Recent advances on Ilex paraguariensis research: minireview.

Ilex paraguariensis dried and minced leaves are made into a brewed tea, prepared in a sui generis manner by large populations in South America, having evolved from a tea drunk by the Guarani ethnic group to a beverage that has a social and almost ritualistic role in some South American modern societies. It is used both as a source of caffeine, in lieu or in parallel with tea and coffee, but also as a therapeutic agent for its alleged pharmacological properties. Although with some exceptions, research on biomedical properties of this herb has had a late start and strongly lags behind the impressive amount of literature on green tea and coffee. However, in the past 15 years, there was a several-fold increase in the literature studying Ilex paraguariensis properties showing effects such as antioxidant properties in chemical models and ex vivo lipoprotein studies, vaso-dilating and lipid reduction properties, antimutagenic effects, controversial association with oropharyngeal cancer, anti-glycation effects and weight reduction properties. Lately, promising results from human intervention studies have surfaced and the literature offers several developments on this area. The aim of this review is to provide a concise summary of the research published in the past three years, with an emphasis on translational studies, inflammation and lipid metabolism. Ilex paraguariensis reduces LDL-cholesterol levels in humans with Ilex paraguariensis dyslipoproteinemia and the effect is synergic with that of statins. Plasma antioxidant capacity as well as expression of antioxidant enzymes is positively modulated by intervention with Ilex paraguariensis in human cohorts. A review on the evidence implicating Ilex paraguariensis heavy consumption with some neoplasias show data that are inconclusive but indicate that contamination with alkylating agents during the drying process of the leaves should be avoided. On the other hand, several new studies confirm the antimutagenic effects of Ilex paraguariensis in different models, from DNA double breaks in cell culture models to mice studies. Novel interesting work has emerged showing significant effect on weight reduction both in mice and in rat models. Some mechanisms involved are inhibition of pancreatic lipase, activation of AMPK and uncoupling of electron transport. Intervention studies in animals have provided strong evidence of anti-inflammatory effects of Ilex paraguariensis, notably protecting cigarette-induced lung inflammation acting on macrophage migration and inactivating matrix-metalloproteinase. Research on the effects of Ilex paraguariensis in health and disease has confirmed its antioxidant, anti-inflammatory, antimutagenic and lipid-lowering activities. Although we are still waiting for the double-blind, randomized prospective clinical trial, the evidence seems to provide support for beneficial effects of mate drinking on chronic diseases with inflammatory component and lipid metabolism disorders.

The polyamines spermine and spermidine protect proteins from structural and functional damage by AGE precursors: a new role for old molecules?

Due to the importance of glycation in the genesis of diabetic complications, an intense search for synthetic new antiglycation agents is ongoing. However, a somewhat neglected avenue is the search for endogenous compounds that may inhibit the process and be a source of protodrugs. Based on their ubiquity, their polycationic nature, their essential role in growth, their relatively high concentrations in tissues, and their high concentrations in sperm, we hypothesized that polyamines inhibit glycation and that might be one of their so far elusive functions. In this study we demonstrate a potent antiglycation effect of physiological concentrations of the polyamines spermine and spermidine. We employed two approaches: in the first, we monitored structural changes on histones and ubiquitin in which polyamines inhibit glycation-induced dimer and polymer formation. In the second we monitored functional impairment of catalytic activity of antithrombin III and plasminogen. Protection is afforded against glycation by hexoses, trioses and dicarbonyls AGE precursors and is comparable to those of aminoguanidine and carnosine.

The botanical extracts of Achyrocline satureoides and Ilex paraguariensis prevent methylglyoxal-induced inhibition of plasminogen and antithrombin III.

Endogenously produced dicarbonyls, such as methylglyoxal (MG), are involved in advanced glycation end-product formation and thus linked to the pathophysiology of diabetic chronic complications. While the search for synthetic new antiglycation agents continues, little attention has been paid to putative antiglycation agents in natural compounds. Given the link between glycation and oxidation, in this work, we study the effects of methylglyoxal on two model systems; plasminogen and antithrombin III (AT III), then we set out to unravel a possible antiglycation effect for extracts of the flavonoid-rich common herbal species Achyrocline satureoides (AS) and Ilex paraguariensis (IP). Using SAR-PRO-ARG-pNA as a specific thrombin substrate, we show that incubation of plasma with MG decreases heparin activation of AT III by up to a 70%, in a dose-dependent manner. A parallel dose-dependent decrease in plasminogen activity reaching more than 50% was shown using D-BUT-CHT-lys-pNA as a plasmin-specific substrate. Extracts of AS and IP display a dose dependent inhibition of the action of the dicarbonyl, already significant at a 1/100 dilution of the herbal infusions. The inhibition was comparable to that obtained by using millimolar concentrations of known AGE inhibitors such as aminoguanidine and carnosine as well as micromolar concentrations of the antioxidant ascorbic acid. We believe our system of whole plasma glycation over 16 h with micromolar concentrations of MG, coupled with the measurement of activities of plasminogen and AT III by specific substrates provides a straightforward, practical method for monitoring the action of putative antiglycation agents. If predictably milder glycated forms of AT III and plasminogen were to be secreted in vivo, the loss of activities shown here could act synergistically to generate hyperthrombicity.

Three different pathways for human LDL oxidation are inhibited in vitro by water extracts of the medicinal herb Achyrocline satureoides.

In this study we investigated the antioxidant properties of one herbal preparation widely used in complementary and alternative medicine in large areas of the world: Achyrocline satureoides (AS), popularly known as "marcela". Although rich in flavonoids, the ethnopharmacological uses of this plant do not include atherosclerosis prevention. Furthermore, no study had been conducted so far exploring the antioxidant activity of Achyrocline satureoides vis-à-vis human LDL oxidation, which is the compelling issue in pinpointing potential cardioprotective new uses for a traditional remedy. We explored the effects of AS extracts on human LDL oxidation, employing 3 different systems which are thought to play a role in oxidation of LDL in the arterial wall: copper, peroxynitrite, and lipoxygenase. Oxidation was monitored by conjugate dienes, TBARS formation and aggregation of apoB using SDS-PAGE. In copper-initiated oxidation a dose dependent inhibition of the initiation and propagation of lipid oxidation is shown by an increase in the lag phase for conjugate diene production which was 60 +/- 15 min in the absence and 120 +/- 20 min in the presence of 4 microg/ml AS extracts (p < 0.001). TBARS production was reduced by 95% after 3 h incubation at 5 microg/ml. Aggregation of apoB was abolished at the same concentrations. SIN-1 (3-morpholinosydnonimine) produces peroxynitrite via generation of NO and O2-. When LDL was incubated in its presence, a milder oxidation was observed as compared with Cu2+, and AS produced over 70% inhibition. Finally, we show a striking dose-dependent inhibitory effect of lipoxygenase conjugate diene production, which is over 95% at AS concentrations of 5 microg/ml. When compared with other antioxidants, AS effect is greater but in the same order of magnitude than that of ascorbic acid and similar to the popular herbal tea Ilex paraguariensis. In all three systems employed an effect is already substantiated at a concentration of the AS extract of 4 microg/ml, which corresponds to a 1/100 dilution of the preparations usually drunk.

Circulating advanced glycation peptides in streptozotocin-induced diabetic rats: evidence for preferential modification of IgG light chains.

As the glycation/glycoxidation hypothesis for the genesis of diabetic complications is achieving widespread acceptance, much attention is being paid to the role of low molecular weight advanced glycation (AGE) adducts, as second generation glycating agents. We set out a study with the objective of attesting the presence of increased amounts of AGE-peptides in the circulation of streptozotocin-induced diabetic rats and to determine the nature of the plasma proteins which are main targets for advanced glycation. AGE (Ex 370/Em 440 nm) and pentosidine fluorescence (Ex 335/Em 385 nm) were significantly higher in plasma from diabetic rats after only one month of hyperglycemia as compared to controls (35 +/- 7 vs 25 +/- 2 AU, p< 0.05 and 54 +/- 14 vs 27 +/- 3 AU, p< 0.01 respectively). AGE-peptides (<10 kDa) were more than two-fold higher in diabetic animals. Immunoblots after SDS-PAGE of plasma proteins showed that AGE-IgG displayed a selective predominant increment in the same animals. When native rat IgG was incubated in the presence of AGE-peptides isolated from diabetic animals, AGE modification was already apparent after only 24 h of incubation, and was particularly important for light chains. AGE-immunoreactive light chains displayed an apparent increase in molecular weight. Aminoguanidine prevented, while copper enhanced AGE binding to IgG light chains. Our data validate the streptozotocin-induced diabetic rat as a model reproducing the presence of circulating AGE-peptides, give evidence that IgG are preferential targets for advanced glycation in plasma and suggest that this modification, mediated by AGE-peptides, can be prevented by aminoguanidine.

Susceptibility to copper-enhanced autoxidation of VLDL+LDL fractions from diabetic patients.

We describe a simplified method for studying the susceptibility to in vitro autoxidation of lipoproteins containing apolipoprotein B. It comprises copper induced autoxidation of VLDL+LDL plasma fractions and an assay for thiobarbituric acid reactive soluble substances. We studied autoxidation of VLDL+LDL in a population of 30 healthy subjects and a population of 30 diabetic patients. No significant difference in susceptibility to autoxidation could be detected. Protection afforded by ascorbic acid amounted to more than 95% at concentrations easily attainable in vivo by oral supplementation. Thiobarbituric acid reactive substances were higher in plasma from diabetic patients: 0.51 +/- 0.25 mumol/l vs 0.35 +/- 0.07 mumol/l for control subjects (p < 0.001). Our data confirm the presence of higher levels of lipid peroxides in diabetic patients, but fail to demonstrate any difference in susceptibility to oxidation of apolipoprotein B containing particles between control and diabetic subjects.

Glycated immunoglobulin M in diabetic patients.

Measurement of glycation levels on isolated immunoglobulin M (IgM), a short half-life protein, could be an index of glycaemic control. We determined glycated IgM levels in a diabetic patients population as compared to control non-diabetic subjects in a cross-sectional and longitudinal study. For that purpose we developed a precipitation method for IgM purification, measured glycation on the purified protein by a nitroblue tetrazolium assay and correlated glycated IgM, glycated haemoglobin (HbA1c) and fructosamine rates. The purification method we developed comprises dextran sulfate-CaCl2, ammonium sulfate and polyethylene glycol precipitations and it allows extraction of IgM free of contaminants as shown by immunoelectrophoresis. Glycated IgM levels were 8.65 +/- 0.15 nmol deoxymorpholinofructose (DOMF) equivalents/mg protein for non-diabetic subjects (n = 30) and 12.08 +/- 0.60 nmol DOMF equivalents/mg protein (n = 67) for diabetic patients. Diabetic patients had then a 40% rise in glycated IgM (p < 0.0005). In a longitudinal study with patients undergoing treatment aimed at improving their metabolic control, glycated IgM levels decreased significantly (p < 0.001) between the day of admission and the fifth day. Average rate of fall was 13% with a range of 3.0 to 22.0% (n = 28). Glycated IgM clearly responds more rapidly than fructosamines which fell by 7.0% and than HbA1c, which showed a rate of fall of 3.9% in the same period. A significant positive correlation was found between these parameters, being stronger between glycated IgM and fructosamines. This method could provide an alternative approach as a short-term marker of glycaemic control for clinical trials.

Polyclonal immunoglobulin M: location of glycation sites.

A four step purification procedure for polyclonal human serum IgM was elaborated, including ultracentrifugation, ammonium sulfate and polyethyleneglycol precipitations and diffusion-exclusion gel chromatography. IgM was glycated in vitro both in the presence of [14C]glucose and with unlabeled glucose. Influence of incubation time up to 10 days and of glucose concentration between 10 and 60 mmol/l were studied. With 10 mmol/l glucose, a molar ratio glucose/IgM of 5.7 was attained in 10 days. Increase of glucose concentration up to 60 mmol/l led to a molar ratio of 16.0. Both basal and in vitro glycation were evaluated by 3H-labeling by gel filtration and Concanavalin A-Sepharose chromatographies. Glycation occurs mainly on the heavy chains (> 85%), particularly on the Fd region.

Efecto del hábito de fumar sobre las lipoproteínas plasmáticas / Effedt of smoking on the plasmas lipoproteins

Las lipoproteínas plasmáticas han sido objeto de estudios tendientes a poner de mainfiesto al tabaquismo como un factor de riesgo en el desarrollo de la aterosclerosis. Con tal fin en este trabajo se estudian las LP en dos poblaciones cuya única diferencia manifiesta es que una de ellas está compuesta por fumadores de más de 20 cigarrillos por día por más de 3 años. Los resultados muestran un aumento de las LP aterogénicas, de la familia LP B y una disminución de las LP antiaterogénicas de la familia LP A1, con un descenso significativo de los índices de riesgo aterogénico. En suma, esto revela un aumento del poder aterogénico metabólico del plasma de los fumadores. Se concluye que las variaciones producidas en un factor de riesgo aterogénico, las LP, por un factor de riesgo coronario, el tabaquismo, llevan a una alteración metabólica de las mismas que acelera el proceso aterosclerótico en los fumadores. Se plantean los posibles mecanismos actuantes

Efecto del hábito de fumar sobre las lipoproteínas plasmáticas / Effedt of smoking on the plasmas lipoproteins

Las lipoproteínas plasmáticas han sido objeto de estudios tendientes a poner de mainfiesto al tabaquismo como un factor de riesgo en el desarrollo de la aterosclerosis. Con tal fin en este trabajo se estudian las LP en dos poblaciones cuya única diferencia manifiesta es que una de ellas está compuesta por fumadores de más de 20 cigarrillos por día por más de 3 años. Los resultados muestran un aumento de las LP aterogénicas, de la familia LP B y una disminución de las LP antiaterogénicas de la familia LP A1, con un descenso significativo de los índices de riesgo aterogénico. En suma, esto revela un aumento del poder aterogénico metabólico del plasma de los fumadores. Se concluye que las variaciones producidas en un factor de riesgo aterogénico, las LP, por un factor de riesgo coronario, el tabaquismo, llevan a una alteración metabólica de las mismas que acelera el proceso aterosclerótico en los fumadores. Se plantean los posibles mecanismos actuantes (AU)