A Comparison Between Characterization And Biological Properties Of Brazilian Fresh And Aged Propolis.

As propolis is a highly valued bee product, we aimed to verify the quality of aged propolis, investigating their phenolic and flavonoid composition, levels of toxic metals, radical scavenging and antimicrobial activities. Samples of fresh and aged propolis of six different beekeepers, from the same geographical location, were investigated in terms of their phenolic and flavonoid composition and levels of Pb, Cd, and Cr, as well as radical scavenging and antimicrobial activities. The two groups of propolis had similar qualitative composition by HPLC-PDA and ESI(-)-MS. Fresh propolis and aged propolis show no differences when average values of extraction yield, flavonoids, EC50, or MIC were compared and both types of propolis showed good antimicrobial activity at low concentrations. Only levels of phenolic compounds were higher in fresh propolis. The propolis samples considered in this study, aged or fresh, had similar qualitative composition, although they were collected in different periods. Samples only differed in their levels of total phenolic content. Moreover, aged propolis conserves significant radical scavenging and antimicrobial properties. We suggest that aged propolis should not be discarded but explored for alternative applications.

Pharmacokinetic And Pharmacodynamic Evaluation Of A Nanotechnological Topical Formulation Of Lidocaine/prilocaine (nanorap) In Healthy Volunteers.

Nanorap is a new nanotechnological formulation for topical anesthesia composed of lidocaine (2.5%) and prilocaine (2.5%). The present study evaluated the pharmacokinetics (PK) of Nanorap. For the determination of lidocaine and prilocaine in human plasma a new method using high-performance liquid-chromatography coupled to tandem mass spectrometry was developed. Nanorap pharmacodynamic (PD) and its physical proprieties were also evaluated. Nanorap was administered by topical application of 2g to healthy volunteers and blood samples were collected for the PK analysis. The drugs were extracted from plasma by liquid-liquid extraction with ether/hexane (80/20, v/v). The chromatography separation was performed on a Genesis C18 analytical column 4 µm (100 x 2.1 mm i.d.) with a mobile phase of methanol/acetonitrile/water (40/30/30, for lidocaine, and 50/30/20, for prilocaine, v/v/v) + 2 mM of ammonium acetate and ropivacaine as internal standard. The drugs were quantified using a mass spectrometer with an electrospray source in the ESI positive mode (ES+) configured for multiple reaction monitoring. The PD of Nanorap was evaluated with the use of a visual analogue scale. Nanorap was characterized by cryofracture. The chromatography run time was 5.5 min for lidocaine and 3.3 min for prilocaine and the lower limit of quantification was 0.05 ng/mL for both drugs. Mean Cmax was 6.62 and 1.72 ng/mL for lidocaine and prilocaine, respectively. Median Tmax was 6.5 hours for both drugs. Nanocapsules had a mean size of 88nm and mean drug association of 92.5% and 89% for lidocaine and prilocaine, respectively. The PD study showed that Nanorap has a sufficient analgesic effect (>30% reduction in pain) after 10 minutes of application. A new simple, selective and sensitive method for determination of lidocaine and prilocaine in human plasma was developed. Nanorap generated safe plasma levels of the drugs and satisfactory analgesic effect.

Metal Ions Bound To The Human Milk Immunoglobulin A: Metalloproteomic Approach.

The presence of calcium, iron, and zinc bound to human milk secretory IgA (sIgA) was investigated. The sIgA components were first separated by two-dimensional polyacrylamide gel electrophoresis and then identified by electrospray ionization-tandem mass spectrometry (ESI MS MS). The metal ions were detected by flame atomic absorption spectrometry after acid mineralization of the spots. The results showed eight protein spots corresponding to the IgA heavy chain constant region. Another spot was identified as the transmembrane secretory component. Calcium was bound to both the transmembrane component and the heavy chain constant region, while zinc was bound to the heavy chain constant region and iron was not bound with the identified proteins. The association of a metal ion with a protein is important for a number of reasons, and therefore, the findings of the present study may lead to a better understanding of the mechanisms of action and of additional roles that sIgA and its components play in human milk.

Anthocyanin And Other Phenolic Compounds In Ceylon Gooseberry (dovyalis Hebecarpa) Fruits.

Ceylon gooseberry is a deep-purple exotic berry that is being produced in Brazil with great market potential. This work aimed to determine major phenolic compounds in this specie by HPLC-PDA-ESI/MS. Samples were collected in two different seasons. Pulp and skin were analyzed separately. Non-acylated rutinoside derivatives of delphinidin (∼60-63%) and cyanidin (∼17-21%) were major anthocyanins tentatively identified. All anthocyanins had higher concentration in skin than in pulp (64-82 and 646-534mg of cyaniding-3-glucoside equivalents/100g skin and pulp, respectively). Moreover, anthocyanin profile changed between sampling dates (p<0.05). Mainly for delphinidin-3-rutinoside which could be a result of season variation. In this specie, non-anthocyanin polyphenols represent less than 35% of total extracted polyphenols. The tentative identification proposed a flavonol and three ellagitannins as major compounds of the non-anthocyanin phenolics fraction. Finally, anthocyanin is the major phenolic class in this fruit and its composition and content are significantly affected by season.

Determination Of The Phenolic Composition From Brazilian Tropical Fruits By Uhplc-ms/ms.

Although Brazil is the third largest fruit producer in the world, several specimens consumed are not well studied from the chemical viewpoint, especially for quantitative analysis. For this reason and the crescent employment of mass spectrometry (MS) techniques in food science we selected twenty-two phenolic compounds with important biological activities and developed an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method using electrospray (ESI) in negative ion mode aiming their quantification in largely consumed Brazilian fruits (açaí-do-Amazonas, acerola, cashew apple, camu-camu, pineapple and taperebá). Multiple reaction monitoring (MRM) was applied and the selection of proper product ions for each transition assured high selectivity. Linearity (0.99580%), precision (CV<20%) and extraction recovery rate (>80%) were satisfactory and showed that the method provides an efficient protocol to analyze phenolic compounds in fruit pulp extracts.

Impact Of Drug Formulation And Free Platinum/cisplatin Ratio On Hypersensitivity Reactions To Cisplatin: Formulation Matters.

Use of cisplatin can induce type I hypersensitivity reactions that may also be linked to the quality of the drug utilized. We observed cases of hypersensitivity that appeared to be associated with the brand of cisplatin used. The aim of this study was to compare two different brands of cisplatin in relation to type I hypersensitivity reactions. Brand A was used in a tertiary care teaching hospital until 2012, and use of brand B started from January 2013, when the first hypersensitivity cases were observed. Patients were categorized based on symptom. Cisplatin of both brands was analysed by high-performance liquid chromatography (HPLC) and high-resolution electrospray ionization mass spectrometry (ESI-(+)-MS) and characterized according to US Pharmacopeia. There were no cases of hypersensitivity associated with the use of cisplatin brand A, whereas four of 127 outpatients that used cisplatin brand B were affected. The two brands were in accordance with the US Pharmacopeia parameters, and there was no significant difference in the total platinum levels between the two brands when analysed by HPLC. However, high-resolution ESI-(+)-MS analyses show that brand B contains approximately 2.7 times more hydrolysed cisplatin than brand A. The increase in the hydrolysed form of cisplatin found in brand B may be the cause of the hypersensitivity reaction observed in a subset of patients. We present the first study of the quality of drugs by high-resolution ESI-(+)-MS. Drug regulatory agencies and manufacturers should consider including measurement of hydrolysed cisplatin as a quality criterion for cisplatin formulations.

Bipolar Disorder: Recent Advances And Future Trends In Bioanalytical Developments For Biomarker Discovery.

In this manuscript we briefly describe bipolar disorder (a depressive and manic mental disease), its classification, its effects on the patient, which sometimes include suicidal tendencies, and the drugs used for treatment. We also address the status quo with regard to diagnosis of bipolar disorder and recent advances in bioanalytical approaches for biomarker discovery. These approaches focus on blood samples (serum and plasma) and proteins as the main biomarker targets, and use various strategies for protein depletion. Strategies include use of commercially available kits or other homemade strategies and use of classical proteomics methods for protein identification based on bottom-up or top-down approaches, which used SELDI, ESI, or MALDI as sources for mass spectrometry, and up-to-date mass analyzers, for example Orbitrap. We also discuss some future objectives for treatment of this disorder and possible directions for the correct diagnosis of this still-unclear mental illness.

Study of C- and O-glycosylflavones in sugarcane extracts using liquid chromatography: exact mass measurement mass spectrometry

The flavonoids present in sugarcane (Saccharum officinarum) extracts were analyzed by liquid chromatography - mass spectrometry (LC-MS), and a study of the fragmentation patterns of selected flavonoids was conducted using orthogonal acceleration time-of-flight electrospray ionization mass spectrometry (ESI-oa-ToF MS). Seven C- and O-glycosylflavones were identified in the extracts, namely, schaftoside, isoschaftoside, luteolin-8-C-(rhamnosylglucoside), vitexin, orientin, tricin-7-O-neohesperidoside and tricin-7-O-glucoside. Of these, five were identified in the absence of direct comparison with their respective standards. The described method also permitted the differentiation of the 6-C and 8-C isomeric flavones, schaftoside and isoschaftoside. The combination of fragmentation data and exact mass measurement showed to be complimentary to the HPLC-UV-MS techniques previously utilized for isomers discrimination in sugarcane studies.

The inducing NO-vasodilation by chemical reduction of coordinated nitrite ion in cis-[Ru(NO(2))L(bpy)(2)](+) complex

The synthesis of [Ru(NO(2)) L(bpy)(2)](+) (bpy = 2,2'-bipyridine and L = pyridine (py) and pyrazine (pz)) can be accomplished by addition of [Ru(NO) L(bpy) 2](PF(6))(3) to aqueous solutions of physiological pH. The electrochemical processes of [Ru(NO2) L(bpy) 2]+ in aqueous solution were studied by cyclic voltammetry and differential pulse voltammetry. The anodic scan shows a peak around 1.00 V vs. Ag/AgCl attributed to the oxidation process centered on the metal ion. However, in the cathodic scan a second peak around-0.60 V vs. Ag/AgCl was observed and attributed to the reduction process centered on the nitrite ligand. The controlled reduction potential electrolysis at-0.80 V vs. Ag/AgCl shows NO release characteristics as judged by NO measurement with a NO-sensor. This assumption was confirmed by ESI/MS(+) and spectroelectrochemical experiment where cis-[Ru(bpy)(2)L(H(2)O)](2+) was obtained as a product of the reduction of cis-[Ru(II)(NO(2)) L(bpy)(2)](+). The vasorelaxation observed in denuded aortic rings pre-contracted with 0.1 mu mol L(-1) phenylephrine responded with relaxation in the presence of cis-[RuII(NO2) L(bpy) 2]+. The potential of rat aorta cells to metabolize cis-[RuII(NO(2)) L(bpy)(2)](+) was also followed by confocal analysis. The obtained results suggest that NO release happens by reduction of cis-[RuII(NO(2)) L(bpy)(2)](+) inside the cell. The maximum vasorelaxation was achieved with 1 x 10(-5) mol L(-1) of cis-[RuII(NO(2)) L(bpy)(2)](+) complex.

Spatial genetic structure of Hymenaea stigonocarpa Mart. ex Hayne assessed with chloroplast microsatellite markers

The use of chloroplast DNA markers (cpDNA) helps to elucidate questions related to ecology, evolution and genetic structure. The knowledge of inter-and intra-population genetic structure allows to design effective conservation and management strategies for tropical tree species. With the aim to help the conservation of Hymenaea stigonocarpa of the Cerrado (Brazilian savanna) in Sao Paulo State, an analysis of the spatial genetic structure (SGS) was conducted in two populations using five universal chloroplast microsatellite loci (cpSSR). The population of 68 trees of H. stigonocarpa in the Ecological Station of Itirapina (ESI) had a single haplotype, indicating a strong founder effect. In turn, the population of 47 trees of H. stigonocarpa in a contiguous area that includes the Ecological Station of Assis and the Assis State Forest (ESA), showed six haplotypes ((n) over cap (h) = 6) with a moderate haplotype diversity ((h) over cap = 0667 + 0094), revealing that it was founded by a small number of maternal lineages. The SGS analysis for the population ESA/ASF, using Moran`s I index, indicated limited seed dispersal. Considering SGS, for ex situ conservation strategies in the population ESA/ASF, seed harvesting should require a minimum distance of 750 m among seed-trees.

Saxitoxins accumulation by freshwater tilapia (Oreochromis niloticus) for human consumption

The accumulation of saxitoxins (STXs) in fish from freshwater aquaculture was investigated for the first time in the present study. Cyanotoxins have been monitored in liver and muscle samples of Oreochromis miloticus by chromatographic methods, both before and after the deputation process. The results show that tilapia can accumulate STXs. Our findings suggest that deputation with clean water is an alternative process to eliminate STXs from fish and, therefore, improve the safety of tilapia for consumers. (C) 2009 Elsevier Ltd. All rights reserved.

Sweet Basil (Ocimum basilicum) Extracts Obtained by Supercritical Fluid Extraction (SFE): Global Yields, Chemical Composition, Antioxidant Activity, and Estimation of the Cost of Manufacturing

In this work, supercritical technology was used to obtain extracts from Ocimum basilicum (sweet basil) with CO(2) and the cosolvent H(2)O at 1, 10, and 20% (w/w). The raw material was obtained from hydroponic cultivation. The extract`s global yield isotherms, chemical compositions, antioxidant activity, and cost of manufacturing were determined. The extraction assays were done for pressures of 10 to 30 MPa at 303 to 323 K. The identification of the compounds present in the extracts was made by GC-MS and ESI-MS. The antioxidant activity of extracts was determined using the coupled reaction of beta-carotene and linolenic acid. At 1% of cosolvent, the largest global yield was obtained at 10 MPa and 303 K (2%, dry basis-d.b.); at 10% of cosolvent the largest global yield was obtained at 10 and 15 MPa (11%, d.b.), and at 20% of cosolvent the largest global yield was detected at 30 MPa and 303 K (24%, d.b.). The main components identified in the extracts were eugenol, germacrene-D, epi-alpha-cadinol, malic acid, tartaric acid, ramnose, caffeic acid, quinic acid, kaempferol, caffeoylquinic acid, and kaempferol 3-O-glucoside. Sweet basil extracts exhibited high antioxidant activity compared to beta-carotene. Three types of SFE extracts from sweet basil were produced, for which the estimated cost of manufacturing (class 5 type) varied from US$ 47.96 to US$ 1,049.58 per kilogram of dry extract.

Regiospecific synthesis of 5-trichloromethyl-1H-pyrazole and 1H-pyrazole-5-carboxylic ester derivatives

Reaction of 1,1,1-trichloro-4-methoxy-3-penten-2-one (1) with hydrazines (2a-h) (NH2NHR, R = H, Me, t-Bu, Ph, 4-NO2-C6H4, C6F5, CO2Me, CONH2) under differing conditions regiospecifically affords different pyrazole derivatives, 3-methyl-5-trichloromethyl-5-hydroxy-4,5-dihydropyrazoles (3a, d-h), 3-methyl-5-trichloromethyl-1H-pyrazoles (4a,b,d-g) and 5-carboxyethyl-3-methyl-1H-pyrazoles (5a-e). The structural assignments were based on the analysis of their H-1/C-13 NMR and ESI-MS data.

Effect of Toasting Intensity at Cooperage on Phenolic Compounds in Acacia (Robinia pseudoacacia) Heartwood

The phenolic composition of heartwood from Robinia pseudoacacia, commonly known as false acacia, before and after toasting in cooperage was studied by HPLC-DAD and HPLC-DAD/ESI-MS/MS. A total of 41 flavonoid and nonflavonoid compounds were identified, some tentatively, and quantified. Seasoned acacia wood showed high concentrations of flavonoid and low levels of nonflavonoid compounds, the main compounds being the dihydroflavonols dihydrorobinetin, fustin, tetrahydroxy, and trihydroxymethoxy dihydroflavonol, the flavonol robinetin, the flavanones robtin and butin, and a leucorobinetinidin, none of which are found in oak wood. The low molecular weight (LMW) phenolic compounds present also differed from those found in oak, since compounds with a beta-resorcylic structure, gallic related compounds, protocatechuic aldehyde, and some hydroxycinnamic compounds are included, but only a little gallic and ellagic acid. Toasting changed the chromatographic profiles of extracts spectacularly. Thus, the toasted acacia wood contributed flavonoids and condensed tannins (prorobinetin type) in inverse proportion to toasting intensity, while LMW phenolic compounds were directly proportional to toasting intensity, except for gallic and ellagic acid and related compounds. Even though toasting reduced differences between oak and acacia, particular characteristics of this wood must be taken into account when considering its use in cooperage: the presence of flavonoids and compounds with beta-resorcylic structure and the absence of hydrolyzable tannins.

Mass Spectrometric Analysis of a Cyclic 7,8-Butanoguanine Adduct of N-Nitrosopyrrolidine: Comparison to Other N-Nitrosopyrrolidine Adducts in Rat Hepatic DNA

The well established rat hepatocarcinogen N-nitrosopytrolidine (NPYR, 1) requires metabolic activation to DNA adducts to express its carcinogenic activity. Among the NPYR-DNA adducts that have been identified, the cyclic 7,8-butanoguanine adduct 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purine-4(3H)-one (6) has been quantified using moderately sensitive methods, but its levels have never been compared to those of other DNA adducts of NPYR in rat hepatic DNA. Therefore, in this study, we developed a sensitive new LC-ESI-MS/MS-SRM method for the quantitation of adduct 6 and compared its levels to those of several other NPYR-DNA adducts formed by different mechanisms. The new method was shown to be accurate and precise, with good recoveries and low fmol detection limits. Rats were treated with NPYR by gavage at doses of 46, 92, or 184 mg/kg body weight and sacrificed 16 h later. Hepatic DNA was isolated and analyzed for NPYR-DNA adducts. Adduct 6 was by far the most prevalent, with levels ranging from about 900-3000 mu mol/mol Gua and responsive to dose. Levels of adducts formed from crotonaldehyde, a metabolite of NPYR, were about 0.2-0.9 mu mol/mol dGuo, while those of adducts resulting from reaction with DNA of tetrahydrofuranyl-like intermediates were in the range of 0.01-4 mu mol/mol deoxyribonucleoside. The results of this study demonstrate that, among typical NPYR-DNA adducts, adduct 6 is easily the most abundant in hepatic DNA. Since previous studies have shown that it can be detected in the urine of NPYR-treated rats, the results suggest that it is a potential candidate as a biomarker for assessing human exposure to and metabolic activation of NPYR.