Proteolytic hydrolysis of cowpea proteins is able to ...

FRIN-05782; No of Pages 6 Food Research International xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Food Research International journal homepage: www.elsevier.com/locate/foodres

Proteolytic hydrolysis of cowpea proteins is able to release peptides with hypocholesterolemic activity Marcelo Rodrigues Marques a, Gustavo Guadagnucci Fontanari a, Daniel Carvalho Pimenta b, Rosana Manólio Soares-Freitas a, José Alfredo Gomes Arêas a,⁎ a b

Laboratory of Food Functional Properties—Nutrition Department, School of Public Health, Universidade de São Paulo—USP, São Paulo, SP, Brazil Laboratory of Biochemistry and Biophysics, Instituto Butantan, Avenida Vital Brasil 1500, São Paulo, SP, Brazil

a r t i c l e

i n f o

Article history: Received 14 January 2015 Received in revised form 7 April 2015 Accepted 12 April 2015 Available online xxxx Keywords: Cowpea bean HMG-CoA reductase Micellar cholesterol solubilization Peptides Chemical compounds studied in this article: Taurocholate (PubChem CID 23666345) Cholestrol (PubChem CID 5997) Phosphatidylcholine (PubChem CID 45266626) NADPH (PubChem CID 5884) Pravastatin (PubChem CID 54687) HMG-CoA (PubChem CID 445127)

a b s t r a c t This study aimed to assess the hypocholesterolemic activity of peptides obtained by in vitro simulated human digestion of cowpea bean proteins; moreover, we have screened the bioactive peptides through chromatographic separation by RP-HPLC of the 3 kDa molecular mass cut-off fraction of hydrolyzed isolated cowpea protein. Micellar solubility of cholesterol was measured after adding 35 μg mL−1 of each fraction on in vitro prepared intestine-like micelles. The inhibiting activity of each fraction (50 μg mL−1) also was tested on the enzyme 3hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). The whole hydrolysate was analyzed by ‘de novo’ mass spectrometry peptide sequencing (RP-HPLC-MS2) and the top score candidate peptide sequences were further analyzed by computational modeling. All collected fractions inhibited the initial HMGCoA reductase activity by 47.8 to 57.1%. They also reduced cholesterol micellar solubilization, with fraction 1 being the most effective (71.7%). The peptide conserved domains may interact with the phosphatidylcholine added in the reaction of the cholesterol micelles. According with a computational prediction, the only peptide able to bind significantly the HMG-CoA reductase was GCTLN. This is the first report of peptide fractions from cowpea bean protein released by human digestion enzymes (pepsin followed by pancreatin) assigned to its cholesterol-lowering effect. These routes may define their action in lipid metabolism. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Cardiovascular diseases, and especially the hypercholesterolemia, are treated based on the synergy of drugs and dietary restrictions. Preferred drugs for treating hypercholesterolemia existing on the market are inhibitors of endogenous synthesis and/or membrane proteins that carry the intestinal absorption of cholesterol. Even with all the medical resources (dietary or pharmacological), the treatments are not fully effective. The human body has several alternative routes which are compensatory, making possible multiple control points. Furthermore, due to side effects and toxicity, there is growing interest in new and powerful natural peptides with similar actions (Boschin, Scigliuolo, Resta, & Arnoldi, 2014a,b; Descamps, De Sutter, Guillaume, & Missault, 2011).

⁎ Corresponding author at: Av. Dr. Arnaldo, 715-2° andar, Cerqueira César, CEP: 01246904, São Paulo, SP, Brazil. Tel.: +55 11 3061 7858. E-mail addresses: [email protected] (M.R. Marques), [email protected] (G.G. Fontanari), [email protected] (D.C. Pimenta), [email protected] (R.M. Soares-Freitas), [email protected] (J.A.G. Arêas).

Food derived peptides released after enzymatic hydrolysis or through fermentation are already known to act favorably on metabolic processes. So far, however, there has been little understanding about how the human body itself is capable of releasing peptides after digestion of food and how these peptides affect biological processes (Ben Khaled et al., 2012; Hsu, 2010; Ramírez et al., 2013; Zhang, Yokoyama, & Zhang, 2012). Cowpea bean seeds have a lot of protein in its composition (≈ 25%), they are widely grown in African countries and they have been explored as grain portfolio in South America and Asia. The protein of cowpea is mostly composed of the globulin fraction (50–70%) with 7S and 11S sedimentation coefficients. The 7S vicilin fraction, also named β-vignina, is considered predominant among globulins. It is consists of two polypeptide chains of 56 and 52 kDa (Derbyshire, Wright, & Boulter, 1976; Falade & Kolawole, 2013; Murray et al., 1983). In previous studies, we reported that protein isolated from the cowpea bean, and other grains such as lupin and amaranth, produced a significant reduction in non-HDL cholesterol in hamsters (Fontanari, Batistuti, da Cruz, Saldiva, & Arêas, 2012; Frota, Mendonca, Saldiva,

http://dx.doi.org/10.1016/j.foodres.2015.04.020 0963-9969/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: Marques, M.R., et al., Proteolytic hydrolysis of cowpea proteins is able to release peptides with hypocholesterolemic activity, Food Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.04.020

2

M.R. Marques et al. / Food Research International xxx (2015) xxx–xxx

Cruz, & Arêas, 2008; Mendonca, Saldiva, Cruz, & Areas, 2009). These effects were observed later in patients with hypercholesterolemia (Frota, Santos-Filho, Ribeiro, & Arêas, 2015). A relationship exists between food peptides and cholesterol metabolism. They can act on the key enzyme of cholesterol synthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase–HMG-CoA reductase) and/or are capable of inhibiting the absorption of dietary cholesterol, inhibiting cholesterol transport proteins or disrupting the cholesterol micelles in the lumen (Dziuba, Iwaniak, & Minkiewicz, 2003; Erdmann, Cheung, & Schroder, 2008; Zhang et al., 2012). This study reports the cholesterol-lowering capacity of distinct peptide fractions from cowpea bean after simulated human digestion and the peptide screening of whole hydrolysate.

2.2. Gastrointestinal digestion simulation

2. Material and methods

2.3. RP-HPLC separation of fractions

The cowpea seeds (Vigna unguiculata L. Walp) BRS-Milênio, were supplied by EMBRAPA (Brazilian Agricultural Research Corporation). The seeds were milled (M 20 universal mill, IKA®, Germany) and sieved (0.42 mm sieve), to generate a cowpea flour.

A liquid chromatograph (Shimadzu—Japan) was used with a LC10AT controller, a pump DGU-14A, coupled to Rheodyne manual injector and detector UV/vis photodiode array SPD-M10A VP. Detection wavelength was set to 220 nm, and spectra of each peak were also recorded in the range of 190–700 nm. The chromatographic separation was carried out on C18 reverse phase column Atlantis 250 × 0.46 mm, particle size 5 μm (Waters—Ireland). The mobile phase gradient was set: solvent A 0.045% trifluoroacetic acid (TFA) in water; solvent B 0.0365% TFA (v/v) in acetonitrile. The sample was eluted with gradient of 5 to 95% buffer B in 55 min, another 5 min to return to the initial conditions and 10 more minutes to stabilize. The flow rate was at 1 mL min−1. The gradient was selected based on the polarity and solubility of different peptides (Swergold & Rubin, 1983; Vijayalakshmi, Lemieux, & Amiot, 1986). The flow cell volume of the detector (10 μL) plus volume of the pipe detector output 750 × 0.5 mm (147 μL) results in delay of 18 s from detection to output to collect manually. Three fractions (peaks) were collected and concentrated and the peptide content was quantified by the method of Lowry (Lowry, Rosebrough, Farr, & Randall, 1951) which is based on chelating the peptide bonds by Cu (II). All three peaks were tested in the biological activities described below.

2.1. Protein preparation Cowpea protein isolate was obtained by conventional methods of alkaline solubilization and isoelectric precipitation of the proteins, as described in a previous study (Frota, Soares, & Arêas, 2008; Frota, Mendonca et al., 2008). The cowpea flour was defatted using hexane (1:6, w/v) during 4 h and dried overnight to evaporate the solvent. The defatted flour was stirred for 2 h at 25 °C in ultrapure water (1:10, w/v), at pH 8.5 by addition of NaOH (1 mol L−1), and was then centrifuged (10.000 × g for 20 min, at 4 °C) to remove insoluble components such as fibers and pigments. The supernatant was then precipitated at pH 4.5 (corresponding to the isoelectric point) by addition of HCl (1 mol L−1) and centrifuged at 10.000 × g for 20 min (4 °C). The insoluble fraction, representing the cowpea protein isolate, was then collected. Targeting improved purity, the protein isolates were defatted again according to Bligh and Dyer method (Bligh & Dyer, 1959). The protein content was analyzed according the method 920.87 from AOAC (Horwitz and Latimer, 2005). To calculate the total nitrogen the conversion factor 6.25 was used. The result of protein content after isolation was about 91.24 ± 0.37% in dry basis.

The protein isolate from cowpea bean was subjected to hydrolysis using an enzyme/substrate ratio of 1:1000 (w/w of protein) in 2% protein solution (w/v) according to Megías et al. (2009) with modifications. This ratio was chosen based on preliminary tests that minimized the presence of peptides from the proteolytic enzymes. Pepsin was used first (37 °C for 2 h, pH = 2) and, subsequently, pancreatin was added to the hydrolysis medium (37 °C for 2 h, pH = 7). The sample was centrifuged (10.000 × g/15 min), the supernatant was collected and filtered through a 3 kDa molecular weight cut-off (MWCO) membrane to isolate the low molecular mass peptides. The peptide mixture from cowpea bean with molecular mass smaller than 3 kDa (named whole hydrolysate) was injected in RP-HPLC for separation.

2.4. In vitro biological activities 2.4.1. Micellar solubility of cholesterol Micellar solubility of cholesterol was measured after adding 35 μg mL−1 of each peptide fraction to a suspension of intestinal

Fig. 1. HPLC profile of cowpea protein hydrolysate and its collected fractions (peaks 1, 2, and 3). The bottom chromatogram is of the pepsin + pancreatin mixture after the same digestion time.


Cholesterol concentration in micelles (% of control)


a b

100

c d 80

60

40

3

2.4.2. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibition assay The HMG-CoA reductase assay kit CS-1090 from Sigma-Aldrich (St. Louis, MO, USA) was used and statin drug—pravastatin—was employed as a positive control. In summary, aliquots containing NADPH (400 μM), HMG-CoA substrate (500 μM) and a buffer pH 7.4 were placed into an UV compatible 96-well plate. The analyses were initiated by the addition of HMG-CoA reductase in each well and incubated in the presence or absence of pravastatin (250 nM), or 50 μg mL−1 peptide concentration of each fraction. The rate of NADPH consumed was monitored by reading the decrease in absorbance at 340 nm. Results were expressed as % of the specific activity of the control enzyme (nmol of NADPH oxidized/min/mg protein) in the absence of drug or peptides.

20

2.5. Peptide screening by “de novo” sequencing 0

micelles prepared in vitro. Micelles were prepared according to the method of Zhang et al. (2012) with modifications (Jesch & Carr, 2006). Sodium phosphate buffer at pH 7.4, containing, taurocholate and NaCl salts, was mixed with cholesterol, linoleic acid and phosphatidylcholine. The supernatant fractions were filtered through a 0.22 μm Millex-GP filter (Millipore, Bedford, MA, USA), and cholesterol concentrations were determined using Amplex® Red Cholesterol Assay Kit. The inhibition ability was calculated using the following equation:

The whole hydrolysate was submitted to a typical proteomic approach. Briefly, previously lyophilized samples were dissolved into 0.5% formic acid (FA) and deposited into the 96 well plate of the SIL-20A auto-sampler for LC-MS analysis in an IT-TOF mass spectrometer system (Shimadzu). Typically, 20 μL sample aliquots were injected and subjected to a binary RP-HPLC (20A Prominence system) separation by a Discovery C18 1.5 (2 × 50 mm) column employing as solvents 0.5% FA (A) and acetonitrile 90%, containing 0.5% FA (B) in a linear gradient of B over A from 0 to 100% in 15 min, under a constant flow rate of 0.2 mL min−1. Instrument control, data acquisition and processing were performed by the LCMS Solution suite (Shimadzu). PEAKS studio 7.0 (Ma et al., 2003) software was used for de novo peptide sequencing as well as proteomic analyses on public databases. All MS/MS assignments were manually revised for correctness as well as the quality of the mass spectra of peptides from nearthreshold identification.

Inhibition ability ð%Þ ¼ ½ðCo ‐ Cs Þ=Co x 100

2.6. Computational prediction

where Co is the cholesterol concentration of original micelles and Cs is the cholesterol concentration after the peptide fraction was added.

The cowpea protein source of the peptides of the whole hydrolysates was confirmed using BLAST® tool. The potential biological activity of the peptides was predicted by using the BIOPEP database (http://

Control

Peak 1

Peak 2

Peak 3

Fig. 2. Effect of peptide fractions on micellar solubility of cholesterol in vitro. The micellar cholesterol concentration at 0 μg mL−1 of peptide is estimated to be 100% (control). Data are mean ± standard deviation. Different letters mean statistical differences for p b 0.05.

Table 1 Peptide sequences screening of the whole hydrolysate and their potential biological activity, physicochemical properties and protein source. Peptidea

Observed molecular ALC score mass/theoretical molecular mass (%) (Da)e

LSEGDL 81 FFGQDGAVVAGSC 77

632.3/632.6 1256.5/1257.3

LLNPDDEQL

73

1055.5/1056.1

MPTTSL GCLTLN GCTLN LLDMKDNKGH

70 63 61 57

648.3/648.7 619.2/619.7 506.2/506.5 1169.5/1170.3

KD LNL LDSLT FFFGQDGGSKGEE

52 51 51 50

261.1/261.2 358.2/358.4 547.2/547.6 1403.5/1404.4

a b c d e

Possible biological activitiesb

Total Iso-electric Net Cowpea protein sourced hydrophobic pointe charge ratio (%)c at pH 7e pH 3.01 pH 3.1

−2 −1

pH 2.87

−3

33.3 50.00 40.00 30.00

pH 6.01 pH 5.33 pH 5.33 pH 7.79

0 0 0 0.1

0 66.66 40.00 23.07

pH 6.75 pH 6.01 pH 3.1 pH 3.83

0 0 −1 −2

ACE inhibitor; stimulating vasoactive substance release 33.3 ACE inhibitor; neuropeptide inhibitor; 53.84 dipeptidyl-aminopeptidase IV inhibitor ACE inhibitor; dipeptidyl-aminopeptidase IV inhibitor; 30.00 Glucose uptake stimulating peptide ACE inhibitor; dipeptidyl-aminopeptidase IV inhibitor; ACE inhibitor ACE inhibitor Glucose uptake stimulating peptide; ACE inhibitor; dipeptidyl-aminopeptidase IV inhibitor; antioxidant. Antioxidant ACE inhibitor None ACE inhibitor; stimulating vasoactive substance release; neuropeptide inhibitor.

Lectin Zeaxanthin epoxidase; glutathione reductase Acetyl-CoA carboxylase carboxyltransferase; phospholipase D alpha 1 Hypothetical chloroplast RF34 Kunitz-type protease inhibitor No significant similarity found Bowman–Birk type proteinase inhibitor No significant similarity found No significant similarity found Lectin Phospholipase D alpha 1; probable non-specific lipid-transfer protein AKCS9

Peptides with PEAKS ALC (average local confidence) score of 50% or greater. Determined using BIOPEP. Calculated the percentage of hydrophobic residues (I, V, L, F, C, M, A, W) in the peptide sequence. Determined using BLAST tool. Determined using Innovagen's peptide property calculator.


4


Table 2 Inhibition of HMG-CoA reductase activity in the presence of the three peptide fractions shown in Fig. 1. Data are mean ± standard deviation. Sample

nmol NADPH oxidized/min/mg peptide

Inhibition (%)

Control Pravastatin (250 nM) Peak 1 Peak 2 Peak 3

617 ± 48 121 ± 19 265 ± 42 309 ± 49 322 ± 31

0.00c 80.4 ± 3.17a 57.1 ± 6.79b 50.0 ± 7.92b 47.8 ± 5.09b

Different super scripted letters in the same column indicate significant difference between means (one-way ANOVA, p b 0.05)

www.uwm.edu.pl/biochemia). The physicochemical properties were predicted using web-based Innovagen's peptide calculator tool (http:// www.innovagen.se/custom-peptide-synthesis/peptide-propertycalculator/peptide-property-calculator.asp) and the prediction of peptide binding sites on protein surfaces was made using PepSite2 beta (http://pepsite2.russelllab.org/) (Petsalaki, Stark, García-Urdiales, & Russell, 2009). 2.7. Statistical analysis Statistical analysis to test the significance of differences (p b 0.05) between conditions was the one-way analysis of variance (ANOVA) through Statistica 11 software for Windows®. 3. Results and discussion Food proteins are decomposed into small fragments during human digestion, resulting peptides and amino acids that are easily absorbed by intestinal mucosa. Cowpea bean protein has been shown to present hypocholesterolemic effect (Frota, Mendonca

et al., 2008; Frota, Santos-Filho, Ribeiro, & Arêas, 2015). Fig. 1 (upper panel) presents the RP-HPLC chromatographic profile of the digested cowpea proteins by physiological proteases. The blank chromatogram, corresponding to the enzymes incubated without substrates, is presented in the lower panel. Peaks 1, 2 and 3 were selected for subsequent biochemical assays. We have investigated their activity in two possible mechanisms to account for the cholesterollowering effect of the parent protein. Fig. 2 shows that the different peaks exhibited different rates of micellar solubility of cholesterol, being Peak 1 the most effective. The significance of hyperlipidemia and hypercholesterolemia suppression due to the insolubilization of cholesterol in the luminal micelles has been emphasized in recent years. This micellization is necessary to facilitate its absorption into intestinal mucosal cells through Niemann–Pick C1-Like 1 (NPC1L1) receptors (Duangjai, Ingkaninan, Praputbut, & Limpeanchob, 2013). Direct evidence documenting the cowpea peptides in the solubility of cholesterol is not available. These findings support our earlier observations which showed that the whole hydrolysate of cowpea (molecular mass smaller than 3 kDa at 1 mg mL−1) had capacity to insolubilize the micellar cholesterol however, it was less effective than separate peaks (≈95% of cholesterol total was maintained into micelles compared to control) (Marques et al., 2015). Our data is the first report showing that micellar solubility of cholesterol is inhibited by specific fractions. The reasons for this interaction are not clear but it may occur a hydrophobic interaction of the peptides with the cholesterol, hindering its solubilization into the micelles. This is in agreement with previous studies performed with other protein hydrolysates, and with the whole hydrolysate of cowpea even after thermal processing (Marques et al., 2015). The hydrophobicity of the cowpea protein can be assessed by its amino acid composition (Chávez-Santoscoy, Gutiérrez-Uribe, & Serna-Saldívar, 2013; Ikeda, Yamahira, Kato, & Ishikawa, 2010; Megias

Fig. 3. Tetramer structure of HMG-CoA reductase enzyme and the predict site of interaction with the peptide GCTLN (A). Predict interaction with S-domain of HMG-CoA reductase monomer C (B). Primary structure of the peptide GCTLN (C).



5

Moreover, Table 1 summarizes that many peptides that can compete with cholesterol take into account its hydrophobicity ratio. Because of this characteristic composition, micellar structures are possibly formed with preferential rearrangement of bile salts around hydrophobic structures, competing with cholesterol. Therefore, more detailed structural studies are necessary to determine how peptides associate with cholesterol and bile acids. The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) is a key enzyme in the human hepatic cholesterol production. For this reason, the knowledge of the rate step of the cholesterol enabled the development of drugs for hypercholesterolemia treatment. Table 2 shows the inhibition of the HMG-CoA reductase activity by the three peptide fractions. Peptides present in all fractions could inhibit significantly HMG-CoA reductase activity, albeit ANOVA test did not show any significant differences between their inhibition rates. Pravastatin, a known inhibitor, used as a positive control, displayed 80% of inhibition. These findings seem to be consistent with others that found peptides derived from food acting as HMG-CoA reductase inhibitors (Pak, Koo, Kasymova, & Kwon, 2005; Pak et al., 2005). Our previous study has demonstrated that the whole hydrolysate was capable to inhibit almost 25% of the velocity of reaction and these posterior findings show that the

et al., 2009). We have previously shown that most of the amino acids from cowpea protein are hydrophobic and neutral (Frota, Soares et al., 2008). De novo peptide sequencing of the active fractions is presented in Table 1 together with the BLAST analyses indicating the probable origin of the peptides, as well as the computational prediction that may help to explain the capacity to insolubilize the cholesterol. The protein source of the cowpea peptides LLNPDDEQL and FFFGQDGGSKGEE, phospholipase D alpha 1 is responsible for the enzymatic catalysis of phosphatidylcholine. For cellular lipid droplets, phosphatidylcholine is preferentially adsorbed at the interface of intestinal micelles due to their amphiphilic property acting such surfactant. They decrease surface tension and provide high bending elasticity increasing micellar emulsion stability with cholesterol. There are different types of forces (Van der Waals, entropic or electrostatic interactions) that are mediated by biochemical or hydrodynamic parameters that it has been suggested as causal interaction between peptides and phosphatidylcholine. Anyway as a result of these interactions, micelles can destabilize by alterations on pressure differences inside and outside of the molecule (de Souza Rocha, Hernandez, Chang, & de Mejía, 2014; Thiam, Farese, & Walther, 2013). Hence, it could conceivably be hypothesized that these conserved domains may interact with the phosphatidylcholine added in the reaction.

Inten. (x100,000) 507.2502

A

3.0 2.5 2.0 1.5

550.8050

1.0

585.3127 590.6162

457.2475 317.6633

0.5

352.3466

279.1531 0.0 275.0

300.0

325.0

350.0

495.2428

414.2433 390.5275 375.0

400.0

425.0

450.0

475.0

500.0

658.3265 634.3312 672.2998 525.0

550.0

575.0

600.0

625.0

650.0

675.0

730.8901 711.8524 700.0

725.0

750.0

775.0 m/z

Inten. (x100,000) 507.2502

B

3.0 2.5 2.0 1.5 1.0

508.2643

0.5 0.0 504.5

505.0

505.5

506.0

506.5

507.0

507.5

508.0

508.5

509.0

509.5

510.0

510.5

m/z

Inten. (x100,000) 347.2035

3.5

y3

3.0

b3

2.5

C b4

262.1149

375.1992

2.0 1.5 1.0

CT-28 CT TL

0.5

177.1095 205.0950

y3-H2O

y2 246.1498

472.2297

303.1647

0.0 50.0

75.0

100.0

125.0

150.0

175.0

200.0

225.0

250.0

275.0

300.0

325.0

350.0

375.0

400.0

425.0

450.0

475.0

500.0 525.0 m/z

Fig. 4. MS profile of retention time 15.46′ showing the measured m/z values (A). Zoomed m/z 507.25 indicating the single charged state (B). Annotated MS2 profile of the fragmentation of m/z 507.25 (C).


6


separate peaks were more effective at same conditions (Marques et al., 2015). This inhibition can be explained by interaction of these peptides with the catalytic domain of the enzyme. Moreover, the reaction also depends on NADPH cofactor binding. HMG-CoA reductase has a second domain that is capable of accepting NADPH, and these peptides can also prevent its binding, lowering the reaction velocity (Istvan & Deisenhofer, 2001). In general, enzyme binding sites are formed by hydrophobic amino acids located deeper inside the molecule. Therefore, cowpea hydrophobic peptides may be internalized and thus occupy both domains (Istvan & Deisenhofer, 2001). A web-based simulation of peptide–enzyme interaction was done with all peptides up to 10 residues to test the theory above. The only peptide able to significantly bind the enzyme HMG-CoA-reductase was GCTLN (p = 0.07605) and the binding occurs upon S-domain of the monomer C near the active site of the enzyme (Fig. 3). Fig. 4 provides MS profile of de novo sequencing of peptide GCTLN. These findings do not rule out other possibilities. The peptide FFGQDGAVVAGSC (Table 1) is part of a conserved domain of zeaxanthin epoxidase and glutathione reductase from cowpea. One of the issues that emerge from these findings is that both enzymes degrade NADPH. Therefore, the HMG-CoA reductase inhibition may be caused by the interaction of the peptide with the cofactor, preventing them from binding. Those mechanisms will be cleared out by kinetic studies of the observed inhibition, as well as isolation of the exact peptide sequences responsible for each of these results. 4. Conclusions This is the first report of cholesterol micellar solubilization and HMG-CoA-reductase inhibition by separated peptide fractions of cowpea bean protein. Moreover, the active peptides could be screened for their biological activity and were de novo sequenced from the total hydrolysate, which simulated the human digestion. The peptide conserved domains may interact with the phosphatidylcholine added in the reaction of the cholesterol micelles. The peptide GCTLN could significantly bind the HMG-CoA reductase and to inhibit the enzyme by changes on its active site. These mechanisms can be the responsible for the cowpea protein cholesterol-lowering effect. The purification of these peptides and the confirmation of these effects by specific sequences may help in the effort to produce alternative ways to combat hypercholesterolemia. Acknowledgments The authors are grateful to FAPESP (Foundation for Research Support of the State of São Paulo, Brazil), grants 2011/04179-0 and 2012/159004, and FINEP (grant 01.09.0278.04, DCP). The authors wish to thank Ms Amanda Caroline C. C. Carlos for collaboration on hydrolysis. The authors and sponsor declare no conflict of interest whatsoever. References Ben Khaled, H., Ghlissi, Z., Chtourou, Y., Hakim, A., Ktari, N., Fatma, M.A., et al. (2012). Effect of protein hydrolysates from sardinelle (Sardinella aurita) on the oxidative status and blood lipid profile of cholesterol-fed rats. Food Research International, 45(1), 60–68. Bligh, E.G., & Dyer, W.J. (1959). A rapid method of total lipid extraction and purification. [Article]. Canadian Journal of Biochemistry and Physiology, 37(8), 911–917. Boschin, G., Scigliuolo, G.M., Resta, D., & Arnoldi, A. (2014a). ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes. Food Chemistry, 145, 34–40. Boschin, G., Scigliuolo, G.M., Resta, D., & Arnoldi, A. (2014b). Optimization of the enzymatic hydrolysis of lupin (lupinus) proteins for producing ACE-inhibitory peptides. Journal of Agricultural and Food Chemistry, 62(8), 1846–1851. Chávez-Santoscoy, R., Gutiérrez-Uribe, J., & Serna-Saldívar, S. (2013). Effect of flavonoids and saponins extracted from black bean (Phaseolus vulgaris L.) seed coats as cholesterol micelle disruptors. Plant Foods for Human Nutrition, 68(4), 416–423. de Souza Rocha, T., Hernandez, L.M.R., Chang, Y.K., & de Mejía, E.G. (2014). Impact of germination and enzymatic hydrolysis of cowpea bean (Vigna unguiculata) on the generation of peptides capable of inhibiting dipeptidyl peptidase IV. Food Research International, 64, 799–809.

Derbyshire, E., Wright, D.J., & Boulter, D. (1976). Legumin and vicilin, storage proteins of legume, seeds15(1), 3–24. Descamps, O.S., De Sutter, J., Guillaume, M., & Missault, L. (2011). Where does the interplay between cholesterol absorption and synthesis in the context of statin and/or ezetimibe treatment stand today? Atherosclerosis, 217(2), 308–321. Duangjai, A., Ingkaninan, K., Praputbut, S., & Limpeanchob, N. (2013). Black pepper and piperine reduce cholesterol uptake and enhance translocation of cholesterol transporter proteins. Journal of Natural Medicines, 67(2), 303–310. Dziuba, J., Iwaniak, A., & Minkiewicz, P. (2003). Computer-aided characteristics of proteins as potential precursors of bioactive peptides. [Article]. Polimery, 48(1), 50–53. Erdmann, K., Cheung, B.W.Y., & Schroder, H. (2008). The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. Journal of Nutritional Biochemistry, 19(10), 643–654. Falade, K.O., & Kolawole, T.A. (2013). Effect of irradiation dose on physical, functional and pasting properties of cowpea (Vigna unguiculata L. Walp) cultivars. Journal of Food Process Engineering, 36(2), 147–159. Fontanari, G.G., Batistuti, J.P., da Cruz, R.J., Saldiva, P.H.N., & Arêas, J.A.G. (2012). Cholesterol-lowering effect of whole lupin (Lupinus albus) seed and its protein isolate. Food Chemistry, 132(3), 1521–1526. Frota, K.M.G., Mendonca, S., Saldiva, P.H.N., Cruz, R.J., & Arêas, J.A.G. (2008a). Cholesterollowering properties of whole cowpea seed and its protein isolate in hamsters. Journal of Food Science, 73(9), H235–H240. Frota, K.M.G., Santos-Filho, R.D., Ribeiro, V.Q., & Arêas, J.A.G. (2015). Cowpea protein reduces LDL-cholesterol and apolipoprotein B concentrations but does not improve inflammatory and endothelial dysfunction biomarkers in adults with moderate hypercholesterolemia. Nutrición Hospitalaria, 31(4), 1611–1619, http://dx.doi.org/10. 3305/nh.2015.31.4.8457 Frota, K.M.G., Soares, R.A.M., & Arêas, J.A.G. (2008b). Composição química do feijão caupi (Vigna unguiculata L. Walp), cultivar BRS-Milênio. Ciência e Tecnologia de Alimentos (Campinas), 28, 470–476, http://dx.doi.org/10.1590/S0101-20612008000200031 Horwitz, W., & Latimer, G.W.E., Jr (2005). Official methods of analysis of the Association of Official Analytical Chemists. Association of Official Analytical Chemists, 18. Hsu, K. -C. (2010). Purification of antioxidative peptides prepared from enzymatic hydrolysates of tuna dark muscle by-product. Food Chemistry, 122(1), 42–48. Ikeda, I., Yamahira, T., Kato, M., & Ishikawa, A. (2010). Black-tea polyphenols decrease micellar solubility of cholesterol in vitro and intestinal absorption of cholesterol in rats. Journal of Agricultural and Food Chemistry, 58(15), 8591–8595. Istvan, E.S., & Deisenhofer, J. (2001). Structural mechanism for statin inhibition of HMGCoA reductase. [Article]. Science, 292(5519), 1160–1164, http://dx.doi.org/10.1126/ science.1059344 Jesch, E.D., & Carr, T.P. (2006). Sitosterol reduces micellar cholesterol solubility in model bile. Nutrition Research, 26(11), 579–584. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193(1), 265–275. Ma, B., Zhang, K., Hendrie, C., Liang, C., Li, M., Doherty-Kirby, A., et al. (2003). PEAKS: Powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 17(20), 2337–2342. Marques, M.R., Soares Freitas, R.A.M., Corrêa Carlos, A.C., Siguemoto, É. S., Fontanari, G.G., & Arêas, J.A.G. (2015). Peptides from cowpea present antioxidant activity, inhibit cholesterol synthesis and its solubilisation into micelles. Food Chemistry, 168, 288–293. Megias, C., Pedroche, J., del Mar Yust, M., Alaiz, M., Giron-Calle, J., Millan, F., et al. (2009). Sunflower protein hydrolysates reduce cholesterol micellar solubility. Plant Foods for Human Nutrition, 64(2), 86–93. Megías, C., Pedroche, J., Yust, M., & d. M., Alaiz, M., Girón-Calle, J., Millán, F., & Vioque, J. (2009). Stability of sunflower protein hydrolysates in simulated gastric and intestinal fluids and Caco-2 cell extracts. Food Science and Technology, 42(9), 1496–1500. Mendonca, S., Saldiva, P.H., Cruz, R.J., & Areas, J.A.G. (2009). Amaranth protein presents cholesterol-lowering effect. Food Chemistry, 116(3), 738–742. Murray, D.R., Mackenzie, K.F., Vairinhos, F., Peoples, M.B., Atkins, C.A., & Pate, J.S. (1983). Electrophoretic studies of the seed proteins of cowpea, Vigna unguiculata (L.) walp109(4), 363–370. Pak, V.V., Koo, M.S., Kasymova, T.D., & Kwon, D.Y. (2005a). Isolation and identification of peptides from soy 11S-globulin with hypocholesterolemic activity. [Article]. Chemistry of Natural Compounds, 41(6), 710–714. Pak, V.V., Koo, M., Lee, N., Oh, S.K., Kim, M.S., Lee, J.S., et al. (2005b). Hypocholesterolemic soybean peptide (IAVP) inhibits HMG-CoA reductase in a competitive manner. [Article]. Food Science and Biotechnology, 14(6), 727–731. Petsalaki, E., Stark, A., García-Urdiales, E., & Russell, R.B. (2009). Accurate prediction of peptide binding sites on protein surfaces. PLoS Computational Biology, 5(3), e1000335. Ramírez, C.M., Rotllan, N., Vlassov, A.V., Dávalos, A., Li, M., Goedeke, L., et al. (2013). Control of cholesterol metabolism and plasma high-density lipoprotein levels by microRNA-144. Circulation Research, 112(12), 1592–1601. Swergold, G.D., & Rubin, C.S. (1983). High-performance gel-permeation chromatography of polypeptides in a volatile solvent—rapid resolution and molecular-weight estimations of proteins and peptides on a column of TSK-G3000-PW. Analytical Biochemistry, 131(2), 295–300. Thiam, A.R., Farese, R.V., Jr., & Walther, T.C. (2013). The biophysics and cell biology of lipid droplets. Nature Reviews Molecular Cell Biology, 14(12), 775–786, http://dx.doi.org/10. 1038/nrm3699 Vijayalakshmi, M.A., Lemieux, L., & Amiot, J. (1986). High-performance size exclusion liquid-chromatography of small molecular-weight peptides from protein hydrolysates using methanol as a mobile phase additive. Journal of Liquid Chromatography, 9(16), 3559–3576. Zhang, H., Yokoyama, W.H., & Zhang, H. (2012). Concentration-dependent displacement of cholesterol in micelles by hydrophobic rice bran protein hydrolysates. Journal of the Science of Food and Agriculture, 92(7), 1395–1401.

.

.

.

.
