**5. Phenolic compounds**

Phenolic compounds are plant-derived secondary metabolites, biosynthesized mainly for protection against stress and oxidative damage and transferred via the nectar to the honey. The intensity of the color of a honey may be associated with the antioxidant strength of the honey. The phenolic, flavonoid, and carotenoid content is increased in darker honeys and reduced in lighter more transparent honey. As a result, biological properties, such as antioxidant activities and antibacterial capabilities, of the honey are related to its color, and darker honey tends to have enhanced properties [4, 15].

The most common phenolic compounds are phenolic acids and flavonoids [16]. Phenolic acids constitute an important class of phenolic compounds with bioactive functions typically found in vegetable products and foods. Also are secondary metabolites required for normal operation of naturally occurring plants. They can be divided into two subgroups according to their structure: the hydroxybenzoic and hydroxycinnamic acids. Acids derived from hydroxybenzoic acids include þ-hydroxybenzoic, vanillic, syringic, salicylic (2-hydroxybenzoate), gallic, and ellagic. These compounds might be existing in soluble form in cells, along with sugars or organic acids, or formed with cells linked to lignins. Hydroxycinnamic acids occur normally in their conjugated form as esters of hydroxy acids such as tartaric acid and shikimic as well as in their pure form, including þ-coumaric, caffeic, ferulic, and sinapic acids. Flavonoids (flavones, flavonols, flavanones, flavanols, anthocyanidin, isoflavones and chalcones) are the largest group of plant phenolic compounds. This group represents over 50% of all naturally occurring phenolic compounds. They are generally distributed in the seeds, bark, leaves, and flowers of plants and trees. In plants, these compounds give protection, against pathogens, herbivores, and UV radiation [5, 17].

The qualitative and quantitative difference in the phenolic profile of honey according to the different botanical sources represents the scientific basis of the two main lines of research about the study of honey phenolic fraction. The first approach is focused on the evaluation of the bioactive properties of honeys while the second approach attempts to attribute the botanical and/or the geographical origin of honey based on the existence and the abundance of at least one or more specific phenolic compounds, thus proposed as chemical marker(s) of origin. The results of these research studies are relevant in both directions; honey of varying botanical origins show a wide range of health-promoting properties like antibacterial, antiinflammatory, antioxidant, and radical-scavenging activity [4, 16, 18]. A wide range of phenolic constituents are present in honey such as quercetin, caffeic acid, caffeic acid phenethyl ester (CAPE), acacetin, kaempferol, galangin, chrysin, pinocembrin, pinobanksin, and apigenin, which have promising effects in the treatment of some diseases [19, 20].

Pinocembrin, pinobanksin, and chrysin are the characteristic flavonoids of propolis and these flavonoids have been found in European [19, 21] and Chilean honey samples [22]. Small amounts of propolis might be incorporated into honey; therefore, propolis flavonoids might contribute to the phenolic composition of honey. In temperate areas, the main sources of propolis are poplar (*Populus*) bud exudates. The identification of propolis-derived compounds like pinocembrin and chrysin could have an important contribution to the phenolic composition and antioxidant activity in corcolen (*Azara petiolaris*) Chilean honey [22].

Antioxidant, antiinflammatory, antimicrobial, antiviral, and antiulcerous action, and the capability for regulating enzymatic browning are some of the principal characteristics of honey primarily attributed to phenolic compounds. The implementation of honey polyphenols has recently gained great interest from the functional food, nutraceutical and pharmaceutical industries. However, the efficacy of polyphenols relies on the preservation of their stability and bioactivity. Phenolic compounds, as well as other organic compounds, are degraded depending on the environmental conditions to which they are subjected. Spanish honey samples were subjected to liquefaction and liquefaction/pasteurization and the phenolic compounds evaluated as to the impact of industrial heat treatment. Phenolic compounds found in these honey samples were caffeic and þ-coumaric acids and flavonoids naringenin, hesperetin, pinocembrin, chrysin, galangin, quercetin, and kaempferol. A significant decrease in the concentration of galangin, kaempferol, myricetin, and þ-coumaric acid was observed after heat treatment [23]. Moreover, some flavonoid glycosides present in honey demonstrate certain instability under slight alkaline conditions and high sensitivity to oxidation in the presence of slight oxidizing agents such as hydrogen peroxide, which is present in honey and is responsible for the degradation verified in the flavonoids analyzed [24, 25].

The complexity of a food matrix like honey implies that the target analytes are usually present in low concentrations, and this demands the adoption of a multistep analytical procedure able to provide a careful measurement of these quantities [16]. Procedures using Amberlite XAD-2 columns for cleaning the complex matrices of honey and isolation of their phenols are often performed. In some cases, this step would reduce the need for sample manipulation and give a sample extract uniformly enriched in all components of interest and free from interfering matrix components. In these procedures, aqueous- acidified honey solutions are passed through the columns to retain phenols in sorbent beds and afterward eluded with methanol [19, 26]. These extracts are widely employed in analytical methods, biological assays, and functional food development, since the presence of sugars gives the entire honey a syrupy texture, which causes difficulties for some analysis and preparations. However, recoveries of phenolic acids and flavonoids extracted from deionized water (pH 2) using Amberlite XAD-2 demonstrated different recovery percentages, probably depending on the structure of the phenols studied. Kaempferol, *p-*coumaric acid, and syringic acid were completely adsorbed, but the recovery of gallic acid, caffeic acid, and quercetin by methanol is much less efficient [13, 27–29].
