Introduction
Anthocyanins (also anthocyans; from Greek: ἀνθός (anthos) = flower + κυανός (kyanos) = blue) are water-soluble vacuolar pigments that may appear red, purple, or blue depending on the pH. They belong to a parent class of molecules called flavonoids synthesized via the phenylpropanoid pathway; they are odorless and nearly flavorless, contributing to taste as a moderately astringent sensation. Anthocyanins occur in all tissues of higher plants, including leaves, stems, roots, flowers, and fruits. Anthoxanthins are clear, white to yellow counterparts of anthocyanins occurring in plants. Anthocyanins are derived from anthocyanidins by adding pendant sugars.
Recent epidemiological and experimental data suggest that fruit and vegetable juices containing various phenolic compounds can reduce the risk of Alzheimer's disease (AD) (Dai Q,2006) . Individuals drinking three or more glasses of fruit or vegetable juice per week were shown to have over 50% lower risk of AD compared to those who had less than one serving per week. Polyphenols, which are abundant in red wine, bilberry and blackcurrant, have been shown to inhibit the formation and extension of β-amyloid (Aβ) fibrils in a dose dependent manner and to destabilize the preformed Aβ fibrils in vitro. These are important observations considering that Aβ, which is released after sequential cleavage of amyloid precursor
protein (APP) by β- and γ-secretases, is a key determinant in AD pathogenesis. Aβ-induced formation of reactive oxygen species (ROS) is also inhibited by flavonols from many plants (e.g., grapes,
blackcurrant and green tea). Moreover, transgenic AD model mice (APPswe) that received pomegranate juice had about 50% less accumulation of soluble Aβ in the hippocampus than sugar water treated
control mice. These mice were also able to learn more quickly and swim faster than the controls in the Morris water maze test(Hartman RE,2006).
Discussion
To investigate the proper role of anthocyanins in enhancing memory and special capabilities, it has been built up a murine model. The programme of work was reviewed by the University of Reading Local Ethical Review Panel and was given a favorable ethical opinion for conduct. Utmost effort was utilized to prevent suffering and minimize the numbers of rats required for this experiment. Four groups of adult, male Wistar (n =8 per group, Harlan, UK) were housed in groups of 2 and maintained on a 12 h light-dark cycle (lights on at 10 a.m.). All rats were 18 months old at the start of the experiment. After the habituation and shaping sessions period animals were administered one of 4 diets for 6 weeks: A) Control diet; B) 2% (w/w) Blueberry diet; C) Anthocyanin Extract and D) Flavanols (2)-epicatechin and (+)- catechin. All diets were kept in a dry and dark place and administered fresh each day to the animals. Food intake was monitored daily (around 10 am) by weighting the amount of foodm administered to each cage and the amount remaining in the cage in the following day. Animal weight was monitored daily. During the supplementation period, all animals were tested once a week on a standard X-maze alternation task.
On average, the blueberry-supplemented group consumed 7.89 mg/day/rat of flavonoids (5.58 mg of anthocyanins; 2.31 mg of total flavanols), the anthocyanin group consumed on average 5.60 mg/
day/rat anthocyanins, and the flavanols group consumed on average 2.31 mg/day/rat flavanols (0.46 mg of (2)-epicatechin and 1.85 mg of (+)-catechin). The results are really interesting.
At baseline, the choice accuracy for all four diet groups was similar, showing approximately 59% accuracy . There was a highly significant difference in performance between the 4 dietary groups (F 1, 25 =7.915, P,0.001), and a highly significant effect of time (F 6, 150 =5.354, P,0.001). Subsequent post hoc Tukey tests examining specific differences in performance between the individual diet groups indicated that there was a significant increase in choice accuracy between the control group and each diet (control vs. blueberry:
P,0.005; control vs. anthocyanin: P,0.01; control vs. flavanol: P,0.005). Although it has been observed an apparent decline in performance in the control group in the first two weeks of intervention in comparison to baseline performance, this was not statistically significance (F 6, 48 = 1.239, NS). As such, the control
group maintained an average score of 60% correct choices throughout the experiment, whilst the blueberry-, anthocyaninand flavanol-diets induced an improvement in choice accuracy
over the course of the intervention period, with all groups achieving between 75–80% choice accuracy by the end of the intervention period. Post-hoc analysis indicated that the significant improvements in choice accuracy observed achieved significance (in relation to baseline) by the 4th week of
supplementation (P,0.05), and were maintained throughout the remainder of the intervention (week 5 and 6, P,0.05)
Effect of 6 weeks blueberry (BB), Anthocyanins Extract (A) and Flavanols (F) on spatial working memory in aged rats (18 months old).Effect of flavonoid-rich diets (BB, A or F) on correct choices (± standard error of the mean) in a X-maze Alternation Task: a significant increase in choice accuracy was observed between the control group and the blueberry group (P<0.005); the control and the anthocyanins groups (P<0.01) and the control and the flavanols groups (P<0.005). Maximum score is 8 correct choices (Control group: Triangle ‘down’; Blueberry group: Square; Anthocyanins group: Circle; Flavanols group: Triangle ‘up’). B) Comparison between animals performance at baseline and following 6 week supplementation with either a Control, Blueberry, Anthocyanins or Flavanol diet. * Indicates a significant increase in number of correct choices in comparison to baseline performance, P<0.05.
Rendeiro C,2013
It is clear that anthocyanins-enriched diet can improve spacial performances. However they can be helpful to avoid amyloid plaque formation as in AD.
Abnormalities in APP processing that lead to increased production of Aβ peptide are known to play a key
role in AD pathogenesis [5]. APP undergoes sequential cleavage byα- or β- and γ-secretases resulting in the generation of p3 fragment and sAPPα (nonamyloidogenic pathway) or a self-aggregating Aβ peptide
consisting of 37 to 42 amino acid residues and sAPPβ (amyloidogenic pathway), respectively. Maintenance of the balance between the amyloidogenic and the nonamyloidogenic pathway is crucial, since
only small changes in this fine equilibrium may initiate pathogenic cascades, ultimately leading to the onset of AD. Polyphenols, which are abundant in bilberry and blackcurrant, have been shown to inhibit the
formation and extension of Aβ fibrils in a dose-dependent manner and to destabilize preformed Aβ fibrils in vitro
In this study( Vepsalainen,2012), researchers tried to determine whether flavonols and anthocyanin-rich extracts from blackcurrant exert neuroprotective activity and affect APP processing under different in vitro stress conditions, such as free radical-mediated toxicity and apoptosis. They induced these stress conditions in human SH-SY5Y neuroblastoma cells stably overexpressing APP751 isoform (SH-SY5Y-APP751) and subsequently investigated the effects of myricetin, quercetin and anthocyanin-rich extracts on cell viability, production of ROS.
Adinistration of anthocyanins enriched extracts resulted in diminished production of ROS which can be responsible for the beginning of the neurodegenerative process of AD.
They induced these stress conditions in human SH-SY5Y neuroblastoma cells stably overexpressing APP751 isoform (SH-SY5Y-APP751) and subsequently investigated the effects of myricetin, quercetin and anthocyanin-rich extracts on cell viability, production of ROS. Quercetin significantly increased the levels of total APP (APPtot) and immature APP (APPim), but not mature APP (APPm), resulting in a decreased ratio of APPm/APPim; anthocyanin-rich extract (86%) concentrations were used to assess alteration in APP
processing. Collectively, these data indicate that under normal growth conditions, the highest quercetin concentration tested (10 ìM) decreased the ratio of APPm/APPim, suggesting that APP maturation is reduced in SH-SY5Y-APP751 cells due to quercetin-related effects. Subsequent studies revealed that Myrecitin, Quercitin and anthocyanin rich extracts significantly reduce ROS production; administration of anthocyanins enriched extracts resulted in diminished production of ROS which can be responsible for the beginning of the neurodegenerative process of AD in a various neuronal cell.
Fig. 1. Myricetin, quercetin and anthocyanin-rich blackcurrant extracts alleviate menadione-induced ROS production in SH-SY5Y-APP751 cells. The cells were treated with 50 μM menadione together with different concentrations of myricetin (A),quercetin (B), or 86% anthocyanin extracts for 60 min ©, after which ROS production was measured. Groups are compared to menadione-treated samples. ***Pb.001, *Pb.05;one-way ANOVA with Bonferroni correction, n=4–6, S.D.
The effects of different polyphenols on neuroprotection and APP processing compounds were also investigated in transgenic AD mice (APdE9). APdE9 mice fed with anthocyanin-rich bilberry or
blackcurrant extracts showed decreased APP C-terminal fragment levels in the cerebral cortex as compared to APdE9 mice on the control diet. Soluble Aβ40 and Aβ42 levels were significantly decreased in bilberry-fed mice as compared to blackcurrant-fed mice. Conversely, the ratio of insoluble 42/40 was significantly decreased in blackcurrant-fed mice relative to bilberry-fed mice. Both berry diets alleviated the spatial working memory deficit of aged APdE9 mice as compared to mice on the control diet.
Conclusions
Findings suggest that the flavonols and anthocyanin-rich blackcurrant extracts studied here exert
protective effects in different in vitro stress conditions,
particularly in menadione induced ROS production. Interestingly, myricetin consistently encompassed
a protective role in all the studied stress conditions, indicating that this flavonol is a potent bioactive compound with a strong antioxidant-like activity. However, the fact that we only observed
moderate alterations in APP processing emphasized the importance of pursuing these studies also in an in vivo model of AD. Long-lasting supplementation of APdE9 mice with bilberry or blackcurrant revealed
beneficial effects on APP and Aβ metabolism, but not tau-related changes. In addition, these supplementations alleviated behavioral abnormalities in a well-characterized AD mouse model.
Based on the present study, it is anticipated that bilberry- and blackcurrant-derived bioactive compounds display beneficial effects on neuroprotection, behavioral outcome and APP processing and Aβ accumulation.
