Determination of Flavonoids, Phenolic Acids, and Xanthines in Mate Tea (Ilex paraguariensis St.-Hil.)

Raw material, different formulations of foods, and dietary supplements of mate demands control of the content of bioactive substances for which high performance thin layer chromatography (TLC), described here, presents simple and rapid approach for detections as well as quantification. Using TLC densitometry, the following bioactive compounds were identified and quantified: chlorogenic acid (2.1 mg/g), caffeic acid (1.5 mg/g), rutin (5.2 mg/g), quercetin (2.2 mg/g), and kaempferol (4.5 mg/g). The results obtained with TLC densitometry for caffeine (5.4 mg/g) and theobromine (2.7 mg/g) show no statistical difference to the content of total xanthines (7.6 mg/g) obtained by UV-Vis spectrophotometry. Thus, TLC remains a technique of choice for simple and rapid analysis of great number of samples as well as a primary screening technique in plant analysis.


Introduction
Mate tea (Sp. yerba mate, Port. Erva-mate) consists of well cut, dried, aerial parts or leaves of Ilex paraguariensis St.-Hil. (Aquifoliaceae) shrub. It contains methylxanthines, mainly caffeine, phenolic acids, and saponins and is used as everyday substitution for coffee (psycho stimulant, analeptic) in the south of Brazil, North of Argentina, Oriental Paraguay, and Uruguay. Besides beneficial effects on cardiovascular system, it has significant antioxidant capacity and can be used for the management of weight in obesity [1][2][3].
Thin layer chromatography (TLC) often represents firstchoice technique for samples of plant origin as results can be easily visualized. This technique has been successfully employed for analysis of flavonoids and phenolic acids in wine, propolis, and different medicinal plants [4]. Several studies comparing TLC and HPLC have shown that there is no statistical difference between these methods, and advantage due to lower cost is given to TLC [5,6].
Determination of xanthines as the main constituents is time consuming titration [7]. Although flavonoids present ubiquitous class of compounds, Bastos et al. [8] did not find any of the analyzed flavonols in mate tea using HPLC technique. Thus, the goal of this work was to identify and quantify polyphenolic and xanthine components of mate densitometrically using HPTLC plates and compare the results obtained with the content of total polyphenols and xanthines based on UV-Vis spectrophotometry.

Experimental Section
2.1. Reagents. All standards were purchased from Sigma Aldrich (Germany) and organic solutions from Kemika (Croatia). The sample of mate powder (dried aerial parts) was obtained from SantoÂngelo, RS, Brazil.

Standard Solutions.
Stock solutions of phenolic acids (chlorogenic, caffeic, ferulic, and p-coumaric acid), flavonoids (rutin, quercetin, naringenin, and kaempferol), and xanthines (caffeine, theobromine) were prepared by dissolving standards with methanol (polyphenols) or water (xanthines) to obtain concentration of 1.0 mg/mL. Standard solutions were prepared by dissolution with methanol to 0.1 mg/mL.        [7,9,10]. Briefly flavonoids were determined after acid hydrolysis (see Section 2.4.) as liberated aglycones spectrometrically at 425 nm as a complex with AlCl 3 in a methanol-ethyl acetate-acetic acid medium. For the purpose of phenolic acids determination 0.2 g of mate powder was refluxed with 190 mL of 50% ethanol on the water bath. Total phenolic acids were determined by measuring the absorbance at 505 nm of the complex formed between phenolic acids and sodium molybdate-sodium nitrite as an equivalent of rosmarinic acid.
Aqueous solution of yerba mate was used for the determination of xanthines. Total xanthines were determined after clarification with lead acetate spectrometrically at 272 nm as an equivalent of theobromine.
2.6. TLC Densitometry. Chloroform : methanol : formic acid in volume ratio 44.1 : 2.5 : 2.15 was used as mobile phase for aglycones, and ethyl acetate : formic acid : acetic acid : water in volume ratio 100 : 11: 11 : 26 was used for glycosides [4,11]. System used for xanthines development was ethyl acetate, methanol and water in volume ratio 40 : 5.4 : 4 [11]. Merck HPTLC plates (Silica Gel 60 F 254 , 10 × 20 cm) were used as stationary phase. Samples were applied using CAMAG Linomate V semiautomatic sample applicator. After development the HPTLC plates were dried and sprayed for the analysis of polyphenolics with 1% ethanolic solution of AlCl 3 ⋅6H 2 O. Plates were recorded at 254 and 366 nm. Identification and quantification were performed by TLC densitometry using CAMAG TLC Scanner 3 and WinCATS software version 1.3.4.

Statistical Analysis.
The results of TLC densitometry and spectrophotometric analysis were compared using Student's t-test. Statistical analysis was performed using Microsoft Office Excel 2003. Hydrolyzed fraction of flavonoids, 2 nonhydrolyzed fraction of phenolic acids used for the spectrophotometric analysis according to the European pharmacopoeia, and nd: not detected, na: not applicable.

Results and Discussion
The total content of flavonoids determined by Christ-Müller's method was 3.0 mg/g (RSD = 0.5%, = 3), phenolic acids determined according to Ph. Eur. method 55.1 mg/g (RSD = 0.6%, = 3), and xanthines by IOCCSC method 7.6 mg/g (RSD = 0.7%, = 3). 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 Table 1 and Figure 1.
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 ( Figure 2). 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 Table 2. 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 [12,13], 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 Table 2 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 [13,14] 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 mg/g, respectively.
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 mg/g ( = 0.16, = 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].

Conclusion
Based on the obtained results, it can be concluded that TLC analysis is an appropriate technique for the analysis of individual polyphenols as well as xanthines. Content of individual methylxanthines corresponds to the spectrophotometric determined total xanthines.
As thin layer chromatography is readily available technique, it could be easily employed for the analysis of polyphenols and xanthines in raw material, different formulations of foods, and dietary supplements of mate.