Total Polyphenol Content and Antioxidant Capacity of Commercially ...

9226 J. Agric. Food Chem., Vol. 56, No. 19, 2008 Anesini et al.Figure 1. Chemical structures of major polyphenols present in tea. (A)Major polyphenolic constituents present in green tea (epicatechin derivatives).(B) Major polyphenolic constituents present in black tea. R ) galloylgroup.ing reactive oxygen and nitrogen species (9, 10). The role offree radicals in injury to small-for-size grafts was investigatedin rat liver explants. A polyphenol extract from Camelliasinensis decreased liver graft injury and increased the survivalof small-for-size liver grafts, most likely by scavenging freeradicals (11). The antioxidant capacity of polyphenols presentin teas has been reported to be both chemoprotective andtherapeutic (12). The ability to inhibit the effects of variousmutagens in cultured cell lines has also been observed (13).They may also work indirectly as antioxidants through theireffects on the activity of transcription factors and enzymes (14).The antioxidant properties of green tea and its constituentcatechins were evaluated in a number of diseases associatedwith reactive oxygen species, such as cancer and cardiovascularand neurodegenerative diseases. Several epidemiological studiesas well as studies in animal models have shown that green teacan confer protection against various cancers such as those ofthe skin, breast, prostate, and lung. Green tea has also beenshown to be hypocholesterolemiant (15), to prevent the developmentof atherosclerotic plaques (16), to have antidiabetic effectsin animal models of insulin resistance, and to have antibacterial,anti-inflammatory, and anti-HIV activities (1, 17-19). Amongage-associated pathologies and neurodegenerative diseases,green tea has been shown to confer significant protection againstParkinson’s disease, Alzheimer’s disease, and ischemic damage(20). The increasing interest in its health benefits has led to theinclusion of green tea in the group of beverages with functionalproperties. The amount of tea polyphenols has been regardedas a quality indicator of tea (21). Information based onparameters, chemical standardization, and biological assays arecomplementary indicators of the quality of tea, regarding itsbiological activities. To date, no studies have been reported onthe phenolic compounds and antioxidant capacity of tea fromArgentina.MATERIALS AND METHODSSamples. Twelve commercial samples, consisting of tea leaf bagsof eight different brands (A-H) were purchased in the supermarket inBuenos Aires city. Three of the samples were green tea (g.t.), and ninewere black tea. Brands A, B, and C had both green tea and black tea.Brands D, E, F, G, and H corresponded to black tea only. For eachcommercial tea sample studied, three bags were sampled.Chemicals and Reagents. For the determination of the total phenoliccontent (TPC), Folin-Ciocalteu’s phenol reagent (Merck ChemicalsArgentina, Buenos Aires), gallic acid (99% purity, Sigma Argentina),and anhydrous sodium carbonate (99% purity, Anedra Argentina) wereused. For the determination of the antioxidant activity, 1,1-diphenyl-2-picrylhydrazyl, linoleic acid, ascorbic acid, and trizma base (SigmaArgentina), and FeCl 2 and ammonium thiocyanate (Merck ChemicalsArgentina) were employed.Extraction of Polyphenols. The method described by the InternationalOrganization for Standardization (ISO) 14502-1 was used (22).Briefly, 0.200 ( 0.001 g of each sample was weighed in an extractiontube, and 5 mL of 70% methanol at 70 °C was added. The extract wasmixed and heated at 70 °C on a vortex for 10 min. After cooling atroom temperature, the extract was centrifuged at 200g for 10 min. Thesupernatant was decanted in a graduated tube. The extraction step wasrepeated twice. Both extracts were pooled and the volume adjusted to10 mL with cold 70% methanol. One milliliter of the extract was dilutedwith water to 100 mL.Determination of Total Polyphenol Content. The total polyphenolcontent (TPC) was determined by spectrophotometry, using gallic acidas standard, according to the method described by the InternationalOrganization for Standardization (ISO) 14502-1 (22). Briefly, 1.0 mLof the diluted sample extract was transferred in duplicate to separatetubes containing 5.0 mL of a 1/10 dilution of Folin-Ciocalteu’s reagentin water. Then, 4.0 mL of a sodium carbonate solution (7.5% w/v)was added. The tubes were then allowed to stand at room temperaturefor 60 min before absorbance at 765 nm was measured against water.The TPC was expressed as gallic acid equivalents (GAE) in g/100 gmaterial. The concentration of polyphenols in samples was derived froma standard curve of gallic acid ranging from 10 to 50 µg/mL (Pearson’scorrelation coefficient: r 2 ) 0.9996).Determination of the Antioxidant Activity by the Ferric ThiocyanateMethod. The antioxidant capacity was determined by the ferricthiocyanate method (FTC) (23). A volume of 0.8 mL of extracts withdifferent TPC was mixed with 0.05 M phosphate buffer at pH 7 and 2.5%linoleic acid in ethanol to obtain 4 mL of solution. The resulting solutionswere incubated at 38.5 °C in a glass flask. Aliquots were removed at regularintervals, and FeCl 2/ammonium thiocyanate solution was added in orderto allow any peroxides resulting from the oxidation of linoleic acid to react,forming a complex that can be detected spectrophotometrically at 500 nm(Shimadzu UV 2101). This step was repeated every 24 h until the control(phosphate buffer plus linoleic acid) reached its maximum absorbancevalue. Therefore, high absorbance values indicated high levels of lino1eicacid oxidation. Phosphate buffer was used as the reaction blank. The totalantioxidant activity was expressed as the average of three independentdeterminations carried out in duplicate. The percentage inhibition of lipidperoxidation of linoleic acid was calculated by applying the followingequation: inhibition of lipid peroxidation (%) ) 100 - [(A s/A 0) × 100],where A 0 is the absorbance of the control reaction (linoleic acid alone,100% peroxidation), and A s is the absorbance obtained in the presence ofthe sample extract or positive control of antioxidant activity (1 mg/mLascorbic acid). The inhibitory concentration 50 (IC 50) values were calculatedfrom data obtained graphically, using a mathematical method based onthe principle of the right-angled triangle: IE 50 ) D - [(A - 50% maxresponse)X]/Y, in which A is the immediately higher response of 50% maxresponse; B is the immediately lower response of 50% max response; D) log concentration corresponding to A response; C ) log concentrationcorresponding to B response; X ) D - C; and Y ) A - B (24).Determination of the Free Radical Scavenging Activity by the1,1-Diphenyl-2-picrylhydrazyl Free-Radical Scavenging Assay. Scavengingactivities of the extracts on the stable free radical DPPH wereassayed using the modified Blois’ method (25), in which the bleachingrate of DPPH is monitored at a characteristic wavelength in presenceof the sample. A volume of 0.1 mL of an aqueous dilution of the extracts