**4.1. The classical colorimetric methods**

The classical spectrophotometry UV/Vis method [124], even with modifications, is still widely used to measure total phenolic content in plant materials. This method is based on the chemical reduction of polyphenols in an alkaline medium to form a blue chromophore complex (phosphomolybdic/phosphotungstic acid) that can be quantified by visible-light spectrophotometry (at 760 765 nm). Many studies have discussed the advantages and disadvantage of using routinely this method to quantify the level of polyphenols, and most of them seems to agree that although they are easy to perform, low cost, rapid and applicable routinely in the most laboratories, they are not accurate. In addition, the reagents used in the method do not react specifically with only polyphenols, and they react with any reducing substance like ascorbic acid, pigments, aromatic amines and sugars [125], and thus, these methods measure the total reducing capacity and not just the polyphenols compounds. Also, their reagents react with some nitrogen-containing compounds such as hydroxylamine and guanidine [126], thiols, many vitamins and some inorganic ions [127] Therefore, many researchers have chosen to use this method only as an indicative tool of total reduction capacity and not for a specific quantification of polyphenol compounds. However, these methods are still considered useful for a quick and prior screening of numerous samples, and for many applications, a simple measure of total amount of polyphenols is enough.

Similar to total polyphenols, total flavonoids can be measured by spectrophotometry methods, and the AlCl<sup>3</sup> method [128, 129] is the most vulgarized method used to determine the total flavonoid content. Vanillin and 4-(dimethylamino)-cinnamaldehyde (DMCA) assays are often used to determine the level of proanthocyanidins, in which the flavonoid catechin is used as standard [130, 131]. Like in total polyphenols, the vanillin or DMCA method can overestimate the amount of total flavonoids present in samples. The proanthocyanidins can also be determined by butanol-HCl [132] and bovine serum albumin (BSA) [133] methods. The butanol-HCl method is based on the cleavage of the flavonoid bonds by hot acid, followed by an auto-oxidation reaction which converts flavan-3-ols into anthocyanidins. The red extract formed has a maximum absorbance at around 550 nm. In the BSA method, the flavonoids complex is dissolved in an alkaline solution (sodium decyl sulphate-triethanolamine) followed by a reaction with ferric chloride solution to form a violet complex with a maximum absorbance at 510 nm.

Another spectrophotometric method widely used in the quantification of polyphenols is the UV/Vis spectrophotometry method to determine the anthocyanin content. The anthocyanins constitute one of the main class of polyphenols largely present in plant samples, particularly in red, blue and black color fruits such as grapes, blueberries, raspberries, redcurrants, blackcurrants, pomegranates and strawberries, among others. The quantification of anthocyanins is in general performed by the differential pH method [134] based on the property of the anthocyanin pigments to change the color with pH, in the wavelength ranging from 490 to 550 nm [134]. The anthocyanins suffer reversible structural modification with a change of pH, and this change allows to estimate spectrophotometrically the total amount of anthocyanins, even in the presence of degraded pigments and other interfering compounds.

All these spectrophotometric methods are considered simple and cheap, but only gives a general estimation about the content of each class of polyphenols but do not allow the quantification of polyphenols individually.
