Biljana Božin

Antioxidant properties of Galium verum L. (Rubiaceae) extracts

The antioxidant properties of methanol extracts of Lady’s Bedstraw (Galium verum L., Rubiaceae) herb from two different localities in Serbia were evaluated. Antioxidant activity was assessed in four different model systems. Free radical scavenging capacity (RSC) was examined by measuring the scavenging activity of extracts on 2,2-diphenyl-1-pycrylhydrazil (DPPH) and hydroxyl radical (OH), as well as on hydrogen peroxide. In addition, the protective effects of lipid peroxidation (LP) in corn oil were evaluated by the TBA-assay using the Fe2+/ascorbate system of induction. The amount of dried extract, the content of total phenolics, flavonoids and chlorophylls was also determined. Extracts from both locations expressed very strong scavenger activity, reducing the DPPH⊙ (IC50=3.10 µg/mland 8.04 µg/ml) and OH radical formation (IC50=0.05 µg/ml and 0.54 µg/ml) and neutralising H2O2 (IC50=4.98 µg/ml and 3.80 µg/ml), in a dose dependant manner. Also, examined extracts showed notable inhibition of LP (IC50=11.69 µg/ml and 19.47 µg/ml). The observed differences in antioxidant activity could be partially explained by the levels of phenolics (2.44–4.65 mg and 4.57–5.16 mg gallic acid equivalents/g dry extract), flavonoids (6.38–10.70 µg and 15.56–17.96 µg quercetin equivalents/g dry extract) and chlorophylls in the investigated Lady’s Bedstraw extracts.

Chemical Composition of Essential Oil and Leaf Anatomy of Salvia bertolonii Vis. and Salvia pratensis L. (Sect. Plethiosphace, Lamiaceae)

The taxonomical relationship between Salvia pratensis and S. bertolonii has been unclear for a long time. Salvia bertolonii has alternatively been considered a synonym, a subspecies, a problematic subspecies and a form of Salvia pratensis. However, both these two species are sometimes used in traditional medicine instead of sage (Salvia officinalis) or as an adulteration for the same drug. In order to confirm the status of S. bertolonii, together with the potential identification characteristics for differentiation from sage, both taxa were analyzed through the analysis of their essential oils, together with the micromorphological characteristics of the leaf surface and the anatomy and morphology of the leaves. The obtained results show that there are clear differences in the quantity of essential oil (0.073% for S. pratensis and 0.0016% for S. berolonii). The major compound in the essential oil of S. pratensis was E-caryophyllene (26.4%) while in S. berolonii essential oil caryophyllene oxide was the major component (35.1%). The micromorphological differences are also pronounced in the leaf indumentum (density and distribution of certain types of non-glandular and glandular trichomes). Clear distinction between the investigated Salvia species is also observed in the leaf anatomy (in S. pratensis leaves are thinner, palisade tissue is made of 1-2 layers of cells, and leaves of S. bertolonii are characterized by 2-3 layers of palisade tissue cells, and consequently thicker).

Impact of origin and biological source on chemical composition, anticholinesterase and antioxidant properties of some St. John's wort species (Hypericum spp., Hypericaceae) from the Central Balkans.

The study shows the influence of the origin of plant material and biological source on the in vitro antioxidant (neutralization of DPPH and OH radical, nitric oxide, and inhibition of lipid peroxidation) and anticholinesterase activity of chemically characterized and quantified ethanol extracts of ten St. John’s wort samples. The investigated samples were: five Hypericum perforatum species representatives collected at different localities, one commercial sample of Hyperici herba purchased at a local market and four Hypericum species autochtonous to the Balkan Peninsula (H. maculatum subsp. immaculatum, H. olympicum, H. richeri subsp. grisebachii and H. barbatum). All the examined extracts exhibited notable antioxidant potential, but in most of the cases indigenous Hypericum species expressed stronger effects compared to the original source of the drug, H. perforatum. The changes in the content of phenolic compounds, especially flavonoids, hyperforin and hypericin, related to the source of the drug affected the investigated activities. Since all of the investigated species have shown prominent inhibition of acetylcholinesterase in vitro activity, they could be further investigated as potential substances in preventing of Alzheimer’s disease.

The influence of essential oil of aniseed (Pimpinella anisum, L.) on drug effects on the central nervous system

Anise (Pimpinella anisum L.; Apiaceae) and its essential oil have been widely used in folk medicine, pharmacy and food industry. Since there are some data about the impact of anise on functions of central nervous system (CNS), the issue of possible interactions with drugs acting in CNS should be considered. This survey aimed to examine the influence of aniseed essential oil (EO) intake on the effects of drugs that act in CNS. The chemical profile of essential oil determined by GC-MS revealed as the main components: trans-anethole (88.49%), γ-himachalene (3.13%), cis-isoeugenol (1.99%), and linalool (1.79%). The effects of codeine, diazepam, midazolam, pentobarbital, imipramine and fluoxetine were tested in mice after 5days of peroral pretreatment with human equivalent dose of aniseed EO (0.3mg/kg). The intake of EO led to significant increase of analgesic effect of codeine. The motor impairment caused by midazolam was enhanced in the group treated by EO. The application of diazepam decreased the number and percentage of entries in open arm in elevated maze plus test in the group pretreated with EO indicating augmented effect of drug on motor activity. EO pretreatment caused significant shortage of pentobarbital induced sleeping time when compared to control. The decrease in antidepressant effect of imipramine and fluoxetine was diminished by the pretreatment with aniseed EO. Based on the results of this study we conclude that concomitant intake of aniseed EO preparations and drugs that act on CNS should be avoided due to potential herb-drug interactions, which also need further clinical confirmation.

Alternative treatment of vaginal infections – in vitro antimicrobial and toxic effects of Coriandrum sativum L. and Thymus vulgaris L. essential oils

The aims of study were to examine the antibacterial potential of two commercial essential oils (EOs) from coriander (Coriandrum sativum L.) and thyme (Thymus vulgaris L.) against vaginal clinical strains of bacteria and yeast and their chemical composition.

Ganoderma: insights into anticancer effects.

The genus Ganoderma includes about 80 species growing on cut or rotten trees. The most commonly used species is Ganoderma ludicum. Biomolecules responsible for the health benefits of Ganoderma are polysaccharides with an immunostimulative effect and triterpenes with a cytotoxic action. For more than 2000 years, it has been used traditionally in the treatment of various pathological conditions and recently, its immunoregulatory, antiviral, antibacterial, antioxidant, hepatoprotective, and anticancer potential has been confirmed. A wide range of Ganoderma extracts and preparations arrest the cell cycle in different phases and consequently inhibit the growth of various types of cancer cells. Extracts containing polysaccharides stimulate immunological reactions through the production of various cytokines and mobilization of immune system cells. In-vivo studies have confirmed the anticancer potential and the antimetastatic effects of compounds originating from Ganoderma. There is also evidence for the chemopreventive action of Ganoderma extracts in bladder, prostate, liver, and breast cancer. The results of clinical studies suggest the combined use of G. lucidum with conventional chemotherapy/radiotherapy, but the methodology and the results of these studies are being questioned. Therefore, a constant need for new clinical trials exists.

Different associations of apoE gene polymorphism with metabolic syndrome in the Vojvodina Province (Serbia)

The metabolic syndrome (MetS) is a polygenic multifactorial metabolic disorder with strong socioeconomic influence. MetS has became a worldwide epidemic, that directly increases the risk of cardiovascular diseases and type 2 diabetes mellitus. The human apoE gene, coding Apolipoprotein E, has three common polymorphisms in human population: e2, e3 and e4, which are proved to be associated with impaired lipid metabolism. The contribution of apoE polymorphism to MetS disorders has not been investigated previously in Vojvodina Province, region with the highest number of obese people in Serbia. The aim of this study was to evaluate apoE gene polymorphism in relation to MetS disorders. The healthy control group of 30 individuals and 63 MetS patients were examined for apoE variants in relation to biochemical and anthropometric parameters. The genotypes were determined by PCR–RFLP. Regarding all parameters, significantly higher values were detected in MetS group compared to control. The MetS group of patients had significantly higher frequency of e4 allele. In addition, positive relation was revealed between e4 allele presence and all measured parameters. It was found that the e4 allele was related with a significantly increased OR of MetS disorders according to the International Diabetes Federation definition. These results suggested that e4 allele may act as a one of determinants for development of metabolic syndrome.

Acute and Chronic Pretreatment with Essential Oil of Peppermint (Mentha × piperita L., Lamiaceae) Influences Drug Effects

The appearance of common and self‐initiative usage of various herbal preparations in everyday practice and life imposes the question of possible interactions with drugs. This survey examined the influence of acute and chronic peppermint oil (PO – Mentha × piperita L., Lamiaceae; prepared as emulsion for oral use) on pentobarbitone‐induced sleeping time, analgesic effect of codeine and impairment of motor coordination caused by midazolam in mice. The chemical profile of essential oil was determined by GC‐MS. Applied doses of PO were 0.1 and 0.2 mL/kg. Chronic PO intake (in both doses) led to significant decrease of analgesic effect of codeine, while acute intake of PO did not change this effect. Acute PO pretreatment in higher dose caused significant prolongation of pentobarbitone‐induced sleeping time, while it was significantly shortened by chronic PO pretreatment at the same dose. Midazolam effect was enhanced and prolonged significantly by chronic PO intake at higher dose, while acute intake of PO did not change this effect. Gut motility was increased only by acute intake of higher PO dose. Regarding the fact that PO produces changes in tested drug effects, the interaction between drugs and phytopreparations containing PO should be additionally followed/confirmed in humans. Copyright

Biochemical Characterization of Helichrysum italicum (Roth) G.Don subsp. italicum (Asteraceae) from Montenegro: Phytochemical Screening, Chemotaxonomy, and Antioxidant Properties

The chemical composition and antioxidant properties of the essential oil and EtOH extract of immortelle (Helichrysum italicum (Roth) G.Don subsp. italicum, Asteraceae) collected in Montenegro were evaluated. The essential oil was characterized by GC/MS analysis, and the content of total phenolics and flavonoids in the EtOH extract was determined using the FolinCiocalteu reagent. The free‐radical‐scavenging capacity (RSC) of both the essential oil and the EtOH extract was assessed with the 2,2‐diphenyl‐1‐pycrylhydrazyl (DPPH) method. Moreover, the inhibition of hydroxyl radical (.OH) generation by the EtOH extract of immortelle was evaluated for the first time here. Neryl acetate (28.2%) and γ‐curcumene (18.8%) were the main compounds in the essential oil, followed by neryl propionate (9.1%) and ar‐curcumene (8.3%). The chemical composition of the oils of the examined and additional 16 selected Helichrysum italicum taxa described in literature were compared using principal component (PCA) and cluster (CA) analyses. The results of the statistical analyses implied the occurrence of at least four different main and three subchemotypes of essential oils. Considering the antioxidant properties, the EtOH extract of immortelle exhibited similar potential as propyl gallate and quercetin, while the essential oil exhibited relatively weak DPPH.‐scavenging capacity.

Effects of selected essential oils on the growth and production of ochratoxin A by Penicillium verrucosum

Abstract Essential oils from oregano (Origanum vulgare L.), mint (Mentha piperita L.), fennel (Foeniculum vulgare Mill.), and pine (Abies alba Mill.) needles and cones, and their active substances thymol, carvacrol, menthol, and anisaldehyde were tested for antifungal activity against Penicillium verrucosum. The lowest minimal inhibitory concentrations (MICs) were achieved for essential oil of oregano, followed by carvacrol, thymol, and menthol. These antifungal components were further investigated, as the main aim of our study was to elucidate the effect of natural antifungals on ochratoxin A production. During 21 days of exposure, the growth of P. verrucosum, and subsequently the production of ochratoxin A, was fully inhibited by thymol at ½ MIC (0.0625 mg mL-1), but menthol at ¼ and ½ MIC (0.1875 and 3750 mg mL-1) showed no growth inhibition. After 21 days of incubation, the greatest inhibitory effect on ochratoxin production (inhibition was 96.9 %) was also achieved with thymol at ¼ MIC (0.0313 mg mL-1). Essential oil of oregano (¼ MIC, 0.2930 μL mL-1) and carvacrol (½ MIC, 0.1953 μL mL-1) stimulate production of ochratoxin A at 13.9 % to 28.8 %, respectively. The observed antifungal effects depended on the agent, the concentration used, and the time of interaction between the agent and P. verrucosum. Our results indicate the possibility of using oregano essential oil as a substitute for artificial preservatives in certain foods, but further research is needed. Izvleček Eterična olja origana (Origanum vulgare L.), mete (Mentha piperita L.), janeža (Foeniculum vulgare Mill.), in borovih (Abies alba Mill.) iglic in storžev, ter njihove sestavine timol, karvakrol, mentol in anisaldehid smo testirali kot snovi z protiglivno aktivnostjo proti plesnim vrste Penicillium verrucosum. Najnižje minimalne inhibitorne koncentracije (MICs) smo določili eteričnemu olju origana, ter karvakrolu, timolu, in mentolu. Rast plesni vrste P. verrucosum je bila popolnoma inhibirana po 21 dneh inkubacije v prisotnosti ½ MIC timola (0,0625 mg mL-1), medtem ko so eterično olje origana (½ MIC, ¼ MIC), timol (¼ MIC) in karvakrol (½ MIC, ¼ MIC) inhibirali rast plesni od 8,3 % do 70.8 %. Dodatek mentola v koncentracijah ¼ MIC in ½ MIC (0.1875 mg mL-1 in 0.3750 mg mL-1) pa ni imel inhibitornega vpliva na rast plesni vrste P. verrucosum. Popolno inhibicijo tvorbe ohratoksina A smo določili le v primeru, ko je bila tudi rast teh plesni popolnoma inhibirana (pri ½ MIC timola). Največjo inhibicijo tvorbe ohratoksina A (96,9 %) po 21 dnevih inkubacije smo določili pri ¼ MIC timola (0.0313 mg mL-1). Pospešeno tvorbo ohratoksina A (od 13,9 % do 28,8 %) pa smo določili pri ¼ MIC eteričnega olja origana (0.2930 mg mL-1) in ½ MIC karvakrola (0.1953 mg mL-1). Protiglivni vplivi so bili odvisni od testirane snovi, njene koncentracije ter časa interakcij med protiglivno snovjo in plesnimi vrste P. verrucosum. Rezultati nakazujejo možnosti uporabe eteričnega olja origana v živilih namesto kemijskih konzervansov. Ker pa so živila kompleksna okolja v katerih je potrebno upoštevati več dejavnikov, so potrebne nadaljnje raziskave.