Journal of Functional Foods 46 (2018) 57–65

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Release of phenolics compounds from Rubus idaeus L. dried fruits and seeds T
during simulated in vitro digestion and their bio-activities
⁎
Yin Qina,1, Lu Wangb,1, Yufeng Liub, Qunying Zhangc, Yongxia Lic, Zhenqiang Wub,
a
School of Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang 550003, PR China
b
School of Biology and Biological Engineering, Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology,
Guangzhou 510006, PR China
c
Guizhou Guishanhong Agricultural Development Co. Ltd., Guiyang 550003, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: To evaluate the bio-availability of raspberries (Rubus idaeus L.), raspberry dried fruits (RDF) and seeds (RDS)
Rubus idaeus L. were subjected to in vitro simulated digestion. In total, 2158.6 mg and 1904.7 mg GAE/100 g DM of phenolics
Phenolics compounds could be released from RDF and RDS, respectively, after four in vitro digestion stages, including oral, gastric,
Simulated in vitro digestion small-intestine and large-intestine digestion. The phenolics were mostly released in the gastric digestion stage for
Antioxidant activity
RDF and in the small-intestine digestion stage for RDS. The results include an analysis of ten major phenolic
α-Glucosidase inhibitory activity
compounds, which showed that ellagic acid pentoside was the major compound released from RDF and RDS
during gastric digestion, whereas ellagic acid was the primary compound released in small-intestine digestion. A
significantly positive correlation existed between the observed bio-activities and the total phenolics/flavonoids
released. Consequently, both raspberry fruits and seeds can be considered as raw materials of functional food
enriched in natural antioxidants.

1. Introduction
Plant fruits, as crucial sources of basic nutrients and phytochemicals
(especially of phenolics and flavonoids), play important roles in the
promotion of human health. Evidence has confirmed that the long-term
intake of phenolics from fruits and vegetables is conducive to the pre-
vention of some chronic diseases, such as reactive oxygen species (ROS)
damage, diabetes, lung diseases, heart diseases, and DNA mutations
(Albishi, John, Al-Khalifa, & Shahidi, 2013; Ismail, Sestili, & Akhtar,
2012; Wang, Huang, Shao, Qian, & Xu, 2012).
Raspberries (Rubus idaeus L.), a member of the family Rosaceae, are
widely distributed in Europe, Asia, and North America (Määttä-
Riihinen, Kamal-Eldin, & Törrönen, 2004; Bobinaitė, Viškelis, &
Venskutonis, 2012). According to previous studies, phenolic com-
pounds exist in the extracts of raspberry species and possess relatively
strong bio-activity, as measured by various antioxidant, anti-diabetes,
and anti-cancer cell lines tests performed in vitro (Bobinaitė, et al.,
2012; Liu, et al., 2002; Wang, & Lin, 2000; Beekwilder et al., 2005).
However, these studies might not present a true reflection of the po-
tential health benefits in the human body, which depends on many
factors, including the release capacity and digestive stability of the fruit
matrix during the human digestion process (Bobinaitė, et al., 2012;
Zafrilla, Ferreres, & Tomás-Barberán, 2001). Many reports have con-
firmed that temperature, pH and related biological enzymes have sig-
nificant effects on the stability and bio-availability of the ingested
phenolics (Hollebeeck, Borlon, Schneider, Larondelle, & Rogez, 2013;
Hur, Lim, Decker, & McClements, 2011). For example, Tagliazucchi,
Verzelloni, Bertolini, & Conte, (2010) reported that anthocyanins have
low bio-availability and stability in the alkaline conditions of the small
intestine. Stanisavljević et al. (2015) determined that some flavonoid
compounds have low release efficiencies during in vitro digestion.
Moreover, not all phenolics and antioxidants ingested can be absolutely
absorbed in the stomach and small intestine. Some of them fail to be
utilized by human body and are directly released through the large
intestine. Consequently, in order to better understand and evaluate
their potential biological properties, it is very important to investigate

Abbreviations: RDF, raspberry dried fruits; RDS, raspberry dried seeds; DM, dried mass; TPC, total phenolics content; TFC, total flavonoids contents; DPPH, 1,1-diphenyl-2-picrylhy-
drazyl; ABTS, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt; FRAP, ferric reducing/antioxidant activity; TPTZ, 2,4,6-tris(2-pyridyl)-s-triazine; Trolox, 6-hydroxy-
2,5,7,8-etramethylchroman-2-carboxylic acid; HPLC-ESI-TOF/MS, high performance liquid chromatography coupled to electrospray ionization-time of flight-mass spectrometry; GIA, α-
glucosidase inhibitory activity; S-intestine, small-intestine; L-intestine, large-intestine
⁎
Corresponding author.
E-mail address: btzhqwu@scut.edu.cn (Z. Wu).
1
The authors contributed equally to this work.

https://doi.org/10.1016/j.jff.2018.04.046
Received 24 January 2018; Received in revised form 27 March 2018; Accepted 21 April 2018
Available online 26 May 2018
1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
Y. Qin et al.
the release efficiency and bio-availability of phenolics and other anti-
oxidants from fruit matrices in the human digestive tract.
Simulated in vitro digestion is a very realistic model to evaluate the
bio-activities of samples and has been widely used in the study of
structural changes, release efficiency, digestibility, and bio-availability
of food nutrients, as well as of the effects of a digestive protocol on
some phytochemicals and antioxidant activities (Papillo, Vitaglione,
Graziani, Gokmen, & Fogliano, 2014; Zheng et al., 2018). To the best of
our knowledge, there is no report on the release efficiency and bio-
availability of phytochemicals and antioxidant components from rasp-
berry fruits and seeds during in vitro digestion. This study aims to ex-
plore the bio-availability of raspberry dried fruits (RDF) and seeds
(RDS) by a simulated in vitro digestion on the release of phenolic
compounds. It may provide useful information to promote effective
utilization of fruit resources and upgrade their commercial values.
2. Materials and method
2.1. Materials and chemicals
Raspberry species were harvested from Guizhou Guishanhong
Agricultural Development Co. Ltd., Guiyang, Guizhou Province, China
(latitude 26.57 N, longitude 106.71E). The seeds were first separated
from the rinsed fresh fruits. The raspberry fruits and seeds were dried at
60 °C for 12 h in a drying oven (Shanghai Shenxian Thermostatic
Equipment DHG-9070B, Shanghai, China), then the raspberry dried
fruits (RDF) and dried seeds (RDS) were ground to powder using a
micromill and passed through a round screen (40-mesh, GB/T6003.1,
Guangzhou, China). Samples were stored at −20 °C until use. Phenolic
standards, 1,1-diphenyl-2-picrylhydrazyl (DPPH, > 99.7%), 2-azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
(ABTS, > 99.7%), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ, > 99.7%), 6-
hydroxy-2,5,7,8-etramethylchroman-2-carboxylic acid (Trolox), α-
amylase (Type VI-B, ≥13 U/mg), pepsin (≥250 U/mg solid, from
porcine gastric mucosa), pancreatin (4 × USP, from porcine pancreas),
bile salts and viscozyme L (4 × USP), α-glucosidase (EC 3.2.1.20), and
p-Nitrophenyl-α-D-glucopyranoside were all purchased from Sigma-
Aldrich (St. Louis, MO, USA). Chromatographic-grade formic acid and
acetonitrile were purchased from Fisher Scientific (Waltham, MA,
USA). Analytical-grade absolute ethyl alcohol, methanol, ethanol,
FeCl3, Folin-Ciocalteu phenol reagent (> 99.8%), potassium persulfate
(K2S2O8, > 99.8%), Na2CO3, potassium hydroxide (KOH), acetic acid
and hydrochloric acid (HCl) were obtained from Guangzhou Reagent
Co. (Guangzhou, China).
2.2. Simulated in vitro digestion
The in vitro simulated digestion procedure was performed based on
a previously reported method with slight modifications (Papillo et al.,
2014; Pinacho, Cavero, Astiasarán, Ansorena, & Calvo, 2015). As de-
scribed in Fig. 1, the in vitro simulated digestion process included four
key stages: oral, gastric, small-intestine and large-intestine digestion. In
the first stage, which was performed in triplicate, 2 g of RDF or RDS
powder was suspended in 20 mL of distilled water (adjust pH to 6.5
using citric acid), then 0.5 mL of α-amylase solution (13 U/mL solution
in 1 mM CaCl2) was added. The mixture was incubated for 10 min be-
fore centrifugation. The residue was resuspended in 20 mL of distilled
water for the gastric digestion stage, and the suspension was adjusted to
pH 2.0 with 6 M HCl before the addition of 105.6 mg of pepsin. The
mixture was then incubated for 1 h and centrifuged. Next, for the si-
mulated small intestine digestion, 18 mL of distilled water was added to
the residue, and the pH was adjusted to 7.4 using 2 M NaHCO3 solution,
followed by the addition of 1 mL of pancreatin (10 mg/mL, dissolved in
0.9 M NaHCO3) and 1 mL bile salts (65 mg/mL, dissolved in 1 M
NaHCO3). Then, the mixture was incubated for 2 h before centrifuga-
tion. For large intestine digestion, the residue was resuspended in
58
Journal of Functional Foods 46 (2018) 57–65
20 mL of distilled water, and 6 M HCl was used to adjust the pH to 4.0.
Viscozyme L (80 μL) was then added and incubated for 16 h before
centrifugation. All of the simulated in vitro digestion procedures above
mentioned were performed in a shaking (180 rpm) water bath at 37 °C.
After each digestion step, the soluble fractions (supernatants) from
centrifugation at 10,000g for 5 min were collected, and evaporated at
45 °C for 5 min, then reconstituted with distilled water to 20 mL and
stored at -20 °C until analysis. The residues were then transferred to the
following digestion step.
2.3. Determination of total phenolics and flavonoids
The total phenolic content was determined according to the method
proposed by Viacava, Roura, and Agüero (2015), with modifications.
Briefly, the reaction system (0.18 mL) containing 0.03 mL of Folin-
Ciocalteu reagent, 0.15 mL of 20% Na2CO3 and 0.1 mL of the soluble
fractions from the above digestion steps or a gallic acid standard so-
lution was incubated at 25 °C for 20 min. Absorbance was measured
using a SpectraMax Gemini microtiter plate reader (Molecular Devices,
Sunnyvale, CA) at a wavelength of 760 nm (A760) with purified water as
the blank. A standard curve was plotted using gallic acid (10–100 μg/
mL) as a standard (R2 = 0.9996). The phenolic contents were all ex-
pressed as mg gallic acid equivalents (GAE) /g sample DM.
The flavonoid content was determined using a previously described
method, with slight modification (Wang, Luo, Wu, & Wu, 2018).
Briefly, 0.1 mL of the soluble fractions from the above digestion steps
was placed in a 2-mL Eppendorf tube. A 70% ethanol solution was
added to make a 0.5 mL solution, to which 30 μL of 5% NaNO2 solution
(w/v, Tianjin, China) was added. After being kept at room temperature
for 5 min, 30 μL of 10% AlCl3 solution was added, and the mixture was
allowed to stand for another 6 min. Then, a total of 0.2 mL of 1 M NaOH
was added, and the total volume was diluted to 1 mL using a 70%
ethanol solution. The solution was thoroughly mixed again and allowed
to stand for 30 min at 35 °C. The absorbance was read at 510 nm using
the 70% ethanol solution as a blank. The flavonoid contents were de-
termined as rutin equivalents based on the standard calibration curves
and expressed as milligrams of rutin equivalents/g of fruit or seed DM
(mg RE/g DM). The rutin calibration curves were prepared with a
standard chemical content between 0.01 and 0.1 mg/mL
(R2 = 0.9997). All reported values are presented as the mean va-
lues ± standard deviation (SD).
2.4. Identification and quantification of major phenolic compounds
The major phenolic compounds in the soluble fractions from the
digestion process were identified and quantified by a HPLC-ESI-TOF/
MS method. The HPLC-ESI-TOF/MS analysis was conducted using an
Agilent 1200 HPLC system equipped with an Aglient Zorbax Eclipse C18
plus column (250 mm × 4.6 mm, 5 μm, Aligent, USA), as well as a
diode array detector (DAD, Aglient) and an ultra-high-resolution
microTOF-QII mass spectrometer with a 40,000 FWHM mass resolution
(maXis, Bruker, Billerica, MA, USA). A binary solvent system consisting
of formic acid solution (0.1%, v/v) as solution A and acetonitrile solu-
tion as solution B was used to separate the phenolic compounds.
Samples were eluted at a flow rate of 0.8 mL/min with a time-course
increasing gradient of solution B to 15% B, for 0–5 min; 15–20% B, for
5–10 min; 20–25% B, for 10–20 min; 25–35% B, for 20–30 min;
35–50% B, for 30–40 min; 80% B, for 40–50 min; and 15% B, for
50–55 min. The temperature of the column was maintained at 30 °C.
The peaks were monitored by wavelength scanning between 200 and
550 nm. Mass spectra in the positive- or negative-ion mode were re-
corded within 60 min. The abundance of ions at m/z 100 to 1000 at a
dwell time of 500 ms was recorded under a selected ion-monitoring
mode. The operation conditions were a drying gas (N2) flow of 6.0 L/
min, an electrospray voltage of 4 kV, and a vaporizer temperature and
voltage of 350 °C and ± 40 V. Peak identification was performed by
Y. Qin et al.
Fig. 1. Graphic representation of a simulated in vitro digestion procedure carried out on raspberry dried fruits (RDF) or dried seeds (RDS).
comparing the mass spectra and fragmentation ions combined with the
retention time, UV–vis spectra, and the chromatography reference data
from authentic standards (Liu et al., 2002; Bobinaitė, et al., 2012). In
the TOF/MS analysis, the mass error tolerance was set as 4 ppm, re-
presenting the systematic error in the measurement. The concentration
of each analyte was calculated based on its corresponding standard
curve, and the content of each analyte was expressed as milligrams per
100 g DM of samples.
2.5. Measurement of antioxidant activities
2.5.1. ABTS radical cation scavenging activity
The scavenging activity of the ABTS radical cation (ABTS+) was
determined according to the method by Wang, Wei, Tian, Shi, and Wu
(2016), with slight adjustments. Briefly, fifty microliters of the soluble
fraction dilutions from the above digestion steps or Trolox was added to
400 μL of the freshly diluted ABTS+ solution (to an absorbance of
0.70 ± 0.02 at 734 nm) and thoroughly mixed. The reaction mixture
was kept at room temperature in the dark for 30 min, and the absor-
bance at 734 nm was subsequently recorded. The ABTS+ scavenging
activity values were expressed as μmol Trolox equivalents (TE)/g
sample in DM and were derived from a standard curve.
2.5.2. DPPH radical scavenging activity
The DPPH radical scavenging activity of sample extractions ob-
tained during the different phases of in vitro digestion was analyzed
according to the methodology described by Brand-Williams, Cuvelier,
and Berset (1995) using the stable radical DPPH. The working solution
contained 50-μL of the soluble fractions dilutions from above digestion
steps and 400 μL of a DPPH-methanol solution (100 μM). The reaction
was performed at 25 °C for 30 min. Then, the absorbance was measured
59
Journal of Functional Foods 46 (2018) 57–65
at a wavelength of 517 nm (A517). The results were expressed in μmol
Trolox equivalents (TE)/g sample in DM and were derived from a
standard curve.
2.5.3. OH− radical scavenging activity
The scavenging activity of OH− radicals in the sample extractions
obtained during the different phases of in vitro digestion was analyzed
according to the methodology described by Liu et al., 2017. The
working solution contained 100-μL of the soluble fractions dilutions
from the above digestion steps, a 100-μL FeSO4 solution (6 mM) and
100-μL of H2O2 (2.4 mM). The reaction was performed at 25 °C for
30 min. The absorbance was measured at a wavelength of 510 nm
(A510). The results were expressed in μmol Trolox equivalents (TE)/g
sample in DM and were derived from a standard curve.
2.5.4. Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was performed according to the method reported by
Wong, Li, Cheng, and Chen (2006). The fresh FRAP reagent was pre-
pared using 50 mL of 0.3 M acetate buffer (5.1 g of sodium acetate in
20 mL of acetic acid, pH 3.6), 5 mL of 20 mM ferric chloride solution
and 5 mL of TPTZ solution (10 mM TPTZ in 40 mM hydrochloric acid),
which was warmed to 35 °C before use. The above extract was diluted
with distilled water, and then 30 μL of the soluble fractions dilutions
from the above digestion steps or Trolox was mixed with 900 μL of
FRAP reagent (freshly prepared). After incubation in the dark at room
temperature for 30 min, the absorbance of the reaction mixture was
read at 593 nm. The FRAP values, expressed in mmol ferrous sulfate
equivalents (Fe(II)SE)/g sample in DM, were derived from a standard
curve.
Y. Qin et al. Journal of Functional Foods 46 (2018) 57–65

Bb
2.6. α-Glucosidase inhibitory activity analysis A 900 RDF
Bc RDS
800

TPC (mg GAE/100 g DM)

α-Glucosidase inhibitory activity was measured as previously de- Ab
scribed (Kim, Jeong, Wang, Lee, & Rhee, 2005) with modifications. 700
Briefly, a mixture containing 50 μL of phosphate buffer (0.1 M, pH 6.8), 600
50 μL of α-glucosidase (1 U/mL), and 50 μL of the soluble fractions Ba Aa
500 Ba
dilutions from above digestion steps (0.05, 0.2, 0.5, 1.0, 2.0, and 4.0 mg
DM/mL) was incubated at 37 °C for 10 min. Next, 100 μL of p-ni- 400 Aa Bc
Aa
trophenyl-α-D-glucopyranoside solution (5 mM) was added and the 300
incubation was continued for another 20 min. The reaction was termi-
200
nated by adding 500 μL of Na2CO3 solution (1 M), and the absorbance Ad
at 405 nm (A405) was measured. The IC50, which was defined as the 100
amount of sample required to inhibit 50% of the enzyme activity, was 0
d l ic ne e
used to represent the inhibition efficiency of the extracts against α- st e Ora st r st i st in
d i ge Ga nt e nte
glucosidase. The α-glucosidase inhibitory activity was calculated as Un S-i L- i
shown in Eq. (1), In vitro digestion

A −Ad ⎤
α−Glucosidase inhibitory activity (%) = ⎡1− c × 100 B 600
⎢
⎣ Aa −Ab ⎥
⎦ (1) Bb RDF
500 RDS

TFC (mg RE/100 g DM)

Bb
where Aa, Ab, Ac, and Ad represent the A405 of the enzyme control
(containing buffer and α-glucosidase), blank (containing buffer only), Ba
400 Ba Aa
reaction mixtures (containing the soluble fractions dilutions, buffer, Ab
and α-glucosidase), and extract control (containing the soluble fractions Aa
300 Aa Ba
dilutions and buffer), respectively.

200
2.7. Statistical analysis

All of the experiments were conducted three times, and the data are 100 Ac
expressed as the mean ± the standard deviation. The data were ana-
lyzed using Statistic 7.1 (StatSoft, Tulsa, OK). One-way analyses of 0 l e e
ed Ora ic
stin
est Ga
str stin
variance (ANOVA) was used to evaluate the significance of the differ-
ndig - i nte - i nte
U S L
ences among the mean values of test levels. Differences with a p
value < 0.05 and a p value < 0.01 were considered to be significant
In vitro digestion
and highly significant, respectively. Fig. 2. The total released phenolic content (A) and flavonoid content (B) from
raspberry dried fruits (RDF) or raspberry dried seeds (RDS) after in vitro di-
3. Results and discussion gestion. Undigested samples were extracted using water. TP, total phenolics
content; TF, total flavonoids content. Different lowercase letters mean statisti-
cally significant differences between the un-digested samples and the digested
3.1. Release of total phenolics and flavonoids during in vitro simulated
samples at different phase for RDF or RDS; Different uppercase letters mean
digestion statistically significant differences between RDF and RDS samples at un-di-
gested or digested phase.
Not all phenolics can be identified and quantified; therefore, the
Folin-Ciocalteu assay is still the most commonly used procedure to
improved the release of total phenolics and total flavonoids, which
determine the total phenolic compounds in food or tea extracts
were enhanced 4.62- and 4.37-fold for RDF, as well as 5.94- and 3.30-
(Cassani, Gerbino, del Rosario Moreira, & Gómez-Zavaglia, 2018;
fold for RDS, respectively. Compared to the chemical extraction (70%
Rahman, de Camargo, & Shahidi, 2018). In the present work, a simu-
ethanol), in vitro digestion produces a 68.3% and 71.6% release of the
lated in vitro digestion model was used to evaluate the amounts of
sum of total phenolics, as well as a 77.2% and 80.4% release of the sum
health-related components released from RDF or RDS into the gastro-
of total flavonoids, for RDF and RDS, respectively.
intestinal tract during digestion. Fig. 2AB displays the released amount
After simulated oral digestion, 459.8 mg GAE/100 g DM and
of total phenolics and total flavonoids before (undigested) and after a
330.24 mg GAE/100 g DM of total phenolics, 280.98 mg RE/100 g DM
simulated in vitro digestion. It was found that the phenolic and flavo-
and 369.59 mg RE/100 g DM of total flavonoids from RDF and RDS
noid compounds of RDF and RDS were significantly released during
were released, respectively. For RDF and RDS, it was found that only
simulated in vitro digestion. The sum of total phenolics obtained in the
20.88% and 17.34% of the total phenolics and 20.60% and 30.12% of
individual digestion phases were 2158.6 mg GAE/100 g DM for RDF
the total flavonoids were released, respectively. Zheng et al. (2018)
and 1904.7 mg GAE/100 g DM for RDS, respectively. The sum of fla-
reported that more than 65% of the total phenolics and flavonoids in
vonoids contents obtained in the individual digestion phases were
Chinese hawthorn were released during simulated oral digestion, which
1364.2 mg RE/100 g DM for RDF and 1223.6 mg RE/100 g DM for RDS.
was not consistent with the present research. However, some studies
The results also showed that the concentrations of total phenolics from
have confirmed that only a small amount of phenolic compounds was
undigested RDF and RDS extracted by water were only 467.3 mg GAE/
released from Arbutus unedo, pomegranate and blackthorn fruit during
100 g DM and 320.4 mg GAE/100 g DM, and the total flavonoids con-
simulated oral digestion (Mosele, Macià, Romero, Motilva, & Rubió,
tents were 310.3 mg RE/100 g DM and 370.35 mg RE/100 g DM. For
2015; Mosele, Macià, Romero, & Motilva, 2016; Pinacho et al., 2015).
undigested RDF and RDS extracted using 70% ethanol, the total phe-
The differences in the released amounts of these active ingredients may
nolics released reached up to 3158.7 mg GAE/100 g DM and 2659.2 mg
be due to the different bioactive compositions and properties among
GAE/100 g DM, respectively. The total flavonoid contents were
varied fruits.
1767.3 mg RE/100 g DM and 1522.5 mg RE/100 g DM, respectively.
After simulated gastric digestion, the total phenolics released were
Therefore, compared to the water extraction, in vitro digestion

60
Y. Qin et al. Journal of Functional Foods 46 (2018) 57–65

886.40 mg GAE/100 g DM from RDF and 678.68 mg GAE/100 g DM

Standard
Standard

Standard

Standard
Standard
Standard
Standard
Standard
from RDS, while the total flavonoids released were 502.50 mg RE/100 g
DM from RDF and 334.66 mg RE/100 g DM from RDS. For RCF, near

Reference

MS/MS,
MS/MS,

MS/MS,

MS/MS,
MS/MS,
MS/MS,
MS/MS,
MS/MS,
MS/MS

MS/MS
41.06% of the total phenolics were released, and 36.84% of the total
flavonoids were released. In both RDF and RDS, the amounts of total
released phenolics and flavonoids during simulated gastric digestion
were significantly higher than those during simulated oral digestion.

Kaempferol-galactoside-glucoside
Moreover, during the simulated gastric digestion, the highest amount of
total phenolics and flavonoids were released from RDF. These results

Quercetin-7-O-glucuronide
were consistent with the previously reported results (Gumienna, Lasik,

Ellagic acid pentoside

Quercetin 3-glucoside
& Czarnecki, 2011). The low pH and the pepsin action in the gastric
digestion were expected to release some phenolic compounds bound to

Chlorogenic acid
Procyanidins B1
carbohydrates, thus lead these bioactive compounds more bioacces-

Compounds

Ellagic acid
Gallic acid

Avicularin
sible. However, for the RDS, the highest amount of total phenolics and
total flavonoids were released in the stage of simulated small intestine

Rutin
digestion. Bohn et al. (2015) have reported many phenolics compounds
may be released during the small intestine digestion. During simulated
large intestine digestion, there was still some release of phenolics and

Error (ppm)
flavonoids for both RDF and RDS. It may be due to that some enzymes
produced by gut microbes commonly existed in the large intestine could

−0.7

−2.5
−2.5
−0.3
−0.7
−0.2
1.2
0.2
0.3

0.2
further cause the release of non-extractable polyphenols (Cassani et al.,
2018).

C45H38O18
C19H14O12
C27H30O16
C27H30O16

C21H20O12
C20H18O11
C21H18O13
C16H17O9

C14H6O8
Formula
3.2. Differences between major phenolic compounds released during in vitro

C7H6O5
digestion

Ten phenolic compounds from RDF and RDS were identified by

Identification of phenolics compositions from RDF and RDS during simulated in vitro digestion using HPLC-ESI-TOF/MS method.

170
354
866
434
610
610
302
464
434
478
Mw
comparing their m/z fragmentation patterns of quasi-molecular ions,
HPLC retention times or their standard chromatographic data (Table 1
and Fig. 3). To investigate the changes in the compositions and contents

435.0557, 287.0563
of the soluble phenolics fractions released from RDF and RDS during in
vitro digestion, the eight main compounds, including gallic acid,
chlorogenic acid, ellagic acid pentoside, ellagic acid, rutin, quercetin-3-
185.0085
185.0085

303.0563

219.1754
98.9212

O-glucoside, avicularin, and quercetin-7-O-glucuronide were quantified

(Table 2 and Fig. 3AB). Compared with undigested samples extracted
by water, a total of eight individual phenolics were identified as re-
191.0121,
291.0155,
303.0142,
449.1254,
465.1081,

303.0500,
193.1221
303.0506

303.0506
leased in small amounts during the simulated oral digestion of RDF and
RDS. However, during simulated gastric digestion, the compounds el-
lagic acid pentoside and rutin were released in significant amounts
353.2410,
579.1502,
435.0564,
611.1254,
611.2732,
303.0101,
465.1037,
435.0924,
479.0829,
169.1221
MS (m/z)

compared with the oral digestion or un-digested (water). The highest

amount of ellagic acid pentoside released reached up to 125.3 mg/100 g
DM for RDF and 32.9 mg/100 g DM for RDS. Moreover, the con-
centration of avicularin, during gastric digestion, is higher in compar-
Molecular ion (m/z)

[M+H]−
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+

ison with those released during oral digestion or un-digested (water). In

[M−H]

the simulated small intestine digestion, ellagic acid and rutin were
massively released, and the highest amount of ellagic acid released was
169.2101
353.2410
579.1502
435.0564
611.1607
611.2732
303.0101
465.1037
435.0924
479.0827

201.2 mg/100 g DM for RDF and 347.7 mg/100 g DM for RDS. Gil-
Izquierdo, Zafrilla, & Tomás-Barberán, (2002) also reported the con-
tents of ellagic acid and some individual flavonoids in strawberry were
significantly increased during the simulated small intestine digestion.
254, 358

After 16 h of simulated large intestine digestion of RDF, some phenolic

λmax (nm)

compounds including ellagic acid and quercetin-7-O-glucuronide were

271
268
352

354
354
351
356
391
353

still released in a high concentration. The others compounds including

215,
210,
254,
204,
254,
254,
256,
254,
262,
253,

gallic acid, quercetin 3-glucoside and avicularin also appear, although

at lower concentrations. Bouayed, Hoffmann, and Bohn (2011) have
reported the effect of intestinal digestive enzyme on the complex food
Retention time (min)

matrix, facilitating the release of flavonoids bound to the matrix after

small intestinal digestion. However, for this stage of digestion of RDS,
only quercetin-7-O-glucuronide and low levels of ellagic acid and gallic
acid were detected in the obtained soluble fractions. Compared with the
13.13
14.25
15.13
17.81
19.76
20.12
24.72
4.23
5.89
7.98

undigested samples extracted using water, simulated gastric digestion

contributed to a 6.74-fold (RDF) and 15.4-fold (RDS) increase in the
release of total ellagic acid pentoside, whereas simulated intestinal di-
Peak No.

gestion contributed to a 34.69-fold (RDF) and 6.15-fold (RDS) increase

Table 1

in the release of total ellagic acid. Compared with the undigested

10
1
2
3
4
5
6
7
8
9

samples extracted using 70% ethanol, simulated gastric digestion

61
Y. Qin et al. Journal of Functional Foods 46 (2018) 57–65

A 1 4 6

21.5 ± 2.4b

58.9 ± 3.5a
12.9 ± 0.2c
L-intestine
8 Standard phenolics
7
2

N.D.
N.D.
N.D.

N.D.
N.D.
9
10

347.7 ± 6.7i
17.8 ± 1.3b
54.3 ± 2.7c

25.6 ± 1.2c

9.8 ± 0.2e
0 4 8 12 16 20 24 28

S-intestine
Time (min)

N.D.

N.D.
N.D.
1 4 7 RDF
10

63.2 ± 3.1d

37.8 ± 1.7d
13.4 ± 1.5a
32.9 ± 1.9c
7.7 ± 0.1a

5.9 ± 0.1c
B

Gastric

N.D.
N.D.
5 89 L-intestine
7
1

81.3 ± 4.5e

47.3 ± 3.1e
2 5 6

9.8 ± 0.8a

3.7 ± 0.1a
89 S-intestine
1 2 5
3 6

N.D.
N.D.

N.D.
N.D.
Oral
1 2 7
4 8 Gastric
3 5 6
1 7 8
2 Oral
3 4
5 6 8 Un-digested
7

10.4 ± 1.2a
76.3 ± 7.1e

56.5 ± 1.7f
Un-digested

2.1 ± 0.3a

3.3 ± 0.2a
0 4 8 12 16 20 24 28

(water)
Time (min)

N.D.

N.D.
N.D.
1 7 RDS

RDS (mg/100 g DM)

The concentrations of the major phenolics compounds of the soluble fractions obtained from simulated in vitro digestion in RDF and RDS.

Un-digested (70%
10

335.5 ± 3.2 h
C

513.5 ± 5.6j
31.4 ± 2.7d
41.2 ± 1.8d
49.5 ± 2.3d

61.5 ± 3.1a
25.4 ± 1.7c
25.4 ± 0.3f
L-intestine

ethanol)
7
1 2 5 6
8 S-intestine
4 5

82.5 ± 2.8 g
1

91.7 ± 2.8b
13.3 ± 0.6a
6 7

3.9 ± 0.1a
5.3 ± 0.1a
8
L-intestine

Gastric
1 2
5 7 Oral
8 N.D.
N.D.
N.D.
1 2 7
5 8 Un-digested
201.2 ± 3.9 h
67.3 ± 4.1d
16.5 ± 1.2b

4.78 ± 0.1b
14.3 ± 1.1b
23.3 ± 1.7c
0 4 8 12 16 20 24 28
S-intestine

Time (min)
N.D.

Fig. 3. HPLC chromatogram of a standard phenolics mixture (A) and the so- N.D.
luble fractions obtained from raspberry dried fruits (B) or dried seeds (C) after
125.3 ± 3.9e
19.8 ± 1.7b

18.2 ± 1.5b

13.3 ± 0.7b

in vitro digestion (280 nm). Peaks: 1, gallic acid; 2, chlorogenic acid; 4, ellagic
97.3 ± 2.6f

Different lowercase letters (a–i) in the same line mean significant difference (P < 0.05).
7.3 ± 1.1b
4.6 ± 0.2b

acid pentoside; 6, rutin; 7, ellagic acid; 8, quercetin 3-glucoside; 9, avicularin;

Gastric

10, quercetin-7-O-glucuronide. Undigested samples were extracted using water.

N.D.
141.2 ± 3.7 g

contributed to a 73.1% (RDF) and 79.9% (RDS) increase in the release

19.7 ± 2.7b
12.5 ± 1.1a
21.5 ± 1.4c

6.1 ± 0.1a
9.4 ± 0.3e

of total ellagic acid pentoside, and simulated intestinal digestion con-

tributed to a 63.7% (RDF) and 67.7% (RDS) increased release of total
N.D.
N.D.
Oral

ellagic acid. The results confirmed that the enzymes produced by gut
microbes have significant impacts on the release of phenolic com-
pounds from RDF or RDS during the intestinal digestion.
132.4 ± 5.2 g

18.6 ± 2.5b
10.5 ± 4.7a
23.6 ± 1.8c
Un-digested

7.9 ± 0.8d
5.8 ± 3.5a
(water)

3.3. Antioxidant activities

N.D.
N.D.

To completely investigate the antioxidant capacity of the digested

RDF (mg/100 g DM)

soluble fractions from RDF and RDS, four anti-oxidant models, in-
Un-digested (70%

−
cluding the scavenging activity of ABTS+ , DPPH and OH free radical
171.3 ± 1.5f
414.5 ± 4.5i

315.7 ± 2.6i
35.8 ± 1.3 g
54.5 ± 1.5d
95.4 ± 1.7b
73.2 ± 2.7e

55.4 ± 3.2e

and the ferric reducing/antioxidant activity. As seen from Fig. 4A–D, on

the whole, the released soluble fractions from RDF possessed sig-
ethanol)

nificantly higher antioxidant activities than those from RDS. In detail,

in comparison of RDF fractions after simulated oral digestion with non-
−
digested fractions extracted by water, the ABTS+ , DPPH and OH va-
Ellagic acid pentoside

Quercetin 3-glucoside

lues and FRAP were slightly changed, measuring only 23.4 μmol TE/g
Chlorogenic acid

glucuronide

DM, 18.4 μmol TE/g DM, 8.7 μmol TE/g DM, and 75.5 mM Fe(II)SE/g
Quercetin-7-O-

DM, respectively. After simulated gastric digestion, the ABTS+

Ellagic acid

, DPPH
Gallic acid

Avicularin
Analytes

and OH free radical and FRAP of the soluble fractions were the highest
Table 2

Rutin

detected levels, measuring at 40.1 μmol TE/g DM, 37.6 μmol TE/g DM,
17.4 μmol TE/g DM, and 166.7 mM Fe(II)SE/g DM. After simulated

62
Y. Qin et al. Journal of Functional Foods 46 (2018) 57–65

A B
45 40 Bb
Bb RDF
ABTS radical scavenging activity
RDF

DPPH radical scavenging activity

40 RDS 35 RDS
Bc
35
Ab 30 Ab
(μmol TE/g DM)

(μmol TE/g DM)

30 Ab Ab
Ba 25
Ba Ba Aa
25 Aa Aa Aa Ba
20 Aa Aa
20
Ac
15
15
Ac 10
10 Ad

5 5

0 l 0
tric l
est
ed Ora tine tine est
ed Ora tric tine tine
dig Gas tes tes dig Gas tes tes
Un- S-in L-in Un- S-in L-in
In vitro digestion In vitro digestion

C D
20 180
RDF Bb
RDF
OH radical scavenging activity

Bb
18

Ferric antioxidant/reducing power

RDS 160 RDS
16 140
(μM TE/g DM)

14
120
Ab
12 Aa
100 Bc
10 Ba Ab
Ba Ba Aa
80 Ab
8 Aa Aa Ba
Aa Aa
60 Ab
6
Ac 40 Ad
4 Ac

2 20

0 0
l tric
l
tric
est
ed Ora tine tine est
ed Ora
Gas
tine tine
dig Gas tes tes dig tes tes
Un- S-in L-in Un- S-in L-in
In vitro digestion In vitro digestion

Fig. 4. The DPPH (A), ABTS+ (B), OH− (C) radical scavenging activity and ferric reducing/antioxidant power (D) of the soluble fractions obtained from raspberry
dried fruits (RDF) or dried seeds (RDS) after in vitro digestion. Different lowercase letters mean statistically significant differences between the un-digested samples
and the digested samples at different phase for RDF or RDS; Different uppercase letters mean statistically significant differences between RDF and RDS samples at un-
digested or digested phase.

small intestine digestion, the ABTS+ , DPPH and OH free radical and
FRAP of the soluble fractions significantly decreased, measuring only
27.0 μmol TE/g DM, 9.9 μmol TE/g DM, 17.2 μmol TE/g DM, and
50.2 mM Fe(II)SE/g DM, respectively.
For the RDS fractions after simulated oral digestion compared with
−
non-digested fractions extracted by water, the ABTS+ , DPPH and OH
values and FRAP were slightly changed, measuring only 20.4 μmol TE/g
DM, 16.8 μmol TE/g DM, 5.8 μmol TE/g DM, and 57.9 mM Fe(II)SE/g
DM, respectively. After simulated gastric digestion, the ABTS+
, DPPH
and OH free radical and FRAP of soluble fractions were all at the
highest detected levels, measuring at 27.1 μmol TE/g DM, 26.1 μmol
TE/g DM, 8.1 μmol TE/g DM, and 70.0 mM Fe(II)SE/g DM. After si-
mulated small intestine digestion, the ABTS+
, DPPH and OH free radical
and FRAP of the soluble fractions significantly decreased, measuring
only 28.3 μmol TE/g DM, 29.3 μmol TE/g DM, 10.1 μmol TE/g DM, and
81.1 mM Fe(II)SE/g DM, respectively.
The results showed that the antioxidant components of RDF and
RDS were mainly released during simulated gastric and small intestine
digestion, with a small release occurring during simulated oral and
large intestine digestion. In contrast, in the apple or hawthorns fruit,
antioxidant compounds were found to be mainly released during sti-
mulated oral and gastric digestion (Papillo et al., 2014; Zheng et al.,
2018). This may be because phenolic compounds exist in different
forms (free, conjugate, and bound forms) in a variety of food materials.
Sun, Chu, Wu, & Liu, (2002) reported that most phenolics in fruits are
in soluble-free form, such as in apples, hawthorns fruit and red grapes,
while plant seeds often contain relatively abundant insoluble-bound
phenolics, providing them with the potential to exert antioxidant ac-
tivity during the intestinal digestion process (Ayoub, de Camargo, &
Shahidi, 2016; Singh, Negi, & Radha, 2013).
3.4. α-Glucosidase inhibitory activity
Many different medicines are available to manage diabetes, but
some side effects are generated from their application. Some alternative
α-glucosidase inhibitors with fewer side effects have been suggested.
Phenolic compounds from some fruits or vegetables have been con-
firmed to have inhibitory activity against α-glucosidase, which is a key
enzyme regulating the absorption of glucose in the small intestine
(Nyambe-Silavwe et al., 2015; Ranilla, Kwon, Apostolidis, & Shetty,
2010). Raspberry fruits have been reported to possess a hypoglycemic
effect, as measured by an in vitro α-glucosidase inhibitory activity assay
(Kirakosyan, Seymour, Gutierrez, & Bolling, 2017; Yang, Xie, Jiang, &
Wei, 2016), but this finding fails to reflect the true conditions of their α-
glucosidase inhibitory activity in the human body. The results are
shown in Fig. 5. The released soluble fractions from RDF possessed
significantly higher α-glucosidase inhibitory activity than did those
from RDS, which may be because the phenolics content of RDF was

63
Y. Qin et al.
3.5 Bc
RDF Bc
RDS
IC50 values (mg DM/mL)
3.0
Acarbose
Bb Bb
2.5 Ad
2.0
Bc
1.5 Ab
Ab
1.0
Aa
0.5 Aa
0.0 l
est
ed Ora tric tine tine
-dig Gas -intes -intes
Un S L
In vitro digestion
Fig. 5. The α-glucosidase inhibitory activity of the soluble fractions obtained
from raspberry dried fruits (RDF) or dried seeds (RDS) after in vitro digestion
and of the positive control, acarbose. Different lowercase letters mean statisti-
cally significant differences between the un-digested samples and the digested
samples at different phase for RDF or RDS and acarbose; Different uppercase
letters mean statistically significant differences between RDF and RDS samples
at un-digested or digested phase.
significantly higher than that of RDS. In detail, for fractions from RDF
after simulated oral digestion compared with non-digested fractions
extracted by water (IC50 = 0.98 mg DM/mL), the α-glucosidase in-
hibitory activity of or RDF changed slightly (IC50 = 1.09 mg DM/mL).
After simulated gastric digestion, the IC50 values were all at the lowest
measured levels at only 0.15 mg DM/mL, which represented the highest
observed α-glucosidase inhibitory activity. After simulated small or
large intestine digestion, the α-glucosidase inhibitory activity of the
soluble fractions significantly decreased, with IC50 values of 1.39 mg
DM/mL and 1.87 mg DM/mL, respectively.
For fractions from RDS after simulated oral digestion compared with
non-digested fractions extracted by water (IC50 = 2.07 mg DM/mL), the
α-glucosidase inhibitory activity of the soluble fractions changed
slightly, with an IC50 value of 2.17 mg DM/mL. After simulated gastric
digestion, the IC50 values were all the highest measured levels at only
3.15 mg DM/mL, which represented the lowest α-glucosidase inhibitory
activity. However, after simulated small intestine digestion, the α-glu-
cosidase inhibitory activity of the soluble fractions was at the strongest
level measured (IC50 = 0.53 mg DM/mL), but the IC50 value of acarbose
α-glucosidase inhibitory activity was only 2.11 mg DM/mL. These re-
sults confirmed that the phenolics released from RDF or RDS after in
vitro digestion have the potential to be used as alternative α-glucosidase
inhibitors to regulate blood glucose in the human body.
3.5. Correlation of bio-activity with the total phenolics or total flavonoids
released
A Pearson’s correlation coefficient analysis was applied to elucidate
the correlation coefficients between the release of total phenolic con-
tents or total flavonoid contents and the bio-activities of RDF and RDS
during stimulated in vitro digestion. The results of the correlation ana-
lyses are shown in Table 3. It was found that there were significant
positive correlations between the total released phenolics or flavonoids
and the bio-activities of RDF. The correlation coefficient results were as
follows: total phenolics vs DPPH radical scavenging activity (r = 0.915,
p < 0.05), total phenolics vs ABTS radical scavenging activity
(r = 0.986, p < 0.01), total phenolics vs OH radical scavenging ac-
tivity (r = 0.942, p < 0.01), total phenolics vs FRAP (r = 0.957,
p < 0.01), total phenolics vs α-glucosidase inhibitory activity
(r = 0.932, p < 0.01), total flavonoids vs DPPH radical scavenging
Journal of Functional Foods 46 (2018) 57–65
Table 3
Pearson’s correlation coefficient analysis between released bioactive composi-
tions and bio-activities from RDF and RDS during simulated in vitro digestion.
Bio-activities RDF RDS
b
TF TP TF TP
DPPH 0.894* 0.915* 0.840** 0.943**
ABTS+ 0.996** 0.986** 0.883** 0.918**
OH− 0.927* 0.942** 0.736* 0.854**
FRAP 0.929* 0.957** 0.734* 0.818**
GIA 0.891* 0.932** 0.672* 0.627*
TP, Total phenolics; TF, Total flavonoids; GIA, α-glucosidase inhibitory activity;
RDF, Raspberry dried fruits; RDS, Raspberry dried seeds
* Correlation was significant at the 0.05 level (two-tailed).
ose
Acarb ** Correlation was significant at the 0.01 level (two-tailed).
activity (r = 0.894, p < 0.05), total flavonoids vs ABTS radical
scavenging activity (r = 0.996, p < 0.01), total flavonoids vs OH ra-
dical scavenging activity (r = 0.927, p < 0.05), total flavonoids vs
FRAP (r = 0.929, p < 0.05), and total flavonoids vs α-glucosidase in-
hibitory activity (r = 0.891, p < 0.05). For RDS, a positive correlation
was also determined between the total released phenolics or flavonoids
and the bio-activities, as follows: total phenolics vs DPPH radical
scavenging activity (r = 0.943, p < 0.01), total phenolics vs ABTS ra-
dical scavenging activity (r = 0.918, p < 0.01), total phenolics vs OH
radical scavenging activity (r = 0.854, p < 0.01), total phenolics vs
FRAP (r = 0.818, p < 0.01), total phenolics vs α-glucosidase inhibitory
activity (r = 0.627, p < 0.05), total flavonoids vs DPPH radical
scavenging activity (r = 0.840, p < 0.01), total flavonoids vs ABTS
radical scavenging activity (r = 0.883, p < 0.01), total flavonoids vs
OH radical scavenging activity (r = 0.736, p < 0.05), total flavonoids
vs FRAP (r = 0.734, p < 0.05), and total flavonoids vs α-glucosidase
inhibitory activity (r = 0.672, p < 0.05).
The data demonstrated that in the simulated digestion process, there
were significantly positive correlations between the total released
phenolic or flavonoid content and the antioxidant or α-glucosidase in-
hibitory activity, especially the ability to scavenge free radicals. These
results further demonstrated that the released phenolics and flavonoids
from RDF and RDS during simulated in vitro digestions play important
roles in the bio-activities of these food sources. The results were con-
sistent with the reporting of Zheng et al. (2018). They demonstrated
that in the simulated digestion process, there are significant linear
correlations between the total phenolic or total flavonoid content re-
leased by Shanlihong and Dajinxing fruits and the related oxygen free-
radical scavenging activity.
4. Conclusions
The release of phenolics or flavonoids from the dried fruits and
seeds of raspberries and their associated bio-activities were different
during simulated in vitro digestion. The total phenolic or flavonoid
compounds released from RDF and RDS were significantly higher after
simulated in vitro digestion compared with those released from un-
digested samples. The quantitative results of ten major phenolic com-
pounds obtained by HPLC-ESI-TOF/MS showed that the most-released
compounds were ellagic acid pentoside during gastric digestion of RDF
and ellagic acid during the S-intestine digestion of RDF and RDS. The
results also verified that the released phenolics and flavonoids play
important roles in the antioxidant activities (the scavenging activities of
DPPH, ABTS+, and OH− free radical, and FRAP) and α-glucosidase
inhibitory activity of the food sources. In summary, simulated in vitro
digestion is a good method to evaluate the bioavailability of compounds
in food matrix. Importantly, both the fruits and seeds of raspberries
were found to be good sources of natural bio-active compounds.
64
Y. Qin et al.
5. Conflict of interest statement
The authors declared no conflict of interest.
Acknowledgements
The work was supported by the Youth Science and Technology
Project of Guizhou Provincial Education Department (KY[2017] 221),
Innovation Groups Major Research Projects of Guizhou Provincial
Education Department (KY[2017]046), and the Science and
Technology Project of Guangdong Province, China (2016A020210011
and 2017B020207003).
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