyanins in food with high oil content, bilberry anthocyanins were acylated with cinnamic acids to address their poor lipid solubility. Structural analyses based on Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and 1H nuclear magnetic resonance analyses indicated that cinnamic acids were efficiently grafted onto 6-OH of glucoside and galactoside and 5-OH of arabinose through an esterification reaction. The higher the dose of the acylating agent, the higher the acylation degree (AD) and the lower the total anthocyanidin content (TAC) of bilberry anthocyanins. An-Ci4 presented the highest AD value (6.61%), and An-Ci3 exhibited the lowest TAC value (50.16 mg/g). After acylating with lipophilic cinnamic acids, the lipid solubility of acylated bilberry anthocyanins significantly improved. The color of the native bilberry anthocyanin solution dissolved in ethyl acetate and dioxane was transparent. By contrast, the acylated anthocyanin solution dissolved in these solvents was unmistakably red. In terms of the antioxidant activity, acylated bilberry anthocyanins demonstrated inferior performance in 2,2-diphenyl-1-picrylhydrazyl (DPPH) clearance but a better inhibition ratio in β-carotene bleaching assay compared with native bilberry anthocyanins. As AD value increased, the DPPH clearance of acylated anthocyanins decreased and their inhibition ratio increased in β-carotene bleaching assay.

Biostimulating Gut Microbiome with Bilberry Anthocyanin Combo to Enhance Anti-PD-L1 Efficiency against Murine Colon Cancer.

Recent advances have revealed the essential role of gut microbiomes in the therapeutic efficiency of immune checkpoint inhibitors (ICIs). Inspired by biostimulation, a method using nutrients to accelerate the growth of soil microorganisms and the recovery of soil microbial consortia, here we propose a bilberry anthocyanin combo containing chitosan and low molecular citrus pectin (LCP), in which LCP–chitosan is used to encapsulate anthocyanins so to enhance its digestive stability and, moreover, modulate the microbiome more favorable for the PD-L1 blockade treatment. Using murine MC38 colon cancer as a model system, we examined the effects of the combo on modulating the gut microbiome and therapeutic efficiency of PD-L1 blockade treatment. It was shown that bilberry anthocyanins enriched the subdominant species, increased both the concentration and the proportion of butyrate in feces and enhanced intratumoral CD8+ T cell infiltrations. The application of the bilberry anthocyanin combo restored the species diversity of gut microbiome decreased by LCP–chitosan and achieved the best control of tumor growth. These preliminary results indicated unprecedented opportunities of probiotics combo in improving the therapeutic efficiency of immune checkpoint inhibitor through manipulating gut microbiome.

Bilberry anthocyanin extracts enhance anti-PD-L1 efficiency by modulating gut microbiota.

The undesirable low response rate is a major hurdle to garnering the maximum potential of immune checkpoint inhibitors in cancer treatments. Recent advances in exploring the effects of intestinal flora on the medical efficacy of immune checkpoint blockade have shed new light on the application of immune checkpoint inhibitors. Inspired by the prebiotic role of anthocyanin-rich extracts, we propose using bilberry anthocyanin extracts to modulate the composition of gut microbiota and eventually, promote the efficiency of immune checkpoint inhibitors. This study demonstrates the effectiveness of orally administered bilberry anthocyanin extracts in enhancing the anti-tumor efficiency of the PD-L1 antibody in the experimental mouse MC38 tumor model. We observed an increase in the fecal abundance of Clostridia and Lactobacillus johnsonii and improved effective community diversity. These findings reinforce the importance of intestinal flora composition and open up unprecedented opportunities in using natural compounds to enhance the efficacy of immune checkpoint inhibitors.

OxiCyan®, a phytocomplex of bilberry (Vaccinium myrtillus) and spirulina (Spirulina platensis), exerts both direct antioxidant activity and modulation of ARE/Nrf2 pathway in HepG2 cells

• OxiCyan® is a phytocomplex based on bilberry and spirulina extracts. • The bilberry anthocyanin moiety exerts a direct effect on ROS quenching on HepG2 cells. • The constituents of spirulina induce ARE/Nrf2 pathway on HepG2 cells. • OxiCyan® is a strong antioxidant at the cellular level and triggers the natural antioxidant cell defense. The antioxidant activity of plant extracts requires compelling evidence of the physiological functions of antioxidants at the cellular level. In order to assess the antioxidant power of the phytocomplex OxiCyan®, a bilberry extract functionalized on spirulina, traditional chemical methods (DPPH and ABTS) were compared to in vitro cellular responses by using HepG2 human hepatocyte carcinoma 85011430 cell lines. OxiCyan® was found to act on HepG2 cells in a dual mode: with a direct effect on ROS scavenging and as a cytoprotective agent via induction of ARE/Nrf2 pathway. Direct ROS scavenging activity of OxiCyan® depended on the anthocyanin moiety provided by bilberry, whereas the gene activation of the ARE/Nrf2 pathway was triggered by spirulina chemical constituents. The comparative analysis of chemical and cellular assays indicated contrasting results. OxiCyan® showed a high antioxidant activity at the cellular level by both scavenging ROS and inducing the gene expression of natural antioxidant cell defense.

Reduction of Aging-Induced Oxidative Stress and Activation of Autophagy by Bilberry Anthocyanin Supplementation via the AMPK-mTOR Signaling Pathway in Aged Female Rats.

Oxidative-stress-induced senescence constitutes a great risk factor for chronic diseases. Therefore, ameliorating oxidative-stress-induced senescence is expected to prevent chronic diseases. The beneficial effects of bilberry anthocyanin (BA) on healthy aging were evaluated using 12 month old, aging female SD rats in this study. The experimental results suggested that consumption of a middle-dose of BA (MBA) appreciably increased the relative liver mass by 7.34% when compared with that of the AC group. Furthermore, BA significantly increased the total antioxidant capacity, total superoxide dismutase activity, and catalase activities; decreased malondialdehyde, serum low-density lipoprotein cholesterol (LDL-C), serum total cholesterol (TC), serum triglyceride (TG), and glycated serum protein (GSP) levels; and reduced TC/high-density lipoprotein cholesterol (HDL-C) and LDL-C/HDL-C ratios. In addition, MBA decreased the activity of fecal bacterial enzymes and increased the content of fecal short-chain fatty acids. The Western blot results showed that MBA significantly upregulated the expression of OCLN, ZO-1, and autophagy-related proteins (ATP6 V0C, ATG4D, and CTSB) in aging rats. Moreover, it also showed that MBA induced the phosphorylation of AMPK and FOXO3a and inhibited the phosphorylation of mTOR, which indicated that bilberry anthocyanin induced autophagy via the AMPK-mTOR signaling pathways. This induction of autophagy further promoted oxidative stress resistance effects and intestinal epithelial barrier function of bilberry anthocyanin in aging female rats.

Bilberry anthocyanin improves the serum cholesterol in aging perimenopausal rats via the estrogen receptor signaling pathway.

With aging, there is an increasing risk for women to develop perimenopause syndrome, which is harmful to women's physical and mental health. The present study investigated the health benefits of bilberry anthocyanin (BA) on aging perimenopausal Sprague-Dawley rats. Rats that entered into perimenopause through natural aging were treated for 8 weeks with BA, and received either a low dose (LD, 35 mg per kg of bodyweight), medium dose (MD, 70 mg per kg of bodyweight), or high dose (HD, 140 mg per kg of bodyweight). The experimental results suggested that all three dosages of BA, especially the high dose, significantly reduced the serum total cholesterol (TC), triglyceride (TG) and low density lipoprotein cholesterol (LDL-C) levels. In addition, BA supplementation markedly reduced the serum malondialdehyde (MDA), effectively increased the activity of hepatic total superoxide dismutase (T-SOD), significantly raised the high density lipoprotein cholesterol (HDL-C), increased the number of estrogen receptors, and effectively up-regulated the expression levels of G protein-coupled receptor 30 (GPR30), protein kinase B (AKT), and extracellular regulated protein kinase 2 (ERK2). In summary, BA has a great effect on improving the serum cholesterol in natural aging perimenopausal rats via the estrogen receptor signaling pathway, and it may be used as a dietary supplement for perimenopause women to decrease the risk of cardiovascular disease.

Bilberry anthocyanin extract promotes intestinal barrier function and inhibits digestive enzyme activity by regulating the gut microbiota in aging rats.

This study was aimed at understanding potential mechanisms regarding bilberry anthocyanin extract consumption and healthy aging and the effects on intestinal barrier function and digestive enzyme activity, through regulating the gut microbiota in aging rats. Medium-dose bilberry anthocyanin extract consumption (20 mg per kg bw per day) was the optimum amount to regulate the intestinal function of aging rats. After consumption, bacteria beneficial to the intestine (Aspergillus oryzae, Lactobacillus, Bacteroides, Clostridiaceae-1, the Bacteroidales-S24-7-group and the Lachnospiraceae_NK4A136_group) were induced to grow, and harmful bacteria (Verrucomicrobia and Euryarchaeota) were inhibited. However, high-dose bilberry anthocyanin extract consumption altered some intestinally beneficial bacteria in an adverse way. There was a correlation between changes in bacterial composition and changes in short-chain fatty acids and the intestinal mucosal barrier. Bilberry anthocyanin extract consumption also decreased the activity of digestive enzymes. Our results suggest that bilberry anthocyanin extract consumption is a potential approach for assisting healthy aging.

Expression of cardiac insulin signalling genes and proteins in rats fed a high-sucrose diet: effect of bilberry anthocyanin extract

ScopeInsulin resistance is associated with impaired cardiac function, but the underlying molecular abnormalities are largely unexplained. Bilberry anthocyanin (BAcn) may be protective, as it appears to potentiate insulin action.MethodsRats were randomly allocated to control, sucrose-fed (SF) or sucrose-fed + BAcn diets (SF-A) for 15 weeks. Cardiac insulin signalling genes and proteins were quantified using reverse transcription quantitative real-time polymerase chain reaction and western blots.ResultsGlucose tolerance was not different with treatment. SF showed lower (p < 0.05) ferric reducing antioxidant power, which increased with BAcn. SF resulted in significantly decreased (p < 0.05) expression of 10 genes: acetyl-coenzyme A carboxylase alpha; V-Akt murine thymoma viral oncogene homolog 1; Bcl2-like 1; cytosine-cytosine-adenosine-adenosine-thymidine/enhancer binding protein; FK506 binding protein 12-rapamycin associated; glycerol-3-phosphate dehydrogenase 1 (soluble); solute carrier family 2 (facilitated glucose transporter), member 1, 4; hexokinase 2; and thyroglobulin. SF-A prevented these changes. Compared to SF-A, SF up-regulated (p < 0.05) complement factor D and phosphoinositide-3-kinase, regulatory subunit1 (α); sterol regulatory element binding transcription factor 1 was down-regulated (p < 0.05). SF increased (p < 0.05) cardiac phospholamban and decreased phosphorylated troponin I, which were not attenuated by BAcn. Compared to control or SF, SF-A resulted in significantly lower (p < 0.05) 5′-AMP-activated protein kinase.ConclusionsSF lowered antioxidant capacity and changed the expression of insulin signalling genes, which were modulated by BAcn.

Retinoprotective Effects of Bilberry Anthocyanins via Antioxidant, Anti-Inflammatory, and Anti-Apoptotic Mechanisms in a Visible Light-Induced Retinal Degeneration Model in Pigmented Rabbits.

Excessive visible light exposure can induce damage to retinal cells and contribute to the development or progression of age-related macular degeneration. In this study we created a model of phototoxicity in pigmented rabbits. Furthermore, we investigated the protective effect of bilberry anthocyanin extract (BAE, Table A1) and explored the possible mechanisms of action in this model. The model of light-induced retinal damage was established by the pigmented rabbits exposed to light at 18,000 lx for 2 h, and they were sacrificed on day 7. After administration of BAE at dosages of 250 and 500 mg/kg/day, retinal dysfunction was significantly inhibited in terms of electroretinograms, and the decreased thicknesses of retinal outer nuclear layer and lengths of the outer segments of the photoreceptor cells were suppressed in rabbits with retinal degeneration. BAE attenuated the changes caused by light to certain apoptotic proteins (Bax, Bcl-2, and caspase-3). The extract increased the levels of superoxide dismutase, glutathione peroxidase, and catalase, as well as the total antioxidant capacity, but decreased the malondialdehyde level in the retinal cells. BAE inhibited the light-induced elevation in the levels of proinflammatory cytokines and angiogenic parameters (IL-1β and VEGF). Results showed that visible light-induced retinal degeneration model in pigmented rabbits was successfully established and BAE exhibited protective effects by increasing the antioxidant defense mechanisms, suppressing lipid peroxidation and proinflammatory cytokines, and inhibiting retinal cells apoptosis.

The role of dietary polyphenols in the management of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is an idiopathic chronic, relapsing inflammation of the bowel which is caused by dysregulation of the mucosal immune system. Polyphenols as the secondary plant metabolites universally present in vegetables and fruits and are the most abundant antioxidants in the human diet. There is evidence demonstrating the beneficial health effects of dietary polyphenols. This review criticizes the potential of commonly used polyphenols including apple polyphenol, bilberry anthocyanin, curcumin, epigallocatechin-3-gallate (EGCG) and green tea polyphenols, naringenin, olive oil polyphenols, pomegranate polyphenols and ellagic acid, quercetin, as well as resveratrol specifically in IBD with an emphasis on cellular mechanisms and pharmaceutical aspects. Scientific research confirmed that dietary polyphenols possess both protective and therapeutic effects in the management of IBD mediated via down-regulation of inflammatory cytokines and enzymes, enhancing antioxidant defense, and suppressing inflammatory pathways and their cellular signaling mechanisms. Further preclinical and clinical studies are needed in order to understand safety, bioavailability and bioefficacy of dietary polyphenols in IBD patients.

Isolation of Bilberry Anthocyanidin 3-Glycosides Bearing ortho-Dihydroxyl Groups on the B Ring by Forming an Aluminum Complex and Their Antioxidant Activity

Bilberry anthocyanin bearing an ortho-dihydroxyl group on the B ring was selectively isolated by complex formation with flavocommelin and aluminum ions (Al(3+)). The interaction between the anthocyanins, flavocommelin, and Al(3+) might have happened in a manner similar to rigid supramolecules, such as commelinin, protocyanin, and other complex pigments. Complex blue pigment (CP, 8.85 ± 0.26 mg) with Al(3+) was formed from 18.6 mg (15 μmol) of bilberry pigment, 18.2 mg (30 μmol, 2.0 equiv) of flavocommelin, and 0.03 mL of 0.5 M aluminum chloride aqueous solution (15 μmol, 1.0 equiv), yielding 36.5 ± 0.7% recovery of anthocyanins in the complex after precipitation by ethanol. Consequently, anthocyanins bearing ortho-dihydroxyl groups on the B rings (delphinidin 3-glycosides, cyanidin 3-glycosides, and petunidin 3-glycosides) predominating (98.0 ± 0.2%) in the complex were selectively isolated in a dose-dependent manner by the addition of Al(3+), increasing the antioxidative activity of the mixture of anthocyanins.

Bog Bilberry Anthocyanin Extract Improves Motor Functional Recovery by Multifaceted Effects in Spinal Cord Injury

The aim of this study was to determine the therapeutic efficiency of bog bilberry anthocyanin extract (BBAE) treatment starting 1 d after spinal cord injury (SCI) in rats and to investigate the underlying mechanism. The BBAE contained cyanidin-3-glucoside, malvidin-3-galactoside and malvidin-3-glucoside. SCI models were induced using the weight-drop method in Sprague-Dawley rats and additionally with sham group (laminectomy only). The animals were divided into four groups: vehicle-treated group; 10 mg/kg BBAE-treated group; 20 mg/kg BBAE-treated group; sham group. BBAE-treated or vehicle-treated group was administered orally at one day after SCI and then daily for 8 weeks. Locomotor functional recovery was assessed during the 8 weeks post operation period by performing a Basso, Beattie, and Bresnahan (BBB) locomotor score test. At the end of study, the animals were killed, and 1.5 cm segments of spinal cord encompassing the injury site were removed for immunohistochemistry, histopathological and western blotting analysis. Immunohistochemistry for GFAP, aggrecan, neurocan and NeuN was used to assess the degree of astrocytic glial scar formation and neuron survival. Immunohistochemistry and western blotting analysis for TNF-α, IL-6, IL-1β was used to evaluate the anti-inflammation effect of BBAE. To evaluate its inhibition effect on the astrocytes, we performed the MTT assay and immunohistochemistry for Ki67 in vitro. Results show that the BBAE-treated animals showed significantly better locomotor functional recovery, neuron death and smaller glial scar formation after spinal cord injury in vivo. In addition, BBAE administration could inhibit astrocyte proliferation in vivo and vitro. Therefore, BBAE may be useful as a promising therapeutic agent for SCI.

Bog Bilberry (Vaccinium uliginosum L.) Extract Reduces Cultured Hep‐G2, Caco‐2, and 3T3‐L1 Cell Viability, Affects Cell Cycle Progression, and Has Variable Effects on Membrane Permeability

Bog bilberry (Vaccinium uliginosum L.) is a blue-pigmented edible berry related to bilberry (Vaccinium myrtillus L.) and the common blueberry (Vaccinium corymbosum). The objective of this study was to investigate the effect of a bog bilberry anthocyanin extract (BBAE) on cell growth, membrane permeability, and cell cycle of 2 malignant cancer cell lines, Caco-2 and Hep-G2, and a nonmalignant murine 3T3-L1 cell line. BBAE contained 3 identified anthocyanins. The most abundant anthocyanin was cyanidin-3-glucoside (140.9 +/- 2.6 microg/mg of dry weight), followed by malvidin-3-glucoside (10.3 +/- 0.3 microg/mg) and malvidin-3-galactoside (8.1 +/- 0.4 microg/mg). Hep-G2 LC50 was calculated to be 0.563 +/- 0.04 mg/mL, Caco-2 LC50 was 0.390 +/- 0.30 mg/mL and 0.214 +/- 0.02 mg/mL for 3T3-L1 cells. LDH release, a marker of membrane permeability, was significantly increased in Hep-G2 cells and Caco-2 cells after 48 and 72 h compared to 24 h. The increase was 21% at 48 h and 57% at 72 h in Caco-2 cells and 66% and 139% in Hep-G2 cells compared to 24 h. However, 3T3-L1 cells showed an unexpected significant lower LDH activity (P < or = 0.05) after 72 h of exposure corresponding to a 21% reduction in LDH release. BBAE treatment increased sub-G1 in all 3 cell lines without influencing cells in the G2/M phase. BBAE treatment reduced the growth and increased the accumulation of sub-G1 cells in 2 malignant and 1 nonmalignant cell line; however, the effect on membrane permeability differs considerably between the malignant and nonmalignant cells and may in part be due to differences in cellular membrane composition.

Effects of casein‐ and whey protein–dextran conjugates on the stability of bog bilberry anthocyanin extract

SummaryBog bilberry (Vaccinium uliginosum) contains a high concentration of anthocyanins. Anthocyanin stability depends on many factors, including pH, temperature and composition of food matrices in a product, such as protein and polysaccharide. The effect of casein–dextran and whey protein–dextran conjugates on the stability of bog bilberry anthocyanin extract (BBAE) was investigated in an accelerated model system. Cyanidin‐3‐glucoside was the main anthocyanin (79%) found in BBAE. The cyanidin‐3‐glucoside content, absorbance and ABTS [2, 2‐Azinobis (3‐ethylbenzothiazoline‐6‐sulfonic acid) diammonium salt] radical scavenging activity of BBAE significantly (P &lt; 0.05) decreased after heating at pH 8.0 and 80 °C for 60 min. The addition of casein–dextran and whey protein–dextran conjugates significantly (P &lt; 0.05) prevented cyanidin‐3‐glucoside from degradation, and increased the absorbance and ABTS radical scavenging activity of heated BBAE. The protection of casein–dextran conjugate on BBAE was significantly higher (P &lt; 0.05) than that of whey protein–dextran conjugate. The results suggest that protein–dextran conjugates have the ability to stabilise BBAE.