Isabella Calzuola

Comparative activity of antioxidants from wheat sprouts, Morinda citrifolia, fermented papaya and white tea

Hydroalcoholic extracts from wheat sprouts, white tea, Morinda citrifolia and fermented papaya were analysed to determine their reducing power and antioxidant activity. The results show that the micromoles of potassium ferricyanide reduced by a quantity of extract corresponding to 1 g of the various dehydrated starting tissues are: 12.91±0.83 (wheat sprouts), 10.66±1.22 (M. citrifolia), 17.06±1.24 (white tea), and 1.05±0.09 (fermented papaya). In addition the results show a strong oxygen superoxide scavenging activity in the extracts from white tea, M. citrifolia and wheat sprouts. The activity of the fermented papaya extract is the lowest. The thin-layer chromatography and UV spectrophotometry of the extracts show in each source a mixture of antioxidant compounds probably belonging to the families of reducing glycosides and polyphenols. The chromatographic pattern of the antioxidant compounds and the UV spectrum are quite different in the various sources.

Wheat sprout extract induces changes on 20S proteasomes functionality

Wheat sprouts contain a very high level of organic phosphates and a powerful cocktail of different molecules such as enzymes, reducing glycosides and polyphenols. The antioxidant properties of wheat sprouts have been widely documented and it has been shown that they are able to protect DNA against free-radicals mediated oxidative damage. Furthermore, we have recently reported on the effects of several polyphenols on 20S proteasomes, underlying the dual role of epigallocatechin-3-gallate as an antioxidant and a proteasome effector in cancer cells. The aim of this study was to investigate the effects of wheat sprout extracts on 20S proteasome functionality. Wheat sprout extracts have been analysed and characterized for their polyphenolic content using the Folin-Ciocalteau reagent and RP-HPLC technique. Comparing our data with a polyphenol standard mixture we identified five different polyphenols: gallic acid, epigallocatechin-3-gallate, epigallocatechin, epicatechin and catechin. The treatment of isolated 20S proteasomes with the extract induced a gradual inhibition of all the tested components, ChT-L, T-L, PGPH and BrAAP, in both the complexes. At low extract concentration a slight activation of the enzyme was evident only for the BrAAP component of the constitutive enzyme and the ChT-L activity of the immunoproteasome. beta-casein degradation rate decreased, particularly with the immunoproteasome. Human Colon adenocarcinoma (Caco) cells, stimulated with 12-O-tetradecanoylphorbol-13-acetate, showed activation of the 20S proteasome activities at short incubation times and an increase in intracellular oxidative proteins. Cells treatment with wheat sprout extract led to proteasome inhibition in unstimulated cells and attenuated the effects mediated by TPA. Finally, exposure to the extract affected the expression levels of pro-apoptotic proteins.

Nutritional relevance of wheat sprouts containing high levels of organic phosphates and antioxidant compounds

Wheat sprouts contain a very high level of organic phosphates and a powerful cocktail of antioxidant molecules. Catalase and peroxidase activity appears very strong. Regarding low-molecular weight antioxidant molecules, TLC and other biochemical analysis show the presence of several classes of antioxidant compounds such as reducing glycosides, polyphenols, and –SH groups. Antioxidant activity has been measured by potassium ferricyanide reduction and by means of superoxide scavenging NBT. It has also been observed that the oral assumption of wheat sprout powder induces in old dogs a significant reduction of senile cataract. These results clearly confirm that the administration of natural antioxidant compounds can prevent or delay senile cataract. Moreover, it is evident that wheat sprout biologically active substances can be at least partially absorbed during the digestion process. Our research is now directed to the recovery of large amounts of wheat sprout biologically active substances that could also be used in the formulation of nutritional complements.

Short acidic peptides isolated from wheat sprout chromatin and involved in the control of cell proliferation. Characterization by infrared spectroscopy and mass spectrometry.

Low molecular weight peptides were isolated from the chromatin of wheat sprouts. Following gel filtration the peptide fraction shows a sharp inhibiting activity on the growth of HeLa cancer cells. Infrared (IR) spectroscopy and mass spectrometry have been utilized to characterize the wheat sprout peptides in an attempt to recognize the peptide sequence involved in the control of cell growth. The quantitative presence of a peptide with MH+=572 appears proportional to the cell growth inhibition activity. This compound has been subjected to extensive mass spectrometry analysis. The automatic computational analysis of the ions of second, third and fourth generations indicate a peptide sequence, AcHis-Asp-Ser-Glu-, that binds at the C-terminal a molecule of ethanolamine. Moreover, the results show that some sequences of the wheat sprout peptide family are present in the peptide fractions isolated from several other tissues, thus supporting the hypothesis of ubiquitous regulatory peptides.

Aging Reversibility: from Thymus Graft to Vegetable Extract Treatment – Application to Cure an Age-associated Pathology

Neonatal thymus graft and thymus calf extract (TME) in vivo treatment exert similar corrective actions on different mouse age-related alterations. The aim of the present paper is to investigate whether a vegetal extract, wheat sprout extract (WESPRE), could mimic the thymus action on recovering age-related alterations and if this extract can cure an age-associated pathology, the cataract in dogs. Present experiments were carried out by using WESPRE and TME in vivo in old mice to check their ability to recover the altered DNA synthesis in hepatocyte primary cultures. Old mice treated with WESPRE and TME showed a recovery of hepatocyte DNA synthesis levels when compared with the old untreated ones. The increase of DNA and protein contents observed in aged animals is reduced by WESPRE treatments to levels observed in young mice hepatocytes. We measured also WESPRE phosphorylation activity by endogenous kinase: it was from 10 to 40 times higher with respect to wheat seeds. Old dogs were orally treated for a month and the lens opacity analysed before and after the treatment. Results showed a reduction from 25 to 40% of lens opacity. The efficacy of wheat sprouts in the recovery of age-related alterations and in treating age-associated pathologies could be due to the contemporary presence of small regulatory acid peptides, a remarkable level of highly energetic phosphoric radicals and antioxidant molecules, peculiarities that may be, to some extent, related to the aging process regulation.

Characterization of Phospholipid Molecular Species and Peptide Molecules in Wheat Sprout Hydroalcoholic Extract

The phospholipid molecular species and the main peptide molecules of wheat sprout hydroalcoholic extract have been fully characterized by normal-phase high performance liquid chromatography coupled online with positive electrospray ionization tandem mass spectrometry. The extract that resulted was rich in phospholipid molecular species formed by the combination of the two essential fatty acids (α-linoleic and α-linolenic). These species accounted for 51.7% of total phosphatidic acid, 47.3% of total phosphatidylethanolamine, 37.7% of total phosphatidylcholine, and 14.1% of total phosphatidylinositol. The last one showed the highest amounts of species containing palmitic acid, thus representing the most saturated phospholipid class. The extract was also shown to contain several peptide sequences with both potential antioxidant domains and interaction sites for phospholipids (i.e., H-Ala-Gly-Ser-Met-Met-Cys-NH2, H-Tyr-Met-Thr-Val-Val-Ala-Cys-NH2, etc.); this latter finding can have a highly positive impact on the poor peptides bioavailability. Because of the presence of essential fatty acids-rich phospholipids and bioactive peptides, wheat sprout hydroalcoholic extract can be considered a potential functional food ingredient.

Interaction of DDSDEEN peptide with N-CAM protein. Possible mechanism enhancing neuronal differentiation

DDSDEEN chromatin peptide, after dansylation, was studied for its ability to bind N-CAM protein. The binding causes a quenching of the Dns-peptide fluorescence emission. Dose- and time-dependent binding of Dns-peptide with N-CAM has been shown. Fluorescence quenching is completely lost if the Dns-peptide is subjected to carboxypeptidase digestion. Moreover the undansylated peptide pEDDSDEEN competes with the DnsDDSDEEN peptide for the binding with the N-CAM protein. The Dns-peptide-N-CAM bond has been related to the peptide biological activity probably involved in the promotion of neuronal differentiation. An attempt to recognize a possible N-CAM binding site for Dns-peptide was performed by alignment of N-CAM from various sources with some sequences that have been previously reported as binding sites for the pEDDSDEEN and DDSDEEN peptides. Interestingly, the alignment of N-CAM from various sources with the peptides WHPREGWAL and WFPRWAGQA recognizes on rat and human N-CAM a unique sequence that could be the specific binding site for chromatin peptide: WHSKWYDAK. This sequence is present in fibronectin type-III domain of N-CAM. In addition molecular modeling studies indicate the N-CAM sequence WHSKWYDAK as, probably, the main active site for DnsDDSDEEN (or pEDDSDEEN) peptide ligand. Accordingly the binding experiments show a high affinity between WHSKWYDAK and DnsDDSDEEN peptides.

Competition between citrate and heptapeptide DDSDEEN binding to DNA in presence of divalent cations

The binding of citrate and acidic peptide DDSDEEN with DNA in the presence of divalent cations is compared. Citric acid shows a higher number of binding sites on the DNA compared to the peptide; this is probably due to the bigger sitric hindrance of the peptide compared to the citric acid for the binding in the DNA grooves. Moreover, DNA preincubated with saturating amounts of citric acid is not available for the binding with successively added peptide. Therefore the peptide and citrate binding sites to some extent overlap.

Biochemical and mass spectrometry recognition of phospholipid–peptide complexes in wheat sprouts extract

Total hydroalcoholic extract of wheat sprouts was treated with 90% cold acetone as a preliminary step directed to separate antioxidant peptides from antioxidant polyphenols. Surprisingly, the addition of acetone causes the formation of a yellow buoyant gelatinous drop that prevailingly contains peptides and phospholipids. In this context, evidences have been presented that support the hypothesis that peptides (and perhaps other active molecules) are complexed with phospholipids. In fact, the MS/MS analysis of some main ions, present in RP HPLC fractions of wheat sprout extract, generates several ions that correspond to molecular weight of phospholipids or phospholipid fragments. Moreover, several ions were detected that correspond to lysophosphatidylcholine or phosphatidylcholine–peptide complexes. The possibility that phospholipids can be complexed with peptides has been discussed in the light of potential involvement in the peptide bioavailability. Copyright

Phenolic Content and Antioxidant Activity in Raw and Denatured Aqueous Extracts from Sprouts and Wheatgrass of Einkorn and Emmer Obtained under Salinity

Total phenolic content (TPC), reducing power (RP), superoxide radical scavenging (RS), and thiobarbituric acid reactive substances (TBARS) production inhibition were measured in raw and denatured aqueous extracts from sprouts and wheatgrass of einkorn and emmer obtained at increasing salinity. Grains were incubated and kept at 0, 25, 50, and 100 mM NaCl until either sprout or wheatgrass stage. Additionally, a recovery treatment was included, in which sprouts obtained at 100 mM NaCl were then transferred at 0 mM NaCl until wheatgrass stage. All parameters (TPC, RP, RS, and TBARS production inhibition) increased with sprouting and were highest in wheatgrass. Salinity increased all parameters, but the effect varied with NaCl concentration, genotype, developmental stage, and plant material processing (raw or denatured). Overall, given the delay and limitation of growth at high NaCl concentration, the best compromise appears to be the application of a moderate salinity (25 to 50 mM NaCl). In denatured extracts, TPC, RP, and RS slightly decreased, and TBARS was not affected, which means that antioxidant activity was mainly related to compounds other than enzymes and peptides, and thus it can be assumed to remain after digestion. Thus, supplementing the human diet with einkorn or emmer sprouts and wheatgrass can actually benefit health.