Extract from <Emphasis Type="Italic">Conyza canadensis</Emphasis> as a modulator of plasma protein oxidation induced by peroxynitrite <Emphasis Type="Italic">in vitro</Emphasis>

The antioxidative activity of the extract from Conyza canadensis in plasma treated with peroxynitrite (ONOO⁻) (0.1 mM) was studied. C. canadensis is known to possess a broad set of pharmacological effects because of content of various antioxidants, antiplatelet and anticoagulant compounds. The aim of our study was to assess if this extract protects plasma proteins against oxidative/nitrative damages induced by ONOO⁻. The plasma components are continuously exposed to reactive oxygen/nitrogen species action. Peroxynitrite evokes oxidative stress and induces undesirable effects in biological systems and causes damage to biomolecules. The extract from Conyza (50–2500 mg/ml) caused a dose-dependent reduction of protein nitration by 90%. The oxidation of plasma proteins was diminished by about 75%. ONOO⁻ oxidized the plasma thiol groups and this process was inhibited by tested extract. The level of reduced protein thiols was increased thrice at the lowest concentration of extract (50 mg/ml). The highest concentration of extract decreased twice the level of protein thiols in reduced forms and increased the homocysteine level about 4.5 times. The obtained results demonstrated that the extract from Conyza possesses antioxidative properties in vitro, protects plasma proteins against toxicity induced by peroxynitrite and has modulating effects on thiol/disulfide redox status.     

Clovamide-rich extract from <Emphasis Type="Italic">Trifolium pallidum</Emphasis> reduces oxidative stress-induced damage to blood platelets and plasma

Numerous plants (including clovers) have been widely used in folk medicine for the treatment of different disorders. This in vitro study was designed to examine the antioxidative effects of the clovamide-rich fraction, obtained from aerial parts of Trifolium pallidum, in the protection of blood platelets and plasma against the nitrative and oxidative damage, caused by peroxynitrite (ONOO⁻). Carbonyl groups and 3-nitrotyrosine in blood platelet and plasma proteins were determined by ELISA tests. Thiol groups level was estimated by using 5,5′-dithio-bis(2-nitro-benzoic acid, DTNB). Plasma lipid peroxidation was measured spectrophotometrically as the production of thiobarbituric acid reactive substances. The results from our work indicate that clovamide-rich T. pallidum extract may reveal the protective properties in the prevention against oxidative stress. The presence of clovamide-rich T. pallidum extract (12.5–100 μg/ml) partly inhibited ONOO⁻-mediated protein carbonylation and nitration. All the used concentrations of T. pallidum extract reduced lipid peroxidation in plasma. The antioxidative action of the tested extract in the protection of blood platelet lipids was less effective; the extract at the lowest final concentration (12.5 μg/ml) had no protective effect against lipid peroxidation. The present results indicate that the extract from T. pallidum is likely to be a source of compounds with the antioxidative properties, useful in the prevention against the oxidative stress-related diseases.     

Red cabbage anthocyanins may protect blood plasma proteins and lipids

The present in vitro study was designed to examine the antioxidative activity of red cabbage anthocyanins (ATH) in the protection of blood plasma proteins and lipids against damage induced by oxidative stress. Fresh leaves of red cabbage were extracted with a mixture of methanol/distilled water/0.01% HCl (MeOH/H₂O/HCl, 50/50/1, v/v/w). Total ATH concentration [μM] was determined with cyanidin 3-glucoside as a standard. Phenolic profiles in the crude red cabbage extract were determined using the HPLC method. Plasma samples were exposed to 100 μM peroxynitrite (ONOO⁻) or 2 mM hydrogen peroxide (H₂O₂) in the presence/absence of ATH extract (5–15 μM); oxidative alterations were then assessed. Pre-incubation of plasma with ATH extract partly reduced oxidative stress in plasma proteins and lipids. Dose-dependent reduction of both ONOO⁻ and H₂O₂-mediated plasma protein carbonylation was observed. ATH extract partly inhibited the nitrative action of ONOO⁻, and significantly decreased plasma lipid peroxidation caused by ONOO⁻ or H₂O₂. Our results demonstrate that anthocyanins present in red cabbage have inhibitory effects on ONOO⁻ and H₂O₂-induced oxidative stress in blood plasma components. We suggest that red cabbage ATH, as dietary antioxidants, should be considered as potentially usable nutraceuticals in the prevention of oxidative stress-related diseases.     

In vitro study of the antioxidative properties of the glucose derivatives against oxidation of plasma components

Oxidative stress has been implicated in the pathogenesis of variety of diseases. Since the endogenous antioxidant defense may be not adequate to counteract the enhanced generation of oxidants, a growing interest in research for exogenous nutrients has been observed. The present study was designed to assess in vitro the antioxidative properties of the glucose derivatives: calcium d-glucarate, d-gluconic acid lactone, and sodium d-gluconate (0.5–3 mM) in the protection of plasma proteins and lipids, against the damage caused by 0.1 mM peroxynitrite (ONOO⁻). Exposure of plasma to ONOO⁻ resulted in carbonyl groups increase, 3-nitrotyrosine (3-NT) formation, reduction in thiol groups, and enhanced lipid peroxidation. d-Gluconic acid lactone and sodium d-gluconate effectively decreased 3-NT formation; the antinitrative action of calcium d-glucarate was less effective. In plasma samples incubated with ONOO⁻ and tested compounds, the level of carbonyl groups was decreased in comparison to plasma samples treated only with ONOO⁻. The level of protein −SH groups and glutathione was significantly higher in the presence of glucose derivatives than in plasma samples treated with ONOO⁻ only. All the tested compounds had the inhibitory effect on the peroxynitrite-induced plasma lipids peroxidation. The results obtained from our work indicate that calcium d-glucarate, d-gluconic acid lactone, and sodium d-gluconate may partly protect plasma proteins and lipids against peroxynitrite-induced damages.     

The protective effects of selenoorganic compounds against peroxynitrite-induced changes in plasma proteins and lipids

Many selenoorganic compounds play an important role in biochemical processes and act as antioxidants, enzyme inhibitors or drugs. The effects of a new selenocompound — bis(2-aminophenyl)-diselenide on oxidative/nitrative changes in human plasma proteins induced by peroxynitrite (ONOO⁻) were studied in vitro and compared with the those of ebselen, a well-known antioxidant. We also studied the role of the tested selenocompounds in peroxynitrite-induced plasma lipid peroxidation. Exposure of the plasma to peroxynitrite (0.1 mM) resulted in an increase in the level of carbonyl groups and nitrotyrosine residues in plasma proteins (estimated using the ELISA method and Western blot analysis). In the presence of different concentrations (0.025–0.1 mM) of the tested selenocompounds, 0.1 mM peroxynitrite caused a distinct decrease in the level of carbonyl group formation and tyrosine nitration in plasma proteins. Moreover, these selenocompounds also inhibited plasma lipid peroxidation induced by ONOO⁻¹ (0.1 mM). The obtained results indicate that in vitro bis(2-aminophenyl)-diselenide and ebselen have very similar protective effects against peroxynitrite-induced oxidative/nitrative damage to human plasma proteins and lipids.     

A Comparative Evaluation of the Response to Peroxynitrite by a Brain Endothelial Cell Line and Control of the Effects by Drug Targeting

The potent oxidant peroxynitrite (ONOO⁻) is formed after the combination of nitric oxide with superoxide and has been closely associated with the pathology of inflammatory disease. In particular, the generation of ONOO⁻ has been linked to central nervous system disorders including Alzheimer’s and Parkinson’s disease, multiple sclerosis and bacterial and viral meningitis. Specifically, ONOO⁻ has been implicated in the loss of blood–brain barrier (BBB) integrity during neuroinflammation, but the precise mechanisms through which the molecule acts to mediate neurovascular breakdown have not been established. The disruptive effects of ONOO⁻ could be mediated by either direct or indirect actions on the endothelial cells that comprise the major component of the BBB. The current study has comparatively assessed the direct toxic effects of ONOO⁻ on the brain endothelial cell line, b.End3 and C6 astrocytoma and NA neuroblastoma preparations. b.End3 cells were relatively resistant to ONOO⁻-induced cell death compared with C6 and NA cultures. The indirect involvement of ONOO⁻ in neuroendothelial disruption was pharmacologically determined via adhesion molecule expression and immunocompetent cell attachment to b.End3 cells. ONOO⁻-targeted drugs, including the selective free radical scavenger, uric acid, the decomposition catalyst 5,10,15,20-tetrakis (4-sulphonatophenyl) porphyrinatoiron (III) (FeTPPS) and the poly(ADP-ribose) polymerase inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-(N,N-dimethylamino) acetamide hydrochloride (PJ34) revealed that ONOO⁻ was only partly involved in E-selectin, ICAM-1 and VCAM-1 expression on b.End3 cells and also cytokine-induced T-lymphocyte attachment to the cell line. The results indicate that ONOO⁻ contributes to b.End3 cell disruption but is not exclusively responsible for the breakdown of neuroendothelial function.     

Hypoxic postconditioning enhances the survival and inhibits apoptosis of cardiomyocytes following reoxygenation: role of peroxynitrite formation

Objectives: Our previous study has shown that slow or “controlled” reperfusion for the ischemic heart reduces cardiomyocyte injury and myocardial infarction, while the mechanisms involved are largely unclear. In this study, we tested the hypothesis that enhancement of survival and prevention of apoptosis in hypoxic/reoxygenated cardiomyocytes by hypoxic postconditioning (HPC) are associated with the reduction in peroxynitrite (ONOO⁻) formation induced by hypoxia/reoxygenation (H/R). Methods: Isolated adult rat cardiomyocytes were exposed to 2 h of hypoxia followed by 3 h of reoxygenation. After 2 h of hypoxia the cardiomyocytes were either abruptly reperfused with pre-oxygenized culture medium or postconditioned by two cycles of 5 min of brief reoxygenation and 5 min of re-hypoxia followed by 160 min of abrupt reoxygenation. Results: H/R resulted in severe injury in cardiomyocytes as evidenced by decreased cell viability, increased LDH leakage in the culture medium, increased apoptotic index (P values all less than 0.01 vs. normoxia control group) and DNA ladder formation, which could be significantly attenuated by HPC treatment applied before the abrupt reoxygenation (P < 0.05 vs. H/R group). In addition, H/R induced a significant increase in ONOO⁻ formation as determined by nitrotyrosine content in cardiomyocytes (P < 0.01 vs. normoxia control). Treatment with the potent ONOO⁻ scavenger uric acid (UA) at reoxygenation significantly decreased ONOO⁻ production and protected myocytes against H/R injury, whereas the same treatment with UA could not further enhance myocyte survival in HPC group (P > 0.05 vs. HPC alone). Statistical analysis showed that cell viability closely correlated inversely with myocyte ONOO⁻ formation (P < 0.01). Conclusion: These data demonstrate that hypoxic postconditioning protects myocytes against apoptosis following reoxygenation and enhances myocytes survival, which is partly attributable to the reduced ONOO⁻ formation following reoxygenation. H.-C. Wang and H.-F. Zhang contributed equally to this study.     

Reactive oxygen species plasmatic levels in ischemic stroke

Oxidative stress is probably one of the mechanisms involved in neuronal damage induced by ischemia-reperfusion, and the antioxidant activity of plasma may be an important factor providing protection from neurological damage caused by stroke-associated oxidative stress. The aim of this study was to investigate the status of oxidative stress, NO and ONOO⁻ levels in patients with atherothrombotic and lacunar acute ischemic stroke and iNOS, eNOS and nitrotyrosine expression in the same patients. Plasma ONOO⁻ levels were significantly higher in patients than in controls while NO decreases in patients in respect to controls. Densitometric analysis of bands indicated that iNOS and N-Tyr protein levels were significantly higher in patients in respect to controls. This study has highlighted a significant NO decrease in our patients compared with controls and this is most probably due to the increased expression of inducible NO synthase by the effect of thrombotic attack. In fact, the constitutive NO isoforms, which produce small amounts of NO, are beneficial, while activation of the inducible isoform of NO, which produces much more NO, causes injury, being its toxicity greatly enhanced by generation of peroxynitrite. The significant ONOO⁻ increase observed in our patients, compared to controls, is most probably due to reaction of NO with O₂^·−. These findings suggest that free radical production and oxidative stress in ischemic stroke might have a major role in the pathogenesis of ischemic brain injury. Peroxynitrite might be the main marker of brain damage and neurological impairment in acute ischemic stroke.     

In vivo and in situ visualization of early physiological events induced by heavy metals in pea root meristem

Heavy metals (HMs) are toxic pollutants, which can negatively affect the physiological processes of plants; moreover, HMs can be present in the food chain endangering people’s health. The aim of this study was to investigate the early physiological events during HM exposure in the root tips of the food plant Pisum sativum L. Ten-day-old pea plants were treated with 100 μM CdCl₂ or CuSO₄, in nutrient solution for 48 h. We studied the rapid formation of different reactive oxygen species (hydrogen peroxide H₂O₂ and superoxide radical O₂^·−) and reactive nitrogen species (nitric oxide NO· and peroxynitrite ONOO⁻) together with membrane damage and cell death in the meristem cells of pea roots using in vivo and in situ microscopic methods. In our experimental system, copper and cadmium induced the formation of H₂O₂ and NO. Two hours of heavy metal treatments resulted in an increased O₂^·− formation; however, later the level of this reactive molecule dramatically decreased. We found that high levels of NO were needed for ONOO⁻ production under HM exposure. A fast loss of membrane integrity and decreased cell viability were detected in root tips of copper-treated plants. The effects of cadmium seemed to be slower compared to copper, but this non-essential metal also caused cell death. We concluded that viability decreased when NO and H₂O₂ levels were simultaneously high in the same tissues. Using the NO scavenger it was also evidenced that NO generation is essential for cell death induction under copper or cadmium stress.     

Anticoagulant activity of sulfated polysaccharide isolated from fermented brown seaweed Sargassum fulvellum

A sulfated polysaccharide with anticoagulant properties was isolated from the fermented brown seaweed Sargassum fulvellum. Freeze-dried S. fulvellum was fermented in an incubator for 10^th week at 25°C to convert seaweed macromolecules into anticoagulant sulfated polysaccharides (ASP). Anticoagulant activity was determined by an activated partial thromboplastin time (APTT) test using citrated human blood plasma. The 8^th week S. fulvellum crude seaweed extract (SWE) exhibited the highest blood anticoagulant activity. Therefore, 8^th week crude SWE was used for purification of ASP by two steps; DEAE cellulose anion-exchange followed by Sepharose 4B chromatography. The isolated ASP showed a single spot on agarose gel electrophoresis, which confirmed the purification status of our ASP. Polyacrylamide gel electrophoresis (PAGE) analysis showed that the molecular mass of the purified ASP was between 8 and 20 kDa. Polysaccharide and sulfate concentrations of the purified ASP were 180 and 29.70 μg mL⁻¹ respectively. ASP recovery was 1.32% (w/w) from the crude polysaccharide applied to the DEAE column. Purified ASP had a pH of 3.86 and was considered an acidic polysaccharide. Moreover, both ASP and heparin showed a relative clotting factor of 27.47 at the concentrations of 180 and 60 μg mL⁻¹ respectively. Therefore, S. fulvellum ASP can be considered a weaker anticoagulant than heparin. Results of the APTT, PT, and TT clotting assays showed that ASP was able to inhibit both intrinsic and extrinsic blood coagulation pathways. Finally, this study established a feasible and simple experimental protocol to isolate anticoagulant from fermented seaweeds leading to potential further development of anticoagulant agent for the pharmaceutical industry.     

Elicitor-induced accumulation of stilbenes in cell suspension cultures of Cayratia trifolia (L.) Domin

Cell cultures of Cayratia trifolia (Vitaceae), a tropical lianas, were maintained in Murashige and Skoog’s medium containing 0.25 mg l⁻¹ NAA, 0.2 mg l⁻¹ kinetin and casein hydrolysate 250 mg l⁻¹. Cell suspension cultures of C. trifolia accumulate stilbenes (piceid, resveratrol, viniferin, ampelopsin), which on elicitation by any of 500 μM salicylic acid, 100 μM methyl jasmonate, 500 μM ethrel and 500 mg l⁻¹ yeast extract, added on the 7th day, were enhanced by 3- to 6-fold (5–11 mg l⁻¹) by the 15th day.     

Influence of extremely-low-frequency magnetic field on antioxidative melatonin properties in AT478 murine squamous cell carcinoma culture

Effects of melatonin, extremely-low-frequency magnetic field (ELF-MF), and their combination on AT478 murine squamous cell carcinoma line were studied. Manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (Cu/ZnSOD), and glutathione peroxidase (GSH-Px) were used as markers of cells antioxidative status, and malondialdehyde (MDA) level was used as a marker of lipid peroxidation. After melatonin treatment, antioxidative enzyme activities were increased and MDA level was decreased. Application of ELF-MF on treated cells caused an increase of both superoxide dismutases activity and MDA level, but influence of ELF-MF on GSH-Px activity was negligible. All enzyme activity in culture medium containing melatonin (10⁻³, 10⁻⁴, 10⁻⁵ M) after exposure to ELF-MF were significantly diminished compared to cells treated only with melatonin. Also MDA levels after combined treatment with melatonin and ELF-MF were significantly decreased. Observed changes were statistically significant (p<0.05). These results strongly suggest that ELF-MF attenuates antioxidative actions of melatonin on cellular level.     

Estimation of fungal biomass in the decaying cones ofPinus densiflora

Fungal biomass in the decaying cones ofPinus densiflora was investigated. Leaching, immobilization and mobilization phases were recognized in the decomposition process of cones. Fungal biomass was estimated by the agar-film technique, using a conversion factor of 0.62 mg dry wt. mm⁻³ of hyphal volume to biomass and a factor of 2.5 for in-efficiencency of homogenization. The fungal biomass was 4.9±2.1 (mean±S.D.) mg dry wt. g⁻¹ dry matter in the cones on the tree, 11±6 mg g⁻¹ in the leaching phase, 19±7 mg g⁻¹ in the immobilization phase and 30±15 mg g⁻¹ in the mobilization phase. It significantly increased after cones had lain on the forest floor, and also in the immobilization phase. The latter result suggests that the fungal biomass contributed to the immobilization of nitrogen in the decomposition process. The ratio of ergosterol content to fungal biomass in the cones was 2.9–8.8 μg mg⁻¹ dry wt., lying in the range of 2–16 μg mg⁻¹ reported for mycelia. This suggested that the estimate of fungal biomass was reasonable. Reduction in this ratio with the dry weight loss in the cones suggested that the proportion of relatively active fungal biomass decreased with the progress of decomposition.     

<Emphasis Type="SmallCaps">l</Emphasis>-carnitine modulates blood platelet oxidative stress

The oxidative stress induced by acute exertion may interfere with blood platelet activation. The beneficial effect of l-carnitine (γ-trimethylamino-β-hydroxybutyric acid) on oxidative stress in blood platelets has not been fully investigated; however, different studies indicate that this compound modulates platelet functions. The aim of our study was to assess the effects of l-carnitine on platelet activation and oxidative/nitrative protein damage (determined by the levels of protein carbonyl groups, thiol groups, and 3-nitrotyrosine residues) in resting blood platelets or platelets treated with peroxynitrite (ONOO⁻, a strong physiological oxidant) in vitro. We also investigated the effects of l-carnitine on the level of platelet glutathione and on the formation of superoxide anion radicals ( O₂ ^{- ·} ) \left( {{\hbox{O}}_2^{ - \bullet }} \right) , lipid peroxidation measured by thiobarbituric acid reactive substances (TBARS) in blood platelets stimulated by thrombin (a strong physiological agonist), and platelet aggregation induced by adenosine diphosphate (a strong physiological stimulator). We have observed that carnitine decreases platelet activation (measured by platelet aggregation, the generation of O₂ ^{- ·} {\hbox{O}}_2^{ - \bullet } , and TBARS production). Moreover, our results in vitro demonstrate that carnitine may protect against oxidation of thiol groups induced by ONOO⁻. Thus, carnitine may have some protectory effects against oxidative changes induced in blood platelets.     

High stilbenes accumulation in root cultures of <Emphasis Type="Italic">Cayratia trifolia</Emphasis> (L.) Domin grown in shake flasks

The Root cultures of Cayratia trifolia (Vitaceae) a tropical lianas, were maintained in liquid Murashige and Skoog’s medium containing 0.5 mg l⁻¹ NAA, 0.1 mg l⁻¹ kinetin with 3% sucrose. These root cultures when grown with 6% sucrose accumulated stilbenes (piceid, resveratrol, viniferin, ampelopsin) in high amounts, which on elicitation by 500 mg l⁻¹ yeast extract, 50 μM salicylic acid (SA), 50 μM methyl jasmonate (MeJa), 500 μM ethrel added at 25th day, increased up to ninefolds (7.1 mg l⁻¹). Addition of alar or phenylalanine along with the elicitors further enhanced the stilbenes content. In the present study, stilbenes accumulation up to 12 folds (9.2 mg l⁻¹) was obtained with SA and alar. The SA was the most effective in increasing the stilbenes contents while less than control values were recorded in the cells treated with MeJa. The roots could be grown up to 2 l flasks. The present work demonstrates that presence of precursor and sucrose during elicitation at an appropriate time combined with growth retardation significantly increased the production of stilbenes in C. trifolia cell cultures.     

Brain mitochondrial dysfunction and oxidative damage in Parkinson’s disease

Complex factors contribute to the appearance of Parkinson’s disease (PD), but with a constant mitochondrial involvement. There are two interdependent conditions in PD: brain mitochondrial dysfunction and brain mitochondrial oxidative damage. Mitochondrial dysfunction and reduced complex I activity are recognized in substantia nigra and in frontal cortex in PD patients. The molecular mechanism involved in the inactivation of complex I is likely accounted by the sum of ONOO⁻ mediated reactions, reactions with free radical intermediates of the lipid peroxidation process and amine-aldehyde adduction reactions. The inhibitory effects on complex I lead synergistically to denaturation of the protein structure and to further increases of O₂⁻ and ONOO⁻ production at the vicinity of complex I. An adaptive response in PD patients has been described with increases in mtNOS activity, mitochondrial mass and mitochondrial biogenesis. Mitochondrial dysfunction in the human frontal cortex is to be considered a factor contributing to impaired cognition in PD.     

Ginsenoside Rd production from the major ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">β</Emphasis>-glucosidase from <Emphasis Type="Italic">Thermus caldophilus</Emphasis>

Under optimum conditions (pH 5, 75°C, and 0.2 U purified enzyme ml⁻¹), 4 mg ginsenoside Rd was produced from 5 mg reagent-grade ginsenoside Rb₁ in 5 ml after 30 min by β-glucosidase from Thermus caldophilus GK24. Using a ginseng root extract containing 1 mg ginsenoside Rb₁ ml⁻¹ and 3.2 mg additional ginsenosides ml⁻¹, 1.23 mg ginsenoside Rd ml⁻¹ was produced after 18 h; the concentrations of ginsenosides Rb₁, Rb₂, and Rc used for ginsenoside Rd production were 0.77, 0.17, and 0.19 mg ml⁻¹, respectively.     

Antioxidant role of astaxanthin in the green alga Haematococcus pluvialis

The green unicellular alga, Haematococcus pluvialis has two antioxidative mechanisms against environmental oxidative stress: antioxidative enzymes in vegetative cells and the antioxidative ketocarotenoid, astaxanthin, in cyst cells. We added a reagent that generates superoxide anion radicals (O₂ ⁻), methyl viologen, to mature and immature cysts of H. pluvialis. Tolerance to methyl viologen was higher in mature than in immature cysts. Mature (astaxanthin-rich) cysts showed high antioxidant activity against O₂ ⁻ in permeabilized cells, but not in astaxanthin-free cell extracts, while immature (astaxanthin-poor) cysts had very low antioxidant activities against O₂ ⁻ in both. The results suggested that astaxanthin accumulated in the cyst cells functions as an antioxidant against excessive oxidative stress. The same levels of antioxidant activities against O₂ ⁻ in both permeabilized cells and cell extracts from vegetative cells suggested the presence of antioxidative enzymes (superoxide dismutase). Received: 13 January 1997 / Received revision: 26 February 1997 / Accepted: 27 March 1997     

Production of sophorolipids from whey

Sophorolipids, obtained by a two-stage process starting from deproteinized whey concentrate using Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214, were compared to products from one-stage processes, using different lipidic compounds as substrates. Results showed that above all carbon source and not cultivation conditions had a distinct influence on the composition of the crude product mixture and therefore on the physicochemical and biological properties of the sophorolipids, such as, for example, surface activity, cytotoxicity and stability against hydrolases. The results were completed by corresponding data for purified mono- and diacetylated (17-hydroxyoctadecenoic)-1′,4′′-lactonized sophorolipids. Crude sophorolipid mixtures showed moderate to good surface active properties (SFT^min 39 mN m⁻¹, CMC 130 mg l⁻¹), water solubilities (2–3 g l⁻¹) and low cytotoxicities (LC₅₀ 300–700 mg l⁻¹). In contrast, purified sophorolipids were more surface active (SFT^min 36 mN m⁻¹, CMC 10 mg l⁻¹), less water soluble (max. 70 mg l⁻¹) and showed stronger cytotoxic effects (LC₅₀ 15 mg l⁻¹). Incubation of crude sophorolipid mixtures with different hydrolases demonstrated that treatment with commercially available lipases such as from Candida rugosa and Mucor miehei distinctly reduced the surface active properties of the sophorolipids, while treatment with porcine liver esterase and glycosidases had no effect. Received: 23 February 1999 / Received revision: 27 May 1999 / Accepted: 28 May 1999     

Effects of aqueous hop (Humulus Lupulus L.) extract on vascular reactivity in rats: Mechanisms and influence of gender and hormonal status

Phytoestrogens, naturally occurring plant compounds having oestrogenic and/or anti-oestrogenic activity, are present in many human foodstuffs including hop. Moderate intakes of isoflavonoid phytoestrogens have been associated with a reduction in cardiovascular diseases incidence. So, it is possible that hop (Humulus Lupulus L.) might similarly contribute to the reported health-beneficial effects of moderate beer consumption. Thus, the purpose of this study was to investigate in vitro effects of aqueous hop extract on thoracic vascular reactivity in Sprague Dawley male and female rats. Endothelium-intact thoracic arterial rings from male rats (MALE, n=8), sham-ovariectomized (Sham OVX) female (n=8) and ovariectomized (OVX) female rats (n=8) were used. We assessed the relaxation induced by aqueous hop extract (10⁻⁹, 10⁻² g/l) in aortic rings precontracted with norepinephrine (10⁻⁷ M), in the absence or in the presence of l-NAME (10⁻⁴ M), indomethacin (10⁻⁵ M), thapsigargin (10⁻⁴ M), iberiotoxin (3.10⁻⁸ M), apamin (3.10⁻⁸ M) and TEA (3.10⁻⁴ M). Aqueous hop extract induced relaxation of endothelium-intact thoracic arterial rings in MALE and Sham OVX rats, whereas a weak effect was observed in OVX rats. This vasorelaxation was strongly inhibited in presence of l-NAME, indomethacin and thapsigargin. These data indicated that aqueous hop extract-induced vasodilation, in male and intact female rats, is mediated by NOS activation, cyclooxygenase products and Ca²⁺ pathways. Moreover, our results suggested that effect of hop in enhancing vascular reactivity was independent of gender but strongly related to hormonal status.