**Phytocompounds Targeting Cancer Angiogenesis Using the Chorioallantoic Membrane Assay**

Stefana Avram, Roxana Ghiulai, Ioana Zinuca Pavel, Marius Mioc, Roxana Babuta, Mirela Voicu, Dorina Coricovac, Corina Danciu, Cristina Dehelean and Codruta Soica

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68506

#### **Abstract**

Cancer is the second cause of mortality worldwide. Angiogenesis is an important process involved in the growth of primary tumors and metastasis. New approaches for controlling the cancer progression and invasiveness can be addressed by limiting the angiogenesis process. An increasingly large number of natural compounds are evaluated as angiogenesis inhibitors. The chorioallantoic membrane (CAM) assay represents an *in vivo* attractive experimental model for cancer and angiogenesis studies as prescreening to the murine models. Since the discovery of tumor angiogenesis, the CAM has been intensively used in cancer research. The advantages of this *in vivo* technique are in terms of low time-consuming, costs, and a lower number of sacrificed animals. Currently, a great number of natural compounds are being investigated for their effectiveness in controlling tumor angiogenesis. Potential reducing of angiogenesis has been investigated by our group for pentacyclic triterpenes, in various formulations, and differences in their mechanism were registered. This chapter aims to give an overview on a number of phytocompounds investigated using *in vitro*, murine models and the chorioallantoic membrane assay as well as to emphasize the use of CAM assay in the study of natural compounds with potential effects in malignancies.

**Keywords:** phytocompounds, tumor angiogenesis, chorioallantoic membrane assay

### **1. Introduction**

Angiogenesis represents the process by which new vessels are formed from preexisting vessels [1] and has important implications associated with tumor growth and metastasis [2]. Studies

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

have shown that neovascularization is essential for tumor survival and growth, whereas in angiogenic absent conditions, tumor may display necrosis or even apoptosis [3, 4]. The angiogenic switch represents the process in which endothelial cells are led to a rapid growth state induced by stimuli secreted by the tumor microenvironment, comprising tumor and stromal cells, extracellular matrix components, immunologic cells, fibroblasts, adipocytes, muscle cells, and pericytes [5]. The switch may also involve downregulation of endogenous inhibitors of angiogenesis such as endostatin, angiostatin, or thrombospondin.

The undergoing of tumor angiogenesis represents a four-step process [6]: (i) tissue basement membrane injury; (ii) migration of endothelial cells, activated by angiogenic factors; (iii) endothelial cell proliferation and stabilization; (iv) continuous angiogenesis induced by angiogenic factors. Therefore, key elements in the angiogenesis process are the endogenous angiogenic factors. The most relevant angiogenic activators, signal mediators, and signaling effects are represented in **Figure 1**.

A class of proteins that is widely responsible for tumor angiogenesis is represented by growth factors, such as the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived endothelial growth factor (PDGF), tumor necrosis factor-α (TNF-α), epidermal growth factor (EGF), placental growth factor (PGF), transforming growth factor (TGF), granulocyte colony stimulating factor (GCSF), hepatocyte growth factor (HGF), angiostatin, and angiogenin [7]. However, VEGF is thought to be the main proangiogenic growth factor, because it induces all four phases of angiogenesis by augmenting vascular permeability, endothelial cell proliferation, endothelial cell migration, and capillary like tube formation [8]. Angiogenic cytokines or other growth factors such as VEGF are expressed under hypoxia conditions or by various oncogenes (e.g., mutant ras, erbB-2/HER2) [9].

As shown in **Figure 1**, after binding the tyrosine kinase specific domain of the receptors, multiple ways of signaling are possible for the angiogenic factors. Important molecular mechanisms involve activation of RAS/RAF1/kinase through the extracellular signal (ERK-1 și-2), inducing proliferation and differentiation; RAS/p38 mitogen-activated kinase (MAPK) and JUN/kinase 1-3 N-terminal, modulating inflammation, apoptosis, and differentiation; phosfatidyl-3-inositol kinase-1 (PI3K) and AKT dependent, regulating cell survival, mammalian receptor for rapamycin (mTOR), highly involved in proliferation and cell growth. Other inductor factors of the signaling pathways of angiogenesis are found in the cytoplasm (e.g., GAB1, SHC, SRC, PI3K, and phosfolipase γ C) [10].

VEGF and its receptors, the VEGFR family, remain intensively researched for targeting angiogenesis in different tumors. At the same time, other angiogenesis suppressing-related targets are being studied for the development of anticancer therapies for tumors resistant to anti-VEGF therapy. A number of therapeutic agents are currently in use for several malignancies: monoclonal antibodies against angiogenic growth factors (e.g., antibody against VEGF, Bevacizumab), inhibitors of angiogenic factors synthesis (e.g., mTOR inhibitor Rapamycin), and inhibitors of angiogenic factor receptors (tyrosine-kinase inhibitors, e.g., imatinib and sorafenib) [11]. Unfortunately, clinical response to the new molecular advances in cancer therapy by targeting angiogenesis is unsatisfactory. Resistance and low survival rates are signaled. New therapeutic approaches with minimal side effects are desired to act by targeting the multiple factors that are activated during tumor progression.

Phytocompounds Targeting Cancer Angiogenesis Using the Chorioallantoic Membrane Assay http://dx.doi.org/10.5772/intechopen.68506 47

have shown that neovascularization is essential for tumor survival and growth, whereas in angiogenic absent conditions, tumor may display necrosis or even apoptosis [3, 4]. The angiogenic switch represents the process in which endothelial cells are led to a rapid growth state induced by stimuli secreted by the tumor microenvironment, comprising tumor and stromal cells, extracellular matrix components, immunologic cells, fibroblasts, adipocytes, muscle cells, and pericytes [5]. The switch may also involve downregulation of endogenous inhibitors of

The undergoing of tumor angiogenesis represents a four-step process [6]: (i) tissue basement membrane injury; (ii) migration of endothelial cells, activated by angiogenic factors; (iii) endothelial cell proliferation and stabilization; (iv) continuous angiogenesis induced by angiogenic factors. Therefore, key elements in the angiogenesis process are the endogenous angiogenic factors. The most relevant angiogenic activators, signal mediators, and signaling

A class of proteins that is widely responsible for tumor angiogenesis is represented by growth factors, such as the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived endothelial growth factor (PDGF), tumor necrosis factor-α (TNF-α), epidermal growth factor (EGF), placental growth factor (PGF), transforming growth factor (TGF), granulocyte colony stimulating factor (GCSF), hepatocyte growth factor (HGF), angiostatin, and angiogenin [7]. However, VEGF is thought to be the main proangiogenic growth factor, because it induces all four phases of angiogenesis by augmenting vascular permeability, endothelial cell proliferation, endothelial cell migration, and capillary like tube formation [8]. Angiogenic cytokines or other growth factors such as VEGF are expressed under hypoxia

As shown in **Figure 1**, after binding the tyrosine kinase specific domain of the receptors, multiple ways of signaling are possible for the angiogenic factors. Important molecular mechanisms involve activation of RAS/RAF1/kinase through the extracellular signal (ERK-1 și-2), inducing proliferation and differentiation; RAS/p38 mitogen-activated kinase (MAPK) and JUN/kinase 1-3 N-terminal, modulating inflammation, apoptosis, and differentiation; phosfatidyl-3-inositol kinase-1 (PI3K) and AKT dependent, regulating cell survival, mammalian receptor for rapamycin (mTOR), highly involved in proliferation and cell growth. Other inductor factors of the signaling pathways of angiogenesis are found in the cytoplasm (e.g.,

VEGF and its receptors, the VEGFR family, remain intensively researched for targeting angiogenesis in different tumors. At the same time, other angiogenesis suppressing-related targets are being studied for the development of anticancer therapies for tumors resistant to anti-VEGF therapy. A number of therapeutic agents are currently in use for several malignancies: monoclonal antibodies against angiogenic growth factors (e.g., antibody against VEGF, Bevacizumab), inhibitors of angiogenic factors synthesis (e.g., mTOR inhibitor Rapamycin), and inhibitors of angiogenic factor receptors (tyrosine-kinase inhibitors, e.g., imatinib and sorafenib) [11]. Unfortunately, clinical response to the new molecular advances in cancer therapy by targeting angiogenesis is unsatisfactory. Resistance and low survival rates are signaled. New therapeutic approaches with minimal side effects are desired to act by targeting

angiogenesis such as endostatin, angiostatin, or thrombospondin.

conditions or by various oncogenes (e.g., mutant ras, erbB-2/HER2) [9].

GAB1, SHC, SRC, PI3K, and phosfolipase γ C) [10].

the multiple factors that are activated during tumor progression.

effects are represented in **Figure 1**.

46 Natural Products and Cancer Drug Discovery

**Figure 1.** Angiogenic factors and signaling pathways involved in angiogenesis mediation. Abbreviations: Akt, RACalpha serine/threonine-protein kinase, ERK1/2, mitogen-activated protein kinase 1/2, FAK, focal adhesion kinase; FGFR, fibroblast growth factor receptor; IGFR, insulin growth factor receptor; MAPK, mitogen-activated protein kinases; NOS, nitric oxide synthase; p38, mitogen-activated protein kinase 11; PDGFR, olated-delivered endothelial growth factor receptor; PI3K, phosphatidylinositol 4,5-bisphosphate 3-kinase; PLCγ, phospholipase C gamma; Smad, Smad protein; Src, proto-oncogene tyrosine-protein kinase; TGFα-R, transforming growth factor α receptor; Tie, angiopoietin receptor; VEGFR, vascular endothelial growth factor receptor.

Based on the preventive effect that healthy diets have on the epidemiology of cancer, medicinal plants, spices, fruits, and vegetables represent an interesting source of phytochemicals. Natural compounds or even plant extracts are now considered important and accessible therapeutic or chemopreventive agents in cancer. In the search of the suitable phytocompounds to test for specific effects, virtual screening methods can be successfully applied in the selection of selective compounds for specific targets [12]. To avoid lack of selectivity, computational filtering schemes can be used [13]. Extensive studies demonstrate the high potential of plant-derived chemicals in controlling tumor angiogenesis with minimal secondary effects and drug resistance, by targeting multiple key pathways in a synergistic manner.

### **2. Experimental models for tumor angiogenesis: focus on the CAM assay**

An important issue in angiogenesis studies is the appropriate choice of the assays. To evaluate the efficacy of potential phytocompounds and to identify potential targets within the angiogenic process, several methods both *in vitro* and *in vivo* can be applied. Each of them having one or more drawbacks, ideally more techniques are to be applied. *In vitro* techniques are used by co-culturing endothelial cell and other tumor microenvironment factors with tumor cells in 2D or even 3D models which facilitate the identification of the involved molecular mechanisms. Despite the advances made in the direction of designing *in vitro* assays, the *in vivo* environment can be difficultly reproduced with such protocols [14]. To better assess the key aspects of tumor angiogenesis and therapeutic approaches, *in vivo* assays can be applied, such as the chick chorioallantoic membrane (CAM), the zebrafish, the sponge implantation, the corneal, or dorsal air sac and tumor angiogenesis models in rodents or rabbits [15]. Several drawbacks can still be cited, especially for the murine models, including high costs, complex technical and surgical abilities, and important quantities of test compounds.

#### **2.1. Chorioallantoic membrane assay**

The chorioallantoic membrane (CAM) assay represents an attractive *in vivo* experimental model for angiogenesis and cancer studies. The advantages of this *in vivo* technique in terms of costs, time, simplicity, reproducibility, and ease of the approval by the ethic committee make it a good prescreening assay to murine models in the research of biological systems and new therapeutic targets. Especially tumor angiogenesis and metastasis protocols benefit for a much shorter time for the tumor to grow and metastasize than the classical animal models.

The limitations of the model include a restricted number of reagents to work with due to low compatibility, nonspecific inflammatory reactions, keratinization of the membrane, and a vascular reaction that interferes with the visualization of vascular modifications. Technical skills may be significant to counteract these limitations [16, 17].

The chorioallantoic membrane is the vascularized respiratory extraembryonic tissue of avian species. First, this biologic system has been used for embryologic, immunological, and tumor grafting studies [18], and more recently, since the discovery of tumor angiogenesis [19], it is intensively applied in cancer research [20]. During the stages of embryo development, the immunologic, nervous, and nociceptive systems are not fully developed [21]. Several types of CAM assay protocols have been developed.

### **2.2. Uses in biological studies**

The method can be applied for bioengineering development, morphology, biochemistry, transplant biology, cancer research, and drug development, but also in immunology, wound healing, tissue repair, or drug toxicity [22, 23]. The possibilities of imaging and evaluation have attracted many research studies. Nutritional therapeutics is an example of products approved by the U.S. Food and Drug Administration (FDA) that were preclinically evaluated in the CAM model [16].

Phytocompounds can be tested in order to evaluate their potential bioavailability, tolerability, and lack of irritation effects. For this purpose, the variations of the HET-CAM protocol can be applied, according the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recommendations published in November 2006 in Appendix G of reference [24]. Our previous evaluations proved its applicability in testing different sets of compounds, i.e., surfactants and aflatoxins [25].

In the attempt of finding new means for cancer chemoprevention, the chorioallantoic membrane assay can be used to test natural compounds that could reduce or inhibit several pathways involved in malignancies, especially pro-inflammatory cytokine activation and excessive angiogenesis. Tumor microenvironment, including inflammation and angiogenesis next to the development of new therapeutic targets for these pathological conditions, is intensively researched on murine models [26]. Previously, we have evaluated mast cell involvement in the angiogenesis process implementing a mastocytoma model on the CAM assay [27], which can be further developed for the evaluation of natural compounds on mast cells as key participants in the tumor microenvironment.

### **2.3. General** *in ovo* **method**

cells in 2D or even 3D models which facilitate the identification of the involved molecular mechanisms. Despite the advances made in the direction of designing *in vitro* assays, the *in vivo* environment can be difficultly reproduced with such protocols [14]. To better assess the key aspects of tumor angiogenesis and therapeutic approaches, *in vivo* assays can be applied, such as the chick chorioallantoic membrane (CAM), the zebrafish, the sponge implantation, the corneal, or dorsal air sac and tumor angiogenesis models in rodents or rabbits [15]. Several drawbacks can still be cited, especially for the murine models, including high costs, complex

The chorioallantoic membrane (CAM) assay represents an attractive *in vivo* experimental model for angiogenesis and cancer studies. The advantages of this *in vivo* technique in terms of costs, time, simplicity, reproducibility, and ease of the approval by the ethic committee make it a good prescreening assay to murine models in the research of biological systems and new therapeutic targets. Especially tumor angiogenesis and metastasis protocols benefit for a much shorter time for the tumor to grow and metastasize than the classical animal models. The limitations of the model include a restricted number of reagents to work with due to low compatibility, nonspecific inflammatory reactions, keratinization of the membrane, and a vascular reaction that interferes with the visualization of vascular modifications. Technical skills

The chorioallantoic membrane is the vascularized respiratory extraembryonic tissue of avian species. First, this biologic system has been used for embryologic, immunological, and tumor grafting studies [18], and more recently, since the discovery of tumor angiogenesis [19], it is intensively applied in cancer research [20]. During the stages of embryo development, the immunologic, nervous, and nociceptive systems are not fully developed [21]. Several types of

The method can be applied for bioengineering development, morphology, biochemistry, transplant biology, cancer research, and drug development, but also in immunology, wound healing, tissue repair, or drug toxicity [22, 23]. The possibilities of imaging and evaluation have attracted many research studies. Nutritional therapeutics is an example of products approved by the U.S. Food and Drug Administration (FDA) that were preclinically evaluated in the CAM model [16]. Phytocompounds can be tested in order to evaluate their potential bioavailability, tolerability, and lack of irritation effects. For this purpose, the variations of the HET-CAM protocol can be applied, according the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recommendations published in November 2006 in Appendix G of reference [24]. Our previous evaluations proved its applicability in testing different sets of com-

In the attempt of finding new means for cancer chemoprevention, the chorioallantoic membrane assay can be used to test natural compounds that could reduce or inhibit several pathways

technical and surgical abilities, and important quantities of test compounds.

**2.1. Chorioallantoic membrane assay**

48 Natural Products and Cancer Drug Discovery

may be significant to counteract these limitations [16, 17].

CAM assay protocols have been developed.

pounds, i.e., surfactants and aflatoxins [25].

**2.2. Uses in biological studies**

*Ex ovo* or *in ovo* techniques are applicable. The *ex ovo* protocol involves the transfer of the egg content on day 3 of incubation into a Petri dish. It facilitates the visualization of the experiment, but the unnatural milieu of development of the embryo is detrimental to the survival rate of the specimens. Therefore, we prefer the *in ovo* protocol and is the type of method described here.

Fertilized eggs are horizontally incubated 7 days prior to use, at 37°C, in a controlled wet atmosphere. On the third day of incubation, in order to detach the chorioallantoic membrane, a volume of 2–3 ml of albumen is aspired through a perforation at the more pointed end of the eggs. The hole is resealed and returned to the incubator. The next day, a window is cut and resealed on the superior side of the shell. The eggs are returned to incubation until the day of the experiment [28]. Generally, 5–10 eggs are used for each test sample. Samples are applied inside a sterile plastic ring on the surface of the membrane. Samples are applied in triplicate. *In ovo* investigation by means of a stereomicroscope is performed throughout the experiment. Photographs are recorded for further analysis (**Figure 2**).

Starting with day 11 of incubation, samples can be considered active on excessive angiogenesis. The rapid growth of the vessels occurs during days 7–11; therefore, applying substances during this interval can be evaluated in terms of antiangiogenic effects. Morphometric evaluation of the angiogenic reaction can be conducted using a 0–5 arbitrary scale, the mean values expressing the vascular density around the site of application [20]. Finally, specimens are sacrificed and membranes are submitted to histological and immunohistological evaluation. On slides with immunohistochemical marked vessels, the mean microvascular density can be determined using the hotspot method, and counting the blood vessels, to calculate an antiangiogenic index, with the aid of the formula: AAI = 1 – NoBVtest/NoBVcontrol, AAI = antiangiogenic index, BV = blood vessels [29].

### **2.4. Tumor angiogenesis model on CAM**

Tumor cells are used on the CAM in order to obtain tumors, to study their microenvironment and the effects that phytochemicals might have. Tumor grafts can be used as well. Usually, cultured cancer cells are inoculated on the surface of the CAM, on day 10 of incubation, after being trypsinized and resuspended in culture medium to final concentrations in the range of 105 –106 ml−1. Cells can be applied directly on the CAM using a plastic ring for localizing the cells or using Matrigel impregnated with cells. Further, test compound solutions diluted with minimal DMSO (dimethyl sulfoxide) concentration in phosphate buffer can be applied on the

**Figure 2.** Chorioallantoic membrane assay—*in ovo* practical approach: incubation of the eggs (a–c); albumen removal, shell opening, and resealing (d–f); visualization of the CAM, sample application, and sample application inside a plastic ring (g–i) [30].

same spot as the cancer cell samples. *In ovo* stereomicroscopic follow-up is performed daily to register the changes in the vascular response around the tumor developing area that will be used for the morphometric analysis. On the final day of the experiment, after sacrificing the embryos, tumor masses are measured; the chorioallantoic membrane, the formed tumors, and some organs suspected to have metastasis are harvested and histologically processed.

In order to observe morphologic changes in the chorioallantoic membrane, hematoxylin eosin staining is analyzed. Different panels of immunohistochemical markers can be further applied: tumor cell markers and specific antibodies for different key proteins involved in the tumor microenvironment (e.g., endothelial cell marker-factor VIII, smooth muscle actin (SMA) marker, vascular endothelial growth factors, and its receptors, mast cells marker— Tryptase, the proliferation marker—Ki67). Results can reveal molecular modifications and serve to vascular density quantification.

Our experience is related to testing phytocompounds and plant extracts for the effect on angiogenesis. Using the angiogenesis method in the rapid stage of CAM development, we found that pentacyclic triterpenes, betulinic (BA) acid, and betulin (Bet) in various formulations with cyclodextrin and in nanoemulsion are potential antiangiogenic compounds, acting differently, both through direct and indirect mechanisms [31, 32]. Immunohistochemical staining for smooth muscle actin (SMA) on the specimens treated with betulin in nanoemulsion, next to blank and control samples, are shown in **Figure 3**. The low expression of the marker in the betulin-treated specimen indicates a minimal implication of pericytes in the angiogenesis process [32]. On the contrary, we found that betulinic acid determined rapid maturation of the vessels and high levels of SMA [31]. We also evaluated triterpenes and other types of natural compounds in melanoma models on CAM, which confirms the inhibitory effect on tumor angiogenesis (data not published).

Most studies that use the CAM assay are evaluated through stereomicroscopy that allows a series of quantitative measurements, and by histologic an immunohistological interpretation. Advances in the evaluation techniques include fluorescence microscopy, confocal microscopy, microCT scanning, and imaging, *in situ* hybridization (ISH), quantitative PCR (qPCR) determination of specific targets [16, 33].

**Figure 3.** Light microscopic evaluation of CAM sections from ID 11 smooth muscle actin marker: (a) blank specimen, ×40, (b) control specimen treated with nanoemulsion, ×40, (c) specimen treated with betulin in nanoemulsion, ×40 [32].

### **3. Phytocompounds targeting cancer angiogenesis:** *in vitro***, on the chorioallantoic membrane assay, in animal model**

same spot as the cancer cell samples. *In ovo* stereomicroscopic follow-up is performed daily to register the changes in the vascular response around the tumor developing area that will be used for the morphometric analysis. On the final day of the experiment, after sacrificing the embryos, tumor masses are measured; the chorioallantoic membrane, the formed tumors, and

**Figure 2.** Chorioallantoic membrane assay—*in ovo* practical approach: incubation of the eggs (a–c); albumen removal, shell opening, and resealing (d–f); visualization of the CAM, sample application, and sample application inside a plastic

In order to observe morphologic changes in the chorioallantoic membrane, hematoxylin eosin staining is analyzed. Different panels of immunohistochemical markers can be further applied: tumor cell markers and specific antibodies for different key proteins involved in the tumor microenvironment (e.g., endothelial cell marker-factor VIII, smooth muscle actin (SMA) marker, vascular endothelial growth factors, and its receptors, mast cells marker— Tryptase, the proliferation marker—Ki67). Results can reveal molecular modifications and

Our experience is related to testing phytocompounds and plant extracts for the effect on angiogenesis. Using the angiogenesis method in the rapid stage of CAM development, we found that pentacyclic triterpenes, betulinic (BA) acid, and betulin (Bet) in various formulations with cyclodextrin and in nanoemulsion are potential antiangiogenic compounds, acting differently, both through direct and indirect mechanisms [31, 32]. Immunohistochemical staining for smooth muscle actin (SMA) on the specimens treated with betulin in nanoemulsion, next

some organs suspected to have metastasis are harvested and histologically processed.

serve to vascular density quantification.

ring (g–i) [30].

50 Natural Products and Cancer Drug Discovery

Chemicals derived from plant sources as well as various types of extracts have been already investigated for their effects on angiogenesis and on cancer. Currently, based on the failure of the approved therapeutics and also by crediting the traditional medicine philosophy that pathologies are imbalances that have to be rebalanced, the idea of multiple targeting through synergetic phytocompounds mixtures is gaining more attention. Extensive research is being dedicated to the understanding of their mechanism and their efficacy using *in vitro* and *in vivo* methods. The most in depth evidence comes from the results on cell cultures. *In vivo* methods also offer other accurate data on their effects. The chorioallantoic membrane assay is being used by more and more researchers for the evaluation of plant-derived chemicals or extracts. Correlations can be made using the results obtained for *in vitro*, animal and CAM assays, which will improve the knowledge and the future analysis to perform for the active compounds. We reviewed here some of the most investigated phytocompounds concerning the results obtained on all the three experimental models (**Table 1**).



**Phytochemical** 

**Compound**

**Chemical structure**

**Plant source** *Curcuma longa*

MiaPaCa-2; BxPC-3; Panc-1;

Angiogenesis

Athymic nude mice xenograft

with prostate cancer cells

Reduced expression of

NFκB-p65, STAT3 and SRC;

52 Natural Products and Cancer Drug Discovery

Reduced expression of

ANXA2 and VEGFR2

[36]

inhibitor on small

capillaries [35]

MPanc-96 prostate cancer

cell lines

Reduced expression of

NF-κB

[34]

Epigallocatechin-

*Camellia sinensis* L.

Hepatocellular carcinoma

Inhibition of the VEGF-

VEGFR axis

[37]

Inhibition of

BGC-823 human gastric

cancer xenograft mice model

Reduction of VEGF

fibroblast growth

factor (FGF) and

inhibition of mean

[39]

branch formation

and tumor weight

of neuroblastomainduced

angiogenesis

[38]

Multiple target

Wistar rats inoculated with

MT-450 at mammary tumor

angiogenesis

inhibitor

cells

Suppression

tumor-induced

lymphangiogenesis

[41]

[40]

Phloroglucinol

Hyperforin

*Hypericum* 

BAE—bovine aortic

endothelial cell

MDA-MB231 human breast

cancer and NIH-3T3 mouse

fibroblast cell

Inhibition of capillary tube

formation; Inhibition of

urokinase and MMP2

[40]

*Vitis vinifera* L.,

YUZAZ6, M14, A375

Significant

C57BL/6 Mice inoculated with

Lewis lung carcinoma cells

Inhibition of

neovascularization

reduction in

angiogenesis in

higher doses

[43]

[44]

melanoma cell lines

Downregulation of VEGF

and upregulation of TSP1

[42]

*Polygonum* 

*cuspidatum* L.

Stilbene

Resveratrol

phytoalexin

derivative

*perforatum* L.

derivative

gallate

L.

*In vitro* **effects**

**Effects on CAM**

*In vivo* **effects**

**class**

Polyphenols

 Curcumin



**Phytochemical** 

**Compound**

**Chemical structure**

*Camelia* 

PC-3 prostate cells

Potent angiogenesis

DMBA-induced experimental

mammary carcinoma in rats

Inhibition of H-ras protein;

inhibition of VEGF and bFGF

54 Natural Products and Cancer Drug Discovery

[54]

inhibitor [53]

Inhibition of VEGF

*sinensis* L.,

*Angelica keiskei* 

[53]

*Momordica* 

*cochinchinensis*,

*Citrus* sp.

cancer cells

Inhibition of TGF-β1-induced

migration; Decreased

expression of MMP2 and

MMP9 proteins

[55]

*Entada africana*,

PC-3 and DU145 prostate

Promising

BALB/cA-nu nude mice

injected with PC-3 prostate

cancer cells and OVCAR-3

ovarian cancer cells

Inhibition of blood vessels

formation; Inhibition of

hemoglobin levels [57]

antiangiogenic

effect

[58]

*Matricaria* 

cancer cells

Inhibition of HIF-1α and

VEGF

LNCaP prostate cancer cells,

HCT-8 colon cancer cells, and

MCF-7breast cancer cells

Inhibition of hypoxia-induced

HIF-1α and VEGF[57]

ACC-M, ACC-2 adenoid

Angiogenesis

BALB/c nude mice injected

with ACC-M cells

Reduction in S6

phosphorylation; Decreased

VEGF; Inhibition of the mTOR

signaling pathway[59]

suppressor [60]

cystic carcinoma cells

and EAhy926 endothelial

hybridoma cell line

Prevention of tube formation;

Downregulation of VEGF

[59]

*Silybum* 

SW480, HT-29 and LoVo

dose-dependent

A/J mice with Urethaneinduced lung tumors

suppressive on

angiogenesis [62]

Inhibition of new microvessels

formation; Decreased levels of

IL-1α,-6, -9, -13, -16, IFN-γ and

TNF-α[63]

colorectal cancer cells

Inhibition of NF-κB;

Reduction of MMP9, COX-2

and VEGF[61]

*marianum* L

Silibinin

*Glycyrrhiza* 

*glabra* L

Isoliquiritigenin

*chamomilla* L

Apigenin

Aspc-1 and panc-1 prostate

Potent angiogenesis

n/a

inhibitor

[56]

Naringenin

**Plant source**

*In vitro* **effects**

**Effects on CAM**

*In vivo* **effects**

**class**

Flavonoids

 Quercetin


### **4. Clinical trials correlation**

**Phytochemical** 

**Compound**

**Chemical structure**

**Plant source**

*In vitro* **effects**

H-460 cells assessed using

Dose-dependent

H-460 athymic nude mice,

tumor growth and survival

*low expression of* 12-LOX,

*VEGF* and *FGFR-2 gene* [73]

56 Natural Products and Cancer Drug Discovery

antiangiogenic

activity

[74]

BrdU assay Significant

antiproliferative and pro

apoptotic; inhibit bFGFinduced HUVEC tube formation in Matrigel

stronger than baicalin [74,

75]

*Scutellaria* 

Growth and survival,

Dose-dependent

Inhibit growth of S180 solid

tumor in mice [76]

antiangiogenic

activity

[74]

MMP-2 expression, inhibit

bFGF-induced HUVEC

tube formation in Matrigel

[74]; increases VEGF

expression by activating the

ERRα/PGC-1α pathway [75]

Antiangiogenic activity in

Significant

Inhibits FGF-2 Induced

angiogenesis in C57BL/6J

mice [77]

antiangiogenic

activity

[78]

primary endothelial cells

HUVEC [77]

Steroids

Withaferin A

*Withania* 

*somnifera*

Dunal

**Table 1.** Common phytocompounds with *in vitro* and *in vivo* antiangiogenic activity.

*baicalensis*

Georgi

Baicalin

**Effects on CAM**

*In vivo* **effects**

**class**

Flavones

Baicalein *Scutellaria* 

*baicalensis*

Georgi

Implementation of clinical trials is vital for the validation and future use of the active phytocompounds as additional therapies to the oncologic protocols or as chemopreventive strategies. These types of experiments are difficult to implement and therefore not many trials are finalized for the evaluation of antiangiogenic effect in cancer. Two of the above-listed phytochemicals (**Table 1**) benefit from large investigations among which some are clinical trials, but the modulation of the angiogenic process does not appear as a distinct evaluation, cancer effects being the first ones to be described.

Most of the controlled clinical trials of curcumin supplementation in cancer patients aimed to determine its feasibility, tolerability, safety, and to provide early evidence of efficacy [79]. For patients with advanced colorectal cancer, oral doses up to 3.6 g/day for 4 months were well tolerated, although the systemic bioavailability of oral curcumin was low [80]. For this dose, trace levels of curcumin metabolites were measured in liver tissue, but curcumin itself was not detected [81]. These findings suggested that oral curcumin is effective as a therapeutic agent in cancers of the gastrointestinal tract. Other trials found that combining curcumin with anticancer drugs like gemcitabine in pancreatic cancer [82], docetaxel in breast cancer [83], and imatinib in chronic myeloid leukemia may confer additional benefits to conventional drugs against different types of cancer.

Green tea made from *Camellia sinensis* L. leaves, originated in China, is one of the most extensively consumed beverages and achieved significant attention due to health benefits against cancer. Representative compounds are polyphenols and catechins with therapeutic potential against cancer [84]. Recent clinical trials proved that green tea extract and epigallocatechin gallate (EGCG) can be active in several forms of cancer. There is an increasing trend to employ green tea extract and EGCG as conservative management for patients diagnosed with less advanced prostate cancer. Combinations of chemopreventive agents should be carefully investigated because mechanisms of action may be additive or synergistic [85]. Several clinical examinations reported different molecular mechanisms regarding green tea beneficial effects against oral cancer chemoprevention [86–88]. Lung cancer induction may also be inhibited by tea polyphenols. Some studies suggest that individuals who never drank green tea have an elevated lung cancer risk compared to those who drank green tea at least one cup per day, and the effect is more pronounced in smokers [88]. Hepatocellular carcinoma (HCC) usually develops in a cirrhotic liver due to hepatitis virus infection. Green tea catechins (GTCs) may possess potent anticancer and chemopreventive properties for a number of different malignancies, including liver cancer. Antioxidant and anti-inflammatory activities are key mechanisms through which GTCs prevent the development of neoplasms, and they also exert cancer chemopreventive effects by modulating several signaling transduction and metabolic pathways where angiogenesis is exacerbated. Several interventional trials in humans have shown that GTCs may ameliorate metabolic abnormalities and prevent the development of precancerous lesions [89].

### **5. Conclusion**

Currently, a great number of natural compounds are being investigated for their potential effectiveness in controlling tumor angiogenesis and therefore the reduction of tumor growth and metastasis. Observing the high number of molecular pathways that are deregulated in tumor angiogenesis and that many phytocompounds are active on several key factors, it is recommendable that more *in vivo* studies should investigate mixture of compounds for broader targeting, having eventually lower secondary effects and resistance. The optimal experimental technique is an important factor in order to get a useful output. More types of assays are always a good choice, including *in vivo* assays. The chorioallantoic membrane protocol is a good candidate for one type of "golden standardized method" in tumor angiogenesis, being a versatile, rapid, easy, and cheap method to apply in the research of phytocompounds. A great number of plant-derived chemicals, alone or in combination, are studied using this method, but standardization, next to applying new analysis techniques will outcome useful data that will be easier translated to clinical trials.

### **Acknowledgements**

This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS—UEFISCDI, project number PN-II-RU-TE-2014-4-2842 to S.A., R.G., I.Z.P. and D.C. Special thanks to the Histology and Angiogenesis Department, University of Medicine and Pharmacy Victor Babes Timisoara, for the technical support and help in setting up the CAM assay.

### **Author details**

Stefana Avram1 , Roxana Ghiulai2 \*, Ioana Zinuca Pavel1 , Marius Mioc2 , Roxana Babuta2 , Mirela Voicu3 , Dorina Coricovac4 , Corina Danciu1 , Cristina Dehelean4 and Codruta Soica2

\*Address all correspondence to: roxana.ghiulai@umft.ro

1 Department of Phamacognosy, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania

2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania

3 Department of Phamacology, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania

4 Department of Toxicology, Faculty of Pharmacy, Victor Babeș University of Medicine and Pharmacy, Timisoara, Romania

### **References**

**5. Conclusion**

58 Natural Products and Cancer Drug Discovery

will be easier translated to clinical trials.

**Acknowledgements**

**Author details**

Stefana Avram1

Voicu3

help in setting up the CAM assay.

, Dorina Coricovac4

and Pharmacy, Timisoara, Romania

Pharmacy, Timisoara, Romania

Pharmacy, Timisoara, Romania

, Roxana Ghiulai2

Medicine and Pharmacy, Timisoara, Romania

\*Address all correspondence to: roxana.ghiulai@umft.ro

Currently, a great number of natural compounds are being investigated for their potential effectiveness in controlling tumor angiogenesis and therefore the reduction of tumor growth and metastasis. Observing the high number of molecular pathways that are deregulated in tumor angiogenesis and that many phytocompounds are active on several key factors, it is recommendable that more *in vivo* studies should investigate mixture of compounds for broader targeting, having eventually lower secondary effects and resistance. The optimal experimental technique is an important factor in order to get a useful output. More types of assays are always a good choice, including *in vivo* assays. The chorioallantoic membrane protocol is a good candidate for one type of "golden standardized method" in tumor angiogenesis, being a versatile, rapid, easy, and cheap method to apply in the research of phytocompounds. A great number of plant-derived chemicals, alone or in combination, are studied using this method, but standardization, next to applying new analysis techniques will outcome useful data that

This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS—UEFISCDI, project number PN-II-RU-TE-2014-4-2842 to S.A., R.G., I.Z.P. and D.C. Special thanks to the Histology and Angiogenesis Department, University of Medicine and Pharmacy Victor Babes Timisoara, for the technical support and

\*, Ioana Zinuca Pavel1

1 Department of Phamacognosy, Faculty of Pharmacy, Victor Babeș University of Medicine

2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Victor Babeș University of

3 Department of Phamacology, Faculty of Pharmacy, Victor Babeș University of Medicine and

4 Department of Toxicology, Faculty of Pharmacy, Victor Babeș University of Medicine and

, Cristina Dehelean4

, Corina Danciu1

, Marius Mioc2

, Roxana Babuta2

and Codruta Soica2

, Mirela


[29] Demir R, Peros G, Hohenberger W. Definition of the 'Drug-Angiogenic-Activity-Index' that allows the quantification of the positive and negative angiogenic active drugs: A study based on the chorioallantoic membrane model. Pathology and Oncology Research. 2011;**17**(2):309-313

[14] Roudsari LC, West JL. Studying the influence of angiogenesis in in vitro cancer model

[15] Staton CA, Reed MWR, Brown NJ. A critical analysis of current in vitro and in vivo angiogenesis assays. International Journal of Experimental Pathology. 2009;**90**(3):195-221

[16] Dupertuis YM, Delie F, Cohen M, Pichard C. In ovo method for evaluating the effect of nutritional therapies on tumor development, growth and vascularization. Clinical

[17] Nowak-Sliwinska P, Segura T, Iruela-Arispe ML. The chicken chorioallantoic membrane model in biology, medicine and bioengineering. Angiogenesis. 2016;**17**(4):779-804 [18] Harris RJ. Multiplication of Rous No. 1 sarcoma agent in the chorioallantoic membrane

[19] Folkman J, Cotran R. Relation of vascular proliferation to tumor growth. International

[20] Ribatti D. The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis

[21] Friend JV, Crevel RW, Williams TC, Parish WE. Immaturity of the inflammatory response of the chick chorioallantoic membrane. Toxicology In Vitro. 1990;**4**(4-5):324-326

[22] Rashidi H, Sottile V. The chick embryo: Hatching a model for contemporary biomedical

[23] Vargas A, Zeisser-Labouèbe M, Lange N, Gurny R, Delie F. The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems.

[24] Scheel J, Kleber M, Kreutz J, Lehringer E, Mehling A, Reisinger K, Steiling W. Eye irritation potential: Usefulness of the HET-CAM under the globally harmonized system of classification and labeling of chemicals (GHS). Regulatory Toxicology and Pharmacology.

[25] Ardelean S, Feflea S, Ionescu D, Năstase V, Dehelean CA. Toxicologic screening of some surfactants using modern in vivo bioassays. Revista Medico-Chirurgicala a Societatii De

[26] Lokman NA, Elder ASF, Ricciardelli C, Oehler MK. Chick chorioallantoic membrane (CAM) assay as an in vivo model to study the effect of newly identified molecules on ovarian cancer invasion and metastasis. International Journal of Molecular Sciences.

[27] (Feflea) Avram S, Cimpean AM, Raica M. Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos. Brazilian Journal of

[28] Ribatti D. The chick embryo chorioallantoic membrane in the study of tumor angiogenesis. Romanian Journal of Morphology and Embryology. 2008;**49**(2):131-135

of the embryonated egg. British Journal of Cancer. 1954;**8**(4):731-736

Review of Experimental Pathology. 1976;**16**:207-248

Advanced Drug Delivery Reviews. 2007;**59**(11):1162-1176

Medici Si Naturalisti Din Iasi Nat. din Iaşi. 2011;**115**(1):251-258

Medical and Biological Research. 2013;**46**(1):52-57.

and Metastasis. Springer Netherlands; 2010

research. Bioessays. 2009;**31**(4):459-465

2011;**59**(3):471-492

2012;**13**(8):9959-9970

systems. Advanced Drug Delivery Reviews. 2016;**97**:250-259

Nutrition Experimental. 2015;**2**:9-17

60 Natural Products and Cancer Drug Discovery


[52] Gu Y, Zhu C-F, Iwamoto H, Chen J-S. Genistein inhibits invasive potential of human hepatocellular carcinoma by altering cell cycle, apoptosis, and angiogenesis. World Journal of Gastroenterology. 2005;**11**(41):6512-6517

[40] Martínez-Poveda B, Quesada AR, Medina MÁ. Hyperforin, a bio-active compound of St. John's Wort, is a new inhibitor of angiogenesis targeting several key steps of the process.

[41] Rothley M, Schmid A, Thiele W, Schacht V, Plaumann D, Gartner M, Yektaoglu A, Bruyère F, Noël A, Giannis A, Sleeman JP. Hyperforin and aristoforin inhibit lymphatic endothelial cell proliferation in vitro and suppress tumor-induced lymphangiogenesis

[42] Trapp V, Basmina P, Papazian V, Lyndsay W, Fruehauf JP. Anti-angiogenic effects of resveratrol mediated by decreased VEGF and increased TSP1 expression in melanoma-

[43] Wang H, Zhou H, Zou Y, Liu Q, Guo C, Gao G, Shao C, Gong Y. Resveratrol modulates angiogenesis through the GSK3β/β-catenin/TCF-dependent pathway in human endo-

[44] Kimura Y, Okuda H. Resveratrol isolated from Polygonum cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in Lewis

[45] López-Jiménez A, García-Caballero M, Medina MÁ, Quesada AR. Anti-angiogenic properties of carnosol and carnosic acid, two major dietary compounds from rosemary.

[46] Rajasekaran D, Manoharan S, Silvan S, Vasudevan K, Baskaran N, Palanimuthu D. Proapoptotic, anti-cell proliferative, anti-inflammatory and anti-angiogenic potential of carnosic acid during 7,12 dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. African Journal of Traditional, Complementary and Alternative

[47] Chakraborty S, Adhikary A, Mazumdar M, Mukherjee S, Bhattacharjee P, Guha D, Choudhuri T, Chattopadhyay S, Sa G, Sen A, Das T. Capsaicin-induced activation of p53-SMAR1 auto-regulatory loop down-regulates VEGF in non-small cell lung cancer to

[48] Min J-K. Capsaicin inhibits in vitro and in vivo angiogenesis. Cancer Research.

[49] Mahmoud AM, Yang W, Bosland MC. Soy isoflavones and prostate cancer: A review of molecular mechanisms. The Journal of Steroid Biochemistry and Molecular Biology.

[50] Krenn L, Paper DH. Inhibition of angiogenesis and inflammation by an extract of red

[51] Fotsis T, Pepper M, Adlercreutz H, Fleischmann G, Hase T, Montesano R, Schweigerer L. Genistein, a dietary-derived inhibitor of in vitro angiogenesis. Proceedings of the National Academy of Sciences of the United States of America. 1993;**90**(7):2690-2694

clover (Trifolium pratense L.). Phytomedicine. 2009;**16**(12):1083-1088

lung carcinoma-bearing mice. The Journal of Nutrition. 2001;**131**(6):1844-1849

International Journal of Cancer. 2005;**117**(5):775-780

62 Natural Products and Cancer Drug Discovery

in vivo. International Journal of Cancer. 2009;**125**(1):34-42

endothelial cell co-culture. Angiogenesis. 2010;**13**:305-315

thelial cells. Biochemical Pharmacology. 2010;**80**(9):1386-1395

European Journal of Nutrition. 2013;**52**(1):85-95

restrain angiogenesis. PLoS One. 2014;**9**(6):e99743

Medicine. 2012;**10**(1):102-112

2004;**64**(2):644-651

2014;**140**:116-132


Factor- B and signal transducers and activators of transcription 3. Cancer Prevention Research. 2009;**2**(1):74-83


[75] Zhang K, Lu J, Mori T, Smith-Powell L, Synold TW, Chen S, Wen W. Baicalin increases VEGF expression and angiogenesis by activating the ERR /PGC-1 pathway. Cardiovascular Research. 2011;**89**(2):426-435

Factor- B and signal transducers and activators of transcription 3. Cancer Prevention

[64] Marimpietri D, Brignole C, Nico B, Pastorino F, Pezzolo A, Piccardi F, Cilli M, Di Paolo D, Pagnan G, Longo L, Perri P, Ribatti D, Ponzoni M. Combined therapeutic effects of vinblastine and rapamycin on human neuroblastoma growth, apoptosis, and angiogen-

[65] Park K-J, Yu MO, Park D-H, Park J-Y, Chung Y-G, Kang S-H. Role of vincristine in the inhibition of angiogenesis in glioblastoma. Neurology Research. 2016; 38(10):871-9. doi:

[66] Michaelis M, Hinsch N, Michaelis UR, Rothweiler F, Simon T, ilhelm Doerr HW, Cinatl J, Cinatl J. Chemotherapy-associated angiogenesis in neuroblastoma tumors. The American

[67] Schirner M, Hoffmann J, Menrad A, Schneider MR. Antiangiogenic chemotherapeutic agents: Characterization in comparison to their tumor growth inhibition in human renal

[68] Dehelean CA, Feflea S, Molnár J, Zupko I, Soica C. Betulin as an antitumor agent tested in vitro on A431, hela and MCF7, and as an angiogenic inhibitor in vivo in the CAM

[69] Chintharlapalli S, Papineni S, Ramaiah SK, Safe S. Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors. Cancer Research.

[70] Chen Q-J, Zhang M-Z, Wang L-X. Gensenoside Rg3 inhibits hypoxia-induced VEGF expression in human cancer cells. Cellular Physiology and Biochemistry.

[71] Xiu Yu JL, Xu H, Hu M, Luan X, Wang K, Fu Y, Zhang D. Ginsenoside Rg3 bile Salt-Phosphatidylcholine-Based mixed micelles: Design, characterization, and evaluation.

[72] Kim J-W, Jung S-Y, Kwon Y-H, Lee J-H, Lee YM, Lee B-Y, Kwon S-M. Ginsenoside Rg3 attenuates tumor angiogenesis via inhibiting bioactivities of endothelial progenitor cells.

[73] Cathcart M-C, Useckaite Z, Drakeford C, Semik V, Lysaght J, Gately K, O'Byrne KJ, Pidgeon GP. Anti-cancer effects of baicalein in non-small cell lung cancer in-vitro and

[74] Liu J-J, Huang T-S, Cheng W-F, Lu F-J. Baicalein and baicalin are potent inhibitors of angiogenesis: Inhibition of endothelial cell proliferation, migration and differentiation.

cell carcinoma models. Clinical Cancer Research. 1998;**4**(5):1331-1336.

assay. Natural Product Communications. 2012;**7**(8):981-985

Chemical and Pharmaceutical Bulletin. 2015;**63**(5):361-368

Cancer Biology & Therapy. 2012;**13**(7):504-515

International Journal of Cancer. 2003;**106**(4):559-565

in-vivo. BMC Cancer. 2016;**16**(1):707

Research. 2009;**2**(1):74-83

64 Natural Products and Cancer Drug Discovery

10.1080/01616412.2016.1211231

2007;**67**(6):2816-2823

2010;**26**(6):849-858

Journal of Pathology. 2012;**180**(4):1370-1377

esis. Clinical Cancer Research. 2007;**13**(13):3977-3988


**Provisional chapter**

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

**Chemical, Antioxidant, and Cytotoxic Properties of** 

Rosa Guzmán‐Gerónimo, Edna Alarcón Aparicio, Oscar García Barradas, Jose Chávez‐Servia and Tania Alarcón‐Zavaleta Oscar García Barradas, Jose Chávez‐Servia and Tania Alarcón‐Zavaleta

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67574

Rosa Guzmán‐Gerónimo, Edna Alarcón Aparicio,

#### **Abstract**

[87] Lee U-L, Choi S-W. The chemopreventive properties and therapeutic modulation of green tea polyphenols in oral squamous cell carcinoma. ISRN Oncology. 2011;**2011**:1-7

[88] Yang X, Thomas DP, Zhang X, Culver BW, Alexander BM, Murdoch WJ, Rao MN, Tulis DA, Ren J, Sreejayan N. Curcumin inhibits platelet-derived growth factor-stimulated vascular smooth muscle cell function and injury-induced neointima formation.

[89] Shimizu M, Shirakami Y, Sakai H, Kubota M, Kochi T, Ideta T, Miyazaki T, Moriwaki H. Chemopreventive potential of green tea catechins in hepatocellular carcinoma.

Arteriosclerosis Thrombosis and Vascular Biology. 2006;**26**

66 Natural Products and Cancer Drug Discovery

International Journal of Molecular Sciences. 2015;**16**(3):6124-6139

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.

**Keywords:** blue corn extract, dietary phytoconstituents, anthocyanin profile, cancer cell lines, cytotoxic activity

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

Molecules derived from natural sources, such as plants, marine organisms, and microorgan‐ isms, 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 phyto‐ constituents have been the focus of scientific studies for cancer prevention [1]. Epidemiological and preclinical research indicates that dietary compounds possess chemopreventive proper‐ ties, for example, garlic consumption has been associated with a lower risk of cancer [2–4]. 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 [8–10].

Mexico is the center of origin and biodiversity of maize (*Zea mays* L.). Species have an exten‐ sive genetic diversity, with 59 different races described with different shapes and colors rang‐ ing 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 infor‐ mation 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 phytochemi‐ cals 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, anthocya‐ nins, and the antioxidant activity of blue corn from the Mixteco race, and to determine its antho‐ cyanin profile and the cytotoxic activity of the anthocyanin fraction in several cancer cell lines.

### **2. Research methods**

#### **2.1. 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

**Figure 1.** Grains of blue maize from Mixteco race.

Spanish acronym) of the National Polytechnic Institute, Oaxaca Unit in Mexico. Maize kernels were grounded and placed in amber bottles for analysis.

#### **2.2. Blue corn extracts**

**1. Introduction**

68 Natural Products and Cancer Drug Discovery

as well as acylated anthocyanins [12, 13].

**2. Research methods**

**2.1. Plant material**

phytochemicals for cancer prevention or therapy [14].

Molecules derived from natural sources, such as plants, marine organisms, and microorgan‐ isms, 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 phyto‐ constituents have been the focus of scientific studies for cancer prevention [1]. Epidemiological and preclinical research indicates that dietary compounds possess chemopreventive proper‐ ties, for example, garlic consumption has been associated with a lower risk of cancer [2–4]. 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 [8–10]. Mexico is the center of origin and biodiversity of maize (*Zea mays* L.). Species have an exten‐ sive genetic diversity, with 59 different races described with different shapes and colors rang‐ ing 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

Even though blue maize is an important part of the Mexican diet, there is little scientific infor‐ mation 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 phytochemi‐ cals from blue maize. It is well known that *in vitro* studies examine preliminary efficacy of

Given the above, the aim of this work is to evaluate the total content of polyphenols, anthocya‐ nins, and the antioxidant activity of blue corn from the Mixteco race, and to determine its antho‐ cyanin profile and the cytotoxic activity of the anthocyanin fraction in several cancer cell lines.

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 Ground blue corn kernels (1:5 p:v) were homogenized for 20 min with ethanol acidified with citric acid 1M (85:15 v:v). This was performed using an ultrasonic homogenizer at a frequency of 20 kHz and 750 W power (Cole‐Palmer Instrument Company, VCX‐750, USA) with a tip diameter of 13 mm at an amplitude of 25 µm with a pulse of 5 s in the 'On' position and 5 s in the 'Off' position. The sample was placed under refrigeration for 24 h and centrifuged at 4000 rpm for 15 min at a temperature of 5°C. The process was repeated twice and the extract was concentrated using a rotary evaporator under vacuum. The conditions of extraction have been included in a patent request, MX/A/20131011202.

### **2.3. Total phenolic content**

For analytical purposes, total polyphenols were evaluated using the colorimetric method pre‐ viously 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 measure‐ ment 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).

### **2.4. 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 deter‐ mined in buffers at pH 1.0 and 4.5. The monomeric anthocyanin content was expressed as cyanidin 3‐glucoside mg/100g.

### **2.5. 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.

### **2.6. 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 sys‐ tem 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.

### **2.7. 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% CO2 atmosphere and 95% humidity. For the assay, the following suspensions were prepared: 5 × 104 cell mL (K‐562, MCF‐7), 7.5 × 104 cell/mL (PC‐3), and 10 × 104 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.

### **3. Results and discussion**

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 measure‐ ment 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

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 deter‐ mined in buffers at pH 1.0 and 4.5. The monomeric anthocyanin content was expressed as

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

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 sys‐ tem 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

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

acid/100 g of fresh weight (mg GAE/100g).

**2.4. Total monomeric anthocyanin content**

cyanidin 3‐glucoside mg/100g.

70 Natural Products and Cancer Drug Discovery

expressed as µmol eq. trolox/g of sample.

**2.7. Cell culture and assay for cytotoxic activity**

**2.6. Isolation and chromatographic analysis of anthocyanins**

range: 50–3000 amu, capillary voltage 3.8 kV, dry gas flow at 4.0 L/min.

**2.5. Antioxidant activity**

### **3.1. 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 antho‐ cyanin 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.

Results for the total content of polyphenols, monomeric anthocyanins, and antioxidant activity were plotted in a polygons graph in order to identify the sample with the best characteristics. **Figure 2** shows that sample CIIDIR‐125 had the largest content of anthocyanins and anti‐ oxidant activity; therefore, it was selected to undergo the anthocyanin profile analysis and biological tests.

### **3.2. Anthocyanin profile of blue corn extract**

**Figure 3** shows the profile of anthocyanins isolated from blue corn using amberlite XAD‐ resin. The MS data analysis for blue corn anthocyanins is summarized in **Table 2**. It shows ions at *m/z* = 287 and 271, suggesting that anthocyanins are derived mainly from cyanidin and pelargonidin. Eight anthocyanins were identified such as: cyanidin‐3‐(3″,6″‐dimalonyl‐gluco‐ side), pelargonidin‐3‐glucoside dimalonate, pelargonidin‐3‐(sinapoyl glucoside)‐5‐glucoside,


1 mg GAE/100g.

2 mg C3G/100 g.

3 µmol TE/g.

**Table 1.** Content of total polyphenols, monomeric anthocyanins and antioxidant activity in blue corn samples.

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‐ cyanins are present in blue corn from the Mixteco race. This could be due to genetic factors, farming practices, weather conditions, and soil type, which have an influence on the chemical composition of maize varieties.

### **3.3. 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 myelog‐ enous 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**).

Chemical, Antioxidant, and Cytotoxic Properties of Native Blue Corn Extract http://dx.doi.org/10.5772/67574 73


**Figure 2.** Polygon graph of total polyphenols, monomeric anthocyanins and antioxidant activity.

**Figure 3.** Anthocyanin profile of blue corn.

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‐ cyanins are present in blue corn from the Mixteco race. This could be due to genetic factors, farming practices, weather conditions, and soil type, which have an influence on the chemical

**Table 1.** Content of total polyphenols, monomeric anthocyanins and antioxidant activity in blue corn samples.

e

**Sample Total polyphenols1 Monomeric anthocyanins2 Antioxidant activity3**

CIIDIR‐02 154.4f,g,h,i 32.5de2 18.5e1 CIIDIR‐12 176.7c,d 48.5<sup>b</sup> 21.2d,e CIIDIR‐54 158.4f,g 31.2<sup>e</sup> 18.6<sup>e</sup> CIIDIR‐107 173.3d,e 53.4<sup>b</sup> 26.7a,b,c CIIDIR‐112 142.8<sup>j</sup> 30.7<sup>e</sup> 18.5<sup>e</sup> CIIDIR‐125 203.2ª 66.9<sup>a</sup> 24.4ab,c,d CIIDIR‐129 164.3e,f 47.5b,c 23.6b,c,d CIIDIR‐131 173.4d,e 53.4<sup>b</sup> 27.8<sup>a</sup> CIIDIR‐167 187.1b,c 32.3<sup>e</sup> 24.4a,b,c,d CIIDIR‐172 147.4g,h,i 28.6ef 22.7c,d CIIDIR‐179 162.1<sup>f</sup> 30.7<sup>e</sup> 21.5d,e CIIDIR‐184 146.5i,j 21.4<sup>f</sup> 20.6d,

CIIDIR‐185 192.9a,b 35.1de 26.8a,b CIIDIR‐189 157.7f,g,h 40.3cd 22.8c,d CIIDIR‐190 148.1g,h,i 33.1de 23.5b,c,d CIIDIR‐197 175.3<sup>d</sup> 35de 26.7a,b,c

Samples with the same letters are not significant statistically (*p* < 0.05).

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 myelog‐ enous 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**).

**3.3. Cytotoxic activity of the anthocyanin fraction from blue corn**

composition of maize varieties.

72 Natural Products and Cancer Drug Discovery

1

2

3 µmol TE/g.

mg GAE/100g.

mg C3G/100 g.

PC3 cells were selected due to their highly aggressive nature. Data showed 2.43% inhibi‐ tion on prostate cancer cells at 500 µg/mL. Previous studies have analyzed the anticancer properties of the anthocyanin fraction from potato extracts in prostate cancer cells (PC‐3) 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


**Table 2.** Chromatographic and mass spectral data of anthocyanins.

research, the anthocyanin profile of blue corn was composed only of cyanidin and pelar‐ gonidin 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**) at the same concentration

**Figure 4.** *In vitro* cytotoxicity of blue corn extract on several human cancer lines.

(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 indi‐ cate 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.

### **Acknowledgements**

The authors would like to thank the SINAREFI (BEI‐MAI‐10‐32) from México.

### **Author details**

research, the anthocyanin profile of blue corn was composed only of cyanidin and pelar‐

*m/z* **Tentative identification**

3 2.8 639–271 Pelargonidin‐3‐(sinapoyl glucoside)‐5‐glucoside 4 3.4 605–271 Pelargonidin 3‐(3″,6‴‐dimalonylglucoside)

6 5.0 654–595–434–271 Pelargonidin 3‐glucoside‐5‐(6″‐acetyl‐glucoside)

7 6.6 519–271 Pelargonidin 3‐(6″‐malonylglucoside) 8 8.4 595–271 Pelargonidin 3,5‐diacetylglucoside

5 3.9 740–519–433–271 Pelargonidin 3‐(6″‐malonyl glucoside)‐5‐(6″ acetyl glucoside)

1 1.2 621–287 Cyanidin‐3‐(3″, 6″‐dimalonyl‐glucoside) 2 1.9 358–271 Pelargonidin‐3‐glucoside dimalonate

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**) at the same concentration

gonidin derivatives; delphinidin was not detected.

**Figure 4.** *In vitro* cytotoxicity of blue corn extract on several human cancer lines.

**Table 2.** Chromatographic and mass spectral data of anthocyanins.

**Peak Retention**

**time (min)**

74 Natural Products and Cancer Drug Discovery

Rosa Guzmán‐Gerónimo1 \*, Edna Alarcón Aparicio1 , Oscar García Barradas2 , Jose Chávez‐Servia3 and Tania Alarcón‐Zavaleta1

\*Address all correspondence to: roguzman@uv.mx

1 Basic Sciences Institute, University of Veracruz, Xalapa, Veracruz, México

2 Services in Analytical Resolution, SARA, University of Veracruz, Xalapa, Veracruz, México

3 The Interdisciplinary Research Center for Integrated Regional Development, Oaxaca Unit, National Polytechnic Institute, Oaxaca, México

### **References**


[15] Singleton VL, Rossi, JA. Colorimetry of total phenolics with phosphomolybdic‐phos‐ photungstic acid reagents. Am J Enol Vitic. 1965;**16**:144‐150.

**References**

2004;**117**:1155‐1160.

76 Natural Products and Cancer Drug Discovery

2009. 116 p.

[1] Kotesha R, Takami A, Espiniza JL. Dietary phytochemicals and cancer chemoprevention:

[2] Mehta M, Shike M. Diet and physical activity in the prevention of colorectal cancer. J

[3] Howes MJ, Simmonds MS. The role of phytochemicals as micronutrients in health and disease. Curr Opin Clin Nutr. 2014;**17**:558‐566. DOI: 10.1097/MCO.0000000000000115.

[4] Li H, Li HQ, Wang Y, Xu HX, Fan WT, Wang ML, Sun PH, Xie XY. An intervention study to prevent gastric cancer by micro‐selenium and large dose of allitridum. Chin Med J.

[5] Priyadarsini RV, Nagini S. Cancer chemoprevention by dietary phytochemicals: promises and pitfalls. Curr Pharm Biotechnol. 2012;**13**:125‐136. DOI: 10.2174/138920112798868610

[6] Wang LS1, Stoner GD. Anthocyanins and their role in cancer prevention. Cancer Lett.

[7] Delgado‐Vargas F, Jiménez AR, Paredes‐López O. Natural pigments: carotenoids, antho‐ cyanins, and betalains—characteristics, biosynthesis, processing, and stability. 1st ed.

[8] Goufo, Trindade H. Rice antioxidants: phenolic acids, flavonoids, anthocyanins, pro‐ anthocyanidins, tocopherols, tocotrienols, γ‐oryzanol, and phytic acid. Food Sci Nutr.

[9] Awika JM1, Rooney LW.. Sorghum phytochemicals and their potential impact on human health. Phytochemistry. 2004;**65**:1199‐1221. DOI: 10.1016/j.phytochem.2004.04.001

[10] Guzmán‐Gerónimo RI, Alarcón‐Zavaleta TM, Oliart‐Ros RM, Meza‐Alvarado JE, Herrera‐Meza S, Chávez‐Servia JL. Blue maize extract improves blood pressure, lipid profiles, and adipose tissue in high‐sucrose diet‐Induced metabolic syndrome in rats. J

[11] Kato T, Mapes C, Mera, Serratos J, Bye R. Origin and diversification of maize: an analyti‐ cal review. National Autonomous University of Mexico. 1st ed. Mexico, D.F.: CONABIO;

[12] Salinas‐Moreno Y, Pérez‐Alonso JJ, Vázquez‐Carrill, G, Aragón‐Cuevas F. Anthocyanins and antioxidant activity in maize grains (Zea mays L.) of chalqueño, elotes cónicos and

[13] Salinas MY, Salas SG, Rubio HD, Ramos LN. Characterization of anthocyanin extracts

[14] Ross NT, Wilson CJ. In vitro clinical trials: the future of cell‐based profiling. Front

Boca Raton, Florida: CRC Press; 2003. 313 p. DOI: 10.1080/10408690091189257

a review. Oncot. 2016;**7**:52517‐52529. DOI: 10.18632/oncotarget.9593

Natl Compr Canc Netw. 2014;**12**:1721‐1726.

2008;**269**:281‐290. DOI: 10.1016/j.canlet.2008.05.020

Med Food. Forthcoming. DOI: 10.1089/jmf.2016.0087

from maize kernels. J Chromatogr Sci. 2005;**43**:483‐487.

Pharmacol. 2014;**5**:1‐6. DOI: 10.3389/fphar.2014.00121

2014;**2**:75‐104. DOI: 10.1002/fsn3.86

bolita races. Agroc. 2012;**46**:693‐706.

