The total content of flavonoids determined by Christ-Müller's method was 3.0
mg/g (RSD = 0.5%, n
= 3), phenolic acids determined according to Ph. Eur. method 55.1
mg/g (RSD = 0.6%, n
= 3), and xanthines by IOCCSC method 7.6
mg/g (RSD = 0.7%, n
Identification of bioactive constituents was based on retention factor, absorption spectra in situ, and, if applicable, color of the band after spraying with aluminium chloride. The identification parameters are presented in and .
Table 1 Identification parameters of polyphenolics and xanthines: retention factor, color of band under 366nm, and spectra after spraying with aluminium chloride (not applicable for caffeine and theobromine).
Identification of chlorogenic acid—overlapping spectra of standards and erva samples.
Times of chromatogram development were 11.5, 24, and 12
min. for aglycones, glycosides, and xanthines, respectively. Out of eight polyphenolics analyzed rutin, and chlorogenic acid, as well as aglycones quercetin, kaempferol, and caffeic acid were identified and quantified. Both xanthines analyzed, caffeine, and theobromine, were determined (). Quantification was based on the area under curve using five bands with different amounts of standard in triplicate. The results of quantification are presented in .
Chromatographic plate (a) and 3D chromatograms (b) of caffeine standard (six tracks from the left and nine from the right) and sample of mate (middle part).
Results of quantification of individual flavonoids, phenolic acids, and xanthines.
Although in the last few years HPLC has been a major method for the analysis of polyphenolics in yerba mate
and detailed MS analysis was done [12
], we have not encountered TLC method in available primary sources. As the TLC is a rather simple technique, we tried to identify and quantify polyphenolic constituents, primarily flavonoids, and phenolic acids of mate
. Bastos et al. [8
] did not detect quercetin, myricetin, and kaempferol in infusion from dried mate
leaves using HPLC. This could be caused by inappropriate selection of solvents for extraction, inadequate time of extraction, or poor hydrolysis of glycosides as aglycones were to be determined. As it can be noticed from the result in most commonly employed acid hydrolysis used for the determination of total flavonoids in aglycone form (Christ-Müller's method) did not provide complete degradation of glycoside rutin.
Although advanced techniques for the separation and quantification of individual polyphenols including LC-MS [13
] are available, we used simple TLC method with HPTLC plates to determine quercetin and kaempferol in yerba mate
in the amounts of 2.2
mg/g and 4.5
The results of TLC analysis concurred with the total content of methylxanthines: 5.4
mg/g for caffeine and 2.7
mg/g for theobromine, while total xanthines were 7.6
= 0.16, α
= 0.01). These results suggest that thin layer chromatography with densitometry could be used for both, identification as well as determination of caffeine and theobromine in yerba mate
having in mind that the most common method only for determination is time-consuming titration [8