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Argania spinosa kernel cakes, obtained from argan oil extraction process, are known to contain large amounts of saponins. Only a few have been characterised previously, due to the use of pure ethanol as extracting solvent. The use of aqueous 50% ethanol improved the extraction of more polar saponins.Identification of polar saponins in kernel cakes of Argania spinosa by liquid chromatography-mass spectrometry and NMR techniques.Defatted kernel cakes were first extracted with ethanol and then twice with 50% aqueous ethanol. Individual crude extracts were analysed with an ion-trap mass spectrometer in negative mode electrospray MS and MS/MS modes. NMR experiments were run under standard conditions at 300 K on a Bruker DRX-600 spectrometer.The LC-MS base peak chromatogram of saponins from pure ethanol extract was dominated by 11 large and several small peaks but the UV chromatogram showed only two peaks, corresponding to the main neutral saponins found previously in Argania: arganine A and B. In 50% aqueous ethanol extracts, numerous other saponins were detected. Many of them were glucuronide oleanane-type triterpene carboxylic acid 3,28-O-bidesmosides (GOTCAB saponins). The assignments of (1) H- and (13) C-NMR spectra of the four most abundant GOTCAB saponins confirmed the MS results.Four GOTCAB saponins were structurally identified by NMR analysis in the 50% aqueous ethanol extract. Furthermore, LC-MS analyses showed the presence of at least 19 additional polar saponins in these kernel cakes.

The phenolic fraction and the crude extract from Tribulus pterocarpus have different biological activity, including antiplatelet–antiadhesive properties. Since it is demonstrated that hyperhomocysteinemia may act as stimulator of blood platelet activation (platelet adhesion, aggregation, and secretion), but various antiplatelet compounds are able to reduce hyperactivation of blood platelets induced by hyperhomocysteinemia. The aim of our present experiments was to investigate in vitro one of the step in platelet activation process – platelet adhesion to collagen induced by the model of severe hyperhomocyateinemia in the presence of the phenolic fraction and the crude extract from T. pterocarpus. Severe hyperhomocysteinemia was induced by reduced form of Hcy in the concentrations 0.1 mM and 1 mM, or using HTL in the concentrations 0.1, 0.5 and 1 μM. Adhesion of blood platelets to collagen was determined according to Tuszynski and Murphy. We observed that the phenolic fraction and the crude extract from T. pterocarpus have the inhibitory effect on platelet adhesion during severe hyperhomocysteinemia. The action of tested phenolic and crude extract was concentration-dependent, but the phenolic fraction was stronger antiadhesive action than the crude extract. We suggest that T. pterocarpus may be good source of antiplatelet compounds during hyperhomocysteinemia.

Clovers (Trifolium) may possess a significant therapeutic potential, but the effects of compounds from these plants on blood platelets and haemostasis have been poorly recognized. The present study was designed to evaluate the antioxidative action of extracts from three species of clovers: Trifolium pratense, Trifolium pallidum and Trifolium scabrum in the protection of human blood platelets in vitro. Platelet suspensions were pre-incubated with crude extract and phenolic fraction of T. pratense or phenolic fractions of T. scabrum and T. pallidum, at the final concentrations of 0.5–50 μg/ml. Then, for the induction of oxidative stress, 100 μM peroxynitrite was added. The antioxidative activity of plant extracts was assessed by measurements of the level of 3-nitrotyrosine, thiol groups and lipid peroxidation products (hydroperoxides and thiobarbituric acid-reactive substances). Despite the significant differences in the composition of the investigated extracts, we observed antioxidative effects of all used mixtures. The presence of Trifolium extracts considerably reduced the peroxynitrite-mediated modifications of proteins and diminished peroxidation of lipids in platelets. Our results indicate on a strong antioxidative activity of the tested extracts-statistically significant effects were found even for the lowest concentrations (0.5 μg/ml) of all extracts. This action may be useful in the protection of blood components, very susceptible to oxidative modifications. The obtained results suggest that the examined clovers are a promising source of compounds, valuable for the protection against oxidative stress-induced damage to blood platelets.

A growing number of reports indicate that some species of clover (Trifolium) may have remarkable medical importance; however, the effects of these plants on blood platelets and hemostasis are inadequately recognized. This work was designed to study the effects of Trifolium pallidum and Trifolium scabrum extracts on the functions of human blood platelets in vitro. Platelet suspensions were preincubated with extracts from aerial parts of T. pallidum (phenolic fraction and clovamide fraction) and T. scabrum (phenolic fraction) at the final concentrations of 12.5, 25, and 50 µg/ml. Then, for platelet activation thrombin (0.1 U/ml), thrombin receptor activating peptide (TRAP; 20 µM), or adenosine diphosphate (ADP; 1 µM) were used. The effects of Trifolium extracts on adhesion of blood platelets to fibrinogen and collagen were determined by enzyme-linked immunosorbent assay (ELISA) method. Platelet aggregation was monitored on a dual-channel Chronolog aggregometer. In these studies, we also compared the action of tested plant extracts with the effects of another antiplatelet plant-derived compound - resveratrol (3,4',5-trihydroxystilbene). The performed assays demonstrated that the tested extracts might influence the platelet functions in vitro. The inhibitory, concentration-dependent effects of all tested extracts on adhesion of thrombin-stimulated platelets to collagen was found. Both extracts from T. pallidum and from T. scabrum reduced the thrombin-induced platelet adhesion to fibrinogen. Furthermore, in the presence of all three extracts, the platelet aggregation induced by thrombin was slightly inhibited. Our results also indicate that the tested plant extracts (at the highest concentrations used of 50 µg/ml), similar to purified resveratrol, inhibit selected steps of platelet activation stimulated by both proteolytic (thrombin) and nonproteolytic agonists (TRAP or ADP). In the comparative studies, T. pallidum and T. scabrum extracts was not found to be more effective antiaggregatory factor, than resveratrol. Extracts from T. pallidum and T. scabrum aerial parts reveal antiplatelet properties: the antiadhesive effect was similar to that of the reference compound resveratrol, whereas the antiaggregant effect was less marked. The results obtained suggest that these plants may be a promising source of natural compounds, valuable in the prevention of the enhanced activity of blood platelets in numerous cardiovascular diseases, observed in menopausal or postmenopausal women.

Elevated concentration of homocysteine (Hcy) in human plasma, defined as hyperhomocysteinemia has been correlated with some diseases, such as cardiovascular, neurodegenerative, and kidney disorders. Homocysteine occurs in human plasma in several forms, including the most reactive form of Hcy - its cyclic thioester - homocysteine thiolactone (HTL), which represents up to 0.29% of plasma total Hcy. It is suggested that Hcy and HTL may also act as oxidants, but various polyphenolic antioxidants are able to inhibit the oxidative damage induced by Hcy or HTL. The aim of our present study was to investigate in vitro oxidative changes in human plasma induced by the model of hyperhomocysteinemia in the presence of the phenolic fractions from selected clovers (Trifolium pallidum and Trifolium scabrum aerial parts). Hyperhomocysteinemia was stimulated by a reduced form of Hcy (final dose 100 μM) or HTL (final dose 1 μM). The aim of our study was also to explain the effect of the phenolic fractions on the coagulation activity of human plasma treated with Hcy and its thiolactone. Tested phenolic fractions significantly inhibited the oxidative stress (measured by the total antioxidant level - TAS) in plasma treated with Hcy or HTL. The phenolic fractions from T. pallidum and T. scabrum also caused a distinct reduction of plasma lipid peroxidation (measured by the level of thiobarbituric acid reactive substance) induced by the model of hyperhomocysteinemia. Moreover, tested fractions modulated the coagulation properties of plasma treated with homocysteine and its thiolactone. It seems that antioxidative activities of the phenolic fractions from T. pallidum and T. scabrum aerial parts may be responsible for their medicinal properties during hyperhomocysteinemia.

The mechanism action of the polyphenol-rich extracts from berries of Aronia melanocarpa (black chokeberry) and from grape seeds in the defence against homocysteine (Hcy) and its derivatives action in blood platelets is still unknown. In this study, the influence of the aronia extract and grape seeds extract (GSE) on the platelet adhesion to collagen and fibrinogen and the platelet aggregation during a model of hyperhomocysteinemia was investigated. The aim of our study in vitro was also to investigate superoxide anion radicals (O 2 −• ) production after incubation of platelets with Hcy, HTL and the aronia extract and GSE during a model of hyperhomocysteinemia (induced by reduced form of homocysteine at final dose of 100 μM) and the most reactive form of Hcy—its cyclic thioester, homocysteine thiolactone (HTL, 1 μM). Moreover, the additional aim of our study was also to establish and compare the influence of the aronia extract, GSE and resveratrol (3,4′,5-trihydroxystilben), a phenolic compound, which has been supposed to be beneficial for the prevention of cardiovascular events, on selected steps of platelet activation. The effects of tested extracts on adhesion of blood platelets to collagen and fibrinogen were determined according to Tuszynski and Murphy. The platelet aggregation was determined by turbidimetry method using a Chrono-log Lumi-aggregometer. We have observed that HTL, like its precursor—Hcy stimulated the generation of O 2 −• (measured by the superoxide dismutase—inhibitable reduction of cytochrome c) in platelets and caused an augmentation of the platelet adhesion and aggregation induced by the strong physiological agonist—thrombin. Our present results in vitro also demonstrated that the aronia extract and grape seeds extract reduced the toxicity action of Hcy and HTL on blood platelet adhesion to collagen and fibrinogen, the platelet aggregation and superoxide anion radicals production in platelets, suggesting its potential protective effects on hemostasis during hyperhomocysteinemia. In the comparative studies, the aronia extract was found to be more effective antiplatelet factors, than GSE or resveratrol during a model of hyperhomocysteinemia. It gives hopes for development of diet supplements, which may be important during hyperhomocysteinemia.

There is a great interest in reducing the methane emission from ruminants as one possible cause of global warming. The aim of the presented study was to determine the effects of xanthohumol, one of the hop (Humulus lupulus) phytofactors, on methane production, microbial population and basic parameters of ruminal fermentation. The experiment was carried out in a batch culture system. The basic substrate (400 mg) consisting of meadow hay and barley meal (60:40) was supplemented with 0 (Control), 0.1, 0.2 or 1.0 mg of xanthohumol. The basic parameters of rumen fermentation and composition of microbial population were measured after 24 h of incubation. For the first time, the results of this in vitro study have demonstrated that xanthohumol is capable to reduce the methane production, even at the lowest dose applied (0.1 mg/400 mg). The observed reduction in methane production by 12-13% was not accompanied by altering the basic rumen fermentation parameters. However, the practical utility of this supplement needs further investigation under long-term in vivo conditions.

TLC-DPPH test belongs to a group of frequently performed assays aimed at detection of compounds with desired activity (effect directed analysis). Despite its popularity a standard procedure has not been elaborated so far causing difficulties in comparing results obtained in different laboratories. Thus the aim of the presented research was an approach to develop a standardized procedure for assessing free radical scavenging properties of plant polyphenols. It was observed that specifically positive adsorbent (silica gel) strengthened the observed result of radical-antioxidant reaction, while polar bonded stationary phase CN-silica weakened it. Based on the observed results it was concluded that the TLC-DPPH assay should be preferably performed on the surface of non-specific adsorbents ( e.g. : RP-18) with the use of n-hexane for DPPH dissolution. It is also proposed to document the results every 5 min after staining, as they change in time.

The revised structures of avenacosides A and B and a new sulfated steroidal saponin isolated from grains of Avena sativa L. were elucidated. Their structures and complete NMR assignments are based on 1D and 2D NMR studies and identified as nuatigenin 3-O-{α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-glucopyranoside}-26-O-β-D-glucopyranoside (1), nuatigenin 3-O-{α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)]-β-D-glucopyranoside}-26-O-β-D-glucopyranoside (2), and nuatigenin 3-O-{α-L-rhamnopyranosyl-(1→2)-[β-D-6-O-sulfoglucopyranosyl-(1→4)]-β-D-glucopyranoside}-26-O-β-D-glucopyranoside (3).

Tribulus is a well-known pharmaceutical herb that has been used for a long time in the traditional Chinese and Indian systems of medicine for the treatment of various diseases. It has been found that the genus Tribulus is rich in biologically active furostane-, cholestane- and spirostane-type steroidal saponins.To develop a rapid, sensitive and accurate method based on liquid-phase extraction followed by high-performance liquid chromatography and electrospray ionisation mass spectrometry (HPLC-ESI-MS) to identify different saponins in three species of the genus Tribulus, and to quantify the compounds that are already known.After extraction from the species studied, the extracts were subjected to HPLC analyses with an XTerra® MS C(18) -column and a binary mobile phase consisting of 0.05% formic acid in water and acetonitrile, and with an ESI-MS detection in the negative ion mode. Data were acquired and processed using the Xcalibur 1.3 software.The results exhibited that the profiles of native steroidal glycosides of both T. pentandrus and T. megistopterus subsp. pterocarpus were very similar to each other, but that of T. parvispinus was remarkably different. The fragmentation patterns provided evidence that the saponins possess spirostane-, cholestane- and furostane-type aglycones. Quantitative analyses suggested that these species are a rich source of steroidal saponins.HPLC-ESI-MS/MS allowed identification of the key compounds without preparative isolation of the components from the crude extract of Tribulus species.

The plant Humulus lupulus is known as the raw material of the brewing industry. Hop cones, rich in polyphenolic compounds and acyl phloroglucides, are widely used to preserve beer and to give it a characteristic aroma and flavor. Hop cones have long been used for medicinal purposes. In particular, hop preparations were mainly recommended for the treatment of sleeping disorders. The antioxidative action of hop cones, however, is poorly understood. The aim of our present study was to investigate in vitro changes in human blood platelets induced by peroxynitrite (ONOO(-), the compound of particular importance for vascular thrombosis and inflammatory process) in the presence of hop cone extract (Humulus lupulus). The antioxidative action of the extract was also compared with the properties of a well-characterized antioxidative commercial monomeric polyphenol, resveratrol (3,4',5-trihydroxystilbene) in a model system in vitro. Various biomarkers of oxidative/nitrative stress, such as carbonyl groups, 3-nitrotyrosine and thiobarbituric acid reactive substances (TBARS) were estimated. The 3-nitrotyrosine formation and carbonyl group generation was assessed by the use of a competition ELISA test and ELISA test, respectively. Tested plant extract (12.5-50 µg/ml), like resveratrol, significantly inhibited protein carbonylation and nitration in the blood platelets treated with ONOO(-) (0.1 mM). The extract from hop cones, like resveratrol, also caused a distinct reduction of platelet lipid peroxidation induced by ONOO(-). The present results indicate that the hope cone extract has in vitro protective effects against ONOO(-), such as induced oxidative/nitrative damage to the human platelet proteins and lipids. However, in comparative studies the extract was not found to be a more effective antioxidant than the solution of pure resveratrol.

A new C-6 flavone glycoside (6), together with seven known compounds, cryptochlorogenic acid (1), chlorogenic acid (2), 2- O- trans-caffeoylhydrocitric acid (3), isovitexin 7-β-D-glucopyranoside (4), 7,4′-dihydroxy-5-methoxyflavone-6- C-β-D-glucopyranoside (5), 3,5- O-dicaffeoylquinic acid (7) and 4,5- O-dicaffeoylquinic acid (8), were isolated from the aerial parts of Knautia arvensis. Their structures were elucidated by extensive spectroscopic methods including 1D- (1H, 13C and TOCSY) and 2D-NMR (DQF-COSY, HSQC, HMBC) experiments, as well as ESIMS analysis. Compounds 1, 3-5 and 8 are reported for the first time in Knautia arvensis.

Acacia nilotica (L.) Del. syn is a species rich in polyphenolic constituents, in which catechins are hypothesized to possess antioxidant properties and to play a role in the anti-inflammatory activity of several plants. Due to the complexity of catechin derivatives, the investigation of this class of natural compounds has been limited by difficulties in their separation. In this paper, rationalization of the phenolics occurring in the 80% EtOH extract of Acacia nilotica pods, on the basis of ESI-MS and ESI-MS/MS profiles, has been proposed. Additionally, an LC-ESI-MS qualitative study has been performed by using a C18 polar endcapped stationary phase. The fragmentation pattern obtained evidenced the presence in A. nilotica pods of galloylated catechin- and gallocatechin derivatives along with galloylated glucose derivatives. The structures were confirmed by NMR, after isolation of the pure compounds. In addition, the radical scavenging activities of extracts and pure compounds were investigated, by using the TEAC assay. Furthermore quantitative analyses were performed by LC-ESI-MS/MS, confirming the interest of this species as a rich source of very strong antioxidant principles.

Bioactive substances found in numerous foods can be successfully and safely used to modify various cellular functions and affect the oxidative stress. Aronia melanocarpa fruits (Rosaceae) are one of the richest plant sources of phenolic substances shown to have anti-inflammatory, antitumor, antioxidative and antiplatelet activities. We investigated antioxidant properties of the extract from berries of A. melanocarpa by the estimation of the selected and other biomarkers of oxidative stress, i.e. the level of 8-epi-prostaglandin F(2) (8-EPI) (by immunoassay kit) and the amount of glutathione (by HPLC method) in control platelets and platelets treated with H(2)O(2). The expression of alpha(IIb)beta(3) (a marker of platelet activation) was measured by flow cytometer. The antioxidative and antiplatelet properties of the tested extract were also compared with the action of a well characterized antioxidative and antiplatelet commercial monomeric polyphenol-resveratrol. The extract from berries of A. melanocarpa (at the highest tested concentration -100 microg/ml) decreased the production of 8-EPI (a marker of lipid peroxidation) in control blood platelets and platelets treated with H(2)O(2) (2 mM). A combined action of the tested plant extract and H(2)O(2) evoked a significant increase of reduced form of glutathione in platelets compared with cells treated with H(2)O(2) only. Moreover, the tested plant extract (at the highest used concentration -100 microg/ml) reduced the expression of alpha(IIb)beta(3) on blood platelets. Comparative studies indicate that the tested plant extract was found to be more reactive in blood platelets than the solution of pure resveratrol.

The nutritional quality of Persian clover (Trifolium resupinatum), an important pasture crop, depends not only on a high protein content but also on the occurrence of animal health and welfare promoting phytochemicals. Nine phenolic constituents present in the aerial parts of this species were isolated and their structures confirmed by NMR and ESI-MS analyses. The compounds included two chlorogenic acids, four quercetin and two kaempferol glycosides, as well as the isoflavone formononetin-7-glucoside. The concentration of isoflavone was low, not exceeding 1.2 mg/g of dry matter. The concentration of flavonols ranged between 5.9 and 11.8 mg/g, depending on the sampling dates, with the highest concentration occurring in the first cut. A similar trend in the concentration was found for chlorogenic acids, which ranged from 2 mg/g in summer to 7.3 mg/g in spring.

In the search of natural compounds inhibiting methane production in ruminants three novel steroidal saponins have been isolated from the aerial parts of Helleborus viridis L. Their structures have been established based on spectral analyses as: (25R)-26-O-β- d -glucopyranosyl-5β-furostan-3β,22α,26-triol 3-O-β- d -glucopyranosyl-(1 → 6)-O-β- d -glucopyranoside, (25R)-26-O-β- d -glucopyranosyl-5α-furostan-3β,22α,26-triol 3-O-β- d -glucopyranosyl-(1 → 6)-O-β- d -glucopyranoside and (25R)-26-O-β- d -glucopyranosyl-furost-5-ene-1β,3β,22α,26-tetraol 1-O-{α- l -rhamnopyranosyl-(1 → 2)-O-[β- d -glucopyranosyl-(1 → 3)]-6-O-acetoxy-β- d -glucopyranoside}.

Three phenolic glycosides 5- O -{[5′′- O - E -(4′′′- O - threo -guaiacylglycerol)-feruloyl]-β-apiofuranosyl-(1→2)-β-xylopyranosyl} gentisic acid, 5- O -[(5′′- O -vanilloyl)-β-apiofuranosyl-(1→2)-β-xylopyranosyl] gentisic acid and 1- O -[ E -(4′′′- O - threo -guaiacylglycerol)-feruloyl]-3- O -β-galacturonopyranosyl glycerol were isolated and identified from the roots of Medicago truncatula together with four known 5- O -β-xylopyranosyl gentisic acid, vicenin-2, hovetrichoside C and pterosupin identified for the first time in this species. Structural elucidation was carried out on the basis of UV, mass, 1 H and 13 C NMR spectral data.

The concentrations of four triterpene saponins present in amaranth seeds were determined with high-performance liquid chromatography. It was shown that the total concentration of saponins in seeds was 0. 09-0.1% of dry matter. In germinating seeds an increase in concentration to 0.18% was observed after 4 days of germination, which remained stable for the next 3 days and later dropped to 0.09%. Highly purified extracts from the seeds were tested for their toxicity against hamsters. The hydrophobic fraction obtained by the extraction of seeds with methylene chloride showed no toxicity; the behavior of tested animals was similar to that of the group given an equivalent dose of rapeseed oil. A crude saponin fraction, containing approximately 70% of pure saponins in the matrix, showed some toxicity; the approximate lethal dose was calculated as 1100 mg/kg of body weight. It is concluded that low contents of saponins in amaranth seeds and their relatively low toxicity guarantee that amaranth-derived products create no significant hazard for the consumer.

In addition to the known soyasaponin I, 3- O -[α- l -rhamnopyranosyl(1→2)-β- d -galactopyranosyl(1→2-β- d -glucuronopyranosyl]-22- O -[β- d -glucuronopyranosyl(1→2)-β- d -glucopyranosyl]soyasapogenol B, soyasaponin II, 3- O -β- d -glucopyranosyl sitosterol, 3- O -β- d -glucopyranosyl stigmasterol and 3- O -α- l -arabinopyranosyl-(1→6)-β- d -glucopyranosyl]oct-1-ene-3-ol, the new saponin, 3- O -α- l -rhamnopyranosyl(1→2)-β- d -galactopyranosyl(1→2)-β- d -glucuronopyranosyl]-22- O -β- d -glucopyranosyl soyasapogenol B ( 2 ) was isolated and characterised from the seed saponins fraction of Trifolium resupinatum . The structural determination is based on spectroscopic methods (including DQF-COSY, HETCOR, TOCSY, ROESY and NOEs experiments).

Yucca schidigera Roezl. (Agavaceae) has been traditionally used to treat a variety of diseases including arthritis and rheumatism. Phenolic constituents isolated from yucca bark, such as resveratrol, trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene, and the yuccaols, have been shown to possess various activities in vitro, such as antioxidant, radical scavenging, iNOS expression inhibitory, and platelet aggregation inhibitory effects. In the present study, the influence of a phenolic-rich fraction from yucca bark and of its main phenolic constituents on key enzymes of arachidonate metabolism was investigated. The fraction and the pure phenolics were shown to inhibit COX-1, COX-2, and LTB 4 formation by 5-LOX in vitro to different extents. The degree of COX-1 inhibition was found to be strongly dependent on the substitution pattern of ring B of the stilbenic moiety. The same trend was observed for the COX-2 inhibitory potential, which was, however, in general much lower for the yuccaols as compared with resveratrol. Resveratrol was also the only compound possessing an LTB 4 formation inhibitory activity. The inhibitory activity on key enzymes of arachidonate metabolism observed in this study might contribute to the explanation of the anti-inflammatory and antiplatelet effects observed for Y. schidigera and its phenolic constituents.

The aim of the present study was to investigate and compare the anti-platelet action of extracts from three different plants: bark of Yucca schidigera, seeds of grape and berries of Aronia melanocarpa (chokeberry). Anti-platelet action of tested extracts was compared with action of well characterized antioxidative and anti-platelet commercial monomeric polyphenol-resveratrol. The effects of extracts on platelet adhesion to collagen, collagen-induced platelet aggregation and on the production of O2-* in resting platelets and platelets stimulated by a strong platelet agonist-thrombin were studied. The in vitro experiments have shown that all three tested extracts (5-50 microg/ml) rich in polyphenols reduce platelet adhesion, aggregation and generation of O2-* in blood platelets. Comparative studies indicate that all three plant extracts were found to be more reactive in reduction of platelet processes than the solution of pure resveratrol. The tested extracts due to their anti-platelet effects may play an important role as components of human diet in prevention of cardiovascular or inflammatory diseases, where blood platelets are involved.

Body mass reduction and regular physical training form part of a strategy to treat disorders of carbohydrate metabolism associated with obesity. Evidence shows that even a slight reduction in body mass may improve carbohydrate tolerance, lipid profile and insulin resistance, reduce insulin levels and finally delay or reduce risk of diabetes mellitus. Multiple studies, including prospective studies confirm the independent protective effects of physical training against future development of type 2 diabetes mellitus. Myocardial infarction is a severe complication of atherosclerosis. Patients with glucose intolerance have a 2-fold higher risk of dying. Impaired glucose tolerance is negatively associated with prognosis in patients after myocardial infarction. Glucose intolerance accompanies hyperinsulinemia, a major indicator of insulin resistance.The aim of the study was to analyze the effect of physical training on hyperinsulinemia/ insulin resistance in patients after myocardial infarction (MI) with impaired glucose tolerance (IGT).31 men aged 37-69 years (mean 51 +/- 7.4) with IGT 3.5 years after MI, in NYHA class I and II participated in the study. Group A (n=16 men) underwent supervised physical training and group B (n=15) received standard information on physical training. Tissue glucose disposal using normoglycemic glucose clamp technique, fasting insulinemia, glycemia during OGTT, lipid profile, BMI and body mass composition were obtained in all patients.The groups were matched for age. There were no differences in BMI, percent fat content, blood pressure, diet, smoking status and pharmacotherapy. Glycemia during baseline OGTT did not differentiate the groups, either. Analysis of insulinemia and glycemia during OGTT at baseline and at 12 weeks after regular physical training showed lower levels of insulinemia and glycemia compared with baseline levels in group A (fasting glycemia 6.41+/-0.46 vs. 4.8+/-0.32 mmol/l, p0.001; fasting insulinemia 59.42+/-38.9 vs. 27.42+/-26.5 umol/l, p0.001). A similar analysis in group B did not reveal any significant differences (fasting glycemia 6.17+/-0.49 vs. 6.18+/-0.46 mmol/ l; fasting insulinemia 61.86+/-48.5 vs. 68.86+/-78.5 umol/l). Mean tissue glucose disposal in the whole study population was 2.96+/-1.03 mg/kg bm/min.The present findings indicate the beneficial effects of regular physical training on the reduction of fasting insulinemia in patients after myocardial infarction with insulin resistance and improve lipid profile. In patients after myocardial infarction standard recommendations concerning physical activity are not well realized. It seems that better compliance, more strict control and motivation of patients can result in better metabolic control.

Horse chestnut extracts are widely used in pharmacy and cosmetic industries. The main active constituents are saponins of oleane type, but seeds of horse chestnut also contain flavonoids, being glycosides of quercetin and kaempferol. Their contribution to the overall activity of the extracts was not clear. In the present work, the main flavonoids from horse chestnut seeds were isolated and their structures established with spectral methods. Seven glycosides were isolated, out of which six ( 2, 3, 4, 7, 11, 13) were previously reported and one ( 9) was identified as a new tamarixetin 3- O- [beta- d-glucopyranosyl(1--3)]- O-beta- d-xylopyranosyl-(1--2)- O-beta- d-glucopyranoside. The structures of three additional compounds 1, 10, and 12, not previously reported, were deduced on the basis of their LC-ESI/MS/MS fragmentation characteristics. A new ultraperformance liquid chromatographic (UPLC) method has been developed for profiling and quantitation of horse chestnut flavonoids. The method allowed good separation over 4.5 min. Thirteen compounds could be identified in the profile, out of which di- and triglycoisdes of quercetin and kaempferol were the dominant forms and their acylated forms occurred in just trace amounts. The total concentration of flavonoids in the powdered horse chestnut seed was 0.88% of dry matter. The alcohol extract contained 3.46%, and after purification on C18 solid phase, this concentration increased to 9.40% of dry matter. The flavonoid profile and their content were also measured in the horse chestnut wastewater obtained as byproduct in industrial processing of horse chestnut seeds. The total flavonoid concentration in the powder obtained after evaporation of water was 2.58%, while after purification on solid phase, this increased to 11.23% dry matter. It was concluded that flavonoids are present in a horse chestnut extract in a relatively high amount and have the potential to contribute to the overall activity of these extracts. Industrial horse chestnut wastewater can be used to obtain quercetine and kaempferol glycosides for cosmetic, nutraceutical, and food supplement industries.

The results of two experiments with wheat grain inoculated with Penicillium verrucosum are reported. In Experiment I, wheat grain, containing 10, 20 and 30% water, was incubated for 2 weeks at 10, 15, 21 and 28 degrees C. In Experiment II, wheat grain, containing 14, 16, 18, 20 and 22% water, was incubated for 2 weeks at 10, 15, and 20 degrees C. At initial moisture content (IMC) of the wheat grain up to 16% neither P. verrucosum growth nor ochratoxin A (OTA) formation were observed. In the range of IMC from 18% to 22% both the fungal growth and OTA synthesis were distinct, and the parameters were higher at higher temperature in the range 10-21 degrees C. A temperature of 28 degrees C was probably too high for proper metabolism of the fungus, including OTA formation. OTA formation was distinctly related to P. verrucosum abundance in the temperature range 10-21 degrees C, expressed both as the counts of fungal colony forming units (CFU) on agar DYSG medium and diameters of the fungal colonies growing around the wheat kernels placed on the surface of DYSG medium. OTA formation and abundance of P. verrucosum were negatively correlated with the percentage of wheat kernels, placed on DYSG medium, with growing colonies of fungi different from P. verrucosum. CFU counts of P. verrucosum on the wheat grain were significantly related to the diameter of the fungal colonies growing around the wheat kernels placed on DYSG medium. The relationship is described by an exponential regression equation.

The phenolic content and composition in leaves of Trifolium pratense (red clover) and T. pratense subsp. nivale (snow clover) grown in Italy were evaluated by means of ultraperformance liquid chromatography (UPLC) coupled with photodiode array and mass spectrometry detectors. Compound identification was based on UV and MS data comparing results with those of reference compounds. Quantitative evaluation of all detected compounds was based on calibration curves obtained with available standards. Several phenolics were identified in both extracts, including clovamide, flavonols and isoflavones as their glycosilated and malonated derivatives. The total phenolic content was higher in red clover (53.7 ± 2.2 mg/g dry weight) than in snow clover (44.4 ± 4.9 mg/g dry weight). Red clover contained higher amounts of clovamide and isoflavones (15.6 ± 0.6 and 24.6 ± 1.6 mg/g dry weight, respectively) than snow clover (8.2 ± 0.1 mg/g and 16.9 ± 0.4 mg/g dry weight, respectively), while flavonols were quantified almost in the same amount in both extracts (13.2 ± 0.6 mg/g and 15.8 ± 0.6 mg/g dry weight in red clover and snow clover, respectively). Red clover was characterized by the presence of quercetin, formononetin and biochanin A derivatives as the most abundant flavonoids, whereas snow clover was characterized by higher amounts of quercetin and prunetin derivatives. This investigation, conducted for the first time on phenolics from T. pratense subsp. nivale, revealed the presence in this plant of several flavonoid derivatives the same as in T. pratense. The higher amount of prunetin in snow clover suggest a possible role of this isoflavone as a chemotaxonomic marker for this subspecies. Moreover, snow clover may represent an interesting new source of natural isoflavones with a different concentration pattern than in red clover.

Partially inconsistent data exist on mutual relations between nontraditional atherosclerotic risk factors, including the magnitude of insulin resistance (IR), as well as on their relevance for atherogenesis in the metabolic syndrome. Subjects exhibiting combined impaired fasting glucose and impaired glucose tolerance (IFG/IGT) are exposed to an exceptionally high risk for atherogenesis and development of type 2 diabetes mellitus. Because of islet Beta-cell dysfunction, the usefulness of commonly used indices of IR is limited in IFG/IGT. Our aim was to assess the relationship between extent of angiographic coronary artery disease (CAD) and nontraditional atherosclerotic risk factors (including IR by a clamp-based golden standard method) in IFG/IGT. Fifty-three subjects (32 men, 21 women; mean age, 55 +/- 11 years) with stable angina, preserved left ventricular systolic function, and IFG/IGT were divided into 3 groups: group A (no coronary stenoses >50%, n = 22), group B (1-vessel CAD, n = 15), and group C (2/3-vessel CAD, n = 16). Insulin sensitivity was quantified by a hyperinsulinemic euglycemic clamp technique and expressed as M. M value, plasma homocysteine (Hcy) level, and asymmetric dimethyl-L-arginine (ADMA)/L-arginine ratio were independent determinants of CAD extent as shown by forward stepwise discriminant function analysis. Compared with group A (M = 32.7 +/- 9.3 micromol/kg fat-free mass [FFM] per minute; Hcy, 8.1 +/- 1.4 micromol/L), lower M and higher Hcy levels were found in group B (M = 16.9 +/- 8.2 micromol/kg FFM per minute, P < .001; Hcy, 11.2 +/- 2.9 micromol/L, P = .003) and C (M = 16.4 +/- 7.8 micromol/kg FFM per minute, P < .001; Hcy, 12.8 +/- 3.9 micromol/L, P < .001). The ADMA/L-arginine ratio was increased in group C (0.0078 +/- 0.0011) compared with group A (0.0063 +/- 0.0013, P = .03) and B (0.0058 +/- 0.0012, P = .01). Multivariate correlates (P < .05) of plasma Hcy concentrations were M (beta = -.34 +/- .12, P = .008), creatinine clearance (beta = -.23 +/- .10, P = .03) and fasting insulin (beta = .25 +/- .12, P = .04). This indicates an additive contribution of IR, plasma Hcy, and elevated ADMA/L-arginine ratio to the extent of angiographic CAD in combined IFG/IGT.

Interactions between the moth Spodoptera littoralis and two of its host plants, alfalfa (Medicago sativa) and cotton (Gossypium hirsutum) were examined, using plants grown under ambient (350 ppm) and elevated (700 ppm) CO2 conditions. To determine strength and effects of herbivore-induced responses assays were performed with both undamaged (control) and herbivore damaged plants. CO2 and damage effects on larval host plant preferences were determined through dual-choice bioassays. In addition, larvae were reared from hatching to pupation on experimental foliage to examine effects on larval growth and development. When undamaged plants were used S. littoralis larvae in consumed more cotton than alfalfa, and CO2 enrichment caused a reduction in the preference for cotton. With damaged plants larvae consumed equal amounts of the two plant species (ambient CO2 conditions), but CO2 enrichment strongly shifted preferences towards cotton, which was then consumed three times more than alfalfa. Complementary assays showed that elevated CO2 levels had no effect on the herbivore-induced responses of cotton, whereas those of alfalfa were significantly increased. Larval growth was highest for larvae fed undamaged cotton irrespectively of CO2 level, and lowest for larvae on damaged alfalfa from the high CO2 treatment. Development time increased on damaged cotton irrespectively of CO2 treatment, and on damaged alfalfa in the elevated CO2 treatment. These results demonstrate that elevated CO2 levels can cause insect herbivores to alter host plant preferences, and that effects on herbivore-induced responses may be a key mechanism behind these processes. Furthermore, since the insects were shown to avoid foliage that reduced their physiological performance, our data suggest that behavioural host plant shifts result in partial escape from negative consequences of feeding on high CO2 foliage. Thus, CO2 enrichment can alter both physiology and behaviour of important insect herbivores, which in turn may to impact plant biodiver

Triterpene saponins from aerial parts of Medicago truncatula cv. Jemalong A-17, M. truncatula Gaertn.var. longispina Urb., and M. truncatula Gaertn. var. truncatula were profiled and quantified using reverse-phase liquid chromatography with on-line photodiode array detection and electrospray ionization mass spectrometry (LC-PDA/ESI/MS/MS). The determination was based on standard curves obtained for the 18 available saponin standards, previously isolated from Jemalong A-17. Aerial parts of all three subspecies contained 17 saponins previously identified and also a substantial amount of astragaloside VIII (3-GlcA-Xyl-Rha soyasapogenol B), not previously reported in M. truncatula. The compositions of saponin mixtures were very similar in the three subspecies with three dominant groups, recognized as zanhic acid, medicagenic acid, and soyasapogenol glycosides. Relative proportions of these three groups were also similar in the three subspecies: var. longispina had 49.5, 48.1, and 2.4%; var. truncatula, 41.5, 53.4, and 5.1%; and Jemalong A-17, 42.1, 56.6, and 1.3% of zanhic acid, medicagenic acid, and soyasapogenol glycosides, respectively. Jemalong A-17 had 30% lower total content of saponins as compared to M. truncatula var. longispina and M. truncatula var. truncatula; in relation to the dry matter, var. longispina contained 0.22%, var. truncatula, 0.22%, and Jemalong A-17, 0.15% dry matter of saponins. If one takes into consideration that this determination was performed on spring-collected samples, it can be concluded that the concentration of saponins in M. truncatula is similar to the concentration in alfalfa (Medicago sativa); the proportions of the three groups of saponins in these species are slightly different from those found in alfalfa, having a higher content of zanhic acid glycosides.

We examined herbivore-induced responses of alfalfa (Medicago sativa) and cotton (Gossypium hirsutum) under different CO2 conditions. Plants were grown under ambient (350 ppm) or elevated (700 ppm) CO2 levels, and were either damaged or undamaged by Spodoptera littoralis larvae. At harvest, growth of undamaged (control) plants was determined, and foliar chemical composition of both undamaged and damaged plants was analyzed. Cotton grew faster overall and showed a greater increase in growth in response to CO2 enrichment than did alfalfa. Elevated CO2 levels increased starch and decreased nitrogen levels in damaged alfalfa and undamaged cotton plants. Alfalfa saponin levels were significantly increased by elevated CO2 and damage. Regarding specific saponins, medicagenic acid bidesmoside (3GlcA,28AraRhaXyl medicagenate) concentrations were reduced by high CO2, whereas zanhic acid tridesmoside (3GlcGlcGlc,23Ara,28AraRhaXylApi Za) levels were unaffected by the treatments. Soyasaponin I (3GlcAGalRha soyasapogenol B) was only detected in minute amounts. Alfalfa flavonoid analyses showed that total flavonoid levels were similar between treatments, although free apigenin increased and apigenin glucoside (7-O-[2-O-feruloyl-beta-D-glucuronopyranozyl (1--2)-O-8-D-glucuronopyranozyl]-4'-O-beta-D-glucuronopyranozide apigenin) decreased in CO2-enriched plants. In cotton, herbivore damage increased levels of total terpenoid aldehydes, gossypol, hemigossypolone, the heliocides H1 and H4, but not H2 and H3, whereas CO2 enrichment had no effect. These results demonstrate that combined effects of CO2 and herbivore damage vary between plant species, which has implications for the competitive balance within plant communities.

Seven new steroidal glycosides named pentandrosides A(1)-G(7) were isolated from the EtOH extract of the aerial parts of Tribulus pentandrus. Pentandrosides A(1)-E(5) possess cholestane aglycones, pentandroside F(6) a furostan-type aglycone and pentandroside G(7) an unusual acyloxypregnane aglycone probably derived from the degradation of a furostan skeleton. Structure elucidation of 1-7 was accomplished through the extensive use of 1D- and 2D NMR experiments including 1H-1H (DQF-COSY, 1D-TOCSY) and 1H-13C (HSQC, HMBC) spectroscopy along with ESIMS and HRESIMS.

The mutagenic activity of yuccaols A, B, and C, trans-resveratrol and trans - 3.3',5.5'-tetrahydroxy -4'-methoxystilbene was tested by the Ames method with Salmonella typhimurium strains TA97, TA98, TA100, TA102 in the absence and presence of metabolic activation (S9 fraction). These phenolic compounds have been isolated and identified from the hark of Yucca schidigera. All of them were found to be non-toxic and non-mutagenic for testing doses in any of the S. typhimurium strains.

8 pages, 4 figures, 6 tables.-- PMID: 15481461 [PubMed].-- Printed version published Aug 20, 2004.-- Presented at the 3rd Meeting of the Spanish Association of Chromatography and Related Techniques and the European Workshop: 3rd Waste Water Cluster, Aguadulce (Almeria), Spain, Nov 19–21, 2003., Six laboratories from four different countries participated in the first European interlaboratory comparison exercise within the framework of the “Fate and toxicity of allelochemicals (natural plant toxins) in relation to environment and consumer” (FATEALLCHEM) European Union Project. The study, organized between November 2002 and March 2003, involved the analyses of seven benzoxazinone derivatives in two standard solutions and one purified extract of root material. Results are reported from the first phase of the study that examined the variability associated with different detection methods and different laboratories. The analytical strategies were based on liquid chromatography (LC) with diode array detection, LC coupled to mass spectrometry (MS) and LC coupled to tandem MS. When data from all laboratories were pooled, the relative standard deviation values ranged from 2 to 14% for the determination of target compounds in standard solutions, and between 19 and 47% for the analysis in root material. Comparison of the three detection techniques leads to the conclusion that MS approaches are the most accurate and precise techniques for the determination of benzoxazinone derivatives at ng/μL level in plant material., This work was financially supported by the programme “Quality of life and management of living resources (1998 to 2002)” of the European Union, FATEALLCHEM Contract No. QLRT-2000-01967, and by the Spanish Ministerio de Ciencia y Tecnología (AGL2001-4952-E).

The herbivore-induced response of alfalfa (Medicago sativa) was examined through assays with Spodoptera littoralis larvae and analyses of important secondary substances. In food preference experiments, larvae preferred young undamaged alfalfa plants over plants that had been damaged by feeding larvae 5 and 7 days earlier. while no difference in feeding preferences could be detected 1. 9. and 14 days after damage. This suggests a peak in the herbivore induced resistance of alfalfa approximately one week after initial damage. The induced resistance in young plants was also shown to be systemic, while older flowering plants failed to show increased resistance after defoliation. Larvae gained weight slower and had lower pupal mass when fed damaged alfalfa than when fed undamaged alfalfa. Levels of total saponins were increased in foliage of damaged alfalfa, and detailed analyses of specific saponin components revealed doubled concentrations of 3GlcA,28AraRhaXyl medicagenate (medicagenic acid bidesmoside) and 3GlcAGalRha soyasapogenol B (soyasaponin I). Levels of the flavonoid apigenin (as free aglycone) also were increased in herbivore damaged plants. The herbivore-induced response of alfalfa was significantly weaker than that of cotton: S. littoralis larvae given a choice of undamaged cotton and undamaged alfalfa preferred to feed on cotton, whereas preferences shifted towards alfalfa when plants were damaged.

Hot pepper fruits (Capsicum annuum L.) var. Bronowicka Ostra have been studied with regard to content of flavonoids and other phenolics. Nine compounds were isolated from pericarp of pepper fruits by preparative HPLC. Their structures were identified by chromatographic (analytical HPLC) and spectroscopic (UV, NMR) techniques. Two of the identified compounds, trans-p-ferulylalcohol-4-O-(6-(2-methyl-3-hydroxypropionyl) glucopyranoside and luteolin-7-O-(2-apiofuranosyl-4-glucopyranosyl-6-malonyl)-glucopyranoside were found for the first time in the plant kingdom. Additionally compounds: trans-p-feruloyl-beta-D-glucopyranoside, trans-p-sinapoyl-beta- D-glucopyranoside, quercetin 3-O-alpha-L-rhamnopyranoside-7-O-beta-D-glucopyranoside, luteolin 6-C-beta-D-glucopyranoside-8-C-alpha-L-arabinopyranoside, apigenin 6-C-beta-D-glucopyranoside-8-C-alpha-L-arabinopyranoside and luteolin 7-O-[2-(beta-D-apiofuranosyl)-beta-D-glucopyranoside] were found for the first time in pepper fruit Capsicum annuum L.

Resveratrol (3,4',5-trihydroxystilbene) has been reported to have a variety of anti-inflammatory, anti-carcinogenic, anti-fungal and anti-platelet effects. It occurs naturally in different medicinal plants. Recently, resveratrol and other related phenolic compounds including trans-3,3',5,5'-tetrahydroxy-4'-methoxystilbene and yuccaols A and C were isolated from the bark of Yucca schidigera. The aim of the present study was to evaluate in vitro the effects of these compounds on platelet aggregation induced by thrombin and ADP. Pretreatment of platelets with resveratrol or other tested phenolics (1-25 microg/ml) slightly reduced platelet aggregation stimulated by 5 microM ADP (P0.05) or 10 microM ADP (P0.005). The comparison of the inhibitory effects of tested compound in thrombin-induced platelet aggregation revealed that phenolic showed even stronger antiplatelet actions than resveratrol. These compounds also had an inhibitory effect on the thrombin-induced enzymatic platelet lipid peroxidation determined as the level of thiobarbituric acid reactive substances.

A new, very unusual, phenolic constituent based on a spiro benzopyran-4-cyclopentan-3-one system has been isolated from Yucca schidigera bark and its structure has been established by ESI-MS and NMR experiments. In particular the relative configuration of the molecule has been defined combining the NMR data with ab initio conformational studies; the minimum energy conformers for the eight possible diastereomers were compared to the NMR experimental data in order to assess the diastereomer fitting of all the J coupling values and the dipolar values.

The concomitant presence of diabetes mellitus and arterial hypertension significantly impairs myocardial function through a direct negative effect on cardiac myocytes, coronary microvessels and precipitation of atherosclerosis in major coronary arteries. The purpose of the present study was to establish to what extent non-insulin dependent diabetes mellitus (NIDDM) modified silent myocardial ischaemia (SMI) in patients with essential hypertension and without documented coronary artery disease (CAD). The study population consisted of 41 patients with essential arterial hypertension associated with NIDDM, treated with diet and oral hypoglycaemic agents (group I) and 40 patients with essential arterial hypertension without diabetes mellitus (group II). Both groups were comparable with respect to age, gender, duration, severity and complications of hypertension. A mean duration of diabetes mellitus in group I was 6.8 years. Conventional and automatic blood pressure and heart rate measurements, continuous ECG recordings, echocardiograms and laboratory tests were obtained in all patients. SMI was more frequent in group I than in group II (29.3% vs 12.5%, P < 0.05). In group I the total duration of SMI was longer (37.3 vs 2.8 min, P < 0.001) and the total number of silent episodes was larger (15.5 vs 2.6, P < 0.001). No inter-group differences were seen in conventional and automatic blood pressure and heart rate measurements. Both groups did not differ significantly in left ventricular mass index (LVMI) or the proportion of patients with left ventricular hypertrophy (LVH) (75.6% vs 60%). Lipid profile in both groups indicated an increased risk of CAD, but without significant differences. In conclusion, in patients with essential arterial hypertension and diabetes mellitus, the incidence and severity of SMI were clearly higher than in hypertensives with normal carbohydrate metabolism. Employment of modern diagnostic techniques in hypertensives permits identification of those at greater risk, which may have further clinical implications.

The fruits of the edible and medicinal Egyptian palm, Medemia argun, were collected from Aswan in Egypt and the essential oil (EO) from fruits and headspace (HS) of the seeds and fleshy mesocarps were analyzed by GC and GC-MS. Results obtained by GC-MS analysis indicated a high variability in the oil and in the headspace from seeds and mesocarps. Sesquiterpene derivatives were the main group of volatiles in the EO from fruits and in the HS from seeds (45.0 and 64.0%, respectively), while oxygenated hydrocarbon derivatives were the main constituents in the HS obtained from fleshy mesocarps (96.5%). The different chemical composition of the headspace obtained from the seeds and mesocarps of M. argun can be correlated with the different roles that the different constituents play in the prevention of dehydration of the fruits in the desert region from where the plant was collected.

The nutritional quality of Persian clover (Trifolium resupinatum), an important pasture crop, depends not only on a high protein content but also on the occurrence of animal health and welfare promoting phytochemicals. Nine phenolic constituents present in the aerial parts of this species were isolated and their structures confirmed by NMR and ESI-MS analyses. The compounds included two chlorogenic acids, four quercetin and two kaempferol glycosides, as well as the isoflavone formononetin-7-glucoside. The concentration of isoflavone was low, not exceeding 1.2 mg/g of dry matter. The concentration of flavonols ranged between 5.9 and 11.8 mg/g, depending on the sampling dates, with the highest concentration occurring in the first cut. A similar trend in the concentration was found for chlorogenic acids, which ranged from 2 mg/g in summer to 7.3 mg/g in spring.

The absorption spectra of twenty-one flavones including glycosides of apigenin, luteolin, chrysoeriol and tricin, nonacylated or acylated with coumaric, ferulic or sinapic acids, were examined to document the influence of acylation on their spectral characteristics. Acylation did not shift the absorption maximum of band II, but the molar absorption coefficients of this band increased 1.3–2.0 fold. Acylation of apigenin and chrysoeriol glycosides did not shift the absorption maximum of band I and the absorption maxima of nonacylated and acylated compounds were at 319–334 nm, but this increased their molar absorption coefficients 1.7–3.0 fold. For luteolin and tricin glycosides, nonacylated compound maxima of absorption of band I were in the range of 342–349 nm. Acylation shifted band I absorption maxima to 329–340 nm and increased their molar absorption coefficient 1.6–1.8 fold. These data document that acylation of flavones leads to significant increase of their absorption in the 280–320 nm region and can be an effective mechanism of protecting plants against UV-B radiation.