CHARLES UNIVERSITY Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Diana Karaščáková Supervisor: prof. RNDr. Lubomír Opletal, CSc. Title of diploma thesis: Biological activity of plants secondary metabolites XXX.; Basic search of selected taxons on anticholinesterase activity As part of the study of the biological activity of secondary metabolites, 7 taxa of higher plants were selected. The study deals with basic research of summary and alkaloid extracts prepared from morphological parts of plants Annona muricata (leaves), Leonotis leonurus (perch), Turnera diffusa (perch), Hamelia patens (perch), Uncaria guianensis (bark), Allamanda cathartica (perch) and Morinda citrifolia (leaves). To elucidate the presence of the major types of secondary metabolites, extracts were prepared and subjected to detection reactions by TLC using ten detection reagents. After detection by Dragendorff's reagent of ethyl acetate extracts, alkaloids were significantly present only in Annona muricata. Alkaloids were not detected in the bark of Uncaria guianensis. Using the Ellman method, the extracts were tested for potential inhibitory activity against human brain cholinesterases, using both recombinant enzymes. No significant active substances were present in any of the measured...

With many natural landscapes undergoing restoration efforts, there is a growing need for the optimization of direct seeding practices. Seeds planted on wildlands are often consumed by rodents leading to reduced plant establishment. Coating seeds in rodent aversive products may prevent seed predation. We tested ten seed-coating formulations containing products expected to deter rodents, namely: ghost and cayenne pepper powders; essential oils from bergamot, neem, and pine; methyl-nonyl-ketone, anthraquinone, activated carbon, beta-cyclodextrin and a blank coating containing no rodent deterrents to serve as a control treatment. Each treatment was applied to Pseudoroegneria spicata (bluebunch wheatgrass) seeds. These seeds germinated similarly to uncoated control seeds unless the coating contained methyl-nonyl-ketone which reduced germination. Seeds were offered to Ord's kangaroo rats (Dipodomys ordii) that strongly avoided the treatments in favor of uncoated control seeds. Notably, the blank coating, lacking active ingredients, still elicited 99% avoidance. However, these results indicated behavior when alternative food sources are readily available, a scenario rare in nature. To address this, a second feeding experiment was conducted to observe D. ordii's behavior under calorie-restricted conditions. D. ordii were subjected to a fast period and then offered only one treatment. Under these conditions, many subjects chose to consume coated seeds, but to a lesser degree than subjects offered control seeds. Seeds coated in ghost pepper, neem oil, and activated carbon reduced consumption by 47-50%. Given these lab results, we would expect these seed-coatings to increase the establishment of native seeds following the direct seeding of wildlands by deterring rodent seed-predation.

Masters of Science / Diabetes mellitus (DM) is well known as a group of systemic metabolic disorders with a considerable mortality rate around the world. Hyperglycemia is the main consequence of DM, which results from the shortage in insulin production or degradation of produced insulin. Other internal and external factors including obesity, oxidative stress, and sedentary lifestyle have been also suggested as the causes of DM. Among the well-known existing types of DM, type 1 and 2 are the most common. Treatment of type 1 necessitates insulin injection, while type II can be controlled by physical exercises, diet control in addition to different synthetic antidiabetic drugs. However, their effectiveness is restricted because of the high cost and unfriendly side effects. There is a significant need for producing alternative and more bioactive antidiabetic drugs from natural sources. Natural products are a well-known source for the discovery of new scaffold for drugs discovery, and South Africa is one of the most important megaflora with a high percentage of endemism. South Africa is home to about 245 species of Helichrysum species, which are generally rich sources of secondary metabolites, especially flavonoids, which give them a great value for global acceptability and wide application in the pharmaceutical fields as antibacterial, anti-inflammatory, antioxidant, and antidiabetic agents. The phytochemical investigation of the methanolic extract of H. cymosum resulted in the isolation and identification of seven pure compounds (1-7). Structural characterization of these isolated compounds was conducted using 1D NMR, in comparison with reported spectroscopic data. The in vitro bio-evaluation of H. cymosum against alpha-glucosidase shown that 5 exhibited the highest alpha-glucosidase inhibitory activity with IC50 value of 13 μM, followed by 7 and 3 with IC50 values of 18.16 μM and 44.4 μM respectively. Additionally, strong total antioxidant capacities were displayed by 6 and 2 as ORAC (122.86 ± 0.7 and 91.70 ± 0.4 μM TE/mL) respectively as well as 5 and 7 as FRAP (1006.34 ± 1.7; 977.79 ± 0.8) μM AAE/g. This is the first scientific report to be carried out on alpha-glucosidase inhibitory activities and antioxidant capacities of H. cymosum constituents. The findings suggest that these compounds might become prominent natural candidates to inhibit alpha-glucosidase as well as oxidative stress related to diabetes with the prospect to be employed in the formulation of diabetes drugs upon further biological studies.

No description available.

<p>U doktorskoj disertaciji je rađena analiza produkcije i procena stabilnosti sekundarnih metabolita cijanobakterija. U analizi pigmenata cijanobakterija kao biomarkera u paleoklimatskoj rekonstrukciji poslužio je nov model analize-AMMI model, koji pruža značajan doprinos odabiru odgovarajućih biomarkera u paleoklimatiskoj rekonstrukciji. Analizom produkcije MOSA i MOMA 15 vodenih i zemlji&scaron;nih cijanobakterija NSCCC sposobnost produkcije MOSA je uočena kod 8, a MOMA kod svih sojeva. Kultivacija u različitim pH vrednostima podloge i različitim temperaturnim uslovima nije pokazala znatan uticaj na produkciju MOSA. Produkcija MOMA je bila izraženija u baznoj sredini (pH 9.0) i na vi&scaron;im temperaturama (30-35 &deg;C). UV svetlost se pokazala kao najznačajniji faktor i inicirala je najveću produkciju MOSA i MOMA kod svih sojeva. Veća koncentracija azota u podlozi nije uticala na povećanje produkcije MOSA kod većine analiziranih sojeva, dok je znatno uticala na povećanje produkcije MOMA kod svih analiziranih sojeva.<br />Analizom produkcije pigmenata 19 lesnih cijanobakterija NSCCC prisustvo fikobilina i MOSA je uočeno u svim analiziranim kulturama, dok sposobnost produkcije MOMA nije uočena u dve kulture lesnih cijanobakterija NSCCC. Tokom posmatranog vremenskog perioda uočena je razgradnja ukupnih fikobilina u kontrolnim uslovima rasta, dok za isti vremenski period nije do&scaron;lo do degradacije MOSA i MOMA u kontrolnim uslovima.<br />Procenom stabilnosti MOSA i MOMA delovanjem abiotičkih faktora (različitih pH i temperaturnih vrednosti podloge, različitog vremena izlaganja UV svetlosti) utvrđeno je da su MOSA i MOMA pokazali izraženu stabilnost na testirane abiotičke faktore. Procenom stabilnosti pigmenata nakon delovanja biotičkih faktora primetna je intenzivnija razgradnja ukupnih fikobilina posmatrano u zavisnosti od vremena, do postizanja potpune degradacije u pojednim kulturama lesnih cijanobakterija, dok je uočeno da su MOSA i MOMA pokazali stabilnu strukturu i u testu biodegradabilnosti nije do&scaron;lo do njihove degradacije.<br />Analizom prisustva MOSA i MOMA u lesnom sedimentu i biolo&scaron;kim lesnim pokoricama njihovo prisustvo je uočeno u svim analiziranim uzorcima. Takođe, kori&scaron;ćenjem LC-MS(/MS) metode utvrđeno je prisustvo scitonemina u 10 terestričnih kultura NSCCC.<br />Analizom toksičnosti i produkcije mikrocistina, cilindrospermopsina i saksitoksina lesnih cijanobakterija NSCCC dobijeni su negativni rezultati. Razvojem novih metoda za detekciju cijanotoksina u terestričnim ekosistemima potrebno je proveriti dobijene rezultate. Procenom stabilnosti mikrocistina referentnog soja Microcystis aeruginosa PCC 7806, uočena je njegova izrazita stabilnost tokom vremenskog perioda od 96 h u kontrolnim uslovima i delovanjem tri bakterijska soja.<br />Shodno dobijenim rezultatima, UV za&scaron;titni pigmenti su mnogo podesniji za paleoklimatsku rekonstrukciju od fikobilina s obzirom da molekuli MOSA i MOMA imaju postojaniju strukturu i da nisu degradirani tokom posmatranih stresnih uslova. Usled nemogućnosti detekcije cijanotoksina lesnih cijanobakterija, i pored izražene stabilnosti mikrocistina referentnog soja, cijanotoksini se ne mogu smatrati biomarkerima cijanobakterija u geolo&scaron;kim istraživanjima.<br />Postavljanje BLOCDUST teorije i otkriće stabilnosti MOSA i MOMA i njihove upotrebe kao pouzdanih biomarkera u paleoklimatskoj rekonstrukciji predstavlja osnovu za mnoga buduća istraživanja od neprocenjivog naučnog značaja, pogotovo u paleoklimatskoj rekonstrukciji lesa. Predloženi scenario se može smatrati osnovom u paleoklimatskoj rekonstrukciji.</p> / <p>This PhD thesis analyzed the production and stability of cyanobacterial secondary metabolites. The results describe the effects of pH, temperature and light source combined with the effects of medium nitrogen content on the production of the MOSA and MOMA compounds of aquatic and soil cyanobacterial strains through AMMI model. The application of the AMMI model represents a significant contribution to the selection of appropriate biomarkers in the paleoclimatic reconstructions because it reveals the increased production of certain secondary metabolites in certain environmental conditions. MOSA compounds were observed in 8 out of 15 soil and aquatic cyanobacterial strains, while MOMA compounds were found in all 15 strains. Results show that exposure to UV light induced a higher synthesis of both pigments. The production of the MOSA compounds was clearly increased by UV irradiation and other treatments did not show a significant impact on its production. The production of MOMA compounds was increased by several stress factors including pH (pH 9.0), temperature (30-35 &deg;C), nitrogen content and UV irradiation. A higher concentration of nitrogen in the medium did not influence the increase in the production of MOSA compounds in most of the analyzed strains, while it significantly influenced the increase in the production of MOMA in all analyzed strains.<br />By analyzing the production of pigments in 19 loess cultures, phycobilins and MOSA were present in all examined loess isolates, while presence of MOMA was not detected in two samples from China. In control conditions, it was observed degradation of phycobilins depending on time, but MOSA and MOMA showed a stable structure.<br />Stability assessment of the MOSA and MOMA by the treatment with abiotic factors (different pH and temperature values of the medium, different time of exposure to UV light) revealed their pronounced stability on tested abiotic factors. Considering unstable structure of phycobilins in the presence of bacterial strains, phycobilins cannot be considered as biomarkers in loess studies. Detected results indicate that MOSA and MOMA have stable core structures resistant to bacterial strains, which makes them potentially good biomarkers for paleoclimatic reconstruction.<br />Moreover, the presence of MOSA and MOMA compounds was confirmed in loess sediment samples and BLC. Also, the LC-MS(/MS) method revealed the presence of scytonemin in 10 terrestrial cyanobacterial cultures.<br />Testing of the toxicity of loess cyanobacterial cultures and their ability to produce microcystins, cylindrospermopsin and saxitoxins, negative results were obtained. The development of new methods for detection of cyanotoxins in terrestrial ecosystems is necessary in order to revise obtained results. By assessing the stability of microcystins of the reference strain of Microcystis aeruginosa PCC 7806, its pronounced stability was observed over a 96 h in control conditions and in the presence of three bacterial strains.<br />Due to MOSA and MOMA narrow environment and organism specificity, as well as its structural stability, these metabolites are designated with a strong potential to be used as a cyanobacterial biomarker in paleoclimatic research. Due to the inability to detect cyanotoxins in loess cyanobacteria, despite the pronounced stability of the microcystin of the reference strain, cyanotoxins cannot be considered as adequate biomarkers of cyanobacteria in geological research.<br />The production of the MOSA and MOMA compounds across examined stress conditions, and further, their presence in loess samples and BLCs indicate the potential of these compounds to be regarded as biomarkers in paleoclimatic research of lacustrine/marine and loess sediments. Setting up the BLOCDUST theory and determining stability of MOSA and MOMA compounds and their aplication as a convinient biomarkers of cyanobacteria in paleoclimatic reconstruction provides the foundation for many future research of invaluable scientific significance, especially in the paleoclimatic reconstruction of loess. The proposed scenario can be considered as one of the basic model for paleoclimatic reconstruction.</p>

The increasing bacterial resistance to available antibiotics requires the search for new antibacterial compounds to be broadened. This study investigated the antimicrobial properties of two secondary metabolites from fluorescent pseudo monads -- syringopeptin 25A, a lipodepsipeptide produced by Pseudomonas syringae pv. syringae, and a rhamnolipid mixture produced by Pseudomonas aeruginosa. The rate of antimicrobial action was determined by monitoring the rate of uptake of propidium iodide during exposure to the compounds. Inhibition was also confirmed by the microbroth dilution method to determine the MI Cs. Both the compounds inhibited growth of Gram-positive organisms, including Mycobacterium smegmatis, staphylococci, and listeria. Inhibition of spore germination was also notable. SP 25A inhibited two multiple antibiotic strains of Staphylococcus aureus subsp. aureus and Enterococcus faecalis, while RLs failed to do so, even at 60 μg/ml. Addition of the compounds together showed a synergistic activity against Listeria monocytogenes. Neither compound was toxic to human cells in vitro at 8 μg/ml. It is postulated that both compounds exert their antimicrobial effect by forming pores in the bacterial cell membrane, but we did not observe a relation between membrane permeabilization and inhibition of growth in each case. At sub-MIC concentrations RLs did cause pores in the membrane of L. monocytogenes, while SP 25A did not. However, RLs did not inhibit cell growth, while SP 25A completely inhibited cell growth. To investigate these effects gene expression was monitored just before treating the cells with the antimicrobials, 30 min after treatment and 120 min after treatment. The gene expression profile was distinct when cells were treated with both the antimicrobials. SP 25A repressed genes related to cell division, intermediary metabolism, transcription, translation, and virulence genes. These effects were not produced when cells were treated with RLs, hence giving indications that even though both the antimicrobials may act on the same site (i.e. the cell membrane), the cellular response was different, which led to different phenotypes for growth. This work indicates that SP 25A holds promise for further development as a therapeutic agent and provides evidence that the proposed pore-forming model alone does not suffice to explain the mode of action of SP 25A.

Plants naturally produce primary and secondary metabolites. Primary metabolites are directly involved with plant growth and metabolic function. Plant secondary metabolites (PSM) were once thought of as metabolic waste products, and more recently viewed as toxins to herbivores. However, ongoing research shows that PSM are beneficial to herbivores at low doses, and PSM aid plants by attracting pollinators, recovering from injury, protecting from ultraviolet radiation, increasing drought tolerance, and aid in defense against pathogens, diseases, and herbivores. Plant secondary metabolites also influence soil nutrient cycling, and can increase the sustainability of agroecosystems. Endophyte-infected tall fescue (Festuca arundinacea Schreb.) , which contains ergovaline, and reed canarygrass (Phalaris arundinacea L.), containing gramine, were studied along with the legumes alfalfa (Medicago sativa L.) which contains saponins, and tannin-containing sainfoin (Onobrychis viciifolia Scop.) and birdsfoot trefoil (Lotus corniculatus L.). This dissertation researches (i) how planting configuration (monocultures vs. two-way mixtures) influences PSM and total N in plants, (ii) how cattle grazing forages containing PSM affects soil quality, nutrient cycling, and PSM, and (iii) how cattle manure from different diets, containing different PSM, influences soil nutrient cycling.

No description available.

Underexplored environments such as soil samples continue to be an untapped source of bacterial strains with great potential to produce secondary metabolites for medicinal applications. As a result, these microorganisms represent a broad and yet unknown reservoir of new strains capable of producing these novel compounds. The current research primarily seeks to perform the isolation, purification, and characterization of secondary metabolites from a soil bacterium (Janthinobacterium lividum TAJX1901). The isolated soil bacterium was successfully cultured on rich media agar plates, followed by extraction using methanol and chloroform. The purification methods utilized include flash column chromatography, preparative thin-layer chromatography, and high-performance liquid chromatography. For structural elucidation, UV-Vis analysis, infrared spectroscopy, and nuclear magnetic resonance spectroscopy were employed. The extraction resulted in a dominant violet pigment soluble in methanol. Results revealed the presence of highly conjugated, polar, and aromatic compounds (violacein or relatives of violacein) and dioctyl phthalate (a contaminant).

Underexplored environments such as soil samples continue to be an untapped source of bacterial strains with great potential to produce potent secondary metabolites for medicinal applications. As a result, these microorganisms represent a broad and yet unknown reservoir of new strains capable of producing these novel natural compounds. Secondary metabolites from microorganisms have been used in antibiotic production, chemotherapy, immunosuppressants, and various industrial applications. The current research primarily seeks to perform the isolation, purification, and characterization of secondary metabolites from a soil bacterium (Janthinobacterium lividum TAJX1901). To achieve these objectives, the soil bacteria was successfully cultured on rich media agar plates followed by liquid–liquid extraction using a solvent mixture of methanol and chloroform(3:1). Various purification methods were utilized, including flash column chromatography, preparative thin layer chromatography, centrifugation, and high-performance liquid chromatography (HPLC ) using different column types and elution methods. For structural elucidation, UV/Vis analysis, infrared spectroscopy, and nuclear magnetic resonance spectroscopy were employed. The extraction resulted in a dominant violet pigment soluble in methanol. Preliminary results reveal the presence of highly conjugated, polar, and aromatic compounds. This work is relevant in the current global search for novel compounds for tackling antibiotic-resistant organisms and treating other diseases and infections.