Bio Activity Antioxidants Extrusion

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Article

BIOACTIVITY OF ANTIOXIDANTS IN EXTRUDED PRODUCTSPREPARED FROM PURPLE POTATO AND DRY PEA FLOURS

Balunkeswar Nayak, Rui Hai Liu, Jose De J. Berrios, Juming Tang, and Christopher Derito

J. Agric. Food Chem., Just Accepted Manuscript DOI: 10.1021/jf200732p Publication Date (Web): 26 May 2011

Downloaded from http://pubs.acs.org on June 3, 2011

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BIOACTIVITY OF ANTIOXIDANTS IN EXTRUDED PRODUCTS1

PREPARED FROM PURPLE POTATO AND DRY PEA FLOURS2

3

Balunkeswar Nayaka, Rui Hai Liub, Jose De J Berriosc, Juming Tanga*, Christopher Deritob,4

5

a Department of Biological Systems Engineering, Washington State University, Pullman, WA6

99163, USA7

8

b Department of Food Science, Stocking Hall, Cornell University, Ithaca, NY 14853, USA.9

10

c Processed Foods Research Unit, WRRC, USDA-ARS, 800 Buchanan Street, Albany, CA11

94710, USA12

13

* 256, LJ Smith Hall, Department of Biological Systems Engineering, Washington State14

University, Pullman, WA 99163, USA; Tel: +1-509-335-2140, Fax: +1-509-335-2722. Email:15

[email protected]

17

Page 1 of 39 Journal of Agricultural and Food Chemistry


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ABSTRACT18

Measuring antioxidant activity using a biologically relevant assay adds important evidence to19

aid understanding the role of phytochemicals based on data from in-vivo and chemical assays of20

extrusion processed purple potato and pea flours. A cellular antioxidant activity assay could21

provide biologically relevant information on bioactive compounds in raw as well as processed22

food products. The objective of this study was to investigate the complete phytochemical23

profiles, antioxidant activity, cellular antioxidant activity, and their contribution to bioactivity in24

purple potato flour, dry pea flour, raw formulations and processed products prepared from the25

above ingredients using extrusion cooking. Free fraction of extracts contributed 68% to total26

phenolics, 64% to total antioxidant activity (ORAC value), and 88% to total flavonoids in purple27

potato flour (PPF). Similarly, extracts in free fraction contributed 87, 86 and 64% to total28

phenolics, total antioxidant activity (ORAC value), and total flavonoids, respectively, in dry pea29

flour (DPF). Quantity of total phenolics, and total flavonoids in purple potato flour, and30

antioxidant activity of purple potato and dry pea flours was comparable to published data.31

However, higher quantity of total flavonoids and lower total phenolics in dry pea flour was32

observed. Caffeic,p-coumaric and ferulic acids were mostly observed in the bound extracts of33

raw formulations as well as in extrudates whereas chlorogenic acid was predominant in free34

extracts. Extruded products prepared from raw formulations using extrusion cooking of the35

above ingredients significantly increased (p < 0.05) the total phenolics, ORAC antioxidant36

activity and flavonoids compared to the raw formulations. Extrusion processing increased the37

Page 2 of 39Journal of Agricultural and Food Chemistry


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KEYWORDS: Purple potato flour; dry pea flour; extrusion; phenolics; antioxidant42

activity; flavonoids; cellular antioxidant activity; bioactivity43

44

INTRODUCTION45

Processed fruits and vegetables are generally believed to have fewer naturally occurring46

antioxidants than fresh product resulting in reduced health benefits. One of the major reasons47

has been measuring antioxidant activity solely focusing on vitamin C levels in foods as an48

indicator for health benefits of processed products (1-4). However, Eberhardt et al. (5) observed49

that vitamin C contributed less than 0.4% to the total antioxidant activity in apples indicating the50

importance of phytochemicals towards total antioxidant activity. Antioxidant compounds derived51

from fruits and vegetables act in preventing formation of reactive oxygen, nitrogen, hydroxyl and52

lipid species either by scavenging free radicals, or by repairing or removing damaged molecules53

(6). These bioactive phytochemicals are proposed to prevent chronic diseases such as54cardiovascular disease, diabetes and certain forms of cancers (7-10). Bioactive phytochemicals55

exist in free, soluble-conjugated, and bound forms (11). Bound phytochemicals, mostly in cell56

wall materials, are difficult to digest in the upper gastrointestine and may be digested by bacteria57

in the colon to provide health benefits and reduce the risk of colon cancer (12). Only a small58

portion of flavonoids absorbed across the intestinal membrane are partly transformed to59

glucuronides and sulfates; however, the majority of the flavonoids are degraded by intestinal60



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Many researchers have reviewed and documented the contributions of phenolic compounds65

including anthocyanins, individual phenolic acids and carotenoids present in white and colored66

potato skin and flesh to antioxidant activity (14-16). Wounding, boiling, baking, drum-drying,67

freeze-drying and microwave cooking of white and colored potatoes had different effects on the68

total phenolic content (17-19). Potato protein hydrolysate inhibited lipid oxidation of beef patties69

by scavenging free radicals (20) and potato peel extracts retarded oxidation in radiation-70

processed lamb meat (21).71

Legumes are rich sources of protein and dietary fiber. Consumption of legumes helps prevent72

osteoporosis (22), certain cancers (23), and reduces body lipid accumulation (24). Various73

effects of thermal processed cool season legumes on antioxidant activity, phenolic content and74

potential health benefits were reported (25, 26). The combination of purple potato and dry pea75

may produce healthy snack foods incorporating natural color along with positive effects on76

human health. Limited investigations have been done on the effect of processing on formulations77

of potatoes and legumes, further to characterize the phytochemical profiles and their78

contributions to antioxidant activities. Metabolism, absorption, and bioactivity of health-79

beneficial phytochemicals could be improved by combining protein and individual80

phytochemicals. Liu (9) reported the additive and synergistic effects of biologically active81

compounds from fruits, vegetables, and whole grains are responsible for their health benefits.82

The objective of this study was to investigate the complete phytochemical profiles that exist83

in free and bound forms as well as their contribution to the total antioxidant activity in the raw84



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assay (37). Cellular antioxidant assay has been used to assess antioxidants, foods and dietary88

supplements due to its capability to consider cell uptake, distribution, and efficiency of89

protection against peroxyl radicals under physiological conditions.90

91

MATERIALS AND METHODS92

Chemicals and Reagents. Folin-Ciocalteu reagent, sodium nitrite, catechin, sodium93

borohydride, chloranil, and vanillin and gallic acid were purchased from Sigma (St. Louis, MO).94

Sodium hydroxide, hexane, aluminum chloride, and acetonitrile were obtained from Fisher95

Scientific (Pittsburgh, PA), while ethyl acetate, trifluoroacetic acid, methanol, hydrochloric acid,96

acetic acid, acetone and ethanol were of analytical grade and were purchased from Mallinckrodt97

Chemicals (Phillipsburg, NJ). Tetrahydrofuran and aluminum chloride were purchased from98

Fisher Scientific (Fair Lawn, NJ).99

100

Preparation of Samples. Purple Majesty potatoes (purchased from SLV Research Center,101

Colorado State University, Fort Collins, CO) were peeled, sliced, blanched and drum dried in the102

school of Food Science pilot plant, Washington State University. Split dry peas (purchased from103

Giustos specialty food, San Francisco, CA ) and drum dried potato flakes were pin milled to104

produce flours at the Processed Foods Research Unit, Western Regional Research Center,105

USDA, Albany, CA. Raw formulations were prepared from purple potato flour (PPF) and dry106



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temperature at 130 C). The extrudates were milled to flour using a coffee grinder and stored at -111

80 C until further use. The flours for routine analysis were stored at -20 C.112

113

Extraction of Free Phenolic Compounds. Free phenolic compounds in the ingredients, raw114

formulations, or extruded flours were extracted using the method previously reported in our115

laboratory (27, 28). Briefly, 1-2 g of flour sample was homogenized with 50 mL of 80% chilled116

acetone for 10 min using a high speed homogenizer (Brinkmann model no PT 10/35117

homogenizer). After centrifugation of homogenized flour at 2500g for 5 min, the supernatant118

was removed and extraction was repeated one more time. Supernatants were pooled, evaporated119

at 45 C to dryness and reconstituted with water to a final volume of 10 mL. The extracts were120

stored at -75 C until use.121

122

Extraction of Bound Phenolic Compounds. Bound phenolics of the ingredients, raw123

formulations, or extruded flours were extracted using the method previously reported in our124

laboratory (11, 28, 29). Briefly, 1-2 grams of flour samples were extracted twice with 80%125

chilled acetone with centrifugation at 2500g for 5 min, and the supernatant was discarded after126

each extraction. The residues were digested with 20 mL of 2 M sodium hydroxide at room127

temperature for 1 h with shaking under nitrogen gas. The mixture was neutralized with an128

appropriate amount of hydrochloric acid and extracted with hexane to remove lipids. The final129

solution was extracted five times with ethyl acetate. The ethyl acetate fraction was evaporated to130



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Determination of Total Phenolics. The total phenolics of each extract was determined using134

methods previously described by Singleton et al. (30) and modified in our laboratory (31, 32).135

Briefly, 400 L of deionized water and 100 L of a known dilution of the extract or standard136

solution were added to a test tube. Folin-Ciocalteu reagent (100 L) was added to the solution137

and allowed to react for 6 min. Then, 1 mL of 7% sodium carbonate solution and 800 L of138

deionized water was added into the test tubes, and the mixture mixed well. The color developed139

for 90 min at room temperature, and absorbance was read at 760 nm using a MRX II DYNEX140

spectrophotometer (DYNEX Technologies, Inc., Chantilly, VA). The measurements for free,141

bound and total phenolics were compared to a standard curve of prepared gallic acid solutions142

and expressed as micrograms of gallic acid equivalents (GAE) per gram dry weight (DW)143

sample SD for triplicate extracts.144

145

Determination of Individual Phenolic Acids. Chlorogenic, caffeic,p-coumaric and ferulic146

acidsin sample extracts were quantified using a reverse phase HPLC procedure employing a147

Supelcosil LC-18-DB, 150 mm x4.6 mm, 3 mm column as reported previously (33). Briefly,148isocratic elution was conducted with 20% acetonitrile in water adjusted to pH 2 with149

trifluoroacetic acid, at a flow rate of 1.0 mL/min, delivered using a Waters 515 HPLC pump150

(Waters Corp., Milford, MA). A Waters 2487 dual wavelength absorbance detector (Waters151

Corp.) was used for UV detection of analytes at 280 nm. Data signals were acquired and152

processed on a PC running the Waters Millennium software, version 3.2 (1999) (Waters Corp.).153



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r2=0.99;p-coumaric acid, r2=0.99; and ferulic acid, r2=0.99). Twenty microliter injections were157

made in each run, and areas were used for all calculations. The selected individual peaks were158

identified by the retention times and co-injection of the pure standards. The method was159

validated by the recovery of chlorogenic acid, and the percentage recovery for chlorogenic acid160

was 96.5 6.22 (n=3).161

162

Determination of Total Flavonoids. The total flavonoid contents of the samples were163

determined using the method of Sodium Borohydride/Chloronil (SBC) total flavonoid assay with164

modifications (34). Briefly, stored sample extracts for total phenolic analysis were thawed and165

added into test tubes (15 150 mm), then dried at 45 C under nitrogen gas, and reconstituted in166

1 mL of THF/EtOH (1:1, v/v). Catechin standards (0.3-10.0 mM) were prepared fresh each day167

before use in 1 mL of THF/EtOH (1:1, v/v). Each test tube with 1 mL of sample solution or 1 mL168

of catechin standard solution had 0.5 mL of 50 mM NaBH4 solution and 0.5 mL of 74.56 mM169

AlCl3 solution added and was then shaken in an orbital shaker (Laboratory-Line Instruments,170

Inc., Melrose Park, IL) for 30 min at room temperature. Then an additional 0.5 mL of NaBH 4171

solution was added into each test tube with continual shaking for another 30 min at room172

temperature. Cold acetic acid solution (2.0 mL of 0.8 M, 4 C) was added into each test tube, and173

the solutions were shaken in the orbital shaker in the dark for 15 min after thorough mixing.174

Then, 1 mL of 20 mM chloranil was added into each tube, which was heated at 95 C with175

shaking for 60 min in a reciprocal shaking bath (Precision Scientific Inc., Chicago, IL). The176


P 9 f 39 J l f A i lt l d F d Ch i t


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mL of 12 M) was added into each tube, and the reaction solutions were kept in the dark for 15180

min after thorough mixing. Aliquots of the final reaction solutions (200 L) were added into each181

well of a 96-well plate after centrifuging for 3 min at 2500g, and absorbances were measured at182

490 nm using a MRX Microplate Reader with Revelation work station (Dynex Technologies,183

Inc., Chantilly, VA). Total flavonoids were expressed as micrograms of catechin equivalents per184

gram of dry weight sample. Data were reported as mean SD for three replicates.185

186

Measurement of Antioxidant Activity (ORAC). The antioxidant activity of the samples187

(ingredients, raw formulations and extruded products) were determined using the oxygen radical188

absorbance capacity (ORAC) assay described by Prior et al. (35) and modified in our laboratory189

(36). Briefly, 20 L of blank, trolox standard, or sample extracts in 75 mM potassium phosphate190

buffer, pH 7.4 (working buffer), was added to triplicate wells in a black, clear-bottom, 96-well191

microplate. The triplicate samples were distributed throughout the microplate and were not192

placed side-by-side, to avoid any effect on readings due to location. In addition, no outside wells193

were used, as use of those wells results in greater variation. A volume of 200 L of 0.96 M194

fluorescein in working buffer was added to each well and incubated at 37 C for 20 min, with195

intermittent shaking, before the addition of 20 L of freshly prepared 119 mM ABAP in working196

buffer using a 12-channel pipetter. The microplate was immediately inserted into a Fluoroskan197

Ascent FL plate reader (ThermoLabsystems) at 37 C. The decay of fluorescence at 538 nm was198

measured with excitation at 485 nm every 4.5 min for 2.5 h. The areas under the fluorescence199




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trolox standards minus the area under the curve for blank. ORAC values were expressed as mean202

micromoles of trolox equivalents (TE) per gram of dry weight sample SD for triplicate data.203

204

Cellular Antioxidant Activity (CAA) assay. The assay for CAA was performed following205

the procedures of Wolfe and Liu (37). Briefly, HepG2 liver cancer cells from the passages of 5206

and 7 were seeded at a density of 6 x 104

in 100 L complete growth medium per well on a 96-207

well microplate in a humidified 5 % CO2 incubator at 37 C. The wells on the boundary of the208

microplate were filled with 200 L of PBS. Twenty-four hours after seeding, the growth medium209

was removed and the wells were washed with PBS. The wells were treated in triplicate with 100210

L of solutions containing different concentrations of antioxidant extracts plus 25 M DCFH-211

DA dissolved in antioxidant treatment media for 1 h at 37 C. Then treatment mediums were212

removed and wells were washed with 100 L of PBS to remove extracellular residues. One213

hundred microliters of 600 M ABAP in oxidant treatment medium (HBSS) was applied to all214

the cells and the microplate was immediately placed into a Fluoroskan Ascent FL plate reader215

(ThermoLabsystems, Franklin, MA) at 37 C. Emission was measured for 1 h at 538 nm after216

excitement at 485 nm in every 5 min. The blank wells contained cells treated with DCFH-DA,217

HBSS and antioxidant extracts without ABAP whereas the control wells contained cells treated218

with DCFH-DA, HBSS and ABAP without antioxidant extracts.219

The area under curve for fluorescence (after subtraction of blank and initial fluorescence220

values) versus time was integrated to calculate the CAA value at each concentration of the221




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where SA is the integrated area under the sample fluorescence vs time curve and CA is the224

integrated area from the control curve. The median effective dose (EC50), i.e. dose required to225

give a 50% inhibition for sample extract, was determined from the median effective plot of226

log(fa/fu) vs log(dose), wherefa is the fraction affected (CAA unit) andfu is the fraction227

unaffected (1 CAA unit) by the treatment. In the experiments, quercetin was used as a228

standard.229

230

Statistical Analyses. All resultswere reported as mean SD for at least three analyses for231

each type of extraction and each extract was analyzed in triplicate. Results were subjected to232

ANOVA, and significance of differences between means were determined using Tukeys233

multiple comparison test run on SAS (version 9.1, SAS Institute Inc., Cary, NC). Correlations234

among various parameters were also investigated using Pearsons correlation coefficient.235

236

RESULTS237

Phenolic Contents. The phenolic contents of the ingredients, raw formulations, and extruded238

products were determined using the Folin-Ciocalteu method and expressed as g GAE/g DW239

sample (Figure 1). Free, bound, and total phenolics in purple potato flour (PPF) were 2008 240

118, 932 66, and 2940 183 g GAE/g DW sample, respectively. Dry pea flour (DPF) had241

366 20 55 2 and 421 19 g GAE/g DW sample of free bound and total phenolics242




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sample, respectively). The bound phenolic contents in the extruded products prepared from 35,246

50 and 65% PPF (175 8, 378 28, and 331 35 g GAE/g DW sample, respectively) were247

significantly lower (p < 0.05) than their raw formulations (415 26, 478 21 and 451 31 g248

GAE/g DW sample, respectively). The total phenolic contents of the extruded products prepared249

from 35, 50 and 65% PPF (2122 53, 3070 62 and 4308 64 g GAE/g DW sample,250

respectively) were significantly higher (p < 0.05) than their raw formulations (1334 51, 2030 251

39 and 2741 120 g GAE/g DW sample, respectively). Table 1 shows the percentage252

contributions of free and bound phenolics to the total phenolic content of raw ingredients and253

extruded products.254

255

Flavonoid Contents. The flavonoid contents of the raw ingredients, formulations and256

extruded products were determined using the SBC assay and were expressed as g catechin eq/g257

DW sample (Figure 2). The free, bound and total flavonoid contents of potato flour were 5495 258

42, 724 23, and 6219 417 g catechin eq/g DW sample, respectively. The free, bound, and259

total flavonoid contents of DPF were 1305 36, 731 71, and 2036 72 g catechin eq/g DW260

sample, respectively. The free flavonoid contents of the extruded products prepared from 35, 50261

and 65% PPF were 2606 220, 2929 153, and 3468 64 g catechin eq/g DW sample,262

respectively, and were significantly higher (p < 0.05) than their raw formulations (1944 100,263

2196 68, and 2890 103 g catechin eq/g DW sample, respectively). Extruded products264

prepared from 35, 50 and 65% PPF had significantly higher (p < 0.05) bound flavonoid contents265




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DW sample) was significantly higher (p < 0.05) than its raw formulation (2217 124 g269

catechin eq/g DW sample). Similarly, extruded products prepared from 50 and 65% PPF (3400 270149 and 3758 89 g catechin eq/g DW sample, respectively) were significantly higher (p


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However, no significant difference (p > 0.05) was observed in the content of totalp-coumaric291

acid in the extruded product prepared from 35% PPF than its raw formulation.292293

Total Antioxidant Activity. The total antioxidant activities of the ingredients, raw294

formulations and extruded products were determined using the ORAC assay and expressed as295

mol TE/g DW sample (Figure 3).The free, bound, and total ORAC values of PPF were 47.82296

1.66, 26.80 0.89, and 74.62 2.53 mol TE/g DW sample, respectively. The free, bound and297

total ORAC values of DPF samples were 8.86 0.88, 1.43 0.07, and 10.29 0.92 mol TE/g298

DW sample, respectively. The free ORAC values of extruded products prepared from 35, 50 and299

65% PPF (45.48 2.34, 75.29 5.32 and 113.83 6.67 mol TE/g DW sample, respectively)300

were significantly higher (p < 0.05) than these of their raw formulations (17.69 1.56, 33.36 301

4.27 and 57.80 1.44 mol TE/g DW sample, respectively). The bound ORAC values of302

extruded products prepared from 35 and 65% PPF (6.08 0.23, and 8.60 1.42 mol TE/g DW303

sample, respectively) were significantly lower (p < 0.05) than these of their raw formulations304

(10.28 0.52, and 12.25 0.64 /g DW sample, respectively). However, there was no significant305

difference (p > 0.05) in the ORAC values of the bound phytochemicals in the extruded products306

prepared from 50% PPF (9.93 0.54 mol TE /g DW sample) formulation compared to its raw307

formulation (11.58 1.74 mol TE /g DW sample). Extruded products prepared from 35, 50 and308

65% PPF had significantly higher (p < 0.05) ORAC values (51.57 2.57, 85.22 5.81 and309

122.43 7.52 mol TE/g DW sample, respectively) than these of their raw formulations (27.98310

gg y



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Cellular Antioxidant Activity. The CAA of raw formulations and extruded products were313

quantified using the protocol of the CAA method and expressed as EC50 in mg/mL and CAA314values in mol quercetin equivalent (QE)/g DW sample (Figure 4). The EC50 value of CAA of315

PPF sample was 52.1 4.0 mg/mL (CAA = 0.084 mol QE/g DW sample) whereas no CAA316

activity was detected in the DPF sample. The EC50 values of CAA of the extruded product317

prepared from 35% PPF (64.7 9.0 mg/mL) and 50% PPF (38.9 7.4 mg/mL) were318

significantly lower (p < 0.05) than those of their raw formulations (424.2 344 and 195.8 10319

mg/mL, respectively). However, no significant difference in the EC50 values of CAA was320

observed between the raw (77.8 19 mg/mL) and extruded products (70.3 4.4 mg/mL)321

prepared from 65% PPF. Similarly, the CAA values of the 35, 50 and 65% PPF raw formulations322

(0.017 0.018, 0.025 0.001, and 0.06 0.015 mol QE/g DW sample, respectively) and323

extruded products (0.56 0.008, 0.128 0.023, and 0.063 0.004 mol QE/g DW sample,324

respectively) followed the same trend as their EC50 values. The EC50 value of quercetin standard325

in all the replicates were in the range of 3.35 4.5 mg/mL, which were similar as reported by326

Wolfe & Liu (37).327

328

Correlation Analyses. Relationships among total phenolics, total ORAC values and CAA329

with individual phenolic acids and flavonoids in free and bound forms were determined using330

Pearsons correlation coefficient (Table 3). Free phenolic content was significantly correlated (p331

< 0.05) to total ORAC values (r2 = 0.98) and CAA values (r2 = 0.66). Similar correlation patterns332



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correlated (p < 0.05) to the total phenolics, ORAC and CAA values. p-Coumaric and ferulic336

acids in the bound and total extracts significantly correlated (p < 0.05) to the total phenolics,337ORAC and CAA values. However, caffeic acid did not show any positive correlation with338

phenolics, ORAC or CAA values. Total ORAC values were significantly correlated (p < 0.05)339

with CAA values (r2 = 0.69).340

341

DISCUSSION342

Phytochemicals in food samples are present in both free and bound form (11, 12). Without343

accounting for bound phytochemicals, the total phytochemical content will be underestimated344

(11). Therefore, studying free and bound phytochemicals provides accurate information on the345

total phytochemicals and the contributions of free, soluble conjugated and bound to the total346

phenolics and their total antioxidant activity. The cellular antioxidant activity assay, which is347

used in this study, is a biologically relevant way to quantify the bioactivity of antioxidants in348

food products as it takes into consideration the cellular uptake, metabolism, and distribution of349

bioactive compounds in the cell (38). The values of cellular antioxidant activities of samples350

were also compared with a chemical assay, ORAC. This study was designed to determine the351

phytochemicals, their relationships and contributions to total antioxidant activity in potato and352

dry pea flours, raw formulations, and processed snack foods applying extrusion technology on353

the raw formulations.354



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Colorado potato breeding program have shown that there was no difference among microwaved,359

boiled or baked Purple Majesty potatoes with skin, but all were slightly less than raw in total360phenolic contents (19). The percentage contribution of free phenolics to the total was 68%361

whereas bound phenolics contributed 32% in potato flour (Table 1). This is similar to the results362

of Chu et al., (40) who reported the contributions of 60 and 40% by free and bound phenolics,363

respectively, to the total phenolics in white potatoes with skin (40). Therefore, the total phenolic364

contents reported in the previous literature citations were underestimated by not including the365

bound phenolics in potatoes. Bound phenolics might be associated with the plant cell walls that366

survive upper gastrointestinal digestion and reach the colon where most of it is released by367

intestinal microflora (11, 12). Total phenolic content in dry pea flour in the present study368

(Figure 1) was 3 5 fold lower than in the reported literature (25, 26). This could be due to (i)369

use of previously milled flours compared to whole raw legumes by other researchers; (ii)370

different solvents for extraction of phenolic compounds (41). Free phenolics in dry pea flour371

contributed more (87%) to the total phenolics than the bound ones (13%). Raw formulations372

from potato and dry pea flours in selected concentrations had 69 84% free phenolics. Extrusion373

cooking of raw formulations increased the percentage contributions of free phenolics to the total374

and decreased the contributions from bound phenolics (Table 1). Increase in the total phenolics375

in the extruded food products (50 60%) rather than in raw formulations could be due to (i)376

breaking of conjugated phenolics into free phenolics, and (ii) leaching of soluble fibers, proteins377

and other non-phenolic soluble components such as mono-, di- and oligosaccharides (26). Han378



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season food legumes were significantly reduced (p < 0.05) when compared to uncooked samples381

(25).382Free and bound phytochemicals in potato flour contributed 64 and 36%, respectively, to the383

total ORAC antioxidant activity, similar to their contributions to the total phenolics. The total384

ORAC value in potato flour was in the range of hydrophilic ORAC values (28.25 250.67 mol385

TE/g DW) of Andean potato cultivars (39). Individual ORAC values of free and bound386

phytochemicals could not be compared due to limited information in the literature. The total387

ORAC value in dry pea flour was comparable to that reported in the literature (42) and had388

similar contributions from free (86%) and bound (14%) phytochemicals as was the case in total389

phenolics (Table 1). Han and Baik (26) reported free and bound phytochemicals contributed 68390

and 32% respectively, to the total TEAC antioxidant activity of yellow peas. Processing of all391

raw formulations using extrusion cooking significantly increased (p < 0.05) the total ORAC392

values of extruded products. Increase in the total ORAC values followed the same pattern as in393

total phenolics in the extruded products. It was also observed that ORAC values of bound394

phytochemicals significantly decreased (p < 0.05) and ORAC values of free phytochemicals395

increased in the extruded products. This phenomenon could be attributed to (i) breaking of396

conjugated phytochemicals to release free phytochemicals (31), (ii) prevention of enzymatic397

oxidation, and (iii) darker colors of the extruded products indicating formation of Maillard398

reaction products having antioxidant properties (43). ORAC values of processed green peas,399

yellow peas and chickpeas were significantly increased (p < 0.05) (27 114%, 12 67% and 25400



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by conventional and pressure boiling (25, 42). An increase in antioxidant activities of sweet corn,403

teas and tomatoes with thermal processing were also reported (4, 31, 44).404Quantities of free flavonoids in potato and dry pea flours were significantly higher (p < 0.05)405

when compared to their bound flavonoids. The total flavonoids of both flours followed similar406

pattern as free flavonoids, because of the larger contribution from free flavonoids (Table 1).407

There is limited literature available to compare the free, bound and total flavonoids in colored408

potatoes (45). The total flavonoids of dry pea flour in our study was higher when compared to409

the reported total flavonoid contents of whole (41, 46) and dehulled yellow peas (47). Quantities410

of free flavonoids increased after extrusion cooking of all the raw formulations. Interestingly, the411

contents of bound flavonoids were also significantly increased (p < 0.05) in the extruded412

products when compared to their raw formulations. Choi et al. (48) reported a significant413

increase of free flavonoids in Shiitake mushrooms after heat treatment at 100 and 121 C when414

compare to raw mushrooms. However, the investigators observed decreases in the bound415

flavonoids after heat treatment. Increase in flavonoid contents in the extrudates could be416

attributed to (i) disruption of plant cell walls providing better extractability, (ii) breaking of417

chemical bonds of higher molecular weight polyphenols and forming soluble low molecular418

weight polyphenol compounds, and (iii) inter-conversion of flavonoids in different forms (49).419

Variyar et al. (50) reported an increase in antioxidant potential of soybeans with the dose of-420

irradiation due to increased levels of genistin (an isoflavone) and degradation products of421

diadzein.422



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carcinogenesis (51).p-Coumaric acid was the major phenolic acid observed in potato flour,426

followed by chlorogenic, caffeic and ferulic acid (Table 2). Most of thep-coumaric and caffeic427acids were observed in the bound phenolic fraction whereas chlorogenic acid was higher in the428

free phenolics. Ferulic acid was observed only in the bound phenolics. Adom and Liu reported429

that more than 93% ferulic acids were present in the bound form in corn, wheat, oats and rice430

(11). The content of free chlorogenic acid we reported in this study was similar to the total431

chlorogenic content in Purple Majesty potatoes found in a Colorado Potato Breeding Program432

(19). Interestingly, our results demonstrated the bound phenolics contributed additional ~20% of433

total chlorogenic acid, which was commonly underestimated in the published literature. Lewis et434

al. (52) and Lachman et al. (53) reported in detail the presence of chlorogenic, caffeic, p-435

coumaric, ferulic and other phenolic acids in colored potatoes. Chlorogenic followed by ferulic436

and caffeic acids were detected mostly in the bound phenolics in dry pea flour whereas p-437

coumaric acid was not detected. Dry pea flour contained only chlorogenic acid in the free438

phenolic fraction. The content of total chlorogenic acid in this study was higher when compared439

to the reported content of whole yellow peas (25). The investigators, however, reported having440

p-coumaric acid along with chlorogenic and gallic acids in yellow peas. A multifold increase in441

chlorogenic acid in free and bound phenolic fractions of formulations rather than raw ingredients442

could be due to the binding ofo-hydroxy phenolic group in chlorogenic acid to protein via the443

bidentate hydrogen bond (54). The mechanism for the binding of chlorogenic acid to sunflower444

proteins involved both hydrogen bonding and covalent linkages between oxidized phenolics and445



22/40

in all extruded products were significantly higher (p < 0.05) when compared to their raw449

formulations (Table 2

). A similar pattern was observed in total chlorogenic acid contents in the450extruded products. Xu and Chang (25) reported significant increases in gallic, chlorogenic and451

total phenolic acids in yellow peas after pressure boiling. Significant decrea117 - 118ses in452

caffeic acid in bound fraction and total phenolics were observed in extruded products when453

compared to their raw formulations whereas those decreases were not detected in free phenolics.454

A decrease in caffeic acid contents with exposure of potato strips to home processing conditions455

was reported (56). Totalp-coumaric and ferulic acid contents were increased in extruded456

products compared to their raw formulations, mostly due to increases in their bound phenolics.457

However, caffeic,p-coumaric and ferulic acids were not detected in free phenolic extractions of458

extruded products. The undetected phenolic acids in extrudates may be due to alkaline hydrolysis459

that partly or completely broke down original phenolic acids. Increases in total chlorogenic, p-460

coumaric and ferulic contents in the extruded products could be attributed to the alkaline461

hydrolysis of caffeic acid present in conjugated phenolics. The reasons for change in the462

individual phenolic acids during extrusion cooking could be explained according to Fleuriet and463

Macheix (57) as (i) oxidative degradation of phenolic acids; (ii) break down of conjugated464

phenolics and release of free phenolic acids; and (iii) formation of complex phenolic acids.465

Increases in the contents of chlorogenic, p-coumaric and ferulic acids and low molecular466

phenolic acids from the breakdown of caffeic acid could have increased the total phenolics in the467

extruded products. Thermal decomposition of caffeic acid generated compounds such as468



23/40

as antioxidant activity. Significant positive correlations among individual phenolic acids except472

caffeic acid to the total phenolics and total ORAC antioxidant activity justify these assumptions.473

Increases in the total phenolics after processing and a strong correlation (r 2 = 0.9847) with474

ORAC antioxidant activity showed that phenolics were mostly responsible for the chemical475

antioxidant activity in the extruded products.476

The bioactivity of phytochemicals in free phytochemical extracts in the raw ingredients, raw477

formulations and extruded products were studied using the cellular antioxidant activity assay.478

Potato flour showed potent cellular antioxidant activity with an EC50 of 52 mg/mL. The lower479

EC50 values indicate higher cellular antioxidant activity. Dry pea flour had no measureable480

cellular antioxidant activity. Cellular antioxidant activities were observed in all the raw481

formulations and their extruded products (Figure 4). Extruded products prepared from 50% PPF482

had higher cellular antioxidant activity and lower EC50 value among the extrudates (p < 0.05),483

and also had much higher cellular antioxidant activity (p < 0.05) and lower EC50 value (p < 0.05)484

than that of its raw formulation. The effect of extrusion on the products prepared from 65% PPF485

was not significantly different (p > 0.05) in the EC50 or CAA values. Similar results were486

observed in the 35% PPF raw formulation; CAA values were increased in the extrudates when487

compared to its raw formulation (Figure 4). There was no specific pattern observed in EC50488

values of extruded products. It could be possible that products extruded from a 50% PPF raw489

formulation had a better composition of potato and dry pea flour for possible additive and490

synergistic effects of phytochemicals responsible for the higher cellular antioxidant activity as491



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when compared to the raw materials, which may be due to extrusion processing increasing the495

amount of bioaccessible phytochemicals as well as increased cellular uptake of these496

phytochemicals.497

In summary, a complete study on the contributions of free and bound phytochemicals to the498

total phenolics, flavonoids, ORAC antioxidant activity, and cellular antioxidant activity in499

Purple Majesty potato and dry pea flours was made. Raw formulations of these ingredients and500

extruded products prepared were analyzed in detail with regard to changes in individual phenolic501

acids, bound and free phytochemicals, and their contributions to ORAC antioxidant activities502

after extrusion. In most of the extruded products, the total phenolics, flavonoids and ORAC503

values increased after extrusion processing. Cellular antioxidants were observed in potato flour,504

raw formulations, and extruded products. Extrusion processing improved the cellular antioxidant505

activity of 50% PPF raw formulation. Measuring the antioxidant activity using the CAA assay in506

extruded food products is important for assessing the bioactivity of processed foods because it is507

more biologically relevant than chemical antioxidant assays (37). Further studies on the508

antioxidant activities of individual phytochemicals and changes during processing are needed to509

understand the detailed mechanism of breaking and formation of total phytochemicals and their510

contribution to bioactivity.511

512

ABBREVIATIONS USED513



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reducing/antioxidant power; ORAC, oxygen radical absorbance capacity; GAE, gallic acid518

equivalent; QE, quercetin equivalents; TE, Trolox equivalents; TEAC, Trolox equivalent519

antioxidant capacity; TRAP, total radical-scavenging antioxidant parameter.520

521

ACKNOWLEDGEMENT522

We gratefully acknowledge the assistance of Jams Pan and Matt, Processed Foods Research523

Unit, WRRC, USDA-ARS, Albany, CA for their assistance and feedback during the extrusion524

experiment. We also acknowledge the US Dry Pea and Lentil Council for their financial support525

for this study.526

527

LITERATURES CITED528

1) Burg, P.; Fraile, P., Vitamin C destruction during the cooking of a potato dish.529

Lebensmittel-Wissenschaft und-Technologie 1995, 28, (5), 506-514.530

2) Lathrop, P. J.; Leung, H. K., Rates of ascorbic acid degradation during thermal531

processing of canned peas.Journal of Food Science 1980, 45, (1), 152-153.532

3) Murcia, M A.; Lpez-Ayerra, B.; Martinez-Tom, M.; Vera, A. M.; Garca-Carmona, F.,533

Evolution of ascorbic acid and peroxidase during industrial processing of broccoli.534

Journal of the Science of Food and Agriculture 2000, 80, (13), 1882-1886.535



26/40

5) Eberhardt, M. V.; Lee, C. Y.; Liu, R. H., Nutrition - antioxidant activity of fresh apples.539

Nature 2000, 405, (6789), 903-904.540

6) Liu, R. H.; Hotchkiss, J. H., Potential genotoxicity of chronically elevated nitric oxide - a541

review.Mutation Research-Reviews in Genetic Toxicology 1995, 339, (2), 73-89.542

7) Cohen, J. H.; Kristal, A. R.; Stanford, J. L., Fruit and vegetable intakes and prostate543

cancer risk.Journal of the National Cancer Institute 2000, 92, (1), 61-68.5448) Lunet, N.; Lacerda-Vieira, A.; Barros, H., Fruit and vegetables consumption and gastric545

cancer: A systematic review and meta-analysis of cohort studies.Nutrition and Cancer-546

an International Journal 2005, 53, (1), 1-10.547

9) Liu, R. H., Potential synergy of phytochemicals in cancer prevention: mechanism of548

action.J. Nutr. 2004, 134, (12), 3479S-3485.549

10) Liu, R. H., Health benefits of fruit and vegetables are from additive and synergistic550

combinations of phytochemicals.Am J Clin Nutr2003, 78, (3), 517S-520.551

11) Adom, K. K.; Liu, R. H., Antioxidant activity of grains.Journal of Agricultural and Food552

Chemistry 2002, 50, (21), 6182-6187.553

12) Liu, R. H., Whole grain phytochemicals and health.Journal of Cereal Science 2007, 46,554

(3), 207-219.555

13) Pietta, P.-G., Flavonoids as antioxidants.Journal of Natural Products 2000, 63, (7),556

1035-1042.557

14) Friedman, M., Chemistry, biochemistry, and dietary role of potato polyphenols. A558



27/40

fleshed potatoes and their relation to antioxidant activity. Food Chemistry 2009, 114 (3),562

836 - 843.563

16) Brown, C. R.; Wrolstad, R.; Durst, R.; Yang, C. P.; Clevidence, B., Breeding studies in564

potatoes containing high concentrations of anthocyanins.American Journal of Potato565

Research 2003, 80, (4), 241-249.566

17) Nayak, B.; Berrios, J. J.; Powers, J. R.; Tang, J.; Ji, Y., Colored potatoes (Solanum567tuberosum L.) dried for antioxidant-rich value-added foods.Journal of Food Processing568

and Preservation (Published online April 1, 2011) 2011.569

18) Reyes, L. F.; Cisneros-Zevallos, L., Wounding stress increases the phenolic content and570

antioxidant capacity of purple-flesh potatoes (Solanum tuberosum L.).Journal of571

Agricultural and Food Chemistry 2003, 51, (18), 5296-5300.572

19) Stushnoff, C.; Holm, D.; Thompson, M. D.; Jiang, W.; Thompson, H. J.; Joyce, N. I.;573

Wilson, P., Antioxidant properties of cultivars and selections from the Colorado potato574

breeding program.American Journal of Potato Research 2008, 85, (4), 267-276.575

20) Wang, L. L.; Xiong, Y. L. L., Inhibition of lipid oxidation in cooked beef patties by576

hydrolyzed potato protein is related to its reducing and radical scavenging ability.577

Journal of Agricultural and Food Chemistry 2005, 53, (23), 9186-9192.578

21) Kanatt, S. R.; Chander, R.; Radhakrishna, P.; Sharma, A., Potato peel extract - a natural579

antioxidant for retarding lipid peroxidation in radiation processed lamb meat.Journal of580

Agricultural and Food Chemistry 2005, 53, (5), 1499-1504.581



28/40

23) Lamartiniere, C. A., Protection against breast cancer with genistein: a component of soy.584

American Journal of Clinical Nutrition 2000, 71, (6), 1705s-1707s.585

24) Pusztai, A.; Grant, G.; Buchan, W. C.; Bardocz, S.; de Carvalho, A. F. F. U.; Ewen, S. W.586

B., Lipid accumulation in obese Zucker rats is reduced by inclusion of raw kidney bean587

(Phaseolus vulgaris) in the diet.British Journal of Nutrition 1998, 79, (2), 213-221.588

25) Xu, B.; Chang, S. K. C., Phytochemical profiles and health-promoting effects of cool-589season food legumes as influenced by thermal processing.Journal of Agricultural and590

Food Chemistry 2009, 57, (22), 10718-10731.591

26) Han, H.; Baik, B. K., Antioxidant activity and phenolic content of lentils (Lens culinaris),592

chickpeas (Cicer arietinum L.), peas (Pisum sativum L.) and soybeans (Glycine max),593

and their quantitative changes during processing.International Journal of Food Science594

and Technology 2008, 43, (11), 1971-1978.595

27) Sun, J.; Chu, Y. F.; Wu, X. Z.; Liu, R. H., Antioxidant and anti proliferative activities of596

common fruits.Journal of Agricultural and Food Chemistry 2002, 50, (25), 7449-7454.597

28) de la Parra, C.; Serna Saldivar, S. O.; Liu, R. H., Effect of processing on the598

phytochemical profiles and antioxidant activity of corn for production of masa, tortillas,599

and tortilla chips.Journal of Agricultural and Food Chemistry 2007, 55, (10), 4177-600

4183.601

29) Adom, K. K.; Sorrells, M. E.; Liu, R. H., Phytochemical profiles and antioxidant activity602

of wheat varieties.Journal of Agricultural and Food Chemistry 2003, 51, (26), 7825-603



29/40

30) Singleton, V. L.; Orthofer, R.; Lamuela-Raventos, R. M., Analysis of total phenols and605

other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent.606

Oxidants and Antioxidants, Pt A 1999, 299, 152-178.607

31) Dewanto, V.; Wu, X. Z.; Liu, R. H., Processed sweet corn has higher antioxidant activity.608

Journal of Agricultural and Food Chemistry 2002, 50, (17), 4959-4964.609

32) Adom, K. K.; Sorrells, M. E.; Liu, R. H., Phytochemicals and antioxidant activity of610milled fractions of different wheat varieties.Journal of Agricultural and Food Chemistry611

2005, 53, (6), 2297-2306.612

33) Okarter, N.; Liu, R. H., Health benefits of whole grain phytochemicals. Critical Reviews613

in Food Science and Nutrition 2010, 50, (3), 193-208.614

34) He, X.; Liu, D.; Liu, R. H., Sodium borohydride/chloranil-based assay for quantifying615

total flavonoids.Journal of Agricultural and Food Chemistry 2008, 56, (20), 9337-9344.616

35) Prior, R. L.; Hoang, H.; Gu, L. W.; Wu, X. L.; Bacchiocca, M.; Howard, L.; Hampsch-617

Woodill, M.; Huang, D. J.; Ou, B. X.; Jacob, R., Assays for hydrophilic and lipophilic618

antioxidant capacity (oxygen radical absorbance capacity (ORAC(FL))) of plasma and619

other biological and food samples.Journal of Agricultural and Food Chemistry 2003, 51,620

(11), 3273-3279.621

36) Wolfe, K. L.; Kang, X.; He, X.; Dong, M.; Zhang, Q.; Liu, R. H., Cellular antioxidant622

activity of common fruits.Journal of Agricultural and Food Chemistry 2008, 56, (18),623

8418-8426.624



30/40

38) Spencer, J. P. E.; El Mohsen, M. M. A.; Rice-Evans, C., Cellular uptake and metabolism628

of flavonoids and their metabolites: implications for their bioactivity.Archives of629

Biochemistry and Biophysics 2004, 423, (1), 148-161.630

39) Andre, C. M.; Ghislain, M.; Bertin, P.; Oufir, M.; Herrera, M. D.; Hoffmann, L.;631

Hausman, J. F.; Larondelle, Y.; Evers, D., Andean potato cultivars (Solanum tuberosum632

L.) as a source of antioxidant and mineral micronutrients. Journal of Agricultural and633Food Chemistry 2007, 55, (2), 366-378.634

40) Chu, Y. F.; Sun, J.; Wu, X. Z.; Liu, R. H., Antioxidant and anti proliferative activities of635

common vegetables.Journal of Agricultural and Food Chemistry 2002, 50, (23), 6910-636

6916.637

41) Xu, B. J.; Chang, S. K. C., A comparative study on phenolic profiles and antioxidant638

activities of legumes as affected by extraction solvents.Journal of Food Science 2007,639

72, (2), S159-S166.640

42) Xu, B.; Chang, S. K. C., Effect of soaking, boiling, and steaming on total phenolic641

contentand antioxidant activities of cool season food legumes. Food Chemistry 2008,642

110, (1), 1-13.643

43) Nicoli, M. C.; Anese, M.; Parpinel, M., Influence of processing on the antioxidant644

properties of fruit and vegetables. Trends in Food Science & Technology 1999, 10, (3),645

94-100.646

44) Manzocco, L.; Anese, M.; Nicoli, M. C., Antioxidant properties of tea extracts as affected647



31/40

45) Christine, E. L.; John, R. L. W.; Jane, E. L., Changes in anthocyanin, flavonoid and650

phenolic acid concentrations during development and storage of coloured potato651

(Solanum tuberosum L) tubers.Journal of the Science of Food and Agriculture 1999, 79,652

(2), 311-316.653

46) Xu, B. J.; Yuan, S. H.; Chang, S. K. C., Comparative analyses of phenolic composition,654

antioxidant capacity, and color of cool season legumes and other selected food legumes.655Journal of Food Science 2007, 72, (2), S167-S177.656

47) Aharon, S.; Hana, B.; Yoram, K.; Ilan, S.; Michal, O.-S.; Shmuel, G., Determination of657

polyphenols, flavonoids, and antioxidant capacity in colored chickpea (Cicer arietinum658

L.).Journal of Food Science 2010, 75, (2), S115-S119.659

48) Choi, Y.; Lee, S. M.; Chun, J.; Lee, H. B.; Lee, J., Influence of heat treatment on the660

antioxidant activities and polyphenolic compounds of Shiitake (Lentinus edodes)661

mushroom. Food Chemistry 2006, 99, (2), 381-387.662

49) Coward, L.; Smith, M.; Kirk, M.; Barnes, S., Chemical modification of isoflavones in663

soyfoods during cooking and processing.American Journal of Clinical Nutrition 1998,664

68, (6), 1486s-1491s.665

50) Variyar, P. S.; Limaye, A.; Sharma, A., Radiation-induced enhancement of antioxidant666

contents of soybean (Glycine max Merrill).Journal of Agricultural and Food Chemistry667

2004, 52, (11), 3385-3388.668

51) Feng, R. T.; Lu, Y. J.; Bowman, L. L.; Qian, Y.; Castranova, V.; Ding, M., Inhibition of669



32/40

52) Lewis, C. E.; Walker, J. R. L.; Lancaster, J. E.; Sutton, K. H., Determination of673

anthocyanins, flavonoids and phenolic acids in potatoes. I: Coloured cultivars of Solanum674

tuberosum L.Journal of the Science of Food and Agriculture 1998, 77, (1), 45-57.675

53) Lachman, J.; Hamouz, K.; Orsak, M., Red and purple potatoes - A significant antioxidant676

source in human nutrition. Chemicke Listy 2005, 99, (7), 474-482.677

54) Mcmanus, J. P.; Davis, K. G.; Beart, J. E.; Gaffney, S. H.; Lilley, T. H.; Haslam, E.,678Polyphenol interactions .1. Introduction - Some observations on the reversible679

complexation of polyphenols with proteins and polysaccharides.Journal of the Chemical680

Society-Perkin Transactions 2 1985, (9), 1429-1438.681

55) Sastry, M. C. S.; Rao, M. S. N., Binding of chlorogenic acid by the isolated polyphenol-682

free 11s protein of sunflower (Helianthusa annuus) seed.Journal of Agricultural and683

Food Chemistry 1990, 38, (12), 2103-2110.684

56) Im, H. W.; Suh, B.-S.; Lee, S.-U.; Kozukue, N.; Ohnisi-Kameyama, M.; Levin, C. E.;685

Friedman, M., Analysis of phenolic compounds by high-performance liquid686

chromatography and liquid chromatography/mass spectrometry in potato plant flowers,687

leaves, stems, and tubers and in home-processed potatoes.Journal of Agricutural and688

Food Chemistry. 2008, 56, (9), 3341-3349.689

57) Fleuriet, A., Macheix, J. J., Phenolics in fruits and vegetables. In Flavonoids in Health690

and Disease, Rice-Evans, C., Packer, L, Ed. CRC Press: Boca Raton, FL, 2003; pp 1 - 42.691

58) Chen, J. H.; Ho, C. T., Antioxidant activities of caffeic acid and its related692



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696



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0

1000

2000

3000

4000

5000

PPF DPF Raw Extruded Raw Extruded Raw Extruded

Ingredients 35% Purple potato

flour formulation

50% Purple potato

flour formulation

65% Purple potato

flour formulation

Phenoliccontents

(ggallicacideq./gsample,D

W)

Free Bound Total

a

b

c

d

cd

d

e

ef ff

f

g

h

ii

jj

j j j k kl

m

697

Figure 1. Phenolic contents of ingredients, raw formulations, and extruded products prepared698

from purple potato flour and dry pea flour using a co-rotating twin screw extruder at 45 g/min699

feed rate, 300 rpm screw speed, 17 % (wet basis) feed moisture, and barrel zones set at700

80/100/110/120/130 C (die temperature at 130 C). Selected formulations for preparing701

extrudates contained x% of purple potato flour (PPF) and (100-x)% of dry pea flour (DPF). Bars702

with different letters are significantly different (p < 0.05).703



35/40

0

1000

2000

3000

4000

5000

6000

7000



flour formulation

50% Purple potato

flour formulation

65% Purple potato

flour formulation

Flavonoidcontents

(gcatechin/gsa

mple,D

W)

Free Bound Total

a

b

ccdcd

de dee

efe

ffg

fgfg

g

iii

kkk l

h

704

Figure 2. Flavonoid contents of ingredients, raw formulations and extruded products prepared705

from purple potato flour and dry pea flour using a co-rotating twin screw extruder at 45 g/min706



extrudates contained x% of purple potato flour (PPF) and (100-x)% of dry pea flour (DPF). Bars709




36/40

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140



flour formulation

50% Purple potato

flour formulation

65% Purple potato

flour formulation

AntioxidantActivity(ORAC)

(molTroloxeq./g

sample,D

W)

Free Bound Total

a

a

b

bcc c

dede

ee

e

f

f f

g hh hhhi i

k

711

Figure 3 Antioxidant activity of ingredients, raw formulations and extruded products prepared712from purple potato flour and dry pea flour using a co-rotating twin screw extruder at 45 g/min713



extrudates contained x% of purple potato flour (PPF) and (100-x)% of dry pea flour (DPF) . Bars716




37/40

0.000

0.050

0.100

0.150

0.200



flour formulation

50% Purple potato

flour formulation

65% Purple potato

flour formulation

CellularAntioxidantActivity

(molquercetineq./gsample,

DW)

0

100

200

300

400

500

EC50value(mg/mL)

CAA

EC50

a

b

d

d

c

c c

D

B C

D

A

CC

718

Figure 4. Cellular antioxidant activity and EC50 value of ingredients, raw formulations, and719

extruded products prepared from purple potato flour and dry pea flour using a co-rotating twin720

screw extruder at 45 g/min feed rate, 300 rpm screw speed, 17 % (wet basis) feed moisture, and721

barrel zones set at 80/100/110/120/130 C (die temperature at 130 C). Selected formulations for722

preparing extrudates contained x% of purple potato flour (PPF) and (100-x)% of dry pea flour723

(DPF). Bars with different small letters are significantly different (p < 0.05) for cellular724

antioxidant activity, whereas capital letters are significantly different (p < 0.05) for EC50.725



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Table 1. Percentage contributions of phytochemicals in free and bound extract of ingredients726

(purple potato flour and dry pea flour), raw formulations, and extruded products# to total727

phenolics, total antioxidant activity and total flavonoids (n=3).728

Total Phenolics Total Antioxidant Activity(ORAC value)

Total Flavonoids

Free(%)

Bound(%)

Free (%) Bound (%) Free(%)

Bound(%)

Purple potato flour (PPF) 68 32 64 36 88 12Dry pea flour (DPF) 87 13 86 14 64 36Raw 69 31 63 37 88 12

35% PPF*Extruded 92 8 88 12 82 18Raw 76 24 74 26 88 12

50% PPFExtruded 88 12 88 12 86 14Raw 84 16 83 17 93 7

65% PPFExtruded 92 8 93 7 92 8

* The formulation contains x% of purple potato flour and (100-x)% of dry pea flour.729

# Extruded products prepared from purple potato flour and dry pea flour using a co-rotating twin730

screw extruder at 45 g/min feed rate, 300 rpm screw speed, 17 % (wet basis) feed moisture, and731

barrel zones set at 80/100/110/120/130 C (die temperature at 130 C).732



39/40

38

Table 2. Quantity of free, bound, and total individual phenolic acids present in extracts of ingredients (purple potato flour and dry pea733

flour), raw formulations and extruded products# (Mean SD, n=3)734

Free(g/g sample, DW)

Bound(g/g sample, DW)

Total(g/g sample, DW)

Chloro Caffeic p-Coum Ferul Chloro Caffeic p-Coum Ferul Chloro Caffeic p-Coum FerulPPF 714 55d 141b 52 0.3b n.d. 202 8c 80155a 1019 34a 109 11b 916 55e 815 56a 1071 35a 109 11bDPF 2.0 1g n.d. n.d. n.d. 236 20b 15 3 f n.d. 22 3d 260 25g 15 3f n.d. 22 3d

Raw 298 24f 101c 61d n.d. 25327b 30645b 33410e 591c 55146f 31646b 34010e 591c35%PPF* Exd 98410c n.d. n.d. n.d. 58160a 776 e 36211d 18812a 156566c 777e 36211e 18812a

Raw 54077e 131b 378c n.d. 25333b 33939b 38754cd 577c 794107e 35339b 42547d 577c50%PPF Exd 2011211b n.d. n.d. n.d. 60960a 16817c 970113ab 17927a 2620170b 16817c 970113ab 17927a

Raw 101142c 234a 11718a n.d. 24321b 30952b 46335c 584c 125463d 33355b 58151c 584c65%PPF Exd 2967267a n.d. n.d. n.d. 57481a 937d 85773b 15921a 3541348a 937d 85773b 15921a

* The formulation contains x% of purple potato flour (PPF) and (100-x)% of dry pea flour (DPF).735

# Extruded products prepared from purple potato flour and dry pea flour using a co-rotating twin screw extruder at 45 g/min feed rate,736

300 rpm screw speed, 17 % (wet basis) feed moisture, and barrel zones set at 80/100/110/120/130 C (die temperature at 130 C).737

aValues in each column with no letters in common are significantly different (p < 0.05). Exd Extruded; Chloro Chlorogenic acid;738

p-Coum Coumaric acid; n.d. not detected.739

ACS Paragon Plus Environment



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39

Table 3. Correlation analysis of phenolics, antioxidant activity (ORAC) and cellular antioxidant activity of ingredients (purple potato740

flour and dry pea flour), raw formulations* and extruded product#.741

Free extract Bound extract Total

PhenolicsTotal

ORACvalue

CAA PhenolicsTotal

ORACvalue

CAATotal

Phenolics

TotalORACvalue

CAA

Phenolics 0.9780(

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