Anthocyanin-rich Berry Extracts Research Articles

Anthocyanin-Rich Berry Extracts Affect SN-38-Induced Response: A Comparison of Non-Tumorigenic HCEC-1CT and HCT116 Colon Carcinoma Cells.

Chemotherapy with irinotecan (CPT-11), the pro-drug of the highly cytotoxic SN-38, is among the standard-of-care treatments for colorectal cancer. To counteract undesired toxic side effects on healthy tissue such as the intestinal epithelium, the use of preparations rich in polyphenols with anti-oxidative and anti-inflammatory properties such as anthocyanins has been proposed. In the present study, the question of whether non-tumorigenic human epithelium cells (HCEC-1CT) can be protected against the cytotoxic impact of SN-38 by anthocyanin-rich polyphenol extracts without compromising the desired therapeutic effect against tumor cells (HCT-116) was addressed. Hence, single and combinatory effects of anthocyanin-rich polyphenol extracts of elderberry (EB), bilberry (Bil), blackberry (BB) and black currant (BC) with the chemotherapeutic drug SN-38 were investigated. Out of the extracts, BB showed the most potent concentration-dependent cytotoxicity alone and in combination with SN-38, with even stronger effects in non-tumorigenic HCEC-1CT cells. In cytotoxic concentrations, BB decreased the level of DNA/topoisomerase I covalent complexes in HCEC-1CT cells below base level but without concomitant reduction in SN-38-induced DNA strand breaks. The herein reported data argue towards an interference of anthocyanins with successful treatment of cancer cells and a lack of protective properties in healthy cells.

In Vitro Inhibitory Potential of Different Anthocyanin-Rich Berry Extracts in Murine CT26 Colon Cancer Cells

Anti-oxidant, -inflammatory, and -carcinogenic activities of bioactive plant constituents, such as anthocyanins, have been widely discussed in literature. However, the potential interaction of anthocyanin-rich extracts with routinely used chemotherapeutics is still not fully elucidated. In the present study, anthocyanin-rich polyphenol extracts of blackberry (BB), bilberry (Bil), black currant (BC), elderberry (EB), and their respective main anthocyanins (cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-sambubioside) were investigated concerning their cytotoxic and DNA-damaging properties in murine CT26 cells either alone or in combination with the chemotherapeutic agent SN-38. BB exerted potent cytotoxic effects, while Bil, BC, and EB only had marginal effects on cell viability. Single anthocyanins comprised of the extracts could not induce comparable effects. Even though the BB extract further pronounced SN-38-induced cytotoxicity and inhibited cell adhesion at 100–200 µg/mL, no effect on DNA damage was observed. In conclusion, anti-carcinogenic properties of the extracts on CT26 cells could be ranked BB >> BC ≥ Bil ≈ EB. Mechanisms underlying the potent cytotoxic effects are still to be elucidated since the induction of DNA damage does not play a role.

Three anthocyanin-rich berry extracts regulate the in vitro digestibility of corn starch: Physicochemical properties, structure and α-amylase

This study aimed to compare the regulatory effects of blue honeysuckle anthocyanins (BHA), blueberry anthocyanins (BBA), and blackcurrant anthocyanins (BCA) on the in vitro digestibility of corn starch in terms of starch physicochemical properties and structure, as well as α-amylase inhibition. The results revealed that adding all three anthocyanins lowered digestibility in the following order: BHA > BCA > BBA. The terminal digestibility (C∞) decreased from 73.84 % to 57.3 % with the addition of 10 % BHA, while the resistant starch (RS) content increased from 4.39 % to 48.82 %. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis indicated that anthocyanins and starch interacted through noncovalent bonds. Differential scanning calorimetry (DSC) analysis showed that the gelatinization enthalpy was dramatically lowered in all three anthocyanin groups, with 10 % BHA producing a 38.58 % drop. Rheological property analysis showed that anthocyanins increased the apparent viscosity and modulus with starch. The interaction between anthocyanin and α-amylase was mainly through the formation of hydrogen bonds and hydrophobic forces. This research provides theoretical guidance for developing low glycemic index (GI) anthocyanin starch-based foods.

Anthocyanin Rich-Berry extracts coated magnetic Fe3O4 bionanocomposites and their antibacterial activity

The relentless evolution of antibiotic-resistant bacteria has made treating bacterial infections with conventional antibiotics increasingly difficult. Consequently, there is an urgent demand for innovative strategies that demonstrate exceptional antibacterial efficiency to effectively combat these resilient pathogens. This study aimed to investigate the efficiency of extraction of anthocyanins-coated iron oxide nanoparticles (Fe3O4 NPs) as an antibacterial agent against Escherichia coli and Staphylococcus aureus-902. The anthocyanins were extracted from three different berries: mulberry, blackberry, and blackcurrant. The Fe3O4 NPs were synthesized using the co-precipitation method with FeCl2·4H2O and FeCl3·6H2O metal precursors. Furthermore, anthocyanins were coated onto the surface of Fe3O4 through a simple sonication process, followed by the freeze-drying, resulting in Fe3O4/bionanocomposites (BNC). The mulberry, blackberry, and black currant-derived anthocyanins coated Fe3O4 were labelled as Fe3O4/MB, Fe3O4/BB, and Fe3O4/BC, respectively. The resulting Fe3O4/BNCs were analyzed using various spectroscopy and microscopic techniques. The XRD results showed that the Fe3O4/BNC formed a face-centred cubic structure, with particle size determined debye-Scherrer’s equation, and the obtained particle size was 15–18 nm. Finally, the obtained Fe3O4/BNC was used as an antibacterial agent against Escherichia coli and Staphylococcus aureus-902. The Fe3O4/BNC exhibited strong antibacterial activity against bacterial species. Thus, this study offers a competitive approach to create recyclable antibacterial materials with specific antibacterial therapeutic properties.

Bilberry extract, its major polyphenolic compounds, and the soy isoflavone genistein antagonize the cytostatic drug erlotinib in human epithelial cells.

Erlotinib (Tarceva®) is a chemotherapeutic drug approved for the treatment of pancreatic cancer and non-small cell lung cancer. Its primary mode of action is the inhibition of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK). Recently, RTK-inhibiting polyphenols have been reported to interact synergistically with erlotinib. Furthermore some anthocyanidins and anthocyanin-rich berry extracts have been reported to inhibit tyrosine kinases, including the EGFR, which raises the question of potential interactions with erlotinib. Polyphenol-rich preparations such as berry- or soy-based products are commercially available as food supplements. In the present study we tested a bilberry extract, its major anthocyanin and potential intestinal degradation products, as well as genistein, with respect to possible interactions with erlotinib. Cell growth inhibition was assessed using the sulforhodamine B assay, while interactions with EGFR phosphorylation were analyzed by SDS-PAGE/western blotting with subsequent immunodetection. Genistein, bilberry extract, delphinidin-3-O-glucoside and delphinidin were found to antagonize erlotinib whereas phloroglucinol aldehyde was found to enhance cytostatic effects of the drug on human epithelial A431 cells. Genistein also antagonized the EGFR inhibitory effects of erlotinib, whereas bilberry anthocyanins showed no significant interactions in this regard. Our data indicate that different polyphenols are potentially able to impair the cytostatic effect of erlotinib in vitro. Genistein interacts via the modulation of erlotinib-mediated EGFR inhibition whereas bilberry anthocyanins modulated the growth-inhibitory effect of erlotinib without affecting EGFR phosphorylation, thus indicating a different mechanism of interference.

Regulation of Glucose Transporter Expression in Human Intestinal Caco-2 Cells following Exposure to an Anthocyanin-Rich Berry Extract

Polyphenols contained within plant tissues are consumed in significant amounts in the human diet and are known to influence a number of biological processes. This study investigated the effects of an anthocyanin-rich berry-extract on glucose uptake by human intestinal Caco-2 cells. Acute exposure (15 min) to berry extract (0.125%, w/v) significantly decreased both sodium-dependent (Total uptake) and sodium-independent (facilitated uptake) 3H-D-glucose uptake. In longer-term studies, SGLT1 mRNA and GLUT2 mRNA expression were reduced significantly. Polyphenols are known to interact directly with glucose transporters to regulate the rate of glucose absorption. Our in vitro data support this mechanism and also suggest that berry flavonoids may modulate post-prandial glycaemia by decreasing glucose transporter expression. Further studies are warranted to investigate the longer term effects of berry flavonoids on the management of glycaemia in human volunteers.

Can anthocyanins improve maintenance therapy of Ph+ acute lymphoblastic leukaemia?

Chemotherapy and tyrosine kinase inhibitors provide high remission rates. However, prognosis of adult patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia (Ph(+) ALL) still remains poor. Because most adults eventually relapse without allogeneic stem cell transplantation, which is not available for all patients, novel strategies are required for relapse prevention. As the integrity of the immune system is essential for the control of remaining leukaemia cells, we compared the efficacy of anthocyanins, imatinib and a DNA-based vaccine as non-immunosuppressant components with 6-mercaptopurine (6-MP) to control minimal residual disease in vitro and in vivo using different leukaemia cell lines and syngeneic mice. Proliferation of Ph(+) ALL was significantly better inhibited by anthocyanin-rich berry extract or imatinib compared with 6-MP. Although anthocyanins induced apoptosis in some leukaemia cell lines, the level of caspase-3, caspase-8 and caspase-9 was significantly lower compared with imatinib and 6-MP. When used as single components, anthocyanins and imatinib mesylate failed to eradicate pre-existing Ph(+) ALL in syngeneic mice, while 6-MP led to 10% and DNA vaccination to 56% survival. Intriguingly, only the combination of DNA vaccination with berry extract but not with the isolated anthocyanin, cyanidin-3-rutinoside or imatinib further increased leukaemia-free and overall survival, and 90% of lethally challenged mice survived. We suggest that induction and enhancement of a leukaemia-specific immunity by DNA vaccination and anthocyanin-rich berry extract can also decrease the relapse rate in patients with Ph(+) ALL. Furthermore, this approach may serve as strategy for maintenance therapy of other malignancies.

Anthocyanin‐rich extracts suppress the DNA‐damaging effects of topoisomerase poisons in human colon cancer cells

The effect of two anthocyanin-rich berry extracts (A, bilberry; B, red grape) on topoisomerases was investigated in a cell-free system and in human HT29 colon carcinoma cells. In parallel, their impact on DNA integrity was determined. The berry extracts suppressed the activity of topoisomerase I at concentrations ≥50 μg/mL. The activity of the topoisomerase II isoform was preferentially diminished (≥1 μg/mL). Within HT29 cells, the extracts were found to act as catalytic inhibitors without stabilizing the cleavable complex. Although topoisomerase activity was inhibited, none of the extracts induced DNA strand breaks up to 50 μg/mL. Moreover, pre- and coincubation of HT29 cells with A (≥1 μg/mL) significantly suppressed (p-value ≤0.001) the strand-breaking effects of camptothecin, whereas B was found to be less effective (1 μg/mL; p-value ≤0.05). Both extracts were found to significantly diminish doxorubicin-mediated DNA strand breaks at concentrations ≥1 μg/mL (p-value ≤0.001). Consistent with these results, the extracts suppressed doxorubicin-mediated enhancement of levels of topoisomerase II covalently linked to DNA in HT29 cells. These results raise the possibility that high intake of berry extracts may protect DNA and thus counteract the therapeutic effectiveness of orally applied topoisomerase poisons during chemotherapy.

Antileukemic Efficacy of Anthocyanins In-Vitro and In-Vivo and In Combination with a DNA Vaccine Against Ph+ Acute Lymphoblastic Leukemia

AbstractAbstract 3267Polychemotherapy has dramatically improved the outcome of patients with acute lymphoblastic leukemia (ALL) and leads to long term remission in 70–80 % of children and 40–50% of adults. However, it is likely that after remission the immune system plays an essential part in the eradication of minimal residual disease. Since chemotherapy compromises the immune system, compounds without immunosuppressant activity should be developed for maintenance therapy in order to reduce the relapse rate. Anthocyanins are a group of naturally occurring phenolic compounds widely available in fruits and vegetables in human diets. They have broad biological activities including antimutagenesis and anticarcinogenesis, which are generally attributed to their antioxidant activities.We studied the effects and the mechanisms of anthocyanin-rich berry extract and the most common types of anthocyanins, cyanidin-3-rutinoside and dephinidin, in several leukemia cell lines. Proliferation was significantly reduced in the murine Ph+ ALL cell line BM185 by berry extract as well as by cyanidin-3-rutinoside and dephinidin which was comparable to the effect seen with imatinib or 6-mercaptourine and methotrexate. In the leukemia cell lines HL-60, Sup-B15 and K562 the anti-leukemia efficacy of berry extract was superior compared to cyanidin-3-rutinoside or dephinidin. Apoptosis was induced by upregulation of caspases 3 and 9.We then evaluated the therapeutic effect of berry extract and cyanidin-3-rutinoside with or without a DNA-based vaccine in a syngeneic Balb/c mouse model. Non-viral minimalistic immunogenically defined gene expression vectors (MIDGE) encoding a BCR-ABLp185 fusion specific peptide, GM-CSF and IL12 were used for in vivo transfection of murine skin. In addition, we used natural DNA-based double stem-loop immunomodulators (dSLIM), containing 6 CpG-motifs and exhibiting an unique structure, as non-specific immune adjuvant. One day after the application of a lethal challenge dose of the syngeneic BCR-ABLp185 expressing leukemia cell line BM185, mice received either i) daily oral applications of berry extract (500mg/kg/d) or ii) intraperitoneal applications of cyanidin-3-rutinoside (50 mg/kg/d) or iii) were immunized twice with the DNA vaccine BCR-ABL/GM-CSF/IL-12/dSLIM intracutaneously. The fourth group received both, cyanidin-3-rutinoside and the DNA-vaccine and the fifth group was treated with berry extract and the DNA-vaccine. While the treatment with berry extracts or cyanidin-3-rutinoside alone had no benefit compared to the control, mice which received the DNA-vaccine showed a significant longer tumor-free and overall survival compared to the control and a survival rate of 56%. Interestingly, the combined treatment with DNA-vaccine and berry extract but not with DNA-vaccine and cyanidin-3-rutinoside further improved the outcome and lead to a significant longer tumor-free and overall survival compared to the DNA-vaccine alone and a survival rate of 90%. In conclusion, we provide data that anthocyanin-rich berry extract combined with a leukemia-specific DNA-vaccine acts synergistically in the treatment of Ph+ ALL. This approach may have implications to optimize maintenance therapy in patients with Ph+ ALL. Disclosures:Schmidt:Mologen AG: Employment. Wittig:Mologen AG: Employment, Membership on an entity's Board of Directors or advisory committees.

Anthocyanin-rich red grape extract impedes adenoma development in the Apc Min mouse: Pharmacodynamic changes and anthocyanin levels in the murine biophase

Purpose Red grape pomace extract (oenocyanin) is a cheap and rich source of anthocyanins, the agents suggested to possess cancer chemopreventive properties. Here the hypothesis was tested that oenocyanin added to the diet can interfere with intestinal adenoma development in the Apc Min mouse, a model of intestinal carcinogenesis linked to an Apc mutation. Methods Mice received oenocyanin (0.3%) in their diet until week 16, when adenoma number and burden were recorded. Expression of Akt and ERK proteins was studied by Western blot in adenomas to discover effects of anthocyanins on cellular signalling via the PI3 and MAP kinase pathways. Levels of anthocyanins were measured by HPLC with visible spectroscopic or mass spectrometric detection. Results In mice which had consumed oenocyanin, overall adenoma burden was halved and adenoma number was marginally reduced when compared with mice on control diet. The proliferation index in colonic adenomatous crypts, as reflected by Ki-67 staining, was significantly decreased from 88.14% in control mice to 75.6 ± 4% in mice on oenocyanin ( P = 0.014). Expression of Akt in small intestinal adenomas from Apc Min mice on oenocyanin was reduced by 54% ( P = 0.003), when compared to controls. Oenocyanin anthocyanins and glucuronide metabolites were found in the urine and intestine but not in plasma. Conclusions The results suggest that oenocyanin may be a viable and economical alternative to anthocyanin-rich berry extracts for chemopreventive intervention. Akt and pErk might be suitable biomarkers of anthocyanin target organ efficacy.

Use of Accurate Mass Full Scan Mass Spectrometry for the Analysis of Anthocyanins in Berries and Berry-Fed Tissues

Anthocyanins in extracts from raspberries and blueberries were analyzed by reversed-phase HPLC coupled to a high-resolution Exactive Orbitrap mass spectrometer (HR-MS) with a resolution of 100,000, operated with an electrospray source in the positive ionization mode. As consumption of anthocyanin-rich berry extracts has been associated with improved cognitive function, brain extracts from European greenfinches ( Carduelis chloris ) that had been fed one blackberry daily for a period of 2 weeks were analyzed by both HPLC with traditional tandem MS in the selected reaction monitoring mode and HPLC-HR-MS. Cyanidin-3-O-glucoside was detected in the brain extracts by both methods, but because of its high level of selectivity, HR-MS was ca. 200-fold more sensitive. A further advantage of HR-MS is that unlike MS-SRM it enables both targeted and nontargeted compounds to be detected and much lower limits of detection are achieved without compromising the selectivity of the analysis.

Berry Phenolic Extracts Modulate the Expression of p21WAF1 and Bax but Not Bcl-2 in HT-29 Colon Cancer Cells

Previous studies have shown that anthocyanin-rich berry extracts inhibit the growth of cancer cells in vitro. The objective of this study was to compare the effects of berry extracts containing different phenolic profiles on cell viability and expression of markers of cell proliferation and apoptosis in human colon cancer HT-29 cells. Berry extracts were prepared with methanol extraction, and contents of the main phenolic compounds were analyzed using HPLC. Anthocyanins were the predominant phenolic compounds in bilberry, black currant, and lingonberry extracts and ellagitannins in cloudberry extract, whereas both were present in raspberry and strawberry extracts. Cells were exposed to 0-60 mg/mL of extracts, and the cell growth inhibition was determined after 24 h. The degree of cell growth inhibition was as follows: bilberry > black currant > cloudberry > lingonberry > raspberry > strawberry. A 14-fold increase in the expression of p21WAF1, an inhibitor of cell proliferation and a member of the cyclin kinase inhibitors, was seen in cells exposed to cloudberry extract compared to other berry treatments (2.7-7-fold increase). The pro-apoptosis marker, Bax, was increased 1.3-fold only in cloudberry- and bilberry-treated cells, whereas the pro-survival marker, Bcl-2, was detected only in control cells. The results demonstrate that berry extracts inhibit cancer cell proliferation mainly via the p21WAF1 pathway. Cloudberry, despite its very low anthocyanin content, was a potent inhibitor of cell proliferation. Therefore, it is concluded that, in addition to anthocyanins, also other phenolic or nonphenolic phytochemicals are responsible for the antiproliferative activity of berries.

Total phenolic content of anthocyanin rich berry extracts reduces reactive oxygen species generated from the xanthine:xanthine oxidase reaction

We have reported previously that extracts from chokeberry (Ch), bilberry (B) and elderberry (E) reduce damage to coronary arteries following either external or internal exposure to reactive oxygen species (ROS) with Ch having the highest efficacy. We examined whether these extracts could reduce ROS generated from a reaction associated with myocardial ischemia and reperfusion. Generation of ROS from the xanthine: xanthine oxidase reaction (X:XO) was assessed through the chemiluminescence of the reaction of ROS generated with luminol in a 37°C controlled well plate luminometer. Extracts at 0.05 mg total anthocyanins (Ta) /L reduced ROS generation from X:XO (Initial RLU C=2379±71, Ch =1479±72, B=1301±72, E=1832±54. p<0.05, E vs Ch, B n=6). When extracts were normalized to the same total phenolic concentration of Ch the reduction of ROS generation was similar (Initial RLU C = 2371±71, Ch= 1423±44, B=1459± 50, E=1201±42). Similar trends were observed when Ta of the extracts was reduced to 0.005 mg Ta/L. We conclude that anthocyanin rich berry extracts can reduce ROS generated from a reaction associated with ischemia reperfusion injury. This effect is most related to the total phenolic concentration in the extracts and indicates that naturally derived polyphenolic compounds may serve as protectants against ROS injury associated with myocardial ischemia. (Supported by grants from the Lutheran Foundation of Indiana and IUSM -REG)

Anti-angiogenic, antioxidant, and anti-carcinogenic properties of a novel anthocyanin-rich berry extract formula.

Edible berry anthocyanins possess a broad spectrum of therapeutic and anti-carcinogenic properties. Berries are rich in anthocyanins, compounds that provide pigmentation to fruits and serve as natural antioxidants. Anthocyanins repair and protect genomic DNA integrity. Earlier studies have shown that berry anthocyanins are beneficial in reducing age-associated oxidative stress, as well as in improving neuronal and cognitive brain function. Six berry extracts (wild blueberry, bilberry, cranberry, elderberry, raspberry seeds, and strawberry) were studied for antioxidant efficacy, cytotoxic potential, cellular uptake, and anti-angiogenic (the ability to reduce unwanted growth of blood vessels, which can lead to varicose veins and tumor formation) properties. We evaluated various combinations of edible berry extracts and developed a synergistic formula, OptiBerry IH141, which exhibited high ORAC (Oxygen-Radical Absorbing Capacity) value, low cytotoxicity, and superior anti-angiogenic properties compared to the other combinations tested. Anti-angiogenic approaches to treat cancer represent a priority area in vascular tumor biology. OptiBerry significantly inhibited both H2O2- and TNF-alpha-induced VEGF (Vascular Endothelial Growth Factor) expression by human keratinocytes. VEGF is a key regulator of tumor angiogenesis. Matrigel assay using human microvascular endothelial cells showed that OptiBerry impaired angiogenesis. In an in vivo model of angiogenesis, OptiBerry significantly inhibited basal MCP-1 and inducible NF-kappaB transcriptions. Endothelioma cells pretreated with OptiBerry showed a diminished ability to form hemangioma and markedly decreased tumor growth by more than 50%. In essence, these studies highlight the novel anti-angiogenic, antioxidant, and anti-carcinogenic potential of a novel anthocyanin-rich berry extract formula, OptiBerry.