Investigation of the antimicrobial and antioxidant activities of the extracts of three selected plant species of the genus Daphne

Ova doktorska disertacija je koncipirana sa ciljem da se ispita fitohemijski sastav i potencijalna biološka aktivnost ekstrakata biljnih vrsta Daphne blagayana L., Daphne cneorum L. i Daphne alpina L. Upotrebom različitih rastvarača izvršena je ekstrakcija grančica i listova. Ispitivanja hemijskog sastava dobijenih ekstrakata obuhvatala su određivanje sadržaja ukupnih fenola, ukupnih flavonoida i HPLC-UV analizu ekstrakata. Ispitivani ekstrakti su pokazali visok sadržaj ukupnih fenola (68,77 ± 0,95 mg EGA/g - 90,26 ± 0,69 mg EGA/g) i flavonoida (24,67 ± 0,35 mg ERU/g - 35,24 ± 0,55 mg ERU/g). HPLC-UV analizom potvrđeno je prisustvo sekundarnih metabolita iz grupa fenola i kumarina: dafnetin, umbeliferon i 4- hidroksibenzoeva kiselina. Ispitivanje bioloških aktivnosti ekstrakata obuhvatalo je određivanje ukupne antioksidativne aktivnosti, određivanje DPPH „skevindžer” aktivnosti, određivanje inhibicije lipidne peroksidacije, Fe2+ helatizaciona aktivnost, određivanje antioksidativne aktivnosti na nivou hidroksil radikala i određivanje antimikrobne aktivnosti (in vitro). Ekstrakti ispoljavaju antioksidativnu aktivnost u svim primenjenim modelima. Takođe, ekstrakti su ispoljili i antimikrobnu aktivnost sa vrednostima minimalnih inhibitornih koncentracija koje su se kretale od 15,62 μg/ml do 125 μg/ml. Na osnovu dobijenih rezultata, može se zaključiti da vrste roda Daphne mogu predstavljati izvor novih, prirodnih, farmakološki aktivnih jedinjenja koja mogu imati potencijalnu primenu u farmaceutskoj, kozmetičkoj i industriji hrane.This doctoral thesis has been conceived with the aim to investigate phytochemical and potential biological activity of extracts obtained from plants species Daphne blagayana L., Daphne cneorum L. и Daphne alpinа L. Using different extraction solvents extracts of twigs and leaves were prepared. The chemical composition of extracts obtained from different parts was analysed. These analyses included the determination of total phenols, total flavonoids and HPLC-UV analysis. All the tested extracts showed the high total phenols (68,77 ± 0,95 mg EGA/g - 90,26 ± 0,69 mg EGA/g) and flavonoids (24,67 ± 0,35 mg ERU/g - 35,24±0,55 mg ERU/g) contents. HPLC-UV analysis was confirmed the presence secondary metabolites which belong to the phenol and coumarin group: daphnetin, umbelliferone and 4-hydroxybenzoic acid. Obtained extracts of tested plants were the subjected of determining the total antioxidant activity, DPPH determination activities, methods of inhibiting lipid peroxidation, the ferrous ion chelating ability, antioxidant activity at the level of hydroxyl radicals, the determination of antimicrobial activity (in vitro). The extracts exhibit antioxidant activity on the basis of all conducted tests. Also, the extracts shows antimicrobials activity with MIC value ranged from 15,62 μg/ml to 125 μg/ml. Based on these results it can be concluded that species from the Daphne genus may represent a source of new, natural, pharmacologically active compounds, which may potentially be used in pharmaceutical, cosmetic and food industry

Phytochemical, Antioxidant and Antimicrobial Profiles of Extracts of Daphne alpina (Thymelaeaceae) L Leaf and Twig from Mt Kopaonik (Serbia)

Purpose: To investigate the phytochemical composition, as well as antioxidant and antimicrobial activities of the leaf and twig extracts of Daphne alpina L. (Thymelaeaceae).Methods: The dry chloroform and methanol extracts of the leaf and twigs of Daphne alpinа were used for analysis. Total phenolic and flavonoid contents were determined by established procedures. Antioxidant potential was investigated by several methods. The antimicrobial properties of the extracts were obtained by microdilution method. High performance liquid chromatography (HPLC) was employed for the identification of the most abundant metabolites, present in D. alpina extracts.Results: The total phenolics of the extracts ranged from 78.98 to 88.98 mg GA/g while total flavonoids were in the range 28.09 to 34.65 mg GA/g of fresh weight. HPLC analysis of the extracts showed the presence 4-hydroxybenzoic acid, 7,8-dihydroxycoumarine and 7-hydroxycoumarine. Total antioxidant capacity ranged from 69.71 μg AA/g for the methanol leaf extract to 73.55 μg AA/g for the chloroform twig extract. All the extracts showed DPPH radical scavenging activity (21.57 - 25.45 μg/mL), inhibitory activity against lipid peroxidation (26.79 - 35.24 μg/mL), ferrous ion chelating ability (21.57 - 45.45 μg/ml) and hydroxyl radical scavenging activity (87.98 - 98.86 μg/mL). Minimum inhibitory concentration (MIC) was in the range 15.62 - 125 μg/mL.Conclusion: The extracts possess moderate antioxidant and antimicrobial activities due probably to the phenolic compounds in the extracts.Keywords: Daphne alpina, Coumarines, 4-Hydroxybenzoic Acid, Phenols, Flavonoids, Antimicrobial Activity, Antioxidant Activit

Risk factors for severe dental anxiety among medical students

Background/Aim. Severe dental anxiety (SDA) is the most severe form of dental anxiety, thus the aim of this study was to determine the factors associated with SDA in students of health-related disciplines. Methods. In this case-control study the cases were students with severe dental anxiety. The study was conducted at the Faculty of Medical Sciences, University of Kragujevac, Serbia. The participants were undergraduate students attending lectures during spring semester 2010/2011 (n = 1,812). A random sample of 800 students was assessed for the association between various risk factors and the severe dental anxiety. The main outcome measures were the data on demographics, dental anxiety, habits concerning oral hygiene, nutrition, general anxiety and (co)morbidity which were collected from the study participants by semi-structured questionnaire. Results. Less frequent visits to the dentist (OR adjusted = 7.02 [2.65; 18.60]) and visiting the dentist only when there is a dental problem (OR adjusted = 8.08 [1.28; 50.93]) were associated with severe dental anxiety. The same was true for improper oral hygiene (OR adjusted = 4.25 [1.16; 15.60]). Factors as changing toothbrush more frequently (OR adjusted = 0.33 [0.14; 0.76]) and having chronic disease (OR adjusted = 0.01 [0.00; 0.09]) were inversely associated with severe dental anxiety. The level of education of students was not associated with severe dental anxiety. Conclusion. Inappropriate oral hygiene, less frequent changes of a toothbrush and less frequent visits to the dentist are important risk factors for severe dental anxiety. [Projekat Ministarstva nauke Republike Srbije, br. 175007

Investigation of the antimicrobial and antioxidant activities of the extracts of three selected plant species of the genus Daphne

Ova doktorska disertacija je koncipirana sa ciljem da se ispita fitohemijski sastav i potencijalna biološka aktivnost ekstrakata biljnih vrsta Daphne blagayana L., Daphne cneorum L. i Daphne alpina L. Upotrebom različitih rastvarača izvršena je ekstrakcija grančica i listova. Ispitivanja hemijskog sastava dobijenih ekstrakata obuhvatala su određivanje sadržaja ukupnih fenola, ukupnih flavonoida i HPLC-UV analizu ekstrakata. Ispitivani ekstrakti su pokazali visok sadržaj ukupnih fenola (68,77 ± 0,95 mg EGA/g - 90,26 ± 0,69 mg EGA/g) i flavonoida (24,67 ± 0,35 mg ERU/g - 35,24 ± 0,55 mg ERU/g). HPLC-UV analizom potvrđeno je prisustvo sekundarnih metabolita iz grupa fenola i kumarina: dafnetin, umbeliferon i 4- hidroksibenzoeva kiselina. Ispitivanje bioloških aktivnosti ekstrakata obuhvatalo je određivanje ukupne antioksidativne aktivnosti, određivanje DPPH „skevindžer” aktivnosti, određivanje inhibicije lipidne peroksidacije, Fe2+ helatizaciona aktivnost, određivanje antioksidativne aktivnosti na nivou hidroksil radikala i određivanje antimikrobne aktivnosti (in vitro). Ekstrakti ispoljavaju antioksidativnu aktivnost u svim primenjenim modelima. Takođe, ekstrakti su ispoljili i antimikrobnu aktivnost sa vrednostima minimalnih inhibitornih koncentracija koje su se kretale od 15,62 μg/ml do 125 μg/ml. Na osnovu dobijenih rezultata, može se zaključiti da vrste roda Daphne mogu predstavljati izvor novih, prirodnih, farmakološki aktivnih jedinjenja koja mogu imati potencijalnu primenu u farmaceutskoj, kozmetičkoj i industriji hrane.This doctoral thesis has been conceived with the aim to investigate phytochemical and potential biological activity of extracts obtained from plants species Daphne blagayana L., Daphne cneorum L. и Daphne alpinа L. Using different extraction solvents extracts of twigs and leaves were prepared. The chemical composition of extracts obtained from different parts was analysed. These analyses included the determination of total phenols, total flavonoids and HPLC-UV analysis. All the tested extracts showed the high total phenols (68,77 ± 0,95 mg EGA/g - 90,26 ± 0,69 mg EGA/g) and flavonoids (24,67 ± 0,35 mg ERU/g - 35,24±0,55 mg ERU/g) contents. HPLC-UV analysis was confirmed the presence secondary metabolites which belong to the phenol and coumarin group: daphnetin, umbelliferone and 4-hydroxybenzoic acid. Obtained extracts of tested plants were the subjected of determining the total antioxidant activity, DPPH determination activities, methods of inhibiting lipid peroxidation, the ferrous ion chelating ability, antioxidant activity at the level of hydroxyl radicals, the determination of antimicrobial activity (in vitro). The extracts exhibit antioxidant activity on the basis of all conducted tests. Also, the extracts shows antimicrobials activity with MIC value ranged from 15,62 μg/ml to 125 μg/ml. Based on these results it can be concluded that species from the Daphne genus may represent a source of new, natural, pharmacologically active compounds, which may potentially be used in pharmaceutical, cosmetic and food industry

Plants from The Genus Daphne: A Review of its Traditional Uses, Phytochemistry, Biological and Pharmacological Activity

Plants have an important role in maintaining people’s health and improving the quality of human life. They are an important component of people’s diet, but they are also used in other spheres of human life as a therapeutic resources, ingredients of cosmetic products, paints and others. The Daphne genus belongs to family Thymeleaceae which includes 44 families with approximately 500 herbal species. The plant species of the genus Daphne are used in the traditional medicine in China and tropical part of Africa for the treatment of various conditions. Previous studies showed significant biological potential of these species as a source of pharmacologically active compounds. This indicates that this genus possess a broad spectrum of biological activity including antimicrobial, antioxidant, analgesic, anti-inflammatory, cytotoxic, anti-ulcerogenic, abortive, hypocholesterolemic and hemostatic effects. Additionally, Daphne plants are the source of valuable bioactive phytochemicals such as coumarins, flavonoids, lignans, steroids and different classes of terpenes. Different parts of the Daphne plants contain specific bioactive metabolites and can represent a source of new, natural, pharmacologically active compounds, which may potentially be used in pharmaceutical, cosmetic and food industries

A synthesis of N-alkyl and N,N-dialkyl O-ethyl thiocarbamates from diethyl dixanthogenate using different oxidants

A novel synthesis of N-alkyl and N,N-dialkyl O-ethyl thiocarbamates from diethyl dixanthogenate and primary and secondary amines, using three oxidizing systems, has been developed on the laboratory scale, and the method using sodium hypochlorite has been applied on a semi-industrial scale. The effect of the oxidizing agents, sodium hypochlorite, in-situ-generated peracetic acid, and the manganese(II) acetate/oxygen system on product purity and yield was studied. The results obtained by use of these three methods were compared with those obtained by reaction of sodium ethyl xanthogenacetate and amines, and of sodium ethyl xanthate with amines in the presence of sulfated nickel zeolite catalyst. The reaction mechanism of sodium hypochlorite oxidation has been established on the basis of isolation of reaction intermediates and determination of their structure by use of Fourier-transform infrared, (1)H and (13)C NMR, and mass spectrometric methods. The suggested sodium hypochlorite and manganese(II) acetate/oxygen systems have many advantages in comparison with commercial and catalytically promoted synthetic methods, because they are new ecologically friendly syntheses

Antimicrobial activity and HPLC analysis of Daphne blagayana L. (Thymelaeceae) extracts

The aim of this study was to investigate the antimicrobial activity and HPLC analysis of the methanol and chloroform extracts of leaves and twigs of endemic species Daphne blagayana collected from the area of Suva Mountain, Serbia. The antimicrobial activity, expressed as the values of MIC, were investigated on the 6 standardized bacterial strains and two strains of fungi. Amracin and ketoconazole are used as standard antibiotics. MIC values of the tested extracts were ranged from 15.62 to 125 μg/ml. The strongest effect of the extracts exhibited against Klebsiella pneumoniae, Staphylococcus aureus and Proteus mirabilis. Results of the HPLC analysis showed the presence of a variety of secondary metabolites, one of which is 7,8-dihydroxycoumarin (daphnetin), which was identified in all the extracts tested. Daphnetin, as biologically active substance, is most responsible for the antimicrobial activity of the tested extracts, but its role can not exclude the influence of other secondary metabolites (phenols and flavonoids) present in the extracts, which may have a synergistic effect

Antioxidant activity of lichen Cetraria aculeata

The aim of the present study is to investigate the antioxidant properties of the lichen Cetraria aculeata. Antioxidant activity of the methanol and ethyl acetate extracts of lichen was tested by different methods including determination of total phenolics content, determination of total antioxidant capacity, DPPH free radical scavenging activity, inhibitory activity towards lipid peroxidation, ferrous ion chelating ability and hydroxyl radical scavenging activity. The extracts of the lichen C. aculeata showed significant antioxidant activity. The methanol extract showed higher values for total phenolics and total antioxidant capacity compared to the ethyl acetate extract, while the ethyl acetate extract demonstrated better results for DPPH radical scavenging, inhibitory activity towards lipid peroxidation, chelating ability and hydroxyl radical scavenging than the methanol extract. This is the first report of the antioxidant properties of Cetraria aculeata growing in Serbia. The results of antioxidant activity indicate the application of this lichen as source of natural antioxidants that could be used as a possible food supplement, in the pharmaceutical industry and in the treatment of various diseases

Antioxidant activity of lichen Cetraria aculeatas

The aim of the present study is to investigate the antioxidant properties of the lichen Cetraria aculeata. Antioxidant activity of the methanol and ethyl acetate extracts of lichen was tested by different methods including determination of total phenolics content, determination of total antioxidant capacity, DPPH free radical scavenging activity, inhibitory activity towards lipid peroxidation, ferrous ion chelating ability and hydroxyl radical scavenging activity. The extracts of the lichen C. aculeata showed significant antioxidant activity. The methanol extract showed higher values for total phenolics and total antioxidant capacity compared to the ethyl acetate extract, while the ethyl acetate extract demonstrated better results for DPPH radical scavenging, inhibitory activity towards lipid peroxidation, chelating ability and hydroxyl radical scavenging than the methanol extract. This is the first report of the antioxidant properties of Cetraria aculeata growing in Serbia. The results of antioxidant activity indicate the application of this lichen as source of natural antioxidants that could be used as a possible food supplement, in the pharmaceutical industry and in the treatment of various diseases

Uporedna analiza oksidativnih postupaka sinteze N-alkil, N,N-dialkil i N-cikloalkil-O-izobutil tionkarbamata

An optimized synthesis of N-alkyl, N,N-dialkyl- and N-cycloalkyl-O-isobutyl thioncarbamates by aminolysis of sodium isobutylxanthogenic acid (NaiBXAc) and primary, secondary and cycloalkyl amines was developed at laboratory scale and applied at semi-industrial level. Studies on dependence of N-n-propyl-O-isopropylthiocarbamate yield and purity with respect to reaction parameters: reaction time and molar ratio of n-propylamine and NaiBXAc, were performed. In such way, optimal reaction conditions for synthesis of N-alkyl, N,N-dialkyl- and N-cycloalkyl- O-isobutyl thioncarbamates, by aminolysis of NaiBXAc, were established. Also, comparative results of thioncarbamates synthesis starting from potassium isobutyl xanthate (KiBX) and corresponding amines in presence of different oxidants (hydrogen peroxide, sodium hypochlorite and new oxidative agent potassium peroxodisulfate) were evaluated. The synthesized compounds have been fully characterized by FTIR, 1H- and 13C-NMR and MS data, elemental analysis and purity have been determined by gas chromatographic method (GC). To our knowledge, ten synthesized thioncarbamates are here characterized for the first time. The synthesized compounds could be used as selective reagents for flotation of copper and zinc ores. The presented methods offer several benefits, namely, high product yields and purity, simple operation, mild reaction conditions without use of hazardous organic solvents, while some of them could be implemented on industrial scale production.U radu je prikazana optimizovana sinteza N-alkil-, N,N-dialkil- i N-cikloalkil-O-izobutiltionkarbamata reakcijom aminolize natrijum-izobutilksantogenacetata (NaiBXAc) i primarnih, sekundarnih i cikloalkilamina. Takođe, dat je pregled uporednih postupaka sinteze tionkarbamata polazeći od kalijum-izobutilksantata (KiBX) i odgovarajućih amina u prisustvu različitih oksidacionih sredstava: vodonik-peroksida, natrijum-hipohlorita i kalijum-peroksodisulfata kao novog oksidacionog sredstva. Struktura sintetisanih jedinjenja potvrđena je FTIR, 1H- i 13C-NMR i MS instrumentalnim metodama i elementarnom analizom, a čistoća je određena gasnom hromatografskom metodom. Deset sintetisanih tionkarbamata su prvi put okarakterisani i predstavljaju nova jedinjenja. Sintetisana jedinjenja mogu naći primenu kao selektivni reagensi za flotaciju rude bakra i cinka. Opisane postupke karakteriše visok stepen konverzije i čistoća proizvoda, blagi reakcioni uslovi bez upotrebe organskih rastvarača kao reakcionog medijuma, a neki su tehnološki primenljivi na industrijskom nivou proizvodnje