Spray-drying Microencapsulation of an Extract from Tilia tomentosa Moench Flowers: Physicochemical Characterization and in Vitro Intestinal Activity.

Silver linden (Tilia tomentosa Moench, TtM) flowers possess several health-promoting properties, especially at the neurological level, such as intestinal relaxation activity associated with specific flavonols, particularly quercetin and kaempferol derivatives. However, such molecules are susceptible to degradation upon different triggers like heat, light and extreme pH values. To overcome the scarce stability of TtM flowers bioactive molecules and make them suitable for developing functional food and supplements, we applied microencapsulation. Spray-drying microencapsulation of TtM flowers extract was performed using three starch-derived wall materials: maltodextrin 12 DE (MD12) and 19 DE (MD19), and OSA-modified starch (OSA-S). The stability of total phenols, flavanols, and antioxidant capacity was monitored for 70 days under accelerated stress conditions (40 °C/70% RH) by HPLC and spectrophotometric methods, and the intestinal contractile activity was tested in a murine model. In comparison to MD12 and MD19, OSA-S stood out for the higher encapsulation efficiency of quercetin and kaempferol glycosides (+ 36-47% compared to MD12 and + 18-24% compared to MD19) and stability thereof (half-life on average + 30% compared to MD12 and + 51% compared to MD19). The intestinal contractile activity of OAS-S powders resulted comparable to the original extract, indicating that flavonols were biologically active and accessible. Our results underly the potential advantages of OSA-S encapsulated formulation as a functional ingredient for the development of nutraceutical products.

Characterization of herbal teas containing lime flowers - Tiliae flos by HPTLC method with chemometric analysis.

Linden trees are a source of food products called lime flowers (Tiliae flos), traditionally used in the form of infusion for the treatment of feverish colds and coughs. Lime flowers should include flowers of Tilia cordata Mill, T.x europaea L., and T. platyphyllos Scop. or a mixture of these. The aim of current research was to establish a fast, sensitive HPTLC (high-performance thin-layer chromatography) method that would allow the differentiation of material obtained from five species of lime occurring in Europe. The fingerprints for distinguishing these species were established, as well as a key for identification based on a visual evaluation of chromatograms. The results obtained were also subjected to chemometric analyses. It was shown that each species contains characteristic compounds i.e. linarin that can be used for their identification. The method developed can, in theory, be introduced for the quality control or authentication of linden flowers on the European market.

Assessment of the anti-pathogenic effects of condensed tannin extracts using scanning electron microscopy.

Two different types of condensed tannins (CTs), which were extracted and purified from tilia (Tilia L.) and black locust (Robinia pseudoacacia), were studied and tested against two kinds of bacteria, including Gram-negative and Gram-positive, avian pathogenic E. coli (APEC) and Staphylococcus epidermidis (S. epidermidis) respectively, by minimal bactericidal concentrations (MBCs) and scanning electron microscopy (SEM). Both CT extracts were significantly effective (p ≤ 0.05) at MBCs of 5-10 mg CT/ml against APEC (Gram-negative), and at 1.25-5 mg CT/ml on S. epidermidis (Gram-positive). This indicated that the CTs were more potent against the Gram-positive than the Gram-negative bacteria. Further, SEM revealed that CTs caused mainly morphological deformations of the bacterial cells and some conjoined cell growth.

Nutlets of Tilia cordata Mill. and Tilia platyphyllos Scop. - Source of bioactive compounds.

The aim of this study was to analyse contents of phytosterols, carotenoids, tocochromanols and fatty acid composition in oil from nutlets of linden. Standard determination methods for these compounds were applied using liquid and gas chromatography. In the analysed oils from small and large-leaved lime the dominant acids included linoleic (53.1 and 54.1%), oleic (18.5 and 22.2%), palmitic (15.3 and 11.8%). Linden seed oil was characterised by very high contents of phytosterols (2-2.5 g/100 g). ß-Sitosterol was the dominant phytosterol (80-83%). Additionally squalene was detected at 806 and 607 mg/100 g, respectively, for Tilia cordata and T.platyphyllos. It was found that linden seed oil contains tocopherols (93%) and tocotrienols (7%). In terms of tocochromanol contents this oil is surpassed only by wheat germ oil. In terms of the α-T equivalent these oils contain 95 (Tilia cordata) and 50.6 mg/100 g vitamin E (Tilia platyphyllos).

A mixture of quercetin 4'-O-rhamnoside and isoquercitrin from Tilia americana var. mexicana and its biotransformation products with antidepressant activity in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The aerial parts of Tilia americana var. mexicana (Malvaceae, formerly Tiliaceae) or "sirimo" are used in Mexican traditional medicine for the relief of mild symptoms of mental stress, commonly referred to as "nerve diseases". Individuals use this plant to fall asleep, to calm states of nervous excitement, headaches, mood disorders, and general discomfort. Recent studies indicated that fractions standardized in their flavonoid content possess antidepressant activity in behavioral assays in mice. The present study aims to focus on the evaluation of the antidepressant effect of the mixture of two flavonoids (FMix), and its interaction with serotonergic drugs. Also, the pharmacological effect of the products of the metabolism of aglycone, quercetin, was evaluated in mice subjected to forced swimming test (FST) and open field test (OFT). MATERIALS AND METHODS: A methanol-soluble extract obtained from leaves of Tilia americana was fractionated in an open column chromatographic separation. One of the fractions contained FMix wich is constituted of the mixture of quercetin 4'-O-rhamnoside (1, 47%) y isoquercitrin (2, 53%). The mice were divided into the several following groups: FMix (0.01, 0.1, 0.5, 1.0, and 2 mg/kg); FMix (1.0 mg/kg) and agonist DOI (2.0 mg/kg); FMix (1.0 mg/kg) and antagonist ketanserin (KET, 0.03 mg/kg) of 5-HT2A receptors; FMix (1.0 mg/kg) and selective agonist 8-OH-DPAT (8-OH, 0.01 mg/kg); FMix (1.0 mg/kg) and antagonist WAY100635 (WAY, 0.5 mg/kg) of 5HT1 receptors; Phloroglucinol (PHL); 3,4-dihydroxy-phenyl acid (DOPAC); p-hydroxyphenyl acetic acid (p-HPAA); and m-hydroxyphenyl acetic acid (m-HPAA) were tested in FST or OFT. RESULTS: FMix induced dependent-dose antidepressant activity and, at the highest dose administered, a sedative effect was also observed. The 8-OH-DPAT, or the DOI, or the KET combination with FMix (1.0 mg/kg) induced a higher antidepressant effect than compounds alone; there was no effect exerted with WAY. The activity on OFT increased only with the FMix and KET combination. At the same time, the products of the aglycone metabolism of quercetin, that is, DOPAC and p-HPAA, decreased the immobility time of the mice in FST at 1.0 mg/kg, and a dose-curve was formed for these. CONCLUSION: The antidepressant effect of FMix could depend, at least in part, on the degradation products of quercetin and with a possible action mode through interaction with the serotoninergic system.

Use of an Animal Model to Evaluate Anxiolytic Effects of Dietary Supplementation with Tilia tomentosa Moench Bud Extracts.

Anxiety disorders are common and complex psychiatric syndromes affecting a broad spectrum of patients. On top of that, we know that aging produces an increase in anxiety vulnerability and sedative consumption. Moreover, stress disorders frequently show a clear gender susceptibility. Currently, the approved pharmacological strategies have severe side effects such as hallucinations, addiction, suicide, insomnia, and loss of motor coordination. Dietary integration with supplements represents an intriguing strategy for improving the efficacy and the safety of synthetic anxiolytics. Accordingly, a recent article demonstrated that glyceric bud extracts from Tilia tomentosa Moench (TTBEs) exert effects that are consistent with anxiolytic activity. However, the effects of these compounds in vivo are unknown. To examine this question, we conducted behavioral analysis in mice. A total of 21 days of oral supplements (vehicle and TTBEs) were assessed by Light Dark and Hole Board tests in male and female mice (young, 3 months; old, 24 months). Interestingly, the principal component analysis revealed gender and age-specific behavioral modulations. Moreover, the diet integration with the botanicals did not modify the body weight gain and the daily intake of water. Our results support the use of TTBEs as dietary supplements for anxiolytic purposes and unveil age and gender-dependent responses.

Polyherbal Formulation Enhancing Cerebral Slow Waves in Sleeping Rats.

Polyherbal medicines are composed of multiple herbs and have traditionally been used in East Asian countries for the remedy of physiological symptoms. Although the effects of polyherbal formulations have been investigated at the molecular and behavioral levels, less is known about whether and how medicinal herbs affect the central nervous system in terms of neurophysiology. We introduced a novel blended herbal formulation that consisted of 35% linden, 21% mulberry, 20% lavandin, 20% butterfly pea, and 4% tulsi. After intraperitoneal administration of this formulation or saline, we simultaneously recorded epidural electrocorticograms (ECoGs) from the olfactory bulb (OB), primary somatosensory cortex (S1), and primary motor cortex (M1), along with electromyograms (EMGs) and electrocardiograms (ECGs), of rats exploring an open field arena. Using the EMGs and OB ECoGs, we segmented the behavioral states of rats into active awake, quiet awake, and sleeping states. Compared to saline, herbal medicine significantly shortened the total sleep time. Moreover, we converted the ECoG signal into a frequency domain using a fast Fourier transform (FFT) and calculated the powers at various ECoG oscillation frequencies. In the sleeping state, a slow component (0.5-3 Hz) of S1 ECoGs was significantly enhanced following the administration of the formulation, which suggests a region- and frequency-specific modulation of extracellular field oscillations by the polyherbal medicine.

Honey as Source of Nitrogen Compounds: Aromatic Amino Acids, Free Nucleosides and Their Derivatives.

The content of selected major nitrogen compounds including nucleosides and their derivatives was evaluated in 75 samples of seven varieties of honey (heather, buckwheat, black locust, goldenrod, canola, fir, linden) by targeted ultra-high performance liquid chromatography-diode array detector - high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QqTOF-MS) and determined by UHPLC-DAD. The honey samples contained nucleosides, nucleobases and their derivatives (adenine: 8.9 to 18.4 mg/kg, xanthine: 1.2 to 3.3 mg/kg, uridine: 17.5 to 51.2 mg/kg, guanosine: 2.0 to 4.1 mg/kg; mean amounts), aromatic amino acids (tyrosine: 7.8 to 263.9 mg/kg, phenylalanine: 9.5 to 64.1 mg/kg; mean amounts). The amounts of compounds significantly differed between some honey types. For example, canola honey contained a much lower amount of uridine (17.5 ± 3.9 mg/kg) than black locust where it was most abundant (51.2 ± 7.8 mg/kg). The presence of free nucleosides and nucleobases in different honey varieties is reported first time and supports previous findings on medicinal activities of honey reported in the literature as well as traditional therapy and may contribute for their explanation. This applies, e.g., to the topical application of honey in herpes infections, as well as its beneficial activity on cognitive functions as nootropic and neuroprotective, in neuralgia and is also important for the understanding of nutritional values of honey.

Comparison of Protein Precipitation Ability of Structurally Diverse Procyanidin-Rich Condensed Tannins in Two Buffer Systems.

The protein precipitation (PP) of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by four procyanidin-rich condensed tannin (CT) samples in both 2-[N-morpholino]ethanesulfonic acid (MES) and a modified Goering-Van Soest (GVS) buffer is described. Purified CT samples examined included Vitis vinifera seed (mean degree of polymerization [mDP] 4.1, 16.5% galloylated), Tilia sp. flowers (B-type linkages, mDP 5.9), Vaccinium macrocarpon berries (mDP 8.7, 31.7% A-type linkages). and Trifolium pratense flowers (B-type linkages, mDP 12.3) and were characterized by 2D NMR (>90% purity). In general, CTs precipitated ALF > LYS ≥ BSA. PP in GVS buffer was 1 to 2.25 times greater than that in MES buffer (25 °C). The GVS buffer system better reflects the results/conclusions from the literature on the impacts mDP, galloylation, and A-type linkages have on PP. Determinations of PP using the MES buffer at 37 °C indicated that some of these differences may be attributed to the temperature at which GVS buffer determinations are conducted. In vitro PP studies using the GVS buffer may offer better guidance when selecting CT-containing forages and amendments for ruminant feeding studies.

A green, accurate and sensitive analytical method based on vortex assisted deep eutectic solvent-liquid phase microextraction for the determination of cobalt by slotted quartz tube flame atomic absorption spectrometry.

Preconcentration of cobalt was carried out with deep eutectic solvent based liquid phase microextraction (DES-LPME) for trace determination by a slotted quartz tube (SQT) attached flame atomic absorption spectrometry (FAAS) system. Choline chloride and phenol in a 1:2 M ratio was used as a green solvent to extract cobalt from the aqueous sample solution. Key parameters influencing the extraction efficiency of cobalt were examined and optimized. Under the conditions optimized, the linear dynamic range was found between 5.0 and 50 µg L-1, and the limits of detection and quantification (LOD and LOQ) were calculated as 2.0 and 6.6 µg L-1, respectively. The detection power of the conventional FAAS was improved upon by 67 folds using the optimized DES-LPME-SQT-FAAS method. The developed analytical method was successfully applied for the determination of cobalt in linden tea samples and the recovery results obtained for different spiked concentrations (20, 30 and 40 µg L-1) were remarkable (≈100%).

Secondary metabolites of Tilia tomentosa Moench inflorescences collected in Central Italy: chemotaxonomy relevance and phytochemical rationale of traditional use.

In this paper, the phytochemical analysis on the inflorescences (flowers and bracts) of a sample of Tilia tomentosa collected from an isolated population in Central Italy, was reported for the first time. Thirteen compounds (oleanolic acid, maslinic acid, apigenin, luteolin, kaempferol, quercetin, kaempferol-3-O-glucoside, quercetin-3-O-glucoside, rutin, tiliroside, ellagic acid, shikimic acid and quinic acid), belonging to several different classes of natural products, were identified on the basis of spectroscopic and spectrometric analysis. This sample was found to be mainly composed by flavonoids, followed by organic acids and pentacyclic triterpenes. The chemosystematic aspects of the identified components were also discussed, together with their pharmacologic relevance with respect to the traditional medicinal uses of this plant material.

Hypoglycemic Activity of Tilia americana, Borago officinalis, Chenopodium nuttalliae, and Piper sanctum on Wistar Rats.

Diabetes mellitus (DM) is considered the epidemic of the 21st century. Traditional medicine uses plants to treat DM; many of these have hypoglycemic effects in both animal models and diabetic patients. Our objective was to evaluate the hypoglycemic activity of Tilia americana, Borago officinalis, Chenopodium nuttalliae, and Piper sanctum on diabetic rats. The methanolic extracts of the plants under study were obtained by Soxhlet extraction. Toxicity was evaluated on Artemia salina; the antioxidant potential was evaluated using the DPPH technique. Hypoglycemic capacity at doses of 250 and 500 mg/kg was tested on Wistar rats with diabetes induced by alloxan (120 mg/kg). The toxicity on A. salina was null for the extracts of B. officinalis and P. sanctum, moderate for T. americana, and highly toxic for C. nuttalliae. The relevant extract of T. americana var. mexicana showed antioxidant activity. Three plants of the studied plants showed hypoglycemic activity: Tilia Americana (p = 0.0142), Borago officinalis (p = 0.0112), and Piper sanctum (p = 0.0078); P. sanctum was the one that showed the greatest reduction in glucose levels at a lower dose.

Magnetic and SEM-EDS analyses of Tilia cordata leaves and PM10 filters as a complementary source of information on polluted air: Results from the city of Parma (Northern Italy).

In this work, both PM10 filters and leaves have been collected, on a daily basis, over a period of five months and compared systematically. Filters were taken from an air-quality monitoring station and leaves from two Tilia cordata trees, both located near the railway station of Parma. SEM-EDS analysis on the surface and across the leaves shows that magnetic particles are almost entirely made of magnetite, and that they are found invariably on the leaves surface. The saturation isothermal magnetic remanence (SIRM) shows that for both filters and leaves the magnetic fraction mainly consists of a low coercivity, magnetite-like phase. The magnetic signals of filter and leaves and atmospheric PM concentrations are compared. The correlation is better for filters, mostly with parameters related to vehicular pollution, and improved for both filters and leaves once data were averaged on a 10 days basis. Filters and leaves equally show an increase in magnetic signal during the fall-winter period together with PM10 content. The comparison between leaves and filters shows that: 1) leaves give a qualitative picture, and in our case they could be used as environmental proxies after averaging the results over multiple days; 2) the correlation with PM10 is weaker, indicating that there is a PM10 contribution from non-magnetic particles, like calcite and clay minerals, pollen and spores; 3) multidomain particles contribution from filters indicates a strong relation with vehicular polluters, suggesting the important role of larger particles; 4) magnetization from leaves and filters are weakly related, due to the different sampling lapse.

The influence of procyanidins isolated from small-leaved lime flowers (Tilia cordata Mill.) on human neutrophils.

Linden flower is a wildly used plant material among patients in the treatment of common cold symptoms and mucosa inflammations. However, the structure and bioactivity of flavan-3-ol derivatives present in infusions from flowers of Tilia cordata have not been studied so far. The aim of current study was to isolate and identify main procyanidins present in the flowers of small-leaved lime and to evaluate their influence on the inflammatory response of human neutrophils ex vivo. The chemical structure of isolated compounds was established by 1D and 2D NMR experiments. The bioactivity of obtained compounds was tested in human neutrophils model. Cytotoxicity and influence of compounds on apoptosis was established by flow cytometry. The levels of produced cytokines were established by ELISA after stimulation with lipopolysaccharide (LPS). The inhibition of the production of reactive oxygen species was checked by luminol-dependent chemiluminescence method after N-formylmethionyl-leucyl-phenylalanine (f-MLP) induction. The phytochemical work resulted in the isolation of 10 compounds. Compounds were identified as oligomeric procyanidins and their precursor epicatechin. The potential anti-inflammatory activity of compounds was evaluated in the concentration range 5-20 µM. All compounds were able to decrease the production of ROS from f-MLP-stimulated neutrophils. Most of compounds were able to inhibit the LPS-induced release of IL-8. Some trimeric and tetrameric derivatives were also able to decrease the production of MIP-1ß. Obtained results partially support the traditional usage of infusion from lime flowers in the treatment of symptoms of inflammation and irritation of mucosa in common cold, pharyngitis and tonsillitis.

Acidic polysaccharide complexes from purslane, silver linden and lavender stimulate Peyer's patch immune cells through innate and adaptive mechanisms.

Three polysaccharide complexes (PSCs) were isolated from the aerial parts of common purslane (Portulaca oleracea L.), and the flowers of common lavender (Lavandula angustifolia Mill.) and silver linden (Tilia tomentosa Moench) by boiling water extraction and ethanol precipitation. The chemical composition and immunomodulating effects of isolated PSCs were characterized. The chemical characterization revealed that the three samples contain mainly pectic polysaccharides. They exhibited ex vivo intestinal immunomodulating activity through the murine Peyer's patch-mediated bone marrow cell proliferation test at 100µg/ml concentration. At the same time, they stimulated ex vivo human blood T-cell populations (CD4+/CD25+ and CD8+/CD25+), phagocytic leukocytes (CD14+ and CD64+ cells) and induced IL-6 production from human white blood cells and Peyer's patch cells. The herbal PSCs stimulated ex vivo ROS production from whole blood phagocytes and showed unspecific in vitro anti-proliferative activity against normal and A549, HeLa and LS180 tumor cells. This is the first report on immunomodulating studies of linden flower pectins and chemical and biological activity characterization of lavender polysaccharides. Our study demonstrates that similarly to purslane, lavender and silver linden herbal materials contain immunomodulating polysaccharides that could be useful for support of compromised immune system.

Pharmacokinetic Study of Biotransformation Products from an Anxiolytic Fraction of Tilia americana.

An anxiolytic fraction of Tilia americana standardized in tiliroside, rutin, quercitrin, quercetin glucoside, and kaempferol was obtained. After oral administration of the fraction, the above-mentioned flavonoids were not detected in plasma over 24 h. However, meta and para hydroxyphenylacetic acid and dihydroxyphenylacetic acid (m-HPAA, p-HPAA and DOPAC) were monitored. These are the biotransformation compounds of the aglycones of kaempferol and quercetin; these aglycones are products of the other flavonoids present in the anxiolytic fraction. The analytical methods (HPLC) for flavonoids and the related compounds (m-HPAA, p-HPAA and DOPAC) were validated, determining the parameters of accuracy, precision, specificity or selectivity, limit of detection, quantification range, and robustness. The pharmacokinetic assay was performed with ICR mice strains, which were given 200 mg/kg of the standardized active fraction. The results of validation of the analytical methods were obtained, allowing it to be established in a validated way that no flavonoids present in the anxiolytic fraction of T. americana were detected in plasma. However, detection and follow up was possible for the serum levels of m-HPAA, p-HPAA, and DOPAC. The three compounds follow a two-compartment model with very similar parameters between m-HPAA and p-HPAA, some being different from the ones characterized in the pharmacokinetics of DOPAC.

Contribution of the phenolic composition to the antioxidant, anti-inflammatory and antitumor potential of Equisetum giganteum L. and Tilia platyphyllos Scop.

Naturally-occurring phytochemicals have received pivotal attention in the last few years, due to the increasing evidence of biological activities. Thus, in the present study, the antioxidant, anti-inflammatory and antitumor potentials of hydroethanolic extracts rich in phenolic compounds obtained from Equisetum giganteum L. and Tilia platyphyllos Scop. were assessed and directly correlated with their content of phenolic compounds, by using HPLC-DAD-ESI/MS analysis. T. platyphyllos showed the higher bioactive potential, evaluated in terms of antioxidant (radical scavenging effects - 105 µg mL-1, reducing power - 123 µg mL-1, ß-carotene bleaching inhibition - 167 µg mL-1, and lipid peroxidation inhibition - 56 µg mL-1), anti-inflammatory (225 µg mL-1 inhibited 50% of nitric oxide production) and antitumor (breast - 224 µg mL-1; lung - 247 µg mL-1; cervical - 195 µg mL-1 and hepatocellular - 173 µg mL-1 carcinoma cells) activity, without having cytotoxic effects (>400 µg mL-1). These biological properties were positively correlated with its content and composition of phenolic compounds. Flavonoid contents were markedly higher than the content of phenolic acids, in both samples, being respectively 50.4 mg g-1 and 11.65 mg g-1 for T. platyphyllos, and 21.7 mg g-1 and 4.98 mg g-1 for E. giganteum. Moreover, while in E. giganteum extract, kaempferol-O-glucoside-O-rutinoside was the most abundant flavonoid, in T. platyphyllos extract protocatechuic acid and (-)-epicatechin were the most abundant phenolic acid and flavonoid, respectively. In relation to their content of phenolic acids, protocatechuic and caffeic acids existed in higher abundance in T. platyphyllos and E. giganteum hydroethanolic extracts, respectively. However, it would be interesting to evaluate the in vivo efficacy of both plant extracts to unveil the involved modes of action and to establish effective therapeutic doses.

Neuroprotective effects of Tilia americana var. mexicana on damage induced by cerebral ischaemia in mice.

Tilia americana var. mexicana (T. americana) is a plant widely used in Mexico for its medicinal properties on the central nervous system. In the present study, we designed a protocol to investigate the neuroprotective effects of non-polar and polar extracts of T. americana on damage induced by cerebral ischaemia in mice. Vehicle or extracts were administered immediately after ischaemia. Functional neurological deficit, survival percentage and infarct area were determined in each experimental group. Results showed that groups treated with non-polar or polar extracts of T. americana had increased survival rate, improved neurological deficits and diminished the infarct area in relation to the ischaemic group. In conclusion, this study confirms the neuroprotective activity of T. americana, suggests a possible synergism between non-polar and polar constituents and supports its potential as a useful aid in the clinical management of stroke.

The novel approach to the biomonitor survey using one- and two-dimensional Kohonen networks.

To compare the applicability of the leaves of horse chestnut (Aesculus hippocastanum) and linden (Tilia spp.) as biomonitors of trace element concentrations, a coupled approach of one- and two-dimensional Kohonen networks was applied for the first time. The self-organizing networks (SONs) and the self-organizing maps (SOMs) were applied on the database obtained for the element accumulation (Cr, Fe, Ni, Cu, Zn, Pb, V, As, Cd) and the SOM for the Pb isotopes in the leaves for a multiyear period (2002-2006). A. hippocastanum seems to be a more appropriate biomonitor since it showed more consistent results in the analysis of trace elements and Pb isotopes. The SOM proved to be a suitable and sensitive tool for assessing differences in trace element concentrations and for the Pb isotopic composition in leaves of different species. In addition, the SON provided more clear data on seasonal and temporal accumulation of trace elements in the leaves and could be recommended complementary to the SOM analysis of trace elements in biomonitoring studies.

Phenolic composition of "bud extracts" of Ribes nigrum L., Rosa canina L. and Tilia tomentosa M.

Liquid preparations such as tinctures and "bud extracts" are typical products widely used in European countries but which have been poorly studied to date. Our research was focused on obtaining a phytochemical characterization of secondary metabolites of black currant, dog rose and silver linden bud extracts and evaluating the changes of selected chemical markers over time. 16 different preparations have been analyzed by the use of HPLC-DAD-MS. Several flavonols were identified in black currant samples including quercetin derivatives (6.7-30.4mg/L). Also hydroxycinnamic acids, mainly p-coumaric acid derivatives (4.1-38.9mg/L) were identified. Flavonols such as glycosides of quercetin and kaempferol, hydroxycinnamic acids, and ellagitannins were detected in dog rose samples, with gallotannins being the main components (up to 1.7g/L). The Tilia tomentosa extracts contained flavonols (quercetin, kaempferol, apigenin derivatives) as principal components with the exception of a single commercial extract with hydroxycinnamic acids as the most abundant metabolites. In this study, without applying accelerated ageing protocols, the stability over time of these liquid preparations was evaluated for up to 10 months and demonstrated negligible variations.