**9. Antioxidant activity assays**

Various assays have been applied to determine honey antioxidant activity. The most com‐ mon ones are colorimetric assays, DPPH (1,1 diphenyl‐2‐picrylhydrazyl), ABTS (2,2′‐azino‐ bis (3‐ethylbenzthiazoline‐6‐sulfonic acid)), FRAP (ferric reducing antioxidant power) and TEAC (Trolox equivalent antioxidant capacity), based on electron transfer, and ORAC (oxygen radical absorbance capacity) assay, based on hydrogen atom transfer and other techniques as voltammetric assays [34, 41, 46]. The total phenolic content is commonly spec‐ trophotometrically determined with a Folin‐Ciocalteu method, sometimes with modification and total flavonoid contents is generally measured by colorimetric assay with aluminum chloride [40, 54].

At the present time, no single available assay for testing the antioxidant capacity provides all the desired information. An evaluation of the overall antioxidant capacity may require mul‐ tiple assays to generate an "antioxidant profile" encompassing reactivity towards both aque‐ ous (DPPH and ABTS) and lipid/organic radicals (ORAC) directly through radical quenching and radical‐reducing mechanisms (DPPH, ABTS, FRAP and ORAC) and indirectly through metal complexing (FRAP) [64].

Gorjanović et al. [41] evaluate hydrogen peroxide sequestration capacity of single bioactive compounds isolated from honey by voltammetric technique. As result, the flavonoids showed the highest hydrogen peroxide scavenging activity among the compounds, followed by phe‐ nolic acids. Activity of predominant honey sugars, fructose, glucose and maltose was found to be three orders of magnitude lower than tested flavonoids, but their contribution to total activity is significant due to their quantity. High hydrogen peroxide scavenging activity has been attributed to some amino acids, aromatic and basic ones, whereas non‐polar amino acids, such as proline, the most prevalent amino acid in honey (0.40–2.2 mg/kg), possess low activity. Although phenolics are minor honey constituents, their antioxidant activity is high enough to correlate between honey hydrogen peroxide scavenge and total phenolic content [41].

Antioxidants *in vitro* assays do not consider physiological conditions such as concentration of intracellular metabolites nor does consider metabolic factors such as bioavailability and enzymatic transformations [58]. The *in vivo* assay, using yeast cells, specifically the specie *Saccharomyces cerevisiae,* represents an alternative to evaluate antioxidant activity. Yeasts are unicellular eukaryotic organisms widely studied and have great similarity with higher mammalian cells, especially in regard to the antioxidant defence system [66]. Because of this, it becomes an interesting biological model to evaluate biological activity related to natural extracts and molecules [66].

The use of *S. cerevisiae* cells as a study model has other important advantages. Its genome is completely elucidated, thereby facilitating the production of genetically modified strains for further studies; adding to this, its low cost of cells maintenance, ease of handling in the laboratory, rapid growth and low rate of spontaneous mutations [67]. Moreover, prelimi‐ nary studies in yeast substituting the use of guinea pigs, rats and mice, certainly speeds up research work.

Furthermore, the *in vivo* assays measure the effect of an antioxidant on cell survival [9]. The biological yeast‐based method can also measure the ability of a compound to induce cellular resistance to the damaging effects of oxidants [10, 11]. The determination of the lipid mem‐ brane integrity is an important parameter in verifying oxidative damage.

**9. Antioxidant activity assays**

chloride [40, 54].

300 Honey Analysis

metal complexing (FRAP) [64].

extracts and molecules [66].

research work.

Various assays have been applied to determine honey antioxidant activity. The most com‐ mon ones are colorimetric assays, DPPH (1,1 diphenyl‐2‐picrylhydrazyl), ABTS (2,2′‐azino‐ bis (3‐ethylbenzthiazoline‐6‐sulfonic acid)), FRAP (ferric reducing antioxidant power) and TEAC (Trolox equivalent antioxidant capacity), based on electron transfer, and ORAC (oxygen radical absorbance capacity) assay, based on hydrogen atom transfer and other techniques as voltammetric assays [34, 41, 46]. The total phenolic content is commonly spec‐ trophotometrically determined with a Folin‐Ciocalteu method, sometimes with modification and total flavonoid contents is generally measured by colorimetric assay with aluminum

At the present time, no single available assay for testing the antioxidant capacity provides all the desired information. An evaluation of the overall antioxidant capacity may require mul‐ tiple assays to generate an "antioxidant profile" encompassing reactivity towards both aque‐ ous (DPPH and ABTS) and lipid/organic radicals (ORAC) directly through radical quenching and radical‐reducing mechanisms (DPPH, ABTS, FRAP and ORAC) and indirectly through

Gorjanović et al. [41] evaluate hydrogen peroxide sequestration capacity of single bioactive compounds isolated from honey by voltammetric technique. As result, the flavonoids showed the highest hydrogen peroxide scavenging activity among the compounds, followed by phe‐ nolic acids. Activity of predominant honey sugars, fructose, glucose and maltose was found to be three orders of magnitude lower than tested flavonoids, but their contribution to total activity is significant due to their quantity. High hydrogen peroxide scavenging activity has been attributed to some amino acids, aromatic and basic ones, whereas non‐polar amino acids, such as proline, the most prevalent amino acid in honey (0.40–2.2 mg/kg), possess low activity. Although phenolics are minor honey constituents, their antioxidant activity is high enough to

correlate between honey hydrogen peroxide scavenge and total phenolic content [41].

Antioxidants *in vitro* assays do not consider physiological conditions such as concentration of intracellular metabolites nor does consider metabolic factors such as bioavailability and enzymatic transformations [58]. The *in vivo* assay, using yeast cells, specifically the specie *Saccharomyces cerevisiae,* represents an alternative to evaluate antioxidant activity. Yeasts are unicellular eukaryotic organisms widely studied and have great similarity with higher mammalian cells, especially in regard to the antioxidant defence system [66]. Because of this, it becomes an interesting biological model to evaluate biological activity related to natural

The use of *S. cerevisiae* cells as a study model has other important advantages. Its genome is completely elucidated, thereby facilitating the production of genetically modified strains for further studies; adding to this, its low cost of cells maintenance, ease of handling in the laboratory, rapid growth and low rate of spontaneous mutations [67]. Moreover, prelimi‐ nary studies in yeast substituting the use of guinea pigs, rats and mice, certainly speeds up Lipid membrane peroxidation constitutes a primary cytotoxic event that triggers a sequence of lesions in the cell. Changes in membranes lead to disorders related to membrane permeability by changing the ionic flow and the flow of other substances, which results in the loss of selectivity for intake and/or outtake of nutrients and toxic substances to the cell, DNA damage and changes in the cell cycle [68, 69]. The Thiobarbituric Acid Reactive Substances (TBARS) assay method [70] measures the extent of lipid degradation by quantify‐ ing malondialdehyde (MDA) formed from the oxidation of triacylglycerols. In this method, the reagent thiobarbituric acid generates adduct with malondialdehyde, which is detectable spectrophotometrically at 532 nm. Besides the aforementioned method, cell viability assays are also employed in assessing oxidative damage in yeast, which evaluates the stress toler‐ ance increase caused by treatment with antioxidant compounds [71]; mitochondrial function assays, since many apoptotic processes start in this organelle [72]; measurement of intracel‐ lular reactive oxygen species formation, using 2,7‐dichlorofluorescein as indicator [71, 73]; protein carbonylation tests [74, 75], which is also formed as consequence of oxidative damage; assessment of energetic metabolism and enzymatic activity associated with the stress response [67, 74], among other methods.

Propolis, as well as honey, is a product of bees derived from the collection of plant flu‐ ids and alike contains phenolic compounds in its composition. Sá et al. [76] evaluated the antioxidant capacity of propolis extracts using a wild‐type (BY4741) *S. cerevisiae* and anti‐ oxidant‐deficient strains (Δctt1, Δsod1, Δgsh1, Δgtt1 and Δgtt2), either to 15 mM menadi‐ one or to 2 mM hydrogen peroxide during 60 min. They observed that all strains, except the mutant Δsod1, acquired tolerance when previously treated with 25 μg/mL of alcoholic propolis extract. Such a treatment reduced the levels of ROS generation and lipid peroxida‐ tion, after oxidative stress. However, cells were drastically affected by direct exposure to H2 O2 , after propolis treatment, survival increased almost three times. The increase in Cu/ Zn‐Sod activity by propolis suggests that the protection might be acting synergistically with Cu/Zn‐Sod.

The antioxidative activities of propolis and its main phenolic compounds, caffeic acid, p‐coumaric acid, ferulic acid and caffeic acid phenethyl ester (all 0.05 g/L), were investigated in the yeast *S. cerevisiae.* After 1 h of yeast cells exposure, their intracellu‐ lar oxidation was measured using 2,7‐dichlorofluorescein. Yeast cells exposed to 96% ethanolic extracts of propolis in DMSO showed 42% decreased intracellular oxidation compared with nontreated cells, with no significant differences seen for the individual phenolic compounds [72].

It is concluded that honey and other bee products possess proven *in vivo* and *in vitro* antioxi‐ dant activity, and this property can be the foundation of the functional properties assigned to them.
