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The Effect of Abiotic Elicitors on the Content of Secondary Metabolites in In vitro Plant Cultures - II. The method of elicitation is used for the increased production of secondary metabolites in in vitro plant cultures. In this study, there the effect of pyridine derivate N-(5-chlorpyridin-2-yl)-4-ethylbenzamide in three concentration - 3.845.10-3 mol/l; 3.845.10-4 mol/l and 3.845.10-5 mol/l for the production of flavonolignans in callus and suspension cultures of Silybum marianum was tested. The evaluation of elicitation effect proceeded after 6, 24, 48, 72 a 168 hours against control samples without the elicitor treatment after 24 and 168 hours. In the same intervals the samples of culture medium were taken and assessed. The cells were cultivated on Murashige and Skoog medium with 10 mg/ml of α-naphtylacetic acid as growth regulator. The obtained and adjusted samples were analysed by the HPLC method. From the parts of silymarin complex were detected silychristin, silybin A, isosylibin A, isosilybin B. Flavonoid taxifolin wasn̕ t detected in any case. In callus culture, there was achieved a maximal production of all detected components of silymarin complex (30.508 μg/g DW) after 168 hours of elicitor treatment in concentration of 3.845.10-4 mol/l, it was about only substance - silychristin. The...

One of the possibilities how to increase the production of secondary metabolites (SM) in plant in vitro cultures is the method of elicitation. The aim of this work was to determine the effect of an abiotic elicitor from the class of pyrazine derivatives 1-benzyl-3-(pyrazin-2-yl)urea on the production of flavonolignans of silymarin complex and flavonoid taxifolin in the plant culture Silybum marianum (L.) Gaertn. Pyrazine derivatives are investigated for their herbicidal properties, so elicitation proceeds by the mechanism of stress inducing effect. Tissue cultures were grown on Murashige and Skoog growth medium with the addition of the growth regulator α-naphthylacetic acid. Elicitation was performed on both callus and suspension cultures. The elicitor was used in three different concentrations: c1 = 100.0 mg/100 ml; c2 = 10.0 mg/100 ml; c3 = 1.00 mg/100 ml. Particular samples were taken after 6, 24, 48, 72 and 168 hours of elicitor effect, control samples after 6, 48 and 168 hours. After drying, the callus and suspension tissues were extracted with methanol and the content of the monitored secondary metabolites was determined by HPLC. It was also tested if SM are released into the growth medium. Flavonolignans silybinin A and silybinin B were not detected in any of the analyzed samples. The...

Tyrimo objektas ir metodai: eksperimentinės augalinio vaistinio preparato Silymarinum – Aconitum 140 mg kietos kapsulės, kurių veiklioji medžiaga yra silimarinas. Kiekybinė jų sudėtis nustatyta ESC metodu. Darbo tikslas: pagrįsti augalinio vaistinio preparato kiekybinį stabilumą validuotu ESC metodu. Darbo uždaviniai: atlikti mokslinės literatūros šaltinių analizę įvertinant pasirinkto vaistinio augalinio preparato savybes bei aktyvių junginių nustatymo metodikas. Optimizuoti ir validuoti ESC metodiką kiekybiniam pasirinkto vaistinio augalinio preparato įvertinimui. Validuotu metodu atlikti eksperimentinių augalinio vaistinio preparato serijų stabilumo kiekybinę analizę. Apdoroti ir įvertinti augalinio vaistinio preparato stabilumo duomenis, remiantis mokslinių gairių nurodytais kriterijais ir gautais rezultatais pagrįsti preparato stabilumą. Išvados: atlikta mokslinės literatūros šaltinių analizė, pagrindžianti gydomąsias augalinio vaistinio preparato Silymarinum – Aconitum 140 mg kietomis kapsulėmis savybes, aktyvių junginių nustatymo metodikas bei taikomus metodo optimizavimo ir validacijos kriterijus. Optimizuotas ESC metodas eksperimentinio augalinio vaistinio preparato Silymarinum – Aconitum 140 mg kietomis kapsulėmis kiekybinei analizei. Validacijos eksperimentų metu pagrįstas metodikos glaudumas ir tiesiškumas. Atlikta kiekybinė eksperimentinio augalinio vaistinio preparato Silymarinum – Aconitum 140 mg kietomis kapsulėmis mėginių analizė. Preparato mėginiuose... [toliau žr. visą tekstą] / Object and methods: experimental herbal medicinal product Silymarinum – Aconitum 140 mg in hard capsules, as the main active component is silimarin. Assay analysis has been performed by HPLC method. Aim: to justify herbal medicinal product quantitative stability by validated HPLC method. Objective: to perform analysis of scientific literature and evaluate characteristics of chosen herbal medicinal product, and analysis methods of active components. Optimize and validate HPLC method for quantitative evaluation of chosen herbal medicinal product. Perform stability indicating quantitative analysis of experimental herbal medicinal product. Evaluate stability data of herbal medicinal product and justify its stability with reference to scientific guidelines and gained results. Results: performed scientific literature analysis justified therapeutic effect of herbal medicinal product Silymarinum – Aconitum 140 mg in hard capsules, evaluated analysis methods of active components and applicable criteria for method optimization and validation. HPLC method for quantitative analysis of experimental herbal medicinal product Silymarinum – Aconitum 140 mh in hard capsules was optimized. Precision and linearity of method was justified during experiments of validation. Assay analysis of herbal medicinal product Silymarinum – Aconitum 140 mg in hard capsules clarified, that amount of total silymarin varies from 136,966 mg to 146,573 mg. Stability testing clarified that herbal medicinal... [to full text]

The aim of this diploma was evaluate the effect of feeding milk thistle (Silybum marianum L.) seed cakes on sensoric quality of broiler chickens meat. The experimental groups of broiler ROSS 308 received feed mixtures containing 0 % (C - control), 5 % (MT5) and 15 % (MT15) of milk thistle seed cakes. The chickens were fattened on conventional deep litter system. The trial lasted 37 days. Sensory analysis of breast and thigh muscle samples was evaluated in special sensory laboratory (Department of Food Technology, Mendel university in Brno) according ISO 8589. Breast meat was significantly (P < 0,05) rated as the best in parameter flavour in C (control) and MT15 group. The thigh meat was evaluated significantly (P < 0,05) best for colour and fibreness parameters in MT15 group. The addition of milk thistle seed cakes do not worsened sensory characteristic of breast or thigh meat of broilers and reflects standard sensory quality traits.

Silybum marianum has been a plant known for millenniums thanks to its pharmacological effects especially in fields of liver, gallbladder or even colon cancer treatment. A complex of effective components is called silymarin. Its amount and structure in milk thistle seeds are very important. That{\crq}s why there{\crq}s a tendency to develop methods which could increase silymarin quantity together with silymarin quality in this herb. This was also the aim of this thesis. One of the possibilities to level up the content of effective components in drugs is to bring this plant in stress. There were realized two ground-plot experiments. The stress agens was acetatonsalicylic acid (ASA) of different concentrations applicated on leaves of these plants, concretely in the concentrations of 10-5 mol.l-1, 10-4 mol.l-1, 10-3 mol.l-1. In the case of the application ASA concentration 10-3 mol.l-1 there was proved an effective action. The increase of effective components in seeds reached approximately 116,5 % ratio compared with control application of distilled water on the Silybum marianum leaves. The silymarin complex was determined by High Performance Liquid Chromatography. The preparation of the extract was practised in different ways {--} using ethyl alcohol extract and distilled water in combination with various temperatures of extracts storage. These methods were compared one another. As the best one has been turned out to be using 60% ethanol concentration for the duration of 96 hours in the storage temperature 20°C.

This thesis is aimed at studying the effect of the addition of milk thistle (Silybum marianum) seed cakes to the diet on metabolism, liver function and performance of laying hens at the beginning of laying. In the experiment, the results were compared between a group of laying hens, which were fed with the basic feed mixture without the addition of milk thistle seed cakes with the experimental group with the addition of milk thistle seed cakes in the feed mixture. It was situated 30 hens breed ISA Brown into the experiment, divided into two groups. The first group was experimental with the addition of milk thistle seed cakes (7 % in the mixture), and the second group was a control. The experiment evaluated egg laying, egg weight, food consumption and the animal weight. During the experiment, blood was twice collected from animals for biochemical examination focusing on the parameters of liver profile. The results of the experiments didn´t show any effects of feeding milk thistle seed cakes on the body weight of laying hens. By evaluating the performance parameters of the laying hens it showed a significantly lower egg laying, but also showed a lower consumption of feed (P < 0.05). At the same time, the data found higher weigh of eggs (P < 0.01) in this group, but the egg production was significantly lower than in the control group. During the examination of biochemical parameters of blood there was a significantly higher concentration of bilirubin, uric acid and cholesterol (P < 0.05) in the blood plasma of laying hens fed by the addition of milk thistle seed cakes. At the second sampling (approximately 2 months) the laying hens that were fed with milk thistle seed cakes have consistently higher concentration of cholesterol (P < 0.05) and lower activity of dehydrogenase, creatine kinase (P < 0.05) and uric acid (P < 0.01) in the blood. In the experiment there was no clear positive or negative effects of the milk thistle seed cakes on the performance parameters and biochemical profile of the blood plasma.

La famille des Astéracées est économiquement très importante, car beaucoup de ses plantessont cultivées principalement pour leurs valeurs alimentaires dont la laitue (Lactuca), lachicorée (Cichorium) et le tournesol (Heliantus annuus) cultivé pour son huile. Une despropriétés typiques de cette famille, dont fait partie le Silybum marianum, est la richesse deses huiles en divers composés naturels bénéfiques pour la santé. Actuellement et pour lesdécennies à venir, les maladies liées à l’âge constituent un problème majeur de santépublique. Prévenir leur apparition ou s'opposer à leur évolution est un objectif majeur. Dansce contexte, les activités cytoprotectrices de l'huile de graines de Silybum marianumoriginaires de différentes régions de la Tunisie ont été étudiées sur le modèled’oligodendrocytes murins 158N en utilisant le 7-cétocholestérol (7KC) comme agentcytotoxique. Le 7KC a été utilisé car il est augmenté dans le plasma, le liquide céphalorachidienet/ou certains tissus (paroi vasculaire et intestinale, rétine, cristallin, cerveau) depatients atteints de maladies liées à l’âge (maladies cardiovasculaires, maladie d'Alzheimer,dégénérescence maculaire liée à l’âge, cataracte) ou de maladies inflammatoires évolutives del’intestin. Le 24S-hydroxycholestérol (24S-OHC) a aussi été utilisé car il est augmenté dans lecerveau aux stades précoces de la maladie d’Alzheimer. Afin de déterminer la composition del'huile de Silybum marianum (acides gras, tocophérols, polyphénols, phytostérols) diversestechniques physicochimiques et de chromatographie liquide et gazeuse ont été utilisées. Lespropriétés anti-oxydantes de l'huile de Silybum marianum ont été définies par les tests KRL,FRAP et DPPH et les propriétés cytoprotectrices par des techniques complémentaires demicroscopie, de cytométrie en flux et de biochimie. Les études sur la compositionphysicochimique des huiles des graines de Silybum marianum de différentes régions de laTunisie (Bizerte, Sousse, Zaghouan) ont montré que ces dernières sont riches en Vitamine E(α-tocophérol) et sont fortement anti-oxydantes. Les acides gras majeurs sont l'acidelinoléique (C18:2) (valeurs comprises entre 57,0% et 60,3%) et l'acide oléique (C18:1) (lesvaleurs se situent entre 15,5% et 22,4%). Trois acides phénoliques ont également étéidentifiés (vanillique, p-coumarique et silybine), avec une prédominance de l'acide vanillique.Sur les cellules 158N, ces huiles atténuent la cytotoxicité du 7KC et du 24S-OHC: perted'adhérence cellulaire, altération de la membrane plasmique, dysfonctionnementmitochondrial, surproduction d’espèces réactives de l’oxygène, induction de l'apoptose(condensation et / ou fragmentation nucléaire, activation de la caspase-3 et clivage PARP) etautophagie (activation de LC3-I en LC3-II). L'atténuation des effets cytotoxiques du 7KC etdu 24S-OHC observée avec les huiles de Silybum marianum est de l'ordre de celle observéeavec l’α-tocophérol utilisé comme témoin positif. La composition chimique et les résultatsobtenus cellules 158N sont en faveur d’effets bénéfiques de l’huile de Chardon-Marie sur lasanté humaine et la prévention de certaines maladies liées à l’âge. / The family Asteraceae is economically very important because many of its plants are grownmainly for their food values including lettuce (Lactuca), chicory (Cichorium) and sunflower(Heliantus annuus) grown for its oil. One of the typical properties of this family, whichincludes Silybum marianum, is the richness of its oils in various natural compounds beneficialto health. Currently and for decades to come, age-related diseases are a major public healthproblem. Preventing their appearance or opposing their evolution is a major objective. In thiscontext, the cytoprotective activities of Silybum marianum seed oil originating from differentarea of Tunisia were studied on the 158N murine oligodendrocyte model using 7-ketocholesterol (7KC) and 24S-hydroxycholesterol (24S-OHC) as a cytotoxic agent. The 7KChas been used because it is increased in plasma, cerebrospinal fluid and / or tissues (vascularand intestinal wall, retina, lens, brain) of patients with age-related diseases (cardiovasculardiseases, Alzheimer's disease, age-related macular degeneration, cataract) or inflammatorybowel disease. The 24S-OHC was used because it is increased in the brain at the early stagesof Alzheimer’s disease. In order to determine the composition of Silybum marianum oil (fattyacids, tocopherols, polyphenols, phytosterols) various liquid and gas chromatographytechniques were used. The anti-oxidant properties of Silybum marianum oil were defined byKRL, FRAP and DPPH tests and cytoprotective properties by complementary microscopy,flow cytometry and biochemistry techniques. Studies on the physicochemical composition ofSilybum marianum seeds oils from different regions of Tunisia (Bizerte, Sousse, Zaghouan)have shown that the latter are rich in vitamin E (α-tocopherol) and are strongly antioxidant.The major fatty acids are linoleic acid (C18:2) (values between 57.0% and 60.3%) and oleicacid (C18:1) (values between 15.5% and 22.4%).. Three phenolic acids have also beenidentified (vanillic, p-coumaric and silybin), with a predominance of vanillic acid. On 158Ncells, these oils attenuate the cytotoxicity of 7KC and 24S-OHC: loss of cell adhesion,alteration of the plasma membrane, mitochondrial dysfunction, overproduction of reactiveoxygen species, induction of apoptosis (nuclear condensation and / or fragmentation). ,activation of caspase-3 and PARP cleavage) and autophagy (activation of LC3-I in LC3-II).The attenuation of cytotoxic effects of 7KC and 24S-OHC observed with Silybum marianumoils is of the order of that observed with α-tocopherol used as a positive control. The chemicalcomposition and results obtained on 158N cells are in favor of the beneficial effects of milkthistle oil on human health and on its ability to prevent some age-related diseases.

Milk Thistle (Silybum marianum) has been one of the best known medici herb for a very long time. Seeds contain biologicky aktive substances taxifoiln, silychristin, silydianin, silybin A, silybin B, isosilybin A and isosilybin B, commonly known as silymarin complex. The aim of this thesis was the influence of elicitors, fertilization and growing technology on this product and its utilizaion. In the first part I focused on history, botanic attributes, agrotechnology, chemical composition and substance efficiency method of determinativ and also on pharmacological onpacts of effective substances. In the practical part I conducted twho small parcel experiments to reveal the impal of elicitors on effective substances. I used two elicitors NanoFYTSi? in 1ml/l concentrantion and N-FENOLMIX? in 0.5ml/l concentration. In the second experiment from my supervisor elicitor NanoFYT Si? in 1ml/l concentraiton and N-FENOL MIX? in 0.5ml/l and elicitor ASA were used in three different concentration: low [10-5 mol/l], medium [ 10-4 mol/l] and high [10-3 mol/l]. In the conclusion I did statistical analysis of the above montioned experiments and compared them with other Publisher experiments. Then I propřed the growing technology and utilization of Milk Thistle.

The aim of this thesis was to study the effect of the acetylsalicylic acid on the stimulation of plant immunity and thus the influence on the content of the active constituents in Silybum marianum L. plants. The main active constituents of Silybum marianum L. seeds are silybine, silydianine, isosilybine, silycristine, usually expressed as silymarine content, and taxifoline. These constituents have antihepatotoxic effect and many different protective effects on numerous organs and cells. Knowledge about stress and elicitation, origin, botanical characterization, growth, development and cultivation of Silybum marianum L. were summarized before the research.The small-plot experiment was set up in Hluboká nad Vltavou in 2013. Plants of Silybum marianum were divided into four groups and then three goups were treated three times during the vegetation with the acetylsalicylic acid of three different concentrations - 10-3 mol.l-1 (high), 10-4 mol.l-1 (medium) a 10-5 mol.l-1 (low). Every single one group was treated with only one concentration of the acetylsalicylic acid. The last group was treated only with water and served as a control group. The preparation of the extracts was being held with using mixture of acetone, methanol and water. The extracts were determined by High Performance Liquid Chromatography.The effects of each chosen concetration of acetylsalicylic acid (high, medium, low) on the active constituents in seeds were not statistically proven compared to seeds without aplication of the elicitor (control group). The ineffectiveness of the elicitor should have been also caused by nonoptimal condition of plants due to various abiotic and biotic stressors.