Light-Emitting Diodes and Liquid System Affect the Caffeoylquinic Acid Derivative and Flavonoid Production and Shoot Growth of Rhaponticum carthamoides (Willd.) Iljin.

Plant in vitro cultures can be an effective tool in obtaining desired specialized metabolites. The purpose of this study was to evaluate the effect of light-emitting diodes (LEDs) on phenolic compounds in Rhaponticum carthamoides shoots cultured in vitro. R. carthamoides is an endemic and medicinal plant at risk of extinction due to the massive harvesting of its roots and rhizomes from the natural environment. The shoots were cultured on an agar-solidified and liquid-agitated Murashige and Skoog's medium supplemented with 0.1 mg/L of indole-3-acetic acid (IAA) and 0.5 mg/L of 6-benzyladenine (BA). The effect of the medium and different treatments of LED lights (blue (BL), red (RL), white (WL), and a combination of red and blue (R:BL; 7:3)) on R. carthamoides shoot growth and its biosynthetic potential was observed. Medium type and the duration of LED light exposure did not affect the proliferation rate of shoots, but they altered the shoot morphology and specialized metabolite accumulation. The liquid medium and BL light were the most beneficial for the caffeoylquinic acid derivatives (CQAs) production, shoot growth, and biomass increment. The liquid medium and BL light enhanced the content of the sum of all identified CQAs (6 mg/g DW) about three-fold compared to WL light and control, fluorescent lamps. HPLC-UV analysis confirmed that chlorogenic acid (5-CQA) was the primary compound in shoot extracts regardless of the type of culture and the light conditions (1.19-3.25 mg/g DW), with the highest level under R:BL light. BL and RL lights were equally effective. The abundant component was also 3,5-di-O-caffeoylquinic acid, accompanied by 4,5-di-O-caffeoylquinic acid, a tentatively identified dicaffeoylquinic acid derivative, and a tricaffeoylquinic acid derivative 2, the contents of which depended on the LED light conditions.

Phytochemistry and Biological Profile of Gaultheria procumbens L. and Wintergreen Essential Oil: From Traditional Application to Molecular Mechanisms and Therapeutic Targets.

Gaultheria procumbens L. is a medicinal plant whose aerial parts (leaves, stems, and fruits) and methyl salicylate-rich essential oil (wintergreen oil) are used in phytotherapy to treat inflammation, muscular pain, and infection-related disorders. This overview summarises the current knowledge about ethnobotany, phytochemistry, pharmacology, molecular mechanisms, biocompatibility, and traditional use of G. procumbens and the wintergreen oil distilled from different plant organs. Over 70 hydrophilic compounds, including methyl salicylate glycosides, flavonoids, procyanidins, free catechins, caffeoylquinic acids, and simple phenolic acids, have been identified in G. procumbens plant parts. Moreover, aliphatic compounds, triterpene acids, and sterols have been revealed in lipophilic fractions. Furthermore, over 130 volatile compounds have been detected in wintergreen oil with dominating methyl salicylate (96.9-100%). The accumulated research indicates that mainly hydrophilic non-volatiles are responsible for the pharmacological effects of G. procumbens, primarily its potent anti-inflammatory, antioxidant, and photoprotective activity, with mechanisms verified in vitro and ex vivo in cellular and cell-free assays. The biological effectiveness of the dominant methyl salicylate glycoside-gaultherin-has also been confirmed in animals. Wintergreen oil is reported as a potent anti-inflammatory agent exhibiting moderate antioxidant and antimicrobial activity in vitro and significant insecticidal and larvicidal capacity. Together, G. procumbens accumulate a diverse fraction of polyphenols, triterpenes, and volatiles with validated in vitro and ex vivo biological activity but with the absence of in vivo studies, especially clinical trials concerning effective dose determination and toxicological verification and technological research, including drug formulation.

Flavonol and A-type procyanidin-rich extracts of Prunus spinosa L. flower exhibit anticoagulant activity through direct thrombin inhibition, but do not affect platelet aggregation in vitro.

Background: Blackthorn flower (Prunus spinosa L.) is a traditional herbal remedy recommended for treating cardiovascular diseases (CVDs). Aim: This in vitro study investigates the effects of flavonol and A-type procyanidin-rich blackthorn flower extracts on the hemostatic system, including the blood plasma coagulation cascade and platelet aggregation. Methods: Six distinct extracts, characterized through various techniques, including LC-MS/MS, were assessed at in vivo-relevant levels (1-50 µg/mL) for their antithrombotic activity. The thrombin, prothrombin, and activated partial thromboplastin times were measured. Additionally, the thrombin enzymatic activity was tested using the chromogenic substrate S-2238 and fibrinogen as the physiological substrate of the enzyme. To gain insights into the mechanism of action, the interactions between the primary extracts' constituents, their potential metabolites, and thrombin were examined in silico. The computational analyses were complemented by in vitro experiments and circular dichroism spectroscopy. The platelet aggregation in human platelet-rich plasma was assessed after ADP or collagen stimulation. Furthermore, the extracts' biocompatibility was tested on human peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs). Results: The extracts slightly prolonged the prothrombin and thrombin times and effectively inhibited the thrombin's enzymatic activity, reducing its amidolytic and proteolytic functions at 50 µg/mL by 91.2% and 74.8%, respectively. In silico molecular docking demonstrated a strong binding affinity of the examined polyphenols and their metabolites to thrombin. Most analytes bound exclusively within the enzyme active site; however, afzelin, kaempferitrin, and procyanidin A2 revealed the affinity to additional binding sites, including exosite I. The structure-activity relationship of flavonols as thrombin inhibitors was studied in vitro. Circular dichroism spectroscopy confirmed that the interactions between thrombin and the compounds (even at 1 µg/mL) induce alterations within the α-helices' secondary structure, resulting in noticeable changes in the enzyme's CD spectrum. On the other hand, the extracts did not influence platelet aggregation. Eventually, their cellular biocompatibility with PBMCs and RBCs was confirmed. Conclusion: The extracts directly inhibit thrombin, a critical serine protease in hemostasis and a prime anticoagulant drug target, and do not exhibit antiplatelet effects. This study enhances the knowledge of the biological activity of blackthorn flowers and supports their traditional use in CVDs.

Nine Years of African Swine Fever in Poland.

(1) Background: African swine fever (ASF) is a highly contagious and fatal haemorrhagic disease in domestic pigs and wild boars, causing significant economic loss to the swine industry in the European Union. The genotype II of African swine fever has spread in many European countries since the virus was detected in 2007 in Georgia. In Poland, the genotype II of the ASF virus was confirmed on 17 February 2014 in the eastern part of the country and appeared to have been transmitted to Poland from Belarus. Poland has been particularly affected by ASF epidemics in the last decade, resulting in a significant decline in the Polish pig population. Wild boars are the main reservoir of the African swine fever virus (ASFV), but human activities such as transportation and illegal animal trade are the primary reasons for the long-distance transmission of the disease. (2) Conclusions: During the nine years of ASF in Poland, multiple measures have been taken to prevent the spread of the virus among the wild boar population via the passive and active surveillance of these animals. With regard to pig farms, the only effective measure for preventing the spread of ASF is the efficient enforcement by state authorities of the biosecurity standards and the farmers' compliance with them.

Chemometrics-Driven Variability Evaluation of Phenolic Composition, Antioxidant Capacity, and α-Glucosidase Inhibition of Sorbus aucuparia L. Fruits from Poland: Identification of Variability Markers for Plant Material Valorization.

Sorbus aucuparia L. (rowan tree) is a widely distributed European plant, valued for its nutritional and medicinal qualities. The medicinal application of rowanberries, relying particularly on their antioxidant and antidiabetic effects, is closely connected with the presence of numerous phenolic compounds. However, the broad geographical occurrence of rowan trees may contribute to fluctuations in fruit composition, influencing their biological properties. This study aimed to identify the constituents most involved in this variability to facilitate effective quality control. The investigation encompassed 20 samples collected from diverse locations across Poland, evaluated in terms of the variation in composition and bioactivity. The UHPLC-PDA-ESI-MSn study identified 45 different constituents, including flavonoids, phenolic acid and flavon-3-ols. The detected compounds were quantitatively assessed by HPLC-PDA, alongside spectrophotometric evaluation of total phenolic content and the content of high-molecular-weight proanthocyanidins (TPA). Additionally, •OH scavenging capacity and α-glucosidase inhibition were included as bioactivity parameters. Chemometric analyses, including hierarchical cluster analysis and principal component analysis, revealed geographically dependent variability, with low to moderate variation observed for most factors (variation coefficients 20.44-44.97%), except for flavonoids (variation coefficients 45-76%). They also enabled the selection of seven constituents and TPA as the key markers of variability and biological activity of rowanberries. These markers could be employed for quality control of the fruits, offering a more efficient and cost-effective approach compared to full phytochemical analysis.

Anti-Diabetic Potential of Polyphenol-Rich Fruits from the Maleae Tribe-A Review of In Vitro and In Vivo Animal and Human Trials.

The Maleae tribe consists of over one thousand species, including many well-known polyphenol-containing fruit crops with wide-ranging biological properties, e.g., apples (Malus), chokeberries (Aronia), pears (Pyrus), quinces (Cydonia, Chaenomeles), saskatoon (Amelanchier), loquats (Eriobotrya), medlars (Mespilus), rowans (Sorbus), and hawthorns (Crataegus). Considering the current interest in the concept of functional foods and the still-insufficient methods of diabetes management, the anti-diabetic potential of fruits has been studied intensively, including those of the Maleae tribe. This paper is the first comprehensive overview of this selected topic, covering articles published from 2000 to 2023 (131 articles in total). The first part of this review focuses on the potential mechanisms of action of fruits investigated so far (46 species), including their effects on tissue-specific glucose transport and the expression or activity of proteins in the insulin signalling pathway. The second part covers the phytocompounds responsible for particular fruits' activity-primarily polyphenols (e.g., flavonols, dihydrochalcones, proanthocyanidins, anthocyanins, phenolic acids), but also polysaccharides, triterpenes, and their additive and synergistic effects. In summary, fruits from the Maleae tribe seem promising as functional foods and anti-diabetic agents; however, their prospects for more expansive pro-health application require further research, especially more profound in vivo trials.

Effect of Light Conditions on Polyphenol Production in Transformed Shoot Culture of Salvia bulleyana Diels.

Various strategies have been used to increase the efficiency of secondary metabolite production in Salvia plants. This report is the first to examine the spontaneous development of Salvia bulleyana shoots transformed by Agrobacterium rhizogenes on hairy roots and the influence of light conditions on the phytochemical profile of this shoot culture. The transformed shoots were cultivated on solid MS medium with 0.1 mg/L of IAA (indole-3-acetic acid) and 1 mg/L of m-Top (meta-topolin), and their transgenic characteristic was confirmed by PCR-based detection of the rolB and rolC genes in the target plant genome. This study assessed the phytochemical, morphological, and physiological responses of the shoot culture under stimulation by light-emitting diodes (LEDs) with different wavelengths (white, WL; blue, B; red, RL; and red/blue, ML) and under fluorescent lamps (FL, control). Eleven polyphenols identified as phenolic acids and their derivatives were detected via ultrahigh-performance liquid chromatography with diode-array detection coupled to electrospray ionization tandem mass spectrometry (UPLC-DAD/ESI-MS) in the plant material, and their content was determined using high-performance liquid chromatography (HPLC). Rosmarinic acid was the predominant compound in the analyzed extracts. The mixed red and blue LEDs gave the highest levels of polyphenol and rosmarinic acid accumulation (respectively, 24.3 mg/g of DW and 20.0 mg/g of DW), reaching two times greater concentrations of polyphenols and three times greater rosmarinic acid levels compared to the aerial parts of two-year-old intact plants. Similar to WL, ML also stimulated regeneration ability and biomass accumulation effectively. However, the highest total photosynthetic pigment production (1.13 mg/g of DW for total chlorophyll and 0.231 mg/g of DW for carotenoids) was found in the shoots cultivated under RL followed by BL, while the culture exposed to BL was characterized as having the highest antioxidant enzyme activities.

The Genus Cuphea P. Browne as a Source of Biologically Active Phytochemicals for Pharmaceutical Application and Beyond-A Review.

Cuphea P. Browne (Lythraceae) is a monophyletic taxon comprising some 240-260 species that grow wild in the warm, temperate, and tropical regions of South and Central America and the southern part of North America. They have been valued as traditional medicinal remedies for numerous indications, including treating wounds, parasitic infections, hypertension, digestive disorders, cough, rheumatism, and pain. Modern pharmacological research provides data that support many of these traditional uses. Such a wide array of medicinal applications may be due to the exceptionally rich phytochemical profile of these plants, which includes bioactive compounds classified into various metabolite groups, such as polyphenols, triterpenes, alkaloids, and coumarins. Furthermore, Cuphea seed oils, containing medium-chain fatty acids, are of increasing interest in various industries as potential substitutes for coconut and palm oils. This review aims to summarize the results of phytochemical and pharmacological studies on Cuphea plants, with a particular focus on the therapeutic potential and molecular mechanisms of the action of polyphenolic compounds (especially flavonoids and tannins), which have been the subject of many recently published articles.

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.

In vitro cytotoxic activity of Cuphea ingrata Cham. & Schltdl. extracts related to the oenothein B content.

Cuphea ingrata is a traditional medicinal plant species of the Lythraceae family. This work reports on the cytotoxic activity of the methanolic extract from the aerial parts of C. ingrata and the n-butanol and ethyl acetate fractions against human skin and prostate cancer cells. The selectivity of action was tested in normal skin keratinocytes HaCaT and prostate epithelial cells PNT2. The ethyl acetate fraction showed the highest activity in all three human skin cancer cell lines: A375, HTB-140, WM793, with IC50 = 15.90; 3.40; 18.75 µg/mL, respectively. To obtain comparative information on the chemical composition, a quantitative analysis of oenothein B was performed using the UHPLC-PDA method. An analysis of its cytotoxic activity was also carried out.

Enzymatically Modified Isoquercitrin: Production, Metabolism, Bioavailability, Toxicity, Pharmacology, and Related Molecular Mechanisms.

Quercetin and its glycosides, such as isoquercitrin or rutin, are among the most ubiquitous flavonoids present in plants. They possess numerous health-promoting properties, whose applicability is, however, limited by poor water solubility and absorption issues. Enzymatically modified isoquercitrin (EMIQ) is an isoquercitrin derivative obtained from rutin via enzymatic transformations that greatly enhance its bioavailability. Due to advantageous reports on its safety and bioactivity, EMIQ is currently gaining importance as a food additive and a constituent of dietary supplements. This review summarizes the thus-far-conducted investigations into the metabolism, toxicity, biological properties, and molecular mechanisms of EMIQ and presents a comprehensive characterization of this valuable substance, which might represent the future of flavonoid supplementation.

Effect of Sucrose Concentration on Rhaponticum carthamoides (Willd.) Iljin Transformed Root Biomass, Caffeoylquinic Acid Derivative, and Flavonoid Production.

Rhaponticum carthamoides (Willd.) Iljin is a rare, pharmacopoeial, and medicinal plant, endemic to Siberia and endangered due to the massive collection of raw material from the natural habitat. The aim of the current study was to estimate the effect of sucrose concentration (0-7%) on R. carthamoides transformed root growth and on caffeoylquinic acid derivative (CQA) and flavonoid production. Sucrose in higher concentrations may induce osmotic stress and thus may affect secondary metabolism in plants. It was revealed that sucrose concentration influenced R. carthamoides transformed root biomass and modified the phenolic compound metabolic pathway. However, the dynamics of both processes varied significantly. The optimal sucrose level was different for biomass accumulation and the biosynthesis of specialized metabolite. The highest dry weight of roots was achieved for 7% sucrose (31.17 g L-1 of dry weight), while 1% sucrose was found to be optimal for phenolic acid and flavonoid production. Considering the dry weight increase and metabolite accumulation, 3% sucrose was revealed to give optimal yields of CQAs (511.1 mg L-1) and flavonoids (38.9 mg L-1). Chlorogenic acid, 3,5-, 4,5-di-O-caffeoylquinic acids, 1,4,5-O-tricaffeoylquinic acid, and a tentatively-identified tricaffeoylquinic acid derivative 1 were found to be the most abundant specialized metabolites among the identified CQAs. Our findings indicate that R. carthamoides transformed roots may be an efficient source of CQA derivatives, with valuable health-promoting activities.

The Inhibition of α-Glucosidase, α-Amylase and Protein Glycation by Phenolic Extracts of Cotoneaster bullatus, Cotoneaster zabelii, and Cotoneaster integerrimus Leaves and Fruits: Focus on Anti-Hyperglycemic Activity and Kinetic Parameters.

Cotoneaster species have gained significant importance in traditional Asian medicine for their ability to prevent and treat hyperglycemia and diabetes. Therefore, in this study, some aspects of the beneficial health effects of hydromethanolic extracts of C. bullatus, C. zabelii, and C. integerrimus leaves and fruits were evaluated, including their influence on α-glucosidase, α-amylase, and nonenzymatic protein glycation. The activity was investigated in relation to the polyphenolic profile of the extracts determined by UV-spectrophotometric and HPLC-PDA-fingerprint methods. It was revealed that all leaf and fruit extracts are a promising source of biological components (caffeic acid pseudodepsides, proanthocyanidins, and flavonols), and the leaf extracts of C. bullatus and C. zabelii contain the highest levels of polyphenols (316.3 and 337.6 mg/g in total, respectively). The leaf extracts were also the most effective inhibitors of digestive enzymes and nonenzymatic protein glycation. IC50 values of 8.6, 41.8, and 32.6 µg/mL were obtained for the most active leaf extract of C. bullatus (MBL) in the α-glucosidase, α-amylase, and glycation inhibition tests, respectively. In the kinetic study, MBL was displayed as a mixed-type inhibitor of both enzymes. The correlations between the polyphenol profiles and activity parameters (|r| > 0.72, p < 0.05) indicate a significant contribution of proanthocyanidins to the tested activity. These results support the traditional use of Cotoneaster leaves and fruits in diabetes and suggest their hydrophilic extracts be promising in functional applications.

Valorisation of the Inhibitory Potential of Fresh and Dried Fruit Extracts of Prunus spinosa L. towards Carbohydrate Hydrolysing Enzymes, Protein Glycation, Multiple Oxidants and Oxidative Stress-Induced Changes in Human Plasma Constituents.

Prunus spinosa fruits (sloes), both fresh and dried, are underexplored dietary components and ethno-phytotherapeutic remedies applied to treat chronic oxidative-stress-related diseases, including diabetes. The present study aimed to evaluate drying-related changes in the antidiabetic potential of sloe extracts and some bioactivity mechanisms, which might be connected with their traditional application. The polyphenol-enriched extracts, prepared by fractionated extraction and phytochemically standardised, i.a., by LC-MS/MS, were tested in vitro using a set of biological and chemical models. The experiments revealed the significant extracts' ability to counteract the generation of advanced glycation end products (AGEs) and inhibit the activity of key glycolytic enzymes, i.e., α-glucosidase and α-amylase. Moreover, they were proved to effectively scavenge multiple oxidants of physiological importance (O2•-, HO•, H2O2, NO•, HOCl), increase the non-enzymatic antioxidant capacity of human plasma (NEAC) under oxidative stress conditions induced by peroxynitrite, and protect plasma proteins and lipids against peroxidation and nitration at in vivo-relevant levels (1-50 µg/mL, equivalent to 0.03-6.32 µg polyphenols/mL). In most cases, the activity of fresh fruit extracts surpassed that of dried-based products. The correlation studies and tests on model compounds proved polyphenols as dominant contributors to the observed effects. Furthermore, the co-occurring representatives of various polyphenolic classes were found to contribute to the biological activity of sloes through additive and synergistic effects. Considering the extraction yield and activity parameters, especially the superior outcomes compared to anti-diabetic drugs aminoguanidine and acarbose in the anti-glycation and α-glucosidase inhibition tests, the methanol-water (75:25, v/v) extract of fresh fruits and its phenolic-enriched fractions revealed the most advantageous potential for functional application.

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.

Polyphenols and Maillard Reaction Products in Dried Prunus spinosa Fruits: Quality Aspects and Contribution to Anti-Inflammatory and Antioxidant Activity in Human Immune Cells Ex Vivo.

Dried Prunus spinosa fruits (sloes) are folk phytotherapeutics applied to treat chronic inflammatory disorders. However, their pharmacological potential, activity vectors, and drying-related changes in bioactive components remain unexplored. Therefore, the present research aimed to evaluate the anti-inflammatory and antioxidant effects of dried sloes in ex vivo models of human neutrophils and peripheral blood mononuclear cells (PMBCs) and establish their main active components. It was revealed that the fruit extracts significantly and dose-dependently inhibited the respiratory burst, downregulated the production of elastase (ELA-2) and TNF-α, and upregulated the IL-10 secretion by immune cells under pro-inflammatory and pro-oxidant stimulation. The slightly reduced IL-6 and IL-8 secretion was also observed. The structural identification of active compounds, including 45 phenolics and three Maillard reaction products (MRPs) which were formed during drying, was performed by an integrated approach combining LC-MS/MS, preparative HPLC isolation, and NMR studies. The cellular tests of four isolated model compounds (chlorogenic acid, quercetin, procyanidin B2, and 5-hydroxymethylfurfural), supported by statistical correlation studies, revealed a significant polyphenolic contribution and a slight impact of MRPs on the extracts' effects. Moreover, a substantial synergy was observed for phenolic acids, flavonoids, condensed proanthocyanidins, and MPRs. These results might support the phytotherapeutic use of dried P. spinosa fruits to relieve inflammation and establish the quality control procedure for the extracts prepared thereof.

The Effect of Standardised Leaf Extracts of Gaultheria procumbens on Multiple Oxidants, Inflammation-Related Enzymes, and Pro-Oxidant and Pro-Inflammatory Functions of Human Neutrophils.

The leaves of Gaultheria procumbens are polyphenol-rich traditional medicines used to treat inflammation-related diseases. The present study aimed to optimise the solvent for the effective recovery of active leaf components through simple direct extraction and verify the biological effects of the selected extract in a model of human neutrophils ex vivo. The extracts were comprehensively standardised, and forty-one individual polyphenols, representing salicylates, catechins, procyanidins, phenolic acids, and flavonoids, were identified by UHPLC-PDA-ESI-MS3. The chosen methanol-water (75:25, v/v) extract (ME) was obtained with the highest extraction yield and total phenolic levels (397.9 mg/g extract's dw), including 98.9 mg/g salicylates and 299.0 mg/g non-salicylate polyphenols. In biological tests, ME revealed a significant and dose-dependent ability to modulate pro-oxidant and pro-inflammatory functions of human neutrophils: it strongly reduced the ROS level and downregulated the release of pro-inflammatory cytokines and tissue remodelling enzymes, especially IL-1ß and elastase 2, in cells stimulated by fMLP, LPS, or fMLP + cytochalasin B. The extracts were also potent direct scavengers of in vivo relevant oxidants (O2•-, •OH, and H2O2) and inhibitors of pro-inflammatory enzymes (cyclooxygenase-2, hyaluronidase, and lipoxygenase). The statistically significant correlations between the tested variables revealed the synergic contribution of individual polyphenols to the observed effects and indicated them as useful active markers for the standardisation of the extract/plant material. Moreover, the safety of ME was confirmed in cytotoxicity tests. The obtained results might partially explain the ethnomedicinal application of G. procumbens leaves and support the usage of the standardised leaf extract in the adjuvant treatment of oxidative stress and inflammation-related chronic diseases.

Polyphenol-Enriched Extracts of Prunus spinosa Fruits: Anti-Inflammatory and Antioxidant Effects in Human Immune Cells Ex Vivo in Relation to Phytochemical Profile.

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.

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.