Phenolic acids of plant origin are recognized as key bioactive compounds with potential for both internal and topical applications. Although some of these phytochemicals are used for skin care and to improve wound healing, oligomeric derivatives of rosmarinic acid (RA) remain poorly characterized in this context. This study aimed to evaluate the anti-inflammatory potential of salvianolic acid H (SA H) and yunnaneic acid B (YA B) in experimental models related to wound-healing, specifically in skin cells (HaCaT keratinocyte and NHDF fibroblast lines), THP1-ASC-GFP monocytes, and human peripheral blood mononuclear cells (PBMCs). Both SA H and YA B reduced pro-inflammatory cytokine release from HaCaT, NHDF, and PBMCs with efficacy comparable to or exceeding that of RA. Analyses of intracellular pathways of inflammatory response revealed that SA H and YA B were also efficient inhibitors of inflammasome formation in THP1-ASC-GFP reporter cells. Furthermore, SA H showed significant inhibitory effects on the activities of cyclooxygenase-2 and 5-lipoxygenase (IC50 = 11.53 µg/mL and 2.41 µg/mL, respectively). None of the examined acids influenced the hemostatic system at concentrations of 1–5 μg/mL. At 50 μg/mL, a slight increase in plasma clotting rate was observed for SA H and RA. These findings indicate that SA H and YA B, two naturally occurring oligomeric derivatives of RA, exert significant anti-inflammatory activity and represent promising agents for further studies on their use to improve wound healing.

Background/Objectives:Rheum officinale, an ethnomedicinal plant, has roots widely employed in modern pharmacological formulations. However, many of its biological activities remain only partly recognized. Furthermore, the metabolome and biological activity of its edible petioles, often considered a waste product, have received limited scientific attention. Methods and Results: The examination of anti-inflammatory properties of both root and petiole extracts (1-50 µg/mL) revealed the inhibition of the pro-inflammatory cytokine release from human peripheral blood mononuclear cells, a reduction in ALOX5 gene expression in human umbilical vein endothelial cells, and the significant inhibition (>60%) of cyclooxygenase-2 and 5-lipoxygenase activities. Importantly, no cytotoxic effects were detected at the tested concentrations. Conclusions: The petiole extract demonstrated anti-inflammatory efficiency comparable to, or exceeding that of the root extract, suggesting that R. officinale petioles could be valuable source of bioactive compounds for future investigations.

Background: Inflammation, endothelial dysfunction, and alterations in blood physiology are key factors contributing to atherosclerosis and other cardiovascular disorders. Hence, modulation of endothelial function and reducing its pro-inflammatory and pro-thrombotic activity is considered one of the most important cardioprotective strategies. This study aimed to evaluate the anti-inflammatory potential of rhubarb extracts isolated from petioles and underground organs of Rheum rhabarbarum L. (garden rhubarb) and R. rhaponticum L. (rhapontic rhubarb) as well as two stilbenoids, typically found in these plants, i.e., rhapontigenin (RHPG) and its glycoside, rhaponticin (RHPT). Methods: Analysis of the anti-inflammatory effects of the indicated rhubarb-derived substances involved different aspects of the endothelial cells’ (HUVECs) response: release of the inflammatory mediators; cyclooxygenase (COX-2) and 5-lipoxygenase (5-LOX) expression as well as the recruitment of leukocytes to the activated HUVECs. The ability of the rhubarb-derived extracts to inhibit COX-2 and 5-LOX activities was examined as well. The study was supplemented with the in silico analysis of major components of the analyzed extracts’ interactions with COX-2 and 5-LOX. Results: The obtained results indicated that the examined plant extracts and stilbenes possess anti-inflammatory properties and influence the inflammatory response of endothelial cells. Biochemical and in silico tests revealed significant inhibition of COX-2, with special importance of rhaponticin, as a compound abundant in both plant species. In addition to the reduction in COX-2 gene expression and enzyme activity, a decrease in the cytokine level and leukocyte influx was observed. Biochemical tests and computational analyses indicate that some components of rhubarb extracts may act as COX-2 inhibitors, with marginal inhibitory effect on 5-LOX.

Rheum rhaponticum L. (rhapontic rhubarb) and Rheum rhabarbarum L. (garden rhubarb) are edible and medicinal rhubarb species used for many centuries in traditional medicine. This work is focused on the biological activity of extracts from petioles and roots of R. rhaponticum and R. rhabarbarum as well as rhapontigenin and rhaponticin, typical stilbenes present in these rhubarbs, in a context of their effects on blood physiology and cardiovascular health. Anti-inflammatory properties of the examined substances were evaluated in human peripheral blood mononuclear cells (PBMCs) and THP1-ASC-GFP inflammasome reporter cells. Due to the coexistence of inflammation and oxidative stress in cardiovascular diseases, the study design included also antioxidant assays. This part of the work involved the assessment of the protective efficiency of the examined substances against the peroxynitrite-triggered damage to human blood plasma components, including fibrinogen, a protein of critical importance for blood clotting and maintaining the haemostatic balance. Pre-incubation of PBMCs with the examined substances (1-50 & mu;g/mL) considerably decreased the synthesis of prostaglandin E2 as well as the release of pro-inflammatory cytokines (IL-2 and TNF-& alpha;) and metalloproteinase-9. A reduced level of secreted apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks in the THP-1-ASCGFP cells was also observed. The examined substances significantly diminished the extent of ONOO?induced oxidative modifications of blood plasma proteins and lipids and normalized, or even strengthened blood plasma antioxidant capacity. Furthermore, a reduction of oxidative damage to fibrinogen, including modifications of tyrosine and tryptophan residues along with the formation of protein aggregates was found.

Ethnopharmacological relevance: Traditional medicine recommends the use of Rheum rhaponticum L. and R. rhabarbarum L. to treat over thirty complaints, including disorders related to the cardiovascular system such as heartache, pains in the pericardium, epistaxis and other types of haemorrhage, blood purification as well as disorders of venous circulation. Aim of the study: This work was dedicated to examining for the first time the effects of extracts from petioles and roots of R. rhaponticum and R. rhabarbarum, as well as two stilbene compounds (rhapontigenin and rhaponticin) on the haemostatic activity of endothelial cells and functionality of blood plasma components of the haemostatic system. Materials and methods: The study was based on three main experimental modules, including the activity of proteins of the human blood plasma coagulation cascade and the fibrinolytic system as well as analyses of the haemostatic activity of human vascular endothelial cells. Additionally, interactions of the main components of the rhubarb extracts with crucial serine proteases of the coagulation cascade and fibrinolysis (i.e. thrombin, the coagulation factor Xa and plasmin) were analyzed in silico. Results: The examined extracts displayed anticoagulant properties and significantly reduced the tissue factor -induced clotting of human blood plasma (by about 40%). Inhibitory effects of the tested extracts on thrombin and the coagulation factor Xa (FXa) were found as well. For the extracts, the IC50 was ranging from 20.26 to 48.11 & mu;g/ml. Modulatory effects on the haemostatic response of endothelial cells, including the release of von Willebrand factor, tissue-type plasminogen activator and the plasminogen activator inhibitor-1, have been also found. Conclusions: Our results indicated for the first time that the examined Rheum extracts influenced the haemostatic properties of blood plasma proteins and endothelial cells, with the prevalence of the anticoagulant action. The anticoagulant effect of the investigated extracts may be partly attributed to the inhibition of the FXa and thrombin activities, the key serine proteases of the blood coagulation cascade.

The Pulmonaria species (lungwort) are edible plants and traditional remedies for different disorders of the respiratory system. Our work covers a comparative study on biological actions in human blood plasma and cyclooxygenase-2 (COX-2) -inhibitory properties of plant extracts (i.e., phenolic-rich fractions) originated from aerial parts of P. obscura Dumort. and P. officinalis L. Phytochemical profiling demonstrated the abundance of phenolic acids and their derivatives (over 80% of the isolated fractions). Danshensu conjugates with caffeic acid, i.e., rosmarinic, lithospermic, salvianolic, monardic, shimobashiric and yunnaneic acids were identified as predominant components. The examined extracts (1–100 µg/mL) partly prevented harmful effects of the peroxynitrite-induced oxidative stress in blood plasma (decreased oxidative damage to blood plasma components and improved its non-enzymatic antioxidant capacity). The cellular safety of the extracts was confirmed in experimental models of blood platelets and peripheral blood mononuclear cells. COX-2 inhibitor screening evidently suggested a stronger activity of P. officinalis (IC50 of 13.28 and 7.24 µg/mL, in reaction with synthetic chromogen and physiological substrate (arachidonic acid), respectively). In silico studies on interactions of main components of the Pulmonaria extracts with the COX-2 demonstrated the abilities of ten compounds to bind with the enzyme, including rosmarinic acid, menisdaurin, globoidnan A and salvianolic acid H.

INTRODUCTION Consumption of fava beans seeds can be harmful to both humans and animals due to the presence of pyrimidine glucosides: vicine and convicine. Therefore, seeds, feed supplements, and protein extracts derived from fava beans should be monitored for the contents of pyrimidine derivatives. OBJECTIVES The main objective of this work was the development and validation of an inexpensive, uncomplicated, quick, and robust method for the quantitation of vicine and convicine in seeds of fava beans. METHODOLOGY Plant material was extracted using a dual-phase extraction system to remove lipophilic contaminants and deactivate residual enzymes. Pyrimidine glucosides together with the internal standard: 5-bromouridine were separated using capillary zone electrophoresis (CZE) on an uncoated fused-silica capillary with alkaline, tetraborate-based electrolyte. Linearity, limits of detection and quantitation, precision, accuracy, recovery, and robustness of the method were investigated. The method was applied for quantitation of pyrimidine glucosides in seeds. RESULTS Validation results suggest that the method is suitable for quantitation of pyrimidine glucosides, although it may not be sensitive enough for low-concentrated samples. Application of the method showed that seeds of the investigated variety of fava beans contain both vicine at 10-15 mg/g of dry weight and convicine at 3-5 mg/g of dry weight. CONCLUSION Developed and validated CZE-ultraviolet (UV) method is suitable for analysis of pyrimidine glucosides in seeds of Vicia faba L. var. minor. It can be used as an inexpensive, greener alternative to more advanced methods while providing equally reliable and accurate results.

The justification for the use of herbal supplements with Pulmonaria officinalis L. extract (POE) in the case of staphylococcal lung colonization/infections characteristic for cystic fibrosis (CF), was examined in vitro. The impact of POE phenolic-rich fraction on the virulence attributes of CF-associated Staphylococcus aureus (S. aureus) clinical strains has been assessed, including pathogen adhesion, biofilm formation on native and protein-conditioned surfaces (mucin, elastin), mature biofilm eradication, staphylococcal protein A expression, α-toxin release, and S. a. adhesion to A549 cells. Cytotoxicity of the extract to lung epithelial cells was also investigated. It was found that POE has bacteriostatic effects at MIC 1–2 mg/mL, recognized as of limited efficacy, but at MIC/subMICs it targeted virulence not viability. It usually decreased S. aureus adhesion and less frequently inhibited biofilm formation on native and protein-conditioned surfaces. Observed effect seems to be related to significant reduction by POE of sortase A activity. However, in some cases POE favored the creation of biofilm by staphylococci and S. aureus adhesion to the lung epithelium was not limited. On the other side POE caused significant decrease of S. a. α-toxin synthesis and slightly weakened the expression of SpA. When used at supraMICs POE eradicated mature biofilm, but in some cases with unsatisfying outcomes. Promisingly, POE has been recognized as a safe product, with no cytotoxicity up to 4 mg/mL. These results reflect the positive, negative or neutral anti-staphylococcal properties of POE. It seems that POE may be beneficial as a prophylactic, but not as a therapeutic or supportive agent in the area of CF—integrative medicine. However, introduction the official recommendations needs further in vivo studies.

Lungwort (Pulmonaria officinalis L., Boraginaceae) is considered to possess therapeutic properties and it has been traditionally used as a remedy against various lung disorders in many countries. Nevertheless, very few data concerning its phytochemical composition are available. This research aims to provide a detailed description of specialized metabolites from the aerial parts of lungwort. Nine previously undescribed and 36 known phenolic compounds were detected in the 50% methanolic extract. Following multistep preparative procedures, structures of newly discovered compounds were determined using one- and two-dimensional techniques of NMR spectroscopy. Among the identified compounds were caffeic acid esters with aliphatic hydroxycarboxylic acids, conjugates of dicaffeic acid with rosmarinic acid, and previously unknown isomers of isosalvianolic acid A and yunnaneic acid E, as well as other lignans. Concentrations of all identified phenolic derivatives in the investigated herbal material were estimated using a method based on liquid chromatography with high-resolution mass spectrometry detection. Seasonal changes in the concentration of metabolites were also investigated using targeted and untargeted metabolomics techniques.

Our work reveals that the aerial parts of Pulmonaria officinalis L. are a new source of yunnaneic acid B. We studied antioxidant activity and cytotoxicity of this compound (1-50 μg/mL) and its contents in various plant extracts. This is the first study confirming the presence of yunnaneic acid B in P. officinalis L. and Pulmonaria obscura Dumort and hence in the Boraginaceae family. Determination of 1,1-diphenyl-2-picrylhydrazyl radical reduction and peroxynitrite-scavenging efficacy in inorganic experimental systems provided EC50 values of 7.14 and 50.45 μg/mL, respectively. Then we examined the antioxidant action of yunnaneic acid B in blood plasma under peroxynitrite-induced oxidative stress in vitro. Yunnaneic acid B effectively diminished oxidative damage to blood plasma proteins and lipids. Furthermore, it was able to prevent the peroxynitrite-induced decrease in nonenzymatic antioxidant capacity of blood plasma. Additionally, cytotoxicity of yunnaneic acid B (at concentrations ≤50 μg/mL) toward peripheral blood mononuclear cells was excluded.

This in vitro study provides a new insight into the phytochemical, antioxidant, antiplatelet and cytotoxic profiles of commercial trans-clovamide and clovamide-rich extracts, isolated from aerial parts of three Trifolium species (i.e. T. clypeatum L., T. obscurum Savi and T. squarrosum L.).Under inorganic experimental conditions, the EC50 for the investigated substances varied between 4.87 (clovamide) and 7.91 μg/ml (T. clypeatum) according to the DPPH• assay. The most effective 1 mM ONOO− scavenger was clovamide (IC50 = 19.29 μg/ml), and the weakest was the T. obsucurum extract (IC50 = 41.79 μg/ml). The antioxidant action of the examined substances (1–50 μg/ml) was also evaluated in blood plasma (under 100 μM ONOO−-induced oxidative stress) using 3-nitrotyrosine, protein thiol groups, lipid hydroperoxides and thiobarbituric acid-reactive substances as biomarkers. All examined substances were more effective in limiting the oxidative damage to blood plasma components than the nitrative damage. Their anti-nitrative action was statistically significant only at the highest concentration (50 μg/ml). Measurements of platelet adhesion and aggregation found the compounds to have moderate antiplatelet properties of up to 20% inhibition of platelet adhesion at concentrations of 1–5 μg/ml. Additionally, clovamide and Trifolium extracts demonstrated no cytotoxicity towards blood platelets or peripheral blood mononuclear cells.

AbstractSaponins are naturally occurring secondary metabolites, which can be widely found in a number of plant families. They can be found in different parts of plants usually as multicomponent mixtures. They are composed of the triterpene or steroidal aglycone to which a number of sugars in the form of one, two, or three sugar chains are attached. Depending on the chemical structure, they show different biological activities. The common feature of these compounds is surface activity, so they can be used as emulsifiers, and in the past, they were used as soap substitutes. Some of them have found application in food industry, whereas the others are being considered as antinutritional substances. Owing to the fact that saponins occur in quite complex mixtures, their separation to individual compounds is still a challenge. Also, their determination with chromatographic techniques creates some problems as they are lacking chromophores allowing ultraviolet (UV) detection. Precolumn derivatization, light scattering detection, and liquid chromatography coupled with mass detector are the methods of choice for their determination.

The essential oils (EOs) and static headspaces (HSs) of in vitro plantlets and callus of Mentha x piperita were characterized by GC-MS analysis. Leaves were used as explants to induce in vitro plant material. The EO yields of the in vitro biomass were much lower (0.1% v/w) than those of the parent plants (2% v/w). Many typical mint volatiles were emitted by the in vitro production, but the callus and in vitro plantelet EOs were characterized by the lack of both pulegone and menthofuran. This was an important difference between in vitro and in vivo plant material as huge amounts of pulegone and menthofuran may jeopardise the safety of mint essential oil. Regarding the other characteristic volatiles, menthone was present in reduced amounts (2%) in the in vitro plantlets and was not detected in the callus, even if it represented the main constituent of the stem and leaf EOs obtained from the cultivated mint (26% leaves; 33% stems). The M. piperita callus was characterized by menthol (9%) and menthone (2%), while the in vitro plantlet EO showed lower amounts of both these compounds in favour of piperitenone oxide (45%). Therefore, the established callus and in vitro plantlets showed peculiar aromatic profiles characterized by the lack of pulegone and menthofuran which have to be monitored in the mint oil for their toxicity.

The effects of two elicitors: jasmonic acid and methyl jasmonate on cell growth as well as on rosmarinic acid accumulation in cell suspension cultures of Mentha × piperita were investigated. The highest rosmarinic acid accumulation 117.95 mg g−1 DW (12% DW) was measured 24 h after addition of 100 μM methyl jasmonate. A similar concentration 110.12 mg g−1 DW was detected 48 h after application of 200 μM jasmonic acid. Those values were nearly 1.5 times higher compared to the control sample, without elicitation. There was no substantial influence of elicitors on rosmarinic acid secretion into the culture media. Extracellular concentrations of rosmarinic acid were similar to the values from the control variants. It was documented that suspension cultures of M. piperita treated with elicitors showed a decrease in biomass accumulation when compared to the control.

1Research Institute of Horticulture, Skierniewice, Poland; 2Graduate School of Science, Osaka Prefecture University, Sakai, Osaka, Japan; 3Faculty of Liberal Arts and Sciences, Osaka Prefecture University, Sakai, Osaka, Japan; 4Laboratory of Horticultural Science, Department of Plant Resources, Faculty of Agriculture, Kyushu University, Fukuoka, Japan; 5Polish Academy of Sciences, Botanical Garden – Center for Biological Diversity Conservation in Powsin, Warszawa, Poland; 6Siedlce University of Natural Sciences and Humanities, Institute of Biology, Department of Plant Physiology and Genetics, Siedlce, Poland

Nine polyphenols in the aerial parts of Mentha longifolia have been separated by chromatographic techniques. Their structures have been confirmed by HPLC/electrospray ionization-MS/MS. The compounds identified included rosmarinic acid, salvianolic acid L, dedihydro-salvianolic acid, luteolin-glucuronide, luteolin-diglucuronide, luteolin-glucopyranosyl-rhamnopyranoside, and eriodictyol-glucopyranosyl-rhamnopyranoside. The extracts of M. longifolia and M. piperita field plants, in vitro plants, callus tissues, and cell suspension cultures were profiled, and their polyphenol composition was compared in different tissues and quantified using ultra-performance column liquid chromatography (UPLC)/triple-quadrupole-MS in the selected-ion recording detection mode. Determination of desired compounds was based on calibration curves obtained for standards, which were previously isolated from M. longifolia aerial parts. The UPLC profiles revealed considerable differences in the synthesis of secondary metabolites among samples coming from field plants, in vitro plants, callus tissues, and cell suspension cultures. Plant tissues coming from field cultivation (for both M. piperita and M. longifolia) contained several phenolic compounds (flavonoids and phenolic acids), whereas plants from in vitro conditions, callus tissues, and suspension cultures contained only a few of them. Rosmarinic acid dominated in all of these samples. These results show that under in vitro conditions, the metabolism of phenolics undergoes a fundamental change.

Introduction Biological Elicitors of Defence Responses in Plants Perception and Transduction of General Elicitors Emerging Techniques to Improve Secondary Metabolites Production, Based on Elicitor Signalling Pathways Conclusion 13. Hairy Root Cultures for Secondary Metabolites Production Laura Pistelli, Annalisa Giovannini, Barbara Ruffoni, Alessandra Bertoli and Luisa Pistelli Abstract Introduction How Agrobacterium rhizogenes Genes Can Affect Plant Tissues Development? RolC Mechanism of Action RolB Mechanism of Action RolD Mechanism of Action RolA and ORF13 Mechanism of Action A. rhizogenes Transformed Medicinal Plants as Farm for Aromatic and Nutraceutical Metabolite Production Conclusion 14. Plant Tissue Cultureâ€â€​An Opportunity for the Production of Nutraceuticals Mariella Lucchesini and Anna Mensuali ̃Sodi Abstract Introduction Why to Cultivate In Vitro Plants for the Production of Secondary Metabolites? In Vitro Technologies for the Cultivation of Nutraceutical Plants The Micropropagation Conclusion 15. Plant Cell Cultures: Bioreactors for Industrial Production Barbara Ruffoni, Laura Pistelli, Alessandra Bertoli and Luisa Pistelli Abstract

Oxidative stress is strictly correlated to the pathogenesis of many diseases, and a diet rich in fruits and vegetables, or adequately integrated, is currently considered to be a protective and preventive factor. This study aimed to analyze the efficacy of a 1 h preincubation with the highest nontoxic dose of a characterized Mentha longifolia extract (80 μg/mL) in protecting human keratinocytes (NCTC2544) from chemically induced oxidative stress (500 μM H2O2 for 2, 16, and 24 h). As reference synthetic pure compounds rosmarinic acid (360.31 μg/mL), a major mint phenolic constituent, and resveratrol (31.95 mg/mL), a well-known antioxidant, were used. Cellular viability was significantly protected by mint, which limited protein and DNA damage, decreased lipid peroxidation, and preserved glutathione and superoxide dismutase activity in the shorter phases of oxidative stress induction, in extents comparable to or better than those of pure compounds. These data suggest that mint use as only a flavoring has to be revised, taking into consideration its enrichment in foodstuff and cosmetics.