HPLC-DAD method for simultaneous determination of gallic acid, catechins, and methylxanthines and its application in quantitative analysis and origin identification of green tea.

The high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) was optimized for the simultaneous determination of 11 compounds, belonging to polyphenols (gallic acid and seven catechins) and methylxanthines (caffeine, theobromine, and theophylline). The results obtained for all the validation parameters of the HPLC-DAD method showed that the method is sensitive enough for routine analysis with basic chromatographic equipment, thus it has a significant potential to be highly applicable in common laboratory practice. The method was used in the analysis of 60 green tea infusions originating from four tea-producing countries. The dataset contributes to enhancing current data on green tea. The analysis of green tea extracts revealed significant differences depending on the origin of the samples. Linear Discriminant Analysis (LDA) was applied to test the accuracy of identification of the origin of the tea samples, based on the chemical composition of tea with a focus on polyphenolic compounds and methylxanthines analysed in this study. Based on cross-validation results, the model showed 93.75 % accuracy in the classification of green tea originating from Japan, China (Mainland), China (Taiwan) and South Korea.

In vitro versus cryo-induced capacitation of bovine spermatozoa, part 3: Compositional and molecular changes to the plasma membrane.

The aim of this study was to assess the level of membrane cryodamage through the levels of selected capacitation and apoptosis-associated proteins, together with compositional membrane changes in capacitated (CAP), cryopreserved (CRYO) and non-capacitated bovine spermatozoa (CRTL). Sperm kinetic parameters were analyzed by the computer assisted sperm analysis (CASA) while the capacitation patterns were examined with the chlortetracycline (CTC) assay. In the case of DNA integrity, sperm chromatin structure assay and aniline blue staining were used. For the quantification of fatty acid content gas chromatography was performed. Using Western blotting the expression of capacitation (protein kinase C - PKC; phospholipases A2 and Cζ - PLA2, PLCζ; soluble adenylyl cyclase 10 - sAC10) and apoptosis-associated (apoptosis regulator Bax; B-cell lymphoma 2 - Bcl-2; caspase 3) proteins were evaluated. Data indicate a significant decline (p < 0.0001) of sperm kinetic parameters and higher occurrence (p < 0.0001) of DNA fragmentation in the CRYO group. CTC assay revealed a significant increase of acrosome-reacted spermatozoa in the CRYO group when compared to others. Compositional changes in the sperm membrane were visible as a notable decline of docosahexaenoic acid (p < 0.0001) associated with a significant decrease of membrane cholesterol (p < 0.05) and proteins (p < 0.0001) in the CRYO group while the amount of palmitic, stearic, oleic, and linoleic acid increased (p < 0.0001) significantly. Protein expression of all capacitation-associated proteins (PKC, PLA2, PLCζ, sAC10) was significantly down-regulated (p < 0.001; p < 0.0001) in the CRYO group. Relative quantification of apoptosis-associated proteins revealed increased Bax and decreased Bcl-2 levels in the CRYO group, except for caspase-3, which remained without significant changes.

Assessment of Sourdough Fermentation Impact on the Antioxidant and Anti-Inflammatory Potential of Pearl Millet from Burkina Faso.

Millet, a gluten-free cereal, has received attention for its environmental friendliness and higher protein content than other grains. It represents a staple food in many African countries, where fermentation is traditionally used for preserving food products and preparing different cereal-based products. This study aimed to assess the impact of sourdough fermentation on bioactive compounds and antioxidant and anti-inflammatory properties of pearl millet from Burkina Faso. Phenolic compounds were investigated spectrophotometrically and by HPLC-DAD. The antioxidant activity of unfermented (MF) and fermented (FeMF) millet was evaluated in vitro by spectrophotometric and fluorometric assays and ex vivo on oxidized human erythrocytes for hemolysis inhibition. Finally, the potential anti-inflammatory effect of FeMF and MF was evaluated on human adenocarcinoma cell line (HT-29) exposed to TNF-α inflammatory stimulus. Results revealed significantly higher levels of polyphenols, flavonoids, and in vitro antioxidant activity following millet fermentation. Notable differences in phenolic composition between FeMF and MF are observed, with fermentation facilitating the release of bioactive compounds such as gallic acid, quercetin, and rutin. A dose-dependent protection against oxidative hemolysis was observed in both FeMF- and MF-pretreated erythrocytes. Similarly, pretreatment with FeMF significantly reduced the levels of inflammatory markers in TNF-α-treated cells, with effects comparable to those of MF. Fermentation with sourdough represents a simple and low-cost method to improve the bioactive compounds content and in vitro antioxidant activity of millet flour with promising nutraceutical potential.

Modulatory effect of pomegranate peel extract on key regulators of ovarian cellular processes in vitro.

In this study, response of ovarian cells (human granulosa cell line HGL5, and human adenocarcinoma cell line OVCAR-3) to short-term pomegranate peel extract (PPE) treatment (for 24 hours in cell culture) was evaluated in vitro. Quantitative and qualitative screening of polyphenols revealed punicalagins α and ß as major polyphenolic components. Total phenolic content (TPC) was 93.76 mg GAE/g d.w. with a high antioxidant activity of 95.30 mg TEAC/g d.w. In OVCAR-3, PPE treatment inhibited the metabolic activity, and increased cyclin-dependent kinase 1 (CDKN1A, p21) level at the highest dose, but not in HGL5. Flow cytometry analysis could not detect any significant difference between proportions of live, dead, and apoptotic cells in both cell lines. Reactive oxygen species (ROS) revealed an antioxidant effect on HGL5, and a prooxidant effect by stimulating ROS generation in OVCAR-3 cells at the higher doses of PPE. However, in contrast to HGL5, PPE treatment decreased release of growth factors - TGF-ß2 and EGF at the highest dose, as well as their receptors TGFBR2 and EGFR in OVCAR-3 cells. PPE also influenced steroidogenesis in granulosa cells HGL5 by stimulating 17ß-estradiol secretion at higher doses. In conclusion, the present study highlighted the bioactive compounds in pomegranate peels and the possible mechanisms of action of PPE, shedding light on its promising role in ovarian cancer (chemo)prevention and/or management.

Sourdough Fermentation Improves the Antioxidant, Antihypertensive, and Anti-Inflammatory Properties of Triticum dicoccum.

The fermentation process has been widely used to improve plant-based foods' nutritional and nutraceutical properties. This study aimed to investigate and compare the impact of sourdough fermentation on the bioactive content and profile, antioxidant and antihypertensive activities, as well as the anti-inflammatory properties of fermented (FS) and non-fermented (NFS) flour from Tuscan Triticum dicoccum wheat (spelt) on tumor necrosis factor-alpha (TNF-α)-inflamed human intestinal epithelial cells (HT-29). FS showed significantly higher total phenolic and flavonoid content, in vitro and ex vivo antioxidant activities, and ACE-inhibitory activities than NFS. Gallic acid was identified by HPLC-DAD as the most representative polyphenol, followed by rutin, trans-ferulic acid, iso-quercitrin, and quercetin, in the fermented spelt sample. Instead, rutin and gallic acid were identified as the predominant compounds in the non-fermented ones. Moreover, FS exhibited a better protective effect on inflamed HT-29 cells by significantly counteracting the TNFα-induced alterations, lowering the expression of IL-8, COX-2, and ICAM-1 inflammatory mediator while enhancing antioxidant enzyme HO-1 gene expression. In conclusion, sourdough fermentation positively affected the nutraceutical and functional properties of spelt, which may represent a valuable ingredient for the formulation of functional foods and a key product for managing hypertension and inflammatory intestinal diseases.

Impact of 3-Year Period as a Factor on the Content of Biologically Valuable Substances in Seeds of White Lupin.

White lupin seed is a unique legume rich in protein and fiber contents, as well as phytochemicals with health potential that contributes to a reduced risk of dyslipidemia, obesity and intestinal dysfunction. This study was focused on the effect of the year on the contents of caffeic acid, 4-hydroxybenzoic acid, trans-ferulic, trans-p-coumaric, quercetin, myricetin, kaempferol, apigenin and genistein, as well as the antioxidant activity and total polyphenols, of seeds of eleven varieties (Lupine albus). The contents of individual phenolic substances were determined by high-performance liquid chromatography-HPLC. The total content of polyphenols and the antioxidant activity were determined spectrophotometrically. The results show that the lowest contents of phenolic acids were found in the seeds from 2018. The caffeic acid and trans-ferulic acid were the most represented among all phenolic acids, during all 3 monitored years (2017, 2018, and 2019). Our results confirm the significant influence of the year of cultivation on the bioactive substances' content in the seeds, and this can be potentially useful for the appropriate selection of locations for lupine growers in Slovakia, taking into account the climatic conditions of the given location. This study provides information about a legume that is underutilized in human nutrition, which may be a valuable source of bioactive substances.

The hypolipidemic, anti-inflammatory and antioxidant effect of Kavolì® aqueous extract, a mixture of Brassica oleracea leaves, in a rat model of NAFLD.

Herein we characterized the bioactive metabolites of the aqueous extract of Kavolì®, a commercial product composed of a mixture of Brassica oleracea leaves, and assessed its potential ameliorating effects in a rat model of non-alcoholic fatty liver disease (NAFLD). Kavolì® extract showed high levels of bioactive compounds and strong in vitro antioxidant activities. Chlorogenic and neochlorogenic acids were identified as the most representative polyphenols. The administration of brassica extract to steatotic rats significantly ameliorated the levels of blood lipids and transaminases, and lipid content and inflammatory markers in liver. Oxidative stress parameters were significantly improved in both liver and brain of steatotic rats. Moreover, plasma and feces levels of short chain fatty acids (SCFAs) were bring back close to control values by Kavolì® treatment, in spite of high fat diet/streptozotocin (HFD/STZ)-induced alterations. The efficacy of Kavolì® in treating hypercholesterolemia, reducing the level of inflammation and cardiovascular disease biomarkers, steatosis and oxidative stress parameters, as well as the ability in modulating SCFAs levels is probably related to the bioactive compounds of the water extract administered to the rat model of NAFLD. In particular, the ameliorating effects are largely attributable to the high content in polyphenols observed in our study.

Changes in Antioxidant Properties and Phenolics in Sweet Potatoes (Ipomoea batatas L.) Due to Heat Treatments.

Processing is one of the most crucial factors affecting polyphenol content in foods. Therefore, the study is aimed at the evaluation of heat treatment effects (microwaving, steaming, baking, and boiling) on the content of chlorogenic acids, total polyphenols, and antioxidant activity of three varieties of sweet potato with different flesh colors (Beauregard­orange-fleshed, O'Henry­white-fleshed, 414-purple­purple-fleshed). According to high performance liquid chromatography analysis, chlorogenic acid was the predominant chlorogenic acid in sweet potatoes. Obtained results also suggested the purple-fleshed variety (414-purple) had significantly (p < 0.05) higher total polyphenol content and thus the highest antioxidant activity. Heat treatment positively influenced the chlorogenic acid content, total polyphenols, and antioxidant activity of sweet potatoes. Among the used methods, steaming had the greatest effect on the chlorogenic acids and total polyphenols, while microwaved samples showed the highest antioxidant activity (DPPH). The content of chlorogenic acids and total polyphenols decreased in the order of steaming > baking > microwaving > boiling > raw. However, the individual varieties differed not only in the flesh color but also in the reaction to the used heat treatment methods. Spearman's correlation coefficient showed a strong correlation between chlorogenic acid and antioxidant activity.

Mercury in scarletina bolete mushroom (Neoboletus luridiformis): Intake, spatial distribution in the fruiting body, accumulation ability and health risk assessment.

In the present work, we focused on two aspects of mercury (Hg) bioconcentration in the above-ground parts of Neoboletus luridiformis. In the first part, we monitored the bioconcentration potential of individual anatomical parts of a particular fruiting body and evaluated the obtained data by the spline interpolation method. In the second part, we focused on assessing the mercury content in 378 samples of N. luridiformis and associated samples of substrates from 38 localities with different levels of Hg content in Slovakia. From the obtained data of Hg content in samples of substrate and fungi, we evaluated ecological indicators (geoaccumulation index - Igeo, contamination factor - Cf a potential ecological risk - PER), bioconcentration indicators (bioconcentration factor - BCF; cap/stipe quotient - Qc/s) and health indicators (percentage of provisional tolerable weekly intake - %PTWI a target hazard quotient - THQ). Based on the Hg distribution results, the highest Hg content was found in the tubes & pores (3.86 mg/kg DW), followed by the flesh of cap (1.82 mg/kg DW). The lowest Hg content was in the stipe (1.23 mg/kg DW). The results of the BCF values indicate that the studied species can be included in the category of mercury accumulators. The results of the ecological indices representing the state of soil pollution pointed out that two localities (Malachov and Nizná Slaná) stood apart from all monitored localities and showed a state of an extremely disturbed environment. This fact was also reflected in the values of Hg content in the fruiting bodies of the studied mushroom species. In the case of the consumption of mushrooms from these localities, it can be stated that long-term and regular consumption could have a negative non-carcinogenic effect on the health of consumers. It was confirmed by the %PTWI (Malachov: 57.8%; Nizná Slaná: 53.2%) and THQ (Malachov: 1.11 Nizná Slaná: 1.02). The locality Cacín-Jelsovec is interesting from the bioconcentration characteristics point of view, where the level of environmental pollution was the lowest (Hg content in the soil was below the background value) compared to other localities, however, the THQ value was the highest (1.29).

Detection of Changes in Total Antioxidant Capacity, the Content of Polyphenols, Caffeine, and Heavy Metals of Teas in Relation to Their Origin and Fermentation.

Tea (Camellia sinensis) is widely sought for beverages worldwide. Heavy metals are often the main aims of the survey of teas, given that the use of agricultural fertilization is very frequent. Some of these may affect the content of bioactive compounds. Therefore, in this study, we analyzed fermented and non-fermented teas of a single plant origin from Japan, Nepal, Korea, and China, and described mutual correlations and changes in the total antioxidant capacity (TAC), and the content of polyphenols (TPC), caffeine, and heavy metals in tea leaves, in relation to the origin and fermentation process. Using UV-VIS spectrophotometry and HPLC-DAD, we determined variations in bioactive compounds' content in relation to the fermentation process and origin and observed negative correlations between TAC and TPC. Heavy metal content followed this order: Mn > Fe > Cu > Zn > Ni > Cr > Pb > Co > Cd > Hg. Given the homogenous content of these elements in relation to fermentation, this paper also describes the possibility of using heavy metals as determinants of geographical origin. Linear Discriminant Analysis showed an accuracy of 75% for Ni, Co, Cd, Hg, and Pb, explaining 95.19% of the variability between geographical regions.

Characterization of the Omija (Schisandra chinensis) Extract and Its Effects on the Bovine Sperm Vitality and Oxidative Profile during In Vitro Storage.

Schisandra chinensis is a woody vine native to China, Korea, and Russia, which has been used as a traditional herbal remedy to treat male infertility. As very little information is available concerning its effects on ejaculated spermatozoa, the aim of this study was to investigate the chemical, antioxidant, and antibacterial properties of the S. chinensis berry (Omija) extract followed by an assessment of its in vitro effects on bovine sperm function and oxidative balance. Phytochemical components of the Omija extract were determined by high performance liquid chromatography. The content of polyphenols, flavonoids, and carotenoids was assessed by spectrophotometric protocols. Antioxidant characteristics of the Omija extract were determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and molybdenum-reducing antioxidant power (MRAP) assays. The disc diffusion method and determination of the minimal inhibitory concentration were applied to study the antibacterial properties of Schisandra. Thirty semen samples were exposed to different concentrations of Omija (1, 5, 10, 25, 50, and 75 µg/mL) for 0, 2, and 24 h. Sperm motility, mitochondrial activity, and superoxide and reactive oxygen species production, as well as total antioxidant capacity and oxidative damage to proteins and lipids were determined. Our data reveal that the Omija extract, particularly at a concentration range within 5-50 µg/mL, exhibited dose-dependent motion-promoting and metabolism-enhancing properties, accompanied by significant antioxidant effects. We may conclude that the biomolecules present in the Omija extract such as schisandrins and phenolic molecules offer protection to critical sperm structures against oxidative insults and/or possible bacterial contamination, leading to a higher preservation of mammalian sperm viability and functional activity.

The effect of roasting on the total polyphenols and antioxidant activity of coffee.

The total polyphenolic content and the antioxidant activity have been analyzed in ground beans of green, light, medium and dark roasted coffee by UV-VIS spectrometry. Water coffee extracts showed the highest levels of polyphenols in green and light roasted coffees where the total polyphenolic content (TPC) ranged from 49.19 ± 0.70 to 74.05 ± 0.28 and from 59.79 ± 1.45 to 38.34 ± 1.26 g GAE.kg-1, respectively. In medium roast samples it ranged from 43.90 ± 3.07 to 74.05 ± 0.28g GAE.kg-1 and in dark roast from 37.44 ± 0.63 to 47.41 ± 0.69 g GAE.kg-1. The total antioxidant capacity (TAC) reached the highest values (DPPH inhibition ranging from 69.08 ± 1.33% to 78.55 ± 0.89%) in light roasted coffees. Dark roasted coffees showed both the lowest content of polyphenols as well as the total antioxidant capacity. In case of TPC, statistically significant differences (P˂0.001) have been identified between green coffee and other roasted degrees. Also, dark coffee showed statistically noticeable differences (P˂0.001) in TPC in relation to other roasted stages. Statistically important difference (P˂0.001) has been discovered between the total antioxidant capacity of dark roasted coffee and other roasting levels. The results demonstrated that roasting process affects both the oxidative activity as well as polyphenolic content.

Impact of cadmium and nickel on ion homeostasis in the yeast Schizosaccharomyces pombe.

Toxicity of heavy metals to living organisms is a worldwide research topic. Although, much has been discovered about cadmium and nickel impact on biological systems, a lot still remains unclear. We used inductively coupled plasma - optical emission spectroscopy to address the question of the effect of two different heavy metals nickel, and cadmium on intracellular ion balance. Increase or decrease of the content of several essential cations including Ca2+, Na+, K+, Mg2+, Cu2+, Fe3+ in the yeast Schizosaccharomyces pombe was determined. Our results revealed that the cell exposure to high nickel and cadmium concentrations led to significant elevation of Ca2+, Na+, Mg2+, Cu2+, Fe3+ levels in the yeast cell, while the content of K+ decreased. Correlation analyses showing in the presence of nickel and cadmium strong positive correlation among each tested element (Ca2+, Na+, Cu2+, Mg2+ and Fe3+) except for K+, demonstrate the significant impact of heavy metal treatment to ion homeostasis of the cell. Our data indicate that acute nickel and cadmium contamination leads to substantial ionome misbalance in yeast.

Wild Italian Prunus spinosa L. Fruit Exerts In Vitro Antimicrobial Activity and Protects Against In Vitro and In Vivo Oxidative Stress.

Polyphenol-rich foods could have a pivotal function in the prevention of oxidative stress-based pathologies and antibacterial action. The purpose of this study was to investigate the in vitro antimicrobial activity, as well as the in vitro and in vivo antioxidant capacities of wild Prunus spinosa L. fruit (PSF) from the southeast regions of Italy. The total phenolic content (TPC) was quantified, and the single polyphenols were analyzed by HPLC-DAD, showing high rutin and 4-hydroxybenzoic acid levels, followed by gallic and trans-sinapic acids. PSF extract demonstrated antimicrobial activity against some potentially pathogenic Gram-negative and Gram-positive bacteria. Besides, we investigated the cellular antioxidant activity (CAA) and the hemolysis inhibition of PSF extract on human erythrocytes, evidencing both a good antioxidant power and a marked hemolysis inhibition. Furthermore, an in vivo experiment with oxidative stress-induced rats treated with a high-fat diet (HFD) and a low dose of streptozotocin (STZ) demonstrated that PSF has a dose-dependent antioxidant capacity both in liver and in brain. In conclusion, the wild Italian Prunus spinosa L. fruit could be considered a potentially useful material for both nutraceutical and food industries because of its antioxidant and antimicrobial effects.

Polyphenolic characterisation of plant mixture (Lisosan® Reduction) and its hypocholesterolaemic effect in high fat diet-fed mice.

Lisosan® Reduction is a plant mixture produced from powder of fermented Triticum aestivum (Lisosan® G), Desmodium adscendens, Malus domestica, Picrorhiza kurroa and Hordeum vulgare. The aim of this study was to characterise the phenolic profile of Lisosan® Reduction and to evaluate the effects of aqueous extract on mice fed a high fat diet (HFD). Syringic acid, trans sinapic acid and neochlorogenic acid were identified by HPLC-DAD to be the dominant polyphenols of Lisosan® Reduction, followed by vitexin, trans p-coumeric acid and trans ferulic acid. Mice treated with aqueous extract of Lisosan® Reduction (60 mg/kg b.w.) showed a significant decrease of serum cholesterol, glucose and triglycerides level and a significant increase of CYP7A1 gene expression, compared to HFD group.

Environmental Contamination by Heavy Metals in Region with Previous Mining Activity.

Due to its status as one of the most contaminated regions in Slovakia, 45 soil and plant samples were collected in the Middle Spis region. In soil, the exchangeable soil reaction, humus content and heavy metals content (Zn, Cd, Cu, Pb, Hg) were determined. Total content of heavy metals (TC-HMs) and content of mobile forms (MF-HMs) in soil, as well as metal content in plants, were determined by atomic absorption spectrometry. The concentration ranges for total Zn, Cd, Cu, Pb and Hg in soil were 33.1-953, 0.65-6.73, 11.0-913, 26.5-165 and 0.28--415 mg/kg, respectively. The overall concentration ranges of these metals in plants of two types (Athyrium filix-femina L. and Poaceae herbs) were 12.4-158.6, 0.10-1.63, 3.34-85.7, 0.09-29.7 and 0.01-12.8 mg/kg, respectively. Despite the values of Zn, Cd, Cu, Pb and Hg content in the soil exceeding limit values, only the Hg content in plants presented an ecological risk.