Anthocyanin Profile of Red Maize Native from Mixteco Race and Their Antiproliferative Activity on Cell Line DU145

Mexico is regarded as the point of origin and biodiversity of maize, which takes the col- ors white, yellow, blue, or red. Red maize in particular owes its coloring to a type of poly-phenolic compounds known as anthocyanins. The aim of this study was to determine the profile anthocyanin of red maize, as well as their antiproliferative activity on prostate cancer cell line DU145. Three samples of red maize were analyzed. Total polyphenols, monomeric anthocyanins, antioxidant activity by DDPH and FRAP were evaluated. The sample of red maize with the highest levels of total polyphenols and monomeric anthocyanins was selected, and its anthocyanin fraction was analyzed by HPLC-ESI-MS. Twenty compounds were detected in the anthocyanin profile, and from these, 12 anthocyanins derived of cyanidin were identified. MTT assay was used to determine the antiprolif - erative activity of the anthocyanin fraction from red maize at different concentrations (7–1000 μg/mL), and a significant antiproliferative activity was observed at 1000 μg/mL. Microscopy analysis showed that the anthocyanin fraction of red maize induced apoptosis in cell lines DU145. This is the first report showing that anthocyanin fraction of red maize possess antiproliferative activity in the DU145 cell line. prostate cancer cells.


Introduction
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 or aleurone layer of the grain [3].
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 found.
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 development of disease has been reported [7].
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][9][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 people [12]. Unfortunately, some cancer tumors are extremely resistant to current therapeutic agents; therefore, the development of new strategies in prevention, treatment, and control of cancer is urgently needed [13].
The scientific community is looking for alternatives, such as the use of natural compounds as chemopreventives or adjuvants. In this regard, red maize form Mixteco race may be an important source of flavonoids; however, the number of studies investigating the antiproliferative activity of this grain is scarce. Given the above, the aim of the present work was to evaluate the antiproliferative activity of purified anthocyanins from red maize in the prostate cancer cell line DU145.

Plant material
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.

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/ C 6 H 8 O 7 (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].

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.

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.

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.

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.

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:

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

Statistical analysis
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.

Contents of total polyphenols, monomeric anthocyanins, and antioxidant activity
In the first part of the present work, the contents of total polyphenols, monomeric anthocyanins, and antioxidant activity by DPPH and FRAP were evaluated. Sample R-14 showed the highest values for total polyphenols ( Table 1). Values of monomeric anthocyanins for red maize were higher than those reported for Chalco and Red Chihuahua maize [20] and lower than those reported for samples of red maize from Mexico City and Puebla, Mexico [21]. The values for antioxidant activity as evaluated by the DPPH method were between 10.

Anthocyanin profile of red maize from Mixteco race
It is widely known that biological properties of anthocyanins depend upon their chemical structure, substitutions, conjugations, and polymerization. Therefore, in the present study, the anthocyanin profile of red maize was analyzed by HPLC-ESI-MS. Figure 2 shows the total ion chromatogram (TIC) of red maize anthocyanins from Mixteco race. A total of 20 different compounds were detected, their retention times (tr) and main ions (m/z) are shown in Table 2. A total of 17 compounds were derived from cyanidin (287 m/z). On the other hand, 15 different compounds were identified in the anthocyanin profile of red maize from Mixteco race ( Table 2), which includes monoglycosilated, acylated anthocyanins, and proanthocyanidins. Regarding the role of anthocyanins in the prevention of prostate cancer, a recent study shows that cyanidin-3-glucoside produces cancer cell apoptosis in line DU145 [22]. After the anthocyanin profile was completed on the selected sample, its antiproliferative activity was tested on cell line DU145. Figure 3 shows the percentage of cell viability after 24 h of treatment with different concentrations of purified anthocyanins from red maize. As seen in the graph, no significant differences were detected between the different concentrations and the control (p ≤ 0.05). This shows that red maize extract had no significant effect on prostate cancer cell line DU145 after 24 h. Figure 4 shows the percentage of cell viability after 48 h of application of different concentrations of purified anthocyanins of red maize, where a significant reduction percentage on cell growth was observed at 1000 μg/mL as compared to the control, corresponding to 35% of cell viability; the same effect was observed in the micrograph (Figure 5), where a cytoplasmic vacuolization is present and the growth of live cells is haltered. Even though there are no reports on the effect of anthocyanins of red maize on cancer cell lines, recent studies show that cyanidin-3-glucoside produces an antiproliferative effect through the activation of caspase-3 on prostate cell lines LnCap and DU145 [22]. It has also been reported that flavonoids from blueberry inhibit the activity of metalloproteinase in DU145 [23]. These data suggest a potential for red maize flavonoids in the chemoprevention of prostate cancer, which is nowadays the first cause of death by cancer.    Figure 6 shows an image obtained by optical microscopy: a DU145 cell culture treated with the maximum concentration of red maize anthocyanins (1000 μg/mL) along with a culture in the absence of anthocyanins; after 48 h of incubation, cell vacuolization is observed in the experimental sample.

Peak T R (min) Fragment ions (m/z)
In summary, red maize from Mixteco race is a rich source of flavonoids such as anthocyanins, and their profile is mainly constituted by cyanidin-derived anthocyanins. These compounds have a potential application in the prevention of prostate cancer, showing antiproliferative activity on cell line DU145. Future research is needed.