Accepted Manuscript

Title: Analysis of the content of bioactive compounds in

selected flours and enriched extruded corn products

Authors: Joanna Kolniak-Ostek, Agnieszka Kita, Anna Peksa,

Agata Wawrzyniak, Jadwiga Hamuka, Maria Jeznach,
Honorata Danilcenko, Elvyra Jariene

PII: S0889-1575(17)30216-8
DOI: http://dx.doi.org/10.1016/j.jfca.2017.08.008
Reference: YJFCA 2967

To appear in:

Received date: 16-11-2015

Revised date: 29-7-2017
Accepted date: 19-8-2017

Please cite this article as: Kolniak-Ostek, Joanna., Kita, Agnieszka., Peksa, Anna.,
Wawrzyniak, Agata., Hamuka, Jadwiga., Jeznach, Maria., Danilcenko, Honorata., &
Jariene, Elvyra., Analysis of the content of bioactive compounds in selected flours
and enriched extruded corn products.Journal of Food Composition and Analysis
http://dx.doi.org/10.1016/j.jfca.2017.08.008

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Analysis of the content of bioactive compounds in selected flours and enriched extruded

corn products

Joanna Kolniak-Osteka, Agnieszka Kita*b, Anna Pksab, Agata Wawrzyniakc, Jadwiga

Hamukac, Maria Jeznachc, Honorata Danilcenkod, Elvyra Jariened

a
Department of Fruit,Vegetable and Plant Nutraceutical Technology, Faculty of

Biotechnology and Food Science, Wrocaw University of Environmental and Life Sciences,

Chemoskiego 37, 51-630 Wrocaw, Poland

b
Department of Food Storage and Technology, Faculty of Biotechnology and Food Science,

Wrocaw University of Environmental and Life Sciences, Chemoskiego 37, 51-630

Wrocaw, Poland
c
Faculty of Human Nutrition and Consumer Sciences, Warsaw University of Life Sciences

(SGGW-WULS), Nowoursynowska 159C, 02-776 Warsaw, Poland

d
Agriculture and Food Sciences Institute, Aleksandras Stulginskis University, Kaunas, Lithuania

Corresponding author:

Tel.: +48 71 3207762, fax: +48 71 32057767

E-mail address: Agnieszka.Kita@wnoz.up.wroc.pl

Highlights

The polyphenols in the 3 flours (Jerusalem artichoke, pumpkin, amaranth) were

identified

Phenolics, carotenoids, antioxidant capacity in flours and snacks were determined

1
 Amaranth and J. artichoke flours increased polyphenol content in corn snacks

Pumpkin flour increased carotenoids (lutein) content in corn snacks

Abstract

The aim of this study was to identify the phenolic compounds in the amaranth,

pumpkin and Jerusalem artichoke and compare the content of phenolic compounds and

carotenoids, as well as antioxidant capacity in flours and extruded corn snacks enriched with

different additives. The addition of 10% flour from pumpkin tissue, Jerusalem artichoke and

amaranth seeds was used during extrusion. Twenty phenolic compounds in amaranth, twenty-

six in Jerusalem artichoke and sixteen in pumpkin were determined in the examined samples

using the UPLC-PDA-Q/TOF-MS method. The addition of flours to snacks increased

phenolics and carotenoids concentration. The highest lutein and -carotene concentration

(262.7 g/100 g) was reported in snacks with the addition of pumpkin flour, while the highest

polyphenolics content (1034.2 mg/100 g) in snacks obtained with Jerusalem artichoke flour.

The use of 10% Jerusalem artichoke and amaranth flours in the corn snacks production may

significantly increase the content of polyphenolic compounds in newly designed products,

while the use of pumpkin flour may increase the content of polyphenols and carotenoids

(mainly lutein) simultaneously affecting the antioxidant capacity of the final product.

Keywords: food composition, food analysis, phenolics, betalains, carotenoids, antioxidant

capacity, amaranth (Amaranthus cruentus L.), pumpkin (Cucurbita maxima L.), Jerusalem

artichoke (Helianthus tuberosus L.), extruded corn snacks

2
 1. Introduction

The expectations of contemporary consumers in relation to health benefits derived

from foods, which they ingest, including cereal products, has led to the search for innovative

products on the snack market. Extruded cereal products have become popular due to their

high stability and durability. However they are often the primary source of energy in the diet,

which makes them suitable for enrichment with bioactive compounds (Tonyali et al., 2013).

Presently, the addition of dried cranberries and blueberries (Camire et al., 2007), leguminous

additives (Anuonye et al., 2010; Diaz et al. 2013; Pastor-Cavada et al., 2011), granules from

purple potatoes (Nayak et al., 2011), omega-3 fatty acids (Paras and Hardeep, 2012), dried

broccoli (Bisharat et al., 2013) as well as tomatoes (Dehghan-Shoar et al., 2010; Tonyali et

al., 2013) to traditional cereal extrudates is proposed and practiced in experiments and tests.

The suggested addition of vegetables, such as tomatoes or broccoli to traditional cereal

extrudates may increase the consumption of carotenoids by consumers, while red or purple

coloured fruits and vegetables increased polyphenols. Carotenoids are a ubiquitous fat soluble

group of isoprenoid pigments, which, besides chlorophyll and anthocyanins, belong to the

most important pigments occurring in nature. Their sources are orange and red fruits and

vegetables as well as green leafy vegetables. Animals and humans cannot synthesise

carotenoids de novo. The biological significance of these compounds is based first of all on

their antioxidant properties. Carotenoids can prevent the formation and activity of reactive

oxygen species, thereby protecting against cancer and cardiovascular disorders. Furthermore,

they regulate gene expression, improve eyesight (lutein and zeaxanthin) and positively impact

on the immune system. Some carotenoids, for example -carotene, are precursors of vitamin

A (Chvez-Juregui et al., 2010; Norfezah et al., 2011; Hock-Eng Khoo et al., 2011; Murphy

et al., 2014).

3
 Polyphenols are a heterogeneous group of secondary metabolites in plants, which are

vital for plant development. Due to the variety of chemical structure, polyphenols are divided

into phenolic acids (hydroxycinnamic, hydroxybenzoic) and flavonoids (Majewska and

Czeczot, 2009). Phenolic compounds are commonly found in both edible and nonedible parts

of plants, and have been reported to have multiple biological effects. It has been suggested

that the possible health benefits of polyphenol consumption are derived from their antioxidant

and anti-inflammatory properties (Borges et al., 2010). They are associated with a reduced

risk of cancer, cardiovascular diseases, atopic dermatitis, diabetes and Alzheimers disease

(Krishnaiah et al., 2011; Conforti et al., 2008). Potential sources of antioxidant compounds

have been investigated in several types of plant material such as fruits, vegetables, leaves,

oilseeds, cereal crops, barks and roots, spices and herbs, and crude plant drugs (Khknen et

al., 1999).

Since the enrichment of foods with antioxidant compounds seems to be useful in

human nutrition and beneficial from the health point of view, it appears worthwhile to

determine the content of carotenoids and polyphenols as well as the antioxidant potential of

newly designed extruded corn products enriched with 10% flour from pumpkin, amaranth or

Jerusalem artichoke as a natural source of carotenoids and/or polyphenols.

The purpose of the present study was to identify the phenolic compounds in the

amaranth, pumpkin and Jerusalem artichoke and compare the content of phenolic compounds

and carotenoids, as well as antioxidant capacity, in flours as well as in extruded corn products

with the addition of 10% of the aforementioned flours.

2. Materials and methods

2.1. Reagents and standards

Formic acid, methanol, kaempferol 3-O-glucoside, isorhamnetin 3-O-rutinoside and 3-O-

glucoside were purchased from Sigma-Aldrich (Steinheim, Germany). Acetonitrile was

4
purchased from Merck (Darmstadt, Germany). Quercetin, quercetin 3-O-glucoside, quercetin

3-O-rutinoside, p-coumaric, ferulic and quinic acid, procyanidin A2 were purchased from

Extrasynthese (Lyon, France). All solvents (e.g. acetonitrile purity: 99.9%, hexane purity:

98.5%, dichloromethane purity: 99.9%, methanol purity: 99.5%) used were of high-

performance liquid chromatography (HPLC) quality and the other chemicals were of

analytical grade. They were purchased from Sigma (St. Louis, MO, USA). The carotenoids

(standards): -carotene C4582, lutein X6250, lycopene L9879 were also purchased from

Sigma (St. Louis, MO, USA).

2.2. Preparation of the experimental flours

Freshly harvested Jerusalem artichoke tubers (Helianthus tuberosus L.) cv. Sauliai,

pumpkin fruits (Cucurbita maxima L.) cv. Karovita, and amaranth seeds (Amaranthus

cruentus L.) cv. Geltoniukiai were used in this experiment. Flours were prepared under the

described laboratory conditions using the material organically grown in Lithuanian farms and

originated from harvest experiment led by Agriculture and Food Sciences Institute at

Aleksandras Stulginskis University in Lithuania in 2011 year. Tubers (25 kg sample) were

brushed, washed and sliced (1.01.5 mm thickness). The peel and seeds of pumpkin fruits

(25 kg sample) were removed and the flesh cut into slices with a thickness of 2-3 mm.

Prepared pieces of raw material uniformly layered in a tray were dried for 24 h at the

temperature of 55oC in a dryer with system forced air flow (Venticell 111Comfort MMM

Medcenter Einrichtungen, Planegg, Germany). The amaranths seeds (15 kg sample) were

dried in the same dryer at 45oC for 48 hours. The dried material was ground on a knife mill

GRINDOMIX GM 200 (Retsch, Haan, Germany) and amaranths mill additionally sieved

through a 200 m nylon sieve. The obtained flours were packed in airtight containers prior to

use. The corn grits (0.75-1.25 mm) used for the experiment were obtained from local retail

market (Bio-Corn Company, Zibice, Poland).

5
2.3. Extruded snacks with flours addition

Extruded corn snacks with the addition of 10% flour from pumpkin tissue, Jerusalem

artichoke or amaranth seeds as well as without additives (control sample) were obtained

industrially in one of processing plants in Lithuania (JSC Naujasis Nevezis, Kaunas,

Lithuania). Extrusion was carried out using high shear single-screw extruder type KESH-

3 (Te , Kirovograd, Ukraine) with a 4.0 mm nozzle. The screw speed was

180 rpm and the temperatures of the barrel were set at 160, 170 and 180 oC for the feed zone,

metering zone, and compression zone, respectively. Samples of 10 kg of snacks were taken

for laboratory analyses. The analyses were carried out in the years 2013-2014.

2.4. UPLCPDAMS analysis

2.4.1 Extraction procedure

For the extraction, a protocol described before by Kolniak-Ostek and Oszmiaski

(2015) was followed. The powder samples of artichoke, pumpkin and amaranth (0.2 g) were

extracted with 5 mL of 38% methanol acidified with 1% of acetic acid and containing 1%

ascorbic acid as an antioxidant. The extraction was performed by incubation for 20 min under

sonication (Sonic 6D, Polsonic, Warsaw, Poland) and with occasional shaking. Next, the

slurry was centrifuged at 19,000 g for 10 min, and the supernatant was filtered through a

hydrophilic PTFE 0.20 m membrane (Millex Samplicity Filter, Merck, Darmstadt,

Germany) and used for analysis.

2.4.2 Identification of phenolics and betalains

Identification of polyphenols of artichoke, pumpkin and amaranth extracts was carried out

using an ACQUITY Ultra Performance LC system equipped with a photodiode array detector

with a binary solvent manager (Waters Corporation, Milford, MA, USA) with a mass detector

G2 Q-Tof micro mass spectrometer (Waters Corporation, Milford, MA, USA) equipped with

an electrospray ionisation (ESI) source operating in negative and positive modes set to the

6
base peak intensity (BPI) chromatograms, and scaled to 12,400 counts per second (cps)

(100%) (Kolniak-Ostek and Oszmiaski, 2015). Separations of individual polyphenols were

carried out using a UPLC BEH C18 column (1.7 m, 2.1 100 mm, Waters Corporation,

Milford, MA, USA) at 30C. The samples (10 mm3) were injected, and the elution was

completed in 15 min with a sequence of linear gradients and isocratic flow rates of 0.45 cm3

min1. The mobile phase consisted of solvent A (0.1% formic acid, v/v) and solvent B (100%

acetonitrile). The program began with isocratic elution with 99% solvent A (01 min), and

then a linear gradient was used for 12 min, lowering solvent A to 0%; from 12.5 to 13.5 min,

the gradient returned to the initial composition (99% A), and then was held constant to re-

equilibrate the column. The analysis was carried out using full-scan, data-dependent MS

scanning from m/z 100 to 2000. Leucine enkephalin was used as the reference compound at a

concentration of 500 pg/L, and a flow rate of 2 L/min, and the [M-H]- ion at 554.2615 Da

and [M+H]+ ion at 556.2771 were detected. The [M-H]-/[M+H]+ ions were detected during a

15 min analysis performed within ESIMS accurate mass experiments, which were

permanently introduced via the LockSpray channel using a Hamilton pump. The lock mass

correction was 1.000 for the mass window. The optimised MS conditions were as follows:

capillary voltage of 2500 V, cone voltage of 30 V, source temperature of 100C, desolvation

temperature of 300C, and desolvation gas (nitrogen) flow rate of 300 dm3/h. Collision-

induced fragmentation experiments were performed using argon as the collision gas, with

voltage ramping cycles from 0.3 to 2 V. Characterisation of the single components was

carried out via the retention time and the accurate molecular masses. Each compound was

optimised to its estimated molecular mass in the negative and positive mode, before and after

fragmentation. The data obtained from UPLCMS were subsequently entered into the

MassLynx 4.0 ChromaLynx Application Manager software (Waters, Manchester, UK). On

the basis of these data, the software is able to scan different samples for the characterised

7
substances. The runs were monitored at the following wavelengths: flavan-3-ols at 280 nm,

phenolic acids at 320 nm, flavonol glycosides at 360 nm and betalains at 475 and 540 nm. The

identification of phenolic compounds was performed using reference compounds selected

based on the principle of structure-related target analyte/standard (chemical structure or

functional group). 3-Caffeoylquinic acid was used to identify quinic acid, caffeoylquinic acid

and di-O-caffeoylquinic acid isomers. Caffeic acid was used to identify caffeic acid and their

derivatives. Ferulic acid was used to identify ferulic acid derivatives. p-Coumaric acid was

used, as well as for its own identification, to identify p-coumarylquinic acid and p-

coumaroylhexose. Procyanidin A2 was used for the identification of A-type procyanidins.

Kaempferol 3-O-glucoside was used for the identification of kaempferol O--L-rhamnoside

and kaempferol 3-O-6-acetylglucoside. Quercetin 3-O-rutinoside and 3-O-glucoside were

used to identify quercetin derivatives. For isorhamnetin derivatives identification,

isorhamnetin 3-O-rutinoside and 3-O-glucoside were used.

2.5. Determination of carotenoids

Carotenoids were extracted and quantified using previously described methods with

modifications (Murkovic et al., 2002). In order to obtain representative samples for analysis,

before the weighing, each product was mixed thoroughly and homogenised (homogeniser

IKA-WERTKE T25 BASIC, 13.500 oscillations/min, Staufen, Germany). Following this, the

products were weighed in the amount of 1.0 g and 20 mL methanol as well as 0.2 mL 2%

alcoholic solution of hydrochinon were added. The prepared samples were homogenised

again to obtain a uniform consistency. The extraction of carotenoids from the samples was

performed with the use of petroleum ether. The ether was added 4-5 times in the amounts of

20 mL depending on the colour intensity of the samples. The ether extracts were poured and

filtered through filter paper into a laboratory cylinder. The collected ether extracts were

8
thoroughly mixed and later evaporated in nitrogen atmosphere on a heated water bath

(Memmert WB22, Schwabach, Germany). The evaporated residue was dissolved in 0.5 mL

hexane. Analysis was conducted with the use of HPLC UV-VIS detector (Gilson Company,

Middleton, WI, USA). Hexane solutions of carotenoids were injected onto the

chromatographic column C18 RP (Vydac 201 TP54, Vydac, Hesperia, CA, USA; 250 x 4.6

mm, 5 m) with a pre-column (10 x 4.6 mm, 5 m) from the same producer. The mobile

phase applied was acetonitrile/hexane/dichloromethane/methanol (50/20/20/10; v/v), with

0.1% BHT as antioxidant, at the flow rate of 1.0 mL/min at the wavelength of 450 nm. The

obtained results were compared to standard curves performed with the use of lutein, -

carotene and lycopene standards and expressed per 100 g of dry weight (DW). Data are

reported as mean value standard deviation (SD) for three measurements. Within-run and between-

run coefficients of variation for carotenoids were <5% and <8%, respectively.

2.6 Determination of total phenolics and antioxidant activity

The total polyphenol content (TPC) of the extracts was determined using Folin-

Ciocalteu colorimetric method as described by Singleton et al. (1999) with modifications. One

mL of studied extract and 1 mL of Folin-Ciocalteu reagents was pipetted into a 100 mL

volumetric flask. After 3 min, 10 mL of 20% aqueous solution of sodium carbonate (Na2CO3)

was added, and the flask was brought to volume with distilled water. The absorbance at 765

nm was measured after 1 hour and the results were expressed as mg of gallic acid equivalents

per 100g of dry weight (DW). Data are reported as a mean value standard deviation (SD)

for six measurements.

The antioxidant activity of polyphenols extracts was determined using the Trolox

equivalent antioxidant capacity (TEAC) with 2,2'-azino-bis-3-ethylbenzothiazoline-6-

sulphonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals as well as the

9
ferric reducing ability of plasma assay (FRAP). The TEAC assay with ABTS radical was

carried out according to Re et al. (1999), while the TEAC with DPPH radical was according

to the procedure described by Yen and Chen (1995). The total antioxidant potential of sample

using the FRAP assay was carried out with the method proposed by Benzie and Strain (1996).

TEAC results are expressed as millimoles of Trolox equivalents per 100 grams of DW of

product. Data are reported as mean value SD for three measurements.

2.7. Statistical analyses

Statistical analysis was performed using Statgraphic software (StatPoint, Cracow,

Poland). Data were subjected to one-way analysis of variance for mean comparison;

significant inter-ingredient differences were calculated according to HSD Tukeys multiple

range test. Data are reported as mean standard deviation (SD). Differences at p < 0.05 were

considered to be statistically significant.

3. Results and discussion

3.1. UPLCPDAMS identification of phenolics and betalains

Tables 1, 2 and 3 and Figures 1, 2 and 3 shows the list of compounds identified in the

Jerusalem artichoke, pumpkin and amaranth extracts, respectively, through UPLC-MS/MS

(with PDA and Q/TOF detectors) experiments, along with their retention times (Rt), and a

comparison with standard reference compounds, when available.

3.1.1. Jerusalem artichoke

Table 1 lists 26 compounds identified in the Jerusalem artichoke. Molecules that were

certainly or putatively identified in negative ion mode belong to the compound group of

10
phenolic acids, flavan-3-ols and flavonols. Compounds 1-4, 7, 8, 12, 13, 15-20 and 26 were

identified in Jerusalem artichoke for the first time.

Compound 1 was identified as quinic acid, based on its [M-H]- ion at m/z 191.0185 and on the

resulting product ions at m/z 173, 127 and 111, which is consistent with the data reported in

the literature for this compound (Santos et al., 2012), and with authentic standard. Compounds

3, 12, 16, 17 and 18 have pseudomolecular ions at m/z 371.0636 that fragmented into m/z 353

and 209, owing to the loss of H2O (18 Da) and hexose (162 Da) residues. Those compounds

were therefore tentatively identified as hydroxyferulic acid hexoside isomers, on the basis of

data in the literature (Bamawa et al., 2013), and compared with authentic standard of ferulic

acid. Compounds 4, 7, 8, 19, 20 and 26 were identified as caffeoylhexose isomers due to their

[M-H]- at m/z 341.1075, which fragmented in m/z 179, owing to the loss of hexose residue

(162 Da). Compounds 6, 10, 23 and 24 with [M-H]- at m/z 353.0879, that fragmented to

generate m/z at 191.0552 (quinate ion), were identified as caffeoylquinic acid hexosides, by

comparing them with the retention times and mass profile of authentic standards of

chlorogenic, cryptochlorogenic and neochlorogenic acids, and with literature data (Fritsche et

al., 2002). Peak 9 had an [M-2H]-2 at m/z 707.1852 and was identified as a caffeoylquinic acid

dimer. The MS/MS fragmentation gave an ion with [M-H]- at m/z 353.0879, owing the loss of

caffeoylquinic acid (353 Da), and fragment with m/z at 191, corresponding to quinic acid.

Compound 11 was identified as caffeic acid based on its [M-H]- ion at m/z 179.0321 and on

the resulting product ion at m/z 135, which is consistent with retention time and mass spectra

of authentic standards, and with the literature data (Snchez-Rabaneda et al., 2003).

Compound 13, with its [M-H]- at m/z 337.0932 that fragmented at m/z 191 and 163,

corresponding to quinic and p-coumaric acid, respectively, was identified as p-

coumaroylquinic acid. Compounds 14, 21, 22 and 25 have pseudomolecular ions at m/z

515.1196 that fragmented in m/z 353 and 335, owing to the loss of hexose (162 Da) and H2O

11
(18 Da) residues. Those compounds were therefore tentatively identified as dicaffeoylquinic

acid isomers, on the basis of literature data (Snchez-Rabaneda et al., 2003). Compound 15

was identified as feruloylquinic acid, based on its [M-H]- ion at m/z 367.1041 and on the

resulting product ion at m/z 191, which is consistent with the data reported in the literature for

this compound (Guarnerio et al., 2012), and with authentic standards of ferulic and quinic

acids.

Compound 2 with its [M-H]- at m/z 1151.3739 and typical fragmentation pattern at m/z 863,

577 and 289 was identified as A-type procyanidin tetramer, on the basis of literature data

(Oszmiaski et al., 2015) and compared with an authentic standard of procyanidin A2. Peak 5

with its [M-H]- at m/z 299.0784 was reported previously in Jerusalem artichoke (Snchez-

Rabaneda et al., 2003), and was therefore identified as chrysoeriol.

3.1.2. Pumpkin

16 compounds identified in the pumpkin extracts, belong to the compound group of phenolic

acids, flavonols, organic acids, amino acids and others. Compounds 1 and 2 were identified

for the first time in pumpkin.

Compounds 1 and 2 with their [M-H]- at m/z 191.0185 and 325.1023, were identified as

quinic acid and p-coumaroylhexose, respectively, based on retention time and characteristic

mass spectra of authentic standards. Compounds 3 and 15 with their [M-H]- at m/z 229.0784

and typical fragmentation pattern at m/z 137 was tentatively identified as hydroxybenzoic acid

hexosides, on the basis of literature data (Iswaldi et al., 2013). Compound 9 was identified as

protocatechuic acid, based on its [M-H]- at m/z 153.0204 and on the resulting product ion at

m/z 109, which is consistent with the data reported in the literature for this compound (Iswaldi

et al., 2013). Compound 10, with [M-H]- at m/z at 163.0406 and with product ion at m/z 119,

was identified as p-coumaric acid, which is consistent with the data reported in the literature

for this compound (Iswaldi et al., 2013), and with an authentic standard. Compound 12 was

12
identified as sinapic acid hexoside, based on literature data (Iswaldi et al., 2013) and on its

[M-H]- at m/z 385.1701, which fragmented at 223, owing to the loss of hexose (162 Da).

Compound 11 had an [M-H]- at m/z 769.2181, and was identified as isorhamnetin 3-

rutinoside-7-rhamnoside. The MS/MS fragmentation gave an ion with [M-H]- at m/z 623

owing the loss of rhamnoside (146 Da), and m/z at 315, owing the loss of rutinoside (308 Da),

which is consistent with literature data (Iswaldi et al., 2013). Compounds 13 and 14 with their

[M-H]- at m/z 489.2101 and 431.1754, which fragmented at m/z 285, were identified as

kaempferol O-(6-O-acetyl-glucoside) and kaempferol O--L-rhamnoside, respectively, based

on literature data (Iswaldi et al., 2013). Compound 16 was identified as quercetin, based on its

[M-H]- ion at m/z 301.2023, which is consistent with the authentic standard of quercetin.

Compounds 4 and 5 with their [M-H]- at m/z 365.1381 and typical fragmentation pattern at

m/z 203 was tentatively identified as tryptophan hexosides, on the basis of literature data

(Iswaldi et al., 2013).

Compound 12 was identified as ispropylmalic acid, based on literature data (Iswaldi et al.,

2013) and on its [M-H]- at m/z 175.0585, which fragmented at 115, owing to the loss of

isopropyl alcohol (60 Da).

Compounds 7 and 8 with their [M-H]- at m/z 293.1230 and fragmentation ion at m/z 131 was

tentatively identified as ethyl 3-hydrobutanoate hexosides, on the basis of literature data

(Iswaldi et al., 2013).

3.1.3. Amaranth

Table 3 shows the list of 20 compounds identified in the amaranth extracts. Molecules that

were certainly or putatively identified in negative ion mode belong to the compound group of

phenolic acids and flavonols and molecules that were identified in positive ion mode belong

to the compound group of betalains. Compounds 1, 2, 5 and 10 were identified for the first

time in amaranth.

13
Compound 1 was identified as quinic acid, based on its [M-H]- ion at m/z 191.0185 and on the

resulting product ions at m/z 173, 127 and 111, which is consistent with the data reported in

the literature for this compound (Santos et al., 2012), and with authentic standard. Compounds

2 and 10 were identified as caffeoylhexose isomers, based on their [M-H]- at m/z 341.1085

and on the resulting product ion at m/z 179, which is consistent with retention time and mass

spectra of authentic standard of caffeic acid. Compounds 3 and 14 with their [M-H]- at m/z

337.0448, which fragmented at m/z 191 and 163, corresponding to quinic and p-coumaric

acid, respectively, was identified as p-coumaroylquinic acid isomers (Kolniak-Ostek et al.,

2013). Compound 5 was identified as p-coumaroylhexose, due to their [M-H]- at m/z

325.0912, which fragmented in m/z 191, owing to the loss of caffeic acid residue (136 Da).

Peak 15 had a pseudomolecular ion at m/z 609.1434 that fragmented in m/z 301, owing to the

loss of rhamnose (146 Da) and glucose (162 Da) residues. This compound was therefore

tentatively identified as quercetin 3-O-rutinoside (rutin), based on its retention time and

compared with those of corresponding authentic standards, and with literature data (Stintzing

et al., 2004). Compound 16 was identified as quercetin 3-O-hexoside based on its [M-H]- at

m/z 463.0843 and the corresponding loss of 162 mass units, which indicates the presence of a

hexose unit. Additionally, the fragmentation pathways observed and the retention times match

those of authentic standards, and with literature data (Kolniak-Ostek et al., 2015, Stintzing et

al., 2004). Compound 20 was identified as feruloylquinic acid, based on its [M-H]- ion at m/z

367.1354, which is consistent with the data reported in the literature for this compound

(Stintzing et al., 2004), and with authentic standards.

Compounds 4 and 6 have an [M+H]+ at m/z 727.1846 and were identified as amaranthine and

its C15-epimer isoamarantine (Cai et al., 2001). The MS/MS fragmentation gave an ion with

[M+H]+ at m/z 551, owing to the loss of glucuronic acid (176 Da), and fragment with m/z at

389, corresponding to the loss of glucose (162 Da). Compounds 7, 8 and 17 have an [M+H]+

14
at m/z 551.1387 and were identified as betanine and isobetanine isomers, respectively

(Stintzing et al., 2004). The MS/MS fragmentation gave an ion with [M+H]+ at m/z 389,

corresponding to the loss of glucose (162 Da). Compound 9 had a pseudomolecular ion at m/z

389.1766 that fragmented in m/z 345, owing to the loss of CO2 (44 Da) residue. This

compound was therefore tentatively identified as betanidin, on the basis of literature data (Jerz

et al., 2008). Compound 11 with its [M+H]+ at m/z 713.1904 ad fragmentation ions at m/z 551

and 389 was tentatively identified as isobetanin glucoside (Wybraniec et al., 2009).

Compound 12 was tentatively identified as isoleucine-isobetaxantin, based on its [M+H]+ at

m/z 325.1190 and literature data (Wybraniec et al., 2009). Compound 13 had an [M+H]+ at

m/z 549.1491 and was identified as neobetanin (Jerz et al., 2008). The MS/MS fragmentation

gave an ion with [M+H]+ at m/z 387, owing the loss of hexose (162 Da), and fragment with

m/z at 343, corresponding to the loss of CO2 (44 Da). Compound 18 was identified as 4'-O-

malonyl-isobetanin, based on its [M+H]+ ion at m/z 637.1697, which is consistent with the

data reported in the literature for this compound (Wybraniec et al., 2009). Compound 19 had

an [M+H]+ at m/z 323.1199 and was identified as isovulgaxantin.

3.2. Carotenoids content

The highest content of carotenoids was found in pumpkin flour (in total 5718 mg 100

g-1 DW) (Table 4), whereby 3/4 of the content was represented by lutein (4307 mg 100 g-1

DW), 17% constituted -carotene (970 mg 100 g-1 DW) and 8% lycopene (442 mg 100 g-1

DW). Corn flour, which dominated in all products and was the basis for the production of

extruded corn snacks, contained only lutein, which was confirmed by amount of lutein in

extruded corn snacks without additives (127 mg 100 g-1 DW). Flour obtained from amaranth

and Jerusalem artichoke, which was a source of beneficial bioactive compounds, among

others polyphenols, was found to contain only small quantities of lutein (respectively 14 or

6.9 mg 100 g-1 DW). It is worth highlighting that the addition of 10% flour from pumpkin to

15
extruded corn snacks brought about an increase in antioxidant potential of the final product as

well as the content of carotenoids (nearly twice), which was the result of the significant

amount of these compounds as well as polyphenols from pumpkin flour (Table 5).

The presence of carotenoids in the designed products, which are searched by

consumers is important due to their health benefits. The awareness of the necessity to

consume vegetables has increased in recent years in both Poland and other European

countries, which is due to their high content of biologically active compounds, such as

carotenoids and polyphenols (Murphy et al., 2014; Tennant et al., 2014). The presence of

these compounds in a product increases its attractiveness and health benefits because of high

antioxidant capacity, which can protect cells and tissues against oxidative damage both in the

aqueous and lipid milieu. For these reasons, attempts have been made to enrich extruded

products with tomato pulp or dried broccoli as sources of carotenoids (Berset et al., 1989;

Dehghan-Shoar et al., 2010; Tonyali et al., 2013) without altering the appearance, taste,

colour, porosity, crispness and overall preferences of the final product. However, the process

of extrusion significantly decrease the content of polyphenolic compounds in the product, but

without any negative impact on the overall antioxidant activity (Tonyali et al., 2013). It was

also found that the addition of 20% tomato pulp to extruded corn products only slightly

increased the content of lycopene in the final product, which is an indication that parameters

applied during extrusion lead to the degradation of carotenoids (Dehghan-Shoar et al., 2010);

this has been demonstrated in our studies as well as studies with -carotene (Berset et al.,

1989). It was also caused that xanthophyls (lutein) were less sensitive to extrusion than

carotenes (Obradovi et al., 2015).

3.3 Total phenolics and antioxidant activity

The results of the Folin-Ciocalteu assay are shown in Table 5. Innovative flours used in snack

processing were rich sources of polyphenol compounds. The highest total phenolics content

16
was measured in Jerusalem artichoke flour, while amaranth flour had a phenolics

concentration that was about 3-fold lower. Flour addition increased polyphenols content in all

of the analysed snacks. The highest increase was observed for snacks with Jerusalem

artichoke flour additive (700%) while the lowest was for amaranth flour (229%). In other

studies it was found that the TPC was also influenced by extrusion conditions. Total phenolics

content in extruded snacks increased with the screw speed and the extruding temperature

(Pksa et al., 2016).

The antioxidant potential of flours and extruded snacks was determined on the basis of the

scavenging activity of the stable free radicals DPPH and ABTS and reducing ability by FRAP

assay (Table 5). Jerusalem artichoke flour which contained the highest level of polyphenols

was characterised by the highest antioxidant activity measured by FRAP (260 mol Trolox

100g-1 DW), while pumpkin tissue flour with highest carotenoids content exhibited the

highest antioxidant activity measured by ABTS and DPPH (189 and 143 mol Trolox 100g-1

DW, respectively). Amaranth flour and snacks with this flour showed the lowest antioxidant

potential among additives tested. Extruded snacks with pumpkin flour exhibited the highest

carotenoids and polyphenols content and had the highest ferric reducing ability (Table 5).

3.4 Benefits of the consumption of bioactive compounds for health

Polyphenols are known to have strong antioxidant activities (Triantis et al., 2005) and there is

a significant correlation between phenolic concentration and free radical scavenging activity,

particularly for the DPPH radical (Kivrak et al., 2009). In combination with other antioxidant

compounds, polyphenols present in fruits and vegetables are documented to be potential

therapeutic agents for neurodegenerative diseases, diabetes, inflammatory diseases and ageing

(Fraga et al., 2010; Molino et al., 2016). For instance, carotenoids are extremely reactive and

consequently unstable due to their long chain of conjugated double bonds. These organic

17
pigments perform diverse biological functions including protection from ultraviolet light and

carcinogenic agents, among others, supporting the immune system and antioxidant activity

(Stahl et al., 2003). Several studies have shown that their consumption lowers the risk of

degenerative and cardiovascular diseases, cataracts and certain types of carcinomas (Provesi

et al., 2011) There also posses anti-allergic and anticoagulation action. The protective effects

of antioxidants could involve their ability to trap or inhibit the formation of free radicals,

donate hydrogen atoms, quench singlet oxygen and chelate metal ions (Kurihara et al. 2002;

Fraser et al., 2004; Stahl et al., 2003).

Increased antioxidative capacity of human plasma is consistently observed in many studies

over the hours following the intake of fruit- and vegetable-based foods. Increased plasma

antioxidant capacity after the consumption of polyphenols and carotenoids could improve the

ability of the body to protect cellular components, like lipids and DNA from oxidative

damage. Epidemiological studies have shown that the regular consumption of fruit and

vegetables is associated with a reduced risk of chronic diseases occurring in the ageing human

population. Ultimately, the health benefits of plant-based foods are attributed to bioactive

phytochemicals, including polyphenols and carotenoids, acting in an additive and synergistic

manner. No single compound can replace the combination of various phytonutrients, playing

their complementary or synergistic roles together.

4. Conclusions

The study showed that the use of 10% Jerusalem artichoke and amaranth flours in

corn snack production may significantly increase the content of polyphenols in novel

designed products. Furthermore, the use of pumpkin flour may simultaneously increase the

contents of carotenoids (mainly lutein) and polyphenolic compounds, affecting the

antioxidant capacity of the final product. Snacks enriched with 10% amaranth flour affected

their content of antioxidant compounds and antioxidant capacity.. This suggests that the use

18
of pumpkin, Jerusalem artichoke and amaranth flours in corn snack production, can be a good

source of bioactive compounds. Whilst only a diverse diet, rich in fruits and vegetables,

could be effective in the prevention of diseases in which free radicals are implicated, valuable

compounds could be applied to great effect in the formulation of functional food.

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgement

The study was co-financed by the National Centre for Research and Development of Poland

through the project EUREKA no. E! 6855 ECORAW/2012-2013.

19
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Figure 1. Segment from 1.00 to 11.00 min of polyphenols LC-DAD chromatogram at 280 nm of Jerusalem
artichoke. (1) Quinic acid; (3) Hydroxyferulic acid hexoside; (4) Caffeoylhexose; (5) Chrysoeriol; (6)
Caffeoylquinic acid; (7) Caffeoylhexose; (8) Caffeoylhexose; (9) Caffeoylquinic acid dimer; (10) Caffeoylquinic
acid; (11) Caffeic acid; (12) Hydroxyferulic acid hexoside; (13) p-Coumaroylquinic acid; (14) Dicaffeoylquinic
acid; (15) Feruloylquinic acid; (16) Hydroxyferulic acid hexoside; (17) Hydroxyferulic acid hexoside; (18)
Hydroxyferulic acid hexoside; (19) Caffeoylhexose; (20) Caffeoylhexose; (21) Dicaffeoylquinic acid; (22)
Dicaffeoylquinic acid; (23) Caffeoylquinic acid; (24) Caffeoylquinic acid; (25) Dicaffeoylquinic acid.

Figure 2. Segment from 1.00 to 11.00 min of polyphenols and other polar compounds LC-DAD chromatogram
at 280 nm of Pumpkin. (1) Quinic acid; (2) p-Coumaroylhexose; (3) Hydroxybenzoic acid hexoside; (4)
Tryptophan hexoside; (5) Tryptophan hexoside; (6) Isopropylmalic acid; (7) Ethyl 3-hydrobutanoate hexoside;
(8) Ethyl 3-hydrobutanoate hexoside; (9) Protocatechuic acid; (10) p-Coumaric acid; (11) Isorhamnetin 3-
rutinoside-7-rhamnoside; (12) Sinapic acid hexoside; (13) Kaempferol O-(6-O-acetyl-glucoside); (14)
Kaempferol O--L-rhamnoside; (15) Hydroxybenzoic acid hexoside; (16) Quercetin.

26
Figure 3. Segment from 1.00 to 11.00 min of polyphenols and betalains LC-DAD chromatogram at 280 nm of
Amaranth. (1) Quinic acid; (2) Caffeoylhexose; (3) p-Coumarylquinic acid; (4) Amaranthine; (5) p-
Coumaroylhexose; (6) Isoamaranthine; (7) Betanin; (8) Isobetanin; (9) Betanidin; (10) Caffeoylhexose; (11)
Isobetanin glucoside; (12) Isoleucine-isoBetaxanthin; (13) Neobetanin); (14) p-Coumarylquinic acid; (15)
Quercetin 3-O-rutinoside; (16) Quercetin 3-O-hexoside; (17) Isobetanin; (18) 4'-O-Malonyl-isobetanin; (19)
Isovulgaxanthin; (20) Feruloylquinic acid.

27
Table 1. Retention times and characteristic ions of phenolic compounds of Jerusalem artichoke
Peak No. Rt (min) [M-H]- (m/z) a MS/MS fragments (m/z)a Tentative indentification
1. 1.44 191.0185 173.0096, 127.9567, 111.0093 Quinic acid b
2. 1.74 1151.3739 863.1915, 577.1349, 289.0717 A-type PA-tetramer b
3. 3.10 371.0636 353.0928, 209.0302 Hydroxyferulic acid hexoside (isomer 1) b
4. 3.28 341.1075 179.0349 Caffeoylhexose (isomer 1) b
5. 3.44 299.0784 Chrysoeriol
6. 3.79 353.0879 191.0552 Caffeoylquinic acid (isomer 1) b
7. 4.58 341.1075 179.0349 Caffeoylhexose (isomer 2) b
8. 4.62 341.0849 179.0349 Caffeoylhexose (isomer 3) b
9. 4.74 707.1852 353.0879 [M-2H]2-, 191.0554 Caffeoylquinic acid dimer b
10. 5.03 353.0879 191.0552 Caffeoylquinic acid (isomer 2) b
11. 5.28 179.0321 135.1075 Caffeic acid b
12. 5.58 371.0597 353.0928, 209.0302 Hydroxyferulic acid hexoside (isomer 2) b
13. 5.75 337.0932 191.0531, 163.0397 p-Coumaroylquinic acid b
14. 5.95 515.1196 353.0879, 335.0750 Dicaffeoylquinic acid (isomer 1) b
15. 6.23 367.1041 191.0553 Feruloylquinic acid b
16. 6.61 371.0597 353.0928, 209.0302 Hydroxyferulic acid hexoside (isomer 3) b
17. 6.80 371.0636 353.0928, 209.0302 Hydroxyferulic acid hexoside (isomer 4) b
18. 7.06 372.0636 353.0928, 209.0302 Hydroxyferulic acid hexoside (isomer 5) b
19. 7.24 341.1075 179.0349 Caffeoylhexose (isomer 4) b
20. 7.58 341.1075 179.0349 Caffeoylhexose (isomer 5) b
21. 7.91 515.1149 353.0928, 191.0543 Dicaffeoylquinic acid (isomer 2) b
22. 8.08 515.1196 353.0928, 191.0543 Dicaffeoylquinic acid (isomer 3) b
23. 8.18 353.0879 191.0543 Caffeoylquinic acid (isomer 3) b
24. 8.26 353.0879 191.0543 Caffeoylquinic acid (isomer 4) b
25. 8.76 515.1149 353.0928, 191.0543 Dicaffeoylquinic acid (isomer 4) b
26. 11.07 341.1075 179.0349 Caffeoylhexose (isomer 6) b
aExperimental data; b Identification confirmed by commercial standards; 3-Caffeoylquinic acid was used to identify quinic
acid, caffeoylquinic acid and di-O-caffeoylquinic acid isomers. Caffeic acid was used to identify caffeic acid and derivatives.
Ferulic acid was used to identify ferulic acid derivatives. p-Coumaric acid was used to identify p-coumaric, p-coumarylquinic
acid and p-coumaroylhexose. Procyanidin A2 was used for identification of A-type procyanidins.

28
Table 2. Retention times and characteristic ions of phenolic and other polar compounds of Pumpkin
Peak No. Rt (min) [M-H]- (m/z) a MS/MS fragments (m/z)a Tentative indentification
Phenolic compounds
1. 1.44 191.0185 Quinic acid b
2. 2.40 325.1023 191.0531, 163.0397 p-Coumaroylhexose b
3. 3.30 299.0784 137.0254 Hydroxybenzoic acid hexoside b
9. 5.54 153.0204 109.0300 Protocatechuic acid
10. 6.61 163.0406 119.0488 p-Coumaric acid b
11. 7.02 769.2181 623.1654, 315.0515 Isorhamnetin 3-rutinoside-7-rhamnoside b
12. 7.12 385.1701 223.0998 Sinapic acid hexoside
13. 8.61 489.2101 285.0359 Kaempferol O-(6-O-acetyl-glucoside) b
14. 8.70 431.1754 285.0360 Kaempferol O--L-rhamnoside b
15. 9.73 299.1846 137.0254 Hydroxybenzoic acid hexoside
16. 10.36 301.2023 Quercetin b
Amino acids
4. 3.79 365.1381 203.081 Tryptophan hexoside
5. 4.44 365.1381 203.081 Tryptophan hexoside
Organic acids
6. 4.62 175.0585 115.0389 Isopropylmalic acid
Other metabolites
7. 4.79 293.1230 131.0709 Ethyl 3-hydrobutanoate hexoside
8. 4.97 293.1230 131.0710 Ethyl 3-hydrobutanoate hexoside
a Experimental data; b Identification confirmed by commercial standards; 3-Caffeoylquinic acid was used to identify quinic
acid. Ferulic acid was used to identify ferulic acid derivatives. p-Coumaric acid was used to identify p-coumaric acid and p-
coumaroylhexose; Kaempferol 3-O-glucoside was used for identification of kaempferol O--L-rhamnoside and kaempferol
3-O-6-acetylglucoside. Quercetin 3-O-rutinoside and 3-O-glucoside were used to identify quercetin derivatives. For
isorhamnetin derivatives identification, isorhamnetin 3-O-rutinoside and 3-O-glucoside were used.

29
Table 3. Retention times and characteristic ions of phenolic and betalain compounds of Amaranth
Peak No. Rt (min) [M-H]- (m/z) a MS/MS fragments (m/z)a Tentative indentification
Phenolic compounds
1. 1.44 191.0185 173.0096, 127.9567, 111.0093 Quinic acid b
2. 1.70 341.1085 179.0349 Caffeoylhexose (isomer 1) b
3. 1.95 337.0448 p-Coumarylquinic acid (isomer 1) b
5. 2.42 325.1023 191.0572, 145.0554 p-Coumaroylhexose b
10. 4.63 341.0849 179.0349 Caffeoylhexose (isomer 2) b
14. 5.95 337.0905 191.0571, 163.0397 p-Coumarylquinic acid (isomer 2) b
15. 6.27 609.1434 301.0255 Quercetin 3-O-rutinoside b
16. 6.89 463.0843 301.0354 Quercetin 3-O-hexoside b
b
20. 10.50 367.1354 Feruloylquinic acid

Peak No. Rt (min) [M+H]+ m/z a MS/MS fragments (m/z)a Tentative indentification
Betalains
4. 2.10 727.1846 551.1387, 389.1765 Amaranthine
6. 3.99 727.1846 551.1387., 389.1765 Isoamaranthine
7. 5.74 551.1387 389.1851 Betanin
8. 4.23 551.1387 389.1851 Isobetanin (isomer 1)
9. 4.49 389.1766 345.1190 Betanidin
11. 4.73 713.1904 551.1387, 389.1851 Isobetanin glucoside
12. 5.20 325.119 Isoleucine-isoBetaxanthin
13. 5.46 549.1491 387.1387, 343.3543 Neobetanin
17. 7.11 551.1387 389.1851 Isobetanin (isomer 2)
18. 7.31 637.1697 551.1387, 389.1765 4'-O-Malonyl-isobetanin
19. 9.10 323.1199 Isovulgaxanthin
aExperimental data; b Identification confirmed by commercial standards; 3-Caffeoylquinic acid was used to identify quinic
acid. Caffeic acid was used to identify caffeic acid derivatives. Ferulic acid was used to identify ferulic acid derivatives. p-
Coumaric acid was used to identify p-coumarylquinic acid and p-coumaroylhexose. Quercetin 3-O-rutinoside and 3-O-
glucoside were used to identify quercetin derivatives.

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Table 4. Carotenoids content of selected flours and enriched corn products.

Product Carotenoids content

(g/100g)

lutein -carotene lycopene

Flours

amaranth 14 1 f - -

pumpkin tissue 4307 98 a 970 23 a 442 8

Jerusalem artichoke 70g - -

Extruded corn snacks

with amaranth flour 155 5 c - -

with pumpkin flour 219 6 b 44 3 b -

with Jerusalem artichoke flour 114 5 e - -

without additives 127 4 d - -

- not detected under the analysis condition Values are means SD of three independent analysis
Mean values within a column with different letters are significantly different at p <0.05.

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Table 5. Total polyphenolic content and antioxidant activity of selected flours and enriched corn products.

Product Polyphenolic Antioxidant capacity

content
(mg/100g) (mol Trolox/100g)

DPPH ABTS FRAP

Flours

amaranth 498.8 5.4 e 134.8 18.2 c 179.8 20.5 b 147.4 16.3 e

pumpkin tissue 803.4 11.2 d 143.0 14.4 b 189.0 19.8 ab 228.7 19.8 cd

Jerusalem artichoke 1370.0 16.2 a 136.6 17.8 bc 178.6 16.1 b 260.0 22.7 b

corn grits 158.4 16.2 h 112.4 11.3 d 153.7 14.2 c 211.0 16.2 d

Extruded corn snacks

with amaranth flour 335.1 4.7 f 149.3 20.1 b 173.8 15.3 b 204.8 19.7 d

with pumpkin flour 975.4 10.6 c 183.3 16.7 a 208.2 21.3 a 364.0 23.8 a

with Jerusalem artichoke flour 1034.2 12.3 b 146.7 15.2 b 177.7 18.6 b 255.4 18.9 b

without additives 146.8 8.2 g 150.0 14.9 b 178.9 16.9 b 233.3 19.3 c

Values are means SD of six independent analysis

Mean values within a column with different letters are significantly different at p <0.05.

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