Identification of the plant origin of the botanical biomarkers of Mediterranean type propolis

Propolis is a honeybee product which bees produce by collecting resins from various botanical sources. The chemical composition of propolis is directly dependant on the availability of resinous plant materials in different geographic regions. This study was undertaken to evaluate the resinous plant sources used by bees to produce Mediterranean type propolis. Although this propolis type has already been the subject of numerous studies, its major botanical source had not yet been identified. In this study, using GC-MS analysis, we identify the resin of the common cypress, Cupressus_sempervirens, as the major plant source of the characteristic diterpene fingerprint profile of Mediterranean propolis.     

Chemical composition of propolis from Canada, its antiradical activity and plant origin

The chemical composition of propolis from two regions in Canada was studied: Boreal forest and the Pacific coastal forest that lie outside the area of distribution of Aigeiros poplars, the usual propolis source plants. In the sample from Victoria, p-hydroxyacetophenone, benzyl hydroxybenzoate and cinnamic acid were the major components, accompanied by significant amounts of dihydrochalcones, which allowed the identification of its plant source: Populus trichocarpa of section Tacamahaca. Three dihydrochalcones were new for propolis. The sample from Richmond was characterized by large amounts of p-coumaric and cinnamic acid, typical for poplars of section Leuce, subsection Trepidae, its plant source was identified as P. tremuloides. Both samples showed a good radical scavenging activity against DPPH. Obviously, the Northern type propolis is a promising potential source of biologically active substances and deserves further investigation.     

Determination of the geographical origin of Brazilian wines by isotope and mineral analysis

In the present research, we studied wines from three different south Brazilian winemaking regions with the purpose of differentiating them by geographical origin of the grapes. Brazil's wide territory and climate diversity allow grape cultivation and winemaking in many regions of different and unique characteristics. The wine grape cultivation for winemaking concentrates in the South Region, mainly in the Serra Gaúcha, the mountain area of the state of Rio Grande do Sul, which is responsible for 90% of the domestic wine production. However, in recent years, two new production regions have developed: the Campanha, the plains to the south and the Serra do Sudeste, the hills to the southeast of the state. Analysis of isotopic ratios of ¹⁸O/¹⁶O of wine water, ¹³C/¹²C of ethanol, and of minerals were used to characterize wines from different regions. The isotope analysis of δ¹⁸O of wine water and minerals Mg and Rb were the most efficient to differentiate the regions. By using isotope and mineral analysis, and discrimination analysis, it was possible to classify the wines from south Brazil.     

Chemical composition of propolis from Canada, its antiradical activity and plant origin

The chemical composition of propolis from two regions in Canada was studied: Boreal forest and Pacific coastal forest that lay outside the area of distribution of Aigeiros poplars, the usual propolis source plants. In the sample from Victoria, p-hydroxyacetophenone, benzyl hydroxybenzoate and cinnamic acid were the major components, accompanied by significant amounts of dihydrochalcones, which allowed the identification of its plant source: Populus trichocarpa of Section Tacamahaca. Three dihydrochalcones were new for propolis. The sample from Richmond was characterized by large amounts of p-coumaric and cinnamic acid, typical for poplars of Section Leuce, subsection Trepidae, its plant source was identified as P. tremuloides. Both samples showed good radical scavenging activity against DPPH. Obviously, Northern type propolis is a promising potential source of biologically active substances and deserves further investigation.     

A reliable quantitative method for the analysis of phenolic compounds in Brazilian propolis by reverse phase high performance liquid chromatography

A sensitive and reliable RP-HPLC method was developed using a C18 CLC-ODS (M) - 4.6x250 mm(2)column and gradient elution for the analysis of phenolic compounds in propolis raw material and its products. A procedure for the extraction of phenolic compounds using aqueous ethanol (90%) with the addition of veratraldehyde as the internal standard (IS) was developed allowing to quantify ten compounds: caffeic acid, coumaric acid, ferulic acid, cinnamic acid, aromadendrin-4'-methyl ether (AME), isosakuranetin, drupanin, artepellin C, baccharin, and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran acid (DCBEN). The developed method gave good detection response and linearity in the range of 20.83-533.33 microg/mL.     

Isolation and structures of two new compounds from the essential oil of Brazilian propolis

Two new and seven known compounds, including terpenoids and aromatic compounds, were isolated from the essential oil of Brazilian propolis. The structures of the new compounds were elucidated as 2,2-dimethyl-8-prenyl-6-vinylchromene (1) and 2,6-diprenyl-4-vinylphenol (2) on the basis of spectroscopic analyses.     

Geographic origin of southern Brazilian wines by carbon and oxygen isotope analyses

We present a method that can differentiate between the varieties of grapes and the vintages of wines and show the relationship between the grapes, the wine and the geographic location. The place of origin and its geographic and climatic characteristics were determined by the isotopic ratios, ¹³C/¹²C of the ethanol and ¹⁸O/¹⁶O of the water content of wine (wine water), for southern Brazil wines. The producing subregions of Pinto Bandeira, Vale dos Vinhedos and Nova Pádua showed differences in the temperature, rainfall and humidity conditions used for the production under microvinification conditions of Merlot and Cabernet Sauvignon varieties, in the harvests of 2005 and 2006. An isotope ratio mass spectrometer coupled to an elemental analyzer was used to measure the ¹³C/¹²C of ethanol and the ¹⁸O/¹⁶O of wine water. Regardless of the grape variety used, it was possible to determine the subregion through measurement of the δ¹⁸O values in both harvests. The altitudes of the different subregions led to statistical differences and demonstrated an influence mainly on the δ¹⁸O values of wine water. The δ¹⁸O value of wine water was determined to be more selective for the determination of the cultivation subregions than the δ¹³C value of the ethanol. The altitude and latitude influenced mainly the δ ¹⁸O values of wine water and the ethanol. The climatic influences are more noteworthy in distinguishing the year of the harvest than the cultivation subregion. Copyright © 2010 John Wiley & Sons, Ltd.     

Optimization of CZE for analysis of phytochemical bioactive compounds

Advantages of CZE such as high efficiency, low cost, short analysis time, and easy implementation result in its wide applications for analysis of phytochemical bioactive compounds (e.g. flavonoids, alkaloids, terpenoids, phenolic acid, saponins, anthraquinones and coumarins). However, several aspects, including sample preparation, separation, and detection have significant effects on CZE analysis. Therefore, optimization of these procedures is necessary for development of the method. In this review, sample preparation such as extraction method and preconcentration, separation factors including buffer type, concentration and pH, additives, voltage and temperature, as well as detection, e.g. direct and indirect UV detection, LIF and MS were discussed for optimization of CZE analysis on phytochemical bioactive compounds. The optimized strategies were also reviewed.     

Direct evidence of crosslinks by metathesis degradation

The degradation of unsaturated polymers by olefin metathesis has already been applied to the investigation of crosslink structures. In the present paper, crosslinks in poly(butadiene-alt-propene), obtained by partial bromination with N-bromosuccinimide and reaction with 1,4-bis(bromomagnesium)butane, were investigated by this method. Metathesis degradation was carried out with (E)-4-octene using the catalyst WCl₆-Sn(CH₃)₄. The low-molecular-weight products were identified by combination of gas chromatography (GC) and mass spectrometry (MS).     

Trace element analysis in CRM of plant origin by inductively coupled plasma mass spectrometry

Three CRMs of plant origin (SRM 1515 Apple Leaves, SRM 1570a Trace Elements in Spinach Leaves, and SRM 1575 Pine Needles) were used for analytical quality assurance of Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Mo, Cd, and Pb determination by ICP-MS. The samples were decomposed using microwave assisted pressurized digestion by HNO₃ and HNO₃ + H₂O₂ mixture and temperature programmed dry ashing. Concentrations of elements in samples were evaluated by external calibration. All samples, blanks, and standards were spiked to 100 μg/L concentration of In and Bi as internal standards. During the measurement, signals of ⁵²Cr, ⁵³Cr, ⁵⁴Fe, ⁵⁵Mn, ⁵⁹Co, ⁶⁰Ni, ⁶²Ni, ⁶³Cu, ⁶⁵Cu, ⁶⁶Zn, ⁹⁵Mo, ¹¹¹Cd, ¹¹²Cd, ¹¹⁵In, ²⁰⁶Pb, ²⁰⁷Pb, ²⁰⁸Pb and ²⁰⁹Bi were monitored. Results of Pb, Cd, Cu, Zn, Mo and Co determination were in good agreement with certified values. In order to obtain accurate results for As at low levels, it was necessary to mathematically correct the analytical signal. This correction effectively eliminates the influence of interfering ArCl⁺ ions. For Cr analysis it is advisable to use as powerful oxidizing conditions during sample decomposition as possible.     

New CZE-DAD method for honeybee venom analysis and standardization of the product

The aim of this study was to develop a new precise and accurate CZE-DAD method for honeybee venom analysis using cytochrome c as an internal standard. The 64.5 cm total length, 56 cm effective length, 75 μm ID, and 360 μm OD uncoated fused-silica capillary was used. The samples were injected into the capillary under a 50-mbar pressure for 7 s. There were 15 kV of electric field across the capillary applied. The current intensity was 26 μA. The separation was carried out at 25 °C. The analysis was run with the normal electrode polarity. The following steps and parameters were taken into account for the validation of the developed method: selectivity, precision, accuracy, linearity, limit of detection and limit of quantitation. All steps of the validation procedure proved that the developed analytical procedure was suitable for its intended purpose. Possibly this was the first study in which several honeybee venom components were separated and five of them were identified by capillary zone electrophoresis. In addition, the developed method was applied for quantitative analysis of 38 honeybee venom samples. The content (relative to the dry venom mass) of analyzed peptides in honeybee venom samples collected in 2002–2007 was as follows: apamine from 0.93% to 4.34% (mean, 2.85 ± 0.79%); mast cell degranulating peptide (MCDP) from 1.46% to 4.37% (mean, 2.82 ± 0.64%); phospholipase A₂ from 7.41% to 20.25% (mean, 12.95 ± 3.09%); melittin from 25.40% to 60.27%, (mean, 45.91 ± 9.78%). The results were compared with the experimental data obtained for the same venom samples analyzed earlier by the HPLC method. It was stated that HPCE and HPLC data did not differ significantly and that the HPCE method was the alternative for the HPLC method. Moreover, using the results obtained principal component analysis (PCA) was applied to clarify the general distribution patterns or similarities of four major honeybee venom constituents collected from two different bee strains in various months and years. PCA has shown that the strain of bee appears to be the only criteria for bee venom sample classification. Strong correlations between apamine, MCDP, phospholipase A₂, and melittin were confirmed. These correlations have to be taken into account in the honeybee venom standardization. The developed method due to its simplicity can be easily automated and incorporated into routine operations both in the bee venom identification, quality control, and standardization of the product.     

Polarographic behavior of flavanoids from propolis and their potential carcinogenity

The electrochemical reduction of 12 flavanoids isolated from propolis and synthetic 7,8-benzoflavone was investigated in anhydrous dimethylformamide (DMF) in the absence or presence of the growth factor α-lipoic acid. In anhydrous conditions α-lipoic acid influences the polarographic reduction of compounds with carcinogenic activity. The same effect is not observed with non-carcinogens. In the presence of α-lipoic acid in the polarographed solution of carcinogen, a new polarographic wave is observed. This wave increases linearly with increasing concentration of α-lipoic acid. This increase was used to evaluate the potential carcinogenity of the compounds examined by polarography. The index of potential carcinogenity tan α was estimated for all flavanoids investigated. The relationship between chemical structure and potential carcinogeneity was analyzed. Values of tan α indicating possible mutagenic or carcinogenic properties were found only for quercetin, rhamnetin and kempferol.     

Direct observation for the cyclization of a diarylcarbonyl oxide

Trapping and laser flash spectroscopic experiments showed that the cyclization of diphenylcarbonyl oxide is turned into a very facile process by introducing p-methoxyl substituent, the lifetime of which is as short as 10⁻⁸ s. The trapping with diphenylsulfide and -sulfoxide suggested the intervention of a dioxirane intermediate.     

Multivariate analysis of nutritional information of foodstuff of plant origin for the selection of representative matrices for the analysis of pesticide residues

Testing safety of foodstuffs of plant origin involves the analysis of hundreds of pesticide residues. This control is only cost-effective through the use of methods validated for the analysis of many thousands of analyte/matrix combinations. Several documents propose representative matrices of groups of matrices from which the validity of the analytical method can be extrapolated to the represented matrices after summarised experimental check of within group method performance homogeneity. Those groups are based on an evolved expert consensus based on the empirical knowledge on the current analytical procedures; they are not exhaustive, they are not objectively defined and they propose a large list of representative matrices which makes their application difficult. This work proposes grouping 240 matrices, based on the nutritional composition pattern equivalence of the analytical portion right after hydration and before solvent extraction, aiming at defining groups that observe method performance homogeneity. This grouping was based on the combined outcome of three multivariate tools, namely: Principal Component Analysis, Hierarchical Cluster Analysis and K-Mean Cluster Analysis. These tools allowed the selection of eight groups for which representative matrices with average characteristics and objective criteria to test inclusion of new matrices were established. The proposed matrices groups are homogeneous to nutritional data not considered in their definition but correlated with the studied multivariate nutritional pattern. The developed grouping that must be checked with experimental test before use was tested against small deviations in food composition and for the integration of new matrices.     

Direct observation of the target cell for jasmonate-type leaf-closing factor: genus-specific binding of leaf-movement factors to the plant motor cell

We report the synthesis of the novel fluorescence-labeled jasmonate glycoside 2 based on β-d-glucopyranosyl 12-hydroxyjasmonate 1, which is a leaf-closing substance of Albizzia julibrissin Durazz. The fluorescence study using 2 revealed that the target cell for 1 is a motor cell. Probe 2 bound to the motor cells of two plants belonging to genus Albizzia. This result suggested that a receptor for 2, which is common among genus Albizzia would be involved in the nyctinastic leaf movement.     

Direct observation of molecular rotation-translation coupling by far-infrared spectroscopy

The differences between the far-infrared absorption of liquid (+)3-methylcyclohexanone and the racemic mixture are interpreted as supporting evidence for the direct observation of rotation-translation coupling.     

Direct observation of substrate-enzyme complexation by surface forces measurement

The substrate-enzyme complexation of heptaprenyl diphosphate synthase was directly investigated using colloidal probe atomic force microscopy (AFM) and a quartz crystal microbalance (QCM) in order to obtain new insights into the molecular mechanism of the enzyme reaction. This enzyme is composed of two dissociable subunits that exhibit a catalytic activity only when they are associated together in the presence of a cofactor, Mg2+, and a substrate, farnesyl diphosphate (FPP). The QCM measurement revealed that FPP was preferentially bound to subunit II in the presence of Mg2+, while the AFM measurement showed that the adhesive force between the subunits was observed only in the presence of both Mg2+ and FPP. This is the first direct demonstration of the specific interaction involved in the enzyme reaction. The dependence of the Mg2+ concentration on the specific interaction between subunits I and II well agreed with that on the enzyme activity of heptaprenyl diphosphate synthase. This indicated that the observed adhesive forces were indeed involved in the catalytic reaction of this enzyme. On the basis of these results, we discussed the processes involved in the substrate-enzyme complexation. The first, the substrate FPP bound to subunit II using Mg2+, followed by the formation of the subunit I-FPP-Mg2+-subunit II complex. Our study showed a very useful methodology for examining the elemental processes of biological reactions such as an enzyme reaction.