Essential oils from plants and in vitro shoot cultures of Hypericum androsaemum L., H. perforatum L. and H. undulatum Schousboe ex. Wild

Tese de doutoramento em Ciências (ramo do conhecimento em Biologia)Apical buds and nodal segments, from plants grown in Nature were used as primary explants to establish in vitro shoot cultures of Hypericum androsaemum and Hypericum undulatum, respectively. Hypericum perforatum shoot cultures were established from nodal segments of axenic seedlings grown from seeds germinated asseptically on MS medium devoid of growth regulators. Shoot multiplication was performed by subculturing nodal segments on MS medium devoid of growth regulators, in the cases of H. perforatum and H. undulatum, and on MS medium supplemented with IAA and KIN, in the case of H. androsaemum. A modified Mg medium was used in parallel with the MS medium both devoid of growth regulators, in the case of H. undulatum. After 60 days on MS basal medium, H. undulatum cultures were characterized by a higher number of shoots and roots, comparing with the cultures grown on Mg basal medium. Essential oils (EO) from in vivo plants and in vitro cultures of H. androsaemum, H. perforatum and H. undulatum were isolated by hydrodistillation in a Clevenger type apparatus and analyzed by Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GC-MS). More than 70 compounds were identified in EO from plants of H. androsaemum L. cultivated in two places (Arouca and Arcos de Valdevez) and harvested with intervals of 2 months, over one year. Seasonal variations on the content of sesquiterpene hydrocarbons, the most represented group of compounds, were registered (42.8-72.7% in plants grown in Arouca and 43.4-78.5% in plants grown in Arcos de Valdevez). A high number of intermediate to long chain n-alkanes and 1-alkenes was recorded in EO of H. androsaemum plants grown in Arouca, in the month of February, as well as plants grown in Arcos de Valdevez, during the spring and in the end of winter. In most of the EO of this species, (Ε)-caryophyllene, β-gurjunene and γ- elemene were the major compounds independently of the experimental field. Ripened seed capsules and stems were the H. androsaemum organs with the highest and the lowest EO contents, respectively. Sesquiterpene hydrocarbons dominated the EO of leaves and stems of H. androsaemum, while monoterpene hydrocarbons dominated the EO of the ripened seed capsules. From the five most represented compounds in the EO of H. androsaemum no one was common to all the three organs (leaves, stems and ripened seed capsules). Almost 80% of the total EO of in vitro shoots of H. androsaemum was represented by sesquiterpene hydrocarbons, with γ-elemene as the only major constituent common to EO from in vitro shoots and from in Nature cultivated plants. Essential oils from aerial parts of H. perforatum plants of two cultivars (common cultivar and cv. ‘Topaz’), grown in two experimental fields and sampled over one year, revealed high levels of sesquiterpene hydrocarbons, and low levels of oxygenated compounds. Germacrene D, (E)-caryophyllene and β-selinene were the major compounds. The highest EO content was found in flowers (~12-17 mg/g of dry biomass), in which sesquiterpene hydrocarbons was the major compound group and 2-methyloctane the most represented compound (22-29%). Alkanes which represented no more than 9% of the total EO from in Nature cultivated plants, was the second major group in the EO of in vitro shoots, in which nnonane accounted for more than 24% of the total EO. Essential oils of plants and in vitro shoots of H. undulatum Schousboe ex Willd had nnonane as the major constituent, accounting for more than 40% in most of them. This compound was that most contributed for the high level of n-alkanes group in the EO of the different plant organs, notwithstanding sesquiterpene hydrocarbons constituted the dominant group. The highest yield of H. undulatum EO was obtained from leaves, followed by ripened seed capsules, flowers and stems. The EO contents observed in in vitro H. undulatum shoots (4.9-10.5 mg/g of dry weight, for MS basal medium and 4.1-9.5 mg/g of dry weight, for Mg basal medium) were higher than those observed in aerial parts of in Nature growing plants. Although variations in the composition of the EO from shoots grown on two different basal media had been registered, over the 60 days of culture, the group of alkanes was the major one independently of the culture conditions. The highest contents of n-nonane were recorded in the EO from shoots grown on Mg basal medium. In order to get hairy root cultures of H. androsaemum, H. perforatum and H. undulatum, the influence of several factors (effect of explant pre-culture, bacterial density, explant wounding, addition of acetosyringone to the bacterial suspension and co-culture medium, as well as co-culture period) were evaluated, using the A. rhizogenes-mediated transformation as the main approach. Notwithstanding the several assays performed, hairy roots production was not achieved in any of the tested explants (leaves, internodal segments and roots).No âmbito do presente trabalho, foram estabelecidas culturas in vitro de Hypericum androsaemum, Hypericum perforatum e Hypericum undulatum. As culturas in vitro de H. androsaemum e H. undulatum foram obtidas, respectivamente, a partir de gemas apicais e segmentos nodais de plantas desenvolvidas na Natureza. Os explantes primários de H. perforatum foram obtidos de plântulas desenvolvidas a partir de sementes germinadas em condições de assépsia em meio de cultura MS sem fitorreguladores. Segmentos nodais foram utilizados na multiplicação de rebentos caulinares em meio MS suplementado com IAA e KIN, no caso de H. androsaemum, meio base MS, no caso de H. perforatum e meios base MS e Mg, no caso de H. undulatum. Após 60 dias de cultura, as plântulas de H. undulatum desenvolvidas em meio MS apresentavam maior número de rebentos e raízes, do que as plântulas obtidas em meio Mg. Os óleos essenciais (OE) de plantas in vivo e culturas in vitro de H. androsaemum, H. perforatum e H. undulatum foram isolados por hidrodestilação em aparelho tipo Clevenger e analisados por Cromatografia Gasosa (CG) e Cromatografia Gasosa acoplada a Espectrometria de Massa (CG-EM). Mais de 70 compostos foram identificados nos OE de plantas de H. androsaemum L., cultivadas em dois locais distintos (Arouca e Arcos de Valdevez), e colhidas com a periodicidade de 2 meses. Ao longo do ano, foram registadas variações de composição traduzidas em variações dos teores percentuais dos grupos de compostos, designadamente dos hidrocarbonetos sesquiterpénicos, grupo maioritário cuja expressão variou entre 42.8% e 72.7% nos OE de plantas desenvolvidas em Arouca e entre 43.4% e 78.5% nos OE de plantas desenvolvidas em Arcos de Valdevez. Nos OE de plantas desenvolvidas em Arcos de Valdevez, durante a Primavera e no final do Inverno foi registada a acumulação de um número superior de n-alcanos, de cadeia intermédia a longa e, de 1-alcenos, ao passo que nos OE de plantas desenvolvidas em Arouca a maior diversidade de n-alcanos de cadeia intermédia a longa e 1- alcenos foi registada em Fevereiro. (Ε)-Cariofileno, β-gurjuneno e γ-elemeno foram os compostos maioritários na maioria das amostras de OE desta espécie, em ambos os campos experimentais. Os teores de OE mais elevados foram registados nos frutos e os mais baixos nos caules desta espécie. Nos OE das folhas e dos caules predominaram os hidrocarbonetos sesquiterpénicos, ao passo que nos OE dos frutos predominaram os hidrocarbonetos monoterpénicos. Dos cinco compostos maioritários dos OE de cada um dos órgãos em estudo, nenhum era comum aos três, reflectindo a dissemelhança entre eles. Cerca de 80% do total dos OE das culturas in vitro de H. androsaemum era constituído por hidrocarbonetos sesquiterpénicos, sendo o γ-elemeno o único composto deste grupo maioritário nos OE dos rebentos caulinares e das plantas cultivadas. Os OE da parte aérea de plantas das cultivares comum e ‘Topaz’ de H. perforatum, cultivadas em dois campos experimentais distintos revelaram teores elevados de hidrocarbonetos sesquiterpénicos e teores baixos de compostos oxigenados. Os compostos maioritários dos OE de ambas as cultivares foram o germacreno D, o (E)-cariofileno e o β- elemeno. Os OE de flores proporcionaram rendimentos superiores (~12-17 mg/g de biomassa seca) aos das partes aéreas das plantas. A sua composição era maioritariamente constituída por hidrocarbonetos sesquiterpénicos embora o composto maioritário fosse um alcano, o 2- metiloctano cujo teor variou de 22% a 29%. O constituinte maioritário dos rebentos caulinares foi o n-nonano (24% do OE), sendo os alcanos o segundo grupo de compostos maioritário neste tipo de culturas in vitro de H. perforatum. Nos OE de plantas e rebentos caulinares de H. undulatum Schousboe ex Willd., o nnonano foi o composto maioritário, constituindo mais de 40% do seu total na maioria das amostras analisadas. De facto, este composto foi o responsável pela elevada expressão percentual do grupo dos n-alcanos nos diferentes órgãos de H. undulatum. Nas folhas, porém, os hidrocarbonetos sesquiterpénicos foram o grupo predominante. Os teores mais elevados de OE foram registados nas folhas, seguindo-se os frutos, flores e caules. Os rendimentos em OE dos rebentos caulinares variaram de 4,9 a 10,5 mg/g de biomassa seca nas culturas mantidas em meio base MS e de 4,1 a 9,5 mg/g de biomassa seca nos rebentos caulinares desenvolvidos em meio base Mg, sendo superiores aos registados para os OE das partes aéreas das plantas. Embora se tenham verificado variações na composição dos OE de rebentos caulinares mantidos nos dois meios de cultura, o grupo dos n-alcanos foi o maioritário em ambos os casos. Os teores mais elevados de n-nonano, composto maioritário, foram registados nos rebentos caulinares desenvolvidos em meio base Mg. Na tentativa de se induzir a formação de hairy roots de H. androsaemum, H. perforatum e H. undulatum foi testada a influência de diversos factores (pré-cultura dos explantes, densidade bacteriana, ferimento dos explantes, adição de acetoseringona à suspensão bacteriana e ao meio de co-cultura, e período de co-cultura) na transformação das referidas espécies mediada por A. rhizogenes A4. Apesar dos diversos ensaios realizados, não se verificou a produção de hairy roots em nenhum dos explantes utilizados (folhas, segmentos internodais e raízes)

The effects of spinal anaesthesia for elective caesarean section on uterine and umbilical arterial pulsatility indexes in normotensive and chronic hypertensive pregnant women: a prospective, longitudinal study

Background: Despite the known effects of neuraxial blockade on major vessel function and the rapid decrease in uterine vascular impedance, it is unclear how the blockade affects the utero-placental circulation in the near-term. We hypothesize that among women with chronic hypertension, a loss of sympathetic tonus consequent to spinal block may cause significant changes in the utero-placental haemodynamics than the changes typical in normal pregnant women. Therefore, the main study objective was to analyse the effect of spinal anaesthesia for caesarean section on uterine and umbilical arterial impedance in pregnant women at term diagnosed with stage-1 chronic hypertension. Methods: A prospective, longitudinal study was performed in singleton pregnant women (203 low-risk and 33 with hypertension) scheduled to undergo elective caesarean section. The mean arterial blood pressure and pulsatility indexes for the uterine and umbilical arteries were recorded before and after spinal anaesthesia was performed using 8–9 mg hyperbaric bupivacaine (5 mg/mL) and 2–2.5 μg sufentanil (5 μg/mL). Multiple linear regression models with errors capable of correlation or with unequal variances were fitted using the generalized least squares. Results: In normotensive women, the mean arterial blood pressure decreased after administering spinal anaesthesia (p < 0.05). The pulsatility index of the uterine and umbilical arteries did not change after spinal anaesthesia. In the hypertensive women, the mean arterial blood pressure (p < 0.05) and uterine artery pulsatility index (p < 0.05) decreased. In both groups, the umbilical artery pulsatility index did not change after spinal anaesthesia. Conclusions: In stage-1 chronic hypertensive pregnant women at term, spinal anaesthesia for caesarean section reduces uterine artery impedance but not umbilical artery impedance. Electronic supplementary material The online version of this article (doi:10.1186/1471-2393-14-291) contains supplementary material, which is available to authorized users

Essencial oil components of Hypericum androsaemum infusions and their nematotoxic effects against Meloidogyne javanica (Treub) Chitwood.

Real Sociedad Española de Química, CITAB -UM, CBMA - UM, FCTUniversidade do Minho - CITAB -UM, CBMA - UMFundação para a Ciência e a Tecnologia (FCT

Anaerobic co-digestion of cork based oil sorbent and cow manure or sludge

Cork, a material with great economic, social and environmental importance in Portugal, is also a good oil sorbent that can be used in the remediation of oil spills. The oil-impregnated cork can be easily removed, but requires further treatment. In the case of vegetable oil spills, anaerobic digestion may be a potential solution. This study aims to evaluate the effect of adding cork contaminated with sunflower oil as co-substrate in anaerobic digestion processes. Biodegradability assays were prepared with cow manure or sludge from a wastewater treatment plant, in the presence of five concentrations of oil-contaminated cork, between 200 and 1000 mg· L-1 as COD. Maximum cumulative methane production increased with the amount of oily cork up to 41 % and 101 % in the assays with manure and sludge, respectively. Sporadic addition of cork contaminated with vegetable oil during anaerobic digestion of manure or sludge increases significantly the methane production of these processes.Programa Operacional Regional do Norte (ON.2 - 0 Novo Norte), QREN, FEDERPortuguese Foundation for Science and Technology (FCT, in the frame of projects FCOMPO 1-0124-FEDER-014784 (FCT: PTDC/EBBEBI/114364/2009

3-Hydroxypyrrolidine and (3,4)-dihydroxypyrrolidine derivatives: inhibition of rat intestinal α-glucosidase

Thirteen pyrrolidine-based iminosugar derivatives have been synthesized and evaluated for inhibition of α-glucosidase from rat intestine. The compounds studied were the non-hydroxy, mono-hydroxy and dihydroxypyrrolidines. All the compounds were N-benzylated apart from one. Four of the compounds had a carbonyl group in the 2,5-position of the pyrrolidine ring. The most promising iminosugar was the trans-3,4-dihydroxypyrrolidine 5 giving an IC50 of 2.97 ± 0.046 and a KI of 1.18 mM. Kinetic studies showed that the inhibition was of the mixed type, but predominantly competitive for all the compounds tested. Toxicological assay results showed that the compounds have low toxicity. Docking studies showed that all the compounds occupy the same region as the DNJ inhibitor on the enzyme binding site with the most active compounds establishing similar interactions with key residues. Our studies suggest that a rotation of ∼90° of some compounds inside the binding pocket is responsible for the complete loss of inhibitory activity. Despite the fact that activity was found only in the mM range, these compounds have served as simple molecular tools for probing the structural features of the enzyme, so that inhibition can be improved in further studies

Addition of electron acceptors stimulates methanogenesis from lipids by anaerobic sludge

Incubation of anaerobic sludge with triolein or oleate in the presence of nitrate or sulphate led to an increased methane production, relatively to incubations without inorganic electron acceptor. Faster methane production was obtained in assays amended with nitrate. Methanogenesis occurred after the reduction of alternative electron acceptors

Exploring syntrophic relationships in the anaerobic biodegradation of lipids and long chain fatty acids

ICBM-3 - 3rd International Conference on Biogas Microbiology (Abstract Book)[Excerpt] Practical knowledge on anaerobic digestion of waste lipids has been improving for several decades, but the microbiology of these processes remains partially undisclosed, with non-cultivated taxonomic groups often detected in anaerobic communities degrading lipids. This work studies the diversity and physiology of anaerobic microorganisms involved in the metabolism of lipids and long chain fatty acids. Anaerobic culturing procedures were applied for the development of enrichment cultures, and combined with next generation sequencing techniques. Enriched microbial communities specialized in the degradation of triolein (0.3 mmol·L-1) and oleate (1 mmol·L-1) were obtained under methanogenic conditions. Oleatedegrading cultures were also developed in the presence of the external electron acceptors ferric hydroxide (75 mmol·L-1) or sulfate (15 mmol·L-1). Three mesophilic sludges from different origins were used as inocula. [...]info:eu-repo/semantics/publishedVersio

Co-cultivation of Thermoanaerobacter strains with a methanogenic partner enhances glycerol conversion

Glycerolrich waste streams produced by the biodiesel, bioethanol and oleochemical industries can be treated and valorized by anaerobic microbial communities to produce methane. As current knowledge of the microorganisms involved in thermophilic glycerol conversion to methane is scarce, thermophilic glyceroldegrading methanogenic communities were enriched. A coculture of Thermoanaerobacter and Methanothermobacter species was obtained, pointing to a nonobligately syntrophic glycerol degradation. This hypothesis was further studied by incubating Thermoanaerobacter brockii subsp. finnii and T. wiegelii with glycerol (10 mM) in pure culture and with different hydrogenotrophic methanogens. The presence of the methanogen accelerated glycerol fermentation by the two Thermoanaerobacter strains up to 3.3 mM day1, corresponding to 12 times higher volumetric glycerol depletion rates in the methanogenic cocultures than in the pure bacterial cultures. The catabolic pathways of glycerol conversion were identified by genome analysis of the two Thermoanaerobacter strains. NADH and reduced ferredoxin formed in the pathway are linked to proton reduction, which becomes thermodynamically favourable when the hydrogen partial pressure is kept low by the hydrogenotrophic methanogenic partner.The authors thank Ruben Gonçalves for preparing the thermophilic biomass and Andreia Salvador for the sup port with the microbial communities’ analysis. This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit, Project SAICTPAC/0040/2015 (POCI-01-0145-FEDER-016403) and BioTecNorte operation (NORTE-01-0145-FEDER 000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Opera cional Regional do Norte. The authors also acknowledge the financial support of FCT and European Social Fund through the grants attributed to C.P. Magalhaes (SFRH/BD/132845/2017) and A.L. Arantes (PD/BD/128030/2016).info:eu-repo/semantics/publishedVersio

Conversion of Cn-unsaturated into Cn-2-saturated LCFA can occur uncoupled from methanogenesis in anaerobic bioreactors

Fat, oils, and grease present in complex wastewater can be readily converted to methane, but the energy potential of these compounds is not always recyclable, due to incomplete degradation of long chain fatty acids (LCFA) released during lipids hydrolysis. Oleate (C18:1) is generally the dominant LCFA in lipid-containing wastewater, and its conversion in anaerobic bioreactors results in palmitate (C16:0) accumulation. The reason why oleate is continuously converted to palmitate without further degradation via β-oxidation is still unknown. In this work, the influence of methanogenic activity in the initial conversion steps of unsaturated LCFA was studied in 10 bioreactors continuously operated with saturated or unsaturated C16- and C18-LCFA, in the presence or absence of the methanogenic inhibitor bromoethanesulfonate (BrES). Saturated Cn-2-LCFA accumulated both in the presence and absence of BrES during the degradation of unsaturated Cn-LCFA, and represented more than 50\% of total LCFA. In the presence of BrES further conversion of saturated intermediates did not proceed, not even when prolonged batch incubation was applied. As the initial steps of unsaturated LCFA degradation proceed uncoupled from methanogenesis, accumulation of saturated LCFA can be expected. Analysis of the active microbial communities suggests a role for facultative anaerobic bacteria in the initial steps of unsaturated LCFA biodegradation. Understanding this role is now imperative to optimize methane production from LCFA.European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement No 323009, and the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), and Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). We also thank the Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO

Effect of sulfate and iron (III) on LCFA degradation by a methanogenic community

[Excerpt] Under anaerobic conditions long chain fatty acids (LCFA) can be converted to methane by syntrophic bacteria and methanogenic archaea. LCFA degradation was also reported in the presence of alternative hydrogenotrophic partners, such as sulfate-reducing bacteria (SRB) and iron-reducing bacteria (IRB), which generally show higher affinity for H2 than methanogens and are more resistant to LCFA [1,2,3]. Their presence in a microbial culture degrading LCFA can be advantageous to reduce LCFA toxicity towards methanogens, although high concentrations of external electron acceptor (EEA) can lead to outcompetition of methanogens and cease methane production. In this work, we tested the effect of adding sub-stoichiometric concentrations of sulfate and iron(III) to methanogenic communities degrading LCFA. (...