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I.R.C.C.S. Neuromed

The aim of this Unit work is to verify the possible beneficial effect of a human intervention trial with anthocyanins in reducing the side effects of radiotherapy in breast cancer patients.
Preliminary bioavailability studies were performed to evaluate the urinary levels achieved in human volunteers following the intake of corn derived anthocyanins administered under different forms, e.g. breakfast bars, prepared through two different procedures, or granular preparation of cob floor administered in stick packs. The latter formulation was ultimately selected as the best in terms of bioavailability for human studies.
An epidemiological study was undertaken as a prelude to the intervention study on subjects within the Moli-sani population. The Moli-sani project is an observational prospective study on a representative sample of nearly 25,000 subjects from a general population in the Molise region (Southern Italy). 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 design of the 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. Three stick packs (~375 mg anthocyanins) were the recommended amount of supplement per day per patient. Cutometric measure of skin parameters will be the primary end-point of the anti-inflammatory effect of anthocyanins, together with levels of circulating markers of inflammation in patients, performed at baseline, at the end of treatment and at late (six and twelve months) follow-up.
Due to delays independent from the investigators’control (ethical committee vacancy, repeated trials to find out the best anthocyanin preparation for human studies and others) the intervention trial could not be completed by the administrative end of the project and is still in progress.
Although the trial will be continued until the foreseen number of patients (300) have been recruited, a preliminary, blind on treatment, analysis of baseline data on the patients already recruited shows that there are no differences in skin parameters between the tumor breast (homolateral) and the controlateral one in each patient. It also appears that there are no differences in breast skin parameters and in other circulating markers in patients randomized to placebo or to the active treatment, suggesting a correct randomization of the sample.The trial results are foreseen within the next eighteen months.

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CRA-ACM, Acireale CT, Sicily, Italy

In the ATHENA project the CRA-ACM was involved in the production of purified anthocyanins from Citrus wastes. An experimental procedure, based on enzymatic treatment and further adsorption on polymeric resins, allowed to obtain an highly concentrated extract rich in anthocyanins from blood orange processing wastes. Moreover a purified fraction of cyanidin-3-glucoside from the raw extract was obtained and employed in the studies on the effects of purified compounds compared to their effects in whole food contexts. Furthermore, verdello lemon (a typical production predominantly performed in the eastern Sicily, at the mountainsides of the Mount Etna, rich in flavanones and other antioxidants because of the particular cultivation technique, called 'forcing') processing wastes were used to develop a method for the recovery of eriocitrin and other flavanones. Finally the expertise of CRA-ACM have been used to produce a standardized new phytoextract in powder form, obtained by properly mixing anthocyanins and other polyphenols from red orange (cv. Moro) processing wastes and eriocitrin and other flavanones from lemon peel (cv. Femminello, verdello blooming). For this new phytoextract an application for patent is going on. This should be conveniently employed for nutra-pharmaceutical purposes.

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DLO, Wageningen, The Netherlands:

The task of P5 within ATHENA was to investigate the potential of transferring the entire anthocyanin biosynthetic pathway from plants into a suitable in vitro production organism. Yeast was chosen and an initial selection of 12 genes was made to perform the work. The goal was to have a production organism which could be exploited to produce relatively pure anthocyanins and to be used as a starting point for further engineering to produce strains each of which accumulates one of a range of differently decorated anthocyanins. This was an ambitious goal and while we did not fully succeed during the project we came a long way and we have learned a great deal about the challenges along the way. Furthermore, thanks to the materials generated and knowledge gained within the project the work has been able to be continued in collaboration with another group and very recent reports suggest that the final goal of an anthocyanin-producing yeast has indeed been achieved. Within ATHENA high yielding strains accumulating flavonoids such as naringenin and dihydrokaempferol have been generated. This was achieved using 6 exogenous plant genes together with a number of additional engineering steps required to maximise flux along the pathway and avoid branches / substrate loss. Through this project we have also benefited greatly and broadened our perspectives through the highly multi-disciplinary nature of the consortium. This has led to a significant number of joint scientific publications as well as several more popular articles. This work is now also a standard component of our conference presentations and for lectures to undergraduates.