The Effects of Berry Extracts on Oxidative Stress in Cultured Cardiomyocytes and Microglial Cells: A Potential Cardioprotective and Neuroprotective Mechanism

Oxidative stress is a key underlying factor in cognitive decline and atherosclerosis. Oxidative stress occurs at the cellular level with an imbalance between reactive oxygen species and reactive nitrogen species and a deficiency in antioxidants. Mounting evidence suggests that berry flavonoids may promote cellular health by exerting antioxidant properties. Black currant and various berry extracts were tested in microglia (BV-2) and cardiomyocyte (HL-1) cell lines to study their biological effects. The principal ingredients in black currant and cranberry extract–delphinidin 3-rutinoside (D3R) and cyanidin 3-glucoside (C3G), were also assessed. A menadione-induced oxidative stressor was used, and its output was quantified to detect oxidative stress (CellROX^TM). Black currant extract had similar antioxidant effects as N-acetylcysteine (NAC) in HL-1 cells with regard to cellular protection, whereas cranberry extract was ineffective. In contrast, cranberry extract was comparable in effectiveness to black currant extract in BV-2 cells. D3R and C3G also reduced oxidative stress similarly to whole berry extracts, which indicates that these ingredients may confer the antioxidant effects of berries. Black currant and cranberry extracts inhibit oxidative stress in microglial and cardiomyocyte cell lines. Black currant extract was more effective in reducing oxidative stress in the HL-1 cells, whereas cranberry extract was comparable in reducing oxidative stress in the BV-2 cells. The results suggest that berry flavonoids exert neuro- and cardioprotective effects.     

Effect of Ultrasound and Enzymatic Mash Treatment on Bioactive Compounds and Antioxidant Capacity of Black,Red and White Currant Juices

Ultrasound treatment is recognized as a potential technique for improvement in the nutritional values of fruit juices. This study was initiated with the objective of evaluating bioactive compounds and some important quality parameters of black (BC), red (RC) and white (WC) currant juices obtained from fruit mash preliminarily treated by enzymes combined with ultrasound. Individual and total phenolic content (TPC), anthocyanins, color parameters, ascorbic acid, antioxidant capacity (TEAC), juice yield, pH, titratable acidity, and soluble solids were investigated. Significant increases in the levels of TPC and antioxidant capacity of sonicated samples were observed. However, ultrasound treatment had no effect on individual phenolic compounds of juices. Sonication of mash before juice pressing did not cause any noticeable changes in ascorbic acid content. Only in the case of WC was an increase in content of vitamin C noticed. The color of juices obtained after treatment was similar to the control sample. It was demonstrated that enzymatic combined with ultrasound treatment of mash for different colored currant fruit did not have any dismissive effect and could even improve some parameters of the juice obtained.     

Evaluation of Anthocyanin Content,Antioxidant Potential and Antimicrobial Activity of Black,Purple and Blue Colored Wheat Flour and Wheat-Grass Juice against Common Human Pathogens

The present study aimed to analyze the antioxidant and antimicrobial activity of anthocyanins extracted from colored wheat flour and wheat-grass juice against human pathogens. The total anthocyanin content and antioxidant potential in colored wheat flour and wheat-grass juice extracts were significantly higher than white flour and wheat-grass juice extracts. Ultra-performance liquid chromatography showed the maximum number of anthocyanin peaks in black wheat, with delphinidin-3-o-galactoside chloride, delphinidin-3-o-glucoside chloride, and cyanindin-3-o-glucoside chloride as the major contributors. Among flour extracts, maximum zones of inhibition against Staphylococcus aureus (MTCC 1934), Pseudomonas aeruginosa (MTCC 1434), Escherichia coli, and Candida albicans (MTCC 227) were produced by black flour extract, having the highest anthocyanin content. It exhibited a minimum microbicidal concentration (MMC) of 200 mg/mL against E. coli and C. albicans; and 100 and 150 mg/mL against S. aureus and P. aeruginosa, respectively. Black and purple flour extracts exhibited a minimum inhibitory concentration (MIC) of 50 mg/mL against S. aureus and P. aeruginosa. White flour extracts did not show MMC against E. coli and C. albicans. Among wheat-grass juice extracts, black wheat-grass was most effective and showed an MIC of 100–150 mg/mL against all pathogens. It exhibited an MMC of 200 mg/mL against S. aureus and P. aeruginosa. Hence, anthocyanin-rich colored wheat could be of nutraceutical importance.     

Camu-Camu Fruit Extract Inhibits Oxidative Stress and Inflammatory Responses by Regulating NFAT and Nrf2 Signaling Pathways in High Glucose-Induced Human Keratinocytes

Myrciaria dubia (HBK) McVaugh (camu-camu) belongs to the family Myrtaceae. Although camu-camu has received a great deal of attention for its potential pharmacological activities, there is little information on the anti-oxidative stress and anti-inflammatory effects of camu-camu fruit in skin diseases. In the present study, we investigated the preventative effect of 70% ethanol camu-camu fruit extract against high glucose-induced human keratinocytes. High glucose-induced overproduction of reactive oxygen species (ROS) was inhibited by camu-camu fruit treatment. In response to ROS reduction, camu-camu fruit modulated the mitogen-activated protein kinases (MAPK)/activator protein-1 (AP-1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor of activated T cells (NFAT) signaling pathways related to inflammation by downregulating the expression of proinflammatory cytokines and chemokines. Furthermore, camu-camu fruit treatment activated the expression of nuclear factor E2-related factor 2 (Nrf2) and subsequently increased the NAD(P)H:quinone oxidoreductase1 (NQO1) expression to protect keratinocytes against high-glucose-induced oxidative stress. These results indicate that camu-camu fruit is a promising material for preventing oxidative stress and skin inflammation induced by high glucose level.     

Inhibition of UVB-mediated oxidative stress and markers of photoaging in immortalized HaCaT keratinocytes by pomegranate polyphenol extract POMx

In recent years there has been an increase in use of botanicals with antioxidant properties as skin photoprotective agents. Pomegranate (Punica granatum L.) fruit possesses strong antioxidant and antiinflammatory properties. Recently, we have shown that pomegranate-derived products rich in anthocyanidins and ellagitannins inhibit UVB-mediated activation of nuclear factor kappa B and modulate UVA-mediated cell proliferation pathways in normal human epidermal keratinocytes. In this study, we evaluated the effect of polyphenol-rich pomegranate fruit extract (POMx) on UVB-induced oxidative stress and photoaging in human immortalized HaCaT keratinocytes. Our data show that pretreatment of HaCaT cells with POMx (10-40 microg mL(-1)) inhibited UVB (15-30 mJ cm(-2))-mediated (1) decrease in cell viability, (2) decrease in intracellular glutathione content and (3) increase in lipid peroxidation. Employing immunoblot analysis we found that pretreatment of HaCaT cells with POMx inhibited UVB-induced (1) upregulation of MMP-1, -2, -7 and -9, (2) decrease in TIMP-1, (3) phosphorylation of MAPKs and (iv) phosphorylation of c-jun, whereas no effect was observed on UVB-induced c-fos protein levels. These results suggest that POMx protects HaCaT cells against UVB-induced oxidative stress and markers of photoaging and could be a useful supplement in skin care products.     

Antioxidant Extract from Cleistocalyx nervosum var. paniala Pulp Ameliorates Acetaminophen-Induced Acute Hepatotoxicity in Rats

The indigenous purplish red fruit, Cleistocalyx nervosum var. paniala (CN), is grown in northern Thailand. The aqueous extract of CN pulp is known to exhibit antioxidant and anticarcinogenic properties. To search for an antioxidant fraction separated from CN, various hydroalcoholic extractions were performed. The acidified ethanolic extract of CN obtained from 0.5% (v/v) citric acid in 80% (v/v) ethanol yielded greater polyphenol content and DPPH radical scavenging activity when compared with other hydroethanolic extracts. Cyanidin-3-glucoside is a major anthocyanin present in the acidified ethanolic extract of CN (AECN). At a dose of 5000 mg/kg bw, an anthocyanin-rich extract was found to be safe when given to rats without any acute toxicity. To examine the hepatoprotective properties of AECN, an overdose of acetaminophen (APAP) was induced in a rat model, while silymarin was used as a standard reference. The administration of AECN at a dose of 300 mg/kg bw for 28 days improved hepatocyte architecture and modulated serum alanine aminotransferase levels in APAP-induced rats. Furthermore, it significantly decreased serum and hepatic malondialdehyde levels but increased hepatic glutathione content, as well as glutathione peroxidase and UDP-glucuronosyltransferase activities. In conclusion, AECN may effectively reduce oxidative stress induced acute hepatotoxicity in overdose APAP-treated rats through the suppression of oxidative stress and the enhancement of the antioxidant system in rat livers.     

Targeting Inflammation by Anthocyanins as the Novel Therapeutic Potential for Chronic Diseases: An Update

Low-grade chronic inflammation (LGCI) and oxidative stress act as cooperative and synergistic partners in the pathogenesis of a wide variety of diseases. Polyphenols, including anthocyanins, are involved in regulating the inflammatory state and activating the endogenous antioxidant defenses. Anthocyanins’ effects on inflammatory markers are promising and may have the potential to exert an anti-inflammatory effect in vitro and in vivo. Therefore, translating these research findings into clinical practice would effectively contribute to the prevention and treatment of chronic disease. The present narrative review summarizes the results of clinical studies from the last 5 years in the context of the anti-inflammatory and anti-oxidative role of anthocyanins in both health and disease. There is evidence to indicate that anthocyanins supplementation in the regulation of pro-inflammatory markers among the healthy and chronic disease population. Although the inconsistencies between the result of randomized control trials (RCTs) and meta-analyses were also observed. Regarding anthocyanins’ effects on inflammatory markers, there is a need for long-term clinical trials allowing for the quantifiable progression of inflammation. The present review can help clinicians and other health care professionals understand the importance of anthocyanins use in patients with chronic diseases.     

Protective activity of an anthocyanin-rich extract from bilberries and blackcurrants on acute acetaminophen-induced hepatotoxicity in rats

Acetaminophen (N-acetyl-p-aminophenol, APAP) overdosage can produce fatal centrilobular hepatic necrosis in humans. The present study attempted to investigate the protective effect of an anthocyanin-rich extract from bilberries and blackcurrants (AE) against APAP-induced acute hepatic damage in rats. Treatment with AE normalised blood activities of glutamate oxaloacetate and glutamate pyruvate transaminase and prevented APAP-induced plasmatic and tissutal alterations in biomarkers of oxidative stress, probably due to various bioproperties of the components of the extract.     

Clinical evaluation of Moro (Citrus sinensis (L.) Osbeck) orange juice supplementation for the weight management

In the last years, several studies have recently evaluated the beneficial effects of red orange juice (Citrus sinensis (L.) Osbeck) and its active components in weight management and obesity. Moro orange is a cultivar of red orange, particularly rich in active compounds such as anthocyanins, hydroxycinnamic acids, flavone glycosides and ascorbic acid, which displays anti-obesity effects in in vitro and in vivo studies. In this clinical study, the effect of a Moro juice extract (Morosil^®, 400 mg/die) supplementation was evaluated in overweight healthy human volunteers for 12 weeks. Results showed that Moro juice extract intake was able to induce a significant reduction in body mass index (BMI) after 4 weeks of treatment (p < 0.05). Moreover, in subjects treated with Moro extract, body weight, BMI, waist and hip circumference were significantly different from the placebo group (p < 0.05). In conclusion, it could be suggested that the active compounds contained in Moro juice have a synergistic effect on fat accumulation in humans and Moro juice extract can be used in weight management and in the prevention of human obesity.     

Blueberry Anthocyanins from Commercial Products: Structure Identification and Potential for Diabetic Retinopathy Amelioration

The aim of the present study was to determine the major anthocyanins of blueberry extracts from northeast China and explore their vision health improvement effects. HPLC-Q-TOF-MS/MS results suggested that six different anthocyanins were accurately identified, among which the Cy-3-glu (C3G) was the most abundant, ranging from 376.91 ± 7.91 to 763.70 ± 4.99 μM. The blueberry extract contained a higher purity of anthocyanins, and the anthocyanosides reached 342.98 mg/kg. The anti-oxidative stress function of C3G on HG-treated ARPE-19 cells were evaluated, and showed that the GSSG level of HG-cells pretreated with 10 μM C3G was significantly decreased, while the Nrf2 and NQO1 gene expression levels were increased. Further molecular docking (MD) results indicated that the C3G displayed favorable binding affinity towards REDD1, and only the B-ring of the C3G molecule displayed binding interactions with the CYS-140 amino acids within the REDD1 protein. It implied that the oxidative stress amelioration effects of C3G on the ARPE-19 cells were related to the REDD1 protein, which was probably via the Nrf2 pathways, although further studies are needed to provide mechanism evidence. The present study provides novel insights into understanding the roles of blueberry anthocyanins in ameliorating oxidative stress-induced BRB damage in the retina.     

Co-Microencapsulated Black Rice Anthocyanins and Lactic Acid Bacteria: Evidence on Powders Profile and In Vitro Digestion

Two multi-functional powders, in terms of anthocyanins from black rice (Oryza sativa L.) and lactic acid bacteria (Lactobacillus paracasei, L. casei 431^®) were obtained through co-microencapsulation into a biopolymer matrix composed of milk proteins and inulin. Two extracts were obtained using black rice flour as a raw material and hot water and ethanol as solvents. Both powders (called P1 for aqueous extract and P2 for ethanolic extract) proved to be rich sources of valuable bioactives, with microencapsulation efficiency up to 80%, both for anthocyanins and lactic acid bacteria. A higher content of anthocyanins was found in P1, of 102.91 ± 1.83 mg cyanindin-3-O-glucoside (C3G)/g dry weight (DW) when compared with only 27.60 ± 17.36 mg C3G/g DW in P2. The morphological analysis revealed the presence of large, thin, and fragile structures, with different sizes. A different pattern of gastric digestion was observed, with a highly protective effect of the matrix in P1 and a maximum decrease in anthocyanins of approximatively 44% in P2. In intestinal juice, the anthocyanins decreased significantly in P2, reaching a maximum of 97% at the end of digestion; whereas in P1, more than 45% from the initial anthocyanins content remained in the microparticles. Overall, the short-term storage stability test revealed a release of bioactive from P2 and a decrease in P1. The viable cells of lactic acid bacteria after 21 days of storage reached 7 log colony forming units (CFU)/g DW.     

Ultrasound-Assisted Deep Eutectic Solvent Extraction of Anthocyanins from Blueberry Wine Residues: Optimization,Identification, and HepG2 Antitumor Activity

Blueberry wine residues produced during the wine-brewing process contain abundant anthocyanins and other bioactive compounds. To extract anthocyanins from blueberry wine residues more efficiently, a novel procedure of ultrasound-assisted deep eutectic solvent extraction (UADESE) was proposed in this work. The extraction process was optimized by response surface methodology coupled with genetic algorithm. The optimum extraction parameters to achieve the highest yield of anthocyanins (9.32 ± 0.08 mg/g) from blueberry wine residues by UADESE were obtained at water content of 29%, ultrasonic power of 380 W, extraction temperature of 55 °C, and extraction time of 40 min. The AB-8 macroporous resin combined with Sephadex LH-20 techniques was used to purify the crude extract (CE) obtained under optimum extraction conditions and analyze the anthocyanins composition by HPLC-ESI-MS/MS. The cyanidin-3-rutinoside with purity of 92.81% was obtained. The HepG2 antitumor activity of CE was better than that of the purified anthocyanins component. Moreover, CE could increase the intracellular reactive oxygen species levels and the apoptosis, and arrest HepG2 cells in the S phases. These findings provided an effective and feasible method for anthocyanins extraction, and reduced the environmental burden of this waste.     

Modeling and analysis of vacuum membrane distillation for the recovery of volatile aroma compounds from black currant juice

A vacuum membrane distillation (VMD) model has been developed and validated with experimental data. The model consists of an extended transport model for the VMD process and is able to predict the effects of concentration and temperature polarization on the overall process performance. To validate the model, first it was tested with few experimental case studies from literature [S. Bandini, G.G. Sarti, Heat and mass transport resistances in vacuum membrane distillation per drop, AIChE J. 45 (7) (1999) 1422–1433; K.W. Lawson, D.R. Lloyd, Membrane distillation. I. Module design and performance evaluation using vacuum membrane distillation, J. Membr. Sci. 120 (1996) 111–121; A.M. Urtiaga, G. Ruiz, I. Ortiz, Kinetic analysis of the vacuum membrane distillation of chloroform from aqueous solutions, J. Membr. Sci. 165 (2000) 99–110]. Then the VMD model has been validated with experimental data collected from the recovery of aroma compounds from black currant [R.B. Jørgensen, A.S. Meyer, C. Varming, G. Jonsson, Recovery of volatile aroma compounds from black currant juice by vacuum membrane distillation, J. Food Eng. 64 (2004) 23–31]. In this work, recovery of 12 characteristic volatile aroma compounds from black currant juice has been studied. The simulated results from the VMD model, in terms of aroma concentration in the permeate have been compared with those obtained from laboratory experiments. The validated model has been used to study the effects of various process and membrane parameters on the concentration factor. The physical properties of various aroma compounds have been predicted using group contribution method as a function of temperature.     

Antiinflammatory effect of Graptophyllum pictum (L.) Griff

The red leaves of Graptophyllum pictum (L.) Griff. (G. pictum) are used in Indonesian folk medicine. The present study was carried out to elucidate the antiinflammatory effect of the ethanol extract obtained from this crude drug. The extract was partitioned between ether and water, and then the water-soluble fraction was extracted with 1-butanol. The 1-butanol-soluble fraction was extracted with chloroform-acetone, hot methanol and water, successively, and the hot methanol-soluble fraction (fr.) was chromatographed (frs. I-III). The antiinflammatory activity of these fractions was investigated on carrageenin-induced edema in rats and acetic acid-induced vascular permeability as well as the writhing symptom in mice. The ethanol extract (p.o.) showed an antiinflammatory activity as well as an analgesic activity and these activities shifted to the water-soluble fraction, 1-butanol-soluble fraction, methanol-soluble fraction and fr. II, successively. It was found that fr. II contained flavonoids. These results suggest that these flavonoids are at least partly responsible for the antiinflammatory effect of the ethanol extract of G. pictum.     

Anthocyanins from Aristotelia chilensis Prevent Olanzapine-Induced Hepatic-Lipid Accumulation but Not Insulin Resistance in Skeletal Muscle Cells

Type 2 diabetes and obesity are major problems worldwide and dietary polyphenols have shown efficacy to ameliorate signs of these diseases. Anthocyanins from berries display potent antioxidants and protect against weight gain and insulin resistance in different models of diet-induced metabolic syndrome. Olanzapine is known to induce an accelerated form of metabolic syndrome. Due to the aforementioned, we evaluated whether delphinidin-3,5-O-diglucoside (DG) and delphinidin-3-O-sambubioside-5-O-glucoside (DS), two potent antidiabetic anthocyanins isolated from Aristotelia chilensis fruit, could prevent olanzapine-induced steatosis and insulin resistance in liver and skeletal muscle cells, respectively. HepG2 liver cells and L6 skeletal muscle cells were co-incubated with DG 50 μg/mL or DS 50 μg/mL plus olanzapine 50 μg/mL. Lipid accumulation was determined in HepG2 cells while the expression of p-Akt as a key regulator of the insulin-activated signaling pathways, mitochondrial function, and glucose uptake was assessed in L6 cells. DS and DG prevented olanzapine-induced lipid accumulation in liver cells. However, insulin signaling impairment induced by olanzapine in L6 cells was not rescued by DS and DG. Thus, anthocyanins modulate lipid metabolism, which is a relevant factor in hepatic tissue, but do not significantly influence skeletal muscle, where a potent antioxidant effect of olanzapine was found.     

Antiangiogenic and Antioxidant In Vitro Properties of Hydroethanolic Extract from açaí (Euterpe oleracea) Dietary Powder Supplement

The Euterpe oleracea fruit (açaí) is a promising source of polyphenols with health-promoting properties. To our knowledge, few studies have focused on the influence of açaí phytochemicals on angiogenesis, with a significant impact on cancer. This study aimed at investigating the phytochemical profile of a purple açaí hydroethanolic extract (AHE) obtained from a commercial dietary powder supplement by high-performance liquid chromatography coupled to diode array detection and electrospray ionization mass spectrometry, and evaluate its in vitro effects on distinct angiogenic steps during vessel growth and on oxidative markers in human microvascular endothelial cells (HMEC-1). The phenolic profile of AHE revealed the presence of significant levels of anthocyanins, mainly cyanidin-3-O-rutinoside, and other flavonoids with promising health effects. The in vitro studies demonstrated that AHE exerts antiangiogenic activity with no cytotoxic effect. The AHE was able to decrease HMEC-1 migration and invasion potential, as well as to inhibit the formation of capillary-like structures. Additionally, AHE increased antioxidant defenses by upregulating superoxide dismutase and catalase enzymatic activities, accompanied by a reduction in the production of reactive oxygen species. These data bring new insights into the potential application of angiogenic inhibitors present in AHE on the development of novel therapeutic approaches for angiogenesis-dependent diseases.     

Lactic Acid Bacteria Simultaneously Encapsulate Diverse Bioactive Compounds from a Fruit Extract and Enhance Thermal Stability

This study develops an innovative cell-based carrier to simultaneously encapsulate multiple phytochemicals from a complex plant source. Muscadine grapes (MG) juice prepared from fresh fruit was used as a model juice. After incubation with inactivated bacterial cells, 66.97% of the total anthocyanins, and 72.67% of the total antioxidant compounds were encapsulated in the cells from MG juice. Confocal images illustrated a uniform localization of the encapsulated material in the cells. The spectral emission scans indicated the presence of a diverse class of phenolic compounds, which was characterized using high-performance liquid chromatography (HPLC). Using HPLC, diverse phytochemical compound classes were analyzed, including flavanols, phenolic acid, hydroxycinnamic acid, flavonols, and polymeric polyphenols. The analysis validated that the cell carrier could encapsulate a complex profile of bioactive compounds from fruit juice, and the encapsulated content and efficiencies varied by the chemical class and compound. In addition, after the heat treatment at 90 °C for 60 min, >87% total antioxidant capacity and 90% anthocyanin content were recovered from the encapsulated MG. In summary, these results highlight the significant potential of a selected bacterial strain for simultaneous encapsulation of diverse phenolic compounds from fruit juice and improving their process stability.     

Analysis of anthocyanins in commercial fruit juices by using nano-liquid chromatography-electrospray-mass spectrometry and high-performance liquid chromatography with UV-vis detector

Nano-LC and conventional HPLC techniques were applied for the analysis of anthocyanins present in commercial fruit juices using a capillary column of 100 μm id and a 2.1 mm id narrow-bore C(18) column. Analytes were detected by UV-Vis at 518 nm and ESI-ion trap MS with HPLC and nano-LC, respectively. Commercial blueberry juice (14 anthocyanins detected) was used to optimize chromatographic separation of analytes and other analysis parameters. Qualitative identification of anthocyanins was performed by comparing the recorded mass spectral data with those of published papers. The use of the same mobile phase composition in both techniques revealed that the miniaturized method exhibited shorter analysis time and higher sensitivity than narrow-bore chromatography. Good intra-day and day-to-day precision of retention time was obtained in both methods with values of RSD less than 3.4 and 0.8% for nano-LC and HPLC, respectively. Quantitative analysis was performed by external standard curve calibration of cyanidin-3-O-glucoside standard. Calibration curves were linear in the concentration ranges studied, 0.1-50 and 6-50 μg/mL for HPLC-UV/Vis and nano-LC-MS, respectively. LOD and LOQ values were good for both methods. In addition to commercial blueberry juice, qualitative and quantitative analysis of other juices (e.g. raspberry, sweet cherry and pomegranate) was performed. The optimized nano-LC-MS method allowed an easy and selective identification and quantification of anthocyanins in commercial fruit juices; it offered good results, shorter analysis time and reduced mobile phase volume with respect to narrow-bore HPLC.     

The RP-HPLC analysis of anthocyanins

Summary Conditions were determined for the separation of a complex set of anthocyanins (free aglycones, mono- and multiglycosides and esterified forms) by HPLC. The optimised gradient elution method was then used to carry out qualitative and quantitative analysis of anthocyanin compounds present in the callus tissue ofRudbeckia hirta L. and the tubular flowers of the soil-based plant. The summary content of anthocyanin pigments and the content of the main pigment was identified in the analysed biomass. The method developed is useful for the purposes of monitoring the process of biosynthesis of anthocyanins in tissues obtained through in vitro cultures. The advantages of the method for anthocyanins and its application to other anthocyanin-rich materials are also discussed.