Flower extracts of Prunus spinosa L. (blackthorn)—a traditional medicinal plant of Central and Eastern Europe indicated for the treatment of urinary tract disorders, inflammation, and adjunctive therapy of cardiovascular diseases—were evaluated in terms of chemical composition, antioxidant activity, potential anti-inflammatory effects, and cellular safety in function of fractionated extraction. The UHPLC-PDA-ESI-MS3 fingerprinting led to full or partial identification of 57 marker constituents (36 new for the flowers), mostly flavonoids, A-type proanthocyanidins, and phenolic acids, and provided the basis for authentication and standardization of the flower extracts. With the contents up to 584.07 mg/g dry weight (dw), 490.63, 109.43, and 66.77 mg/g dw of total phenolics (TPC), flavonoids, proanthocyanidins, and phenolic acids, respectively, the extracts were proven to be rich sources of polyphenols. In chemical in vitro tests of antioxidant (DPPH, FRAP, TBARS) and enzyme (lipoxygenase and hyaluronidase) inhibitory activity, the extracts effects were profound, dose-, phenolic-, and extraction solvent-dependent. Moreover, at in vivo-relevant levels (1–5 μg/mL) the extracts effectively protected the human plasma components against peroxynitrite-induced damage (reduced the levels of oxidative stress biomarkers: 3-nitrotyrosine, lipid hydroperoxides, and thiobarbituric acid-reactive substances) and enhanced the total antioxidant status of plasma. The effects observed in biological models were in general dose- and TPC-dependent; only for protein nitration the relationships were not significant. Furthermore, in cytotoxicity tests, the extracts did not affect the viability of human peripheral blood mononuclear cells (PBMC), and might be regarded as safe. Among extracts, the defatted methanol-water (7:3, v/v) extract and its diethyl ether and ethyl acetate fractions appear to be the most advantageous for biological applications. As compared to the positive controls, activity of the extracts was favorable, which might be attributed to some synergic effects of their constituents. In conclusion, this research proves that the antioxidant and enzyme inhibitory capacity of phenolic fractions should be counted as one of the mechanisms behind the activity of the flowers reported by traditional medicine and demonstrates the potential of the extracts as alternative ingredients for functional products supporting the treatment of oxidative stress-related pathologies cross-linked with inflammatory changes, especially in cardiovascular protection.

Dry leaf extracts of eastern teaberry (Gaultheria procumbens L.) were evaluated as a source of bioactive phytocompounds through systematic activity testing and phytochemical profiling. The antioxidant efficiency was tested using five complementary in vitro models (DPPH; FRAP; linoleic acid (LA) peroxidation assay; O2•− and H2O2 scavenging tests) in parallel with standard antioxidants. The 75% methanol extract and its diethyl ether, ethyl acetate (EAF), n-butanol and water fractions exhibited the dose-dependent responses in all assays, with the highest capacities found for EAF (DPPH EC50 = 2.9 μg/mL; FRAP = 12.8 mmol Fe 2+ /g; IC50 for LA-peroxidation = 123.9 μg/mL; O2•− SC50 = 3.9 μg/mL; H2O2 SC50 = 7.2 μg/mL). The EAF had also the highest anti-inflammatory activity in the inhibition tests of lipoxygenase and hyaluronidase (60.14% and 21.83% effects, respectively, at the concentration of 100 μg/mL). Activity parameters of the extracts correlated strongly with the levels of total phenolics (72.4-270.7 mg GAE/g), procyanidins, and phenolic acids, whereas for flavonoids only moderate effects were and HPLC-PDA studies led to the identification of 35 polyphenols with a procyanidin A-type trimer, quercetin 3-O-glucuronide, isomers of caffeoylquinic acids, and (-)-epicatechin being the dominant components. Significant activity levels, high phenolic contents and high extraction yields (39.4%-42.5% DW for defatted and crude methanol extracts, respectively) indicate the value of eastern teaberry leaves as bioactive products.

The present study investigated the phenolic profile and biological activity of dry extracts from leaves of C. bullatus, C. zabelii and C. integerrimus—traditional medicinal and dietary plants—and evaluated their potential in adjunctive therapy of cardiovascular diseases. Complementary UHPLC-PDA-ESI-MS3, HPLC-PDA-fingerprint, Folin-Ciocalteu, and n-butanol/HCl assays of the extracts derived by fractionated extraction confirmed that they are rich in structurally diverse polyphenols (47 analytes, content up to 650.8 mg GAE/g dw) with proanthocyanidins (83.3–358.2 mg CYE/g) dominating in C. bullatus and C. zabelii, and flavonoids (53.4–147.8 mg/g) in C. integerrimus. In chemical in vitro tests of pro-inflammatory enzymes (lipoxygenase, hyaluronidase) inhibition and antioxidant activity (DPPH, FRAP), the extracts effects were dose-, phenolic- and extraction solvent-dependent. The most promising polyphenolic extracts were demonstrated to be effective antioxidants in a biological model of human blood plasma—at in vivo-relevant levels (1–5 µg/mL) they normalized/enhanced the non-enzymatic antioxidant capacity of plasma and effectively prevented peroxynitrite-induced oxidative/nitrative damage of plasma proteins and lipids. As demonstrated in cytotoxicity tests, the extracts were safe—they did not affect viability of human peripheral blood mononuclear cells. In conclusion, Cotoneaster leaves may be useful in development of natural-based products, supporting the treatment of oxidative stress/inflammation-related chronic diseases, including cardiovascular disorders.

Abstract:The antioxidant efficiency of 70% aqueous methanolic extracts from the leaves of twelve selected Cotoneaster Medik. species was evaluated using four complementary in vitro tests based on SET-(single electron transfer) and HAT-type (hydrogen atom transfer) mechanisms (DPPH, FRAP, O 2 ‚´a nd H 2 O 2 scavenging assays). The samples exhibited the dose-dependent responses in all assays with activity parameters of EC 50 = 18.5-34.5 µg/mL for DPPH; 0.9-3.8 mmol Fe 2+ /g for FRAP; SC 50 = 27.7-74.8 µg/mL for O 2 ‚´; and SC 50 = 29.0-91.3 µg/mL for H 2 O 2 . Significant linear correlations (|r| = 0.76-0.97, p < 0.01) between activity parameters and total contents of phenolics (5.2%-15.4% GAE) and proanthocyanidins (2.1%-15.0% CYE), with weak or no effects for chlorogenic acid isomers (0.69%-2.93%) and total flavonoids (0.28%-1.40%) suggested that among the listed polyphenols, proanthocyanidins are the most important determinants of the tested activity. UHPLC-PDA-ESI-QTOF-MS analyses led to detection of 34 polyphenols, of which 10 B-type procyanidins, 5 caffeoylquinic acids and 14 flavonoids were identified. After cluster analysis of the data matrix, the leaves of Cotoneaster zabelii, C. splendens, C. bullatus, C. divaricatus, C. hjelmqvistii and C. lucidus were selected as the most promising sources of natural antioxidants, exhibiting the highest phenolic levels and antioxidant capacities, and therefore the greatest potential for pharmaceutical applications.

The fresh fruits of Prunus spinosa L., a wild plum species, are traditionally used for dietary purposes and medicinal applications in disorders related to inflammation and oxidative stress. This study aimed to investigate the phytochemical composition of the fruits in the function of fractionated extraction and evaluate the biological potential of the extracts as functional products in two models of human immune cells ex vivo. Fifty-seven phenolic components were identified in the extracts by UHPLC-PDA-ESI-MS3, including twenty-eight new for the analysed fruits. Fractionation enabled the enrichment of polyphenols in the extracts up to 126.5 mg gallic acid equivalents/g dw total contents, 91.3 mg/g phenolic acids (caffeoyl-, coumaroyl-, and feruloylquinic acids), 41.1 mg/g flavonoids (mostly quercetin mono-, di- and triglycosides), 44.5 mg/g condensed proanthocyanidins, and 9.2 mg/g anthocyanins (cyanidin and peonidin glycosides). The hydroalcoholic extract and phenolic-enriched fractions of the fruits revealed significant ability to modulate pro-oxidant, pro-inflammatory, and anti-inflammatory functions of human neutrophils and peripheral blood mononuclear cells (PBMCs): they strongly downregulated the release of reactive oxygen species, TNF-α, and neutrophils elastase, upregulated the secretion of IL-10, and slightly inhibited the production of IL-8 and IL-6 in the cells stimulated by fMLP, fMLP+cytochalasin B, and LPS, depending on the test. Correlation studies and experiments on the pure compounds indicated a significant contribution of polyphenols to these effects. Moreover, cellular safety was confirmed for the extracts by flow cytometry in a wide range of concentrations. The results support the traditional use of fresh blackthorn fruits in inflammatory disorders and indicate extracts that are most promising for functional applications.

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Excessive activity of NADPH oxidase (Nox) is considered to be of importance for the progress of diabetic nephropathy. The aim of the study was to elucidate the effect of melatonin, known for its nephroprotective properties, on Nox activity under diabetic conditions. The experiments were performed on three groups of animals: (i) untreated lean (?/+) Zucker diabetic fatty (ZDF) rats; (ii) untreated obese diabetic (fa/fa) ZDF rats; and (iii) ZDF fa/fa rats treated with melatonin (20 mg/L) in drinking water. Urinary albumin excretion was measured weekly. After 4 wk of the treatment, the following parameters were determined in kidney cortex: Nox activity, expression of subunits of the enzyme, their phosphorylation and subcellular distribution. Histological studies were also performed. Compared to ?/+ controls, ZDF fa/fa rats exhibited increased renal Nox activity, augmented expression of Nox4 and p47(phox) subunits, elevated level of p47(phox) phosphorylation, and enlarged phospho-p47(phox) and p67(phox) content in membrane. Melatonin administration to ZDF fa/fa rats resulted in the improvement of renal functions, as manifested by considerable attenuation of albuminuria and some amelioration of structural abnormalities. The treatment turned out to nearly normalize Nox activity, which was accompanied by considerably lowered expression and diminished membrane distribution of regulatory subunits, that is, phospho-p47(phox) and p67(phox) . Thus, it is concluded that: (i) melatonin beneficial action against diabetic nephropathy involves attenuation of the excessive activity of Nox; and (ii) the mechanism of melatonin inhibitory effect on Nox is based on the mitigation of expression and membrane translocation of its regulatory subunits.

The work presents the results of an investigation into the molecular background of the activity of Cotoneaster fruits, providing a detailed description of their phytochemical composition and some of the mechanisms of their anti-inflammatory and antioxidant effects. GS-FID-MS and UHPLC-PDA-ESI-MS3 methods were applied to identify the potentially health-beneficial constituents of lipophilic and hydrophilic fractions, leading to the identification of fourteen unsaturated fatty acids (with dominant linoleic acid, 375.4–1690.2 mg/100 g dw), three phytosterols (with dominant β-sitosterol, 132.2–463.3 mg/100 g), two triterpenoid acids (10.9–54.5 mg/100 g), and twenty-six polyphenols (26.0–43.5 mg GAE/g dw). The most promising polyphenolic fractions exhibited dose-dependent anti-inflammatory activity in in vitro tests of lipoxygenase (IC50 in the range of 7.7–24.9 μg/U) and hyaluronidase (IC50 in the range of 16.4–29.3 μg/U) inhibition. They were also demonstrated to be a source of effective antioxidants, both in in vitro chemical tests (DPPH, FRAP, and TBARS) and in a biological model, in which at in vivo-relevant levels (1–5 μg/mL) they normalized/enhanced the nonenzymatic antioxidant capacity of human plasma and efficiently protected protein and lipid components of plasma against peroxynitrite-induced oxidative/nitrative damage. Moreover, the investigated extracts did not exhibit cytotoxicity towards human PMBCs. Among the nine Cotoneaster species tested, C. hjelmqvistii, C. zabelii, C. splendens, and C. bullatus possess the highest bioactive potential and might be recommended as dietary and functional food products.