Optimization and Validation of a Simple Spectrophotometric Based DPPH Method for Analysis of Antioxidant Activity in Aerated, Semi-Aerated and Non-Aerated Tea Products ()
1. Introduction
Tea (Camellia sinensis L.), a widely consumed beverage in the world, comes in a variety of forms, scents and flavors [1]. Green (un-aerated), oolong (semi-aerated) and black (aerated) teas are made from the shoots of tea plants at different degrees of aeration [2] [3]. These beverages have been receiving much attention in the last decade because of their inherent beneficial components, mainly polyphenols [4]. Many biological functions of tea polyphenols have been associated with antioxidant activity [5], anti-mutagenic effects [6], cardiovascular diseases [7] and anti-obesity prevention [8]. Furthermore, epi-gallocatechin gallate (EGCG) a polyphenol found in large amounts inun-aerated tea is reported to have anti-HIV activity when bound to cluster of differentiations (CD4) receptor [9]. 2,2-diphenyl-1-picrylhydrazyl (DPPH; C18H12N5O6, M = 394.33) free radical scavenging method present popular, quick, easy, and affordable approaches for determining antioxidant capacity in material of biological origin [10]. The method was first suggested by Blois (1958) using cysteine as model antioxidant [11]. Brand-Williams et al., 1995 and Bond et al., 1997 introduced modifications to the earlier method of Blois where they applied the term “EC50” (efficient concentration) for the interpretation of results from DPPH method. “EC50” referred to substrate concentration that caused 50% depletion in the DPPH colour [12] [13]. The disadvantage of this factor was that it showed an inverse relation to the substrate antioxidant activity [14]. Furthermore, to overcome the disadvantage, Brand-Williams applied the term “antiradical power” (ARP) the inverse of EC50 and hence, larger the ARP, the more efficient is the antioxidant [15]. In an effort to improve reporting on findings of the DPPH assy, another term that was used to describe the DPPH scavenging capacity was “antiradical efficacy” (AE), with higher AE value indicating higher antioxidant activity [16]. Researchers over the years have worked to improve on determination of the antioxidant potential of a substrate through the DPPH assay since different literatures have presented discrepancies. Recently, Bryan Brummelhaus de Menezes et al. through intensive literature search, highlighted two main problems. First, was the absolute determination of the fifty percent inhibitory concentration (IC50) despite the initial concentration of DPPH used and secondly, the determination of the percentage of scavenged DPPH radical (DPPH_), due to the missing measure of the absorbance of the molecular DPPH (DPPH-H) formed in the reaction. This team of researchers observed that the model which is currently being used to calculate the DPPH concentration can lead to an overestimation of up to 7%, as it does not take into account the small contribution of the reaction products. Their work demonstrated that to address the first problem IC50 value needs to be expressed as a function of the DPPH mol number used in the reaction while the reported mathematical model would correct the overestimation of the DPPH concentration [17].
Generally, the principle of DPPH method, is reduction by an antioxidant through the transfer of a proton “plus” electron (a hydride equivalent) resulting in color loss of the DPPH [18]. The DPPH free radical gives a strong absorption maximum at 517 nm and is purple in color [19]. The color turns from purple to yellow as the molar absorptive of the DPPH radical at 517 nm decreases from 9660 to 1640 µM−1 cm−1 [20]. During this process the DPPH radical odd electron becomes paired with an electron donated from the antioxidant and hydrogen atom to form the reduced DPPH-H [21] (Figure 1).
Figure 1. DPPH radical reaction scheme against a hydrogen donor antioxidant molecule.
To appreciate the antioxidant property of any sample, the color loss cannot always be taken to completion; therefore, the weight of sample is adjusted so that approximately 50% of DPPH color is lost [22]. This requires a pre-knowledge of the sample or, alternatively, preliminary tests to decide the most appropriate sample weight. Other than DPPH, several techniques have been used for antioxidant measurements [23]. In total peroxyl radical trapping (TRAP) method, thermal decomposition of AAPH produces luminal derived radicals and from it CL signal [24]. TRAP measurement is dependent on period at which sample quench the signal due to the presence of antioxidants [25]. In ABTS technique, interaction is between an antioxidant and pre-generated ABTS•+ radical cation that is easily quantified due to bleaching of absorption spectrum characteristic maxima at 414, 417, 645, 734, and 815 nM. [26]
The instrumentation commonly used for anti-oxidant capacity profiling involves spectroscopy, electrochemical and chromatography [27]. In the spectrometric techniques, radical, radical cation or complex reacts with an antioxidant molecule which donates a hydrogen atom [28]. The techniques involves well defined steps including, initiation (LH + R∙ → L∙ + RH); propagation (L. + O2→ LOO.LOO. + LH → L. + LOOH); branching (LOOH→LO. + HO., 2LOOH → LOO. + LO. + H2O) and termination (LO. + LO., LOO. + LOO., LO. + LOO.) [29]. The electrochemical methods for determination of antioxidant activity are based on cyclic, differential pulse, square wave voltammetry and coulometry [30]. These methods are sensitive, rapid and simple [31]. Evaluation involves direct measurement of electrons transferred by an antioxidant [32]. Chromatographic assays for antioxidant activity determination easily eliminate interferences from photosynthetic pigments occurring naturally in plant extracts [33]. The DPPH-HPLC enables the separation of the DPPH free radical peak and its reduced form (DPPH-H) from other components in the analyte [34]. However, simultaneous analysis of antioxidants and evaluation of their activity is more complicated [35]. Therefore, post-column reaction between the antioxidant and DPPH has been used to enhance efficiency of these methods [36].
In this study attempt has been made to optimize and validate a simple, quick, easy, reliable and affordable DPPH method for use in routine analysis of antioxidant activity in tea and related products. For this purpose, the method has been used to generate anti-oxidant activity levels in cultivars of high, medium and low total catechin contents. Additionally, processed tea products were profiled using the steps of the method. The finding of the study suggests that, the steps of the method are adequate enough to determine antioxidant activity in tea and related products.
2. Materials and Methods
2.1. Sample Collection
The samples required for analysis using the optimized and validated method were collected at the Timbilil estate fields (0˚22'S, 35˚21'E) with an altitude of 2180 meters (mean above sea level) of Kericho County in Kenya. Two leaves and a bud were hand plucked fresh for the cultivars of TRFK895/22, Ejulu, TRFK895/7, AHP S15/10, TRFK914/39, TN14-3, D99/10, TRFK6/8, ST832, ST831, ST830, ST543, ST536, TRFK306/4, TRFK306/3, TRFK306/2, TRFK306/1, TRFK91/1, TRFK660/1, TRFK597/4, TRI306/FLD/GB/2, TRI306/FLD/GB/3, TRI306/FLD/ GB/4, TRI306/FLD/GB/5, TRI306/FLD/GB/6 and TRI306/FLD/GB/3. The different orthodox tea grades of BO-OP1, GO-FP, BO-FOF, BO-FP, GO-OP1, GO-FOP, GO-OPA, GO-FOF, GOFBOE, BO-FBOB and BO-FOP were sourced from selected tea processing factories. The cut, tear and curl (CTC) tea grades of PD, D1, F1, PF1 and BP1 were collected from Kipkebe and Matara tea factories. The samples were kept in well labeled aluminium lined khaki bags for preservation before determination.
2.2. Chemicals and Reagents
The DPPH (2,2-Diphenyl-1-picrylhydrazyl) and HPLC grade methanol were purchased from Sigma-Aldrich Chemie GmbH, Germany through Kobian local suppliers. Distilled water was generated in the laboratory. Spectrophotometric measurements were done using a Shimadzu 1800 series spectrophotometer with 1 cm quertz cuvettes.
2.3. Sample Preparation
The hand plucked fresh two leaves and a bud were sorted for high quality material in each of the cultivars above. The leaf was then steamed for 1 min before being microwaved. The microwaved leaf and the different tea grade samples were finely ground using an electric coffee miller (AR40 Molineux) and packed in aluminium lined brown paper sachets ready for use in antioxidant activity analytical determinations.
2.4. Sample Moisture Content Determination
2.0 ± 0.01 g of the sample was weighed into aluminium dishes and heated in an oven at 103˚C ± 2.0˚C for 4 hours to obtain constant weight. The samples were weighed again and moisture content was then determined by subtracting the final weight from the initial weight, and expressed as a percent. The moisture content obtained was used to determine the sample dry matter content necessary for standardizing the sample weights during analysis.
2.5. Preparation of 0.06 mM DPPH Working Solution
0.0024 g of DPPH was accurately weighed and transferred into a 100 mL volumetric flask. 80% methanol (v/v) in distilled water was added to above half full mark. The mixture was stirred on a magnetic stirrer until all the DPPH dissolved and then toped to the mark with 80% methanol. The DPPH is sensitive to light and hence must be prepared in light controlled environment. Prepare fresh every day.
2.6. Optimization of the Method
Optimization in analytical chemistry involves designating a set of experiments in order to establish the proper conditions for carrying out a method, to achieve the best possible responses and ensure ideal analytical characteristics. For each parameters considered, the focus is to identify the factors for optimization, measure performance under the set conditions, establish trends and visualize improvement. The optimized conditions in this study were, sample weight, extraction solvent concentration, extraction temperature, extraction time, ideal sample extract concentration for DPPH reaction and sample extract-DPPH mixture incubation time.
2.7. Validation of the Method
2.7.1. Precision of the Method
The precision of the method was investigated by preparing one sample solution containing the target level of analyte. Ten replicates of this sample solution were analyzed with the mean, standard deviation and relative standard deviation (RSD %) determined. From the measured standard deviation (SD) and mean value, precision as relative standard deviation (% RSD) was calculated.
(1)
2.7.2. Linearity of the Method
Six different sample concentrations were prepared in triplicate and determined for antioxidant activity using the conditions of the method. The mean anti-oxidant activity in each of the five concentrates was used to generate equations for the regression line and correlation coefficient (R2) in order to establish linear relationship of the method.
2.7.3. Accuracy of the Method
Accuracy of the analytical method was determined by preparing quality control (QC) materials of gallic acid and a non-aerated tea sample of TRI 6/8 cultivar in 10 mL of 80% (v/v) methanol in distilled water. The anti-oxidant activity (AA) of the QC materials was accurately determined against the DPPH solution. The QC material was used as the standard to spike the tea sample in order to determine the recovery percent of the method.
(2)
2.7.4. Limit of Detection (LOD) and Quantitation (LOQ) of the Method
LOD and LOQ were obtained from the standard deviation (σ) of the blank response (n = 6) and slope (S) of calibration curve using the formula 3.3 σ/S and 10 σ/S, respectively.
2.8. Application of the Method
All the samples were evaluated for anti-oxidant activity using the optimized and validated conditions of the method.
2.9. Statistical Determinations
Statistical analysis was carried out using SAS® V 9.1 for windows statistical software. ANOVA was used to determine the means, coefficient of variation (CV) and Least Significance Difference (LSD). The probability limit was set at p ≤ 0.05 significant levels. Results of the parameter determined were expressed as a mean of the triplicate determination.
3. Results and Discussion
3.1. Optimization of the Method
The influences of sample weight and solvent concentration for the method were tested. The sample weight was optimized on the basis of a 0.06 mM DPPH concentration. From literature search, DPPH concentration of 0.06 mM is extensively used in analytical methods where DPPH is the scavenged free radical [37]. The tea sample weight was evaluated in the range of 0.001 - 1.000 g. It was found out that a 0.0100 ± 0.0001 g sample weight contained in a 10mL extracting solvent and diluted fivefold was optimal for the method. Plank et al., 2012 while working on a collaborative study on food and beverage reaction with DPPH, suggested that for antioxidant property of any sample, the DPPH color loss cannot always be taken to completion; therefore, the weight of sample is adjusted so that approximately 50% of DPPH color is lost [22]. A medium total catechins cultivar of ST 543 was selected as reference material for moderating the DPPH depletion by sample antioxidants to obtain a consistent end point of approximately 50 ± 2AA%. Methanol was used as the extraction solvent due to its high extraction efficiency in extracting both lipophilic and hydrophilic molecules [38]. The solvent concentration range was optimized at 50% - 100% methanol: water (v/v) and 80% methanol was found ideal since it gave a good yield of the antioxidants. A study by Chigayo et al., 2016 concluded that 80% methanol was the best solvent to use for extraction of phytochemicals and antioxidant properties of different solvent extracts of Kirkia wilmsii tubers [39]. Furthermore, elevated temperature of 70◦C for the reaction mixture gave improved antioxidant activity in a short extraction time of 10 min. Finally, a spectrophotometric scan of DPPH colour change in a DPPH-sample matrix, was done over a period of 90 min (Figure 2) in order to establish the reaction time end point. It was observed that after 30 min, recorded change in absorbance levels had little or no change.
![]()
Figure 2. 0.06 mM DPPH depletion curve against 0.2 mg/mL tea sample in 80% methanol.
3.2. Validation of the Method
The proposed method was validated as per the Association of Official Analytical Chemists (AOAC) guidelines [40]. Tests for precision, linearity, accuracy, LOD and LOQ for the method were carried out according to earlier adopted procedures in Section 2.7 above.
3.2.1. Precision of the Method
The precision of the validated method was expressed as percent relative standard deviation %RSD (CV). The results showed an RSD of less than 2% (Table 1). This demonstrates that the present method is sufficiently precise for samples tested and can be used to get good predictive antioxidant activity (AA) results in tea.
Table 1. Precision studies.
n |
Maximum AA% |
Minimum AA% |
Mean AA% |
SD |
%RSD (CV) |
10 |
49.43 |
47.22 |
48.30 |
0.819 |
1.695 |
3.2.2. Linearity of the Method
Well ground microwaved samples of cultivar ST 543 was accurately weighed to generate concentrations of 0.025, 0.038, 0.050, 0.083, 0.125 and 0.250 mg/g sample on dry weight basis in 10 mL of 80% methanol in distilled water. Antioxidant activity against DPPH was determined using the conditions of the optimized and validated method before a calibration curve of percent antioxidant activity against concentration was generated (Table 2). The linear regression data for the calibration curve showed good linear relationship over the concentration range of 0.025 - 0.250 mg/g sample dry.wt. Additionally, the linear equation was found to be Y = 200.7X + 5.25 (r2 = 0.997). This showed that the method met the requirements of validation based on this parameter and hence fit for application during analysis.
Table 2. Linearity studies.
Concentration(mg/g sample dry.wt) |
% AA (mean ± SD)n = 6 |
%RSD |
0.025 |
9.62 ± 0.14 |
1.46 |
0.038 |
12.40 ± 0.16 |
1.30 |
0.050 |
14.88 ± 0.28 |
1.88 |
0.083 |
22.91 ± 0.70 |
3.06 |
0.125 |
31.65 ± 0.42 |
1.33 |
0.250 |
54.67 ± 1.27 |
2.32 |
Regression equation |
Y = 200.7X + 5.25 (r2 = 0.997) |
|
3.2.3. Accuracy of the Method
Among known polyphenols, gallic acid (GA), a naturally occurring low molecular weight tri-phenolic compound has emerged as a strong antioxidant. Therefore, it was used as the QC material for spiking known concentrations of the tea sample to evaluate recovery of the method. The mean antioxidant activity (n = 3) for pre-analyzed sample, GA standard and GA spiked sample were 48.40, 18.25 and 65.58%respectively (Table 3). The recovery for the method was found to be 94%a demonstration that the method is satisfactory for the intended purpose and adequate for routine analysis.
Table 3. Accuracy studies.
n |
Sample type |
% AA (mean ± SD) |
%RSD |
3 |
Pre-analyzed sample |
48.40 ± 0.83 |
1.72 |
3 |
GA standard |
18.25 ± 0.30 |
1.64 |
3 |
GA spiked sample |
65.58 ± 0.83 |
1.30 |
|
Recovery % |
|
94 |
3.2.4. Limit of Detection (LOD) and Quantitation (LOQ) of the Method
Limit of detection (LOD) is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value whereas the quantitation limit (LOQ) of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The LOD found was 0.0017 mg/mL whereas LOQ was 0.0053 mg/mL. The low levels indicate that the method is capable of detecting significantly low antioxidant activity in tea matrices and tea based products. Moreover, the conditions of the validated method can be used in making decisions in tea related product formulations which would otherwise require very low levels of tea lacing to enhance flavor and strength.
3.3. Procedures of the Optimized and Validated Method
3.3.1. Sample Extraction
Finely ground tea test samples were weighed (0.0100 ± 0.0001) g into extraction tubes using analytical balance (BL-3200 HL, Shimadzu, Japan). 5.0 mL of hot methanol/water (8:2 v/v) extraction mixture was dispensed into the extraction tubes containing the sample before being stoppered. Mixing was done with the help of a vortex mixer (MV-1000, Taiwan Region) for 1 min and the mixture was placed in a water bath set at 70˚C. The heating continued for 10 min, mixing on the vortex mixer at the intervals of 5 min and 10 min. The extraction tubes from the water bath were removed, allowed to cool to room temperature and the stoppers removed. The tubes were placed in a centrifuge at 3500 rpm for 10 min after which the supernatant was carefully decanted into graduated tubes. The extraction steps were repeated resulting in two extracts. The two extracts were combined and made to 10 mL with cold methanol/water (8:2 v/v). The extracted sample was subjected to DPPH assay.
3.3.2. DPPH Assaying
The sample extract at room temperature (20˚C - 25˚C) was diluted fivefold withmethanol/water (8:2 v/v) and vortexed. A 0.1 mL of the diluted sample aliquot was well mixed with 3.9 mL of 0.06 mM DPPH. The working solution (Ws) was a mixture of 0.1 mL methanol/water (8:2 v/v) and 3.9 mL of 0.06 mM DPPH. Both working solution and sample/DPPH mixture were vortexed for 0.5 min before being incubated at 25˚C for 30 min in the dark. Absorbance (ABS) was then recorded at 517 nm using a Shimadzu 1800 series spectrophotometer. Methanol/water (8:2 v/v) mixturewas used as the blank.
The calculation of the sample antioxidant activity was done using the equation:
(2)
3.4. Applications of the Optimized and Validated Method
With the development of functional foods having beneficial effects on human health, the interest of scientists, consumers and industrialists in raw materials rich in antioxidants has increased considerably over the last few years. Moreover, consumers require more accurate information on the composition of the food which they eat. Natural antioxidants such as phenolic compounds have been reported to possess beneficial bioactivities due to their capacity to act as antioxidants. In tea varietal type, processing and storage are factors that have considerably affected the antioxidant activity in the products.
3.4.1. Variation in Levels of Antioxidant Activity with Varietal Type
Using the Validated DPPH Method
The procedures of the optimized and validated DPPH method for determination of antioxidant activity in aerated, semi-aerated and non-aerated tea products were used in twenty six (26) selected cultivars of KALRO-TRI germplasm in Kenya. The antioxidant activity of the cultivars is as shown in Table 4.
Table 4. Variation in levels of antioxidant activity with varietal type.
Cultivars |
Mean AA% |
Cultivars |
Mean AA% |
TRFK306/1 |
62.60edf |
306/FLD/GB/3 |
50.19f |
TRFK306/2 |
62.03b |
306/FLD/GB/4 |
50.58f |
TRFK306/3 |
60.79f |
306/FLD/GB/5 |
50.23f |
TRFK306/4 |
58.49c |
306/FLD/GB/6 |
44.91g |
TRFK91/1 |
61.73b |
306/FLD/GB/13 |
59.91cb |
TRFK660/1 |
60.88cb |
Mean |
56.24 |
TRFK597/4 |
68.61a |
CV% |
2.99 |
TRFK895/7 |
54.76d |
LSD (p < 0.005) |
2.75 |
TRFK895/22 |
54.42d |
|
|
AHP S15/10 |
58.49c |
|
|
TRFK914/39 |
54.40d |
|
|
TRFK6/8 |
59.65cb |
|
|
Ejulu |
61.76b |
|
|
BBK TN14/3 |
60.88cb |
|
|
D99/10 |
60.66cb |
|
|
ST 536 |
47.09j |
|
|
ST 543 |
50.93f |
|
|
ST 830 |
53.73ed |
|
|
ST 831 |
61.76b |
|
|
ST 832 |
61.45b |
|
|
306/FLD/GB/2 |
51.14ef |
|
|
Means with the same letters are not significantly different at p < 0.005.
Cultivars TRI 306/1, 306/2, 306/3, 306/4, 91/1, 660/1 and 597/4 are purple pigmented. Furthermore, in addition to the individual phenolic biochemicals of the green pigmented tea cultivars, they are rich in anthocyanins and GHG a special polyphenol. These attributes are replicated in the cultivars of 306/FLD/GB/3, 4, 5, 6 and 13 second generation cultivars of the TRI 306 series. The remaining cultivars are all green pigmented with the cultivars ST536, 543, 830, 831, and 832 having their origin from China. The antioxidant activity ranged from 44.91% - 68.61%. TRFK597/4 had the highest antioxidant activity while 306/FLD/GB/6 gave the lowest levels. Different Kenyan tea cultivars have different polyphenolic composition which impacts on their unique biochemical qualities [41]. Antioxidant activity of any tea product is a function of its total polyphenol content. Thus, the higher the total polyphenol content in a product, the higher the antioxidant activity [42]. Additionally, a study carried out by Cleverdon et al. while comparing total polyphenol content in infusions of black, green, red rooibos, chamomile and peppermint over different steep times gave an observation that the absolute polyphenol content and prediction of total antioxidant capacity was dependent on the type of tea product used [43]. The optimized and validated method unequivocally distinguishes the different tea cultivars in terms of their antioxidant levels and hence can be used in routine analyses.
3.4.2. Variation in Levels of Antioxidant Activity with Processed Tea
Type Using the Validated DPPH Method
Differently processed teas were sourced from selected factories across the tea growing regions in Kenya. The orthodox tea grades of BO-OP1, GO-FP, BO-FOF, BO-FP, GO-OP1, GO-FOP, GO-OPA, GO-FOF, GOFBOE, BO-FBOB and BO-FOP were processed as aerated, semi aerated and unaerated according to the optimized manufacture conditions in each of the different factories. Additionally, the cut, tear and curl (CTC) grades of PD, D1, F1, PF1 and BP1 were fully aerated before drying and grading accordingly. All the teas were subjected to the procedures of the optimized and validated DPPH method in determining their antioxidant capacity. The antioxidant activity of the teas is as shown in Table 5.
Table 5. Variation in levels of antioxidant activity with processed tea type.
Tea grade |
Mean AA% |
Tea grade |
Mean AA% |
Kipkebe PD |
36.48ji |
Matara BP1 |
38.95hg |
Kipkebe D1 |
37.98hgi |
Matara PF1 |
30.37hgi |
Kipkebe F1 |
31.08k |
Matara PD |
32.27k |
Kipkebe PF1 |
38.12hg |
Mean |
44.71 |
Kipkebe BP1 |
42.18f |
CV% |
2.27 |
Kangaita BO-OP1 |
46.22e |
LSD (p < 0.005) |
2.12 |
Kangaita GO-FP |
61.22c |
|
|
Gitugi BO-FOF |
39.47g |
|
|
Gitugi BO-FP |
38.05hgi |
|
|
Kiru GO-OP1 |
57.77c |
|
|
Kiru GO-OPA |
55.83d |
|
|
Itumbe GO-FOP |
65.61ba |
|
|
Itumbe BO-OPA |
37.00hji |
|
|
Itumbe GO-FOF |
64.12b |
|
|
Itumbe GO-FBOE |
66.71a |
|
|
Michimikuru BO-FBOB |
39.12g |
|
|
Mutunwa BO-FOP |
35.49j |
|
|
Matara D1 |
37.23hgi |
|
|
Means with the same letters are not significantly different at p < 0.005.
Aerated CTC teas in both Kipkebe and Matara tea factories ranged from 30.37% - 42.18%. The lowest antioxidant activity in black orthodox teas was 37.00% in Itumbe BO-OPA while the highest was 46.22% in Kangaita BO-OP1. Green orthodox teas had generally high antioxidant activity (55.83% - 66.71%) with Kangaita GO-FP being the highest.
Studies have shown that green tea has higher levels of polyphenols when compared to black tea [44]. However, the type and amount of flavonoids they contain differ [45]. For example, green tea contains a much higher amount of epigallocatechin-3-gallate (EGCG), whereas black tea is a rich source of theaflavins [46]. The findings on variation of antioxidant activity with different tea grades in the present study congruent well with work by Erol and Velioglu, 2010 on antioxidant activity of different grades of Turkish black tea [47]. Furthermore, differences in the total antioxidant potential obtained in green, black and earl grey teas, have demonstrated that not only the production method (fermentation or its absence), but also individual technological processes utilized by different manufacturers, as well as the origin of the tea itself, can impact the antioxidant properties of the final product which reaches the consumer [48].
4. Conclusion
This optimized and validated spectrophotometric DPPH method for analysis of antioxidant activity in aerated, semi-aerated and non-aerated tea products is simple, accurate, precise, reproducible and sensitive. The validation procedure confirms that this is a reliable method for determination of antioxidant activity in tea and related products. Furthermore, the method is adequate for application in routine analysis. Additionally, it is a proven alternative for companies and universities since it is quick, does not have extra costs with solvents or inert gas flows, and does not require intense training for operation.