Monika A. Olszewska

Antioxidant activity of inflorescences, leaves and fruits of three Sorbus species in relation to their polyphenolic composition

The antioxidant potential of 70% methanolic extracts from the inflorescences, leaves and fruits of Sorbus aucuparia, S. aria and S. intermedia was evaluated using three in vitro test systems (DPPH: 2,2-diphenyl-1-picryl hydrazyl; TEAC: trolox equivalent antioxidant capacity and FRAP: ferric reducing/antioxidant power). High activities found were expressed as micromoles trolox equivalents per gram dry weight and were in the range of 86.9–956.2 for the DPPH test, 65.3–577.2 for the TEAC assay and 221.1–1915.2 for the FRAP method. Significant linear correlations (R 2 in the range of 0.75–0.98) between these values and the contents of total phenolics, total flavonoids, total proanthocyanidins and chlorogenic acid isomers showed that the listed phenolic compounds are determinants of the antioxidant capacity tested. As the superior activity and the highest phenolic levels (11.83% of total phenolics calculated as gallic acid equivalents, 4.35% of chlorogenic acid isomers, 5.01% of proanthocyanidins and 1.28% of flavonoid aglycones) were found for S. aucuparia inflorescence, this plant material has the greatest potential as a source for natural health products.

Profiling of Phenolic Compounds and Antioxidant Activity of Dry Extracts from the Selected Sorbus Species

The antioxidant efficiency of dry extracts from inflorescences and/or leaves of seven Sorbus species was studied using four in vitro tests of SET (single electron transfer) and HAT-type (hydrogen atom transfer) mechanisms. The 70% methanol extracts and its diethyl ether, ethyl acetate, n-butanol and water fractions were tested in parallel with the phenolic standards, e.g., caffeic acid, quercetin, BHA, BHT, and Trolox®. The SET-type activity of the extracts depended primarily on the extraction solvent. The most valuable extracts were n-butanol and ethyl acetate ones, which activity was high in the DPPH (EC50 = 3.2–5.2 μg/mL), TEAC (2.8–4.0 mmol Trolox®/g), and FRAP (9.8–13.7 mmol Fe2+/g) tests, and strongly correlated with the total phenolic levels (39.6–58.2% of gallic acid equivalents). The HPLC-PDA analysis of the extracts led to the identification of chlorogenic acid, isoquercitrin, hyperoside, rutin, quercetin 3-O-sophoroside, and sexangularetin 3-O-β-D-glucopyranoside as the main components. Apart from flavonoids and hydroxycinnamic acids, proanthocyanidins have also a significant impact on the SET-type activity. The HAT-reactivity of the extracts in the linoleic acid peroxidation test (IC50 = 36.9–228.3 μg/mL) depended more strongly on the plant tissue than on the extraction solvent, and its correlation with the phenolic content was weak. Both SET and HAT-type activity of the most potent Sorbus extracts was comparable with the activity of the standards, indicating their great potential as effective sources for health products.

Establishment of Hairy Root Cultures of Rhaponticum carthamoides (Willd.) Iljin for the Production of Biomass and Caffeic Acid Derivatives

The aim of the study was to obtain transformed roots of Rhaponticum carthamoides and evaluate their phytochemical profile. Hairy roots were induced from leaf explants by the transformation of Agrobacterium rhizogenes strains A4 and ATCC 15834. The best response (43%) was achieved by infection with A4 strain. The effects of different liquid media (WPM, B5, SH) with full and half-strength concentrations of macro- and micronutrients on biomass accumulation of the best grown hairy root line (RC3) at two different lighting conditions (light or dark) were investigated. The highest biomass (93 g L−1 of the fresh weight after 35 days) was obtained in WPM medium under periodic light. UPLC-PDA-ESI-MS3 and HPLC-PDA analyses of 80% aqueous methanol extracts from the obtained hairy roots revealed the presence of eleven caffeoylquinic acids and their derivatives and five flavonoid glycosides. The production of caffeoylquinic acids and their derivatives was elevated in hairy roots grown in the light. Only light-grown hairy roots demonstrated the capability for the biosynthesis of such flavonoid glycosides as quercetagetin, quercetin, luteolin, and patuletin hexosides. Chlorogenic acid, 3,5-di-O-caffeoylquinic acid and a tentatively identified tricaffeoylquinic acid derivative were detected as the major compounds present in the transformed roots.

Polyphenolic Profile, Antioxidant and Anti-Inflammatory Activity of Eastern Teaberry (Gaultheria procumbens L.) Leaf Extracts.

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 Fe2+/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 observed. Comprehensive UHPLC-PDA-ESI-MS3 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.

Activity-guided isolation and identification of free radical-scavenging components from various leaf extracts of Sorbus aria (L.) Crantz

Nine phenolics were obtained from the leaves of Sorbus aria (L.) Crantz by activity-directed isolation: isorhamnetin 3-O-β-glucopyranoside (1), astragalin (2), isoquercitrin (3), hyperoside (4), kaempferol 3-O-β-glucopyranoside-7-O-α-rhamnopyranoside (5), quercetin 3-O-β-glucopyranoside-7-O-α-rhamnopyranoside (6), rutin (7), chlorogenic acid (8) and neochlorogenic acid (9). The isolates were identified by spectral methods (UV, 1H- and 13C-NMR, COSY, HMQC and HMBC), and their free radical-scavenging activity was tested using the l,l-diphenyl-2-picrylhydrazyl (DPPH) method. The antioxidant potential of the different extracts obtained in the fractionation process was evaluated using the DPPH test in relation to the HPLC contents of the isolates 1–9, total phenolics and total proanthocyanidins. Among the analytes tested, superior activity was expressed by isoquercitrin (3, EC50 = 2.76 mg L−1) and the ethyl acetate extract (EC50 = 2.99 mg L−1). Five strongly active isolates 3, 6, 7, 8 and 9 were found to be major components and to be principally responsible for the radical-scavenging activity of S. aria extracts.

Phenolic Profile and Antioxidant Potential of Leaves from Selected Cotoneaster Medik. Species

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, O2•− and H2O2 scavenging assays). The samples exhibited the dose-dependent responses in all assays with activity parameters of EC50 = 18.5–34.5 µg/mL for DPPH; 0.9–3.8 mmol Fe2+/g for FRAP; SC50 = 27.7–74.8 µg/mL for O2•−; and SC50 = 29.0–91.3 µg/mL for H2O2. 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.

Assessment of the content of phenolics and antioxidant action of inflorescences and leaves of selected species from the genus Sorbus sensu stricto.

In order to find new sources of natural antioxidants, the antioxidant potential of 70% methanolic extracts from the inflorescences and leaves of 16 species from the genus Sorbus s.s. was evaluated using two complementary in vitro test systems: the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assay and the AAPH [2,2′-azobis-(2-amidinopropane)dihydrochloride]-induced linoleic acid (LA) peroxidation test. The radical-scavenging capacities of the extracts towards the DPPH radical were in the range of 0.25−0.86 millimolar Trolox® equivalents/g dry weight. They were significantly correlated (r = -0.8089, p < 0.001) with the results of the LA-peroxidation test, indicating the Sorbus extracts to be universal antioxidants. Significant linear correlations were also found between the different antioxidant potentials and total phenolic contents as estimated by the Folin-Ciocalteu method and further verified by serial determinations of proanthocyanidins, chlorogenic acid isomers and flavonoids (|r| in the range of 0.71−0.95, p < 0.001). Cluster analysis of the data matrix identified the ten samples (inflorescences of S. aucuparia, S. pohuashanensis, S. decora, S. koehneana, S. commixta, S. gracilis, and S. sitchensis, and the leaves of S. wilfordii, S. pogonopetala, and S. gracilis) exhibiting the highest antioxidant activity and total phenolic levels and therefore the greatest potential as effective sources for natural health products.

Metabolite Profiling of Eastern Teaberry (Gaultheria procumbens L.) Lipophilic Leaf Extracts with Hyaluronidase and Lipoxygenase Inhibitory Activity

The phytochemical profile and anti-inflammatory activity of Gaultheria procumbens dry lipophilic leaf extracts were evaluated. Forty compounds were identified by GC-MS, representing 86.36% and 81.97% of the petroleum ether (PE) and chloroform (CHE) extracts, respectively, with ursolic acid (28.82%), oleanolic acid (10.11%), methyl benzoate (10.03%), and methyl salicylate (6.88%) dominating in CHE, and methyl benzoate (21.59%), docosane (18.86%), and octacosane (11.72%) prevailing in PE. Three components of CHE were fully identified after flash chromatography isolation and spectroscopic studies as (6S,9R)-vomifoliol (4.35%), 8-demethyl-latifolin (1.13%), and 8-demethylsideroxylin (2.25%). Hyaluronidase and lipoxygenase inhibitory activity was tested for CHE (IC50 = 282.15 ± 10.38 μg/mL and 899.97 ± 31.17 μg/mL, respectively), PE (IC50 = 401.82 ± 16.12 μg/mL and 738.49 ± 15.92 μg/mL), and nine of the main constituents versus heparin (IC50 = 366.24 ± 14.72 μg/mL) and indomethacin (IC50 = 92.60 ± 3.71 μg/mL) as positive controls. With the best activity/concentration relationships, ursolic and oleanolic acids were recommended as analytical markers for the extracts and plant material. Seasonal variation of both markers following foliar development was investigated by UHPLC-PDA. The highest levels of ursolic (5.36–5.87 mg/g DW of the leaves) and oleanolic (1.14–1.26 mg/g DW) acids were observed between August and October, indicating the optimal season for harvesting.

Metabolite profiling and antioxidant activity of Prunus padus L. flowers and leaves

Six phenolics were obtained from the leaves of Prunus padus by activity-guided isolation: isorhamnetin 3-O-β-xylopyranosyl-(1 → 2)-β-galactopyranoside (1), astragalin (2), hyperoside (3), quercetin 3-O-β-xylopyranosyl-(1 → 2)-β-galactopyranoside (4), quercetin 3-O-β-xylopyranosyl-(1 → 2)-β-glucopyranoside (5) and chlorogenic acid (6). The antioxidant potential of 70% methanolic extracts from the flowers and leaves collected over the growing season was evaluated using the 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging and 2,2′-azobis-(2-amidinopropane) dihydrochloride (AAPH)-induced linoleic acid (LA) peroxidation tests in relation to the contents of the isolates 1–6, total phenolics, total proanthocyanidins and total quercetin. The IC50 values were expressed in gram dry weight per gram of DPPH or LA, respectively, and were in the range of 1.42−2.42 for the DPPH test and 1.78−4.92 for the LA peroxidation, with superior activity found for the flowers and the autumn leaves. Significant linear correlation of these values to the sum of proanthocyanidins and compounds 1–6 (R 2 > 0.87) showed that the listed phenolics are synergists of the tested activity.

Bioactivity Potential of Prunus spinosa L. Flower Extracts: Phytochemical Profiling, Cellular Safety, Pro-inflammatory Enzymes Inhibition and Protective Effects Against Oxidative Stress In Vitro

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.