München, Techn. Univ., Diss., 2003.

Microbial oral diseases such as caries and periodontitis are among the most frequent human infections. Conventional chemical antiseptics used for their treatment and prevention often produce adverse side-effects, which restrict their long-term use. Although plants are considered as perspective sources of novel antimicrobial compounds, little is still known about their inhibitory properties against oral pathogens and about their safety while used on a daily basis. The objective of this study was to investigate in vitro growth-inhibitory effects of ethanol Generally Recognised as Safe (GRAS) plant and supercritical CO2 hop extracts on planktonic cultures of cariogenic, periodontal and candidal human pathogens, namely Aggregatibacter actinomycetemcomitans, Bifidobacterium dentium, B. longum, Candida albicans, Eikenella corrodens, Fusobacterium nucleatum subsp. vincentii, Lactobacillus salivarius subsp. salivarius, Porphyromonas gingivalis, Streptococcus mutans, S. salivarius subsp. salivarius and S. sobrinus using the broth microdilution method. The findings showed that ethanol extracts of all 109 GRAS plant species inhibited the growth of at least one microorganism under study with minimum inhibitory concentration (MIC) equal or lower than 4096 ug/mL. However, only six of them possessed very interesting antiseptic potential against the oral pathogens with MIC < 100 ug/mL. Additionally, three GRAS plant species showed good inhibitory activity with MIC = 128 ug/mL. As far as the particular results are concerned, the best antiseptic effect was observed for both Humulus lupulus CO2 supercritical and ethanol extracts that inhibited the growth of all microorganisms at MICs higher or equal to 8 ug/mL and MICs higher or equal to 16 ug/mL, respectively. Hence, the CO2 supercritical extraction proved to be superior for extraction of active constituents of H. lupulus. Further, the ethanol extracts of Capsicum annuum and C. frutescens showed significant antiseptic potential against S. sobrinus and S. salivarius (MICs from 64 to 128 ug/mL). The oral streptococci were also inhibited by Zanthoxylum clava-herculis (MIC higher or equal to 64 ug/mL), Helichrysum angustifolium and Myristica fragrans (MIC higher or equal to 128 ug/mL) which further showed the antimicrobial activity against F. nucleatum (MIC = 64 ug/mL). Interesting inhibitory effects exhibited an extract of Punica granatum against C. albicans (MIC = 128 ug/mL) and F. nucleatum (MIC = 64 ug/mL). Moreover, the growth of F. nucleatum was inhibited by extracts of Pimenta officinalis and Thea sinensis (MIC = 128 ug/mL). The chemical analysis of the CO2 supercritical H. lupulus extracts revealed that alpha and beta bitter acids represented the two major groups of constituents. Cohumulone was the predominant compound of the alpha acids, whereas colupulone was the prevalent constituent of the beta acids. Our results suggest that the six GRAS plant species, namely C. annuum, C. frutescens, H. lupulus, M. fragrans, P. granatum and Z. clava-herculis have good potential to become new safe antiseptic agents that might be used for incorporation into oral care products such as toothpastes and mouthrinses.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Barbora Gorová Supervisor: Assoc Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Title of thesis: Iron-chelating properties of fruit extracts of various elderberries Inorganic iron is the major food source of iron in humans. It plays role in many biochemical reactions. Thus, iron metabolism disorders can lead to different diseases associated with lack of iron or iron overload. One of the possible treatment modalities of the latter represents the administration of iron chelators. Elderberry, Sambucus nigra L. (Adoxaceae), has been used in traditional medicine. The fruits of elderberry are a rich source of cyanidin-based anthocyanins as the main component. There are important differences, both in chemical and physical properties between several cultivars of elderberry. Anthocyanins might interact with metals in the gastrointestinal tract by formation of chelates. However, data on metal interactions with anthocyanins are sparse. The main aim of this study was to perform the analysis of interaction of iron with elderberry fruit extracts as a rich and cheap source of anthocyanins with cyanidin as the aglycon. In this in vitro study ten elderberry fruit exctracts were tested for iron chelating activities...

This thesis focuses on the preparation of selected alcohol extracts of elderberry and their use in cosmetology. In the theoretical part are described the botanical properties of the elderberry (Sambucus nigra). There are introduced and characterized bioactive compounds and toxic compounds contained in all vegetative parts of the plant. Attention is mostly focused on compounds with antioxidant properties. The theoretical part also contains a description of basic analytical methods, which are used in the practical part to characterize compounds contained in elder flowers. The practical part focuses on the optimization of the preparation of alcohol extracts with the aim to prepare the extract with the greatest possible content of polyphenols with respect to economy and production costs. Factors such as selection of the appropriate solvent, solvent to plant material ratio, solvent temperature, and extraction time are examined. Based on the total polyphenol concentration measured spectrophotometrically by the Folin-Ciocalteu method, the most suitable solvent is 60% ethanol, the ratio of plant material to solvent is 1:8, the laboratory temperature of solvent and the extraction time of 24 hours.Under these conditions, an ethanol extract is prepared, analyzed and determined selected properties, such as refractive index and density. Rutin, chlorogenic, caffeic and ferulic acids are identified by HPLC with a DAD detector. The pH value of alcohol extract is 5.743 ± 0.006, the refractive index is 1.365 ± 0.02 at 22.7 ° C and the density is 0.9288 gcm-3. By liquid chromatography the concentration of chlorogenic acids is quantified as 0.104 gl-1, caffeic acid as 0.086 gl-1 and ferulic acid as 0.060 gl-1. The extract is incorporated into cosmetic products, that are used in balneology. Prepared products are toilet soap and bath salt.

Chemical substances are transferred between bumblebees of the species Bombus terrestris during their mating behavior. These substances are present mainly on the cuticle. The aim of this thesis was to identify and quantitatify differences in the amount of the compounds among defined groups of bumblebees - mated, non-mated and attempting to mate. Cuticular extracts were obtained by washing of the whole bumblebees bodies in hexane. These extracts were analyzed by using a two-dimensional gas chromatography with the time of fly mass spectrometr (GCxGC-TOF/MS). Cuticular hydrocarbons were identified and semi-quantified. Double bounds locations were determined in heptacosene and nonacosene. Five compounds were differed in the examined groups - β-springene, α-springene, dihydrofarnesyl-oleate, palmityl-palmitate and palmityl- palmitoleate. By comparing of the amount of these compounds differences between defined groups of bumblebees were determined. Analysis of the cutilar extracts showed that mated and non-mated bumblebees can be distinguished. Furthermore, there were statistically significant differences in the amounts of identified compounds in all defined groups of bumblebees. Key words: two-dimensional gas chromatography, Bombus terrestris, cutilar extract

The thesis deals with the extraction of antioxidants from elder flowers and the way of storage obtained extract. The elder flowers which are obtained by cutting trees as a waste product and after their processing it could be used as an ingredient to cosmetics products for body and face skin or in a spa like additive to baths. In theoretical part are information about black elder, its botanical characteristics, utilization and about substances contained in it. There is also a chapter about lyophilization as a method which was used for concentration of extract. The last chapter describes theoretical information about liquid chromatography because this method was used for analysis of contents of extract. Experimental part describes optimalization steps in preparation of extract. It was found that the biggest content of polyphenolic compounds was when the proportion between flower and water was 1:10, optimal temperature of water for extraction is 100 °C and it was also found that the time of extraction don´t have any influence on content of polyphenols. Thanks to the lyophilization it was obtained dry light brown powder which is stable for long time. The lyophilizate was tested for some physical properties like pH, refractive index, solubility etc. There was also determined antioxidant activity by DPPH method and there was found quenching activity is 64,9 %. In determining of heavy metals in sample was found trace amounts of lead and chrome. By liquid chromatography was determined content of polyphenols, caffeic acid was in an amount 59,6 mgl1, chlorogenic acid 398 mgl1 and ferulic acid wasn´t detected. All of analysis was for 1% solution of extract because to cosmetic it could be just this amount to addition. In the last step was prepared a skin lotion with elder flower extract and it was tested for stability also was prepared an ointment from pork lard.

The peppermint (Mentha piperita), the salvia (Salvia officinalis), the lemon balm (Melissa oficinalis), the camomile (Matricaria chamomilla) and the valerian (Valeriana officinalis) are herbs with healing effects. Their ethanol extracts are used for food fortification or in a cosmetic industry. Substances contained in herbal extracts were analyzed in this thesis. The extracts were prepared by maceration. Next, they were analyzed by using a gas chromatography and a mass spectrometry. The aim of this thesis was an optimalization of the method used for the analysis of sensorially active substances. Then, herbal extracts were analyzed and concentration of sensorially active substances was determined.

Für diese Arbeit wurden in-vitro-Studien an kaninen Mammatumorzellen und humanen follikulären B-NHL-Zellen durchgeführt. Es wurden erstmals die migratorischen Eigenschaften einer kaninen Mammatumorzelllinie (CMT-U-27, infiltrierendes duktuläres Karzinom mit Metastasen) und deren Beeinflussbarkeit untersucht. Die Experimente zeigten einen leichten promigratorischen Einfluss von Noradrenalin und EGF; mit dem Tumorpromotor PMA wurde eine sehr deutliche Stimulation der Migration erreicht. Es konnte gezeigt werden, dass Iscador® M in der Lage ist, die PMA-stimulierte Migration der CMT-U-27-Zellen zu hemmen, wobei die spontane, Matrix-induzierte Zellmigration unbeeinflusst blieb. Zwischen der migratorischen Aktivität von Tumorzellen und ihrer Fähigkeit Metastasen zu bilden, besteht ein Zusammenhang. Durch einen inhibierenden Effekt auf die Migration von Tumorzellen könnte eine Progression der Erkrankung durch Metastasenbildung verhindert oder zumindest verzögert werden. IL-6 dient als prognostischer Faktor bei lymphoproliferativen Erkrankungen der B-Zellen und kann sich negativ auf den Krankheitsverlauf bei B-Lymphompatienten auswirken. Unterstützend zu den guten in-vivo Erfahrungen in der Lukasklinik in Arlesheim, Schweiz, sollte in dieser Arbeit untersucht werden, ob Iscador® P bei follikulären B-NHL-Zelllinien (DoHH2, WSU-NHL, Sc-1) einen autokrinen IL-6-Loop auslösen kann. Dafür wurden die Zellen über definierte Zeiträume mit verschiedenen Iscador® P-Konzentrationen inkubiert. Es wurden Proben entnommen und durchflusszytometrische Messungen auf membrangebundenen IL-6R und gp130, Messungen der mRNA-Expression von IL-6, IL-6R und gp130 mittels Real-Time-RT-PCR und ELISA-Messungen zur Bestimmung von IL-6, löslichem IL-6R (sIL-6R) und löslichem gp130 (sgp130) durchgeführt. Es konnte kein Einfluss von Iscador® P auf die Oberflächen- oder Genexpression von IL-6 und den dazugehörigen Rezeptorkomponenten gp130 und IL-6R nachgewiesen werden. Zudem wird kein IL-6, sgp130 oder sIL-6R von den Zellen freigesetzt. Damit kann ein autokriner IL-6-Loop und ein durch Freisetzen von sIL-6R ermöglichtes Transsignalling bei den in dieser Arbeit untersuchten follikulären B-NHL-Zelllinien ausgeschlossen werden. In Wachstumskurven konnte gezeigt werden, dass Iscador® P den proliferativen Einfluss von IL-6, bei gleichzeitiger Inkubation, aufhebt. Genexpressionsmessungen der apoptoserelevanten Gene bcl-2, bax, bcl-xl und bad mittels Real-Time-RT-PCR zeigten bei den WSU-NHL-Zellen, dass die Apoptose über ein Verschieben des bcl-2/bax-Quotienten ausgelöst wird, welches mittels durchflusszytometrischer Analyse bestätigt werden konnte. Im Gegensatz dazu konnte bei Sc-1 Zellen keine Verschiebung des bcl﷓2/bax-Quotienten in Richtung Induktion der Apoptose beobachtet werden, sodass die bei dieser Zelllinie beobachtete Hemmung der IL-6 mediierten Proliferation durch Iscador® P, durch einen anderen, bislang noch nicht geklärten, Mechanismus ablaufen muss.

This diploma thesis deals with the determination of conditions for the preparation of elderberry extract so that the prepared extract retains as many biologically active substances as possible and the way of preserving this extract. Furthermore, the work deals with the transmittance of already specific phenolic acids, namely chlorogenic acid, coffee and ferulic skin, through the Franc diffusion cells. In the theoretical part of the thesis there is a taxonomic classification, description and use of elderberry. Furthermore, significant active substances, in particular phenolic acids, are mentioned. beneficial effects on the skin. The anatomy of the skin and the problem of the penetration of phenolic acids through the skin are described in detail. Finally, a description of high performance liquid chromatography is provided. The experimental part is devoted to verification of optimal conditions for preparation of extract. The best option is to extract the flowers with water at 100 ° C for 10 minutes in a ratio of 1 part by weight of dry flowers and 10 parts by volume boiling water. The ratio of flowers and stems to the phenolic acid content of the extract was also investigated. In the flower extract and its thickened version, the skin permeability was examined by transdermal absorption. The aim of this experiment was to find out if and how much the above mentioned phenolic acids go through the skin. As skin membrane, pork skin from the auricle was used. All determinations of the above phenolic acids in all samples were carried out by liquid chromatography.

In the last years, the edible packaging has been gaining popularity due to the potential usage as the packaging for food instead of synthetic materials. The edible packaging can positively affect the packaged food by the active properties or due to its intelligent properties can monitor the changes in food. In theory, the legislation about the packaging of food and edible packaging was described. The substances for edible films preparation and additives, which can improve the properties of the edible films, were described too. In this part, the undesirable microorganisms occur in food and the chapters about active and intelligent properties of the edible films were summarized. The main aim of the thesis was the practical preparation of edible packaging/films made from furcellaran, gelatin hydrolysate with the addition of water dried or fresh rosemary extracts in concentrations 5 %, 10 %, and 20 %. In the experimental part, the physical analysis was done – textural parameters such as tensile strength and elongation at break, water content, and thickness. In the chemical analysis the total polyphenols content, determination of polyphenols by HPLC, antioxidant activity, optical properties and antimicrobial activity of the prepared edible films were done. There were found out, that the total polyphenols content and antioxidant activity were the highest in the 20%DR (TPC: 6,79 ± 0,06 mg gallic acid/g; DPPH: 87,84 ± 0,07 %). Also, the 20%DR and 20%FR changed color in different pH (3 and 12). The antimicrobial activity was negative. The thesis showed that the addition of natural extracts to edible films can increase the polyphenols content and they can also work as the UV protector.