Vesna Stanković

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.

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.

Serum albumin binding analysis and toxicological screening of novel chroman-2,4-diones as oral anticoagulants.

Two chroman-2,4-dione derivatives, namely 2a and 2f, were tested as in vivo anticoagulants by seven days of continuous per os application to adult male Wistar rats in a concentration of 20 mg/kg of body weight. Derivatives were selected from a group of six previously intraperitoneally applied compounds on the basis of presenting remarkable activity in a concentration of 2 mg/kg of body weight. The derivatives 2a and 2f are VKORC1 inhibitors, and comparison of the absorption spectra, association, and dissociation constants suggested that the compounds will be bound to serum albumin in the same manner as warfarin is, leading to transfer towards the molecular target VKORC1. After oral administration, the compounds proved to be anticoagulants comparable with warfarin, inasmuch as the measured prothrombin times for 2a and 2f were 56.63 and 60.08 s, respectively. The INR values of 2a and 2f ranged from 2.6 to 2.8, recommending them as useful therapeutics in the treatment of patients suffering from thromboembolic events and atrial fibrillation. The high percentage of binding and high binding affinity of 2a and 2f towards serum albumin reduced the risk of induced internal bleeding. Several kinds of toxicity studies were performed to investigate whether or not 2a and 2f can cause pathological changes in the liver, kidneys, and DNA. The catalytic activity of serum enzymes, concentration and catalytic activity of liver and kidney oxidative stress markers and enzymes, respectively, as well as the observed hepatic and renal morphological changes indicated that the compounds in relation to warfarin induced irrelevant hepatic toxicity, no increment of necrosis, and inconsiderable oxidative damage in the liver and kidneys. Estimation of DNA damage using the comet assay confirmed that 2a and 2f caused no clinically significant genotoxicity. The higher activity and lower toxicity of 2f recommended this compound as a better drug candidate than 2a.

Neuroendocrine Markers-Useful Predictors of Therapeutic Responses in Non-resectable Non-small Cell Lung Cancer

Objective: This study correlates the neuroendocrine (NE) markers neuron-specific enolase (NSE), chromogranin A (CGA), and synaptophysin (SYN) with the therapeutic responses and the survival times of patients with non-resectable non-small cell lung cancer (NSCLC). Method: One hundred twenty-six patients (male 64%, female 36%) with histopathologically confirmed NSCLC, treated with combination chemotherapy and radiotherapy in 1 center, underwent immunohistochemical analyses. Results: Thirty-nine patients (31%) were positive for NSE, 24 (19%) for Chr A, and 27 (21%) for SYN. Favorable therapeutic responses (partial and complete responses [CR]) were significantly greater in patients expressing NSE, Chr A, and SYN (P<0.05). There was a significant correlation between the therapeutic response and the percentage of NE-positive tumor cells (P<0.05). Tumors with greater than 25.5% NSE-positive cells can be predicted to have a favorable therapeutic response (94% sensitivity, 100% specificity). Patients with multiple markers were also more likely to have a favorable response compared to those with only 1 marker (59% compared with 38%) (P<0.05). Those with positive NE expression had significantly better 1- and 2-year survival compared with non-expressers (60% compared with 18%, P<0.001, and 33% compared with 0% P=0.000, respectively). Conclusion: Patients expressing the NE markers NSE, Chr A, and SYN, and those with a larger proportion of positive tumor cells have better therapeutic responses and longer survival times.

Summer savory (Satureja hortensis L.) extract: Phytochemical profile and modulation of cisplatin-induced liver, renal and testicular toxicity

The aim of our study was to examine the potential ameliorating effect of the methanolic extract of Satureja hortensis L. (summer savory) aerial parts against cisplatin-induced oxidative damage in renal, hepatic, and testicular tissues. S. hortensis methanol extract at the doses of 50, 100 and 200 mg/kg of body weight were orally administered to Wistar rats once daily for 10 days. Toxicity was induced by intraperitoneal injection of a single dose of cisplatin (7.5 mg/kg of body weight) on the 5th day of the experiment. Applied treatment with S. hortensis extract restored tissue morphology, ameliorated levels of serum parameters for liver, renal and testes function, tissue oxidative stress parameters, and increased Bcl-2/Bax ratio as an indicator of apoptosis in experimental animals caused by application of cisplatin. UHPLC/DAD/HESI-MS/MS analysis revealed that S. hortensis extract was rich in phenolic compounds with rosmarinic acid (24.9 mg/g) as the main compound, followed by caffeic acid (1.28 mg/g) and naringenin (1.06 mg/g). Our findings suggest that S. hortensis may be a valuable source of dietary and pharmacologically important phenolic compounds, especially rosmarinic acid, in pharmaceutical and functional food formulations in order to maintain normal health conditions or as a remedy in various diseases caused by oxidative damage.

Interplay Between the Immunohistochemical Expression of P53 and the Proliferation Index in the Keratinocyte Tumors of the Skin

Abstract P53 is important for cell cycle regulation, and its overexpression is seen in malignant tumors. We examined correlation between p53 expression and cell proliferation, and its role in the pathogenesis of keratinocyte skin tumors. We used biopsies from patients with squamous cell carcinoma, actinic keratosis and keratoacanthoma. We examined crosssections stained with HE and using anti-cytokeratin, antip53 and anti-Ki67 antibodies. Expression of p53 is found in 87, 85% of SCC, in 83. 3% of AK and 13. 4% KA. The high index of p53 expression was higher in SCC and AK compared to KA. We also observed a positive correlation between the expression of p53 and localization of the tumors. The largest proportion of subjects with AK and SCC has a high index of p53 expression on photoexposed region. We also observed that p53 expression correlates with age whereby in AK p53 expression increases with age. The high index of proliferation is most frequent in SCC and KA. Also at AK we found a strong correlation between a moderate proliferation index and tumor localization in photoexposed region. Between the proliferation index and p53 expression we observed a significant positive correlation only in SCC. Proliferation index and the expression of p53 are useful for the differentiation of precursor keratinocyte lesions and skin carcinoma. High p53 expression has been associated with the aging and significantly correlates with the exposure to UV radiation in SCC and AK. High expression of p53 in AK and SCC supports the importance of this oncoprotein in carcinogenesis of the skin.