Wild-Growing and Conventionally or Organically Cultivated Sambucus nigra Germplasm: Fruit Phytochemical Profile, Total Phenolic Content, Antioxidant Activity, and Leaf Elements.

European elder or elderberry (Sambucus nigra L., Viburnaceae) is a plant species with known high pharmaceutical and nutritional value. However, the Greek native germplasm of S. nigra has not been adequately utilized to date as in other regions. This study evaluates the fruit antioxidant potential (total phenolic content and radical scavenging activity) of wild-growing and cultivated germplasm of Greek S. nigra. In addition, nine cultivated Greek S. nigra genotypes were evaluated regarding the effects of fertilization (conventional and organic) on fruit phytochemical and physicochemical potential (total flavonoids, ascorbic acid content, pH, total soluble solids, and total acidity), as well as on the antioxidant potential (total phenolic content and radical scavenging activity) of fruits and leaves. Additionally, an analysis of macro- and micro-elements in the leaves of the cultivated germplasm was performed. The results demonstrated comparatively higher total phenolic contents of fruits of cultivated germplasm. The genotype was the decisive factor in the fruits' phytochemical potential and leaves' total phenolic content of cultivated S. nigra germplasm. Similarly, fertilization regime effects were found to be genotype-dependent, affecting fruit phytochemical and physicochemical attributes. The trace element analysis results were similar, with genotypes varying significantly in their concentrations of macro- and micro-elements. The current work builds on previous domestication attempts for Greek S. nigra, providing new data on the phytochemical potential of this important nutraceutical species.

Synergistic effect of peptide inhibitors derived from the extracellular and intracellular domain of αIIb subunit of integrin αIIbß3 on platelet activation and aggregation.

αIIbß3, the major platelet integrin, plays a central role in hemostasis and thrombosis. Upon platelet activation, conformation of αIIbß3 changes and allows fibrinogen binding and, subsequently, platelet aggregation. It was previously shown that a lipid-modified platelet permeable peptide, which corresponds to the intracellular acidic membrane distal sequence 1000LEEDDEEGE1008 of αIIb (pal-K-LEEDDEEGE or pal-K-1000-1008), inhibits thrombin-induced human platelet aggregation, by inhibiting talin association with the integrin. YMESRADR, a peptide corresponding to the extracellular sequence 313-320 of αIIb, is also a potent platelet aggregation inhibitor by mimicking the effect of a clasp between the head domains of αIIb and ß3. The aim of the present study was to investigate the synergistic effect of the intra- and extracellular- peptide inhibitors on platelet aggregation, as well as on the phosphorylation of two signaling proteins, focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Platelet preincubation with Pal-K-LEEDDEGE followed by YMESRADR showed a synergistic inhibitory activity on platelet aggregation. Platelet incubation with threshold inhibitory concentrations of both peptides provoked almost the total inhibition of aggregation, PAC-1 binding, and fibrinogen binding, but not P-selectin exposure on activated platelets' surface. Like RGDS peptide, this mixture inhibits FAK phosphorylation whose phosphorylation is well known to be consecutive to the aggregation (postoccupancy events). However, in contrast to RGDS peptide that enhances ERK phosphorylation and activation, the mixture of threshold inhibitory concentrations of Pal-K-LEEDDEEGE and YMESRADR inhibits ERK phosphorylation. We suggest that the use of the intracellular in combination with the extracellular peptide inhibitor, acting with a non-RGD-like mechanism, may provide an alternative way to antagonize integrin αIIbß3 activation.

Palmitoylated peptide, being derived from the carboxyl-terminal sequence of the integrin αIIb cytoplasmic domain, inhibits talin binding to αIIbß3.

The αIIb cytoplasmic domain of platelet integrin αIIbß3 contains an unorganized acidic membrane-distal (1000)LEEDDEEGE(1008) region. We have shown that a platelet permeable peptide corresponding to the above region the palmitoyl-K-LEEDDEEGE (pal-K-1000-1008) inhibits platelet aggregation induced by thrombin or by pal-K-989-995, a palmitoylated peptide corresponding to the membrane-proximal αIIb cytoplasmic domain (989)KVGFFKR(995). We now tested the anti-aggregatory activity of (i) a lipid-modified scrambled acidic peptide (pal-K-GDDEELEEE), (ii) two smaller peptides derived from the acidic amino sequence: palmitoyl-K-(1000)LEEDDE(1005) (pal-K-1000-1005) and palmitoyl-K-(1005)EEGE(1008) (pal-K-1005-1008) and (iii) lipid-modified palmitoyl-acidic peptides with alanine (Ala) substitution at residues 1001, 1003, 1004 and 1005 and one peptide with a double Ala substitution at residues 1001 and 1004 of the 1000-1008 sequence. All the peptides tested showed an inhibitory activity, however, the palmitoylated peptide with the natural and the whole acidic sequence, being the most active. Our results suggest that the whole acidic sequence, rather than some specific amino acids, contributes to the aggregation inhibitory activity. The inhibitory peptide, pal-K-1000-1008, inhibited the association of talin with αIIbß3 in thrombin-activated platelets, as demonstrated by co-immunoprecipitation experiments, while the scrambled peptide was inefficient. We suggest that, by interacting with αIIb cytoplasmic domain, pal-K-1000-1008 has an anti-aggregatory inhibitory activity due to a specific inhibition of talin binding to αIIbß3.