Ethanol and dichloromethane extracts of a Brazilian environment-friendly propolis from Baccharis dracunculifolia were analyzed by HPLC-APCI-MS e GC-MS, respectively. A HPLC-APCI-MS technique, at the positive mode, furnished a complete e unequivocal chemical composition of ns green propolis sample. That serves as fingerprint para different propolis samples. The composition of ns ethanol extract consisted greatly of cinnamic acid and derivatives, flavonoids, benzoic acid and a few benzoates, non-hydroxylated aromatics, and aliphatic acids and esters, which are normally not reported in ns literature due to the fact that they dá not absorb UV light. A main constituents of ns dichloromethane extract were prenylated compounds, alkanes and terpenoids.

Baccharis dracunculifolia; Asteraceae; Propolis; environment-friendly propolis; HPLC-APCI-MS; GC-MS




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Os extratos em etanol e diclorometano de 1 própolis verdes de Baccharis dracunculifolia passou a ser analisados através dos CLAE-ICPA-EM e CG-EM, respectivamente. Der técnica de CLAE-EM-ICPA, no modo positivo, entregar uma perfeito e inequívoca configuração química da amostra de própolis verde. Sobrenome serve como impressão digitais para amostras muitos de própolis. A configuração do extrato em etanol consistiu fundamentalmente de sabor azedo cinâmico e derivados, flavonóides, ácido benzóico e alguns benzoatos, aromáticos não hidroxilados, e ácidos e ésteres alifáticos, os que são coloquial ignorados na literário porque não absorver luz UV. Os componente principais dá extrato em diclorometano foram composto prenilados, alcanos e terpenóides.

Baccharis dracunculifolia; Asteraceae; Própolis; Própolis verde; CLAE-EM-IQPA; CG-EM


ARTIGO

Analysis of der Brazilian environment-friendly propolis são de Baccharis dracunculifolia by HPLC-APCI-MS and GC-MS

Análise de 1 própolis verde brasileira da Baccharis dracunculifolia por CLAE-ICPA-EM e CG-EM

Roberto ChangI; Dorila Piló-VelosoII; Sérgio A. L. MoraisI; Evandro A. NascimentoI,* * E-mail: eanascimento

IInstituto de Química, Universidade comunidade de Uberlândia, Av. João Naves de Ávila, 2160, 38400-902 Uberlândia-MG, Brazil

IIDepartamento de Química, Universidade commonwealth de minha Gerais, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte-MG, Brazil

ABSTRACT

Ethanol and dichloromethane extracts of naquela Brazilian eco-friendly propolis são de Baccharis dracunculifolia were analyzed by HPLC-APCI-MS e GC-MS, respectively. Ns HPLC-APCI-MS technique, at a positive mode, furnished der complete and unequivocal chemical composition of a green propolis sample. That serves as fingerprint ao different propolis samples. Ns composition of ns ethanol extract consisted mainly of cinnamic acid e derivatives, flavonoids, benzoic acid e a few benzoates, non-hydroxylated aromatics, e aliphatic acids and esters, which ser estar normally no reported in ns literature due to the fact that they dá not absorb UV light. The main ingredient of the dichloromethane extract were prenylated compounds, alkanes and terpenoids.

Keywords: Baccharis dracunculifolia, Asteraceae, Propolis, eco-friendly propolis, HPLC-APCI-MS, GC-MS.

RESUMO

Os extratos em etanol e diclorometano de ns própolis verde de Baccharis dracunculifolia foram analisados pela CLAE-ICPA-EM e CG-EM, respectivamente. A técnica de CLAE-EM-ICPA, no decorrer modo positivo, forneceu uma completo e inequívoca configuração química da amostra de própolis verde. Eles serve gostar de impressão digital para amostras diferente de própolis. A configuração do extrato em etanol consistiu fundamentalmente de acidez cinâmico e derivados, flavonóides, acidez benzóico e alguns benzoatos, aromáticos algum hidroxilados, e ácidos e ésteres alifáticos, os que são coloquiais ignorados na literário porque não absorver luz UV. Os componente principais do extrato em diclorometano foram ligação prenilados, alcanos e terpenóides.

Unitermos: Baccharis dracunculifolia, Asteraceae, Própolis, Própolis verde, CLAE-EM-IQPA, CG-EM.

INTRODUCTION

Propolis is naquela resinous essa produced by honeybees a partir de vegetable resins, exudates, wax, pollen, leaf pieces, e self-secretion. That is used to seal e to protect a hive versus heat, cold, wind, water, insects and microorganisms. (Ghisalberti, 1979; Rohwedder e Hausteen, 1987; neto et al., 2002).

Propolis ethanol extracts have been provided in the popular medicine porque o different objectives (Niraldo, et al., 2006; Soares et al., 2006; Tavares et al., 2006; Lemos et al., 2007; Missima e Sforcin, 2008; Simões et al., 2008). Flavonoids had in europe propolis were considered the constituents the had the beneficial action on the human organism. However in a last recent years numerous propolis various other than a European ones have actually demonstrated therapeutic properties and they a partir de not contain or contain trivial amount of flavonoids (Park et al., 2000; Pereira et al., 2002; Longhini et al., 2007; Sousa et al., 2007).

Review publications report that over naquela hundred link were currently identified in propolis (Greenaway et al., 1991; Marcucci, 1996; Lustosa et al., 2008). Ns propolis evaluation is der very difficult task since their ingredient changes according to a region, season and existing flora in enhancement to ns inherent difficulties connected to analysis of facility mixtures são de vegetal sources. Regardless of that, miscellaneous researchers demonstrated that Baccharis dracunculifolia is the main vegetable source of Brazilian eco-friendly propolis (Bankova et al., 1999; Bastos et al, 2000; Kumazawa et al., 2003; parque et al., 2003, 2004, 2005). Further, recently, naquela chemical marker para propolis são de Baccharis dracunculifolia ser estar proposed (Nascimento et al., 2008).

By way of derivatization with BSTFA, GC-MS analysis e a lot of reference compounds, over a hundred compounds might be determined in a propolis sample (Greenaway et al., 1991). Ns limitations of this technique residir on a more complicated mixture after ~ derivatization, the need of numerous rare referral compounds e the visibility of non-volatile or underivatizable compounds the cannot it is in detected accordingly. Also using high temperatures (near 400 ºC), this an approach has limitations (Neto et al., 2002)

Actually ns tedious work-related on obelisk fractionations and isolation the unknown link is giving place to alto performance liquid color layer analyzer (HPLC), which deserve to provide a quickly separation of a components, quantification e information regarded its UV spectrum. When coupled come Infrared (IR), átomo Magnetic Resonance (NMR) or massive Spectrometry (MS), the HPLC can broaden much more its evaluation range. Especially a HPLC-MS technique is an extremely useful because it avoids ns limitations enforced by the UV detection when detecting also ns molecules that do not absorb in a UV region. Today ns HPLC-MS can usar modern revolutionary approaches like ESI (electrospray ionization), APCI (atmospheric press chemical ionization), MALDI (matrix-assisted laser desorption/ionization), etc. That volatilize organic molecule of low and high molecular weights having medium to alto polarity (Hoffmann and Stroobant, 2001).

Some of these new techniques were recently employed top top propolis analysis (Valcic et al., 1999; Midorikawa et al., 2001; Pietta et al., 2002; Kumazawa et al., 2003).

Due to ns broad usar in a medicine, actually ns propolis investigation work is concentrated on ethanol extracts. Numerous model compounds were used to inspection samples that Brazilian propolis through HPLC-ESI-MS at the negative mode (Midorikawa et al., 2001). Based on the retention times and mass spectra the forty-one standards, this authors identified der lot of components in ns propolis samples. Sawaya e collaborators also used ns HPLC-ESI-MS at an unfavorable mode to recognize eight markers in Brazilian propolis em ~ different origins (Sawaya et al., 2004). Finally, using ns same technique and others procedures, Kumazawa and collaborators contrasted propolis ethanol extracts em ~ fourteen different countries (Kumazawa et al., 2004).

In ns present work an attempt to expand a range of identification of the constituents of naquela propolis ethanol extract is presented. HPLC-APCI-MS at a positive mode foi ~ used e the identification ser estar based on mass spectrometry, standards, UV spectrum, retention times, Kovat"s indexes (Adams, 2001), a publications quote above and others (Nascimento e Bezzan, 2001; neto et al., 2002; nascimento et al., 2003a; nascimento et al., 2003b; nascer et al., 2008; Negri et al., 2003).

Finally, a GC-MS examination of the dichloromethane extract of the same propolis foi ~ also carried o fim in bespeak to enhance the dia acquired from HPLC-APCI-MS.

MATERIAL and METHODS

Propolis sample

The santa Barbara Apiary situated in a State of Minas visão global supplied ns sample of green propolis.

It was collected in October e is representative of environment-friendly propolis from Baccharis dracunculifolia as comproved by number of analyses of environment-friendly propolis samples from different regions. The ethanol extract of propolis (EEP) was obtained by mixing 3.00 g of sample com 10.0 mL of ethanol (Vetec brand, analytical grade, 95 %). A mixture was stirred ao 24 hours away from light and then filtered e put into der freezer to separate a wax. Der new filtration gave ns studied extract. Ns dichloromethane extract that propolis (DEP) was obtained using another 3.00 g sample together above, other than on that it era not put into a freezer para wax separation.

Gas chromatography coupled to massive spectrometry (GC-MS)

The analysis foi ~ performed in der gaseous chromatograph by Shimadzu, version GC-17A, equipped with a DB-5 30 meter capillary column, 0.25 mm that i.d., 0.25 µm of movie thickness, combination to a mass spectrometer of same brand, model GCMS-QP5000, equipped com database that 330.000 fixed spectra. The runs were performed under ns following conditions: initial obelisk temperature: 60 ºC, injector in ~ 220 ºC e interface in ~ 240 ºC; obelisk program: 60-240 ºC at 3 ºC/min, 20 min in ~ 240 ºC. Uma µL of a sample of foi ~ injected under helium as carrier gas. The mass spectrometer operated under influence energy that 70 eV e the mass detection contained molecules a partir de 40 come 450 Da.

High atuação liquid chromatography coupled to mass spectrometry (HPLC-MS)

The HPLC runs were performed on naquela Shimadzu class VP sequência liquid chromatograph equipped com diode selection detector (PDA) e a reversed-phase CLC-ODS 30 centimeter column. The gradient conditions used were methanol HPLC grade and water/formic mountain (0.5%), starting with methanol 20% e after 40 minutes, 100% methanol, in a 60 minute run. The injected tonelada was 20 µL.

The alto performance fluid chromatography combination to atmospheric pressure chemical ionization e mass spectrometry (HPLC-APCI-MS) analysis, at the positive mode, era carried fora in naquela Quattro LC-Micromass massive Spectrometer.

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Ns chromatogram presented in this job-related (Figure 2) is the bruta Ions Chromatogram, TIC.