Grapefruits have shown a multitude of health promoting properties owing to their secondary metabolites. Modulation of production systems to increase the levels of nutrient content (secondary metabolites) in fruits and vegetables is a topic of intense scientific debate. The goal of this present research is to understand the influence of production systems and storage on grapefruit secondary metabolites and to identify and purify potentially bioactive grapefruit secondary metabolites. The first and second studies encompass the optimization of extraction procedures for the accurate quantification of flavanones and vitamin C respectively. The grapefruit flavanones were best extracted using two times dimethyl sulfoxide with the grapefruit sample volumes. Three percent meta phosphoric acid is the best extraction solvent and 5mM of TCEP is the best reducing agent for the quantification of vitamin C in grapefruit. The optimized extraction procedures were used for the quantification of grapefruit flavanoids and vitamin C. The third and fourth studies encompass the influence of production systems (organic or conventional) and storage on various groups of grapefruit secondary metabolites and their antioxidant properties. Vitamin C, limonoids and flavonoids were found to be higher in organic grapefruits compared to conventional grapefruits in the November 2008 harvest. However, there were no significant differences observed in the above mentioned secondary metabolites in the February 2010 harvest. In general, during storage the vitamin C losses were minor while limonoids and carotenoids losses were significant. In the 2010 sample, flavonoid levels increased during storage. The total phenolics and total antioxidant (DPPH) showed trends similar to flavanones during storage. The results of these studies suggest that organic production (might have) caused a small increase in the levels of a few secondary metabolites. However, it was the harvest season that had a greater impact that probably masked the effect of production systems in the 2010 sample. The fifth study focused on the isolation and purification of grapefruit minor bioactive compounds. Seven coumarins and two polymethoxy flavones including Meranzin and pranferin were purified from grapefruit byproducts such as grapefruit oil and peels using solvent partitioning and flash chromatography. The purified dihydroxy bergamottin was used as a standard in the quantification of coumarins from organic and conventional grapefruits. In future, pure coumarins, especially meranzin and pranferin, produced using various isolation techniques needs to be studied to understand the mechanism of drug interaction.

Decreases in risk of chronic disease associated with diets high in fruits and vegetables have been observed. Difficulty in determining the active ingredients responsible for the beneficial effects of dietary plant intake comes in part from the lack of pre-clinical pharmaceutical characterization of compounds thought to play a role. Flavonoids are a class of low molecular weight secondary metabolites present in plants. Pinocembrin, pinostrobin, and liquiritigenin are three chiral flavonoid implicated in the chronic disease prevention seen in high plant diets. To further the pre-clinical development of novel compounds for the potential prevention of chronic disease, stereospecific analytical methods of detection and quantification were developed for pinocembrin, pinostrobin, and liquiritigenin in biological matrices. These methods were used to elucidate the enantiospecific pharmacokinetics of all three compounds in the rat. Additionally, pinocembrin, pinostrobin, and liquiritigenin enantiomers were quantified in multiple marketed natural health products and dietary supplements. Finally, the compounds were screened for activity in several in vitro pharmacodynamic assays with roles in chronic disease pathology to assess for potential stereopecific pharmacologic behaviour. The methods were successfully used to determine the stereospecific pharmacokinetics, pharmacodynamics, and content analysis of selected marketed products. Stereopecific differences were observed in several instances. Further stereospecific studies are needed, especially in the field of toxicokinetics.

No description available.

Artemisinin is a potent antimalarial sesquiterpene lactone produced and stored in the glandular trichomes (GLTs) of Artemisia annua. Although they produce no artemisinin, nor any of the precursor compounds, A. annua roots appear to have a regulatory effect on production of the terpene in leaves. However, more information is needed to define the role of the roots in artemisinin production in the plant. Grafting among three cultivars was used to measure phenotypic responses: SAM, and #15 cultivars both have GLTs, but produce artemisinin at 1.49% and 0.57% DW, respectively; GLS cultivar produces neither GLTs nor artemisinin. Compared to ungrafted plants, all self-grafts, e.g. SAM/SAM (scion/rootstock), increased scion artemisinin probably from grafting stress. SAM/#15 grafts yielded less artemisinin than SAM/SAM, but more than either #15/#15 or ungrafted #15 and SAM suggesting rootstock inhibition of the scion. SAM/SAM also had more artemisinin than #15/SAM, which was also greater than either #15/#15 or ungrafted #15 and SAM. The #15/SAM graft also produced more artemisinin than SAM/#15, and with the other grafting results suggested that SAM roots were stimulating artemisinin production in the #15 scion. There was no appearance of either GLTs or artemisinin when GLS scions were grafted to SAM indicating that GLTs had to be present to receive putative signals from SAM rootstocks. Furthermore, artemisinic acid and arteannuin B were only present in SAM scions and not scions of #15 suggesting a block in one of the side pathways of artemisinin biosynthesis. Other artemisinic metabolites, total flavonoids, and GLTs numbers were also measured. The various phenotypes were analyzed several months after grafting indicating a persistent change and suggesting a possible epigenetic alteration of the scion. This study will provide fundamental information regarding the role that roots play in the production of artemisinin in the shoots of A. annua.

Introduction: Lactase, in addition to its role in the digestion of lactose available in milk and dairy products, is implicated in the metabolism of a range of phenolic phytochemicals in the gut. Experiments with Caco-2 cells have shown that these cells which mimic the intestinal mucosa indicate that quercetin glucosides and quercetin aglycone (widely consumed in onions and apples) block glucose uptake from the gut by competing with glucose for the sodium-dependent SGLT-1 and sodium-independent GLUT-2 transporters respectively (Johnston et al., 2005a, Schulze et al., 2015). It has been suggested that dietary phenolics that block glucose uptake from the gut may reduce the risk of type 2 diabetes. However, the ability of quercetin glucosides to block SGLT-1 is lost or reduced when the glucoside moiety is cleaved off during lactase hydrolysis. It is currently unknown if lactose-tolerant individuals deglycosylate quercetin to a greater extent than lactose-intolerant individuals and therefore are less able to reduce glucose uptake from the intestine. The aim of in vitro study was to model human gut condition for glucose transport by using Caco-2 cell models and to model role of human intestinal LPH by incubation of Caco-2 cells with quercetin flavonoids and purified β-galactosidases and in vivo was to investigate whether lactose-tolerant and lactose-intolerant subjects show differences in the uptake of glucose. Methods: Caco-2 cells were cultured in DMEM full medium in 24 well plates. Thereafter, glucose uptake assay was conducted by using 3H-glucose in the presence and absence of sodium, to assess the effect of flavonoids such as phloridzin, quercetin 4'-glucoside, quercetin 3,4'-diglucoside, quercetin 3'-glucoside, and quercetin aglycone on glucose uptake. Transwell inserts were also used to demonstrate the bidirectional permeability through Caco-2 monolayers, transport of glucose from apical (SGLT-1) to basolateral side (GLUT-2). β-galactosidase enzyme assay was conducted by using β-galactosidase from Aspergillus oryzae, Caco-2 cells were treated with 100 μM quercetin glucosides, 25% w/v onion extract and β-galactosidase in order to model the hydrolysis of flavonoids by lactase in the small intestine. HPLC was carried out to determine if quercetin glucosides are found in onion extract and test whether β-galactosidase is active and result in deglycosylation of substrates such as individual quercetin glucosides and quercetin glucosides in onion extract. For the clinical study, lactose intolerance was identified by the hydrogen breath test (Gastrolyzer), and blood glucose levels were measured by taking finger-prick blood samples in several intervals (0, 15, 30, 60, 90, 120) minutes using an EKF glucose analyser. Results: Findings from the current in vitro research confirm that phloridzin is an inhibitor of sodium-dependent conditions (SGLT-1) transporter with 80% reduction, this therefore was used as a positive control. Quercetin 4'-glucoside and quercetin 3,4'-diglucoside at (100πM) significantly decreased the uptake of glucose in the presence of sodium with up to 75% reduction compared to control p < 0.01. However, no significant glucose inhibition was found from these quercetin glucosides in the absence of sodium condition (p > 0.5), whilst quercetin aglycone significantly inhibited the glucose uptake with 50% reduction compared to control at significance levels of (p = 0.02). HPLC data identified quercetin 3,4'-diglucoside and quercetin 4'-glucoside with RT = 4.082 min and 11.392 min in the onion extract by showing peaks at similar ranges with RT= 4.114 min and 11.385 min with their standards, and the concentration of quercetin 4'-glucoside was measured as the highest level (42μg/ml) in onion extract compared to 3,4'-glucoside and quercetin 3-glucoside. Further HPLC illustrated that, after incubation of quercetin glucosides and onion extract with β-galactosidase Aspergillus oryzae for 20, 40 and 60 minutes, the peaks occurred at similar RT =16.453 min and 16.441 min respectively in accordance with standard quercetin (RT=16.239 min), suggesting the deglycosylation of these compounds with β-galactosidase from Aspergillus oryzae. According to findings from the clinical study, reduction of peak glucose levels by an onion meal was higher in lactose-intolerant people than lactose-tolerant people (44.2% versus 19.3%, p = 0.042). Also, the area under the blood glucose curve was reduced more in lactose-intolerant people compared to lactose-tolerant people, however was not statistically significant (54.5% versus 42.1%, p = 0.425). Discussion: Our result suggests that quercetin 4'-glucoside and quercetin 3,4'-diglucosides and onion extract were the main inhibitors of glucose uptake in sodium-dependent conditions (SGLT-1). Whereas, quercetin aglycone inhibited GLUT-2 glucose transport on Caco-2 cell monolayers under sodium independent conditions. Our findings were in accordance to several previous studies (Boyer et al., 2005, Kwon et al., 2007, Schulze et al., 2015). Notably, in vitro studies were conducted to model whether the in vivo study is likely to succeed or not. Findings from our human study showed that glucose uptake was blocked by the onion solution and a diet containing quercetin glucosides (onion meal) may be of greater benefit for glycaemic control in lactose-intolerant people than in lactose-tolerant people.

Living cells manufacture and degrade thousands of chemical compounds in vivo. To do this cells rely on the activities of thousands of different protein catalysts distributed in aqueous interior compartments. Over the past several decades studies have shown that the thermodynamic and kinetic properties of most proteins, including enzymes, are different in vivo as compared to in vitro. Based on in vitro studies metabolic pathways have traditionally been thought to consist of intermediates randomly diffusing between soluble enzymes and are still portrayed as such in many biochemistry textbooks. A large number of metabolic pathways however are now known to exist as enzyme complexes due to molecular crowding effects in vivo. These differences have contributed to the controversy that surrounds explanations of how metabolic pathways are spatially organized and regulated in the living cell. The organization of enzymes in vivo is now thought to play a significant role in normal cellular physiology but evidence of this role, beyond intermediate channeling, is lacking. The long term goal of this work is to develop an experimental model and test the validity of theories concerning the spatial arrangement of enzymes in regulating metabolic pathways. The studies described in this dissertation have been focused on understanding how living cells organize metabolic pathways. I have examined some of the theoretical aspects of enzyme-enzyme interactions by modeling the complex formed by mitochondrial malate dehydrogenase and citrate synthase. These studies show that MDH and CS may bind in a specific orientation that facilitates the direct transfer of oxaloacetate from MDH to CS through a molecular channel. During these studies it was determined that A. thaliana does not encode stilbene synthase (STS), which catalyzes the first step in a pathway that competes with flavonoid biosynthesis in other plant species. Moreover, it was shown that flavonols are not required for pollen viability in A. thaliana as they are in maize and petunia. I also describe a novel method to clone fragments of DNA without ligase using the polymerase chain reaction (PCR). To establish an experimental model I have used a variety of techniques to analyze interactions between enzymes in the well-characterized flavonoid biosynthetic pathway in Arabidopsis thaliana. Evidence is presented that indicates that the first four enzymes in this pathway form a complex. Collectively this work suggests that the structural organization of enzymes into complexes is an important aspect of cellular metabolism and might directly impact the relative levels of specific compounds that are synthesized in vivo. / Ph. D.

Lucie Ptáčníková, The volume of alkaloids and flavonoids in Fontinalis antipyretica, Thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, thesis tutor: PharmDr. Jan Martin, PhD., Hradec Králové, 2012, 55 pages. Thesis called "Content of alkaloids and flavonoids in Fontinalis anitpyretica" establishes alkaloids and flavonoids and identifies one substance of these alkaloids in the above-mentioned water moss. The main purpose of this thesis was to obtain samples of Fontinalis antipyretica from selected locations in the Czech Republic (Ledce, Rychnovek, Kunvald). Next purpose was to determine alkaloids and flavonoids of collected material, isolated one substance of alkaloid character and verify identity of selected alkaloid. The content of alkaloids and flavonoids was determined by using the high-performance liquid chromatography (HPLC). The results show that β - carboline alkaloid harmin was determined by consensus UV spectrum and retention time with standard. Consequently flavonoids called myricetin and hyperosid was determined. Identity of β - carboline alkaloid harmin was verified by using NMR analysis. Keywords: alkaloids - harmalol - harmin - Fontinalis antipyretica - flavonoids

In addition to AtCPL1-4, the genome of Arabidopsis thaliana encodes a large number of putative acid phosphatases. The predicted Arabidopsis SCP1-like small phosphatases (SSP) are highly homologous to the catalytic domain of eukaryotic RNA polymerase II carboxyl terminal domain (pol II CTD) phosphatases. Among the family members, SSP4, SSP4b and SSP5 form a unique group characterized by long N-terminal extensions. These three SSPs showed similar and ubiquitous gene expression. SSP4 and SSP4b were localized exclusively in the nuclei, while SSP5 accumulated both in the nucleus and cytoplasm. In vitro observation revealed that SSP4 and SSP4b dephosphorylated the pol II CTD-PO4 at both Ser2 and Ser5 in the conserved heptad repeats; however, SSP5 dephosphorylated only Ser5 of CTD-PO4. These results indicate that Arabidopsis SSP family encodes active CTD phosphatases similarly to animal SCP1 family proteins and plant CPLs family proteins, but with distinct substrate specificities. ssp mutants did not exhibit phenotypic abnormalities under normal growth conditions. However, ssp5 single mutants and ssp4 ssp4b ssp5 triple mutants showed enhanced sensitivity to ABA and glucose during seed germination. Yet, increased ABA-inducible gene expressions were not distinguishable in triple mutants compared to wild type plants upon ABA treatment. Unlike the ssp mutations, the cpl1 mutation strongly induced RD29A expression in response to cold, ABA and NaCl treatments. Thus, the cpl1 mutant is an ideal genetic background for an inducible gene expression system, in which the detrimental effect to host plants caused by a conventional constitutive expression could be avoided. Production of flavonoid such as anthocyanins in Arabidopsis is relatively easy to monitor and is regulated by transcription factors such as PAP1. PAP1 activates the expression of multiple enzymes in the anthocyanin biosynthesis pathway; however, high level of flavonoid production could cause vegetative growth retardation. To optimize flavonoid accumulation, a three-component system was designed consisting of a cold inducible RD29A-PAP1 expression cassette, a feedforward effector RD29A-CBF3, and a mutation in host repressor CPL1. Transgenic cpl1 plants containing both homozygous PAP1 and CBF3 transgenes produced 30-fold higher level of total anthocyanins than control plants upon cold treatment. LC/MS/MS analysis showed the flavonoid profile in cold-induced transgenic plants resembled that of previously reported pap1-D plants but were enriched for kaempferol derivatives. Furthermore, PAP1 and environmental signals synergistically regulate flavonoid pathway to produce a flavonoid blend that has not been produced by PAP1 overexpression or cold treatment alone. These results delineate the usability of the three-component inducible system in plant metabolic engineering.

published_or_final_version / Anatomy / Master / Master of Philosophy

EVALUATION OF PHYTOCHEMICAL COMPOSITION OF PLANTS OF GENUS HAWTHORN (CRATAEGUS L.) GROWING IN LITHUANIA INTRODUCTION For the last fifteen years, the attention of doctors and their patients on drugs and food supplements of herbal origin has been continuously increasing. The growing interest in raw plant materials is expanding the possibilities of use of these materials in the industries of medicine, cosmetics and food as well as encouraging assessment and description of natural resources using new modern methods. Scientific research on natural herbal resources has bee receiving special attention in the European Union. In recent years, a number of important documents have been published in the European Union, which declare the aims of ensuring the use and quality of natural herbal resources, such as Good Agricultural Practice document, Points to consider on Good agricultural and collection practice for starting materials of herbal origin, a long term vision of the future of herbal biotechnology in Europe Plants for the Future. Cardiovascular system diseases constitute one of the most urgent health problems. The range of effective herbal preparations for the target phytotherapy of cardiovascular system diseases is not wide. Hawthorn preparations are a promising drug for the treatment of cardiovascular system diseases, whose pharmacological effects have been substantiated by scientific studies. Throughout the territory of Lithuania, three kinds of hawthorn can be found growing... [to full text]