Why proanthocyanidins elute at increasing order of molecular masses when analysed by normal phase high performance liquid chromatography? Considerations of use.

Although it is widely known that proanthocyanidins elute at an increasing order of molecular masses when analysed by normal phase high performance liquid chromatography (NP-HPLC), there is no a consistent explanation of the mechanisms of their separation until now. Therefore, the aim of the present study was to give a reliable response to this question, using a complex procyanidin-rich grape seed extract. For this, an off-column static simulation of extract injection and a fragmented-column dynamic procyanidin location tests were studied to show their precipitation in an aprotic solvent, besides another off-column static simulation and multiple contact dynamic solubilisation tests to confirm procyanidin redissolution in an aprotic/protic solvent system. The results showed that separation of procyanidins in the aprotic/protic solvent system of Diol-NP-HPLC was governed by precipitation/redissolution mechanism, that could be extended to all known plant proanthocyanidin homopolymers, including hydrolysable tannins, if they are able to accomplish this condition. However, separation of monomer species, namely catechins and some hydroxybenzoic acids, was based on classic adsorption/partition mechanism. Other factors, such as analyte solubility, chromatographic conditions and sample preparation, that affect the viability of proanthocyanidin analysis by NP-HPLC were stand out and guidelines for its durable and reproducible use were defined.

Olive Leaf as a Source of Antibacterial Compounds Active against Antibiotic-Resistant Strains of Campylobacter jejuni and Campylobacter coli.

Campylobacter spp. are the main cause of bacterial gastroenteritis worldwide, and broiler chicks are the main vector of transmission to humans. The high prevalence of Campylobacter in poultry meat and the increase of antibiotic resistant strains have raised the need to identify new antimicrobial agents. For this reason, the aim of the current study was to evaluate the antibacterial activity of two extracts of olive leaf against antibiotic-resistant Campylobacter strains (C. jejuni and C. coli) isolated from poultry food chain. The extracts of olive leaf (E1 and E2) were markedly different in their chemical compositions. While E1 was composed predominantly of highly hydrophilic compounds such as hydroxytyrosol and hydroxytyrosol glucosides (14,708 mg/100 g), E2 mainly contained moderately hydrophilic compounds, with oleuropein (20,471 mg/100 g) being prevalent. All Campylobacter strains exhibited similar antibiotic profiles, being resistant to ciprofloxacin and tetracycline. E1 showed strong antibacterial activity and reduced bacterial growth from 4.12 to 8.14 log CFU/mL, depending on the strain. Hydroxytyrosol was the main compound responsible, causing the inhibition of growth of Campylobacter strains at low concentrations (0.1-0.25 mg/mL). E2 demonstrated a lower antibacterial effect than E1, reducing growth from 0.52 to 2.49 log CFU/mL. The results of this study suggest that the optimization of the composition of olive-leaf extracts can provide improved treatment results against Campylobacter strains.

Influence of In Vitro Gastric Digestion of Olive Leaf Extracts on Their Bioactive Properties against H. pylori.

The aim of this work was to evaluate the influence of in vitro gastric digestion of two olive leaf extracts (E1 and E2) on their chemical composition and bioactive properties against Helicobacter pylori (H. pylori), one of the most successful and prevalent human pathogens. HPLC-PAD/MS analysis and anti-inflammatory, antioxidant, and antibacterial activities of both olive leaf extracts were carried out before and after their in vitro gastric digestion. The results showed that gastric digestion produced modifications of the chemical composition and bioactive properties of both olive leaf extracts. The main compounds in the extract E1 were hydroxytyrosol and its glucoside derivatives (14,556 mg/100 g), presenting all the identified compounds a more polar character than those found in the E2 extract. E2 showed a higher concentration of less polar compounds than E1 extract, with oleuropein (21,419 mg/100 g) being the major component. Gastric digestion during the fasted state (pH 2) induced an overall decrease of the most identified compounds. In the extract E1, while the anti-inflammatory capacity showed only a slight decrease (9% of IL-8 production), the antioxidant properties suffered a drastic drop (23% of ROS inhibition), as well as the antibacterial capacity. However, in the extract E2, these changes caused an increase in the anti-inflammatory (19% of IL-8 production) and antioxidant activity (9% of ROS inhibition), which could be due to the hydrolysis of oleuropein and ligustroside into their main degradation products, hydroxytyrosol and tyrosol, but the antibacterial activity was reduced. Gastric digestion during fed state (pH 5) had less influence on the composition of the extracts, affecting in a lesser degree their anti-inflammatory and antioxidant activity, although there was a decrease in the antibacterial activity in both extracts similar to that observed at pH 2.

Olive-Leaf Extracts Modulate Inflammation and Oxidative Stress Associated with Human H. pylori Infection.

Helicobacter pylori (H. pylori) is one of the major human pathogens and the main cause of pathological damages that can progress from chronic gastritis to gastric cancer. During the colonization of gastric mucosa, this bacterium provokes a strong inflammatory response and subsequent oxidative process, which are associated with tissue damage. Therefore, the objective of this research was to evaluate the ability of two olive-leaf extracts (E1 and E2) to modulate the inflammatory response and oxidative stress in H. pylori-infected human gastric AGS cells. The obtained results showed that both extracts significantly decreased interleukin-8 (IL-8) secretion and reactive oxygen species (ROS) production in human gastric AGS cells. Both extracts also showed antibacterial activity against different H. pylori strains. HPLC-PAD-MS characterization demonstrated that extract E1 was mainly composed of highly hydrophilic compounds, such as hydroxytyrosol (HT) and its glucosides, and it was the most effective extract as an antibacterial agent. In contrast, extract E2 was composed mostly of moderately hydrophilic compounds, such as oleuropein (OLE), and it was more effective than extract E1 as an anti-inflammatory agent. Both extracts exhibited similar potential to decrease ROS production. These results show the importance of standardizing the extract composition according to the bioactive properties that should be potentiated.

Pre-Treatment with Grape Seed Extract Reduces Inflammatory Response and Oxidative Stress Induced by Helicobacter pylori Infection in Human Gastric Epithelial Cells.

Helicobacter pylori (H. pylori) is a pathogenic bacteria identified as a potential risk factor for gastritis, gastric ulcers and gastric cancer. During the stomach colonization, H. pylori triggers a strong inflammatory response and subsequent oxidative stress, which are associated with tissue damage. For this reason, it is of particular interest to develop alternative natural tools that enable modulation of the associated damaging immune response. With this purpose, we obtained grape seed extract (GSE) from sweet (not fermented) food grade seeds. The aim of our study was to investigate the effect of GSE and its two enriched procyanidins fractions (OPC and PPC) on the inflammatory process and oxidative stress produced by different H. pylori strains in human gastric epithelial cells (AGS). Anti-inflammatory activity was evaluated by measuring the level of interleukin-8 (IL-8) secretion. IL-8 production was significantly reduced in H. pylori-infected human gastric epithelial cells pre-treated with GSE or its enriched fractions when compared with non-pre-treated infected cells (from 21.6% to 87.8%). Pre-treatment with GSE or its fractions significantly decreased intracellular reactive oxygen species (ROS) production in AGS cells after infection, depending on the H. pylori strain. Our results also showed that GSE and its fractions demonstrate antibacterial activity against all strains of H. pylori used in the study. This work demonstrates the effectiveness of GSE enriched in procyanidins against the main events associated with H. pylori infection.

Extracts from the edible insects Acheta domesticus and Tenebrio molitor with improved fatty acid profile due to ultrasound assisted or pressurized liquid extraction.

Extracts from the edible insects Acheta domesticus and Tenebrio molitor were obtained by ultrasound-assisted extraction (UAE) and pressurized-liquid extraction (PLE) using ethanol (E) or ethanol:water (E:W). Extraction yield, fatty acid profile, nutritional impact and cholesterol content were determined and compared with the initial insects. The highest extraction yield corresponded to PLE-T. molitor extracts. A decrease in total saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) contents, and an increase in the total polyunsaturated fatty acid (PUFA) content were observed for both UAE-E:W insect extracts, due to an enrichment in linoleic acid. The lipid indices (PUFA/SFA ratio, atherogenic and thrombogenic indices) for both UAE-E:W extracts were significantly improved compared with the initial insects. Although either extraction procedure led to cholesterol enrichment, the UAE-E:W conditions favoured the lowest concentration. Therefore, insects extracts with improved fatty acid profile can be selectively obtained, being UAE-E:W conditions preferred from the nutritional point of view.

Characterization, antioxidant activity, and inhibitory effect on pancreatic lipase of extracts from the edible insects Acheta domesticus and Tenebrio molitor.

Extracts from the edible insects Acheta domesticus and Tenebrio molitor were obtained by ultrasound-assisted extraction (UAE) and pressurized-liquid extraction (PLE) using ethanol (E) or ethanol:water (E:W). Characterization by GC-MS was performed and total phenolic compounds (TPC), antioxidant activity (DPPH) and pancreatic lipase inhibitory capacity were assayed. Most extracts, mainly ethanolic extracts, predominantly presented lipids as free fatty acids, followed by aminoacids, organic acids, carbohydrates, hydrocarbons and sterols. The UAE-E:W extracts were different, being characterized by organic acids for A. domesticus, or aminoacids for T. molitor. All the extracts exhibited antioxidant activity, which correlated with TPC values, being the E:W extracts the most effective. All the extracts showed inhibitory activity of lipase, although those from T. molitor and extracted by PLE were the most effective. Therefore, bioactive insect extracts can be selectively obtained by advanced methods of extraction, being aqueous ethanol preferred for antioxidant activity and PLE for inhibitory lipase activity.

Procyanidin-Rich Extract from Grape Seeds as a Putative Tool against Helicobacter pylori.

Strains of Helicobacter pylori (H. pylori) resistant to various antibiotics have increased in recent years. In this context, the search for new therapeutic approaches is crucial. The aim of the present study was to demonstrate the antibacterial activity of a procyanidin-rich extract obtained from food-grade winery grape seeds against 14 H. pylori strains and elucidate its phenolic composition. Ten strains (71.4%) showed resistance to at least some of the tested antibiotics, while four isolates (28.6%) were susceptible to all antibiotics. Resistance to more than one class of antibiotics was observed in six strains (42.9%). The extract was able to inhibit the growth of all H. pylori strains in a range of a minimum inhibitory concentration (MIC) from 0.015 mg/mL to 0.125 mg/mL, confirming also the existence of a strain-dependent effect. The phenolic composition determined by reverse phase high pressure liquid chromatography, photodiode array, and mass spectrometry detection (RP-HPLC-PAD-MS) analysis revealed the presence of 43 individual compounds and allowed the quantification of 41 of them, including seven procyanidin tetramers, seven procyanidin pentamers, and six galloylated procyanidin dimers, trimers, and tetramers. The extract was composed mainly by catechin and procyanidin oligomers with a total amount of 5.801 mg/100 g, which represent 92% of the total individual phenolic content. Among them, the most abundant were catechins (2.047 mg/100 g), followed by procyanidin dimers (1.550 mg/100 g), trimers (1.176 mg/100 g), tetramers (436 mg/100 g), and pentamers (296 mg/100 g) that represent 35, 27, 20, 8, and 5%, respectively of the total flavanol constituents. The composition profile information may help to improve the production process of useful antibacterial extracts against H. pylori.

Exploring the Extracellular Macromolecular Composition of Crude Extracts of Penicillium rubens Strain 212 for Elucidation Its Mode of Action as a Biocontrol Agent.

Penicillium rubens strain 212 (PO212) acts as an inducer of systemic resistance in tomato plants. The effect of crude extracellular extracts of PO212 on the soil-borne pathogen Fusarium oxysporum f. sp. lycopersici has been evaluated. Evidence of the involvement of soluble, thermo-labile, and proteinase-inactivated macromolecules present in PO212 crude extracts in the control of Fusarium vascular disease in tomato plants was found. Proteomic techniques and the availability of the access to the PO212 genome database have allowed the identification of glycosyl hydrolases, oxidases, and peptidases in these extracellular extracts. Furthermore, a bioassay-guided fractionation of PO212 crude extracellular extracts using an integrated membrane/solid phase extraction process was set up. This method enabled the separation of a PO212 crude extracellular extract of seven days of growth into four fractions of different molecular sizes and polarities: high molecular mass protein fraction >5 kDa, middle molecular mass protein fraction 5-1 kDa, low molecular mass metabolite fraction, and nutrients from culture medium (mainly glucose and minerals). The high and middle molecular mass protein fractions retained disease control activity in a way similar to that of the control extracts. Proteomic techniques have allowed the identification of nine putatively secreted proteins in the high molecular mass protein fraction matching those identified in the total crude extracts. Therefore, these enzymes are considered to be potentially responsible of the crude extracellular extract-induced resistance in tomato plants against F. oxysporum f. sp. lycopersici. Further studies are required to establish which of the identified proteins participate in the PO212's action mode as a biocontrol agent.

Evaluation of an Integrated Ultrafiltration/Solid Phase Extraction Process for Purification of Oligomeric Grape Seed Procyanidins.

The effectiveness of a preparative integrated ultrafiltration/solid-phase extraction (UF/SPE) process for purification of oligomeric procyanidins (OPCs) from a crude grape seed extract (GSE) was studied for the first time. The separation of OPCs from polymeric procyanidins (PPCs) by UF was very efficient. The membrane showed an acceptable filtration flux of 6 to 3.5 L/h·m2 at 0.5 bar of transmembrane pressure and 95% recovery of its water flux after chemical cleaning. The process was scalable to a pilot scale. The separation of very polar and ionic species from OPCs by SPE (XAD7HP and XAD16 resins) was also very good, but both adsorbents lost their retention capacities quickly, due probably to irreversible retention of OPCs/PPCs. Even though the global purification of OPCs by the integrated UF/SPE process allowed the recovery of 24.2 g of highly purified OPCs (83% purity) from 14.4 L of crude grape seed extract, the use of these adsorbents for further purification of the OPCs was very limited.