A new high performance liquid chromatographic method has been established for simultaneous determination of chlorogenic acid, caffeic acid and caffeine in hydroalcoholic and aqueous extracts ofIlex paraguariensis. Analytical curves showed good linear regression in the concentration ranges 0.49-7.8 μg/mL for chlorogenic acid, 0.25-3.9 μg/mL for caffeic acid and 0.244-7.8 μg/mL for caffeine. Reduction of the DPPH· radical was used to determine the antioxidant capacity of the extracts. Our method for the simultaneous determination of chlorogenic acid, caffeic acid and caffeine was highly sensitive, having lower detection and quantitation limits than other papers that used similar methodology.
INTRODUCTIONA free radical is any chemical species capable of independent existence that contains one or more unpaired electrons (Halliwel, Gutteridge, 1999). The role of freeradical reactions in biology has become an area of intense interest. It is generally accepted that free radicals play an important role in the development of tissue damage and pathological events in living organisms (De Groot, Noll, 1987;Schinella et al., 2000). The consumption of plant foods is associated with a reduced risk of chronic diseases (cancer and cardiovascular), in part because of substances with antioxidant capacity, and those that modulate enzyme activity and gene expression (Day et al., 2004).Antioxidative properties have been found in leaf infusions of yerba mate (Ilex paraguariensis) (Chandra, Mejia, 2004). Mate is a popular tea in South America, where it is frequently drunk instead of coffee as a stimulating drink. It is greenish in color and contains caffeine, tannins and several vitamins such as B1 (thiamine), B2 (riboflavin), B5 (pantothenic acid), C, E, β-carotene, sucrose, fructose, folic acid, trigonelline, choline and many polyphenolic compounds (flavonoids and chlorogenic acid) (Gugliucci, Stahl, 1995). Much experimental work (in vivo and in vitro) has been performed to evaluate the antioxidant capacity of yerba mate. The leaf extract of I. paraguariensis was shown to be absorbed and to be active in vivo when healthy patients drank it, and one hour later blood was collected and the LDL content analysed in relation to lipid peroxidation (Gugliucci, 1996). The Ilex paraguariensis aqueous / ascorbate system and that induced by the CCl 4 /NADPH system, in both cases detected by the thiobarbituric acid assay (Schinella et al., 2000) For the simultaneous determination of CGA (chlorogenic acid) and CFA (caffeic acid) (Figure 1) by HPLC, various sets of chromatographic conditions have been used in recent work: in Li et al. (2004), the mobile phase was a mixture of methanol and 0.2 mol/L acetate buffer (pH 3.6) (15:85, v/v) flowing at 1.0 mL/min, the detection wavelength (λ) being set at 300 nm; in Li et al. (2005), methanol:water:acetic acid (19:81:1.5, v/v) was used, flowing at 1.0 mL/min, with λ at 240 nm, and in Pellati et al. (2005), aqueous phosphoric acid solution (0.1%) and acetonitrile (90:10 v/v), flowing at 1.5 mL/min, with the...