In vitro study of the antihypertensive, antioxidant and antiproliferative activities of peptides obtained from two varieties of Phaseolus coccineus L

ABSTRACT Three biological activities were assessed for peptides obtained from two ayocote bean varieties, antioxidant and antihypertensive properties and antiproliferative activity against MDA breast cancer cells. For the peptides obtained from the ayocote bean black variety the highest antihypertensive activity was obtained with low-molecular weight peptides, principally with those between 5 and 10 kDa. However, for the peptides obtained from ayocote bean purple variety, the highest antihypertensive activity was achieved with peptides with a molecular weight ≥10 kDa, while that the higher antioxidant activity was noted for peptides obtained from ayocote bean purple variety. All peptides from both purple and black ayocote beans varieties inhibited MDA cells proliferation; however, the IC50 was only reached with high concentrations of peptides (2000 or 5000 µg*mL −1) and exposure times of 12 or 72 h. The obtained results reveal a potential relationship among the antihypertensive, antioxidant activities and anticancer properties of the peptides.


Introduction
The continuing increase in the incidence of breast cancer represents a current public health problem; however, an early detection and management strategy (Gernaat et al., 2018) has permitted approximately three million people to survive breast cancer in the last five years worldwide. In addition to several cancer types, such as chronic diseases, another adult chronic disease that affects people´s health and increases the risk of death is cardiovascular disease (CVD), and 17.5 million people have died from this disease in recent years (Castro-Juárez et al., 2018). Previous studies have reported high comorbidity between breast cancer and cardiovascular disease (Abdel-Rahman et al., 2018), which could be attributed to complications generated by cardiovascular disease in cancer patients and difficulty in the selection of cancer treatments. On the other hand, some cancer treatments generate cardiovascular complications; thus, these treatments are not administered, creating a vicious disease cycle (Mehta et al., 2018). Some studies indicate that breast cancer patients may have a higher risk of CVD than the general population, and an increase in the mortality index of approximately 24% in patients over 65 years old has been observed and attributed to this phenomenon (Bradshaw et al., 2016;Colzani et al., 2011). The increase in comorbidities is mainly associated with some breast cancer treatments, such as the use of anthracycline-based therapies, trastuzumab and radiation therapy, which are efficient in cancer treatment and reduce the mortality index (Chavez-MacGregor et al., 2013;Dalfardi et al., 2014;Gernaat et al., 2018;Rehammar et al., 2017); however, the association between diseases is increased in patients with breast cancer. Thus, oncological treatment represents a significant challenge (Abdel-Rahman et al., 2018). Considering this situation, several reports with a wide variety of breast cancer treatments have been published, which limits the ability of doctors to identify and provide accurate recommendations to decrease the CVD risk in patients with breast cancer, particularly regarding the timing of interventions (Bradshaw et al., 2016).
On the other hand, a correlation between antioxidant activity and the prevention of some cancer types has been reported that apparently is due to the presence of phenolic compounds that can contribute to the death induction of cancer cells through a pro-oxidant effect; for example, genistein acts synergistically with 5-fluorouracil, promoting the death of colon cancer cells (Aydin et al., 2006). In another study, resveratrol, an antioxidant present in red wine, inhibited the proliferation of prostate cancer cells by altering mitogenesis (Diaz et al., 2012). As noted above, phenolic compounds potentially increase the efficacy of anticancer drugs, allowing dose reductions (Agbor et al., 2006). Consequently, an alternative in the prevention, control or treatment of cancer is the use of compounds of vegetable origin, particularly proteins, protein fractions or peptides, which have beneficial therapeutic effects on health in addition to their nutritional value and physicochemical characteristics as demonstrated by several studies (Banan-Mwine et al., 2017;Chakrabarti et al., 2018;Hou et al., 2019;Mada et al., 2020;Okagu et al., 2021). For these reasons, interest in natural products as sources of nutraceutical compounds has increased in response to the need to search for new alternatives for cancer therapy without compromising cardiovascular health (Barac et al., 2015;Lenihan et al., 2010). For the above reasons, the focus of this research was to determine the presence of potentially functional peptides from the ayocote bean protein isolate generated by a simulated gastrointestinal digestive system to obtain peptides similar to those released in a physiological digestion process using proteinases obtained from animal tissue (pancreatin and trypsin). This study also seeks to evaluate the anticancer, antihypertensive and antioxidant activities of the peptides obtained from protein isolates from two varieties of ayocote beans (black and purple).

Vegetal material
Two varieties of ayocote bean (black and purple) were obtained from Zacatlan Puebla, Mexico with moisture values of 9.37 ± 0.208% and 9.62 ± 0.113%, respectively. The seeds were selected, and any extra material was removed. Then, the beans were ground using a domestic coffee grinder. The dried ayocote bean powder was packed in PVC bags and stored in an LG Model GR-452SH refrigerator (LG electronics, México) at 4°C until use.

Preparation of protein isolates
Protein isolates from the defatted (by the ethereal extract method: AOAC method 9020.39) ayocote bean powder samples were prepared by the method described by Teniente-martínez et al. (2019). The defatted powder was dispersed in distilled water (1:20) and homogenized by magnetic stirring, adjusting the pH to 11.8 with NaOH (0.1 N).
Then, the solution was centrifuged at 6000 rpm for 30 min at a temperature of 4°C and the supernatant was collected.
Then, the pH of the collected supernatant was adjusted to pH 4 (HCl; 0.1 N), and the precipitated protein was recovered by centrifugation at 3000 × g for 30 min. Then, the recovered protein was dried using a forced convectiondrying oven (Binder, Model FD115-UL, USA) at a temperature of 50°C for approximately 6 h.

Enzymatic hydrolysis
The protein isolates were hydrolyzed by sequential treatment with pepsin (P7012, Sigma) and pancreatin (P1750, Sigma) according to the method of Mora-Escobedo et al. (2009). The protein isolate was suspended in distilled water to prepare the protein substrate (44 mg/mL). The protein solution was adjusted by the addition of 1 N of HCl to a pH of 2 and a temperature of 37°C for 60 min. A measured amount of pre-suspended pepsin in distilled water and adjusted to hydrolysis pH conditions was then added to the substrate to obtain an enzyme/protein ratio of approximately 4.5% (AU/w). Later, the solution was adjusted by the addition of 0.9 M NaHCO3 to a pH of 5.3. Then, a solution of pancreatin with an enzyme/protein ratio of approximately 4.5% (AU/w) was added. The mix was gently homogenized, and the pH was adjusted to 7.5 using 1 N NaOH and a temperature of 37°C The reaction mixture was maintained for 120 min. The hydrolysis reaction was stopped by heat treatment of the reaction mixture to 90°C for 10 min. All procedures were performed in a 100 mL glass reactor.

Determination of the degree of hydrolysis (DH)
The degree of hydrolysis was obtained as the protein solubility in trichloroacetic acid (TCA) according to Kim et al. (1990). A 10-mL aliquot of hydrolysate was solubilized in 10% TCA solution. After 15 min, the sample was centrifuged at 12,000 × g for 15 min. The nitrogen content of the hydrolysate and the supernatant of the sample treated with TCA were analyzed using the Kjeldahl method (Cunniff, 1995). The calculation of the degree of hydrolysis (DH) was carried out with the following formula 1: where DH is the degree of hydrolysis; N 2 is nitrogen; and TCA is trichloroacetic acid.

Peptide fractionation by ultrafiltration
The protein hydrolysate was further fractionated by ultrafiltration with a stirred cell and disc membrane system (Millipore Amicon, Model 8050). The protein hydrolysate solution was first separated by a 30-kDa molecular weight cutoff (MWCO) membrane in the cell at 75 psi and 4°C The separation generated two peptides, stream permeate (designated P30) and retentate 1 (R30). R30 was dialyzed against deionized water at 4°C for 1 h and lyophilized. Then, P30 was separated using a 10-kDa MWCO membrane to obtain the two peptides, stream permeate (designated P10) and retentate 10 (R10). R10 was dialyzed against deionized water at 4°C for 1 h and lyophilized; P10 was separated using a 5-kDa MWCO membrane to obtain the two peptides, stream permeate (designated P5) and retentate 5 (R5). R5 was dialyzed against deionized water at 4°C for 1 h and lyophilized; P5 was separated using a 3-kDa MWCO membrane to obtain the two peptides, stream permeate (designated P3) and retentate 3 (R3). Finally, P3 was separated by a 1-kDa MWCO membrane and generated a permeate (designated P1) and a final retentate (R1). For satisfactory separation, each retentate was passed through the same membrane twice before being separated with the next membrane. The resulting six peptides (R30, R10, R5, R3, R1 and P1) were each freeze-dried. The resulting powder was milled in a mortar, sealed in glass bottles, and stored at 4°C until use.

In vitro evaluation of the inhibitory activity of angiotensin converting enzyme (ACE)
ACE inhibitory activity was measured using the method of Chang-Bum et al. (2012). A sample solution of 50 µL with 50 µL of ACE solution (Sigma-Aldrich, A2580) (25 mU/mL) was preincubated at 37°C for 10 min, and the mixture was incubated with 150 µL of substrate (8.3 mM HHL in 50 mM sodium borate buffer containing 0.3 M NaCl at pH 8.3) for 30 min at the same temperature. The reaction was terminated by the addition of 250 µL of 1.0 M HCl. The resulting hippuric acid was extracted with 0.5 mL of ethyl acetate. After centrifugation (1000 × g, 15 min), 200 µL of the upper layer was transferred into a test tube and evaporated at room temperature for 2 h in a vacuum. Hippuric acid was dissolved in 1.0 mL of distilled water, and the absorbance was measured at 228 nm using a UV spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The IC 50 value (µg/mL) was defined as the concentration of inhibitor required to inhibit 50% of the ACE activity.
The percentage of inhibition was calculated using the formula 2: The terms in the equation are defined as follows: A-Absorbance of the negative control, which contains substrate and enzyme.
B-Absorbance of the blank, which includes an inhibitor (enalapril 2 mg*mL −1 ) and substrate.
C-Absorbance obtained from the samples, which include enzymes, substrate and sample.

Determination of antioxidant activity (DPPH method)
The DPPH method, which employs the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich, St. Louis, MO, USA), was used according to Brand-Williams et al. (1995). To determine the IC 50 values, concentration profile studies were performed in the range of 5 to 100 mg of sample*mL −1 of distilled water. Each sample was vortexmixed for 2 min and then centrifuged at 1000 × g for 20 min, and 0.1 mL of the supernatant was placed in vials. Then, 3.9 mL of DPPH in methanol (6x10 −5 M) was added. The reduction of absorbance was determined at 515 nm from time 0 and then every 10 min until the reaction was completed using a spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).
The inhibition (% I) of DPPH radicals was calculated using the formula 3: The terms in the equation are defined as follows: A 0 -The absorbance of the control (0.1 mL of distilled water and 3.9 mL of DPPH in methanol).
A s -The absorbance of the test sample. The IC 50 values were calculated from the graphs generated by the percentage of inhibition of DPPH versus sample concentrations. IC 50 denotes the concentration of the compound required to inhibit the initial DPPH value of 50% to an absorbance of 515 nm.

Total phenolic content (TPC)
The TPC of each sample was quantified using Folin-Ciocalteu methodology (Singleton et al., 1999). The extraction of phenolic compounds was performed using 0.5 mg of each sample (fresh or dry) to which 2 mL of methanol (at 80%) containing 1% HCl was added. The prepared dissolution was placed in an orbital shaker (Eberbach Corporation, Ann Arbor, MI, USA) (200 rpm) for 2 h at room temperature. The mixture was centrifuged at 1000 × g for 15 min in a centrifuge (Hermle Z200A, Germany), and the supernatant was placed in vials. The precipitate was used to perform a second extraction under the same conditions. The resulting supernatants for both extractions were mixed and used to perform the quantification of TPC. An aliquot of the extract (100 μL) was mixed with 0.75 mL of Folin-Ciocalteu reagent (Hycel of México SA de CV, México) (previously diluted in distilled water) and was left to stand at room temperature for 5 min, after which 0.75 mL of sodium bicarbonate was added at a concentration of 6%. The absorbance was then read at 725 nm after 90 min of incubation at room temperature. The results were expressed as gallic acid equivalents (GAE) in milligrams per gram of sample using a gallic acid (Sigma-Aldrich, St. Louis, MO, USA) standard curve.

Cell culture
MDA cells (origin: breast; histopathology: adenocarcinoma of the breast) were obtained from the Cancer National Institute (Mexico City, Mexico). MDA cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U*mL −1 penicillin and 100 μg*mL −1 streptomycin in a 5% CO 2 humid atmosphere at 37°C Cells were washed with DMEM containing 1% FBS 24 h before experiments and replated onto 96-well plates.

Anticancer activity assay
Cell viability was measured by using the MTT assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases (Hansen et al., 1989). MDA cells (25 cells*μL −1 ) were treated with various concentrations of peptides from purple or black ayocote beans (1000-5000 µg*mL −1 ) at different contact times (3, 6, 12, 24, 48 and 72 h) at 37°C Then, the medium was incubated with 10 μL of 5 mg *mL −1 MTT solution for 3 h. After the culture medium was removed by vacuum aspiration, 100 μL of dimethyl sulfoxide was added to each well to dissolve the formazan. Absorbance was measured at 540 nm using a microplate reader (EPOCHH, BioTek, BioTek Instruments, Inc., USA). Cell viability was expressed as a percentage of the value in the control cultures.

Statistical analysis
The quantitative data are expressed as the mean ± standard deviation, and analysis of variance (ANOVA) was performed followed by Tukey's test. SAS software was used for data analysis, and all experimental determinations were performed in triplicate.

Protein isolate yield and degree of hydrolysis
The protein isolate yield and the degree of hydrolysis of both ayocote bean varieties tested were reported in our previously published study (Teniente-martínez et al., 2019). For the protein isolate yield of the black bean variety, the highest recovery percentage of protein was obtained (90.80%), whereas a slightly lower concentration of protein of the protein isolate of the purple bean variety was obtained (86.60%). A similar degree of hydrolysis for both varieties (73%) was also reported.

Inhibition of the angiotensin converting enzyme (ACE)
The capacity to inhibit the angiotensin converting enzyme (ACE) of the peptides at two doses (3 mg and 20 mg) from both purple and black bean varieties reported as percentages are shown in Table 1. These concentrations in preliminary studies (data not shown) were the only concentrations that showed ACE inhibition activity.
A higher number of peptides with antihypertensive activity and higher antihypertensive activity were obtained for the black beans (Table 1); however, only sample R3 showed 6% higher antihypertensive activity compared with the control at a concentration of five mg. However, with an increase to 20 mg of peptide, an inhibitor saturation response was observed in most peptide groups with some level of inhibition; thus, no ACE inhibition activity was observed. However, in those peptide groups without ACE inhibition with a peptide concentration of 3 mg, the increase in the concentration of the peptide groups to a level of 20 mg (R30 and R5) yielded an ACE inhibition percentage greater than that of enalapril (control).
These results indicate that the groups of peptides generated from the black beans have a higher proportion of aromatic amino acids than those obtained from the purple beans as it has been shown that these amino acids enhance the binding peptide-active site of ACE, causing their inhibition (Cheung et al., 1980). High concentrations of ACE inhibitors peptides obtained from soy, fish, egg, meat and milk have been reported (Gallegos-Tintoré et al., 2013).
The increase in the concentration of the peptide tested influenced the ACE inhibition percentage. The increase in the peptide promoted interactions between the peptides and restricted the interaction with ACE. However, in R30 and R5, the increase in ACE inhibition is an inverse process that allows better interactions between the peptide and ACE. Thus, ACE was almost completely inhibited with the R5 sample obtained from black bean.

Determination of antioxidant activity
The results obtained are shown in Table 2 and are expressed as IC 50 values, which represent the sample peptide concentration required to inhibit 50% of the oxidizing agent (DPPH). In general, all peptides obtained from the black beans showed lower IC 50 values than the peptides obtained from the purple beans. The results indicate that peptide with lower molecular weights obtained from both bean varieties have lower antioxidant activities with a linear tendency.
Apparently, the lower concentration required to reach the IC 50 in the peptide obtained from the purple beans is due to the high concentration of hydrophobic amino acids (valine, leucine, tryptophan, histidine, proline, tyrosine, methionine and cysteine) reported (Ajibola et al., 2011;Chuan-He et al., 2009), which, according to some authors, are mainly responsible for antioxidant activity given that these amino acids have the ability to inhibit oxidation by participating in reactions involving different oxidizing species (Gallegos-Tintoré et al., 2013). On the other hand, it has been reported that extensive hydrolysis of proteins could result in a decrease in their antioxidant activity because the amino acids in their free form are not effective as antioxidant agents (Saito et al., 2003). It is not possible to identify one only factor that influences antioxidant activity due to the variability in the structure and hydrophobicity of each peptide and the relationship that exists in the amino acid composition sequence (Saito et al., 2003).

Determination of total phenolic compound content (TPC)
The results obtained are shown in Table 3. Similar to the results obtained for the antioxidant activity, we found that the peptides greater than 10 kDa had higher total phenolic content than those of lower molecular weight with the peptides of purple bean presenting the highest concentration. Again, the R30 exhibited the highest concentration. In the peptides with high molecular weights, high concentrations of amino acids with aromatic groups are present (tryptophan, tyrosine and phenylalanine); these amino acids are similar in structure to phenolic compounds (Sánchez-Mendoza et al., 2016). In addition, these peptides are also rich in lysine, which contains a phenolic group. Thus, these amino acids act as proton donors to stabilize free radicals and react with the Folin-Ciocalteu reagent (Muñoz-Bernal et al., 2017;Singleton & Rossi, 1965). On the other hand, other amino acids can also react with the reagent, such as sulfur amino acids (methionine and cysteine) and Valores seguidos por letras minúsculas diferentes en la misma columna son significativamente distintas de acuerdo a la prueba de Tukey a una p ≤ .05. Valores seguidos por letras mayúsculas diferentes en la misma fila son significativamente distintas de acuerdo a la prueba de Tukey a una p ≤ .05. histidine through its imidazole group (Elias et al., 2008;Loganayaki et al., 2011;Muñoz-Bernal et al., 2017). The results obtained indicate that in the peptides with high molecular weight, a direct correlation potentially exists between the concentration of amino acids capable of reacting with Folin-Ciocalteu reagent and the antioxidant activity.

Effect of the ayocote bean variety and exposure time on MDA cell proliferation
The results of the peptides from black and purple beans on the inhibition of MDA cell proliferation are shown in Figures 1 and 2, respectively. The peptides R30 and R10 obtained from both bean varieties (black and purple) did not affect MDA cell proliferation at any concentration or with 24 h of exposure time.
The number of peptides that inhibited MDA cell proliferation obtained from the black beans was greater than the number of peptides obtained from the purple beans. Thus, only the peptides obtained from black beans showed some inhibition against the proliferation of MDA cells at exposure times of 3, 6, 12, 48 and 72 h and concentrations of 3000 and 5000 μg*mL −1 .
The high molecular weight peptides (R30 and R10) obtained from the black beans showed three times more inhibition of MDA cell proliferation compared with peptides with low molecular weights (R5, R3 and P1) at two concentrations (3000 and 5000 μg*mL −1 ) and 72 h of exposure ( Figure 1). Similar results were obtained in a previous study performed with the SiHa cell line, demonstrating that peptides with molecular weights <10 kDa exhibited reduced inhibition of SiHa cell proliferation compared with peptides with molecular weights ≥10 kDa (Teniente-martínez et al., 2019).
The results obtained are similar to those reported by López-Sánchez et al. (2010), who tested the inhibitory effect of peptides ≥30 kDa obtained from Phaseolus acutifolius on MCF-7 breast cancer cell proliferation. On the other hand, it has also been demonstrated that human milk peptides with a molecular mass ≥10 kDa showed antiproliferative activity against MCF-7 cells (Nekipelaya et al., 2008). The results obtained are consistent with those published in the literature that indicate that effectiveness against cancer cells of peptides varies depending on the protein source (vegetal or animal), subtype (cultivar or race), type of cancer cell tested and molecular mass of the peptides. Thus, the results on the antiproliferative activity of peptides of ayocote beans differed. Compared with our previous study using SiHa cells (Teniente-martínez et al., 2019), the antiproliferative activity was reduced in MDA cells exposed to ayocote black bean peptides with a molecular weight >10 kDa. However, when peptides with a weight molecular weight ≤10 kDa were Figure 2. Anti-Proliferative activity of different peptides from purple ayocote bean protein hydrolysate against the MDA cell line. R30, peptides with molecular weight >30 kDa; R10, peptides with molecular weight ≤30 kDa and >10 kDa; R5, peptides with molecular weight ≤10 kDa and >5 kDa; R3, peptides with molecular weight ≤5 kDa and >3 kDa; R1, peptides with molecular weight ≤3 kDa and >1 kDa; and P1, peptides with molecular weight ≤1 kDa. Different letters above the bars indicate significant difference (p ≤ .05). Figura 2. Actividad antiproliferativa de diferentes péptidos obtenidos de proteína de frijol ayocote morado contra líneas célulares MDA. R30, péptidos de masa molecular >30 kDa; R10 péptidos de masa molecular ≤30 kDa y >10 kDa; R5, péptidos de masa molecular ≤10 kDa and >5 kDa; R3, péptidos de masa molecular ≤5 kDa y >3 kDa; R1, péptidos de masa molecular ≤3 kDa y >1 kDa; P1, péptidos de masa molecular ≤1 kDa. Letras diferentes arriba de las barras indican diferencias significativas (p ≤ .05).
tested, greater antiproliferative activity was noted for MDA cells compared with SiHa cells.
On the other hand, for peptides from purple ayocote bean (Figure 2), only the peptides with a molecular weight ≥30 kDa showed adequate antiproliferative activity on MDA cells at a concentration of 5000 µg*mL −1 and an exposure time of 48 h.
The maximal inhibitory concentration to reach the IC 50 of each peptides from the black and purple bean hydrolysates is shown in Table 4. In both black and purple peptides, only those peptides with molecular weight >30 (R30) reached the IC 50 . However, a lower concentration of peptides from the black ayocote bean hydrolysate (2000 μg*mL −1 ) was required to reach the IC 50 compared with those from the purple ayocote bean hydrolysate (5000 μg*mL −1 ).
The peptides with molecular weights less than 30 kDa obtained from the ayocote bean hydrolysate generally did not achieve the IC 50 with all concentrations and exposure times tested. The IC 50 was only reached using peptides with molecular weights between 10 kDa and 30 kDa obtained from the black ayocote bean hydrolysate at a concentration of 2000 μg*mL −1 and exposure time of 72 h.According to these results, the peptides obtained from the purple bean hydrolysates are considered the most effective to achieve the IC 50 given that the exposure time required to reach the IC 50 for the peptides obtained from the purple bean hydrolysates (12 h) was less than the exposure time required with peptides obtained from the black bean hydrolysates (72 h) although the concentration required is higher (5000 μg*mL −1 ).The IC 50 results in MDA cells were different from those reported for SiHa cells in a previous study (Teniente-martínez et al., 2019) using the same ayocote bean varieties. However, the results obtained were similar to those reported by González-Montoya et al. (2016). These researchers found that when peptides with molecular weights >10 kDa obtained from germinated soybean were used in MDA-MB-231 and MCF-7 cells, concentrations of 1519 µg*mL −1 and 2000 µg*mL −1 , respectively, were required to reach the IC 50 .

Conclusion
Based on the results obtained in this study, all peptides showed antihypertensive activity. The highest values were obtained with the peptides between 5 kDa and 10 kDa (R5) obtained from the black bean hydrolysate, whereas the highest antihypertensive activity was obtained with peptides with molecular weights ≥10 kDa (R10 and R30) from the purple bean hydrolysate.
Increased antioxidant activity (1.7 times more on average) was observed with the peptides obtained from the purple bean hydrolysate compared with the peptides obtained from the black bean hydrolysate. All peptides obtained from both purple and black bean hydrolysates showed some level of inhibition of MDA cell proliferation. However, the IC 50 was only reached with high concentrations of peptides (2000 µg*mL −1 or 5000 µg*mL −1 ) and exposure times of 12 or 72 h. The peptides obtained from both purple and black bean hydrolysates exhibit adequate antihypertensive, antioxidant and antiproliferative activities, and these activities are influenced by the variety and molecular weight of the peptides. Table 4. Antiproliferative activity IC 50 values (µg*ml −1 ) of ayocote bean peptides from both varieties on the MDA cell line.