**3. Results and discussion**

#### **3.1. 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 10.5 and 12.7 μmol ET/g, whereas antioxidant activity by FRAP recorded 2.77 and 2.79 μmol ET/g. This suggests that red maize samples are a potential source of phenolic compounds with antioxidant properties. On the other hand, sample R-14 was selected to evaluate antioxidant profile and antiproliferative activity on prostate cancer cell line DU145, since this particular sample showed the highest values for total polyphenols and monomeric anthocyanins.


**Table 1.** Total polyphenols, monomeric anthocyanins, and antioxidant activity of red maize from Mixteco race.

#### **3.2. 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.

#### **3.3. Antiproliferative activity**

**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 2.** Anthocyanin profile of red maize from Mixteco race.


**Table 2.** Identity of anthocyanins detected in red maize from Mixteco race.

**3.2. Anthocyanin profile of red maize from Mixteco race**

tested on cell line DU145.

**3.3. Antiproliferative activity**

398 Flavonoids - From Biosynthesis to Human Health

days the first cause of death by cancer.

**Figure 2.** 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

**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 nowa-

**Figure 3.** Percentage of cell viability after 24 h of treatment with red maize anthocyanins. Columns show the mean value and standard deviation of three independent experiments.

**Figure 4.** Percentage of cell viability after 48 h of treatment with red maize anthocyanins. Columns show the mean value and standard deviation of three independent experiments.

**Figure 5.** Microscopy of cancer cells DU145 after 48 h application of different concentrations of purified anthocyanin extract from red maize.

**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.

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.

Anthocyanin Profile of Red Maize Native from Mixteco Race and Their Antiproliferative Activity on Cell Line DU145 http://dx.doi.org/10.5772/67809 401

**Figure 6.** Microscopy of DU145 cells incubated with 1000 μg/ml of red maize anthocyanins for 48 h. (A) Control culture; (B) culture treated with anthocyanins (1000 μg/mL).
