Cichorium intybus L. "hairy" roots as a rich source of antioxidants and anti-inflammatory compounds.

The present study aimed to determine the bioactive profile of various extracts of Cichorium intybus L. "hairy" roots. In particular, the total content of flavonoids as well as the reducing power, antioxidant and anti-inflammatory activity of the aqueous and ethanolic (70%) extracts were evaluated. The total content of flavonoids the ethanolic extract of the dry "hairy" root reached up to 121.3 mg (RE)/g, which was twofold greater than in the aqueous one. A total of 33 diverse polyphenols were identified by the LC-HRMS method. The experimental results showed a high amount of gallic (6.103 ± 0.008 mg/g) and caffeic (7.001 ± 0.068 mg/g) acids. In the "hairy" roots, the presence of rutin, apigenin, kaempferol, quercetin, and its derivatives was found in concentrations of 0.201±0.003 - 6.710±0.052 mg/g. The broad spectrum of pharmacological activities (antioxidant, anti-inflammatory, antimutagenic, anticarcinogenic, etc.) of the key flavonoids identified in the chicory "hairy" root extract was predicted by the General Unrestricted Structure-Activity Relationships algorithm based on in the substances detected in the extract. The evaluation of the antioxidant activity showed that the EC50 values of the ethanol and the aqueous extracts were 0.174 and 0.346 mg, respectively. Thus, the higher ability of the ethanol extract to scavenge the DPPH radical was observed. The calculated Michaelis and inhibition constants indicated that the ethanolic extract of C. intybus "hairy" roots is an efficient inhibitor of soybean 15-Lipoxygenase activity (IC50 = 84.13 ± 7.22 µM) in a mixed mechanism. Therefore, the obtained extracts could be the basis of herbal pharmaceuticals for the therapy of human diseases accompanied by oxidative stress and inflammation, including the pandemic coronavirus disease COVID-19.

Effect of Priestia endophytica on the metabolites accumulation in chicory and lettuce plants cultivated in vitro.

The influence of the culture medium without bacterial cells, obtained after the cultivation of endophytic bacteria Priestia endophytica UCM B-5715, on the growth and synthesis of some metabolites in lettuce and chicory seedlings under in vitro conditions was studied. Bacteria were cultivated in liquid LB medium at 37 ºC for 24 h with periodic stirring. The culture fluid was separated from the cell biomass. For preparing the test solution, the supernatant was sterilized by filtration through a filter with a pore diameter of 0.2 µm (Sartorius, Minisart) and diluted with sterile distilled water. The 20% culture fluid (30 µl/plant) was applied to 3-day-old seedlings. In 28 days root and shoot weights of treated chicory plants were 54.3 ± 6.9 and 260.0 ± 20.2 mg, respectively (8.0 ± 0.7 and 91.4 ± 7.0 mg for the control plants). Total flavonoid content and antioxidant activity increased only in chicory plants after the addition of the test solution. Significant changes in the metabolism of treated plants were detected. In the treated lettuce plants asparagine content increased compared to the control (90 vs 22 µg/g, p < 0.1). The median content of fructose was also higher in treated lettuce and chicory plants (1469 vs 73 µg/g and 2278 vs 1051 µg/g). Therefore, the use of culture fluid obtained after the cultivation of P. endophytica UСM B-5715 stimulated the growth of lettuce and chicory plants, affecting the synthesis of some compounds in single-treated plants. These results indicate the potential of compounds excreted during bacterial growth to create natural growth stimulators.

Morphology-Controlled Green Synthesis of Magnetic Nanoparticles Using Extracts of 'Hairy' Roots: Environmental Application and Toxicity Evaluation.

Magnetic nanoparticles (MNPs) were "green" synthesized from a FeCl3/FeSO4/CoCl2 mixture using ethanolic extracts of Artemisia tilesii Ledeb 'hairy' roots. The effect of chemical composition and reducing power of ethanolic extracts on the morphology, size destribution and other features of obtained MNPs was evaluated. Depending on the extract properties, nanosized magnetic materials of spherical (8-11 nm), nanorod-like (15-24 nm) and cubic (14-24 nm) shapes were obtained via self-assembly. Microspherical MNPs composed of nanoclusters were observed when using extract of the control root line in the synthesis. Polyhedral magnetic nanoparticles with an average size of ~30 nm were formed using 'hairy' root ethanolic extract without any additive. Studied samples manifested excellent magnetic characteristics. Field-dependent magnetic measurements of most MNPs demonstrated a saturation magnetization of 42.0-72.9 emu/g with negligible coercivity (∼0.02-0.29 emu/g), indicating superparamagnetic behaviour only for solids with a magnetite phase. The synthesized MNPs were minimally aggregated and well-dispersed in aqueous medium, probably due to their stabilization by bioactive compounds in the initial extract. The nanoparticles were tested for magnetic solid-phase extraction of copper (Cu), cadmium (Cd) and arsenic (As) pollutants in aqueous solution, followed by ICP-OES analysis. The magnetic oxides, mainly magnetite, showed high adsorption capacity and effectively removed arsenic ions at pH 6.7. The maximum adsorption capacity was ~150 mg/g for As(III, V) on the selected MNPs with cubic morphology, which is higher than that of previously reported adsorbents. The best adsorption was achieved using Fe3O4-based nanomaterials with low crystallinity, non-spherical form and a large number of surface-localized organic molecules. The phytotoxicity of the obtained MNPs was estimated in vitro using lettuce and chicory as model plants. The obtained MNPs did not exhibit inhibitory activity. This work provides novel insights on the morphology of "green" synthesized magnetic nanoparticles that can be used for applications in adsorption technologies.

Study of Superoxide Dismutase Activity in Long-Term Cultivated Artemisia and Althaea "hairy" Roots.

The aim of the study was to evaluate the long-term effect of Agrobacterium rhizogenes genes transfer on plant antioxidant system by the study of superoxide dismutase (SOD) activity in "hairy" roots of Artemisia and Althaea spp plants. PCR analyses revealed stability of the transformation and presence of bacterial rol B and rol C genes in the "hairy" roots after 4-6 years from the transformation event. SOD activity in the roots of untransformed in vitro cultivated plants used for the initiation of "hairy" roots growth was in the range of 45.8 ± 8.7 U/µg (Althaea officinalis) and 275 ± 97.1 U/µg (Artemisia ludoviciana). After a long-term in vitro cultivation more than half of tested "hairy" root lines (54%) showed a significant increase in SOD activity values compared to untransformed roots. The highest SOD activity values of "hairy" root lines (24-fold increase) were founded in A. officinalis (1105 ± 174 U/µg) and A. dracunculus (1356 ± 402 U/µg). The increase of the activity was found also in "hairy" roots of A. vulgaris (up to 375 ± 28.2 U/µg, sevenfold increase), A. ludoviciana (1001 ± 191 U/µg, 3.6-fold increase), and A. tilesii (438 ± 104 U/µg, 1.6-fold increase). The results of our study indicate that transformation by wild-type A. rhizogenes not harboring any foreign genes implementing in SOD activity regulation can often stably activate plant antioxidant enzyme system. This effect, observed in the "hairy" roots of five plant species in 4-6 years of the transformation event, obviously, should be taken into account in works aimed at creating transgenic plants by Agrobacterium-mediated transformation.

Biosynthesis of magnetite and cobalt ferrite nanoparticles using extracts of "hairy" roots: preparation, characterization, estimation for environmental remediation and biological application.

The "green" synthesis of magnetite and cobalt ferrite nanoparticles (Fe3O4-NPs and CoFe2O4-NPs) using extracts of Artemisia annua L "hairy" roots was proposed. In particular, the effect and role of important variables in the 'green' synthesis process, including the metal-salt ratio, various counter ions in the reaction mixture, concentration of total flavonoids and reducing power of the extract, were evaluated. The morphology and size distribution of the magnetic nanoparticles (MNPs) depended on the metal oxidation state and ratio of Fe(iii) : Fe(ii) in the initial reaction mixture. MNPs obtained from divalent metal salts in the reaction mixture were non-uniform in size with high aggregation level. Samples obtained by the FeCl3/FeSO4 mixture with a ratio of Fe(iii) : Fe(ii) = 1 : 2 showed an irregular shape of the nanoparticles and high aggregation level. MNPs obtained by the FeCl3/FeSO4/CoCl2 mixture showed a regular shape with slight aggregation, and were in the nanosize range (10-17 nm). Thus, this mixture as a metal-precursor was used for MNP biosynthesis in the subsequent experiments. The XRD data showed that the magnetic specimens contained mainly spinel type phase. The data of EDX and XPS analysis indicated that the product of the "green" synthesis was magnetite with some impurities, owing to the obtained ratio of Fe : O being similar to the theoretical atomic ratio of magnetite (3 : 4). The Fe3O4-NP samples were superparamagnetic with high magnetization (until 68 emu g-1). The Co-containing MNPs demonstrated low ferromagnetic properties. The MNPs with pure magnetite phase, very good magnetization and uniform size distribution (ca. 12-14 nm) were prepared by the "hairy" root extract characterized by the highest amount of total flavonoids. According to the FTIR data, the synthesized Fe3O4-NPs had a core-shell like structure, in which the core was composed of Fe3O4, and the shell was formed by bioactive molecules. The presence of several organic compounds (such as flavonoids or carboxylic acids) plays a key role in the suppression of Fe3O4-NP aggregation without addition of a stabilizing agents. Synthesized Fe3O4-NP samples effectively removed Cu(ii) and Cd(ii) with the maximum adsorption capacity, reaching 29.9 mg g-1 and 33.5 mg g-1, respectively. It is probable that the presence of organic components in extracts plays an important role in the adsorption properties of biosynthesised MNPs. The obtained MNPs were successfully applied to the removal of heavy metal ions in the environmental water samples. Fe3O4-NPs also negatively affected plant growth in the case of using "hairy" roots as a test model, and the greatest inhibitory activity (99.56 wt%) was possessed by MNPs with high magnetic properties.

Agrobacterium rhizogenes-mediated transformation enhances the antioxidant potential of Artemisia tilesii Ledeb.

Plants belonging to the genus Artemisia L. have been used for medicinal purposes since ancient times. These aromatic plants produce and accumulate a wide range of potent secondary metabolites, many of which have shown antioxidant, antiparasitic, antimicrobial, anti-inflammatory, and even anticancer activities. Enhanced biosynthesis of these compounds is a prerequisite for comprehensive studies of their therapeutic properties and cost-efficient use. Transformation of plants with Agrobacterium rhizogenes native root locus (rol) genes is a promising approach to increase the biosynthesis of plant secondary metabolites. The aim of the present study was to evaluate the effects of A. rhizogenes-mediated transformation on the flavonoid contents in hairy roots of medicinal herb A. tilesii Ledeb. Transgenic A. tilesii hairy root lines were analyzed for stable integration of the rolB and rolC transgenes into the plant genome, total flavonoid contents, antioxidant activities of extracts, and the spatiotemporal expression of two flavonoid biosynthetic genes, phenylalanine ammonialyase (PAL) and chalcone synthase (CHS). The flavonoid contents of A. tilesii directly correlated with the antiradical activity and reducing power of their respective lines, with the greatest antioxidant activity found in the plants with the highest level of total flavonoids. Furthermore, all hairy root lines demonstrated altered expression of plant native PAL and CHS genes. Most importantly, A. rhizogenes-mediated transformation enhanced the biosynthesis of natural antioxidants in A. tilesii, producing almost twice the amount of flavonoids than controls. These findings provide an opportunity for the identification of the bioactive molecules in A. tilesii extracts and their potential health benefits.

'Hairy' root extracts as source for 'green' synthesis of silver nanoparticles and medical applications.

The research was focused on the synthesis of silver nanoparticles (AgNPs) using extracts from the "hairy" root cultures of Artemisia tilesii Ledeb. and Artemisia annua L. The effect of operational parameters such as type of solvent, temperature of extraction, flavonoids concentration, and reducing power of the wormwood "hairy" root extracts on the particle size and yield of the resultant nanoparticles is reported for the first time. From the studied solvents, a water-ethanol mixture with a concentration of 70 vol% was found to be the best for the extraction of flavonoids from all "hairy" root cultures. The total flavonoid contents in A. annua and A. tilesii "hairy" root extracts were up to 80.0 ± 0.9 and 108 ± 4.4 mg RuE per g DW, respectively. Identification of flavonoids was confirmed by UPLC-ESI-UHR-Qq-TOF-MS analysis. Luteolin-7-ß-d-glucopyranosid, isorhamnetin 3-O-glucoside, baicalein-7-O-glucuronide, apigenin-7-O-glucoside, quercetin, sitosterol, caffeoylquinic, galic, chlorogenic and caffeic acids were founded in the extracts. These extracts demonstrated the high reducing activities. Spherical, oval and triangular nanoparticles with effective sizes of 5-100 nm were observed. The TEM data revealed great differences in the shapes of NPs, obtained from the extracts from different root clones. The clustered and irregular NPs were found in the case of using ethanol extracts, mostly aggregated and having the size of 10-50 nm. The sizes of AgNPs decreased to 10-30 nm in the case of using aqueous extracts obtained at 80 °C. Biosynthesized AgNPs showed surface plasmon resonance in the range of 400-450 nm. The antimicrobial activity of the as-produced AgNPs was studied by disc diffusion method on Gram-positive (Staphylococcus aureus ATCC 25923 (F-49)), Gram-negative (Pseudomonas aeruginosa ATCC 27853 (F-51), Escherichia coli ATCC 25922 (F-50)) and Candida albicans ATCC 88-653 strains. It was found that the nanoparticles in some cases possessed the greater ability to inhibit microorganism growth compared to 1 mM AgNO3 solution. The colloidal solutions of the obtained AgNPs were stable in the dark for 12 months at room temperature. Thus, the A. annua and A. tilesii "hairy" root extracts can be used for obtaining of bioactive AgNPs.

Flavonoid content and antioxidant activity of Artemisia vulgaris L. "hairy" roots.

We investigated the effect of Agrobacterium rhizogenes-mediated transformation on antioxidant activity of Artemisia vulgaris "hairy" roots. It appeared that transformation may increase flavonoid content as well as DPPH-scavenging activity and ability to reduce Fe3+ as compared to the non-transformed plants. Some "hairy" roots accumulated flavonoids up to 73.1 ± 10.6 mg RE/g DW (while the amount of flavonoids in the leaves of non-transformed plants was up to 49.4 ± 5.0 mg RE/g DW). DPPH-scavenging activity of some "hairy" root lines was 3-3.8 times higher than such one of the roots of the control plants. The Fe3+-reducing power of most transgenic root extracts exceeded such power of the extracts of the roots of the control plants. The decrease in SOD activity was found in the most "hairy" root lines compared to the control roots. The increase of flavonoid content correlated with the increase of ability of extracts to scavenge DPPH*- radical and Fe3+ - reducing power. No correlation between SOD activity of extracts and concentration of flavonoids was found (p ≥ 0.2).Thus, transformation has led to the alteration in flavonoid accumulation and antioxidant activity in A. vulgaris "hairy" roots. Transgenic roots with high-antioxidant properties can be selected after A. rhizogenes-mediated transformation.

Study of artemisinin and sugar accumulation in Artemisia vulgaris and Artemisia dracunculus "hairy" root cultures.

We studied the effect of genetic transformation on biologically active compound (artemisinin and its co-products (ART) as well as sugars) accumulation in Artemisia vulgaris and Artemisia dracunculus "hairy" root cultures. Glucose, fructose, sucrose, and mannitol were accumulated in A. vulgaris and A. dracunculus "hairy" root lines. Genetic transformation has led in some cases to the sugar content increasing or appearing of nonrelevant for the control plant carbohydrates. Sucrose content was 1.6 times higher in A. vulgaris "hairy" root lines. Fructose content was found to be 3.4 times higher in A. dracunculus "hairy" root cultures than in the control roots. The accumulation of mannitol was a special feature of the leaves of A. vulgaris and A. dracunculus control roots. A. vulgaris "hairy" root lines differed also in ART accumulation level. The increase of ART content up to 1.02 mg/g DW in comparison with the nontransformed roots (up to 0.687 mg/g DW) was observed. Thus, Agrobacterium rhizogenes-mediated genetic transformation can be used for obtaining of A. vulgaris and A. dracunculus "hairy" root culture produced ART and sugars in a higher amount than mother plants.