Chemical analysis of Cabernet Sauvignon and muscadine wines. Constituent polyphenolic components in Cabernet Sauvignon (A,C,E) and muscadine (B,D,F) wines were analyzed by reverse phase HPLC using a C18 column. (A,B) Detection of phenolic acid compounds at 280 nm. (C,D) Detection of flavonoids at 370 nm. (E,F) Detection of anthocyanins at 520 nm. (G) Identification of polyphenols corresponding to peaks detected in panel (A-F) based on spectroscopic interpretations

Chemical analysis of Cabernet Sauvignon and muscadine wines. Constituent polyphenolic components in Cabernet Sauvignon (A,C,E) and muscadine (B,D,F) wines were analyzed by reverse phase HPLC using a C18 column. (A,B) Detection of phenolic acid compounds at 280 nm. (C,D) Detection of flavonoids at 370 nm. (E,F) Detection of anthocyanins at 520 nm. (G) Identification of polyphenols corresponding to peaks detected in panel (A-F) based on spectroscopic interpretations

No detectable changes in α-, β- and γ-secretase activities in the brain of Tg2576 mice in response to muscadine treatment. α–, β-, and γ-secretase enzymatic activities were assessed in cerebral cortex brain specimens from the same 14 month old Tg2576 animals used in the behavioral studies in Fig. 2 and neuropathology assessments in Fig. 3. (A) α-secretase activity, (B) β-secretase activity, and (C) γ-secretase activity in Tg-muscadine and Tg-control mice. (A-C). Bar graphs represent group mean (± SEM), n = 6–8 per group.

Muscadine treatment significantly reduced Aβ neuropathology in Tg2576 mice. Tg2576 mice were assessed for indexes of AD-type amyloid burden in the brain in response to muscadine treatment using stereological technologies; the same 14 month old Tg2576 mice used for behavioral assessments in Fig. 2 were used in this study. (A) Assessments of amyloid neuritic plaque density in cerebral cortex and in the hippocampal formation of brain specimens from muscadine-treated and control, non-treated Tg2576 mice. (A, inset) Control studies confirming muscadine treatment did not modulate expression of the holo-AβPP in the brain (cerebral cortex). Bar graphs represent means ± SEM., n = 6–8 per group; * P < 0.05 vs. non-treated control Tg2576 group (2-tailed Student's t test). (B) Representative micrograph of brain specimen stained for amyloid neuritic plaques in muscadine-treated (Tg-musc.) or in control, non-treated (Tg-cont.) Tg2576 mice.

Muscadine treatment significantly attenuated the accumulation soluble HMW Aβ species in the brain of Tg2576 mice. The contents of HMW Aβ oligomeric species in cerebral cortex brain specimens from muscadine-treated or control, non-treated 14 month old Tg2576 mice were assessed by independent dot-blot (A) and western blot studies (B). (A) Immunodetection of HMW Aβ oligomeric species in the cerebral cortex and in the hippocampal formation using the oligomer-specific antibody A11 antibody in an dot blot assay. (A, inset) Representative A11-immunoreactive dot-blot analysis of cortical and hippocampal formation brain specimens. Abbreviations: C: control, non-treated Tg2576 mice; M: muscadine-treated Tg2576 mice. (B) Western blot analysis in which HMW Aβ oligomeric species are resolved by gel electrophoresis followed by immunodetection using a pan-Aβ 6E10 antibody. (B, Inset) Representative western blot analysis of muscadine-treated and control non-treated Tg2576 mice. Immunoreactive holo-AβPP and relatively less abundant HMW Aβ species are identified. In (A, B), bar graphs represent means ± SEM., n = 6–8 per group; * P < 0.05 vs. non-treated control Tg2576 group (2-tailed Student's t test).

Muscadine polyphenols exert anti-Aβ oligomerization bioactivity in vitro. (A) Muscadine wine interferes with aggregation of synthetic Aβ_1–42 peptides into HMW oligomer Aβ species, in vitro. Synthetic Aβ_1–42 peptides were aggregated in the absence or in the presence of muscadine wine. Aβ species were then resolved by molecular size, transblotted onto a nitrocellulose membrane, followed by immunodetection of Aβ peptides using and the 6E10 antibody. Lane 1 represents non-aggregated Aβ_1–42 peptides; Lane 2, aggregated Aβ_1–42 peptides; Lane 3, Aβ_1–42 peptides aggregated in the presence of 1.2% ethanol (the same amount of ethanol presented in the aggregation assay in the presence of muscadine in Lane 4); Lane 4, Aβ_1–42 peptides aggregated in the presence of 1 μl muscadine wine. (B) Application of an independent PICUP assay to explore the role of muscadine wine and its polyphenolic components to interfere with aggregations of synthetic Aβ_1–40 peptides. The PICUP assay is designed to explore the initial protein-protein interactions that are necessary for the formation of HMW Aβ oligomeric species. Aβ aggregates are stabilized by photo-cross-linking. Monomeric and multimeric Aβ species are separated by molecular size and visualized by silver staining. Lane 1 represents molecular weight marker; Lane 2, non-aggregated Aβ_1–40 peptide; Lane 3, aggregated Aβ_1–40 peptide; Lane 4, Aβ_1–40 peptide aggregated in the presence of muscadine wine (2 μl of the wine); Lane 5, Aβ_1–40 peptide aggregated in the presence of a polyphenolic extract from 2 μl of the muscadine wine. The position of Aβ_1–40 monomer, dimmer, trimer and 4-mer are as indicated.

Muscadine treatment improves spatial memory function in Tg2576 mice. Assessments of spatial memory behavioral functions of 14 month old muscadine-treated (Tg-muscadine) and control, gender- and age-matched non-treated (Tg-control) Tg2576 mice using the Morris water maze (MWM) protocol. (A) Learning trial hidden-platform acquisition curves. Tg-muscadine group performed significantly better than the control, non-treated group (Tg-control) [2-way ANOVA analysis of Tg-muscadine vs. Tg-control groups for muscadine treatment (p < 0.05, F = 4.24, DFn = 1, DFd = 84) and for training days (p < 0.05, F = 6.43, DFn = 6, Dfd = 84)]. (B) Probe trial conducted 24 hours after completion of hidden-platform training. Muscadine-treated Tg2576 mice exhibited a significantly higher preference for the target platform compared to control, non-treated Tg2576 mice (p < 0.05, 2-tailed Student t test). (C) Visible-platform learning curves. There is no significant difference in visible platform performance among Tg-muscadine compared to Tg-control mice. (D) Average swimming speed. There is no significant difference in swimming ability of muscadine-treated animals compared to control, non-treated animals. In (A-D) Values represent group mean (+SEM); n = 7–9 mice per group.