Stress response in tomato as influenced by repeated waterlogging.

Introduction: Plants respond to water stress with a variety of physiological and biochemical changes, but their response varies among species, varieties and cultivars. Waterlogging in tomato reduces plant growth, degrade chlorophyll and increase concentration of oxidative parameters. Priming can alleviate stress in plants caused by waterlogging enabling plants to be more tolerant to an additional stress in the current or even subsequent generation. The aim of this study was to evaluate tomato genotypes for their sensitivity to waterlogging stress applied during early vegetative growth and at full flowering stage. Materials and methods: The study included two local genotypes, Trebinjski sitni (GB1126) and Zuti (GB1129), and the reference variety Novosadski jabucar (NJ), which is the variety most commonly used in Serbia and Bosnia and Herzegovina. The activity of class III peroxidase (POX), hydrogen peroxide (H2O2) content and malondialdehyde (MDA) content were measured spectrophotometrically, and for quantification of individual phenolic compounds, targeted approach was adopted, using UHPLC/DAD/(-)HESI-MS2 instrument (Dionex UltiMate 3000 UHPLC system with a DAD detector, configured with a triple quadrupole mass spectrometer TSQ Quantum Access Max (Thermo Fisher Scientific, Germany)). Results and discussion: Oxidative parameters (H2O2 and MDA) exhibited an increase in content in leaves of tomato plants that underwent waterlogging stress compared to control plants. Moreover, oxidative parameters showed positive correlation with proteins and phenolics content. The obtained correlations can indicate that one of the response strategies of tomato plants to waterlogging is the increased synthesis of proteins and phenolic compounds. The POX activity was not correlated with other parameters except with the polyphenols. A positive correlation was shown between POX activity and the content of phenolic compounds, indicating their independent roles in the removal of ROS. Changes in the phenolic profiles after the exposure of plants to waterlogging stress are recorded, and these changes were more severe in leaves and fruits of GB1129 and NJ genotypes than in GB1126. Thus, genotype GB1126 is the most efficient in maintaining the phenolic profiles of leaves and fruits, and therefore of the nutritive and organoleptic qualities of fruits following the exposure to waterlogging. Also, genotype GB1126 exhibited the ability to maintain the content of oxidative parameters during waterlogging at certain growth stages, implying certain waterlogging tolerance. Conclusion: Waterlogging triggered stress memory but not at all growth stages. The most pronounced stress memory was obtained in fruit samples in the phase of full fruit maturity on the 1st truss. This study shed light on the defense mechanisms of tomato plants to repeated waterlogging stress from the perspectives of the changes in the composition of major phenolics, and pointed to the 5-O-caffeoylquinic acid and rutin as the chemical markers of the waterlogging stress tolerance in tomato. However, it remains to be determined whether this modulation has a positive or negative effect on the overall plant metabolism. Further investigations are needed to fully elucidate the benefits of waterlogging pretreatment in this context.

Chemical profiling of Anthriscus cerefolium (L.) Hoffm., biological potential of the herbal extract, molecular modeling and KEGG pathway analysis.

This study was designed to investigate chemical composition and biological activities of the Anthriscus cerefolium methanolic extract. Chemical characterization of the extracts was performed by LC-HRMS/MS analysis. Antimicrobial activities of the extract were investigated on six bacteria and eight fungi while antioxidant activity was assessed by six different assays. Anti-enzymatic activity of the methanolic extract was tested on five enzymes associated with therapy of neurodegenerative diseases and diabetes mellitus type 2. Cytotoxic properties of the extract were tested on human immortalized keratinocytes (HaCaT) and tumor cell lines (SiHa, MCF7, HepG2). Anti-inflammatory activity of the extract was assessed on bacteria mediated inflammation model using HaCaT cell line. Molecular docking studies of enzymes and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis were performed. The results showed that the obtained extract was rich in phenolic compounds (a total of seventy-two were identified), with malonyl-1,4-O-dicaffeoylquinic acid and 3,5-O-dicaffeoylquinic acid dominating in the sample. The extract expressed antimicrobial, antioxidant, anti-enzymatic, cytotoxic and anti-inflammatory properties. The identified compounds demonstrated strong binding to the acetylcholinesterase (AChE) and to a lesser extent, to the butyrylcholinesterase (BChE), glucosidase, amylase, and modestly, to tyrosinase. KEGG pathway analysis has shown that the certain phenolic compounds may be related to anti-tumor, anti-inflammatory and anti-microbial activities of the extract. The data obtained suggest that phenolic compounds of the extract and their mixtures should be considered for future research as ingredients in pharmaceutical and nutraceutical formulations.

Antibacterial Activities of Agaricus bisporus Extracts and Their Synergistic Effects with the Antistaphylococcal Drug AFN-1252.

Agaricus bisporus, commonly known as the button mushroom, has attracted attention for its biological properties, including antimicrobial activities. Here, we evaluated the efficacy of ethanolic and acetonic extracts from white and brown A. bisporus against different bacterial strains, including antibiotic-resistant strains. Bioautography and principal component analysis identified the most active antibacterial compounds for each of the tested bacteria and indicated the main markers responsible for the strain-specific effects. In addition, the mushroom extracts demonstrated a synergistic impact when combined with the antistaphylococcal antibiotic AFN-1252.

Patterns of Genetic Variation of Nepeta nuda L. from the Central Balkans: Understanding Drivers of Chemical Diversity.

Nepeta nuda L., a notable medicinal species in the tradition of the Balkan region, is a rich source of bioactive iridoids and phenolics previously described as high-resolution taxonomical classifiers for the genus Nepeta. However, their potential in investigating intra-species differentiation is here described for the first time. The aim was to recognize the sources of natural chemical diversity and their association with the genetic variability both within and among N. nuda populations in the Central Balkans. Chemical diversity was assessed from methanol extracts and essential oils through untargeted and targeted metabolomics using state-of-the-art analytical tools, covering a broad spectrum of compounds that represent the N. nuda metabolome. We found that chemodiversity primarily resides within populations of N. nuda, and similar results were obtained at the DNA level using microsatellite markers. The low genetic and chemical differentiation of the studied N. nuda populations implies that their metabolomic profiles may be less influenced by geographic distance and variable environmental conditions within the Central Balkans, as they are under the pivotal control of their genetic backgrounds. Screening the distribution of the major bioactive compounds belonging to phenolics (phenolic acids and flavonoids) and iridoids (both aglycones and glycosylated forms), within and among N. nuda populations, is able to guarantee mass spectrometry-based tools for the selection of elite representative genotypes with practical importance. The knowledge acquired will allow us to delve deeper into the molecular background of N. nuda chemical diversity, which is the course of our further work.

A systematic UHPLC Q-ToF MS approach for the characterization of bioactive compounds from freeze-dried red goji berries (L. barbarum L.) grown in Serbia: Phenolic compounds and phenylamides.

The aim of this study was to identify and characterise different classes of bioactive compounds from freeze-dried red goji berries (RGB) grown in Serbia, using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC Q-ToF MS). In addition, this study aims to demonstrate the importance of applying the advanced UHPLC Q-ToF MS technique in the identification of various biocompounds. The analysis showed the presence of 28 phenolic compounds, 3 organic acids, and 26 phenylamides. The 2-O-ß-d-glucopyranosyl-l-ascorbic acid (AA-2ßG) was identified by UHPLC Q-ToF MS and quantified by standardised UHPLC-DAD method. Most of the compounds detected were derivatives of caffeic acid and ferulic acid, followed by quercetin derivatives. Among the phenylamides, several glucosylated caffeoyl and/or dihydrocaffeoyl derivatives of spermidine and spermine were characterized, confirming their recent characterization. Some glycosylated/non-glycosylated putrescine derivatives and caffeoyl-dihydrocaffeoyl-feruloyl spermidines were identified in goji berriesfor the first time. Their tentative structures and fragmentations were proposed.

Cytokinins enhance the metabolic activity of in vitro-grown catmint (Nepeta nuda L.).

The phytohormones cytokinins are essential mediators of developmental and environmental signaling, primarily during cell division and endophytic interactions, among other processes. Considering the limited understanding of the regulatory mechanisms that affect the growth and bioactivity of the medicinal plant Nepeta nuda (Lamiaceae), our study aimed to explore how cytokinins influence the plant's metabolic status. Exogenous administration of active cytokinin forms on in vitro N. nuda internodes stimulated intensive callus formation and de novo shoot regeneration, leading to a marked increase in biomass. This process involved an accumulation of oxidants, which were scavenged by peroxidases using phenolics as substrates. The callus tissue formed upon the addition of the cytokinin 6-benzylaminopurine (BAP) acted as a sink for sugars and phenolics during the allocation of nutrients between the culture medium and regenerated plants. In accordance, the cytokinin significantly enhanced the content of polar metabolites and their respective in vitro biological activities compared to untreated in vitro and wild-grown plants. The BAP-mediated accumulation of major phenolic metabolites, rosmarinic acid (RA) and caffeic acid (CA), corresponded with variations in the expression levels of genes involved in their biosynthesis. In contrast, the accumulation of iridoids and the expression of corresponding biosynthetic genes were not significantly affected. In conclusion, our study elucidated the mechanism of cytokinin action in N. nuda in vitro culture and demonstrated its potential in stimulating the production of bioactive compounds. This knowledge could serve as a basis for further investigations of the environmental impact on plant productivity.

Exploring traditional and modern approaches for extracting bioactive compounds from Ferulago trachycarpa.

For more than two millennia, Ferulago species have been revered as therapeutic herbs, maintaining their significance in present-day folk medicine practices. Therefore, the present study was conducted to investigate the phytochemical composition, inhibitory effects on metabolic enzymes, and possible therapeutic applications of F. trachycarpa, specifically focusing on its efficacy in diabetes management, anticholinergic effects, and antioxidant capabilities. The current investigation comprised an evaluation of a range of extracts acquired via conventional and modern methodologies, such as soxhlet (SOX), maceration (MAC) accelerated solvent extraction (ASE), homogenizer-assisted extraction (HAE), supercritical fluid extraction (SFE), microwave-assisted extraction (MW), and ultrasound-assisted extraction (UAE). Various techniques were employed to assess their antioxidant capacity and enzyme inhibition. Furthermore, the research utilized ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) to ascertain the principal phenolic compounds that are responsible for the antioxidant capacity observed in the various F. trachycarpa extracts. Among these, extracts from HAE, ASE, and MW revealed the most promise across all methodologies tested for their antioxidant potential. Furthermore, SFE and MAC extracts inhibited the most enzymes, including cholinesterases, tyrosinase, α -amylase, and α -glycosidase, indicating their potential as efficient natural treatments for several health-related issues.

A novel source of biologically active compounds - The leaves of Serbian herbaceous peonies.

In order to gain further insight into how various extraction techniques (maceration, microwave-, and ultrasound-assisted extractions) affect the chemical profile and biological activities of leaf extracts from Paeonia tenuifolia L., Paeonia peregrina Mill., and Paeonia officinalis L., this research was performed. The targeted chemical characterization of the extracts was achieved using the Ultra-High-Performance-Liquid-Chromatography-Linear-Trap-Mass-Spectrometry OrbiTrap instrumental technique, while Fourier Transform Infrared Spectroscopy was conducted to investigate the structural properties of the examined leaf extracts. According to the results, the species P. officinalis, Bozurna locality as the origin of the plant material, and microwave-assisted extraction produced the maximum polyphenol yield, (491.9 ± 2.7 mg gallic acid equivalent (GAE)/mL). The ethanolic extracts exhibited moderate antioxidant activity as evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) and phosphomolybdenum tests. With MIC values of 0.125 mg/mL, the leaf extracts produced by ultrasound-assisted extraction and maceration (Deliblato sands and Bogovo gumno) had the best antibacterial activity against Pseudomonas aeruginosa and Salmonella Typhimurium. Ultrasound-assisted extraction has proven to produce the most effective antimicrobial agents. Inhibitory potential towards glucosidase, amylase, cholinesterases, and tyrosinase was evaluated in enzyme inhibition assays and molecular docking simulations. Results show that leaves of P. tenuifolia L. obtained by ultrasound-assisted extraction had the highest acetylcholinesterase and butyrylcholinesterase inhibitory activity. Namely, the complexity of the polyphenol structures, the extraction method, the used locality, and the different mechanisms of the reactions between bioactives from leaf extracts and other components (free radicals, microorganisms, and enzymes) are the main factors that influence the results of the antioxidant tests, as well as the antibacterial and enzyme-inhibitory activities of the extracts. Hydroxymethyl-phenyl pentosyl-hexoside and acetyl-hydroxyphenyl-hexoside were the first time identified in the leaf extract of the Paeonia species. Due to their proven biological activities and the confirmed existence of bioactive compounds, leaf extracts may find use in foodstuffs, functional foods, and pharmaceutical products.

Phytochemical Analysis, Biological Activities, and Molecular Docking Studies of Root Extracts from Paeonia Species in Serbia.

Without being aware of their chemical composition, many cultures have used herbaceous peony roots for medicinal purposes. Modern phytopreparations intended for use in human therapy require specific knowledge about the chemistry of peony roots and their biological activities. In this study, ethanol-water extracts were prepared by maceration and microwave- and ultrasound-assisted extractions (MAE and UAE, respectively) in order to obtain bioactive molecules from the roots of Paeonia tenuifolia L., Paeonia peregrina Mill., and Paeonia officinalis L. wild growing in Serbia. Chemical characterization; polyphenol and flavonoid content; antioxidant, multianti-enzymatic, and antibacterial activities of extracts; and in vitro gastrointestinal digestion (GID) of hot water extracts were performed. The strongest anti-cholinesterase activity was observed in PT extracts. The highest anti-ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical potential was observed in PP extracts, whereas against DPPH (2,2-diphenyl-1-picrylhydrazyl radicals), the best results were achieved with PO extracts. Regarding antibacterial activity, extracts were strongly potent against Bacillus cereus. A molecular docking simulation was conducted to gather insights into the binding affinity and interactions of polyphenols and other Paeonia-specific molecules in the active sites of tested enzymes. In vitro GID of Paeonia teas showed a different recovery and behavior of the individual bioactives, with an increased recovery of methyl gallate and digallate and a decreased recovery of paeoniflorin and its derivatives. PT (Gulenovci) and PP (Pirot) extracts obtained by UAE and M were more efficient in the majority of the bioactivity assays. This study represents an initial step toward the possible application of Paeonia root extracts in pharmacy, medicine, and food technologies.

UHPLC-MS/MS analysis, cytotoxic, enzyme inhibition, and antioxidant properties of Lantana camara L. extracts obtained by conventional and nonconventional methods.

Lantana camara is widely known as a garden plant, but its use for various medicinal purposes is widespread in traditional medicine. In the frame of this study, L. camara was subjected to several different extraction techniques, including supercritical carbon dioxide extraction, accelerated solvent extraction (ASE), homogenizer-assisted extraction, microwave-assisted extraction, ultrasound-assisted extraction, maceration, and Soxhlet extraction. The investigation encompasses the analysis of the chemical composition alongside assessments of biological activities, such as antioxidant and enzyme-inhibition potential and cytotoxicity of the obtained extracts. The obtained results showed that the extract obtained by accelerated-solvent extraction was the richest in the content of total phenols and of individual compounds. Of the 17 components identified in total, hispidulin was detected in the highest concentration (5.43-475.97 mg/kg). In the antioxidant assays, the extracts obtained by accelerated-solvent and microwave extraction possessed the highest level of antioxidant and antiradical protection. All obtained extracts showed enzyme-inhibitory action on amylase, glucosidase, tyrosinase, and cholinesterase, showing a high potential for application against diseases induced by excessive activity of these enzymes. Cytotoxic analysis was performed on normal and tumor cells, whereby the obtained IC50 values were in the range of 7.685-79.26 µg/mL, showing the high cytotoxicity of the obtained extracts. Using Z score analysis, ASE resulted in an optimal combination of tested quality characteristics of the L. camara extracts.

Potential application of green extracts rich in phenolics for innovative functional foods: natural deep eutectic solvents as media for isolation of biocompounds from berries.

The health-promoting effects of berries have attracted attention due to the possible application of their extracts as functional ingredients in food products. Natural deep eutectic solvents (NADESs) are a new generation of environmentally friendly solvents for the extraction of natural products, and they are green alternatives to organic solvents, and they can improve the solubility, stability, and bioavailability of isolated biocompounds. In this study, an efficient eco-friendly method was used for the extraction of phenolic compounds from different berries: chokeberries, blueberries, and black goji berries with a range of eutectic solvents consisting of hydrogen bond acceptors (HBAs) such as choline chloride, L-proline, L-glycine, and L-lysine and hydrogen bond donors (HBDs) such as malic, citric, tartaric, lactic and succinic acids, glucose and glycerol. The obtained results indicated the ability of NADESs towards selective extraction of phenolics; the eutectic system choline chloride : malic acid showed selective extraction of anthocyanins, while choline chloride : glycerol and choline chloride : urea showed selectivity towards flavonoids and phenolic acids. The methodology for screening of the NADES extraction performance, which included chromatographic profiling via high-performance thin layer chromatography combined with chemometrics and spectrophotometric essays, allowed effective assessment of optimal eutectic solvents for isolation of different groups of phenolics. Great antioxidant and antimicrobial activities of extracts, along with the green nature of eutectic solvents, enable NADES berry extracts to be used as "green-labelled" functional foods or ingredients.

Broccoli, Amaranth, and Red Beet Microgreen Juices: The Influence of Cold-Pressing on the Phytochemical Composition and the Antioxidant and Sensory Properties.

The aim of this study was to analyze in detail the phytochemical composition of amaranth (AMJ), red beet (RBJ), and broccoli (BCJ) microgreens and cold-pressed juices and to evaluate the antioxidant and sensory properties of the juices. The results showed the presence of various phenolic compounds in all samples, namely betalains in amaranth and red beet microgreens, while glucosinolates were only detected in broccoli microgreens. Phenolic acids and derivatives dominated in amaranth and broccoli microgreens, while apigenin C-glycosides were most abundant in red beet microgreens. Cold-pressing of microgreens into juice significantly altered the profiles of bioactive compounds. Various isothiocyanates were detected in BCJ, while more phenolic acid aglycones and their derivatives with organic acids (quinic acid and malic acid) were identified in all juices. Microgreen juices exhibited good antioxidant properties, especially ABTS•+ scavenging activity and ferric reducing antioxidant power. Microgreen juices had mild acidity, low sugar content, and good sensory acceptability and quality with the typical flavors of the respective microgreen species. Cold-pressed microgreen juices from AMJ, RBJ, and BCJ represent a rich source of bioactive compounds and can be characterized as novel functional products.

Extractions of aerial parts of Hippomarathrum scabrum with conventional and green methodologies: Chemical profiling, antioxidant, enzyme inhibition, and anti-cancer effects.

Hippomarathrum scabrum L. is an endemic medicinal plant in Turkey; however, there have been few studies investigating the phytochemistry and biological properties of these plants has not been investigated. The aim of this work is to determine the chemical composition of different extracts (extracts obtained by using supercritical carbon dioxide extraction, accelerated solvent extraction, homogenizer-assisted extraction, microwave-assisted extraction, and ultrasound-assisted extraction from Hippomarathrum scabrum L., and evaluate their biological properties. The analysis revealed that 5-O-caffeoylquinic acid, rutin, and isorhamnetin 3-O-rutinoside were the main bioactive compounds. The extract obtained by accelerated extraction contains the highest concentration of 5-O-Caffeoylquinic acid (7616.74 ± 63.09 mg/kg dry extract) followed by the extract obtained by homogenizer-assisted extraction (6682.53 ± 13.04 mg/kg dry extract). In antioxidant tests, all extracts expressed significant antioxidant activity. Also, cytotoxic and anticancer effects of these plant extracts were detected in the human prostate cancer cell line. Intrinsic apoptotic genes were up-regulated and anti-apoptotic genes were down-regulated in human prostate cancer cells after inhibition concentration dose treatment. The findings are promising, and suggest the use of these plant extracts could be used as natural sources with different biological activities, as well as anticancer agents.

Paeonia peregrina Mill Petals as a New Source of Biologically Active Compounds: Chemical Characterization and Skin Regeneration Effects of the Extracts.

Paeonia peregrina Mill. is a perennial herbaceous plant species, known for the medicinal value of all of its plant parts, although the chemical composition of the petals is unknown. This study aimed to determine the chemical fingerprint of the petals and also establish the optimal extraction parameters, extraction medium, and extraction method for petals collected from different localities in Serbia. The optimization was performed in order to acquire extracts that are rich in the contents of total polyphenol content (TPC) and total flavonoid content (TFC), and also exhibit strong antioxidant activity. In addition, the influence of the extracts on several human skin pathogens was evaluated, as well as their ability to aid wound closure and act as anti-inflammatory agents. Both the extraction medium and the applied technique significantly influenced the skin-beneficial biological activities, while methanol proved to be a more favorable extraction medium. In conclusion, the extraction conditions that yielded the extract with the richest phenolic content with satisfactory biological potential varied between the assays, while the most promising locality in Serbia for the collection of P. peregrina petals was Pancevo (South Banat).

Monofloral Corn Poppy Bee-Collected Pollen-A Detailed Insight into Its Phytochemical Composition and Antioxidant Properties.

The aim of this study was to compile a detailed phytochemical profile and assess the antioxidant properties of bee-collected pollen (PBP) obtained from corn poppy (Papaver rhoeas L.) plants. To achieve this, a lipid fraction was prepared for quantifying fatty acids using GC-FID. Extractable and alkaline-hydrolysable PBP fractions (obtained from a defatted sample) were used to determine the qualitative and quantitative profiles of phenolic compounds, phenylamides and alkaloids using UHPLC/Q-ToF-MS. Additionally, various spectrophotometric assays (TAC, FRP, CUPRAC, DPPH⦁) were conducted to evaluate the antioxidant properties. Phenolic compounds were more present in the extractable fraction than in the alkaline-hydrolysable fraction. Luteolin was the predominant compound in the extractable fraction, followed by tricetin and various derivatives of kaempferol. This study presents one of the first reports on the quantification of tricetin aglycone outside the Myrtaceae plant family. The alkaline-hydrolysable fraction exhibited a different phenolic profile, with a significantly lower amount of phenolics. Kaempferol/derivatives, specific compounds like ferulic and 5-carboxyvanillic acids, and (epi)catechin 3-O-gallate were the predominant compounds in this fraction. Regarding phenylamides, the extractable fraction demonstrated a diverse range of these bioactive compounds, with a notable abundance of different spermine derivatives. In contrast, the hydrolysable fraction contained six spermine derivatives and one spermidine derivative. The examined fractions also revealed the presence of seventeen different alkaloids, belonging to the benzylisoquinoline, berberine and isoquinoline classes. The fatty-acid profile confirmed the prevalence of unsaturated fatty acids. Furthermore, both fractions exhibited significant antioxidant activity, with the extractable fraction showing particularly high activity. Among the assays conducted, the CUPRAC assay highlighted the exceptional ability of PBP's bioactive compounds to reduce cupric ions.

Natural extracts against agricultural pathogens: A case study of Celtis australis L.

Plant extracts and other plant products have been used as an alternative to synthetic fungicides or an additional way to reduce their use. The choice of plant extracts and their application depends on their functional characteristics, availability, cost-effectiveness, and their impact on phytopathogens, and also on the environment. Therefore, the present study aims to assess the potential of Celtis australis methanolic extracts as source of compounds with antifungal activity. Methanolic extracts prepared from leaves and unripe mesocarps of C. australis collected from different localities of Montenegro (Podgorica-PG, Donja Gorica-DG, and Bar-BR) were evaluated for their phenolic compounds' composition as well as antifungal and cytotoxic properties. Obtained results revealed that extracts contain various bioactive constituents including phenolic acids, flavonoids, and their derivatives. The predominant phenolic acid was ferulic acid, identified in leaf samples from DG (187.97 mg/100 g dw), while isoorientin was the most abundant phenolic compound found in all examined samples. Regarding antifungal potential of the tested samples, all but one (prepared from mesocarp BR) possessed higher activity than Previcur, a commercial systemic fungicide intended to control seedlings. In vitro studies on HaCaT cell line showed that the extracts had no toxic effect toward the tested cell line. These results lead to the conclusion that methanolic extracts of C. australis can become an alternative to the use of synthetic fungicides in agriculture. Those extracts represent natural biodegradable fungicides and enable more efficient control of pathogenic fungi.

Leaves micromorphology, chemical profile, and bioactivity of in vitro-propagated Nepeta cyrenaica (Lamiaceae).

INTRODUCTION: The endemic species Nepeta cyrenaica Quézel & Zaffran, native to northeastern Libya, is valued as an important honey-bearing plant. OBJECTIVES: This study was aimed to examine the micromorphology, phytochemistry, and bioactivity of in vitro-propagated N. cyrenaica for the first time. MATERIALS AND METHODS: The leaf indumentum was examined using light and scanning electron microscopy and further characterised for histochemistry. The chemical composition of essential oil (EO) was performed using GC-MS analysis, while dichloromethane (DCM), methanol (ME), ethanol (ET), and aqueous (AQ) extracts were analysed using qualitative and quantitative LC/MS analyses. The antioxidant activities of EO and extracts were assessed using three parallel assays, while enzyme-inhibiting effects were evaluated against four enzymes. RESULTS: The leaves bear various types of glandular trichomes, with lipophilic secretion predominating. The main EO component of EO was 1,8-cineole. A considerable number of phenolics and iridoids were tentatively identified in the ME extract. Quantitative LC/MS analysis confirmed that ferulic acid, rosmarinic acid, and epigallocatechin gallate were present in the highest amount in the extracts, in which three iridoids were also quantified. Although the ME extract contained the highest amount of polyphenolics and iridoids, the DCM extract showed the best overall biological potential. Additionally, EO exerted the strongest acetylcholinesterase and tyrosinase inhibition. CONCLUSION: This study demonstrated that the endemic N. cyrenaica can be efficiently grown under in vitro conditions, where it develops various glandular trichomes that are thought to secrete and/or accumulate bioactive compounds with valuable medicinal potential.

Solvent System-Guided Extraction of Centaurium spicatum (L.) Fritch Provides Optimized Conditions for the Biological and Chemical Characteristics of the Herbal Extracts.

Spiked centaury (Centaurium spicatum) is a well-known medicinal plant from the Mediterranean region with various bioactivities, but there are no studies addressing the use of different solvent systems to improve its pharmacological potential. Nine extraction procedures were adapted to study the effects of solvent composition on the content of bioactive compounds in C. spicatum extracts and on corresponding bioactivities. Targeted metabolomics was performed to obtain information on the chemical composition of extracts. Ethanol-water-based extraction procedures were the most efficient in isolating polyphenols, while less polar butanol extract contained the highest amount of iridoids. Antioxidant potential analysis revealed stronger activity in extracts with higher polyphenol content. Bacillus cereus and Staphylococus aureus were designated as the most sensitive bacterial strains to the activity of extracts, while among the micromycetes tested, Penicillium funiculosum was the most susceptible strain. Butanol extract showed antivirulence potential on Candida albicans morphological transition from yeast to hyphal form, and selected extracts were effective against biofilm formation in two Candida species. All the extracts tested in this study showed no cytotoxic activity to immortalize human skin keratinocyte cell line (HaCaT), whereas extracts obtained by ethanol-water extraction stand out for their potent wound healing effects. Moreover, the influence of the extraction solvent system on various bioactivities of C. spicatum is reported herein for the first time. Overall, the results presented in this study promote the use of C. spicatum as a source of natural products with potential antioxidant, wound healing, and antimicrobial applications that are potentially safe for human use.

Recovery of Polyphenolic Compounds and Vitamins from the Stinging Nettle Leaves: Thermal and Behavior and Biological Activity of Obtained Extracts.

Stinging nettle (SN) is an extraordinary plant from the Urticaceae botanical family. It is well-known and widely used in food and folk medicine to treat different disorders and diseases. This article aimed to study the chemical composition of SN leaves extracts, i.e., polyphenolic compounds and vitamins B and C, because many studies ascribed high biological potency to these compounds and their significance in the human diet. Besides the chemical profile, the thermal properties of the extracts were studied. The results confirmed presence of many polyphenolic compounds and vitamins B and C. It also showed that the chemical profile closely correlated with the applied extraction technique. The thermal analysis showed that analyzed samples were thermally stable up to about 160 °C. Thermal degradation of samples UAE, MAE, and MAC took place in four steps, and sample SE in three steps. Altogether, results confirmed the presence of health-beneficial compounds in stinging nettle leaves and indicated the possible application of its extract in pharmaceutical and food industries as both a medicinal and food additive.

Variation in the chemical profiles of three foxglove species in the central Balkans.

The aim of this study was to determine intra- and interspecies variation in the qualitative and quantitative composition of methanol-soluble metabolites in the leaves of three Digitalis species (D. lanata, D. ferruginea, and D. grandiflora) from the central Balkans. Despite the steady use of foxglove constituents for human health as valuable medicinal products, populations of the genus Digitalis (Plantaginaceae) have been poorly investigated to describe their genetic and phenetic variation. Following untargeted profiling using UHPLC-LTQ Orbitrap MS, by which we identified a total of 115 compounds, 16 compounds were quantified using the UHPLC(-)HESI-QqQ-MS/MS approach. In total, 55 steroid compounds, 15 phenylethanoid glycosides, 27 flavonoids, and 14 phenolic acid derivatives were identified across the samples with D. lanata and D. ferruginea showing a great similarity, while 15 compounds were characteristic only for D. grandiflora. The phytochemical composition of methanol extracts, considered here as complex phenotypes, are further examined along multiple levels of biological organization (intra- and interpopulation) and subsequently subjected to chemometric data analysis. The quantitative composition of the selected set of 16 chemomarkers belonging to the classes of cardenolides (3 compounds) and phenolics (13 compounds) pointed to considerable differences between the taxa studied. D. grandiflora and D. ferruginea were found to be richer in phenolics as compared to cardenolides, which otherwise predominate in D. lanata over other compounds. PCA revealed lanatoside C, deslanoside, hispidulin, and p-coumaric acid to be the main compounds contributing to the differences between D. lanata on one side and D. grandiflora and D. ferruginea on the other, while p-coumaric acid, hispidulin, and digoxin contribute to the diversification between D. grandiflora and D. ferruginea. However, quantitative variation in the metabolite content within species was faint with mild population diversification visible in D. grandiflora and particularly in D. ferruginea. This pointed to the highly conserved content and ratio of targeted compounds within the analyzed species, which was not severely influenced by the geographic origin or environmental conditions. The presented metabolomics approach might have, along with morphometrics and molecular genetics studies, a high information value for further elucidation of the relationships among taxa within the genus Digitalis.