Sanja Matić

Hepatoprotective effects of Gentiana asclepiadea L. extracts against carbon tetrachloride induced liver injury in rats

This study is an attempt to evaluate the hepatoprotective activity of Gentiana asclepiadea L. against carbon tetrachloride-induced liver injury in rats. Methanol extracts of aerial parts (GAA) and roots (GAR) of G. asclepiadea at doses of 100, 200, and 400mg/ kg b.w. were orally administered to Wistar rats once daily for 7 days before they were treated with CCl(4). The hepatoprotective activity of the extracts in this study was compared with the reference drug silymarin. In CCl(4) treated animals, GAA and GAR significantly decreased levels of serum transaminases, alkaline phosphatase and total bilirubin, and increased the level of total protein. Treatment with the extracts resulted in a significant increase in the levels of catalase, superoxide dismutase and reduced glutathione, accompanied with a marked reduction in the levels of malondialdehyde, as compared to CCl(4) treated group. The histopathological studies confirmed protective effects of extracts against CCl(4)-induced liver injuries. No genotoxicity was observed in liver cells after GAA treatment, while GAR showed only slight genotoxic effects by comet assay. Phytochemical analysis revealed the presence of sweroside, swertiamarin and gentiopicrin in high concentrations in both extracts. It could be concluded that the use of G. asclepiadea extracts in the treatment of chemical-induced hepatotoxicity.

In vitro antioxidant activity of selected 4-hydroxy-chromene-2-one derivatives-SAR, QSAR and DFT studies.

The series of fifteen synthesized 4-hydroxycoumarin derivatives was subjected to antioxidant activity evaluation in vitro, through total antioxidant capacity, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), hydroxyl radical, lipid peroxide scavenging and chelating activity. The highest activity was detected during the radicals scavenging, with 2b, 6b, 2c, and 4c noticed as the most active. The antioxidant activity was further quantified by the quantitative structure-activity relationships (QSAR) studies. For this purpose, the structures were optimized using Paramethric Method 6 (PM6) semi-empirical and Density Functional Theory (DFT) B3LYP methods. Bond dissociation enthalpies of coumarin 4-OH, Natural Bond Orbital (NBO) gained hybridization of the oxygen, acidity of the hydrogen atom and various molecular descriptors obtained, were correlated with biological activity, after which we designed 20 new antioxidant structures, using the most favorable structural motifs, with much improved predicted activity in vitro.

Methanol extract from the stem of Cotinus coggygria Scop., and its major bioactive phytochemical constituent myricetin modulate pyrogallol-induced DNA damage and liver injury

The present study was undertaken to investigate the hepatoprotective effect of the methanol extract of Cotinus coggygria Scop. in rats exposed to the hepatotoxic compound pyrogallol. Assessed with the alkaline version of the comet assay, 1000 and 2000mg/kg body weight (bw) of the extract showed a low level of genotoxicity, while 500mg/kg bw of the extract showed no genotoxic potential. Quantitative HPLC analysis of phenolic acids and flavonoids in the methanol extract of C. coggygria showed that myricetin was a major component. To test the hepatoprotective effect, a non-genotoxic dose of the C. coggygria extract and an equivalent amount of synthetic myricetin, as present in the extract, were applied either 2 or 12h prior to administration of 100mg/kg bw of pyrogallol. The extract and myricetin promoted restoration of hepatic function by significantly reducing pyrogallol-induced elevation in the serum enzymes AST, ALT, ALP and in total bilirubin. As measured by the decrease in total score and tail moment, the DNA damage in liver was also reduced by the extract and by myricetin. Our results suggest that pro-surviving Akt activity and STAT3 protein expression play important roles in decreasing DNA damage and in mediating hepatic protection by the extract. These results suggest that myricetin, as a major component in the extract, is responsible for the antigenotoxic and hepatoprotective properties of the methanol extract of C. coggygria against pyrogallol-induced toxicity.

Combining molecular docking and 3-D pharmacophore generation to enclose the in vivo antigenotoxic activity of naturally occurring aromatic compounds: myricetin, quercetin, rutin, and rosmarinic acid.

Considering the controversial results concerning the antimutagenicity of some phenolic compounds recorded in the literature, the antigenotoxic effects of four selected phenolic compounds, myricetin, quercetin, rutin, and rosmarinic acid, against DNA damage induced by alkylation with ethyl methanesulfonate (EMS), were evaluated in Drosophila melanogaster males using the sex-linked recessive lethal (SLRL) test. To assess the protective effects against DNA damage, D. melanogaster males were exposed to a monofunctional alkylating agent EMS in concentration of 0.75 ppm, 24 h prior to one of the selected phenolic compounds in the concentration of 100 ppm. The possible differences in mechanisms of protection by selected compounds were determined by molecular docking, after which structure-based 3-D pharmacophore models were generated. EMS induced considerable DNA damage as shown by significant increase in the frequency of germinative mutations. The frequency decreased with high significance (p<0.001***) after post-treatments with all selected phenolic compounds. Further, docking analysis revealed EMS pre-bond conformations against guanine and thymine as a necessary condition for alkylation, after which resulting O⁶-ethylguanine and O⁴-ethylthimine were docked into the active site of O⁶-alkylguanine-DNA alkyltransferase to confirm that particular lesions are going to be repaired. Finally, myricetin and quercetin protected dealkylated nucleotides from further EMS alkylation by forming the strong hydrogen bonds with O⁶-guanine and O⁴-thymine via B ring hydroxyl group (bond lengths lower than 2.5 Å). On the other side, rutin and rosmarinic acid encircled nucleotides and by fulfilling the EMS binding space they made an impermeable barrier for the EMS molecule and prevented further alkylation.

Biochemical and pharmacological evaluation of 4-hydroxychromen-2-ones bearing polar C-3 substituents as anticoagulants.

The objective of this study was to investigate in vitro and in vivo anticoagulant activity of sixteen 4-hydroxycoumarin derivatives bearing polar C-3 scaffolds. The activity was evaluated by measuring prothrombin time. Enhanced anticoagulant activity in vitro was observed for all tested compounds. Upon successive administration of 0.5 mg/kg of body weight to adult Wistar rats, over a period of five days, four derivatives (2b, 4c, 5c and 9c) presented anticoagulant activity in vivo. The most active compound was 2b, with PT = 30.0 s. Low or non-toxic effects in vivo were determined based on the catalytic activity of liver enzymes and the concentration of bilirubin, iron and proteins. Metabolic pathways of the most active compounds in vivo were determined after GC/MS analysis of collected rat urine samples. The excretion occurs by glucuronidation of 7-hydroxy forms of tested derivatives. In vivo results were described using PLS-based CoMFA and CoMSIA 3D-QSAR studies, which showed CoMFA-SE (q(2) = 0.738) and CoMSIA-SEA (q(2) = 0.763) to be the statistically most relevant models. Furthermore, molecular docking and DFT mechanistic studies performed on the rat VKORC1 homology model revealed interactions between the 4-OH coumarin group in the form of phenolic anion and the Cys135 catalytic site in the transition state.

Study of genotoxicity and antigenotoxicity of the Cotinus coggygria Scop. methanol extract by Drosophila melanogaster sex-linked recessive lethal test

The genotoxic and antigenotoxic effects of Cotinus coggygria Scop. methanol extract was investigated using the Drosophila sex-linked recessive lethal (or SLRL) test. The results presented here show that the methanol extract of Cotinus coggygria in a concentration of 5% and artificial chemical agent ethyl methanesulfonate EMS (0.75 ppm) induce recessive lethal mutations on X-chromosome on Drosophila melanogaster in all broods (I, II and III). Post-treatment with lower concentration of the methanol extract of Cotinus coggygria (2%) was effective in reducing genotoxicity of mutagen.

Synthesis and toxicological studies of in vivo anticoagulant activity of novel 3-(1-aminoethylidene)chroman-2,4-diones and 4-hydroxy- 3-(1-iminoethyl)-2H-chromen-2-ones combined with a structure-based 3-D pharmacophore model

Eight synthesized 3-(1-aminoethylidene)chroman-2,4-diones and 4-hydroxy-3-(1-iminoethyl)-2H-chromen-2-ones were evaluated as in vivo anticoagulants by intraperitoneal application to adult male Wistar rats in order to examine their pharmacological potential, evaluate ther toxicity and propose the mechanism of action. Two of them, 2f and 2a, in concentration of 2mg/kg of body weight, presented remarkable activity (PT=130s; PT=90s) upon seven days of continuous application. The results of rat serum and liver biochemical screening, as well those of histopathological studies, proved the compounds to be non-toxic. Activity of the compounds was further examined on the molecular level. Here, molecular docking studies were performed to position the compounds in relation to the active site of VKORC1 and determine the bioactive conformations. Docking results suggested a non-covalent mode of action during which the proton transfer occurs from Cys135 SH towards 4-carbonyl group of anticoagulant. All crucial interactions for anticoagulant activity were confirmed in generated structure-based 3-D pharmacophore model, consisted of hydrogen bond acceptor and hydrophobic aromatic features, and quantified by a best correlation coefficient of 0.97.

Chemical composition, antioxidant and antigenotoxic activities of different fractions of Gentiana asclepiadea L. roots extract.

The aim of this study was to evaluate the antioxidant and antigenotoxic activities of chloroform, ethyl acetate and n-butanol fractions obtained from Gentiana asclepiadea L. roots methanolic extract. The main secondary metabolites sweroside, swertiamarin and gentiopicrine were quantified in G. asclepiadea root extracts using HPLC-DAD analysis. Amount of total phenols, flavonoids, flavonols and gallotannins was also determined. The antigenotoxic potential of extracts from roots of G. asclepiadea was assessed using the standard in vivo procedure for the detection of sex linked recessive lethal mutations in Drosophila melanogaster males treated with ethyl methanesulfonate (EMS). The results showed that the most abundant secoiridoid in G. asclepiadea roots was gentiopicrine and its content in the n-butanol fraction (442.89 mg/g) was the highest. Among all extracts, ethyl acetate fraction showed the highest antioxidant activity, as well as total phenolics (146.64 GAE/g), flavonoids (44.62 RUE/g), flavonols (22.71 RUE/g) and gallotannins (0.99 mg GAE/g) content. All the fractions showed antioxidant activity using in vitro model systems and the results have been correlated with total phenolics, flavonoids, flavonols and gallotannins content. In addition to antioxidant activity, G. asclepiadea root extract fractions possess an antigenotoxic effect against DNA damage induced by alkylation with EMS. The antioxidant activity exhibited by G. asclepiadea depended on the phenolic compounds content of the tested extracts, while there was no significant difference in the antigenotoxic potential between fractions.

In vitro and in vivo assessment of the genotoxicity and antigenotoxicity of the Filipendula hexapetala and Filipendula ulmaria methanol extracts.

ETHNOPHARMACOLOGICAL RELEVANCE The two species of Filipendula genus, Filipendula hexapetala Gilib. and Filipendula ulmaria (L.) Maxim are a traditional herbal medicine widely used to treat haemorrhoids, diarrhoea, fever, rheumatism and arthritic pain, kidney problems, to stop bleeding, and the common cold, as well as food supplements. However, no scientific study has been performed to validate genotoxic and/or antigenotoxic potentials of these two Filipendula species. AIM OF THE STUDY The aim of the present study was to examine the genotoxic and possible in vitro and in vivo DNA protection potential of methanol extracts of F. hexapetala and F. ulmaria. MATERIALS AND METHODS The genotoxicity of different concentrations of F. hexapetala and F. ulmaria methanol extracts from roots and aerial parts (20, 40 and 80 mg/ml), mixed with standard food for Drosophila, was evaluated in vivo in the anterior midgut of Drosophila melanogaster using a modified alkaline comet assay. The protective effects of the highest dose of extracts were observed in somatic cells of third-instar larvae against ethyl methanesulphonate (EMS)-induced genotoxicity. Also, DNA protection activity of methanol extracts from F. hexapetala and F. ulmaria (100, 200, and 400 μg/ml) against hydroxyl radical-induced DNA damage was determined under in vitro conditions. RESULTS The results showed that methanol extracts from the root and aerial part of F. hexapetala at a concentration of 20mg/ml indicated the absence of genotoxicity. Also, there were no statistically significant differences in total scores between any of the groups treated with F. ulmaria root extract and the negative control group, while F. ulmaria aerial part extract possess weak genotoxic effects depending on the concentrations. The percentage reduction in DNA damage was more evident in the group of larvae simultaneously treated with EMS and the highest dose of F. hexapetala root or aerial part extracts and F. ulmaria root extract (91.02, 80.21, and 87.5%, respectively) and less expressive in the group simultaneously treated with F. ulmaria aerial part extract (54.7%). F. hexapetala root and aerial part extracts and F. ulmaria root extract possess strong capabilities to protect DNA from being damaged by hydroxyl radicals. CONCLUSIONS It can be concluded that F. hexapetala root and aerial part extracts and F. ulmaria root extract demonstrated the absence of genotoxic activity. The extracts appeared to have antigenotoxic effect, reducing the levels of DNA damage induced by EMS by more than 80%. Also, F. hexapetala root and aerial part extracts and F. ulmaria root extracts could effectively protect against hydroxyl radical-induced DNA damage.

Genotoxic potential of Cotinus coggygria Scop. (Anacardiaceae) stem extract in vivo

The intention was to evaluate the possible in vivo genotoxic potential in different cell-types, of a methanol extract obtained from the plant stem of Cotinus coggygria Scop., using the sex-linked recessive lethal (or SLRL) test and alkaline comet assay. The SLRL test, revealed the genotoxic effect of this extract in postmeiotic and premeiotic germ-cell lines. The comet assay was carried out on rat liver and bone marrow at 24 and 72 h after intraperitoneal administration. For genotoxic evaluation, three concentrations of the extract were tested, viz., 500, 1000 and 2000 mg/kg body weight (bw), based on the solubility limit of the extract in saline. Comet tail moment and total scores in the group treated with 500 mg/kg bw, 24 and 72 h after treatment, were not significantly different from the control group, whereas in the groups of animals, under the same conditions, but with 1000 and 2000 mg/kg bw of the extract, scores were statistically so. A slight decrease in the comet score and tail moment observed in all the doses in the 72 h treatment, gave to understand that DNA damage induced by Cotinus coggygria extract decreased with time. The results of both tests revealed the genotoxic effect of Cotinus coggygria under our experimental conditions.