Reagents and chemicals
HPLC grade acetonitrile was purchased from BDH (BDH Chemicals Ltd. Poole, England). β-hydroxyethyltheophylline, caffeine, gallic acid, epicatechin, (−)-epigallocatechin gallate, Folin Ciocalteu reagent, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and aluminium chloride were purchased from Sigma Chemicals, USA. Sodium nitrite was purchased from Riedel De Haen Ag, Wunstorfer Strasse 40, SEELZE1, D3016, Germany. Crush, Tear, Curl (CTC) low grown pure Ceylon black tea was obtained from Danduwangalawatta Tea factory, Millawitiya, Kuruwita, Sri Lanka.
Equipment
Shimadzu UV-1601 UV Visible spectrophotometer (Shimadzu Corporation, Japan) was used to read the absorbance. HPLC was performed with Shimadzu LC 10AS solvent delivery system equipped with UV/VIS variable wavelength detector Shimadzu SPD 10A (Shimadzu Corporation, Japan) and an integrator Shimadzu C-R8A (Shimadzu Corporation, Japan). Chromatographic resolution of components in tea was achieved on betasil phenyl HPLC column (2.1 x 150 mm) from Thermo scientific. Samples were injected with a syringe loading injector fitted with a 100 μl loop.
Shimadzu Libror AEG-220 analytical balance (Shimadzu Corporation, Japan) was used to prepare standard solutions. Purified deionized water was obtained from Labconco Water Pro-PS UV ultra filtered water system (Labconco Corporation, Missouri) and distilled water was obtained by Aquatron A4S water system. Micro-centrifugation was performed using a BioFuge-Pico D-37520 centrifuge (Heraeus Instruments, Germany).
Preparation of tea brew
Tea brew was prepared according to the conventional method. Deonized water (500 ml) was boiled in a glass beaker placed on a hot plate. At the onset of boiling, heating was terminated and the tea leaves (5.0 g) were added to boiled water. The beaker was then covered with a watch glass. Magnetic stirrer was used at a constant speed to maintain a homogenous sample. A volume of 1.0 ml was withdrawn at different time intervals (0, 1, 2, 4, 6, 8, 10, 12, 14, 20 min) and centrifuged. The supernatant was assayed for their phenolic and flavonoid content by spectrophotometry. Gallic acid, caffeine, epicatechin and epigallocatechin gallate were quantified by Reversed Phase High Pressure Liquid Chromatography (RP-HPLC). Antioxidant activity was assayed by DPPH radical scavenging and Ferric reducing Antioxidant Power (FRAP) methods.
Determination of phenolic content
Total phenolic content was determined by Folin Ciocalteu method [26]. Samples (25 μl) were diluted up to 1500 μl with deionized water. Folin Ciocalteu’s reagent (1 N, 250 μl) was added to the samples (500 μl), and the mixture was allowed to stand at room temperature for 2 min. Sodium carbonate solution (10 %, 1.25 ml) was then added and incubated for 45 min in the dark at room temperature. The absorbance of the resulting solution was measured at 760 nm against a blank prepared with deionized water. Calibration curves were constructed with gallic acid and (−)-epigallocatechin gallate (EGCG) standards. The total phenolic content was expressed as gallic acid equivalents (GAE) mg/g of tea leaves as well as EGCG equivalents mg/g of tea leaves. Tea samples brewed independently were analyzed in replicates (n = 6).
Determination of flavonoid content
The flavonoid content was measured by the aluminum chloride colorimetric assay [27]. Tea brew (25 μl) collected at different time intervals were diluted with deionized water up to 500 μl and mixed with sodium nitrite (5 %, 30 μl). After 5 min aluminium chloride (10 %, 30 μl) was added to the mixture followed by sodium hydroxide (1 M, 200 μl) at the 6th minute. The final volume was adjusted to 1000 μl with deionized water and absorbance was measured at 510 nm against a blank prepared with deionized water replacing the tea brew. Calibration curve was plotted using EGCG standards and flavonoid content was expressed as EGCG equivalents mg/g of tea leaves. Tea brew prepared independently were analyzed in replicates (n = 6).
Antioxidant capacity by 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical assay
Free radical scavenging ability of tea samples collected at different time intervals and authentic samples of tea constituents (gallic acid, caffeine, epicatechin and epigallocatechin gallate) was assayed by DPPH radical scavenging method with slight modifications [28]. Test samples (50 μl) were diluted up to 1000 μl with deionized water. DPPH reagent prepared in absolute ethanol (100 μM, 950 μl) was added to the test sample (50 μl) and the mixture was allowed to stand for 30 min in the dark. The scavenging activity was quantified by measuring the absorbance at 517 nm. Deionized water was used as the blank. The control was prepared by mixing deionized water (50 μl) with DPPH (950 μl). Results were expressed as percentage scavenging of DPPH radical calculated using the following equation:
$$ \%\ \mathrm{Scavenging}\ \mathrm{of}\ \mathrm{DPPH}\ \mathrm{free}\ \mathrm{radical}=\frac{\mathrm{Abs}.\ \mathrm{of}\ \mathrm{control}-\mathrm{Abs}.\ \mathrm{of}\ \mathrm{sample}}{\mathrm{Abs}.\ \mathrm{of}\ \mathrm{control}} \times 100\% $$
Percentage scavenging of DPPH radical against time was plotted. Tea brew prepared independently was analyzed in replicates (n = 6) for antioxidant activity. L-Ascorbic acid was used as the standard antioxidant. The effective concentration needed to scavenge 50 % of the DPPH radical with respect to the control (EC50) was calculated for each of the tea constituents and the standard antioxidant.
Antioxidant capacity by ferric reducing antioxidant power (FRAP) assay
The ferric ion reducing power of the samples collected at different time intervals was determined according to Sharma and Kumar (2011) with slight modifications [29]. Samples (50 μl) were diluted up to 1000 μl with deionized water. The test sample (100 μl) was mixed with phosphate buffer (0.2 M, pH 6.6, 250 μl) and potassium ferricyanide (1 %, 250 μl). The mixture was incubated at 50 °C for 20 min. Trichloroacetic acid (10 %, 250 μl) was added and the samples were centrifuged at 6500 rpm for 10 min. The supernatant was mixed with deionized water and ferric chloride (0.1 %) at a ratio of 1:1:2 respectively. The samples were vortexed and absorbance was measured at 700 nm. The reagent blank was prepared by replacing tea sample with deionized water. L-ascorbic acid was used as the standard antioxidant. The antioxidant capacity was expressed as Ascorbic acid equivalent reducing power (mg/g of tea leaves).
Determination of Gallic acid, Caffeine, Epicatechin (EC) and (−)-Epigallocatechin gallate (EGCG) using Reversed Phase High Pressure Liquid Chromatography
Tea brew was diluted as necessary for the quantification of gallic acid, epicatechin and (−)-epigallocatechin gallate and caffeine. Samples (100 μl) were mixed with the internal standard, β–hydroxyethyltheophilline (10 μg/ml, 100 μl) and centrifuged (2000 rpm, 5 min). The supernatant (25 μl) was injected onto the HPLC column. The mobile phase used was isocratic elution system consists of 8 % acetonitrile, 1 % glacial acetic acid and 91 % deionized water at a flow rate of 0.5 ml/min. The peaks were detected at 280 nm. Standards were prepared with a mixture of gallic acid, caffeine, EC and EGCG (2.5 – 25 μg/ml) in deionized water. Calibration curve was constructed using peak area ratio of gallic acid, caffeine, EC and EGCG (ratio of peak area of the relevant standard to that of the internal standard) against the concentration. The HPLC method was validated for accuracy, intraday and interday precision, linearity, limit of detection (LOD) and limit of quantitation (LOQ) according to the guidelines provided [30].
Evaluation of kinetics of releasing phytochemicals from tea leaves
Kinetics of solid liquid extraction of polyphenols, flavanoids, gallic acid, caffeine, EC and EGCG mixtures were studied.
Second-order rate law for extraction of compounds from tea leaves is considered [31];
$$ \frac{dc}{dt}={k}_1{\left(c-{c}_{\infty}\right)}^2 $$
(1)
k1 = the second-order extraction rate constant (g μg−1 min−1)
C∞ = the extraction capacity (concentration of tea constituents at saturation in g L−1)
C = the concentration of tea constituents in the solution at any time (g L−1), t (min)
By considering the boundary condition t = 0 to t and C = 0 to C, the integrated rate law for a second-order extraction was obtained.
$$ c=\frac{c_{\infty}^2{k}_1t}{1+{c}_{\infty }{k}_1t} $$
(2)
By linear transformation of the above equation, the rate constant k
1
can be determined by fitting the experimental data.
$$ \frac{t}{c}=\frac{1}{k_1{c}_{\infty}^2}+\frac{t}{c_{\infty }} $$
(3)
Statistical analysis
Results are presented as mean ± standard deviation (Mean ± SD) of six independent experiments. Statistical analysis, student’s t-test and calculation of Pearson’s correlation coefficient (r) were performed using Microsoft Excel. Value of p < 0.05 was considered as significant.
