Biotransformation of Anthocyanins From Two Purple Fleshed Sweet Potato Accessions in a Dynamic Gastrointestinal System 2016 Food Chemistry

7/24/2019 Biotransformation of Anthocyanins From Two Purple Fleshed Sweet Potato Accessions in a Dynamic Gastrointestin

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Biotransformation of anthocyanins from two purple-fleshed sweetpotato accessions in a dynamic gastrointestinal system

Stan Kubow a,, Michle M. Iskandar a, Kebba Sabally a, Behnam Azadi a, Shima Sadeghi Ekbatan a,Premkumari Kumarathasan b, Dharani Dhar Das b, Satya Prakash c, Gabriela Burgos d, Thomas zum Felde d

a School of Dietetics and Human Nutrition, McGill University, 21111 Lakeshore, Ste. Anne de Bellevue, QC H9X 3V9, CanadabAnalytical Biochemistry and Proteomics Laboratory, Mechanistic Studies Division, Healthy Environments and Consumer Safety Branch, Health Canada, 50 Colombine Drwy,

Ottawa, ON K1A 0K9, Canadac BioMedical Engineering Department, McGill University, 3775 University Street, Room 311, Montreal, QC H3A 2B4, Canadad International Potato Center (CIP), Avenida La Molina 1895, La Molina, Apartado Postal 1558 Lima, Peru

a r t i c l e i n f o

Article history:

Received 1 April 2015

Received in revised form 26 June 2015

Accepted 29 June 2015

Available online 30 June 2015

Keywords:

Ipomoea batatasL.

Purple fleshed sweet potato

Anthocyanins

Bioaccessibility

Biotransformation

Human gastro-intestinal model

a b s t r a c t

Cooked, milled purple-fleshed sweet potato (PFSP) accessions, PM09.812 and PM09.960, underwent

digestion in a dynamic human gastrointestinal (GI) model that simulates gut digestive conditions to

study the bioaccessibility and biotransformation of anthocyanins. Matrix-assisted laser desorption

ionization time-of-flight mass spectrometry showed accession-dependent variations in anthocyanin

release and degradation. After 24 h, more anthocyanin species were detected in the small intestinal vessel

relative to other vessels for accession PM09.960 whereas more species appeared in the ascending colonic

vessel for accession PM09.812. The ferric reducing antioxidant power was increased in the small

intestinal vessel for PM09.960 and in the ascending colonic vessel for accession PM09.812, corresponding

to the appearance of a majority of anthocyanins for each accession. These results show that intestinal

and colonic microbial digestion of PFSP leads to an accession-dependent pattern for anthocyanin

bioaccessibility and degradation.

2015 Elsevier Ltd. All rights reserved.

1. Introduction

Purple fleshed sweet potatoes (PFSP) contain a significant con-

tent of anthocyanins comparable to that of other high anthocyanin

containing fruits and vegetables such as grapes, plums, sweet cher-

ries, raspberries and eggplant (Truong et al., 2010). Anthocyanins

from PFSP have been shown to exhibit stronger radical-

scavenging activity than anthocyanin pigments from red cabbage,

elderberry, grape skin and purple corn (Kano, Takayanagi, &

Harada, 2005).

Despite their high in vitro antioxidant properties, there is lim-

ited research regarding how digestive processes of PFSP affect their

antioxidant activity and anthocyanin structures, which could be

altered during gastrointestinal (GI) digestion. During GI digestive

processes involving pH changes, digestive enzymes and microbial

metabolism, anthocyanins are released from the food matrix and

undergo extensive degradation that can affect their bioactivities.

Furthermore, anthocyanin metabolites could also be bioavailable

for absorption and exert protective antioxidant and

anti-inflammatory effects on intestinal disorders such as inflam-

matory bowel disease (Wu, Xu, Dong, He, & Yu, 2011) and colon

cancer (Lim et al., 2013).

In vitro GI models used to evaluate digestion of polyphenols

such as anthocyanins have utilized dual enzyme pepsinpancre-

atin digestion to simulate gastric and small intestinal tract diges-

tion that could also be coupled with batch colonic reactors with

human gut microflora to assess the impact of human microbial

metabolism (Alminger et al., 2014; Tarko, Duda-Chodak, & Zajac,

2013). Such studies have shown major variations in anthocyanin

stability of different foods during digestive processes, which

appear to be highly dependent on food matrix composition and

structure. For example, anthocyanin-rich extracts of red cabbage

showed extensive degradation under the mild alkaline conditions

of pancreatic digestion whereas most anthocyanins of raw red

cabbage reached colonic vessels intact to be extensively degraded

by bacteria (Pods?dek, Redzynia, Klewicka, & Koziokiewicz,

2014).

http://dx.doi.org/10.1016/j.foodchem.2015.06.105

0308-8146/2015 Elsevier Ltd. All rights reserved.

Corresponding author.

E-mail addresses: [email protected] (S. Kubow), michele.iskandar@mail.

mcgill.ca (M.M. Iskandar), [email protected] (K. Sabally), behnam.azadi@

mcgill.ca (B. Azadi), [email protected] (S. Sadeghi Ekbatan),

[email protected] (P. Kumarathasan), [email protected].

ca(D.D. Das),[email protected](S. Prakash),[email protected](G. Burgos),

[email protected](T. zum Felde).

Food Chemistry 192 (2016) 171177

Contents lists available at ScienceDirect

Food Chemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m
http://dx.doi.org/10.1016/j.foodchem.2015.06.105mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.foodchem.2015.06.105http://www.sciencedirect.com/science/journal/03088146http://www.elsevier.com/locate/foodchemhttp://www.elsevier.com/locate/foodchemhttp://www.sciencedirect.com/science/journal/03088146http://dx.doi.org/10.1016/j.foodchem.2015.06.105mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.foodchem.2015.06.105http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://crossmark.crossref.org/dialog/?doi=10.1016/j.foodchem.2015.06.105&domain=pdfhttp://-/?-


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To date, studies of anthocyanin digestion have not utilized

computer-controlled dynamic multistage continuous digestion

models that can better simulate in vivo conditions (Alminger

et al., 2014). The dynamic in vitro digestion system mimics the

in vivo dynamics of transit during digestion and considers the vary-

ing microbial or digestive conditions in different segments of the

GI tract including the ascending colon, transverse colon and

descending colon. Adjustment of the pertinent pH for each colonic

region leads to differences in growth of corresponding microbial

communities that results in alterations in the metabolic activity

of the colonic bioreactors (Blanquet-Diot et al., 2009; Martoni

et al., 2007; Molly, Vande Woestyne, De Smet, & Verstraete, 1994).

In the present study, a computer controlled dynamic human GI

model was used to evaluate the effect of digestion on the release

and biotransformation of anthocyanins present in cooked, milled

and freeze-dried samples of two PFSP accessions (PM09.812 and

PM09.960) conserved in the in-trust germplasm collection at

International Potato Center (CIP). Following digestion of the sweet

potato accessions in the GI model, samples from all the compart-

ments of the GI model (stomach, small intestine, ascending, trans-

verse and descending colon) were assessed for anthocyanin

profiles using matrix assisted laser desorption ionization time of

flight mass spectrometry (MALDI-TOFTOF-MS) and measured

for their antioxidant capacity via the ferric reducing antioxidant

power (FRAP) assay.

2. Materials and methods

2.1. Plant material

Two PFSP accessions: PM09.812 (CIP number: 109264.3) and

PM09.960 (CIP number: 109270.2) were used for this study

(Fig. 1). The two CIP breeding clones with the same mother but dif-

ferent fathers were selected for their good yield and deep purple

colors (PM09.812: female (SM07.048, CIP107791.1) male

(SM07.524; CIP107805.1); PM09.960: female (SM07.048;CIP107791.1) male (SM07.626; CIP107809.3)). The two acces-

sions were grown in San Ramon, Junin, Peru and harvested in

October 2013. Ten roots per accession were collected and brought

to the Quality and Nutrition Laboratory of CIP in Lima, Peru where

they were cooked. Unpeeled roots of each accession were placed in

stainless steel pots containing boiling water. One control for each

accession was used to determine the cooking time required.

Roots were considered as cooked when a stainless steel stick could

penetrate the control roots easily. Following cooking, the roots

were peeled, representatively sampled, freeze dried and milled

through 40 mesh. Freeze dried and milled samples of each acces-

sion were sent to McGill University.

2.2. Computer controlled dynamic human gastrointestinal model

The components of the simulated human GI model involved five

reactors, the last three of which contain microbiota of a different

part of the human GI tract. In order of sequence (reactors 15):

the stomach (V1), small intestine (V2; duodenum, jejunum and

ileum), the ascending (V3), the transverse (V4) and the descending

colon (V5). The system is fully computer-controlled and has been

validated using enumeration procedures, short chain fatty acid

production patterns, enzymatic activities, gas production, and by

microorganism-associated activities (Blanquet-Diot et al., 2009;

Martoni et al., 2007; Molly et al., 1994).

Five healthy, non-smoking individuals with no history of GI dis-

ease or antibiotic use in the previous 6 months provided fecal sam-

ples for the study. Fecal samples were freshly collected, pooled,and readily used to prepare the fecal solution. The system

underwent a 2-week stabilization period to allow bacterial com-

munities from the diluted human fecal samples to grow and

stabilize.

During and following stabilization, the microbial ecosystem

was sustained by feeding the system with 300 mL of sterile med-

ium (set at pH 2 before autoclaving and stored at 37 C) every

8 h. The composition of the GI nutrient solution essential for bacte-

rial survival is shown inTable 1.

The fermentation vessels were maintained anaerobic by purg-

ing the headspace with oxygen-free nitrogen and stirring continu-

ously on magnetic stirrers. The temperature of the simulator was

kept at 37 C. Upon entering vessel 2 (small intestine), pancreatic

juice supplemented with bile (12 g/L NaHCO3, 0.9 g/L pancreatin;

SigmaAldrich) and 6 g/L Oxgall (Difco) were added to neutralize

PM09.812

PM09.960

Fig. 1. The purple-fleshed sweet potato accessions PM09.812 and PM09.960.

Table 1

Composition of the GI nutrient solution essential for bacterial survival.

Component Concentration (g/L)

Arabinogalactan 1

Pectin 2

Starch 3

Xylan 1

Glucose 0.4

Yeast extracts 3

Peptone 1

Mucin 4

Cysteine 0.5

Source:Molly et al. (1994).

172 S. Kubow et al./ Food Chemistry 192 (2016) 171177


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stomach acidity. In this way, the pH of the first two vessels was set

by the input of either supply medium or pancreatic juice. Vessels 3,

4 and 5 (representing the colon) were pH-controlled between 5.6

and 5.9; 6.1 and 6.4; and 6.6 and 6.9, respectively. The pH was

measured with a probe connected to a pH meter and was automat-

ically adjusted by adding 0.2 M NaOH or 0.5 M HCl.

The cooked, freeze-dried, and milled samples of each accession

(18.15 g) were digested in the GI model. The amount provided to

the GI model was based on the fact that only a small amount of

PFSP is included in the human diet. Considering that the accessions

used in this study have around 35% dry matter, the amount pro-

vided to the GI model represent approximately 50 g of each acces-

sion on a fresh weight basis.

On the day of treatment the freeze-dried sweet potato samples

were incorporated into the GI food solution and subjected to 24 h

digestion by the GI model. Samples were collected from all the

compartments of the GI model before addition of the

freeze-dried potato (T= 0 control) and after 8 h (T= 8) and 24 h

(T= 24) of digestion of the freeze-dried potato. One day of treat-

ment was followed by a 3-day washout period, during which the

system was fed the control GI nutrient solution without potato.

Each treatment lasted 24 h and sampling was performed at 0, 8

and 24 h throughout the day of treatment (Fig. 2).

For each day of treatment, an aliquot (20 mL) was removed

from vessels 1 through 5, and stored at 80 C for later analysis

and characterization of polyphenolic content. To prevent photode-

composition of the polyphenols, all of the digestive compartments

and collection vessels were wrapped in tin foil.

2.3. Fecal water (FW) preparation

Fecal water (FW) was prepared by collecting samples of the

contents of vessels 1 through 5 and centrifuging them at 200gfor

20 min; the supernatants were stored at 80 C until use for

MALDI-TOFTOF-MS and antioxidant capacity analyses.

2.4. Solid phase extraction procedure for the FW samples

Solid phase extraction was performed based on the method of

Mullen, Edwards, Serafini, and Crozier (2008) with modifications.

Equal amounts of FW and phosphate buffer pH 6 (5 mL:5 mL) were

applied to a 100 mg/3 mL of C18-E cartridges, which were condi-

tioned with 300lL of 5% formic acid and equilibrated with:

300lL of 5% formic acid in methanol. The cartridges were washed

twice with 300 lL of 5% formic acid and dried for 3 min. Bound

anthocyanins were then eluted using two folds of 300lL of 5%

formic acid in methanol. The eluted samples were dried under

nitrogen and stored at 80 C until MALDI-TOFTOF-MS analysis.

2.5. MALDI-TOFTOF-MS analysis

MALDI-TOFTOF-MS analysis was performed based on the

method of Kumarathasan et al. (2014) with modifications. Dried

samples were re-solubilized in 50% acetonitrile/0.1% trifluoroaceticacid (TFA)/H2O prior to MALDI-TOFTOF-MS analysis, 1 lL of sam-

ple was spotted in duplicates at the center of the sample location

on a 384/600 Anchor Chip target plate (Bruker Daltonics). One lL

of matrix solution (0.5 mg/mL a-cyano-4-hydroxycinnamic acid

in 50% acetonitrile/0.1% TFA/H2O) was added on top of the sample

spot and was mixed by pulling and releasing the liquid with the

help of the pipette tip. The sample spots were allowed to dry by

evaporation in open air. An on-target washing of the sample spots

was carried out by placing 2.5 lL of cold 1% TFA in water on the

dried sample spot, and removing the liquid after 10 s. Washed

spots were dried in open air and the mass spectral profiles were

recorded using Autoflex III time-of-flight mass spectrometer

(Bruker Daltonics, Bremen, Germany) equipped with a Smart

Beam laser emitting at 355 nm wavelength, a 1 GHz samplingrate digitizer, a pulsed ion extraction source, and a TOFTOF-MS

analyzer. The instrument calibration was done using external cali-

bration standards (Bruker Daltonics). Detection was carried out in

a reflectron positive mode. In a typical experiment, a composite

spectrum (total of 1000 shots) was obtained by summation of five

200-shots of individual spectra. The sampling sites were selected

randomly for every sample in order to obtain homogenous acquisi-

tion. The acquisition of mass spectra was carried out using the Flex

Control 3.3 software whereas further processing was done using

Flex Analysis 3.3 software. Anthocyanins in the samples were iden-

tified based on their masses and information obtained from litera-

ture. The anthocyanins were quantified using the external

standard, keracyanin chloride (cyanidin-3-O-rutinoside chloride)

(KCC-Eq).

2.6. Ferric reducing ability of plasma (FRAP) assay

The ferric reducing ability of plasma (FRAP) antioxidant capac-

ity assay was used to determine the total antioxidant potential in

the supernatant of the fecal water obtained from the gut model.

Briefly, the electron-donating capacity of an antioxidant is mea-

sured by the change in absorbance at 593nm when a

blue-colored Fe2+-tripyridyltriazine (Fe2+TPTZ) compound was

formed from a colorless oxidized Fe3+ form (Benzie & Strain,

Time 0h:

Collection ofcontrol

samples fromall vessels

Followed byinjection ofGI foodcontainingpotato meal

Time 8h:

Collection ofsamples fromall vessels

Followed bysecondinjection ofGI foodcontainingpotato meal

Time 16h:

Thirdinjection ofGI foodcontainingpotato meal

Time 24h:

Collection ofsamples fromall vessels

2 weeks:

Stabilization

period:

Injection ofGI food every8 hours

Fig. 2. Schematic representation of gut model experiments timeline and sample collection.

S. Kubow et al. / Food Chemistry 192 (2016) 171177 173
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1996). A standard curve was prepared using solutions of ferrous

sulfate at concentrations ranging from 0.1 to 10 mM. The reagent

was prepared with 10:1:1 ratio of 300 mM acetate buffer pH 3.6:

10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution: 40 mM HCl at

50 C, and 20 mM FeCl36H2O solution. Once the FRAP working

solution was prepared it was immediately incubated for 10 min

at 37 C. Thirty lL H2O, 10 lL standards or samples, and 200 lL

FRAP working solution were added into a 96-well plate. After

30 min incubation absorbance was read at 593 nm in a microplate

reader (Infinite PRO 200 series, Tecan Group).

3. Results and discussion

3.1. Anthocyanin profiles in digested PM09.812 and PM09.960 sweet

potatoes

As many anthocyanin compounds are not generally commer-

cially available as standards for identification and since the iden-

tity of individual anthocyanins using UVvisible spectra is not

definitive, identification of individual anthocyanins was based on

MALDI-TOFTOF-MS data using information obtained from litera-

ture pertaining to sweet potato anthocyanins. Previous studies

have shown that the principal anthocyanin structure of sweet

potatoes is anthocyanidin 3-acyl-sophoroside-5-glucoside, with

the acyl substitute being primarily caffeoyl, hydroxybenzoyl, or

feruloyl residues (Montilla, Hillebrand, & Winterhalter, 2011),

which corresponds to the primary anthocyanin structures

observed in the gut model vessels of the sweet potato samples

(Table 2). The anthocyanin profiles in the stomach have been well

demonstrated to remain stable after 2 h under simulated gastric

conditions (McDougall, Dobson, Smith, Blake, & Stewart, 2005),

which is likely due to the stability of the flavylium cation form of

anthocyanin at low pH (Clifford, 2000).

In the stomach vessel (V1) after 24 h, the major anthocyanins

observed in both accessions have a general anthocyanidin

3-acyl-rutinoside-5-glucoside structure that differs composition-

ally between the two accessions (Table 2). There were two major

anthocyanidins in PM09.812 in the form of peonidin and

pelargonidin with p-coumaroyl and feruloyl as the acyl residue

whereas peonidin was the primary anthocyanidin present in the

sweet potato accession PM09.960, which contained a feruloyl acyl

substitute. A total of eight acylated anthocyanins were identified in

V1 of the pigmented sweet potato PM09.812 with petunidin,

peonidin, pelargonidin, or cyanidin bases (Table 2). Non- and

mono-acylated petunidin species and mono-acylated peonidin

species accounted for 35% and 36% of total anthocyanins, respec-

tively. Mono-acylated cyanidin and pelargonidin species accounted

for 16% and 13% of total anthocyanins, respectively. The sweet

potato PM09.960 in V1 contained seven acylated anthocyanins

on pelargonidin, peonidin or cyanidin bases. Mono- and

di-acylated peonidin species accounted for 64% of total antho-

cyanins. Mono- and di-acylated cyanidin species and monoacy-

lated pelargonidin species accounted for 17% and 19% of total

anthocyanins, respectively. The above findings coincide with previ-

ous literature, which indicates that peonidin amounts are generally

higher in PFSP than cyanidin-based anthocyanins (Lee, Park, Choi,

& Jung, 2013).

Peonidin 3-(600-p-coumaroyl-sophoroside)-5-glucoside and

petunidin-3-p-coumaroyl-rutinoside-5-glucoside with m/z values

of 771 and 933, respectively, were the major anthocyanins seen

in V1 of the sweet potato accession PM09.812, accounting for

54%of the total anthocyanin content in V1. The sweet potato acces-

sion PM09.812 also contained lesser quantities of the petunidin

anthocyanidin in V1 in the form of petunidin-3-rutinoside-5-gluco

side (m/z 787) and the peonidin anthocyanidin in the form of

peonidin 3-p-coumaroyl-rutinoside-5-glucoside. Cyanidin antho-

cyanins were seen in relatively lower amounts in the form of

cyanidin 3-p-hydroxy-benzoyl-sophoroside-5-glucoside (m/z

893). Smaller quantities of pelargonidin derivatives were also

observed in V1 including pelargonidin-procyanidin-rutinoside

and pelargonidin-feruloyl-rutinoside (m/z933).

Table 2

Proposed identification of anthocyanin peaks in pigmented sweet potatoes and their concentration in the samples exposed to human simulated intestinal digestion (mg/L).a

(m/z)b Proposed compoundc Sweet potato A: PM09.812 Sweet potato B: PM09.960

V1d V2 V3 V4 V5 V1 V2 V3 V4 V5

725 Pelargonidin-procyanidin-rutinoside 22e 34 31 26 26 35 25

731 Cyanidin 3-p-hydroxy-benzoyl-sophoroside-5-glucoside 26 31 32 28 25 33 26 62 21

741 Fragment of pelargonidin 3-feruloyl-sophoroside-5-glucoside 44 38 23 25 21 33 29 21

745 Peonidin 3-p-hydroxy-benzoyl-sophoroside-5-glucoside 35 45 27 26 63 27 85 24

755 Pelargonidin-feruloyl-rutinoside 55 42 31 24 25 42 41 54 69 21

757 Pelargonidin 3-sophoroside-5-glucoside 40 38 40 33 36 23

771 Peonidin 3-(600-p-coumaroyl-sophoroside)-5-glucoside 179 33 27 23

773 Cyanidin 3-sophoroside-5-glucoside 35

773 Cyanidin 3-(600-caffeoyl-sophoroside)-5-glucoside 43 32 24 50 41 32 23

787 Petunidin-3-rutinoside-5-glucoside 64 38 23 27 50 49 26 23

801 Fragment of Peonidin 3-(600-feruloyl-sophoroside)-5-glucoside 59 32 36 27 35

887 Pelargonidin-3-(p-coumaroyl)-rutinoside-5-glucoside 40 39 25

893 Cyanidin 3-p-hydroxy-benzoyl-sophoroside-5-glucoside 75 32

907 Peonidin 3-p-hydroxy-benzoyl-sophoroside-5-glucoside 32 34

917 Peonidin-3-p-coumaroyl-rutinoside-5-glucoside 40 33

933 Pelargonidin 3-feruloyl-sophoroside-5-glucoside 154 31

935 Cyanidin 3-caffeoyl-sophoroside-5-glucoside 30 63

963 Peonidin 3-(600-feruloyl-sophoroside)-5-glucoside 43

1055 Cyanidin-3-(600-caffeoyl-600-p-hydroxy-benzoyl-sophoroside)-5-glucoside 24

1065 Cyanidin 3-(600,600-dicoumaryl-sophoroside)-5-glucoside 55

1069 Peonidin 3-caffeoyl-p-hydroxybenzoyl-sophoroside-5-glucoside 37 27 28

1095 Cyanidin 3-feruloyl-p-coumaryl-sophoroside-5-glucoside 26

1127 Peonidin 3-feruloyl-p-caffeoyl-sophoroside-5-glucoside 148

a Expressed as cyanidin 3-glucoside equivalents.b Determined by MALDI-TOFTOF-MS analysis.c Identification based on previous literature data (Kim et al., 2012; Montilla et al., 2011; Mori et al., 2010; Tian et al., 2005).d

V1 = Stomach; V2 = Small intestine; V3 = Ascending colon; V4 = Transverse colon; V5 = Descending colon.e KCC-Eq values


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Peonidin 3-feruloyl-p-caffeoylsophoroside-5-glucoside(m/z1127)

was the primary anthocyanin noted in V1 from the sweet potato

accession PM09.960that contributed to 54%of thetotalanthocyanin

content. Thesweet potato accession PM09.960also contained lesser

quantities of the pelargonidin anthocyanidin in V1 in the form of p

elargonidin-feruloyl-rutinoside (m/z 755) and a trace amount of

pelargonidin in the form of pelargonidin 3-feruloyl-sophorosi

de-5-glucoside (m/z741). Smaller amounts of cyanidin (cyanidin

3-p-hydroxy-benzoyl-sophoroside-5-glucoside (m/z 893)) and

cyanidin-3-(600-caffeoyl-600-p-hydroxy-benzoyl-sophoroside)-5-

glucoside (m/z 1055) and peonidin compounds peonidin 3-p-

hydroxy-benzoyl-sophoroside-5-glucoside (m/z907) and peonidin

3-caffeoyl-p-hydroxy-benzoyl-sophoroside-5-glucoside (m/z 1069)

were also observed. Cyanidin-3-(600-caffeoyl-600-p-hydroxy-benzoy

l-sophoroside)-5-glucoside (m/z 1055) and its deacylated form

cyanidin-3-p-hydroxy-benzoyl-sophoroside-5-glucoside (m/z 893)

were observed in the stomach.

The two major anthocyanins of PM09.812 observed in V1,

peonidin 3-(600-p-coumaroyl-sophoroside)-5-glucoside (m/z 771)

and petunidin-3-p-coumaroyl-rutinoside-5-glucoside (m/z 933),

appeared to be completely broken down during simulated human

intestinal digestion in the small intestine vessel (V2). In contrast,

for PM09.960, both of the above anthocyanins only first appeared

in the ascending colon vessel (V3), which could signify that

microbial metabolism allowed for the release of these antho-

cyanins from the sweet potato food matrix of PM09.960. In V2 of

PM09.812, pelargonidin-3-feruloyl-sophoroside-5-glucoside was

digested to form the fragment of m/z 741. Pelargonidin-3-

(p-coumaroyl)-rutinoside-5-glucoside (m/z 887), also known as

pelanin, was present only in the V3 vessel for PM09.812 and V2

and V4 vessels for PM09.960.

For both sweet potato accessions, pelargonidin-feruloyl-rutino

side with a mass of 755 was present in significant amounts in all

vessels (V1V5). The concentration of this anthocyanin showed a

trend to decrease from V1 to V5 for PM09.812, but showed a differ-

ent pattern in PM09.960 with a tendency to increase in V3 and V4

and a subsequent decrease in V5. The anthocyanin pelargonidin-procyanidin-rutinoside with am/zof 725, was found in signif-

icant amounts in all digestion vessels from the digestion of

PM09.812 but was only present in V2 and V4 vessels for

PM09.960. The biphasic phenomenon of a drop and a subsequent

increase in concentrations of various anthocyanins seen during

simulated human gut digestion in the present work has been noted

previously (Aura et al., 2005). Such findings could be related to

enzymatic release from the food matrix, de-conjugation and subse-

quent re-conjugation of the anthocyanins, microbial anthocyanin

degradation during digestive enzyme and microbial metabolism

and anthocyanin release and re-incorporation into the fecal matrix

that has a high binding capacity for anthocyanins and their

metabolites. Alternatively or in addition, the lack of detection of

certain metabolites at trace levels (KCC-Eq values


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resulting from gut microbial metabolism to generate secondary

metabolites. For the PFSP accession PM09.812, similar drops in

antioxidant activity were observed across the vessels of the gut

model; however, the FRAP values were lower for V1 and V2 at

24 h than at 8 h, a different observation than for accession

PM09.960, suggesting a degradation of antioxidant compounds

within the GI nutrient solution over the length of the digestionexperiment (24 h).

The increase in antioxidant capacity that was observed is sup-

portive of the suggestion that unabsorbed anthocyanins as well

as their microbial metabolites can contribute to antioxidant capac-

ity of human fecal fluid. An assessment of the total antioxidant

activity of feces obtained from 14 healthy volunteers showed a

high radical scavenging antioxidant capacity that was almost

20-fold greater than that detected in the plasma (Garsetti,

Pellegrini, Baggio, & Brighenti, 2000). The production of secondary

metabolites from gut microbial metabolism of polyphenols could

exert significant post-absorptive physiological effects. For exam-

ple, only microbial metabolites and none of the parent/original

polyphenols in pomegranate juice were detected in the plasma

or urine of healthy volunteers after they consumed pomegranatejuice daily for 5 days (Cerd, Espn, Parra, Martnez, &

Toms-Barbern, 2004). Unabsorbed anthocyanin compounds have

been implicated with enhanced colorectal health and protection

against colorectal cancer via antioxidant mechanisms (Lala et al.,

2006).

4. Concluding remarks

To our knowledge, this is the first study to report regarding the

effects of digestion on anthocyanins via the human simulated

dynamicin vitro multistage continuous digestion model involving

the ascending colon, transverse colon and descending colon ves-

sels. Although some anthocyanins appear to be stable under simu-

lated upper intestinal tract conditions in the present work, severalanthocyanins were unstable in the intestinal vessel. This

observation is likely due to their chemical decomposition at neu-

tralpH (Woodward, Kroon, Cassidy, & Colin, 2009), which is known

to occur before their subsequent exposure to colonic microbial

metabolism and first pass intestinal and hepatic metabolism

(Fang, 2014). Anthocyanins are well known to be subject to micro-

bial degradation (Kim et al., 1998). Thus, microbial metabolism can

account for the anthocyanin degradation in the present study as

seen by diminished concentrations of several anthocyanin species

in the colonic vessels, particularly evident in V5. Accession differ-

ences in the appearance of anthocyanin species were evident as

PM09.812 showed the appearance of a greater number of antho-

cyanin species in V3, whereas V2 showed the greatest presence

of anthocyanin species for the PM09.960 accession. This finding

could be indicative of cultivar differences in other food compo-

nents that might affect anthocyanin bioaccessibility. The two

accessions showed a relatively low quantity of anthocyanin species

noted in the descending colon vessel V5, which could signify

extensive anthocyanin breakdown in the multistage reactor diges-

tion model used in this study which is an advantage compared to

single batch reactors where there is accumulation of microbial

metabolites with prolonged incubation.

Both sweet potato samples demonstrated an increase in FRAP

antioxidant capacity measures over a 24 h time course, which is

supportive of the concept that antioxidant activities of unabsorbed

anthocyanins and their metabolites protect intestinal cells against

reactive oxygen species (ROS) generated within the gut and atten-

uate ROS-mediated gut inflammatory conditions (Glvez et al.,

2001).

In summary, the present study shows that the release of antho-

cyanins from the food matrix of sweet potatoes under simulated

gastric, intestinal and colonic digestions is an accession dependent

process. There are significant losses in the variety of anthocyanin

species detected as digestion proceeds from the intestine and

colon, which differs according to the sweet potato accession.

These findings suggest that cultivar-based variations in other food

components can affect anthocyanin release during digestive pro-

cesses, which can also impact on their antioxidant capacities inthe small intestine.

Conflict of interest

The authors declare that they have no conflict of interest in the

research.

Acknowledgments

This research was supported by the CGIAR Research Programon

Agriculture for Nutrition & Health (CRP-A4NH) and the Discovery

Grant Program from the Natural Sciences and Engineering

Council of Canada (S.K.). The authors would like to thank Dr.Wolfgang Grneberg (CIP) for providing the accessions, Dr.

Gordon Prain (CIP) for enabling the collaboration between CIP

and McGill University, and Mr. Federico Diaz, M.Sc. (CIP) for per-

forming the field trial in Peru.

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0.00

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