Brazilian journal-2000-30.p65

Metilxantinas em Amostras de Chá Preto e Commercial samples of black tea, mate tea and other types of tea were submitted to different extraction methods: decoction, ultrasonic and microwave assisted extraction in order to improve the extraction of methyl xanthines (caffeine, theobromine and theophylline). The microwave-assisted extraction appeared to be, statistically, more efficient than the other extraction methods. Chromatographic analysis (HPLC) revealed that extracts from black tea were richer in caffeine than green tea and mate tea, whereas a higher theobromine content was found in CCS - Bloco H - Cidade Universitária - CEP 21941-590 “chimarrão” mate. The ratio of the caffeine/theobromine chromatographic signals was a characteristic of the type of tea and it could be used as a chemical marker in quality control.
Amostras comerciais de chá preto, de chá mate e de outros tipos de chás foram submetidas a diferentes técnicas de extração assim como: decocção, extração com auxílio de ondas ultrassônicas e de microondas como melhoramento na extração de metil xantinas (cafeína, teobromina e teofilina). A técnica de extração com o auxílio de microondas mostrou ser, estatisticamente, mais eficiente que as demais. Análise cromatográfica (CLAE) revelou que os extratos provenientes de chá preto são mais ricos em cafeína do que os de chá verde e chá mate, enquanto o maior teor de teobromina foi encontrado no chimarrão. A razão dos sinais cromatográficos relativos à cafeína e à tebromina é uma característica marcante do tipo de chá e pode ser empregada como marcador químico no controle de qualidade de produtos correlatos.
Metil xantinas; Thea sinensis; Ilex paraguariensis; Técnicas de extração; Microonda / Methyl xanthine; Thea sinensis; Ilex paraguariensis; Extraction techniques; Microwave.
Recebido / Received: 17/03/1999. Aprovado / Approved: 13/01/2000.
Xanthine Percentages in CommercialSamples of Black Tea and Mate Tea (MC1 and MC2), black tea (BT1 and BT2) and green tea (GT1and GT2). Each sample (2,0g) was submitted to the followingwater (200mL) extraction techniques: decoction at 80ºC (DEC), Tea has been a popular beverage since early times.
extraction using ultrasonic waves at 80ºC (USH), microwave- Interest in the real potential of teas has increased recently assisted extraction (MAE). The extraction time was one minute because of their medicinal properties. Black tea and mate tea and it was performed in duplicate. The extracts obtained were are widely used by the Brazilian population. Herb mate or filtered and analyzed by HPLC. An Ultrasonic wave apparatus mate is a powdered or ground product of the leaves and roots Elma 9332 and a domestic microwave oven Prosdócimo were of Ilex paraguariensis, or their aqueous extracts. “Chimarrão” is used for the USH and MAE extractions, respectively.
a common beverage consumed in southern Brazil, prepared with dried herb mate, but toasted mate is commonly used throughout the Brazilian territory. Tea is a product consisting of the young leaves and shoots of Thea species (Thea sinensis and others). Green tea is a product obtained from the dried plant, while black tea is obtained from the dried and fermented Liquid chromatography was carried out using a Shimadzu apparatus having two pumps (Shimadzu modelLC-10AD), an ultraviolet variable wavelength detector Methyl xanthines (caffeine and theobromine) are very (Shimadzu model SPD 10 A), set at 272nm and having a 0.2 well distributed in nature; they are found in many kinds of absorbance unit full scale, a Rheodyne injection valve with food as well as in black tea and mate tea. These substances act on the central nervous system and produce various psychic a fixed loop of 20mL, a pré-column of Nova-Pak C-18, an phenomena, such as increasing the perception capacity analytical column of Alltech RP-18 Lichosphere, 5mm, 250 (GRAHAM, 1978). The diuretic property of xanthines is also x 4.6mm ID and a mobile phase (1.0mL min-1) of methanol/ very important and, among them, theophylline is the most water, increasing from 0% to 16% methanol with linear effective, followed by theobromine (COSTA, 1994). Mate tea segments of 10 min; and from 16% to 70% methanol with and black tea are known as stimulants. Black tea has bactericidal linear segments of 25 min. Samples (20mL) of tea filtered properties, mainly against bacteria of the mouth tract.
through Millipore 0.45 mm PVDF, were analysed by HPLCusing external standardization to quantify the methyl Due to the high consumption of mate tea and black tea xanthines present. The conditions were the same for all by the Brazilian population, quality control of these beverages has become necessary. Decoction is the most frequently experiments. In addition, linearity of the instrumentation employed extraction method for tea preparation. However, was verified from the calibration curve in the range from with the increased use of the microwave oven to prepare tea, 10 to 100mg mL-1. In each case, a good correlation an investigation of the extracts obtained using this technique coefficient was obtained (theobromine 0.9998, is required (LACERDA, 1992). In the official methodology for theophylline 0.9999 and caffeine 0.9978). The methyl xanthine analysis, gas chromatography is recommended chromatographic resolution in the methyl xanthine (AOAC, 1995). Nevertheless, high performance liquid aborbance region was very good, considering that the chromatography (HPLC) is an analytical method adequate for samples were crude extracts (Figure 1 a-f).
the qualitative and quantitative evaluation of methyl xanthines (TRUGO et al., 1983). Such compounds are present in the aqueous extracts as true chemical markers and they do not require extensive or complex techniques for sample preparation The aim of this work was to compare the yields of crude extracts as well as the percentages of methyl xanthines The percentages of theobromine, theophylline and (theobromine, theophylline and caffeine) in commercial samples caffeine in the samples analyzed varied with the type of tea.
of black tea, mate tea and other samples of tea, using different The results, shown in Table 1, indicate that the extracts from extraction techniques (decoction, ultrasonic and microwave BT were richer in caffeine than those from GT, MT and MC, assisted extractions) in order to perform their quality control.
while the highest theobromine content in the samplesanalyzed was found in MC. The analysis also revealed higherlevels of theophylline and theobromine in BT than in GT, withthe exception of theophylline in GT2 (Table 1). Under the conditions used, theophylline was not detected in MT andMC samples. Each sample of tea showed similar chromatographic patterns, regardless of the extractionmethod, indicating that microwave and ultrasonic assisted Two trade marks of each tea type were commercially extractions only influenced the results quantitatively, not obtained: toasted mate (MT1 and MT2), mate tea “Chimarrão” Xanthine Percentages in CommercialSamples of Black Tea and Mate Tea FIGURE 1. Typical HPLC-UV chromatogram patterns of aqueous extracts from xanthine containing plants. Chromatographic conditions: mobile phase (1.0mL min-1) methanol/water, increasing from 0% to 16% methanol with linear segments of 10 min; and from 16% to 70% methanol with linear segments of 25 min; column RP-18 Lichosphere, 5µm, 250 x 4.6mm ID. UV detection at 272nm. a) MT1; b) MT2; c) MC1; d) MC2; e) GT1; f) GT2; g) BT1; h) BT2. Theobromine at 17 min. (1), Theophylline at 21 min. (2), Caffeine at 29 min. (3).
TABLE 1. Theobromine, theophylline and caffeine contents of tea samples obtained by extraction using microwave (MAE), decoction at 80ºC (DEC), extraction using ultrasonic wave at 80ºC (USH). Means and coefficients of variation (CV) for determinations in duplicate Xanthine Percentages in CommercialSamples of Black Tea and Mate Tea The ratios of the caffeine/theobromine chromatographic ratio for a given product did not differ significantly and it may signal integrations of the HPLC-UV chromatograms for the represent a potential chemical marker for the quality control different teas revealed higher values for Camelia sinensis teas of black tea, mate tea and similar products. Thus, determination of the caffeine/theobromine values for commercial samples of GT (32.1 – 35.8) and BT (17.1 – 27.0), than for Ilex paraguariensis black tea and mate tea, even in the beverage state, is a useful teas MT (5.2 – 5.7) and MC (3.0 – 4.6) (Table 2). It is interesting tool to determine whether the genera Camelia or Ilex were to note that, independent of the extraction method used, the the real source of the products (Figure 1 a-f).
TABLE 2. Ratios of caffeine/theobromine integrated chromatographic signals. Means and Coefficients of Variation (CV).
To evaluate the results obtained with the different did not occur with USH/DEC (0.513). In spite of the contents of extraction methods, the significance paired t test (MILLER, theobromine and theophylline being systematically higher MILLER, 1993) was used to compare the averages of two for the extracts obtained by MAE, the values obtained in the t- samples. The paired t-test was calculated for MAE/DEC, MAE/ test were below the critical value, indicating that there was no USH and USH/DEC (Table 3) considering each difference in methyl xanthine contents. For caffeine, MAE differed significant difference amongst the extraction methods.
significantly from the other two extraction methods, as shown However, this should not be taken into consideration, because by the values calculated in the paired t-test (MAE – DEC = 5.24 of the high dispersion of the averages, leading to high e MAE – USH = 4.08), exceeding the critical value (2.36). This TABLE 3. Differences in theobromine, theophylline and caffeine contents in tea samples obtained by the MAE, DEC and USH methods. Statistical paired t-test results.
There are seven degrees of freedom. Critical value 2.36 (P=0.05. two tail). The MAE-DEC caffeine value (0.573) for BT2 was rejected.
Xanthine Percentages in CommercialSamples of Black Tea and Mate Tea The microwave-assisted extraction coupled with AOAC, Official Methods of Analysis 18 ed. Association of Official HPLC-UV analysis has been shown to be an important tool Analytical Chemists, Washington, D.C., 1995.
to evaluate qualitatively and quantitatively the methyl COSTA, A. F. Farmacognosia, 4.ed., Fundação Calouste Gulbenkian, xanthines theobromine, theophylline and caffeine in crude aqueous extracts from Ilex paraguariensis and Camelia sinensis. This methodology can be used to validate DIÁRIO OFICIAL DA UNIÃO (DOU), portaria nº 363 - 23 de julho de 1996, Ministério da Saude, Governo do Brasil.
GRAHAM, O.M. Caffeine its identity, dietary sources, intake and biological effects, Nutr. Rev., 36:97-102, 1978.
LACERDA, M.E.G. Otimização dos Processos de Extração de Plantas. Dissertação (Mestrado em Química de Produtos Naturais) - Nucleo de Pesquisas de Produtos Naturais, UFRJ, 1992.
Special acknowledgment to the technician João Marcelo MILLER, J.C., MILLER, J.N. Stastictics for Analytical Chemistry. 3 Lanzilotti and to Prof. Jorge H. Petrestski of Coordenação de Pesquisa e Desenvolvimento Tecnológico da Escola Técnica TRUGO, L.C., MACRAE, R., DICK, J. Determination of Purine Federal de Química do Rio de Janeiro - Brasil for obtaining and Alkaloids and Trigonelline in Instant Coffee and Other Beverages optimizing the chromatograms and also to the Brazilian National Using High Performance Liquid Chromatography, J. Sci. Food


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