Phytochemical and Bioactivity Studies on Hedera helix L. (Ivy) Flower Pollen and Ivy Bee Pollen.

Bee pollen, known as a 'life-giving dust', is a product of honeybees using flower pollen grains and combining them with their saliva secretions. Thus, flower pollen could be an indicator of the bee pollen botanical source. Identification of bee pollen sources is a highly crucial process for the evaluation of its health benefits, as chemical composition is directly related to its pharmacological activity. In this study, the chemical profiles, contents of phenolic marker compounds and pharmacological activities of Hedera helix L. (ivy) bee pollen samples from Türkiye and Slovenia, as well as ivy flower pollen grains, were compared. High-performance thin-layer chromatography (HPTLC) analyses revealed that pollen samples, regardless of where they were collected, have similar chemical profiles due to the fact that they have the same botanical origins. Marker compounds afzelin, platanoside and quercetin-3-O-ß-glucopyranosyl-(1→2)-ß-galactopyranoside, common to both bee pollen and flower pollen, were isolated from bee pollen, and their structures were elucidated by nuclear magnetic resonance (NMR) and mass spectrometry (MS). These three compounds, as well as chlorogenic acid and 3,5-dicaffeoylquinic acid (found in flower pollen), were quantified using high-performance liquid chromatography (HPLC) analyses. In vitro tests and effect-directed analyses were used to evaluate the xanthine oxidase inhibition and antioxidant activity of the marker compounds and extracts from flower pollen and bee pollen. This is the first report comparing chemical profiles and related bioactivities of the flower pollen and bee pollen of the same botanical origin, as well as the first report of the chemical profile and related bioactivities of ivy flower pollen.

The Role of Isoflavones in the Prevention of Breast Cancer and Prostate Cancer.

This narrative review summarizes epidemiological studies on breast cancer and prostate cancer with an overview of their global incidence distribution to investigate the relationship between these diseases and diet. The biological properties, mechanisms of action, and available data supporting the potential role of isoflavones in the prevention of breast cancer and prostate cancer are discussed. Studies evaluating the effects of isoflavones in tissue cultures of normal and malignant breast and prostate cells, as well as the current body of research regarding the effects of isoflavones attained through multiple modifications of cellular molecular signaling pathways and control of oxidative stress, are summarized. Furthermore, this review compiles literature sources reporting on the following: (1) levels of estrogen in breast and prostate tissue; (2) levels of isoflavones in the normal and malignant tissue of these organs in European and Asian populations; (3) average concentrations of isoflavones in the secretion of these organs (milk and semen). Finally, particular emphasis is placed on studies investigating the effect of isoflavones on tissues via estrogen receptors (ER).

Resveratrol Food Supplement Products and the Challenges of Accurate Label Information to Ensure Food Safety for Consumers.

The food supplement market is growing as many consumers wish to complement their nutrient intake. Despite all the regulations in place to ensure food supplements safety, there are still many cases of irregularities reported especially connected to internet sales. Twenty resveratrol food supplement products sold on the Slovenian market were evaluated on their compliance of declared vs. determined resveratrol content, as well as the compliance of labels with the European Union (EU) and Slovenian regulatory requirements. Both the ingredient contents and food information are important parts of food safety. Analyses of 20 food supplements performed using high-performance thin-layer chromatography (HPTLC) coupled with densitometry showed that 95% of products had contents different from what was declared and 55% of products contained higher contents than declared. In 25% of the products the determined content per unit exceeded the maximum level (150 mg/day) specified in EU novel food conditions for food supplement with trans-resveratrol. Evaluation of the 20 food supplement labels included mandatory and voluntary food information, food supplement information, novel food information, health claims and nutrition claims. Most labels contained the necessary information, but multiple errors were observed ranging from typos to misleading practices. From a food safety perspective there is still a lot of improvement needed in the field of food supplements.

Extraction of Polyphenols and Valorization of Fibers from Istrian-Grown Pomegranate (Punica granatum L.).

Pomegranate fruit is an ancient fruit that is used not only because of its deep-red color and tasty arils but also due to the health benefits of its extracts. Pomegranate is a valuable source of bioactive compounds, including colorful anthocyanins and other polyphenols. The main objective of the present study was to gain comprehensive knowledge of the phenolic composition and antioxidative activity of a new pomegranate cultivar, grown in Northwest Istria, a part of the North Adriatic coastal area. Various parts of the pomegranate fruit parts were extracted in 70% ethanol or water. Total phenolic content and antioxidative capacity were respectively determined with Folin-Ciocalteu reagent and ABTS radical. Phenolics were examined and analyzed with TLC, LC-MS, and HPLC. Pomegranate juice was prepared from red arils and after thermal treatment, the stability of anthocyanins was monitored for several months to understand the effect of storage. The highest total phenolics were determined in ethanol pomegranate peel extracts (30.5 ± 0.6 mg GAE/g DM), and water peel extracts exhibited the highest antioxidative activity (128 ± 2 µg TE/g DM). After five months of storage of thermally treated pomegranate juice, 50-60 percentage points increase in anthocyanin degradation was observed. Pomegranate peel was further tested as a sustainable inedible food source for papermaking. Due to the low content of cellulose and the high percentage of extractives, as well as a distinguished texture and appearance, the paper made from pomegranate peel is best suited for the production of specialty papers, making it particularly interesting for bioactives recovery, followed by material restructuring.

Polyphenolic and Chemical Profiles of Honey From the Tara Mountain in Serbia.

This study presents a detailed characterization of 27 honey samples from the Tara Mountain region in Serbia using different comprehensive techniques and methods. The types of the honey samples were defined as monofloral (4 samples), honeydew (5 samples) and polyfloral (18 samples) honey based on determined polyphenol content, antioxidant activity, electrical conductivity and melissopalynological analyses. Physicochemical parameters such as pH (4.13-4.94), diastase activity (24.20-41.70 DN), acidity (14.60-29.70 meq/kg), content of 5-(hydroxymethyl)furfural (in range below 5, up to 16.90 mg/kg), sucrose (0.20-3.90 g/100 g), and moisture content (15.01-19.23%) confirmed the required quality of the honey samples. Sensory analysis revealed honey characteristics favorable to consumers. Analyses of 19 phenolic compounds using ultra-high-performance liquid chromatography with a diode-array detection and triple quadrupole mass spectrometry (UHPLC-DAD-MS/MS) revealed six phenolic acids and 13 other compounds from the group of flavonoids and their glycosides. In all the samples the highest content was determined for p-coumaric acid, followed by caffeic acid and pinocembrin. Besides total phenolic content and radical scavenging activity, antimicrobial activity was also examined. Most honey samples showed bactericidal activity against Staphylococcus aureus and bacteriostatic activity against Escherichia coli, while none of the honey samples inhibited the growth of Candida albicans. Chemometric analyses were applied for an in-depth study of the results to further evaluate the characteristics of the honey samples studied. Principal component analysis (PCA) was used for assessing the differences in physicochemical parameters, polyphenols content and antioxidant capacity between honey samples. The unrooted cluster tree was used to group the samples based on the melissopalynological analyses.

High-performance thin-layer chromatography - antibacterial assay first reveals bioactive clerodane diterpenes in giant goldenrod (Solidago gigantea Ait.).

The present work introduces a high-performance thin-layer chromatography (HPTLC)-direct bioautography method using the Gram-positive plant pathogenic bacterium, Rhodococcus fascians. The screening and isolation procedure comprised of a non-targeted high-performance thin-layer chromatography-effect-directed analysis (HPTLC-EDA) against Bacillus subtilis, B. subtilis subsp. spizizenii, R. fascians, and Aliivibrio fischeri, a targeted HPTLC-mass spectrometry (MS), and bioassay-guided column chromatographic (preparative flash and semi-preparative HPLC) fractionation and purification. The developed new separation methods enabled the discovery of four bioactive cis-clerodane diterpenes, solidagoic acid H (1), solidagoic acid E (2), solidagoic acid I (3), and solidagoic acid F (4), in the n-hexane extract of giant goldenrod (Solidago gigantea Ait.) leaf for the first time. These compounds were identified by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. The initially used HPTLC method (chloroform - ethyl acetate - methanol 15:3:2, V/V/V) was changed (to n-hexane - isopropyl acetate - methanol - acetic acid 29:20:1:1, V/V/V/V) to achieve the separation of the closely related isomer pairs (1-2 and 3-4). Compounds 1 and 3 exhibited moderate antibacterial activity against the Gram-positive B. subtilis subsp. spizizenii and R. fascians bacterial strains in microdilution assays with half-maximal inhibitory concentration (IC50) values in the range of 32.3-64.4 µg/mL. The mass spectrometric fragmentation of the isolated compounds was interpreted and their previously published NMR assignments lacking certain resonances were completed.

Antioxidant and Antimicrobial Biofoil Based on Chitosan and Japanese Knotweed (Fallopia japonica, Houtt.) Rhizome Bark Extract.

A 70% ethanol(aq) extract of the rhizome bark of the invasive alien plant species Japanese knotweed (JKRB) with potent (in the range of vitamin C) and stable antioxidant activity was incorporated in 1% w/v into a chitosan biofoil, which was then characterized on a lab-scale. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay confirmed the antioxidant activity of the JKRB biofoil upon contact with the food simulants A, B, C, and D1 (measured half-maximal inhibitory concentrations-IC50) and supported the Folin-Ciocalteu assay result. The migration of the antioxidant marker, (-)-epicatechin, into all food simulants (A, B, C, D1, D2, and E) was quantified using liquid chromatography hyphenated to mass spectrometry (LC-MS). Calculations showed that 1 cm2 of JKRB biofoil provided antioxidant activity to ~0.5 L of liquid food upon 1 h of contact. The JKRB biofoil demonstrated antimicrobial activity against Gram-positive bacteria. The incorporation of JKRB into the chitosan biofoil resulted in improved tensile strength from 0.75 MPa to 1.81 MPa, while elongation decreased to 28%. JKRB biofoil's lower moisture content compared to chitosan biofoil was attributed to the formation of hydrogen bonds between chitosan biofoil and JKRB compounds, further confirmed with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The JKRB biofoil completely degraded in compost in 11 days. The future upscaled production of JKRB biofoil from biowastes for active packaging may support the fights against plastic waste, food waste, and the invasiveness of Japanese knotweed, while greatly contributing to the so-called 'zero-waste' strategy and the reduction in greenhouse gas emissions.

Flavan-3-ols and Proanthocyanidins in Japanese, Bohemian and Giant Knotweed.

Flavan-3-ols and proanthocyanidins of invasive alien plants Japanese knotweed (Fallopia japonica Houtt.), giant knotweed (Fallopia sachalinensis F. Schmidt) and Bohemian knotweed (Fallopia × bohemica (Chrtek & Chrtkova) J.P. Bailey) were investigated using high performance thin-layer chromatography (HPTLC) coupled to densitometry, image analysis and mass spectrometry (HPTLC-MS/MS). (+)-Catechin, (-)-epicatechin, (-)-epicatechin gallate and procyanidin B2 were found in rhizomes of these three species, and for the first time in Bohemian knotweed. (-)-Epicatechin gallate, procyanidin B1, procyanidin B2 and procyanidin C1 were found in giant knotweed rhizomes for the first time. Rhizomes of Bohemian and giant knotweed have the same chemical profiles of proanthocyanidins with respect to the degree of polymerization and with respect to gallates. Japanese and Bohemian knotweed have equal chromatographic fingerprint profiles with the additional peak not present in giant knotweed. Within the individual species giant knotweed rhizomes and leaves have the most similar fingerprints, while the fingerprints of Japanese and Bohemian knotweed rhizomes have additional peaks not found in leaves. Rhizomes of all three species proved to be a rich source of proanthocyanidins, with the highest content in Japanese and the lowest in Bohemian knotweed (based on the total peak areas). The contents of monomers in Japanese, Bohemian and giant knotweed rhizomes were 2.99 kg/t of dry mass (DM), 1.52 kg/t DM, 2.36 kg/t DM, respectively, while the contents of dimers were 2.81 kg/t DM, 1.09 kg/t DM, 2.17 kg/t DM, respectively. All B-type proanthocyanidins from monomers to decamers (monomers-flavan-3-ols, dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers and decamers) and some of their gallates (monomer gallates, dimer gallates, dimer digallates, trimer gallates, tetramer gallates, pentamer gallates and hexamer gallates) were identified in rhizomes of Bohemian knotweed and giant knotweed. Pentamer gallates, hexamers, hexamer gallates, nonamers and decamers were identified for the first time in this study in Bohemian and giant knotweed rhizomes.

(-)-Epicatechin-An Important Contributor to the Antioxidant Activity of Japanese Knotweed Rhizome Bark Extract as Determined by Antioxidant Activity-Guided Fractionation.

The antioxidant activities of Japanese knotweed rhizome bark extracts, prepared with eight different solvents or solvent mixtures (water, methanol, 80% methanol(aq), acetone, 70% acetone(aq), ethanol, 70% ethanol(aq), and 90% ethyl acetate(aq)), were determined using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay. Low half maximal inhibitory concentration (IC50) values (2.632-3.720 µg mL-1) for all the extracts were in the range of the IC50 value of the known antioxidant ascorbic acid at t0 (3.115 µg mL-1). Due to the highest extraction yield (~44%), 70% ethanol(aq) was selected for the preparation of the extract for further investigations. The IC50 value calculated for its antioxidant activity remained stable for at least 14 days, while the IC50 of ascorbic acid increased over time. The stability study showed that the container material was of great importance for the light-protected storage of the ascorbic acid(aq) solution in a refrigerator. Size exclusion-high-performance liquid chromatography (SEC-HPLC)-UV and reversed phase (RP)-HPLC-UV coupled with multistage mass spectrometry (MSn) were developed for fractionation of the 70% ethanol(aq) extract and for further compound identification, respectively. In the most potent antioxidant SEC fraction, determined using an on-line post-column SEC-HPLC-DPPH assay, epicatechin, resveratrol malonyl hexoside, and its in-source fragments (resveratrol and resveratrol acetyl hexoside) were tentatively identified by RP-HPLC-MSn. Moreover, epicatechin was additionally confirmed by two orthogonal methods, SEC-HPLC-UV and high-performance thin-layer chromatography (HPTLC) coupled with densitometry. Finally, the latter technique enabled the identification of (-)-epicatechin. (-)-Epicatechin demonstrated potent and stable time-dependent antioxidant activity (IC50 value ~1.5 µg mL-1) for at least 14 days.

Off-line multidimensional high performance thin-layer chromatography for fractionation of Japanese knotweed rhizome bark extract and isolation of flavan-3-ols, proanthocyanidins and anthraquinones.

A methodology based on off-line multidimensional thin-layer chromatography was developed for isolation of several secondary metabolites from bark of Japanese knotweed (Fallopia japonica Houtt.) rhizomes. Successive fractionation steps using PLC silica gel and HPTLC silica gel or HPTLC cellulose plates in combination with various developing solvents enabled isolation of (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, procyanidin B1, procyanidin B2, procyanidin B3, proanthocyanidin B dimer gallate, emodin, emodin-8-O-glucoside and emodin-8-O-malonyl-glucoside. Their identity was confirmed by HPTLC, HPTLC-MSn and for most of them also by 1H NMR and 2D NMR analyses. To the best of our knowledge emodin-8-O-malonyl-glucoside, procyanidins B1 and B2 were for the first time isolated from this plant material. HPTLC and HPTLC-MSn analyses were also performed as support of fractionation/isolation process, leading to first detection of some compounds in bark of Japanese knotweed rhizomes and Japanese knotweed rhizomes in general: procyanidins B1 and B2, methyl derivatives of emodin bianthrone and emodin bianthrone-hexose, resveratrol-malonyl-hexoside and taxifolin derivatives. Characterization of flavan-3-ols and proanthocyanidins was facilitated by post-chromatographic derivatization of the corresponding chromatographic zones with 4-dimethylaminocinnamaldehyde (DMACA) detection reagent.

Extraction of Anthraquinones from Japanese Knotweed Rhizomes and Their Analyses by High Performance Thin-Layer Chromatography and Mass Spectrometry.

Anthraquinones (yellow dyes) were extracted from Japanese knotweed rhizomes with twelve extraction solvents (water; ethanol(aq) (20%, 40%, 60%, 70% and 80%), ethanol, 70% methanol(aq), methanol, 70% acetone(aq), acetone and dichloromethane). The obtained sample test solutions (STSs) were analyzed using high-performance thin-layer chromatography (HPTLC) coupled to densitometry and mass spectrometry (HPTLC-MS/MS) on HPTLC silica gel plates. Identical qualitative densitometric profiles (with anthraquinone aglycones and glycosylated anthraquinones) were obtained for STSs in all the solvents except for the STS in dichloromethane, which enabled the most selective extractions of anthraquinone aglycones emodin and physcion. The highest extraction efficiency, evaluated by comparison of the total peak areas in the densitograms of all STSs scanned at 442 nm, was achieved for 70% acetone(aq). In STS prepared with 70% acetone(aq), the separation of non-glycosylated and glycosylated anthraquinones was achieved with developing solvents toluene-acetone-formic acid (6:6:1, 3:6:1 and 3:3:1 v/v) and dichloromethane-acetone-formic acid (1:1:0.1, v/v). Non-glycosylated anthraquinones were separated only with toluene-acetone-formic acid, among which the best resolution between emodin and physcion gave the ratio 6:6:1 (v/v). This solvent and dichloromethane-acetone-formic acid (1:1:0.1, v/v) enabled the best separation of glycosylated anthraquinones. Four HPTLC-MS/MS methods enabled the identification of emodin and tentative identification of its three glycosylated analogs (emodin-8-O-hexoside, emodin-O-acetyl-hexoside and emodin-O-malonyl-hexoside), while only the HPTLC-MS/MS method with toluene-acetone-formic acid (6:6:1, v/v) enabled the identification of physcion. Changes of the shapes and the absorption maxima (bathochromic shifts) in the absorption spectra after post-chromatographic derivatization provided additional proof for the detection of physcion and rejection of the presence of chrysophanol in STS.

Simplified LC-MS Method for Analysis of Sterols in Biological Samples.

We developed a simple and robust liquid chromatographic/mass spectrometric method (LC-MS) for the quantitative analysis of 10 sterols from the late part of cholesterol synthesis (zymosterol, dehydrolathosterol, 7-dehydrodesmosterol, desmosterol, zymostenol, lathosterol, FFMAS, TMAS, lanosterol, and dihydrolanosterol) from cultured human hepatocytes in a single chromatographic run using a pentafluorophenyl (PFP) stationary phase. The method also avails on a minimized sample preparation procedure in order to obtain a relatively high sample throughput. The method was validated on 10 sterol standards that were detected in a single chromatographic LC-MS run without derivatization. Our developed method can be used in research or clinical applications for disease-related detection of accumulated cholesterol intermediates. Disorders in the late part of cholesterol synthesis lead to severe malformation in human patients. The developed method enables a simple, sensitive, and fast quantification of sterols, without the need of extended knowledge of the LC-MS technique, and represents a new analytical tool in the rising field of cholesterolomics.

Oleamide, a Bioactive Compound, Unwittingly Introduced into the Human Body through Some Plastic Food/Beverages and Medicine Containers.

The purpose of the study was to investigate the migration of oleamide, a polymer lubricant, and a bioactive compound, from various plastic, marketed containers for food/beverages and medicines into polymer contact liquid. Methanol, food/medicine simulants or real samples were used to extract polymer leachables and extractables. Migrated oleamide into polymer contact liquids was determined by ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS). The concentration of oleamide in the extracts of medicinal and insulin syringes was 7351 ng mL-1 and 21,984 ng mL-1, respectively. The leachates of intravenous (i.v.) infusion bottle, medicinal and insulin syringes contained 17 ng mL-1, 12 ng mL-1 and 152 ng mL-1, respectively. Oleamide in the extracts of dummies ranged from 30 to 39 ng mL-1, while in the leachates of baby bottles, from 12 to 23 ng mL-1. Leachates of soft drink bottles contained from 6 to 15 ng mL-1 oleamide, milk bottles from 3 to 9 ng mL-1, liquid yogurt bottles 17 ng mL-1 and water bottles from 11 to 18 ng mL-1. Bottled real matrices of oil and milk contained oleamide in the range from 217 to 293 ng mL-1. Moreover, the source of migrated oleamide (e.g., containers, caps, other parts) was identified. Oleamide is listed in the current EU regulations without a specific migration limit. Accordingly, these values are considered of no concern, unless future toxicological studies prove the opposite.

Interference of oleamide with analytical and bioassay results.

During sample preparation and analysis, samples are coming in contact with different labware materials. By four unrelated analytical (phytochemical and pharmaceutical) case-studies and employing different analytical techniques, we demonstrated the potential misinterpretation of analytical results due to the use of contaminants-leaching labware during sample handling. Oleamide, a common polymer lubricant and a bioactive compound, was identified as a main analytical interference, leaching from different labware items into solvents, recognised as chemically compatible with the tested polymer material. Moreover, anti-inflammatory effect of oleamide at 100 µg mL-1 and considerable pro-inflammatory effect of the plastic syringe extractables (containing oleamide) at the same level were shown in a TLR4-based bioassay. Taking these results into account, together with the fact that oleamide can be a compound of natural origin, we would like to notify the professional public regarding the possible erroneous oleamide-related analytical and bioassay results due to the use of oleamide-leaching labware. Researchers are alerted to double check the real source of oleamide (labware or natural extract), which will prevent further reporting of false results. Analysis of procedural blanks with de-novo developed UHPLC-ESI-MS method is, among some other strategies, proposed for detection of oleamide interference and avoidance of misleading results of certain analyses.

Establishing the chromatographic fingerprints of flavan-3-ols and proanthocyanidins from rose hip (Rosa sp.) species.

The profile of flavan-3-ols and proanthocyanidins in five different Rosa species (R. canina, R. glutinosa, R. rubiginosa, R. multiflora, and R. spinosissima) was estimated on high performance thin layer chromatography cellulose plates. Differences in flavanol and proanthocyanidin profiles of the extracts were evident, among which Rosa spinosissima stood out with catechin as the only detected flavanol and red zones as indication of anthocyanins. Furthermore, the elution solvent for thin layer chromatography with mass spectrometry analyses of glycosylated flavan-3-ols and proanthocyanidins was optimized, enabling identification of catechin, (epi)catechin hexoside, proanthocyanidin dimer, and proanthocyanidin dimers and trimers hexosides. A total of 15 flavanols and their derivatives were identified using ultra-high-performance liquid chromatography with linear trap quadrupole-Orbitrap mass analyzer and epicatechin, gallocatechin, and proanthocyanidin trimer were identified only using this technique. However, proanthocyanidin trimer trihexoside was identified only by thin-layer chromatography with mass spectrometry. To establish the relationships between the flavanols and proanthocyanidins composition of rose hip and their origin, principal component analysis was performed on the entire set of liquid chromatography/mass spectrometry data. Both principal components' scores plots showed that Rosa spinosissima could be considered as an outlier. Our study demonstrated that flavanol and proanthocyanidin profiles of different rose hips depend on the geographical origin rather than on the cultivar and genotype.

Leaves of Invasive Plants-Japanese, Bohemian and Giant Knotweed-The Promising New Source of Flavan-3-ols and Proanthocyanidins.

This is the first report on identification of all B-type proanthocyanidins from monomers to decamers (monomers-flavan-3-ols, dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, and decamers) and some of their gallates in leaves of Japanese knotweed (Fallopia japonica Houtt.), giant knotweed (Fallopia sachalinensis F. Schmidt) and Bohemian knotweed (Fallopia × bohemica (Chrtek & Chrtkova) J.P. Bailey). Flavan-3-ols and proanthocyanidins were investigated using high performance thin-layer chromatography (HPTLC) coupled to densitometry, image analysis, and mass spectrometry (HPTLC-MS/MS). All species contained (-)-epicatechin and procyanidin B2, while (+)-catechin was only detected in Bohemian and giant knotweed. (-)-Epicatechin gallate, procyanidin B1 and procyanidin C1 was only confirmed in giant knotweed. Leaves of all three knotweeds have the same chemical profiles of proanthocyanidins with respect to the degree of polymerization but differ with respect to gallates. Therefore, chromatographic fingerprint profiles of proanthocyanidins enabled differentiation among leaves of studied knotweeds, and between Japanese knotweed leaves and rhizomes. Leaves of all three species proved to be a rich source of proanthocyanidins (based on the total peak areas), with the highest content in giant and the lowest in Japanese knotweed. The contents of monomers in Japanese, Bohemian and giant knotweed were 0.84 kg/t of dry weight (DW), 1.39 kg/t DW, 2.36 kg/t, respectively, while the contents of dimers were 0.99 kg/t DW, 1.40 kg/t, 2.06 kg/t, respectively. Giant knotweed leaves showed the highest variety of gallates (dimer gallates, dimer digallates, trimer gallates, tetramer gallates, pentamer gallates, and hexamer gallates), while only monomer gallates and dimer gallates were confirmed in Japanese knotweed and monomer gallates, dimer gallates, and dimer digallates were detected in leaves of Bohemian knotweed. The profile of the Bohemian knotweed clearly showed the traits inherited from Japanese and giant knotweed from which it originated.

Phenolic Composition Influences the Health-Promoting Potential of Bee-Pollen.

Information on compositional, nutritional and functional properties of bee-pollen, as a health-promoting food, is essential for defining its quality. Concerning the nutritional importance of phenolic compounds, the aim of this study was to determine the phenolic profile and antioxidant activity of twenty-four bee-pollen samples collected from different regions of Serbia. High-performance thin-layer chromatographic (HPTLC) fingerprinting was used for profiling of bee-pollen samples according to the botanical type. HPTLC hyphenated with image analysis and a pattern recognition technique confirmed the grouping of samples caused by the specific phenolic composition of pollens of different botanical origin. Flavonoid glycosides in bee-pollen samples were identified by applying ultra-high-performance liquid chromatography (UHPLC) coupled with linear ion trap-Orbitrap mass spectrometry (LTQ Orbitrap MS). Eight out of twenty-seven flavonol glycosides were identified in bee-pollen samples for the first time. All analyzed bee-pollen samples showed a high number of phenolic compounds which may have therapeutic potential.

Japanese and Bohemian Knotweeds as Sustainable Sources of Carotenoids.

Japanese knotweed (Fallopia japonica Houtt.) and Bohemian knotweed (Fallopia x bohemica) are invasive alien plant species, causing great global ecological and economic damage. Mechanical excavation of plant material represents an effective containment method, but it is not economically and environmentally sustainable as it produces an excessive amount of waste. Thus, practical uses of these plants are actively being sought. In this study, we explored the carotenoid profiles and carotenoid content of mature (green) and senescing leaves of both knotweeds. Both plants showed similar pigment profiles. By means of high performance thin-layer chromatography with densitometry and high performance liquid chromatography coupled to photodiode array and mass spectrometric detector, 11 carotenoids (and their derivatives) and 4 chlorophylls were identified in green leaves, whereas 16 distinct carotenoids (free carotenoids and xanthophyll esters) were found in senescing leaves. Total carotenoid content in green leaves of Japanese knotweed and Bohemian knotweed (378 and 260 mg of lutein equivalent (LE)/100 g dry weight (DW), respectively) was comparable to that of spinach (384 mg LE/100 g DW), a well-known rich source of carotenoids. A much lower total carotenoid content was found for senescing leaves of Japanese and Bohemian knotweed (67 and 70 mg LE/100 g DW, respectively). Thus, green leaves of both studied knotweeds represent a rich and sustainable natural source of bioactive carotenoids. Exploitation of these invaders for the production of high value-added products should consequently promote their mechanical control.

High performance thin-layer chromatography-mass spectrometry methods on diol stationary phase for the analyses of flavan-3-ols and proanthocyanidins in invasive Japanese knotweed.

We developed the first four HPTLC methods for the separation of proanthocyanidins according to degree of polymerization on HPTLC diol F254S plates. Acetonitrile, ethyl acetate, ethyl acetate-formic acid (9:0.1, v/v) and toluene-acetone-formic acid (3:6:1, v/v) were used as developing solvents and 4-dimethylaminocinnamaldehyde (DMACA) as the detection reagent. Each of these methods enables separation of standards of procyanidin dimers from procyanidin trimer (procyanidin C1) and separation of B-type dimers (procyanidins B1, B2, B3) from A-type dimers (procyanidins A1, A2). Based on these HPTLC methods we developed four new HPTLC-MS/MS methods for analyses of proanthocyanidins on HPTLC diol F254S plates and we identified B-type proanthocyanidins from monomers up to decamers in crude extracts of invasive Japanese knotweed (Fallopia japonica Houtt., Polygonum cuspidatum Sieb. & Zucc.) rhizomes. Monomers, monomer gallates, dimers, dimer gallates, dimer digallates, trimers, trimer gallates, tetramers, tetramer gallates, pentamers, pentamer gallates, hexamers, hexamer gallates, heptamers, octamers, nonamers and decamers were tentatively identified in Japanese knotweed rhizomes using developing solvents acetonitrile and toluene-acetone-formic acid (3:6:1, v/v). Ethyl acetate enabled separation from monomers up to hexamer gallates and ethyl acetate-formic acid (9:0.1, v/v) from monomers up to hexamers. During the five hours of stability testing of (-)-epicatechin and procyanidin B2 standards on HPTLC diol plates developed with all solvents we observed enhanced absorption at 280 nm. This was a totally unexpected phenomenon. This new discovery confirmed what we reported in our previous study on HPTLC silica gel. Enhanced absorption is influenced by the developing solvent (more than 30%), the stationary phase (up to 24%) and the light (up to 15%).