Roberta Petlevski

Effect of ‘antidiabetis’ herbal preparation on serum glucose and fructosamine in NOD mice

The antihyperglycemic effect of the Antidiabetis herbal preparation ((Myrtilli folium (Vaccinium myrtillus L.), Taraxaci radix (Taraxacum officinale Web.), Cichorii radix (Cichorium intybus L.), Juniperi fructus (Juniperus communis L.), Centaurii herba (Centaurium umbellatum Gilib.), Phaseoli pericarpium (Phaseolus vulgaris), Millefollii herba (Achillea millefolium L.), Morii folium (Morus nigra L.), Valeriane radix (Valleriana officinalis L.), Urticae herba et radix (Urtica dioica L.)), patent No. P-9801091 Zagreb, Croatia was investigated. Two extracts were prepared: ethanol extract (extract 1), and ethanol extract from which ethanol was evaporated on a rotatory evaporator at a temperature of 45 degrees C (extract 2). Extract 1 and extract 2 were administered (in experiment 1) to alloxan-induced non-obese diabetic (NOD) mice in the same dose of 20 mg/kg. Blood glucose was determined before, and 10, 30, 60 and 120 min after the preparation administration. Extract 1 and extract 2 decreased the level of blood glucose by 10 and 20%, respectively, of the initial value (at 0 min, mean = 22.6 +/- 8.3 mmol/l). Serum levels of glucose and fructosamine were determined in NOD mice, NOD mice administered extract 2 in a dose of 20 mg/kg of extract 2, and NOD mice administered acarbose in a dose of 25 mg/100 g chow, in order to verify the hypoglycemic action of extract 2 (in experiment 2). Extract 2 and acarbose were admixed to the chow. The duration of treatment was 7 days. Significantly lower glucose (P < 0.05) and fructosamine (P < 0.001) levels were recorded in extract 2 treated NOD mice as compared with NOD mice. Study results showed extract 2 to significantly decrease the level of glucose and fructosamine in alloxan induced NOD mice. Our future studies will be focused on the search of active principles of the extracts.

Comparison of in vitro toxicity of silver ions and silver nanoparticles on human hepatoma cells.

Scientific information on the potential harmful effects of silver nanoparticles (AgNPs) on human health severely lags behind their exponentially growing applications in consumer products. In assessing the toxic risk of AgNP usage, liver, as a detoxifying organ, is particularly important. The aim of this study was to explore the toxicity mechanisms of nano and ionic forms of silver on human hepatoblastoma (HepG2) cells. The results showed that silver ions and citrate‐coated AgNPs reduced cell viability in a dose‐dependent manner. The IC50 values of silver ions and citrate‐coated AgNPs were 0.5 and 50 mg L−1, respectively. The LDH leakage and inhibition of albumin synthesis, along with decreased ALT activity, indicated that treatment with either AgNP or Ag ions resulted in membrane damage and reduced the cell function of human liver cells. Evaluation of oxidative stress markers demonstrating depletion of GSH, increased ROS production, and increased SOD activity, indicated that oxidative stress might contribute to the toxicity effects of nano and ionic forms of silver. The observed toxic effect of AgNP on HepG2 cells was substantially weaker than that caused by ionic silver, while the uptake of nano and ionic forms of silver by HepG2 cells was nearly the same.

The glutathione S-transferase polymorphisms in a control population and in Alzheimer's disease patients.

Abstract In this study, we investigated the role of glutathione S-transferase P1 (GSTP1) polymorphisms in the pathogenesis of Alzheimers disease (AD). We genotyped the GSTP1 polymorphisms in exon 5 (A313G) and exon 6 (C341T) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 56 Croatian patients with AD and 231 controls. Distributions and frequencies of GSTP1 genetic variants were not statistically different between AD patients and healthy controls. Higher frequencies of the mutant genotypes were observed in AD patients (13% for both A313G and C341T) when compared with control subjects (7% for A313G and 8% for C341T), but association of GSTP1 GG (OR 2.057, 95% CI 0.796–5.315, p=0.094) and TT (OR 1.691, 95% CI 0.669–4.270, p=0.514) genotypes with an increased risk of AD was not confirmed by statistical analysis. The frequencies of GSTP1 alleles (A, B, C, D) did not significantly differ between AD patients and controls and they were indicated as follows: 52.7%, 15.2%, 12.5% and 19.6% for AD cases and 58.4%, 14.1%, 14.1% and 13.4% for controls. The estimation of the GSTP1 haplotype distribution showed that GSTP1*A/GSTP1*B and GSTP1*A/GSTP1*C haplotypes were less frequent, while GSTP1*B/GSTP1*B and GSTP1*C/GSTP1*D haplotypes were more frequent in AD patients than in controls. In conclusion, the involvement of GSTP1 alleles in individual susceptibility to AD was not confirmed as statistically significant in the tested Croatian Caucasian population. A possible role of GSTP1 in the complex etiopathogenesis of AD is further discussed, based on observed differences in haplotype distribution and higher frequencies of mutant genotypes in AD patients.

GSTP1, GSTM1 and GSTT1 genetic polymorphisms and total serum GST concentration in stable male COPD

Abstract The aim of this study was to test the hypothesis that glutathione- S-transferase (GST) genotypes were associated with COPD. GSTP1, GSTM1 and GSTT1 genotypes were determined by DNA methods and GST activity spectrophotometrically in older male Caucasian Croats (non- -smokers, ex-smokers, and smokers) with stable COPD (n = 30) and sex/age matched controls (n = 60). The distribution of GSTP1 genotypes and alleles in controls vs. COPD showed a statistical difference (p < 0.05). The odds ratio of CC/CT+TT (wild type GSTP1 exon 6 vs. joint heterozygous and mutant homozygous GSTP1 exon 6) was 10.000 and statistically different (p = 0.002). In this study, the GSTP1 mutant genotype of exon 5 (GG), as well as GSTP1 mutant and heterozygous genotypes of exon 6 (TT and CT), were suggested to be genetic contributors to COPD susceptibility. Null GSTM1, null GSTT1 and joint GSTM1/GSTT1 null genotypes were not disease associated. Serum GST was not associated with GST genotypes and COPD or smoking history in our study subjects. Conclusions drawn from the study should be further supported and clarified by studies with larger sample sizes.

Chemical Composition, Antioxidant and α-Glucosidase-Inhibiting Activities of the Aqueous and Hydroethanolic Extracts of Vaccinium myrtillus Leaves

Vaccinium myrtillus (bilberry) leaf is traditionally used in southeastern Europe for the treatment of diabetes. In the present study, the ability of bilberry leaf extracts to inhibit carbohydrate-hydrolyzing enzymes and restore glutathione concentration in Hep G2 cells subjected to glucose-induced oxidative stress was investigated. A comprehensive analysis of the antioxidant activity of two bilberry leaf extracts was performed. The aqueous extract showed excellent total antioxidant and chelating activity. Its antioxidant activity in the β-carotene-linoleic acid assay was very good, reaching the activity of the antioxidant standard BHA (93.4 ± 2.3% vs. 95.1 ± 2.4%, respectively). The hydroethanolic extract (ethanol/H2O, 8:2, v/v), on the other hand, was a better radical scavenger and Fe2+ reducing agent. Furthermore, the aqueous extract was able to efficiently increase glutathione concentration in Hep G2 cells subjected to glucose-induced oxidative stress and restore it to the levels observed in non-hyperglycaemic cells. The hydroethanolic extract strongly inhibited α-glucosidase, with the IC50 statistically equal to the antidiabetic drug acarbose (0.29 ± 0.02 mg/mL vs. 0.50 ± 0.01 mg/mL, respectively). Phytochemical analysis revealed the presence of quercetin and kaemferol derivatives, as well as chlorogenic and p-coumaric acid. The study results indicate that V. myrtillus leaf may have promising properties as a supporting therapy for diabetes.

Polymorphism 4G/5G of the plasminogen activator inhibitor 1 gene as a risk factor for the development of allergic rhinitis symptoms in patients with asthma

Plasminogen activator inhibitor-1 (PAI-1) is a glycoprotein which has a role in tissue remodelling after inflammatory processes. The objective is to investigate the frequency of PAI-1 gene polymorphism (4G/5G) in patients with a lung ventilation dysfunction in asthma and allergic rhinitis. Genomic DNA was isolated and genotypes of polymorphism of PAI-1 4G/5G and ABO were determined using the methods of RT-PCR and PCR-SSP. Study group includes 145 adult patients diagnosed with chronic asthma, with all clinically relevant parameters and the laboratory markers of pO2, IgE and eosinophils in sputum and nasal swab. In the processing of data, appropriate statistical tests (Kolmogorov–Smirnov test, median, interquartile ranges, χ2 and Mann–Whitney U tests) were used. Patients with symptoms of allergic rhinitis were significantly younger and had an almost four time higher levels of IgE (P = 0.001), higher pO2 (P = 0.002) and PEF (P = 0.036), compared to those who do not have these symptoms. Genotype PAI 4G/4G is significantly more common in patients with allergic rhinitis (28.1% vs. 16.1%; P = 0.017) compared to the genotype 5G/5G. Carriers of the genotype 4G/5G also have a borderline statistical significance. There were no statistically significant difference in the incidence of allergic rhinitis in the carriers of any ABO genotypes. The frequency of PAI genotype 4G/4G is significantly more common in patients with allergic rhinitis. The results suggest that the carriers of at least one 4G allele are at a higher risk for developing symptoms of allergic rhinitis in asthma.

Effect of Betula pendula Leaf Extract on α-Glucosidase and Glutathione Level in Glucose-Induced Oxidative Stress

B. pendula leaf is a common ingredient in traditional herbal combinations for treatment of diabetes in southeastern Europe. Present study investigated B. pendula ethanolic and aqueous extract as inhibitors of carbohydrate hydrolyzing enzymes, as well as their ability to restore glutathione concentration in Hep G2 cells subjected to glucose-induced oxidative stress. Phytochemical analysis revealed presence of rutin and other quercetin derivatives, as well as chlorogenic acid. In general, ethanolic extract was richer in phenolic substances than the aqueous extract. Furthermore, a comprehensive analysis of antioxidant activity of two extracts (determined by DPPH and ABTS radical scavenging activity, total antioxidant activity, and chelating activity as well as ferric-reducing antioxidant power) has shown that ethanolic extract was better radical scavenger and metal ion reductant. In addition, ethanolic extract effectively increased cellular glutathione levels caused by hyperglycemia and inhibited α-glucosidase with the activity comparable to that of acarbose. Therefore, in vitro research using B. pendula plant extracts has confirmed their antidiabetic properties.

Antioxidant Activity of Ipomoea batatas L. Lam. Leaf Grown in Continental Croatia and Its Effect on Glutathione Level in Glucose-Induced Oxidative Stress

Ethanolic and aqueous extract of Ipomoea batatas (L.) Lam. leaf grown in Croatia were prepared. Antioxidant activity of the extracts, as well as their effect on intracellular glucose-induced oxidative stress, was determined. Antioxidant activity was assayed by DPPH radical scavenging activity, reducing power, activity in β-carotene-linoleic acid assay, and superoxide dismutase-like activity. In addition to being richer in phenols and flavonoids than aqueous extract, ethanolic extract also demonstrated superior antioxidant activity in all the assays. In a concentration of 10 μg/ml, both extracts were able to significantly increase intracellular glutathione levels.

Protective effect of the flavonoid myricetin on glucose induced oxidative stress in Hep G2 cells

Myricetin is a naturally occurring flavonol with hydroxyl substitutions at the 3, 5, 7, 3, , 4, and 5, positions and has a hypoglycaemic and hypotrigyceridemic effect in diabetes mellitus. Hyperglicemia in diabetes can induce oxidative stress via several mechanisms. These include glucose autoxidation, the formation of advanced glycation end-products (AGE), and activation of the polyol pathway. Other circulating factors such free fatty acids and leptin, also contribute to increased reactive oxygen species (ROS). The major roles of GSH ( -glutamiylcysteinylglycine) are to maintain the intracellular redox balance and to eliminate ROS in cells. The aims of this study were 1) to investigate the effect of the flavonoid myricetin (Sigma) on the concentration of total glutathione (GSH) in Hep G2 cells and 2) to determine whether this flavonoid could protect thi cells against glucose-induced oxidative stress. Hep G2 cells was supplemented with with 0.5  M and 1.0  M of myricetin for 4 hours or 0.5  M and 1.0  M of myricetin plus 20  M glucose for same time. Concentration of GSH in cells was determined by Cayman, s GSH assay kit with enzymatic recycling method, using glutathione reductase. Exposure the Hep G2 cells to 0.5  M of myricetin for 4 hours at 37 degrees C resulted in significant increased of the GSH level (p 0.05) when compared with control cells. This data suggest that major features of glucose-induced hepatotoxicity are partially mediated by oxidative stress, and that myricetin in low concentration (0.5  M) protect Hep G2 cells against glucose toxicity affecting the glutathione level.