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.

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.

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.

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.).

TLC determination of flavonoids from different cultivars of Allium cepa and Allium ascalonicum.

This study comprises the optimization and validation of a new TLC method for determination of flavonols in the bulbs of seven cultivars of onions and shallots. Separation was performed on RP-18 plates with the solvent mixture tetrahydrofuran/water/formic acid (40+60+6, V/V/V) as a mobile phase. The method was evaluated for precision, linearity, LOD, LOQ, accuracy and robustness. Chromatographic analysis of the extracts revealed the presence of three main flavonols, quercetin, quercetin-4'-O-glucoside and quercetin-3,4'-O-diglucoside in the majority of analyzed cultivars. The content of flavonols in the analyzed extracts of onion bulbs varied from 123 ('Exihibition') to 1079 mg kg-1 fresh mass (fm) ('Hybing') in edible parts, and from 1727 ('Hyline') to 28949 mg kg-1 fm ('Red Baron') in outer scales. The bulbs of two shallot cultivars contained 209 ('Ambition') and 523 mg kg-1 fm ('Matador') of flavonols in edible parts and 5426 and 8916 mg kg-1 fm in outer scales, respectively.

2D LC HEART CUTTING ON-LINE OF PHENOLIC COMPOUNDS FROM THREE SPECIES OF THE GENUS SALIX.

The 2D LC heart-cutting on-line system was elaborated and employed to the analysis of simple phenols and polyphenols occurring in willow barks. Using the test-set of 52 compounds, the conditions of chromatographic separation in each dimension were optimized. The worked-up system was based on RP-separation in both dimensions and the use of different elution profiles on the first- and second-dimensional columns: gradient and multistep gradient elution, respectively. In all analyses the UV detector was used. Under optimized separation conditions slightly modified in respect to chemical composition of the each analyzed MeOH extracts from three willow barks: Salix daphnoides, S. puiputea and S. sachalinensis 'Sekka' the differences in phenolie acid and flavonoid compositions were revealed.

Effect of enoxaparin and onion extract on human skin fibroblast cell line - therapeutic implications for the treatment of keloids.

CONTEXT: Keloids and hypertrophic scars are hyperproliferative skin disorders resulting in abnormal wound healing. In the prevention and treatment of keloids and hypertrophic scars, ointments containing heparin and onion extract are very popular. Their therapeutic effects, however, are still controversial and the mechanism of action is not fully understood. OBJECTIVE: The aim of this study was to assess the effect of enoxaparin and dry onion extract on proliferation, apoptosis and ß1 integrin expression in human fibroblasts. MATERIALS AND METHODS: Fibroblast human cell lines (46 BR.1 N) were treated for 48 h with various concentrations of enoxaparin sodium (20, 100, 500 µg/mL) and/or onion [Allium cepa L. (Alliaceae)] extract (50, 250, 1000 µg/mL). The cell proliferation was evaluated by [(3)H]-thymidine incorporation assay. Furthermore, the expression of ß1 integrin and apoptosis was determined by flow cytometry. RESULTS AND DISCUSSION: The results demonstrate that enoxaparin and onion extract inhibited the proliferation of human fibroblasts. Almost complete inhibition of cell proliferation was achieved by enoxaparin in 500 µg/mL concentration (91.5% reduction). The onion extract at a concentration of 250 µg/mL also strongly inhibited the proliferation of cells (50.8% reduction). Depending on concentration, enoxaparin and onion extract induced apoptosis (500 and 1000 µg/mL, respectively) and, depending on concentration, downregulated the expression of ß1 integrin on human fibroblasts. CONCLUSION: This work points at possible mechanism of action of enoxaparin and onion extract, when administered in the treatment of patients with keloids and hypertrophic scars.

HPLC of flavanones and chalcones in different species and clones of Salix.

The SPE-HPLC method was developed to determine an isosalipurposide (5) and its derivative, 6"-O-p-coumaroyl ester (6) in the bark of eight taxa (I-VIII) belonging to three species of the genus Salix and originating from a natural habitat or cultivated for pharmaceutical purposes. The chalcones were separated by HPLC under gradient elution with the concentration of ACN increasing from 20% to 50% in 0.1% aqueous H3PO, (tG 15 min). The content of both compounds was determined by an external standardization with the use of isoliquiritigenin (7) as a reference substance - a commercially available chalcone, and also isosalipurposide (5) and its derivative, 6"-O-p-coumaroyl ester (6). The latter compound was isolated from the bark of Salix daphnoides (IV) by a CC and semi-preparative HPLC and its structure was elucidated by MS and NMR spectra. It was stated that 6"-O-p-coumaroylisosalipurposide (6), in addition to isosalipurposide (5), is a characteristic flavonoid for the S. daphnoides species. Moreover, the presence of these two chalcones was confirmed in the bark of S. acutifolia (I). Differences were observed in the results obtained from a quantitative analysis due to the type of reference substance used. The content of chalcones was varied and dependent on the species selected for analysis, namely from 22.01/21.08 mg/g in S. daphnoides clone 1095 (III) to 2.47/2.44 mg/g in S. daphnoides (II), collected from a natural habitat. Isosalipurposide (5) was determined in all the investigated species and clones of Salix, besides a number of naringenin derivatives. Separation of all flavonoids: flavanones - naringenin (1), naringenin (+)-5-O-glucoside (2), (-)-5-O-glucoside (3), 7-O-glucoside (4) and chalcones (5 and 6) was performed under gradient elution with the same solvents and changes in ACN concentration from 2% to 37% (tG 60 min). The total amount of flavanones ranged from 4.69 mg/g in S. purpurea clone 1132 (VII) to 41.93 mg/g in S. purpuea (VIII) from Herbapol Wroclaw.

Chromatographic analysis of simple phenols in some species from the genus Salix.

INTRODUCTION: Salicis Cortex, made from willow bark is a herbal remedy, which is standardised based on the content of salicin, a compound with analgesic and antiphlogistic properties. However, clinical trials suggest that other compounds also present in Salicis Cortex can contribute to the pharmacological effects. OBJECTIVE: To characterise the composition of phenolic acids in the barks of different species and clones from the genus Salix by use of chromatographic methods--HPTLC and HPLC. METHODOLOGY: The phenolic acid composition was analysed by MGD (multiple gradient development)-HPTLC technique. The separation was performed on HPTLC Diol plates with gradient elution using a mixture of chloroform:hexane:ethyl acetate with increasing concentration of ethyl acetate from 10 to 25%. Derivatisation with thymol reagent was employed for the first time for specific detection of phenolic acids containing methoxyl groups. RESULTS: The presence of all phenolic acids previously reported in the genus Salix was confirmed, namely p-hydroxybenzoic, vanillic, cinnamic, p-coumaric, ferulic and caffeic acids. Furthermore, pyrocatechol as a constituent of willow bark was revealed. The highest concentration of this compound was observed in the S. purpurea bark (2.25 mg/g). CONCLUSION: The presence of a relatively high content of pyrocatechol in Salix species may raise doubts about the safe application of this herbal medicine.

Chemosystematic value of the essential oil composition of Thuja species cultivated in Poland-antimicrobial activity.

In the framework of the correlation between chemotaxonomy and chemical analysis studies, the chemical composition of the essential oils of four varieties of Thuja species cultivated in Poland - T. occidentalis 'globosa', T. occidentalis 'aurea', T. plicata and T. plicata 'gracialis' - were investigated by GC and GC-MS. Thirty-one compounds were identified from T. occidentalis 'globosa', representing 96.92% of the total oil; twenty-seven from T. occidentalis 'aurea' (94.34%); thirty-one from T. plicata (94.75%); and thirty compounds from T. plicata 'gracialis' (96.36%). The main constituents in all samples were the monoterpene ketones alpha- and beta-thujone, fenchone and sabinene, as well as the diterpenes beyerene and rimuene. The chemosystematic value of the total ketone content of all samples (which varied from 54.30-69.18%) has been discussed and investigated. The constituents, beyerene and the mixture of alpha- and beta-thujone, were isolated from the oils and tested against six Gram-positive and -negative bacteria and three pathogenic fungi. The oils of the two T. plicata species exhibited significant antimicrobial activity, while the mixture of alpha- and beta-thujone showed very strong activity as well.

SPE-HPTLC of procyanidins from the barks of different species and clones of Salix.

A SPE-HPTLC method was developed for the qualitative and quantitative analysis of procyanidin B(1) in willow barks. The chromatography was performed on HPTLC silica gel layer with the mobile phase chloroform-ethanol-formic acid (50:40:6 v/v/v), in the Automatic Developing Chamber-ADC 2. The methanol extracts from willow barks were purified by SPE method on RP-18 silica gel columns with methanol-water (7:93 v/v) as the eluent. The presence of procyanidin B(1) was revealed in the majority of investigated willow barks. The content of procyanidin B(1) varied from 0.26 mg/g in the extract of Salix purpurea clone 1067-2.24 mg/g in the extract of Salix alba clone 1100. The method was validated for linearity, precision, LOD, LOQ and repeatability.

Chromatographic analysis of salicylic compounds in different species of the genus Salix.

The separation of nine phenol glycosides--salicin, salicortin, 2'-acetylsalicortin, populin, tremulacin, salidroside, triandrin, picein and helicin--by normal phase (NP), reversed phase (RP) HPLC techniques and a coupling of NP and RP monolithic silica columns was studied. Among the above nine compounds only five--salicin, populin, tremulacin, salidroside and triandrin--were resolved in an NP system with a mobile phase comprising hexane/isopropanol/methanol (87:12:1, v/v/v). Optimized separation was performed with two coupled monolithic silica columns of different polarity (bare silica and RP-18). The method was applied to verify the presence of salicylic compounds and other phenolic derivatives in the bark of six species from the genus Salix, namely S. purpurea, S. daphnoides clone 1095, S. alba clone 1100, S. triandra, S. viminalis, and S. herbacea. Gradient elution with a mobile phase composed of acetonitrile and water containing 0.05% of trifluoroacetic acid, with increasing acetonitrile concentration from 3% to 48%, was chosen as optimal. For the selective detection of the salicylic compounds, an evaporative light scattering detector was employed along with a UV detector. The differences in the composition of phenols in the different plant materials were confirmed. Additionally, it must be emphasized that for the first time the presence of 2'-acetylsalicortin was revealed in S. alba clone 1100. Furthermore, an SPE-HPLC method was developed for the rapid analysis of the salicin content, analyzed as free and total fraction, in willow barks. The determined concentrations of total salicin varied from 25.4 mg/g in S. alba clone 1100 to 96.47 mg/g in S. daphnoides clone 1095.

Evaluation of biologically active compounds in roots and rhizomes of Rhodiola rosea L. cultivated in Lithuania.

Salidroside and rosavins (rosavin, rosarin, and rosin), biologically active compounds of Rhodiola rosea L., were analyzed in dried roots and rhizomes of the plants cultivated in Lithuania and naturally growing in Altai geographical region in Russian Federation. The quantitative analysis of the aqueous-ethanolic extracts prepared by percolation was performed employing solid-phase extraction and thin-layer chromatography followed by densitometric determination. Similar amounts of salidroside were found in the extracts (1.35-1.62 mg/mL), irrespective of the origin of the crude plant; however, the differences in the profile of rosavins were substantial with higher content of total rosavins in the extracts obtained from the plant cultivated in Lithuania (7.4 vs. 4.2 mg/mL). It was demonstrated that extracts prepared with 70% (v/v) ethanol contained low concentration of salidroside (0.16-0.17 mg/mL), while the extraction of rosavins with 70% (v/v) ethanol was more efficient compared to 40% (v/v) ethanol.