Black bean anthocyanin-rich extracts as food colorants: Physicochemical stability and antidiabetes potential

Accepted Manuscript Black bean anthocyanin-rich extracts as food colorants: physicochemical stability and antidiabetes potential Luis Mojica, Mark Berhow, Elvira Gonzalez de Mejia PII: DOI: Reference:

S0308-8146(17)30336-9 http://dx.doi.org/10.1016/j.foodchem.2017.02.124 FOCH 20677

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

26 November 2016 22 February 2017 25 February 2017

Please cite this article as: Mojica, L., Berhow, M., Gonzalez de Mejia, E., Black bean anthocyanin-rich extracts as food colorants: physicochemical stability and antidiabetes potential, Food Chemistry (2017), doi: http://dx.doi.org/ 10.1016/j.foodchem.2017.02.124

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Black bean anthocyanin-rich extracts as food colorants: physicochemical stability and antidiabetes potential

Luis Mojicaab, Mark Berhowc and Elvira Gonzalez de Mejiaa*

a

Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, IL, 61801, United States.

Tecnología Alimentaria, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A. C., CIATEJ, 44270 Guadalajara, México. b

c

United States Department of Agriculture, Agricultural Research Service, 1815 North University Street, Peoria, IL 61604, United States.

* E-mail: [email protected] Tel: 217-244-3196 Fax:+1-217-265–0925:

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Running title: Optimized black bean anthocyanins stability and antidiabetes potential

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ABSTRACT:

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Black beans contain anthocyanins that could be used as colorants in foods with associated health

13

benefits. The objective was to optimize anthocyanins extraction from black bean coats and

14

evaluate their physicochemical stability and antidiabetes potential. Optimal extraction conditions

15

were 24% ethanol, 1:40 solid-to-liquid ratio and 29 ˚C (P < 0.0001). Three anthocyanins were

16

identified by MS ions, delphinidin-3-O-glucoside (465.1 m/z), petunidin-3-O-glucoside (479.1

17

m/z) and malvidin-3-O-glucoside (493.1 m/z). A total of 32 mg of anthocyanins were quantified

18

per gram of dry extract. Bean anthocyanins were stable at pH 2.5 and low-temperature 4˚C

19

(89.6%), with an extrapolated half-life of 277 days. Anthocyanin-rich extracts inhibited α-

20

glucosidase (37.8%), α-amylase (35.6%), dipeptidyl peptidase-IV (34.4%), reactive oxygen

21

species (81.6%), and decreased glucose uptake. Black bean coats are a good source of

22

anthocyanins and other phenolics with the potential to be used as natural-source food colorants

23

with exceptional antidiabetes potential.

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KEYWORDS: anthocyanins; antidiabetes potential; black bean coats; extraction optimization;

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natural pigments; ROS inhibition

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CHEMICAL COMPOUNDS: Acarbose (PubChem CID: 41774); DCFDA (PubChem CID:

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6711158); delphinidin-3-O-glucoside (PubChem CID: 443650); malvidin-3-O-glucoside

30

(PubChem CID: 443652); 2-NBDG (PubChem CID: 163790); petunidin-3-O-glucoside

31

(PubChem CID: 176449); phloretin (PubChem CID: 4788); sitagliptin (PubChem CID: 4369359).

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1.0 INTRODUCTION

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Anthocyanins represent the largest group of phenolic pigments and the most important group

36

of water-soluble pigments in plants, responsible for colors in fruits, vegetables, cereal grains, and

37

flowers (Shipp & Adbel-Aal, 2010). They are formed by two or three chemical units: an aglycon

38

base or flavylium ring (anthocyanidin), sugars, and possibly acylation groups. Cyanidin,

39

delphinidin, petunidin, peonidin, pelargonidin, and malvidin are the most frequently occurring

40

anthocyanidins, which may be glycosylated or acylated by different sugars and aromatic or

41

aliphatic acids on their aglycon unit to yield anthocyanins in the plant (Bueno, Sáez-Plaza,

42

Ramos-Escudero, Jiménez, Fett, & Asuero, 2012).

43

Anthocyanins are very unstable and susceptible to degradation. Its color stability is affected

44

by pH, their chemical structure, concentration, storage temperature, light, oxygen, and the

45

presence of enzymes, flavonoids, proteins and metal ions (Hernandez-Herrero & Frutos, 2014;

46

Castaneda-Ovando, Pacheco-Hernandez, Paez-Hernandez, Rodrıguez, & Galan-Vidal, 2009).

47

Anthocyanins are usually stable at pH 1 to 4 and degrade above pH 7. At pH 1, the predominant

48

structure corresponds to the flavylium cation, conferring red and purple colors, whereas, at

49

values between pH 2 and 4, blue quinoid bases predominate. Some of the ways to optimize

50

anthocyanin stability during storage are to increase anthocyanin concentration, remove oxygen

51

and inactivate enzymes (Castaneda-Ovando, Pacheco-Hernandez, Paez-Hernandez, Rodrıguez,

52

& Galan-Vidal, 2009).

53

Anthocyanins are usually located in the seed coat of common beans. In previous studies, we

54

found that Negro-Otomi cultivar (black bean), had the highest anthocyanin concentration (2.5

55

mg/g coat) (Mojica, Meyer, Berhow, & de Mejía, 2015) among other 14 common bean cultivars.

56

Anthocyanins may provide anti-inflammatory and antidiabetes benefits since they inhibit pro-

3

57

inflammatory cytokines, decrease their production, and prevent β-cell dysfunction (de Mejia, &

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Johnson, 2013). The potential antidiabetes mechanism of action of anthocyanins and other

59

polyphenols from berries or other food sources can be classified into two groups: insulin-

60

dependent and insulin-independent. The insulin-dependent mechanism involves the improvement

61

of pancreatic β-cell function (reducing oxidative stress, increasing insulin production, reducing

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β-cell apoptosis and promoting β-cell proliferation), and enhancing tissue sensitivity (changes in

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peripheral tissue in inflammation and oxidative stress). On the other hand, the insulin-

64

independent mechanism involves the blockage of starch degrading enzymes and the reduction in

65

glucose absorption (inhibition of α-glucosidase, α-amylase, and glucose transporters SGLT1 and

66

GLUT2); and changes in energy metabolism status (AMP-activated protein kinase) (Edirisinghe

67

& Burton-Freeman, 2016; Castro-Acosta, Lenihan-Geels, Corpe, & Hall, 2016). Furthermore,

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anthocyanins have a wide range of health benefits for the human body such as antioxidant,

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anticancer, anti-cardiovascular disease, and hepatoprotective activity (Hu, Zheng, Li, & Suo,

70

2014).

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Consumers may have a preference towards natural pigments versus synthetic colorants due to

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their perception of being a healthier and safer option. Besides, anthocyanins exert a wide range

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of colors and hydro-solubility, making them an important alternative as a food pigment. Also,

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these anthocyanins could promote important health benefits when consumed. Therefore, the

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objective of this study was to optimize the extraction conditions of anthocyanins from black bean

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coats, evaluate their shelf-life and thermal stability at different pHs and temperatures and

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evaluate their antidiabetes potential.

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2.0. MATERIALS AND METHODS

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2.1. Materials

82

Black bean (Phaseolus vulgaris L.) “Negro Otomi” cultivar was obtained from INIFAP

83

research center in Mexico. The 7-up cherry beverage (Dr Pepper Snapple Group) contained

84

artificial flavors and red 40 among other ingredients. Chemicals used for extraction were all ACS

85

grade and purchased from Sigma-Aldrich (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA).

86

All solvents for chromatographic techniques were of HPLC-grade. For sample preparation, five

87

kg of black beans were soaked in drinking potable water (1:2 beans/water ratio), at room

88

temperature for 16 h; the hulls (coats) were manually removed from cotyledons and dried at 50ºC

89

in a conventional oven, ground in a commercial blender, sieved in mesh 40 (Advantech, USA

90

standard testing sieve), mean particle size 0.420 mm and stored in a double plastic bag at 4ºC

91

until analysis (not more than one month). The yield obtained was 100 g of coats per kg of

92

processed beans. The cotyledons were used for the production of bioactive peptides (Mojica,

93

Gonzalez de Mejia, Granados-Silvestre, & Menjivar, 2017). Enzymes human dipeptidyl

94

peptidase IV (EC 3.4.14.5), α-glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20), α-

95

amylase (EC 3.2.1.1), acarbose, phloretin, and sitagliptin were purchased from Sigma-Aldrich

96

(St. Louis, MO). DPPIV-GLO® protease assay kit was purchased from Promega (Madison, WI).

97

2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) was purchased

98

from ThermoFisher (Carlsbad, CA). Human colon epithelial cells Caco-2 [Caco2]

99

(ATCC®HTB-37], Eagle’s Minimum Essential Medium (EMEM), and 0.25% (w/v) trypsin-0.53

100

mM EDTA were purchased from American Type Culture Collection (ATCC) (Manassas, VA,

101

USA). Penicillin-streptomycin was purchased from Corning Inc. (Corning, NY, USA). Fetal

5

102

bovine serum (FBS) was purchased from Hyclone (Thermo Scientific Hyclone, Logan, UT,

103

USA).

104 105

2.2. Physicochemical stability

106

2.2.1 Optimization of extraction of anthocyanins from bean coat by response surface

107

methodology (RSM)

108

Anthocyanins and total polyphenols were extracted from bean coats using either only water

109

or two different concentrations of ethanol (0, 12.5 and 25%) in acidified water with 2% formic

110

acid (pH = 2.0). Extractions were performed by stirring coat beans and the respective solution at

111

600 rpm for two h at different temperatures (4, 22 and 40 °C), and different solid-to-liquid ratios

112

(1:30, 1:40 and 1:50). After extraction, the mixtures were filtered using Whatman No. 1 filter

113

paper. All extracts were immediately analyzed for total monomeric anthocyanins, color, and total

114

polyphenols.

115

Response surface methodology was used to optimize the extraction of anthocyanin and

116

polyphenols by using functional relationships between the dependent variable and the

117

independent variables as previously reported (Khuri, & Mukhopadhyay, 2010). Factorial 3^3

118

experimental design was used with three independent randomized replications. Ethanol

119

concentration (x1), solid-to-liquid- ratio (x2) and extraction temperature (x3) were chosen for

120

independent variables. The range and center point values with actual and coded values of

121

variables used for the optimization of anthocyanins and total polyphenols extraction from black

122

bean coat were coded levels -1, 0, +1, for ethanol concentration (x1, %), 0, 12.5 and 25; for

123

solid-to-liquid ratio (x2, mL/g), 1:30, 1:40 and 1:50; and for extraction temperature (x3, °C), 4,

124

22 and 40. Anthocyanin concentration and total polyphenols were selected as the responses for

6

125

the combination of the independent variables as presented in Table 1. The variables were coded

126

according to the following equation:
=


 −
 ∆


127

Where x is the coded value; xi, the corresponding actual value; x0, the actual value at the center

128

of the domain; and ∆x, the increment of xi corresponding to a variation of 1 unit of x. The

129

polynomial second-degree equation is described below:



 

 





 =  +  
 +  
 + +   

    

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2.2.2 Anthocyanins extraction and measurement of total anthocyanin concentration

132

Anthocyanin-rich extracts (AE) were obtained from black bean coats using the parameters

133

found from the optimization analysis previously mentioned. Three consecutive extractions were

134

performed to evaluate the percent recovery of anthocyanins and polyphenols after extraction with

135

24% ethanol in acidified water (2% formic acid), 1:40 solid-to-liquid ratio and 29˚C during two h.

136

Ethanol was removed using a rotary vacuum evaporator at 40˚C. Anthocyanin extracts were

137

freeze-dried in a Labconco Freeze Dryer 4.5 (Kansas, MO). The obtained powder was recovered

138

and stored at -20˚C in capped 50 ml falconTM tubes surrounded with parafilm until analysis.

139

Total monomeric anthocyanins were determined by the pH differential method as previously

140

reported (Lee, Durst, & Wrolstad, 2008). Samples were diluted to a factor of 1:5 using two

141

buffers (pH 1.0, 0.25 M KCl buffer and pH 4.5, 0.40 M sodium acetate buffer). Two hundred

142

microliters of diluted solutions at each pH were transferred to a 96-well plate, and the absorbance

143

was read at 520 and 700 nm using a Synergy 2 multiwell plate reader (Biotek, Winooski, VT).

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144

The total monomeric anthocyanin concentration was calculated as cyanidin-3-O-glucoside (C3G)

145

equivalents per L as below:

146

Total monomeric anthocyanins (mg/L) =  ××× × 0.45  × 

147

Where: A = (A520 – A700) at pH1.0 – (A520 – A700) at pH4.5; MW = 449.2 143 g/moL for

148

C3G; D = dilution factor; PL = constant path length 1 cm; ε = 26900 L/moL-cm the molar

149

extinction coefficient for C3G, 1000= conversion factor from grams to milligrams and 0.45=

150

conversion factor from the established method to the plate reader method. Final concentrations

151

were expressed as mg C3G equivalents per g of dry weight, DW.

152 153

2.2.3 Measurement of total polyphenol concentration

154

Total polyphenols were measured using the Folin-Ciocalteu method adapted to a microassay

155

(Heck, Schmalko, & de Mejia. 2008). Samples were diluted to a factor of 1:10 with deionized

156

water. Fifty microliters of these diluted samples, standard or blank (deionized water) were placed

157

in a 96-well plate and then added with 50 µL of 1N Folin-Ciocalteu’s phenol reagent. After 5

158

min, 100 µL of 20% Na2CO3 were added and the mixture was incubated for 10 min. The

159

absorbance was read at 690 nm using a Synergy multiwell plate reader (Biotek, Winooski, VT)

160

and the results were expressed as mg gallic acid equivalents (GAE) per g of DW.

161 162

2.2.4. Analysis of anthocyanins by LC-ESI-MS and HPLC

163

Sample treatment for HPLC and LC-ESI-MS analysis was performed using a standardized

164

method reported by Berhow (2002). Between 0.05 and 0.1 g of coats were placed in a capped

165

vial with 2–5 mL of methanol (100%). The vials were sonicated for 15 min, and allowed to stand

166

overnight. After another brief sonication, a portion of this extract was filtered through a 0.45 µm

8

167

filter into an auto sampler vial. Samples were extracted in three independent replicates.

168

Anthocyanin solutions were run on a Thermo Electron LTQ Orbitrap Discovery Mass

169

Spectrometer -- a linear ion trap (LTQ XL) MS, coupled to a high precision electrostatic ion trap

170

(Orbitrap) MS with a high energy collision (HCD) cell -- with an Ion Max electrospray

171

ionization (ESI) source, and a Thermo Scientific ACCELA series HPLC system (ACCELA 1250

172

UHPLC pump, ACCELA1 HTC cool stack autoinjector, and a ACCELA 80 Hz PDA detector);

173

all running under Thermo Scientific Xcalibur 2.1.0.1140 LC-MS software. As reported

174

previously by Mojica et al. (2015) standard curves were prepared on ranges 1 to 40 nanomoles

175

from pure standards. Extinction coefficients were calculated from a linear regression formula

176

based on four different nanomolar concentrations of anthocyanins aglycone standards

177

(ChromaDex®, Irvine, CA) injected and determined their respective mAbs areas. The molar

178

extinction coefficient of delphinidin (2.1 x 10-7 L mol−1 cm−1), malvidin (1.7 x 10-7 L mol−1

179

cm−1) and petunidin (7.3 x 10-7 L mol−1 cm−1) were used to quantify anthocyanin glycoside

180

concentrations by the following formula:

181

µg/mg or mg/g = mAbs (area) * extinction coefficient (nM/mAbs) / injection volume (µL) *

182

total volume of extract (µL) * MW of anthocyanins glucoside (µg/nM)/ sample weight (mg)

183 184

Different columns, aqueous mobile phases and gradients were used for LC-MS analysis

185

compared to the method used for the quantification. A smaller column was needed on the LC-

186

MS system due to lower flow rates required and used in an optimal MS system to get the best

187

MS response. For the identification of compounds, the column was a 3 mm × 150 mm Inertsil

188

reverse phase C-18, ODS 3, 3 µm column (Metachem, Torrance, CA). For anthocyanins, the MS

189

was typically run with the ESI probe in the positive mode. The initial column conditions were 5%

9

190

methanol and 0.2% acetic acid in water, at a flow rate of 0.25 mL per min. The eluate was

191

monitored at 520 nm on the PDA. After a delay of 2 min, the column was developed to 100%

192

methanol with a linear gradient over 60 min. For the quantification, the column used was an

193

Inertsil ODS-3 reverse phase C-18 column (5 µ, 250 × 4.6mm from Varian). The initial column

194

conditions were 2% acetonitrile and 0.5% acetic acid in water, at a flow rate of 1 ml per min. The

195

eluate was monitored at 520 nm. After injection, the column was held at the initial conditions for

196

2 min, and then developed to 100% acetonitrile in a linear gradient over 60 min. Once the

197

identification was completed, standards were used on the analytical system to determine both

198

retention time and quantitation (Mojica, Meyer, Berhow & de Mejia, 2015).

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2.2.5 Color measurements

201

The color was measured using a Color flex Hunter Lab Colorimeter (Reston, VA). The

202

instrument was calibrated as the manufacturer recommended and the following parameters were

203

used: L*, a*, and b*; observer/illuminant: 10° and D65 and path length: 1 cm. Briefly, three mL

204

of extracts were placed in a disposable Petri dish and the color parameters L*, a* and b*

205

measured and recorded as indicated by the International Commission on Illumination (CIE),

206

brightness (L*), redness (+a*), greenness (−a*), yellowness (+b*), and blueness (−b*). Color

207

squares were generated by converting L*, a* and b* values to R, G and B values using the color

208

converter website (http://colormine.org/ convert/rgb-to-lab) and Microsoft PowerPoint Software.

209

Color parameters Chroma (C*), Hue (h˚) and ∆E* were calculated using the L*, a*, and b*

210

values. Chroma (C* = [(a*)2 + (b*)2]1/2 indicates color purity or saturation (high values are

211

more vivid) and hue angle (H° = arctan (b*/a*) indicates sample color, they were additionally

10

212

calculated (Anton, Ross, Beta, Gary Fulcher & Arntfield, 2008). Color difference was calculated

213

as ∆E = [(∆L*)2 + (∆a*)2 +(∆b*)2]1/2.

214 215

2.2.6. Shelf-life studies

216

Extracted anthocyanins were stored in the dark for five weeks to assess degradation kinetics.

217

The experiment was performed using different pHs found in different commercially available

218

beverages such as pH 2.5 (soda), 3.0 (sparkling flavored water), 3.5 (energy drink) and 4.3 (iced

219

tea), and two storage temperatures, refrigeration (4˚C) and room temperature (22˚C).

220

evaluated parameters were anthocyanin concentration and variation of a* color parameter.

The

221 222

2.2.7. Anthocyanins and color shelf-life stability and degradation kinetics study

223

Anthocyanin solutions were prepared using 1 mg/mL of the anthocyanin dry powder extract.

224

Solutions were prepared under aseptic conditions, stirred until dissolved, filtered and then added

225

into sterile 15 mL falconTM tubes (three independent tubes were prepared for each condition of

226

time, temperature and pH). The pHs were adjusted according to the pH in commercial beverages

227

using 2% formic acid. Anthocyanin and color were measured at time zero, and the independent

228

aliquots were stored for five weeks at 4 and 22°C to be evaluated every week.

229 230

2.2.8. Anthocyanin and color thermal stability study

231

Anthocyanin solutions at different pHs (2.5, 3.0, 3.5, and 4.3) were exposed to 70, 80 and

232

90 °C in a water bath for five h and sampled each hour. After exposure at specific temperature

233

and time conditions, samples were removed from the water bath and placed in an ice bath to

11

234

minimize further degradation. These conditions were selected based on shelf-life accelerated

235

storage studies (Kirca, & Cemeroglu, 2003).

236 237

2.2.9 Reaction kinetics and Arrhenius model

238

Shelf-life and thermal stability data on anthocyanin concentration and individual

239

anthocyanins were plotted using the first order reaction rate kinetics using the following equation:

240

ln At = ln A0 – kt

241

where At is the total monomeric anthocyanin or anthocyanin abundance at time t, A0 is the total

242

monomeric anthocyanin or anthocyanin abundance at time zero; k is the reaction rate constant in

243

h-1 and t is the time of heating in hours.

244

Activation energy for total monomeric anthocyanin and individual anthocyanins was calculated

245

using the Arrhenius equation: ln ! = ln " −

#$ 1 ( ) % (

246

where k is the reaction rate constant, A is the Arrhenius pre-exponential factor, Ea is the

247

activation energy (kJ/moL), R is the gas constant (8.314 J/moL-K), and T is the temperature in K.

248 249

2.3. Biological activity

250

2.3.1. α-Glucosidase inhibition biochemical assay

251

For the α-glucosidase assay, in a 96-well plate, either 50 µL of anthocyanin solutions (1 mg

252

AE/mL in 0.1 M sodium phosphate buffer, PBS, pH 6.9), or purified anthocyanins (100 µM

253

malvidin, 100 µM delphinidin) or (1 mM acarbose) positive control were added to 100 µL of 1

254

U/mL α-glucosidase solution (0.1 M PBS pH 6.9) and incubated for 10 min. A 50 µL aliquot of a

255

5 mM p-nitrophenyl-α-D-glucopyranoside solution (0.1 M PBS pH 6.9) was added to each well, 12

256

incubated at 25°C for 5 min and absorbance read at 405 nm (Johnson, Lucius, Meyer & de Mejia,

257

2011). Results were presented as percent inhibition per mg/mL of AE, or per 100 µM of

258

malvidin, or per 100 µM of delphinidin.

259 260

2.3.2. α-Amylase inhibition biochemical assay

261

For the α-amylase assay, 500 µL of anthocyanin solutions (1 mg AE/mL buffer), purified

262

anthocyanins (100 µM malvidin, 100 µM delphinidin), or (1 mM acarbose) positive control

263

were added to 500 µL of 13 U/mL α-amylase solution (type VI-B from porcine pancreas in 0.02

264

M sodium phosphate buffer pH 6.9) and incubated in test tubes at 25°C for 10 min before 500 µL

265

of 1% soluble starch solution (previously dissolved in sodium phosphate buffer and boiled for 15

266

min) was added to each tube and incubated for another 10 min. Finally, one mL of dinitro

267

salicylic acid reagent was added, and the tubes were placed in 100 °C water bath for 5 min. The

268

mixture was diluted with ten mL of distilled water and absorbance read at 520 nm. Results were

269

presented as percent inhibition per mg/mL of AE, per 100 µM of malvidin, or per 100 µM of

270

delphinidin.

271 272

2.3.3. Dipeptidyl peptidase IV (DPP-IV) inhibition biochemical assay

273

DPP-IV inhibition was measured using the DPP-IVGLO® Protease Assay (G8351, Promega,

274

Madison, WI). A 50 µL of DPP-IVGLO® reagent was added to a white-walled 96-well plate

275

containing either 50 µL of blank, 40 µL enzyme control or 40 µL anthocyanin solutions.

276

Anthocyanin solutions were prepared in buffer (100 mM Tris, pH 8.0, 200 mM NaCl, 1 mM

277

EDTA) at a concentration of 1 mg AE/mL buffer; purified anthocyanins (100 µM malvidin, 100

278

µM delphinidin) or positive control 5 µM sitagliptin (SIT). The blank contained only buffer and

279

DPP-IVGLO® reagent, while the enzyme control and the samples contained buffer, DPP13

280

IVGLO® reagent and 10 µL purified DPP-IV human enzyme (10 ng/mL). Luminescence was

281

measured after mixing and incubating for 30 min using a Synergy2 multiwell plate reader

282

(Biotek Instruments, Winooski, VT). Percent inhibition was calculated from the blank and

283

enzyme control for each sample. Results are presented as percent inhibition per mg AE /mL, or

284

per 100 µM of malvidin, or per 100 µM of delphinidin.

285 286 287

2.3.4. Caco-2 cell proliferation Caco-2 cells (HTB-37 from ATCC, Manassas, VA) were subcultured using Eagle's

288

Minimum Essential Medium (EMEM) ATCC ® 30-2003 media supplemented with 20% FBS, 1%

289

penicillin-streptomycin, and 1% sodium pyruvate. Cells were maintained at 37°C in 5% CO2/95%

290

air using a CO2 Jacketed Incubator (NuAIRE DH Autoflow, Plymouth, MN). Cell proliferation

291

was measured using a CellTiter 96® AQueous One Solution Proliferation Assay kit (Promega,

292

Madison, WI). Anthocyanin rich extract from black bean coat at a concentration of 1 mg/ml,

293

malvidin isolated and purified from black bean at a concentration of 100 µM (purity > 85%) and

294

delphinidin isolated and purified from black bean at a concentration of 100 µM (purity > 85%)

295

showed cell viability > 80% within all treatments.

296 297

2.3.5. Glucose uptake in vitro

298

Caco-2 cells were seeded in 24-well plates at the density of 2 ×105 cells/well. The medium

299

was changed every two days, and the culture was carried out for 13 days. For uptake studies,

300

Caco-2 cells were placed in glucose-free media for 2 h, then exposed to 400 µL glucose-free

301

media containing anthocyanin solutions (1 mg AE/mL), purified anthocyanins (100 µM malvidin,

14

302

100 µM delphinidin), media only as control and a fluorescent D-glucose derivative, 2-[N-(7-

303

nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose

304

fluorescence readings were taken after 30, 60 and 180 min at 37 °C. Glucose uptake was

305

stopped by washing three times with two-fold volume of ice-cold PBS. Fluorescent intensity was

306

measured by a Synergy2 multi-well plate reader (Biotek, Winooski, VT) at 485 nm excitation

307

and 535 nm emission filter. The cells were lysed in 100 µL RIPA lysis buffer and protein

308

concentration was measured using DC protein assay (Bio-Rad Laboratories, Hercules, CA).

309

Results were expressed as a percentage of glucose uptakes relative to the untreated control and

310

normalized to protein concentration per mg/mL of AE, or per 100 µM of malvidin, or per 100

311

µM of delphinidin.

(2-NBDG)

(100

µM)

and

312 313

2.3.6. Reactive oxygen species assay (ROS)

314

Independent cell treatments were performed in 96 well plates for the ROS inhibition assay,

315

using the cellular ROS detection assay kit (Abcam®, ab113851, Cambridge, MA). Caco-2 cells

316

(1 x 104 cells/well) were treated with 100 µM H2O2, 2’,7’- dichlorofluorescein diacetate

317

(DCFDA) (25 µM), anthocyanin solutions (1 mg AE/mL), purified anthocyanins (100 µM

318

malvidin, 100 µM delphinidin) or phloretin (PHL (100 µM). Followed by four h of incubation,

319

after this period the plate was read in the Synergy2 multi-well plate reader (Biotek, Winooski,

320

VT) with excitation wavelength at 485 nm and emission wavelength at 535 nm. The results were

321

expressed as a percentage of fluorescence inhibition relative to the untreated control per mg/mL

322

of AE, or per 100 µM of malvidin, or per 100 µM of delphinidin.

323 324

2.3.7. Computational docking 15

325

Docking calculations of anthocyanins (delphinidin-O-glucoside, malvidin-O-glucoside and

326

petunidin-O-glucoside) and positive controls (sitagliptin or acarbose), and enzyme crystal

327

structures [α-glucosidase (3AJ7), α-amylase (1B2Y) and DPP-IV (3W2T] were carried out using

328

DockingServer following the methodology previously reported by (Mojica & de Mejia, 2016).

329

2.4. Statistical Analysis

330

Each assay was run in triplicate, and all analyses were performed in three independent

331

replicates. The data obtained were analyzed using one-way ANOVA to compare experimental to

332

control values using SAS version 9.4 Software or JMP 8.0 (Cary, NC); statistical differences

333

among independent variables were determined using the Proc GLM procedure and Tukey

334

Posthoc Test (P < 0.05). RSM analysis was performed using the Proc Rsreg procedure.

335

Correlation among parameters measured was performed using the GraphPad Prism software

336

(Version 5.02; GraphPad Software, Inc.; San Diego, CA).

337 338

3.0 RESULTS

339

3.1. Physicochemical stability

340

3.1.1. Optimization of extraction of anthocyanins and total polyphenols from black bean

341

coats

342

Table 1 shows the conditions used to extract phenolic compounds and optimize the

343

extraction yield. A combination of twenty-seven treatments was used to model the equation

344

using the RSM method. Multiple regression analysis of anthocyanin concentration from black

345

bean coat showed that the test variables were related by the second-degree polynomial equation

346

(eq 1).

16

347

Y= 0.0586 + 0.0268x1 + 0.0074x2 + 0.027x3 - 0.0001x1x2 + 0.00023 x1x3 + 0.0000065x2x3 +

348

0.000118x12 + 0.000522x22 - 0.002089x32 (eq. 1)

349

Similarly, by applying the multiple regression analysis, total polyphenols concentration and

350

the other independent variables were related to the dependable variables by the second-degree

351

polynomial equation (eq 2).

352

Y= 2.73 + 0.174829x1 - 0.00977x2 + 0.1590x3 -0.000395x1x2+ 0.00372x1x3 + 0.000469x2x3 +

353

0.00319x12 + 0.000522x22 - 0.002089x32 (eq. 2)

354

Y in equations 1 and 2 represents anthocyanin or total polyphenols concentration, x1 ethanol

355

concentration, x2 solid-to-liquid ratio, and x3 extraction temperature. For the optimization of the

356

extraction of anthocyanins and total polyphenols from black bean coats, it was found that the

357

total model was significant for both parameters (P < 0.0001). For anthocyanins, ethanol

358

concentration and temperature were highly significant (P < 0.0001) and solid-to-liquid ratio

359

presented a P-value of 0.047. On the other hand, for total polyphenols also ethanol concentration

360

and temperature were highly significant (P < 0.0001), and solid-to-liquid ratio presented a P-

361

value of 0.049.

362

Figure 1 shows the three-dimensional response surface plots. Figure 1 ABC shows a

363

tendency of increasing the anthocyanins extraction as the concentration of ethanol and

364

temperature increased. Regarding the solid-to-liquid ratio, there was a tendency of increasing the

365

extraction as the ratio was smaller. For anthocyanin extraction, the optimal conditions found

366

were ethanol 24%, the solid-to-liquid ratio of 1:40 and 29 ˚C. Similarly, for total polyphenol

367

extraction (Figure 1 D, E, F), there was an increment in the extraction when ethanol

368

concentration and temperature increased. On the other hand, the solid-to-liquid ratio showed a

369

small increase in total polyphenol concentration when the solution tended to be more diluted.

17

370

The optimal conditions for total polyphenol extraction from the optimization procedure were 23%

371

ethanol, the solid-to-liquid ratio of 1:40 g coat/mL and temperature of 30 ˚C.

372

The highest anthocyanins and polyphenols concentrations were obtained at 40 ˚C with a

373

solid-to-liquid ratio of 1:50. On the other hand, the lowest anthocyanins concentration was

374

extracted at 4˚C with 1:30, solid-to-liquid ratio. This condition also generated the lowest

375

concentration of total polyphenols. The difference between highest and lowest anthocyanin

376

concentration was about 4.5 fold yield while the difference between highest and lowest

377

polyphenol concentration was about 4.8 fold. In general, a tendency was observed of increasing

378

the yield of anthocyanins and total polyphenols when ethanol concentration and temperature

379

increased. Regarding the solid-to-liquid ratio, more anthocyanins were extracted when the

380

solvent proportion increased. Samples with higher anthocyanin yield tended to have a higher

381

Chroma value, color intensity, and lower Hue value, or color tone. This was related to the angle

382

on the chromaticity diagram and the low values of Hue positioned in an intense red zone.

383

Correlations of these parameters were performed (Supplemental Figure 1 A-F) and positive

384

correlations were found for the concentration of anthocyanins and total polyphenols (P < 0.0001,

385

r = 0.99). In addition, the anthocyanin concentration positively correlated with delphinidin

386

concentration, a known biologically active phenolic compound (P < 0.0001, r = 0.73).

387

Anthocyanins revealed a significant positive correlation with Chroma and significantly negative

388

correlation with Hue (P < 0.0001 and r = 0.894 and r = -0.797, respectively). Similarly, the total

389

polyphenols concentration correlated positively with Chroma and negatively with Hue (P <

390

0.0001, r = 0.087 and r = -0.900, respectively).

391 392

3.1.2. Extraction yield and characterization

18

393

The maximum extraction yield of total phenolic compounds and anthocyanins in solution

394

was 17.3 mg GAE/g DW and 1.7 mg C3GE/g DW respectively. Figure 2 B presents the

395

characterization and quantification of dried anthocyanin extract using LC-ESI-MS and HPLC.

396

Three main anthocyanins were found by their respective ions [M+] 465.1 for delphinidin 3-O-

397

glucoside, [M+] 479.12 for petunidin 3-O-glucoside and [M+] 493.13 for malvidin 3-O-

398

glucoside. The three main anthocyanins were quantified by HPLC using pure standards. A total

399

of 32 mg of anthocyanins were quantified per gram of dry extract. Petunidin-3-O-glucoside

400

represented the highest proportion of 56% of the total, followed by delphinidin-3-O-glucoside

401

with 34% and malvidin-3-O-glucoside with 10% of the total. A representative HPLC

402

chromatogram and chemical structures are shown in Figure 2 AC.

403 404

3.1.3. Shelf stability of black bean anthocyanins

405

Table 2 shows the degradation kinetics values for anthocyanins and a* color parameter,

406

which is related with the redness of the sample. The anthocyanin stability was higher at 4˚C and

407

pH 2.5 with a half-life of 277 days; the projected shelf-life was around 2.8 years (Anderson, &

408

Scott, 1991). In contrast, at 22˚C, the half-life was only 43 days. The half-life of the

409

anthocyanins decreased as the pH increased from 2.5 to 4.3 at 22 ˚C from 43 days to 16 days,

410

respectively. In contrast, 4 ˚C and pH 3.5 offered a protective effect to anthocyanins showing a

411

half-life of 172 days, compared to pH 3.0 with a half-life of 139 days and 56 days at pH 4.3.

412

Similarly, for a* color parameter, refrigeration offered a protective effect compared to room

413

temperature. For redness stability, pH 3.5 improved the stability and half-life at 4 ˚C (151 days)

414

compared with 124, 50 and 45 days at pH 2.5, 3.0 and 4.3, respectively. Table 2 shows the color

19

415

of anthocyanins in solutions at different pHs using 1 mg/mL of dry extract at time zero and five

416

weeks of storage in comparison to cherry flavor soda.

417 418

3.1.4. First order reaction kinetics and Arrhenius model for black bean anthocyanins and

419

a* color parameter

420

Reaction rate constants and half-lives for anthocyanins and a* color parameter are

421

summarized in Supplemental Table 1 for anthocyanins exposed to high temperatures (70, 80

422

and 90 ºC) and different pHs for 5 h. Anthocyanins and a* color followed a first order kinetics

423

for thermal degradation. Plots of anthocyanin concentration indicated that degradation followed a

424

first-order reaction kinetic (Supplemental Figure 2 A-D). The highest reaction rate constants

425

were observed at 90 ˚C and pH 4.3 (0.387/h). The increase in the temperature had an increase in

426

the rate constants; however, pH 3.0 and 3.5 showed lower thermal degradation by having lower k

427

values compared to pH 2.5. The longer half-lives (11.29 h, 3.86 h, and 2.5 h) were for pH 3.0 at

428

70, 80 and 90 ˚C, respectively. On the contrary, pH 4.3 showed the shortest half-lives for all

429

temperatures (1.84 h, 2.11 h and 1.79 h, respectively). The highest Q10 change in the reaction

430

rate constant for 10˚C of temperature was observed at pH 2.5 with a value of 2.99 indicating that

431

thermal degradation was three-fold higher in this temperature range and pH, while the lowest Q10

432

was found at pH 4.3 (1.18) for 80 to 90 ˚C change. Arrhenius modeling of anthocyanin

433

degradation showed a temperature and pH dependent change with activation energies of 84.76,

434

78.32, 75.28 and 55.92 kJ/mol and regression coefficients R2 of 0.97, 0.95, 0.97 and 0.88 at pH

435

2.5, 3.0, 3.5 and 4.3, respectively.

436

Regarding the color parameter a*, there was a tendency to increase the reaction rate k as the

437

temperature and the pH increased; however, similarly to degradation of anthocyanin, pH 3.0

438

seemed to have a protective effect. The highest k value was found at 90 ˚C and pH 4.3 (1.618/h), 20

439

and the lowest k value was found at 70 ˚C and pH 2.5 (0.097/h). As expected the half-lives of

440

anthocyanin solutions decreased as the temperature and pH increased; from 7.1 h at 70 ˚C and

441

pH 2.5 to 0.43 h at 90 ˚C and pH 4.3. The Q10 change in reaction rate constant k values remained

442

around two fold for pH 2.5, 3.0 and 3.5. Whereas pH 4.3 presented Q10 values around 1.5 for

443

both increases of temperature 70-80 ˚C and 80-90 ˚C. The Arrhenius modeling also showed

444

temperature dependent color a* degradation; however, pH 3.5 presented the highest activation

445

energy (79.6 kJ/mol) compared to 76.34, 68.19 and 41.22 kJ/mol for pH 2.5, 3.0 and 4.3,

446

respectively, correspondingly with R2 values of 0.99 for all the treatments.

447 448

3.1.5. Chroma, Hue, and ∆E* color changes

449

The color of the anthocyanin solutions showed different trends depending on pH during the

450

evaluated times and temperatures. In Supplemental Figure 3 A-D the color parameter Chroma

451

showed no statistical differences (P < 0.05) among temperatures; however, for pH 3.5 and 4.3

452

there was an increasing tendency on the Chroma value with time. On the other hand, hue color

453

increased when increasing the temperature from 70 to 90˚C at pH 2.5; at pH 4.3 there was no

454

change in the hue value between time and temperature (Supplemental Figure 3 E-H).

455

Supplemental Figure 4 A-C shows the total change in color (∆E*) at different pHs in

456

comparison with time zero at 70, 80 and 90 ˚C. The pH 2.5 showed the highest color changes at

457

80 and 90˚C; pHs 3.0, 3.5 and 4.3 showed a slight increase on ∆E* after two h of heating. In

458

general, the highest color change was observed at 90 ˚C. While the color was more stable at 70

459

˚C, after three h of heating, not significant (P > 0.05) changes were observed at pH 2.5 and 3.5.

460 461

3.2 Biological potential

21

462

3.2.1. α-Glucosidase, α-amylase and dipeptidyl peptidase IV inhibition, glucose uptake and

463

reactive oxygen species inhibition

464

Figure 3 A shows the inhibitory potential of α-glucosidase enzyme of the AE (1 mg/mL)

465

38.7% and purified anthocyanins malvidin (100 µM) 42.8% and delphinidin (100 µM) 44.5%,

466

presenting no significant differences among them. However, positive control acarbose inhibition

467

was significantly higher (60.9%) (P<0.05). Figure 3 B presents the inhibition potential of

468

anthocyanins to inhibit α-amylase enzyme. AE showed higher potential to inhibit the enzyme

469

than purified anthocyanins malvidin and delphinidin (35.6, 29.6 and 24.2%, respectively) with

470

no significant differences (P > 0.05). These values were lower than the positive control acarbose

471

66.8% (P < 0.05). DPP-IV enzyme anthocyanins inhibition is showed in Figure 3 C. Higher

472

inhibition potential was for positive control sitagliptin (99.6%). Moreover, purified anthocyanins

473

malvidin and delphinidin (82.4 and 78.8%, respectively) showed higher inhibition potential than

474

AE (34.4%) (P < 0.05). For glucose uptake, time of exposure 60 and 180 min presented a

475

significant reduction in glucose uptake compared to untreated control (P < 0.05). However, no

476

statistical differences were detected among anthocyanins treatments. Contrary, after 30 min

477

exposure to anthocyanins treatments, glucose uptake results were significantly different among

478

treatments. At this time malvidin showed the highest decrease in glucose uptake (55.2%)

479

compared to untreated control, followed by delphinidin (37.1 %) and AE (5.2%) that presented

480

the lowest reduction in glucose uptake with no differences compared to untreated control (P >

481

0.05) (Figure 3 D). The inhibition of oxygen reactive species formation results are showed in

482

Figure 3 E. Most potent inhibitor was malvidin (91.2%) (P < 0.05) followed by delphinidin

483

(83.4%), AE (81.6%) and control phloretin (66.2%).

484

22

485

3.2.2. Computational docking

486

Figure 4 ADG presents a representative pose of delphinidin interacting with the enzymes α-

487

glucosidase, α-amylase and DPP-IV. Figure 4 BEH displays α-glucosidase, α -amylase and

488

DPP-IV enzymes catalytic sites interacting with the anthocyanin delphinidin. Figure 4 CFI

489

presents the predicted free energy of binding and the inhibition constant (Ki). For α-glucosidase,

490

positive control acarbose presented lower free energy value (-6.41 kcal/mol) and Ki (20 µM)

491

compared to the three anthocyanins present in AE (malvidin-3-O-glucoside, delpinidin-3-O-

492

glucoside, and petunidin-3-O-glucoside). Besides, those compounds still show good potential to

493

interact with the enzyme mainly by polar interactions, hydrogen bonds, and hydrophobic

494

interactions. Similarly, α-amylase inhibition potential was higher for positive control acarbose

495

(free energy, -8.30 kcal/mol; Ki, 0.82 µM). Moreover, delphinidin presented outstanding affinity

496

for the enzyme (free energy, -7.35 kcal/mol; Ki, 4.08 µM) mainly by polar interactions,

497

hydrophobic interactions, π-π interactions and cation-π interactions. For the inhibition of DPP-IV,

498

the positive control sitagliptin presented the higher inhibition potential (free energy, -11.01

499

kcal/mol; Ki, 0.008 µM). Anthocyanins malvidin, delphinidin, and petunidin presented averaged

500

free energy values -5.14 kcal/mol. Furthermore, malvidin present higher affinity for the enzyme.

501

Principal potential interaction with the enzyme was hydrogen bonds, polar interactions,

502

hydrophobic interactions, π-π interactions and cation-π interactions.

503 504

4.0. DISCUSSION

505

In this study, we optimized the conditions to extract anthocyanins and other phenolic compounds

506

from common bean coats and evaluate their shelf and thermal stability at different pHs and

507

conditions, as well as, their antidiabetes potential. Anthocyanins stability is highly affected by

23

508

pH, which varies in different food systems. At optimal extraction conditions, the anthocyanin-

509

rich extract concentration of anthocyanins was 1.7 mg C3GE/g DW. This value is lower than the

510

one reported by Mojica et al. (2015) for Negro Otomi cultivar, probably due to different

511

extraction methods since the investigators used acidified ethanol 85:15 ethanol:HCl.

512

Optimization conditions were used to extract the coat pigments that were freeze-dried for easy

513

handling; anthocyanins enriched dry powder (1 mg/mL) was used to prepare the solutions at

514

different pHs that were found in commercially available beverages. The enriched anthocyanins

515

powder represented 26.4% of yield from the bean coat after three consecutive extractions; 32.8

516

mg/g dry extract with three identified main anthocyanins (delphinidin-O-glucoside, petunidin-O-

517

glucoside, and malvidin-O-glucoside). These results are in agreement with previous findings on

518

anthocyanin composition in Mexican black bean cultivars (Mojica, Meyer, Berhow, & de Mejía,

519

2015; Aguilera, Mojica, Rebollo-Hernanz, Berhow, de Mejía, & Martín-Cabrejas, 2016).

520

Several factors affect anthocyanin stability, besides pH and temperature which are the most

521

significant factors; processing and storage conditions, pressure, light, O2, enzymes, ascorbic acid,

522

sulfur dioxide, sulfite salts, metal ions, sugars and some co-pigments contribute to their

523

degradation (Hernandez-Herrero, & Frutos, 2014; Hou, Qin, Zhang, Cui, & Ren, 2013; Zoric,

524

Dragovic-Uzelac, Pedisic, Kurtanjek, Garofulic, 2014). All of these factors can cause oxidation

525

and cleavage of covalent bonds that generate colorless smaller molecules (Zoric, Dragovic-

526

Uzelac, Pedisic, Kurtanjek, Garofulic, 2014).

527

Stability plays a fundamental role in evaluating natural compounds with potential as

528

colorants. Some studies reported that during refrigeration anthocyanin increased their stability.

529

This important parameter is evaluated by the half-life (t1/2) that represents the time needed for 50%

530

of their degradation (Hou, Qin, Zhang, Cui, & Ren, 2013). Shelf-life stability was monitored

24

531

during five weeks at 4 ºC and 22 ºC, and anthocyanins were more stable under refrigeration

532

conditions and low pH. This is in agreement with previously reported data (Kirca, & Cemeroglu,

533

2003; Hou, Qin, Zhang, Cui, & Ren, 2013). For example, Liu et al. (2014) evaluated the stability

534

of anthocyanins from Chinese red radish and quantified their half-life using different fruit juice

535

models at 4 °C. Their results were lower (130.9 - 259.1 days) compared with 277 days of black

536

bean anthocyanins in our study. Regarding color, Hernandez-Herrero & Frutos, (2014) reported

537

that the color of grape and plum peel remained stable during eight weeks of storage at 6 ˚C and

538

23 ˚C. We observed the highest stability of a* color parameter at pH 3.5 (151.1 days). This effect

539

may be influenced by the proportion of the chemical forms of anthocyanins such as the flavylium

540

cation and the quinoidal. These forms are affected by pH and may be playing important role in

541

providing stability to the system under storage conditions. The stability of anthocyanins can be

542

influenced by the ring B substituents and the presence of additional hydroxyl or methoxyl groups

543

which decrease the aglycon stability in neutral media. However, aglycons, monoglycosides, and

544

mostly, diglycosides derivatives are more stable in neutral pH conditions. This performance is

545

explained because the sugar molecules avoid the degradation of instable intermediaries into

546

phenolic acids and aldehydes (Castaneda-Ovando, Pacheco-Hernandez, Paez-Hernandez,

547

Rodrıguez, & Galan-Vidal, 2009). Similar to our results, Jie et al. (2013) reported that purple-

548

fleshed sweet potato anthocyanins showed higher thermal stability at pH 3.0 to 4.0 compared to

549

lower pHs.

550

During the thermal stability assays, all anthocyanin solutions followed first-order reaction

551

kinetics. The energy of activation (Ea) is a thermodynamic parameter that indicated thermal

552

stability in which the higher Ea represented the most stable. Zoric et al. (2014) reported values of

553

Ea ranging from 42 to 55 kJ/mol on single anthocyanins from Marasca paste, with cyanidin-3-O-

25

554

glucoside the most stable. When comparing the results of the current study with the literature,

555

only pH 4.3 presented a lower Ea value. Q10 indicates the degradation rate; the higher the value,

556

the more temperature dependent is the reaction. The Q10 values in the current study were higher

557

than the values reported by Liu et al. (2014) 1.70 from 70-80 ˚C and 1.48 from 80-90 ˚C in an

558

apple juice model. The color of the solutions was evaluated by the color parameter a* which is

559

related to the redness on the chromaticity dimensions of the samples. Black bean anthocyanins

560

a* color parameters were more stable than plum puree anthocyanins when their activation energy

561

(27.78 kJ/mol) (Ma, Lei, Yang, Wang, Zhao, & Zu, 2012) was compared; this value was lower

562

than the Ea from all pHs of bean extracts.

563

Common bean coats represent approximately 10% of the total seed; coats contain the highest

564

concentration of phenolic compounds in beans. In general, common beans are consumed as a

565

whole food. However, there is an increasing market of processed common beans as ingredients

566

for the food industry (Mojica, Chen, & de Mejia, 2015).

567

Beyond the potential technological application of anthocyanins as pigments in the food

568

industry, its use as beverage natural colorant present the advantage of their multiple health

569

benefits associated with their consumption. For instance, these compounds can decrease

570

oxidative stress, protect against coronary heart disease, exert anti-inflammatory and anti-

571

carcinogenic activities and can help to control obesity and diabetes (He, & Giusti, 2010).

572

Moreover, dietary polyphenols have been associated with a prebiotic effect on microbiota

573

modulation with beneficial health effects (Faria, Fernandes, Norberto, Mateus, & Calhau, 2014).

574

Anthocyanin technological functionality as food colorants and their potential bioactivity

575

make these natural occurring compounds more attractive for consumers. This dual functionality

576

potentiates its application as a food additive. Anthocyanin-rich extract and purified anthocyanins

26

577

delphinidin and malvidin showed potential to inhibit enzymes used as molecular targets of

578

diabetes. Several fruits and vegetables contain important amounts of anthocyanin and other

579

phenolic compounds. However, most research have been performed around berries and their

580

polyphenols antidiabetes potential (Edirisinghe & Burton-Freeman, 2016; Guo, Yang, Tan, Jiang,

581

& Li, 2016; Alzaid, Cheung, Preedy, & Sharp, 2013; Castro-Acosta, Lenihan-Geels, Corpe, &

582

Hall, 2016; Crozier, Jaganath, & Clifford, M, 2009; Jennings, Welch, & Spector, 2014).

583

Anthocyanin-rich extract showed potential to inhibit starch degrading enzymes (α-

584

glucosidase and α-amylase) in around 40% at the concentration used to color a beverage, 1

585

mg/mL. This represents an important diminution in free glucose to be absorbed. Moreover, this

586

polyphenolic extract also presented potential to decrease glucose uptake at the gastrointestinal

587

level, when compared to untreated control. Also, these bioactive compounds from beans

588

inhibited DPP-IV, an important enzyme metabolically related to insulin secretion. Potential of

589

anthocyanins in black bean coats to inhibit α-glucosidase, α-amylase and DPP-IV in biochemical

590

assays was corroborated using molecular docking to predict the interactions of the anthocyanins

591

in the catalytic cavity of the enzymes. In silico results showed good potential to inhibit the

592

enzymes mentioned above. Moreover, DPP-IV docking results coincide with reports of the

593

interaction of berry wine anthocyanins and the enzyme (Johnson, de Mejia, Fan, Lila, and

594

Yousef, 2013). Furthermore, the anthocyanin-rich extract showed outstanding potential to

595

decrease generation of reactive oxygen species (>80%). This important antioxidant potential

596

could protect β-cells and improve their function. The sum of the potential bioactivities of the

597

anthocyanin-rich extract contributes to their antidiabetes potential. Moreover, results coincide

598

with other studies performed with anthocyanin-rich foods such as berries. Similarly to our results,

599

Da Silva et al. (2008) reported that Brazilian strawberry extracts from various species

27

600

significantly inhibited α-glucosidase activity up to 70% in a dose-dependent manner using a

601

Caco-2 model. Moreover, using the same Caco-2 model, Johnson et at. (2005) found that dietary

602

polyphenols showed an effect on glucose transporters SGLT1 and GLUT2 and decrease glucose

603

absorption. Cohort studies showed that high consumptions of anthocyanins and anthocyanin

604

containing foods are associated with a lower risk for type-2 diabetes (T2D) in US population.

605

Moreover, higher ingestions of delphinidin, malvidin and petunidin are associated to

606

low homeostatic model assessment insulin resistance (HOMA-IR) and lower fasting serum

607

insulin levels (Jennings, Welch, & Spector, 2014). This supports longitudinal observations of

608

T2D risk and suggests that anthocyanins may reduce T2D risk (Castro-Acosta, Lenihan-Geels,

609

Corpe, & Hall, 2016; Guo, Yang, Tan, Jiang, & Li, 2016). Health effects associated with food

610

anthocyanins could partially be attributable to metabolites of parent anthocyanin compounds.

611

Due to their relatively short half-life, their metabolites, including phase I and II compounds

612

(glucuronic, sulfur or methyl derivatives) Castro-Acosta, Lenihan-Geels, Corpe, & Hall, 2016;

613

Crozier, Jagannath, & Clifford, M, 2009).

614

Edirisinghe and Burton-Freeman (2016) recommend the incorporation of berry extracts rich

615

in anthocyanins and polyphenols to drinks, cereal bar among other food products to provide their

616

associated health benefits. In this sense, anthocyanin-rich extracts from black bean could be used

617

as a natural colorant, with important health benefits for consumers. This research sets precedent

618

data for the technological and biological potential of black bean coat anthocyanin-rich extracts.

619

Future research is needed to standardize and validate its use as a bifunctional food ingredient.

620 621

5.0 CONCLUSION

28

622

Extraction using food grade ethanol was technically feasible to obtain stable anthocyanins

623

from black bean coats. At pH 2.5 and refrigeration temperatures of storage (4 ˚C) anthocyanins

624

stability was promoted up to (t1/2) 277 days; moreover under same conditions following the Q

625

rule, the projected shelf-life was around 2.8 years. Anthocyanins have outstanding potential to be

626

used as food pigments; however, stability and feasibility are one of the main challenges to

627

overcome. In addition, black bean anthocyanin-rich extract exert important biological potential

628

that may contribute to modulate markers of diabetes. Black beans are a good source of natural

629

pigments that could replace synthetic colorants commonly used in the food industry increasing

630

the potential health benefits associated with the consumption of anthocyanins.

631 632

6.0 ABBREVIATIONS USED

633

AE: anthocyanin-rich extract; b*: yellowness/blueness; C*: Chroma; C3GE: cyaniding-3-

634

glucoside equivalent;; DW: dry weight; DPP-IV: dipeptidyl peptidase IV; Ea: Arrhenius

635

activation energy; ∆E*: change of color; GAE: gallic acid equivalent; h˚: Hue; HDC: high

636

energy collision; K: first-order kinetic rate; Ki: inhibition constant;

637

mass/charge; PHL: phloretin; Q10: change in the reaction rate constant for 10˚C; RSM: response

638

surface methodology; tR; retention time; t1/2 half-life; a*: redness/greenness; t0: time zero; T2D:

639

type-2 diabetes; ROS: reactive oxygen species.

640 641 642 643

L*:lightness;

m/z:

7.0 ACKNOWLEDGEMENTS Author Luis Mojica was supported by a scholarship from Consejo Nacional de Ciencia y

644

Tecnología CONACyT-Mexico. Thanks to Andy Tan for his technical support.

645 646 647

8.0. CONFLICT OF INTEREST Authors declare no conflict of interest. 29

648 649 650

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α-glucosidase while diverse phenolic composition and concentration. Food Research

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diabetic peptides from black bean (Phaseolus vulgaris L.) proteins, their characterization

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Evaluation of the hypoglycemic potential of a black bean hydrolyzed protein isolate and

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its pure peptides using in silico, in vitro and in vivo approaches. Journal of Functional

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35. Shipp, J., & Adbel-Aal, E. M. (2010). Food applications and physiological effects of

762

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Kinetics of the degradation of anthocyanins, phenolic acids and flavonols during heat

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766

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767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783

35

784

Figure Captions

785

Figure 1. Surface response methodology for the extraction of anthocyanins and total

786

polyphenols from black bean coats as affected by ethanol concentration, solid-to-liquid ratio and

787

temperature. A. Solid-to-liquid ratio vs % ethanol for anthocyanins; B. Temperature vs %

788

ethanol for anthocyanins; C. Temperature vs solid-to-liquid ratio for anthocyanins; D. Solid-to-

789

liquid ratio vs % ethanol for polyphenols; E. Temperature vs % ethanol for polyphenols; F.

790

Temperature vs solid-to-liquid ratio for polyphenols.

791

Figure 2. A. Representative HPLC chromatogram of the anthocyanin extract; B. Anthocyanin

792

characterization and concentration of individual anthocyanins; B. Chemical structure of

793

identified anthocyanins.

794

Figure 3. Biological potential of black bean coat anthocyanins rich extracts. A. Inhibition of α-

795

glucosidase; B. Inhibition of α-amylase; C. Inhibition of DPP-IV; D. Glucose uptake inhibition.

796

E. Oxygen reactive species inhibition.

797

Figure 4. Representative in silico molecular docking diagrams exemplifying the interaction of

798

delphinidin-3-O-glucoside with α-glucosidase, α-amylase, and DPP-IV. ADF. Presents best

799

pose of delphinidin-3-O-glucoside interacting inside α-glucosidase, α-amylase and DPP-IV

800

enzymes; BEH. Displays the catalytic sites α-glucosidase, α -amylase and DPP-IV enzymes,

801

showing the interaction of the anthocyanin delphinidin-3-O-glucoside with the amino acids in the

802

catalytic cavity; CFI. Shows the predicted free energy of binding and the inhibition constant

803

(Ki) of the enzymes α-glucosidase, α-amylase and DPP-IV enzymes with the anthocyanins

804

identified in black bean coats (malvidin-3-O-glucoside, delpinidin-3-O-glucoside, and petunidin-

805

3-O-glucoside).

36

806

Supplemental Figure 1. Correlation parameters of anthocyanins and total polyphenols with

807

color parameters Hue and Chroma. A. Anthocyanins vs. total polyphenols; B. Anthocyanins vs.

808

delphinidin glucoside; C. Anthocyanins vs. chroma; D. Polyphenols vs. chroma; E.

809

Anthocyanins vs. hue; F. Polyphenols vs. hue.

810

Supplemental Figure 2. First order reaction kinetics of anthocyanins at different pHs and

811

temperatures for black beans anthocyanins. A. pH 2.5; B. pH 3.0; C. pH 3.5; D. pH 4.3.

812

Supplemental Figure 3. Chroma and hue variations at different pHs and temperatures as time

813

progresses. A. Chroma and pH 2.5; B. Chroma and pH 3.0; C. Chroma and pH 3.5; D. Chroma

814

and pH 4.3; E. Hue and pH 2.5; F. Hue and pH 3.0; G. Hue and pH 3.5; H. Hue and pH 4.3.

815

Supplemental Figure 4. Color change of the anthocyanin solutions; effect of pH, time and

816

temperature. A. 70 ˚C; B. 80 ˚C; C. 90 ˚C.

817 818

37

Table 1. Anthocyanin and total polyphenols concentrations, and color parameters of black bean extracts obtained under different extracting conditions. Anthocyanins

Total Polyphenols

Chroma

Hue

C*

h˚

3.63±0.62k

7.08±1.03l

25.01±3.98a

0.67±0.08jklm

6.25±1.29hijk

10.49±1.16lijkl

14.38±0.74cdefgh

4

1.03±0.17defgh

9.95±2.13efg

16.24±1.99bcdef

5.81±0.17kl

1:40

4

0.44±0.05lm

4.205±0.76jk

8.33±1.63kl

21.14±4.48ab

12.5

1:40

4

0.70±0.04jkl

6.40±1.02hijk

11.87±1.75hijk

12.74±0.90defghi

6

25

1:40

4

1.06±0.11defg

10.15±1.57ef

15.69±2.04bcdefgh

4.67±0.19l

7

0

1:50

4

0.46±0.02klm

4.49±0.39jk

7.13±1.32 l

24.74±3.33a

8

12.5

1:50

4

0.73±0.02ijkl

6.65±0.39hijk

10.47±0.91lijkl

13.23±0.66cdefghi

9

25

1:50

4

1.07±0.12def

10.31±1.61ef

14.37±1.40defgh

5.15±0.68l

10

0

1:30

22

0.64±0.08jkl

5.34±0.18ijk

12.11±0.21ghijk

15.57±0.32cdef

11

12.5

1:30

22

1.02±0.05defgh

9.02±0.67efgh

17.07±0.47abcde

11.72±0.17efgihj

12

25

1:30

22

1.49±0.10abc

14.12±1.82bcd

20.36±0.70a

8.53±0.91lijkl

13

0

1:40

22

0.69±0.12jkl

5.86±0.16ijk

10.57±0.43lijkl

17.63±0.42bcd

14

12.5

1:40

22

1.12±0.04de

9.83±0.89efg

15.97±0.56bcdefg

11.38±0.01efghij

15

25

1:40

22

1.54±0.05ab

14.45±1.46abc

19.37±0.69ab

6.96±0.25jkl

16

0

1:50

22

0.75±0.14hijk

6.22±0.22hijk

10.15±0.02jkl

18.19±0.57bc

17

12.5

1:50

22

1.15±0.14de

9.92±0.15efg

14.02±0.38efghi

11.46±0.33efghij

18

25

1:50

22

1.54±0.01ab

14.78±0.66ab

17.64±0.43abcde

4.73±0.08l

19

0

1:30

40

0.76±0.01ghijk

6.38±0.29hijk

12.85±0.82fghij

15.01±0.27cdef

20

12.5

1:30

40

1.25±0.13bcd

11.72±0.59cde

17.79±0.55abcde

11.62±0.22efghij

21

25

1:30

40

1.63±0.11ab

15.87±1.50ab

18.36±1.73abcd

9.33±1.34hijkl

22

0

1:40

40

0.82±0.09fghik

7.05±0.22ghij

12.38±0.23fghij

14.87±0.16cdefg

23

12.5

1:40

40

1.22±0.08dc

11.13±0.25de

18.74±1.41abc

9.69±1.85ghijkl

24

25

1:40

40

1.64±0.04a

16.1±0.70ab

15.81±3.67bcdefgh

10.52±3.91fghijk

25

0

1:50

40

0.88±0.17efghij

7.73±0.55fghi

12.06±0.20ghijk

16.41±0.67bcde

26

12.5

1:50

40

1.24±0.08bcd

11.57±0.03cde

15.10±0.03cdefgh

11.55±0.54efgihj

27

25

1:50

40

1.70±0.01a

17.33±0.15a

17.23±0.48a

5.27±0.46 l

Temp °C

Treat.

EtOH

S/L

mg C3GE/g DW

1

0

1:30

4

0.38±0.04m

2

12.5

1:30

4

3

25

1:30

4

0

5

mg GAE/g DW

Color

Data represent the mean ± SD from at least three independent replicates. Values within a column followed by different letters are significant at p < 0.05; Treatment; EtOH: ethanol concentration; S/L: solid-to liquid ratio; C3GE: cyaniding 3 glucoside equivalents; GAE: gallic acid equivalents; DW: dried weight C*: chroma = sqrt (a*2 + b*2); h˚: hue angle = sqrt (a*2 + b*2).

Table 2. Degradation rate, half-life and color a* of black bean anthocyanin and color a* parameter at refrigeration and room temperature and Red, Green, Blue (RGB) color parameters of commercial cherry soda and anthocyanin extract solutions (1 mg/mL) at different pHs after five weeks of storage. Parameter Rate (k, d-1)

Anthocyanins pH 3.0 pH 3.5 0.034 0.028 0.127 0.130

pH 4.3 0.086 0.304

277.2

139.8

172.6

56.0

43.4

37.94

37.17

15.89

pH 3.0

pH 3.5

Temperature pH 2.5 4˚C 0.017 22˚C 0.113

Half-life(t1/2, 4˚C days) 22˚C

Week 5

t0

4˚C

22˚C

pH 2.5

Color a* Parameter Rate (k, d-1)

Temperature pH 2.5 4˚C 0.039 22˚C 0.144

Half-life (t1/2, 4˚C days) 22˚C

pH 3.0 0.095 0.307

pH 3.5 0.032 0.172

pH 4.3 0.106 0.630

124.6

50.7

151.1

45.7

33.6

15.75

60.7

7.6

pH 4.3

k: rate constant; d: days; t1/2: half-life; t0: time zero. Standard deviation of each value and statistical analysis cannot be added to the table because the degradation rate and half-life values were calculated with the slope of the plotted curves of all the data obtained (de Mejia et al., 2015).

7-Up Cherry

Predictec value Ant, mg C3GE/ g DW

Ant, mg C3GE/ g DW

B

E Polyphenols mg GAE/ g DW

D

Predictec value

Polyphenols mg GAE/ g DW

Polyphenols mg GAE/ g DW

A C

Predictec value F Ant, mg C3GE/ g DW

Predictec value Predictec value

Predictec value

Relative abundance (mAU)

A

160000 140000 120000

Delphinidin 3-O-glucoside

100000 80000

Petunidin 3-O-glucoside

60000 40000

Malvidin 3-O-glucoside

20000 0 0

20

30

40

50

60

Retention time (min)

B Anthocyanins Delphinidin 3-O-glucoside Petunidin 3-O-glucoside Malvidin 3-O-glucoside Total

C

10

Concentration mg/g dry weight 11.15 ± 0.44 18.32 ± 0.88 3.35 ± 0.32

1st Ion 2nd Ion (m/z) (m/z) 465.1 [M]+ 391.28[M]+ 479.1[M]+ 391.28[M]+ 493.1[M]+ 391.28[M]+

tR (min) 9.94 11.12 12.23

Chemical formula C21H21O12 C22H23O12 C23H25O12

32.82 ± 1.64

Delphinidin 3-O-glucoside

Petunidin 3-O-glucoside

Malvidin 3-O-glucoside

PubChem CID: 443650

PubChem CID: 176449

PubChem CID: 443652

A

B

C

D

E

Α-Glucosidase

DPP-IV

α-Amylase D)

G)

A)

B)

E)

C)

F)

Compound/inhibitor Malvidin-3-O-glucoside

Est. Free Energy of Binding (kcal/mol) -4.78

Delphinidin-3-O-glucoside -4.77 Petunidin-3-O-glucoside -4.10 Acarbose -6.41

Est. Inhibition Constant, Ki (µM) 314.30 317.93 980.47 20.00

Est. Free Energy of Binding (kcal/mol) Compound/inhibitor -6.30 Malvidin-3-O-glucoside Delphinidin-3-O-glucoside -7.35 -6.10 Petunidin-3-O-glucoside -8.30 Acarbose

H)

I) Est. Inhibition Constant, Ki (µM) 24.30 4.08 33.51 0.82

Compound/inhibitor Malvidin-3-O-glucoside Delphinidin-3-O-glucoside Petunidin-3-O-glucoside Sitagliptin

Est. Free Energy Est. Inhibition of Binding Constant, Ki (kcal/mol) (µM) -5.48 -5.00 -4.95 -11.01

96.31 215.46 235.66 0.008

854 855 856 857 858 859 860

Highlights • • • • •

Extraction of anthocyanins from black bean coats was optimized using RSM Black bean anthocyanins rich extracts presented good shelf-life stability Black bean anthocyanins inhibit α-glucosidase, α-amylase, DPP-IV and glucose uptake Black bean anthocyanins have technological functionality and antidiabetes potential Anthocyanins from black beans are natural-source colorants for the food industry

861

41