Nazli Arda

Some medicinal plants as immunostimulant for fish

Immunostimulant effects of the dietary intake of various medicinal plant extracts on fish, rainbow trout (Oncorhynchus mykiss), were investigated. For this purpose fish were fed with diets containing aqueous extracts of mistletoe (Viscum album), nettle (Urtica dioica), and ginger (Zingiber officinale). Food containing lyophilized extracts of these plants as 0.1 and 1% was used at a rate of 2% of body weight per day for three weeks. At the end of the experimental period, various parameters of non-specific defence mechanisms, including extracellular and intracellular respiratory burst activities, phagocytosis in blood leukocytes and total plasma protein level were examined. Specific growth rates (SGRs) and condition factors (CFs) of the fish were also measured. Plant materials tested for immunostimulatory food additives caused an enhanced extracellular respiratory burst activity (P<0.001) compared to the control group. Especially the rainbow trout fed with a diet containing 1% aqueous extract of powdered ginger roots for three weeks exhibited a significant non-specific immune response. Phagocytosis and extracellular burst activity of blood leukocytes were significantly higher in this group than those in the control group. All plant extracts added to fish diet increased the total protein level in plasma except 0.1% ginger. The highest level of plasma proteins was observed in the group fed with 1% ginger extract containing feed.

Antioxidant activity of whey protein fractions isolated by gel exclusion chromatography and protease treatment.

Whey proteins were isolated from whey powder by a combination of gel exclusion chromatography and protease (pepsin or trypsin) treatment. Whey solution (6g/dl) was applied to Sephadex G-200 column chromatography and three fractions were obtained. Gel electrophoresis (SDS-PAGE) was used to identify the fractions; the first one contained immunoglobulins and bovine serum albumin, the second contained beta-lactoglobulin and alpha-lactalbumin whereas the third fraction contained small peptides. We have also subjected the whey filtrate to proteases (pepsin and trypsin). Treatment with proteases showed that beta-lactoglobulin can be obtained after hydrolysis of the second fraction with pepsin. When the whey filtrate was treated with pepsin and then applied to Sephadex G-200 column chromatography three fractions were obtained; the first one was bovine serum albumin, the second was beta-lactoglobulin and the third fraction contained small peptides. After trypsin treatment only two fractions were obtained; the first one was serum albumin and the second fraction was an alpha-lactalbumin rich fraction. We have determined the antioxidant activity of the fractions using an assay based on the measurement of superoxide radical scavenging activity. Our results showed that among the three fractions, the first fraction had the highest superoxide radical scavenging activity. Also, protease treatment of the second fraction resulted in an increase in the antioxidant activity.

Intracellular scavenging activity of Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) in the fission yeast, Schizosaccharomyces pombe

The ability of Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), a water-soluble vitamin E analogue, to prevent oxidative damages is well characterized, but the mechanisms underlying it remain unclear. The protective effect of Trolox pre-treatment on H2O2-induced toxicity might be attributed to the decreased cellular permeability to H2O2 or in vitro scavenging activity of Trolox, induction of antioxidant enzymes or the direct scavenging activity of Trolox. The results obtained rule out the first and second possibilities and intracellular scavenging activity was found to be the mechanism whereby Trolox confers protection. This was confirmed by measuring protein oxidation (levels), and the observed decrease in proteasomal activity indicated that the decrease in protein carbonyls was due to Trolox scavenging activity rather than proteasome activation. In conclusion, the intracellular scavenging activity of Trolox is a key protective mechanism against H2O2. These findings obtained in Schizosaccharomyces pombe, a good model organism for eukaryotic cells, can be used as standard protocols for investigating the antioxidant activity of pure or complex potential antioxidants.

Evaluation of the molecular mechanisms of a palladium(II) saccharinate complex with terpyridine as an anticancer agent.

Metal-based compounds represent promising anticancer therapeutic agents. In this study, the mechanism of action of a novel metal-based drug, a palladium(II) (Pd) complex ([PdCl(terpy)](sac)·2H2O, terpy=2,2′:6′,2′′-terpyridine and sac=saccharinate), was elucidated. The tested compound induced cytotoxicity in nine different human cancer cell lines that originated from various organs, suggesting a broad spectrum of activity. The IC50 values were significantly higher for noncancerous cells when compared with cancer cells. We found that cells treated with the Pd(II) complex exhibited increased caspase 3/7 activities and condensed/fragmented nuclei, as demonstrated by nuclear staining and DNA laddering. Morphological features, such as cellular shrinkage and blebbing, were also observed, indicating that apoptosis was the primary mechanism of cell death. Pd(II) treatment induced DNA double-stranded breaks both in vitro and in vivo, potentially accounting for the source of stress in these cells. Although caspase 3/7 activities were elevated after Pd(II) treatment, silencing or using inhibitors of caspase 3 did not block apoptosis. Other molecules that could potentially play a role in Pd(II)-induced apoptosis, such as p53 and Bax, were also tested using silencing technology. However, none of these proteins were essential for cell death, indicating either that these molecules do not participate in Pd(II)-induced apoptosis or that other pathways were activated in their absence. Hence, this new molecule might represent a promising anticancer agent that exhibits cytotoxicity in p53-mutant, Bax-mutant, and/or caspase 3-mutant cancer cells.

Phenols of virgin olive oil protects nuclear DNA against oxidative damage in HeLa cells.

Oxidative DNA damage is an inescapable consequence for cells constantly exposed to oxidative stress derived from normal metabolic processes and from environmental factors. Phenolic compounds, which have strong antioxidant activity, prevent DNA damage by protecting the cells against harmful effects of oxidative stress. In this study, the effect of virgin olive oil phenolic extract (OOPE) was investigated on H2O2-induced mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in HeLa cells. DNA damage was assessed in mitochondria and two nuclear regions by using quantitative PCR (QPCR) assay. The cells were pre-treated with non-cytotoxic doses of OOPE for 4 h, and DNA damage was determined. OOPE alone does not change the steady-state level of DNA damage. The oxidative stress generated with 750 μM H2O2 caused two times greater damages in mtDNA compared to nDNA, which included the nonexpressed β-globin region (1.507±0.110 lesions/10 kb) and the expressed APEX1 gene (1.623±0.243 lesions/10 kb) with respect to the control region. When cells were preincubated with OOPE for 4 h, nDNA damage under stress condition was completely inhibited; however, mtDNA damage was not affected by this procedure. These results suggest that OOPE has a protective effect against nDNA damage in HeLa cells.

Essential fatty acid components and antioxidant activities of eight Cephalaria species from southwestern Anatolia

The hexane extracts of eight Cephalaria (Dipsacaceae) species, which were collected from southwestern Anatolia, were obtained by Soxhlet apparatus. The fatty acids were derived to methyl esters and determined by gas chromatography/flame ionization detector (GC/FID) and gas chromatography/mass spectrometry (GC/MS) systems. The dominant fatty acid components and maximum percentages were detected as myristic [in C. joppica (17.48 %)], palmitic [in C. cilicica, C. elmaliensis, C. isaurica, C. scoparia (19.51 %), and C. gazipashaensis], linoleic [in C. joppica (33.02 %), C. elmaliensis, C. dipsacoides, and C. gazipashaensis], α-linolenic (ALA) [in C. cilicica, C. elmaliensis, C. isaurica, C. scoparia, C. lycica, and C. gazipashaensis (47.95 %)] and oleic [in C. isaurica and C. dipsacoides (40.66 %)] acids. The antioxidant activity of all hexane extracts was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric thiocyanate (FTC), and thiobarbituric acid (TBA) methods. The results indicate that hexane extracts of Cephalaria species possess considerable antioxidant activity. The highest radical scavenging activity was detected in C. isaurica (IC50 = 741 μg/mL). The most effective species on lipid peroxidation are C. lycica and C. gazipashaensis in FTC and TBA assays, respectively. This study reveals that Cephalaria species are attractive sources of fatty acid components, especially the essential ones, as well as of effective natural antioxidants.

Chemical characterization and biological activities of the genus Tanacetum (Compositae)

The genus Tanacetum has been used as medicinal plants for over 2000 years. Interest in the genus has been stimulated by its biological activities, particularly as insect antifeedants, antitumor and antimicrobial activities due to its sesquiterpenoid constituents. The genus Tanacetum is represented by c.a.70 species in the worldand by 44 in Turkey. It is an Asia centered genus whichis widespread in the Northern Hemisphere and temperate regions. Tanacetum species contain mainly sesquiterpenoids and flavonoids, whereas the other terpenoids and phenolic compounds are rarely found. Sesquiterpenoids which are the main constituents of the genus, supposed to be bioactive principles of the plants. Flavonoids and essential oils are also pointed out as active substances in some species. On the other hand,there is a confusion on the systematic position and classification of several species of Asteraceae, therefore chemotaxonomy of the species will help the systematic studies. Since the importance of sesquiterpenes, sesquiterpene lactones and flavonoids from the chemosystematic and the biological point of views, especially the chemistry and the biological activities of these compounds will be reviewed in this chapter, whilethe essential oils and the acetylenic compounds will not be mentioned.

Hydrogen peroxide-induced oxidative damages in Schizosaccharomyces pombe

Oxidative stress causes damage to proteins, lipids and nucleic acids, and thereby compromises cell viability. Some of the oxidative stress markers in an eukaryotic model organism, fission yeast Schizosaccharomyces pombe, were evaluated in this study. Intracellular oxidation, protein carbonyls, lipid peroxidation and reduced glutathione (GSH) levels were investigated in H2O2-treated and non-treated control cells. It was observed that increased H2O2 concentration proportionally lowered the cell number and increased the intracellular oxidation, lipid peroxidation and protein carbonyl levels in S. pombe. A dose-dependent decrease in GSH level was also detected. The fission yeast S. pombe is best known for its contribution to understanding of eukaryotic cell cycle control. S. pombe displays a different physiology from Saccharomyces cerevisiae in several ways and is thus probably more closely related to higher eukaryotes. The purpose of this study was to provide some data about the effects of hydrogen peroxide on the proteins and lipids in the fission yeast. The data obtained here is expected to constitute a basis for the further studies on redox balance and related processes in yeast and mammalian cells.

Evaluation of apoptotic molecular pathways for smooth muscle cells isolated from thoracic aortic aneurysms in response to oxidized sterols

Oxysterols, oxygenated derivatives of cholesterol, are found abundantly in the plasma and atherosclerotic plaques, a common risk factor for thoracic aortic aneurysms (TAAs). Among the oxysterols, namely 7-ketocholesterol (7-KC) and 25-hydroxycholesterol (25-OHC), lead both to induction of reactive oxygen species (ROS) in cells and to apoptosis in smooth muscle cells (SMCs) probably due to increased oxidative stress. Since loss of SMCs through apoptosis is a major event in TAA formation, it is important to understand the molecular pathways of apoptosis in response to ROS in TAAs. Very little is known about the effect of oxysterols on TAA SMCs. Therefore, we investigated molecular pathways participating in the oxysterol induced cell death of TAAs. Our results showed that TAA SMCs died mainly as a result of apoptosis as suggested by cellular shrinkage, blebbing, DNA condensation/fragmentation in response to oxysterol treatment. There was no significant difference in oxysterol induced cell death between TAA and control SMCs. Addition of antioxidant molecules prevented cell death, hence ROS appears to be involved in the apoptosis of these cells. While oxysterol treatment increased caspase 3 activity, cell death was not rescued in its absence. Efficient silencing of other targets including apoptotic proteins (p53, Bax), and survival proteins (Akt1, Akt2) showed that apoptosis can occur through p53, and Bax independent pathways. Silencing Akt1 or Akt2 did not lead to further cell death. These results indicate that oxysterols can induce several cell death pathways in TAA SMCs.

Comparison of antioxidant capacity, protein profile and carbohydrate content of whey protein fractions.

Whey is used as an additive in food industry and a dietary supplement in nutrition. Here we report a comparative analysis of antioxidant potential of whey and its fractions. Fractions were obtained by size exclusion chromatography, before and after enzymatic digestion with pepsin or trypsin. Superoxide radical scavenging, lipid peroxidation inhibition and cupric ion reducing activities of different fractions were checked. Peptides were detected by SDS-PAGE and GC-MS was used to determine carbohydrate content of the fractions. All samples showed antioxidant activity and the second fraction of the trypsin hydrolysate showed the highest superoxide radical scavenging activity. CUPRAC value of this fraction was two-times higher than that of whey filtrate. The first fraction of the pepsin hydrolysate was the most effective inhibitor of lipid peroxidation. Each sample exhibited a different polypeptide profile. Different percentages of carbohydrates were identified in whey filtrate and in all second fractions, where galactose was the major component.