Effects of Fruit Juice Processing and Human Metabolism on the Functionality of Anthocyanins for Cardiovascular Health

People who consume the highest quantities of fruits and vegetables appear to be more protected against heart disease than those who consume lower quantities. Evidence suggests that this protective effect is in part the result of substances in the fruits and vegetables called polyphenols. In recent years, berries and berry derived juices and wines have been promoted as especially healthy foods as they are high in a particular class of polyphenol called anthocyanins. These anthocyanins are reported to have activities that benefit the heart and blood vessels. Cardiovascular diseases (CVD) and specifically stiffness of the arteries results from accumulated damage to blood vessel walls. There is a single layer of cells that lines the blood vessels which is sensitive to agents/compounds within the blood. When this layer is damaged as a result of injury or chronic disease, it loses its ability to maintain normal blood vessel function and becomes prone to processes that lead to heart disease. Anthocyanins and anthocyanin containing foods have been shown to have direct protective effects on this cell layer, thus restoring proper function to the blood vessels. However, the anthocyanins in the foods we eat often become altered during standard food processing and storage conditions, an effect that is believed to negatively alter their function relative to those in raw fruits or vegetables. As well, when we eat anthocyanins they become modified by our bodies, resulting in drastic changes to their original form. Previous experiments have used unaltered or original forms of anthocyanins to explore how these compounds affect the cells in our bodies and blood vessels. However, no studies have explored the true activity of anthocyanins as they exist within our bodies, as altered products in our circulatory system resulting from changes during processing and digestion. The effects of these alterations on the disease fighting properties of anthocyanins are currently unknown and could be greater, different or impartial to what we currently perceive. The aim of the present program of research is to identify the actions of pure anthocyanins relative to their altered products of processing and digestion on CVD risk. In order to determine their functions, we must first identify their forms in the body after we eat them. We will identify changes that occur to anthocyanins (cyanidin-3-glucoside, the most abundant anthocyanin in nature) in common fruit juices on the UK market, during standard processing and storage conditions. We will also feed human participants a pure anthocyanin (cyanidin-3-glucoside) in order to trace its path and alteration through the body. We will then study the effects of the identified compounds on CVD risk by exploring their activities on the cells (cultured-cells) lining our blood vessels. With this study we hope to prove the usefulness of anthocyanins as a dietary treatment for the prevention of cardiovascular disease, using the relevant compounds found in the body; thus providing informed advice on the health benefits of anthocyanins. The results of this study are also relevant to agricultural industries as levels of anthocyanins in food crops can be easily increased using breeding strategies and pre and post harvest manipulation. This project is particularly relevant to the processed food and beverage industry, as although the alteration of anthocyanins during food processing has generally been considered of negative consequence, the proposed research could establish this as a neutral or potentially beneficial outcome; providing valuable evidence to support the use of fruit juices for the delivery of beneficial components for health. This proposal will also generate findings that may be useful for future studies aimed at investigating the relative activity of other dietary polyphenols, such as those found in coffee, tea, wine or chocolate.

We hypothesise that the bioactivity of dietary anthocyanins is not directly related to their parent structures, but mediated by metabolites of their degradation products, whose mechanistic activities have yet to be explored. Preliminary evidence in our lab (pilot data) suggests that the majority of anthocyanins are likely present in the blood as metabolites of phenolic acid and aldehyde derivatives, of which we aim to investigate their mechanisms of action. In this proposal we plan to profile and identify structural changes that occur to anthocyanins during standard processing and storage conditions of common fruit juices on the UK market known to be high in anthocyanins. We will conduct human and cell based investigations to identify the bioavailable structures and cardiovascular mechanisms of action of anthocyanins using synthesised pure C-3-G and its respective degradation products and metabolites. A human 48h stable-isotope recovery trial will be conducted utilising 13C-labeled C-3-G to determine the pharmacokinetics of the parent compounds and their metabolites. Labelled compounds will be identified in the serum, urine, faeces and expired air. Anthocyanins will be quantified using HPLC- and GC-MS and isotope ratio GC-MS. Vascular mechanisms of action will be identified in a series of cell (HUVEC) studies using synthesised degradation products and metabolites as identified in the processed juices and human intervention. Gene and protein expression of eNOS, NADPH-oxidase, endothelin-1, IL-6, TNF-a, and VCAM will be determined using RT-PCR, ELISAs' and western blotting. This study will provide new insight into the true cardiovascular mechanisms of action of anthocyanins, in addition to detailing absorption, metabolism and pharmacokinetics of the physiological relevant bioactives. In general, this experimental approach provides a model for defining the active molecules responsible for the health benefits of all flavonoids in processed foods.

This proposal fulfills the main objectives of DRINC, including improvement of human health and quality of life, providing evidence to benefit and increase opportunities for crops and crop production and providing research required to fulfil the increasing appetite of the public for healthier foods. The findings from this project will provide potential evidence on the benefits to public health of consuming processed fruit products; which is important given the current low intake of fresh fruits and vegetables in the UK diet. This will have implications for policy makers such as the FSA, as data provided could establish juices as having equivalent or greater health benefits compared to fresh fruit, indicating an elevated importance in the context of the current 5-a-day policy. The impact of processing on food bioactivity as explored in this proposal may identify dietary constituents that could be exploited as novel bioactives with potential functional-food, pharmaceutical and agricultural implications. Establishing the bioactivity of anthocyanin degradation products will offer commercial benefit and enhance the economic competitiveness of the UK, through providing evidence required to market products with known contents of these bioactive components as 'functional-foods' for health and well being. Providing proof of the health benefits of anthocyanin degradation products also holds industrial and agricultural benefit as anthocyanin levels in food crops could be easily modified using pre and post harvest manipulation or plant breeding or genetic approaches to produced increased concentrations of anthocyanins in plants, and therefore increased levels of degradation products in processed foodstuffs. The information generated in this project with provide critical early stage data on bioavailability and bioactivity to inform further studies to support potential health claims around the bioactivity of processed foods. Establishing levels of bioactive degradation products in juices will help estimate optimal dietary levels for human interventions and assist in designing future projects exploring levels of degradation products in other processed foods, establishing safety and bioactivity in cell/animal models and ultimately conducting large-scale, long-term dietary interventions to determine effects on chronic disease outcomes and ultimately providing proof of functionality. This proposed research programme has significant industrial impact and Coca-Cola Europe, Nestlé and GSK have expressed interest, in addition to acknowledging its potential usefulness in marketing and manufacturing (refer to attached statements of industrial support). Having strong support from industry establishes the relevance and importance of this project and improves the likelihood that the research will have impact on manufacturing and processing methods, product development and marketing strategies. This project will provide potential training and opportunities for two Postdoctoral Fellows and one PhD student to work in an experienced and multi-disciplinary team and develop advanced professional skills in nutrition, cell biology, analytical biochemistry and synthetic chemistry. This diverse skill-set is rarely seen within any one of these respective fields and will provide an invaluable foundation for a career in academia, industry or the pharmaceutical research sector. The outcomes and impact of the project will be disseminated through participation at planned workshops (i.e., BBSRC/DRINC consortium, BNF, FSA), industry led seminars (i.e, Leatherhead Foods International training conferences, and supporting industrial partners, including GSK, Nestlé, Cocoa-Cola Europe and Unilever), public presentations and workshops, media briefings, via the websites of the applicants, and to the scientific community through the production of high quality scientific papers and presentations at national and international conferences.