GU J Sci, Part C, 10(4): 682-690 (2022)

Gazi University

Journal of Science
PART C: DESIGN AND TECHNOLOGY
http://dergipark.gov.tr/gujsc

Determination of Total Antioxidant Capacities as Ascorbic Acid Equivalent of

Ozan YAĞMUROĞLU1*
1
National Defence University, Turkish Air Force Academy, Department of Chemistry, 34149, İstanbul, Türkiye

Abstract
Article Info
In this study, the Digital Image-Based Colorimetric Detection Method developed by Bakırdere et
Research article al. was used to find the TAC (Total Antioxidant Capacity) value of tea samples from different
Received: 7.09.2022 brands. To determine the total amount of antioxidants in tea samples, the CUPRAC (cupric ion
Revision: 22.10.2022
reducing antioxidant capacity) method, which is widely used in antioxidant determination, was
Accepted: 24.10.2022
combined with a digital image-based colorimetric detection system. To use in our study, a box
with opaque wood material measuring 24 cm x 19 cm x 17 cm (width/length/depth) was designed
Keywords and manufactured. In the analysis, the oxidation reaction between the chromogenic copper(II)-
neocuproine (Cu(II)-Nc) reagent and antioxidants was utilized. The color change that occurs as a
CUPRAC result of the oxidation was calculated using an application on smartphones. In our study, analyzes
Tea
were performed on 4 different brand tea extract samples (tea A, tea B, tea C, tea D) to determine
Total antioxidant capacity
(TAC) the total antioxidant capacity of ascorbic acid equivalent. The TAC values for ascorbic acid
Smartphone equivalent in tea extract samples were found as 380 ± 8 mg/L (tea A), 402 ± 4 mg/L (tea B),
Digital image 213 ± 3 mg/L (tea C), 232 ± 4 mg/L (tea D) using the digital image-based colorimetric detection
systems.

1. INTRODUCTION

The oxygen molecule is a critical molecule for aerobic organisms. It has an important role in the formation
and continuation of life. However, the oxygen molecule causes the formation of free radicals in the
respiration mechanism and metabolism processes. Free radicals are atoms or molecules with one or more
unpaired electrons in their outer orbitals. Free radicals can have a positive or negative charge. They are not
stable structures. They are also very reactive despite their short half-life. They can interact very easily inside
the cell [1-3].
The main conditions that cause free radical formation in the body can be listed as follows; UV rays, various
drugs, fat oxidation, immunological reactions, alcohol, redox reactions, stress, alcohol, and smoking. Free
radicals formed in the body are generally hydrogen peroxide, hydroxy radical, superoxide anion,
peroxynitrite, hypochlorous acid, and alkoxyl radical [4,5]. Free radicals formed in living metabolism affect
the formation and progression rate of diseases such as heart diseases, cancer, diabetes, lung diseases, liver
diseases, and aging-related diseases. It is very important to neutralize free radicals in the body to prevent
the formation of these diseases or to reduce the rate of progression. Antioxidants play a role in preventing
the harmful effects of free radicals [6,7].
Antioxidants in our metabolism are used to prevent the formation of existing free radicals or to eliminate
their destructive effects by interacting with existing free radicals. Antioxidants are also the main component
of anti-aging products. In addition to being produced by the body, antioxidants can also be taken from the

*Corresponding author, e-mail: [email protected] DOI:10.29109/gujsc.1172357

outside through food. The main antioxidants taken by food are vitamins (C, A, and E), flavonoids,
carotenoids, and polyphenols [8]. Ascorbic acid (vitamin C) is a water-soluble chemical with antioxidant
properties. Because of endiol group in its structure, it shows acidic properties as well as reducing properties.
It cannot be synthesized by the human body. For this reason, ascorbic acid is taken into the body from the
outside through food [9].
Due to the critical role of antioxidants in preventing the extremely negative effects of free radicals in the
body, it is necessary to know the total antioxidant capacity (TAC), which reveals the total amount of all
antioxidants in the samples. TAC value is one of the criteria used in the processes of determining the quality
of foods [10,11].
There are many methods with different scientific approaches to determine the total amount of antioxidants
in samples. Total antioxidant capacity (TAC) determination techniques can be grouped under two headings
as methods based on electron transfer (ET) and methods based on hydrogen atom transfer (HAT) reactions.
While the HAT method is based on competitive kinetic reactions, the color change is used in ET-based
methods [12]. The Cupric ion-reducing antioxidant capacity (CUPRAC) method is among the methods of
determining antioxidant capacity based on electron transfer [13].
In the CUPRAC method, firstly, copper(II)-neocuproin complex (Cu(II)-Nc) is obtained as a result of the
reaction between 2,9-dimethyl-1,10-phenanthroline (Neocuproin-Nc) and Cu(II). The synthesized complex
is reduced to copper(I)-neocuproin [Cu(I)-Nc] chelate in the presence of an antioxidant. During this
reduction event, the maximum absorbance occurs at 450 nm. Using this approach, the total antioxidant
capacity in the samples can be determined [14].
This method is frequently used to determine the TAC values of different antioxidant sources [15,16].
Studies on the determination of antioxidant levels in herbal tea [17], human serum [18], tea [19], and apricot
[20] samples by using the CUPRAC technique and using different analysis methods are reported in the
literature.
Colorimetric analysis methods developed by utilizing the color change that occurs as a result of the
interaction of the analyte with different chemicals in sample analysis have been used for a long time.
Recently, with the developing technology, color changes in the sample environment have started to be
detected by digital imaging methods [21,22].
The analysis method developed using this imaging technique is called Digital Image Colorimetry (DIC). In
digital image colorimetry (DIC), analyses can be performed using RGB (red-green-blue) values in digital
images obtained with smartphone cameras, portable cameras, and similar imaging methods. Colors can be
evaluated in terms of three basic components (RGB). Each component can be expressed numerically with
a number between 0 and 255 [23-26].
In this study, the total antioxidant capacity (TAC) value in tea extract samples belonging to different brands
was determined by digital image-based colorimetric detection using the imaging box developed within the
scope of the study as the analysis device. The CUPRAC assay was used to determine the TAC value in tea
extract samples. The TAC value was calculated as the equivalent antioxidant capacity of ascorbic acid.
2.MATERIALS AND METHODS

2.1. Instrumentation

In the sample preparation procedures, Shimadzu ATX224R analytical balance was used for weighing. pH
measurements were conducted with the Ohaus Starter 3100 pH-ion meter. KUDOS SK 3310 HP ultrasonic
water bath was used for homogenization in sample preparation processes. In the study carried out, Daihan
MSH-20A Magnetic Stirrer with Analog Heater and DLAB MX-S Vortex were employed for mixing
processes. A Huawei (Android 9.0) smartphone with 16 megapixels and an f/2.2 lens aperture was used for
digital imaging for colorimetric analysis. The flash lamb of the smartphone was not used during digital
imaging. To use in our study, a box with opaque wood material measuring 24 cm x 19 cm x 17 cm
(width/length/depth) was designed and manufactured (Figure 1). The inner surface of the box is produced
in white color to ensure that the light inside is reflected homogeneously in all directions. 12 V white LED
strips of equal length are attached to the ceiling and side surfaces of the box. A cutout with a diameter of
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2.2 cm was drilled on the cover of the box for the placement of the smartphone's camera. To ensure stability
and optimization during the analysis, a sample placement part was made in the box, sized to fit 10 mm
quartz cuvettes. The Color Detector smartphone application was used to determine the RGB values during
the analysis.

Figure 1. Schematic representation of the designed colorimetric analysis box

Neocuproine standards and Ascorbic acid were obtained from Sigma-Aldrich, Germany. Ethanol was used
as a solvent in the stock standard solution to be used during the study for ascorbic acid. The dilution of the
standard solution was done with ethanol. As part of the modified CUPRAC analysis, 1.0 x 10 −2 M Cu(II)
solution was prepared by dissolving CuCl2 in deionized water. A standard solution of 9.5 x 10−3 M
neocuproine (Nc) was obtained using ethanol as solvent. 1.0 M ammonium acetate (NH4Ac) buffer solution
was prepared by using the solution of NH4Ac in deionized water. NH4Ac, ammonia solution (25%), CuCl2,
and Na2HPO4 were all obtained from Merck, Germany. During the study, ultrapure water obtained from
the Elektro-Mag Laboratory Water Drop M4 system was used for sample/standard preparation and cleaning
of all glassware.
2.3. Digital Image-Based Cuprac Method

In our study, the CUPRAC method described by Bakırdere [23] was used. As part of the CUPRAC method,
1.0 mL of 9.5 × 10−3 M solution of neocuproine and 1.0 mL of 1.0 × 10−2 M copper(II) were added to a
centrifuge tube. Then, 1.0 mL of 1.0 M pH 7.5 ammonium acetate buffer was added to the same tube to
adjust the pH of the medium. Finally, 0.4 mL of standard solution was added to the centrifuge tube. The
sample volume was made up to 4 mL with deionized water. Colorimetric analysis studies were carried out
using the digital imaging box (Figure 1) designed with the last sample obtained. During the analysis,
attention was paid to taking the image from the same area in the sample cuvette. Measurements were
captured three times for each standard/sample.
2.4. Procedure

Tea extract samples of four different brands were taken from a local market in İstanbul. In the continuation
of the study, these teas will be mentioned as tea A, Tea B, Tea C, and Tea D. Using the tea brewing method
recommended by the tea companies in their packages, 2 grams of tea sample was added into 125 mL of
boiled distilled water. It waited for 15 minutes for the teas to brew. 0.4 mL samples were prepared by
diluting the prepared stock tea extract samples 15 times. Then, digital image-based colorimetric analysis of
tea extract samples belonging to four different brands was performed. TAC values in the tea samples were
calculated by using the results obtained as a result of the analysis and the calibration graph drawn.
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3.RESULTS

The values defined by Bakırdere et al. were used as optimum conditions for variables within the scope of
the study [23]. Considering the effect of the concentrations of the reagents on the formation reaction of the
Cu(II)-Nc complex, 10 mM Cu(II) and 9.5 mM neocuproine were used. Another parameter that affects the
complexation reaction is the pH of the medium. A buffer with a pH value of 7.5 was used within the scope
of the study. The duration of the oxidation reaction between the synthesized Cu(II)-Nc complex and the
antioxidants was determined as 45 minutes. When the R, G, and B color channels used in monitoring the
antioxidant concentration in the sample medium are compared, the B channel is used because it has more
linear data [23].
3.1. Analytical Figures of MERIT

The analytical performance of the digital image-based colorimetric system based on the CUPRAC assay,
which will be used within the scope of our study, was evaluated. For this purpose, colorimetric analysis of
samples containing different concentrations of ascorbic acid was performed to obtain a calibration plot. The
blue intensity values obtained as a result of the colorimetric analysis of the samples based on digital imaging
are shown in Table 1, and the calibration graph drawn using these values is shown in Figure 2.
Table 1. Blue intensity value of samples at different concentrations

Concentration
Blue Intensity
(mg/L)

1 102

2 80

3 61

4 44
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Figure 2. Calibration plot of varying blue intensity versus increasing concentration of ascorbic acid
While creating the calibration plot, the intensity of the blue color, one of the RGB color tones, was used as
the analysis parameter. When different concentrations of ascorbic acid are added to the Cu(II)/Nc complex,
a change in the intensity of the blue color occurs. During the analysis, three digital images were taken for
each sample and the color intensity of four different points of each image was measured. As a result of the
measurement results, linearity was observed in the range of 1.0-4.0 mg/L. The correlation coefficient of the
drawn calibration plot was calculated as 0.9962. Limits of quantification (LOQ) and detection (LOD) values
were found to demonstrate the accuracy and sensitivity of the analytical method developed. Eight repetitive
absorbance measurements of the lowest concentration in the calibration plot were performed to calculate
the LOD and LOQ values. Using these measurement results, the standard deviation (SD) value was
calculated. Calculations were made using the 3SD/m (m=slope of the calibration plot) formula for the LOD
value and the 10SD/m formula for the LOQ value. As a result of the calculations, the limit of detection
value for the analytical method we developed was 0.4 mg/L, and the limit of quantification value was 1.3
mg/L. The percent relative standard deviation value (%RSD) of the analytical method developed was found
to be 2.3%. This value shows that the method has high reproducibility.
3.2. TAC Analysis of Different Brands of Tea

Within the scope of TAC analysis, four different brands of tea were purchased. These teas are coded as tea
A, tea B, tea C, and tea D. Analyzes were carried out by taking 0.4 mL of the tea extract samples prepared
as described in the procedure section. The digital images obtained during the analysis of tea A, tea B, tea
C, and tea D are shown in Figure 3.
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Figure 3. Digital images of different brands of tea extract samples

Analyzes based on the blue color intensity of teas belonging to different brands were carried out with digital
imaging techniques. Digital-image-based colorimetric analysis results of tea extract samples are given in
Table 2.
Table 2. Colorimetric analysis results of tea extract samples

TAC Value
Samples Blue Intensity
(mg/L)

Tea A 59 380 ± 8

Tea B 56 402 ± 4

Tea C 85 213 ± 3

Tea D 83 232 ± 4

When the results in Table 2 are examined, it is seen that the tea with the highest antioxidant content among
the four different tea brands is Tea B. Tea B is followed by Tea A, Tea D, and Tea C, respectively.
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4.CONCLUSIONS

In our study, TAC values of teas belonging to different brands were determined by using color change due
to the presence of antioxidants during the CUPRAC assay. The color change in the analysis environment
was followed by the digital image-based colorimetric method. Within the scope of this study, a completely
original digital image-based colorimetric analysis box was designed and manufactured. The developed
colorimetric analysis box is made of opaque material. Its inner surface is covered with white-colored
material to ensure the homogeneous spread of light around the sample. Analyzes were carried out using a
smartphone as the imaging device, thanks to a cutout on the front surface of the colorimetric analysis box.
With the developed colorimetric analysis box, firstly, the analysis of ascorbic acid samples with known
concentrations was carried out using the CUPRAC assay. A calibration curve was drawn using the
measurement results and a linear region was observed in the range of 1-4 ppm ascorbic acid. Antioxidant
capacities of tea extracts were calculated as ascorbic acid equivalents using the determined linear region.
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REFERENCES

[1] Cheeseman, K. H., & Slater, T. F. (1993). An introduction to free radical biochemistry. British
Medical Bulletin, 49(3), 481–493. https://doi.org/10.1093/oxfordjournals.bmb.a072625

[2] Phaniendra, A., Jestadi, D. B., & Periyasamy, L. (2015, January 1). Free Radicals: Properties,
Sources, Targets, and Their Implication in Various Diseases. Indian Journal of Clinical Biochemistry.
Springer India. https://doi.org/10.1007/s12291-014-0446-0

[3] Wu, J. (2020, September 1). Tackle the free radicals damage in COVID-19. Nitric Oxide - Biology
and Chemistry. Academic Press Inc. https://doi.org/10.1016/j.niox.2020.06.002

[4] Kopáni, M., Celec, P., Danišovič, L., Michalka, P., & Biró, C. (2006, February). Oxidative stress
and electron spin resonance. Clinica Chimica Acta. https://doi.org/10.1016/j.cca.2005.05.016

[5] Halliwell, B., & Aruoma, O. I. (1991, April 9). DNA damage by oxygen-derived species Its
mechanism and measurement in mammalian systems. FEBS Letters. https://doi.org/10.1016/0014-
5793(91)80347-6

[6] Gong, Y., Huang, X. Y., Pei, D., Duan, W. D., Zhang, X., Sun, X., & Di, D. L. (2020). The
applicability of high-speed counter current chromatography to the separation of natural
antioxidants. Journal of Chromatography A, 1623. https://doi.org/10.1016/j.chroma.2020.461150

[7] Romera-Castillo, C., & Jaffé, R. (2015). Free radical scavenging (antioxidant activity) of natural
dissolved organic matter. Marine Chemistry, 177, 668–676.
https://doi.org/10.1016/j.marchem.2015.10.008

[8] Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1997). Antioxidant properties of phenolic
compounds. Trends in Plant Science. Elsevier Ltd. https://doi.org/10.1016/S1360-1385(97)01018-2

[9] Buettner, G. R. (1993). The Pecking Order of Free Radicals and Antioxidants: Lipid Peroxidation,
α-Tocopherol, and Ascorbate. Archives of Biochemistry and Biophysics, 300(2), 535–543.
https://doi.org/10.1006/abbi.1993.1074

[10] Cárdenas, A., Gómez, M., & Frontana, C. (2014). Development of an electrochemical cupric
reducing antioxidant capacity method (CUPRAC) for antioxidant analysis. Electrochimica Acta, 128, 113–
118. https://doi.org/10.1016/j.electacta.2013.10.191

[11] Zhao, W., Xiang, Y., Xu, J., He, X., & Zhao, P. (2020). The reversible surface redox of polymer
dots for the assay of total antioxidant capacity in food samples. Microchemical Journal, 156.
https://doi.org/10.1016/j.microc.2020.104805

[12] Cao, G., Sofic, E., & Prior, R. L. (1997). Antioxidant and prooxidant behavior of flavonoids:
Structure-activity relationships. Free Radical Biology and Medicine, 22(5), 749–760.
https://doi.org/10.1016/S0891-5849(96)00351-6

[13] Apak, R., Güçlü, K., Özyürek, M., & Karademir, S. E. (2004). Novel total antioxidant capacity
index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the
presence of neocuproine: CUPRAC method. Journal of Agricultural and Food Chemistry, 52(26), 7970–
7981. https://doi.org/10.1021/jf048741x

[14] Özyürek, M., Bektaşoĝlu, B., Güçlü, K., Güngör, N., & Apak, R. (2010). A novel hydrogen
peroxide scavenging assay of phenolics and flavonoids using cupric reducing antioxidant capacity
(CUPRAC) methodology. Journal of Food Composition and Analysis, 23(7), 689–698.
https://doi.org/10.1016/j.jfca.2010.02.013

[15] Karadirek, Ş., Kanmaz, N., Balta, Z., Demirçivi, P., Üzer, A., Hizal, J., & Apak, R. (2016).
Determination of total antioxidant capacity of humic acids using CUPRAC, Folin-Ciocalteu, noble metal
690 Ozan YAĞMUROĞLU / GU J Sci, Part C, 10(4):682-690 (2022)

nanoparticle- and solid-liquid extraction-based methods. Talanta, 153, 120–129.

[16] Prenesti, E., Berto, S., Gosmaro, F., Fisicaro, P., Bagnati, M., & Bellomo, G. (2020). Measurement
uncertainty evaluation of the Total Antioxidant Capacity of human plasma tested by the CUPRAC-BCS
method. Measurement: Journal of the International Measurement Confederation, 152.
https://doi.org/10.1016/j.measurement.2019.107289

[17] Apak, R., Güçlü, K., Özyürek, M., Esin Karademir, S., & Erçǧ, E. (2006). The cupric ion reducing
antioxidant capacity and polyphenolic content of some herbal teas. International Journal of Food Sciences
and Nutrition, 57(5–6), 292–304. https://doi.org/10.1080/09637480600798132

[18] Apak, R., Güçlü, K., Özyürek, M., Esı̊ n Karademı̊ r, S., & Altun, M. (2005). Total antioxidant
capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: The CUPRAC
method. Free Radical Research, 39(9), 949–961. https://doi.org/10.1080/10715760500210145

[19] Krylova, E., Gavrilenko, N., Saranchina, N., & Gavrilenko, M. (2016). Novel Colorimetric Sensor
for Cupric Reducing Antioxidant Capacity (CUPRAC) Measurement. In Procedia Engineering (Vol. 168,
pp. 355–358). Elsevier Ltd. https://doi.org/10.1016/j.proeng.2016.11.120

[20] Güçlü, K., Altun, M., Özyürek, M., Karademir, S. E., & Apak, R. (2006). Antioxidant capacity of
fresh, sun- and sulphited-dried Malatya apricot (Prunus armeniaca) assayed by CUPRAC, ABTS/TEAC
and folin methods. International Journal of Food Science and Technology, 41(SUPPL. 1), 76–85.
https://doi.org/10.1111/j.1365-2621.2006.01347.x

[21] Catelani, T. A., Bittar, D. B., Pezza, L., & Pezza, H. R. (2019). Determination of amino acids in
gym supplements using digital images and paper platform coupled to diffuse reflectance spectroscopy and
USB device. Talanta, 196, 523–529. https://doi.org/10.1016/j.talanta.2018.12.052

[22] Zamora-Garcia, I., Correa-Tome, F. E., Hernandez-Belmonte, U. H., Ayala-Ramirez, V., &
Ramirez-Paredes, J. P. (2021). Mobile digital colorimetry for the determination of ammonia in aquaculture
applications. Computers and Electronics in Agriculture, 181.
https://doi.org/10.1016/j.compag.2020.105960

[23] Borahan, T., Girgin, A., Atsever, N., Zaman, B. T., Chormey, D. S., & Bakırdere, S. (2022).
Development of a double-monitoring method for the determination of total antioxidant capacity as ascorbic
acid equivalent using CUPRAC assay with RP-HPLC and digital image-based colorimetric
detection. European Food Research and Technology, 248(3), 707–713. https://doi.org/10.1007/s00217-
021-03923-7

[24] Lin, B., Yu, Y., Cao, Y., Guo, M., Zhu, D., Dai, J., & Zheng, M. (2018). Point-of-care testing for
streptomycin based on aptamer recognizing and digital image colorimetry by smartphone. Biosensors and
Bioelectronics, 100, 482–489. https://doi.org/10.1016/j.bios.2017.09.028

[25] Masawat, P., Harfield, A., & Namwong, A. (2015). An iPhone-based digital image colorimeter for
detecting tetracycline in milk. Food Chemistry, 184, 23–29.
https://doi.org/10.1016/j.foodchem.2015.03.089

[26] Silva, A. F. S., & Rocha, F. R. P. (2020). A novel approach to detect milk adulteration based on the
determination of protein content by smartphone-based digital image colorimetry. Food Control, 115.
https://doi.org/10.1016/j.foodcont.2020.107299