Browsing by Subject "HPLC"

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  • Mgbeahuruike, Eunice Ego; Fyhrquist, Pia; Vuorela, Heikki; Julkunen-Tiitto, Riitta; Holm, Yvonne (2018)
    Piper guineense is a food and medicinal plant commonly used to treat infectious diseases in West-African traditional medicine. In a bid to identify new antibacterial compounds due to bacterial resistance to antibiotics, twelve extracts of P. guineense fruits and leaves, obtained by sequential extraction, as well as the piperine and piperlongumine commercial compounds were evaluated for antibacterial activity against human pathogenic bacteria. HPLC-DAD and UHPLC/Q-TOF MS analysis were conducted to characterize and identify the compounds present in the extracts with promising antibacterial activity. The extracts, with the exception of the hot water decoctions and macerations, contained piperamide alkaloids as their main constituents. Piperine, dihydropiperine, piperylin, dihydropiperylin or piperlonguminine, dihydropiperlonguminine, wisanine, dihydrowisanine and derivatives of piperine and piperidine were identified in a hexane extract of the leaf. In addition, some new piperamide alkaloids were identified, such as a piperine and a piperidine alkaloid derivative and two unknown piperamide alkaloids. To the best of our knowledge, there are no piperamides reported in the literature with similar UV absorption maxima and masses. A piperamide alkaloid-rich hexane leaf extract recorded the lowest MIC of 19 mu g/mL against Sarcina sp. and gave promising growth inhibitory effects against S. aureus and E. aerogenes as well, inhibiting the growth of both bacteria with a MIC of 78 mu g/mL. Moreover, this is the first report of the antibacterial activity of P. guineense extracts against Sarcina sp. and E. aerogenes. Marked growth inhibition was also obtained for chloroform extracts of the leaves and fruits against P. aeruginosa with a MIC value of 78 mu g/mL. Piperine and piperlongumine were active against E. aerogenes, S. aureus, E. coli, S. enterica, P. mirabilis and B. cereus with MIC values ranging from 39-1250 mu g/mL. Notably, the water extracts, which were almost devoid of piperamide alkaloids, were not active against the bacterial strains. Our results demonstrate that P. guineense contains antibacterial alkaloids that could be relevant for the discovery of new natural antibiotics.
  • Christodoulou, Maria (Helsingin yliopisto, 2019)
    The rapid emergence of drug resistant pathogens prevents effective treatment of diseases and threatens the lives of millions of people. Similarly, resistance to chemotherapeutic agents has been observed in several types of cancer. Therefore, screening for novel antimicrobial and antileukemic substances is urgently needed. Screening microorganisms for bioactive molecules has resulted in the discovery of several substances that are currently used for disease treatment. Cyanobacteria represent an ancient group of oxygenic, photosynthetic prokaryotes that produce a variety of functionally diverse and structurally complex natural compounds, some of which have already been used as source of inspiration in drug development process. In this study, I evaluated the antimicrobial and antileukemic potential of filamentous cyanobacteria against Gram-positive, Gram-negative and fungal potential pathogens and acute myeloid leukemia (AML) MOLM-13 cell line. Extracts showing antibiotic and/or antileukemic activity were subjected to reversed-phase HPLC and individual fractions were re-evaluated for their ability to kill the abovementioned pathogens and/or induce cell death in MOLM-13 cell line. Metabolites present in the active HPLC fractions were analysed by UPLC/ESI/Q-TOF and elemental compositions were obtained. Identification of metabolites was accomplished by searching online databases for compounds with identical elemental composition. Chemical structures were further confirmed by comparing mass spectrometry data with publicly available data. New bioactive metabolites, new variants of known metabolites and a number of yet unidentified metabolites exhibiting antimicrobial and/or antileukemic activity are reported herein. In detail, novel metabolites belonging to aromatic polyketides and their new variants were present in the active fractions of Nostoc sp. CENA69. The cyanobacterium Aliinostoc sp. CENA513 produced the recently discovered metabolite nocuolin A, a compound with antimicrobial and antiproliferative properties, lipids and unidentified lipidic compounds that showed only bactericidal activity against B. cereus. Interestingly, none of the abovementioned strains had any effect on the growth of Gram-negative pathogens. Planktothrix agardhii UHCC 0018 and Anabaena sp. UHCC 0187 strains showed only antileukemic activity. The majority of the bioactive fractions deriving from these two strains contained either lipids or pigments and their derivatives. The remaining active HPLC fractions of these two strains contained a great number of unidentified compounds. Further studies are required to identify the unknown compounds and purify the novel metabolites and antibacterial lipids. The results presented herein clearly show that Cyanobacteria are an emerging source of bioactive metabolites that can be used in drug development process or act as a source of inspiration for the production of novel synthetic drugs.
  • Marik, Tamás; Szekeres, András; Andersson, Maria A.; Salkinoja-Salonen, Mirja; Tyagi, Chetna; Leitgeb, Balázs; Vágvölgyi, Csaba; Druzhinina, Irina S.; Kredics, László (Springer International Publishing, 2017)
    Sustainability in Plant and Crop Protection
    Filamentous fungi are producers of a large number of secondary metabolites with wide spectra of biological effects. Among them, peptaibols represent a group of compounds produced mainly by members of the mycotrophic filamentous fungal genus Trichoderma. A simple peptaibol characterization strategy including purification and structural elucidation steps was applied to examine the peptaibol production of three strains from the Longibrachiatum section of genus Trichoderma, T. aethiopicum TUCIM 1817, T. novae-zelandiae TUCIM 4158 and T. pseudokoningii TUCIM 1277, all deriving from natural forest habitats (disturbed semiforest, native Notophagus forest and the bark of Beilschmiedia tawa, respectively). After the solid phase clean-up of culture extracts, mass spectrometric analysis of peptaibols produced by the examined strains was performed by on-line reversed-phase high performance liquid chromatography coupled to electrospray ionization ion trap mass spectrometry. All three examined species produced 20-residue trichobrachin-like compounds, some of which are known from the literature, while others proved to be different from any peptaibols reported so far. The spectra of the peptaibols produced by these isolates were entirely different from each other. The largest amount of peptaibols consisting of four yet unknown compounds was produced by T. pseudokoningii TUCIM 1277, while ten and eight new, trichobrachin-like compounds were detected from T. aethiopicum TUCIM 1817 and T. novae-zelandiae TUCIM 4158, respectively. Feline fetal lung cell proliferation inhibition tests and membrane damage bio-assay with boar sperm cells revealed that although T. novae-zelandiae TUCIM 4158 produced the least amount of peptaibols, its compounds were the most inhibitory to mammalian cells.
  • Outinen, Katri (University of Helsinki, 1996)
  • Gürbüz, Göker (Helsingfors universitet, 2010)
    The literature review elucidates the mechanism of oxidation in proteins and amino acids and gives an overview of the detection and analysis of protein oxidation products as well as information about ?-lactoglobulin and studies carried out on modifications of this protein under certain conditions. The experimental research included the fractionation of the tryptic peptides of ?-lactoglobulin using preparative-HPLC-MS and monitoring the oxidation process of these peptides via reverse phase-HPLC-UV. Peptides chosen to be oxidized were selected with respect to their amino acid content which were susceptible to oxidation and fractionated according to their m/z values. These peptides were: IPAVFK (m/z 674), ALPMHIR (m/z 838), LIVTQTMK (m/z 934) and VLVLDTDYK (m/z 1066). Even though it was not possible to solely isolate the target peptides due to co-elution of various fractions, the percentages of target peptides in the samples were satisfactory to carry out the oxidation procedure. IPAVFK and VLVLDTDYK fractions were found to yield the oxidation products reviewed in literature, however, unoxidized peptides were still present in high amounts after 21 days of oxidation. The UV data at 260 and 280 nm enabled to monitor both the main peptides and the oxidation products due to the absorbance of aromatic side-chains these peptides possess. ALPMHIR and LIVTQTMK fractions were oxidatively consumed rapidly and oxidation products of these peptides were observed even on day 0. High rates of depletion of these peptides were acredited to the presence of His (H) and sulfur-containing side-chains of Met (M). In conclusion, selected peptides hold the potential to be utilized as marker peptides in ?-lactoglobulin oxidation.
  • Woiwode, Ulrich; Ferri, Martina; Maier, Norbert M.; Lindner, Wolfgang; Lämmerhofer, Michael (2018)
    Abstract A cardinal requirement for effective 2D-HPLC separations is sufficient complementarity in the retention profiles of first and second dimension separations. It is shown that retention and enantioselectivity of chiral selectors derived from cinchona alkaloids can be conveniently modulated by structural variation of the carbamate residue of the quinine/quinidine carbamate ligand of such chiral stationary phases (CSP). A variety of aliphatic and aromatic residues have been tested in comparison to non-carbamoylated quinine CSP. Various measures of orthogonality have been utilized to derive the CSP that is most complementary to the tert-butylcarbamoylated quinine CSP (tBuCQN CSP), which is commercially available as Chiralpak QN-AX column. It turned out that O-9-(2,6-diisopropylphenylcarbamoyl)-modified quinine is most promising in this respect. Its implementation as a complementary CSP for the separation of amino acids derivatized with Sanger’s reagent (2,4-dinitrophenylated amino acids) in the first dimension combined with a tBuCQN CSP in the second dimension revealed successful enantiomer separations in a comprehensive chiral×chiral 2D-HPLC setup. However, the degree of complementarity could be greatly enhanced when simultaneously the absolute configurations were exchanged from quinine to quinidine in the chiral selector of the first dimension separation resulting in opposite elution orders of the enantiomers in the two dimensions. The advantage of such a chiral×chiral over achiral×chiral 2D-HPLC setup, amongst others, is the perfect compatibility of the mobile phase because in both dimensions the identical eluent can be used.
  • Sillanpää, Meri (Helsingin yliopisto, 2021)
    The literature study of this thesis focuses on the different analytical methods used to analyse amino acids in food and beverage samples. Amino acids are essential organic molecules and their concentrations in foods and beverages constitute, inter alia, the product’s nutritional value, quality, freshness, and flavour. Amino acid analysis of foodstuff has various applications, which exploit several analytical methods. These reviewed methods are founded on academic articles published during the past two decades. This literature review discusses the different sample matrixes, sample preparation methods, ways to derivate analytes, and different separation and detection methods utilized in the recent amino acid studies. The experimental part of this thesis was a modification of L-asparagine and L-aspartic acid test (L-Asp/L-AspAc) in Thermo Fisher Scientific Oy industrial R&D laboratory. An enzymatic photometric method is used to determine L-Asp/L-AspAc amino acids in food samples. The modification process entailed pre-testing of several candidate methods, from which the most suitable one was selected. The feasibility of the chosen test was affirmed before verification and validation of the modified test.
  • Touzani, Soumaya; Imtara, Hamada; Katekhaye, Shankar; Mechchate, Hamza; Ouassou, Hayat; Alqahtani, Ali S.; Noman, Omar M.; Nasr, Fahd A.; Fearnley, Hugo; Fearnley, James; Paradkar, Anant; ElArabi, Ilham; Lyoussi, Badiaa (2021)
    The biological activities of propolis samples are the result of many bioactive compounds present in the propolis. The aim of the present study was to determine the various chemical compounds of some selected propolis samples collected from Palestine and Morocco by the High-Performance Liquid Chromatography-Photodiode Array Detection (HPLC-PDA) method, as well as the antioxidant and antibacterial activities of this bee product. The chemical analysis of propolis samples by HPLC-PDA shows the cinnamic acid content in the Palestinian sample is higher compared to that in Moroccan propolis. The results of antioxidant activity demonstrated an important free radical scavenging activity (2,2-Diphenyl-1-picrylhydrazyl (DPPH); 2,2 '-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and reducing power assays) with EC50 values ranging between 0.02 +/- 0.001 and 0.14 +/- 0.01 mg/mL. Additionally, all tested propolis samples possessed a moderate antibacterial activity against bacterial strains. Notably, Minimum Inhibitory Concentrations (MICs) values ranged from 0.31 to 2.50 mg/mL for Gram-negative bacterial strains and from 0.09 to 0.125 mg/mL for Gram-positive bacterial strains. The S2 sample from Morocco and the S4 sample from Palestine had the highest content of polyphenol level. Thus, the strong antioxidant and antibacterial properties were apparently due to the high total phenolic and flavone/flavonol contents in the samples. As a conclusion, the activities of propolis samples collected from both countries are similar, while the cinnamic acid in the Palestinian samples was more than that of the Moroccan samples.
  • Nykänen, Tina (Helsingfors universitet, 2013)
    Rhazya stricta Decne. is a small evergreen shrub belonging to the Apocynaceae family. The plant grows in South Asia and the Middle East, and in these areas it is used in traditional medicine. All parts of the plant are used in different preparations for a variety of purposes such as infections, bowel diseases, itching and diabetes. R. stricta synthesizes about a hundred different alkaloids, of which only a fraction has been studied closer. Some of the analyzed alkaloids have showed some interesting pharmacological properties such as antibacterial and cytotoxic properties. Because it is often both economically and ecologically unsustainable to cultivate or to collect large amounts of medicinal plants from nature, cell cultures have been developed from plants. The properties and synthesized substances of the cell cultures can be analysed and modified in laboratories. In the experimental part of this work, a system was developed for alkaloid extraction, fractionation and isolation from dried cell material from cultured R. stricta hairy root-cells. The goal was to develop a functioning system that eventually enables identification of the alkaloids synthesized by the cultured cells under given conditions. Alkaloids were extracted from 26 g of dried and ground cell mass. The fractionation of the alkaloids was performed with medium pressure liquid chromatography (MPLC) and the fractions were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The alkaloids were purified by horizontal TLC and preparative HPLC. Ion-pair chromatography was used for analyzing the extract, fractions and purified alkaloids. Five components from two fractions were eventually isolated. One of the components was tentatively identified as vincanine, but further analyzes have to be performed to identify all components reliably. In total, hairy root-cells seem to synthesize approximately 20 alkaloids with variable polarity.
  • Hakala, Kati P. (2020)
    Fungal disease late blight (Phytophthora infestans) causes considerable damage to potato crops worldwide. Fluazinam is a widely used pesticide employed against the late blight in potato cultivation. It ends up into soil during spraying and at the end of the growing season when potato foliage is incorporated into the soil. Nevertheless, there is very little literature about behaviour of fluazinam in soil, especially in the conditions that exist in Finland. Therefore, in the preparation of user guidelines, studies made elsewhere are used. From the environmental risks point of view, behaviour of fluazinam in Finnish conditions should be known better. Soils in the boreal zone are characterised by low pH and low temperatures that delay microbiological decomposition and they are typically high in organic matter and saturated by water for long periods in autumn, winter and spring. A prerequisite for assessing the environmental risk of fluazinam is knowledge of its sorption and desorption tendency as well as its degradation rate in boreal conditions. This information is needed, because more aggressive strains of Phytophthora infestans have spread to northern latitudes, increasing the need to use fungicides. In this study, a specific and repeatable high-performance liquid chromatography method utilizing a diode array detector was developed to determine the presence of fluazinam in soil. This method differs from most of the methods found in the literature, which used gas chromatography or gas chromatography-mass spectrometry as an instrument for analysing fluazinam. The method consists of acetonitrile extraction, clean-up with solid-phase extraction and separation using a mobile phase consisting of 70% acetonitrile and 30% water (v v-1), including 0.02% acetic acid. The method was successfully applied to various laboratory experiments and to soil samples collected from potato fields in which fluazinam had been used. In the systematic experiments carried out in controlled conditions, performed with both the fluazinam standard and the commercial product Shirlan®, the effect of soil organic matter on the fluazinam degradation was tested, as well as the persistence of fluazinam in the boreal zone soils for a maximum of one year. The major outcomes of the laboratory experiments were that fluazinam degradation was enhanced by the presence of soil organic matter and even after one year of incubation, more than half of the added fluazinam was recovered. Additionally, soil samples were collected from intensively cultivated potato fields. Over half of these field samples contained varying concentrations of fluazinam, but no substantial accumulation of fluazinam was detected.
  • Hirvisaari, Laura (Helsingfors universitet, 2012)
    Estradiol is a female sex hormone which is metabolized to two different catechol estradiols. 2-hydroxyestradiol (2-OHE2) is normally the major catechol estradiol metabolite but breast cancer patients have increased amounts of genotoxic 4-hydroxyestradiol (4-OHE2) and it arises to predominant metabolite with these patients. These catechol estradiols can form reactive quinones that can bind to DNA and lead to mutations and finally cause cancer. Catechol-O-methyl transferase can add methyl groups and UDP-glucuronosyl transferase (UGT) glucuronic acid groups to catechol estradiols. These phase II enzymes play important role in the inactivation of catechol estradiols because only non-conjugated catechol estradiols can be oxidized to quinones. The aim of this study was to find out which human UGTs catalyze glucuronidation of 2-OHE2 or 4-OHE2, how many different glucuronides are formed and in which part of the substrate glucuronic acid is added. To answer these questions chromatography methods for 2-OHE2 and 4-OHE2 glucuronides were developed using HPLC. Eleven UGT-enzymes glucuronidate 2-OHE2. UGTs 1A1, 1A7 and 1A10 form two different glucuronides and UGTs 1A3, 1A8, 1A9, 2A1, 2A2, 2A3, 2B7 and 2B15 form only the second glucuronide. It was possible to detect three different glucuronides for 4-OHE2 but the amount of the first glucuronide was under quantification limit. UGT1A10 catalyzed the formation of the second glucuronide and UGTs 1A7, 1A8, 1A9, 2B7 and 2B15 catalyzed the formation of the last glucuronide. One aim of the study was to find out which part of the substrate is glucuronidated but this aim was not achieved because suitable standards were not available.
  • Pulkkinen, Marjo; Coda, Rossana; Lampi, Anna-Maija; Varis, Jutta; Katina, Kati; Piironen, Vieno (2019)
    Vicine and convicine may be removed from faba bean by hydrolysis to the corresponding aglycones, divicine and isouramil. For total elimination of their toxicity, further degradation of the aglycones should be shown. The aim of the study was to investigate hydrolysis of vicine and convicine using the enzymatic activity in faba bean in flour suspensions and selected lactic acid bacteria used as starters for faba bean fermentation. In addition, the effect of acidity on the stability of vicine and convicine was investigated. Sourdoughs were used in a baking process to obtain breads as final products. Vicine, convicine, and their aglycones were analyzed using reversed phase high pressure liquid chromatography with UV detection (RP-HPLC-UV). Incubation of the suspensions showed rather small vicine and convicine losses. Acidity itself did not cause losses under the conditions studied, apart from that of convicine at low pH. In sourdough fermentation with strains of Lactobacillus plantarum and Pediococcus pentosaceus, losses of vicine and convicine were dependent on the fermentation temperature and the β-glucosidase activity of the starter. Compared to fermentation at 20 °C, more intense acidification at 25 °C resulted in decrease of vicine up to 85% and convicine up to 47%. Levels of vicine and convicine in breads were comparable to levels in sourdoughs. Furthermore, the aglycones were not detected from breads.
  • Ranta, Merja; Ketola, Raimo (2016)
    A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the analysis of seven common sartans (candesartan, eprosartan, losartan, olmesartan, telmisartan, valsartan, and losartan carboxylic acid (EXP3174), the active metabolite of losartan) in postmortem blood samples with liquid-liquid extraction. Detection was accomplished by triple-quadrupole MS in the selected reaction monitoring mode. The validation procedure showed low limits of quantitation of 5 ng/mL for all compounds with good accuracy and precision. The matrix effect was evaluated from the variation of peak areas and concentrations. The method shows some matrix effect but the effect was efficiently compensated by using an internal standard method. The method was applied to the analysis of sartans in postmortem blood samples, and produced 534 positive findings during 2014-2015. Nine deaths were encountered where the concentration of a sartan was very high, indicating intoxication together with other causes of death or multi-drug intoxication. The quantitative results indicate that sartans do not show significant postmortem redistribution; thus concentrations higher than the therapeutic range can be considered as being toxic or fatal.
  • Tiilikainen, Saija (Helsingfors universitet, 2016)
    Prolyl oligopeptidase (PREP) is a serine protease which is extensively present in the mammalian system and especially abundant in the brain. Despite the long research history of PREP its physiological function has remained unclear. PREP has been suggested to regulate the functions of many bioactive peptides by hydrolysis and on the other hand to participate in several intracellular processes probably via direct protein-protein interactions. One of the functions suggested for PREP is the regulation of the brain neurotransmitter systems and based on, for instance, the location in the brain PREP has been connected to both excitatory and inhibitory neurotransmitter systems. The literature review of this thesis first describe the brain neurotransmitter systems associated to PREP in general with some examples of diseases related to their malfunctions. In addition the structure of PREP and its location in the brain, both subcellular and cellular levels, and in distinct neurotransmitter systems, are presented, after which the different proposed functions for PREP are reviewed. The aim of the experimental part of this thesis was to investigate the effects of PREP on the brain neurotransmitter concentrations in the mouse nigrostriatal pathway and also to mouse motor behavior. The main research methods were microdialysis, tissue assays and cylinder test. The study was composed of two sections with five week duration each. The first section was performed with wild-type mice expressing naturally PREP and the second section with PREP-knockout (ko) mice and their wild-type littermates. The mice were injected unilaterally above the substantia nigra with adeno associated (AAV1) hPREP viral vector or with AAV1-eGFP (green fluorescent protein) viral vector as a control treatment. The cylinder test was carried out before the injection, and two and four weeks afterwards. Microdialysis was used to study the actions of PREP on the extracellular concentrations of dopamine (DA) and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), gamma-aminobutyric acid (GABA) and 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of serotonin (5-HT). In addition to the baseline assay the concentrations were measured after two amphetamine treatments (10 and 30 µM) administered via the microdialysis probe. The probe guide cannulas were inserted to mice striatums 1-2 weeks before the microdialysis measurement. In the end of the experiment the tissue concentrations of DA, DOPAC, HVA, 5-HT and 5-HIAA were measured from striatum and substantia nigra. Both the microdialysis and tissue sample concentrations were quantified with high performance liquid chromatography. In the first study section the PREP enzyme activity was also determined from striatum. Neither the complete deprivation nor over-expression of PREP in the nigrostriatal pathway had clear or consistent effects on the levels of neurotransmitters studied when compared to naturally occurring PREP expression. When comparing the differences between control treated groups of PREP-ko and littermate mice, a greater amphetamine stimulated DA-levels was seen in the former group proposing negative regulatory influence of PREP. In both study sections the tissue assay results were difficult to interpret due to observed responses also with AAV1-eGFP control treatment in comparison with untreated side of the brain. This was seen as a lower DA- and DOPAC-levels in substantia nigra and thus the meaning of the changes caused by PREP treatment is hard to comprehend. The results of the cylinder test may implicate some protective effect of the PREP-ko-genotype against viral vector injections in general. Then again the existence of compensatory mechanisms is possible when using knockout animals and thus the genotype differences are hardly ever unequivocal. The results of this thesis do not suggest outright regulatory effects of PREP on the neurotransmitter functions in the mouse nigrostriatal pathway although the confirmation of this requires further studies, especially in regard to GABAergic and glutamatergic systems. Studies should include a scale of different behavioral tests of motor activity and repeated microdialysis experiment with some defining method changes. The possible function and mechanisms of PREP as a regulator of neurotransmitter intake or release is rationale to study at molecular level with applicable methods.
  • Zhou, Xiao (Helsingin yliopisto, 2014)
    Divicine and isouramil are the causative agents of favism. The stability of divicine is of vital importance in faba bean detoxification. The literature review of this thesis focused on the hydrolysis of vicine to divicine, the oxidation processes of divicine and its stability studies. The main aims of this thesis were to produce divicine using the hydrolysis of vicine with ?-glucosidase, and study the effects of nitrogen/air atmosphere, reducing agent, pH and temperature on the stability of divicine. The identities of other compounds observed in vicine hydrolysis were also to be investigated. In addition, convicine was also hydrolyzed in the extracts and pure convicine fractions. Vicine and convicine were co-extracted from dehulled faba bean flour and were separated with preparative HPLC-MS. The extracts and the pure vicine and convicine fractions were hydrolyzed with ?-glucosidase to yield divicine and isouramil. The identities of the compounds formed during vicine fraction hydrolysis were studied by MS. In the following stability studies, the pure vicine fractions were hydrolyzed with ?-glucosidase under nitrogen and in the presence of (+)-sodium L-ascorbate. Moreover, the fractions were firstly hydrolyzed under air, next, the formed divicine was incubated at pH 3.0 or 5.0 at 20 or 37 ºC. An analytical HPLC method was used to study the changes during hydrolysis and stability tests. It was found that higher ?-glucosidase concentration and longer incubation period resulted in higher hydrolysis degrees of vicine and convicine. Further, vicine was hydrolyzed more rapidly at pH 3.0 than 5.0. Vicine was hydrolyzed to divicine. Divicine further generated two compounds, named compound 1 and compound 2 in this thesis. Their corresponding retention times and absorption maxima were: 2.15 min, 282 nm; 1.79 min, 262 nm; and 1.94 min, 210 nm. Compound 1 was directly generated from divicine. It was possibly oxidized divicine, but its characterization with MS failed in this study. Compound 1 decomposed to compound 2 at pH 5.0 at 20 ºC, but at pH 3.0 at 20 ºC, divicine might directly decompose to compound 2. Only one compound (named compound 3) was formed during convicine hydrolysis, and its retention time and absorption maximum were 2.50 min and 280 nm. Divicine and compounds 1, 2, and 3 were not stable, they finally decomposed to non-UV absorbing substances. Divicine was more stable under nitrogen than under air, and in the presence of (+)-sodium L-ascorbate than without its presence. Divicine decomposed similarly at pH 3.0 and 5.0 at 37 ºC, but at 20 ºC, divicine was more stable at pH 5.0. At both pH values, the stability of divicine was increased at 20 ºC compared with 37 ºC.
  • Aly, Ashraf A.; Bräse, Stefan; Hassan, Alaa A.; Mohamed, Nasr K.; Abd El-Haleem, Lamiaa E.; Nieger, Martin (2020)
    The manuscript describes the synthesis of new racemic and chiral linked paracyclophane assigned asN-5-(1,4(1,4)-dibenzenacyclohexaphane-1(2)-yl)carbamoyl)-5'-(1,4(1,4)-dibenzenacyclohexaphane-1(2)-yl)carboxamide. The procedure depends upon the reaction of 5-(1,4(1,4)-dibenzenacyclohexaphane-1(2)-yl)hydrazide with 5-(1,4(1,4)-dibenzenacyclohexaphane-1(2)-yl)isocyanate. To prepare the homochiral linked paracyclophane of a compound, the enantioselectivity of 5-(1,4(1,4)-dibenzenacyclohexaphane-1(2)-yl)carbaldehyde (enantiomeric purity 60% ee), was oxidized to the corresponding acid, which on chlorination, gave the corresponding acid chloride of [2.2]paracyclophane. Following up on the same procedure applied for the preparation of racemic-carbamoyl and purified by HPLC purification, we succeeded to obtain the targetSp-Sp-N-5-(1,4(1,4)-dibenzenacyclohexaphane-1(2)-yl)carbamoyl)-5'-(1,4(1,4)-dibenzenacyclohexaphane-1(2)-yl)carboxamide. SubjectingN-5-(1,4(1,4)-dibenzenacyclohexaphane-1(2)-yl)hydrazide to various isothiocyanates, the corresponding paracyclophanyl-acylthiosemicarbazides were obtained. The latter compounds were then cyclized to a new series of 5-(1,4(1,4)-dibenzenacyclohexaphane-1(2)-yl)-2,4-dihydro-3H-1,2,4-triazol-3-thiones. 5-(1,4(1,4)-Dibenzenacyclohexaphane-1(2)-yl)-1,3,4-oxadiazol-2-amines were also synthesized in good yields via internal cyclization of the same paracyclophanyl-acylthiosemicarbazides. NMR, IR, and mass spectra (HRMS) were used to elucidate the structure of the obtained products. The X-ray structure analysis was also used as an unambiguous tool to elucidate the structure of the products.
  • Kontti, Arttu (Helsingfors universitet, 2014)
    Parkinson's disease causes changes in the basal ganglia GABAergic neurotransmission in addition to the well-known dopaminergic changes. These GABAergic modulations may cause somed of the symptoms not responding well to the standard dopaminergic medication. Neurotrophic factors are a group of endogenous proteins showing promise as a future treatment for Parkinson's disease. They are known to have neuroprotective and neurorestorative effects on the dopaminergic cells. Their effects to the GABAergic cells are still mostly unknown. Intrastriatal injection of GDNF to rats caused significantly slower weight gain compared to CDNF, MANF one week after stereotaxic operation (p=0,002 for CDNF vs. GDNF and p<0,001 for MANF vs. GDNF). Difference to the vehicle (phosphate buffered saline) used as a negative control was not statistically significant (p=0,055). Three weeks after the operation the differences between the treatment groups were no longer statistically significant. Because of problems with the separation in analysis, microdialysis samples remain still to be analysed. To help the analysis of GABA in the future we determined the analytical parameters of the analytical apparatus. We also defined differences in probe permeability between 1 mm and 2 mm probes and between old and new batches. GABA analysis was performed with a HPLC-fluorometric detection of o-phtaldialdehyde-derived GABA. Detection limit for old apparatus was 7,2 nM and for new apparatus 6,2 nM in a sample of 15 µl (0,11 pmol and 93 fmol respectively). Quantification limits defined were 22 nM and 19 nM (0,33 pmol and 0,28 pmol) for the old and the new apparatus, respectively. Upper limit of quantification was estimated to be 246 nM (3,7 pmol). Probes had significant differences in permeability between 1 mm and 2 mm probes, as well as between batches. The variance of permeability of 1 mm probes was estimated to be approximately twofold compared to the 2 mm probes. Furthermore the permeability of 1 mm probes varied between batches significantly. An average of permeability of the old batch was 34 % lower than that of a new batch (p<0,001).
  • Moslova, Karina (Helsingin yliopisto, 2020)
    Ribonukleiinihapot (RNA) toimivat kriittisissä biologisissa tehtävissä geneettisen tiedon välittäjinä solun sisällä. Sen lisäksi niillä on merkittävä rooli perintöaineksen säilymisessä ja siirtymisessä sekä katalyyttisten toimintojen ylläpitämisessä. RNA-modifikaatioiden on katsottu toimivan ylimääräisenä tiedonvälittäjäkerroksena solufysiologian säätelemisessä nukleotidisekvenssien koodauksessa. Tämän Pro gradun kirjallisuusosassa esitellään viimeisten viiden vuoden ajalta (2014-2019) vakiintuneet tekniikat sekä ohjelmistot RNA-modifikaatioiden tutkimuksissa. RNA-modifikaatioiden monimuotoisten biologisten toimintojen selvittäminen perustuu tarkkaan detektointiin, kvantitointiin, ja modifikaatioiden kartoitukseen. Näytteen esikäsittely mm. kemiallinen leimaaminen on tärkeässä roolissa onnistuneen analyysin kannalta. Työssä kerrotaan mm. massaspektrometrisistä (MS) identifioinneista ja rakennetutkimuksista sekä sekvenssointtitekniikoiden hyödyntämisestä. Useiden viime vuosien aikana menetelmien kehittämisessä on edistytty merkittävästi. Erityisesti MS datan käsittelyyn soveltuvia ohjelmistoja on luotu tandemmassaspektrien tulkitsemiseksi. Massaspektrien käsittelyn monipuolistaminen on johtanut uusiin lähestymistapoihin. joista voidaan saada kvalitatiivista ja kvantitatiivista tietoa RNA-modifikaatioista, usein jopa sekvenssispesifisyyden tasolla. Koska RNA-tutkimus on vielä varhaisessa vaiheessa, se vaatii paljon tutkimustyötä. Uusien menetelmien, tekniikoiden ja ohjelmistojen kehittymisen myötä ymmärrys RNA-modifikaatioiden olemassaolosta, tarkoituksesta, toiminnasta ja vaikutuksesta elämään kasvaa. Työn kokeellisen osion aiheena oli derivatisoida lähettiribonukleiinihappo (mRNA) fluorisoivaksi ja kehittää analyyttinen erotusmenetelmä kapillaarielektroforeesilla (CE) laserindusoitua fluoresenssidetektoria (LIF) käyttäen. Fluoreskeiini-5-isotiosyanaatti (FITC) valittiin derivatisointireagenssiksi sen monipuolisen käytettävyyden vuoksi ja helpon saatavuuden takia. Edellisestä huolimatta on yleisesti tiedossa FITC:n nopea hajoaminen, mutta tähän asti ei ole raportoitu, kuinka nopeasti FITC todellisuudessa muuttuu reagointikyvyttömäksi. Menetelmän kehitysvaiheessa käytettiin RNA-tyyppisiä biologisia molekyylejä, kuten BSA ja aminohappoja. Työssä testattiin erilaisia puskureita eri pH-arvoilla ja pitoisuuksilla. Lisäksi optimoitiin injektiopaine ja -aika sekä virta käyttäen CE-laitteistoa varustettuna diodirividetektorilla (CE-UV). Koska työn päätavoitteena oli erotusmenetelmän kehittäminen, derivatisointireaktion toimivuuteen ei heti kiinnitetty tarpeeksi huomiota. Menetelmä optimoitiin leimatuille aminohapoille ja BSA:lle. Lopuksi siirryttiin viimeistelemään menetelmä CE-LIF:llä. Derivatisointituotteiden tunnistus tehtiin ESI-TOF-MS:lla. Siinä yhteydessä huomattiin FITC:n nopea hajoaminen. Monien haasteellisten kokeilujen jälkeen päädyttiin keskittyä FITC:n stabiiliuden selvittämiseen. FITC:ä myydään samalla cas-numerolla kahta eri konformeeria ja tämän lisäksi vielä eri cas-numerolla kahta isomeeria. Näillä kaikilla on hieman erilaiset fysikaaliset ominaisuudet, jotka selvitettiin tässä työssä. Tutkittiin FITC:n eri konformeerien stabiilisuutta erilaisissa liuottimissa ja kahdessa lämpötilassa kymmenen päivän ajan käyttäen ESI-TOF-MS-laitetta sekä positiivisella että negatiivisella polariteetilla. Tutkimuksessa standardoidulla derivatisointiproseduurilla testattiin FITC-kemikaalin avulla LIF-detektorin toimintaa. Kuitenkin mRNA-derivatisointiin vaaditaan kalliit leimauskitit, joita ei tässä vaiheessa ollut mahdollista hankkia. Pysyminen alkuperäisessä suunnitelmassa ja optimoida CE-LIF -metodi fluoresoivalla FITC-reagenssilla lähetti-RNA:n leimaamiseen ei aikataulullisesti onnistunut. Siten projekti painottui tämän kokeellisen työn osalta derivatisoinnin optimointiin ja FITC-kemikaalien kinetiikkaan. Sen vuoksi projektissa tehtiin perusteellinen pohjatyö tuleville tutkimuksille. Kokeellisesti onnistuttiin leimaamaan nukleotidiemäksiä, jotka ovat lähetti-RNA:n perusosia. Selvitettiin FITC:n hajoamisnopeus ja liuottimet, joita FITC-tutkimuksissa on käytetty ja joita tulee käyttää, jotta reaktio onnistuu. Leimaus onnistui p-kinoidille. Saatujen tulosten perusteella on mahdollista laskea FITC:n optimaalinen määrä onnistuneen reaktion kannalta. Lisäksi osoitettiin, että leimausreaktio on herkkä, kun käytetään optimaalista puskuria ja optimipitoisuuksia. Jatkotutkimuksissa myös FITC-liuoksen valmistamiseen pitää kiinnittää paljon huomiota.