Kateřina Valentová

Antioxidant and antiviral activities of silybin fatty acid conjugates

Two selective acylation methods for silybin esterification with long-chain fatty acids were developed, yielding a series of silybin 7-O- and 23-O-acyl-derivatives of varying acyl chain lengths. These compounds were tested for their antioxidant (inhibition of lipid peroxidation and DPPH-scavenging) and anti-influenza virus activities. The acyl chain length is an important prerequisite for both biological activities, as they improved with increasing length of the acyl moiety.

The effect of Smallanthus sonchifolius leaf extracts on rat hepatic metabolism

Smallanthus sonchifolius (yacon), originating from South America, has become popular in Japan and in New Zealand for its tubers which contain β-1,2-oligofructans as the main saccharides. The plant is also successfully cultivated in Central Europe in the Czech Republic in particular. Its aerial part is used in Japan and in Brazil as a component in medicinal teas; while aqueous leaf extracts have been studied for their hypoglycemic activity in normal and diabetic rats. We have already demonstrated the high content of phenolic compounds in yacon leaf extracts and their in vitro antioxidant activity. In this paper, we present the effects of two organic fractions and two aqueous extracts from the leaves of S. sonchifolius on rat hepatocyte viability, on oxidative damage induced by tert-butyl hydroperoxide (t-BH) and allyl alcohol (AA), and on glucose metabolism and their insulin-like effect on the expression of cytochrome P450 (CYP) mRNA. All the extracts tested exhibited strong protective effect against oxidative damage to rat hepatocyte primary cultures in concentrations ranging from 1 to 1000 μg/ml, reduced hepatic glucose production via gluconeogenesis and glycogenolysis at 1000 μg/ml. Moreover, the effects of the organic fractions (200 and 250 μg/ml) and to a lesser extent, the tea infusion (500 μg/ml) on rat CYP2B and CYP2E mRNA expression, were comparable to those observed with insulin. The combination of radical scavenging, cytoprotective and anti-hyperglycemic activity predetermine S. sonchifolius leaves for use in prevention and treatment of chronic diseases involving oxidative stress, particularly diabetes.

Protectivity of Blue Honeysuckle Extract against Oxidative Human Endothelial Cells and Rat Hepatocyte Damage

The effect of Lonicera caerulea L. (blue honeysuckle) phenolic fraction (18.5% anthocyanins) on cell viability and against oxidative damage in low density lipoproteins (oxLDL), in rat microsomes and in primary cultures of rat hepatocytes and human umbilical vein endothelial cells (HUVEC), was tested. The phenolic fraction was nontoxic to rat hepatocytes and HUVEC at tested concentrations (1-1000 microg/mL) and time intervals up to 24 h inclusive. Phenolic fraction inhibited rat liver microsome peroxidation, induced by tert-butyl hydroperoxide (tBH), with IC(50) values of 160 +/- 20 microg/mL. The fraction at 0.5, 1.0, and 2.0 microg/mL delayed LDL oxidation, induced by Cu(2+), by 130 +/- 20%, 200 +/- 30%, and 400 +/- 10%, respectively. The treatment of HUVEC with oxidatively modified LDL induced an increase in lactate dehydrogenase (LDH) leakage and thiobarbituric acid reactive substances (TBARS) formation, and resulted in lower formazan formation from 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) uptake, most pronounced for 200 microg/mL (24 h oxidation) after 2 h of incubation. The protective effect of the phenolic fraction against cell damage caused by oxLDL was noted at 0.1 microg/mL for HUVEC and against tBH at 1000 microg/mL for both HUVEC and hepatocytes. The observed protective effects were probably due to the antioxidant properties of L. caerulea constituents, mainly anthocyanins. Microsome peroxidation and LDL oxidation inhibition results provide promising perspectives into the prevention of some oxidative stress-associated diseases. Other data are important in in vitro systems but seem to be accidental in vivo.

The in vitro biological activity of Lepidium meyenii extracts

The biological activity of methanolic and aqueous extracts from dehydrated hypocotyls of Lepidium meyenii (Brassicaceae, vernacular name “maca”), was studied on rat hepatocytes and human breast cancer MCF-7 cells. The extracts did not exhibit cytotoxicity in hepatocyte primary cultures up to 10 mg/ml as measured by the MTT viability test, and lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) leakage. Moreover, after 72 h, extracts inhibited LDH and AST leakage from the hepatocytes. When hepatocytes were intoxicated by t-butyl hydroperoxide, neither extract prevented oxidative damage. Both extracts showed weak antioxidant activity in the DPPH radical scavenging test with IC50 values of 3.46 ± 0.16 and 0.71 ± 0.10 mg/ml, for aqueous and methanolic extracts, respectively. Thus, the observed effect on spontaneous enzyme leakage is probably mediated through mechanisms other than antioxidant activity. Both methanolic and aqueous extracts have shown estrogenic activity comparable with that of silymarin in MCF-7 cell line. Maca estrogenicity was exhibited in the range from 100 to 200 μg of extract per ml. The findings in the present study show that maca does not display in vitro hepatotoxicity. In contrast, a slight cytoprotective effect, probably not mediated by antioxidant capacity, was noted. Maca extracts exhibited estrogenic activity comparably to the effect of silymarin in MCF-7 cells.

Flavonolignan 2,3-dehydroderivatives: Preparation, antiradical and cytoprotective activity

The protective constituents of silymarin, an extract from Silybum marianum fruits, have been extensively studied in terms of their antioxidant and hepatoprotective activities. Here, we explore the electron-donor properties of the major silymarin flavonolignans. Silybin (SB), silychristin (SCH), silydianin (SD) and their respective 2,3-dehydroderivatives (DHSB, DHSCH and DHSD) were oxidized electrochemically and their antiradical/antioxidant properties were investigated. Namely, Folin-Ciocalteau reduction, DPPH and ABTS(+) radical scavenging, inhibition of microsomal lipid peroxidation and cytoprotective effects against tert-butyl hydroperoxide-induced damage to a human hepatocellular carcinoma HepG2 cell line were evaluated. Due to the presence of the highly reactive C3-OH group and the C-2,3 double bond (ring C) allowing electron delocalization across the whole structure in the 2,3-dehydroderivatives, these compounds are much more easily oxidized than the corresponding flavonolignans SB, SCH and SD. This finding was unequivocally confirmed not only by experimental approaches, but also by density functional theory (DFT) calculations. The hierarchy in terms of ability to undergo electrochemical oxidation (DHSCH~DHSD>DHSB>>SCH/SD>SB) was consistent with their antiradical activities, mainly DPPH scavenging, as well as in vitro cytoprotection of HepG2 cells. The results are discussed in the context of the antioxidant vs. prooxidant activities of flavonolignans and molecular interactions in complex biological systems.

Synthesis and Antiangiogenic Activity of New Silybin Galloyl Esters

The synthesis of various silybin monogalloyl esters was developed, and their antiangiogenic activities were evaluated in a variety of in vitro tests with human umbilical vein endothelial cells (HUVECs). A structure-activity relationship (SAR) study found the regioselectivity of the silybin galloylation to be highly significant. Silybin (as an equimolar mixture of two diastereomers A and B) exhibited quite poor antiangiogenic activities, whereas its B stereoisomer is more active than silybin A. The galloylation of phenolic OH groups of natural silybin (a mixture of both isomers) leads to increases in their antiangiogenic activities, which is more apparent with the 7-OH than the 20-OH. In contrast, gallates at aliphatic OH groups either had a comparable activity to the parent compound or are even worse than silybin, which was observed in the case of 3-O-galloylsilybin. The most effective compound from this series (7-O-galloylsilybin) has also been prepared from stereochemically pure silybins A and B to evaluate the effect of stereochemistry on the activity. As with silybin itself, the B isomer of 7-O-galloylsilybin was more active than the A isomer.

Metabolic Profiling of Phenolic Acids and Oxidative Stress Markers after Consumption of Lonicera caerulea L. Fruit

This study investigated the effect of one-week consumption of 165 g/day fresh blue honeysuckle berries (208 mg/day anthocyanins) in 10 healthy volunteers. At the end of intervention, levels of benzoic (median 1782 vs 4156), protocatechuic (709 vs 2417), vanillic (2779 vs 4753), 3-hydroxycinnamic (143 vs 351), p-coumaric (182 vs 271), isoferulic (805 vs 1570), ferulic (1086 vs 2395), and hippuric (194833 vs 398711 μg/mg creatinine) acids by LC/MS were significantly increased in the urine. Clinical chemistry safety markers were not altered. Oxidative stress markers, erythrocyte glutathione peroxidase (0.73 vs 0.88 U/g Hb) and catalase (2.5 vs 2.8 μkat/g Hb) activities, and erythrocyte/plasma thiobarbituric acid reactive substance (522 vs 612/33 vs 38 μmol/g Hb/protein) levels were significantly increased, without change in plasma antioxidant status. Nonsignificant changes of advanced oxidation protein products and oxidized LDL were observed. The results provide a solid base for further study of metabolite excretion and antioxidant parameters after ingestion of anthocyanins.

The silymarin composition… and why does it matter???

The extract from milk thistle (Silybum marianum (L.) Gaertn. (Asteraceae)), known as silymarin, contains a variety of flavonolignans and displays antioxidant, anti-inflammatory, immunomodulatory and hepatoprotective properties. As silybin is the main component of silymarin, the literature mainly focuses on this compound, ignoring all other components. This leads to problems in reproducibility of scientific results, as the exact composition of silymarin is often unknown and can vary to a certain degree depending on the processing, chemo-variety of the plant used and climatic conditions during the plant growth. There are studies dealing with the analytical separation and quantification of silymarin components as well as studies focused on silymarin content in clinically used drugs, in various plant parts, seasons, geographic locations etc. However, no comparison of detail flavonolignan profiles in various silymarin preparations is available to date. Also, as a result of the focus on the flavonolignans; the oil fraction, which contains linoleic, oleic and palmitic acids, sterols, tocopherol (vitamin E) and phospholipids, has been neglected. Due to all these factors, the whole plant is used e.g. as animal feed, the leaves can be eaten in salads and seed oil, besides culinary uses, can be also utilized for biodiesel or polymer production. Various HPLC separation techniques for the determination of the content of the flavonolignans have been vastly summarized in the present review.

Chemo-Enzymatic Synthesis of Silybin and 2,3-Dehydrosilybin Dimers

Divalent or multivalent molecules often show enhanced biological activity relative to the simple monomeric units. Here we present enzymatically and chemically prepared dimers of the flavonolignans silybin and 2,3-dehydrosilybin. Their electrochemical behavior was studied by in situ and ex situ square wave voltammetry. The oxidation of monomers and dimers was similar, but adsorption onto the electrode and cell surfaces was different. A 1,1-diphenyl-2-picrylhydrazyl (DPPH) and an inhibition of microsomal lipoperoxidation assay were performed with same trend of results for silybin and 2,3-dehydrosilybin dimers. Silybin dimer showed better activity than the monomer, while on the contrary 2,3-dehydrosilybin dimer presented weaker antioxidant/antilipoperoxidant activity than its monomer. Cytotoxicity was evaluated on human umbilical vein endothelial cells, normal human adult keratinocytes, mouse fibroblasts (BALB/c 3T3) and human liver hepatocellular carcinoma cell line (HepG2). Silybin dimer was more cytotoxic than the parent compound and in the case of 2,3-dehydrosilybin its dimer showed weaker cytotoxicity than the monomer.

Biosafety and antioxidant effects of a beverage containing silymarin and arginine. A pilot, human intervention cross-over trial

The study objective was to investigate the potential of a beverage containing silymarin and L-arginine to alter basic physiological and urodynamic parameters in 22 normal healthy men aged 38-59 years. The volunteers drank 500 ml/day beverage without silymarin and L-arginine for 10 days followed, after a 7-day washout period, by the beverage with 400mg silymarin and 295 mg L-arginine for 10 days. Blood and urine samples were collected on days 0, 10 and 27. The beverages were well-tolerated with no adverse effects. Most of the biochemical, hematological and urodynamic parameters remained unchanged. Total antioxidant capacity, total level of antioxidants, lipoperoxidation products (malondialdehyde), advanced oxidation products of proteins in plasma and glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase levels in erythrocytes were not influenced. Serum γ-glutamyl transferase, malondialdehyde level and activity of glutathione S-transferase in erythrocytes were lowered at day 27 and the concentration of total plasma SH-groups was higher on day 10. Using an ex vivo system, we found that silymarin/silybin at 10-100 μM is able to adsorb onto human erythrocytes and the complexes displayed antioxidant properties as studied using ex situ square-wave voltammetry. The trial showed that silymarin in vivo may protect erythrocytes against oxidative damage.