The Anti-platelet and Anticoagulant Potential of Isorhamnetin and Its New Derivatives Isolated From Sea Buckthorn Fruits in Whole Blood

Blood platelets play a crucial role in hemostasis, the process responsible for keeping blood flowing in the circulatory system. However, unnecessary platelet activation can lead to aggregation at the site of atherosclerotic plaque rapture and the formation of a thrombus, which promotes atherothrombotic diseases. Various dietary components, such as phenolic compounds, are known to demonstrate antiplatelet and anticoagulant properties, and it is possible that these could form an important element in the prophylaxis and therapy of cardiovascular diseases. Our present study examines the biological activity of isorhamnetin (compound 1) and two isorhamnetin derivatives, compound 2 (3-O-beta-glucoside-7-O-alpha-rhamnoside) and compound 3 (3-O-beta-glucoside-7-O-alpha-(3”’-isovaleryl)-rhamnoside), isolated from the phenolic fraction of sea buckthorn fruit, against human washed blood platelets and human whole blood in vitro. The anti-platelet and anticoagulant potential was determined using (A) flow cytometry, (B) the thrombus-formation analysis system (T-TAS) and (C) colorimetry. The tested flavonoids demonstrated anticoagulant and anti-platelet potential, including anti-adhesive activity, with these effects being more intense in compound 2 than isorhamnetin. Compound 2 inhibited GPIIb/IIIa and P-selectin expression on blood platelets from whole blood, and demonstrated anti-adhesion properties in washed blood platelets and anti-coagulant potential in whole blood, measured by T-TAS.

A Study on the Clustering of Extra Virgin Olive Oils Extracted from Cultivars Growing in Four Ionian Islands (Greece) by Multivariate Analysis of Their Phenolic Profile, Antioxidant Activity and Genetic Markers

Background: The phenolic fraction of extra virgin olive oil (EVOO) has disease preventive and health-promoting properties which are supported by numerous studies. As such, EVOO is defined as a functional food. The aim of the present study was to characterize the phenolic profile of olive oil from cultivars farmed in the Ionian Islands (Zakynthos, Kefalonia, Lefkada, and Kerkyra) and to investigate the association of phenols to antioxidant activity, which is central to its functionality. Furthermore, the study investigates whether multivariate analyses on the concentration of individual biophenolic compounds and genetic population diversity could classify the olive oil samples based on their geographic origin. Methods: Phenols were determined in 103 samples from different Ionian Island tree populations by 1H nuclear magnetic resonance (NMR), and sample antioxidant activity was measured by their capacity to reduce the free radical 2,2-diphenyl-1-picrylhydrazyl) (DPPH). Genetic diversity was measured by estimating Nei’s population genetic distance using 15 reproducible bands from random amplified polymorphic DNA (RAPD) genotyping. Results: Principal component analysis (PCA) of the secoiridoid concentrations clustered samples according to cultivar. Clustering based on genetic distances is not concordant with phenolic clustering. A cultivar effect was also demonstrated in the association between the concentration of individual phenols with DPPH reducing activity. Conclusions: Taken together, the study shows that the olive oil phenolic content defines “cultivar-specific phenolic profiles” and that environmental factors other than agronomic conditions contribute more to phenotype variance than genetics.

Development of Analytical Strategies for the Determination of Olive Fruit Bioactive Compounds Using UPLC-HRMS and HPLC-DAD. Chemical Characterization of Kolovi Lesvos Variety as a Case Study

In this study, an overall survey regarding the determination of several bioactive compounds in olive fruit is presented. Two methodologies were developed, one UPLC-Q-TOF-MS method for the determination of olive fruit phenolic compounds and one HPLC-DAD methodology targeting the determination of pigments (chlorophylls and carotenoids), tocopherols (α-, β, -γ, δ-) and squalene. Target and suspect screening workflows were developed for the thorough fingerprinting of the phenolic fraction of olives. Both methods were validated, presenting excellent performance characteristics, and can be used as reliable tools for the monitoring of bioactive compounds in olive fruit samples. The developed methodologies were utilized to chemical characterize the fruits of the Kolovi olive variety, originating from the island of Lesvos, North Aegean Region, Greece. Twenty-five phenolic compounds were identified and quantified in Kolovi olives with verbascoside, hydroxytyrosol, oleacein and oleomissional found in significantly high concentrations. Moreover, 12 new bioactive compounds were identified in the samples using an in-house suspect database. The results of pigments analysis suggested that Kolovi variety should be characterized as low pigmentation, while the tocopherol and squalene content was relatively high compared to other olive varieties. The characterization of Kolovi olive bioactive content highlighted the high nutritional and possible economic value of the Kolovi olive fruit.

Antiproliferation Effects of Nanophytosome-Loaded Phenolic Compounds From Fruit of Juniperus Polycarpos Against Breast Cancer in Mice Model: Synthesis, Characterization and Therapeutic Effects

Background: This research was performed to synthesize nanophytosomes-loaded high phenolic fraction (HPF) from Juniperus polycarpos fruit extract and investigate its antiproliferation effects against breast cancer in mice model. Results: The nanophytosomes-loaded HPF from Juniperus polycarpos fruit extract was synthesized. The mice trial was conducted to determine the possible toxic effects of the synthesized nanophytosomes. The anticancer, pro-apoptotic, and antioxidative activities of the nanophytosomes were determined. The nanophytosomes-loaded HPF had a spherical structure with a size of 176 nm and a polydispersity index coefficient of 0.24. The in-vivo study manifested that nanophytosomes-loaded HPF significantly improved weight gain and food intake compared to the negative control group (p<0.05). The nanophytosomes-loaded HPF significantly enhanced the expression of bax (3.4-fold) and caspase-3 (2.7-fold) genes but reduced bcl2 (3.6-fold) gene expression in tumor cells. The average tumor size was significantly decreased in mice treated with nanophytosomes-loaded HPF (p<0.05). The expression of GPX (2.3-fold) and SOD (2.7-fold) antioxidants in the liver of mice supplemented with nanophytosomes-loaded HPF was significantly developed compared to the negative control (p<0.05). The nanophytosomes-loaded HPF did not show toxicity on normal cells. Conclusion: Our results indicated that nanophytosomes-loaded HPF might be a potential anticancer agent for the breast cancer treatment.

Bioactive compounds from Lactarius deterrimus interfere with the invasive potential of gastric cancer cells

Stomach cancer is the 4th most common cancer diagnosed worldwide. Despite intensive research on its etiopathology, its treatment strategies have not changed in the last 50 years. Mushrooms have recently attracted much attention as the source of bioactive compounds that can potentially complement cancer therapies. Here, we extracted a phenolic fraction from Lactarius deterrimus and analyzed its composition and bioactivity against the gastric cancer (AGS) cells. The complexity of L. deterrimus compounds was revealed by an HPLC assay, and was accompanied by cytostatic, cytotoxic and anti-invasive effects of the L. deterrimus extract (LDE). These are illustrated by inhibition of the AGS cells’ proliferation, metabolic activity and motility, and by induction of the cytoskeleton rearrangements. Apparently, these effects are exerted via activation of intracellular oxidative stress and decreased ATP production in AGS cells that could not be compensated by induction of autophagy. Less severe LDE effects were seen on physiology of normal gastric fibroblasts; however, inhibition of their motility indicates that LDE can interfere with gastric cancer development via an effect on stromal cells. Along with the observed synergy of LDE and cisplatin/5-fluorouracil effects on AGS cells, our data show the potential of LDE for supplementation of the gastric cancer therapy.

Valorization of Wastewater from Table Olives: NMR Identification of Antioxidant Phenolic Fraction and Microwave Single-Phase Reaction of Sugary Fraction

The table olive industry is producing a huge amount of wastewater, which is a post-processing cost and an environmental concern. The present study aims to valorize this processing by-product to obtain a value-added product, thereby enhancing resource efficiency and contributing to achieving sustainable development goals (SDGs). In this sense, a chemical reaction-based platform was developed to obtain valuable components, such as levulinic acid (LA) and 5-hydromethylfurfural (HMF). The products were then analyzed using NMR identification of the antioxidant phenolic fraction and microwave single-phase reaction of the sugary fraction. According to the results, the highest concentration of phenolic compounds does not correspond to the sample directly obtained from NaOH treatment (S1), indicating that water washing steps (S2–S5) are fundamental to recover phenolic substances. Moreover, glucose was presented in the sugary fraction that can be transformed into levulinic acid by a single-phase reaction under microwave irradiation. The information provided in this manuscript suggests that the wastewater from the olive processing industry can be valorized to obtain valuable products.

Exploring Gunnera tinctoria: From Nutritional and Anti-Tumoral Properties to Phytosome Development Following Structural Arrangement Based on Molecular Docking

Gunnera tinctoria, an underexplored invasive plant found in Azores, Portugal, was studied regarding its nutritional, antioxidant, and antitumoral properties. Higher antioxidant activity was found in baby leaves, followed by adult leaves and inflorescences. A phenolic fraction of the plant was enriched using adsorbent resin column chromatography (DiaionTM HP20LX, and Relite EXA90). Antitumoral effects were observed with the enriched fractions in breast (MCF-7) and pancreatic (AsPC-1) cancer cell lines, being more pronounced in the latter. To improve protection and membrane absorption rates of phenolic compounds, nano-phytosomes and cholesterol-conjugated phytosomes coated with natural polymers were loaded with the enriched fraction. The particles were characterized, and their physiochemical properties were evaluated and compared. All samples presented anionic charge and nanometer size in relation to the inner layer and micrometer size regarding the external layers. In addition, the molecular arrangement of phenolics within both types of phytosomes were studied for the first time by molecular docking. Polarity and molecular size were key factors on the molecular arrangement of the lipid bilayer. In conclusion, G. tinctoria showed to be an interesting source of nutrients and phenolic compounds with anti-tumoral potential. Moreover, phytosome loading with these compounds can increase their stability and bioavailability having in view future applications.

The Possibility of Using Paulownia elongata S. Y. Hu × Paulownia fortunei Hybrid for Phytoextraction of Toxic Elements from Post-Industrial Wastes with Biochar

The potential of the Paulownia hybrid for the uptake and transport of 67 elements along with the physiological response of plants cultivated in highly contaminated post-industrial wastes (flotation tailings—FT, and mining sludge—MS) was investigated. Biochar (BR) was added to substrates to limit metal mobility and facilitate plant survival. Paulownia could effectively uptake and translocate B, Ca, K, P, Rb, Re and Ta. Despite severe growth retardation, chlorophyll biosynthesis was not depleted, while an increased carotenoid content was noted for plants cultivated in waste materials. In Paulownia leaves and roots hydroxybenzoic acids (C6-C1) were dominant phenolics, and hydroxycinnamic acids/phenylpropanoids (C6-C3) and flavonoids (C6-C3-C6) were also detected. Plant cultivation in wastes resulted in quantitative changes in the phenolic fraction, and a significant drop or total inhibition of particular phenolics. Cultivation in waste materials resulted in increased biosynthesis of malic and succinic acids in the roots of FT-cultivated plants, and malic and acetic acids in the case of MS/BR substrate. The obtained results indicate that the addition of biochar can support the adaptation of Paulownia seedlings growing on MS, however, in order to limit unfavorable changes in the plant, an optimal addition of waste is necessary.

Mechanistic Insights into the Inhibition of SARS-CoV-2 Main Protease by Clovamide and Its Derivatives: In Silico Studies

The novel coronavirus SARS-CoV-2 Main Protease (Mpro) is an internally encoded enzyme that hydrolyzes the translated polyproteins at designated sites. The protease directly mediates viral replication processes; hence, a promising target for drug design. Plant-based natural products, especially polyphenols and phenolic compounds, provide the scaffold for many effective antiviral medications, and have recently been shown to be able to inhibit Mpro of SARS-CoV-2. Specifically, polyphenolic compounds found in cacao and chocolate products have been shown by recent experimental studies to have strong inhibitory effects against Mpro activities. This work aims to uncover the inhibition processes of Mpro by a natural phenolic compound found in cacao and chocolate products, clovamide. Clovamide (caffeoyl-DOPA) is a naturally occurring caffeoyl conjugate that is found in the phenolic fraction of Theobroma Cacao L. and a potent radical-scavenging antioxidant as suggested by previous studies of our group. Here, we propose inhibitory mechanisms by which clovamide may act as a Mpro inhibitor as it becomes oxidized by scavenging reactive oxygen species (ROS) in the body, or becomes oxidized as a result of enzymatic browning. We use molecular docking, annealing-based molecular dynamics, and Density Functional Theory (DFT) calculations to study the interactions between clovamide with its derivatives and Mpro catalytic and allosteric sites. Our molecular modelling studies provide mechanistic insights of clovamide inhibition of Mpro, and indicate that clovamide may be a promising candidate as a drug lead molecule for COVID-19 treatments.