Role of Phytohormones in Biomass and Polyphenol Accumulation in Salvia bulleyana In Vitro Culture.

Salvia bulleyana is a plant native to the Chinese Yunnan Province. This species has been used in traditional Chinese medicine as a substitute for Danshen (the roots of Salvia miltiorrhiza). The aim of our study was to establish an effective system for propagating S. bulleyana shoots to obtain large amounts of material rich in bioactive compounds. Phytohormones were used to regulate shoot growth and regeneration potential and influence plant secondary metabolism. The shoot tips were incubated on a Murashige and Skoog agar medium supplemented with 0.1 or 0.5 mg/L IAA (indole-3-acetic acid) and the cytokinins benzylaminopurine (BAP), meta-topoline (M-T), 6-benzylaminopurine riboside (RBAP), N-benzyl-9-(2-tetrahydropyranyl)-adenine (BPA) or kinetin, (K) at concentrations of 0.5, 1 or 2 mg/L. It was observed that the type and concentration of growth regulator significantly influenced the regeneration potential of S. bulleyana shoots. The highest multiplication rate was obtained when 0.1 mg/L IAA and 2 mg/L BPA were used. Under these conditions, 100% of shoot tips formed buds and almost seven buds/shoot per explant were obtained after five weeks. Meanwhile, the highest biomass was found for shoots growing on a medium supplemented with 0.1 mg/L IAA and 1 mg/L M-T: 1.2 g of fresh weight and 0.17 g of dry weight. However, a medium with 0.1 mg/L IAA and 2 mg/L RBAP was most favorable for bioactive phenolic acid content, with a total polyphenol level (37.7 mg/g dw) 4.5 times higher than in shoots grown on medium without growth regulators (8.23 mg/g dw). Finally, optimal conditions were selected by TOPSIS (technique for order of preference by similarity to the ideal solution); the culture of S. bulleyana grown on an MS medium containing 0.1 mg/L IAA and 1 mg/L M-T was found to be the most efficient for polyphenol accumulation and can be used for the production of medicinally relevant compounds.

Valorization of the Photo-Protective Potential of the Phytochemically Standardized Olive (Olea europaea L.) Leaf Extract in UVA-Irradiated Human Skin Fibroblasts.

Leaves of Olea europaea are a by-product of the olive oil industry and a dietary supplement with acknowledged antioxidant and anti-inflammatory activity but underestimated photoprotective potential. We investigated the protective effects of the LC-PDA-MS/MS standardized ethanol-water extract of olive leaves (OLE), containing 26.2% total phenols and 22.2% oleuropein, with underlying mechanisms against the UVA-induced oxidative damage in human dermal fibroblasts. Hs68 cells were pre-treated (24 h) with OLE (2.5-25 µg/mL) or the reference antioxidants, quercetin and ascorbic acid (25 µg/mL), followed by irradiation (8 J/cm2). OLE significantly reduced the UVA-induced DNA damage and reactive oxygen species (ROS) overproduction and increased the thioredoxin reductase (TrxR) expression and post-radiation viability of fibroblasts by inhibiting their apoptosis. Both intrinsic and extrinsic apoptotic signaling pathways appeared to be inhibited by OLE, but the activity of caspase 9 was the most reduced. We hypothesized that the TrxR up-regulation by OLE could have prevented the UVA-induced apoptosis of Hs68 cells. In addition, a significant decrease in UVA-induced secretion levels of tumor necrosis factor (TNF-α) and interleukin-2 (IL-2) was shown in human lymphocyte culture in response to OLE treatment. In summary, our results support the beneficial effect of OLE in an in vitro model and indicate its great potential for use in the cosmetic and pharmaceutical industry as a topical photoprotective, antioxidant, and anti-inflammatory agent.

In Vitro Strategy for the Enhancement of the Production of Bioactive Polyphenols in Transformed Roots of Salvia bulleyana.

The underground parts of Salvia bulleyana, a rare Chinese plant species, have long been used in traditional Chinese medicine. The Rhizobium rhizogenes-transformed root culture obtained from this plant might be a promising novel source of valuable phenolics, including rosmarinic acid. The present study identifies for the first time, the optimal growth conditions of S. bulleyana hairy roots regarding production efficiency. The comprehensive optimization comprised cultivation in different basal media (B5, SH, MS, and WP) with full- and half-strength macro- and microelements, different vitamin contents (full, half, one-quarter part, and without) and sucrose concentrations (2, 3, 4, 5%), and under different light conditions: in dark, under blue LED (λ = 430 nm), red LED (λ = 670 nm), mixed blue and red LED (30%:70%), and white LED (390-670 nm). Hairy root growth and bioactive compound accumulation were also detailed every five days over the 50-day culture cycle. The optimal conditions were determined using a technique for order preference by similarity to the ideal solution (TOPSIS). The most efficient combination for root growth and polyphenol content was found to be ½SH liquid medium with half vitamin concentration and 3% sucrose when grown in the dark. The biomass yield during the growth cycle was 6.1 g (fresh weight-FW) and 0.92 g (dry weight-DW) on one Erlenmeyer flask: a 14.3-fold increase in FW and 16.1-fold increase in DW in relation to the inoculum. The highest mean total phenolic content was 93.6 mg/g DW including about 70 mg/g DW rosmarinic acid, reached on day 40 of culture; compared to roots of two-year-old plants grown under field conditions, the total phenolic acid content was four times higher and rosmarinic acid eight times higher. The obtained results place the investigated culture among the best hair root cultures for rosmarinic acid production.

The Antioxidant, Cytotoxic and Antimicrobial Potential of Phenolic Acids-Enriched Extract of Elicited Hairy Roots of Salvia bulleyana.

Hairy root cultures are valuable sources of a range of phytochemicals. Among them, Salvia bulleyana root culture is a promising source of polyphenols, especially rosmarinic acid (RA), a phenolic acid depside with pleiotropic activity and a wide application in medicine and cosmetology. The aim of the study was to enhance the culture productivity by finding suitable elicitation protocol and to determine its biological potential in terms of antioxidant, anticancer and antimicrobial properties. The total content of phenols and the levels of particular constituents in root extracts were analyzed using HPLC-PDA. Among four elicitors tested (yeast extract; methyl jasmonate, MJA; trans-anethol; and cadmium chloride), MJA was found to be the most effective. The greatest boost in phenolic production (up to 124.4 mg/g dry weight) was observed after three-day treatment with MJA at 100 µM, with an almost 100% improvement compared to the controls (non-treated root culture). The hydromethanolic extract from the elicited culture exhibited strong antioxidant activity with IC50 values of 11.1 µg/mL, 6.5 µg/mL and 69.5 µg/mL for DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)) and superoxide anion radical, respectively. Moreover, in concentrations of 0.5-5 mg/mL the extract inhibited the growth of LoVo, AGS and HeLa cell lines, but was safe for the L929 cells up to the concentration of 5 mg/mL. The extract also exhibited moderate antimicrobial activity. Thus, the results confirmed that elicitation can be a beneficial strategy for increase the phenolic acid biosynthesis in hairy roots of S. bulleyana, and that such a highly productive culture can show significant biological potential.

Cytokinin-Based Tissue Cultures for Stable Medicinal Plant Production: Regeneration and Phytochemical Profiling of Salvia bulleyana Shoots.

Salvia bulleyana is a rare Chinese medicinal plant that due to the presence of polyphenols lowers the risk of some chronic diseases especially those related to the cardiovascular system. The present study examines the organogenic competence of various combinations and concentrations of plant growth regulators to develop an efficient protocol for in vitro regeneration of S. bulleyana via leaf explants, maintaining the high production of active constituents. The purpose of the study was also to assess the possibilities of using a cytokinin-based regeneration to effectively produce therapeutic compounds. The adventitious shoot formation was observed through direct organogenesis on media with purine derivatives (meta-topolin, mT and benzylaminopurine, BAP), and through indirect organogenesis on media with urea derivatives (tidiazuron, TDZ and forchlorfenuron, CPPU). The highest regeneration frequency (95%) with 5.2 shoots per explant was obtained on leaves cultured on Murashige and Skoog (MS) medium containing 0.1 mg/L naphthalene-1-acetic acid (NAA) and 2 mg/L BAP. Following inter simple sequence repeat (ISSR) marker-based profiling, the obtained organogenic shoot lines revealed a similar banding pattern to the mother line, with total variability of 4.2-13.7%, indicating high level of genetic stability. The similar genetic profile of the studied lines translated into similar growth parameters. Moreover, HPLC analysis revealed no qualitative differences in the profile of bioactive metabolites; also, the total polyphenol content was similar for different lines, with the exception of the shoots obtained in the presence of CPPU that produced higher level of bioactive compounds. This is the first report of an effective and rapid in vitro organogenesis protocol for S. bulleyana, which can be efficiently employed for obtaining stable cultures rich in bioactive metabolites.

Novel insight into biological activity and phytochemical composition of Sorbus aucuparia L. fruits: Fractionated extracts as inhibitors of protein glycation and oxidative/nitrative damage of human plasma components.

Sorbus aucuparia L. is a source of edible fruits appreciated for their nutritional and medicinal properties. In this work some bioactivity mechanisms were evaluated, which might be connected with the traditional application of rowanberries in cardiovascular complications of diabetes. With the use of a panel of chemical and biological in vitro models the rowanberry extracts were proved to significantly inhibit the formation of advanced glycation end products, neutralise multiple oxidants generated in vivo, increase the non-enzymatic antioxidant capacity of human plasma and protect plasma components (proteins and lipids) against oxidative/nitrative damage at in vivo-relevant levels (1-5 µg/mL). Moreover, the extracts were found safe in cytotoxicity tests on the peripheral blood mononuclear cells. The comprehensive phytochemical profiling of the extracts (RP/HILIC-UHPLC-PDA-ESI-MS3, HPLC-PDA, and UV-spectrophotometric methods) led to the identification of 51 phenolics, including caffeic and ferulic acids pseudodepsides (34 compounds, prevailing isomers of chlorogenic acid and cynarin, total content up to 269.4 mg/g), flavonols (mostly quercetin glycosides, up to 5.8 mg/g), flavan-3-ol derivatives (proanthocyanidin oligomers and polymers, up to 17.0 mg/g), and simple phenolic acids. The experiments on model constituents of the extracts and correlation studies were used to evaluate contribution of polyphenols to the observed effects. Taking into account the possible additive and synergistic effects, the co-occurrence of various compounds was indicated as partly responsible for biological activity of the fruits. Considering both the composition and activity parameters, the methanol-water (1:1, v/v) extract and its concentrated phenolic fractions appeared to be the most advantageous for biological application.

The Effects of Prunus spinosa L. Flower Extracts, Model Polyphenols and Phenolic Metabolites on Oxidative/Nitrative Modifications of Human Plasma Components with Particular Emphasis on Fibrinogen In Vitro.

Oxidative post-translational modifications of fibrinogen (a multifunctional blood plasma protein essential for hemostasis) are associated with the pathogenesis of cardiovascular disorders (CVDs). Prunus spinosa flower is a herbal medicine used in an adjuvant treatment of CVDs and rich in polyphenolic antioxidants. In the present study, phytochemically standardized P. spinosa flower extracts, their primary native polyphenols and potential phenolic metabolites were evaluated in vitro for their protective effects on fibrinogen (isolated and in the human plasma matrix) using a panel of complementary methods (SDS-PAGE, western blot, C-ELISA, fluorometry, FRAP, TBARS). The results revealed that the tested analytes at in vivo relevant levels (1-5 µg/mL) considerably reduced the structural changes in the fibrinogen molecule under the oxidative stress conditions induced by peroxynitrite. In particular, they diminished the oxidation and/or nitration of amino acid residues, including tyrosine and tryptophan, as well as the formation of high molecular weight aggregates. The decrease in the levels of 3-nitrotyrosine was about 13.5-33.0% and 58.3-97.1% at 1 µg/mL and 50 µg/mL, respectively. The study indicated that low molecular weight polyphenols were crucial for the protective activity of the extracts toward fibrinogen and other human plasma components. The investigated model compounds effectively protected total plasma proteins and lipids against oxidative damage (by reducing the levels of 3-nitrotyrosine and thiobarbituric acid-reactive substances and normalizing/enhancing the non-enzymatic antioxidant capacity of plasma). The work provides insight into the role of native and metabolized polyphenols as contributory factors to the systemic activity of blackthorn flower extracts within the circulatory system.

Lipophilic extracts from leaves, inflorescences and fruits of Prunus padus L. as potential sources of corosolic, ursolic and oleanolic acids with anti-inflammatory activity.

Chloroform extracts from leaves, inflorescences and fruits of Prunus padus were analysed for anti-inflammatory activity and accumulation of corosolic (CA), ursolic (UA) and oleanolic (OA) acids. The analytes were identified and quantified by GC-MS and UHPLC-PDA. Their total levels depend on plant material type and harvesting time, and varied from 0.25 mg/g DW in fruits, through 0.76-1.09 mg/g DW in flowers, to 1.41-4.54 mg/g DW in leaves. Significant variation in the leaf analytes contents was observed during vegetation with the peak amounts in autumn, which indicated the optimal harvesting season. The plant extracts inhibited pro-inflammatory enzymes (lipoxygenase and hyaluronidase) in a concentration-dependent manner, and their activity parameters correlated with the levels and activity of pure triterpene acids, especially CA and UA. The results of the comparison with the positive controls (heparin, indomethacin, dexamethasone) might partly support the application of P. padus in anti-inflammatory therapies, reported by traditional medicine.

Establishment of hairy root cultures of Salvia bulleyana Diels for production of polyphenolic compounds.

This study was to obtain stable transformed roots of Salvia bulleyana using A. rhizogenes strain A4 and then evaluate their phytochemical profile and selected the most productive clone. Our results indicated that the type of explant and medium used for bacterium and explant incubation had an influence on the frequency of hairy root formation. The best response was obtained on leaves infected with bacteria cultivated on YMB medium supplemented with acetosyringone. Of the four selected transformed root clones, after five-week cultivation in Woody Plant (WP) medium, the highest growth indexes were demonstrated for line C1: i.e. 13 for fresh and 15 for dry weight (81.4 and 8.2 g/l fresh and dry weight, respectively). The qualitative analysis of hydromethanolic extracts of hairy roots of S. bulleyana using UPLC-PDA-ESI-MS/MS method showed the presence of 10 polyphenolic compounds including predominant rosmarinic acid (RA), its derivatives (hexoside and methyl rosmarinate), caffeic acid, its derivatives and several salvianolic acids: K, E and F. Their production varied among the four root clones studied; the highest RA (39.6 mg/g dry weight) and total polyphenol (48.9 mg/g dry weight) level were found in the roots of C4 clone. These values were significantly higher than those of the roots of plants grown for several years under field conditions. The transformation of the obtained root cultures was confirmed by polymerase chain reaction using aux1, aux2, rolB, rolC and rolD primers.

The Effect of Standardised Flower Extracts of Sorbus aucuparia L. on Proinflammatory Enzymes, Multiple Oxidants, and Oxidative/Nitrative Damage of Human Plasma Components In Vitro.

Polyphenol-rich plant extracts might alleviate the negative impact of oxidative stress and inflammation, but careful phytochemical standardisation and evaluation of various mechanisms are required to fully understand their effects. In this context, flower extracts of Sorbus aucuparia L.-a traditional medicinal plant-were investigated in the present work. The LC-MS/MS profiling of the extracts, obtained by fractionated extraction, led to the identification of 66 constituents, mostly flavonols (quercetin and sexangularetin glycosides with dominating isoquercitrin), pseudodepsides of quinic and shikimic acids (prevailing isomers of chlorogenic acid and cynarin), and flavanols (catechins and proanthocyanidins). Minor extract components of possible chemotaxonomic value were flavalignans (cinchonain I isomers) and phenylamides (spermidine derivatives). As assessed by HPLC-PDA and UV-spectrophotometric studies, the extracts were polyphenol-abundant, with the contents up to 597.6 mg/g dry weight (dw), 333.9 mg/g dw, 382.0 mg/g dw, and 169.0 mg/g dw of total phenolics, flavonoids, proanthocyanidins, and caffeoylquinic acids, respectively. Their biological in vitro effects were phenolic-dependent and the strongest for diethyl ether, ethyl acetate, and n-butanol fractions of the methanol-water (7 : 3, v/v) extract. The extracts showed significant, concentration-dependent ability to scavenge in vivo-relevant radical/oxidant agents (O2 ∙-, OH∙, H2O2, ONOO-, NO∙, and HClO) with the strongest effects towards OH∙, ONOO-, HClO, and O2 ∙- (compared to ascorbic acid). Moreover, the extracts efficiently inhibited lipoxygenase and hyaluronidase (compared to indomethacin) but were inactive towards xanthine oxidase. At in vivo-relevant levels (1-5 µg/mL), they also effectively protected human plasma components (proteins and lipids) against ONOO--induced oxidative damage (reduced the levels of 3-nitrotyrosine, lipid hydroperoxides, and thiobarbituric acid-reactive substances) and normalised/enhanced the total nonenzymatic antioxidant capacity of plasma. In cytotoxicity tests, the extracts did not affect the viability of human PBMCs and might be regarded as safe. The results support the application of the extracts in the treatment of oxidative stress-related pathologies cross-linked with inflammatory changes.

Multifunctional Phytocompounds in Cotoneaster Fruits: Phytochemical Profiling, Cellular Safety, Anti-Inflammatory and Antioxidant Effects in Chemical and Human Plasma Models In Vitro.

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 ß-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.

Polyphenol-Rich Extracts from Cotoneaster Leaves Inhibit Pro-Inflammatory Enzymes and Protect Human Plasma Components against Oxidative Stress In Vitro.

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.

Lignans From Forsythia x Intermedia Leaves and Flowers Attenuate the Pro-inflammatory Function of Leukocytes and Their Interaction With Endothelial Cells.

Aim of the study: Taking into account that overactivated leukocytes are an important factor in the development of many chronic diseases, we investigated the activity of phytochemically characterized (HPLC-DAD-MSn) extracts from forsythia leaves and flowers on the pro- and anti-inflammatory functions of leukocytes (effects on IL-1ß, IL-8, TNF-α, and TGFß release) and their adherence to endothelial cells. Using bio-guided fractionation, we isolated the active compounds and determined their biological activity, and we included the positive control quercetin. Methods: The effect on IL-1ß, TNF-α, IL-8, and TGF-α production by leukocytes was measured by enzyme-linked immunosorbent assay (ELISA). The surface expression of adhesion molecules was analyzed with flow cytometry, and the neutrophil attachment to the endothelial cells was assessed fluorimetrically. The effects on p38MAPK, ERK1/2 and JNK phosphorylation were determined using western blots. Results: Leaf extracts had the effect of decreasing TNF-α production in neutrophils and monocyte/macrophage cells. The bio-guided fractionation led to the isolation of the following lignan aglycones: (+)-pinoresinol, (+)-epipinoresinol, (-)-matairesinol, (+)-phillygenin, and (-)-arctigenin. Only phillygenin was able to stimulate the anti-inflammatory function of macrophages by inducing TGF-ß release and IL-10 receptor surface expression. Arctigenin, phillygenin, and a metabolite produced by the gut microbiota, enterolactone, decreased TNF-α and IL-1ß production and neutrophil adhesion to endothelial cells, probably by attenuating the p38 and ERK kinase pathways. Conclusion:Forsythia x intermedia is a valuable source of active lignans, which may be potential candidates for treating inflammatory diseases that are associated with the excessive production of cytokines such as TNF-α and IL-1ß.

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.

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.

New validated high-performance liquid chromatographic method for simultaneous analysis of ten flavonoid aglycones in plant extracts using a C18 fused-core column and acetonitrile-tetrahydrofuran gradient.

An HPLC method of high resolution has been developed and validated for the simultaneous determination of ten prominent flavonoid aglycones in plant materials using a fused-core C18-silica column (Ascentis® Express, 4.6 mm × 150 mm, 2.7 µm). The separation was accomplished with an acetonitrile-tetrahydrofuran gradient elution at a flow rate of 1 mL/min and temperature of 30°C. UV spectrophotometric detection was employed at 370 nm for flavonols (quercetin [QU], myricetin [MY], isorhamnetin [IS], kaempferol [KA], sexangularetin [SX], and limocitrin [LM]) and 340 nm for flavones (apigenin [AP], acacetin [AC], chrysoeriol [CH], and luteolin [LU]). The high resolution of critical pairs QU/LU (10.50), QU/CH (3.40), AP/CH (2.51), SX/LM (2.30), and IS/KA (2.70) was achieved within 30.3 min. The observed column back pressure was less than 4300 psi, thus acceptable for conventional HPLC equipment. The method was sensitive enough having LODs of 0.115-0.525 ng and good linearity (r > 0.9999) over the test range. The precision values, expressed as RSD values, were <7.5%, and the accuracy was in the range of 95.3-100.2% for all analytes except MY (73.8%). The method was successfully employed for the determination of flavonoids in several medicinal plants, such as Ginkgo biloba, Betula pendula, and a variety of Sorbus species.

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 (EC(50) = 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-ß-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 (IC(50) = 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.

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-ß-glucopyranoside (1), astragalin (2), isoquercitrin (3), hyperoside (4), kaempferol 3-O-ß-glucopyranoside-7-O-α-rhamnopyranoside (5), quercetin 3-O-ß-glucopyranoside-7-O-α-rhamnopyranoside (6), rutin (7), chlorogenic acid (8) and neochlorogenic acid (9). The isolates were identified by spectral methods (UV, (1)H- and (13)C-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, EC(50) = 2.76 mg L(-1)) and the ethyl acetate extract (EC(50) = 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.

Variation in the phenolic content and in vitro antioxidant activity of Sorbus aucuparia leaf extracts during vegetation.

Seasonal variation in the antioxidant activity and content of phenolic compounds was studied for the 70% methanol extracts of Sorbus aucuparia leaves harvested monthly over the full course of the growing season. The antioxidant potential of the extracts was evaluated using two complementary in vitro tests: the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assay and the AAPH [2,2'-azobis-(2-amidinopropane)dihydrochloride]-induced [corrected] linoleic acid (LA) peroxidation test. The radical-scavenging capacities of the extracts towards the DPPH radical were in the range of 0.40 to 0.57 millimolar Trolox equivalents/g dry weight of the leaves. They were significantly correlated (r = -0.8480, p < 0.05) with the results of the LA-peroxidation test, indicating the S. aucuparia leaf 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 0.81-0.97, p < 0.05). As the best antioxidant capacities and the highest phenolic contents were found for the leaf samples harvested during the three summer months (June, July and August), this period could be considered to be optimal for cost-effective production of natural health products. For the leaf samples collected in July, the values of EC50 and IC50 for the two antioxidant tests were 2.02 and 93.45 µg [corrected] phenolics/mL, respectively. These antioxidant capacities were found to be higher or comparable to those of synthetic and natural phenolic antioxidants, such as BHT (butylated hydroxytoluene), BHA (butylated hydroxyanisole), TBHQ (tert-butylhydroquinone), quercetin and Trolox.

In vitro antioxidant activity and total phenolic content of the inflorescences, leaves and fruits of Sorbus torminalis (L.) Crantz.

The antioxidant potential of 70% methanolic extracts from the inflorescences, leaves and fruits of Sorbus torminalis (L.) Crantz was evaluated using three in vitro test systems: the DPPH (2,2-diphenyl-1-picryl-hydrazyl) and the ABTS [2,2-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid)] free radical scavenging assays, and the AAPH [2,2'-azobis-(2-amidinopropane) dihydrochloride]-induced [corrected] linoleic acid (LA) peroxidation test. The results were compared with the activity of the extracts obtained from the model antioxidant Sorbus species (Sorbus aucuparia L.), and also with the activity of phenolic standards such as quercetin, Trolox [(+/-)-6-hydroxy-2,2,7,8-tetramethylchroman-2-carboxylic acid], BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene) and TBHQ (tert-butylhydroquinone). The radical scavenging capacities of the S. torminalis extracts towards the DPPH radical were in the range of 62.0-244.1 micromolar Trolox equivalents/g d.w. of plant material. They were significantly (p < 0.05) correlated with the results of the ABTS test (r = 0.8535), and with the chain-breaking activities determined in the LA-peroxidation test (r = 0.9831). In comparison with the synthetic standards, the free radical scavenging capacity of the Sorbus extracts was remarkably higher than their chain-breaking activity. Both kinds of antioxidant effects of the extracts were significantly (R2 > 0.8097, p < 0.05) influenced by the total phenolic content (TPC) as determined by the Folin-Ciocalteu method. The plant tissues derived from S. torminalis exhibited lower antioxidant potentials than those of S. aucuparia by a factor of 1.5-3.2, partially due to the lower TPC levels (multiplicity factors of 1.2-1.9). After the original antioxidant capacities of the extracts were recalculated according to the TPC levels, the resulting antioxidant capacities of the phenolic fractions in the S. torminalis extracts were lower than those from S. aucuparia by a factor of 1.1-1.6, suggesting that the distinctive chemistry of the phenolic constituents also influences the antioxidant power of the two species.