Chemical, Antioxidant, and Cytotoxic Properties of Native Blue Corn Extract Chemical, Antioxidant, and Cytotoxic Properties of Native Blue Corn Extract

In recent years, natural products such as dietary phytoconstituents have been the focus of scientific studies for cancer prevention. Among these are polyphenols, which have shown anticancer properties. Pigmented cereals such as blue maize are a rich source of polyphenols such as anthocyanins. Therefore, the aim of this work is to determine the chemical composition and cytotoxic activity of blue maize extract in several cancer cell lines. The total polyphenol content, total anthocyanins, and antioxidant activity of 16 blue corn samples from the Mixteco race were analyzed. From these, the sample with the highest content of polyphenols, anthocyanins, and antioxidant activity was selected and its anthocyanin fraction was isolated using an amberlite column and analyzed by means of HPLC‐ESI‐MS. The total polyphenol content ranged from 142.8 to 203.2 mg GAE/100g. The total anthocyanin contents varied between 19.02 and 66.92 mg C3G/100g. The antiox‐ idant activity ranged from 18.5 to 27.8 µmol TE/g. The anthocyanin profile showed eight different compounds, mainly acylated anthocyanins. Cytotoxicity of blue corn extract on cancer cell lines was determined at concentrations of 100 and 500 µg/mL using the SRB assay. A cytotoxic effect was mainly observed on SKLU‐1 and HTC‐15 cell lines. pelargonidin 3‐(3″,6 ‴ ‐dimalonylglucoside), pelargonidin 3‐(6″‐malonyl glucoside)‐5‐(6″ acetyl glucoside), pelargonidin 3‐glucoside‐5‐(6″‐acetyl‐glucoside), pelargonidin 3‐(6″‐malonylglu‐ coside), and pelargonidin 3,5‐diacetylglucoside. The data indicates that only acylated antho‐ This to and which influence on obtain deeper knowledge on specific molecular targets of blue corn and to ensure the safe use of these active compounds as therapeutic agents on lung and colon cancer.


Introduction
Molecules derived from natural sources, such as plants, marine organisms, and microorganisms, have become important sources of active compounds in the development of drugs for the treatment of human chronic diseases. In recent years, natural products such as dietary phytoconstituents have been the focus of scientific studies for cancer prevention [1]. Epidemiological and preclinical research indicates that dietary compounds possess chemopreventive properties, for example, garlic consumption has been associated with a lower risk of cancer [2][3][4]. In addition, supplementation of dietary phytochemicals for chemoprevention is gaining increased attention due to their chemical diversity, biological activity, and good availability.
Currently, more than 1000 dietary compounds belonging to different chemical classes have shown potential chemopreventive activities [5]. Among dietary constituents, polyphenols such as anthocyanins have demonstrated to exert many biological activities including anticancer properties [6]. From the chemical standpoint, anthocyanins are phenolic substances that belong to the group of flavonoids derived from the 2-phenylbenzopyrilic cation found in nature in a glycosylated or acylated form [7]. These compounds are particularly abundant in pigmented cereals such as red, purple, and black rice, black sorghum, and red, blue, or purple maize [8][9][10].
Mexico is the center of origin and biodiversity of maize (Zea mays L.). Species have an extensive genetic diversity, with 59 different races described with different shapes and colors ranging from white to yellow, red, purple, and blue [11]. Pigmented maize genotypes are used in the production of tortillas, tamales, atoles, and other traditional Mexican foods. These maize varieties have been the focus of scientific studies because they are a rich source of polyphenols such as anthocyanins. Recent data indicate that blue maize contains monomeric anthocyanins as well as acylated anthocyanins [12,13].
Even though blue maize is an important part of the Mexican diet, there is little scientific information regarding its anthocyanin profile and anticancer properties. Chemical composition is a factor that must be considered in the selection of blue maize genotypes due to its impact on biological activity, and thus its potential applications for the treatment of disease such as cancer. For this reason, prior to embarking on cancer phytochemical trials, it is important to carry out a preclinical research in order to evaluate the potential application of phytochemicals from blue maize. It is well known that in vitro studies examine preliminary efficacy of phytochemicals for cancer prevention or therapy [14].
Given the above, the aim of this work is to evaluate the total content of polyphenols, anthocyanins, and the antioxidant activity of blue corn from the Mixteco race, and to determine its anthocyanin profile and the cytotoxic activity of the anthocyanin fraction in several cancer cell lines.

Plant material
Sixteen samples of blue maize from the Mixteco race (Figure 1) were donated by the Interdisciplinary Research Center for Integral Regional Development (CIDIIR as per the Spanish acronym) of the National Polytechnic Institute, Oaxaca Unit in Mexico. Maize kernels were grounded and placed in amber bottles for analysis.

Total phenolic content
For analytical purposes, total polyphenols were evaluated using the colorimetric method previously described by Folin-Ciocalteu and modified by Singleton and Rossi [15]. In this study, 0.2 mL of the extract was mixed with 3.0 mL of distilled water and 0.2 mL of Folin-Ciocalteu reagent. Next, a calcium carbonate saturated solution of 0.75 mL was added. Then the mixture was incubated for 60 min at 37°C in darkness; absorbance was read at 750 nm. This measurement was compared to a standard curve prepared with a gallic acid solution (20-120 mg/L) (Sigma Chemical). The total phenolic content was expressed as milligram equivalents of gallic acid/100 g of fresh weight (mg GAE/100g).

Total monomeric anthocyanin content
Monomeric anthocyanin content was evaluated using the differential pH method [16]. Absorbance was measured in a UV-VIS spectrophotometer (Perkin Elmer, Inc., Shelton, CT, USA). For the analysis, samples were diluted in potassium chloride buffer (pH 1.0) and sodium acetate buffer (pH 4.5). The difference in absorption at 510 and 700 nm was determined in buffers at pH 1.0 and 4.5. The monomeric anthocyanin content was expressed as cyanidin 3-glucoside mg/100g.

Antioxidant activity
The antioxidant assay was performed according to the DPPH (2,2-Diphenyl-1-picrylhydrazyl) method [17]. Trolox was used to make a calibration curve (100-800 µmol). About 2.9 mL of DPPH solution was mixed with 0.1 mL of blue corn extract, and then kept in the dark for 1 h. The sample was incubated for 30 min and it absorbance was read at 517 nm. The result was expressed as µmol eq. trolox/g of sample.

Isolation and chromatographic analysis of anthocyanins
For the isolation of anthocyanins, a column was packed with amberlite XAD-7 preconditioned with 5% acetic acid [18]. Then, 1 mL of the blue corn concentrated extract was placed into the column and eluted with acidified ethanol (5% acetic acid). The eluate was then concentrated to dryness using a Buchi rotary evaporator (Heidolph Digital Laborota pump 4011) coupled to Pimo Vacum Buchi V-700. Anthocyanins were analyzed by HPLC-ESI-MS. The HPLC system was coupled to a Brüker MicrOTOF II spectrometer. The column was C-18 ZORBAX eclipse plus column with 100 mm × 2.1 mm, 3.5 µm. The isocratic elution was done with a mix of methanol:water (2:8 v:v). Mass spectra analysis was carried out in negative ion mode, scan range: 50-3000 amu, capillary voltage 3.8 kV, dry gas flow at 4.0 L/min.

Cell culture and assay for cytotoxic activity
Prostate cancer cell lines (PC-3), neoplastic myelogenous leukemic cell lines (K-562), human colon cancer cell lines (HCT-15), human breast cancer cell lines (MCF-7), and lung cancer cell lines were provided by the National Cancer Institute (NCI), USA, and the Center of HIV/ AIDS Services Center in Mexico City. Cytotoxicity of blue corn extract on tumor cells was determined at different concentrations (50, 100, and 500 µg/mL), using the protein-binding dye sulforhodamine B (SRB) assay in microculture to determine cell growth [19]. Cell lines were cultured in RPMI-1640 (Sigma Chemical Co., Ltd., St. Louis, MO, USA), supplemented with 10% of fetal bovine serum purchased from Invitrogen Corporation, 2 mM l-glutamine, 100 IU/mL, penicillin G, 100 mg/mL streptomycin sulfate, and 0.25 mg/mL amphotericin B (Gibco). They were maintained at 37°C in a 5% CO 2 atmosphere and 95% humidity. For the assay, the following suspensions were prepared: 5 × 10 4 cell mL (K-562, MCF-7), 7.5 × 10 4 cell/mL (PC-3), and 10 × 10 4 cell/mL (HCT-15, SKLU-1); 100 µL of these cells in suspension were seeded in 96-well micro-titer plates and incubated in order to achieve cell attachment to the plates. After 24 h of incubation, 100 µL of each test compound and positive substances (Cisplatin) were added to each well. After 48 h of incubation, adherent cell cultures were fixed 'in situ' by adding 50 mL of cold 50% (wt/vol) trichloroacetic acid (TCA) and incubated for 60 min. at 4°C. The supernatant was discarded and the plates were washed three times with water and then air-dried. Cultures fixed with TCA were stained for 30 min. with 100 mL of 0.4% SRB solution. Protein-bound dye was extracted with 10 µmol of unbuffered tris base and the optical densities were measured by an Ultra Microplate Reader (Elx808, BIO-TEK Instruments, Inc.) with a test wavelength of 515 nm.

Total content of polyphenols, monomeric anthocyanins, and antioxidant activity
The first aim of this research is to evaluate the total content of polyphenols, anthocyanins, and the antioxidant activity of blue corn extracts. Ethanol acidified with citric acid was used in the preparation of the extracts, since organic acids decrease the decomposition of anthocyanins during the following concentration step [20].
The total polyphenol content was observed in the range of 143-203 mg equivalent of gallic acid/100 g sample ( Table 1), while the concentration of monomeric anthocyanins varied from 21 to 69 mg cyanidin-3-glucoside/100 g sample. In this study, the total polyphenol and anthocyanin levels were lower than values previously reported for American and Mexican blue corn [21]. Antioxidant activity evaluated with the DPPH method showed values between 18.5 and 26.8 µmol/100 g.

Cytotoxic activity of the anthocyanin fraction from blue corn
In this study, the SRB assay was used to evaluate cytotoxic activity, and it was selected in order to avoid any interference of anthocyanins in the final reading. The effect of the blue corn extract at different concentrations on the prostate cancer cell line (PC3), neoplastic myelogenous leukemic cell line (K562), human colon cancer cell line (HCT-15), human breast cancer cell line (MCF-7), and lung cancer (SKLU-1) are shown in Figure 4. Generally speaking, it was observed that for blue maize extract, the percentage of growth inhibition of cancer cell lines improved with increased concentration; the analysis indicates that the blue corn extract causes growth inhibition in all cancer cell lines in a dose-dependent manner (Figure 4). showing cytotoxicity [22]. It has been reported that the anthocyanin profile has an effect on anticancer activity. For example, pomegranate extract has an abundance of delphinidin derivatives, a compound with anticancer activity on human prostate [23]. In the present   research, the anthocyanin profile of blue corn was composed only of cyanidin and pelargonidin derivatives; delphinidin was not detected.
In addition, the analysis indicates that the blue corn extract showed higher inhibition of cellular growth in MCF-7 cancer cells than SKLU-1 (Figure 4)   (500 µg/mL). Anthocyanin-rich extracts of cereals such as black rice and black sorghum have also showed the anticancer effects on MCF-7 cells. On the other hand, reports on the effects of anthocyanins on SKLU-1 cells are scarce; a study performed on kenaf seed extract showed cytotoxic activity toward SKLU-1 [24].
Interestingly, blue corn extract was able to inhibit 50.9% of lung cancer cells at 500 µg/mL, which suggests a potential for application in lung cancer treatment, one of the five cancer types most frequently diagnosed in male population worldwide; however, studies in vivo (animal experiments) and clinical trials are needed. In this regard, studies on blueberries report the presence of anthocyanins in lung tissue of mice fed with this anthocyanin-rich fruit (5% w/w) for 10 days, suggesting that fruits, vegetables, and cereals such as blue corn may be an important source of chemopreventive dietary components [25].
Likewise, blue corn extract also inhibited the growth of neoplastic myelogenous leukemic cells (K562) and colon cancer (HCT-15) cell lines on 46.7 and 62 % at 500 µg/mL, respectively. Given the above, the blue corn extract was more effective on the growth inhibitory activity on HCT-15 colon cancer cell lines as compared to other cancer cell lines. Current statistics indicate that in 2012 colorectal cancer was the third most common cancer in the world. For this reason, there is an increasing interest for chemoprevention as a cancer prevention strategy. Dietary agents such as anthocyanins have been explored for their chemopreventive effects against colon cancer [26]. In vitro data obtained in this research provides information for the future application of blue corn extract as a chemopreventive agent in colon cancer.
In summary, blue corn possesses antioxidant properties and its anthocyanin profile is constituted solely by acylated anthocyanins. These results are particularly important since corn is the basis of the Mexican diet; they suggest that corn anthocyanins may have anticancer activity. Further research is necessary to obtain deeper knowledge on specific molecular targets of blue corn and to ensure the safe use of these active compounds as therapeutic agents on lung and colon cancer.