An ethanol-based process to simultaneously extract and fractionate carotenoids from Mauritia flexuosa L. Pulp

Mauritia vinifera (buriti) is a palm tree that grows wild in different areas of Brazil, particularly in the Amazonian region. The buriti oil is rich in carotenoids, especially in β-carotene. The growing interest in other natural sources of β-carotene has stimulated the industrial use of buriti as a raw material for pulp oil extraction. Most processes are based on the conventional technologies, involving drying and pressing the pulp for oil recovery and further separation of carotenoids in a liquid phase using organics solvents. In the present work, the ethanol-based process was evaluated for simultaneous carotenoids recovering and fractionating from buriti pulp. The raw material and ethanol, 1:4 ratio, were placed in an erlenmeyer flask and maintained at 30rpm for 1 hour in a temperature-controlled bath at 65ºC. The mixture was filtered under vacuum and cooling at 10ºC to allow for the separation of the solvent in two phases. Carotenoids composition, determined by HPLC, has indicated a β-carotene concentration about 12 times greater in the lower phase than in the upper phase. The profile of the carotenoids in the denser phase is quite similar to that of raw buriti oil, and the concentration of total carotenoids is 40% higher than that of the original raw oil, making the ethanol-based process particularly attractive for industrial applications.

Composição de carotenoides em passifloras do cerrado

O objetivo foi avaliar, por HPLC, a composição qualitativa e quantitativa de carotenoides em maracujás do cerrado. Frutos procedentes de acessos nativos de quatro espécies (Passiflora cincinnata, P. nitida, P. setacea e P. edulis) foram analisados, utilizando, como referência, o maracujá-amarelo comercial (P. edulis). As polpas de maracujá apresentaram neoxantina, violaxantina, cis-violaxantina, anteraxantina, luteína, zeaxantina, β-criptoxantina, poli-cis-caroteno, prolicopeno, cis-ζ-caroteno, trans-ζ-caroteno, trans-β-caroteno, 13-cis-β-caroteno e fitoflueno. Em geral, os teores de carotenoides entre as espécies e entre os acessos da mesma espécie foram significativamente diferentes. A espécie P. edulis apresentou o maior número de carotenoides, com diferença entre os acessos. Em um acesso de P. edulis comercial, foi encontrado o trans-β-caroteno como o carotenoide principal (7,8±0,8 µg g-1) e no outro o trans-ζ-caroteno (11,4±0,4 µg g-1). Dois acessos de P. edulis nativos do Cerrado apresentaram cis-ζ-caroteno como carotenoide majoritário (6,28±0,15 µg g-1 e 12,1±0,7 µg g-1, casca amarela e roxa, respectivamente). O perfil de carotenoides em frutos de espécies de maracujá apresentou diversidade de composição, com potencial de uso para melhoramento genético para agregar maior valor ao produto e estimular o seu consumo.

Carotenoids profile and total polyphenols in fruits of Pereskia aculeata Miller

Pereskia aculeata Mill. (Ora-pro-nóbis) is a native cactaceae from tropical America, whose leaves have high protein content. In Brazil it is found in all territorial extension between the states of Bahia and Rio Grande do Sul. Most studies have focused on chemical characterization of the leaves of this specie. The objective was to assess the carotenoids profile and the total polyphenols present in the fruits of P. aculeate. Carotenoids were determined by HPLC-PAD (high performance liquid chromatography - photodiode array detector), total polyphenols were determined by Folin-Ciocalteu and vanillin methods. Trans-β-carotene was the main carotenoid, followed by α-carotene, lutein and other minor carotenoids. It was found 64.9 ± 1.1 mg.100g-1 of gallic acid equivalent, 14.8 ± 0.2 mg.100g-1 of catechin equivalent. Carotenoid identification of P. aculeate fruits are presented here by the first time and indicate that these fruits can be researched as source of bioactive substances, especially antioxidant and provitamin A carotenoids.

Päällystetyn graafisen kartongin hajuominaisuudet

Diplomityön tavoitteena oli selvittää elintarvikepakkauksiin soveltuvan graafisen kartongin potentiaalinen hajun aiheuttaja. Lisäksi tavoitteena oli vähentää päällystetyn tasakoosteisen (SBS) kolmikerroskartongin hajutasoa keskittyen päällystyspastakomponentteihin sekä massaosaston jälkeen annosteltaviin kemikaaleihin. Kirjallisuusosassa tarkasteltiin eri kartonkilajeja, päällystyspastoja ja sen sisältämiä komponentteja, pääpainon ollessa kuitenkin kartongin hajuominaisuuksiin vaikuttavien tekijöiden selvittämisessä. Lisäksi luotiin katsaus kartonkien aistinvaraisiin ja instrumentaalisiin määritysmenetelmiin. Instrumentaalisista määritysmenetelmistä käsiteltiin headspace –kaasukromatografia (HSGC) sekä korkeanerotuskyvyn nestekromatografia yhdistettynä massaspektrometriin (HPLC/MS). Kokeellisen osan alussa kartoitettiin päällystyskemikaalien sekä liima- ja retentioaineiden haihtuvat yhdisteet HSGC- ja HPLC/MS –tekniikoiden avulla. Kaasukromatografisesti analysoidut kokonaishaihtuvien tasot eivät poikenneet normaaleista tasoista, joten jouduttiin siirtymään HPLC/MS –menetelmään. HPLC/MS –tekniikalla on päästy tutkimaan herkästi haihtuvien typpiyhdisteiden amiinipitoisuuksia. Kemikaalimittausten perusteella vaihdettiin potentiaaliset hajua aiheuttavat kemikaalit, kovete ja synteettinen paksuntaja, markkinoilla oleviin vaihtoehtoisiin kemikaaleihin ja suoritettiin pilot -koeajo sekä tehdasmittakaavaiset koeajot. Työssä perehdyttiin myös varastoinnin aiheuttamiin vaikutuksiin eri kartonkilaatujen aistinvaraisissa ominaisuuksissa. Päällystyspastan kemikaaleista suuren hajukuorman aiheuttavat kovete, dispergointiaine, päällystyspigmentit ja lateksit sekä synteettinen paksuntaja. Kovetteen annostelumäärä päällystyspastoihin on alhainen, mutta sen sisältämä ammoniakkipitoisuus on kuitenkin huomattavan suuri. Kovete ohjaakin lopputuotteen ammoniakkipitoisuutta. Merkittävän metyyliamiinikuorman sisältää dispergointiaine. Sen vaikutus lopputuotteeseen voidaan olettaa vähäiseksi johtuen kemikaalin pienestä määrästä pastassa. Päällystyspigmenttien ja lateksien korkea metyyliamiinikuorma voi puolestaan aiheuttaa lopputuotteeseen huomattavan hajukuorman johtuen niiden suurista annostelumääristä. Eri synteettisille paksuntajille tehtyjen tutkimusten mukaan niiden hajukuormat olivat hyvin samankaltaisia ja vaikutus lopputuotteen kokonaishajutasoon jäi pieneksi. Aistinvaraisesti tarkasteltuna eri kartonkilaaduille saadaan eri hajutasoja. Elintarvikepakkauksiin soveltuvan graafisen kartongin hajutasot olivat korkeita muihin kartonkilaatuihin nähden niin tuoreena kuin varastoinnin jälkeen. Erityisesti tuoreen kartonkinäytteen metyyliamiinipitoisuus ylitti hajukynnyksen. Käytetty lateksi, kovete ja synteettinen paksuntaja näyttävät muodostavan niin tiiviin hajua koteloivan päällystekerroksen kartongin pinnalle, että hajua vapautuu pitkällä aikavälillä. Myös varastointi tiiviinä pakkauksena estää amiinien haihtumisen. Tutkimusten mukaan ammoniakkivapaalla PZC –kovetteella saadaan alennettua graafisen kartongin ammoniakkikuormaa huomattavasti. Tämä on vaikuttamassa myös lopputuotteen hajutasoon, jota on mahdollisuus alentaa käyttämällä ammoniakkivapaata kovetetta.

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η6-p-cym)RuCl(PPh3)2]+, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N′-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.

High Throughput Purification of Combinatorial Libraries by Preparative LC/MS

Viime vuosien nopea kehitys on kiihdyttänyt uusien lääkkeiden kehittämisprosessia. Kombinatorinen kemia on tehnyt mahdolliseksi syntetisoida suuria kokoelmia rakenteeltaan toisistaan poikkeavia molekyylejä, nk. kombinatorisia kirjastoja, biologista seulontaa varten. Siinä molekyylien rakenteeseen liittyvä aktiivisuus tutkitaan useilla erilaisilla biologisilla testeillä mahdollisten "osumien" löytämiseksi, joista osasta saatetaan myöhemmin kehittää uusia lääkeaineita. Jotta biologisten tutkimusten tulokset olisivat luotettavia, on syntetisoitujen komponenttien oltava mahdollisimman puhtaita. Tämän vuoksi tarvitaan HTP-puhdistusta korkealaatuisten komponenttien ja luotettavan biologisen tiedon takaamiseksi. Jatkuvasti kasvavat tuotantovaatimukset ovat johtaneet näiden puhdistustekniikoiden automatisointiin ja rinnakkaistamiseen. Preparatiivinen LC/MS soveltuu kombinatoristen kirjastojen nopeaan ja tehokkaaseen puhdistamiseen. Monet tekijät, esimerkiksi erotuskolonnin ominaisuudet sekä virtausgradientti, vaikuttavat preparatiivisen LC/MS puhdistusprosessin tehokkuuteen. Nämä parametrit on optimoitava parhaan tuloksen saamiseksi. Tässä työssä tutkittiin emäksisiä komponentteja erilaisissa virtausolosuhteissa. Menetelmä kombinatoristen kirjastojen puhtaustason määrittämiseksi LC/MS-puhdistuksen jälkeen optimoitiin ja määritettiin puhtaus joillekin komponenteille eri kirjastoista ennen puhdistusta.

Urinary and plasma catecholamines and metanephrines in dogs with pheochromocytoma, hypercortisolism, nonadrenal disease and in healthy dogs.

BACKGROUND: Diagnosis of pheochromocytoma (PC) is based on a combination of clinical suspicion, finding an adrenal mass, increased plasma, and urine concentrations of catecholamine metabolites and is finally confirmed with histopathology. In human medicine, it is controversial whether biochemically testing plasma is superior to testing urine. OBJECTIVES: To measure urinary and plasma catecholamines and metanephrines in healthy dogs, dogs with PC, hypercortisolism (HC), and nonadrenal diseases (NAD) and to determine the test with the best diagnostic performance for dogs with PC. ANIMALS: Seven PC dogs, 10 dogs with HC, 14 dogs with NAD, 10 healthy dogs. METHODS: Prospective diagnostic clinical study. Urine and heparin plasma samples were collected and stored at -80°C before analysis using high-pressure liquid chromatography (HPLC) coupled to electrochemical detection or tandem mass spectrometry were performed. Urinary variables were expressed as ratios to urinary creatinine concentration. RESULTS: Dogs with PC had significantly higher urinary normetanephrine and metanephrine : creatinine ratios and significantly higher plasma-total and free normetanephrine and plasma-free metanephrine concentrations compared to the 3 other groups. There were no overlapping results of urinary normetanephrine concentrations between PC and all other groups, and only one PC dog with a plasma normetanephrine concentration in the range of the dogs with HC and NAD disease. Performances of total and free plasma variables were similar. Overlap of epinephrine and norepinephrine results between the groups was large with both urine and plasma. CONCLUSION AND CLINICAL IMPORTANCE: Measurement of normetanephrine is the preferred biochemical test for PC and urine was superior to plasma.

Marjojen flavonoidien uuttaminen

Flavonoidit ovat kasvien polyfenolisia sekundäärimetaboliitteja, jotka koostuvat 15-hiilisestä C6-C3-C6-perusrungosta. Niillä on havaittu useita terveyden kannalta edullisia ominaisuuksia kuten antioksidatiivisia, antikarsinogeenisia, antiallergeenisia, anti-inflammatorisia, estrogeenisia sekä antimikrobiaalisia vaikutuksia. Kasvimateriaalia käyttävän elintarviketeollisuuden sivuvirrat ovat flavonoidi-pitoisten jakeiden arvokkaita raaka-aineita. Tässä työssä tarkoituksena oli tutkia marjojen puristusjäännöksen sisältämien flavonoidien uuttamista ja uuttautumiseen vaikuttavia tekijöitä kuten liuottimen koostumusta ja määrää, uuttolämpötilaa ja -aikaa sekä liuottimen lisäaineita. Uutteet analysointiin HPLC:lla käyttäen ODS-2-kolonnia ja gradienttieluointia. Komponentit detektoitiin niille tyypillisillä UV-VIS-aallonpituuksilla ja identifioitiin vertaamalla flavonoidiprofiililtaan tunnettujen marjojen kromatogrammeihin. Antosyaanien kvantitatiiviseen analysointiin käytettiin spektrofotometrista pH-eromenetelmää ja tulokset ilmoitettiin syanidiini-3-glukosidina. Uutteiden kestävyyttä tutkittiin säilyttämällä näytteitä eri olosuhteissa sekä lisäämällä niihin uuton saantoon ja komponenttien säilyvyyteen vaikuttavia aineita. Useimmat tutkituista hapoista paransivat flavonoidien saantoa ja uutteiden säilyvyyttä, mutta esim. askorbiinihapon lisääminen nopeutti tiettyjen uutteen sisältämien flavonoidien tuhoutumista.

Processamento de frutos de abacaxizeiro cv smooth cayenne: perfil de açúcares e ácidos dos sucos e composição nutricional da farinha de cascas

O desenvolvimento de tecnologias que permitam o aproveitamento integral dos frutos é de grande importância para as indústrias. Uma possibilidade de valorização da cultura do abacaxi seria o estímulo ao processamento do fruto para a obtenção do suco associado ao aproveitamento das cascas para a elaboração de farinha, produto de mercado crescente. Um dos fatores de grande importância para a aceitação do suco é o sabor, que se encontra diretamente relacionado ao perfil de ácidos e açúcares presentes. Nesta linha, este trabalho visou a processar frutos de abacaxi cv Smooth Cayenne colhidos em dois estádios de maturação para a obtenção e a caracterização do suco e da farinha de cascas. Os sucos obtidos foram analisados em HPLC para o perfil de açúcares e ácidos orgânicos. As farinhas de casca foram analisadas para rendimento, umidade, cinzas, fibras solúvel e insolúvel, lipídeos, proteína e carboidratos totais. Os resultados obtidos mostraram que frutos em estádio mais avançado de maturação produzem sucos com maior teor de açúcares e menor teor de ácidos, sendo o principal ácido o cítrico, e os açúcares frutose, glicose e sacarose. As farinhas de abacaxi apresentam-se como fontes de fibras insolúveis.

Secondary metabolismin in Annonaceae: potencial source of drugs

Several species of Annona (Annonaceae) are used in traditional Mexican medicine by their anti-anxiety, anticonvulsant and tranquilizing properties. It has been reported that the alkaloids isolated from some species of the Annona have affinity to serotonergic 5-HT1A receptors and modulate dopaminergic transmission, which is involved in depressive disorders. In this review it is showed the results of the antidepressant-like effect of an alkaloid extract from the aerial parts of Annona cherimola (TA) in mice. The antidepressant-like effect was evaluated in the forced swimming test. To elucidate a possible mechanism of action, experiments of synergism with antidepressant drugs, such as imipramine (IMI), clomipramine (CLIMI), and fluoxetine (FLX), were carried out. The neurotransmitter content (DA: dopamine, 5HT: serotonin and its metabolites, HVA: homovanillic acid and 5HIAA:5-hydroxyindoleacetic) in the whole brain of mice were also determined by HPLC method. The results showed that repeated treatment with TA produced antidepressant-like effects in mice. This effect was not related to an increase in locomotor activity. Administration of TA facilitated the antidepressant effect of IMI and CLIMI as well as increased the turnover of DA and 5-HT. The alkaloids: 1,2-dimethoxy-5, 6.6 to 7-tetrahydro-4H-dibenzoquinoline-3,8,9,10-tetraol, anonaine, liriodenine, and nornuciferine were the main constituents of TA.

Brown algal phlorotannins: Improving and applying chemical methods

Phlorotannins are the least studied group of tannins and are found only in brown algae. Hitherto the roles of phlorotannins, e.g. in plant-herbivore interactions, have been studied by quantifying the total contents of the soluble phlorotannins with a variety of methods. Little attention has been given to either quantitative variation in cell-wall-bound and exuded phlorotannins or to qualitative variation in individual compounds. A quantification procedure was developed to measure the amount of cell-wall-bound phlorotannins. The quantification of soluble phlorotannins was adjusted for both large- and small-scale samples and used to estimate the amounts of exuded phlorotannins using bladder wrack (Fucus vesiculosus) as a model species. In addition, separation of individual soluble phlorotannins to produce a phlorotannin profile from the phenolic crude extract was achieved by high-performance liquid chromatography (HPLC). Along with these methodological studies, attention was focused on the factors in the procedure which generated variation in the yield of phlorotannins. The objective was to enhance the efficiency of the sample preparation procedure. To resolve the problem of rapid oxidation of phlorotannins in HPLC analyses, ascorbic acid was added to the extractant. The widely used colourimetric method was found to produce a variation in the yield that was dependent upon the pH and concentration of the sample. Using these developed, adjusted and modified methods, the phenotypic plasticity of phlorotannins was studied with respect to nutrient availability and herbivory. An increase in nutrients decreased the total amount of soluble phlorotannins but did not affect the cell-wall-bound phlorotannins, the exudation of phlorotannins or the phlorotannin profile achieved with HPLC. The presence of the snail Thedoxus fluviatilis on the thallus induced production of soluble phlorotannins, and grazing by the herbivorous isopod Idotea baltica increased the exudation of phlorotannins. To study whether the among-population variations in phlorotannin contents arise from the genetic divergence or from the plastic response of algae, or both, algae from separate populations were reared in a common garden. Genetic variation among local populations was found in both the phlorotannin profile and the content of total phlorotannins. Phlorotannins were also genetically variable within populations. This suggests that local algal populations have diverged in their contents of phlorotannins, and that they may respond to natural selection and evolve both quantitatively and qualitatively.

Migration of antimony from polyethylene terephthalate used in mineral water bottles.

The influence of storage time and temperature on Sb migration from PET bottles into mineral water was studied in short-term tests lasting up to 15 days and long-term studies lasting up to 220 days. Samples purchased were stored in three different coloured bottles: clear (CL), light blue (LB) and dark blue (DB). Sb migration was assayed by HG-AFS for total determination and HPLC-ICP-MS for speciation analysis. Migration studies showed that waters stored at 4 and 20 oC were not subject to Sb migration. At 40 oC there was a significant increase in Sb concentration, although the maximum limit established by the European Union (5.0 ug/L) was not exceeded, whereas at 60 oC samples were subject to considerable Sb migration after 30 days of storage. In this case, the maximum limit established by the European Union was exceeded and both Sb (V) and Sb (III) were detected.

Development, physical-chemical stability and release studies of four alcohol-free spironolactone suspensions for use in pediatrics

Dissolution studies have become of great significance because, in most cases, drug dissolution is the rate-limiting step in the absorption process. As occurs with solid oral dosage forms, heterogeneous disperse systems (suspensions) could also have some problems with their in vitro dissolution. The objective of this study was to evaluate influence of the excipients on the release of spironolactone from four alcohol free suspensions (pharmaceutical compounding) of spironolactone 5 mg/mL suitable for pediatric use. Also the comparison of the physical and chemical stability of the suspensions stored at 4, 25 and 40 ºC over a 60- day period has been studied. Rheological behavior, particle size, a prediction of long-term physical stability, pH and assay of spironolactone by HPLC were assessed at prefixed times. The dissolution profile of each suspension was determined and compared with that of the commercial tablets. A microbiological study of the best formula was also performed. Chemically, the four spironolactone suspensions were stable for 60 days stored at three temperatures; Suspension IV had optimum pH values and the highest recovery percentage. In terms of physical stability, sedimentation occurred in Suspension IV and flotation of spironolactone in Suspensions I, II and III. Suspension III had the highest viscosity and the slowest drug release. Suspension IV was also microbiologically stable for 60 days. In conclusion, Suspension IV had the best properties and the least suitable form was Suspension III, as its high viscosity made it difficult to achieve homogeneous redispersion, and it had the slowest dissolution profile.

L-tyrosine and nitric oxide synergize to prevent cytotoxic effects of superoxide.

We found previously that the nitric oxide donor DEA/NO enhanced lipid peroxidation, DNA fragmentation, and cytotoxicity in human bronchial epithelial cells (BEAS-2B) when they were cultured in LHC-8 medium containing the superoxide-generating system hypoxanthine/xanthine oxidase (HX/XO). We have now discovered that DEA/NO's prooxidant action can be reversed by raising the L-tyrosine concentration from 30 to 400 microM. DEA/NO also protected the cells when they were cultured in Dulbecco's Modified Eagle's Medium (DMEM), whose standard concentration of L-tyrosine is 400 microM. Similar trends were seen with the colon adenoma cell line CaCo-2. Since HPLC analysis of cell-free DMEM or LHC-8 containing 400 microM L-tyrosine, DEA/NO, and HX/XO revealed no evidence of L-tyrosine nitration, our data suggest the existence of an as-yet uncharacterized mechanism by which L-tyrosine can influence the biochemical and toxicological effects of reactive nitrogen species.

Discriminação quiral por CLAE em carbamatos de polissacarídeos: desenvolvimento, aplicações e perspectivas

The importance of chromatographic separation of enantiomers has been acknowledged. This review surveys the development, application and perspectives of polysaccharide carbamates as chiral stationary phase for HPLC.