Dissertations / Theses on the topic 'Vaccinium Myrtillu'

Sun radiation consists of several spectrum regions, depending on the wavelengths, such as ultraviolet, visible and infrared portions. The ultraviolet rays (UV) are associated with both positive and negative effects on humans’ health. Ultraviolet light is used in the process of disinfection, it shows immunoregolatory activity and it stimulates vitamin D and serotonin production. It is well known that artificial sources of UVR are employed in the health field for sterilization procedures and for therapeutical purposes, such as treatment of dermatological pathologies like psoriasis, vitiligo and icterus neonatorum, as well as for aesthetic purposes (sunbed). In the last decade, besides these positive effects, UV side effects on skin have been demonstrated by different studies which correlated the intense and prolonged exposure to UV light with skin cancer development. Based on wavelength, UV spectrum can be divided into three segments: UVA (320-400 nm), UVB (280-320 nm) and UVC (100-280 nm). Radiation in the UVC range, considered to be the most cytotoxic and harmful, is largely absorbed by the stratospheric ozone layer, so UV regions mainly involved in photocancerogenesis development are considered to be ultraviolet radiation A (UVA) and ultraviolet radiation B (UVB). The amount and the UVA/UVB ratio that reach the Earth’s surface is influenced by a lot of environmental factors as latitude, seasons, weather/atmospheric conditions and the time of day. UVA is by far the most abundant solar UV radiation that reaches the Earth (about 90-95% of total UV radiation), penetrates the human epidermis down to the proliferative basal cells and even further to the derma and mediate biological events generating reactive oxygen species (ROS) in cells. ROS oxide other cellular constituents, particularly carbohydrates, lipids, proteins and DNA. Interactions between ROS and DNA induce formation of single strand breaks (SSBs), DNA-proteins crosslink and oxidized bases. The most abundant oxidized base is the guanine that produces the 8-oxoguanine, considered as promoter of carcinogenesis. UVB represents only 5% of total UV radiation that reaches the Earth. Besides it acts mainly at the epidermis basal layer of the skin, it is considered extremely damaging because of its high energy. UVB photons are directly absorbed by DNA and cause the formation of cyclobutane pyrimide dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), which in turn can lead to apoptotic events or carcinogenesis development. Also tryptophan and tyrosine are modified by UVB inducing the alteration of structure and function of proteins. Moreover, UVB is able to produce reactive oxygen species (ROS) and reactive nitric species (RNS). Cells have developed defense mechanisms and endogenous repair systems in order to reduce genotoxic damage UV-induced: complexes of antioxidant enzymes which take advantage of the presence of a metal to inactivate reactive oxygen species, and DNA repair systems like the base excision repair system (BER), used for oxidized bases, and the nucleotide excision repair (NER), used to repair photolesions like CPD and 6-4 PP. Recently, several molecules have been studied for their ability to give a contribution to these antioxidant defenses. For these reasons, there is a considerable interest in the concept of the use of phytochemicals and micronutrients present in the diet such as carotenoids, vitamin E and C, and polyphenols. This last group of compounds are well known for their several biological properties, such as their antioxidant activity, their ability to enhance the activity of DNA repair systems and also their skills in inflammatory and apoptotic events modulations. Several epidemiological studies have demonstrated their biological effects and their efficiency in the prevention and treatment of several chronic diseases like cardiovascular, respiratory, neurodegenerative and inflammatory diseases and cancer. So, in view of these reasons, the aim of my study was double: first, to confirm literature data regarding the different mechanisms used by UVA and UVB rays to cause damage in various models, then to investigate whether different plant-derivatives compounds, all belonging to the pholiphenols category, were able to prevent UVA- and UVB-induced damage. I reproduced the experiments on in vitro and ex vivo models. In vitro models consist of two human keratinocytes cell lines (HaCaT and NCTC 2544 cells), generally used for routine toxicological tests and photogenotoxic evaluations. Both cell lines are non tumorigenic, but showed a different degree of differentiation, higher in HaCaT cells compared to NCTC 2544. The ex vivo model consist of a three-dimensional model of organotypic human skin cultures useful to reproduce the physiological conditions. I tested the efficiency of three compounds: a water soluble extract of Bilberry only in the in vitro models (Vaccinium Myrtillus extract, rich in polyphenols and considered to be the plant with the highest content of anthocyanins), a Thymus Vulgaris leaf extract (Thyme specie) and the synthetic Thymol (its major component), used in comparable dosages, in both models. All experimental models were pretreated in serum free medium for 1 hour with each compound and then irradiated with UVA or UVB. Control samples were treated in the same way, but not UV exposed. In order to confirm mechanisms of UV damage and to compare results obtained in the experimental models, I performed several tests, each with a specific endpoint. I evaluated the intracellular redox status, quantifying the ROS formed and the lipid peroxidation (malondialdeide levels, MDA), immediately after the end of UVA and UVB exposure and only in the in vitro models; after, I performed some genotoxic tests (the alkaline comet test to detect single and double strand breaks in single cells, the immunostaining of the histone H2AX phosphorilated to detect only double strand breaks and the micronucleous test to detect alterations to mitotic apparatus or to chromosomes); finally I investigated effects of rays on apoptotic events, using the Annexin V test and evaluating the alteration of mitochondrial membrane potential UV-induced. After, using this same assays, I tested the protective effect of Vaccinium Myrtillus extract, thymol and Thymus Vulgaris L. extract. Vaccinium Myrtillus’ effect was evaluated only in the in vitro models, the protection of thymol and Thymus Vulgaris L. extract in both models (in vitro and ex vivo). Results obtained confirmed the different machanisms of action of UVA and UVB in all models. Particularly, confirming the different mechanisms of damage used by UVA and UVB rays. My results showed the more oxidant UVA effect and the more genotoxic and apoptotic UVB effect. Three natural compounds showed their different protective effect against UVA- and UVB- induced damage, in in vitro models. Moreover, thymol and Thymus Vulgaris L. extract reduced damage UV-related both in the NCTC 2544 and HaCaT cell lines but also in the ex vivo model. Vaccinium Myrtillus extract, thymol and Thymus Vulgaris L. extract reduced, to a different extent, UVA-caused damage, on account of their antioxidant properties. Compounds revealed their ability to reduce also UVB-induced damage. The efficiency against UVB damage was lower compared to the one against UVA rays. Probably the protection was related with the ability of pholyphenols, and because of my compounds, to repair DNA damage UVB-induced increasing the expression of genes involved in NER system (specifically involved in UVB-induced damage repair). Vaccinium Myrtillus, thymol and Thymus Vulgaris L. extract showed also their protection in the apoptotic assays, avoiding the increase of extremely damaged cells which could lead to mutagenesis development. Results obtained in my thesis activity should be considered as preliminary data useful to investigate, in detail, mechanisms used by the three natural compounds to prevent genotoxic damage UVA- and UVB-induced. Moreover, it should be very interesting to investigate the bioavailability and the permeability of tested compounds in order to use them as food supplements for a diet enriched in antioxidants (systemic effect) or in sunscreens (topical effect).

Plusieurs techniques de separation sont appliquees pour isoler les differentes classes de composes phenoliques a partir d'un jus fermente de myrtilles vaccinium myrtillus. L'etude des acides phenoliques des flavanols libres et condenses, des flavanols proprement dits et des anthocyanes faiblement et fortement condensees. Une tentative pour definir l'etat de la structure generale des anthocyanes condensees est effectuee d'apres les resultats experimentaux et les donnees bibliographiques. Parallelement, des methodes d'analyse rapides sont appliquees directement sur les jus, frais et fermentes en vue d'une estimation qualitative et quantitative des differentes formes d'anthocyanes presentes et d'une etude comparative de l'effet de certains facteurs

Mustikka (Vaccinium myrtillus L.) on Vaccinium-sukuun kuuluva kasvi, joka kasvaa Euroopan, Aasian ja Pohjois-Amerikan pohjoisosissa. Vaccinium-sukuun kuuluu mustikan lisäksi muun muassa karpalo ja kanadanmustikka. Mustikka sisältää paljon terveydelle hyödyllisiä hivenaineita, vitamiineja ja antioksidantteja, minkä vuoksi sitä voidaan pitää funktionaalisena elintarvikkeena. Iso osa mustikan terveyshyödyistä on liitetty fenoleihin kuuluviin antosyaaneihin, joilla on antioksidanttisten vaikutusten lisäksi muunlaisiakin vaikutusmekanismeja, kuten solusignalointiin, geeniekspressioon, DNA:n korjausmekanismeihin ja soluadheesioon liittyviä toimintoja. Antosyaaneilla on osoitettu olevan mm. soluja suojaavia, antimikrobisia, syöpää estäviä, hermostoa suojaavia sekä lihavuutta ehkäiseviä vaikutuksia. Tämän tutkielman tarkoituksena on koota yhteen tämänhetkistä tietämystä antosyaanin rakenteesta, toimintamekanismeista sekä terveysvaikutuksista.

Subject of research: different parts – roots, stems, leaves, berries – of bilberries (Vaccinium myrtillus L.), growing near the A1 Vilnius – Kaunas – Klaipėda highway (the 94th kilometre, right side of the road). Objective of research: to determine the amount of lead in different parts of bilberries. Methodology: for research the samples of bilberries were picked near the A1 Vilnius – Kaunas – Klaipėda highway at the 15 – 20 km distance from Kaunas, at 10, 15, 20, 25, 30, 35, 40 and 70 m distance from highway. For experiment – at 100 m distance from highway, bilberries were replanted in laboratory and were grown in the soils of different lead concentration four months. The soil was sprinkling with lead nitrate (Pb(NO3)2) solution of different concentration. The samples of plants were desiccate. The mineralization of different part the plants was carried out, as well as the analysis of soil was made (pH, ratio of hygroscope humidity, concentration of lead in soil). The amounts of lead in plants and soil were determined by the method of atomic absorption spectrometer. Research carried out in accordance with legally approved methodologies. For the statistical evaluation of differences the ANOVA and STATISTICA has been applied. Results of research: by carrying out the research, the amount of lead in different parts of the plants was determined; the dependence of lead amount in different parts of the plant upon the lead amount in solution was analysed; it was evaluated the... [to full text]

La myrtille et l’airelle rouge, deux arbrisseaux de la famille des Ericacées, sont consommées comme des aliments, boissons et suppléments alimentaires pour leur valeur nutritionnelle et leur richesse en polyphénols antioxydants. Dans les plantes, la qualité et la quantité de composés phénoliques sont influencées par les parties morphologiques de la plante à utiliser. En particulier, les composés phénoliques des végétaux exercent leur activité antioxydante dans la protection des lipides alimentaires et le compartiment gastrique a été proposé comme le site majeur pour le stress oxydatif lié au régime alimentaire. L’objectif général de cette thèse était d’étudier les variations saisonnières des composés phénoliques d’extraits de feuilles, branches et fruits de la myrtille et de l’airelle rouge ainsi que l’activité antioxydante de ces extraits. Pour cette étude, des extraits aqueux et hydroéthanoliques (fruits uniquement) des échantillons collectés en mai, juillet et septembre pendant les années 2013-2014 ont été obtenus par extraction assistée par microondes.Les analyses qualitatives et quantitatives par UPLC / MS des extraits de la myrtille ont montré la présence de dérivés de l’acide caféique et de l’acide p-coumarique et des glycosides de flavonols dans les feuilles tandis que des oligomères de flavanols étaient aussi présents dans les branches, et ce dans des quantités élevées. La thioacidolyse a révélé de faibles degrés de polymérisation (2-4) et l’(-)-épicatéchine comme unité principale des flavan-3-ols. Il existe une très bonne corrélation entre la Somme des Composés phénoliques par UPLC et la Teneur en Polyphenols Totaux ou l’activité antioxydante dans le test DPPH, excepté pour les feuilles du mois de mai. Ces dernières sont relativement riches en dérives de l’acide p-coumarique. Les effets de la saison apparaissent plus marqués pour les feuilles qui présentent une plus grande activité antioxydante et teneur en polyphénols en juillet et septembre. Ces paramètres sont optimaux en juillet pour les branches de myrtille. La période de de cueillette peut être définie en fonction des structures phénoliques désirées.Dans l’airelle rouge, la présence prédominante de monomères et oligomères de flavanols et de glycosides de quercétine a été identifiée dans toutes les parties morphologiques. Les proanthocyanidines contiennent la (+)-catéchine et la (-)-épicatéchine comme unités d'extension et terminale. De plus, la teneur en polyphénols totaux (méthode de Folin, UPLC) a montré une augmentation légère mais significative de mai à septembre pour les feuilles et les branches. Cette augmentation a été confirmée pour l'activité antioxydante dans le test DPPH pour les feuilles et les branches en 2014.L’activité antioxydante des extraits de myrtille et d’airelle rouge lors de l’inhibition de l’oxydation lipidique (accumulation de diènes conjugués) a été évaluée dans des conditions in vitro simulant la digestion. Tout d'abord, l'inhibition de l’oxydation lipidique a été conduite sur des émulsions huile de tournesol-dans-eau stabilisées par la sérum albumine bovine (BSA) ou des phospholipides d’œuf (PL), qui simulent l’état physique des lipides alimentaires lors de la digestion gastrique. L’oxydation a été initiée par la metmyoglobine, une forme de fer apportée par la viande rouge. Dans les deux modèles d’émulsions, les extraits aqueux des branches et des feuilles et l’extrait hydroethanolique de fruit de myrtille sont des inhibiteurs plus efficaces de l'oxydation lipidique durant la première phase de digestion (pH 5) que durant la seconde phase (pH 3). D’autre part, un extrait de feuilles de myrtille a été testé dans un modèle complet de digestion in vitro statique (étapes orale, gastrique et intestinale). L'oxydation lipidique, rapide lors de la l’étape gastrique (systèmes BSA et PL) et puis plus lente lors de l'étape intestinale (système PL), a été totalement inhibée par l'extrait de feuilles de myrtille
Bilberry and lingonberry, two shrubs of the Ericaceae family, are consumed as food, beverage and dietary supplements due to their nutritional value and richness in antioxidant polyphenols. In plants, the quality and quantity of phenolic compounds are influenced by the parts of the plant to be used. In particular, plant phenolic compounds provide antioxidant activity in the protection of dietary lipids from oxidation and the gastric compartment has been proposed as a major site for diet-related oxidative stress. The aim of this thesis is to simultaneously assess the seasonal variations of phenolic compounds in leaves, stems, and fruits of bilberry and lingonberry extracts, as well as their antioxidant activity. For this study, aqueous and hydroethanolic (only fruits) extracts of bilberry and lingonberry samples collected in May, July and September during the years 2013-2014 were obtained under microwave-assisted extraction.In bilberry extracts, qualitative and quantitative analyses by UPLC/MS showed the presence of caffeoyl derivatives, p-coumaroyl derivatives, and flavonol glycosides in leaves whereas in stems, flavanol oligomers were additionally identified in significant amounts. Thioacidolysis revealed low degrees of polymerization (2-4) and (-)-epicatechin as the main flavan-3-ol unit. The sum of the phenolic compounds by UPLC was highly correlated with the Total Polyphenol Content and the antioxidant activity in the DPPH test for all the extracts except those of May leaves. The latter were relatively richer in p-coumaric acid derivatives. Seasonal effects were more marked for leaves which exhibited higher antioxidant activities and phenolic contents in July and September when these parameters were maximum in July for bilberry stems. The harvest period can be refined based on the desired phenolic structures. For lingonberry, the predominant presence of monomers and oligomers of flavanols and quercetin glycosides was found in all the morphological parts. Proanthocyanidins contain (+)-catechin and (-)-epicatechin as both extension and terminal units. The sum of the phenolic compounds by UPLC was less correlated with the Total Polyphenol Content and the antioxidant activity in the DPPH test than in bilberry. Furthermore, the total phenolic content (Folin method, UPLC) showed a slight but significant increase from May to September for both leaves and stems. This increase was confirmed for the antioxidant activity by the DPPH test for both leaves and stems in 2014.The antioxidant activity of bilberry and lingonberry extracts against lipid oxidation (formation of lipid-derived conjugated dienes) was evaluated under in vitro simulated digestion conditions. Firstly, the inhibition of lipid oxidation was performed using sunflower oil-in-water emulsions stabilized by bovine serum albumin (BSA) or egg yolk phospholipids (PL), both emulsifiers mimicking dietary components. Oxidation was initiated by metmyoglobin, a form of dietary iron from red meat. In both emulsion models, aqueous extracts from stems and leaves and the hydroethanolic fruit extract of bilberry proved to be more efficient inhibitors of lipid oxidation in the early phase of digestion (pH 5) than during the second phase (pH 3). Secondly, a bilberry leaf extract was tested in the inhibition of lipid oxidation in a complete static in vitro digestion model (oral, gastric and intestinal phases). The fast lipid oxidation in the gastric step (BSA and PL systems) and the slower lipid oxidation in the intestinal step (PL system) were totally inhibited by the bilberry leaf extract

Abstract Flavonoids are a class of secondary metabolites in plants that are involved in many important functions. Various flavonoid compounds have also been reported to be beneficial for human health. Bilberry (Vaccinium myrtillus L.) is the characteristic field layer species in boreal forests and the fruits of bilberry are rich in anthocyanin pigments, a subclass of flavonoids. In the present work, flavonoid biosynthesis was examined in different tissues of bilberry. The focus was on the developing fruits of wild type and natural color mutants of bilberry, and on effect of solar radiation on flavonoid biosynthesis in bilberry leaves. For the isolation of RNA for gene expression analysis, a method was optimized for different tissues of bilberry. The cDNA fragments of five genes from the flavonoid pathway, coding phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavanol 4-reductase and anthocyanidin synthase, were isolated from bilberry using polymerase chain reaction technique, sequenced, and labelled with dioxigenin-dUTP label. These homologous, bilberry-specific probes were used for determining the expression of the flavonoid pathway genes in bilberry fruits, flowers and leaves with a modified non-radioactive method developed in the course of the study. The anthocyanins, catechins, proanthocyanidins, flavonols and hydroxycinnamic acids in fruits, leaves and different fractions of bilberry were identified and quantified with high-performance liquid chromatography combined with a diode array detector and mass spectrometer. The results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. A correlation between flavonol and anthocyanin biosynthesis was detected in bilberry and also in previous literature collected from flavonol and anthocyanin analyses from other fruits. Accordingly, models for the connection between flavonol and anthocyanin synthesis in fruit species were suggested. Activation of the expression of flavonoid pathway genes and accumulation of flavonoids and hydroxycinnamic acids was detected in leaves growing under direct solar radiation, compared to the shadow leaves of the same plants. Based on the results, it is suggested that cyanidin of anthocyanins and flavonol quercetin play a predominant role in the defence against high solar radiation in Vaccinium leaves. The results give new information about the biosynthesis of flavonoids in bilberry at the gene level, in addition to the information of the composition and content of flavonoids during fruit development and in different parts of the bilberry plant. Also, new information was obtained of the roles of flavonoids in protecting plants from excess solar radiation.

In Scotland, bilberry is well known as a member of the dwarf shrub community on moorlands, which provide valuable habitats for wildlife. Recently it has been envisaged that bilberry populations could play an important role in improving the dwarf shrub vegetation in degraded moorland areas. This study was therefore undertaken to investigate the above-ground growth dynamics of bilberry with emphasis on responses to grazing by sheep and seedling establishment. The leaf production of bilberry was increased by higher nitrogen availability. High nitrogen supply also led to greater activation of dormant buds. Abortion of buds was less when nitrogen was continuously supplied. The reproductive capacity of bilberry was shown to be decreased by herbage removal. In contrast flowering was increased following supply of high nitrogen in glasshouse conditions. Almost all the flowers developed into fruits in the field, but a very low production of fruits was reported from bilberry plants that were grown in the glasshouse. Results of a series of laboratory experiments on seed production, germination, seed storage and evaluation of a moorland seed bank confirmed that the scanty seedling recruitment of bilberry was due to the limited production of 'germinable' seeds in berries and risks imposed by the environment after dispersal particularly when seeds are in the litter layer and/or soil. This could suggest that limited picking of bilberry fruits would not have a detrimental effect on establishment of bilberry populations in moorlands. Therefore, areas could be identified which were suitable for public access and berry exploitation in Scotland. The berry production was estimated approximately to be about 3220-9660 tones per year in this zone.

The influence of pollution such as sulphur dioxides and heavy metals on forest ecosystems is commonly known and widely described in literature. However, there is not enough information about the long term influence of pollution on undergrowth which increases the natural resistance of forest ecosystems and protects forest soil against the destructive pollution. Vaccinium myrtillus L. and Vaccinium vitis-idaea L. examinations are the basis for the full understanding of long term effects of pollution on the whole forest ecosystem (they constitute supplementation of knowledge about the reactions of forest ecosystems to air and soil pollution). They will allow to determine the reasons of taking some remedial actions in case of highly polluted environment and they are the basis of the integrated environmental protection systems. Seven sites located on former coniferous forest have been included in the research. Three of them are in the area o f industrial influence of Katowice Steelworks in Dąbrowa Górnicza and three consecutive are located within the area Miasteczko Śląskie Zinc Smelter. Kokotek was the reference site located within Lubliniec forestry. The purpose of the paper was to prove the thesis that phenotype diversification at Vaccinium myrtillus L. and Vaccinium vitis-idaea L. population exposed at industrial pollution takes place. The following have been included in the detailed purposes: • comparison of the degree of load with heavy metals and sulphur in soil environment of different habitats within the zone of industrial emission; • determination of changes in the total concentration of heavy metals and sulphur of soil over the years and determination of differences in the concentration of heavy metals and sulphur in different depths in upper soil layers on the areas of various degradation degree; • estimation of differences in the load of different Vaccinium myrtillus L. and Vaccinium vitis-idaea L. populations with heavy metals and sulphur; • comparison of the content of heavy metals in different Vaccinium myrtillus L. and Vaccinium vitis-idaea L. organs from the areas within the industrial emission zone and comparison of their biology under anthropopression. The analysis has proved that on the areas affected by long term activities of heavy industry, despite the application of systems limiting industrial emissions, the level of soil pollution with heavy metals remains at high level. The highest heavy metals cumulation has been recorded on the upper layer of soil (0-5cm) which indicates anthropogenic origin of this pollution. It is reflected in the decrease of soil enzymatic activity on this area which is connected with the presence of soil microorganisms. Despite the increase of pH in soil caused mainly by significant lowering of acidic deposition, the concentration values of heavy metals in plants still exceed the toxic level. The examined populations reacted slightly different to stress connected with soil toxicity. In the direct neighborhood of emissions the difference in the size of leaves has been shown. In Vaccinium vitisidaea L. population the reduction of leaves has been recorded and in Vaccinium myrtillus L. the growth at the simultaneous increase of the amount of chlorophyll and carotenoids at both examined species has been recorded. Both examined species located in the zone of the highest load of heavy metals had the highest mass of overground plant parts. In case of both examined bilberries populations, which grew in the neighborhood of Miasteczko Śląskie Zinc Smelter, on the areas of high concentration of heavy metals in soil, they showed significantly higher aboveground shoot mass than in the remaining ones. On the area close to Katowice Steelworks, where concentrations of heavy metals was lower and the examined populations were exposed at their influence for a shorter period of time, this effect has not appeared. It seems to confirm the thesis that the gigantism effect at the examined species is caused by the long term exposure at high concentrations of heavy metals in soil. The influence of two plants on the layout of the examined pollution also has been noticed. Vaccinium myrtillus L. and Vaccinium vitis-idaea L. populations located in the zone of their direct influence showed accidental layout. On the remaining examined areas both species were characterized with cluster distribution. The conducted analysis show the diversifying influence of industrial pollution on the spatial structure of the examined populations. The highest biomass density was noticed at Vaccinium myrtillus L. and Vaccinium vitis-idaea L. populations from the reference site.

Os efeitos deletérios causados pela radiação ultravioleta (UV) e o aumento significativo no diagnóstico de câncer de pele, confirmam a necessidade de um progresso significativo na pesquisa de produtos fotoprotetores eficazes e seguros, para proteção eficaz da pele. As formulações atuais associam filtros UV, orgânicos e inorgânicos, com eficácia limitada e que podem acarretar reações cutâneas adversas. O extrato de Vaccinium myrtillus L. (EVM) apresenta em sua composição compostos fenólicos, como as antocianinas, que possuem atividade antioxidante. O objetivo deste estudo foi avaliar formulações fotoprotetoras contendo extrato de Vaccinium myrtillus L. com amplo espectro de ação e concentrações reduzidas de filtros UV. As emulsões contendo ou não EVM e filtros orgânicos (octocrileno e metileno bis-benzotriazolil tetrametilbutilfenol) e inorgânico (dióxido de titânio) foram avaliadas e consideradas estáveis perante parâmetros pré-definidos. O perfil de segurança foi determinado pelo método in vitro Hen´s Egg Test - Chorioallantoic Membrane (HET-CAM) e o potencial antirradicalar do EVM foi confirmado pelo teste de DPPH• A formulação fotoprotetora acrescida de 5,0% de EVM foi avaliada quanto à eficácia fotoprotetora in vivo e in vitro e não demonstrou aumento significativo no FPS quando comparada à formulação que continha somente os filtros solares e, no ensaio referente à hidratação da pele, não houve alteração nos dados, segundo teste de eficácia clínica. Os resultados obtidos sugeriram que mesmo não apresentando aumento no valor de FPS in vivo, a formulação fotoprotetora contendo o extrato, poderia atuar como agente antioxidante evitando os danos provocados pela radiação UV.
The deleterious effects caused by ultraviolet radiation (UV) and the significant increase in the diagnosis of skin cancer confirm the need for significant progress in researching effective and safe sunscreen products for effective skin protection. Current formulations associate UV, organic and inorganic filters with limited efficacy that can lead to adverse skin reactions. Vaccinium myrtillus extract (EVM) contains in its composition polyphenols compounds, such as anthocyanins, which have been antioxidant activity. The objective of this study was to evaluate sunscreens containing EVM with broad action spectrum and reduced concentrations of UV filters. Emulsions containing or not EVM and organic (octocrylene and methylene bis-benzotriazolyl tetramethylbutylphenol) and inorganic (titanium dioxide) filters were evaluated and considered stable under pre-defined parameters. The safety of the emulsion was assessed was determined by the in vitro Hen´s Egg Test - Chorioallantoic Membrane method (HET-CAM) and the antiradical potential of EVM was confirmed by the DPPH• test. The sunscreen formulation containing EVM 5.0% was photoprotective efficacy evaluated in vivo and in vitro and did not show a significant increase in SPF when compared the formulation only with UV filters and no improves skin hydration, according to clinical efficacy assay. The results of the study suggest that even without increase SPF in vivo, the sunscreen formulation containing the extract may show antioxidant activity avoiding damage caused by UV.

In Norway today no commercial exploitation of the wild growing Norwegian bilberry (Vaccinium myrtillus) exists. The market in other regions of Europe, North- and South-America is based on the utilization of cultivated Vaccinium species. V.myrtillus shows generally a higher content of biochemical compounds with health-beneficial properties. As a consequence of an increasing demand for healthier food, the Nordic Bilberry project started in 2008 with the major goal to find superior clones adapted for different regions with effective production of phytochemicals. In addition, a 4-years Norwegian Bilberry project aiming at cultivation and yield potential aspects, was launched the same year (NFR project no. 184797). The presented master projected was affiliated to this project focusing on phytochemical and genetic diversity in Bymarka. It is known that life history traits of a plant species influence the clonal diversity and structure within populations. In this thesis different phytochemical methods were used for detection of total phenolics, anthocyanins and antioxidants in berry and plant material from 4 different areas in the geographic restricted area Bymarka, were a total of 80 individuals were collected. Average values detected for total phenols were 490 mg/100 g, 155 mg/ 100 g for anthocyanins and 4 mmol/ 100 g for antioxidants. The results showed little variation among clones in the restricted area. 16 primer pairs for 16 microsatellite loci were tested but only 4 (NA741, NA961, CA421 and CA483) turned out to be of good quality. These microsatellite loci were used to estimate genetic variability within and between populations. Little genetic variation was detected between the different plots, and populations had similar levels of within-population genetic variation. The highest diversity both genetic and phytochemically was found in plot D with berries without any wax layer. This plot was also more genetically different from the other populations than the berries in the same location/area/plot with a wax layer, though no significant differences was found in FST, Heterozygosity observed, Heterozygosity expected or allelic richness (P<0.05). The result from this thesis could be used further for improvement of breeding strategies and selection of cultivars with high phenolic contents for production of quality food.

This diploma thesis deals with the influence of different storage methods on the stability of anthocyanins in the fruits of blueberries. The theoretical part describes blueberry (Vaccinium myrtillus L.), anthocyanin pigments and their stability based on the studied literature. Moreover, an overview of the most common methods for their determination in plant materials is given. The methodical part depicts the methods of determining the content and the extent of the degradation of anthocyanins in blueberries using the different ways of storage. Anthocyanin dyes contained in the blueberries were subjected to acid hydrolysis and their content was expressed in the form of free aglycones (anthocyanidins). During the initial analysis, the measurement of the content of anthocyanidins in the fresh blueberries was done. Furthermore, a sample preparation was carried out applying the different storage methods. Three storage variants were tested, dried samples at 80 ° C, lyophilized and frozen samples. During the eight-month storage, five further measuring of the degradation rate of anthocyanins in time was performed. All samples were analyzed in triplicates. A mass spectrometer 3200 QTRAP with triple quadrupole and ESI ionization, coupled with a liquid chromatograph, type UHPLC was used for the analysis. Afterward, the values were averaged and presented using graphs and tables. The results of the thesis pointed out that the least intrusive variant of preserving the sample was lyophilisation, especially than for the first two months of storage. The amount of the anthocyanins did not changed during this period. Moreover, also the freezing variant was a suitable method for preserving the sample. Freezing was assessed as the favorable method for preserving the sample for longer periods since the value has decreased in the relation to time more gradually and during the final collection have shown higher values than the variant of the lyophilized blueberries. The variant of dried blueberries was the least stable compared to the previous variants. The rapid decline of anthocyanins occurred already during drying, depending on the time the values continued to decline In the variant of drying, petunidin was evaluated as the most stable anthocyanidin, while kyanidin and delphinidin were determined as the least stable. Delphinidin was determined to be the most stable anthocyanin in the variant of both freezing and lyophilization whilst the least stable the anthocyanins were petunidin in the variant of freezing and cyanidin in the variant of lyophilization.

Abstract The aim was to study the effects of drought and indirect drought-related stresses on anthocyanin content in bilberry (Vaccinium myrtillus L.). Anthocyanin content was also studied in relation to developmental stage (juvenile vs. mature leaves, previous vs. current-year stems). It was hypothesised that drought-stressed plants accumulate anthocyanins, but their direct role in osmotic regulation was questioned. Direct drought was created by preventing water supply. Freezing-induced dehydration was accomplished by removal of snow. Effects of summertime chilling (+2 °C during active growth, +5/0 °C during frost hardening) on water status were tested. The trace metal Nickel (Ni) was applied to the soil, as Ni may interfere with plant water relations at the rhizospheric level. Salt (Na+) was added to the soil to observe salt-induced disturbance in osmotic balance and ion homeostasis. Tissue water content (TWC) decreased and anthocyanin level increased under direct drought, especially in the mature leaves. The freezing-stressed plants contained the same TWC and anthocyanin levels in mid-winter compared to plants that overwintered below snow. The freezing-stressed plants had lower TWC and a similar anthocyanin level in early spring, and lower TWC and anthocyanin level in late spring than plants that overwintered below snow. In the summer and autumn following snow removal, current-year stems of freezing-stressed plants had the same TWC, but higher levels of anthocyanins than current-year stems of plants that overwintered below snow. New growth was thus affected by the freezing stress experienced by previous-year stems. Chilling had no effect on anthocyanins. Although TWC decreased and anthocyanin level increased from active growth to the beginning of frost hardening, no increase was observed during frost hardening. Ni did not cause drought stress in the aboveground shoots, but anthocyanin level decreased in the aboveground shoots along with Ni accumulation in the belowground stems. Na+ increased TWC in the belowground stems, but decreased TWC and anthocyanin level in the aboveground stems. It is proposed that anthocyanins do not have direct role in osmotic regulation, or in the development of freezing tolerance. It is suggested that the increase in anthocyanin level under direct drought stress is mainly due to the photoprotection of chlorophylls by anthocyanins. This is supported by two facts: (1) At increased anthocyanin level in the juvenile leaves, chlorophyll a was stabilized despite continuing drought stress, and (2) after the initial peak in the mature leaves, the accumulation of anthocyanins ceased although the drought became more severe. As chlorophyll a decreased in the mature leaves due to senescence, there was less demand for such high levels of anthocyanins.

Plusieurs etudes ont montre que les especes sempervirentes dominent les communautes vegetales habitant les sols a faible valeur nutritive, typiquement pauvres en azote. De nombreuses etudes ont tente d'identifier les mecanismes physiologiques associes au maintien des feuilles persistantes et facilitant l'adaptation a une croissance en condition d'azote limitante. Ces etudes sont passees en revue dans la premiere partie de cette these, soulignant le role primordial que pourrait jouer le cycle interne de l'azote dans cette adaptation. La deuxieme partie de cette these presente trois etudes experimentales comparant certains aspects du cycle interne de l'azote de deux especes natives de milieux pauvres en azote : vaccinium myrtillus l. (feuilles caduques) et v. Vitis-idaea l. (feuilles persistantes). L'isotope stable 1 5n a ete utilise pour differencier l'azote provenant des reserves de celui provenant directement de l'absorption racinaire. Les resultats de ces etudes montrent que v. Myrtillus etait plus capable de tirer avantage d'une forte disponibilite en azote que v. Vitis-idaea. A l'oppose, la croissance de v. Vitis-idaea etait plus forte que celle de v. Myrtillus lorsque la disponibilite en azote etait faible. Cette difference entre les deux especes n'etait pas attribuee a une difference dans l'utilisation des reserves azotees mais a une differente dynamique de remobilisation de ces reserves vers les nouvelles pousses. Chez v. Myrtillus, la remobilisation etait rapide et courte, tandis que chez v. Vitis-idaea elle etait lente et longue. D'autre part, les contraintes morphogenetiques imposees sur la reponse de croissance a l'azote etaient plus severes chez v. Vitis-idaea que chez v. Myrtillus. Les resultats suggerent aussi que la part des reserves, formees a partir d'azote absorbe pendant la saison precedente, differait entre les deux especes et dependait de la relative disponibilite en azote pendant la saison precedente et anterieurement. Finalement, la force de puits exercee par les nouvelles pousses sur les flux d'azote provenant de la remobilisation des reserves a ete alteree par debourgeonnement partiel. Les resultats de cette manipulation suggerent que les flux d'azote sont dependants de la taille des reserves, plutot que de la force de puits, pourvu qu'il y ait un puits.

Bilberry (Vaccinium myrtillus) is native to Europe and North America and constitutes an important nutritional resource for both humans and animals. Over the years, a series of chemical analyses have revealed several health-beneficial compounds in bilberry, and with the current demand of the berries mainly covered by Sweden and Eastern Europe, there has become an increasing desire to cultivate bilberry in Norway. In order for such cultivation to be successful an increased knowledge about bilberry is seen as essential and thus several studies have investigated the chemical composition of the berry. However, the underlying genetic diversity and the variation between populations in biochemical compounds remain to be thoroughly investigated. Therefore, the aim of present study was to investigate the differences in biochemical composition between populations of bilberry distributed on a latitudinal gradient, and estimate the level of genetic variation within and among the populations. This in order to examine whether biochemical composition was reflected by observed levels of genetic variation.Bilberries from four Norwegian populations at three regions differing in latitude were analysed for content of total phenolics (TPH), total anthocyanins (ACY) and antioxidant activity (FRAP). Furthermore, metabolic profiling was performed by gas chromatography-mass spectrometry (GC-MS) to reveal biochemical differences between the populations in content of sugars, acids and some simple phenolics. Multivariate statistics were performed and revealed a clustering of samples from the two locations in Mid-Norway, and a clustering of the northern with the southern population. In addition, there were found significant differences in some of the compounds between the populations.Genetic analyses using four microsatellites were carried out to examine whether metabolic differences between populations were reflected by genetic differentiation. Despite the significant differences between populations in the concentrations of some metabolites no significant genetic differentiation was found. Thus, it seems that the variation in biochemical compounds discovered among populations could be environmentally induced differences on a similar genetic background. However, due to the limited number of working microsatellites and the fact that these molecular markers are neutral, there is still a possibility that the genetic differences causing compound concentrations to differ could be so minor as to remain undetected. Hence, further studies utilizing more microsatellite markers or new state-of-the-art molecular techniques are needed to determine whether this result holds and is valid also for genetic variation in coding parts of the genome.

Dans une première partie, nous nous sommes penchés sur la caractérisation des principaux constituants du fruit de bluet (Vaccinium cortymbosum) afin d'établir la carte d'identité biochimique des acides carboxyliques, sucres simples, protéines, pectines, constituants d'arome, composés phénoliques (anthocyanines, phénols simples, esters et hétérosides phénoliques), cations ainsi que des activités enzymatiques: invertase, sucrose synthase, sucrose phosphate synthase. Ces résultats devraient aider à une meilleure maitrise des transformations technologiques des fruits. La manifestation d'une composante brune, au cours de la conservation de jus de bluet, nous a amenés dans une deuxième partie, à approfondir le processus de brunissement enzymatique au travers de deux activités polyphénol oxydasiques: péroxydases (POD) et polyphénol oxydases (PPO). L’activité péroxydasique des POD dépend étroitement d'un processus générateur de peroxyde d'hydrogène. Il en ressort que la contribution des POD au brunissement enzymatique des fruits est assujettie aux réactions lentes d'autooxydation de divers substrats, avec génération de peroxyde d'hydrogène. Le broyage des fruits frais conduit à un brunissement intense imputable à la fois à l'acide chlorogénique (AC), aux pigments anthocyaniques, à l'oxygène et à l'activité PPO qui a été partiellement caractérisée. Afin de mieux cerner les mécanismes réactionnels nous avons eu recours à des systèmes modèles parfaitement définis. La cafeyl o-quinone nous a permis d'une part de déterminer la stœchiométrie des réactions quinone/pigments anthocyaniques et d'autre part de faciliter la purification des produits de la réaction en vue de déterminer leurs structures. Les résultats obtenus contribuent à mieux comprendre les mécanismes du brunissement et le rôle des différentes structures mises en jeu. Il en ressort que les PPO exercent une activité déterminante pour la transformation des composés polyphénoliques des fruits tandis que les POD ne semblent manifester qu'une activité limitée

In the work described in this thesis I studied how increases in nitrogen (N) inputs may affect plant community structure in boreal forest understorey vegetation. These phenomena were investigated in N fertilization experiments and along a national N deposition gradient. After five years of N additions, large changes in understorey vegetation composition were observed in the fertilization study. In plots that received 50 kg N ha'1 year"1 (N2), the abundance of the dominant species, Vaccinium myrtillus, decreased on average by 32 %. No decrease was observed in control plots during the same period. In contrast, the grass Deschampsia flexuosa responded positively to increased N input, being on average more than five times as abundant in the N2 treatments as in controls. Also an increase was seen in the incidence of disease caused by the parasitic fungus Valdensia heterodoxa on leaves of V. myrtillus following N additions. The parasite was on average nearly twice as abundant in N2 plots than in control plots. This could be explained by increased N concentrations in host plant tissue. Disease incidence also increased following experimental additions of glutamine to leaf surfaces of V. myrtillus, suggesting a causal connection between plant N concentration and performance of the fungus. The parasite also played a key role in the observed changes in understorey species composition. D. flexuosa was more abundant in patches in which V. myrtillus was severely affected by V heterodoxa. This suggests that V heterodoxa mediates the increased abundance of D. flexuosa following increased N additions. The fungus mediates changes in the composition of understorey vegetation mainly by increasing light availability via premature leaf loss of V. myrtillus. The incidence of disease due to the parasite was on average higher in large than in smaller N-treated plots, indicating that the response to N fertilization is spatially scale dependent. This shows that using small plot sizes in experiments that simulate changed environmental conditions may be problematic, as important interactions may be underestimated. Comparison of the occurrence of understorey species between regions with different rates of N deposition revealed that the occurrence of the two dwarf shrubs V. myrtillus and V. vitis- idaea was lower in regions with high N deposition compared to regions with low deposition. The opposite pattern was found for V heterodoxa. This is consistent with expectations from N fertilization experiments. For D. flexuosa no differences in occurrence were found between the different regions investigated. The effects on vegetation and mycorrhizal fungi observed following N additions were also found to be long lasting. Nine years after termination of the fertilization, no signs of recovery were detected, and nearly 50 years after termination characteristic signs of N fertilization were found among bryophytes and mycorrhizal fungi. This suggests that the time needed for re-establishment of the original biota following N-induced changes may be substantial.

Diss. (sammanfattning) Umeå : Umeå universitet, 2002, härtill 5 uppsatser

digitalisering@umu

Die Ergebnisse verschiedener in vitro Untersuchungen und Tierstudien deuten darauf hin, dass Anthocyane zur Prävention und Therapie von intestinalen Erkrankungen wie akutem Durchfall oder chronisch entzündlichen Darm¬erkrankungen (CED) sowie Darmkrebs geeignete Naturstoffe sind. Mit bis zu 780 mg/100 g Frischgewicht weisen Heidelbeeren (Vaccinium myrtillus L.) besonders hohe Anthocyan¬¬gehalte auf. In getrockneter Form sind diese Beeren ein bewährtes Therapeutikum in der Volksheilkunde und werden als pharmazeutische Droge Myrtilli fructus zur Unterstützung der Therapie unspezifischer akuter Durchfall¬erkrankungen eingesetzt. Diese traditionellen Anwendungen hat man jüngst in einer Studie am Modell der experimentellen murinen DSS-Colitis aufgegriffen, in welcher ein therapeutischer Effekt von getrockneten Heidelbeeren (gHB) nachwiesen wurde. Ob die Anthocyane oder andere Inhaltsstoffe verantwortlich für die beobachteten Wirkungen sind, ist noch unbekannt. Ein erstes Ziel der vorliegenden Arbeit war es deshalb, gHB zu charakterisieren, indem potentielle Wirkkomponenten anhand analytischer Inhalts-stoffbestimmung identifiziert und bei der Trocknung ablaufende Prozesse untersucht wurden: I. Nach extraktiver Probenaufarbeitung wurden Anthocyanprofil und -gehalt von gHB-Handelsware mittels HPLC-ESI-MS/MS und HPLC-UV/Vis bestimmt. Dabei zeigte sich, dass sich das Anthocyanprofil der untersuchten gHB, die in oben genanntem DSS-Colitis-Modell verwendet worden waren, von dem frischer Wildheidelbeeren (V. myrtillus L.) unterschied. Die gHB wiesen zusätzlich eine Delphinidin-hexose-pentose sowie verschiedene Anthocyanpentosen auf, wie sie für die Heidelbeerart V. arctostaphylos L. charakteristisch sind. Infolgedessen ist davon auszugehen, dass V. arcto-staphylos L.-Beeren zur Herstellung der gHB verwendet wurden. In den untersuchten gHB wurde ein Anthocyangehalt von 384 ± 39 mg/100g TM bestimmt. Im Vergleich zu frischen V. myrtillus L.- sowie zu V. acto¬staphylos L.-Früchten wiesen die gHB damit einen deutlich geringeren Anthocyan-gehalt auf. II. Als momomere Nicht-Anthocyan-Polyphenole wurden verschiedene phenolische Säuren, Quercetinglycoside sowie das Aglycon Quercetin mittels HPLC-ESI-MS/MS bzw. HPLC-DAD identifiziert und quantifiziert. Chlorogensäure stellte dabei mit 147 ± 5 mg/100 g TM die Haupt¬kompo¬nen-te dar. Der Gesamtgehalt an monomeren Nicht-Anthocyan-Poly¬phenolen von 288 ± 8 mg/100 g TM war mit dem der Anthocyane vergleich¬bar. III. Kondensierte Gerbstoffe (Procyanidine) in gHB wurden mittels Thiolyse erfasst. Mit 2,23 ± 0,05 g/100 g TM war ihr Anteil dreimal höher als die Summe der monomeren Polyphenole, Anthocyane, phenolische Säuren und Flavonole. IV. Mit 44 ± 2 g/100 g TM waren Ballaststoffe die dominierende Nährstoff¬gruppe in gHB. Zur Untersuchung der Zusammensetzung dieser Fraktion wurden nach Hydrolyse Neutralzucker als Alditolacetate mittels HRGC-MS bzw. HRGC-FID analysiert sowie Uronsäuren photo¬metrisch bestimmt. Derart identifizierte Hauptbausteine der Zellwandpolysaccharide waren Glucose, Xylose sowie Uronsäure. Als Rückstand nach Hydrolyse wurde gravi¬-metrisch das sog. Klason-Lignin erfasst. Zellwandpoly¬saccharide und Klason-Lignin waren mit jeweils ca. 36 % in der Ballaststofffraktion enthalten und stellten deren Haupt¬komponenten dar. V. Als Indikator für die thermische Belastung bei Trocknung der gHB wurde 5 Hydroxymethylfurfural mit HPLC-MS/MS und HPLC-DAD nachgewiesen und quantifiziert. Der ermittelte Gehalt von 7,9 ± 0,2 mg/100 g TM lag in einem für getrocknete Früchte üblichen Bereich. VI. Um den Einfluss der Trocknung auf den Polyphenolgehalt direkt zu verfolgen, wurden frische Kulturheidelbeeren (V. corymbosum L.) bei 30°C, 50°C und 70°C im Umlufttrocken¬schrank bis zur Gewichtskonstanz ge-trocknet. Vor und nach Trocknung wurden Polyphenole mittels HPLC-MS/MS und HPLC-DAD bzw. HPLC-UV/Vis untersucht. Trocknung bei 30°C führte zu einer leichten Zunahme im Anthocyangehalt, wohingegen der Temperaturanstieg auf 50°C sowie 70°C mit einem deutlichen Anthocyan-abbau verbunden war. Phenolische Säuren und Flavonole erwiesen sich als thermostabiler; ein geringer Abbau wurde nur bei Chlorogensäure beobachtet. VII. Die Stabilität der Anthocyanine Cyanidin-3-galactosid und Cyanidin-3-glucosid wurde in einem in vitro Modell untersucht, das die Bedingungen in der Pflanzenzelle während der Trocknung von Früchten simulierte. Der dabei beobachtete Anthocyanabbau wies eine Reaktionskinetik 1. Ordnung auf. Die Reaktionsgeschwindigkeit war stark von der Temperatur abhängig, der Zuckerrest hatte dagegen keinen Einfluss. Als Abbauprodukt wurde Protocatechusäure mittels HPLC-DAD-MS/MS identifiziert. Neben der Menge der mit der Nahrung aufgenommener Anthocyane ist deren Verfügbarkeit am Wirkort die Grundlage für potentielle Effekte. Während der Passage durch den Gastrointestinaltrakt (GIT) unterliegen Anthocyane bekanntlich einem chemischen und mikrobiellen Abbau, wodurch die effektive Wirkkonzentration stark vermindert wird. Colon-Targeting, bei dem Wirkstoffe durch Verkapselung vor einem chemischen und enzymatischen Abbau im oberen GIT geschützt werden, stellt eine Möglichkeit dar, die Verfügbarkeit von Anthocyanen für direkte Effekte im Dickdarm zu erhöhen. Die Zielsetzung im zweiten Teil der Arbeit beinhaltete daher die Herstellung und Charakterisierung von im Lebens¬mittelbereich zugelassenen Formulierungen für das Colon-Targeting von Anthocyanen. Für diese Studien dienten kommerziell aus Wildheidelbeeren V. myrtillus L. gewonnene Extrakte (Anthocyangehalte von 65 bis 84 g/100g) als Anthocyanquelle. I. Zur Charakterisierung der Freisetzungseigenschaften der hergestellten Colon-Targeting-Formulierungen wurde ein in vitro und ex vivo Modell entwickelt und angewendet, das durch aufeinanderfolgende Inkubation der Materialien in Magen¬saft¬simulanz (3 h bei pH 2) und Ileostomie- (4 h bei pH 6,3) sowie Colo-stomieflüssigkeit (15 h bei pH 6,2) die Passage des humanen GIT simulierte. Freigesetzte Anthocyangehalte wurden mittels HPLC-DAD erfasst. II. Im Labormaßstab wurde der Heidelbeerextrakt mittels Eintropf¬technik nach dem Prinzip der ionotropen Gelierung mit Calciumionen in eine Matrix aus amidiertem Pektin verkapselt. Durch anschließendes hydrophobes Coating mit Schellack wurde die vorzeitige Freisetzung der Pigmente aus den Anthocyan-Pektin-Kugeln (APK) P4BRT im Magensaft¬simulanz deutlich ver¬ringert, sodass mit Schellack gecoateten APK P4BSch im genannten Modell-System ein Anthocyantransport in den Dickdarm erreicht wurde. III. Aufgrund seiner Säureunlöslichkeit wurde auch die Eignung von Kollagen aus dem Meeresschwamm Chondrosia reniformis Nardo als Verkapselungsmatrix unter¬sucht. Es gelang, Heidelbeerextrakt in dieses Material zu verkapseln. Trotz partieller Magensaftresistenz waren die Formulierungen jedoch aufgrund von Degradation im simulierten Dünndarm nicht zum Colon-Targeting geeignet. IV. Zur Bereitstellung von mit Schellack gecoateten APK für in vivo Studien war eine Steigerung der Produktionsrate vom Gramm- in den Kilogramm-Maßstab erforderlich, was durch Einsatz der „Laminar Jet Break-up“-Technologie realisiert wurde. APK P4BG wurden mit dieser Methode ebenfalls durch iono-trope Gelierung einer anthocyanhaltigen Pektinlösung in Gegenwart von Calcium¬¬¬¬ionen hergestellt. Im Unterschied zum Labormaßstab war der Einsatz von Glycerin als Weichmacher in der Formulierung erforderlich. Das anschlie-ßende Coating der so hergestellten APK P4BG erfolgte mittels Wurster-Technologie unter Verwendung von wässriger Schellack¬lösung. Materialien mit Coatinglevel von 8, 15 bzw. 19 % w/w (P4BGwSch8, P4BwSch15 und P4BwSch19) wurden erzeugt. Die Formulierungen wiesen Anthocyangehalte von 2,2 bis 2,6 g/100g auf. Im vorgestellten in vitro und ex vivo Inkubations-Modell zeigten un¬modifizierte APK P4BG eine vorzeitige Anthocyan-Freisetzung in Magensaft¬simulanz infolge schneller Ablösung der sehr gut wasserlöslichen Pigmente von der Oberfläche. Dieser Effekt wurde durch Coating mit Schellack in Abhängigkeit vom Coatinglevel verringert. Das Material P4BGwSch19 stellte das am besten geeignete Colon-Targeting-System dar, da mit diesem Anthocyane im simulierten Magen und Dünndarm zurückgehalten und im Dick-darm freigesetzt wurden. V. Um das Auflösungsverhalten der Schellackschicht zu optimieren, wurden APK P4BG mit wässriger Schellacklösung gecoatet, welcher der wasserlösliche Poren¬bildner Hydroxypropylmethylcellulose (HPMC) in Konzen¬trationen von 5 % bzw. 15 % (w/w, bezogen auf Schellack) zugesetzt war. Die so herge¬stell-ten gecoateten APK HPMC5 und HPMC15 zeigten eine verbesserte Magen¬¬saft-resistenz im Vergleich zum nur mit Schellack gecoateten Material. Aufgrund des mit 5 % vergleichweise geringen HPMC-Anteils ähnelte das weitere Freisetzungsverhalten von HPMC5 dem von P4BGwSch19. Mit HPMC15 wurde bei beschleunigter Anthocyan-Freisetzung in Ileo¬stomie¬flüssigkeit ein vollstän¬diger Abbau des Materials in Colostomie¬flüssigkeit erzielt. VI. Der in vivo Effekt des entwickelten anthocyanhaltigen Colon-Targeting-Systems P4BGwSch19 bei entzündlichen Darmerkrankungen wurde am Modell der murinen DSS-Colitis untersucht. Im Gegensatz zur Gabe der identischen Dosis an unverkapseltem Heidelbeerextrakt resultierte die orale Zufuhr der mit Schellack gecoateten APK in keinem signifikanten Unterschied in Colonlänge, histolog¬ischem Score sowie IL6- und IFNγ-Sekretion im Vergleich zu Placebo- und Kontrollgruppen. Eine unzureichende Eignung des verwendeten murinen Modells für Freisetzungsstudien könnte hierfür verantwortlich sein
Results of various in vitro tests and animal models indicate the potential of anthocyanins as natural substances in the prevention and therapy of intestinal diseases such as acute diarrhea or chronic inflammatory bowel disease. Bilberries (V. myrtillus L.) possess notably high anthocyanin contents up to 780 mg/100g fresh weight. After drying these berries represent a proven remedy in folk medicine. In addition, they are applied as pharmaceutical drug Myrtillus fructus to support the therapy of unspecific acute diarrheal diseases. Recently, this traditional usage was picked up in a model study on experimental murine DSS colitis that demonstrated a therapeutic effect of dried bilberries (DBB). However, so far it is not known whether anthocyanins or other ingredients account for the observed results. Thus, the first object of the present work was to characterize DBB by identification of potential active compounds with analytical methods as well as by analysis of processes occurring during drying. I. After extractive sample preparation the anthocyanin profil and the content of commercially available DBB were determined by HPLC-ESI-MS/MS and HPLC-UV/Vis. It was shown that anthocyanin profil of the analyzed DBB, used in the above mentioned DSS colitis model, differed from that of fresh bilberries (V. myrtillus L.). DBB additionally contained a delphinidin hexose pentose as well as several anthocyanin pentoses, characteristic for the bilberry species V. arctostaphylos L. Thus, it is assumed that berries of V. arctostaphylos L. were used for manufacturing of DBB. The analyzed DBB revealed an anthocyanin content of 384 ± 39 mg/100g dry matter (dm). In comparison to fresh V. myrtillus L. as well as V. arctostaphylos L. fruits the berries showed a significantly reduced anthocyanin content. II. Phenolic acids, quercetin glycosides as well as the aglycon quercetin were identified as monomeric non anthocyanin like polyphenols and quantified by HPLC-ESI-MS/MS and HPLC-DAD, respectively. With 147 ± 5 mg/100g dm chlorogenic acid represented the dominating compound. The overall content of non-anthocyanin like polyphenols of 288 ± 8 mg/100g was similar to the anthocyanin content. III. Condensed tannins (procyanidins) in DBB were analyzed by thiolysis. With 2.23 ± 0.05 g/100g dm the content of condensed tannins was found to be three times higher compared to the sum of monomeric polyphenols (anthocyanins, phenolic acids, flavonols). IV. With 44 ± 2 g/100g dm dietary fiber was the dominant nutrient group in dried bilberries. To characterize the composition of this fraction the neutral sugars were analyzed after acid hydrolysis and derivatization to alditol acetates by HRGC-MS and HRGC-FID, respectively. Uronic acids were investigated photo-metrically. Main cell wall constituents identified by this way were glucose, xylose as well as uronic acid. As the residue after acid hydrolysis Klason-Lignin was determined by gravimetric technique. Cell wall polysaccharide as well as Klason-Lignin were enclosed each with approximately 36 % in the fiber fraction, hence representing the dominating compounds. V. 5-(Hydroxymethyl)furfural was detected as an indicator of thermal treatment during drying of the DBB and quantified by HPLC-MS/MS and HPLC-DAD. The content of 7.9 ± 0.2 mg/100g found is characteristic for dried fruits. VI. In order to directly investigate the influence of drying on the polyphenol content, fresh bilberries V. corymbosum L. were dehydrated in a drying cabinet at 30°C, 50°C as well as 70°C to constant weight. Before and after drying polyhenols were analyzed by HPLC-MS/MS as well as HPLC-DAD and HPLC-UV/Vis, respectively. During drying at 30°C a slight increase of anthocyanin content was found. Rise of temperature to 50°C and 70°C was connected with clear anthocyanin degradation. Phenolic acids and flavonols possessed accelerated thermo resistance. A slight degradation was determined only for chlorogenic acid. VII. Stability of anthocyanins cyanidin-3-galactoside and cyanidin-3-glucoside was studied in an in vitro model simulating the conditions in the plant cell during drying of fruits. The observed anthocyanin degradation showed a reaction kinetic of 1st order. Reaction speed had a strong temperature dependency. In contrast, the type of sugar residue had no influence. Protocatechuic acid was identified by HPLC-DAD-MS/MS as degradation product. Besides the daily amount of anthocyanins ingested, their availability at the site of action represents the basis of the pigments’ potential effects. As it is gener¬al¬ly known, during passage of the gastrointestinal tract (GIT) anthocyanins are chemically and microbiologically degraded. This results in a severe decrease of the active dose. Colon-targeting, to protect the of active compounds from chemical and enzymatic decomposition in the upper GIT by encapsulation is a possibility to increase the availability of anthocyanins for direct effects in the colon. Thus, the objective in the second part of this work was the preparation and characterization of formulations for dietary colon-targeting of anthocyanins. In these studies, commercially manufactured extracts from bilberries V. myrtillus L. (anthocyanin content from 65 to 84 g/100g) were applied as source of anthocyanins. I. For characterization of the release properties of the designed colon-targeting-systems an in vitro and ex vivo model was used mimicking the transit of the human GIT by consecutive incubation in simulated gastric fluid (3 h, pH 2), ileostomy fluid (4 h, pH 6,3) as well as colostomy fluid (15 h, pH 6,2). The content of anthocyanins released were analyzed by HPLC-DAD. II. In laboratory scale bilberry extract was encapsulated in a matrix of amidated pectin by ionotropic gelation in the presence of calcium ions. Conventional dripping method was applied. By additional hydrophobic coating with shellac, premature release of pigments from anthocyanin pectin beads (APB) P4BRT in simulated gastric fluid was significantly reduced. Thus, an anthocyanin transport in the colon was demonstrated with shellac coated APB P4BSch in the applied model system. III. Due to its insolubility in acid media, applicability of marine sponge collagen from Chondrosia reniformis nardo as a matrix for encapsulation was also studied. We succeeded in encapsulating bilberry extract into this material. Despite partial resistance towards degradation in the simulated stomach the materials were not suitable for colon-targeting as they were degraded in ileostomy fluid. IV. To provide shellac coated ABP for in vivo tests an increase in production rate from the gram to the kilogram scale was necessary. It was realized by application of laminar jet break up-technology for manufacturing of APB P4BG by ionotropic gelation of an anthocyanin pectin solution in the presence of calcium ions. In contrast to laboratory scale, the usage of glycerol as a plasticizer in the material was necessary. Subsequent coating of these APB P4BG was performed by Wurster technique with aqueous shellac solution. Formulations with coating levels of 8, 15 and 19 % w/w, respectively, were prepared showing anthocyanin contents from 2.2 to 2.6 g/100g. In the mentioned in vitro and ex vivo model system unmodified APB P4BG showed premature anthocyanin release in simulated gastric fluid due to fast dissolution of the highly soluble pigments from the surface. This effect was reduced by shellac coating in relation to the coating level. Material P4BGwSch19 represented the most suitable colon-targeting-sys¬tem, as anthocyanins were retarded in the simulated stomach as well as the ileum and were released in the colon. V. To optimize dissolution properties of the shellac film, ABP were coated with aqueous shellac solution containing the water soluble pore former hydroxypropyl methylcellulose in concentrations of 5 and 15 % (w/w, based on shellac), respectively. Compared to shellac coated materials these formulations HPMC5 and HPMC15 exhibited an improved gastric resistance. Due to the comparatively low HPMC content of 5 % the release properties of HPMC5 were similar to that of P4BGwSch19. HPMC15 showed in connection with accelerated anthocyanin release in ileostomy fluid material a complete degradation in colostomy fluid. VI. The in vivo effect of the designed anthocyanin colon targeting-system P4BGwSch19 was studied in the model of murine DSS colitis. In contrast to application of the same dose of unmodified bilberry extract, feeding of shellac coated APB resulted in no significant difference of colon length, histological score as well as IL6- and IFNγ-secretion compared to placebo and control groups. As a consequence of this inconsistency, it has to be checked whether the murine model used is suitable for studying release properties

Der Ernährung wird eine wichtige Rolle bei der Entstehung von entzündlichen und bösartigen Erkrankungen des Darmtraktes zugesprochen. So ist bekannt, dass fettreiche Ernährung mit hohem Fleischverzehr das Auftreten derartiger Krankheiten begünstigt. Auf der anderen Seite weiß man, dass sekundäre Pflanzeninhaltsstoffe wie Polyphenole und Flavonoide sowohl in vitro als auch im Tierversuch chemopräventive Effekte zeigen. Besondere Bedeutung scheint den Anthocyanen zuzukommen. Dabei ist bislang ungeklärt, wie hoch der Anteil an Anthocyanen nach dem Verzehr von z. B. Früchten ist, der in den Dickdarm gelangt. Es war ein Ziel der vorliegenden Arbeit, diese Fragestellung an Hand eines einschlägigen Beispiels, d.h. der Anthocyane aus Heidelbeeren (Vaccinum mytillus L.), zu beantworten. Wir führten hierzu eine Interventionsstudie mit Ileostomie-Probanden durch. Nach polyphenolfreier Ernährung über 24 Stunden verzehrten fünf Probanden je 300 g Wildheidelbeeren. Der Ileostoma-Ausfluss wurde danach in zeitlichen Abständen gesammelt, sofort tiefgefroren und nach extraktiver Probenaufarbeitung mittels HPLC-DAD und HPLC-ESIpos-MS/MS untersucht. Quantitative Bestimmungen erfolgten via HPLC-DAD, wobei die jeweilige Kalibrierung mit eigens aus Heidelbeeren isolierten authentischen Anthocyanreferenzen durchgeführt wurde. Durchschnittlich sind 46 % der aufgenommen Anthocyanmenge im Ileostoma-Ausfluss wieder gefunden worden. Der ausgeschiedene Anteil war abhängig von der Struktur des Aglycons und des jeweiligen Zuckerrestes. Malvidin-3-O-arabinosid (Mv-3-O-ara) (42) wurde zu 85.1% der aufgenommenen Menge im Dünndarmausfluss detektiert, Cyanidin-3-O-glucosid (Cy-3-O-glc) (6) hingegen nur zu 28.3%. Das Maximum der Ausscheidung lag zwischen zwei und vier Stunden. Insgesamt war erkennbar, dass Glucoside am stärksten metabolisiert oder aufgenommen wurden. Stabiler waren die Galactoside; Arabinoside [ausgenommen Delphinidin-3-O-arabinosid (12)] zeigten die größte Stabilität. Diese Tendenz wurde in Modellversuchen, bei denen verschiedene Anthocyane mit polyphenolfreiem Dünndarmausfluss inkubiert wurden, bestätigt. Stabilitätsuntersuchungen der freien Aglycone ergaben Abhängigkeiten vom Substitutionsmuster der Anthocyanidine am B-Ring; methoxylierte Strukturen erwiesen sich weitaus stabiler als hydroxylierte. Zusammenfassend ist erstmals festzustellen, dass ein beachtlicher Teil der applizierten Anthocyane unter physiologischen Umständen in den Dickdarm gelangt und dort zur Prävention von Darmerkrankungen beitragen könnte. Da aber auf Grund der gewonnenen Ergebnisse auch ein beachtlicher Teil der Anthocyane nicht wieder gefunden wurde und aus der Literatur bekannt ist, dass sich nur sehr geringe Mengen im Urin nachweisen lassen, stellt sich die Frage über den Verbleib dieser Anthocyananteile. Eine Strategie, dieses Problem experimentell anzugehen, beruht in der Anwendung markierter Substrate, z. B. 14C-markierter Anthocyanidine. Im Rahmen der vorliegenden Arbeit wurden demzufolge [4-14C]-Pelargonidinchlorid (35) und [4-14C]-Delphinidin-chlorid (36) (auf Grundlage entsprechender nicht markierter Versuche) synthetisiert. Beide Synthesen gingen von 1,3,5-Trihydroxybenzol (37) aus. In einer Vilsmeyer-Reaktion wurde mittels [Formyl-14C]-dimethylformamid markierter 2,4,6-Trihydroxy-benzaldehyd (38) gewonnen. Nach Benzoylierung erhielt man den für die weiteren Schritte benötigten, radioaktiv markierten Baustein 2-Benzoyl-4,6-dihydroxybenzalde-hyd (39). [4-14C]-Pelargonidinchlorid (35) erhielt man durch Kondensation von [Formyl-14C]-2-benzoyl-4,6-dihydroxybenzaldehyd (39) mit ω,4-Diacetoxyacetophenon (17), welches ausgehend von Methoxybenzol (22) in einer dreistufigen Synthese gewonnen wurde. [4-14C]-Delphinidinchlorid (36) wurde durch Kondensation von 2-Benzoyl-4,6-dihydroxybenzaldehyd (39) mit ω,3,4,5-Tetraacetoxyacetophenon (20) erhalten. Die Synthese des Intermediates (20) erfolgte ausgehend von 3,4,5-Trihydroxybenzoesäure (30) via 3,4,5-Triacetoxybenzoesäure (31), deren Acylchlorid (32), anschließender Umsetzung zum Diazoketon (33) und Gewinnung von ω,3,4,5-Tetraacetoxyacetophenon (20), aus dem [4-14C]-Delphinidinchlorid in zwei Schritten zugänglich war. Mit der damit erstmals gewährleisteten Bereitstellung 14C-markierter Anthocyanidine ist der Weg für zukünftige Tierversuche zur Ermittlung der Verteilung von Anthocyanidinen im Körper geebnet
Nutrition is supposed to play an essential role in the pathogenesis of inflammatory and malignant diseases of the gastrointestinal tract. It is well known that fat-rich nutrition favors the occurrence of such diseases. On the other hand, it is known that secondary plant components such as polyphenols and flavonoids show both in vitro and in vivo chemopreventive effects. Anthocyans seem to be of particular importance. It is still unknown how many anthocyans reach the colon after consumption, e.g., of fruits. One of the aims of this work was to answer this question by means of a relevant example, i.e. anthocyans from blueberry fruit (Vaccinum mytillus L.). For this purpose, we performed an intervention study with ileostomy patients. After a polyphenol free diet over a period of 24 hours five test subjects were each given 300 g of wild blueberries. The ileostomy discharge was collected in different intervals, immediately frozen and analyzed after extractive sample processing by means of HPLC-DAD analysis. The samples were quantified via HPLC-DAD, whereby the respective calibration was accomplished with authentic anthocyan references isolated from blueberries. In average 46% of the anthocyans were retrieved in the ileostomy discharge. The excreted amount depended on the structure of the aglycons and the respective sugar residue. In the ileostomy discharge 85.1 % of malvidin-3-O-arabinoside (Mv-3-O-ara) (42) was detected but only 28.3% cyanidin-3-O-glucoside (Cy-3-O-glc) (6). The maximum of the excretion was between two and four hours. Altogether it was to be observed that glucosides were metabolized or absorbed most of all. The galactosides were more stable. Arabinosides [excluding delphinidin-3-O-arabinoside (12)] showed the greatest stability. This tendency was confirmed in model reaction systems for which isolated anthocyans were incubated with polyphenol free ileostomy discharge. The stability of the available aglycones depended on the substitution pattern of the B-ring of the anthocyanidins. Methoxylated anthocyanins showed a by far greater stability than hydroxylated. Summarizing, it is shown for the first time that a considerable part of the consumed anthocyans reach the colon under physiological circumstances and could aid in the prevention of diseases of the colon. As however, due to the obtained results a considerable part of the anthocyans was not retrieved and it is known from literature that only very small quantities were found in urine, the question remains about the fate of the rest of the anthocyanidins. A strategy to approach this problem experimentally is based in the application of marked substrates, e.g. 14C-labelled anthocyanidins. In the range of the work at hand [4-14C]-pelargonidin chloride (35) and [4-14C]-delphinidin chloride (36) were synthesized on the basis of the corresponding non-marked synthesis. Both syntheses begin on the basis of 1,3,5-trihydroxybenzole (37). With a Vilsmeyer reaction with [formyl-14C]-dimethylformamide labelled 2,4,6-trihydroxybenzaldehyd (38) was prepared. After benzoylation, the for the next steps needed radioactive labelled [formyl-14C]-2-benzoyl-4,6-dihydroxybenzaldehyd (39) was obtained. [4-14C]-Pelargonidinchlorid (35) was received by condensation of ω,4-Diacetoxy-acetophenone (17) synthesized from methoxybenzol in a three step synthesis (22) with [formyl-14C]-2-benzoyl 4,6-dihydroxybenzaldehyd (39). The yield of (35) was 0.2 mg with a specific activity of 33.6 µCi/mg. [4-14C]-Delphinidin chloride (35) (0.33 mg with a specific activity of 30.4 µCi/mg) was obtained by condensation of 3,4,5-tetraacetoxyacetophenone (20) with [formyl-14C]-2-benzoyl 4,6-dihydroxybenzaldehyde (39). The synthesis of the intermediate (20) from which (35) was prepared in two steps starting from 3,4,5-trihydroxybenzoic acid via 3,4,5-triacetoxybenzoic acid (31), its acid chloride (32) with subsequent reaction to the diazoketone (33) and yielding of ω,3,4,5-triacetoxy acetophenone (20). In synthesizing 14C-labelled anthocyanidins for the first time the way for future animal experiments for the determination of the distribution of anthocyanidins in the body was opened

10 Abstract All plants live in symbiosis with fungal endophytes - they can form mutualistic, commensal or parasitic symbioses. Symbiosis of root endophytes, called for their darkly pigmented and septated hyphae dark septate endophytes (DSE), and plants has been often overlooked although its role for plant communities can be very important. Despite their ubiquitous presence in roots of terrestrial and also aquatic plants the influence of DSE on their host plants is still unresolved. Results of previous studies are inconsistent - some reported that DSE have positive effects on their host plant growth and some negative. The main reason for this inconsistency might be their complicated taxonomy and difficult identification of different cryptic species which are morphologically indistinguishable. We were able to complete a unique collection of the most common DSE species, mainly members of the Phialocephala fortinii - Acephala applanata species complex. The collection includes the most common DSE species isolated from roots of forest plant communities. The goal of my thesis was to describe behaviour of DSE in roots of typical forest plants and elucidate their physiological influence on host plants. In vitro resynthesis experiments were used to observe root colonization patterns. Nutrient flow between the plant...

The seasonal changes in freezing resistance and water relations in four dwarf shrub species of Ericaceae family: Vaccinium vitis-idaea, V. myrtillus, V. uliginosum and Oxycoccus palustris in two study areas differing in altitude were studied in this work. To establish the freezing resistance, the plants were sampled eight times, to determine parameters of the water relations, the plants were sampled six times during the growing season of 2009. For the freezing resistance examination, ice nucleation temperature (exotherm) and 50 per cent lethal temperature (LT50) were found. The water relations were described using the parameters derived from the pressure-volume curves: osmotic potential at full turgor, osmotic potential at zero turgor, cytoplasmic water fraction, relative water content at turgor loss and bulk modulus of elasticity. An important seasonal variability in the freezing resistance was found. The highest levels of the freezing resistance were reached in the spring and in the autumn, the lowest levels were found in the summer. There were also changes in the used mechanisms of the freezing resistance - for most of the year, the plants were tolerant, they showed the avoidance mechanism only in the summer. The seasonal changes in the water relation parameter were less unambiguous. The most of...

In this thesis has been mapped the current condition and expansion of the vegetation with a focus on bilberry (Vaccinium myrtillus) between 2000 and 2012 in large-scale protected area Praděd by using the methods of remote sensing. For the mapping of the total surface area of 144 hectares was used supervised and unsupervised classification of image data. From the results arised that a species of Vaccinium myrtillus has spread out to vegetation above tree line.