# The Effect of Brewing Time on the Antioxidant Activity of Tea Infusions
## 4 Discussion
It could be an effect of anthocyanins degradation under the influence of high temperature and oxidation processes, which could also explain why the anthocyanins content decreased in green leaf tea and hibiscus flower tea infusions in our study. When the temperature increases, the anthocyanin structure shifts towards the colourless methanol pseudo-base and chalcone forms [36]. If the temperature is reduced to room temperature (or lower), the methanol pseudo-base form can be converted back into red cationic form (the desirable colour of anthocyanin foods), but the chalcone form is difficult to convert into the cationic ones, so this type of heat degradation of anthocyanins is irreversible [4]. Elevated temperatures can also cause monomeric anthocyanins to polymerise, resulting in products browning [37]. The structure of anthocyanins changes due to temperature rise as sugar particles attached to anthocyanins are released and converted into aglycone (anthocyanidin) [38]. The release of sugar is followed by the separation of A, B and C rings, which induces the formation of two new compounds: phloroglucinol aldehyde and 4-Hydroxybenzoic acid, which, in turn, contributes to colour change [38]. In our study, the anthocyanins content of hibiscus flower infusion decreased by 33% after 10 minof brewing, and by 50% after 15 min. Hibiscus contains mostly anthocyanins such as delphinidin-3-sambubioside, cyanidin-3-glucoside and delphinidin-3-glucoside [34]. Studies showed that the rate at which delphinidin-3-sambubioside degrades is higher than that of cyanidin-3-sambubioside, and depends on temperature rise [37]. The degradation of these anthocyanins produces gallic acid and protocetachuic acid, which is accompanied by colour change. [PERSON] et al. [39] indicates that some anthocyanin compounds are also lost and/or oxidised in processes associated with tea manufacturing. Studies by [PERSON] and [PERSON] [31] demonstrated that the capacity of inhibiting DPPH was not determined by brewing time (five and ten minutes) for black, green, white, yellow, oolong and Pu-erh teas. [PERSON] et al. [23] observed the highest activity of DPPH in black tea and green tea after three minutes of brewing. The study by [PERSON] et al. [27] showed that oolong tea brewed for five minutes at 100 \({}^{\circ}\)C revealed the highest anti-oxidant activity to DPPH. However, [PERSON] et al. [29] demonstrated that teas brewed for over 15 min contain less DPPH and ABTS. Similarly, [PERSON] et al. [40] noted the highest anti-oxidant activity to DPPH and ABTS free radicals after 5 min of brewing green tea and black tea, and the lowest after 30 min of brewing. [PERSON] et al. [41] found that a five minute brewing time was sufficient to obtain tea infusions (green + oolong + Pu-erh) with the highest antioxidant activity, reduced for longer brewing times. We discovered that, for most infusions, brewing time extended to 15 min reduced the capacity of DPPH to free radicals away in infusions, whereas antioxidant activity to ABTS did not depend on brewing time. Although both indicators describe the free radical capturing capacity, the results are not identical. This is due to the fact that DPPH is a stable nitrogen-centred radical that bears no similarity to the highly reactive superoxide radicals involved in lipid peroxidation, while ABTS reacts with hydrophilic and lipophilic compounds [23]. Polypphenols strongly tend to polymerise, but when the degree of polymerisation exceeds the critical value, the availability of hydroxyl groups in reactions with radicals decreases, which reduces the antioxidant activity of tea infusions [42]. Therefore, the significant decline in antioxidant activity to DPPH with time and at higher temperatures can be associated with the loss of polyphenic components in infusions. It is also possible that the structure and solubility of -phenolic compounds in water, leaf or flower size, and the porosity of tea bags, do have an effect [23].