Species from the genus Globularia L. have been used as healing agents for various ailments, with utilization of Globularia alypum L. being most frequently reported. The aim of this study was to evaluate the antidiabetic, antioxidant, anti-inflammatory, antibacterial and anticancer potential of G. alypum and three related species, G. punctata Lapeyr., G. cordifolia L. and G. meridionalis (Podp.) O.Schwarz, in relation to their phytochemical compositions. Globularin and verbascoside were identified using LC-PDA-ESI-MSn as the major metabolites of G. alypum with known biological activities. G. alypum demonstrated the greatest α-glucosidase inhibitory activity and DPPH radical scavenging activity (IC50 = 17.25 μg/mL), while its anti-inflammatory activity was not significantly different from those of related species. All investigated species showed considerable antibacterial activity against methicillin-resistant Staphylococcus aureus in the broth microdilution method (MIC = 1.42–3.79 mg/mL). G. punctata also showed antibacterial activities against Escherichia coli (MIC = 1.42 mg/mL), Bacillus subtilis (MIC = 1.89 mg/mL), B. cereus (MIC = 2.84 mg/mL) and Enterococcus faecalis (MBC = 5.68 mg/mL). G. punctata, G. cordifolia and G. meridionalis showed greater anticancer potential than G. alypum. Obtained results indicate investigated Globularia species could serve as sources of diverse bioactive molecules, with G. punctata having the greatest antibacterial potential.

Colorectal cancer (CRC) is the third most common cancer worldwide. The high mortality from CRC is mainly related to metastasis affecting distant organs and their function. Dissemination of tumor cells from the primary tumor and hematogeneous spread are considered crucial in the formation of tumor metastases. The analysis of circulating tumor cells (CTCs) and CTC clusters in the blood can be used for the early detection of invasive cancer. Moreover, CTCs have a prognostic significance in the monitoring of a malignant disease or the response to chemotherapy. This work presents an overview of the research conducted on CTCs with the aim of finding suitable detection systems and assessing the possibility of clinical applications in patients with CRC.

Drug-specific therapeutic approaches for colorectal cancer (CRC) have contributed to significant improvements in patient health. Nevertheless, there is still a great need to improve the personalization of treatments based on genetic and epigenetic tumor profiles to maximize the quality and efficacy while limiting cytotoxicity. Currently, CEA and CA 19-9 are the only validated blood biomarkers in clinical practice. For this reason, laboratories are trying to identify new specific prognostics and, more importantly, predictive biomarkers for CRC patient profiling. Thus, the unique landscape of personalized biomarker data should have a clinical impact on CRC treatment strategies and molecular genetic screening tests should become the standard method for diagnosing CRC. This review concentrates on recent molecular testing in CRC and discusses the potential modifications in CRC assay methodology with the upcoming clinical application of novel genomic approaches. While mechanisms for analyzing circulating tumor DNA have been proven too inaccurate, detecting and analyzing circulating tumor cells and protein analysis of exosomes represent more promising options. Blood liquid biopsy offers good prospects for the future if the results align with pathologists’ tissue analyses. Overall, early detection, accurate diagnosis and treatment monitoring for CRC with specific markers and targeted molecular testing may benefit many patients.

Drug-specific therapeutic approaches for colorectal cancer (CRC) have contributed to a significant improvement in the health status of patients. However, a great need to improve personalization of treatments based on genetic and epigenetic tumor profiles to maximize quality and efficacy while limiting cytotoxicity remains. Currently, CEA and CA 19- 9 are the only validated blood biomarkers in clinical practice. For this reason, laboratories are trying to identify new specific prognostic and, more importantly, predictive biomarkers for CRC patient profiles. Thus, the unique landscape of personalized biomarker data should have a clinical impact on CRC treatment strategies and molecular genetic screening tests should become the standard method for CRC diagnosis, as well as detection of disease progression.

ABO blood group is a risk factor for several cancers, but it is not clear yet whether the risk of breast cancer is greater in particular ABO blood type carriers. The aim of this casecontrol study was to examine the correlation between ABO blood group genotypes, estrogen receptor (ER), progesterone receptor (PR) and HER2 status as tumor grade markers (I-III), and the occurrence of breast cancer. The research included 59 patients with invasive breast cancer and 80 asymptomatic, healthy women, blood donors. Genomic DNA was isolated using QIAampDNA Blood Mini Kit (QIAGEN, Germany). Genotyping was performed using in-house polymerase chain reaction with sequence-specific primers (PCR-SSP) method. Comparison of genotypes and phenotypes of ABO blood groups between patients and control group yielded p>0.05. There was no statistical significance of correlation between ABO genotypes/phenotypes in either patient group or control group. Testing the significance of different tumor grade occurrence, and ER, PR and HER2/neu status showed no statistical significance in the occurrence of a particular tumor grade, or in ER, PR and HER2/neu status as tumor markers in O1A1 genotype compared to non-O1A1 genotypes. Our study results confirmed that there was no correlation between ABO blood type genotypes/phenotypes and breast cancer in study groups.

The aim of this study was to compare zinc (Zn) level in EDTA, heparin, citrate plasma and serum. The blood samples from the same healthy volunteers (n = 10) were sampled by use of different anticoagulants (EDTA, heparin and citrate) or with no anticoagulant. Zn level was determined by flame atomic-absorption spectrometer (AAnalyst 800, PerkinElmer, USA). Zn level in EDTA, heparin, citrate plasma and serum was in reference range (0.7 to 1.5 mu g mL(-1)), and the difference between sample groups was not observed (P= 0.079). However, correlation was observed only between Zn level in heparin plasma and citrate plasma (r(s) = 0.72; P = 0.016) and citrate plasma and serum (r(s)= 0.76; P = 0.012). In conclusion, in Zn blood analysis, sample collection step is important and should be observed when planning study and when comparing the results of Zn level from various studies.

Olive leaf is reputed to have many beneficial health-related properties, often attributed to the iridoid glycoside oleuropein. In this work, the composition and antioxidant activity of oleuropein-rich olive leaf extracts prepared by ultrasonication were investigated. In addition, the influence of the extracts on Hep G2 cells subjected to glucose-induced oxidative stress was examined. The prepared extracts acted as antioxidants rich in oleuropein and other polyphenols. Among them, the extract prepared at 20°C using 100% ethanol was the most effective radical scavenger, antioxidant and metal chelator. It also contained the largest amount of oleuropein and other phenolic constituents. The extract prepared at 20°C using 70% ethanol, on the other hand, showed the best yield among the investigated extracts. Furthermore, it was able to preserve the cell-membrane integrity of Hep G2 cells subjected to glucose-induced oxidative stress, as well as to maintain intracellular concentration of glutathione and the activity of glutathione S-transferase on the levels comparable to those observed in non-stressed cells. The presented results demonstrate that oleuropein-rich olive leaf extracts can effectively ameliorate the consequences of glucose-induced oxidative stress in liver cells.

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, χ 2 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.

Naringenin is a flavanone, a type of flavonoid that is considered to have a bioactive effect on human health as antioxidant, free-radical scavenger, anti-inflammatory, carbohydrate metabolism promoter, and immune system modulator. This study was initiated to determine in which of the examined concentrations at the range 1-1000 μM of the naringenin has the protective effect on high glucose-induced toxicity in Hep G2 cells. The treatment of Hep G2 cells with 30 mM glucose significantly increased the levels of hepatic functional markers such as AST, ALT, LDH and GGT, decreased cell viability (MTT test), and decreased the activity ofhepatic antioxidative enzyme: glutathione peroxidase (GPx), when compared with norma! glucose level (p<0.05). We showed, that naringenin at concentration 1 to 100 μmol/L had hepatoprotective effect on Hep G2 cells under hyperglycemic condition, but at a concentration of 1000 μmol/L this effect is lost and naringenin showed toxic effect.

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.

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.

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. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 679–692, 2016.

Increased activity of sucrase, one of the intestinal alpha-glucosidase founded in diabetes mellitus. Inhibition of sucrase activity, plays a major role in preventing rise in postprandial glucose level in diabetics. On peer-reviewed literature could be found regarding investigation the effect of mixture plant proteins, Mw 3 – 15 kDa (MPP), isolated from Astragali radix – Astragalus membranaceus Fisch., Foenugraeci semen – Trigonella foenum graecum L., Cichorii radix – Cichorium Intybus L. and Urticae radix and herba – Urtica dioica L. on sucrase activity. This plants are used in traditional medicine of treatment of diabetes mellitus. The aim of this study was to determine activity of sucrase in small intestinal homogenates of NOD diabetic mice on feeding with and without MPP in chow. In mice diabetes was induced by i.v. injection of aloxan- monohydrate (75 mg/kg b.m.) seven days before treatment with MPP. The proteins (Mw 3 – 15 kDa), were isolated from ethanol extract, each plants separately, by gel filtration method on Sephadex G-25 column. Eluted fraction which highest absorbance on 280 nm were pooled, dialyzed, lyophilized and mixed (MPP) and before treatment in mice solvent in sterile PBS. After seven days of treatment diabetic NOD mice with MPP (1, 8 g/d), the small intestine was removed and divided into three segments, from pylorus to duodenum, and two equal lengths of the jejunum and ileum and homogenized in cold 0.14M KCl. Specific sucrase activity was determined using method of Dahlquist et. al., by sucrose as substrate. We confirmed the increased specific sucrase activity in the intestine of diabetic NOD mice. Our results also indicate that MPP have strongly inhibitory potential on intestinal sucrase activity (p<0.05) in diabetic mice. Conclusions drawn from this study should be further supported and our future experiments will be focused on determining the amino acid sequence of each protein from MPP.

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.

The impact of glutathione S-transferases (GSTs) detoxification pathway on complex pathogenesis and heterogeneity of clinical findings in multiple sclerosis (MS), particularly the exact correlation between indicators of clinical severity and different GST genotypes, has not yet been fully elucidated. The aim of the study was to assess the relationship between disability level in multiple sclerosis (estimated by Kurtzke Expanded Disability Status Scale), disease progression (estimated by Multiple Sclerosis Severity Score), the level of brain atrophy and lesion load (determined by MRI) and detoxification status (analyzing glutathione S-transferase P1, GSTP1, genotype profile), in a group of 58 MS patients and 68 age/gendermatched controls. The results present the first evidence on significantly higher frequency of GSTP1 C341T polymorphism (C-T transition) in healthy subjects compared to MS patients, suggesting it may act as a moderating factor in developing MS clinical phenotype. Gender-dependent distribution of the C341T polymorphism was found in both MS patients and controls, with higher frequency of C-T transition in females. In addition, preliminary data showed higher proportion of male MS patients with higher median MSSS scores, as well as lower brain atrophy level and lesion load in MS patients carrying the C341T mutation. Observed gender difference in distribution of the C341T polymorphism in MS patients, as well as in disease progression, suggests that GSTP1 detoxification pathway occurs in a gender-dependent manner and could therefore add to clinical severity in male MS patients.

Pelargonium radula (Cav.) L'Hér is a plant with hypoglycaemic properties originating from South Africa. Since oxidative stress plays a major role in the development of diabetic complications, the aim of this study was to evaluate if P. radula, besides its glucose-lowering properties, can have additional beneficial effects on diabetes. For preparation of extracts fresh or dried leaves were extracted at 20°C with either ethanol or water. Since P. radula is commercially cultivated for its essential oil, the residues obtained after steam distillation of essential oil from dry or fresh leaves were also included in the investigation. Content of phenols (total phenols, flavonoids and tannins) was determined in the extracts. Antioxidant activity was established as radical scavenging and chelating activity, reducing power and activity in β-carotene-linoleic acid assay. To establish possible intracellular effects of the extracts, lactate dehydrogenase (LDH) leakage was determined in Hep G2 cells with glucose induced oxidative stress. All the extracts were found to possess antioxidant activities and were able to reduce LDH leakage from Hep G2 cells. In addition to having excellent extraction yields the residues obtained after steam distillation of essential oil showed the most pronounced antioxidant activity in our study. The results of the present study suggest that P. radula leaf might be useful as complementary therapy in diabetes.

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.

In ethnomedicine, olive leaf is used as antidiabetic agent. It is well known that hyperglycemia produces oxidative stress in organism. Oxidative stress has deleterious effects on many organs and tissues, hepatic cells being one of the main targets. Therefore, we have investigated the effects of the olive leaf extracts on the levels of extracellular lactate dehydrogenase (LDH) as well as intracellular glutathione (GSH) and glutathione S-transferase (GST) in Hep G2 cells in hyperglycemic conditions. Moreover, antioxidant activity, content of phenolic compounds and -amylase inhibitory activity of the extracts was investigated. The extract were prepared using 70% ethanol at 25 C (E1) and 100% ethanol at 20 C and 60 C (E2 and E3, respectively). The prepared extracts were rich in phenolic acids, flavonoids and other polyphenols as determined spectrophotometrically. In addition, all the extracts demonstrated notable antioxidant activities. However, neither of the extracts has shown any -amylase inhibitory activity. The activity of the extracts in HepG2 cells was dependant on the method used for its preparation. E1 has shown membrane protective properties as seen in lower amount of extracellular LDH, even in comparison to non-hyperglycemic conditions. Moreover E1 and E3 were able to restore GSH concentration to the levels of non-hyperglycemic conditions. Finally, all the investigated extracts were capable of increasing the GST activity in Hep G2 cells. It can be concluded that olive leaf and its extracts can offer benefits as adjuvant therapy in diabetes.