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AnThocyanin and polyphenol bioactives for Health Enhancement through Nutritional Advancement

Final Report Summary - ATHENA (AnThocyanin and polyphenol bioactives for Health Enhancement through Nutritional Advancement)

Executive Summary:
Anthocyanins are health promoting dietary polyphenols that protect against cardiovascular disease, cancer and obesity in preclinical studies with animals. The ATHENA project will examine how good dietary anthocyanins are in protecting against chronic disease, by addressing the following questions:
• Benefits and risks: What is the dose response to anthocyanin phytonutrients? Are anthocyanins from different food sources equivalent? How well do anthocyanins perform in promoting health compared to other polyphenol phytonutrients such as stilbenes, isoflavones and epicatechins?
• Mechanisms of action: What are the mechanisms of action of polyphenol phytonutrients in combating chronic diseases? How do anthocyanins limit weight gain/fat development? How do dietary anthocyanins offer cardioprotection? How do dietary anthocyanins slow the progression of cancers?
• Food or Pharma: Supplements or extracts of polyphenols do not appear to promote health as well as when they are consumed in whole foods. What is the influence of nutritional context on the efficacy of polyphenol phytonutrients? Does nutritional context influence the bioavailability of polyphenol phytonutrients?
• Roles in human: Do dietary anthocyanins afford protection against cardiovascular disease, cancer and other chronic diseases in human?
It is the uniqueness of the interactions that ATHENA will promote, that will innovate and give the most significant added-value to Europe. ATHENA will bring together groups with widely different expertise from across Europe, so that the consortium will be able to achieve significant progress towards addressing the Grand Challenge of Chronic Disease. It will impact the health, well being and quality of life across Europe.

Project Context and Objectives:
In previous EU-funded projects members of the current consortium have expanded fundamentally the understanding of how polyphenols can promote health and combat a range of chronic diseases in a dietary context. The principal achievement of these research projects was to instate anthocyanins among the health promoting dietary polyphenols that are effective in offering cardioprotection, protection against cancer and limitation of weight gain from obesity-inducing, high fat diets, in preclinical studies with animals. The impact of this research is already being felt in terms of dietary recommendations and messages in the popular press. However, the progress made so far marks only the foundations of the understanding required for the role of dietary polyphenols in promoting health and combating chronic disease. Our investigations need to be extended to human studies, and a number of new questions need to be addressed so that the understanding can be formulated into effective, accurate dietary recommendations and preventive medicine strategies.
A multi-disciplinary team of researchers will work together to address the objectives of ATHENA in four research-orientated work packages co-ordinated through a management-orientated work package which will also be responsible for dissemination of the results of the project to the public.
Overall goal and scientific and technological objectives
The overall objective of the ATHENA project is to provide a robust scientific foundation for improved dietary recommendations that include foods with high levels of anthocyanins and related polyphenols to promote health and to protect against chronic disease. Investigations need to be extended to human studies, and a number of questions have arisen that need to be addressed for understanding to be formulated into effective, accurate dietary recommendations and preventive medicine strategies.
The specific objectives are:
Benefits and risks: To determine what is the dose response to anthocyanin phytonutrients. To determine whether anthocyanins from different food sources are equivalent. To determine how well anthocyanins perform in promoting health compared to other polyphenol phytonutrients such as stilbenes, isoflavones and epicatechins.
Mechanisms of action: To determine the mechanisms of action of polyphenol phytonutrients in combating chronic diseases. To determine how anthocyanins limit weight gain/fat development. To determine how dietary anthocyanins offer cardioprotection. To determine how dietary anthocyanins slow the progression of cancers.
Food or Pharma: Supplements or extracts of polyphenols do not appear to promote health as well as when they are consumed in whole foods. We aim to determine the influence of nutritional context on the efficacy of polyphenol phytonutrients and whether nutritional context influences the bioavailability of polyphenol phytonutrients.
Roles in human: To explore whether dietary anthocyanins afford protection against cardiovascular disease, cancer and other chronic diseases in human.
Projected achievements of the project
1. The ATHENA project will define the dose response relationships between dietary anthocyanins (in different food contexts) and the promotion of health in preclinical studies and identify whether there is a possibility of excessive consumption of dietary anthocyanins.
2. It will identify whether anthocyanins from different food sources promote health to the same or different extents in preclinical animal studies.
3. It will determine whether dietary anthocyanins are comparable in their abilities to promote health to other better known but more nutritionally restricted polyphenols in preclinical animal studies.
4. It will develop models defining the mechanisms whereby dietary anthocyanins offer cardioprotection, limit cancer progression and limit weight gain on high-fat diets using nutrigenomics.
5. It will define the impact of anthocyanins in the diet on the epigenome, and establish the relationship between the nutrition of the mother and the predisposition of her offspring to chronic diseases.
6. It will compare the mechanisms of activity of dietary anthocyanins in promoting health with those of other better known but more nutritionally restricted polyphenols at the molecular, cellular and whole organism levels.
7. It will define whether anthocyanins supplied in purified forms can work as effectively as anthocyanins in a food context in promoting health in preclinical studies.
8. It will define whether the food context of polyphenol phytonutrients has a significant impact on the bioavailability of these bioactives.
9. It will determine whether longer term dietary interventions with anthocyanin-enriched food can offer protection against cardiovascular disease in human.
10. It will determine whether supplementation of diets with anthocyanin-enriched foods can limit the degree of oxidative DNA damage following radiation therapy in breast cancer patients.
11. It will determine whether there is a genetic component to the response to dietary polyphenol phytonutrients in patients with a predisposition to cardiovascular disease and contribute towards personalised medical and health programs.
12. It will take advantage of the synergies arising from the multidisciplinary interactions within the project, such that each group will benefit from the diverse yet complementary expertise, tools and resources available from the participating experts in plant biotechnology, genetics, organic chemistry, food technology, nutrition, experimental medicine and clinical epidemiology to make real advances in an area of enormous strategic relevance for Europe.
13. It will provide robust scientific data for new food formulations and dietary recommendations for healthy living and improved quality of life. It will contribute significantly to meeting the Grand Challenge of chronic, non-communicable diseases.

Project Results:
Main Science and Technology Results/Foregrounds of the ATHENA project:

Protective effects of dietary anthocyanins against cardiovascular disease

The ATHENA project has defined the dose response relationships between dietary anthocyanins (in different food contexts) and the promotion of health in preclinical studies. Epidemiological studies have shown an inverse relationship between consumption of anthocyanins (ACN) and risk of disease, particularly cardiovascular diseases (CVD). Supplementation of animal diets with anthocyanin-rich foods and extracts has been shown to reduce atherosclerosis, improve vascular function and alter gene expression. However, these studies cannot prove that anthocyanins cause the observed effects because these foods and extracts contain many other potentially bioactive components apart from anthocyanins. The major challenge that has prevented researchers from directly linking anthocyanins in foods with beneficial effects in humans is the lack of suitable control foods (i.e. similar foods that lack anthocyanins). Such foods also facilitate studies of the underlying mechanisms by which anthocyanins cause their observed positive effects on health.
An animal feeding study using genetically modified ‘purple tomatoes’ that accumulate high levels of anthocyanins and equivalent anthocyanin-free, red tomatoes was carried out to allow direct assessment of the ability of dietary anthocyanins to affect CVD risk. Increasing concentrations of purple tomatoes (2.5 5 and 10%) were used in chows containing a total of 10% w/w tomato and the effects on atherosclerotic aortal plaque, and lipid metabolism were compared to a red tomato-only supplemented chow and a no tomato chow. This novel, highly controlled dietary intervention in the ApoE-/- mouse model assessed the dose-response effects of anthocyanins from tomato on atherosclerotic plaque size and plasma lipid parameters.

The 10% purple tomato diet caused a 45% mean reduction in aortic lesion size compared to the 10% red tomato control diet (p<0.001). Further, there was a very clear linear dose-response (R2=0.999 p<0.0001 using average data) showing that the dietary anthocyanins had beneficial effects by reducing atherosclerosis in this mouse model, and that the positive effects did not seem to plateau out at higher doses, nor were there any indications of toxicity. Additionally, all purple tomato diets caused up to 53% reduction in total cholesterol in plasma compared to the 10% red tomato group. Mice fed with the two highest doses of purple tomatoes also showed significant mean reductions in LDL and a mean increase in HDL and ApoA-I in plasma. In conclusion, we have shown, for the first time, that consumption of tomato anthocyanins significantly reduces atherosclerosis and improves the plasma lipid profile in the ApoE-/- mouse model. These studies have also revealed that the beneficial effects of dietary anthocyanins do not even plateau with increasing doses, in preclinical trials, giving no evidence of toxicity.

Further analysis of the samples from animals in this trial have given mechanistic insight into the effects of dietary anthocyanins, in that they appear to stimulate the reverse cholesterol transport pathway although lipids do not accumulate in the liver, but rather are metabolised to bile acids and excreted in the faeces.

High anthocyanin corn diets offer cardioprotection and improved levels of omega-3 polyunsaturated fatty acids in rats tested with the Langerdorff apparatus for ex vivo ischaemia/reperfusion injury. However, anthocyanins provided by purple tomatoes did not alter the metabolism of polyunsaturated fatty acids significantly in mice. It is likely that too few animals were included in this study, since small (non-significant) increases in DHA levels were detected with the highest doses of purple tomatoes.

Taken together, the data from the ATHENA project show that anthocyanins from different food sources promote health to similar extents in preclinical animal studies, particularly promoting cardioprotection and reducing the risk markers for cardiovascular disease.
The ATHENA project has also examined whether dietary anthocyanins are comparable in their abilities to promote health to other better known but more nutritionally restricted polyphenols in preclinical animal studies.
These ‘comparative nutrition’ studies represent a ‘first’ in nutritional interventions, because never before has it been possible to compare the effects of anthocyanins to other, more restricted, dietary polyphenols in a common food matrix. During ATHENA we have engineered high resveratrol, high flavonol, high flavonol plus high anthocyanin and high genistin (isoflavone) tomato lines, that have allowed us to address this question for the first time.

Protective effects of anthocyanins compared to other polyphenols

Rodent diets with different polyphenol classes (Flavonol, Resveratrol, Isoflavone) and a mixed content polyphenol (anthocyanin & flavonol) were prepared that contained a total amount of phenolics that was equivalent to the most effective dose of anthocyanin seen in the dose-response animal study. The diets were also prepared so that the pellets contained the same total amount of tomato (10%) by mixing with a low polyphenol tomato powder prepared from Moneymaker tomatoes. ApoE-/- mice were given the diets and allowed feeding ad libitum for 17 weeks, sacrificed and the atherosclerotic plaque area in the aortic sinus was measured.
Analysis of body weight gain and pellet consumption during the feeding period showed no difference between the different diets, indicating that the diets were isoenergetic and that the mice showed no preference for any of the diets that were prepared for the study. Therefore any change in plaque area observed could not be due to a difference in dietary intake.

Analysis of atherosclerotic plaque area in the aortic sinus showed that the mixed polyphenol diet had the greatest effect on plaque area, reducing it by 31% when compared to a low polyphenol tomato diet. The resveratrol diet was also effective and reduced plaque area by 26% compared to the control tomato diet. Neither the high flavonol nor high isoflavone diets proved effective in reducing atherosclerotic plaque area significantly. As the flavonol diet had no effect on plaque area, the reduction in plaque area seen in the anthocycanin+ flavonol diet was presumably due to the anthocyanin content of the diet as observed in the anthocyanin dose response ApoE-/- study.

In summary, effective treatments included diets supplemented with resveratrol-enriched tomatoes and indigo (high flavonol and high anthocyanin) tomatoes. Since there was no effect at all of flavonol-enriched diets, the positive indigo results must reflect the bioactivity of the anthocyanins. These data fit very well with the dose response analysis of the purple tomatoes. The detailed analysis of this comparative trial, which is still to be completed, will reveal whether or not dietary anthocyanins positively impact atherosclerosis by the same mechanism as resveratrol or whether the two bioactives work independently. Currently our data indicate that the primary mechanism for the positive effects of dietary anthocyanins is likely on reverse cholesterol transport. While we did not observe significantly increased levels of omega-3 PUFAs in response to the high-anthocyanin tomatoes, it could be that anthocyans also positively impact long chain poly unsaturated fatty acid metabolism, as suggested by the data from purple corn (Toufektsian et al., 2010). The mechanisms of activity of dietary anthocyanins in promoting health may operate at the molecular, cellular and whole organism levels, as may those of other better known but more nutritionally restricted polyphenols.

Protective effects of anthocyanins against weight gain and cancer

The ATHENA project has shown that anthocyanins in the diet impact the epigenome of animals on a high fat diet.

However, despite there being significant changes in H3K4me3 patterns in the genomes of animals supplemented with purple corn, similar changes were also found when animals were fed a diet supplemented with yellow corn. Consequently it was concluded that changes in the methylation of the genome of animals on a high fat diet were more likely the result of the corn supplement than the anthocyanin content. However, this conclusion does not negate the positive effects of anthocyanins on CVD risk factors not their antitumorigenic properties. The data from ATHENA suggest strongly that the primary mechanism of action of anthocyanins does not involve epigenetic changes.

The mechanisms of action of anthocyanins on the viability of breast cancer cells have also been studied during the ATHENA project. Two breast cancer cell lines were used for these assays, the estrogen-responsive, low metastasis line MCF7 and the estrogen receptor negative, highly metastatic MDA-MB-231 breast cancer cell line. Extracts were prepared from high delphinidin purple tomatoes and from high pelargonidin pink tomatoes and effects on cell viability/ proliferation, the cell cycle and apoptosis were measured using Wst-1 cell proliferation assays, flow cytometry and Annexin V/propidium iodide staining assays, respectively. Effects of extracts on a non-cancerous breast epithelial cell line were also undertaken, along with assays of the effects of purified anthocyanins on cell viability/proliferation.

Analysis of breast cancer cell lines by FACS showed that 24 h exposure to 1% WT tomato extract had no effect on the cell cycle distribution of the cells. This result confirmed the findings of the cell metabolic activity experiment where WT tomato extract did not interfere with the survival or growth of either MCF-7 or MDA-MB-231 cells. In contrast, treatment with extracts from high anthocyanin tomatoes resulted in a dramatic alteration of the cell cycle profile of both cell lines. 3 h exposure to purple tomato extracts (high delphinidin) reduced the MCF-7 cell population in G0/G1 significantly and resulted in an increased population the S and G2 phases. After 6 h treatment, the cell cycle of purple tomato extract-treated MCF-7 cells was severely disturbed; a sub-G1 population, indicative of highly apoptotic cells, had formed and the populations in the G1, S and G2 phases were significantly decreased. After 6 h treatment, cell debris accounted for approximately 90% of the cell sample, indicative of the severely cytotoxic effects of the high anthocyanin tomato extracts. No cell cycle data could be recorded beyond the 6 h time point as no intact cells remained in the sample.

In MDA-MB-231 cells, there was a significant increase in the cells in the G1 population and the formation of a sub-G1 peak representative of late apoptotic cells induced by purple, high anthocyanin tomato extract. As a result of the severe cytotoxicity of purple tomato extracts no more cell cycle data could be collected beyond the 6 h time point due to the absence of sufficient intact cells to generate reliable data.
Analysis of cell death using Annexin V/propidium iodide staining showed that WT tomato extract did not induce apoptosis in either cell line confirming previous observations made in the cell proliferation/viability assays. A significant induction of apoptosis in both breast cancer cell lines was observed after as little as 3 h of incubation with medium supplemented with 1% purple tomato extract. MDA-MB-231 responded more quickly to high-del treatment than MCF-7 cells; at the 3 h time point less than 25% (p<0.001) of MDA-MB-231 cells were healthy compared to more than 50% (p<0.05) of MCF-7 cells. However, after 6 h exposure there was no difference in the apoptotic response between the two cell lines. After 6 h of treatment ~25% of MCF-7 and MDA-MB-231 cells were undergoing early apoptosis and almost 75% of cells had reached a late apoptotic stage. The percentage of cells undergoing cell death was further increased with prolonged incubation periods, and after the maximum exposure period of 24 h, more than 90% of cells were in late apoptosis and the remaining cells in the early stages of apoptosis. The amount of cell debris, representative of severely damaged cells that have been excluded from the apoptosis analysis, increased dramatically with extract exposure times. High pelargonidin tomato extracts similarly promoted apoptosis of both breast cancer cell lines.

Our results showed that extracts of high anthocyanin tomatoes have far more bioefficacy in reducing breast cancer cell viability than purified anthocyanins. The high anthocyanin extracts have significant effects on the cell cycle of breast cancer cell lines (both estrogen responsive and non-responsive) and the major result of exposure of cells to low levels of these extracts was to stimulate apoptosis, such that after 24h or less more than 90% of the cells are in late-stage apoptosis. Both high delphinidin and high pelargonidin extracts had similar effects on apoptosis suggesting that there is little difference conferred by the class of anthocyanin in the extracts. The promotion of apoptosis by these extracts suggests strongly that the delay in cancer progression and extended life span observed in p53-/- mice fed a diet supplemented with high-anthocyanin purple tomatoes may result from the ingested anthocyanins promoting apoptosis to complement the loss of activity of the tumour-supressor, p53 protein (Butelli et al., 2008).

Mice are protected from ischemia/reperfusion after 5 weeks of diet with purple corn so we tested the ability of anthocyanins to prevent the cardiotoxic effects of doxorubicin (Dox), a chemotherapic agent used for the treatment of breast cancer. To this purpose, we assessed the survival of mice on sub-lethal doses of Doxorubicin fed with diets supplemented with yellow or purple corn. The analyses showed that Dox significantly reduced viability of cardiomyocytes to roughly 50% of the control levels, whereas pretreatment with either yellow or purple corn extracts maintained cell viability at roughly 88%. Short term survival was improved in animals fed toxic levels of Dox and the purple corn supplemented diet compared to the yellow corn-supplemented diet although the longer term survival was equal for both corn supplemented diets.

Neither yellow nor purple corn extracts affected the antitumoral effect of Dox on MCF-7 breast cancer cells. Subcellular studies suggested that a purple corn-supplemented diet prevents DOX-induced cardiac histopathological alterations. Subcellular studies suggested that a purple corn-supplemented diet prevents DOX-induced cardiac histopathological alterations.

Overcoming the limited bioavailability of dietary anthocyanins

The ATHENA project investigated the effects of food processing on bioavailability of anthocyanins from different foods. A major finding was that the total anthocyanin recovery from fresh black mulberry fruit was significantly higher than from processed juice. This higher anthocyanin bioavailability in fresh fruit compared to juice samples may be explained by there being higher amounts of anthocyanin compounds accessible in fruit compared to juice. On the other hand, the recoveries of total monomeric anthocyanins from both fruit and juice samples were much lower than the recoveries obtained for total phenolic compounds and total flavonoid compounds. This is consistent with other reports, that anthocyanins are distinct from other flavonoid groups in terms of their apparent low bioavailability. In yellow and purple figs, loss of anthocyanins was observed as a result of drying and indeed, in the dialysed fraction after intestinal digestion, the anthocyanins were reduced significantly compared to fresh figs and no anthocyanins were observed in dried figs. In contrast, black mulberry molasses had significant anthocyanin contents which were not released by simulated gastric digestion although an increase in anthocyanins from plum leather was observed after gastric digestion. Such increases may be explained by the effect of hydrolysis on the release of some bound anthocyanins from plum leather.

A number of trials were performed to enhance the bioavailability of anthocyanins in foods using selected enzymes (β-glucosidase and glycosyl transferase), either in purified form or from different food sources (eg almonds), in selected test foods (chocolate, tomato, fruit juice, etc.). However, none of these experiments gave promising results. Since enzyme treatments did not provide significant improvements, further studies investigating the effects of food matrix and co-digestion of some common food materials were undertaken to measure the effects on bioavailability of anthocyanins and other polyphenols. A study was performed to determine the effects of co-consumption of dried fruits with nuts. The effect of co-digestion of commonly consumed dried fruits together with nuts on stability and recovery of total anthocyanins following simulated gastric digestion was investigated. Consumption of walnuts together with figs and apricots as well as hazelnuts with figs and raisins significantly increased the total anthocyanin content of the soluble fraction following gastric digestion. A similar condition was observed when dried apricots and raisins were consumed together with hazelnuts and similar effects were observed mixing dried fruits with almonds, showing that co-digestion with nuts (walnuts, almonds or hazelnuts) increased the bioavailability of anthocyanins from dried fruit. These results suggest that consuming mixtures of dried fruit and nuts, either directly or in food mixtures (for example chocolate) can improve the bioaccessibility and therefore the bioavailability of anthocyanins, and potentially the nutritional value of the food.

During the ATHENA project, methods were developed for anthocyanin production in microbial systems and in plant cell systems. Limited success was achieved in yeast-based production systems, but considerable progress was made on anthocyanin production systems in plant cell cultures. An efficient stable production system for novel anthocyanins was set up using tobacco cell suspension cultures and is currently being trialled for scale-up production in fermenters. Work on developing this industrial biotechnology platform has been funded by an ERA-NET Industrial Biotechnology grant awarded to P3 (co-ordinator) and P1 partners from the ATHENA Consortium, in collaboration with additional European partners from Italy and Norway. This new project, ANTHOPLUS, incorporates a pathway to commercialisation provided by a European SME (PlantMetaChem).

Participants on the ATHENA project have also worked hard to determine whether the positive health beneficial effects of anthocyanins, observed in preclinical trials with animals, are matched by similar positive effects in human. ATHENA participants have undertaken two epidemiological studies and two longer-term intervention studies.

Protective effects of dietary anthocyanins in humans

An observational study on 495 volunteers to evaluate dietary habits was conducted, with particular focus on anthocyanin and polyphenol consumption, and such dietary habits were correlated to a number of phenotypes associated with the risk of cardiovascular disease. All eligible individuals were genotyped with an array containing 200K highly informative genome-wide tag-SNPs. The genotypic data, further enriched through imputation, were analysed to validate candidate genes and SNPs reported in the literature for HDL and LDL and to identify Gene*Environment (ie Gene*Diet) interactions with reported diets (no restriction vs. vegetarian) and with anthocyanin intake.
We identified Gene*Diet interactions for 2 candidate genes and 3 candidate SNPs for HDL and for 3 candidate genes and 3 candidate SNPs for LDL. We also identified Gene*Anthocyanin interactions for 1 candidate gene and 4 candidate SNPs for HDL and for 1 candidate gene for LDL.

In addition, an integrated platform, called the Dietary Monitoring Solution was established enabling the collection of phenotypic, genetic and lifestyle information, linked to an mHealth application tool. The data collection solution, validated in the context of the epidemiological study, allows maintaining anonymised information and supports a number of features making it particularly well-suited for multicentre studies. The mHealth application was designed to translate knowledge generated from research into personalized prevention programmes and to support patient/citizen adherence to such programs.

A second epidemiological study was undertaken as a prelude to the intervention study on subjects within the Moli-sani population. An investigation was performed to calculate the average intake of anthocyanins and other dietary polyphenols in a general population living in the same area from where the breast cancer patients would be recruited, in order to administer them an anthocyanin supplement well above the average dose introduced by a daily diet. Additionally a preliminary study was undertaken to verify that anthocyanin ingestion was associated with a reduced inflammatory state, as measured by a newly described inflammatory score in this same population. These studies showed that the anthocyanin dosage delivered by three stick packs of anthocyanin extracts from corn would deliver levels of anthocyanins significantly above the average daily intake for the Moli-sani population, and that polyphenol intake is associated with low-grade inflammation in the Moli-sani population.

The first intervention study aimed to compare the effect of anthocyanin-rich blood orange juice with a standard (no anthocyanin) blonde orange juice on markers of cardiovascular disease. Participants aged between 25 and 84 years of age were recruited based on their waist measurement (men ≥94cm and women ≥80cm). They were required to consume 500mL of either blood or blonde juice each day for 28 days in a two way cross over study. Prior to each part of the intervention there was a 2 week ”run in period” where participants were asked to avoid consuming foods rich in anthocyanins. After the 28 day intervention period, there was a 3 week wash out period after which the participants were asked to then drink the other juice for 28 days. The 500mL of blood orange juice contained approximately 50mg of anthocyanins, whereas the standard juice contained none. Eighteen subjects were recruited to the study.

Blood samples were collected for the preparation of plasma and peripheral blood mononuclear cells (PBMCs) for the analysis of anthocyanin metabolite concentrations, transcriptomics and CVD risk markers. Other measurements included pulse wave velocity, central blood pressure, glucose, insulin, high sensitivity C reactive protein, interleukin-6 and endothelin-1 concentrations. Inspection of the preliminary data suggests that there is a general trend of a small reduction in the blood levels of cholesterol & LDL-C for juice A compared to juice B although there is no evidence for effects on CVD risk markers. These preliminary data suggest there is a promising possibility that with more participants, the study might provide evidence of a reduction in LDL-C and cholesterol for high anthocyanin juice A relative to juice B.

The design of the second intervention study was a double blind, randomised, placebo-controlled clinical trial, in which patients with breast cancer scheduled for radiotherapy were assigned to a diet supplemented with an anthocyanin-rich preparation. Anthocyanin extracts from purple corn (or extracts from yellow corn), were provided in stick packs to be dissolved in water to provide anthocyanin-rich beverages for the intervention studies. For the intervention study, it was calculated that 19200 stick packs would be needed from both the B1 Pl1 purple corn and the b1 pl1 yellow corn. Overall, about 2100 kg of yellow corn cobs and 2200 kg of purple corn cobs were produced. Corn cobs were ground by Agrindustria and transferred to SVEBA Srl to perform a semi-industrial hydro-alcoholic extraction to deliver the concentrated extracts in a food-grade powdered form using spray drying techniques. Specifically, 18,200 stick packs of each type were produced. Each stick pack obtained from purple corn contained 125 mg of anthocyanins, whereas no anthocyanins were present in the yellow corn stick packs. The compositional analysis of stick packs showed that yellow and purple formulations were similar in composition and that they essentially consisted of carbohydrates (mainly maltodextrins) and proteins. Three stick packs (~375 mg anthocyanins) were the recommended amount of supplement per day per patient. As the average daily amount of anthocyanins ingested with the diet was 158 ± 90 mg/day in Moli-sani women, the dose administered in the intervention trial was more than double the average and higher than the average ± 1 SD.

Although the trial will be continued until the foreseen number of patients (300) have been recruited, a preliminary analysis of data on the patients already participating (92) has been undertaken, without communicating the results to the clinicians, patients or operators, who remain blind about the treatments. The analysis needs to be considered “confidential” until the end of the study and supplementations are referred to only as A and B.

It appears that at baseline there are no differences in skin parameters between the tumor breast (homolateral) and the controlateral ones. It also appears that there are no differences in skin parameters of homolateral breast from patients randomized to treatment A or to treatment B, taking into account the net values of homolateral breast minus controlateral breast values.

The melanima and erythema indexes are variable in the different areas of the breast (quadrants), but, as for the elasticity skin parameters, no differences were observed between the tumor breast (homolateral) and the controlateral ones, neither between homolateral breast values from patients randomized to treatment A nor to treatment B. The melanin index at baseline in the homolateral quadrant QSI was different between the two treatment groups, but only when the crude index, and not when net index (subtracted by the controlateral value) was considered.

The cutometer data related to skin firmness (R0) and elasticity (R2) show a non-significant decrease of values (lower values demonstrate lower elastic properties) after radiotherapy, a finding biologically consistent with the radiotherapy treatment. The net elasticity (R5) data show a trend towards increasing values (higher values demonstrate higher elastic properties) after radiotherapy. Consideration of these data should take into account changes in extravascular water content occurring after radiotherapy, which may affect net elasticity parameters. Supplementations with A or B show some modification trends, different in different breast quadrants. Increasing the number of patients to the target of 300 might show these differences to be significant, and confirm that there are differences in the protective effects of the two supplements. No significant differences are apparent for the different supplements (A or B) on the toxicity index of radiotherapy.

Conclusions: The ATHENA project has provided compelling evidence for the cardio-protective effects of dietary anthocyanins, both in absolute terms and relative to other dietary polyphenols. It has provided strong evidence that the major effects of consuming high levels of anthocyanins are on lipid metabolism, principally the reverse cholesterol pathway but also on the endogenous synthesis of long chain omega-3 polyunsaturated fatty acids. ATHENA has also demonstrated that high anthocyanin diets can slow the progression of soft tissue carcinoma (as determined by life span in p53-/- mice) and that they promote apoptosis in human breast cancer cell lines, perhaps suggesting promotion of apoptosis as their mechanism of life promotion in cancer prone animal models.

Human epidemiological studies have suggested an inverse correlation between anthocyanin consumption in healthy volunteers and HDL/LDL risk factors for cardiovascular disease. Human intervention studies suggest that positive impacts on plasma LDL levels and HDL/LDL ratios and anthocyanin consumption are also seen in humans. Dietary anthocyanins may also offer some protection against radiation damage from radiotherapy for breast cancer treatment.

Potential Impact:
We wish to develop a web platform (blog/discussion forum) for information and dialogue with the general public about beneficial anthocyanins from plants in food.
Our target audience is specifically the general public and we anticipate using this platform for both dissemination of evidence-based information and for understanding and responding to the questions of consumers on this topic. The over-arching idea behind the platform is that it will provide evidence-based information by academics for public good. This ideal is what the general public, largely, think should be the remit of publically-funded institutions. Core to this is that information should be free to access and free from any commercial interests promoting specific products or biasing the information offered.
Our initial focus will be on the results of the EU-funded Collaborative Project ATHENA, co-ordinated by Cathie Martin. ATHENA has accumulated a considerable body of evidence on the beneficial effects of dietary anthocyanins. Although much of this evidence has been published in scientific journals and has been the subject of many press releases, the benefits of dietary anthocyanins have not yet permeated the public psyche, primarily because the food industry has been slow to respond to the waves of new information on dietary anthocyanins, particularly their impact on limiting the progression of cancers and reducing the risk of cardiovascular disease. This inertia may have been due to the wide distribution of anthocyanins in plants which means that there is little opportunity for strong IP positions in providing anthocyanin-enriched foods, or for exclusive market shares. Anthocyanin-rich food is also relatively expensive to offer as an ingredient in processed foods. Despite this, the information from the ATHENA project should be made available to the general public, in an attractive and accessible format to enable consumers to make evidence-based dietary choices to improve their health and reduce their risk of chronic disease. A publically-oriented, public good forum could overcome the barriers imposed by the lack of innovation of the food industry, and could eventually lead to persuading the food industries to adopt these important, new, nutritional concepts, in response to consumer demand.
Our objective is to create a platform that is easily accessible to the general public. Therefore, it needs to be:
1) Interactive – questions and enquiries from the general public need to get responses. There will also be opportunities for video content and live web-chats via written platforms such as Disqus, or video software such as Google Hangouts.
2) Academic – free, evidence-based information and discussion for public good
3) International. Experience shows that public dissemination works only in the language of each country. Fortunately, ATHENA includes participants from the UK, Netherlands, Germany, France, Italy and Turkey. We can translate articles and respond to queries in six languages based just on those already participating in the project. Our idea would be to recruit students from the participating ATHENA groups and hire Spanish and Chinese freelancers to cover operating the web forum in all major languages, from the outset. We will remunerate contributors on a per article/posting basis.
4) Continuously updated. A well-defined content strategy will result in a large number of short, informative, fully illustrated articles before the launch so that we have material to refresh the forum on a weekly basis. We have drafted examples of the level at which the articles would be pitched and the type of formatting we envisage.
5) Overall editorial control will remain within the founding group, so that responses to enquiries can be screened for scientific accuracy, appropriate citation of evidence and liability, prior to posting. Screening will need to be multilingual and we will ask the international staff of the participating institutions for assistance in these oversight activities.
6) Such a platform, although initially limited to dissemination of the outputs of one project will demonstrate the credentials of the participants in public engagement and public dialogue. The platform will be of particular relevance as a model for demonstrating impact for nationally-funded projects and for future applications to the Horizon 2020 program, for which public dialogue is a priority.
7) We will look to engage a science communication graduate on an externally-funded fellowship to extend the remit of the web forum once it is up and running. We will also look to engage correspondents with special interests in food. This medium is the preserve of the young and should be driven by them to achieve maximum outreach. By improving the eating habits of the young, we are most likely to have a lasting impact on the health of the general public and the prevention of chronic diseases.

Can such a platform generate revenue or leverage further funding?
Beyond the question of whether or not this should be the objective of public engagement activities, it is possible that once the platform has significant numbers of followers, activities that generate income through crowdsourcing could be undertaken. An example is a collection of nutritious recipes rich in anthocyanins, which could be made available to the public for a modest price through the website or through Amazon. A second example is a compendium of foods rich in anthocyanins and suppliers of these foods.
We anticipate that we will gain a better understanding of the opinions and questions of the general public on the topic of nutritional security, diet and health. We anticipate that the platform will offer a vanguard example of science communication that can be used in support of pathways to impact for national grant applications and for public engagement activities in applications to the Horizon 2020 program. We anticipate that this form of public engagement will become a beacon with which to train other scientists of all ages how to communicate their science to the general public. We anticipate that the platform will allow a new role for publically-funded researchers and institutions to emerge; that of offering evidence-based information to the general public free from commercial interests for public good and to help people to enjoy a better quality of life.

ATHENA communication and dissemination activities aimed at maintaining contact among the project’s participants, as well as disseminating information regarding anthocyanins and flavonoids and, in a broader way, healthy eating to the general public. Decision makers and industry representatives have been targeted, as results from laboratories have started to emerge. Press involvement has been considered a crucial way to communicate throughout the project.

The ATHENA web site has been established under the registered domain name

and is updated on a regular basis by the Science Communication Unit at the Istituto Neurologico Mediterraneo NEUROMED. The site contains information about the Project, its aims and the methods used. Visitors can find information about anthocyanins and antioxidants in general, their effects on health and their role in a healthy diet. Moreover, news from participating laboratories, and interviews with researchers involved in ATHENA are provided to inform and attract a general public. A “lively” part of the site is offered by original science news provided by the Science Communication Unit and is based on scientific literature in the field of antioxidants, diet and health.

The web site has had extremely little downtown time, with a maximum of 48 consecutive hours of no service during the third reporting period. It ranks on Google as follows:
#1 for keywords “Athena antioxidants”
#1 for keywords “Athena flavonoids”
#3 for keywords “Athena polyphenols”
#2 for keywords “polyphenols anthocyanins Europe”

Maintaining contacts between partners
The web site, provided with a registration system and a reserved areas, allows partners to add scientific material useful for other participants. The members area also contains presentations and documents regarding ongoing work and results.

The newsletters in English have been shared amongst partners. They feature news about the project, interviews with researchers and reports on ATHENA meetings. Newsletters can be accessed by web site visitors in two formats: low res, to be viewed and printed only for personal use, and hi-res, that could be used by partners for professional printing. Partners receive them also by e-mail. Until now, Athena newsletters are composed of interviews and reports provided by Communication staff. A slight shift in newsletter’s content has occurred, including more stories coming from participant labs, plus editorials and comments about research in the field of anthocyanins, and antioxidants in general.

Press releases
After the highly successful world-wide press campaign about “Unlocking the secrets of blood oranges”, ATHENA’s themes have been demonstrated to capture the attention of media and general public. The Science Communication Unit is keeping an eye on ATHENA undergoing developments in order to provide full assistance in spreading news about new results. A successful press release was issued following the cultivation of the high anthocyanin, purple tomatoes in Canada in the spring of 2013, their processing into a purple juice and their import into the UK for analysis for regulatory consideration and for human intervention studies (D5.8)

Partners always in touch
The web site is provided with a registration system allowing partners to add scientific material useful for other participants. Presentations from the project meetings are already available in the members’ area.

EXPO 2015: ATHENA held its final meeting in Milan 27th to 29th May 2015, linked to the Milan EXPO 2015 Food for life. ATHENA partners participated in three EXPO workshops:

1). “Water, Food Security and Environmental Sustainability” at AQUAE, Venice June 6th-7th 2015 Organiser Chiara Tonelli (P2). The results of the ATHENA project were presented by P1, Cathie Martin
2) Feeding the planet, energy for life: Relationship between health, food and the environment. Workshop organised by the Science Europe Scientific Committee for the Life, Environmental and Geo Sciences. Milan May 12th to 13th 2015. P1 was a presenter and workshop participant.
3) The science behind feeding the world, healthily. Genotype and Environment Interactions (as part of Expo Milano 2015" Feeding the Planet, Energy for Life"). Venue: Università Degli Studi Di Verona, Verona, Italy 3rd to 6th June 2015. P1, Cathie Martin, was a plenary speaker and participant in this workshop.

Full details of all ATHENA dissemination activities have been uploaded to the reporting section of the EC participants portal.

Use of Foreground (i.e. results from the project e.g. in academic research or commercially)

Commercial Exploitation of R&D Results: The spin-out company, Norfolk Plant Sciences (NPS), was assigned the patent on the high anthocyanin and high flavonol tomatoes. They supported patent filing in Europe, the USA and in India, and the filing has been granted in the USA in 2015. They have grown the purple tomatoes (backcrossed into a processing variety) under notification in Canada in 2013, and prepared juice on a pilot scale for commercial release in the USA. NPS was also assigned IP for producing cold-independent Valencia blood orange varieties, on which they have filed IP. NPS is currently undertaking notification for cultivation under permit and juice production by the Food and Drugs Authority (FDA). Commercialisation activities (planting, juicing and sales) are anticipated in the USA in 2017. Confidential: No, Exploitable products: foods and drinks Sector: Food Timetable: 1-2 years; Patents, 2, one granted in the USA, Owner, P1.

A second spin-out company, Persephone Bio, has been founded to incorporate tomato extracts enriched in different polyphenols into skin care products. The company is partnering in sponsoring a BBSRC – iCASE industrial PhD studentship on applications of tomato extracts to human skin biopsies to determine their effects on wound healing and aging. Confidential: No, Exploitable products: cosmetics, skin creams, Sector: Cosmetics Timetable: 1-2 years; Patents, 1, Owner, P1.

Interactions between SMEs producing high anthocyanin corn biscuits and Partner 2 (UMIL) may lead to future stronger interactions based on the commercial production of the corn biscuits by Airfood. UMIL was granted Community Plant Variety Rights on the B1 Pl1 purple corn (decision n. 33449). Confidential: No, Exploitable products: foods and drinks Sector: Food Timetable: 1-2 years; Owner, P2.

B1 Pl1 purple corn cobs have been used to develop a functional supplement (N-Ossid, Coswell Srl) already sold and distributed in Italian national supermarkets. This functional supplement is based on a commercial formulation, containing dry extracts from purple corn, red grape, blueberry and Echinacea, with an antioxidant capacity 14.5-fold higher than blueberries. Confidential: No, Exploitable products: foods and drinks Sector: Food Timetable: current; Owner, P2.

Furthermore, UMIL licensed the cultivation of B1 Pl1 purple corn to two companies (Azienda Agricola Frettoli and Azienda agricola Arioli Angelo) to produce purple polenta. In particular, Frettoli has already produced purple biscuits and polenta flour that were presented at EXPO 2015 and these new products are already on the market in Italy. Confidential: No, Exploitable products: foods and drinks Sector: Food Timetable: current; Owner, P2.

The expertise of CRA-ACM (P9) has been used to develop a method for the recovery of anthocyanins and other polyphenols from red orange processing wastes and eriocitrin and other flavanones from lemon peel. A standardized powder of a new phytoextract, obtained by properly mixing the red orange and the lemon extracts, has been obtained. An application for a patent in the next year is intended for a new nutraceutical supplement. Inventers will be Paolo Rapisarda; Simona Fabroni; Margherita Amenta; Gabriele Ballistreri.
Confidential: No, Exploitable products: health and well-being Sector: Neutraceuticals Timetable: 2-4 years; Patents, 1, Owner, P9.

Tobacco cell cultures which produce high levels of different anthocyanins have been delivered as part of the ATHENA deliverables. These cultures laid the foundations for a successfull ERA-Industrial Biotechnology application to P3 (Co-ordinator) and P1 plus two additional partners from Europe. This project (ANTHOPLUS) lays a pathway through scale-up, purification and marketing to commercialisation as analytical standards for quality control analysis and labeled anthocyanins for nutritional studies. Confidential: No, Exploitable products: natural chemicals as analytical standards Sector: Food, food additives Timetable: 2-5years; Owner, P1.

KOS developed an integrated platform, called the Dietary Monitoring Solution (DMS), to collect phenotypic, genetic and lifestyle information linked to a mHealth application tool providing personalized dietary indications. Confidential: Yes, Exploitable products: Dietary Monitoring Solution. Sector: Neutrigenomics Timetable: 1-2 years; Owner, P11.

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