Study and evaluation antioxidant activity of dietary supplements with green tea extract

The aim of the work is to determine and evaluate the antioxidant activity of dietary supplements with green tea extract, as well as to introduce conditional terms of the level of AOA. Materials and methods. The object of the study was dietary supplements of different manufactures: “Green Tea Extract” of Natural Sources, USA (DS1), “Extract of green tea” of Elit-Pharm, Ukraine (DS2) and “Green tea” of Pharmakom, Ukraine (DS3). Potentiometric measurements were conducted by pH meter Hanna 2550 (Germany) with a combined platinum electrode EZDO 5010. Epigallocatechin-3-O-gallate ˃98.0 % (Sigma Aldrich), K3[Fe(CN)6], K4[Fe(CN)6], NaHPO4, KH2PO4 were analytical grade. Results. It was established that a value of the antioxidant activity of DS1 was 36.51 mmol/tab, DS2 – 29.78 mmol/tab, and DS3 – 16.67 mmol/tab. DS1 had the highest value of the antioxidant activity, which correlated with the content of catechins (r2 = 0.9314). According to the proposed conditional terms of antioxidant activity dietary supplements, DS1 and DS2 possessed a low level of antioxidant activity, whereas DS3 corresponded to very low level of antioxidant activity. Conclusions. It was found that the studied dietary supplements with green tea extract have antioxidant activity, which correlates with the content of catechins.

Актуальные вопросы фармацевтической и медицинской науки и практики. 2021. Т. 14, № 2(36). С. 215-219 In light of the widespread degradation of environmental situa tion, a decrease in the content of nutrients in food, an increase in the negative impact of living in stressful conditions of a modern urban metropolis, interest in antioxidant substances that reduce the negative impact and consequences of all these processes is inexorably increasing. Antioxidants take part in the system of the defense mechanism of the human body, which main aim is combating pathologies and disea ses associated with the attack of free radicals and reactive oxygen, helping to inhibit oxidative processes. The benefit of antioxidants (AO) bases on their healing counteraction to free radicals in particular, oxidative stress in general, as well as in promoting the prevention and treatment of a number of cancers, improving human health, and increasing life [1].
More and more and more, it is possible to find in the market several new products claiming their antioxidant properties. Indeed, these are becoming an important and emerging para meter to assess the quality of the product. It has observed a high demand and interest of people on dietary supplements with green tea extract on account of their high values of antioxidant activity (AOA) [2]. Green tea has a significant high antioxidant potential owing to the presence in leaves flavon-3-ols or other name catechins [3]. These compounds differ from other poly phenols in that both carbonyl and hydroxyl groups are absent in the 4 positions. It is the most recovered of the flavonoids and therefore has the greatest antioxidant potential [4].
The major catechins in green tea are epigallocatechin-3-gal late (EGCG), epicatechin-3-gallate, epigallocatechin, and epicatechin. EGCG is the major catechin in green tea and accounts for 50-80 % representing 200-300 mg/brewed cup of green tea [5]. EGCG is with inhibitory effects in many aspects of abnormal changes, such as antioxidant, an ticancer, anti-inflammatory, anticollagenase, and antifibrosis effects, appearing in its wide functional range [6]. Although, the amount of tea catechins has the highest level of AOA: it is 25-100 times higher than that of α-tocopherol and ascorbate under comparable conditions, and epigallocatechin-3-O-gal late is one of the most powerful known plant AO [7].
However, today there is no regulation documents in which a universal method would be used for determining and as sessing the level of AOA of test samples, such as a dietary supplement, drug, cosmetic product, alcoholic beverages. Also, there are no specific conditional terms for evaluating AOA, which does not allow to evaluate, compare the obtained values of AOA and conduct quality control of products with antioxidant property.

Aim
The aim of our study is to determine and evaluate the anti oxidant activity of dietary supplements with green tea extract, as well as to introduce conditional terms of the level of AOA.
Potentiometric measurements were conducted by pH meter Hanna 2550 (Germany) with a combined platinum electrode EZDO 5010. Weighing was carried out using digital analytical balance АN100 (AXIS, Ukraine) with d = 0.0001 g.
Epigallocatechin-3-O-gallate ˃98.0 % (Sigma Aldrich), The initial potential of the mediator solution was measured after the initial one was established, 1.00 mL of the aliquot of the prepared solutions was added and a final potential was measured. The difference (∆E) between the initial (E 0 ) and final (E 1 ) potentials were found. The shift of potential was explained by the change of the ratio of oxidized and reduced forms of the mediator system as a result of the following chemical reaction: where AO -antioxidant; AO ox -oxidized form of antioxi dant; a, b -stoichiometric coefficients.

+ α
In order to calculate in mmol/tab the expression 2 was used: Statistical analysis was performed in Microsoft Excel 2010 with the accepted significance level α = 0.05. Results were expressed as mean ± confident interval from five measurements.

Results
The level of AOA of DS1 was exceptionally high and contained 36.51 ± 0.46 mmol/tab, DS2 had 29.78 ± 0.35 mmol/tab, and DS3 -16.67 ± 0.29 mmol/tab. These values were found by expression 1. In addition, the values of AOA of dietary sup plements were calculated in mmol/100 mL in order to classify them by offered conditional terms, in this case, expression 2 was used. As result, the DS1 had 73.02 mmol/100 mL, DS2 -59.56 mmol/100 mL and DS3 -33.28 mmol/100 mL. In our previous research [8], the total content of catechins was established by absorption spectrophotometry. It was found that the total content of catechins was 146.80 ± 1.36, 79.0 ± 0.88, 28.0 ± 0.75 mg in DS1, DS2, and DS3, respec tively. The results of the study are in Table 1.
For elaborating conditional terms, the levels of АОА of EGCG were determined for following concentrations: 0.5, 0.1, 0.01, 0.001, 0.0001 и 0.00001 mol/L, the results of the study are shown in Table 2.

Discussion
According to the obtained data it easy to see that DS1 had the highest value of the antioxidant activity, second place took DS2 and last one -DS3.
For evaluation of the antioxidant potentials of the in vestigated dietary supplements, the potentiometric assay was applied. This method was chosen for the following reasons: firstly, it does not request expansive reagents and equipment, secondly, the method is quite sensitive, precise and fastness in comparison with other assays of evaluation AOA and moreover potentiometric method enables a prompt screening of the antioxidant capacity of a series of organic biocompounds even in complex or colored samples [9]. Although, potentiometric assay represents a single electron transferbased method.
A simple linear regression analysis was used to investigate the correlation between the content of catechins and values of AOA. As shown in Figure 1, there was a significant positive correlation (r 2 = 0.9314) between the content of catechins and values of AOA.
Today, there are no uniform criteria for assessing antioxi dant activity, which in turn complicates the understanding of what is good and what is bad. It is also impossible to compare the results of determining the AOA of the analyzed objects and to carry out quality control by the parameter of AOA. Hence, it was decided to create conditional terms denoting the AOA of the analyzed samples. Epigallocatechin-3-O-gal late was chosen as the "gold" standard for creating these conditional terms and this choice is associated with a reason that in many comparative studies to determine the value of AOA of antioxidants [10][11][12], EGCG had the highest ability to scavenge free radicals. According to created conditional terms of АОА, dietary supplements DS1 and DS2 possess a low AOA, whereas "Green tea" has very low AOA.

Conclusions
1. This study reported that all investigated dietary supple ments with green tea extract had antioxidant activity.
2. The greatest value of antioxidant activity was found in the dietary supplement, which had the highest content of catechins.
3. The created conditional terms can be used in evaluating the antioxidant activity of products of the pharmaceutical, food, and cosmetology industries.
Prospects for further research. Introduction of regulation documents of determination antioxidant activity in products that associated with antioxidant activity and labeling informa tion about level and conditional term of antioxidant activity.