Natasa Simin

Phenolic Compounds in Field Horsetail (Equisetum arvense L.) as Natural Antioxidants

In this paper, the study of antioxidant activity and phenolic composition of three different extracts (EtOAc, n-BuOH and H2O) of field horsetail (Equisetum arvense L.) is presented. The antioxidant activity has been evaluated measuring the total reducing power (expressed by Ascorbate Equivalent Antioxidant Capacity - AEAC), inhibition of lipid peroxidation, and free radical scavenging capacity (RSC) towards 2,2-diphenyl-1-picrylhydrazyl (DPPH radical) and nitric oxide (NO), respectively. In addition, the total flavonoid content (TFC) and phenolic constituents of each extract have been determined. The results obtained show that the highest RSC regarding both DPPH and NO radicals is expressed by EtOAc extract (EC50=2.37 μg/mL and EC50=90.07 μg/mL, respectively), and the lowest by H2O extract (EC50=37.2 μg/mL and EC50>333.33 μg/mL, respectively). n-BuOH extract showed the highest total reducing power (AEAC=13.40 μg/mL). Differences in the phenolic composition of examined extracts are found comparing the HPLC chemical profiles. Although, isoquercitrin is the main flavonoid in both EtOAc and n-BuOH extracts, a considerable amount of di-E-caffeoyl-meso-tartaric acid was presented in the n-BuOH extract. In H2O extract high content of phenolic acids and low percentage of flavonoids were detected.

Chemical Composition, Antioxidant and Antibacterial Properties of Achillea collina Becker ex Heimerl s.l. and A. pannonica Scheele Essential oils

The in vitro antioxidant and antimicrobial activities of two Achillea millefolium (Adanson) Koch s.l species essential oils (A. collina Becker ex Heimerl s.l. and A. pannonica Scheele, Asteraceae) originating from the Golija and Radan mountains (Serbia) were investigated. The chemical profiles of the essential oils were evaluated by GC-MS. Antioxidant activity was assessed as free radical scavenging capacity (RSC) towards 2,2-diphenyl-1-picrylhydrazil (DPPH) radicals, together with effects on lipid peroxidation (LP). Antibacterial activity was examined on 21 bacterial strains. Based on the chemical composition of the essential oil, A. collina s.l. from Mount Golija was classified as a chamazulene chemotype (tetraploid). The high percentage of oxygenated monoterpenes and absence of azulene in the essential oil obtained from A. pannonica from Radan pointing that this population is octaploid. Essential oil of A. pannonica expressed stronger antimicrobial activity on almost all tested bacteria. Furthermore, this essential oil expressed higher scavenging effects on DPPH radical (IC50 = 0.52 comparing to 0.62 μg/mL). Only in the LP evaluation, essential oil of A. collina s.l. from Golija exhibited stronger antioxidant activity (IC50 = 0.75 comparing to 2.12 μg/mL).

Antimicrobial activity of Eucalyptus camaldulensis essential oils and their interactions with conventional antimicrobial agents against multi-drug resistant Acinetobacter baumannii.

ETHNOPHARMACOLOGICAL RELEVANCE Traditional herbal medicine has become an important issue on the global scale during the past decade. Among drugs of natural origin, special place belongs to essential oils, known as strong antimicrobial agents that can be used to combat antibiotic-resistant bacteria. Eucalyptus camaldulensis leaves are traditional herbal remedy used for various purposes, including treatment of infections. The aim of this study was to determine antimicrobial potential of two E. camaldulensis essential oils against multi-drug resistant (MDR) Acinetobacter baumannii wound isolates and to examine possible interactions of essential oils with conventional antimicrobial agents. MATERIALS AND METHODS Chemical composition of essential oils was determined by gas chromatography-mass spectrometry analysis (GC-MS). MIC values of essential oils against A. baumannii strains were estimated by modified broth microdilution method. The components responsible for antimicrobial activity were detected by bioautographic analysis. The potential synergy between the essential oils and antibiotics (ciprofloxacin, gentamicin and polymyxin B) was examined by checkerboard method and time kill curve. RESULTS The dominant components of both essential oils were spatulenol, cryptone, p-cimene, 1,8-cineole, terpinen-4-ol and β-pinene. The detected MICs for the E. camaldulensis essential oils were in range from 0.5 to 2 μl mL(-1). The bioautographic assay confirmed antibacterial activity of polar terpene compounds. In combination with conventional antibiotics (ciprofloxacin, gentamicin and polymyxin B), the examined essential oils showed synergistic antibacterial effect in most of the cases, while in some even re-sensitized MDR A. baumannii strains. The synergistic interaction was confirmed by time-kill curves for E. camaldulensis essential oil and polymyxin B combination which reduced bacterial count under detection limit very fast, i.e. after 6h of incubation. CONCLUSIONS The detected anti-A. baumannii activity of E. camaldulensis essential oils justifies traditional use of this plant. The proven E. camaldulensis essential oil synergistic interactions with conventional antibiotics could lead to the development of new treatment strategies of infections caused by MDR A. baumannii strains in the term of antibiotic dose reduction.

Synergistic effect of Myrtus communis L. essential oils and conventional antibiotics against multi-drug resistant Acinetobacter baumannii wound isolates.

Acinetobacter baumannii is a rapidly emerging, highly resistant clinical pathogen with increasing prevalence. In recent years, the limited number of antimicrobial agents available for treatment of infections with multi-drug resistant (MDR) strains reinforced tendency for discovery of novel antimicrobial agents or treatment strategies. The aim of the study was to determine antimicrobial effectiveness of three Myrtus communis L. essential oils, both alone and in combination with conventional antibiotics, against MDR A. baumannii wound isolates. The results obtained highlighted the occurrence of good antibacterial effect of myrtle oils when administered alone. Using checkerboard method, the combinations of subinhibitory concentrations of myrtle essential oils and conventional antibiotics, i.e. polymixin B and ciprofloxacine were examined. The results proved synergism among M. communis L. essential oils and both antibiotics against MDR A. baumannii wound isolates, with a FIC index under or equal 0.50. Combination of subinhibitory concentrations of essential oils and ciprofloxacin most frequently reduced bacterial growth in synergistic manner. The similar has been shown for combination with polymyxin B; furthermore, the myrtle essential oil resulted in re-sensitization of the MDR wound isolates, i.e. MICs used in combination were below the cut off for the sensitivity to the antibiotic. Time-kill curve method confirmed efficacy of myrtle essential oil and polymyxin B combination, with complete reduction of bacterial count after 6h. The detected synergy offers an opportunity for future development of treatment strategies for potentially lethal wound infections caused by MDR A. baumannii.

Biomarkers of oxidative stress in amniotic fluid and complications in pregnancy

Objective. In this study, we tried to determine whether the activities of the primary antioxidant enzymes are detectable in amniotic fluid and whether they can be used as early biomarkers of complications in pregnancy such as pregnancy-induced hypertension (PIH), gestational diabetes mellitus (GDM), and bacterial vaginosis. Methods. This was a prospective study in which amniotic fluid was taken between 16 and 19 week of gestation. In all, 161 pregnant women were divided into two groups: study group – patients with the treated local infection, PIH, and GDM, and control group – healthy pregnant women. Levels of reduced glutathione (GSH) and activities of supeoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSHR), glutathione S-transpherase (GST), xanthine oxidase (XOD) and lipid peroxidation (LP) were determined spectrophotometrically in amniotic fluid samples. Results. Concentration of malondialdehyde varied greatly between investigated groups. XOD and SOD activities, though very low, were present in amniotic fluid samples. Also, enzymes of glutathione cycle and GSH concentrations were detectable and showed certain variations. Conclusion. Parameters of oxidative stress in amniotic fluid could be altered in certain pathological conditions. Their use as clinical biomarkers is limited due to great variations of amniotic fluid volume between patients which gives favor to hemolysate or serum of pregnant women.

The antiinflammatory, gastroprotective and antioxidant activities of Hieracium gymnocephalum extract

The present study investigated the antiinflammatory, gastroprotective and antioxidant activities of a CH2Cl2 extract of western Balkan endemic Hieracium gymnocephalum Griseb. ex Pant. (Compositae). The carrageenan‐induced rat paw oedema test was used as an experimental model for screening the antiinflammatory activity. The extract was administrated p.o. in doses of 25, 50, 100 and 200 mg/kg to rats and its effects compared with indomethacin, used p.o. as a reference drug. The results showed that the investigated extract reduced the oedema in a concentration‐dependent manner. The obtained antiinflammatory effect was 5.9%, 11.7%, 31.2% and 44.1% at doses of 25, 50, 100 and 200 mg/kg, respectively, being statistically significant at a dose of 100 mg/kg. Indomethacin had a strong antiinflammatory effect of 73.4% at a dose of 8 mg/kg, but caused large gastric lesions. When the plant extract in the highest tested dose (200 mg/kg) was concomitantly given with indomethacin, the antiinflammatory effect was slightly enhanced, but the gastric lesions were significantly reduced. The antioxidant activity of the H. gymnocephalum extract, investigated using DPPH radical assay, OH‐radical assay and TBA‐test, was not substantial. Copyright

Binary and Tertiary Mixtures of Satureja hortensis and Origanum vulgare Essential Oils as Potent Antimicrobial Agents Against Helicobacter pylori

Essential oils possess strong antimicrobial activity, even against multiresistant Helicobacter pylori. Available therapies against H. pylori infection have multiple disadvantages, indicating a great need for a development of new therapeutics. The purpose of this study was to develop a potent natural product based anti‐H. pylori formulation. First, anti‐H. pylori activity of nine essential oils was determined, after which the most active oils were mixed in various ratios for further testing. Satureja hortensis, Origanum vulgare subsp. vulgare and O. vulgare subsp. hirtum essential oils expressed the highest activity (MIC = 2 μL mL−1). Their binary and ternary mixtures exhibited notably higher antimicrobial activity (MIC ≤ 2 μL mL−1). The most active was the mixture of S. hortensis and O. vulgare subsp. hirtum oils in volume ratio 2:1, which expressed 4 times higher activity than individual oils (MIC = 0.5 μL mL−1). According to GC‐MS, both oils in the mixture were characterized by high content of phenols (48–73%), with carvacrol as the main carrier of antimicrobial activity. Presented in vitro study pointed out binary mixture of S. hortensis and O. vulgare subsp. hirtum essential oils in volume ratio 2:1 as promising candidate for further in vivo studies targeting H. pylori infection. Copyright

Erythropoietin in amniotic fluid as a potential marker in distinction between growth restricted and constitutionally small fetuses.

Abstract Objective: To determine if there is any difference in amniotic fluid erythropoietin (EPO) concentration between fetuses small for gestational age (SGA) and appropriate for gestational age (AGA), and between the constitutionally small (CSF) and growth-restricted (GRF) fetuses. Methods: EPO concentrations in the amniotic fluid samples were determined by EpoELISA test in 38 pregnancies with SGA and 15 pregnancies with AGA fetuses. In the SGA group we measured Ponderal index (PI) and skin-fold thickness (SFT). If PI and/or SFT were below 10th percentile the neonate was GRF. If both PI and SFT were above 10th percentile the neonate was CSF. Results: Higher levels of EPO were detected in the SGA in comparison to the AGA fetuses (p < 0.01). EPO concentration was higher in GRF compared to CSF (p < 0.05). The EPO cut-off level between SGA and AGA was 6.81 IU/L (sensitivity 92.3%; specificity 73.3%), and between GRF and CSF was 9.8 IU/L (sensitivity 81%; specificity 80%). Conclusion: The preliminary results of this study suggest that amniotic fluid erythropoietin concentration is elevated in growth-restricted fetuses and could potentially be used for distinction between growth restricted and constitutionally small fetuses. Confirmation of these results on a larger group of pregnant women is needed.

Chemical Composition and Immuno-Modulatory Effects of Urtica dioica L. (Stinging Nettle) Extracts

The purpose of this work was to determine the chemical profile of stinging nettle and to provide an insight into the mechanisms by which it ameliorates the immune response. Qualitative and quantitative liquid chromatography tandem mass spectrometry analyses indicated that phenolic acids (5‐O‐caffeoylquinic acid as dominant) and flavonol glycosides (rutin, isoquercitrin, and kaempferol 3‐O‐glucoside) are present in the aerial parts, while lignans (secoisolariciresinol, 9,9′‐bisacetyl‐neo‐olivil and their glucosides) were detected in the root. Herb and root extracts expressed selective inhibition toward cyclooxygenase and lipoxygenase branches in human platelets: root extracts were better at inhibiting thromboxane production, while herb extracts were more specific toward inhibition of 12‐lipoxygenase pathway. Stinging nettle extracts mildly increased monocyte chemoattractant protein‐1 and growth‐related oncogene release from nonstimulated intestinal epithelial cells, stimulating MyD88/NF‐κB/p38 signaling, hence preserving the epithelial integrity and enhancing intestinal steady‐state defense. Additionally, root extract reduced lipopolysaccharide‐induced monocyte chemoattractant protein‐1/growth‐related oncogene secretion and cyclooxygenase‐2 expression in intestinal epithelial cells, thus showing the potential protective effect against tissue damage caused by inflammation processes. These observations suggest that stinging nettle is an interesting candidate for the development of phytopharmaceuticals or dietary supplements for cotreatment of various inflammatory diseases, particularly inflammatory bowel diseases. Copyright

Mid-pregnancy interleukins levels in serum and amniotic fluid as predictors of preterm delivery.

Abstract Objectives: To find out if determination of IL-4, IL-13 and IL-6 in amniotic fluid (AF) and serum in mid-pregnancy can be used as predictors of preterm delivery. Material and methods: The research has comprised 88 pregnant women at 16-24 weeks of gestation, who were subjected to early amniocentesis. Levels of interleukins were determined by ELISA tests. Pregnant women had been monitored untill termination of pregnancy and divided in two groups: 68 term and 20 preterm deliveries. Results: IL-13 was not detectable at all in serum and amniotic fluid samples. IL-4 was not detectable in serum samples of both groups of women and it was detectable only in small number (20 % - 27 %) of AF samples. There was no statistically significant difference (p=0.665) in the mean values of AF IL-4 levels between the examined groups of women. Detectability for IL-6 was very low in serum specimens, while in AF it was detectable in 100% of cases and its levels was significantly higher (p<0.001) in preterm delivery group. Conclusions: The results obtained in this study suggest that the AF mid-pregnancy levels of IL-6 higher than 132 pg/ml may indicate preterm delivery.