Characterization of Secondary Metabolites of Leaf Buds from Some Species and Hybrids of Populus by Gas Chromatography Coupled with Mass Detection and Two-Dimensional High-Performance Thin-Layer Chromatography Methods with Assessment of Their Antioxidant Activity.

Poplars provide medicinal raw plant materials used in pharmacy. Leaf buds are one of the herbal medicinal products collected from poplars, having anti-inflammatory and antiseptic properties, but there are no quality standards for their production and there is a need to determine their botanical sources. Therefore, the chemical compositions of the leaf buds from four species and varieties of poplars, Populus balsamifera, P. × berolinensis, P. × canadensis 'Marilandica', and P. wilsonii were investigated and compared using gas chromatography coupled with mass detection (GC-MS) and two-dimensional high-performance thin-layer chromatography (2D-HPTLC) in order to search for taxa characterized by a high content of biologically active compounds and with a diverse chemical composition that determines their therapeutic effects. The presence of 163 compounds belonging to the groups of flavonoids, phenolic acids derivatives, glycerides, and sesquiterpenes was revealed. Moreover, the conditions for the separation and identification of biologically active compounds occurring in analyzed leaf buds using 2D-HPTLC were optimized and used for metabolomic profiling of the studied poplars, enabling their fast and simple botanical identification. The total phenolic (TPC) and flavonoid (TFC) contents of examined extracts were determined and their antioxidant capacities were estimated by spectrophotometric DPPH, ABTS, and FRAP assays. Based on the analysis of phytochemicals and antioxidant activity, P. × berolinensis buds were selected as the raw plant material for medicinal purposes with the highest content of active compounds and the strongest antioxidant activity.

Anti-Helicobacter pylori Biofilm Extracts from Rubus idaeus and Rubus occidentalis.

Helicobacter pylori infections are still an important health problem and are directly related to the development of gastric ulcer, gastric adenocarcinoma, mucosal lymphoid tissue lymphoma, and diabetes. At the same time, the number of substances/drugs effective against these bacteria is limited due to increasing resistance. Raw plant materials from various species of the Rubus genus-fruits and shoots-have shown antimicrobial activity in numerous studies against different bacteria, including H. pylori in a planktonic form. Research carried out on a model using fragments of intravenous infusions and triphenyl tetrazolium chloride (TTC) as a dye showed that the shoot extract of Rubus idaeus 'Willamette', the fruit extract of R. idaeus 'Poranna Rosa', R. idaeus and R. idaeus 'Laszka', and R. occidentalis Litacz' prevent the formation of biofilm by H. pylori. Active concentrations inhibiting biofilm formation were 6.65 mg/mL for shoots and 16.65 mg/mL for fruits. However, in the resulting biofilm, the extract from the shoots of R. idaeus 'Willamette' and the fruit of R. idaeus 'Poranna Rosa' at a concentration of 16.65 mg/mL was active against living bacteria, and the remaining extracts showed such activity at a concentration of 33.3 mg/mL. In studies on the interaction of the extract with antibiotics on biofilm, the extract from the shoots of R. idaeus 'Willamette' showed synergy with doxycycline and levofloxacin, additivity with amoxicillin and clarithromycin, and neutrality with metronidazole. H. pylori biofilm research was carried out in a newly elaborated research model-culture on fragments of intravenous infusions with the addition of TTC as a marker of living bacterial cells. The research results may constitute the basis for the development of new combination therapies for the treatment of H. pylori infections, including its resistant strains. The proposed new biofilm research model, which is cheap and effective, may allow testing of new substances that are potentially more effective against H. pylori and other biofilm-forming bacterial strains.

HPLC-DAD-MS Characterization, Antioxidant Activity, α-amylase Inhibition, Molecular Docking, and ADMET of Flavonoids from Fenugreek Seeds.

Fenugreek (Trigonella foenum-graecum) has a great beneficial health effect; it has been used in traditional medicine by many cultures. Likewise, the α-amylase inhibitors are potential compounds in the development of drugs for the treatment of diabetes. The beneficial health effects of fenugreek lead us to explore the chemical composition of the seeds and their antioxidant and α-amylase inhibition activities. The flavonoid extraction from fenugreek seeds was achieved with methanol through a Soxhlet apparatus. Then, the flavonoid glycosides were characterized using HPLC-DAD-ESI-MS analysis. The antioxidant capacity of fenugreek seed was measured using DPPH, FRAP, ABTS, and CUPRAC assays. Finally, the α-amylase inhibition activity was carried out using in vitro and in silico methods. The methanolic extract was found to contain high amounts of total phenolics (154.68 ± 1.50 µg GAE/mg E), flavonoids (37.69 ± 0.73 µg QE/mg E). The highest radical-scavenging ability was recorded for the methanolic extract against DPPH (IC50 = 556.6 ± 9.87 µg/mL), ABTS (IC50 = 593.62 ± 9.35 µg/mL). The ME had the best reducing power according to the CUPRAC (A 0.5 = 451.90 ± 9.07 µg/mL). The results indicate that the methanolic extracts of fenugreek seed best α-amylase inhibition activities IC50 = 653.52 ± 3.24 µg/mL. Twenty-seven flavonoids were detected, and all studied flavonoids selected have good affinity and stabilize very well in the pocket of α-amylase. The interactions between the studied flavonoids with α-amylase were investigated. The flavonoids from fenugreek seed present a good inhibitory effect against α-amylase, which is beneficial for the prevention of diabetes and its complications.

Biotechnological strategies for controlled accumulation of flavones in hairy root culture of Scutellaria lateriflora L.

Accumulation of medicinally important flavones and acteoside was evaluated in Scutellaria lateriflora hairy root cultures subjected to different experimental strategies - feeding with precursors of phenolics biosynthesis (phenylalanine, cinnamic acid, and sodium cinnamate), addition of elicitors (chitosan, jasmonic acid) and Amberlite XAD-4 and XAD-7 resins and permeabilization with dimethyl sulfoxide (DMSO) and methanol. The production profile of S. lateriflora cultures changed under the influence of the applied strategies. Hairy roots of S. lateriflora were found to be a rich source of wogonoside or wogonin, depending on the treatment used. The addition of sodium cinnamate (1.0 mg/L) was the most effective approach to provide high production of flavonoids, especially wogonoside (4.41% dry weight /DW/; 566.78 mg/L). Permeabilization with DMSO (2 µg/ml for 12 h) or methanol (30% for 12 h) resulted in high biosynthesis of wogonin (299.77 mg/L and 274.03 mg/L, respectively). The obtained results provide new insight into the selection of the optimal growth conditions for the production of in vitro biomass with a significant level of flavone accumulation. The data may be valuable for designing large-scale cultivation systems of hairy roots of S. lateriflora with high productivity of bioactive compounds - wogonin or wogonoside.

Evaluation of the Content of Micro- and Macroelements in Raspberries Depending on the Species, Cultivar Variety, and Geographical Environment.

The study aimed to analyse the macro- and micro-nutrient content in fruits of Rubus species (R. idaeus, R. occidentalis, R. chamaemorus, and R. chingii) and their varieties or hybrids from different regions. Flame atomic absorption spectrometry with deuterium background correction was used to measure concentrations of nine essential elements (K, Mg, Ca, Na, Mn, Fe, Cr, Zn, and Cu) and two heavy metals (Pb, Cd). Chemometric analysis compared the elemental profiles. Results confirmed raspberries as a rich source of macroelements (K, Mg) and microelements (Zn, Cu, Mn, Cr). The 'Bristol' cultivar consistently had the highest Fe content regardless of origin. Cr presence was observed in black raspberries for the first time. Previously observed relationships like K-Na antagonism and Cr/Zn, Fe/Zn synergism were found in raspberry fruits. Factor and cluster analyses demonstrated species and geographical diversity among Polish raspberry samples and clear separation of R. chingii from China. Raspberry fruits, due to the rich complex of polyphenols, are classified as superfoods, and the content of bioelements determined in them guarantees coverage of the daily requirement for macro- and microelements (RDA depending on the element: 5.6-204% for R. idaeus, 8.8-469, 4% for R. occidentalis, and 1.4-67.2% for R. chamaemorus), finally confirming this opinion.

LC-MS/MS Evaluation of Pyrrolizidine Alkaloids Profile in Relation to Safety of Comfrey Roots and Leaves from Polish Sources.

Comfrey (Symphytum officinale L.) has a long tradition of use in the treatment of musculoskeletal disorders. However, due to hepatotoxic pyrrolizidine alkaloids (PAs), the EMA restricts the use of comfrey root (CR) to external use only and for short periods of time. Recent studies indicate a low permeability of PAs across the skin, calling into question the safety of topical application of products containing comfrey preparations. The aim of our work was to develop and validate an HPLC method enabling the separation of isomeric PAs from comfrey and, on this basis, to assess the potential toxicity of CR and comfrey leaf (CL) obtained from various Polish sources. The qualitative and quantitative analysis of PAs via HPLC-MS/MS was performed in MRM mode. The results obtained confirmed a lower content of PAs in CL than in CR and showed a wide variation in the composition of PAs in CR, with a much more stable profile of PAs in CL. Factor analysis confirmed that CRs and CLs differ in PA content, which is influenced by the growth conditions and geographical origin. The determined concentrations of PAs prove that in some CRs available on the Polish herbal market, the content of PAs may exceed the daily dose considered safe.

Urolithins Modulate the Viability, Autophagy, Apoptosis, and Nephrin Turnover in Podocytes Exposed to High Glucose.

Urolithins are bioactive compounds generated in human and animal intestines because of the bacterial metabolism of dietary ellagitannins (and their constituent, ellagic acid). Due to their multidirectional effects, including anti-inflammatory, antioxidant, anti-cancer, neuroprotective, and antiglycative properties, urolithins are potential novel therapeutic agents. In this study, while considering the future possibility of using urolithins to improve podocyte function in diabetes, we assessed the results of exposing mouse podocytes cultured in normal (NG, 5.5 mM) and high (HG, 25 mM) glucose concentrations to urolithin A (UA) and urolithin B (UB). Podocytes metabolized UA to form glucuronides in a time-dependent manner; however, in HG conditions, the metabolism was lower than in NG conditions. In HG milieu, UA improved podocyte viability more efficiently than UB and reduced the reactive oxygen species level. Both types of urolithins showed cytotoxic activity at high (100 µM) concentration. The UA upregulated total and surface nephrin expression, which was paralleled by enhanced nephrin internalization. Regulation of nephrin turnover was independent of ambient glucose concentration. We conclude that UA affects podocytes in different metabolic and functional aspects. With respect to its pro-survival effects in HG-induced toxicity, UA could be considered as a potent therapeutic candidate against diabetic podocytopathy.

Phyllanthus amarus shoot cultures as a source of biologically active lignans: the influence of selected plant growth regulators.

This is the first comprehensive study of the influence of plant growth regulators (PGRs) on the development of shoots and accumulation of biologically active lignans-phyllanthin and hypophyllanthin, in the shoot culture of P. amarus Schum. & Thonn. (Euphorbiaceae) obtained by direct organogenesis. The following PGRs were included in the experiments-cytokinins: kinetin (Kin), 6-benzylaminopurine (BAP), 2-isopentenyladenine (2iP), 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea, thidiazuron (TDZ) and auxin, indole-3-butyric acid (IBA) and used at various concentrations. Depending on PGRs and their concentrations, differences in the culture response and lignan accumulation were observed. The highest content of the investigated compounds was found in the shoot culture grown on Murashige and Skoog's (MS) medium supplemented with Kin 0.25 mg/L. The sum of phyllanthin and hypophyllanthin was ~ 10 mg/g of dry weight (DW), which was similar or even higher than that in the plant material obtained from natural conditions. The results of the research provide new data on the selection of the optimal growth medium for the production of plant material with a significant level of phyllanthin and hypophyllanthin biosynthesis. The obtained data may also be valuable in designing systems for large-scale cultivation of P. amarus shoots with high productivity of hepatoprotective lignans.

Studies on the Polyphenolic Composition and the Antioxidant Properties of the Leaves of Poplar (Populus spp.) Various Species and Hybrids.

The chemical composition in terms of flavonoid and salicylic compounds of leaves from 6 species and 3 hybrids of poplars (Populus) was identified with the use of TLC and HPLC-DAD/ESI-MS methods. Chromatographic analyses were carried out with 21 standard compounds including salicylic compounds (2), phenolic acids (3) and flavonoids (16). Moreover, on the basis of the obtained chromatographic data from the HPLC-DAD/ESI-MS and TLC separations, the presence of salicortin, tremulacin and chlorogenic acid was confirmed, depending on the analyzed poplar species or hybrid. The content of salicylic compounds was determined by HPLC-UV method and expressed on salicin as free and total fraction. Total flavonoid content was determined by spectroscopic method as quercetin equivalent. Significant qualitative and quantitative differences in the chemical composition of the analyzed leaves were demonstrated. The highest concentration of flavonoids (8.02 mg/g) was found in the leaves of Populus nigra, while the highest content of salicylic compounds (47.14 mg/g) was found in the leaves of P.×berolinensis. The antioxidant and xanthine oxidase inhibition properties of extracts from poplar leaves were investigated by TLC bioautography. It has been shown that the richest set of compounds with antioxidant properties are present in the leaves of P. alba, P.×candicans and P. nigra.

Potential of Silver Nanoparticles in Overcoming the Intrinsic Resistance of Pseudomonas aeruginosa to Secondary Metabolites from Carnivorous Plants.

Carnivorous plants are exemplary natural sources of secondary metabolites with biological activity. However, the therapeutic antimicrobial potential of these compounds is limited due to intrinsic resistance of selected bacterial pathogens, among which Pseudomonas aeruginosa represents an extreme example. The objective of the study was to overcome the intrinsic resistance of P. aeruginosa by combining silver nanoparticles (AgNPs) with secondary metabolites from selected carnivorous plant species. We employed the broth microdilution method, the checkerboard titration technique and comprehensive phytochemical analyses to define interactions between nanoparticles and active compounds from carnivorous plants. It has been confirmed that P. aeruginosa is resistant to a broad range of secondary metabolites from carnivorous plants, i.e., naphthoquinones, flavonoids, phenolic acids (MBC = 512 µg mL-1) and only weakly sensitive to their mixtures, i.e., extracts and extracts' fractions. However, it was shown that the antimicrobial activity of extracts and fractions with a significant level of naphthoquinone (plumbagin) was significantly enhanced by AgNPs. Our studies clearly demonstrated a crucial role of naphthoquinones in AgNPs and extract interaction, as well as depicted the potential of AgNPs to restore the bactericidal activity of naphthoquinones towards P. aeruginosa. Our findings indicate the significant potential of nanoparticles to modulate the activity of selected secondary metabolites and revisit their antimicrobial potential towards human pathogenic bacteria.

Cytotoxic activity of standardized extracts, a fraction, and individual secondary metabolites from fenugreek seeds against SKOV-3, HeLa and MOLT-4 cell lines.

CONTEXT: Trigonella foenum-graecum L. (Fabaceae) has many therapeutic properties and anticancer potential. OBJECTIVE: The cytotoxic activities of standardized extracts and a fraction from fenugreek seeds and their compounds (sapogenins, flavone C-glycosides, alkaloid trigonelline) against human cancer SKOV-3, HeLa and MOLT-4 cells were evaluated. MATERIALS AND METHODS: Fenugreek seeds were extracted with 70% methanol (A) or water (B). Furthermore, the seeds were purified with petroleum ether and chloroform and next extracted with methanol to obtain fraction (C). The quantitative analysis of saponins and flavonoids in the extracts was done with HPLC methods. The extracts (5-120 µg/mL) and compounds (1-50 µg/mL) were tested on the cells by MTT assay and RTCA system. The effect of a fraction on ROS production, mitochondrial membrane potential and caspase-3/7 activity in HeLa and SKOV-3 cells was also evaluated by flow cytometry. RESULTS: The strongest cytotoxic activity on cancer cells showed the fraction C (IC50 was 3.91 ± 0.03 for HeLa, 3.97 ± 0.07 for SKOV-3, and 7.75 ± 0.37 for MOLT-4) with the highest content of steroidal saponins (163.18 ± 11.03 µg/mg) and flavone C-glycosides (820.18 ± 0.05 µg/mg). The fraction significantly increased ROS production (up to four times higher than in keratinocytes as control) and caspases activity in the cells. The examined flavonoids did not exhibit the cytotoxic activity in contrast to yamogenin, tigogenin, and diosgenin. CONCLUSIONS: The obtained results complement the data on the cytotoxic activity of Foenugraeci Semen and synergistic effect of flavonoids and saponins complex contained in the plant.

2D LC as a tool for standardization of Foenugraeci semen extracts containing compounds with anti-Helicobacter pylori activity.

The on-line heart-cutting two-dimensional liquid chromatography method with the use of a diode array detector and a mass spectrometer (LC-LC-DAD-ESI-MS) was established and validated for quantitation of C-glycosylflavones in fenugreek seeds (Foenugraeci semen, Trigonella foenum-graecum L.). The first- (1D) and second- (2D) dimensional separations were performed on Kinetex C-18 columns with different diameters, respectively, and gradient (1D) and isocratic elution (2D). Finally, 17 compounds were separated, 13 of which were quantified by 1D separation and 4 compounds by 2D separation. As a result, it was pointed out that fenugreek seeds of Polish origin can be considered as a rich source of C-glycosylflavones. Antibacterial activity against Helicobacter pylori of standardized 70% methanol extract from fenugreek seeds has been demonstrated, in contrast to the inactive aqueous extract. Anti-H. pylori activity of the 70% methanol extract can be related to a higher concentration of C-glycosylflavones. This is the first report on the bactericidal activity of vitexin, diosgenin, tigogenin and sarsasapogenin against H. pylori and the bacteriostatic activity of orientin against this bacterium.

Iris pseudacorus as an easily accessible source of antibacterial and cytotoxic compounds.

Iris pseudacorus is one of the most widespread iris species and possesses complex secondary metabolites. Our study showed that its rhizomes are abundant with phenolic compounds of which 80 % belong to the tannin group. Methanolic extracts from garden cultured iris rhizomes possessed antibacterial activity against human Gram positive Staphylococcus aureus and Enterococcus faecalis and Gram negative Pseudomonas aeruginosa and Klebsiella pneumoniae pathogens including clinical isolates resistant to commercially available antibiotics. Moreover the extract from rhizome, in concentration 3.125 mg dry weight/mL, containing gallocatechin (1), effectively combats S. aureus biofilm. The same rhizome extract acts against human cancer cell lines, especially against estrogen positive MCF-7 breast cancer cell line (IC50 = 11.75 µg/mL). In vitro culture of excised, anatomical roots of I. pseudacorus excreted three antistaphylococcal compounds into the plant medium, detected by using TLC-overlayer bioautography. By the use of HPLC-DAD-ESIMS system 2 active compounds were identified as 5,7,4'-trihydroxy-6,3'-dimethoxy-isoflavone (7) and unknown dimethoxy-dihydroxy-isoflavone (9). I. pseudacorus as a non-edible plant might be considered to be new, easy accessible, non-wood source of biologically active polyphenolics.

Application of targeted 2D planar chromatography in the control of ginkgolic acids in some herbal drugs and dietary supplements.

Two-step targeted 2D planar chromatographic method (2DTLC) was used in the determination of ginkgolic acids in pharmaceuticals and dietary supplements. The choice of the extraction method and the separation technique was guided by the formulation type (capsule, tablet, tincture) with expected low amounts of ginkgolic acids in the analyzed herbal samples. Separation of ginkgolic acids C15:1 and C17:1 on HPTLC RP18 WF254s was preceded by its separation from the sample matrix on TLC Si60 F254s. Mobile phases consisted of acetonitrile/water/formic acid (80:20:1, V/V/V) and n-heptane/ethyl acetate/formic acid (20:30:1, V/V/V), resp. Identification of separated compounds was based on 2D-TLC co-chromatography with reference substances and off-line 2D-TLC x HPLC-DAD-ESI-MS analysis. Quantification of ginkgolic acids C15:1 and C17:1 was conducted densitometrically. Among the analyzed products, the presence of ginkgolic acids was confirmed only in herbal drugs containing 60 % ethanolic tinctures of Ginkgo biloba leaves. The use of TLC in the quantification of ginkgolic acids C15:1 and C17:1 in ginkgo extracts was described for the first time.

Identification of Flavonoids and Bufadienolides and Cytotoxic Effects of Kalanchoe daigremontiana Extracts on Human Cancer Cell Lines.

Kalanchoe species are well-known medicinal plants used in traditional medicine as anti-inflammatory and analgesic remedies. Recently, it has been reported that Kalanchoe plants have cytotoxic properties; however, data on traditional use of these plants in tumor treatment are extremely limited. Kalanchoe daigremontiana is one of the most popular species cultivated in Europe, and it is used, among other things, as a remedy in treating skin injuries and wounds. Studies on the biological activity of this species are scarce, and there is a lack of data on the cytotoxic activity of K. daigremontiana extracts on epithelial cancer cells in the literature. In our present study, we analyzed the phytochemical composition of K. daigremontiana ethanol extract and fractions-water and dichloromethane-by the HPLC-DAD-ESI-MS method and estimated cytotoxic activity of the extracts on human adenocarcinoma (HeLa), ovarian (SKOV-3), breast (MCF-7), and melanoma (A375) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, real-time cell analyzer (RTCA), and flow cytometry. We identified 6 bufadienolide compounds and 19 flavonoids, mostly kaempferol, quercetin, isorhamnetin, and myricetin glycosides, of which only 3 flavonoids have been identified in K. daigremontiana to date. Other flavonoids that were characterized in our study have not yet been found in this plant. The ethanol extract and water fraction of K. daigremontiana did not show significant cytotoxic activity on the tested cell lines. In contrast, the dichloromethane fraction showed the strongest activity against all cell lines with IC50 values of ≤ 10 µg/mL. The results indicated that this activity is mainly due to the presence of bersaldegenin-1,3,5-orthoacetate.

TLC-densitometric analysis of allantoin in Symphytum officinale L. roots.

A TLC-densitometric method for determination of allantoin in Symphytum officinale root was developed. Densitometric quantification of allantoin was carried out on TLC Si60 plates with butanol-50 % methanol/formic acid, 66.5:33.2:0.3 (V/V/V) as developing solvent, at a wavelength of 190 nm. The method was preliminarily validated in terms of specificity, linearity, precision, limit of detection, limit of quantification, recovery and robustness. The results of TLC quantification were compared with HPLC analysis carried out on a HILIC Luna NH2 100A column, with mobile phase consisting of acetonitrile/water 80:20 (V/V) and UV detection at 190 and 210 nm. Allantoin content was determined in two herbal products and it varied from 0.94 to 2.09 %, depending on the producer, and was in agreement with literature reports.

Qualitative and quantitative HPLC-ELSD-ESI-MS analysis of steroidal saponins in fenugreek seed.

Fenugreek seeds are known as a source of various compounds, the most common of which are steroidal saponins. However, despite the growing interest in this plant material as a healing agent, spice and dietary supplement ingredient, the composition of Polish fenugreek seeds remains unknown. Therefore, the steroidal saponin complex in the seeds of T. foenum-graecum cultivated in Poland was qualitatively and quantitatively analyzed by the HPLC-ELSDESI-MS method. Two C-18 columns connected in series were used for the first time in analysis of fenugreek saponins and ELS detector parameters were optimized. A total of 26 furostanol saponins were revealed, of which 24 were tentatively identified. The HPLC-ELSD method developed for quantitative analysis was preliminarily validated and the determined amount of steroidal saponins in Polish fenugreek seeds was 0.14 %.

Anti-inflammatory and antioxidative effects of the buds from different species of Populus in human gingival fibroblast cells: Role of bioflavanones.

BACKGROUND: Poplar leaf-buds (Populi gemmae) are used traditionally as anti-inflammatory agents to the treatment of skin injuries or cough. They differ in their diverse chemical composition and different types of activities, whose mechanisms are not fully recognized. PURPOSE: Evaluation and comparison of anti-inflammatory activity of leaf-buds extracts from Populus nigra, P. × berolinensis and P. lasiocarpa and flavanones - pinocembrin and pinostrobin towards human gingival fibroblasts (HGF-1) pro-inflammatory stimulated by silver nanoparticles (AgNPs). Determination of antioxidant activity associated with anti-inflammatory properties by means of bioautographic TLC tests. METHODS: Phytochemical analysis was performed by TLC and videodensitometry analysis. The extracts were standardized on the pinocembrin and pinostrobin content. Bioautography was performed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and riboflavin-light blue tetrazolium chloride (riboflavin-light-NBT) radicals to assess the extracts and both flavanones radical scavenging properties as well as potential inhibition of xanthine oxidase (XO) activity. The protective effects of poplar buds extract and flavanones - pinocembrin and pinostrobin on HGF-1 line exposured to AgNPs were investigated by analysis of interleukin 6 (IL-6) and interleukin IL-1ß (IL-1ß) level measured by ELISA kit. The messenger ribonucleic acid (mRNA) of both cytokines was determined by real-time quantitative PCR. The involvement of cyclooxygenase 2 protein (COX-2) was studied using Western blot analysis. RESULTS: The presence of several flavanones and phenolic acids, which have radical scavenging properties, was revealed in all of the bud poplar extracts analyzed. Treatment with particular flavanones or extracts from buds of P. × berolinensis and P. nigra decreased the IL-6 and IL-1ß release in HGF-1 cells and down-regulation of mRNA for both cytokines was observed. The COX-2 protein expression was demonstrated for pinocembrin and P. × berolinensis buds. These effects were not observed for buds from P. lasiocarpa not containing of flavonoids. CONCLUSION: The potential protective role of pinocembrin and pinostrobin and extracts from buds P. nigra and P. × berolinensis against AgNPs induced inflammation and cytotoxicity in HGF-1 cells is disclosed. In addition, the antioxidant properties of poplar bud extracts have been demonstrated. P. × berolinensis buds showed the highest activity in both the in vitro model and in the bioautographic tests.

TLC determination of some flavanones in the buds of different genus Populus species and hybrids.

Flavonoids in the buds of eight Populus species and hybrids were detected and compared with the aid of an optimized TLC method. Separation of 17 flavonoid aglycones belonging to different groups, namely, flavones, flavonols, flavanones and flavanonols, previously described as constituents of poplar buds, was performed on silica gel plates using a hexane/ethyl acetate/formic acid (60:40:1.3, V/V/V) mixture as the mobile phase. Pinocembrin and pinostrobin were found in the majority of analyzed poplar buds. For quantitative analysis of both compounds, two TLC evaluation modes, densitometric and videodensitometric, were compared and the established methods were validated. Concentrations of flavanones in some extracts differed slightly or significantly due to the analyzed plant matrix complexity and the TLC evaluation mode applied. Poplar buds rich in flavanones originated from P. × canadensis 'Robusta' (1.82 and 2.23 g per 100 g, resp.) and P. balsamifera (1.17 and 2.24 g per 100 g, resp.).