Chalcones-Features, Identification Techniques, Attributes, and Application in Agriculture.

This review article is a comprehensive and current overview on chalcones, covering their sources, identification methods, and properties with a particular focus on their applications in the agricultural sector. The widespread use of synthetic pesticides has not only led to increased resistance among weeds and pests, resulting in economic losses, but it has also raised significant health concerns due to the overuse of these chemicals. In line with the European Green Deal 2030 and its Farm to Fork strategy, there is a targeted 50% reduction in the use of chemical pesticides by 2030, emphasizing a shift towards natural alternatives that are more environmentally sustainable and help in the restoration of natural resources. Chalcones and their derivatives, with their herbicidal, fungicidal, bactericidal, and antiviral properties, appear to be ideal candidates. These naturally occurring compounds have been recognized for their beneficial health effects for many years and have applications across multiple areas. This review not only complements the previous literature on the agricultural use of chalcones but also provides updates and introduces methods of detection such as chromatography and MALDI technique.

Spiro-Flavonoids in Nature: A Critical Review of Structural Diversity and Bioactivity.

Based on the literature data from 1973 to 2022, this work summarizes reports on spiro-flavonoids with a spiro-carbon at the center of their structure and how this affects their isolation methods, stereochemistry, and biological activity. The review collects 65 unique structures, including spiro-biflavonoids, spiro-triflavonoids, spiro-tetraflavonoids, spiro-flavostilbenoids, and scillascillin-type homoisoflavonoids. Scillascillin-type homoisoflavonoids comprise spiro[bicyclo[4.2.0]octane-7,3'-chromane]-1(6),2,4-trien-4'-one, while the other spiro-flavonoids contain either 2H,2'H-3,3'-spirobi[benzofuran]-2-one or 2'H,3H-2,3'-spirobi[benzofuran]-3-one in the core of their structures. Spiro-flavonoids have been described in more than 40 species of eight families, including Asparagaceae, Cistaceae, Cupressaceae, Fabaceae, Pentaphylacaceae, Pinaceae, Thymelaeaceae, and Vitaceae. The possible biosynthetic pathways for each group of spiro-flavonoids are summarized in detail. Anti-inflammatory and anticancer activities are the most important biological activities of spiro-flavonoids, both in vitro and in vivo. Our work identifies the most promising natural sources, the existing challenges in assigning the stereochemistry of these compounds, and future research perspectives.

Phytochemicals Derived from Agricultural Residues and Their Valuable Properties and Applications.

Billions of tons of agro-industrial residues are produced worldwide. This is associated with the risk of pollution as well as management and economic problems. Simultaneously, non-edible portions of many crops are rich in bioactive compounds with valuable properties. For this reason, developing various methods for utilizing agro-industrial residues as a source of high-value by-products is very important. The main objective of the paper is a review of the newest studies on biologically active compounds included in non-edible parts of crops with the highest amount of waste generated annually in the world. The review also provides the newest data on the chemical and biological properties, as well as the potential application of phytochemicals from such waste. The review shows that, in 2020, there were above 6 billion tonnes of residues only from the most popular crops. The greatest amount is generated during sugar, oil, and flour production. All described residues contain valuable phytochemicals that exhibit antioxidant, antimicrobial and very often anti-cancer activity. Many studies show interesting applications, mainly in pharmaceuticals and food production, but also in agriculture and wastewater remediation, as well as metal and steel industries.

Pioneering Metabolomic Studies on Diaporthe eres Species Complex from Fruit Trees in the South-Eastern Poland.

Fungi from the genus Diaporthe have been reported as plant pathogens, endophytes, and saprophytes on a wide range of host plants worldwide. Their precise identification is problematic since many Diaporthe species can colonize a single host plant, whereas the same Diaporthe species can inhabit many hosts. Recently, Diaporthe has been proven to be a rich source of bioactive secondary metabolites. In our initial study, 40 Diaporthe isolates were analyzed for their metabolite production. A total of 153 compounds were identified based on their spectroscopic properties-Ultraviolet-visible and mass spectrometry. From these, 43 fungal metabolites were recognized as potential chemotaxonomic markers, mostly belonging to the drimane sesquiterpenoid-phthalide hybrid class. This group included mainly phytotoxic compounds such as cyclopaldic acid, altiloxin A, B, and their derivatives. To the best of our knowledge, this is the first report on the metabolomic studies on Diaporthe eres species complex from fruit trees in the South-Eastern Poland. The results from our study may provide the basis for the future research on the isolation of identified metabolites and on their bioactive potential for agricultural applications as biopesticides or biofertilizers.

Comparison of Phenolic Metabolites in Purified Extracts of Three Wild-Growing Herniaria L. Species and Their Antioxidant and Anti-Inflammatory Activities In Vitro.

The work is aimed at phytochemical characterization and In Vitro evaluation of antioxidant actions, anti-inflammatory effects, and cytotoxicity of purified extracts from three rupturewort (Herniaria L.) species, i.e., Herniaria glabra (HG), H. polygama (HP), and H. incana herb (HIh). The total phenolic content established in the purified extracts (PEs) of HIh, HP, and HG was 29.6, 24.0, and 13.0%, respectively. Thirty-eight non-saponin metabolites were identified using LC-HR-QTOF-ESI-MS; however, only 9 were common for the studied Herniaria species. The most abundant phenolic compound in HG-PE was narcissin (7.4%), HP-PE shared 3 major constituents, namely cis-2-hydroxy-4-methoxycinnamic acid 2-O-ß-glucoside (cis-GMCA, 5.8%), narcissin (5.4%), and rutin (5.3%). Almost half of HIh phenolic content (14.7%) belonged to oxytroflavoside A (7-O-methylkaempferol-3-O-[3-hydroxy-3-methylglutaryl-(1→6)]-[α-rhamnopyranosyl-(1→2)]-ß-galactopyranoside). Antioxidant properties of the Herniaria PEs were evaluated employing an experimental model of human blood plasma, exposed to the peroxynitrite-induced oxidative stress. The assays demonstrated significant reduction of oxidative damage to protein and lipid plasma components (estimated by measurements of 3-nitrotyrosine, protein thiol groups, thiobarbituric acid-reactive substances), and moderate protection of its non-enzymatic antioxidant capacity. Anti-inflammatory properties of the Herniaria PEs were evaluated In Vitro as inhibitory effects against cyclooxygenases (COX-1 and -2) and concanavalin A-induced inflammatory response of the peripheral blood mononuclear cells (PBMCs). None of the studied plants showed inhibitory effects on COXs but all purified extracts partly reduced the release of interleukin 2 (IL-2) and tumor necrosis factor-alpha (TNF-α) from PBMCs, which suggested their prospective ability to up-regulate inflammatory response of the cells. The purified extract from H. glabra turned out to be the most efficient suppressor of PBMCs' inflammatory response. Additionally, cytotoxicity of purified Herniaria extracts on PBMCs was ruled out based on In Vitro studies.

The Influence of High-Intensity Ultrasonication on Properties of Cellulose Produced from the Hop Stems, the Byproduct of the Hop Cones Production.

The goal of this work is to evaluate the hop stems, a byproduct of hop cones production, as a potential source of cellulose. Hop stems contain up to 29% of cellulose. The cellulose isolation was conducted through the thermochemical treatment. After high-speed blending, the cellulose was characterized by 67% of crystallinity degree obtained from X-ray diffraction and median diameter of 6.7 nm obtained from atomic force microscopy imaging. The high-intensity ultrasonication (HIUS) was applied to reach further disintegration of cellulose fibers. The longer HIUS treatment resulted in decrease in crystallinity degree even up to 60% and decrease in the fiber diameter up to 4 nm. The Fourier transform infrared spectroscopy (FTIR) spectra showed that HIUS treatment led to changes in intermolecular hydrogen bonds. The stability of cellulose dispersions versus length of HIUS treatment was monitored over 14 days with back dynamic light scattering and laser Doppler electrophoresis methods. Obtained results are evidence that the hop stems are a potential source of cellulose and that it is possible to obtain stable dispersions after HIUS treatment. This was the first time that the properties of hop cellulose have been described so extensively and in detail after the use of HIUS treatment.

Neuroprotective Effect of Yucca schidigera Roezl ex Ortgies Bark Phenolic Fractions, Yuccaol B and Gloriosaol A on Scopolamine-Induced Memory Deficits in Zebrafish.

Y. schidigera contains a number of unusual polyphenols, derivatives of resveratrol and naringenin, called spiro-flavostilbenoids, which have potent in vitro anti-inflammatory, antioxidant, and moderate cholinesterase inhibitory activities. To date, these compounds have not been tested in vivo for the treatment of neurodegenerative diseases. The aim of the present study was to evaluate the effects of both single spiro-flavostilbenoids (yuccaol B and gloriosaol A) and phenolic fractions derived from Y. schidigera bark on scopolamine-induced anxiety and memory process deterioration using a Danio rerio model. Detailed phytochemical analysis of the studied fractions was carried out using different chromatographic techniques and Nuclear Magnetic Resonance (NMR). The novel tank diving test was used as a method to measure zebrafish anxiety, whereas spatial working memory function was assessed in Y-maze. In addition, acetylcholinesterase/butyrylcholinesterase (AChE/BChE) and 15-lipooxygenase (15-LOX) inhibition tests were performed in vitro. All pure compounds and fractions under study exerted anxiolytic and procognitive action. Moreover, strong anti-oxidant capacity was observed, whereas weak inhibition towards cholinesterases was found. Thus, we may conclude that the observed behavioral effects are complex and result rather from inhibition of oxidative stress processes and influence on cholinergic muscarinic receptors (both 15-LOX and scopolamine assays) than effects on cholinesterases. Y. schidigera is a source of substances with desirable properties in the prevention and treatment of neurodegenerative diseases.

Serjanic Acid Glycosides from Chenopodium hybridum L. with Good Cytotoxicity and Selectivity Profile against Several Panels of Human Cancer Cell Lines.

Two triterpene saponins, including a novel serjanic acid derivative, were isolated from Chenopodium hybridum L. (Amaranthaceae) aerial parts. Their structures were elucidated by a combination of spectroscopic methods (MS, 1D and 2D NMR). Both compounds were evaluated for cytotoxicity and selectivity on skin, prostate, gastrointestinal, thyroid and lung cancer cells. Their effect was dose and time-dependent with varied potency, the highest against prostate PC3 and melanoma WM793, where IC50 was lower than the reference drug doxorubicin. Structure-activity relationship is briefly discussed.

Phytochemical Profile, Antioxidant Activity, and Cytotoxicity Assessment of Tagetes erecta L. Flowers.

Tagetes erecta L. is a popular ornamental plant of the Asteraceae family, which is widely cultivated not only for its decorative use, but also for the extraction of lutein. Besides carotenoid representatives, which have been extensively studied, other important classes of secondary metabolites present in the plant, such as polyphenols, could exhibit important biological activities. The phytochemical analysis of a methanolic extract obtained from T. erecta inflorescences was achieved using liquid chromatography-mass spectrometry (LC-MS) techniques. The extract was further subjected to a multistep purification process, which allowed the separation of different fractions. The total extract and its fractions contain several polyphenolic compounds, such as hydroxybenzoic and hydroxycinnamic acid derivatives, flavonols (especially quercetagetin glycosides), and several aglycons (e.g., quercetin, patuletin). One of the fractions, containing mostly quercetagitrin, was subjected to two different antioxidant assays (metal chelating activity and lipoxygenase inhibition) and to in vitro cytotoxicity assessment. Generally, the biological assays showed promising results for the investigated fraction compared to the initial extract. Given the encouraging outcome of the in vitro assays, further purification and structural analysis of compounds from T. erecta extracts, as well as further in vivo investigations are justified.

Electrospray ionization mass spectrometry characterization of ubiquitous minor lipids and oligosaccharides in milk of the camel (Camelus dromedarius) and their inhibition of oxidative stress in human plasma.

The aim of this study was to characterize minor lipids in methanol fraction extracted from raw camel milk after loading it on a water-preconditioned short C18 open column and fractionating with a gradient of methanol/water. The C18 column showed high fractionation efficiency of minor lipids, such as glycosphingolipids, lipopolysaccharides, or oligosaccharides, when compared with other constituents, in particular polysaccharides, proteins, and free fatty acids. Liquid chromatography electrospray ionization tandem mass spectrometry in negative ion mode was used to identify 21 new glycosphingolipids, lipopolysaccharides, and oligosaccharides. Electrospray ionization tandem mass spectrometry was qualified to provide relevant data for recognizing the molecular mass, glycosylation sequences, and structure of saccharide moieties for the revealed compounds. The sequence of combinations of one selected lipopolysaccharide, which was considered the backbone of the remaining lipopolysaccharides, was confirmed in a density functional theory study. The obtained results showed that the tested fraction is a rich source of glycosphingolipids, lipopolysaccharides, and oligosaccharides with antioxidant activity.

Norditerpenoids with Selective Anti-Cholinesterase Activity from the Roots of Perovskia atriplicifolia Benth.

Inhibition of cholinesterases remains one of a few available treatment strategies for neurodegenerative dementias such as Alzheimer's disease and related conditions. The current study was inspired by previous data on anticholinesterase properties of diterpenoids from Perovskia atriplicifolia and other Lamiaceae species. The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition by the three new natural compounds-(1R,15R)-1-acetoxycryptotanshinone (1), (1R)-1-acetoxytanshinone IIA (2), and (15R)-1-oxoaegyptinone A (3)-as well as, new for this genus, isograndifoliol (4) were assessed. Three of these compounds exhibited profound inhibition of butyrylcholinesterase (BChE) and much weaker inhibition of acetylcholinesterase (AChE). All compounds (1-4) selectively inhibited BChE (IC50 = 2.4, 7.9, 50.8, and 0.9 µM, respectively), whereas only compounds 3 and 4 moderately inhibited AChE (IC50 329.8 µM and 342.9 µM). Molecular docking and in silico toxicology prediction studies were also performed on the active compounds. Natural oxygenated norditerpenoids from the traditional Central Asian medicinal plant P. atriplicifolia are selective BChE inhibitors. Their high potential makes them useful candidate molecules for further investigation as lead compounds in the development of a natural drug against dementia caused by neurodegenerative diseases.

The effect of total and individual alfalfa saponins on rumen methane production.

BACKGROUND: Ten varieties of alfalfa (Medicago sativa L.) were evaluated for saponin content. Two of the most promising varieties were chosen so that their effect on rumen fermentation and methane production could be studied. Initially, four Hohenheim gas tests (HGT) were performed to test the effect of increased levels of total saponin extracted from the two alfalfa cultivars (Kometa and Verko) - either as fresh material or ensiled - on the total bacteria, total protozoa, methane emission, and selected methanogenic population. Afterwards, seven particular saponins were extracted from fresh alfalfa of the Kometa variety and tested in 24 h batch fermentation culture experiments. RESULTS: The ensiled forms of both the Verko and Kometa alfalfa varieties seem to be good sources of saponin, capable of reducing methane production (P < 0.05) without negatively affecting the basic fermentation parameters. Of the two evaluated varieties, Kometa was the most effective, and the saponins extracted from its roots 3-Glc,28-Glc Ma, medicagenic saponin, and 3-Glu Ma showed the most evident effect (P = 0.0001). The most promising aerial alfalfa saponin in mitigating methane production was soysaponin I K salt (P = 0.0001). Three mixtures of saponins were tested and all were found to mitigate methane production; however, one mixture (MIX 1) did so only to a very small extent. CONCLUSION: Saponins have been observed to have differing effects depending on their source; however, the mode of action of saponins depends on their direct or probable indirect effect on the microorganisms involved in methane production. © 2019 Society of Chemical Industry.

The protein quality control system manages plant defence compound synthesis.

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondary metabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Here we show that, in the legume Medicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membrane-anchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

Cytotoxic Cardenolides from the Leaves of Acokanthera oblongifolia.

Three previously undescribed cardenolides, acovenosigenin A 3-O-α-L-acofriopyranoside (1: ), 14-anhydroacovenosigenin A 3-O-[ß-D-glucopyranosyl-(1″→4')-O-α-L-acofriopyranoside] (2: ), and 14-anhydroacovenosigenin A 3-O-[ß-D-glucopyranosyl-(1″→4')-O-α-L-acovenopyranoside] (3: ), together with the two already known ones, 14-anhydrodigitoxigenin 3-O-ß-D-glucopyranoside (4: ) and acospectoside A (5: ), were isolated from the leaves of Acokanthera oblongifolia. The influence of cardenolides 1:  - 3: and acovenoside A (found in the Acokanthera genus) on three cancer cell lines (HT29, HCT116, and AGS) was also investigated. The most promising results, in comparison with oxaliplatin, were obtained for compound 1: , which was found to be highly cytotoxic for all tested cell lines, HT29 (IC50 = 63.49 nM), HCT116 (IC50 = 67.35 nM), and AGS (IC50 = 80.92 nM). Unfortunately, 1: also showed similar toxicity towards normal lymphocytes (IC50 = 98.03 nM).

Effects of berry seed residues on ruminal fermentation, methane concentration, milk production, and fatty acid proportions in the rumen and milk of dairy cows.

Strawberry (SB), black currant (BC), and raspberry seed (RB) residues were used in 3 experiments to study their effects on ruminal fermentation, methane concentration, and fatty acid (FA) proportions in the ruminal fluid and milk of dairy cows. Initially, a batch fermentation in vitro study (experiment 1) was performed to investigate the effects of the 3 berry residues on basic ruminal fermentation parameters. Total volatile fatty acid concentrations, including acetate, propionate, and butyrate, increased in the BC group compared with other treatments. Based on the preliminary in vitro results, 2 consecutive in vivo experiments were conducted using 4 Polish Holstein-Friesian cows fitted with rumen cannulas (experiment 2) and 30 lactating Polish Holstein-Friesian dairy cows (experiment 3) in a replicated 2 × 2 crossover design. Cows in both experiments received a partial mixed ration (PMR) in 2 variants: (1) a control diet of PMR + 2 kg of concentrate (control); (2) PMR + 2 kg of BC seed residues (BC). The BC diet did not mitigate ruminal methane production. Ruminal fermentation (experiment 2) was not affected by the BC diet; however, the concentrations of C18:1 trans-11 and C18:2 cis-9,trans-11 increased significantly by 91 and 131%, respectively. Likewise, concentrations of total trans C18:1 and total monounsaturated FA in ruminal fluid were increased significantly by BC seed residues. In experiment 3, BC significantly increased milk fat C18:1 trans-11, C18:2 cis-9,trans-11, n-3, n-6, and polyunsaturated FA concentrations without affecting milk production performance. In conclusion, the amount (2 kg/d) of BC used in this study did not adversely affect ruminal fermentation or milk production and composition. However, using BC increased proportions of unsaturated FA and conjugated linoleic acid in milk. Although dietary BC did not exert a strong methane inhibition effect, it could represent an inexpensive alternative concentrate to improve beneficial FA in milk without negative effects on rumen fermentation and production parameters in dairy cows. Incorporation of berry seed residues in diets would be profitable economically and nutritionally for dairy cattle production.

Yuccalechins A-C from the Yucca schidigera Roezl ex Ortgies Bark: Elucidation of the Relative and Absolute Configurations of Three New Spirobiflavonoids and Their Cholinesterase Inhibitory Activities.

The ethyl acetate fraction of the methanolic extract of Yucca schidigera Roezl ex Ortgies bark exhibited moderate acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity (IC50 47.44 and 47.40 µg mL-1, respectively). Gel filtration on Sephadex LH-20 and further RP-C18 preparative HPLC of EtOAc fraction afforded 15 known and 3 new compounds, stereoisomers of larixinol. The structures of the isolated spirobiflavonoids 15, 26, and 29 were elucidated using 1D and 2D NMR and MS spectroscopic techniques. The relative configuration of isolated compounds was assigned based on coupling constants and ROESY (rotating-frame Overhauser spectroscopy) correlations along with applying the DP4+ probability method in case of ambiguous chiral centers. Determination of absolute configuration was performed by comparing calculated electronic circular dichroism (ECD) spectra with experimental ones. Compounds 26 and 29, obtained in sufficient amounts, were evaluated for activities against AChE and BChE, and they showed a weak inhibition only towards AChE (IC50 294.18 µM for 26, and 655.18 µM for 29). Furthermore, molecular docking simulations were performed to investigate the possible binding modes of 26 and 29 with AChE.

Chemical Profile and Antioxidant Activity of Zinnia elegans Jacq. Fractions.

Zinnia elegans (syn. Zinnia violacea) is a common ornamental plant of the Asteraceae family, widely cultivated for the impressive range of flower colors and persistent bloom. Given its uncomplicated cultivation and high adaptability to harsh landscape conditions, we investigated the potential use of Z. elegans as a source of valuable secondary metabolites. Preliminary classification of compounds found in a methanolic extract obtained from inflorescences of Z. elegans cv. Caroussel was accomplished using HR LC-MS techniques. The extract was then subjected to solid-phase extraction and separation using Sephadex LH-20 column chromatography, which resulted in several fractions further investigated for their antioxidant properties through lipoxygenase inhibition and metal chelating activity assays. Moreover, following additional purification procedures, structures of some active ingredients were established by NMR spectroscopy. The investigated fractions contained polyphenolic compounds such as chlorogenic acids and apigenin, kaempferol, and quercetin glycosides. Antioxidant assays showed that certain fractions exhibit moderate 15-LOX inhibition (Fr 2, IC50 = 18.98 µg/mL) and metal chelation (e.g., Fr 1-2, EC50 = 0.714-1.037 mg/mL) activities as compared to positive controls (20.25 µg/mL for kaempferol and 0.068 mg/mL for EDTA, respectively). For Fr 2, the 15-LOX inhibition activity seems to be related to the abundance of kaempferol glycosides. The NMR analyses revealed the presence of a kaempferol 3-O-glycoside, and a guanidine alkaloid previously not described in this species.

Structural and quantitative changes of saponins in fresh alfalfa compared to alfalfa silage.

BACKGROUND: Alfalfa (Medicago sativa) is an important legume used in animal feed. The primary objective of the present study was to analyze and trace the individual saponins in fresh and ensiled alfalfa of ten varieties, with the aim of evaluating the structural changes that saponins undergo during ensiling. A secondary objective was to examine whether any of the changes in saponin content were associated with changes in the basic nutrient composition of the ensiled alfalfa. RESULTS: The total saponin concentration increased when the fresh alfalfa was processed into silage. Three of the zanhic acid glycosides were degraded substantially, whereas the other two increased in all the tested varieties. Five of the seven medicagenic acid glycosides were not changed, whereas the remaining saponins completely disappeared. Finally, two glycosides of soyasapogenol B displayed an inverse relationship to each other. CONCLUSION: The nutrient content of fresh material and silages remained relatively constant, with some variations in the crude protein content of the selected alfalfa varieties. The total concentration of saponins increased two- to three-fold after ensilation over the levels present in fresh alfalfa material. The increases in saponin concentration showed a negative linear relationship between protein on a dry matter basis and saponin content. These findings are significant because no known published data have shown this transformation in the ensiling of alfalfa. © 2018 Society of Chemical Industry.

Tentative Characterization of Polyphenolic Compounds in the Male Flowers of Phoenix dactylifera by Liquid Chromatography Coupled with Mass Spectrometry and DFT.

Phoenix dacylifera is an ancient palm species rich in (poly)phenols. These phenolic compounds were tentatively identified by using liquid chromatography coupled with ion spray mass spectrometry in tandem mode (LC/MS/MS) with negative ion detection. Negative identification of the compounds was based on their retention times and mass spectra in full scan mode (MS), and in different MS/MS modes. For the first time, complete hypothesis, and routs for both p-coumaroylshikimic acids (CoSA) and caffeoylshikimic acids (CSA) were suggested and confirmed by Density Fonctional Theory (DFT) study. Notably, of the 53 compounds characterized, 19 hydroxycinnamates derivatives were tentativelycharacterized in male flowers of date palm and 15 of them were recorded for the first time. In addition, five organic acids, six B-type proanthocyanidins, two anthocyanidin and 21 flavonoid derivatives have been tentatively characterized. Identification of B-type proanthocyanidins were based on the diagnostic ions resulting from heterocyclic ring fission (HRF) and retro-Diels-Alder (RDA) reaction of flavan-3-ol provided information on the hydroxylation pattern and the type of inter-flavan bond proanthocyanidins. The sequence of proanthocyanidins was detected through ions extracted from quinone methide (QM) cleavage of the inter-flavan bond.

The anti-adhesive and anti-aggregatory effects of phenolics from Trifolium species in vitro.

The present in vitro study includes a comparative evaluation of anti-platelet (anti-thrombotic) properties of plant phenolics, isolated from nine different clover (Trifolium) species. The analysis covered phenolic fractions isolated from T. alexandrinum L., T. fragiferum L., T. hybridum L., T. incarnatum L., T. pallidum Waldst et Kit., T. resupinatum L. var. majus Boiss, T. resupinatum L. var. resupinatum, T. scabrum L., and T. pratense L. (red clover). The inhibitory effects of plant preparations (1-50 µg/ml) on hemostatic functions of blood platelets were assessed by measurements of thrombin- or ADP-induced platelet adhesion to fibrinogen, platelet aggregation in platelet-rich plasma (activated with ADP or collagen), and by the determination of PF-4 secretion from platelet α-granules. The influence of T. phenolics on arachidonic cascade in blood platelets was also determined. T. resupinatum var. majus, T. resupinatum var. resupinatum, and T. scabrum had the strongest anti-platelet effects. These preparations displayed the most evident anti-adhesive and anti-aggregatory effects in response to all of the used agonists: thrombin (0.2 U/ml), ADP (10 µM), and collagen (2 µg/ml), and their inhibitory properties were also confirmed by an analysis of PF-4 secretion. T. scabrum and some of other examined clover species possess significantly higher concentrations of both isoflavones and other bioactive phenolics, when compared to red clover. The obtained results suggest that these clovers contain substances with potent anti-platelet properties.