1. Introduction
Yerba mate (Ilex paraguariensis Saint Hilare) is an indigenous bush from the subtropical forest of South America. Since the XV century, yerba mate has been consumed in Argentina, Paraguay, Uruguay and Brazil. Natives from this region, guaraníes, macerated the leaves to drink the infusion as a medicine for several diseases (rheumatics, intestinal and other disorders). Nowadays, this energetic beverage is also a source of vitamins and minerals and is included in several codices and worldwide pharmacopeias, like Argentine Food Code, Latinamerican Food Code, British Herbal Pharmacopoeia, etc. One of the distinctive characteristics of yerba mate is its high
concentration of antioxidants, mainly caffeoyl derivatives and flavonoids, which are very important for health care. These substances counteract the action of free radicals, which are responsible for early aging processes and degenerative diseases. The main role of antioxidants is to delay or prevent the oxidation of the substrate, either in food or, after assimilation, in the human organism. The incorporation of natural antioxidants into food products is the common trend to both extend shelf life and supply extra health benefits. However, these additives often show little stability and their production and incorporation costs should be considered. For this purpose, quick and reliable measurement methods for its antioxidant power are required. Several authors found evidence that yerba mate extracts prevent DNA oxidation and in vitro LDL lipoperoxidation ([1,2]). Gugliucci and Stahl [3] attributed to yerba mate an antiatherogenic effect after showing the protection of LDL particles towards oxidation in vitro and in vivo. Lunceford and Gugliucci [4] proved that aqueous yerba mate extracts inhibit the formation of glycation end-products which lead to complications in diabetic patients. Anesini, Ferraro and Filip [5] proved the peroxidase-like activity of yerba extracts and also their chemoprotective and antioxidant capacity.
Lyophilization is a widely used process in the food industry although it could modify the antioxidant composition of some natural extracts, leading to nutritional losses. On this regard, Rodriguez de Sotillo, Hadley and Holm [6] found that caffeic acid was degraded and gallic acid content was increased after freeze-drying antioxidant extracts of potato waste, without changes in Total Phenolic Content (TPC). Several authors have reported the polyphenol composition and the antioxidant activity of yerba mate extracts, measured by different methods ([7,8]). However, comparisons between liquid and lyophilized extract compositions obtained by different techniques have not yet been established for this plant material.
The objective of this work was to characterize liquid and lyophilized yerba mate extracts by spectrophotometric and chromatographic (HPLC) methods to find a relationship between the analytical techniques used. The relatively novel photoluminiscent method was also evaluated and compared. The effects of the lyophilization process on the polyphenols profile and the antioxidant activity of the extract were also analyzed.
2. Materials and Methods
2.1. Preparation of Extracts
Extracts were obtained from commercial yerba mate (Ilex paraguariensis) samples (“La Merced, de campo”, Las Marías, Corrientes, Argentina); 2 g of yerba mate with 100 ml of distilled water were placed in a thermostatic bath (Haake, Germany) at 100˚C for 40 min. Once obtained, the extracts were filtered, kept in dark flasks and immediately cooled in an ice bath until analyzed. Liquid samples were frozen at −20˚C during 24 h, transferred to a −80˚C freezer for 24 h, and finally, freeze-dried (Heto FD4, Denmark) for 48 h at −50˚C under vacuum. The powders were stored in hermetic flasks in a dessecator. The yield of the whole process was calculated as the weight of lyophilized sample (g) obtained from the extracted liquor, per gram of initial dried yerba mate. The UV spectra of liquid extract thoroughly diluted and lyophilized, dissolved in distilled water to reach an equivalent concentration, were determined from 200 to 800 nm in a spectrophotometer (Shimadzu, UV-mini 1240, Japan). The solubility of lyophilized yerba mate extracts was determined dissolving 100 mg of freeze-dried extract in 1 ml of distilled water. The suspensions were stirred, left to rest for 24 h and centrifuged at 1090 g for 10 min at 25˚C (Beckman Coulter, Avanti J-25, USA).
2.2. Chromatographic Analysis
Chromatographic analysis was performed in an HP 1100 liquid chromatograph (Hewlett Packard, US) equipped with a binary pump, thermostated column compartment, auto injector, degasser and diode array detector (DAD) connected to an HP workstation. A Zorbax 300 SB-C18 column (250 × 4.6 mm, i.d.), packed with 5 mm particles and connected to a guard column, was utilized. The mobile phases “A” and “B” consisted of a mixture of water, methanol and formic acid (79.7/20/0.3) and a mixture of methanol and formic acid (99.7/0.3), respectively. A staggered gradient elution program at 0.9 ml/min prepared as follows: 0% B/15 min, 10% B/15 min; 30% B/10 min; 60% B/10 min; 80% B/2 min, was employed. Finally, the mobile phase composition returned to 0% B in 5 min, and this composition was maintained for 10 min to equilibrate the original solvent composition of the stationary phase. Rutin, quercetin, kaempherol, caffeine, chlorogenic acid, gallic acid and caffeic acid were used as standards for identification. Their retention times, DAD spectra stored in the library and the extract samples spiked with each standard were used for identification purposes. Stock solutions of each standard (0.25 mg/ml) were prepared in 50% methanol-water, bubbled with nitrogen and stored in the refrigerator until use. Calibration curves at four different concentration levels were performed. Each level was tested by triplicate. Based on the absorption maxima, the wavelengths selected for the calibration curves were 280 nm for caffeine, 330 nm for chlorogenic and caffeic acids and 360 for rutin. Fresh samples of liquid extract were transferred to a syringe-driven filter (MillexGS, 0.22 um) and then were analyzed; for comparison purposes, equivalent amounts of lyophilized extract were solubilized in water. Results were expressed as mg of polyphenol compound/g of dried yerba mate.
2.3. Total Polyphenol Determination
Total polyphenol content was determined by the FolinCiocalteau method (TPCFC). This test is based on the oxidation of phenolic groups with phosphormolybdic and phosphotungstic acids. A green-blue complex with absorption between 725 and 750 nm is obtained after a given reaction time. Two milliliters of Na2CO3 (2% w/v) (Anedra, Argentina) were mixed with 200 ml of the yerba mate liquid extract, left 2 for min in darkness and finally 200 ml of Folin-Ciocalteau reagent (Anedra, Argentina, 1:1) were added. The absorbance of these samples was measured at 725 nm in a spectrophotometer (Beckman DU 650, USA) after 30 min time reaction. TPCFC was also determined on the reconstituted samples of lyophilized yerba mate extracts, to analyze the effect of the drying process. Gallic acid (Sigma-Aldrich, US) and chlorogenic acid (Fluka, US) were used as standards. Results were expressed as mg standard equivalent/g yerba d.b. (dried basis).
2.4. Antioxidant or Antiradical Activity
The photochemiluminescence (PCL) inhibition capacity of samples and pure compounds (chlorogenic and caffeic acids, rutin and caffeine) was determined as described by Popov and Lewin [9]. An automated PCL inhibition capacity analyzer system (Photochem, Analytik Jena AG, Jena, Germany) was used. Liquid extract, lyophilized extract and pure compounds, either diluted or dissolved in mili-Q water, were analyzed using the kit for integral Antioxidative Capacity of Water-soluble substances (ACW). Fresh samples of liquid extract were transferred to a syringe-driven filter (Millex-GS, 0.22 um) and then were analyzed; for comparison purposes equivalent amounts of lyophilized extract were solubilized in water. Results were expressed as mg of ascorbic acid equivalents per g of dried yerba mate, in the case of extracts, or per g of compound, in the case of standards.
Antiradical activity was determined by using DPPH· (Sigma-Aldrich, US) as a free radical. The method was adapted from Brand-Williams, Cuvelier and Berset [10] and is based on the reaction of specific compounds or vegetal extracts with the radical in an ethanolic solution. DPPH· reduction is followed by measuring the decrease of absorbance at 517 nm while the reaction occurs. Different concentrations of yerba mate extract were tested: 0.47 - 15.0 and 0.65 - 21.0 mg yerba mate/ml for liquid and lyophilized samples, respectively. A volume of 100 µl of each sample was added to 3.9 ml of DPPH· ethanol solution (25 mg DPPH·/ml ethanol). The decrease in absorbance was determined every 0.5 min for 10 min, and then every 15 min until the reaction reached a plateau. Gallic and chlorogenic acids were used as standard compounds for antiradical activity determination, as well.
EC50, the amount of yerba mate extract needed to decrease the initial DPPH· concentration to 50%, and TEC50, the time necessary to reduce the radical to this concentration, were determined according to Sánchez-Moreno, Larrauri and Saura-Calixto [11].
2.5. Relationship between TPCFC and Antioxidant Activity
Analysis of antioxidant activity with DPPH· radical involves a higher economic cost and assay time. Thus, to find an easy way to characterize the antiradical activity of extracts during production, a possible linear relationship between Folin-Ciocalteau and DPPH· methods was studied. Linear models were fit to experimental data using a statistic program SYSTAT INC (Evanston, US).
3. Results and Discussion
3.1. Preparation of Extracts
Water was selected as the solvent for obtaining the yerba mate extracts, mainly to facilitate their use as food additive, although other solvents may be more efficient [12]. Besides being a green solvent, no further evaporation step is necessary. Process yield, including the lyophilization step, was 32.7% on dry basis of yerba mate. Yield values between 25% and 33% were also reported [8]. Depending on the industrial processing step (green leaves, zapecado, drying, and forced aging) of the vegetal material, yield values between 31% and 36% were obtained [13]. Powder yerba mate extracts showed a water solubility of 96% and a moisture content of 3.8%. Similarly, Sinija, Mishra and Bal [14] reported a moisture content of 3% - 5% for freeze-dried tea extracts in an instant beverage product.
3.2 Characterization of Liquid and Lyophilized Extracts
3.2.1. Yerba Mate Composition
Both UV spectra for liquid and lyophilized samples were similar and corresponded to the typical spectrum of chlorogenic acid or its derivatives [15]. UV spectra showed a maximum absorbance at 325 nm corresponding to chlorogenic acid. Lower values were obtained for lyophilized yerba mate extracts compared to liquid ones.
Both extracts were also analyzed by HPLC, as can be observed in Figure 1, chlorogenic acid (tr = 11.92 min), caffeic acid (tr = 12.88 min), caffeine (tr = 15.36 min) and rutin (tr = 36.48 min) were identified, whereas quercetin, kaempferol and gallic acid were not detected in the analyzed samples. Also, several non-identified peaks were found. DAD spectra analysis revealed that their chemical structures have a high concordance with that of chlorogenic acid. Because of the lack of commercial standards, a tentative identification of those peaks was made according to a European patent (ES 2 267 182 T3). In the mentioned work, using HPLC-MS, peaks eluting before chlorogenic acid (tr = 6 and 12.1 min) were identified as chlorogenic acid isomers and peaks eluting after (tr =
Figure 1. Chromatographic profiles (HPLC) of liquid extract and lyophilized extract dissolved in water:methanol (280 nm).
35.7, 36.3 and 38.7 min) were identified as dicaffeoylquinic esters like the 3,4;3,5 and 4,5 dicaffeoylquinic acids. From now, these 5 peaks will be referred as “chlorogenic related compounds”. Heck, Schmalko and González de Mejía [8] identified these compounds using liquid chromatography coupled to mass spectrometry (HPLCMS).
Dugo et al. [12] employed a comprehensive two-dimensional liquid chromatography (LC × LC) system, finding those derivatives among 26 different compounds. Some authors ([16,17]) identified forty-two chlorogenic acids, which were detected and characterized to regioisomeric level on the basis of their fragmentation pattern in tandem MS spectra.
Caffeic acid could not be quantified due to its low content together with the fact that the chromatographic peak appeared as a shoulder that could not be separated from the neighboring peak (Figure 1).
Table 1 shows the amounts of chlorogenic acid and related compounds, rutin and caffeine found in the liquid and lyophilized extracts of yerba mate samples. The amount of chlorogenic acid was similar (p > 0.05) in both types of samples. However, the amounts of chlorogenic acid related compounds, rutin and caffeine in the lyophilized sample were lower than in the liquid extract (p < 0.05). Detected amounts of chlorogenic acid and rutin (Table 1) were similar to those found by Filip, López,