Evaluation of Chemical Profile and Biological Properties of Extracts of Different Origanum vulgare Cultivars Growing in Poland.

This research studied the phenolic content compared with the antioxidant properties of various O. vulgare (Lamiaceae) cultivars grown in Poland. The research results in this paper indicate that the dominant ingredient in all oregano cultivars was rosmarinic acid, known for its strong antioxidant properties. The highest amounts of rosmarinic acid (87.16 ± 4.03 mg/g dm) were identified in the O. vulgare spp. hirtum (Link) Ietsw. Other metabolites identified in the studied extracts include luteolin O-di-glucuronide-O-di-pentoside (30.79 ± 0.38 mg/g dm in the 'Aureum' cultivar), 4'-O-glucopyranosyl-3', 4'-dihydroxy benzyl-protocatechuate (19.84 ± 0.60 mg/g dm in the 'Margerita' cultivar), and p-coumaroyl-triacetyl-hexoside (25.44 ± 0.18 mg/g dm in the 'Margerita' cultivar). 'Hot & spicy' and 'Margerita' cultivars were characterized by the highest activity in eliminating OH• and O2•- radicals. Extracts from Greek oregano had the highest ability to scavenge DPPH radicals and chelate iron ions. This research has also provided new evidence that oregano has anti-migratory, cytotoxic properties and influences the viability of gastric cancer cells (the highest cytotoxicity was attributed to the 'Hot & spicy' cultivar, which performed the worst in antioxidant properties tests). Extracts from the tested cultivars at a concentration of 0.625% effectively inhibited the growth of S. aureus and P. aeruginosa bacteria. It seems that the oregano grown in Poland is of good quality and can be successfully grown on a large scale if the appropriate use is found.

Phytochemical Composition and Antimicrobial Properties of New Lavandula angustifolia Ecotypes.

The purpose of this study was to characterize ethanol extracts from leaves and flowers of two ecotypes (PL-intended for industrial plantations and KC-intended for cut flowers) of Lavandula angustifolia Mill. The plant was cultivated in 2019 in southern Poland as part of a long-term research plan to develop new varieties resistant to difficult environmental conditions. The collected leaves and flowers were used to prepare ethanol extracts, which were then analyzed in terms of phytochemical composition and antioxidant, bactericidal, and fungicidal properties. Using UPLC techniques, 22 compounds belonging to phenolic acids and flavonoids were identified. UPLC test results indicated that ethanol extracts from leaves and flowers differ in phytochemical composition. Lower amounts of phenolic acids and flavonoids were identified in leaf extracts than in flower extracts. The predominant substances in the flower extracts were rosmarinic acid (829.68-1229.33 µg/g), ferulic acid glucoside III (810.97-980.55 µg/g), and ferulic acid glucoside II (789.30-885.06 µg/g). Ferulic acid glucoside II (3981.95-6561.19 µg/g), ferulic acid glucoside I (2349.46-5503.81 µg/g), and ferulic acid glucoside III (1303.84-2774.17 µg/g) contained the highest amounts in the ethanol extracts of the leaves. The following substances were present in the extracts in trace amounts or at low levels: apigenin, kaempferol, and caftaric acid. Leaf extracts of the PL ecotype quantitatively (µg/g) contained more phytochemicals than leaf extracts of the KC ecotype. The results obtained in this study indicate that antioxidant activity depends on the ecotype. Extracts from the PL ecotype have a better ability to eliminate free radicals than extracts from the KC ecotype. At the same time, it was found that the antioxidant activity (total phenolic content, ABTS•+, DPPH•, and FRAP) of PL ecotype leaf extracts was higher (24.49, 177.75, 164.88, and 89.10 µmol (TE)/g) than that determined in flower extracts (15.84, 125.05, 82.35, and 54.64 µmol (TE)/g). The test results confirmed that leaf and flower extracts, even at low concentrations (0.313-0.63%), significantly inhibit the growth of selected Gram-negative and Gram-positive bacteria and Candida yeasts. Inhibition of mold growth was observed at a dose extract of at least 1 mL/100 mL.

UPLC-ESI-TQD-MS/MS Identification and Antioxidant, Anti-Inflammatory, Anti-Diabetic, Anti-Obesity and Anticancer Properties of Polyphenolic Compounds of Hawthorn Seeds.

Hawthorn seeds are a by-product of fruit processing and due to the scale of processing of this raw material, they can be an important source of bioactive compounds. This work is the first report on the phenolic composition of hawthorn seeds and their antioxidant, anti-inflammatory, antidiabetic, antiobesity and anticancer activities. In the isolated phenolic fraction of six seed species, 23 phenolic compounds were identified using the UPLC-ESI-TQD-MS/MS method, the key ones of which included the B-type procyanidin dimer. The seeds of the tested species showed high antioxidant activity (mainly by scavenging O2•- and OH• radicals), anti-inflammatory (mainly through LOX inhibition), anti-diabetic, anti-obesity and anti-cancer, with the highest activity against colon cancer cells (Dld-1 line), showing no activity against healthy colon epithelial cells (CCD841CoN). This activity was significantly dependent on the analyzed hawthorn species and, according to PCA analysis, on the content of flavan-3-ols. These discoveries provided the theoretical basis for the possibility of industrial use of hawthorn seeds.

Chemical Profiling, Bioactive Properties, and Anticancer and Antimicrobial Potential of Juglans regia L. Leaves.

The aim of this study was to assess the biological potential of the polyphenolic fraction isolated from J. regia leaves, collected in the Subcarpathian region (Poland). The phenolic profile was determined using the UPLC-PDA-MS/MS method. Biological activity was determined by evaluating the antioxidant, anticancer, antibacterial, and antifungal effects. Prior to this study, the purified polyphenolic fraction was not been tested in this regard. A total of 40 phenolic compounds (104.28 mg/g dw) were identified, with quercetin 3-O-glucoside and quercetin pentosides dominating. The preparation was characterized by a high ability to chelate iron ions and capture O2•- and OH• radicals (reaching IC50 values of 388.61, 67.78 and 193.29 µg/mL, respectively). As for the anticancer activity, among the six tested cell lines, the preparation reduced the viability of the DLD-1, Caco-2, and MCF-7 lines the most, while in the antibacterial activity, among the seven tested strains, the highest susceptibility has been demonstrated against K. pneumoniae, S. pyogenes, and S. aureus. Depending on the needs, such a preparation can be widely used in the design of functional food and/or the cosmetics industry.

Phenolic Profile and Antioxidant, Antibacterial, and Antiproliferative Activity of Juglans regia L. Male Flowers.

Juglans regia L., walnut, is a large, long-living tree, cultivated in temperate climates around the world. It is highly appreciated for its nutritional kernels and high-quality timber. Its barks, leaves, and husk are used as dyes and in folk medicine as herbal remedies for several diseases. From a biological and chemical standpoint, relatively little is known about the male flowers of the tree. Therefore, the aim of the study was to evaluate the phenolic profile as well as in vitro antioxidant, antimicrobial, and antiproliferative activity of male Juglans regia L. flowers. Phenolic content was determined by UPLC/PDA/MS/MS analyses; antioxidant activity was assessed by five different methods; antimicrobial activity was evaluated against the six most common pathogenic strains of Gram-positive and Gram-negative bacteria, and antiproliferative properties were assessed against six cell lines. Most of the analyses carried out in this study were performed for the first time for this raw material. J. regia flower extract was characterized by a strong ability to scavenge DPPH˙ free radicals, hydroxyl radicals, and chelating metal ions. Among the examined bacterial strains and neoplastic lines, the strongest antimicrobial activity was shown against S. aureus, L. monocytogenes, and B. cereus, and cytotoxic activity against breast cancer, glioblastoma, and astrocytoma cells. Male J. regia flowers have also been found to be a rich source of phenolic compounds. The content of polyphenols in the extract was 4369.73 mg/100 g d.w., and 24 compounds from the group of flavonoids, phenolic acids, and juglunosides were identified. Additionally, a strong correlation between the content of polyphenols and the antioxidant capacity and cytotoxic activity was observed. This is why the tested J. regia flowers are an excellent source of effective natural antioxidant, antibacterial, and chemopreventive compounds that have potential to be used in the pharmaceutical or food industries.

Polyphenolic Compounds of Crataegus Berry, Leaf, and Flower Extracts Affect Viability and Invasive Potential of Human Glioblastoma Cells.

Crataegus contains numerous health-promoting compounds that are also proposed to have anti-cancer properties. Herein, we aimed at a contemporaneous evaluation of the effects of polyphenol-rich extracts of berries, leaves, and flowers of six Crataegus species on the viability and invasive potential on the highly aggressive human glioblastoma U87MG cell line. The treatment with the extracts evoked cytotoxic effects, with the strongest in the berry extracts. All extracts not only promoted the apoptosis-related cleavage of poly (ADP-ribose) polymerase 1 (PARP1) but also substantially inhibited the activity of pro-survival kinases, focal adhesion kinase (FAK), and protein kinase B (PKB; also known as Akt), thus indicating the suppression of proliferative and invasive potentials of the examined glioblastoma cells. The qualitative and quantitative characterization of the extracts' content was also performed and revealed that amongst 37 polyphenolic compounds identified in the examined Crataegus extracts, the majority (29) was detected in berries; the leaf and flower extracts, exerting milder cytotoxic effects, contained only 14 and 13 compounds, respectively. The highest polyphenol content was found in the berries of C. laevigata x rhipidophylla x monogyna, in which flavan-3-ols and phenolic acids predominated. Our results demonstrated that a high content of polyphenolic compounds correlated with the extract cytotoxicity, and especially berries were a valuable source of compounds with anti-cancer potential. This might be a promising option for the development of an effective therapeutic strategy against highly malignant glioblastomas in the future.

The Content of Phenolic Acids and Flavonols in the Leaves of Nine Varieties of Sweet Potatoes (Ipomoea batatas L.) Depending on Their Development, Grown in Central Europe.

The aim of the study was the qualitative and quantitative analysis of the bioactive components present in the leaves of 9 sweet potato cultivars grown in the moderate climate in Poland, which were harvested at different growth stages according to the BBCH (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie) scale (14, 51, 89). It was found that sweet potato leaves contained 7 polyphenolic compounds, including 5 chlorogenic acids-neochlorogenic acid (5-CQA), chlorogenic acid (3-CQA), 4-cryptochlorogenic acid (4-CQA), 34-di-O-caffeoylqunic acid (3,4-CQA), 3,5-di-O-caffeoylqunic acid (3,5-CQA)-and 2 flavonoids, quercetin-3-O-galactoside (Q-3-GA) and quercetin-3-O-glucoside (Q-3-GL). Their content depended on the genotype of the examined cultivars and on the stage of leaf development. The mean content of the identified polyphenolic compounds in the examined cultivars ranged from 148.2 to 14.038.6 mg/100 g-1 DM for the leaves harvested at growth stage 14 according to the BBCH scale. In the case of leaves harvested at BBCH stage 51, the concentration of polyphenolic compounds ranged from 144.76 to 5026.8 mg/100 g-1 DM and at BBCH stage 89 from 4078.1 to 11.183.5 mg/100 g-1 DM. The leaves of the Carmen Rubin cultivar collected at stage 14 contained the highest amount of polyphenolic compounds, while Okinava leaves had the highest amount of these compounds at stage 51. The highest content of polyphenolic compounds in leaves at BBCH growth stage 89 was found in the Radiosa variety. The highest concentration levels were found for 3-CQA at all stages of leaf development. Significant correlations between polyphenol content and antioxidant activity measured by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing/antioxidant power (FRAP) were found. The results of this experiment revealed that the growth stages and genetic properties of cultivars have a very significant influence on the content of phenolic acids and flavonols in sweet potato leaves. The results are innovative and can have a practical application, as the knowledge of the content of the substances under study makes it possible to determine the optimal management practice of sweet potato leaf harvest in order to obtain more top-quality raw material.

Immunohistochemistry annotations enhance AI identification of lymphocytes and neutrophils in digitized H&E slides from inflammatory bowel disease.

BACKGROUND AND OBJECTIVE: Histologic assessment of the immune infiltrate in H&E slides is vital in diagnosing and managing inflammatory bowel diseases, but these assessments are subjective and time-consuming even for those with expertise. The development of deep learning models to aid in these assessments has been limited by the paucity of image data with reliably annotated immune cells available for training. METHODS: To address these challenges, we developed a pipeline that automates the neutrophil and lymphocyte labeling in ROIs from digital H&E slides. The data included ROIs extracted from 19 digitized H&E slides and the same slides restained with immunohistochemistry. Our pipeline first delineates each nucleus in H&E ROIs. Using the colorimetric features of the immunohistochemical stains (red: neutrophils, green: lymphocytes) in the immunohistochemistry ROIs, each cell was labeled as a neutrophil, a lymphocyte, or another cell. The labels were then transferred to the corresponding H&E ROIs by image registration, and the ROI registration accuracy was assessed by the median target registration error resulting in a labeled dataset. The newly formed dataset (NeuLy-IHC) comprising 519 ROIs with 235,256 labeled cells (74,339 lymphocytes, 16,326 neutrophils and 144,591 other cells) was used to train the HoVer-Net(NeuLy) model. The performance of HoVer-Net(NeuLy) measured by DICE coefficient (segmentation accuracy) and F1-scores (classification accuracy), was compared to those achieved by HoVer-Net(MoNuSAC) and SMILE(MoNuSAC) publicly available models trained on cancer-containing ROIs from the MoNuSAC dataset with manual cell labeling and pathologists' annotations. RESULTS: The 1.0 µm median target registration error of ROIs observed was low demonstrating robust transferring of cellular labels from immunohistochemistry ROIs to H&E ROIs. In the test set comprising 76 NeuLy-IHC and 78 MoNuSAC ROIs, the HoVer-Net(NeuLy) achieved a DICE coefficient of 0.861 and F1-sores of 0.827, 0.838, and 0.828, for neutrophils, lymphocytes, and other cells, respectively, outperforming the HoVer-Net(MoNuSAC)'s and SMILE(MoNuSAC)'s DICE coefficient and F1 scores for each cell category. CONCLUSIONS: We attribute the improved performance of HoVer-Net(NeuLy) to the larger number of immune cells in the NeuLy-IHC dataset (in total 5x more, including 21x more neutrophils) than in the MoNuSAC dataset. Despite being trained on data from inflammatory bowel disease specimens, our model maintained robust performance when tested on previously unseen data derived from cancer specimens. The NeuLy-IHC set provides opportunities for training accurate models to quantify the inflammatory infiltrate in digital histologic slides.

Antioxidant and Antiproliferative Activities of Phenolic Extracts of Eriobotrya japonica (Thunb.) Lindl. Fruits and Leaves.

Increasing interest in new sources of secondary metabolites as biologically active substances has resulted in an advanced study of many plant species. Loquat (Eriobotrya japonica (Thunb.) Lindl. = Rhaphiolepis bibas (Lour.) Galasso & Banfi, Rosaceae family), an evergreen, subtropical fruit tree, native to China and Japan, but cultivated in southern countries of Europe, is a species commonly used in folk medicine and may be an excellent source of bioactive compounds. Therefore, the aim of the present study was to evaluate the profile of the phenolic constituents of E. japonica fruits and leaves originating from Tuscany (Italy), as well as their in vitro antioxidant and chemopreventive activities on human cancer cell lines breast adenocarcinoma (MCF-7), colon adenocarcinoma (Caco-2 and HT-29), and glioblastoma (U87MG). Results revealed that the extract of leaves displayed higher antioxidant and anticancer potential than the fruit extract and contained 25 individual phenolic compounds that have been characterized and quantified by the UPLC-PDA-MS method. The antiproliferative activity was correlated with the content of polyphenolic compounds indicating that both fruits and leaves are a good source of antioxidants and may be exploited as nutraceuticals enriching food or as components for the cosmetic/pharmaceutical industry.

Phytochemical Screening and Bioactive Properties of Juglans regia L. Pollen.

Pollen is one of the major by-products of the walnut tree, yet it is poorly investigated. Thus, the aim of this study was to investigate the total phenolics, flavonoids, antioxidant, anticancer potential, and polyphenol profile of pollen obtained from male Juglans regia flowers. A total of 24 phenolic compounds were identified in Juglans regia pollen and all of them were reported for the first time for this raw material. The content of polyphenols was 408.03 mg/100 g dry weight (dw) and the most abundant components were quercetin 3-O-sophoroside and 4',5,7-trihydroxy-3,6-dimethoxyflavone-7-O-beta-D-glucoside. The concentration of these compounds, as well as the total content of polyphenols and flavonoids, strongly determined the antioxidant and cytotoxic activity of Juglans regia pollen. Antioxidant action using the ABTS and CUPRAC methods had the values of 3.35 and 0.32 mmol TE/g dw, respectively. In turn, in the tests of chelating ability of ferrous ion, O2•- and OH- radical scavenging activity, of which the results were expressed as IC50, the values were equal to 335.01, 459.31, and 92.89 µg/mL, respectively. Among the six cancer cell lines, the strongest effect was demonstrated for Caco-2 (140.65 µg/mL) and MCF-7 (140.98 µg/mL) cells. The results provide valuable and previously unpublished data on the polyphenol composition and biological potential of Juglans regia pollen.

Walnut Male Flowers (Juglans regia L.) as a Functional Addition to Wheat Bread.

The aim of this study is to assess the effect of adding dried and ground walnut male flowers to wheat flour on selected physicochemical and antioxidant properties of the obtained wheat bread. The control sample was bread without the addition of walnut male flowers. In the tests, the addition of flowers was used in the amounts of 0.5%, 1.0%, 1.5%, 2.0% and 2.5% of the weight of the flour used in the control sample. Bread dough was prepared according to the direct method with the use of yeast. The parameters of the baking process of the control bread and of the test bread were calculated. The selected parameters determining the quality of the obtained bread, including the loaf volume, the specific volume, the crumb porosity, the crumb texture and the color were measured. The antioxidant potential and total polyphenol content were tested both in the dough and in the bread. The test results were analyzed statistically. It was found that the partial replacement of wheat flour with walnut male flowers resulted in a significant reduction in the loaves' volume and in the darkening of both the color of the dough and the crumb. The value of the L* parameter decreased with the increasing addition of walnut flowers. The lowest values of this parameter were found to be 53.87 (crust) and 39.94 (crumb) in the sample with 2.5% addition. The average volume of the loaves ranged from 565 cm3 (0.5%) to 675 cm3 (control). The use of the additive in the amount of 2.5% caused significant changes in the values of most of the examined parameters of the bread crumb texture. The addition of walnut male flowers to the flour had a significant effect on the antioxidant potential and the total polyphenol content of the tested doughs and breads. In the case of bread, the total polyphenol content value ranged from 96.90 mg GAE/g dw to 208.08 mg GAE/g dw. Similarly, the antioxidant potential increased with increasing walnut male flower supplementation. The antioxidant potential determined by the ABTS and FRAP methods ranged from 1.42 mmol TE/g dw to 2.02 mmol TE/g dw and from 0.08 mmol TE/g dw to 0.33 mmol TE/g dw, respectively. The obtained research results prove the application potential of walnut flowers in the design of food, with beneficial pro-health properties, and are an example of the use of plant by-products in the process of food enrichment.