**2. Research methods**

#### **2.1. Plant material**

**1. Introduction**

394 Flavonoids - From Biosynthesis to Human Health

or aleurone layer of the grain [3].

development of disease has been reported [7].

found.

Cereals are still the most important food source worldwide. Maize, sorghum, millet, wheat, rice, barley, oats, teff, and quinoa account for a large amount of the human diet. Maize (*Zea mays* L.) is the third crop by volume cultivated in the world and has a great economic importance, since it is used for animal and human foods as well as a raw material in the production of several industrial products [1]. There are over 59 maize races in Mexico [2] with different shapes and colors, such as white, yellow, blue, and red. In recent years, there has been a growing interest in pigmented varieties form the scientific point of view, since they are sources of phenolic compounds. Approximately 60% of polyphenols are flavonoids, which are regarded as the most important group of phenolic compounds. Among these are anthocyanins, the chemical compounds responsible for the color of red maize, which are located in the pericarp

Anthocyanins are generally regarded as the most important pigments in nature. These water-soluble compounds belong to the flavonoid family and can be found in the colors blue, red, purple, and dark violet [4]. From the chemical standpoint, anthocyanins in nature take the form of glycosides, whose aglycone is known as anthocyanidin. Anthocyanidins are made up of a benzopyril system (A–C) and a phenolic ring (B). Particular differences between anthocyanins depend upon the number of hydroxyl groups, the nature and number of glycosides attached to the molecule, the position of the point of attachment, and to the nature and number of aromatic acids joined to the glycoside in the molecule [5]. Since each anthocyanidin may be glycosylated and acylated by different acids at different points, there is a large number of chemical combinations [6]. Furthermore, the simplest or monomeric anthocyanins may react between themselves, producing more complex structures of higher molecular weights known as polymeric anthocyanins. They may also react with other compounds, and therefore, in nature more than 600 different anthocyanins are

On the other hand, there is a growing scientific interest on pigmented maize races such as red, due to their content of anthocyanins. Experts recommend the consumption of these because of their strong free-radical scavenging activity and chelation of divalent cations, which is given by their phenolic structure, double bonds of their rings, and hydroxyl groups. Anthocyanins may also modulate enzymes related to oxidative stress, and their preventive action in the

Several epidemiological studies also reveal that the consumption of flavonoid-rich foods is associated with a lower risk of neurodegenerative diseases such as cancer [8–10]. Cancer is one of the main causes of death worldwide, causing 7.6 million deaths only in 2008. For 2030, this number of cancer victims is expected to increase to 11 million. Lung, breast, colorectal, stomach, and prostate cancers cause the majority of cancer deaths [11]. The treatment of this disease depends on the type of cancer as well as on the subtype or stage of the patient and is currently based on hormones, chemotherapy, radiotherapy, pharmaceuticals, and nanotherapy. However, these treatments are aggressive, highly expensive, and unattainable for many Three samples of red maize from the Mixteco race (**Figure 1**), cultivated in the Mixteca region of the State of Oaxaca, Mexico, were used in the present study.

#### **2.2. Preparation of red maize extract**

To prepare the extracts, 200 g of red maize was ground using a coffee grinder (Krups model GX410011V) in order to reduce particle size and increase contact surface area. Then 500 mL of EtOH/ C6 H8 O7 (85:15 1 M) were added, and the mix was thermosonicated in an ultrasonic homogenizer (Cole-Palmer, modelo VCX-750) for 30 min at 40% amplitude, pulse 05:05). Afterward, the extracts were left to stand for 24 h at 4°C and then centrifuged at 4000 rpm for 20 min at 5°C. Extracts were concentrated in a rotavapor (Büchi Rotavapor R-205, Büchi Vacuum Controller V-800, Büchi Heating Bath B-490) at 28°C under vacuum, and finally samples were lyophilized and stored at −20°C. This process was carried out in the dark [14].

**Figure 1.** Samples of red maize from Mixteco race.

### **2.3. Analysis of total polyphenols**

Total polyphenols were analyzed by the method described by Folin and Ciocalteu and modified by Singleton and Rossi [15], which is based on an oxidation-reduction reaction and phenolic compounds react with the Folin-Ciocalteu reagent under alkaline conditions, resulting in a blue coloration that is evaluated by spectrophotometry. Previous to the analysis, a calibration curve with gallic acid was made (12 mg EAG/100 mL) and read at 730 nm. Results were expressed as mg equivalent of gallic acid/L.

#### **2.4. Determination of monomeric anthocyanins**

Quantification of anthocyanins was carried out by the pH differential method described by Giusti and Wrolstad [16]. The amount of anthocyanins was expressed as mg of cyanidin 3-glucoside/L of sample, and each sample was analyzed by triplicate.

### **2.5. Antioxidant activity by DPPH**

The DPPH (1,1-diphenyl-2-picrylhydrazyl) method, as reported by Brand-Williams et al. [17], was used to determine antioxidant activity. A standard calibration curve (100–800 μmol) of trolox was used as reference. A measure of 2.9 mL of DPPH were vigorously mixed with 0.1 mL of each extract and then kept in the dark for 30 min at 25°C. Absorbance was read at 517 nm. Results were expressed as μmol equivalent of trolox/g of simple. All determinations were made by triplicate.

#### **2.6. Antioxidant activity by FRAP**

Analysis of antioxidant activity by FRAP (Ferric reducing antioxidant power) was carried out according to Benzie and Strain [18]. A standard trolox calibration curve (100–800 μmol) was used as reference. About 3 mL of FRAP reagent were mixed with 0.1 mL of each extract, then incubated for 5 min at 37°C, and the absorbance was read at 593 nm. Results were expressed in μmol equivalent of trolox/g of simple. All determinations were made by triplicate.

#### **2.7. Isolation of anthocyanins**

Purification of anthocyanins was made in the dark, using a chromatographic column packed with amberlite XAD-7.1 g of lyophilized red maize extract that was placed per column, and two solutions were added as mobile phases: solution A: H<sup>2</sup> O/acetic acid (95:5 v:v) and solution B: EtOH/acetic acid (95:5 v:v). Fractions of anthocyanins were collected in ambar glass bottles and concentrated in a rotavapor (Büchi Rotavapor R-205, Büchi Vacuum Controller V-800, Büchi Heating Bath B-490) at 28°C under vacuum. Samples were lyophilized and stored at −20°C [14].

#### **2.8. Analysis of anthocyanins by high-performance liquid chromatography with electrospray ionization mass spectrometry (HPLC-ESI-MS)**

Anthocyanin analysis was carried out in a high-performance liquid chromatographer (Agilent model 1200) coupled with a mass spectrometer (Bruker model Esquire 6000), equipped with electrospray and ion trap with a nitrogen nebulizer 15 psi, nitrogen as drying gas at a rate of 7 L/min, drying temperature of 300°C, 500 m/z target, and 50–1000 m/z scan. A Zorbax Eclipse plus C-18 column (2.1 mm × 100 mm × 3.5 μm) was used. The mobile phase consisted of acetonitrile as solvent A and 2% aqueous acetic acid solution as solvent B. The gradient was 7:93 (A:B) at 0 min, 35:65 (A:B) at 80 min, and 100% A in 35 min. Flow rate was 0.2 mL/min. Test time was of 40 min [14].

#### **2.9. Evaluation of cell proliferation in vitro**

DU-145 cells were seeded into 96-well plates at 5000 cells per well and incubated with different extract concentrations. The MTT ( (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay was performed after treatment time (24–48 h) [19]. Roswell Park Memorial Institute (RPMI) 1640 culture medium was replaced with 50 μL of MTT (5 mg/mL), and the cells were incubated for 4 h at 37°C in darkness. Formazan crystals were dissolved in 200 μL of DMSO (dimethyl sulfoxide). Absorbance was read at 570 nm on a micro plate reader (Awareness Technology, Stat Fax 4200 model).

#### **2.10. Statistical analysis**

**2.3. Analysis of total polyphenols**

396 Flavonoids - From Biosynthesis to Human Health

**2.5. Antioxidant activity by DPPH**

**2.6. Antioxidant activity by FRAP**

**2.7. Isolation of anthocyanins**

solutions were added as mobile phases: solution A: H<sup>2</sup>

**electrospray ionization mass spectrometry (HPLC-ESI-MS)**

were made by triplicate.

were expressed as mg equivalent of gallic acid/L.

**2.4. Determination of monomeric anthocyanins**

3-glucoside/L of sample, and each sample was analyzed by triplicate.

Total polyphenols were analyzed by the method described by Folin and Ciocalteu and modified by Singleton and Rossi [15], which is based on an oxidation-reduction reaction and phenolic compounds react with the Folin-Ciocalteu reagent under alkaline conditions, resulting in a blue coloration that is evaluated by spectrophotometry. Previous to the analysis, a calibration curve with gallic acid was made (12 mg EAG/100 mL) and read at 730 nm. Results

Quantification of anthocyanins was carried out by the pH differential method described by Giusti and Wrolstad [16]. The amount of anthocyanins was expressed as mg of cyanidin

The DPPH (1,1-diphenyl-2-picrylhydrazyl) method, as reported by Brand-Williams et al. [17], was used to determine antioxidant activity. A standard calibration curve (100–800 μmol) of trolox was used as reference. A measure of 2.9 mL of DPPH were vigorously mixed with 0.1 mL of each extract and then kept in the dark for 30 min at 25°C. Absorbance was read at 517 nm. Results were expressed as μmol equivalent of trolox/g of simple. All determinations

Analysis of antioxidant activity by FRAP (Ferric reducing antioxidant power) was carried out according to Benzie and Strain [18]. A standard trolox calibration curve (100–800 μmol) was used as reference. About 3 mL of FRAP reagent were mixed with 0.1 mL of each extract, then incubated for 5 min at 37°C, and the absorbance was read at 593 nm. Results were expressed

Purification of anthocyanins was made in the dark, using a chromatographic column packed with amberlite XAD-7.1 g of lyophilized red maize extract that was placed per column, and two

EtOH/acetic acid (95:5 v:v). Fractions of anthocyanins were collected in ambar glass bottles and concentrated in a rotavapor (Büchi Rotavapor R-205, Büchi Vacuum Controller V-800, Büchi Heating Bath B-490) at 28°C under vacuum. Samples were lyophilized and stored at −20°C [14].

Anthocyanin analysis was carried out in a high-performance liquid chromatographer (Agilent model 1200) coupled with a mass spectrometer (Bruker model Esquire 6000), equipped with electrospray and ion trap with a nitrogen nebulizer 15 psi, nitrogen as drying gas at a rate

O/acetic acid (95:5 v:v) and solution B:

in μmol equivalent of trolox/g of simple. All determinations were made by triplicate.

**2.8. Analysis of anthocyanins by high-performance liquid chromatography with** 

Three independent repetitions were made for the cell culture experiments. In order to determine significant differences among data, ANOVA (analysis of variance) tests were performed followed by Tukey (*p* < 0.05) multiple range tests.
