Biological activity of total lipids from red and white wine/must.

Wine is an essential component of the Mediterranean diet, and it is thought to exert a protective effect against coronary heart disease. Although many efforts have been made to determine the protective compounds in wines, their exact nature and how they are involved in the protection mechanisms are still unclear. In this study, total lipids, total polar lipids, and total neutral lipids of five wines and three musts were tested in vitro for their ability to induce washed rabbit platelet aggregation and/or to inhibit platelet activating factor (PAF) induced aggregation. The results showed that the biological activity of wine/must total lipids can be attributed mainly to total polar lipids. In the red wine Cabernet Sauvignon, we fractionated total neutral lipids, total polar lipids, and pigments by HPLC. Each fraction was tested in vitro for its biological activity. Structural data of the most active fractions, based on biological, chemical, and spectral methods, are also presented.     

Optimization of lipase-catalyzed synthesis of acetylated tyrosol by response surface methodology

The ability of a noncommercial immobilized lipase from Staphylococcus xylosus (SXLi) to catalyze the transesterification of tyrosol and ethyl acetate was investigated. Response surface methodology was used to evaluate the effects of the temperature (40-60 degrees C), the enzyme amount (50-500 UI), and the ethyl acetate/hexane volume ratio (0.2-1) on the tyrosol acetylation conversion yield. Two independent replicates were carried out under the optimal conditions predicted by the model (reaction temperature 54 degrees C, enzyme amount 500 UI, and volume ratio ethyl acetate/hexane 0.2). The maximum conversion yield reached 95.36 +/- 3.6%, which agreed with the expected value (96.8 +/- 3.7%). The ester obtained was characterized by spectroscopic methods. Chemical acetylation of tyrosol was performed, and the products were separated using HPLC. Among the eluted products from HPLC, mono- and diacetylated derivatives were identified by positive mass spectrometry. Tyrosol and its monoacetylated derivative exert similar antiradicalar activity on 2,2-diphenyl-1-picrylhydrazyle.     

Separation of biologically active lipids from red wine

A number of lines of evidence suggest that red wine exerts a protective effect against coronary heart disease, but the nature of the protective compounds is unclear and the mechanism is incompletely understood. In this study, total lipids of a Greek red wine were separated into neutral and polar lipids. Polar lipids were further separated into glyco- and phospholipids, which were fractionated by HPLC. Each lipid fraction was tested in vitro for its ability to inhibit platelet-activating factor (PAF) and thrombin-induced washed rabbit platelet aggregation and/or to cause platelet aggregation. A significant number of glyco- and phospholipids that exerted the above biological activities were detected. Structural data of an active phosphoglycolipid are also provided. trans-Resveratrol demonstrated also a dose-dependent inhibition of PAF-induced platelet aggregation along with the already reported inhibitory activity against thrombin and adenosine-5'-diphosphate. Because it has already been reported that PAF is involved in atheromatosis generation, the existence of PAF inhibitors in red wine may contribute to the protective role of red wine against atherosclerosis.     

Structure and function of the oxidation products of polyphenols and identification of potent lipoxygenase inhibitors from Fe-catalyzed oxidation of resveratrol

Polyphenols have recently attracted much attention as potent antioxidants and related bioactive substances. These potent antioxidative polyphenols are very oxidizable due to their chemical properties, and their oxidation products must accumulate in the oxidizing foods when they are contained as the active ingredients. In this investigation, 30 polyphenols and related phenolics were oxidized with oxygen in the presence of a catalytic amount of Fe ions. Piceatannol, catechin, epicatechin, hydroxytyrosol, carnosol, and carnosic acid were oxidized very quickly. Sinapic acid, caffeic acid, chlorogenic acid, rosmarinic acid, gallic acid, propyl gallate, α-tocopherol, quercetin, and nordihydroguaiaretic acid were moderately oxidized. Protocatechuic acid, syringic acid, taxifolin, resveratrol, gentisic acid, secoisolariciresinol, and ellagic acid were oxidized for 19-20 days; however, their oxidation was very slow and did not complete. The other phenolics were not oxidized. The obtained oxidation products were next subjected to a lipoxygenase inhibition assay and the results compared to those of the corresponding phenols. Very interestingly, the oxidation product from resveratrol showed a high inhibitory activity, whereas resveratrol itself had no activity and its oxidation efficiency was low. To clarify the inhibition principle of the oxidation product, an LC-MS analysis was carried out on the oxidation product. The analytical results showed that they are the oligomeric and degraded compounds of resveratrol. Among them, the structures of three dimeric compounds were successfully identified, and their activity data clarified that the closed ring dimers were potent lipoxygenase inhibitors, whereas the opened ring dimer was not. It should be noted that resveratrol had almost no lipoxygenase inhibitory activity, contrary to some researchers' findings.     

New lipophilic tyrosyl esters. Comparative antioxidant evaluation with hydroxytyrosyl esters

New lipophilic esters of tyrosol, a naturally occurring phenol with interesting biological properties, have been synthesized in good yields by a chemoselective procedure, using lipase from Candida antarctica or p-toluenesulfonic acid as catalysts. Their antioxidant activities have been evaluated by the Rancimat test in lipophilic food matrices, as well as by FRAP and ABTS assays in methanolic solutions, and compared with those of previously synthesized hydroxytyrosyl esters. Free tyrosol, hydroxytyrosol, butylhydroxytoluene, and alpha-tocopherol were used as standards. All methods used for the antioxidant activity evaluation emphasized the high influence of the ortho-diphenolic structure on the antioxidant capacity, tyrosol and its derivatives being less active than hydroxytyrosol and its analogues and even less than BHT and alpha-tocopherol. In addition, the Rancimat test revealed a lower activity for ester derivatives than for their respective reference compounds (HTy or Ty), in agreement with the polar paradox. On the other hand, FRAP and ABTS methods reported an opposite behavior between the synthetic esters and their respective references. Thus, hydroxytyrosyl esters were more active than HTy, whereas tyrosyl esters were less active than Ty. The length and nature of the acyl side chain did not seem to play an important role in the antioxidant activity of either the hydroxytyrosyl or tyrosyl ester series, since no significant differences were observed among them.     

Biologically active lipids with antiatherogenic properties from white wine and must

Wine can be considered an integral part of the Mediterranean diet. Evidence from epidemiological and experimental studies suggests a protective effect against the development of coronary heart disease with moderate consumption of wine and especially red wine. The exact nature of the protective effect remains to be established. However, mechanisms including low-density lipoproteins oxidation, inhibition of platelet aggregation, modification of eicosanoid metabolism, and endothelium-dependent relaxation of blood vessels are recognized as contributory. In this study, a new approach has been examined, based on previous reports that platelet-activating factor (PAF) is involved in atherogenesis. An attempt was made to detect key components in wine/must that through inhibition of PAF action may contribute to the protective role of white wine/must against atherosclerosis. More specifically, polar lipids from four wines and three musts were fractionated by thin-layer chromatography, and fractions were tested in vitro for their ability to inhibit PAF and thrombin-induced washed rabbit platelet aggregation and/or to cause platelet aggregation. On the basis of the above results, a white wine from the principal Greek grape Rompola and its respective must with and without extra yeast were chosen for further high-performance liquid chromatography separation. A significant number of biological active lipids were detected, and structural data for the most active lipids are provided.     

Does white wine qualify for French paradox? Comparison of the cardioprotective effects of red and white wines and their constituents: resveratrol, tyrosol, and hydroxytyrosol

It is generally believed that the French paradox is related to the consumption of red wine and not other varieties of wine, including white wine or champagne. Some recent studies have indicated that white wine could also be as cardioprotective as red wine. The present investigation compares the cardioprotective abilities of red wine, white wine, and their principal cardioprotective constituents. Different groups of rats were gavaged with red wine, white wine, resveratrol, tyrosol, and hydroxytyrosol. Red wine and its constituent resveratrol and white wine and its constituents tyrosol and hydroxytyrosol all showed different degrees of cardioprotection as evidenced by their abilities to improve postischemic ventricular performance, reduce myocardial infarct size and cardiomyocyte apoptosis, and reduce peroxide formation. It was discovered in this study that although each of the wines and their components increased the enzymatic activities of the mitochondrial complex (I-IV) and citrate synthase, which play very important roles in oxidative phosphorylation and ATP synthesis, some of the groups were more complex-specific in inducing the activity compared to the other groups. Cardioprotective ability was further confirmed by increased expression of phospho-Akt, Bcl-2, eNOS, iNOS, COX-1, COX-2, Trx-1, Trx-2, and HO-1. The results of this study suggest that white wine can provide cardioprotection similar to red wine if it is rich in tyrosol and hydroxytyrosol.     

Champagne wine polyphenols protect primary cortical neurons against peroxynitrite-induced injury

White wines are generally low in polyphenol content as compared to red wines. However, Champagne wines have been shown to contain relatively high amounts of phenolic acids that may exert protective cellular actions in vivo. In this study, we have investigated the potential neuroprotective effects of Champagne wine extracts, and individual phenolics present in these extracts, against peroxynitrite-induced injury. Organic and aqueous Champagne wine extracts exhibited potent neuroprotective activity against peroxynitrite-induced injury at low concentrations (0.1 microg/mL). This protection appeared to be in part due to the cellular actions of individual components found in the organic extracts, notably tyrosol, caffeic acid, and gallic acid. These phenolics were observed to exert potent neuroprotection at concentrations between 0.1 and 10 microM. Together, these data suggest that polyphenols present in Champagne wine may induce a neuroprotective effect against oxidative neuronal injury.     

Effect of postharvest ultraviolet irradiation on resveratrol and other phenolics of cv. Napoleon table grapes

In the skin of cv. Napoleon table grapes, the anthocyanins malvidin 3-glucoside (and its acetyl and p-coumaroyl derivatives), cyanidin 3-glucoside, peonidin 3-glucoside, cyanidin 3-glucoside, petunidin 3-glucoside, and delphinidin 3-glucoside were identified by HPLC-DAD-MS. In addition, quercetin 3-glucoside and 3-glucuronide, caffeoyltartaric, piceid, and resveratrol were also detected. The content of most phenolics remained quite constant during postharvest refrigerated storage (10 days at 0 degrees C) while the resveratrol derivatives increased 2-fold. Postharvest treatments of grapes with UVC and UVB light induced a large increase in resveratrol derivatives (3- and 2-fold, respectively). This means that a serving of mature Napoleon grapes (200 g) provides approximately 1 mg of resveratrol, which is in the range of the amount supplied by a glass of red wine. This can be increased to 2 or 3 mg of resveratrol per serving in grapes that have been irradiated with UVB or UVC, respectively. These results show that refrigerated storage and UV irradiation of table grapes can be beneficial in terms of increasing the content of potentially health-promoting phenolics.     

Influence of phenolic constituents from Yucca schidigera bark on arachidonate metabolism in vitro

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.     

Isolation and identification of antiplatelet aggregatory principles from the leaves of Piper lolot

The methanolic extract of Piper lolot, having shown potent inhibitory activity on platelet aggregation induced by arachidonic acid (AA) and platelet activating factor (PAF), was subjected to activity-guided isolation to yield twelve new amide alkaloids, piperlotine A-L (1-12), along with twenty-nine known compounds. Their structures were elucidated on the basis of spectroscopic analysis. The isolated compounds were tested for their inhibitory activity on the rabbit platelet aggregation. The compounds piperlotine A (1), piperlotine C (3), piperlotine D (4), piperlotine E (5), 3-phenyl-1-(2,4,6-trihydroxyphenyl)propan-1-one (21), 3-(4-methoxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one (22), 1-trans-cinnamoylpyrrolidine (24), sarmentine (26), pellitorine (27), methyl 3-phenylpropionate (32), and (10S)-10-hydroxypheophorbide a methyl ester (40) showed potent antiplatelet aggregation activity.     

Antioxidant properties of chlorogenic acid and its alkyl esters in stripped corn oil in combination with phospholipids and/or water

In bulk oil, it is generally thought that hydrophilic antioxidants are more active than lipophilic antioxidants. To test this hypothesis, the antioxidant activity of phenolics with increasing hydrophobicity was evaluated in stripped corn oil using both conjugated diene and hexanal measurements. Chlorogenic acid and its butyl, dodecyl, and hexadecyl esters were used as model phenolic antioxidants with various hydrophobicities. Results showed that hydrophobicity did not correlate well with antioxidant capacity. The combination of chlorogenic acid derivatives with dioleoylphosphatidylcholine (DOPC) and/or water was also studied to determine if the physical structure in the oil affected antioxidant activity. DOPC alone made hexadecyl chlorogenate a less effective antioxidant, but it did not change the antioxidant capacity of chlorogenic acid. In contrast, the combination of DOPC and water (～400 ppm) renders chlorogenic acid a less active antioxidant, whereas it does not change the activity of hexadecyl chlorogenate. These results show, in bulk oil, that intrinsic parameters such as the hydrophobicity of lipophilized phenolics do not exert a strong influence on antioxidant capacity, but they can be highly influential if potentialized by extrinsic factors such as physical structures in the oil.     

Lipid fractions with aggregatory and antiaggregatory activity toward platelets in fresh and fried cod (Gadus morhua): correlation with platelet-activating factor and atherogenesis

Cod (Gadus morhua) is a popular part of the diet in many countries on both sides of the North Atlantic; in most cases it is consumed fried. In this study, total lipids of cod muscle were separated into neutral and polar lipids, which were further fractionated by HPLC. The lipid fractions were tested in vitro, against washed rabbit platelets, for the probable existence of lipid compounds that either exhibit an action similar to that of platelet-activating factor (PAF) or inhibit the action of PAF. The platelet bioassay was used to evaluate total lipids, total polar lipids, and total neutral lipids, before any further separation. Detection of these compounds in fresh and fried cod could be used to evaluate the nutritional value of this important fish. The in vitro biological study of lipids showed that in fresh cod lipid fractions, ranges of PAF-like and anti-PAF-like activities were present, whereas in fried cod lipid fractions, both neutral and polar, anti-PAF activities were mainly observed. Because it has already been reported that PAF is involved in atheromatosis generation, the existence of PAF inhibitors in cod may contribute to the possible protective role of fish, in this case cod, against atherosclerosis.     

Antioxidant and antiulcerative properties of phenolics from Chinese quince, quince, and apple fruits

To evaluate the health benefits of Chinese quince and quince phenolics, their antioxidant properties and antiulcerative activity were investigated in comparison with apple phenolics as a reference. The strength of antioxidant activity and DPPH radical scavenging activity of these fruit phenolics varied according to different in vitro evaluation systems, whereas the antioxidative property of rat blood increased in all rats orally administered phenolics. Ferulic acid and isoferulic acid were detected as major metabolites in rats given apple phenolics, quince phenolics, and 5-caffeoylquinic acid standard. (-)-Epicatechin and its 3'-O-methyl ether could be detected in rats administered apple phenolics and (-)-epicatechin standard. In the ethanol-induced gastric ulcer, pre-administration of Chinese quince and quince phenolics suppressed the occurrence of gastric lesions in rats, whereas apple phenolics seemed to promote ulceration. The trend of myeloperoxidase activity was similar to that of the ulcer index. The results showed that Chinese quince and quince phenolics might have health benefits by acting both in blood vessels and on the gastrointestinal tract.     

Antithrombotic lipid minor constituents from vegetable oils. Comparison between olive oils and others

Many epidemiological studies suggest that vegetable oils and especially olive oil present a protective effect against atherosclerosis. In this study, total lipids (TL) of Greek olive oils and seed oils of four kinds, namely, soybean, corn, sunflower, and sesame oil, were separated into total polar lipids (TPL) and total neutral lipids (TNL) via a novel extraction procedure. TPL and TNL of olive oil were fractionated by HPLC for further study. Each lipid fraction from HPLC separation along with TL, TPL, and TNL lipid samples from oils were tested in vitro for their capacity to induce or to inhibit washed rabbit platelet aggregation. Comparison between olive and seed oils supports the superiority of olive oil as high levels of platelet activating factor (PAF) antagonists have been detected, mainly in TPL. In addition, the structure of the most active fraction from olive oil was elucidated, as a glycerol-glycolipid. Because it has already been reported that PAF plays a pivotal role in atherogenesis, the existence of PAF agonists and antagonists in vegetable oils may explain their protective role against atherosclerosis.     

Multivariate analysis of tronchuda cabbage (Brassica oleracea L. var. costata DC) phenolics: influence of fertilizers

A field experiment was carried out to investigate the effect of fertilization level on the phenolic composition of tronchuda cabbage ( Brassica oleracea L. var. costata DC) external and internal leaves. Eight different plots were constituted: a control without fertilization, one with organic matter, and six experiments with conventional fertilizers (nitrogen, boron, and sulfur, two levels each). The phenolic compounds were analyzed by reversed-phase HPLC-DAD. External and internal leaves revealed distinct qualitative composition. In the internal leaves were found 15 phenolics (5 kaempferol and 10 cinnamic acid derivatives), whereas the external leaves presented 3- p-coumaroylquinic acid and 13 kaempferol derivatives. Principal component analysis (PCA) was applied to assess the relationships between phenolic compounds, agronomical practices, and harvesting time. Samples obtained with conventional practices were quite effectively separated from organic samples, for both types of leaves. In general, samples developed without any fertilization presented the highest phenolics amounts: external and internal leaves contained 1.4- and 4.6-fold more phenolic compounds than the ones that received conventional fertilizer, respectively, and the internal leaves presented 2.4 times more phenolics than the ones grown with organic amendment. Additionally, samples from organic production exhibited higher total phenolics content than those from conventional practices, collected at the same time. Samples harvested first were revealed to be distinct from the ones collected later. The results show that it is possible to grow tronchuda cabbage without excess fertilizers, with highest amounts of phenolics and reduced environment contamination.     

Herbicidal pyridyl derivatives of aminomethylene-bisphosphonic acid inhibit plant glutamine synthetase

A series of aminomethylene-bisphosphonic acid derivatives, previously synthesized and shown to be endowed with herbicidal properties, were evaluated as potential inhibitors of plant glutamine synthetase. The cytosolic form of the enzyme was partially purified from rice cultured cells and assayed in the presence of millimolar concentrations of the compounds by means of three different assay methods, respectively measuring the hemibiosynthetic, the transferase, and the full biosynthetic reactions. Several compounds were found to exert a remarkable inhibition, with I(50) values similar to those obtained under the same conditions with a well-established inhibitor of glutamine synthetase, the herbicide phosphinothricin. Contrary to the reference compound, enzyme kinetics accounted for a reversible inhibition mechanism. The biological activity of the most active derivatives was further characterized by measuring free glutamine levels in cell suspension rice cultures following treatment with the inhibitors. Results confirmed their ability to interfere in vivo with nitrogen metabolism. A preliminary analysis of structure-activity relationship allowed it to be hypothesized that steric rather than electronic factors are responsible for the inhibitory potential of these compounds.     

Structural basis for antioxidant activity of trans-resveratrol: ab initio calculations and crystal and molecular structure

From the experimental crystal structure and ab initio calculations on resveratrol and its derivatives, structural features of mechanistic importance are described. The molecular structure reveals the relative coplanarity of the trans-stilbene skeleton, and the molecular packing in the solid state shows an extensive hydrogen bond network that elucidates the flip-flop motion of the three hydroxyl groups that alternately form and break H bonds with each of the neighboring phenolic oxygens. The dynamic behavior provoked by the alternation of hydrogen bond formation and breaking can result in the ready mobility of up to three hydrogen atoms per resveratrol molecule that can be transferred to reactive oxidants that are rich in electron density. In addition, theoretical studies confirm the planarity of resveratrol as well as for half of the molecule of a condensation dimeric derivative of resveratrol, trans-sigma-viniferin. Furthermore, these studies show the p-4'-OH group to be more acidic compared to the other two m-OH groups. These features correlate with the biological activity of resveratrol as an antioxidant and support earlier studies showing H-atom transfer to be the dominant mechanism by which phenolic antioxidants intercept free radicals.     

Resveratrol is a potent inhibitor of the dioxygenase activity of lipoxygenase

Resveratrol is a naturally occurring phytoalexin, present in grapes and other food products, with important antioxidant properties. Although still under debate, it is generally assumed that resveratrol has protective effects against heart diseases and probably tumor development. Lipoxygenase is a dioxygenase with peroxidase activity involved in the synthesis of mediators in inflammatory, atherosclerotic, and carcinogenic processes. Lipoxygenase activity is also involved in the generation of flavors and aromas in foods from animal or vegetal sources. The results presented here show that resveratrol was a potent inhibitor of the dioxygenase activity of lipoxygenase, with an IC(50) = 13 microM. Simultaneously, resveratrol was oxidized by the peroxidase activity of lipoxygenase with a V(max) = 0.28 microM min(-1) and a k(M) = 16.6 microM. Furthermore, oxidized resveratrol was as efficient a lipoxygenase inhibitor as in its reduced form. From the data obtained it can be concluded that both resveratrol and its oxidized form can act as inhibitors of the dioxygenase activity of lipoxygenase. In contrast, the hydroperoxidase activity of lipoxygenase was not inhibited by resveratrol. These results suggest that resveratrol may be used as an antioxidant food additive and as a pharmacological agent to prevent the generation of eicosanoids involved in pathological processes.     

Polyphenol glucosylating activity in cell suspensions of grape (Vitis vinifera)

Stilbenes are phenolic molecules that have antifungal effects in the plant and antioxidant and anti-cancer effects when consumed in the human diet. Glycosylation of stilbenes increases their solubility and may make them more easily absorbed by the intestine. We have found an activity in extracts of cultured cells of Vitis vinifera (cv. Gamay Freaux) that glucosylates the stilbene resveratrol to form piceid. The Km for UDP-Glucose was 1.2 mM, and the Km for resveratrol was 0.06 mM, values similar to those of other phenolic glucosyltransferases. We investigated the resveratrol glucosylating activity of the enzyme extracted from cells grown under different light treatments (dark, visible light, light + ultraviolet (UVC) radiation) and found the activity to be unaffected or slightly reduced. In contrast, UVC light strongly stimulated extractable quercetin glucosyltransferase activity. These results, combined with analysis of phenolic compounds extracted from the differently treated cells, suggest that the resveratrol glucosyltransferase is distinct from the glucosyltransferase(s) active on other phenolics.